Oct 02 2012

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After you install SRS Net Connect 3.1, you must register for a Net Connect account to obtain the activation kit. The activation kit contains your customized customer certificate and activation script. If you already have an activation kit, you do not need to obtain another kit.

Creating a Net Connect Account

If you have already registered as an SRS Net Connect 3.1 user, you can use that username and password to get the activation kit. If you have not registered, perform the following steps:

1. If you have not registered as a Net Connect user, go to to access SRS Net Connect 3.1.

2. At the Welcome page, click Sign Up to create a new root administrator user account. A Net Connect root administrator controls the company profile, views reports, and creates, modifies, and deletes users, user groups, and system groups. More than one person can be a root administrator.

3. Read the agreement, click the check box, and click Accept.

4. At the Sign Up - Account Creation page, complete the required fields to add yourself as a root administrator and click Submit.

5. At the Sign Up Create Company page, click Create Company. To download the installation bundle and use SRS Net Connect 3.1, you must add your company.

6. At the New Company page, complete the required fields. The Company Name field must be less than 80 characters. The Short Name must be less than 20 characters; it cannot be edited later since this is how the company information is stored in the database.

7. A default system group is created using your company short name, and as you install SRS Net Connect 3.1 on new monitored systems, the systems are added to this system group. You can change this later by clicking System Grouping. The last field, Update Mode, defines how software updates are delivered to your monitored system. Select one of the following:

    • Auto - If SRS Net Connect 3.1 detects that your system needs a software update, a Non-Critical alert appears in the Software column on the Monitoring Status Summary page and the update is downloaded. If you have the Client Listener field set to Yes, the update is automatically installed. If the Client Listener is set to No, you must manually install the update by typing the following: . See the SunSM Remote Services Net Connect 3.1 Customer Operations Guide for instructions on running srsinstall in polling mode to check for updates every 20 seconds.

For both Auto and Review settings, you can get email notification about failed software updates by selecting the Net Connect Event Provider on the Notifications page in the SRS Net Connect 3.1 application. See the SunSM Remote Services Net Connect 3.1 Customer Operations Guide for instructions on email notification.

    • Review - If SRS Net Connect 3.1 detects that your system needs a software update, a Non-Critical alert appears in the Software column on the Monitoring Status Summary page. You can initiate the software download to your system by drilling down in the Software column and approving the update for delivery. If you have the Client Listener field set to Yes, the update is automatically installed. If the Client Listener is set to No, you must manually install the update.
    • Never - Software updates are not delivered to your system and a yellow icon appears when a component is out of date.

8. Click Save to display the Download SRS Net Connect page.

9. Click Welcome and then click SRS Net Connect Home to display the SRS Net Connect Home page.

 

Downloading the Activation Kit

You must have an SRS Net Connect username and password to get the activation kit. Perform the following steps to get the activation kit:

1. Log into the SRS Net Connect 3.1 application.

2. On the SRS Net Connect Home page, click Follow Solaris Net Connect Activation Guide and print it.

3. Click Download Activation Kit, as shown in FIGURE 2-1.

 

Click Download Activation Kit on the SRS Net Connect Home Page.


Note - If you did not install SRS Net Connect 3.1 from the Solaris 9 4/04 Extra Value CD, you can also get the installation package by clicking Download SRS Net Connect on the SRS Net Connect Home page.



4. If your company uses an HTTP proxy to connect to the Internet, type the URL or IP address of the HTTP proxy. At HTTP Proxy Port, type the port number it uses. Leave these fields blank if you have a direct connection to the Internet and do not use an HTTP proxy.

5. To allow software updates to be automatically installed on the monitored system, click Yes at Enable Client Update Listener. To disable software update installation, click No. The updates include new software versions and messages. If you click Yes at Client Listener, you should also set the Update Mode field in the New Company page to Auto or Review to automatically send a Non-Critical alert about an update.

6. At the Download Activation Kit section, click Download to save the compressed 53 KByte file to your monitored system. FIGURE 2-2 shows the Quick Download SRS Net Connect page.

 

The Quick Download Page Contains Two Download Buttons.

7. At the Save As dialog box, specify a directory on the monitored system where you want to perform the installation and click OK. If you put the installation file in the directory, the files are deleted if your system reboots, so the directory is a more permanent location. A configuration file and a security certificate are automatically generated for you and included in your activation kit. The configuration file is saved in the directory when the software is installed on the monitored systems. See the SunSM Remote Services Net Connect 3.1 Customer Installation Guide for details on the default configuration values included in the activation kit.

Running the Activation Kit Script

After you download the activation kit tar file, you must extract it and run the activation script on each monitored system. The activation script contains the following items:

  • Customer certificate (
  • Activation script and supporting directories ()

The activation script performs the following actions:

  • In the directory where you extract the tar file, three directories are created to store the proxy messages and core files:

1. The directory - Contains a program called to support globalization in the activation script

2. The directory - Assists the activation script with shell functions

3. The directory - Contains globalization support strings

These directories are not used after the activation is successful, and you can delete them if you wish.

  • The proxy and each provider is configured and activated.
  • The file and the files are both removed.
  • The program is run to build the SRSQueueStore space.
  • The file is updated to include an entry for the proxy process. The file is refreshed to start the process.
  • If you chose to enable automatic software upgrades, an entry is made in the file.

If you have a previous version of the Net Connect 3.x event provider installed, the activation script detects the file archive directory and copies the SRSQueueStore directory to the DISK_STORE_BASE directory. A new file called is created and the file is updated.



Note - If you have a previous version of the CST provider installed, the SUNWcstu package is installed. The CST lock file () is removed to allow CST to start on a reboot. You can start CST by typing the
# command.



Running the Script

Run the activation script by performing the following steps on each monitored system:

1. Open a terminal window and log into the monitored system as the root user.

2. Copy the file you downloaded in Downloading the Activation Kit to each monitored system that will run SRS Net Connect 3.1.

3. Change to the directory where you copied the activation kit tar file. This should be a directory that is owned and readable by the root user.

4. Uncompress and extract the activation kit tar file by typing:

#

5. Run the activation script from the directory where you uncompressed and extracted it by typing:

#

The activation script verifies the following:

    • You are logged in as a root user.
    • The monitored system is running with the required Solaris patches. Log into the SunSolveSM program at to get the latest version of a required patch.

6. If you running the script for the first time, you must provide a user account when the following text appears:

SRS Net Connect 3 executes programs as the user specified in answer to the following questions. The user and their group must already exist on the system and it is highly recommended that this user account be a locked account that denies interactive login. Type the account identifier. Note: the account must already exist. [?,q].

Type the user name and press Return. If you have a previous version of Net Connect installed, the activation script automatically detects the user name and group you used to install Net Connect.

7. After you type the user name, choose a group name when the following text appears:

  Type the group name to be used with this account identifier. Note: This group must already exist and the account identifier specified above must be a member of this group. Available groups: staffType the group name: [?,q]

If the user account has only one group associated with it, this step is skipped. Type the group name and press Return.

8. SRS Net Connect requires space to build and store messages. You must specify a directory for queue space when the following text appears:

  SRS Net Connect 3 requires space to build and store messages that are sent in response to system management monitoring events. The default file system location for this queue space is: /var This directory currently has the following characteristics: Filesystem kbytes used avail capacity Mounted on /dev/dsk/c0t0d0s5 492422 184319 258861 42% /var Type the name of the file system where queue space should be allocated [/var]

Press Return to accept the default directory, or type a different directory (for example, ).

9. You must also specify the size for the DISK_STORE_SIZE value when the following text appears:

Amount of space(MB) to allocate for the SRS Net Connect queue store. Type the maximum size that the proxy may use: [20]

Press Return to accept the default size of 20 MBytes.

10. If you run the proxy through a SOCKS server, you must provide the path to the SOCKS server when the following text appears:

  The SRS Net Connect proxy may be run via a SOCK daemon process. If you want the proxy to use the SOCKS proxy, type the fully-qualified path to the 'runsocks' or equivalent program. This adds the run-time library for the SOCKS libraries to the LD_LIBRARY_PATH. If you do not want to run via the SOCKS daemon, enter 'none' at the prompt. Enter the path to the runsocks program or 'none': [none]

Type the path to the SOCKS server or press Return to accept the default value of none.

11. You can enable automatic software upgrades, so new software is automatically installed on your monitored system. The updates include new software versions and messages. The following text appears:

  SRS Net Connect can automatically install software updates that are received by the proxy. Would you like to enable automatic updates? [y,n,?,q] y

Press Return to automatically install software updates or type .

The activation script checks the user's ability to interact with cron. The SRS Net Connect 3 activation complete message indicates a successful SRS Net Connect activation. Log into SRS Net Connect 3.1 to view your monitored systems. See the SunSM Remote Services Net Connect 3.1 Customer Operations Guide for instructions on creating system groups and user groups.

Testing the Installation

After you run the activation script, the monitored system and its providers run within three minutes. Wait at least 30 minutes before retrieving system reports from SRS Net Connect 3.1. Delta reports require at least two sets of data to compare.

Perform the following steps after you finish the installation:

1. Log into the monitored system as the root user.

2. Verify which SRS Net Connect 3.1 providers are installed by typing:

#

For example, the following text indicates that you have successfully installed the configuration provider (SUWsrscp), Sun RAS System Analysis (SUNWsrsep), and the reboot provider (SUNWsrsrp):

  1 9 * * 5 /opt/SUNWsrscp/bin/config_pvr_runner config_pvr 003.001.001 IM-NC_ENG- config_pvr /tmp/config_pvr 756000 text/plain 1 9 04 * * /opt/SUNWsrsep/bin/eras_pvr_runner eras_pvr 003.001.001 IM-NC_ENG-eras_pvr /tmp/eras_pvr 3348000 application/x-gtar 0-59 * * * * /opt/SUNWsrsrp/bin/gmt_time > /var/opt/SUNWsrsrp/latest

3. Verify that the SRS Net Connect 3.1 providers and proxy are running by typing:

#

The following providers and proxy processes should be running continuously:

    • trend_pvr
    • event_pvr
    • ssha_pvr
    • srsproxy

4. Verify that the monitored system can communicate with the SRS Net Connect Data Center. Change to the directory and type:

#

The -p indicates ping mode, and verifies that it can route to the Data Center.

5. Validate that the hardware alarm provider is working by typing:

#

This causes an error message to be logged in the file, which is detected by the hardware alarm provider. The hardware alarms provider sends a Fan Warning alarm.

6. Check the syslog file for warnings. The log is located in the file.

7. Log into SRS Net Connect 3.1 to verify that the alarm appears.

Copyright © 2004, Sun Microsystems, Inc. All rights reserved.

Источник: https://docs.oracle.com/cd/E19683-01/916-1586/chapter2.html

Step by Step guide on SAP Support Backbone Update and Enabling Note Assistant for Digitally Signed SAP Notes

As you are already aware by now, SAP has updated the support backbone infrastructure, to ensure that its critical infrastructure is up to date and secure, and will switch off the legacy infrastructure on January 1st, 2020.

Due to the increasing demand placed on the support backbone, SAP has updated the infrastructure to continue to provide us with the support we require. As part of this process, the way in which systems connect to SAP has been redesigned to include the following changes:

  • The HTTPS protocol is now used instead of RFC.
  • A technical communication user handles the data transfer instead of generic users.
  • There is no generic inbound interface.
  • Applications send data asynchronously unless the data is sent manually.

You would have been seeing warning messages in SAP systems, SNOTE transaction, EWA reports and SAP Service marketplace notes download sections, to update your systems to support SAP Backbone update.

This blog will explain in detail actions which are required to be configured in all SAP ABAP systems before January 2020 to ensure smooth connectivity SAP Support Backbone.

It is advisable to go through the whole blog before starting the implementation and open the SAP notes and guides referred in this blog as SAP updates the notes on regular basis.

    • What is SAP’s Support Backbone?
    • Configurations and Implementations of Snotes for TCI Enablement based on system releases
    • Setup of connections ( Either RFC or HTTP or Download Service) to SAP Support Backbone based on system versions
    • Enable Note Assistant for digitally signed SAP Notes
    • Defined the right procedure to download SAP Notes based on system releases
    • Defined the file type for downloading SAP Note
    • SDCCN, ANST, RFC Configuration changes
    • Reference links and SAP notes

What is SAP’s Support Backbone?

SAP’s Support Backbone is the central infrastructure located at SAP to provide technical support to our customers. 

The SAP Support Backbone has been updated.

The legacy infrastructure remains in place to allow a safe transition for customers.

You need to switch the communication of SAP Solution Manager and Focused Run to the new infrastructure before January 2020 to ensure continuous connectivity.

To get a list of all systems in your landscape which are not yet ready and need to be switched to the new support backbone connectivity  refer link

It will take you to the Early Watch Alert Work space and pre-selects the filter for “Backbone Connectivity”. As a result, you get a list of all systems (at least those who send EarlyWatch Alert reports to SAP) which are not ready yet and need to be connected to the updated support backbone infrastructure.

You then can drill down further to understand which action needs to be taken:

Refer below landing page for details on support backbone connectivity update. Landing Page for Support Backbone Connectivity Update

Note: This list will show all the systems which are not ready to connect to the updated support backbone. If you would like to see systems which are ready (green alerts), remove the filtering category and search for the text string “HTTPS -> SAP”. If this produces too many results, you can also use the search term “backbone”.

Impact of SAP Support Backbone Update and Required actions 

As a result of the update, the following systems actions are mandatory:

  • SAP Solution Manager systems

    When you switch to the new communication channels to enable the exchange of data with the updated SAP Support Backbone the following is required:

    Scenario 1:  You are on SAP Solution Manager 7.2

    Upgrade to SAP Solution Manager SP07 or higher (*Upgrade to SP08 or higher is recommended)

    Scenario 2:  You are on SAP Solution Manager 7.1

    Upgrade to the latest SAP Solution Manager 7.2 support package

Please note: For SAP partners (PartnerEdge Sell, VAR, CCC, PCOE), SP08 or higher is required.

  • Focused Run for SAP Solution Manager systems

    The new communication channels in Focused Run 2.0 enable the exchange of data with the updated SAP Support Backbone. All Focused Run customers need to upgrade to Focused Run 2.0. Focused Run 1.0 systems will not be able to communicate with the SAP Support Backbone after January 1st, 2020.

    Information on upgrading Focused Run can be found in the Focused Run Expert Portal.

    Remark: Focused Run 1.0 will enter its Customer-Specific Maintenance phase on November 23rd 2019.

  • ABAP systems with direct connectivity to the support backbone
    All customers with ABAP based SAP systems need to react to the SAP Support Backbone update to ensure connectivity of their SAP systems to the SAP Support Backbone.

1. Enabling Note Assistant for Transport Based Correction Instructions

2. Enabling Note Assistant for Digitally Signed SAP Notes

3. Setting up connections to SAP Support Backbone

4. Defining Procedure and File Types to Consume Digitally Signed SAP Notes

5. SDCCN direct connectivity/ Indirect connectivity update, ANST update, SAP RFC destinations update

Step by Step process to prepare managed system to support SAP Support Backbone Update 

 

Preparation and Prerequisite:

  1. Request a Technical Communication User

Connections using generic users will not work anymore after January 1st, 2020. For this purpose, customers need to ensure that all connections use a technical communication user in all systems which have connectivity to SAP (this includes all systems directly sending EWA data to SAP and all systems where SAP Note Assistant is being used on).

We must request a technical communication user for the systems ( Refer SAP Note 2174416) . (You cannot convert a regular S-user into a technical communication user.) The technical communication user is required, for example, to download digitally signed SAP Notes from Note Assistant (transaction SNOTE). Technical communication users cannot be used to log on in dialog mode, and their passwords do not expire.

After you have requested a technical communication user, it is generally available within 24 hours.

Hint: If  preparing an SAP Solution Manager system for the support backbone update, this step is automatically covered there and can be skipped in all managed systems.

Technical communication users can be requested via this app.

     2. If the system to be prepared for SAP’s Support Backbone Update is not directly connected to the backbone, no further action is required.

For all other systems, including SAP Solution Manager systems, Focused Run systems, and ABAP systems, with direct connectivity to the support backbone, you have to be on the following plug-in levels:

    • ST-PI 2008_1_7xx SP20 and higher, or ST-PI 740 SP10 and higher

    • ST-A/PI 01T* SP01 and higher

      3. Strongly recommended that you upgrade your SAP Solution Manager systems to Release 7.2, Support Package Stack 8 or higher.

1. Enabling Note Assistant for Transport Based Correction Instructions

It is strongly recommended that you enable Note Assistant to work with transport-based correction instructions (TCIs). However, to ensure that your systems can continue to communicate with the support backbone, it is sufficient that they can work with digitally signed SAP Notes.

The TCI is a new way to deliver ABAP correction instructions to customers in a flexible manner. A TCI bundles a stack of correction instructions in one transport request that can be installed using the SNOTE transaction.

The TCI reduces the installation time because it requires no pre or post installation steps.

SAP Note : 2187425 – Information about SAP Note Transport based Correction Instructions (TCI)

The TCI enablement in SNOTE are available in the following Support Packages of their respective SAP_BASIS releases. If a system is in any of the following SPs or above SPs, then, implementing the bootstrap is not needed:

Note: All higher releases (752 and above) has the enablement from SP00 itself.

For the releases listed in the table above, if you are on lower SPs, perform the following steps:

2.1 Update to Support Package Manager (SPAM) version 70 or higher.
2.2 Implement the list of notes given below, based on the SAP_BASIS release of your system. Doing this enables SNOTE for TCI.

TCI SNOTE Table

After applying all the prerequisite SAP notes as per your system release apply the bootstrap SAP note to enable TCI in your system.

Procedure for Implementing the Bootstrap SAP Note

Download the Bootstrap TCI: 

In our scenario below we are in Basis level 740

a) Search and open the relevant bootstrap SAP Note xxxxxxx from SAP One Support Launchpad. For more information on relevant bootstrap SAP Notes, refer to the above TCI notes table.

b) Choose Correction Instructions and select SAP_BASIS Software Component.


c) On the Correction Instruction view, select the relevant software component version (on the left side) and choose Download (on the right side). Save the SAR file into your directory.

d) Log on into client 000 of the ABAP system you want to install the TCI.

e) Upload the bootstrap TCI SAP Note to the system: Call transaction SPAM, then choose Support Package > Load Packages > From Front End and navigate to the directory, into which you downloaded the SAR file (for example, K70003CPSAPBASIS.SAR).

Define queue:

a) In transaction SPAM, display the new support packages.

b) From the OCS Package Directory: New Packages view, select the respective bootstrap TCI SAP Note.


c) To define the TCI queue, click the Calculate Queue button.

Click No

Note: If you receive the following message “Not allowed Support Package is already applied,” the import of TCI is not required as the required changes are already available in the system.

d) Import the TCI queue: Click the Import queue button to import the TCI queue and complete the steps.

Continue to import and complete

e) Confirming the queue: Once the queue is imported, you are prompted with the next action – to go to the SAP Note Assistant (transaction SNOTE) and download (Even if the Note is available download it again) and implement the bootstrap note; that the status of the SAP Note is completely implemented. Doing this will automatically confirm the SPAM queue.

Note: If your system is not connected to the Online Service System (OSS) confirming the queue is not possible. In this case, you first download the SAP Note from the SAP Support Portal and upload SAP Note xxxxxxx (for example, SAP Note 2446868 for 700 release) using the Note Assistant tool and then confirm the queue.

Verify the SAP Note Status: Verify if the status of the SAP Note xxxxxxx (for example, SAP Note 2446868 for 700 release) is set to Completely Implemented.

Preparing Note Assistant (Transaction SNOTE) is also called as bootstrapping.

Once the SNOTE is bootstrapped, any SAP Note containing TCI can be implemented in the same way as implementing any other SAP Note.

Note: If you are on SPAM version 70 and above: the bootstrap note is transportable, you need not apply the bootstrap note in each system. You can apply the bootstrap note 1995550 in Dev system and you can move the TR to Quality and Production.

Please follow the below sequence :

  1. Create 1st TR and capture all pre-requsite notes & release the TR.
  2. Create 2nd TR for locking bootstrap note & release the TR.
  3. Create 3rd TR for implementing digitally signed note TCI (*which will be explained in next section below) and finally release the 3rd TR.

Move these TRs in sequence to Quality and Production.

2. Enabling Note Assistant for Digitally Signed SAP notes

SAP recognizes a security threat during upload of SAP Note into customer landscape. The SAP Note can get modified maliciously and the customer can upload unknowingly the maliciously modified SAP Note into their landscape.

Therefore, SAP delivers all SAP Notes having ABAP corrections with digital signature to protect SAP Notes with increased authenticity and improved security.

SAP strongly recommends uploading or download only digitally signed SAP Notes. The digital signature verification feature is enabled for both uploading or downloading of SAP Notes.

 

The SNOTE is enabled to work with Digitally Signed SAP Notes from the following SPs of their respective releases of SAP_BASIS software component.

Note: All higher releases (753 and above) has the enablement from SP00 itself.

For the releases listed in the table above, if you are on lower SPs, perform the following steps:

Prerequisites

  1. You have to implement the SAP Note 2408073 and SAP Note 2546220 for uploading digitally signed SAP Note and digital signature verification.
  1. You have to implement the SAP Note 2508268 for downloading digitally signed SAP Note.

(OR)

1. Implement  TCI NOTE “2576306 – Transport-Based Correction Instruction (TCI) for                      Download of Digitally Signed SAP Notes” containing the SAP Notes 2408073, 2546220 and         2508268

While uploading the TCI package if there is a failure in signature verification please refer to the SAP Note 2520826 for solution.

“SAP recommends Implementing SAP Note 2576306 instead of applying the above individual SAP Notes.”

Follow below steps to implement the SAP Note with the corresponding TCI in your development system, use the Note Assistant via transaction SNOTE.

Note: Warnings about objects without directory entry can be ignored, since TCIs can contain deletions.

Import the adjustment transport into your productive systemto implement the TCI in productive system.

Procedure for implementing an SAP Note containing TCI

Download the SAP Note (which is also a TCI note)

Download the TCI package:
a) Search and open the relevant SAP Note xxxxxxx in SAP ONE Support Launchpad

b) Choose Correction Instructions and select the relevant software component.


c) On the Correction Instruction view, select the relevant software component version (on the left side) and choose Download (on the right side). Save the SAR file into your system.

d) Log on into client 000 of the ABAP system you want to install the TCI.

e) Upload the TCI SAR file in the Note Assistant, Choose Goto > Upload TCI

Note: Alternatively, you can upload the TCI SAR archive (for example, K700005CPSAPBASIS.SAR) through Support Package Manager or SAINT from front end to your development system

To do so, call transaction SPAM or SAINT in client 000 and choose Support Package > Load Packages > From Front End in transaction SPAM or Installation Package > Load Package > From Front End in transaction SAINT.

Note: If Upload TCI is not available in SNOTE transaction, then execute Report RSLANG20(Refer to SAP Notes 110910 and 48624).  After this step, launch SNOTE and choose Goto > Upload TCI

Implement the SAP Note xxxxxxx as any other SAP Note in Note Assistant

Copy the corrections to Transport request and proceed

Download TCI correction for rollback

Upload rollback TCI in SNOTE and extract it.

Continue to implement the TCI note 2576306

If you get error message: 2258238 snote data not available, apply Snote 2258238  and continue to implement 2576306

Now all the required TCI notes are applied in the ABAP development system and copied in transport request, which can be moved to production system.

Required Authorizations 

If the verification of digital signature for an SAP Note fails, the Note Assistant tool logs the security event in the application server using log object (CWBDS). To view the application logs, you should have authorization to the S_APPL_LOG authorization object.

3. Setting up connections to SAP Support Backbone

The tables below provide an overview of the RFC connections that were previously used to connect to the support backbone, along with the new HTTPS connections.

Note: For SAP Solution Manager, some destinations are created as part of task list SAP_SUPPORT_HUB_CONFIG. 

 

Once the prerequisite step of implementing 2576306 – Transport-Based Correction Instruction (TCI) for Download of Digitally Signed SAP Notes is completed, follow below connection setup based on your system version

1> RFC procedure for SAP_BASIS release 700 to 731 only

  • SAP ABAP systems with lower SAP Releases (= lower than SAP Kernel 7.42 Patch Level 400) who want to download SAP notes or uses software components  of ST-PI and ST-A/PI will still use RFC connection SAPOSS or SAPSNOTE, but changes with that RFC connection`s SAPOSS or SAPSNOTE are mandatory!
  • This is the default procedure for all releases of SAP_BASIS until end of 2019.
  • From January 1 st 2020 the following will be enforce:

> This procedure will be the default option for SAP_BASIS releases 700 to 731 only.

>  As system kernel version is below 742, we can use SAPOSS connection but we need                          to make below changes.

> Generic user (OSS_RFC) will not be allowed in RFC destinations SAPOSS/SAPSNOTE.

>  Only customer S user (recommended is Technical Communication User) will be allowed.

>  SAPRouter string to the SAP Service and Support target system of the SAP infrastructure                   can be:

target host: /H/X1/S/S1/H/X2/S/3299/H/oss001.wdf.sap.corp with

X1 = IP address of saprouter on SAP customer side
X2 = IP address of sapservX
S1 = TCP port of SAPRouter on customer side

Possible settings for sapservX could be:

sapserv1 (194.117.106.129) Internet VPN connection
sapserv2 (194.39.131.34) Internet SNC connction
sapserv3 (147.204.2.5) SAP customers from Germany will typically use that settings
sapserv4 (204.79.199.2)SAP customers from US region will typically use that settings
sapserv5 (194.39.138.2) SAP customers from Japan will typically use that settings
sapserv7 (194.39.134.35) SAP customers from region APJ (Asia Pacific) inclusive                                              New Zealand and Australia will typically use  that settings
sapserv9 (169.145.197.110) SAP customers from region APJ (Asia Pacific) inclusive                                          New Zealand and Australia will typically use  that settings
sapserv10 (203.13.159.37) SAP customers from China will typically use that settings.

> There is no change in logon group, you can use 1_PUBLIC, 2_JAPANESE, EWA

>  It is mandatory to use “oss001.wdf.sap.corp” and Load Balancing and a Technical                               Communiation User in your RFC connection to SAP Backbone.

RFC destinations SAPOSS/SAPSNOTE will not work in ABAP systems on SAP_BASIS release 740 and above. Instead HTTPS communication should be used.

2> HTTPS procedure for SAP_BASIS release 740 and above

  • For system higher than 740, mandatory protocol is HTTPS so we need to configure RFC accordingly and make relevant changes so SAP Notes gets download using HTTPS protocol instead of RFC protocol i.e. SAPOSS
  • Destinations to SAP Support Portal and SAP Note Download needs to be defined (SM59). Use S user (recommended Technical Communication User) in the H and G type destinations
  • HTTPS encryption and communication path needs to be configured
  • By following recommended destination names, configuration can be reused in other scenarios

Either manual or automatic configuration of HTTP connection setup can be followed. For manual step follow “Digital Signature.pdf” attached to SAP Note 2576306

In our scenario we will see automatic setup.“SNOTE 2738426 for Automated Configuration of new Support Backbone Communication”HTTPS prerequisites can be configured in ABAP Task Manager (STC01) by executing automated Task List SAP_BASIS_CONFIG_OSS_COMM.This task list contains common configuration steps for the ABAP task manager, and automatically creates the required connections to the support backbone.Technical Communication User and SAP Router string needs to be prepared before execution

Check SPAM version – minimum level 70, Implemented TCI note 2738426Execute task list ‘New OSS Communication’:Run transaction STC01 to open Task Manager for Technical Configuration

Select task list ‘SAP_BASIS_CONFIG_OSS_COMM’ – New OSS Communication

Task 1: Check CommonCryptoLib.

Task 2: We need to set parameter ssl/client_ciphersuites and parameter value for enabling highest TLS protocol version with BEST-OPTION.

Check for the parameter, if it already exists with the required value no change is required if not we need to set parameter ssl/client_ciphersuites value.

Before Configuration of parameter: value of ssl/client_ciphersuites parameter is not set

Added profile parameter value as ssl/client_ciphersuites = 150:PFS:HIGH::EC_P256:EC_HIGH,

Restart the system after adding/changing parameter.

For more information, read SAP Note 510007 – Setting up SSL on Application Server ABAP

Task 3: Check certificate for SSL Client

Checked in transaction STRUST, no certificates exist.

Obtain certificates from certificate provider websites or use below link.

Add the required certificates and save it.

Note: Import client certificate in SSL Client (Standard) or SSL Client (Anonymous), but relative option needs to selected while running task list otherwise you will get error while running task list. I have imported all the above client in SSL client (standard)

Task 4: Create HTTP connection

Maintain the fields Technical Communication User, Password and Router String (optional, if required), press ‘Return’, ‘Save’ and ‘Back’

Task 5: Change the user in RFC connection SAPOSS with technical user

Task 6: Select Restart of ICM if required, optional

Execute the task list by pressing button ‘Start/Resume Task List run in Dialog (F8)’

Click on the detailed log icon of each task to see the results of the task execution

Checked in transaction SM59, new connection destinations are created by the execution of above tasks list.

Following RFC’s created by Digital Signed process are working fine (test is good):

SAP-SUPPORT_PORTAL (Status HTTP response is 200)
SAP-SUPPORT_PARCELBOX (Status HTTP response is 200)
SAP-SUPPORT_NOTE_DOWNLOAD (Status HTTP response is 404)

3> Download Service application for SAP_Basis Release 700 onwards

  • Available for SAP_BASIS release 700 onwards
  • Any ABAP system having download service can be used as download system.

The SAP NetWeaver download service allows you to download files directly into your SAP NetWeaver Application Server ABAP system from any SAP destination addressed through a URL.

The most important use case for the SAP NetWeaver download service is downloading from SAP file shares connected to the SAP Support Portal and the download of SAP Notes with all their dependencies and relevant SAP Notes transport-based correction instructions (TCIs).

The downloading of files from SAP file shares is only possible after a successful login to the respective SAP Support Portal system with an S-user authorized for the file download.

With below scenario we will explain what to configure to use download service. We have taken download system as our Solution Manager 7.2 system in our scenario.

Implement SAP note 2554853 SAP Netweaver Download service for SAP Notes

The following information describes how to configure the SAP NetWeaver download service to your needs.

Authorizations and Roles:

To carry out the following configuration tasks and to use the SAP NetWeaver download service, you require specific authorizations and roles.

Configuring the SAP NetWeaver download service involves the following steps:

 

Step 1: You set up the connection to the SAP Support Portal:

1.You maintain the S-user configuration using the transaction SDS_CONFIGURATION.

2. You configure the client certificates.

The following root certificates must be registered for the SSL client SSL Client                (Standard) in the SAP NetWeaver system:

  1. To import certificates, call transaction STRUST and, under SSL client SSL Client (Standard), choose Import certificate.
  2. On the File tab page, browse to the downloaded certificate files and import the certificates by choosing .
  3. Save your changes.

    The Certificate List is now updated with the new certificates.

If the SAP NetWeaver download service fails during the download from these locations, see SAP Note 2456654 Information published on SAP site.

If errors occur, restart the ICM Monitor using transaction SMICM. This restarts ICM services and reloads all certificates in your system.

3. You adapt the proxy settings.

Procedure

1.Adapting settings for a global proxy server

  • Call transaction SM59 and choose Extras HTTP Proxy Configuration.

  • On the Gobal Settings tab page, make sure that a proxy server exists and specify destinations that should not be accessed using the proxy server.

  • On the HTTPS Protocol tab page, enter the connection information for the proxy server and choose OK.

No Proxy settings are active in our environment, if you have proxy active please make the corrections and save

2. Adapting settings for a local proxy server

  • Call transaction SDS_CONFIGURATION in change mode.
  • On the Proxy Settings tab page, enter the connection information for the proxy server and choose OK

If you have proxy settings available enter them and save.

4. You configure the HTTPS service.

Procedure

1. Check if the HTTPS service is configured.

Call transaction SMICM (ICM Monitor) and choose Goto Services.
Check if an entry for the HTTPS protocol exists and is set to active.

2. If no active entry exists, choose one of the following options:

Create a non-permanent entry that is valid until the next restart.

To create a new entry, choose Service Create, enter the required information for an                            HTTPS protocol and choose Create Service.

To activate an existing but inactive HTTPS entry, select the entry and choose Service                          Activate.

Create a permanent entry.

Call transaction RZ10.

Choose the default or instance profile entry and create a new parameter entry                                      icm/server_port_<number> by choosing Extended maintenace Change Parameter                              Create.

Example: icm/server_port_2 PROT=HTTPS, PORT=44300, PROCTIMEOUT=300, TIMEOUT=300

3. To enable the download of SAP Notes from https://apps.support.sap.com, call transaction RZ10 and create the profile parameter ssl/client_ciphersuites with the value 918:PFS:HIGH::EC_P256:EC_HIGH.

 

Step 2: You set up the download directory.

The logical file DOWNLOAD_SERVICE_DIR is defined and delivered by default. It points to the /usr/sap/trans/EPS/in directory in UNIX nomenclature. This path is specified in the definition of the logical path DOWNLOAD_SERVICE_PATH.

If the target directory fits your system, you can use the default logical file DOWNLOAD_SERVICE_DIR. You can also adjust the directory to which the logical path DOWNLOAD_SERVICE_PATH is pointing to your target directory, or you can create your own logical file paths, assignments of physical paths to logical paths and logical file names.

Procedure

Adjusting the physical path assignment of the default logical path

1.Call transaction FILE and select the DOWNLOAD_SERVICE_PATH entry in the Create a logical file path table.

2. Go to Assignment of Physical Paths to Logical Path and adapt the physical path according to your target directory or operating system, respectively.

3. Save your changes.

(OR)

Defining a new logical file

1. Call transaction FILE, choose New Entries, and specify a logical file path.
2. Under Assignment of Physical Paths to Logical Path, assign a physical path.
3. Go to Logical File Name Definition, Cross-Client, double-click your logical file, assign your new logical file path to it, and save your changes

 

Step 3.You maintain execution parameters using the transaction SDS_CONFIGURATION.

1. Creating a user-specific execution parameter

On the Execution Parameters tab page, choose Create Entry.

In the upcoming dialog box, for all fields the hard-coded system defaults are set. Enter here                the respective values for your configuration. If you want to create a system default execution              parameter, enter all values except the user name.

2. Changing an existing execution parameter

On the Execution Parameters tab page, select the entry that you want to change and choose            Change Entry.

Enter your changes.

You can change all parameters but the user name.

Confirm your changes by choosing Continue and then Save.

3. Deleting an execution parameter

On the Execution Parameters tab page, select the entry that you want to delete and choose          Delete Entry.

The entry is removed from the Execution Parameters table.

 

Step 4. You set up the SL protocol service.

Prerequisites

You only need to perform this step if the following situations apply:

The release version of your SAP NetWeaver Application Server ABAP is 7.4 or higher.
The SL protocol is used.

Procedure

Call transaction SICF.

Set the Hirarchy Type to SERVICE and choose Execute.

Expand the nodes under default_host and navigate to the following service trees:

SL protocol: <defaulthost>/sap/bc/rest/SLProtocol

REST protocol: <defaulthost>/sap/bc/rest

Ensure that the services are active.

Once the connection to SAP support backbone is decided and configured, we can use them to consume digitally Signed SAP Notes by customizing in report RCWB_SNOTE_DWNLD _PROC_ CONFIG .

4. Defining Procedure and File Types to Consume Digitally Signed SAP Notes

Following are the two modes through which you can consume the digitally signed SAP Notes:

  • How to Upload Digitally Signed SAP Notes Using SNOTE Transaction
  • How to Download Digitally Signed SAP Notes Using SNOTE Transaction

1.How to Upload Digitally Signed SAP Notes Using SNOTE Transaction

Digitally signed SAP Notes are available from SAP ONE Support Launchpad. You can upload the digitally signed SAP Notes into the SNOTE transaction as follows:

Procedure

  1. Download the digitally signed SAP Note from SAP ONE Support Launchpad
  2. Run the SNOTE transaction
  3. From the menu bar, choose Goto -> Upload SAP Note

 

2.How to Download Digitally Signed SAP Notes Using SNOTE Transaction

Based on your SAP NetWeaver version, you have the following ways to download SAP Notes into your system.

Download Procedures for NetWeaver 700 to 731:

  1. Download service
  2. RFC (Enabled by default)

Download Procedures for NetWeaver 740 and later:

  1. Download service
  2. HTTP
  3. RFC (Enabled by default until end of 2019.

To directly download the digitally signed SAP Notes using SNOTE transaction, proceed as follows:

Depending upon the settings defined in the Customization, the digitally signed SAP Notes are downloaded.

1. Defining Procedure for Downloading SAP Note (RCWB_SNOTE_DWNLD _PROC_ CONFIG)

2. Defining File Type for Downloading SAP Note (RCWB_UNSIGNED_NOTE _CONFIG) Note

Defining Procedure for Downloading SAP Note (RCWB_SNOTE_DWNLD _PROC_ CONFIG)

With the introduction of digitally signed SAP Notes, various procedures or modes are offered for downloading the SAP Notes. You use this report to define a procedure based on your requirement for downloading the SAP Note.

The report RCWB_SNOTE_DWNLD_PROC_CONFIG is used for customizing the different procedures.

If you are on the SPS level where the feature is delivered or implemented the TCI 2576306, this activity can be performed through IMG customization (IMG > SAP NetWeaver Implementation Guide > Application server > Basis Services > SNOTE )

This is a one time set up. If required, you can change the settings in this report at any given point in time.

RFC procedure for download of digitally signed SAP Note

If you choose this option, the system uses RFC destination SAPOSS or SAPSNOTE, whichever is applicable, to download the digitally signed SAP Note.

By default, the system uses the RFC option when no other option is selected.

->Starting 1st January 2020, downloading SAP Note using RFC procedure will no longer be supported for NetWeaver 740 and higher. You need to choose a download procedure between Download Service Application or HTTP Protocol.

HTTPS procedure for download of digitally signed SAP Note

If you choose below option, the system uses the HTTPS protocol to download the digitally signed SAP Note.

When you run this report RCWB_SNOTE_DWNLD_PROC_CONFIG using the transaction SE38, following are the various procedures offered in the report to download the SAP Note. Select HTTPS and save configuration.

Validation:

Try to download one SNOTE and check the logs.

Logs will now contain Digitally Signed SAP note is downloaded using HTTPS as below.

Note: When using this option we faced situation where no proper messages are displayed like below, to solve it follow manual action in 2508268 snote

 

After maintaining above message numbers with text we were able to read the SNOTE log texts properly.

Download of digitally signed SAP Note using Download Service application

If you choose this option, the system uses the Download Service application to download the digitally signed SAP Note.

The download service can be present in the same system that you are using to download the digitally signed SAP Note or in another system. For example, the SAP Solution Manager can be used as the download service system. Ensure that you have established the RFC connection, of type 3, to the download service system.

Advantage: Associated Transport based Correction Instruction (TCI) packages and prerequisite SAP
Notes are downloaded automatically

For example, assume you have an SAP Note and that SAP Note has around 20 prerequisite SAP Notes. When you try to download the SAP Note, the 20 prerequisite SAP Notes also get downloaded automatically. Whereas in the other two options (RFC and HTTP Protocol), the prerequisite SAP Notes get downloaded during the implementation of the present SAP Note

In report RCWB_SNOTE_DWNLD_PROC_CONFIG, choose download service

On the Download Service System the RFC destination has been set to NONE and click save.

Validation:

Tried to download one SNOTE and check the logs.

Logs will now contain Digitally Signed SAP note is downloaded using Download Service as below.

Note: Refer below snotes if you face any issues while downloading SNOTE with download service.

2803658 – After configuring the Netweaver Download Service for SAP Notes, attempting to download a note gives Error I:SCWNL810 NONE.

2608378 – Download fails when downloading a high number of Notes

2618713 – Timeout during download of SAP Notes via SAP Download Service

 

Defining File Type for Downloading SAP Note (RCWB_UNSIGNED_NOTE _CONFIG)

Report RCWB_UNSIGNED_NOTE_CONFIG was used to set “Do not download unsigned SAP Note”

In future when you try to upload any SNOTE which is not digitally signed choosing this option will not allow that SNOTE to be implemented in our system.

 

5. SDCCN direct connectivity/ Indirect connectivity update, ANST update, SAP RFC update

 

SDCCN Configuration Update:

After you have upgraded to the latest version of ST-A/PI, you must specify new HTTP connections in Service Data Control Center.

Below is automatic option we get to migrate tasks in SDCCN.

Click on migrate tasks, and validate  RFC destinations in task specific settings in SDCCN are migrated.

In below scenario we explain how to manually make configuration changes in SDCCN,

Goto Settings-> task specific

Add destination SAP-SUPPORT_PORTAL and Remove destination SDCC_OSS

Delete all tasks that have the target SAP (O02).

Create the tasks again. The new tasks will use new destination SAP-SUPPORT_PORTAL or SAP-SUPPORT_PARCELBOX depending on the task type.

 

ANST Configuration Update:

In ANST transaction, settings change RFC Destination from SAPOSS to SAP-SUPPORT-PORTAL if the SAPOSS connection throws error while downloading SAP notes using ANST transaction.

 

Update of RFC connection to SAP Support Backbone:

In a SAP ABAP system the following RFC connections to SAP Service and Support backbone infrastructure can exist:

RFC connections:

  • SAPOSS
  • SAPNET_RFC
  • SAPNET_RTCC
  • SDCC_OSS
  • CUPOSS
  • OSSNOTE
  • SAP-OSS
  • SAP-OSS-LIST
  • SAP-OSS-LIST-O01
  • SM_SP_<customer number>

If you connect to SAP Support Backbone infrastructure with an RFC connection not listed here, identify and check the RFC connection. To identify such an RFC connection to OSS, consider using transaction SE16 in your ABAP system and table rfcdes.

  • RFC connection SAPOSS or SAPSNOTE to SAP Service and Support backbone infrastructure needs to be updated before January 2020; other RFC connections similar to RFC connection SAPOSS or SAPSNOTE are normally not necessary anymore
  • It is mandatory to replace the existing generic user`s (like OSS_RFC) in a RFC connection like SAPOSS or SAPSNOTE with a Technical Communication User
  • Please check the settings of RFC connection SAPOSS (or SAPSNOTE) and change it, if necessary.
  • You still can use logon group 1_PUBLIC or EWA or 2_JAPANESE
  • The servers behind the 3 logon groups are configured identically. Japanese customers of SAP SE can still use logon group 2_JAPANESE. Other customers can use either logon group 1_PUBLIC or EWA
  • You still can use target system “OSS”
  • You still can use message server “oss001.wdf.sap.corp”. Please do NOT change that setting with “oss001.wdf.sap.corp” !
  • Please use Load Balancing and set the flag to “yes”. It is mandatory to use flag “yes”.

Reference Links and SAP Notes:

 

->FAQ – Digitally Signed SAP Notes – 2537133 

-> Cheat Sheet for enabling SNOTE for Digitally Signed SAP Notes and for TCI

2737826 – SAP Support Backbone Update / upcoming changes in SAP Service and Support Backbone interfaces (latest) in January 2020.

2740667 – RFC connection SAPOSS to SAP Service & Support backbone will change (latest) in January 2020

2836302 – Automated guided steps for enabling Note Assistant for TCI and Digitally Signed SAP Notes

2392726 – How to unlock a Support Hub User (Technical Communication User)

2508268 – Download of Digitally Signed SAP Notes in SNOTE

2732094 – ANST – Implementing SOAP Based ANST Note Search

2690656 – New communication channel to SAP Backbone for transaction SDCCN

2174416 – Creation and activation of users for the Support Hub Communication

2554853 – SAP NetWeaver download service for SAP Notes

2783798 – SNOTE log messages displayed improperly after enabling Digitally Signed SAP Notes

2603877 – Exception handling corrected in download of digitally signed SAP Note for callers other than SNOTE

https://help.sap.com/viewer/9d6aa238582042678952ab3b4aa5cc71/7.4.19/en-US/48b41a66fc096ff4e10000000a42189b.html

Источник: https://blogs.sap.com/2019/11/14/step-by-step-guide-on-sap-support-backbone-update-and-enabling-note-assistant-for-digitally-signed-sap-notes/
Analyze dual patch results.

  1. (a)

    I-VM relationship can be derived from the stepwise command voltages as shown in Figure 2a. Plot I readings from E1 against their corresponding VM readings from electrode 2. Determine the reversal potential of the current by extrapolating the data point on the I-VM curve.

  2. (b)

    Plot the conductance-voltage (G-VM) curve by dividing the current amplitude change (ΔI) by the measured voltage step (ΔVM) at each potential. As shown in Figure 4f.

? TROUBLESHOOTING

Step 7, when GΩ is difficult to establish, check the following possibilities:

  1. a)

    Brain slice quality is always the first thing to be considered. Ensure the preparation procedure was followed strictly; otherwise a new preparation is needed.

  2. b)

    The difficulty in achieving GΩ seal increases with animal age, start your practice with P10 mice is recommended to familiarize the procedure. Young adult mice from P21 - P30 can be used in most of experiments, unless the study targets on an age related subject.

  3. c)

    Make sure to apply and maintain a small positive pressure before the electrodes touching to the bath solution in step 5.

  4. d)

    Apply the suction as gentle as possible and increase it in a stepwise manner until the membrane is ruptured.

  5. e)

    Ensure electrode solution is freshly prepared with correct pH and osmolality.

If the seal in the first electrode is lost when the second electrode approaches the cell, pay attentions to the following:

  1. a)

    Focus on the cell when the second electrode approaches the cell. Move the first electrode to follow the cell if the cell moves.

  2. b)

    Adjust slice anchor and hold more tightly if too loose.

  3. c)

    The deeper the targeted cell beneath the surface of slice, the more likely to encounter a cell movement during the handling the second electrode to approach the cell. Select a relatively shallow cell, ~50 μm is recommended.

  4. d)

    Approach the cell gently. Avoid larger movements of the second electrode, including approaching, going up or down. If the tip of the second electrode is too shallower or deeper than cell body, withdraw the second electrode gently, adjust the position on the top of slice and re-approach.

Step 8, apply suction pulses either by mouth or use of a syringe. Try following options:

  1. a)

    In our experience, a relatively strong and fast pulse works well to rupture membrane and achieve a relatively stable Ra.

  2. b)

    Keep the same or increase the suction pressures in the repetitive rupture attempt, release pressure immediately if membrane resistance in seal test drops suddenly.

  3. c)

    Keep a small pressure and click “Zap”, and release the pressure after Zap. Repeat Zap if necessary.

  4. d)

    If the seal resistance is higher than 30 MΩ, indication of Rt >30 MΩ, repeat above steps may decrease the Rt of whole-cell recording.

Step 9, Ra tends to increase during recording (Figure 2f), do the following to minimize it:

  1. a)

    Low resistance electrodes, i.e., 2.0-2.5 MΩ, has been demonstrated to be feasible and is recommended [18]; as such, the Rt can be physically reduced to the lowest workable range.

  2. b)

    Empirically, a relatively stronger suction pressure for whole-cell break-through results in a high percentage of low and stable Ra whole-cell recordings.

  3. c)

    To ensure that the brain slice is anchored securely in the chamber; this prevents potential slice movement resulting from perfusion pulsatile.

  4. d)

    In general, the older the animal the more difficulty to achieve a long-lasting stable Ra, use young adult animals unless the experiment examining an ageing related question.

  5. e)

    Use mechanically stable micromanipulators.

  6. f)

    We routinely discard recordings with high Ra (or Rt) from data analysis.

Timing

Step 1, slice preparation: 30 min

Step 2–3, slice recovery and SR101 staining: at least 1 h

Step 4, transfer slice to recording chamber and re-equilibration: 20 min

Step 5, loading both electrodes: 10 min

Step 6, select astrocyte: 5 min

Step 7, approach the cell and form GΩ seals: 5–30 min

Step 8, break through to form dual patch recording: 3–10 min

Step 9, recording: 30 min - 4 h, depending on the experimental design

Step 10, Analysis: variable, depending on required data.

Anticipated results

Dual patch recording was once considered to be a time-consuming technique which required considerable skill. Facilitated by the use of advanced equipment, specifically PatchStar micromanipulators and software (Scientifica, UK), a Polychrome V imaging system (Till Photonics, Germany), and a MultiClamp 700A/700B amplifier (Molecular Devices, Sunnyvale, CA), this recording mode can be performed routinely, studying 3–8 cells in a 6-hour experimental day. For experienced researcher, the dual patch single astrocyte recording can be achieved within 10 min after the cell being visual identified. Should all the steps be followed carefully, successful dual patch single astrocyte recording can be readily achieved. In some of the experiments, successful dual patch recordings recording had lasted up to 4 hours in our designed experiment. Thus, dual patch is an ideal technique for the future study of functional channels, receptors, and electrogenic transporters in astrocytes.

References

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Download references

Acknowledgments

This work was sponsored by grants from the National Institute of Neurological Disorders and Stroke (RO1NS062784 to MZ) and the National Natural Science Foundation of China (81000491to GX). We thank Ms. Judith A. Enyeart and Ms. Kelly E. Crowe for their assistance in manuscript preparation, Mr. Randall Carpenter for critical reading of manuscript.

Author information

Affiliations

  1. Department of Neuroscience, The Ohio State University Wexner Medical Center, Columbus, OH, 43210, USA

    Baofeng Ma, Wei Wang, John J Enyeart & Min Zhou

  2. Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, P.R. China

    Guangjin Xu

Источник: https://molecularbrain.biomedcentral.com/articles/10.1186/1756-6606-7-18

Welcome to Atiz Support

Following these steps:

Precaution

- A power failure during the firmware writing operation may disable the camera.

» Use a fully charged Battery Pack or dedicated AC Adapter Kit (Optional) for the firmware update.
» Do not shut off the power during the firmware writing operation.
» Do not open the card slot cover during the firmware writing operation.

- Do not press any camera buttons during the firmware writing operation.

(1) Prepare the items required to update the firmware.

1. Camera body

2. Dedicated Battery Pack (The battery pack must be fully charged) or dedicated AC Adapter Kit (Optional) ***

3. Memory card (64MB or more, 64GB or less)

4. Firmware update file (you can download it from Canon's Website)

(2) Create the firmware update file.

1. Download the zipped self-extracting file from Canon's Website.

2. Extract the downloaded file, and create the firmware update file.

- How to Extract the Firmware Update File

Windows
When you double-click the downloaded file, the following screen appears.
Click [OK], and the downloaded file will be extracted, and the firmware update file created.

Macintosh
The downloaded file will automatically self-extract, creating the firmware update file. In case the downloaded file does not automatically self-extract, double-click the downloaded file.

3. Check the size of the firmware update fiIe
If the file size does not match, then download the firmware update file again

- How to Confirm the File Size

Windows
Right-click the icon of the firmware update file and select the [Properties] command from the pop-up menu that appears.

Macintosh
Select the icon of the firmware update file, and then select the [Get Info] command from the[File] menu.

4. The name and size of the firmware update file can be checked on the Website.

*** In case of using the memory card reader, follow the procedure from Step (3) onward. In case of not using the memory card reader, follow the procedure from Step (4-1) onward.

(3) Copy the firmware update file to the memory card.

1. Insert a memory card that has been formatted in the Camera into the memory card reader.

2. Copy the firmware update file to the first window that appears when the memory card is opened (the root directory).

3. Remove the memory card from the card reader.

*When removing the memory card, be sure to do so as described in the documentatIon for the computer or the card reader

*If the firmware update file is placed in subfolder of the memory card, the camera will not see it.

4. Rotate the Mode Dial to select mode (or any mode other than Fully-Automatic Modes).

5. Insert the memory card with the firmware into the camera.

6. Turn the Power Switch

7. Rotate the Main Dial and the Quick Control Dial to select the "Firmware Ver.x.x.x" item at the bottom of the "Set-up 3 (Yellow)", and then press the

8. The firmware update screen will appear.
Turn the Quick Control Dial, select [OK], and then press the

(4-1) Connect the camera and the computer.

1. Rotate the Mode Dial to select <P> mode (or any mode other than Fully-Automatic Modes).

2. Insert a memory card that has been formatted in the camera into the camera.

3. Connect the camera and the computer with the USB cable, and then set the camera’s power switch to <ON>.

(4-2) Start the firmware update.

1. Start EOS Utility.

2. Click the [Camera settings I Remote shooting] button.

3. Click [], and then click the [Firmware Ver. X.X.X].

4. The firmware update screen will appear on the computer screen Click [OK].

5. A window for selecting files appears, so select the firmware update file, and then click [Open].

6. A confirmation window appears, so click [OK].

7. A window with cautionary messages about the update appears, so click [OK].

8. Follow the procedure from Step (5) onward on the camera.

(5) Update the firmware.

1. The screen will appear on the camera’s LCD monitor.

2. If you press the

3. The message will appear during the update.

4. When the update is completed, the message will appear on the camera’s LCD monitor.

5. Complete the firmware update by pressing the

The firmware update is now completed.

When the firmware update operations are finished, turn the camera

Format the memory card before using it again.

Verifying the firmware Version

1. Turn the Power Switch <ON>, and press the menu button

2. Rotate the Main Dial and the Quick Control Dial, and you will see the “Firmware Ver.X.X.X” at the end of the settings shown in Set-up 3 (Yellow).

3. This is the currently installed firmware version number.

Note: Select P mode (or any mode other than Fully-Automatic Modes). The version of the firmware will not appear in the Fully-Automatic Modes.

If an ERROR message appears during the firmware update

If this screen appears, remove the battery and check to make sure that there are no problems with the battery capacity or with the firmware update file on the memory card.

If there are no problems, repeat the update operations again.

If the problem persists, please contact the Canon Customer Support Center in your region.

Источник: https://www.atiz.com/support/

Nitrate-functionalized patch confers cardioprotection and improves heart repair after myocardial infarction via local nitric oxide delivery

Abstract

Nitric oxide (NO) is a short-lived signaling molecule that plays a pivotal role in cardiovascular system. Organic nitrates represent a class of NO-donating drugs for treating coronary artery diseases, acting through the vasodilation of systemic vasculature that often leads to adverse effects. Herein, we design a nitrate-functionalized patch, wherein the nitrate pharmacological functional groups are covalently bound to biodegradable polymers, thus transforming small-molecule drugs into therapeutic biomaterials. When implanted onto the myocardium, the patch releases NO locally through a stepwise biotransformation, and NO generation is remarkably enhanced in infarcted myocardium because of the ischemic microenvironment, which gives rise to mitochondrial-targeted cardioprotection as well as enhanced cardiac repair. The therapeutic efficacy is further confirmed in a clinically relevant porcine model of myocardial infarction. All these results support the translational potential of this functional patch for treating ischemic heart disease by therapeutic mechanisms different from conventional organic nitrate drugs.

Introduction

Cardiovascular disease (CVD) is a leading cause of mortality and morbidity worldwide, and ischemic heart disease accounts for approximately half of all deaths1. Coronary artery obstruction results in the occurrence of myocardial ischemia, which causes the death of non-regenerative cardiomyocytes and the subsequent formation of fibrotic scars. Strategies for the treatment of myocardial infarction (MI) in the clinic involve vasodilation and antiplatelet therapeutics2,3, among which organic nitrate derivatives are the first choice and have been utilized for more than a century. The therapeutic effects of organic nitrates have been ascribed to the bioactive molecule nitric oxide, which is generated as a metabolic product through both enzymatic and non-enzymatic pathways4.

As a versatile signaling molecule, nitric oxide (NO) plays critical roles in regulating cardiovascular homeostasis. Dysfunction of the NO signaling pathway is always correlated with the increased morbidity of MI5. In this regard, exogenous supplementation with NO can prevent infarction formation6,7,8 by relaxing vascular tone, inhibiting platelet aggregation9,10, and modulating the inflammatory response11, adding a cardioprotective effect12.

Recently, significant progress has been made in the development of innovative therapeutic strategies for MI, including both cell-based13,14 and biomaterial-based15,16,17,18 approaches, and some of them are undergoing pre-clinical or clinical trials19. Cardiac patches represent an important type of strategy that has received increasing attention for the treatment of the ischemic myocardial injury. Cardiac muscle patches of clinically relevant sizes have been successfully developed from human induced-pluripotent stem cells (hiPSCs) by different research groups20,21. Acellular cardiac patches are another kind of therapy that treat MI by restricting ventricular dilatation, preventing adverse left ventricular remodeling, and improving contractile function that is directly attributable to the elastic (such as polyurethane) or viscoelastic properties22,23. In addition, biomaterial-based patches can also act as carriers to deliver biological moieties with therapeutic functions, including miRNA, proteins, and so on refs. 24,25.

In the present study, we designed a NO cardiac patch based on an original and different concept, wherein the nitrate moiety was covalently bound to biodegradable poly(ε-caprolactone) (PCL), thus transforming small-molecule drugs into functional biomaterials. The biodegradability and biocompatibility of the material guarantees that this patch can be directly implanted onto the heart, and the implanted patch demonstrated local NO delivery to infarcted myocardium that is controlled by the ischemic microenvironment, a vast improvement and advantage over glyceryl trinitrate (GTN) patches. Site-specific delivery of NO provided effective cardioprotection, thus markedly ameliorating heart function and attenuating adverse remodeling. The therapeutics efficacy was further confirmed in a clinical-relevant porcine MI model, with the intent to establish the substantial clinical viability and improvement of the NO cardiac patches.

Results

Fabrication and characterization of nitrate-functionalized cardiac patches

PCL oligomers with both ends capped with nitrates (PCL-ONO2) were first synthesized by acylation of PCL-diol (Mn = 2000) with 4-bromobutanoyl chloride, followed by substitution of the terminal bromide with AgNO3 (Supplementary Fig. 1), and the structure of PCL-Br and PCL-ONO2 was verified by 1H NMR and 13C NMR spectra (Supplementary Figs. 2, 3). Then, PCL-ONO2 was blended with high molecular weight PCL (Mn = 80,000) at a ratio of 1:9 (m/m) and processed into a fibrous mat by electrospinning (Fig. 1a). As a control, plain PCL patches were prepared by the same protocol. The scanning electron microscope (SEM) image showed a homogenous fibrous structure with an average fiber diameter of 0.69 μm and an average pore size of 3.4 μm, which is similar to those of the PCL patch (Fig. 1b and Supplementary Table 1). Mechanical properties were first characterized by tensile testing, and the NO patch demonstrated moderately decreased tensile strength and elongation rate due to the incorporation of low molecular weight PCL-ONO2 (Fig. 1c and Supplementary Table 1). The addition of PCL-ONO2 also improved surface hydrophilicity with a decreased contact angle (Fig. 1d).

a Schematic illustration of the fabrication of nitrate-functionalized cardiac patch and application for the treatment of myocardial infarction. After blending PCL-ONO2 with high molecular weight PCL, a nitrate-functionalized cardiac patch was fabricated through electrospinning, and implanted to cover the infarcted myocardium. Under ischemic microenvironment, nitric oxide was generated from the nitrate-functionalized patch to offer therapeutics for MI. b Representative images of the nitrate-functionalized cardiac patch (scale bar, 5 mm) and its microstructure demonstrated by SEM (scale bar, 20 μm). Three independent tests were repeated with similar results. Characterization of the nitrate-functionalized patch in terms of mechanical properties (c) and water contact angle (d). Data are expressed as mean ± SD, n = 3 independent repeats.

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When the patch was implanted subcutaneously in rats, the total NOx within the patch was reduced in a controlled manner, and the residual NOx exceeded 26.6% after 28 days of implantation (Supplementary Fig. 4).

Local delivery and site-specific accumulation of NO in the infarcted myocardium

A nitrate-functionalized cardiac patch was further utilized for the treatment of rat MI, and regional NO generation in the ischemic heart was first evaluated by electron paramagnetic resonance (EPR) assay using ferrous N-diethyl dithiocarbamate (Fe-DETC) as the spin-trapping reagent (Fig. 2a)26. The resultant NO adduct (DETC)2Fe–NO exhibited a characteristic triplet EPR signal (aN = 12.78 G, giso = 2.041) at room temperature (Fig. 2b). The quantitative data indicated that in the distal region, there was no detectable difference in NO levels among the various groups (Fig. 2c). In the infarcted myocardium, NO levels were moderately enhanced in both MI and MI + PCL groups compared to the sham group due to endogenous NO production by activated macrophages (Supplementary Fig. 5). The NO level was markedly elevated after NO patch treatment, which was significantly (p < 0.001) higher than that after PCL patch treatment (Fig. 2d), suggesting exogenous NO generation from NO patches in infarcted myocardium. As a comparison, the NO patch was also administered to the hearts of rats without MI; the NO level was much lower than that in NO patch-treated MI hearts (Fig. 2d). All these results support the augmented NO generation from nitrate-functionalized patches in the ischemic heart because of the hypoxic and acidic microenvironment that has been investigated extensively before27. Next, site-specific accumulation of NO in the infarcted myocardium was detected by near-infrared (NIR) fluorescence imaging using the NO-sensitive probe DAC-S, developed by Sasaki et al.28 (Fig. 2e, f, Supplementary Figs. 6–8), and regional accumulation of NO was clearly demonstrated in the infarcted zone after NO patch treatment compared to the MI group without any treatment (Fig. 2f).

ad NO-patch was applied for the treatment of MI in rats, and NO accumulation in the myocardium was measured by EPR assay. One day after surgery, cardiac tissues were collected and divided into infarcted part and distal part, which were subjected to EPR analysis, respectively. a Schematic illustration of EPR analyses. b Representative EPR spectra reflecting NO generation in the infarcted myocardium in the presence of (DETC)2Fe. NO levels in the distal (c) and infarcted (d) myocardium were determined by quantitation of (DETC)2Fe–NO complex using 2,2,5,5-tetramethyl piperidine 1-oxyl (TEMPO). Data are expressed as mean ± SEM, n = 6 animals for each group. Significant differences were detected by two-tailed one-way ANOVA with Tukey’s multiple comparisons test. e, f NO accumulation in the myocardium was detected by imaging of the infracted myocardium with NO-sensitive fluorescent probe. The principle for the detection of NO by the probe was shown in (e). The probe DAC-S was non-fluorescent; NO induced a significant fluorescence turn-on response at 805 nm. Representative NIR fluorescence images of infarcted myocardium in different locations were shown in (f). LAD: left anterior descending artery; EPR: electron paramagnetic resonance; DETC(Fe2+): ferrous N-diethyl dithiocarbamate; NO: Nitric oxide.

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Mechanism of biotransformation of nitrate-functionalized patch into NO

The biotransformation of nitrate-functionalized patch into NO proceeds through a stepwise process (Fig. 3a), which is different from organic nitrates that directly liberate into vasculature and release NO via intracellular metabolism by specific enzymes. Nitrate-functionalized polymers cannot cross the cell membrane; instead, they first hydrolyze to release nitrate anion through a non-enzymatic process as demonstrated by an in vitro assay (Fig. 3b). Nitrate anion is the major hydrolysis product during early stage because of the relatively slow degradation of bulk PCL (Supplementary Fig 9). The released nitrate anion can further be metabolized to NO through the nitrate-nitrite-NO reduction pathway under the catalysis of various endogenous enzymes29,30. The conversion of nitrate and nitrite has been observed in PBS buffer with the addition of tissue homogenates from the hearts, which confirmed the presence of relevant reductases (Fig. 3c, d).

a Schematic diagram illustrates the biotransformation pathway of PCL-ONO2 into NO via a stepwise pathway. b In vitro release of NO3 from nitrate-functionalized patch in PBS buffer (pH = 5.4 and 7.4) through the non-enzymatic hydrolysis of PCL-ONO2 (n = 3). c Reduction of NO3− into NO2 in the presence of heart homogenate (10 mg/mL) (n = 3 independent repeats). d Reduction of NO2 into NO in the presence of heart homogenate (10 mg/mL) (n = 3 independent repeats). e, f The levels of pO2 (n = 4 animals per group) and pH (n = 3 animals each group) in sham operated or infarcted myocardium were measured. Data are expressed as mean ± SEM.

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More importantly, the step-by-step transformation is responsive to the pathologic microenvironment of the ischemic heart. First, hypoxia occurred due to the obstruction of blood flow. Following left anterior descending (LAD) ligation, cardiac partial pressure of oxygen (pO2) decreased (Fig. 3e, Supplementary Fig 10), indicating obvious hypoxia. In addition, ischemia also leads to the local accumulation of H+ with pH of about 5.7 as determined by 31P NMR (Fig. 3f, Supplementary Fig. 11). As a result, acidotic microenvironment accelerates the non-enzymatic hydrolysis of nitrate-functionalized PCL (Step 1), which is a crucial step in determining the whole process of NO biotransformation (Fig. 3b). The conversion of nitrate and nitrite by the reductase within the heart tissue has also been enhanced under the acidotic and reduced conditions as reported before27.

In addition, the local and sustained generation of NO by the nitrate-functionalized patches efficiently abolishes side effects, such as hypotension observed in the utilization of organic nitrate drugs by oral or transdermal delivery route due to systemic NO delivery (Supplementary Fig. 12)31,32.

Local NO delivery provides a protective effect in cardiac ischemia

Nagar-Olsen staining was performed to detect cardiac injury at an early stage, in which the damaged myocardium was stained red. After NO patch treatment, the staining area was significantly diminished, suggesting relieved cardiac injury (Fig. 4a). Cell apoptosis at both border and infarcted zones was also decreased, as illustrated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (Fig. 4b, Supplementary Fig. 13). These data support the cardioprotection conferred by the NO patch in MI.

a Representative Nagar-Olsen staining images showing ischemic damaged myocardium, which was stained in red (scale bar, 1 mm); the injury severity was further evaluated by measuring the staining area (n = 5 animals per group). b TUNEL staining was performed to detect apoptotic nucleus (scale bar, 40 μm), and the quantity of TUNEL positive nucleus in the ischemic border zone and infarcted zone was determined, respectively (n = 5 animals per group). c Relative myocardial blood flow was measured through intraventricular perfusion of fluorescent microspheres (n = 3 animals per group). d The activity of mitochondrial complex I and II as well as the hydrogen peroxide levels in cardiac tissues were measured one day post-surgery (n = 5 animals per group). Data are expressed as mean ± SEM. Significant differences were detected by two-tailed one-way ANOVA with Tukey’s multiple comparisons test.

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To further explore the underlying mechanism, myocardial blood flow (MBF) was evaluated through intraventricular perfusion of fluorescent microspheres33. Treatment with the NO patch after LAD artery ligation efficiently restored blood perfusion in the border zone in contrast to treatment with the PCL patch (Fig. 4c and Supplementary Fig. 14). Moreover, implantation of NO patches elevated pO2 levels in MI hearts, suggesting improved oxygen distribution and perfusion status. Implantation of NO patches to non-infarcted hearts resulted in negligible changes to pO2 in the myocardium (Supplementary Fig. 10).

The cardioprotective function of NO has been attributed to the vital role it plays in the mitochondrial respiratory12. Hence, we detected the activities of mitochondrial complex I/II. We found that both the activities of mitochondrial complexes I and II were suppressed after NO patch treatment (Fig. 4d), and consequently, the level of hydrogen peroxide (H2O2) in cardiac tissues was reduced (Fig. 4d). Endogenous NO may cause S-nitrosylation (SNO), a type of posttranslational modification that can regulate protein function and cellular signaling. It has been accepted that the enhanced SNO of mitochondrial complex I inhibits its activity, thereby limiting ROS generation and providing cardioprotection12,34,35. In this study, we also observed that treatment with NO-patch increased the level of S-nitrosylated proteins in MI heart (Supplementary Fig 15). These results implied that NO delivery by NO patches quenched ROS production from mitochondria, which was beneficial for reducing cell apoptosis and ameliorating cardiac injury.

Local NO delivery modulates cardiac inflammation

The inflammatory response after patch implantation was first evaluated by histology analysis; haematoxylin and eosin (H&E) staining demonstrated decreased leukocyte infiltration after NO patch treatment compared to PCL patch treatment 3 days post-surgery (Supplementary Fig. 16). In addition, robust cardiomyocyte survival in the cardiac tissues was detected in NO patch-treated hearts. Macrophage infiltration in the infarcted hearts was further investigated by immunostaining with CD68, and the number of infiltrated macrophages was significantly (p < 0.001) decreased in the NO patch-treated hearts compared to the MI and PCL patch-treated hearts (Fig. 5a).

a Representative images showing macrophage infiltration in infarcted heart by immunostaining with CD68 (scale bar, 40 μm) as well as the corresponding quantitative analysis. b, c Macrophage polarization was further detected by immunostaining targeting CD86 and CD206, the maker of M1 and M2 phenotype, respectively (scale bar, 100 μm). d The expression of pro-inflammatory and anti-inflammatory cytokines was evaluated by RT-PCR. Data are expressed as mean ± SEM, n = 5 animals per group. Significant differences were detected by two-tailed one-way ANOVA with Tukey’s multiple comparisons test. α-SA: α-sarcomeric actin; CD: cluster of differentiation; IL-10(interleukin-10); Arg-1(arginase-1); IL-1β(interleukin-1 β); TNF-α(tumor necrosis factor-α).

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The modulatory effect of local NO delivery on macrophage polarization was further evaluated by immunostaining targeting CD86 and CD206, markers of M1 macrophages and M2 macrophages, respectively. The results showed that after NO patch treatment, the number of M2 (anti-inflammatory and reparative) macrophages was significantly increased, while the quantity of M1 (pro-inflammatory) macrophages was decreased (Fig. 5b, c). Moreover, the expression of IL-1β and TNF-α, two classic pro-inflammatory cytokines of M1 macrophages, was also decreased, while the expression of Arg1 and IL-10, anti-inflammatory cytokines of M2 macrophages, was significantly upregulated after NO patch administration (Fig. 5d). Collectively, local NO delivery demonstrated a modulatory effect on inducing macrophages into the M2 phenotype, thus prohibiting inflammation and promoting tissue repair and regeneration.

Local NO delivery improves cardiac function in MI rats

Echocardiography assessment demonstrated that the NO patch effectively improved cardiac function with enhanced values of ejection fraction (EF) and fractional shortening (FS) 1-day post-surgery, and the improvement lasted over 4 weeks. In contrast, the PCL patch showed only a temporary supportive effect (1 day) that was eventually diminished at 28 days; both the EF and FS were similar to those in MI rats, which exhibited obvious implications of heart failure (Fig. 6a). Consistently, the increased dimensions of the left ventricle (LVIDd and EDV) in the MI and PCL patch-treated groups also indicated the occurrence of dilated cardiomyopathy, one common cause of congestive heart failure (Fig. 6a).

a Cardiac echo measurement was performed at different time points post-surgery, and cardiac function indicators of left ventricular-ejection fraction (LV-EF), left ventricular-fractional shortening (LV-FS), left ventricular internal diameter at end diastole (LVIDd) and left ventricular end-diastolic volume (LV-EDV) were evaluated accordingly. Data are expressed as mean ± SEM, n = 5 animals per group. Significant differences were detected by two-tailed two-way ANOVA with Tukey’s multiple comparisons test. LV-EF: p = 0.0254 (Day 1, PCL patch vs MI), p = 0.0199 (Day 1, NO-patch vs MI), p = 0.0017 (Day 28, NO-patch vs MI), p = 0.0006 (Day 28, NO-patch vs PCL patch); LV-FS: p = 0.0377 (Day 1, PCL patch vs MI), p = 0.0306 (Day 1, NO-patch vs MI), p = 0.0025 (Day 28, NO-patch vs MI), p = 0.0011 (Day 28, NO-patch vs PCL patch); LVIDd: p = 0.0271 (Day 28, NO-patch vs PCL patch); LVEDV: p = 0.0173 (Day 28, NO-patch vs MI, p = 0.0028, NO-patch vs PCL patch). bg Four weeks after surgery, the hearts were collected for histological analysis. b Masson trichrome staining was performed with five paraffin sections evenly separated from cardiac apex to base; infarcted size, thickness of infarcted left ventricular wall (LV-infarction) from S1–S5 and interventricular septum (IVS) were measured accordingly. Scale bar, 5 mm. c Data are expressed as mean ± SEM, n = 5 animals for each group. Significant differences were detected by two-tailed one-way or two-way ANOVA with Tukey’s multiple comparisons test. **p < 0.01, ****p < 0.0001 vs MI, ####p < 0.0001 vs PCL patch. LV infarct thickness: p = 0.0046 (S3, PCL patch vs MI), p < 0.0001 (S3–S5, NO-patch vs MI; S4–S5, NO-patch vs PCL patch). d WGA staining to show cardiomyocyte hypertrophy that was characterized with increased cross-section area (scale bar = 40 μm). e Interstitial collagen deposition in the heart was detected by Sirius red staining (scale bar = 40 μm). f Hearts collected 3 days after surgery were evaluated by immunostaining with CD31, and CD31 positive capillaries were quantified. Scale bar, 60 μm. g Hearts collected 28 days after surgery were evaluated by immunostaining with α-SMA, and α-SMA positive arterioles were quantified. Scale bar, 60 μm. dg Quantitative data are expressed as mean ± SEM, n = 5 animals for each group. Significant differences were detected by two-tailed one-way ANOVA with Tukey’s multiple comparisons test.

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The long-term effect of the NO patch on cardiac remodeling after ischemic injury was further investigated by histological analysis. Masson trichrome staining showed that treatment with the NO patch significantly (p < 0.01) reduced the infarct size and sustained the thickness of the interventricular septum (IVS). Concurrently, evidently increased thickness of the infarcted left ventricular wall was observed in sections distal to the apex due to the increased cardiomyocyte survival in the infarcted zone (Fig. 6b, c). The cross-section area of cardiomyocytes at the distal area of the infarction was further determined by WGA staining, and the NO patch group showed marked suppression of cardiomyocyte expansion compared to both the MI and PCL patch groups (Fig. 6d). Moreover, intensive collagen deposition was observed in MI and PCL patch-treated rats, showing typical pathological changes of heart failure (Fig. 6e).

Rebuilding of the vasculature favors regeneration and repair of the infarcted myocardium. In this study, local NO delivery effectively promoted angiogenesis with markedly enhanced capillary density at an early stage (day 3) (Fig. 6f). Neovascularization at 28 days was further evaluated by immunostaining targeting α-smooth muscle actin (α-SMA), a marker of functional arterioles; the number of α-SMA+ blood vessels distributed at the infarcted border zone was significantly (p < 0.05) higher in the NO patch group than in the MI and PCL patch groups (Fig. 6g).

To implement the research in a clinically applicable setting, we further introduced the NO-patch to a chronic model that underwent 4 weeks MI, and the therapeutics were evaluated (Fig. 7a). As shown by the results of Masson trichrome staining (Fig. 7b, c) and functional measurements (Fig. 7d, e), we concluded that implantation of NO-patch 4 weeks after MI effectively sustained cardiac function and suppressed cardiac dilation. These results further confirmed the benefit roles of NO-patch in a clinically applicable MI model.

a Schematic image showing the NO-patch treatment for a chronic rat MI model. b Representative Masson trichrome staining images of the heart 4 weeks after different treatments. Scale bar, 5 mm. c The infarct size was calculated according to Masson staining. de. Quantitative data of echocardiography for measurement of cardiac function, and representatively, the left ventricular (LV) ejection fraction (LV-EF) and fraction shortening (LV-FS) were shown. Data are expressed as mean ± SEM, n = 6 animals for each group. Significant differences were detected by two-tailed one-way (b) or two-way (d and e) ANOVA with Tukey’s multiple comparisons test. LAD: left anterior descending artery.

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Local NO delivery improves cardiac function in a porcine model of MI

The therapeutic efficacy of nitrate-functionalized cardiac patches was further evaluated in a clinically relevant porcine model of cardiac ischemia/reperfusion (I/R) injury (Fig. 8a, Supplementary Fig. 17a). First, the induction of MI was confirmed by electrocardiography (ECG) recording with an obvious ST-segment elevation after I/R (Supplementary Fig. 17b). At the same time, markedly increased levels of cardiac injury markers, serum levels of cardiac troponin I (cTnI) and Creatine kinase-MB (CK-MB), were detected (Supplementary Fig. 17c). Then, the NO patch was implanted to treat pigs with MI, and survival curves of MI pigs with or without NO patch treatment were plotted individually. I/R caused a mortality rate of 50% in the model group (I/R only), while the survival rate was efficiently enhanced after NO patch treatment (Fig. 8b). More importantly, the ejection and contraction functions were ameliorated, as demonstrated by the significantly (p < 0.05) enhanced EF and FS, together with decreased ESV and LVIDs 28 days post-surgery (Fig. 8c).

a Experimental schedule for the treatment of MI by epicardial implantation of NO patches. b Survival rates of pigs after MI with or without NO-patch treatment were recorded. NO patches improved survival of MI pigs. c Representative echo images reflecting cardiac function, and accordingly, left ventricular-ejection fractions (LV-EF), fraction shortening (LV-FS), left ventricular internal diameter at end diastole (LVIDs) and volume at diastole (ESV) were assessed (n = 4 pigs in I/R group, n = 6 pigs in NO-patch group). d Representative MRI images obtained on day 1 and 7 (left, end systole; right, end diastole) and LV-EF as well as cardiac output (CO) were measured on day 7 accordingly (n = 3 for each group). Data are expressed as mean ± SEM. Significant differences were detected by two-tailed student‘s t test at indicated time points.

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Cardiac magnetic resonance imaging (cMRI), a gold standard for accurate functional assessment in the clinic, was further performed to monitor cardiac performance and function. The results indicated marked enhancement in EF and cardiac output (CO) in NO patch-treated pigs compared to I/R injured pigs, confirming the improved cardiac function (Fig. 8d).

Local NO delivery attenuates adverse remodeling in MI pigs

To further elucidate tissue regeneration and remodeling after I/R injury, Masson trichrome staining was first performed in five sections (S1–S5) acquired from each heart (Fig. 9a), and accordingly, the fibrotic area in each section was measured (Fig. 9b). The area under the curve (AUC) was calculated to indicate the total scar size (Fig. 9c). From these results, we can learn that NO patch treatment strikingly decreased the infarct size. In addition, reduced infarct size was observed in NO patch-treated hearts through cMRI based on late gadolinium enhancement (LGE-cMRI) (Fig. 9d).

ac Masson trichrome staining was performed in sections acquired from five planes from cardiac apex to base (S1-S5, scale bar, 10 mm), and fibrotic area in each section was measured accordingly (b), the area under curve (AUC) was calculated to reflect the infarcted size (c) (n = 4 pigs in I/R group, n = 3 pigs in NO-patch group). d Representative LGE-cMRI images demonstrating infarcted size 7 days post-surgery, and the infarcted zone was circled and quantified. e, f Neovascularization was detected by immunostaining with CD31 and α-SMA, and correspondingly the quantity of capillaries and arterioles was determined. g Cardiomyocyte proliferation was detected by co-staining with Ki67 and cardiomyocyte marker α-SA, and double positive proliferating cardiomyocytes were quantified (n = 4 pigs in I/R group, n = 6 pigs in NO-patch group). Scale bar = 100 μm. Data are expressed as mean ± SEM. Significant differences were detected by two-tailed student‘s t test. I/R: ischemia/reperfusion; AUC: area under curve.

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Next, neovascularization in the infarcted myocardium was detected by immunostaining for CD31 and α-SMA (Fig. 9e, f), and the results confirmed enhanced blood vessel regeneration (both capillary and arteriole) after NO treatment. Moreover, cardiomyocytes in the border zone of I/R injured hearts with NO patches also demonstrated pronounced Ki67 labeling (Fig. 9g), indicating increased proliferation by local delivery of nitric oxide.

Discussion

Organic nitrates represent a conventional class of NO-donating agents that have been used to treat CVDs (ischemic heart disease, heart failure, and hypertension) for many years4. After uptake, organic nitrates first experienced denitration and were metabolized to nitrate (NO3) or nitrite (NO2) depending on the structure of them29. The bioactivation is mediated by either non-enzymatic (endogenous reductants) or enzymatic reactions. Several enzymatic reactions are involved in GTN bioactivation, including aldehyde dehydrogenase 2 (ALDH2), xanthine oxidoreductase, and the cytochrome P45036.

Currently, there is accumulating evidence demonstrating that inorganic nitrate represents an important reservoir for NO in the body4,37. Under specific conditions, inorganic nitrate can be metabolized to generate NO through the nitrate–nitrite–NO reduction pathway. Inorganic nitrate is found in considerable amounts in the diet, and many green vegetables such as spinach or beetroot are particularly rich in nitrate. Recently, supplementation of inorganic nitrate has been attempted by different groups with the aim of providing therapy for CVDs because it is free of tolerance phenomenon. Promising outcomes have been reported, including improved revascularization in chronic ischemia38, and stabilized atherosclerotic plaques39.

It has been widely accepted that nitrate can be reduced to nitrite mainly by commensal bacteria in the digestive system. In addition, there are several recently discovered mammalian enzymes with nitrate reductase activity. Nitrite can then be further reduced to bioactive NO and consequently demonstrate direct vasoactive effects through various enzymatic pathways40.

Among the different types of endogenous reductases for nitrite, myoglobin has received increasing attention in the cardiovascular system. It has been reported that deoxygenated myoglobin expressed in the heart can reduce nitrite to nitric oxide and thereby contribute to cardiomyocyte NO signaling during ischemia41. This effect has been confirmed in myoglobin knockout mice; that is, administration of nitrite reduced MI in myoglobin+/+ mice with cardiac ischemia-reperfusion (I/R) injury, whereas the cardioprotective effect was abolished in myoglobin−/− mice40. Gladwin et al. also found that there is relatively little myoglobin present in lung tissue. Studies in the heart have revealed that 60% to 70% of NO generation from nitrite is mediated by the nitrite reductase activity of deoxymyoglobin, whereas in the lung, xanthine oxidoreductase (xOR) plays a role, with >70% of nitrite reduction mediated by xOR42.

In this regard, nitrate and nitrite, previously considered inert oxidation products of NO metabolism, are now recognized as an important circulating reservoir of NO. On the other hand, in vivo transformation efficiency of inorganic nitrate is influenced by many factors, including the tissue, the pH, oxygen tension and redox status43. In addition, a major health concern is that higher doses of nitrite derived from dietary nitrate may cause severe methemoglobinemia. It may also lead to an increased cancer risk due to the formation of the carcinogen substances N-nitrosamines from ingested nitrate and nitrite44. As a result, the nitrate level in drinking water is strictly regulated in many countries4.

In our study, EPR measurements clearly demonstrated that NO generation from nitrate-functionalized patches was ischemia-responsive; the NO signal in ischemic hearts was markedly elevated compared to that in non-ischemia hearts, with only marginal enhancement detected. This suggests that a low level of NO was generated from the nitrate-functionalized patch in a balanced physiological environment, whereas under ischemic conditions, acidosis occurs as a result of anaerobic metabolism and H+ accumulation45,46 and to a greater extent with the disturbance in the redox that tends to be reductive because of the lack of oxygen species47. Besides, in situations of hypoxia, when oxygen-dependent NO synthases become dysfunctional, nitrite reduction is instead greatly enhanced48. These pathological events create the appropriate microenvironment that accelerates NO generation from nitrate-functionalized patches27.

The underlying mechanism for the protective role of NO in cardiac ischemia has been extensively investigated during the past two decades37. Generally, the therapeutic efficacy can be explained from the following aspects.

First, mitochondria have been viewed as important targets in the cardioprotection provided by nitric oxide49,50. Nitric oxide (NO) has been known for many years to bind to cytochrome C oxidase, the terminal acceptor in the mitochondrial electron transport chain, in competition with oxygen51. This competition results in an inhibition of respiration that is more potent when oxygen tension is decreased.

The protective mechanism has been further elucidated by the fact that NO inhibits the activity of complex I in the mitochondrial electron transport chain through S-nitrosation of critical thiols on it, thereby limiting mitochondrial ROS generation35. This further prevents mitochondrial permeability transition pore opening and cytochrome c release52. Our results also demonstrated the inhibitory effect of NO on complex I as well as ROS generation. As a result, cardiomyocyte apoptosis was efficiently attenuated at an early stage in the nitrate patch-treated group. More prominent protective effects, including suppressed adverse cardiac remodeling, reduced myocardial infarct size, and ameliorated heart function, have been observed after long-term treatment by functional cardiac patches6.

Another protective effect of NO has also been attributed to the activation of ATP-sensitive potassium channels (KATP channels). NO–cGMP-dependent signaling leads to the opening of mitochondrial KATP channels and reduced calcium overload43. The activity of mitochondrial respiratory chain complex II was also evaluated, since it has been proposed as an important regulator of mKATP activity. The reduced complex II activity after nitrate-functionalized patch treatment in this study is consistent with the trend reported by another group53.

Finally, inflammatory modulation is another function of NO. Inflammation is complicated in MI, leading to myocardial injury and severe fibrosis. In this study, NO delivery by nitrate-functionalized cardiac patches modulated macrophage polarization into the M2 phenotype, which can promote wound healing and tissue repair. Furthermore, NO delivery downregulated the expression of pro-inflammatory cytokines while upregulating pro-repair cytokines involving IL-10, which is an important mediator of tissue repair.

Tolerance represents a major limitation of some organic nitrates utilized in the clinic, especially glyceryl trinitrate (GTN). GTN is the most commonly utilized treatment for various ischemic and congestive cardiac diseases54,55,56, although there are also organic nitrates (such as pentaerythritol tetranitrate; PETN) that are devoid of the effects of tolerance emergence57,58. Nitrate tolerance is a complex phenomenon and yet to be clearly elucidated. In general, the typical consensus is that nitrate tolerance is mainly caused by oxidative inactivation of the reductase activity of aldehyde dehydrogenase-2 (ALDH2), which results in decreased GTN bioactivation, and the resultant diminished levels of nitrite (NO2) and nitric oxide (NO). Recently, Mochly-Rosen et al. have also reported that activators of ALDH2 such as Alda-1 may help to inhibit cardiac injury induced by GTN tolerance59,60.

In contrast, as a macromolecular nitrate, PCL-ONO2 demonstrates a specific biotransformation pathway that is different from that of small-molecule drugs. First, polymers undergo non-enzymatic hydrolysis to release nitrate anion locally in infarcted myocardium. Then, the generated nitrate anion is reduced to release NO via the nitrate–nitrite–NO sequential pathway. Since the phenomenon of tolerance is not exhibited with the consumption of inorganic nitrate/nitrite61, it is reasonable to speculate that PCL-ONO2 developed in this study is free from tolerance because the process of biotransformation is different from that of classic organic nitrates. In addition, nitrate-functionalized PCL does not require additional activators (such as Alda-1), although relevant experiments are required to validate this hypothesis.

Collectively, the stepwise biotransformation of nitrate-functionalized polymers proceeds slowly under physiologic conditions and could be accelerated by microenvironment of the ischemic heart. It enables the local generation of inorganic nitrate in a sustained manner and ischemia-enhanced NO transformation that not only guarantees the conversion efficiency but also abolishes disadvantages associated with the systemic nitrate administration as mentioned before. Besides, the nitrate-functionalized polymer and the cardiac patch have a vast improvement and advantage over organic nitrates administrated by transdermal patch or other routes wherein small molecular drugs rapidly release into the vasculature and the bioconversion to NO mainly occurs through intravascular metabolism62. The short half-life of NO (~6 s) restricts its entry into the tissue/organs, thus the therapeutics is mainly based on hemodynamic effects of NO8. Compared to organic nitrates, local generation of NO from nitrate-functionalized patches remarkably abolishes adverse effects, including serious hypotension caused by the vasodilation of systemic vasculature when higher doses of organic nitrates are administrated via oral or transdermal pathways. Local NO delivery can further provide non-hemodynamic effects on the ischemic myocardium, including cardioprotection and immunomodulation. As a result, different NO conversion pathways lead to the divergent releasing profiles that bring on different therapeutic efficacy finally (Fig. 10).

Left: Organic nitrates administrated by transdermal patch or other routes often release into the vasculature rapidly and transform to NO through intravascular metabolism. Due to the short half-life of NO the therapeutics is mainly based on hemodynamic effects through dilation of vascular smooth muscle. Right: Compared to small molecular organic nitrates, local NO delivery by nitrate-functionalized patches can further provide non-hemodynamic effects on the ischemic myocardium, including cardioprotection and immunomodulation.

Full size image

Furthermore, a head-to-head comparison study has been performed in order to demonstrate the advantages and disadvantages of nitrate-functionalized patches through systemic comparison with both organic nitrate, isosorbide mononitrate (iso-mono) and inorganic sodium nitrate (NaNO3) that have been reported previously by utilizing the rat cardiac ischemia/reperfusion(I/R) model. Ten days after treatment, cardiac histology and function were analyzed. Results show that after intake of sodium nitrate there was a reduction in fibrotic area and infarct size (Supplementary Fig. 18a–c). However, it did not show a positive effect on the cardiac function (Supplementary Fig. 18d, e). In contrast, treatment with isosorbide mononitrate and NO-patch significantly improved the outcomes of cardiac I/R injury, the fibrotic area as well as the infarct size were reduced (Supplementary Fig. 18a–c), and the cardiac ejection function was improved (Supplementary Fig. 18d, e). However, serious cardiac arrest was observed in rats (3/7) after gavage of isosorbide mononitrate which could be caused by hypovolemic shock. As intake of isosorbide mononitrate resulted in whole-body vasodilation and hypotension, which turns to be the risk factor of hypovolemia. Besides, the amount of administrated iso-mono (0.15 mmol) is higher than that of PCL-ONO2 present in the NO patch (0.25 μmol). As a result, despite the comparable therapeutic efficacy, we believe that NO-patch implantation is a promising strategy for cardioprotection and cardiac repair in terms of biotransformation efficiency and safety due to the local NO generation that is responsive to ischemia microenvironment after MI.

The application of our patch will be primarily focused on usage during coronary artery bypass grafting surgery (CABG), wherein the implanted patch played a significant role in repairing the damaged myocardium after revascularization as a consequence of ischemia and ischemia-reperfusion injury (IRI). NO-releasing coatings have been employed in the development of drug-eluting stents9,10 for coronary heart diseases, and that some of these have entered into pre-clinical trials. However, based on the limited data that has been reported up until now, the therapeutic efficacy of NO-releasing stents is far from satisfactory because of intimal hyperplasia representing the major adverse event following the complete release of loaded NO-drug in longer-term studies63. In light of this report, it was long-term NO delivery that proved to be a main challenge. This is because existing drug-eluting systems have a finite depot of drug and will lose therapeutic function once the depot in depleted64. Instead, surface modification of bare stents by immobilizing relevant enzymes to strengthen endogenous NO generation65 or catalyze the bioactivation of endogenous NO donors10, such as S-nitrosothiol, may represent a new paradigm for the future of NO-releasing stents.

The relationship between NO and cardiomyocyte proliferation has not been well explored in this study. We indeed detected increased numbers of Ki67+, PH3+, Aurora B+ cardiomyocytes after NO-patch treatment, however, there is no actual division as shown by the formation of midbody observed in Aurora B staining, therefore further studies on the relationship of NO with cardiomyocyte mitosis and cardiac regeneration are needed to reach a consensus conclusion.

In summary, a nitrate-functionalized cardiac patch has been successfully fabricated. The functional patch demonstrated site-specific delivery of NO into the infarcted myocardium under ischemic conditions. In a rat model of MI, administration of NO patches demonstrated optimized therapeutic efficacy, including reduced cardiac injury at an early stage, suppressed adverse cardiac remodeling and ameliorated heart function after long-term treatment, confirming the protective role provided by locally released NO. The translational potential of nitrate-functionalized patches was further evaluated in a porcine ischemia/reperfusion MI model. Cardiac magnetic resource imaging (cMRI) measurements and echography detection reflected remarkably enhanced cardiac function after NO patch implantation, while reduced infarct size and attenuated adverse remodeling were also observed in NO patch-treated hearts.

Methods

Synthesis of PCL-ONO2

All chemicals and reagents were purchased from Sigma-Aldrich (China-mainland), Energy Chemical (China-mainland), and Alfa Aesar (China-mainland), and used directly without further purification. Silica gel (200-300 mesh, Qingdao) was used for column chromatography. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance-400 FT nuclear magnetic resonance spectrometer. Chemical shifts were reported relative to the reference chemical shift of the NMR solvent. The following splitting abbreviations were used: s = singlet, d = doublet, dd = doublet doublet, t = triplet, m = multiplet.

PCL-ONO2 was first synthesized via two steps starting from PCL-diol (Mn = 2000). Briefly, the solution of PCL-diol (5 g, 2.5 mmol) in dry DCM (50 mL) at 0 °C was added sequentially dry TEA (2.07 mL, 15.0 mmol) and 4-bromobutanoyl chloride (1.855 g, 10.0 mmol). The resulting mixture was gradually warmed to room temperature and continued to be stirred for 24 h under argon atmosphere. After completion, the solvent was removed under reduced pressure to give the crude product which was purified by silica column chromatography, eluting with DCM/MeOH (100:4), to give intermediate PCL-Br (3 g) in 60 % yield. 1H NMR (CDCl3, 400 M Hz): 3.45 (t, 4H, -CH2-Br), 2.50 (t, 4H, -CO-CH2-), 2.17 (m, 4H, -CH2-CH2-CH2-), 4.06 (t, 35H), 2.31 (t, 35H), 1.65 (m, 70H), 1.38 (m, 35H), the characteristic signals at 4.06, 2.31, 1.65, and 1.38 ppm corresponded to -CH2- protons of PCL backbone, whereas the new peaks at 3.45, 2.50 and 2.17 ppm were assigned respectively to the protons of -CH2-Br, -CO-CH2- and -CH2- in the 4-bromobutanoyl ester unit. 13C NMR (CDCl3, 101 MHz): 173.54, 64.14, 34.11, 32.71, 28.34, 27.77, 25.52, 24.57, the characteristic signals at 173.48, 64.09, 34.08, 28.31, 25.49 and 24.53 ppm were from PCL backbone, whereas the new peaks at 32.71, 27.77 ppm were assigned respectively to -CH2-Br, -CH2-CH2-Br in the 4-bromobutanoyl ester unit (Supplementary Fig. 2). To a stirred solution of PCL-Br (2 g, 1.0 mmol) in anhydrous acetonitrile (20 mL) was added AgNO3 (1.5 g, 10.0 mmol) at 0 °C. Then the mixture was stirred at room temperature for 24 h. AgNO3 was removed by organic filter and the organic phase was washed with water for three times, dried over Na2SO4 and evaporated under reduced pressure to give the desirable compound as pale waxy solid (1.7 g) with a yield of 85%. 1H NMR (CDCl3, 400 M Hz): 4.52 (t, 4H, -CH2-ONO2), 2.50 (t, 4H, -CO-CH2-), 2.17 (m, 4H, -CH2-CH2-CH2-), 4.06 (t, 35H), 2.31 (t, 35H), 1.65 (m, 70H), 1.38 (m, 35H), the new peaks at 4.52 was assigned to the protons of -CH2-ONO2 in the 4-(nitrooxy)butanoyl ester unit. 13C NMR (CDCl3, 101 MHz): 173.48, 72.05, 64.09, 34.07, 28.30, 27.75, 25.49, 24.53, the characteristic signals at 173.48, 64.09, 34.07, 28.30, 25.49, 24.53 ppm were from PCL backbone, whereas the new peaks at 72.05, 27.75 ppm were assigned respectively to -CH2-ONO2, -CH2-CH2-ONO2 in the 4-(nitrooxy)butanoyl ester unit. (Supplementary Fig. 3).

Synthesis of NO probe (DAC-S)

NO sensitive probe synthesized according to a reported method30. Briefly, 4-Amino-3-nitrophenol (100 mg, 0.65 mmol) and NaH (60% in mineral oil) (30 mg, 0.65 mmol) were dissolved in anhydrous DMF (20 mL). The mixture was stirred at room temperature for 20 min under an argon atmosphere. Then a solution of IR-783 (200 mg, 0.27 mmol) in anhydrous DMF (10 mL) was added to the reaction via a syringe. The reaction mixture was further stirred for 6 h. The mixture was extracted with ethyl acetate twice (30 mL x 2), and the combined organic phase was washed with water (20 mL), brine (20 mL) and dried over Na2SO4. After removal of the solvent under reduced pressure, the crude product was purified by silica gel chromatography with 30% CH3OH in DCM to afford the intermediate (IR-NO2) as a dark green solid (180 mg, 80%).1H NMR (400 MHz, CD3OD): δ 7.89 (d, 2H, J = 14.3 Hz), 7.61 (d, 1H, J = 2.9 Hz), 7.08-7.31 (m, 9H), 6.97 (d, 1H, J = 9.3 Hz), 6.12 (d, 2H, J = 14.3 Hz), 4.04 (t, 4H, J = 6.3 Hz), 2.78 (t, 4H, J = 7.0 Hz), 2.67 (t, 4H, J = 5.9 Hz), 1.80-1.96 (m, 10H), 1.33 (s, 12H). 13C NMR (101 MHz, MeOD): δ 172.21, 163.43, 149.77, 142.20, 141.66, 141.06, 130.14, 128.43, 124.80, 124.61, 122.15, 121.96, 121.09, 110.77, 108.90, 99.98, 50.40, 48.91, 43.54, 35.57, 26.83, 25.82, 23.89, 22.22, 21.08. MS (FAB+): 845 (M – Na+ + 2H+) (Supplementary Fig. 8). To a solution of IR-NO2 (100 mg, 0.12 mmol) in MeOH (4 mL) and concentrated HCl (0.6 mL) was added SnCl2-H2O (450 mg, 2.0 mmol). The solution was stirred at room temperature overnight under an argon atmosphere, then neutralized with 2 N NaOH. The precipitate was removed by filtration and the filtrate was concentrated under reduced pressure and the crude product was purified by silica gel chromatography with 35% CH3OH in DCM to afford the desired product as a dark green solid (40 mg, 40%).1H NMR (400 MHz, CD3OD): δ 8.07 (d, 2H, J = 14.2 Hz), 7.06-7.28 (m, 8H), 6.68 (d, 1H, J = 8.4 Hz), 6.55 (d, 1H, J = 2.9 Hz), 6.31 (dd, 1H, J = 8.4, 2.9 Hz), 6.15 (d, 2H, J = 14.2 Hz), 4.11 (t, 4H, J = 5.5 Hz), 2.87 (t, 4H, J = 6.7 Hz), 2.73 (t, 4H, J = 5.8 Hz), 1.82-2.14 (m, 10H), 1.41 (s, 12H), 13C NMR (101 MHz, MeOD): δ 172.22, 172.02, 171.75, 142.54, 142.39, 142.25, 141.11, 140.89, 128.47, 124.57, 122.65, 122.55, 122.01, 110.54, 99.22, 63.98, 50.49, 48.88, 43.42, 27.34, 26.89, 25.84, 23.94, 22.27, 21.11. MS (FAB+): 815 (M – Na+ + 2H+) (Supplementary Fig. 9).

Fabrication and characterization of nitrate-functionalized cardiac patch

Briefly, high molecular weight PCL (PCL80K) was mixed with PCL-ONO2 (PCL2K) at blending ratios of 9/1 (w/w). The mixture was dissolved in mixed chloroform/methanol (5:1, v/v) by sufficient stirring to obtain homogeneous solution with final concentrations of 10 wt% (w/v). The electrospun mats were fabricated under the following processing conditions: needle tip–collector distance = 15 cm, solution flow rate = 2 mL/h, and voltage = 15 kV. The as-prepared mats were dried in vacuum at room temperature for more than 3 days in order to remove the residual solvents sufficiently.

The surface morphology of electrospun mats was observed under a field emission scanning electron microscopy (SEM; Quanta 200, Czech) at an accelerating voltage of 10 KV. The surface was sputter-coated with gold before observation.

The surface water contact angle of electrospun cardiac patch was measured by the sessile drop method using a Harke-SPCA goniometer (Beijing,China). Samples were adhered on glass slice by double-face adhesive tape and put onto the sample stage. The images were recorded continuously after water dropping on film 10 s at room temperature. The average values were obtained from 5 measurements at different positions.

Mechanical tensile testing was performed on an Instron universal tensile tester (model 5865). The electrospun mats were cut into specimens in rectangular shape with dimension of 15 × 10 × (∼0.6) mm. The distance between two grips was set as 10 mm. A tensile test was performed at ambient temperature with crosshead speed of 10 mm/min. Each test was repeated on 3 specimens.

In vitro degradation

Briefly, 10 mg/mL nitrate-functionalized materials were incubated in PBS buffer at 37 °C, then the production of nitrate and nitrite anions was evaluated according to the Griess methods using Total Nitric Oxide Assay Kit (Beyotime Biotechnology, S0023). The optical density was measured at 540 nm, and the amount of nitrite was determined using sodium nitrite as a reference standard.

In vitro NO release detection

Briefly, 10 mg/mL nitrate-functionalized PCL and 5 μM nitric oxide fluorescence probe DAF-FM DA were co-incubated in heart tissue homogenate (10 mg/mL) at 37 °C. Then, the fluorescent intensity of the reaction mixture was measured at different time points. Data were acquired through microplate reader with, λex = 490 nm and λem = 520 nm.

Subcutaneous NO release detection

Circular samples (6 mm in diameter) were implanted subcutaneously into the dorsal skin of the rat after recording the weight, and at pre-determined time points, the implants were recollected. Then the surrounding tissue was peeled off, followed by washing with sterile saline and air dried. The residual NOx content was assessed by using chemiluminescence NO analyzer (NOA) (Seivers 280i, Boulder, CO). In brief, the samples were immersed in 5 mL of Vanadium (III) chloride (50 mM/L). The generated NO gas was diffused in the test solution and transported to NO analyzer by a stream of N2 (g). The calculation of the generated NO was based on the calibration curves of the NOA, which was described in detail elsewhere66.

Rat MI model

Male Sprague-Dawley (SD) rats (8-week old) were used according to the Animal Use Guidelines. All procedures for rat experiments were approved by the Animal Care and Use Committee of Nankai University. To ensure comparable severity of injury and injury size between groups, rats without pre-determined assignment received LAD ligation at a position 2 mm below the cross-junction of the left atrial appendage and the pulmonary conus, and patch implantation was performed randomly by a surgeon blind to the groups. The rat model of acute MI was established as described previously. Rats were anesthetized via intraperitoneal injection of 10% chloral hydrate (350 mg/kg), followed by fixation to a heating pad (37 °C) at supine position. Endotracheal intubation was performed and rats were ventilated with a mechanical respirator (Hallowell EMC Microvent I) setting at 110 breaths per minute with a tidal volume of 6 ml. After removing the fur covering the chest, an oblique incision of skin from xiphoid towards mid-axilla was made. The muscles were separated by blunt operation without cutting to expose thorax. Then the heart was exposed through a thoracotomy between the third intercostal and the LAD was ligated with 6-0 suture. Cardiac ischemia was confirmed by the observation of pallor and cyanosis in left ventricular. Immediately after LAD ligation, cardiac patches (4 mm × 6 mm) were fixed closely to cover the infarcted myocardium with 8-0 suture. Then the incision was closed and the animals were allowed to recover from anesthesia. Rats in MI group only received LAD ligation without patch implantation, while sham operated rats only experienced thoracotomy without LAD ligation and patch implantation. Buprenex (0.01 mg/kg) and Carpofen (5.00 mg/kg) were administrated to relief pain and distress after procedures.

To create a chronic injury model, MI was induced in rats, and 4 weeks later, echo was measured and followed by the secondary open chest surgery to implant patches.

Rat cardiac ischemia/reperfusion model

Rat model of cardiac ischemia/reperfusion was induced by ligation of left ascending artery for 30 min, followed by removal of the suture. After closing the incision, the rat was allowed to recover. Then the isosorbide mononitrate (75 mg/kg/day)67 or sodium nitrate (100 mg/kg/day) dissolved in sterilized saline was medicated through gavage, twice a day at an interval of 12 h. For the patch group, the patch was implanted immediately after reperfusion.

In vivo NO release detection

Electron paramagnetic resonance (EPR) assay was employed to measure the levels of NO in heart tissues as previously26. Briefly, one day after surgery, the rats were anesthetized by I.P. injection of 10% chloral hydrate (350 mg/kg). DETC sodium salt (500 mg/kg, Sigma-Aldrich) was then administrated by intraperitoneal injection in distilled deionized water (250 mM). Five minutes later, ammonium ferrous sulfate (50 mM) citrate solution (250 mM) was injected subcutaneously (2 ml/kg). After 1 h, the hearts were collected and immediately dissected into infarcted part and distal part, which were frozen separately in liquid nitrogen. Subsequently, the frozen heart tissues were crumbled into small pieces in homogenizer tubes and extracted with ethyl acetate (200 μL) immediately. The ethyl acetate extract was concentrated and transferred to 50 μL capillary tube and measured on x-band EPR at room temperature (23 ± 1 °C). The following acquisition parameters were used: modulation frequency, 100 KHz; microwave power, 10 mW; modulation amplitude, 2 G; number of scans, 30. The double integrated area of EPR spectrum was calibrated into concentrations of NO-Fe(DETC)2 using TEMPO as a standard. EPR spectral simulation was conducted by the WINSIM program.

Ex vivo NIR imaging

NO sensitive probe (DAC-S) was dissolved in sterile saline to make a working solution of 10 μM. One day after surgery, the rats were anesthetized again. After intubation and thoracotomy, the heart was exposed, and DAC-S probe was injected into myocardium at three sites involving the infarcted core and both border zones. The injection volume is 10 μL in each site. After 1 h, the heart was collected and cut into pieces for ex vivo imaging. NO specific signal was detected by using CRI Maestro noninvasive fluorescence imaging system. All images were acquired through 780–950 nm range in 10 nm steps using the “ICG” filter with excitation at 735 nm (exposure time 1000 ms; n = 3 for each group).

Cardiac function measurement

Transthoracic echocardiograph was performed by using the Vevo 2100 Imaging System equipped with a 20 MHz transducer (FujiFilm VisualSonics, Inc. Toronto, Canada) by researchers not knowing the groups as previously described68. The base line of cardiac function of rats was recorded three days before surgery. When measuring the cardiac function, all animals were anesthetized by isoflurane inhalation mixed with oxygen, and M-mode images were acquired. Then the left ventricular internal diameter at end-diastole (LVIDd) and systole (LVIDs) were measured, and accordingly, the LV-ejection fraction (LV-EF), LV-fractional shortening (LV-FS), LV end-diastole volume (LV-EDV), and LV end-systole volume (LV-ESV) were calculated as indicators of LV function and structure. All measurements were recorded from 5 continued cardiac cycles.

Measurement of myocardial blood flow

Myocardial blood flow was measured by using method of fluorescent microspheres perfusion69. Briefly, 500000 microspheres of 15μm in diameter that labeled with red fluorescence (Molecular Probes, Invitrogen, CA, USA) were injected into left ventricular over more than 20 s. Forty-five minutes later, the hearts were removed and fixed in 4% paraformaldehyde over 24 h. For the histological determination of myocardial blood flow, fixed hearts were immersed in 30% glucose for more than 48 h and subsequently embedded with OCT compound. Frozen sections in 20 μm thickness were made. The number of fluorescent microspheres in the ischemic border zone was counted under a fluorescent microscope to indicate the relative blood perfusion volume.

Mitochondrial complex activity measurement

Enzyme activities of mitochondrial respiration chain complex I and complex II were measured according to the operation manual (Solarbio, Beijing, China). Heart tissues harvested at day 1 were first made into 10% homogenate, and the mitochondrion was isolated after centrifuge and sonication. After mixing with its corresponding substrate solution, the activities of complex I and complex II were detected spectrophotometrically, respectively.

The tissue level of H2O2 was measured with 10% homogenates by using Hydrogen Peroxide/Peroxidase assay kit (Nanjing Jiancheng Bioengineering Institute, China).

Detection of NO generation by macrophages

For imaging the endogenous NO in RAW264.7 murine macrophages, the cells were pre-treated with 100 ng/mL LPS for 12 h in DMEM medium at 37 °C, and then incubated with 5 µM NO fluorescence probe DAF-FM DA (Solarbio, Beijing, China) for 2 h at 37 °C in the same medium. Images were acquired through Olympus Fluo View™ FV1200 confocal microscope with band path of 500–600 nm upon excitation at 488 nm.

Measurement of pO2

To measure cardiac pO2 levels, rats were ventilated and anesthetized with inhalation of isoflurane. A heating pad was used to keep the body temperature to be 37 °C. After expose of the heart, OxyLite probes were punctured into the myocardium and readouts were taken for one minute after stabilization. The mean values were used to indicate the level of pO2.

Measurement of pH

Cardiac pH levels was analyzed with 31P magnetic resonance spectroscopy, one day after surgery, the rats were anesthetized by I.P. injection of 10% chloral hydrate (350 mg/kg). The hearts were collected and immediately dissected into infarcted part, prepare into homogenate with D2O for detection.

Measurement of S-nitrosylated proteins

One day after patch implantation, heart sections were used for detection of S-nitrosylation by using commercially available kit (S-Nitrosylated protein detection kit, Cayman, 10006518). To proceed with this assay, heart sections were firstly incubated with blocking reagent to block free thiols in the samples, and then followed by reduction and labeling with maleimide-biotin as well as fluorescein-avidin, S-nitrosylated proteins can be localized.

Histological analysis

Histological analysis were performed by researchers blind to the groups. At indicated time points, rats were anesthetized via intraperitoneal injection of 10% chloral hydrate (350 mg/kg) following sedation with inhalation of isoflurane and fixed in surgery plate in supine position. Hearts were fixed through trans-cardiac perfusion with saline followed by 4 % paraformaldehyde (PFA), subsequently, the heart samples were immersed into 4% PFA and fixed over 24 h. Latterly, the heart samples were embedded into paraffin blocks and cut into 5 μm thickness sections. For frozen sections, heart samples need to be immersed in 30 % glucose over 48 hours after fixation with PFA, and then embedded with OCT. The frozen sections were also 5 μm in thickness, and stored at −20 °C. Hematoxylin-Eosin (H&E) staining, Masson trichrome staining, Nagar-Olsen staining and Sirus Red staining were performed with paraffin sections following a standard protocol. The cell apoptosis was detected by using DeadEnd™ Fluorometric TUNEL System (Promega, G3250) according to the operating manual, after which the sections were incubated with cardiomyocyte marker α-sarcomeric actin (α-SA) (Abcam, ab9465). DAPI was used for nucleus staining. For immunofluorescence staining, frozen sections were washed three times with PBS, and then blocked with goat serum at room temperature for 1 h. Antibodies against α-SA(Abcam, ab9465, 1:100), CD31 (Abcam, ab28364, 1:100), α-SMA (Boster, BM0002, 1:500), CD68 (Abcam, ab201340, 1:100), CD86 (Abcam, ab234000, 1:100), CD206 (Abcam, ab64693, 1:100), Ki67 (Abcam, ab16667, 1:200) diluted in goat serum were then dropped to cover sections and incubated overnight at 4 °C. Then the sections were washed three times with PBS, followed by incubating with Alexa Fluor 594-conjugated goat anti-mouse IgG (Abcam, ab150116, 1:200) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Abcam, ab150077, 1:200) for 2 h at room temperature. After washing with PBS, DAPI-containing fluoromount-G (Southern Biotech, 0100-20) was used to mounting.

qRT-PCR

The rats were sacrificed three days after surgery through CO2 inhalation, and the hearts were collected. The muscle tissues of left ventricular in the border zone were dissected and frozen in liquid nitrogen, after which the total RNA was extracted using Trizol reagent (Invitrogen, Grand Island, NY). RNA yield was determined by using NanoDrop spectrophometer (NanoDrop Technologies). First-strand cDNA was synthesized using ologo dT primers with reverse transcriptase (TransGen Biotech, China). Subsequently, real-time qPCR was performed on a CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA). The relative expression of mRNA of interest was expressed as 2−(△△CT) and normalized to housekeeping gene GAPDH. Each independent experiment was performed in triplicate. The primer sequences used in this study are listed in Supplementary Table 2.

Porcine MI model

All procedures were approved by the Ethics Committee of Shanghai Jiaotong University Animal Department. Pigs weighing 7–8 kg were used in this study. Briefly, intramuscular injection of Zoletil was performed to induce anesthesia. After orotracheal intubation, 2% isoflurane was supplied to maintain anesthesia during the surgery. Electrocardiogram (ECG), arterial oxygen saturation, blood pressure, and heart rate were monitored throughout the operation. After a thoracotomy in the left chest, the heart was exposed and ischemia was introduced by circling the LAD before the branch of second diagonal coronary arteries, and the suture was loosed to achieve reperfusion after 90 min ischemia. Cardiac patch (20 mm × 15 mm) was implanted immediately after ischemia in the experimental group, while I/R injured pigs without patch implantation were used as the control group.

To confirm the cardiac injury, at the preliminary models, blood samples were drawn before and after surgery, and plasma cTnI and CK-MB levels were measured.

Echocardiography

To detect the cardiac function, transthoracic echocardiography (ECG) was performed before and after surgery by using an echocardiographic system (Phillips CX50) equipped with a S5-1 sector array transducer as previously described. The short-axis M-mode images were presented and dimensions of left ventricle at both diastole (LVIDd) and systole (LVIDs) were measured, and accordingly, the ejection fraction (EF), fraction shortening (FS) and end-systole volume (ESV) were calculated.

3T-cMRI acquisition

MRI measurement was performed by operators not knowing the assignment of the groups. Anesthesia was achieved by applying an intramuscular injection of a mix of tiletamine hydrochloride and zolazepam hydrochloride (Zoletil®50, Virbac S.A., France). The studies were performed on a 3.0T-CMR system (MR750, GE Healthcare, Waukesha) by operators blinded to the study medication. Animals were positioned in a head-first supine position with a flexible phased-array surface coil placed over the chest. ECG gating was used to acquire still images of the heart. The following dedicated CMR sequences were acquired in all cases: “cine” (b-SSFP) imaging sequence to assess wall motion and cardiac function, late gadolinium enhancement to assess the extent of myocardial necrosis. All cMRI assays followed the same sequences. First, scout images (T1-TFE sequence) were obtained to localize the true axes of the heart and define a field of view involving the whole heart. Afterward, the b-SSFP cine imaging was performed in both horizontal and vertical long axes (4- chamber and 2-chamber views) and in multiple contiguous short-axis images covering the whole LV. In the short-axis cine sequence 30 cardiac phases of every slice were acquired to guarantee a correct evaluation of the wall motion and heart function. Thereafter, a gadolinium-based contrast agent was injected intravenously (Gd-GTPA, Magnevist®, Berlex Laboratories Inc., Wayne, New Jersey, USA) at a dose of 0.1 mmol/Kg. The early gadolinium enhancement sequence was acquired 1 min after the administration of the contrast. The late gadolinium enhancement (LGE) sequences were obtained 10 min after administration of the contrast.

All CMR images were analyzed using dedicated software (Circle CVI42 Calgary) by a CMR-trained radiologist blinded to the study medication. Briefly, LV cardiac borders were traced in each image of the cardiac phases representing the end diastole and end systole to obtain the left ventricle end-diastolic- and end-systolic volumes (LVEDV and LVESV, respectively) and LVEF. Infarct size (necrosis) was quantified from the extent of myocardial enhancement in the LGE CMR sequence (see below for the detailed parameters).

Histological analysis

Four weeks after surgery, pigs were anesthetized by inhalation of isoflurane, and pentobarbital solution (390 mg/mL, 1 mL/5 kg) was injected to euthanasia. Heart tissues were harvested for histological analysis. To measure infarct size, pig hearts were cut into five evenly separated slices from cardiac apex to base, and each slice was used for paraffin section. Masson staining was performed with all the sections, while immunostaining was performed with the middle one. Masson staining and immunostaining were performed according to a standard protocol in our lab. Briefly, paraffin sections were dewaxed and rehydrated, followed by hematoxylin staining and acid ponceau staining. After differentiation with 1% phosphomolybdic acid solution, brilliant green staining was introduced. Then the sections were dehydrated and mounted with neutral balsam. In immunostaining, antigen retrieval was performed by boiling sections in citrate buffer (0.01 M, pH 6.0), followed by washing and blocking, the primary antibodies was incubated overnight at 4 °C. Then the corresponding fluorescein-conjugated secondary antibodies were dropped and incubated for 2 h at room temperature. Images were acquired under Zeiss Axio Imager M2 Advanced Microscope Platform. More than ten images acquired in each animal were used for quantification.

Date acquisition and statistics

Data acquisition were performed by investigators who are blind to the groups, and the related data sets were acquired and proceeded with Image J version 1.8.0, Zeiss Axio Imager M2 Advanced Microscope Platform, CRI Maestro noninvasive fluorescence imaging system, Circle CVI42 Calgary software, OxyLite monitoring System, Excel 2016(Microsoft), Vevo 2100 Imaging System(FujiFilm VisualSonics), Image Pro Plus 6.0. All statistical analyses were performed through commercial software GraphPad Prism 7, and data are presented as mean ± SD or mean ± SEM. Normal distribution was first checked, and comparisons between two groups were performed by Student‘s t test, while for multiple group comparison, one-way or two-way ANOVA analyses was performed. For all the statistical analyses, significance was accepted at p < 0.05.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The data supporting the findings from this study are available within the manuscript and the supplementary information. Any remaining raw data are available from the corresponding author upon reasonable request. Source data are provided with this paper.

References

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Источник: https://www.nature.com/articles/s41467-021-24804-3

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After you install SRS Net Connect 3.1, you must register for a Net Connect account to obtain the activation kit. The activation kit contains your customized customer certificate and activation script. If you already have an activation kit, you do not need to obtain another kit.

Creating a Net Connect Account

If you have already registered as an SRS Net Connect 3.1 user, you can use that username and password to get the activation kit. If you have not registered, perform the following steps:

1. If you have not registered as a Net Connect user, go to to access SRS Net Connect 3.1.

2. At the Welcome page, click Sign Up to create a new root administrator user account. A Net Connect root administrator controls the company profile, views reports, and creates, modifies, and deletes users, user groups, and system groups. More than one person can be a root administrator.

3. Read the agreement, click the check box, and click Accept.

4. At the Sign Up - Account Creation page, complete the required fields to add yourself as a root administrator and click Submit.

5. At the Sign Up Create Company page, click Create Company. To download the installation bundle and use SRS Net Connect 3.1, you must add your company.

6. At the New Company page, complete the required fields. The Company Name field must be less than 80 characters. The Short Name must be less than 20 characters; it cannot be edited later since this is how the company information is stored in the database.

7. A default system group is created using your company short name, and as you install SRS Net Connect 3.1 on new monitored systems, the systems are added to this system group. You can change this later by clicking System Grouping. The last field, Update Mode, defines how software updates are delivered to your monitored system. Select one of the following:

    • Auto - If SRS Net Connect 3.1 detects that your system needs a software update, a Non-Critical alert appears in the Software column on the Monitoring Status Summary page and the update is downloaded. If you have the Client Listener field set to Yes, the update is automatically installed. If the Client Listener is set to No, you must manually install the update by typing the following:. See the SunSM Remote Services Net Connect 3.1 Customer Operations Guide for instructions on ef download - Activators Patch srsinstall in polling mode to check for updates every 20 seconds.

For both Auto and Review settings, you can get email notification about failed software updates by selecting the Net Connect Event Provider on the Notifications page in the SRS Net Connect 3.1 application. See the SunSM Remote Services Net Connect 3.1 Customer Operations Guide for instructions on email notification.

    • Review - If SRS Net Connect 3.1 detects that your system needs a software update, a Non-Critical alert appears in the Software column on the Monitoring Status Summary page. You can initiate the software download to your system by drilling down in the Software column and approving the update for delivery. If you have the Client Listener field set to Yes, the update is automatically installed. If the Client Listener is set to No, ef download - Activators Patch must manually install the update.
    • Never - Software updates are not delivered to your system and a yellow icon appears when a component is out of date.

8. Click Save to display the Download SRS Net Connect page.

9. Click Welcome and then click SRS Net Connect Home to display the SRS Net Connect Home page.

 

Downloading the Activation Kit

You must have an SRS Net Connect username and password to get the activation kit. Perform the following steps to get the activation kit:

1. Log into the SRS Net Connect 3.1 application.

2. On the SRS Net Connect Home page, click Follow Solaris Net Connect Activation Guide and print it.

3. Click Download Activation Kit, as shown in FIGURE 2-1.

 

Click Download Activation Kit on the SRS Net Connect Home Page.


Note - If you did not install SRS Net Connect 3.1 from the Solaris 9 4/04 Extra Value CD, you can also get the installation package by clicking Download SRS Net Connect on the SRS Net Connect Home page.



4. If your company uses an HTTP proxy to connect to the Internet, type the URL or IP address of the HTTP proxy. At HTTP Proxy Port, type the port number it uses. Leave these fields blank if you have a direct connection to the Internet and do not use an HTTP proxy.

5. To allow software updates to be automatically installed on ef download - Activators Patch monitored system, click Yes at Enable Client Update Listener. To disable software update installation, click No. The updates include new software versions and messages. If you click Yes at Client Listener, you should also set the Update Mode field in the New Company page to Auto or Review to automatically send a Non-Critical alert about an update.

6. At the Download Activation Kit section, click Download to save the compressed 53 KByte file to your monitored system. FIGURE 2-2 shows the Quick Download SRS Net Connect page.

 

The Quick Download Page Contains Two Download Buttons.

7. At the Save As dialog box, specify a directory on the monitored system where you want to perform the installation and click OK. If you put the installation file in the directory, the files are deleted if your system reboots, so the directory is a more permanent location. A configuration file and a security certificate are automatically generated for you and included in your activation kit. The configuration file is saved in the directory when the software is installed on the monitored systems. See the SunSM Remote Services Net Connect 3.1 Customer Installation Guide for details on the default configuration values included in the activation kit.

Running the Activation Kit Script

After you download the activation kit tar file, you must extract it and run the activation script on each monitored system. The activation script contains the following items:

  • Customer certificate (
  • Activation script and supporting directories ()

The activation script performs the following actions:

  • In the directory where you extract the tar file, three directories are created to store the proxy messages and core files:

1. The directory - Contains a program called to support globalization in the activation script

2. The directory - Assists the activation script with shell functions

3. The directory - Contains globalization support strings

These directories are not used after the activation is successful, and you can delete them if you wish.

  • The proxy and each provider is configured and activated.
  • The file and the files are both removed.
  • The program is run to build the SRSQueueStore space.
  • The file is updated to include an entry for the proxy process. The file is refreshed to start the process.
  • If ef download - Activators Patch chose to enable automatic software upgrades, an entry is made in the file.

If you have a previous version of the Net Connect 3.x event provider installed, the activation script detects the file archive directory and copies the SRSQueueStore directory to the DISK_STORE_BASE directory. A new file called is created and the file is updated.



Note - If you have a previous version of the CST provider installed, the SUNWcstu package is installed. The CST lock file () is removed to allow CST to start on a reboot. You can start CST by typing the
# command.



Running ef download - Activators Patch Script

Run the activation script by performing the following steps on each monitored system:

1. Open a terminal window and log into the monitored system as the root user.

2. Copy the file you downloaded in Downloading the Activation Kit to each monitored system that will run SRS Net Connect 3.1.

3. Change to the directory where you copied the activation kit tar file. This should be a directory that is owned and readable by the root user.

4. Uncompress and extract the activation kit tar file by typing:

#

5. Run the activation script from the directory where you uncompressed and extracted it by typing:

#

The activation script verifies the following:

    • You are logged in as a root user.
    • The monitored system is running with the required Solaris patches. Log into the SunSolveSM program at to get the latest version of a required patch.

6. If you running the script for the first time, you must provide a user account when the following text appears:

SRS Net Connect 3 executes programs as the user specified in answer to the following questions. The user and their group must already exist on the system and it is highly recommended that this user account be a locked account that denies interactive login. Type the account identifier. Note: the account must already exist. [?,q].

Type the user name and press Return. If you have a previous version of Net Connect installed, the activation script automatically detects the user name and group you used to install Net Connect.

7. After you type the user name, choose a group name when the following text appears:

  Type the group name to be used with this account identifier. Note: This group must already exist and the account identifier specified above must be a member of this group. Available groups: staffType the group name: [?,q]

If the user account has only one group associated with it, this step is skipped. Type the group name and press Return.

8. SRS Net Connect requires space to build and store messages. You must specify a directory for queue space when the following text appears:

  SRS Net Connect 3 requires space to build and store messages that are sent in response to system management monitoring events. The default file system location for this queue space is: /var This directory currently has the following characteristics: Filesystem kbytes used avail capacity Mounted on /dev/dsk/c0t0d0s5 492422 184319 258861 42% /var Type the name of the file system where queue space should be allocated [/var]

Press Return to accept the default directory, or type a different directory (for example, ).

9. You must also specify the size for the DISK_STORE_SIZE value when the following text appears:

Amount of space(MB) to allocate for the SRS Net Connect queue store. Type the maximum size that the proxy may use: [20]

Press Return to accept the default size of 20 MBytes.

10. If you run the proxy through a SOCKS server, you must provide the path to the SOCKS server when the following text appears:

  The SRS Net Connect adobe after effects 2015.3 download - Crack Key For U may be run via a SOCK daemon process. If you want the proxy to use the SOCKS proxy, type the fully-qualified path to the 'runsocks' or equivalent program. This adds the run-time library for the SOCKS libraries to the LD_LIBRARY_PATH. If you do not want to run via the SOCKS daemon, enter 'none' at the prompt. Enter the path to the runsocks program or 'none': [none]

Type the path to the SOCKS server or press Return to accept the default value of none.

11. You can enable automatic software upgrades, so new software is automatically installed on your monitored system. The updates include new software ef download - Activators Patch and messages. The following text appears:

  SRS Net Connect can automatically install software updates that are received by the proxy. Would you like to enable automatic updates? [y,n,?,q] y

Press Return to automatically install software updates or type.

The activation script checks the user's ability to interact with cron. The SRS Net Connect 3 activation complete message indicates a successful SRS Net Connect activation. Log into SRS Net Connect 3.1 to view your monitored systems. See the SunSM Remote Services Net Connect 3.1 Customer Operations Guide for instructions on creating system groups and user groups.

Testing the Installation

After you run the activation script, the monitored system and its providers run within three minutes. Wait at least 30 minutes before 3D Coat 4.9.74 Crack + Serial Number Free Download system reports from SRS Net Connect 3.1. Delta reports require at least two sets of data to compare.

Perform the following steps after you finish the installation:

1. Log into the monitored system as the root user.

2. Verify which SRS Net Connect 3.1 providers are installed by typing:

#

For example, the following text indicates that you have successfully installed the configuration provider (SUWsrscp), Sun RAS System Analysis (SUNWsrsep), and the reboot provider (SUNWsrsrp):

  1 9 * * 5 /opt/SUNWsrscp/bin/config_pvr_runner config_pvr 003.001.001 IM-NC_ENG- config_pvr /tmp/config_pvr 756000 text/plain 1 9 04 * * /opt/SUNWsrsep/bin/eras_pvr_runner eras_pvr 003.001.001 IM-NC_ENG-eras_pvr /tmp/eras_pvr 3348000 application/x-gtar 0-59 * * * * /opt/SUNWsrsrp/bin/gmt_time > /var/opt/SUNWsrsrp/latest

3. Verify that the SRS Net Connect 3.1 providers and proxy are running by typing:

#

The following providers and proxy processes should be running continuously:

    • trend_pvr
    • event_pvr
    • ssha_pvr
    • srsproxy

4. Verify that the monitored system can communicate with the SRS Net Connect Data Center. Change to the directory and type:

#

The -p indicates ping mode, and verifies that it can route to the Data Center.

5. Validate that the hardware alarm provider is working by typing:

#

This causes an error message to be logged in the file, which is detected by the hardware alarm provider. The hardware alarms provider sends a Fan Warning alarm.

6. Check the syslog file for warnings. Ef download - Activators Patch log is located in the file.

7. Log into SRS Net Connect 3.1 to verify that the alarm appears.

Copyright © 2004, Sun Microsystems, Inc. All rights reserved.

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3 Use a commercially available Vibratome, e.g., Pelco 1500, to prepare 250 μm thickness coronal hippocampal slices from mouse brain with a standard procedure we and others described before [5, 16]. All solutions should be continuously bubbled with 95%O2/5%CO2.

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Following these steps:

Precaution

- A power failure during the firmware writing operation may disable the camera.

» Use a fully charged Battery Pack or dedicated AC Adapter Kit (Optional) for the firmware update.
» Do not shut off the power during the firmware writing operation.
» Do not open the card slot cover during the firmware writing operation.

- Do not press any camera buttons during the firmware writing operation.

(1) Prepare the items required to update the firmware.

1. Camera body

2. Dedicated Battery Pack (The battery pack must be fully charged) or dedicated AC Adapter Kit (Optional) ***

3. Memory card (64MB or more, 64GB or less)

4. Firmware update file (you can download it from Canon's Website)

(2) Create the firmware update file.

1. Download the zipped self-extracting file from Canon's Website.

2. Extract the downloaded file, and create the firmware update file.

- How to Extract the Firmware Update File

Windows
When you double-click the downloaded file, the following screen appears.
Click [OK], and the downloaded file will be extracted, and the firmware update file created.

Macintosh
The downloaded file will automatically self-extract, creating the firmware update file. In case the downloaded file does not automatically self-extract, double-click the downloaded file.

3. Check the size of the firmware update fiIe
If the file size does not match, then download the firmware update file again

- How to Confirm the File Size

Windows
Right-click the icon of the firmware update file and select the [Properties] command from the pop-up menu that appears.

Macintosh
Select the icon of the firmware update file, and then select the [Get Info] command from the[File] menu.

4. The name and size of the firmware update file can be checked on the Website.

*** In case of using the memory card reader, follow the procedure from Step (3) onward. In case of not using the memory card reader, follow the procedure from Step (4-1) onward.

(3) Copy the firmware update file to the memory card.

1. Insert a memory card that has been formatted in the Camera into the memory card reader.

2. Copy the firmware update file to the first window that appears when the memory card is opened (the root directory).

3. Remove the memory card from the card reader.

*When removing the memory card, be sure to do so as described in the documentatIon for the computer or the card reader

*If the firmware update file is placed in subfolder of the memory card, the camera will not see it.

4. Rotate the Mode Dial to select mode (or any mode other than Fully-Automatic Modes).

5. Insert the memory card with the firmware into the camera.

6. Turn the Power Switch

7. Rotate the Main Dial and the Quick Control Dial to select the "Firmware Ver.x.x.x" item at the bottom of the "Set-up 3 (Yellow)", and then press the

8. The firmware update screen will appear.
Turn the Quick Control Dial, select [OK], and then press the

(4-1) Connect the camera Avid Pro Tools 2021.12 Crack + Activation Code Free Download the computer.

1. Rotate the Mode Dial to select <P> mode (or any mode other than Fully-Automatic Modes).

2. Insert a memory card that has been formatted in the camera into the camera.

3. Connect the camera and the computer with the USB cable, and then set the camera’s power switch to <ON>.

(4-2) Start the firmware update.

1. Start EOS Utility.

2. Click the [Camera settings I Remote shooting] button.

3. Click [], and then click the [Firmware Ver. X.X.X].

4. The firmware update screen will appear on the computer screen Click [OK].

5. A window for selecting files appears, so select the firmware update file, and then click [Open].

6. A confirmation window appears, so click [OK].

7. A window with cautionary messages about the update appears, so click [OK].

8. Follow the procedure from Step (5) onward on the camera.

(5) Update the firmware.

1. The screen will appear on the camera’s LCD monitor.

2. If you press the

3. The message will appear during the update.

4. When the update is completed, the message will appear on the camera’s LCD monitor.

5. Complete the firmware update by pressing the

The firmware update is now completed.

When the firmware update operations are finished, turn the camera

Format the memory card before using it again.

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1. Turn the Power Switch <ON>, and press the menu button

2. Rotate the Main Dial and the Quick Control Dial, and you will see the “Firmware Ver.X.X.X” at the end of the settings shown in Set-up 3 (Yellow).

3. This is the currently installed firmware version number.

Note: Select P mode (or any mode other than Fully-Automatic Modes). The version of the firmware will not appear in the Fully-Automatic Modes.

If an ERROR message appears during the firmware update

If this screen appears, remove the battery and check to make sure that there are no problems with the battery capacity or with the firmware update file on the memory card.

If there are no problems, repeat the update operations again.

If the problem persists, please contact the Canon Customer Support Center in your region.

Источник: https://www.atiz.com/support/
Rupture the patch of membrane isolated inside the electrode tip by negative pressure pulses to establish the whole-cell configurations. No obvious change in holding current shift associated with a decrease of seal test resistance to < 30 MΩ is an indication of successful formation of whole-cell configuration. Repeat this for the second electrode. After both whole-cell configurations are established, wait at least 5 min to allow ion equilibration between electrode solution and intracellular cytoplasm

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3 thoughts on “Ef download - Activators Patch”

  • JAI SHREE RAM says:

    Everyone has different perspective in solving problems. If you are willing to do something different be focused, think differently, act differently and feel differently.

  • Heather Morgan says:

    В @WheatleyOffDaGoopВ  What gave you mental illness?

  • harlem alvarez says:

    Dude after updating trakers my speed went form 400kb/s to 14mb/s

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