text:
\section{Introduction}

Since its initial specification, segment routing\xspace (SR) has attracted attention from big vendors in the networking industry such as Cisco \cite{cisco-depl}, Juniper \cite{juniper-depl}, Huawei \cite{huawei-depl} \cite{srv6-deployments}, Nokia \cite{nokia-depl}, and Arrcus \cite{arrcus-depl}.
SR is a technique which leverages the source-routing principle, allowing a sender or an intermediate node to specify (in part) the path a packet takes on its way.
In addition, SR brings many promises \cite{cisco-sr}, one important benefit being the fact that there is no need to keep per-application and per-flow state, as all the necessary information is stored in the packet itself.
It also enables software-defined networking (SDN) and the selective use of different network appliances such as firewalls or snort~\cite{Abdelsalam2018DemoCO}.
Another benefit of SR mentioned by networking companies is its support of resilience techniques such as Topology Independent Loop-free Alternate Fast Re-route (TI-LFA) \cite{ietf-rtgwg-segment-routing-ti-lfa-08}, which in a failure scenario allows quickly shifting traffic to a backup path.

Moreover, many networking vendors claim to be working on adding SR-support into their products.
They also started offering support to their clients to integrate SR into their networks.
Such clients include SoftBank \cite{softbank-press}, Line Corporation \cite{line-corp}, MTN Uganda \cite{srv6-deployments}, Indosat Ooredoo \cite{indosat-press}, Rakuten \cite{rakuten-press}, Bell Canada \cite{bell-press}, China Unicom \cite{china-unicom-press}, China Telecom \cite{srv6-deployments}, Iliad \cite{iliad-press}, and CERNET2 \cite{srv6-deployments}.

Recently, segment routing\xspace also started to attract the attention of the research community, with papers describing how new functions can be implemented on top of segment routing\xspace \cite{tulumello2020micro} \cite{mayer2019network} \cite{paolucci2016interoperable} \cite{bhatia2015optimized}, review standardization activities, and review articles dedicated to segment routing\xspace \cite{ventre2020segment}.

Furthermore, since SR is a relatively new technology compared to e.g., \@ MPLS \cite{rfc3031}, it is not yet as mature.
Thus, network operators engage in lively discussion about the benefits and possible downsides such as security implications of large-scale SR deployment, especially SR over IPv6 (SRv6) \cite{nanog-mails}.
Security issues may also arise from the uninformed use of SRv6 in a network \cite{ietf-topicbox}.
Moreover, the IETF community is also looking into security issues related to SRv6 deployment \cite{li-spring-srv6-security-consideration-07}.
Therefore, in this paper we aim to investigate the current state of SRv6 deployment in the wild.

\section{Background and Related Work}

Segment routing\xspace is based on the concept of source routing, i.e., \@ it allows a source node or an intermediate node to predetermine (part of) the path taken towards a destination \cite{rfc8402}.
Segment routing\xspace, however, goes beyond a pure list of forwarding instructions:
It can chain services and obtain complex behaviors as a solution for service differentiation.
A \textit{segment} is the basis for segment routing\xspace.
It is composed of a \textit{locator} (i.e., \@ a unique identifier of the network node where the instruction should be executed), an \textit{instruction} the node executes, either topological (i.e., \@ forwarding) or requiring a service to be executed, and optional \textit{arguments} for the instruction.
Segments can be recognized by their SR Segment Identifiers (SIDs).
We can chain segments into a list which connects the \textit{ingress node} (i.e., \@ headend, where the packet becomes ``SR-aware'') to the \textit{egress node} (i.e., \@ endpoint, either the final destination of the packet or the node where the SR capabilities are removed from a packet).
This list of segments is called an \textit{SR path}.
The set of nodes a packet travels between the ingress and egress nodes constitutes an \textit{SR domain}.
In \Cref{fig:srv6topology} we depict these concepts in an example topology.

\begin{figure}[t!]
\centerline{\includegraphics[width=0.9\columnwidth]{figures/sr-diag.pdf}}
\caption{Segment routing\xspace topology example showing SR domain, ingress, egress, SR path, and segments \cite{juniper-doc}.}
\label{fig:srv6topology}
\end{figure}

Furthermore, an \textit{SR policy} describes how traffic is handled within an SR domain \cite{srpolicy}.
A policy is identified through the tuple \texttt{<headend, color, endpoint>}.
The color is used to differentiate between policies that have the same headend and endpoint.
Note that a policy can have multiple SR paths available (e.g., \@ for redundancy).
In this case a preference determines which SR policy is being picked.

In order to distribute SR policies we can make use of BGP update messages \cite{srbgp}.
BGP speakers can propagate candidate paths for SR policies by making use of Color Extended Communities.

On the protocol level, segment routing\xspace works on top of MPLS or IPv6.
Due to the long history of using MPLS in networking \cite{rfc3270} \cite{rfc2702} \cite{rfc3031} \cite{809383}, SR-MPLS is thought to be more mature than SR over IPv6 \cite{mpls-nanog}.
In this paper we focus on the prevalence of SR implemented on top of IPv6, i.e., \@ \textit{SRv6}.
This is done through a new type of routing header (next header value 43) called \textit{Segment Routing Header}, with routing type 4 \cite{rfc8754}.
The details regarding this header can be seen in \Cref{fig:srv6header}.
Further details of SRv6 are described in the corresponding RFC~\cite{rfc8986}.

Lately, researchers started exploring the capabilities and implications of segment routing\xspace.

Ventre et al.\xspace conduct a comprehensive survey on the introduction, motivation, and evolution of SR, SR-MPLS, and SRv6 \cite{ventre2020segment}.
The authors provide a classification of current SR activities SR.
They conclude that SR's potential is not yet fully leveraged.
For example, they find a lack of works focusing on failure monitoring, but they seem confident that the network programming capabilities of SR will get more attention in the coming years.

Tulumello et al.\xspace propose ideas on how to make SRv6 more efficient by reducing the size of SIDs \cite{tulumello2020micro}.
Instead of representing only one segment with a single SID, they encode up to six micro instructions, represented through micro SIDs.
This drastically reduces the SIDs needed to be present in the SRH, thus reducing the packet overhead of SRv6 containing many SIDs.

Mayer et al.\xspace present ideas on how SRv6 can be integrated in an IoT and cloud infrastructure leading to a new distributed processing model \cite{mayer2019network}.
They theorize an abstract computing machine, which they call an SR-IoT Computing Machine which can be programmed with its own instruction set.
Thus, by building their own toolchain and leveraging the capabilities of segment routing\xspace, the authors treat the whole Cloud-IoT network (i.e., without differentiation between core and edge) as a single logical machine.
This way a developer focuses on the application logic, while the owner of the infrastructure enforces management policies.

Lebrun and Bonaventure describe their experience implementing SRv6 in the Linux kernel \cite{lebrun2017implementing}.
In addition, they analyze the performance impact of SRH insertion.
They find that the performance overhead for SR entry points with their implementation for Linux 4.10 is limited to 15\%.

To the best of our knowledge this work is the first conducting an empirical analysis of the SRv6 landscape in the wild.

\begin{figure}[t!]
\centerline{\includegraphics[width=0.9\columnwidth]{figures/srh.pdf}}
\caption{SRv6 header structure.}
\label{fig:srv6header}
\end{figure}

\section{Emulation}
\label{sec:emulation}

Before analyzing the deployment of SRv6 in BGP and with traceroute measurements, we set up a controlled emulation environment in a lab.
We use IPMininet \cite{ipmininet}, a Python library that extends Mininet \cite{mininet}, enabling the use of SRv6 and BGP.
IPMininet directly uses the Linux kernel's implementation of segment routing\xspace.
The setup is simple, yet complex enough to emphasize the traces that SRv6 may leave in an multi-AS environment.
We worked in an IPv6-only setting.
\Cref{fig:srv6emulation} shows our emulation topology.
We use one main AS---namely AS1---with 4 routers (i.e., \@ r2, r3, r4, and r5), forming an iBGP full mesh.
AS1 also contains one host system: h2, connected to r5.
Moreover, we set up a smaller AS---namely AS2---containing only one router (i.e., \@ r1).
There is also a host in AS2 (i.e., \@ h1) which is connected to the router r1.
The edge routers r1 and r2 are exchanging reachability information through an eBGP session.
Note that all routers run both OSPF and BGP.
Finally, we enable SRv6 on all the nodes and set up r2 as an SR entry point which enforces a simple SR policy: when receiving a packet destined to h2, r2 should forward it through r4.

\begin{figure}[htbp]
\centerline{\includegraphics[width=0.9\columnwidth]{figures/paper-topo.pdf}}
\caption{SRv6 emulation topology showing two ASes, one of which contains an SR domain with an SR path. The arrows show a packet's path.}
\label{fig:srv6emulation}
\end{figure}

This policy can be implemented by choosing one of the two SRv6 modes: \texttt{H.Insert} (called \texttt{inline} in the Linux Kernel implementation) or \texttt{H.Encap} (\texttt{encap} in Linux).
The \texttt{encap} mode indicates that the incoming packet will be encapsulated into another IPv6 packet (i.e., \@ IPv6-in-IPv6) at the ingress node (i.e., \@ r1).
The source of the outer packet is set to r2 and the destination address is set to the next segment (i.e., \@ r4 in our case, which is also our egress node).
Moreover, the entry point will add a segment routing header (SRH).
Note that in our example here, the segment r4 in the SRH can be omitted if a reduced SRH is being used \cite{rfc8754}.
The egress node will strip the packet of the outer IPv6 layer and forward it to the original destination (i.e., \@ h2).

The \texttt{inline} mode, however, aims to modify the IPv6 layer of the original packet.
It changes the destination address, adds an SRH, but leaves the source address untouched.
This means that an ICMPv6 message (e.g., \@ due to Hop Limit exceeded) will reach the original source \cite{lebrun2017implementing}, allowing detection of SRv6 deployment with traceroute.
Our findings confirm this.
We send \texttt{traceroute} probes from h1 to h2, capturing all the traffic at h1.
When using \texttt{encap}, no packet contained traces of SRv6, but when using \texttt{inline}, we can see SRHs in the ICMPv6 packets returned from the SR domain.
This is a promising finding, as various parties claim to implement and use \texttt{H.Insert} and \texttt{H.Insert.Red} (i.e., \@ reduced insertion) \cite{srdeployment}.

\textbf{To summarize:} Our emulation experiments show that it is possible to detect SRv6 deployment with traceroute, if SRv6 is implemented with inline mode.

\section{Investigating BGP Route Collectors}

One possible source of SRv6 leakage is BGP.
We inspect BGP data from 10 popular route collectors from RIPE RIS and RouteViews (see \Cref{tab:comms_rcs} for a list of RCs) in September 2021.

We find that Color Extended BGP Communities \cite{rfc5512} can be used to steer traffic according to various SR policies \cite{colorcisco,srbgp}.
Thus we explore data from BGP archives looking for these types of communities.
This can be done by detecting the specific bytes in the Type and Sub-Type fields of extended communities: \texttt{0x03} and \texttt{0x0b}.
Our search for these byte combinations does not yield results in the BGP collector data.

Next, we also search for other communities that may be an indicator of SRv6 usage, directly in the BGP data.
Then, we collect a list of ASes that belong to organizations that claim to be using SR (we will call them SR-suspect ASes).
We then follow a simple procedure for inspecting whether these SR-suspect ASes have specific communities in common.
First, we extract all BGP communities that appear with at least one SR-suspect AS on the AS path.
Subsequently, we extract the communities that never appear with SR-suspect ASes on the path.
Finally, we make a difference of both of these sets of BGP communities and obtain a list of communities which only appear in BGP announcements with SR-suspect ASes on the path.
We then count in how many SR-suspect ASes each community appears, aiming to find a community used by as many SR-suspect ASes as possible.
However, each community we find is only used by a small number of ASes.
As can be seen in \Cref{tab:comms_rcs}, the majority of communities is used by only a single AS, whereas a small number is used by two or more ASes.
We further inspect the cases where BGP communities are seen in announcements with more than one SR-suspect AS.
We find that these cases are almost all related to sibling ASes, i.e., \@ two ASes with a different ASN but operated by the same organization.
Only at \texttt{rv4} we find three communities within a single BGP announcement with two suspect ASes on the AS path.
We find that those communities are related to tagging \cite{softbankcomm} and peer selection \cite{nttcomm}, and not SRv6.

\begin{figure}[t!]
\centerline{\includegraphics[width=0.8\columnwidth]{figures/packet-struct-encap.pdf}}
\caption{Packet structure and content for \texttt{H.Encap} behavior at routers r1 and r3.}
\label{fig:encap}
\end{figure}

\begin{figure}[t!]
\centerline{\includegraphics[width=0.8\columnwidth]{figures/packet-struct-inline.pdf}}
\caption{Packet structure and content for \texttt{H.Insert} behavior at routers r1 and r3.}
\label{fig:inline}
\end{figure}

Moreover, we investigate whether the communities found only for SR-suspect ASes are location communities \cite{silva2022automatic}.
We find that between 25\% to 50\% of all communities from \Cref{tab:comms_rcs} are identified by the BGP location community database \cite{locationcomm} as location communities.
This shows that a non-negligible portion of these communities is very unlikely to be used for SR.

Furthermore, we inspect BGP path attributes as potential indicators of SR deployment.
These include \texttt{BGP Prefix-SID}, \texttt{BGP-LS Attribute} and \texttt{Tunnel Encapsulation}.
The \texttt{BGP Prefix-SID} attribute \cite{rfc8669} is a BGP extension used for signaling information about BGP Prefix Segment Identifiers, thus it is a good indicator of SR deployment.
\texttt{BGP-LS} \cite{rfc7752}, on the other hand, is used for more general purposes, yet it has been extended \cite{rfc9085} to support carrying SR information between ASes via BGP.
This also applies to the \texttt{Tunnel Encapsulation} attribute \cite{rfc9012}, which permits carrying Prefix-SID-related information in one of its Sub-TLVs.
We search the BGP collector archives for interesting path attributes, but we only find generic attributes (e.g., \@ \texttt{AGGREGATOR}, \texttt{ATOMIC\_AGGREGATE}, \texttt{AS\_PATHLIMIT}) and no SR-specific attributes.

\begin{table}[!t]
    \centering
    \caption{Unique BGP community values seen only at suspect ASes (\# comms), seen only at multiple suspect ASes (\# multiple comms), and seen only at multiple suspect ASes while ignore sibling communities (\# non-sibling comms).}
    \label{tab:comms_rcs}
    \resizebox{\columnwidth}{!}{
    \begin{tabular}{lrrr}
       \toprule
       Collector & \# comms & \# multiple comms & \# non-sibling comms \\
       \midrule
       rv2       & 208      & 14                & 0 \\
       rv4       & 257      & 23                & 3 \\
       rv5       & 0        & 0                 & 0 \\
       rv6       & 97       & 9                 & 0 \\
       rv-amsix  & 190      & 8                 & 0 \\
       rrc00     & 257      & 16                & 0 \\
       rrc01     & 206      & 11                & 0 \\
       rrc04     & 151      & 5                 & 0 \\
       rrc05     & 171      & 8                 & 0 \\
       rrc06     & 89       & 1                 & 0 \\
       \bottomrule
    \end{tabular}
    }
\end{table}

\textbf{To summarize:} We analyze data collected by 10 BGP collectors, looking for attributes that could indicate SRv6 deployment.
We find no such attributes.
Furthermore, we conduct a thorough investigation of BGP Communities, especially Color Extended Communities, aiming to find a pattern that may indicate SRv6 usage.
Again, we do not find any relevant signal.
This means that these attributes are either properly filtered by BGP speakers on the path \cite{krenc2021level} or the egress routers of SRv6 deployments.

\section{Tracerouting for SRv6}

In order to reveal SRv6 deployment in the Internet, we ran several traceroute measurements.
Further, we want to understand if we can identify SRv6 in networks which claim to have deployed it \cite{srdeployment}.
Companies claiming to deploy \texttt{H.Insert} and its variations are especially valuable to us, since those might leak information in traceroute (cf. \@ \Cref{sec:emulation}).
Those companies are Iliad, SoftBank, Line Corporation, China Unicom, China Telecom, and CERNET2.
We use PeeringDB \cite{peeringdb} and BGPView \cite{bgpview} to collect AS numbers related to these organizations.
We then use the WHOIS database and CAIDA's Routeviews BGP data \cite{routeviews} to get IPv6 prefixes for the identified ASes.
Finally, we generate 100 random addresses for each prefix resulting in \sk{213.8} addresses in total.

Next, we use Yarrp \cite{beverly2016yarrp} to run traceroute towards each of these addresses.
Before conducting active measurements we incorporate proposals by Partridge and Allman \cite{partridge2016ethical} and Kenneally and Dittrich \cite{kenneally2012menlo} and follow best measurement practices \cite{durumeric2013zmap} by using dedicated servers, informative rDNS names, a website with information, and maintaining a blocklist.
We did not receive any complaints while conducting the measurements.
We run the measurements from a single vantage point at MPI-INF, which might influence the traceroute paths we cover.
We send two types of probes: SR-unaware probes and SR-enabled probes.
The former are simply regular traceroute probes, the latter sends probes containing a segment routing\xspace header.
We modify Yarrp in order to send SR-enabled probes.
Sending SR-enabled probes allows us to check if SRv6-enabled routers modify the list of segments in the SRH, which would be visible in the returned quoted ICMPv6 packet.
We conduct both types of measurements for various transport protocols, as shown in \Cref{tab:traceroutes}.
We target three types of addresses:
(1)~ We take a random sample of 10 million addresses from the IPv6 Hitlist \cite{gasser2018clusters} (marked as \textit{hitlist} in \Cref{tab:traceroutes}). (2)~ We generate random addresses within prefixes of SR-suspect ASes (marked as \textit{suspect} in {tab:traceroutes}).
(3)~ We generate random addresses for each BGP-announced prefix.
Even though we see SRHs being returned from the SR-enabled measurements, those are not modified at all and therefore do not leak any SRv6 deployment.
Moreover, our other measurements do not return any SRH and therefore do not leak SRv6 deployment in the wild.

\begin{table}[!t]
    \centering
    \caption{Overview of traceroute measurements.}
    \label{tab:traceroutes}
    \begin{tabular}{llclc}
       \toprule
       Date & Type & SRH sent & Targets & SRv6 leaked \\
       \midrule
       2021-11-09 & TCP SYN & \xmark & hitlist & \xmark \\
       2022-02-08 & TCP6 SYN & \xmark & suspect & \xmark \\
       2022-02-09 & TCP6 SYN & \cmark & suspect & \xmark \\
       2022-02-10 & ICMPv6 & \xmark & prefix & \xmark \\
       2022-02-10 & TCP6 ACK & \xmark & prefix & \xmark \\
       2022-02-10 & TCP6 SYN & \xmark & prefix & \xmark \\
       2022-02-10 & UDP6 & \xmark & prefix & \xmark \\
       2022-02-15 & ICMPv6 & \xmark & prefix & \xmark \\
       2022-02-15 & TCP6 ACK & \xmark & prefix & \xmark \\
       2022-02-17 & TCP6 ACK & \xmark & prefix & \xmark \\
       \bottomrule
    \end{tabular}
\end{table}

\textbf{To summarize:} We conduct traceroute measurements to identify SRv6 deployments in the wild, but do not see any traces of SRv6.
Either companies do not use SRv6's inline mode as they claim or they filter SRHs from the returned ICMPv6 packets.

\section{Conclusion}

Segment routing\xspace is a protocol which has its roots in the source routing space.
It brings many promises, including SDN-readiness, fast rerouting, and statelessness.
Given its relatively young age, experts are concerned to the security implications it brings.
Therefore, in this paper we tried to measure the SRv6 deployment leakage of segment routing\xspace in the wild.
We searched for SRv6 in the real world, by exploring BGP collector archives and actively probing addresses of organizations that claim to be using SRv6.
While analyzing the BGP data, we looked for path attributes that may be indicators of segment routing\xspace and tried to find a correlation between communities and segment routing\xspace usage.
Unfortunately, we found no trace of segment routing\xspace.
BGP speakers may be filtering such attributes, or egress routers themselves may carefully pick what kind and which part of announcements are propagated to neighbors.
Finally, we conducted a traceroute measurement campaign in order to identify leaked SRv6 deployments in the Internet, but we could not find any trace.
Organizations either do not use the inline mode as they claim or they carefully filter segment routing\xspace traces from the returned packets.
Another possibility is that operators might use SRv6 exclusively for 5G deployments, which are likely to be more firewalled than other types of networks, possibly resulting in fewer SRv6 leaks.
We release data \cite{3.19HAUU_2022} and analysis code \cite{codegithub} to be used by fellow researchers looking into investigating SRv6 deployment in the future.

\noindent\textbf{Future work:}
We will continue to leverage BGP collector data and traceroutes in the future to see if SRv6 leaks occur.
Moreover, we plan to extend the BGP analysis to cover more route collectors and longer timespans.
Additionally, we will increase the target set of our traceroute analysis to discover well hidden SRv6 deployments.
Finally, we plan to set up a hardware testing lab in order to extend our emulation to bare-metal devices.

\noindent\textbf{Acknowledgments:}
The authors acknowledge the partial financial support by the Federal Ministry of Education and Research of Germany in the program of ``Souverän. Digital. Vernetzt.'' joint project 6G-RIC (16KISK027).

\balance

\bibliographystyle{IEEEtran}

redpajama_set_name: RedPajamaArXiv


a. One that a judge or presiding officer raps to signal for order.
b. One that an auctioneer raps to mark the end of a transaction.
2. A maul used by masons in fitting stones.
To bring about or compel by using a gavel: "The chairman ... tries to gavel the demonstration to an end" (New Yorker).
Tribute or rent in ancient and medieval England.


Among biological scientists, they are the true nomenklatura, a small and far-flung tribe dedicated to the coherent naming of all living things, past and present.
Gathered in Paris last week, the world's leading taxonomists feted the brilliant and vainglorious Swedish naturalist who, 250 years ago, single-handedly created the system of classification they still use.
But beneath the festive air the proceedings were troubled, on at least two counts.
Carl Linnaeus -- who called the sample-collecting students he dispatched across the globe in the 18th century his "apostles" -- was a creationist born a century before Charles Darwin.
Today many biologists are clamouring for a new approach to cataloging the planet's flora and fauna that goes beyond morphology and takes evolution into account.
A dozen competing theories have cropped up in the last decade, and at least one of them, called PhyloCode, has gained serious traction.
In other scientific disciplines, new ideas elbowing out old ones is a normal and essential process. But in taxonomy, renewal poses a special problem: how can you replace plant and animal names used for two-and-a-half centuries without causing chaos?
"If we did not have a system of classification that was hierarchical and had names that could be easily retrieved we would be helpless," American biologist Edward O. Wilson explained in an interview.
Taxonomists themselves can poke fun at their sometimes arcane bickering as to whether a new discovery is a species in its own right or a subset of one already on the books.
"We have 'lumpers' and 'splitters'," said Richard Pyle, a zoologist and fish specialist at the Bishop Museum in Hawaii, and an officer in the International Commission on Zoological Nomenclature (ICZN).
"Splitters want to draw the lines around a specimen tightly, while lumpers will say, 'no, that's just a slight variation'. And there is nothing in this system which tells you who is right -- it is purely subjective," he said.
But beyond growing internecine quarrels, an even deeper and shared malaise coursed through the proceedings.
It stemmed from a gnawing sense of urgency, and realisation that what Wilson calls "the great Linnean enterprise" -- the mapping of the Earth's entire biosphere -- has barely begun.
Amid predictions that the impact of human activities -- especially climate change -- could wipe out thirty percent or more of all species on Earth by century's end, the scientists charged with finding and defining those species are feeling the heat.
"If you want to preserve a vanishing species, or combat a medically dangerous one, or use it in some beneficial way as a crop, you need to know what you are dealing with," said Andrew Polaszek, a scientist at the Natural History Museum in London and outgoing Executive Secretary of the ICZN.
"That name is the key to all the knowledge that has been accumulated about that organism."
While most people might assume that the job of cataloging Earth's living organisms is nearly complete, the opposite is true.
Some 90 percent of bird species are known, more than 80 percent of flowering plants. But scientific understanding of bacteria and other micro-organisms is "shockingly incomplete," said Wilson, a towering figure in the field of biology, and one of the first to probe the concept of biodiversity.
"Two hundred and fifty years after Linnaeus, we have still only classified as few as 10 percent of the organisms living on Earth," he said.
There are an estimated 1.5 million types of fungi, but only 60,000 are known. Nematodes, including parasitic pinworms and hookworms, comprise the most abundant phylum in the animal kingdom -- probably four or five million strong -- but only 80,000 have been classified.
"In dealing with the living world, we are mostly flying blind," said Wilson. "Trying to diagnose the health of an ecosystem -- a lake or a forest -- to save or stabilise it is like a doctor treating a patient while only knowing ten percent of the organs."
In an effort to catapult the current classification system into the 21st century, a number of taxonomists have launched Zoobank, a Web-based registry of organism names. Some 1.8 million species are listed so far.
"The registry will be the central place where everyone can go look to see what is going on in the rest of the world," said Pyle, who described it as the "most profound change in taxonomy since Linnaeus."
But even this seemingly common-sense step has created controversy, pitting advocates of Internet-based, open-access publishing against traditional and powerful publishers.
Under the current system, a new species does not officially exist until the scientific report of its discovery appears in print.
We are giving away two this week!


Doughnuts make the world go 'round.
Xist Publishing creates books that inspire discovery and delight in young children. We're dedicated to helping children discover a lifetime love of reading, no matter what form it takes.


Since I had been too busy and only posted 1 food review last month, I decided to post 2 food reviews today to make up for the lack of recent updates.
Today I will be reviewing some pre-made packed Japanese Mochis I bought from Maruyu supermarket about 3 weeks ago, however they were not made in Japan, they were actually manufactured by a Taiwanese company called Royal Family. I have bought pre-made mochis from this company before and had even reviewed about their matcha mochi back in 2010. In my opinion, they make some of the best packed Japanese mochis and since I am familiar with this company, I did not hesitate to buy 2 packets of their peanut and taro mochis at Maruyu.
the texture and consistancy of the mochis were extremely soft and stretchy, so much so that they stretched as I tried to lift them out of the plastic tray. The filling was taro paste, but I was pleasantly surprised by how well the flavour of the taro was executed into the mochi, although the taro flavour from the filling wasn't too strong, but it was still substantial enough, and the taro flavour infused into the soft mochis further enhanced the overall flavour. The mochis were also coated with a faint layer of icing sugar.
Overall, these taro mochis were very good, among the many taro sweets & snacks I have tasted over the years with dismal to mediocre results, these taro mochis from Royal Family are among the rare ones which I thought were actually well executed overall.
compared to the taro mochis, the texture of these peanut mochis was less soft and they were slightly chewer. The filling was peanut paste which had a pleasant substantial nutty flavour, and the addition of the peanut powder coating the mochis further enhanced the overall rich peanut flavour.
However I do have 2 very minor complaints about these peanut mochis, firstly I thought there wasn't enough peanut paste filling, I would've prefered to have just a little more filling, and I still prefer the softer and stretchier texture of the taro mochis. But like I mentioned before, these are just very minor disappointments.
Well to be honest, the main reason why I bought these 2 packets of mochi was because they were so cheap! Maruyu was selling them for only AU$2.50 for 2 packets! This just goes to show the stark contrast in price differences between authentic Japanese snacks and snacks manufactured in other asian countries. Snacks from other Asian countries tend to sell for dirt cheap, but authentic Japanese snacks & confectioneries (meaning snacks that are actually made in Japan) in comparison usually costs at least twice as much. The truth is, Japanese snacks are not expensive at all, at least not in Japan, in Japan they are actually very cheap. But once they are exported overseas, their prices suddenly sky-rocket, this is because it's more expensive to import products from Japan. So overseas shops need to significantly increase their prices for the Japanese snacks they sell in order to make a profit from them...thus, authentic Japanese snacks are much less affordable & consumed overseas than other Asian snacks. Which is a pity, because Japan make alot of very yummy snacks, but they are just too expensive for the average overseas person to buy on a regular basis.
And I'm sure I have mentioned before in a previous post that authentic Japanese snacks are also much less readily available overseas than other asian snacks. Although, many of Japan's largest snack & confectionery manufacturers such as Glico and Meiji do have branches in other parts of the world, so they still do make alot of snacks for the overseas market, but these snacks are manufactured by the various overseas branches of these companies. For example, most of Glico's famous 'Pocky' and 'Pretz' biscuit stick snacks you find in Asian groceries overseas are actually manufactured by the company's Shanghai branch in China and Bangkok branch in Thailand. Hence I do not really consider them to be "authentic" Japanese snacks, since they are not made in Japan, even though they are still manufactured by Japanese companies. But this also means that these Japanese snacks made in the overseas branches are much cheaper than their original Japanese counterparts.
Bottom line is that Japanese snacks ARE actually popular overseas...it's just that most of these familiar Japanese snacks that people overseas tend to buy are actually not made in Japan, as they are much more affordable than their original counterparts which ARE made in Japan.
But for me personally, I love my 'Pocky' and 'Pretz' snacks, regardless of whether they are made by Glico's Japan branch or overseas branches!
I do apologise for my long rant, I'm sure some of you might have found it to be a bit tedious to read! XD well, before I bore you any further, I better stop my rant...it's already almost 11:40pm here in Australia so I think it's bedtime now for me as I still need to go to work tomorrow.
Until next time, good night everyone! Oyasumi!
I love Japanese-style sweets & Wagashi such as Daifuku and Dango (japanese sweet dumplings on skewers), and Maruyu sells a range of different flavours of daifuku from Kido. But on the day I went to Maruyu, this blueberry & custard cream double pack was the only type on sale that day because it was nearing its 'best before' date. And since I had never tried any product from this particular manufacturer before, I decided to play safe and bought this cheaper pack.
Since there's 2 different flavours in this pack of daifuku, I will review both flavours separately.
basically the round outer mochi (rice cake) has 2 layers; the first white layer of mochi and the outer layer of mochi was infused with some subtle blueberry flavor and tinged with light pastel purple, I suspect a little bit of food colouring was used. But despite that little drawback, I loved the mochi layers of the daifuku, they were soft and chewy with just the perfect consistency and texture. And the sweetness of the mochi itself was not too strong, it was actually quite mild, and the blueberry flavor was subtle too.
The filling stuffed inside those blueberry daifukus was blueberry jam, which I did not expect, I had initially expected the filling to be some kind of blueberry flavoured paste. The fact that the filling was blueberry jam was quite a disappointment for me, because I don't like any kind of fruit jam, since I've always thought they were a bit too sweet for my taste, I also don't like the lumpy consistency of jam. What's even worse was that the blueberry jam filling was so sweet, it just dominated the whole product, much of the sweetness and blueberry flavor came from the jam filling. The strong flavor of the jam was just too overpowering, so the balance of flavor between the mochi and the filling was inconsistent.
Overall, this blueberry daifuku was quite a letdown for me, I love the mochi layers just fine, everything about the mochi itself was perfect, it had the right consistency and texture, so there is nothing to fault the mochi. What really ruined the daifuku was that overpowering blueberry jam filling, it was just too sweet for me. After consuming just 1 piece of the blueberry daifuku, I gave the rest to my fiance, because I could not tolerate the overwhelming sweetness of the blueberry jam anymore and the fact that I dislike fruit jams in general.
this custard daifuku also had a white layer of mochi, with a clear outer mochi layer, the filling inside is custard cream of course, and for me, this was a vast improvement from the blueberry daifuku. Again, the consistency and texture of the mochi layers were perfect; soft and chewy with subtle amounts of sweetness. The custard filling was also very nice, it was creamy and was not overly sweet. Compared to the blueberry daifuku, the flavours and sweetness of the mochi and the custard filling was much more balanced.
Overall these custard cream daifukus were much better than the blueberry ones for me, like I pointed out before, the sweetness and flavours of the custard daifukus were more balanced, and so were the consistencies and textures. I happily devoured all the custard daifukus in the pack and was rather disappointed when I realised there were none left! I wanted more!
Seriously, I wish the manufacturer Kido would pack their blueberry and custard cream daifukus separately, I would gladly devour a whole pack of their custard daifukus! but when my hunger is calling and I'm snacking on food, I can seriously do without those blueberry daifukus.
So here's my new LONG overdue food review, hope you all enjoy reading it I still have several more snacks I bought at Maruyu to review, so hopefully my next food review will be posted by the end of this week.
the beauty with these little Kyoto candies is not really their taste, but their appearance, just by looking at them you can see that they are sophisticated and difficult to make, mainly due to all the different patterns and pictures embedded in the center of these candies. I was fortunate to have been able to witness with my very own eyes the making process of these candies, and I have to say, there's really no other reaction you can have but to stare at these dedicated candy makers at work in awe. These candies are made into long candy sticks and then cut into little pieces, but it takes a lot of effort to make just one candy stick, and they are made by carefully molding one layer at a time into shape and it's time-consuming. Another reason why it takes so long to make these little candies is that these are all carefully handmade, unlike many commercial confectioneries in the world these days which are machine-made. Then again, these Kyoto candies are not commercial candies, these are specialty candies, even in Japan these candies are mostly given as gifts, even though they are still reasonably cheap in terms of cost.
As for the taste, well, that would be the downside to these Kyoto candies, with all their beautiful colors, sophisticated designs and patterns and grueling hours spent into making them, these candies taste rather bland and monotonous, the only flavour you'll taste is more or less sugar. So that's a disappointment, how much of a disappointment depends on the individual though.
But as I emphasized before, what really makes these Kyoto candies special is their appearance, all the little colorful layers carefully molded into place one by one to create the patterns/pictures and borders, and then cut into little pieces, they're really like little pieces of artwork. It takes alot of dedicated time and hard work to master this kind of candy-making.
The main reasons I gave these candies such a high rating are because of their pretty appearances, colours and complexity, and also taking into consideration the so many grueling hours and efforts the candy makers invest into carefully handmaking these little candies. I would have given these Kyoto candies the highest rating if it wasn't for the bland and monotonous taste.
Ok, now that I've cleared all the Pocky collaborative releases with Mart out of the way, let's move on to a different brand snack.
During my latest shopping errand at my local asian grocery yesterday, I picked up this pack of dorayaki made by Marukyo, the same company that produced the Shirakiku Marukyo Funwariyaki I reviewed about back in February.
Unlike the Funwariyaki, these are truly Dorayaki, I made sure I read the label!
each pack contained 6 individually wrapped dorayaki cakes, these dorayaki were really no different to the Marukyo Funwariyaki, same texture same taste, the cake patties were really soft and with mild amount of sweetness. And the sweetness from the azuki bean paste filling was quite subtle too, which leads me to believe that just like for the company's funwariyakis, either very little or no sugar was added to the azuki paste during the production process. Further more, the azuki bean paste was not too fine, this is because the makers at Marukyo did not overly pound the azuki beans to make the paste, which left some of the skin of the beans intact, and that in turn helped to add texture to the azuki paste. The lovely flavor of azuki from the paste was strong, due to the lacking of sugar added in the paste, so the full flavor & potential of those lovely azuki beans was able to shine through. Therefore you're able to taste that lovely natural taste of azuki beans from the paste, and it complemented well with the mild sweetness of the soft cake patties.
However, the one little draw-back about these dorayakis is that I thought the texture of the cake patties was just a little bit too dry for my palate personally, but that's merely my own opinion and is just a tiny wee dent on what is an overall great snack.
Overall, these dorayakis are fantastic sweets for anyone who craves a snack that's not too sweet.
I went shopping with some friends last Saturday at Chinatown and around the Sydney CBD area and decided to drop by at one of our favorite Japanese restaurants in the area; Kura restaurant for takeaway. I ordered one of my usuals the Sushi deluxe (you can read about my review about Kura's sushi deluxe here) and a serve of Taiyaki with matcha ice cream for dessert.
Taiyaki is a popular Japanese-style fish-shaped cake made from either pancake batter or waffle batter and traditionally has azuki (red bean) paste filling...although nowadays, there's more modern variety of fillings available, such as custard fillings and chocolate, but you will usually still find Taiyaki with azuki paste fillings.
I was a little disappointed that some parts of the batter skin was burnt, obviously the chef had slightly over-baked the Taiyaki, it's not exactly culinary pleasantness when you see dark burnt patches on your food, except maybe if it's for a BBQ, but not when you're baking Japanese sweets like Taiyaki. It brought out that unpleasant burnt taste, but luckily it was only very mild. The batter was rather plain and tasteless and there was not enough azuki paste filling for my liking, the flavor of the azuki paste was surprisingly rather bland as well, meaning the flavor was very subtle.
Overall, these Taiyaki were disappointing, I have tried plenty of Taiyaki from other restaurants and vendors over the years which were baked MUCH better with just the right amount and balance of batter and filling, and were baked for just the right amount of time and with stronger flavors.
I've always enjoyed dining at Kura restaurant and their food are mostly delicious at reasonable prices and the service is always good. However, their Taiyaki is just one of the few disappointments I fault them on. This was the first time I've ever ordered Taiyaki from Kura restaurant, and frankly, I wouldn't want to waste my money on them anymore. But Kura does serve plenty of other food which are really delicious that showcases the talent of its chefs.
If you want to know the address of Kura restaurant located in Haymarket Sydney, please refer to this entry.
I went to my local asian grocery early this morning to buy some groceries and came across this pack of Shirakiku Marukyo Funwariyaki in the snack aisle. Initially I thought these were Dorayaki; a popular Japanese-style cake that consists of 2 small soft cake patties resembling pancakes with various kinds of fillings (usually azuki bean paste) sandwiched between.
These cakes look exactly like Dorayaki, however it states on the package as 'Funwariyaki' This is the first time I have ever heard of 'Funwariyaki' and I have not been able to find any info about it online, so I am not sure whether Funwariyaki is a identical spin-off of Dorayaki, or 'Funwariyaki' is just a novelty name the manufacturer came up with for this product.
like Dorayaki, the texture of the 2 cake patties were soft and moist enough like sponge cakes and had very subtle hints of sweetness. The filling was azuki (red bean) paste made from 100% azuki beans grown in the northern Japanese island of Hokkaido, so you know they're the freshest and of the highest quality azuki beans. The sweetness of the azuki paste was also rather mild but still full of lovely azuki flavor. The subtle amount of sweetness of the azuki paste leads me to believe that very little, or none at all, sugar was added to the paste during the cooking & preparation process, to utilize the flavor of the azuki beans to its fullest potential.
Overall these 'Funwariyaki' were lovely without being overly sweet...even for people who have the sweetest of tooth, often want a snack that's not dominated by sweetness, and these Shirakiku Marukyo Funwariyaki are an ideal treat when you want to indulge on sweets that are not too sweet.
There seems to also be a matcha-flavored Funwariyaki by Shirakiku Marukyo, in which matcha is infused into the cake patties. I would love to try that one too, but I have not seen the matcha Funwariyaki in any of the asian groceries I've been to. So for the moment, it seems the only way to get my hands on the matcha flavor is to order it online, several online stores that sell asian sweets seems to have it in stock.
What I love about Japanese sweets is that Japan tends to put only about half as much sugar into their sweets & confectionery as western countries, without degrading the quality in any way.
Bought these yesterday during my frequent grocery trip to Tong Li, Japanese matcha (green tea) mochi (again), but this time it's made by a different company, this one is from Bamboo House, another Taiwanese company that specializes in Japanese Mochi.
firstly, I love the beautiful print on the box! The glutinous rice molds of each piece of mochi was very soft and chewy like it should be with a nice pastel green color while the filling was matcha paste.
Overall these were really nice & fresh...although I would've liked these a bit better if they had a bit more matcha flavor in them. I still prefer the matcha mochi from Royal Family...but these were still good though.
My second review on peanut mochi! I love Mochi, or any kinds of Wagashi...in case any of you don't know, Wagashi are traditional Japanese sweets normally eaten with tea, they are a staple during Japanese tea ceremonies.
I loved these, the glutinous rice molds of each piece were lovely, soft & chewy, as they should be. The peanut paste filling and dusted peanut powder surrounding each piece really brought out a pleasant peanut flavor, not too strong, but not subtle either.
These were much better than the single serve peanut mochi from Nice Bakery I had back in August.
because she bought me a mango mochi (which btw, was cho oishii so!) from the same bakery the other day that looked exactly the same from outer appearance, I had assumed that it was another mango mochi...until I took my first bite & instantly felt the crunch & tasted the flavor of chopped beautifully roasted peanuts (and quite a large amount of it too). The filling also contained quite a large amount of white sugar to balance out the savory taste of the peanuts. The glutinous rice mold of the mochi was soft & chewy and beautifully sweet, yet not too overpowering. And I love the addition of the shredded coconut sprinkled around the mochi.
Overall it was quite good...my only complaint about it is that I did not like the addition of the sugar in the filling, I felt it was unnecessary. It made the mochi taste even more sweet than it needed to be.
the jelly was thick with beautiful bright orange/yellow color, but I thought it was a bit overly sweet & the manga flavor was a bit too subtle. But overall, it was decent.


Thanks to Bakerella's awesome idea, we got a little crafty around here on Easter. Since my kids didn't actually see the pictures over on Bakerella's blog, they were satisfied with mom's on-the-fly substitutions: plain ol' foil instead of festive cupcake liners, boxed cake mix instead of homemade and, well, my pictures instead of Bakerella's. Sorry kids. What you don't know won't hurt you.
Just look at 'em. These innocent cupcakes have no idea what they're in for.
each with their own unique je ne sais quoi.
Jude took great care assembling his, even though he hates cupcakes.
And Shep's turned out to be pretty darn tasty.
Oh, these are just adorable!! What a good idea – and even more fun than Bakerella's Why wait?? Use up that leftover Easter candy, and let the kids bring them to school for a special Spring treat!!
Oh my gosh, those kiddos are darn cute!! And your photography, incredible!!!
Cute idea. The kids all look like they were having a great time! Adorable!
Oh my goodness, how adorable! Thank you for making me smile! Great idea.
What a GREAT idea! Maybe next year I will have to try that with my kiddos, hopefully all 5 of them! I love the picture of Shepard with green allover his face.
Ooooh, those look yummy! Thanks for the idea.
So cute! Jude hates cupcakes?
Why?? He likes cake right? I think I remember you posting his Thomas cake…or was that Shepherd's? Sorry!
My oldest (biological) won't eat most veggies despite the fact that his father and I eat them ALL! What's up with that?
Looks like a wonderful and tasty time!
What a fun & cute activity. Looks like it was a hit!
Those were a great idea. I like it.
So cute!! Thanks for a great idea for May day!! Somehow I can turn that into flower baskets…right??
BTW, just ordered my first shirt and can not wait to get it!!!
Next year I'm coming to your house.
LOVE these!!! Kiara saw the pics & said, "Momma, let's do THAT! RIGHT NOW!!!" lol!!
Looks like the kids had lots of fun too! Great photos!
Ok, can your kids get any cuter??? So darn precious!
SO cute!! What festive little baskets and precious little kiddos! Happy Easter!!
How cute! And I love the look of suger induced bliss on all of those adorable little faces.
Great pictures of your adorable kiddos, Stefanie.
I contemplated making the cupcakes as well for my student at school, but I could not find the green suger that Bakerella used so I didn't do them. Ended up making the chick cake balls. Didn't think to use plain ole sugar. DUH!!!!
I love good family days!!!
OML these are awsome. I miss baking these days life gets so busy.


Artist-Info - Melinda Stickney-Gibson - Butters Gallery Ltd.
New Yorker Melinda Stickney-Gibson has a truly dedicated following. She is known nation-wide for her unique and challenging brand of abstract painting. While obliquly narrative, her work is brutally honest and speaks to the soul.


If you are looking for the best value prescription eyeglasses online, then you here is a Great Discovery: www.ZenniOptical.com. ZenniOptical.com sells a wide range of stylish prescription glasses that suits anyone taste. They carry a wide selection of frames that have single vision lens, sun-sensor (potochromic) lens, tinted sunglasses lens, bifocal lens and progressive lens. When you buy your prescription eyeglasses from ZenniOptical.com, you get it at low low prices compared to other places. They are able to keep their prices low because they sell only their own manufactured frames direct to the customer, with no middlemen and virtually no advertising budget.The Great Discovery: www.ZenniOptical.com has helped many persons who badly needed prescription glasses but were unable to afford it a chance at seeing better again. What's more, all the eyeglasses that they sell come with full UV protection, anti scratch coating, quality hard eyeglass case, and a microfiber lens cleaning cloth. ZenniOptical.com sells rimless frames, half rim frames, and full rim frames too. Some of their frames are made of pure Titanium or Aluminum Alloy. Come and be a part of the Great Discovery: www.ZenniOptical.com by getting your prescription eyeglasses online. At $8 for a Complete Single Vision Eyeglass, you would be thinking illogically if you do not get your prescription eyeglasses at ZenniOptical.com.


text:
Alfonso Pompella
University of Pisa, Italy
Richard A. DeFazio
University of Michigan, United States
Hongyan Sun
City University of Hong Kong, Hong Kong, SAR China
Front. Pharmacol., 23 September 2016
Sec. Experimental Pharmacology and Drug Discovery
Volume 7 - 2016 | https://doi.org/10.3389/fphar.2016.00315
An NBD Derivative of the Selective Rat Toxicant Norbormide as a New Probe for Living Cell Imaging
Claudio D'Amore1†, Genny Orso2†, Fabio Fusi3, Mario A. Pagano4, Giovanni Miotto5, Alessia Forgiarini6, Sara De Martin4, Giulia Castellani4, Giovanni Ribaudo4, David Rennison7, Margaret A. Brimble7, Brian Hopkins7,8, Alessandro Ferrarese4 and Sergio Bova4*
1Department of Surgical and Biomedical Sciences, University of Perugia, Perugia, Italy
2IRCCS "E. Medea", Conegliano, Italy
3Department of Life Sciences, University of Siena, Siena, Italy
4Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padua, Italy
5Department of Molecular Medicine, University of Padua, Padua, Italy
6Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy
7School of Chemical Sciences, University of Auckland, Auckland, New Zealand
8Landcare Research, Lincoln, New Zealand
Norbormide (NRB) is a unique compound that acts directly on rat vascular myocytes to trigger a contractile process, through an as yet unknown mechanism, which results in the selective contraction of rat peripheral arteries. To gain insight into the mechanisms involved in NRB rat-selective activity, we investigated the subcellular distribution of NRB-AF12, a nitrobenzoxadiazole (NBD)-derivative of NRB, in living NRB-sensitive and NRB-insensitive cells. In both cell types, NRB-AF12 localized to the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, lysosomes, and endosomes; however, in NRB-sensitive cells, the fluorescence also extended to the plasma membrane. NRB-AF12 was rapidly internalized into the cells, could easily be washed out and then reloaded back into the same cells, all with a high degree of reproducibility. Cells exposed for 24 h to NRB-AF12 did not show apparent signs of toxicity, even at concentrations of the dye (10 μM) much higher than those required for fluorescence labeling (500 ηM). The distribution pattern of NRB-AF12 fluorescence was near identical to that of ER-Tracker® (Er-Tr), a fluorescent derivative of glibenclamide, a known KATP channel blocker. Displacement tests did not demonstrate, but at the same time did not rule out the possibility of a common target for ER-Tr, NRB-AF12, NRB, and glibenclamide. On the basis of these results we hypothesize a common target site for NRB-AF12 and ER-Tr, and a similar target profile for NRB and glibenclamide, and propose NRB-AF12 as an alternative fluorescence probe to ER-Tracker. Furthermore, NRB-based fluorescence derivatives could be designed to selectively label single cellular structures.
Norbormide (NRB, see Figure 1A for structure) is a unique vasoactive compound endowed with species-specific vasoconstrictor activity that targets the peripheral blood vessels of the rat (Clarke, 1965; Roszkowski, 1965; Bova et al., 1996). It also provokes a vasorelaxant effect on arteries of several non-rat species, as well as on rat large-caliber vessels (Roszkowski, 1965; Poos et al., 1966; Cavalli et al., 2004). NRB is a mixture of up to eight stereoisomers, four endo-isomers and four exo-isomers; among them, only the endo-isomers demonstrate vasocontractile and rat toxicant activities (Poos et al., 1966), whereas both endo- and exo-isomers have vasodilatatory activity in rat aorta and in non-rat arteries (Cavalli et al., 2004). The contractile effect of NRB on rat peripheral blood vessels is endothelium-independent, and is of myogenic nature as confirmed by its action on single rat caudal artery myocytes (Fusi et al., 2002; Cavalli et al., 2004). NRB binding sites and the molecular mechanisms involved in its biological effects in vascular tissue have yet to be fully characterized; it has been proposed that NRB-induced vasoconstriction may result from an interaction with a phospholipase C-coupled receptor (Bova et al., 2001), whereas its relaxant effect may be caused by an inhibitory action on plasma membrane voltage-dependent L-type Ca2+ channels (Fusi et al., 2002). NRB has also been shown to activate the mitochondrial permeability transition pore (mPTP) in isolated rat mitochondria, but not in mitochondria obtained from either guinea-pigs or mice. This suggests that the effect on the mPTP, like NRB-vasoconstriction, is also species-specific. However, since this effect could also be induced in mitochondria isolated from different organs of the rat (Ricchelli et al., 2005; Zulian et al., 2007, 2011), unlike vasoconstriction, mPTP activation is not tissue-specific. It is therefore unclear whether NRB-induced activation of the mPTP constitutes a role in the vasoconstrictor effect of NRB.
Figure 1. Chemical structures of (A) Norbormide and (B) its fluorescent derivative NRB-AF12. (C) Live cell imaging of LX2 hepatic stellate cells fluorescently labeled with 500 ηM NRB-AF12 for 30 min. Magnification 40x; scale bar 20 μm. (D) LX2 cells stained with NRB-AF12 were fixed and imaged by confocal microscopy. Magnification 60x; scale bar 20 μm.
Correspondingly, addressing the localization of NRB could help characterize the molecular mechanisms underlying the species- and tissue-selective actions of NRB; the original primary objective of this study was therefore to develop a fluorescent derivative of NRB (NRB-AF12, see Figure 1B for structure) in order to compare its subcellular distribution in NRB-sensitive and NRB-insensitive cells. However, since preliminary results indicated a strong overlapping fluorescent distribution between NRB-AF12 and that of ER-Tracker® (ER-Tr), a commercially available fluorescent probe widely used as a selective marker for endoplasmic reticulum (ER), we extended our goals to include a comparison of the staining profiles of the two dyes. The results indicate that NRB-AF12 and ER-Tr show a near identical pattern of fluorescence distribution in both NRB-sensitive and NRB-insensitive cells, and that this fluorescence profile differs between the two cell types. Furthermore, we found that both dyes label not only ER (as would be expected with ER-Tr), but also Golgi apparatus and mitochondria. In addition, unlike ER-Tr, NRB-AF12 was also able to label lysosomes and endosomes.
To a solution of endo-NRB (1.0 g, 2.0 mmol) in DMF (6 ml) was added NaH (160 mg, 4.0 mmol, 60% w/w in oil), and the resulting mixture stirred at room temperature for 15 min, then at 80°C for a further 15 min A solution of 4-[(tert-butoxycarbonyl)amino]butyl methanesulfonate (Cao et al., 2016) (0.59 g, 2.2 mmol) in DMF (2 ml) was added, and the resulting mixture stirred at 100°C for 4 h. The reaction mixture was then allowed to cool, diluted with water and extracted with EtOAc. The separated organic phase was washed with brine, dried over anhydrous Na2SO4, filtered and the solvent removed in vacuo. Purification by column chromatography (hexane/EtOAc, 1:1) afforded N-4′-[(tert-butoxycarbonyl)amino] butyl-5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide as an endo-exclusive mixture of four stereoisomers (white solid; 1.09 g, 1.60 mmol, 80%). 1H NMR (400 MHz, CDCl3) δ 1.30-1.68 (13H, m, C(CH3)3 and NCH2CH2CH2CH2NH), 3.05-3.20 (2H, m, NCH2CH2CH2CH2NH), 3.24-3.70 (4.2H, m, H-2, H-3, W/H-4 and NCH2CH2CH2CH2NH), 3.85–3.90 (0.4H, m, U/H-1 and V/H-1), 3.91-3.95 (0.4H, m, Y/H-4), 4.10–4.13 (0.1H, m, U/H-4), 4.26–4.30 (0.3H, m, V/H-4), 4.42–4.48 (0.6H, m, W/H-1 and Y/H-1), 5.55–5.67 (1.7H, m, OH and V/H-6 and Y/H-6), 6.02–6.04 (0.1H, m, U/H-6), 6.05–6.07 (0.2H, m, W/H-6), 6.70–7.62 (16H, m, ArH), 8.66–8.39 (2H, m, αPyr). To a solution of N-4′-[(tert-butoxycarbonyl)amino]butyl-5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide (0.34 g, 0.5 mmol) in DCM (10 ml) was added TFA (2 ml), and the mixture stirred at room temperature for 3 h. The solvent was then removed in vacuo to afford N-4′-aminobutyl-5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide trifluoroacetate as an endo-exclusive mixture of four stereoisomers [colorless gum (quant.)], which was used without further purification. To a solution of N-4′-aminobutyl-5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide trifluoroacetate (100 mg, 0.14 mmol) in DCM (4 ml) was added DIPEA (73 μl, 0.42 mmol), and the mixture stirred at room temperature for 30 min. NBD-Cl (4-chloro-7-nitrobenzofurazan) (28 mg, 0.14 mmol) was added and the resulting mixture stirred at room temperature for a further 6 h. The mixture was then diluted with DCM, washed with saturated aq. NaHCO3, then brine, and the separated organic phase dried over anhydrous Na2SO4, filtered and the solvent removed in vacuo. Purification by column chromatography (DCM) afforded 5-(α-hydroxy-α-2-pyridylbenzyl)-N-4′-[(7″-nitro-2″,1″,3″-benzoxadiazol-4"-yl)amino]butyl-7- (α-2- pyridylbenzylidene)-5-norbornene-2,3-dicarboximide (NRB-AF12) as an endo-exclusive mixture of four stereoisomers (orange solid; 50 mg, 0.067 mmol, 48%). M.p. 108-112°C; 1H NMR (400 MHz, CDCl3/MeOD 50:1 v/v) δ 1.61–1.84 (4H, m, NCH2CH2CH2CH2N), 3.34–3.73 (6.2H, m, NCH2CH2CH2CH2N, H-2, H-3 and W/H-4), 3.89–3.98 (0.7H, m, V/H-1 and Y/H-4), 3.99–4.02 (0.1H, m, U/H-1), 4.18–4.24 (0.4H, m, V/H-4 and U/H-4), 4.28–4.38 (0.6H, m, Y/H-1 and W/H-1), 5.56–5.62 (0.7H, m, V/H-6 and Y/H-6), 6.07–6.09 (0.1H, m, U/H-6), 6.12–6.14 (0.2H, m, W/H-6), 6.14–6.24 (1H, m, H-5″), 6.79–7.75 (16H, m, ArH), 8.37–8.65 (3H, m, αPyr and H-6″).
Fluorescence Spectra of NRB-AF12
Fluorescence spectra of NRB-AF 12 were obtained by dissolving the dye in water (for details on the methodology see Figure S1 in the Supplementary Material)
LX2 (a human immortalized hepatic stellate cell line), HSC-T6 (a rat immortalized hepatic stellate cell line), and HeLa (a human cervical carcinoma cell line) cells were cultured at 37°C in an atmosphere of 5% CO2 in Dulbecco's Modified Eagle Medium (Euroclone - Milan, Italy) supplemented with 10% Fetal Bovine Serum (Euroclone), 2 mM L-Glutamine and antibiotics. HepG2 cells (a human hepatocarcinoma cell line) were cultured at 37°C in an atmosphere of 5% CO2 in Eagle's Minimum Essential Medium (Euroclone) containing 10% Fetal Bovine Serum, 2 mM L-Glutamine and antibiotics.
VSMC Isolation
Smooth muscle cells were freshly isolated from the rat tail main artery under the following conditions: a 5-mm long piece of artery was incubated at 37°C for 40–45 min in 2 ml of 0.1 mM Ca2+ external solution containing 20 mM taurine, 1.35 mg/ml collagenase (type XI), 1 mg/ml soybean trypsin inhibitor, and 1 mg/ml BSA, which was lightly bubbled with a 95% O2–5% CO2 gas mixture to gently agitate the enzyme solution. Cells were stored in 0.05 mM Ca2+ external solution containing 20 mM taurine and 0.5 mg/ml BSA at 4°C under normal atmosphere, and were used for experiments within 2 days following isolation (Fusi et al., 2016).
All cell lines tested were stained immediately before imaging. Briefly, cells were trypsinized, counted and seeded in glass bottom μ-Dishes (Ibidi - Munich, Germany) at 2 × 104 cells/dish, and incubated for 24 h at 37°C in an atmosphere of 5% CO2. Cells were rinsed with Dulbecco's Modified Eagle Medium without phenol red (Euroclone) and loaded with 500 ηM NRB-AF12, 500 ηM ER-Tr red or green (#E34250 and #E34251; Thermo Fisher), and 500 ηM pHrodo red (#P10361; Thermo Fisher) at 10 μg/ml. In another experiment, LX2 cells were seeded in glass bottom μ-Dishes at 2 × 104 cells/dish and incubated for 24 h at 37°C in an atmosphere of 5% CO2. Cells were transfected with either 500 ηg of MitoDS-red or KDEL-red vector using Lipofectamine LTX Reagent (Invitrogen), according to the manufacturer specifications. At 24-h post-transfection, cells were stained with either 500 ηM NRB-AF12 or 500 ηM ER-Tr green. Cells were mounted in a Okolab microscope stage heated chamber warmed to 37°C. All images were collected with a Yokogawa CSU-X1 spinning disk confocal on a Nikon Ti-E inverted microscope equipped with a Plan Apo 60x NA 1.4 objective. Probes were excited with a 488 and 561 nm Lasers. To separate the individual emissions a dichroic filter 405/488/561/635-25 (Semrock, Rochester, NY) followed by a 440-40/521-21/607-34/700-45 filter (Semrock, Rochester, NY) were used and subsequently filtered further using band-pass filters (Semrock BP-525/30-25 and BP-607/36-25 for the green and red channels, respectively). Images were acquired with an Andor Technology iXon3 DU-897-BV EMCCD camera. For time-lapse experiments, images were collected every 10 s, using an exposure time of 500 ms.
To evaluate cell retention of the probe post-fixation/permeabilization, LX2 cells (2 × 104) were plated on a 12 mm glass coverslip placed in a well of a 24 well-plate and incubated for 24 h at 37°C in an atmosphere of 5% CO2. The cells were then treated with 500 ηM NRB-AF12 for 30 min, fixed with 4% paraformaldehyde for 15 min at room temperature and immediately mounted on microscope slides, or fixed with 4% paraformaldehyde, permeabilized with either 0.1% Triton X-100 (10 min at room temperature) or ice cold methanol (5 min at −20°C), and subsequently mounted.
In order to evaluate NRB-AF12 co-localization with endoplasmic reticulum, Golgi apparatus and with the sulfonylurea receptor (SUR) subunits of the ATP-sensitive potassium channels, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and permeabilized with ice cold methanol for 5 min at −20°C. ER was highlighted with antibodies directed against the ER resident protein calreticulin (1:200; Abcam #ab2907); Golgi apparatus was highlighted with antibodies directed against the Golgi apparatus structural protein GM130 (1:500; BD Biomedsciences #610822); SUR2 subunits of KATP channel were highlighted using either anti-SUR2A (1:750; Abcam #ab174629) or anti-SUR2B (1:750; Abcam #ab174631) antibodies. All primary antibodies were incubated for 90 min at room temperature. Following a PBS wash, cells were incubated with Cy™3 goat anti-rabbit IgG fluorescent secondary antibody (1:500; Jackson ImmunoResearch #111-165-003) or with Cy™3 goat anti mouse IgG fluorescent secondary antibody (1:500; KPL #072-01-18-06) for 60 min at room temperature in the presence of either 500 ηM NRB-AF12 or 500 ηM ER-Tr green. Finally, coverslips were mounted on microscope slides using Mowiol 40–88 (Sigma Aldrich). Confocal images were acquired with a Laser Scanning Confocal Microscope (LSCM) D-Eclipse C1 SHV Nikon system equipped with a Nikon Eclipse E600 microscope and 3 laser diode modules (488/543/637 nm), using a CFI Plan Apochromat 60x NA 1.4 objective and analyzed using NIS Elements software (Nikon) and the open-source platform for biological-image analysis Fiji software.
Drosophila strain W[1118], stock number 5905 was obtained from Bloomington stock center (Indiana University, USA) and used to perform live imaging experiments. Fly larvae having reached the third instar stage whilst feeding on standard food were dissected in hemolymph-like HL3 saline (70 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 20 mM MgCl2, 10 mM NaHCO3, 5 mM trehalose, 115 mM sucrose, 5 mM sodium HEPES, pH 7.2). For live tissue imaging ER-Tr red and NRB-AF12 were added to the medium of dissected larvae and images acquired using a Nikon eclipse C1 confocal microscope and a Nikon Fluor 60x NA 1.00 water objective.
1 × 105 LX2 cells, 1 × 105 HepG2 cells and 5 × 104 HSC-T6 cells were plated in 12 well-plates in complete culture medium and incubated for 24 h at 37°C. Cells were then treated with increasing doses (0.1, 1 and 10 μM) of NRB, NRB-AF12, glibenclamide and ER-Tr for 24 h. Cell viability was assessed by the Trypan Blue exclusion method. Briefly, cells were trypsinized, resuspended in complete medium, diluted 1:2 with a 0.4% Trypan Blue solution and finally counted using a hemacytometer under a light-microscope.
Hazards Related to the Use of NRB and Its Derivatives for Research Purposes
In HSNO (Hazardous Substances and New Organisms) terms, a substance is considered hazardous if it triggers any one of the threshold levels for any of the hazardous properties listed in the USA Environmental Protection Agency (EPA) guidelines, one of which is toxicity. NRB is extremely toxic to rats and as such is classified as an "extremely hazardous chemical" in the US and elsewhere. However, one of the key features of NRB is that its toxic activity is uniquely limited to rats, having little or no activity in any other species tested (including humans). In fact NRB's lethal activity is even further restricted as it seems only to be toxic to species within the Rattus genus, with other rat genera being little or not affected. As a consequence, the NRB and the NRB derivatives reported in this research paper poses little or no hazard to the researchers, and as such no special conditions or safety precautions are required to work with these substances other than those required to conform to standard laboratory practices.
All values are expressed as means ± standard error (SE) of n observations/group. Analysis of co-localization was performed using Pearson's correlation coefficient. Comparisons of more than two groups were made with a one-way ANOVA using post-hoc Tukey's test. Comparison of two groups was obtained by the Student's t-test for unpaired data when appropriate. Differences were considered statistically significant at values of P < 0.05.
The fluorescence spectra of NRB-AF12 are shown in Figure S1 in Supplementary Material.
The distribution of NRB-AF12 was initially evaluated in NRB-insensitive cells.
Figure 1C shows phase contrast microscopy images and conventional epifluorescence microscopy images of living LX2 cells incubated with 500 ηM NRB-AF12. The images clearly show a meshwork distribution of the fluorescence with a sharp demarcation of the nuclei and an accumulation in perinuclear areas; moreover, NRB-AF12 did not stain either the nuclei or the plasma membrane. NRB-AF12 internalization into the cell was fast, commencing after only a few seconds and reaching completion within a few minutes, following its addition to the incubation medium. A similar fluorescence pattern was also observed in fixed LX2 cells (Figure 1D), however, the NRB-AF12 signal was not retained post-permeabilization (data not shown). The sub-cellular distribution of NRB-AF12 was investigated in more detail in LX2 cells using confocal microscopy in combination with fluorescent probes and/or antibodies targeted to specific subcellular structures. To evaluate the distribution of NRB-AF12 in the ER, LX2 cells were transfected with the ER-reporter red fluorescent protein (RFP)-KDEL plasmid and subsequently loaded with the dye. The results, reported in Figures 2A,D, show that the NRB-AF12 signal significantly overlapped with that of (RFP)-KDEL (Pearson's Coefficient: 0.21 ± 0.10; n ≥ 10), but a larger staining distribution of NRB-AF12 was observed which indicated other targets in addition to ER. Similar results were observed in LX2 fixed cells labeled with NRB-AF12 and immunostained for the ER resident protein Calreticulin (Pearson's Coefficient: 0.3 ± 0.06; n ≥ 10) (Figures 2B–D). We also compared the staining pattern of NRB-AF12 with that of ER-Tr, a commercial fluorescent probe widely used to label ER. Surprisingly, co-labeling of LX2 cells with NRB-AF12 and ER-Tr resulted in a strong overlap between fluorescent dyes (Figure 2C), confirmed by the high rate of co-localization index (Pearson's coefficient: 0.75 ± 0.02; n ≥ 15). This remarkable similarity between the two dyes was further supported by the observation that ER-Tr, as well as NRB-AF12, were not selective for the ER, as demonstrated by co-staining LX2 cells with ER-Tr plus either RFP-KDEL (Figure 3A) or Calreticulin (Figure 3B) (Pearson's coefficient: 0.23 ± 0.1 and 0.29 ± 0.03, respectively for RFP-KDEL and Calreticulin; n ≥ 10; Figure 3C).
Figure 2. (A) Live cell imaging of LX2 cells transfected with ER-reporter RFP-KDEL plasmid and subsequently stained with NRB-AF12. (B) Immunostaining of LX2 cells for the endoplasmic reticulum marker calreticulin; cells were counter-labeled with NRB-AF12. (C) Confocal live cell imaging of LX2 cells fluorescently labeled with 500 ηM NRB-AF12 and 500 ηM ER-Tr red. Insets show magnification of the pictures. Magnification 60x; scale bar 10 μm. (D) Quantification of NRB-AF12 co-localization with RFP-KDEL, Calreticulin and ER-Tr is shown as Pearson's coefficient. (n ≥ 10).
Figure 3. (A) Live cell imaging of LX2 cells transfected with ER-reporter RFP-KDEL plasmid and subsequently stained with ER-Tr. (B) Immunostaining of LX2 cells for the endoplasmic reticulum marker calreticulin; cells were counter-labeled with NRB-AF12. Magnification 60x; scale bar 10 μm. (C) Quantification of ER-Tr co-localization with RFP-KDEL and Calreticulin is shown as Pearson's coefficient (n ≥ 10).
The NRB-AF12 and ER-Tr subcellular distribution patterns and co-localization rates were confirmed using other NRB-insensitive cells (i.e., HepG2, HSC-T6, and HeLa cells; Figures 4A–C), and using NRB-sensitive cells such as freshly isolated rat caudal artery myocytes (Figure 4D). Interestingly, in this latter cell type, both fluorescent compounds stained not only intracellular structures, but also the plasma membrane. Co-localization rates of NRB-AF12 and ER-Tr in HepG2, HSC-T6, HeLa and primary rat vascular smooth muscle cells is reported in Figure 4E as Pearson's coefficient. These results indicates that NRB-AF12 labels intracellular structures with a pattern of distribution that is apparently very similar to that of ER-Tr, and is not confined to ER.
Figure 4. Representative live cells microphotographs showing co-localization between NRB-AF12 (left column) and ER-Tr red (mid column) in (A) HepG2, (B) HSC-T6, (C) HeLa, and (D) primary rat vascular smooth muscle cells (VSMC) from caudal artery. For each panel, merged fluorescence is shown in right column. Magnification 60x; scale bar, 10 μm. (E) Quantification of NRB-AF12 and ER-Tr co-localization is shown as Pearson's coefficient (n ≥ 15).
We then investigated, using LX2 cells, which non-ER subcellular organelles were labeled by NRB-AF12, and compared the results with those obtained with ER-Tr, focusing our attention on the Golgi apparatus, mitochondria, lysosomes, and endosomes.
The Golgi apparatus was investigated by co-staining fixed cells with either NRB-AF12 or ER-Tr, and antibodies directed against the Golgi structural protein GM130 (α-GM130). Data obtained, shown in Figure 5A, indicates a co-localization of NRB-AF12 and α-GM130 (Pearson's coefficient: 0.53 ± 0.02; n ≥ 10; Figure 5C). Similar results were obtained when α-GM130 was used in combination with ER-Tr (Pearson's coefficient: 0.58 ± 0.02; n ≥ 10; Figures 5B,C).
Figure 5. LX2 fixed cells immunostained for the Golgi apparatus structural protein GM130; cells were counter-labeled with (A) NRB-AF12 or (B) ER-Tr green. Magnification 60x; scale bar 10 μm. (C) Co-localization measure is shown as Pearson's coefficient (n ≥ 10).
Mitochondrial NRB-AF12 and ER-Tr localization was investigated in living LX2 cells transfected with the mitochondrial marker MitoDS-red plasmid, and then loaded with either NRB-AF12 or ER-Tr. As depicted in Figure 6, both dyes labeled mitochondria to similar levels (Pearson's Coefficient: 0.56 ± 0.05 and 0.54 ± 0.03, respectively for NRB-AF12 and ER-Tr; n ≥ 10).
Figure 6. Live cell imaging of liver myofibroblasts transfected with the mitochondrial marker Mito-Ds red and then stained using (A) NRB-AF12 or (B) ER-Tr green. Magnification 60x; scale bar 10 μm. (C) Co-localization of NRB-AF12 and ER-Tr within mitochondria is reported as Pearson's coefficient (n ≥ 10).
Next, we investigated whether NRB-AF12 and ER-Tr could label endosomes and lysosomes. For this purpose, we performed live cell imaging experiments in LX2 cells using a commercial fluorescent probe which has been previously demonstrated to highlight vesicles involved in endocytosis. As shown in Figure 7, we could not find any staining of endocytic vesicles when cells were loaded with ER-Tr (Pearson's coefficient: −0.33 ± 0.05 and −0.43 ± 0.04, respectively for endosomes and lysosomes; n ≥ 10). In contrast, NRB-AF12 showed an appreciable co-localization rate with both endosomal and lysosomal vesicles (Pearson's coefficient 0.25 ± 0.13 and 0.15 ± 0.15, respectively; n ≥ 10). Interestingly, the data revealed a high index of dispersion of the co-localization coefficients (CI 95%: −0.05 – 0.55 and −0.19 – 0.5, for endosomes and lysosomes respectively; n ≥ 10), indicating that not all endosomes/lysosomes vesicles were stained by the dye.
Figure 7. Confocal live cell microphotographs of LX2 cells stained with the fluorescent dye for endosomal (empty arrowhead) and lysosomal (full arrowhead) compartments pHrodo red and (A) NRB-AF12 or (B) ER-Tr green. Magnification 60x; scale bar 10 μm. (C,D) Co-localization of NRB-AF12 and ER-Tr Green with endosome and lysosome vesicles is shown as Pearson's coefficient (*p < 0.05; n ≥ 10).
Since NRB-AF12 and ER-Tr showed the same cellular labeling pattern, we hypothesized a common target for these compounds. ER-Tr probes are conjugates of glibenclamide bearing different fluorophores, BODIPY FL (ER-Tr green) and BODIPY TR (ER-Tr red). Glibenclamide is an antidiabetic drug belonging to the class of sulfonylureas that binds the sulfonylurea receptor (SUR) subunits of ATP-sensitive potassium channels (Proks et al., 2002). Accordingly, we therefore explored the likelihood of whether NRB could also target these SURs by analyzing the co-localization between NRB-AF12 and the 2 major subunits SUR2A and SUR2B. We focused our attention on these SUR isoforms because of their intracellular distribution (Bao et al., 2011) and the role of the plasmalemmal SUR2B in the regulation of vascular contraction (Morrissey et al., 2005; Teramoto, 2006; Jackson, 2008; Tinker et al., 2014). Our results indicate that in LX2 cells NRB-AF12 co-localized with both SUR2A and SUR2B, with a greater correlation for the latter (Pearson's coefficient: 0.28 ± 0.02 and 0.36 ± 0.01, respectively for SUR2A and SUR2B; n ≥ 10) (Figures 8A,C,E); comparable results were also obtained with ER-Tr (Pearson's coefficient: 0.22 ± 0.04 and 0.32 ± 0.02, respectively for SUR2A and SUR2B; n ≥ 10) (Figure 8B,D,F). It is worth noting that the cellular distribution of NRB-AF12 and ER-Tr fluorescence was larger than that observed with SUR2, suggesting non-SUR2 additional targets for both dyes.
Figure 8. (A,B) Confocal pictures of LX2 fixed cells immunostained for the KATP channels subunit SUR2A and counter-labeled with (A) NRB-AF12 or (B) ER-Tr green. (C,D) Immunostaining of LX2 cells for SUR2B subunit of KATP channels; cells were counter-labeled with (C) NRB-AF12 or (D) ER-Tr green. Insets show magnification of the pictures. Magnification 60x; scale bar 10 μm. (E,F) Pearson's coefficient showing co-localization rate of (E) NRB-AF12 or (F) ER-Tr green with either SURs isoforms (n ≥ 15).
The overlapping fluorescence distributions of AF-12 and ER-Tr prompted us to investigate whether NRB and glibenclamide could compete with their respective fluorescent derivatives in LX2 cells. To this end, LX2 cells were pre-treated with high concentrations (100 μM) of either NRB or glibenclamide, and then loaded with either NRB-AF12 or ER-Tr. Interestingly, pre-treatment with the non-labeled compounds failed to prevent fluorescent labeling following addition of the fluorescent derivatives (data not shown).
To summarize the results concerning the cellular distribution of AF-12, we show in Figure 10 a single living LX2 cell, in which are detailed the subcellular organelles labeled by the dye.
We also explored the fluorescence features of NRB-AF12 in intact tissues of dissected larvae of Drosophila melanogaster. Third instar larvae were labeled with NRB-AF12 and ER-Tr dissolved in the hemolymph-like HL3 saline, added to the medium following dissection and imaged with an immersion objective. As shown in Figure 9, a bright fluorescence was observed when larvae were treated with the fluorescent probes: In vivo live imaging of D. melanogaster muscle fibers (Figure 9A) and tracheal system (Figure 9B) revealed a sarcoplasmic reticulum distribution of the dyes, with NRB-AF12 and ER-Tr fluorescence significantly overlapping, as was observed in cell culture (Johnson et al., 2015). Finally, we investigated some physical properties of NRB-AF-12 such as penetration, internalization rate, retention time, washout time-course in living cells, as well as the induction of cytotoxicity, in order to establish the usefulness and reliability of the dye. Video 1 in Supplementary Material, shows that in living LX2 cells AF-12 is internalized in minutes and is retained in the cells for at least 3 h following its removal from the culture medium. Furthermore, re-exposure of the cells to dye, gave the same results, in terms of fluorescence distribution, compared to the first exposure. NRB-AF12 was apparently not toxic in both human and rat cell lines, even at concentrations much higher than those (500 nM) utilized for labeling (Figures 11B–D).
Figure 9. Confocal live imaging of Drosophila (A) muscles and (B) tracheal system labeled with NRB-AF12 (green) and ER-Tr red (red). Both fluorescent dyes, added to the dissected larva in HL3 physiological solution, show an overlapping pattern of distribution as highlight by the yellow signal of the merged images (*: muscle; white arrowhead: trachea; N: nucleus). Magnification 60x; scale bar 10 μm.
This study was undertaken to verify if a fluorescent derivative of NRB could furnish information about the cellular target/s involved in the selective rat-specific and tissue-selective action of unlabeled NRB. To this end, we attached the NBD fluorophore to the NRB molecule. The NBD group is known to bleach very rapidly in respect to other commercially available fluorophores, nevertheless we decided to use NBD because of its relatively small size, in the attempt to minimize the risk of inducing a distortion of the chemical-physical properties of NRB. We compared the fluorescence distribution of NBD-NRB (NRB-AF12) observed in NRB-sensitive cells (freshly isolated rat caudal artery myocytes) with that observed in NRB-insensitive cells (several cultured cell lines). Preliminary experiments conducted in our lab indicated that NRB-AF12 has the same species- and tissue-specific contractile properties of unlabeled NRB (manuscript in preparation), and therefore is a reliable tool for the purpose of this work.
There are two key observations:
1) In both NRB-sensitive and NRB-insensitive cells NRB-AF12 distributes intracellularly; however, only in the sensitive cells is fluorescence detected in the plasma membrane.
2) In all the cells investigated, NRB-AF12 fluorescence showed a pattern of distribution similar to that of ER-Tr.
Given that the cellular labeling patterns of NRB-AF12 and ER-Tr are near identical, it could be proposed that the two dyes share a common cellular target/s. ER-Tr is a BODIPY derivative of glibenclamide, a widely used antidiabetic drug belonging to the sulfonylurea family. Glibenclamide is a non-selective inhibitor of ATP-sensitive potassium channels (Proks et al., 2002), a multimeric protein complex consisting of four pore-lining inward rectifier α subunits (Kir6.1/2) and four regulatory sulfonylurea-binding β subunits (SUR1/2A/2B) that belong to the ATP-binding cassette family (Yokoshiki et al., 1998; Zerangue et al., 1999; Shi et al., 2005; Ng et al., 2010). Co-assembly of different Kir and SUR subunits generates ion channel combinations with different channel properties, and with different cell compartment or tissue distribution (Yokoshiki et al., 1998), such as the SUR1/Kir6.2 pancreatic type, the SUR2A/Kir6.2 cardiac type and the SUR2B/Kir6.1 vascular smooth muscle type. The ER-Tr and NRB-AF12 fluorescence characteristics may therefore reflect the cellular distribution of KATP channels. Inhibition of plasma membrane ATP-sensitive potassium channels leads to an increased intracellular K+ concentration, plasma membrane depolarization, opening of voltage-dependent calcium channels, and increased intracellular Ca2+ concentration (Ko et al., 2008) that, in vascular smooth muscle, triggers the contractile process. It has been reported that vascular smooth muscle plasma membrane selectively expresses SUR2B subunits of KATP channels (Morrissey et al., 2005; Teramoto, 2006; Tinker et al., 2014). These data, together with the finding (this study) that NRB-AF12 localizes to the plasma membrane only in NRB-sensitive cells, might explain the species- and tissue-selective activity of NRB, by hypothesizing an inhibitory effect of the drug on rat-specific SUR2B-containing KATP channels expressed on the plasma membrane of the rat peripheral artery myocytes. In contrast to this hypothesis is the fact that neither unlabeled NRB nor glibenclamide are able to displace NRB-AF12 or ER-Tr from their binding site, which presents the prospect of a different/additional site of action from/to KATP channels. It also raises the question as to whether the fluorescently labeled compounds have the same binding characteristics of the corresponding unlabeled compounds. However, the lack of displacement is in itself not sufficient to rule out the possibility that NRB-AF12 does bind to KATP channels, with there being at least two plausible explanations: (1) NRB-AF12 and NRB could bind to different sites within the SUR2B moiety, thus allowing the attachment of both compounds; (2) the fluorophore group of NRB-AF12 may have multiple binding sites preventing its displacement by unlabeled NRB. The existence of more than one binding site, at least for NRB-AF12, is supported by the fact that this dye, in contrast to ER-Tr, was able to label both endosomes and lysosomes. As with SUR2-containing channels, NRB-AF12 appears to be similarly targeted to the endosomal/lysosomal pathway, supporting the idea that this compound can be recycled by endocytosis as has been shown for the KATP channel subunits (Bao et al., 2011).
Live cell imaging represents an important technique in the study of biological processes; the use of fluorescent proteins or dyes provides an important tool for the in vivo visualization, in space, and time, of virtually any cellular mechanism or structure, avoiding artifacts or sample alteration that could occur with fixation methods. In this study we show that NRB-AF12 is endowed with features that make this dye an eligible prototype of new fluorescent probes, alternatives to ER-Tr, for live cell imaging (Figure 10). Furthermore, in NRB-sensitive single rat vascular myocytes, this dye, due to its constriction effect (manuscript in preparation), could also be employed to study the morphologic changes that occur in labeled intracellular organelles, as well as in the plasma membrane, during the contractile process. Whatever the binding site/s for NRB-AF12, this dye shows a cellular distribution near identical to that of ER-Tr in real-time fluorescent imaging using different cultured cell lines and freshly isolated rat caudal artery myocytes. NRB-AF12 labeling was also observed in fixed and permeabilized cells, allowing its use in immunocytochemistry co-localization experiments. In all cells tested, NRB-AF12 stained not only ER, but also other subcellular compartments such as Golgi apparatus, mitochondria, and organelles involved in the endocytic pathway (i.e., endosomes and lysosomes). NRB-AF12 internalization rate was extremely fast, since cell incubation with the fluorescent compound resulted in a well-defined meshwork staining of the cellular structures within a few minutes, and could be observed up to 3 h following its removal from the culture medium (Figure 11A). Another useful feature is that NRB-AF12 exhibited no apparent cytotoxicity, a serious disadvantage that often occurs using fluorescent probes for live cell imaging. Cell viability experiments demonstrated the dye to be biocompatible, having no harmful effects in the different cell lines (both human and rat) after 24-h incubation, even at concentrations (10 μM) much higher than those (500 ηM) needed to visualize cellular organelles (Figures 11B–D). Furthermore, the lack of cytotoxicity of NRB-AF12 allows the same sample to be repeatedly stained with the dye (for example at different time points within an experiment), without morphological alteration of cell structures or cell death; concurrently, the ability of NRB to effectively label its cellular target is preserved on re-exposure to the dye (Figure 11A).
Figure 10. Confocal live imaging of a LX2 cell stained with NRB-AF12 showing subcellular distribution of the fluorescent probe. Magnification 100x; scale bar 5 μm.
Figure 11. (A) LX2 cells were incubated for 30 min with NRB-AF12 or ER-Tr green; after replacing the staining solution with probe-free medium, evaluation of NRB-AF12 and ER-Tr extent of fluorescent signal and photostability was performed. (B) LX2, (C) HSC-T6, and (D) HepG2 cells were treated for 24 h with increasing doses (0.1, 1 and 10 μM) of NRB, NRB-AF12, glibenclamide or ER-Tr. Compounds toxicity was evaluated by trypan blue exclusion test of cell viability. NT, not treated cells.
Finally, imaging experiments performed in dissected larvae of D. melanogaster demonstrated NRB-AF12 adaptability in complex biological systems. The live imaging of fly tissues showed that NRB-AF12 maintain the same subcellular distribution of ER-Tr also in vivo, supporting the theory of a common target. Moreover, we observed a significant signal in the Drosophila tracheal system where Drosophila Sur (Dsur) is highly expressed (Nasonkin et al., 1999). Dsur, encodes a glibenclamide-sensitive potassium channel and has been identified as the functional orthologue of the mammalian SUR2 (Kim and Rulifson, 2004). On the basis of these favorable properties, we propose NRB-AF12 as an alternative fluorescent dye to ER-Tr, and a new improved tool for labeling intracellular organelles.
In conclusion, this study presents evidence to demonstrate that NRB-AF12, a fluorescent derivative of the selective rat toxicant NRB, has a highly similar fluorescence distribution to ER-Tr, a fluorescent derivative of glibenclamide, a known KATP channel inhibitor. On the basis of these results we hypothesize common cellular binding sites for NRB and glibenclamide that may potentially lead to common biological properties. Furthermore, we propose NRB-AF12 as a prototype for the development of new fluorescent probes selective for intracellular structures/organelles. Studies are in progress in our lab to investigate these aspects.
CD and GO contributed equally to this work. SB, CD, and MP conceived the study. AFe, MB, DR, BH, and GR designed and performed synthesis of NRB-AF12. CD, GO, GM, FF, AFo, GC, and SD designed and performed experiments. SB, CD, and GO analyzed and interpreted data. SB, CD, GO, DR, and BH wrote the manuscript. All authors approved the final version of the manuscript.
This study was supported by the University of Padova, project n. 148125/14.
We thank Prof. Stefano Fiorucci (University of Perugia) for kindly gifting LX2 and HSC-T6 cells and Prof. Giuseppe Zagotto (University of Padova), for the determination of the fluorescence spectra of NRB-AF12.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fphar.2016.00315
Bao, L., Hadjiolova, K., Coetzee, W. A., and Rindler, M. J. (2011). Endosomal KATP channels as a reservoir after myocardial ischemia: a role for SUR2 subunits. Am. J. Physiol. Heart Circ. Physiol. 300, H262–H270. doi: 10.1152/ajpheart.00857.2010
Bova, S., Cima, L., Golovina, V., Luciani, S., and Cargnelli, G. (2001). Norbormide: a calcium entry blocker with selective vasoconstrictor activity in rat peripheral arteries. Cardiovasc. Drug Rev. 19, 226–233. doi: 10.1111/j.1527-3466.2001.tb00067.x
Bova, S., Trevisi, L., Debetto, P., Cima, L., Furnari, M., Luciani, S., et al. (1996). Vasorelaxant properties of norbormide, a selective vasoconstrictor agent for the rat microvasculature. Br. J. Pharmacol. 117, 1041–1046.
Cao, Y., Min, C., Acharya, S., Kim, K.-M., and Cheon, S. H. (2016). Design, synthesis and evaluation of bitopic arylpiperazinephenyl-1,2,4-oxadiazoles as preferential dopamine D3 receptor ligands. Bioorg. Med. Chem. 24, 191–200. doi: 10.1016/j.bmc.2015.12.002
Cavalli, M., Omiciuolo, L., Cargnelli, G., Cima, L., Hopkins, B., and Bova, S. (2004). Distribution of the vasoconstrictor and vasorelaxant effects of norbormide along the vascular tree of the rat. Life Sci. 75, 2157–2165. doi: 10.1016/j.lfs.2004.04.022
Clarke, E. G. (1965). Identification of norbormide, a new rattus specific rodenticide. J. Pharm. Pharmacol. 17, 126.
Fusi, F., Manetti, F., Durante, M., Sgaragli, G., and Saponara, S. (2016). The vasodilator papaverine stimulates L-type Ca2+ current in rat tail artery myocytes via a PKA-dependent mechanism. Vascul. Pharmacol. 76, 53–61. doi: 10.1016/j.vph.2015.11.041
Fusi, F., Saponara, S., Sgaragli, G., Cargnelli, G., and Bova, S. (2002). Ca(2+) entry blocking and contractility promoting actions of norbormide in single rat caudal artery myocytes. Br. J. Pharmacol. 137, 323–328. doi: 10.1038/sj.bjp.0704877
Jackson, W. F. (2008). Vanishing act: protein kinase C-dependent internalization of adenosine 5′-triphosphate-sensitive K+ channels. Hypertension 52, 470–472. doi: 10.1161/HYPERTENSIONAHA.108.112425
Johnson, A. E., Shu, H., Hauswirth, A. G., Tong, A., and Davis, G. W. (2015). VCP-dependent muscle degeneration is linked to defects in a dynamic tubular lysosomal network in vivo. eLife 4:e07366. doi: 10.7554/eLife.07366
Kim, S. K., and Rulifson, E. J. (2004). Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells. Nature 431, 316–320. doi: 10.1038/nature02897
Ko, E. A., Han, J., Jung, I. D., and Park, W. S. (2008). Physiological roles of K+ channels in vascular smooth muscle cells. J. Smooth Muscle Res. 44, 65–81. doi: 10.1540/jsmr.44.65
Morrissey, A., Rosner, E., Lanning, J., Parachuru, L., Dhar Chowdhury, P., Han, S., et al. (2005). Immunolocalization of KATP channel subunits in mouse and rat cardiac myocytes and the coronary vasculature. BMC Physiol. 5:1. doi: 10.1186/1472-6793-5-1
Nasonkin, I., Alikasifoglu, A., Ambrose, C., Cahill, P., Cheng, M., Sarniak, A., et al. (1999). A novel sulfonylurea receptor family member expressed in the embryonic Drosophila dorsal vessel and tracheal system. J. Biol. Chem. 274, 29420–29425.
Ng, K.-E., Schwarzer, S., Duchen, M. R., and Tinker, A. (2010). The intracellular localization and function of the ATP-sensitive K+ channel subunit Kir6.1. J. Membr. Biol. 234, 137–147. doi: 10.1007/s00232-010-9241-x
Poos, G. I., Mohrbacher, R. J., Carson, E. L., Paragamian, V., Puma, B. M., Rasmussen, C. R., et al. (1966). Structure-activity studies with the selective rat toxicant norbormide. J. Med. Chem. 9, 537–540.
Proks, P., Reimann, F., Green, N., Gribble, F., and Ashcroft, F. (2002). Sulfonylurea stimulation of insulin secretion. Diabetes 51 (Suppl. 3), S368–S376. doi: 10.2337/diabetes.51.2007.S368
Ricchelli, F., Dabbeni-Sala, F., Petronilli, V., Bernardi, P., Hopkins, B., and Bova, S. (2005). Species-specific modulation of the mitochondrial permeability transition by norbormide. Biochim. Biophys. Acta 1708, 178–186. doi: 10.1016/j.bbabio.2005.03.002
Roszkowski, 1965###Roszkowski, A. P. (1965). The pharmacological properties of norbormide, a selective rat toxicant. J. Pharmacol. Exp. Ther. 149, 288–299.
Shi, N.-Q., Ye, B., and Makielski, J. C. (2005). Function and distribution of the SUR isoforms and splice variants. J. Mol. Cell. Cardiol. 39, 51–60. doi: 10.1016/j.yjmcc.2004.11.024
Teramoto, N. (2006). Physiological roles of ATP-sensitive K+ channels in smooth muscle. J. Physiol. 572(Pt 3), 617–624. doi: 10.1113/jphysiol.2006.105973
Tinker, A., Aziz, Q., and Thomas, A. (2014). The role of ATP-sensitive potassium channels in cellular function and protection in the cardiovascular system. Br. J. Pharmacol. 171, 12–23. doi: 10.1111/bph.12407
Yokoshiki, H., Sunagawa, M., Seki, T., and Sperelakis, N. (1998). ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells. Am. J. Physiol. 274(1Pt 1), C25–C37.
Zerangue, N., Schwappach, B., Jan, Y. N., and Jan, L. Y. (1999). A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels. Neuron 22, 537–548.
Zulian, A., Petronilli, V., Bova, S., Dabbeni-Sala, F., Cargnelli, G., Cavalli, M., et al. (2007). Assessing the molecular basis for rat-selective induction of the mitochondrial permeability transition by norbormide. Biochim. Biophys. Acta 1767, 980–988. doi: 10.1016/j.bbabio.2007.04.002
Zulian, A., Sileikytė, J., Petronilli, V., Bova, S., Dabbeni-Sala, F., Cargnelli, G., et al. (2011). The translocator protein (peripheral benzodiazepine receptor) mediates rat-selective activation of the mitochondrial permeability transition by norbormide. Biochim. Biophys. Acta 1807, 1600–1605. doi: 10.1016/j.bbabio.2011.08.007
Keywords: norbormide, ER tracker, fluorescent probes, sulphonylurea receptors, LX2 cells
Citation: D'Amore C, Orso G, Fusi F, Pagano MA, Miotto G, Forgiarini A, De Martin S, Castellani G, Ribaudo G, Rennison D, Brimble MA, Hopkins B, Ferrarese A and Bova S (2016) An NBD Derivative of the Selective Rat Toxicant Norbormide as a New Probe for Living Cell Imaging. Front. Pharmacol. 7:315. doi: 10.3389/fphar.2016.00315
Received: 13 July 2016; Accepted: 01 September 2016;
Published: 23 September 2016.
Alfonso Pompella, University of Pisa, Italy
Richard Anthony DeFazio, University of Michigan, USA
Hongyan Sun, City University of Hong Kong, China
Copyright © 2016 D'Amore, Orso, Fusi, Pagano, Miotto, Forgiarini, De Martin, Castellani, Ribaudo, Rennison, Brimble, Hopkins, Ferrarese and Bova. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Sergio Bova, sergio.bova@unipd.it

redpajama_set_name: RedPajamaCommonCrawl