We have uploaded the verified vector information for the published Drosophila KCR constructs to Zenodo. Files can be assessed under: https://zenodo.org/records/15074206

The file contains vector maps for the below constructs:

pJFRC7_20xUAS_HcKCR1_AAA_YFP
pJFRC7_20xUAS_HcKCR1_C29D_YFP
pJFRC7_20xUAS_HcKCR1_ET_YFP
pJFRC7_20xUAS_HcKCR1_GS_YFP
pJFRC7_20xUAS_HcKCR2_AAA_YFP
pJFRC7_20xUAS_HcKCR2_ET_YFP
pJFRC7_20xUAS_HcKCR2_GS_YFP
pJFRC7_20xUAS_WiChR_ET_YFP

These were reported in:
Ott, S., Xu, S., Lee, N. et al. Kalium channelrhodopsins effectively inhibit neurons. Nat Commun 15, 3480 (2024). https://doi.org/10.1038/s41467-024-47203-w

Also:
Kalium channelrhodopsins effectively inhibit neurons in the small model animals
Stanislav Ott, Sangyu Xu, Nicole Lee, Ivan Hee Kean Hong, Jonathan Anns, Danesha Devini Suresh, Zhiyi Zhang, Xianyuan Zhang, Raihanah Harion, Weiying Ye, Vaishnavi Chandramouli, Suresh Jesuthasan, Yasunori Saheki, Adam Claridge-Chang
bioRxiv 2024.01.14.575538; doi: https://doi.org/10.1101/2024.01.14.575538

Fly constructs and genetics
UAS-KCR1-ET, UAS-KCR2-ET, UAS-KCR1-GS and UAS-WiChR transgenic lines were generated by de novo synthesis (Genscript) of Drosophila codon-optimized HcKCR insert sequences 43 (Genbank #MZ826861 and #MZ826862) or the WiChR sequence 45 (Genbank #OP710241) as eYFP fusions. After Sanger sequencing verification (Genscript), the fragments were cloned into an pJFRC7-20XUAS-IVS-mCD8::GFP vector (Addgene plasmid # 26220), replacing the mCD8::GFP insert via restriction enzyme digest (XhoI, Xba I). For UAS-KCR1-GS, a 3×GGGGS sequence was used to link the opsin with the fluorophore. For the KCR-ET and WiChR constructs, an AAA linker sequence was used as the starting point, to which two modifications were made: (1) an FCYENEV motif was added to the C terminus of eYFP to boost protein export from the endoplasmic reticulum and prevent potential aggregate formation 51; and (2) a KSRITSEGEYIPLDQIDINV trafficking signal from Kir 2.1 52 was added to the linker at C terminus of the opsin to boost protein expression 10. The KCR1-C29D variant 45 was obtained by site-directed mutagenesis of the KCR1-ET sequence, where the cysteine at position 29 was replaced by aspartic acid (Genscript). The synthesized constructs were injected into flies and targeted to attP1 or attP2 insertion sites on the second or third chromosomes respectively and the transgenic progeny were balanced either over CyO or TM6C (BestGene). Expression was verified by imaging of eYFP fluorescence with a Leica TCS SP8 STED confocal microscope. Opsin transgenic flies were crossed with relevant Gal4 driver lines to produce F1 offspring for use as test subjects. Driver Gal4 lines and UAS-opsin responder lines were each crossed with an otherwise wild-type w1118 line and the F1 progeny (e.g. UAS-KCR1-ET/+ or elav-Gal4/+) were used as control subjects.

Our lab has identified a previously unknown parallel function of dopamine neurons involved in olfactory memory in Drosophila. While these neurons were known to be crucial for memory formation, we demonstrate they simultaneously drive immediate attraction and aversion behaviors, independent of their memory-related function.

Through optogenetic manipulation, we found that sensory neurons essential for olfactory memory were not required for dopamine-driven immediate responses. We identified two key neuronal populations: a broad network of dopaminergic neurons that influenced behavior through dopamine, glutamate, and octopamine signaling, and a more specific cluster that drove attractive responses. Notably, inhibiting this latter group caused flies to display active avoidance, highlighting its role in ongoing behavioral control.

This work reveals how dopaminergic systems can coherently guide both immediate responses and memory formation, advancing our understanding of the neural circuits underlying learning and behavior.

The study was published in PLOS Biology.

Update: Now published in Nature Communications.

The Claridge-Chang lab evaluated the utility of the new kalium channelrhodopsins to suppress behavior and inhibit neural activity in Drosophila, C. elegans, and zebrafish. In direct comparisons with ACR1, a variety of KCRs with enhanced plasma-membrane trafficking displayed excellent potency, and with improved properties that include reduced toxicity and superior efficacy in putative high-chloride cells.
This comparative analysis of behavioral inhibition between chloride- and potassium-selective silencing tools establishes KCRs as next-generation optogenetic inhibitors for in vivo circuit analysis in behaving animals.
Read more here.

Date: 22 Jun 2023
Location: DUKE-NUS MEDICAL SCHOOL, Outram Campus, SG
Company: National University of Singapore
Job DescriptionThe Signature Research Programme in Neuroscience and Behavioural Disorders (NBD) is focused on understanding the structure and function of the nervous system, and the neural mechanisms underlying human neurological, psychiatric and ophthalmological disorders.
 
Controlling neuronal activity with light is an important method for the analysis of neuronal circuits. The Ministry of Education has funded a five-year project to develop and use optogenetic tools. This exciting project aims to conduct optogenetic experiments in Drosophila, looking at the circuit function of neuromodulatory cells. The Claridge-Chang Lab at Duke-NUS Medical School (https://www.claridgechang.net/) is seeking to appoint a Research Fellow with a strong background in neuroscience, to join this project. We would be especially interested in scientists with experience in optogenetics, fly genetics, light microscopy, brain imaging, neuronal circuit mapping, molecular cloning, electrophysiology, and/or cell culture.
 
The selected candidate will perform a variety of research activities which include planning, organising and conducting research studies within the overall scope of a re-search project under the supervision of the Principal Investigator or his/her designate, including but not limited to the following: -
 

• Perform tasks and support all aspects of the research project.
• Conduct accurate monitoring, documenting and reporting of experimental results and/or research findings.
• Assist in writing the draft of research papers and review articles.
• Assist to provide guidance and support to other researchers, as well as under-graduate and graduate students.
• Assist with housekeeping of the laboratory area, general maintenance tasks and animal stock maintenance.
• Maintain safety protocols, order/prepare common reagents and maintain laboratory inventory.
• Perform other related duties incidental to the work described therein.

Job Requirements

• PhD in Neuroscience (or other related fields with a keen interest in neuroscience). 
• Possess laboratory experimental skills including the relevant skill sets to perform the following techniques - behavior analysis, Drosophila experiments, imaging, cell culture, electrophysiology, molecular biology (sub-cloning methods including restriction digests, PCR and gel electrophoresis). 
• Possess skills in microscopy such as laser confocal microscopy, image analysis (e.g. ImageJ/FIJI).
• Have skills in biological data analysis such as Python, R or similar analysis methods.
• Prior experience in scientific sourcing & procurement would be advantageous. 
• Proven knowledge, skills and abilities with excellent written and oral communication skills.
• A self-motivated individual who is able to work independently, prioritise, multi-task and work collaboratively in a research environment of diverse work-force.

Please email [email protected] with your CV.  
We regret that only shortlisted candidates will be notified.

Research Assistant -
ACC-RA-DART-202307
Cell-Specific Pharmacology in Neuroscience (FILLED)
The Signature Research Programme in Neuroscience and Behavioural Disorders (NBD) at Duke-NUS Medical School is focused on understanding the structure and function of the nervous system, and the neural mechanisms underlying human neurological, psychiatric and ophthalmological disorders.
The Claridge-Chang Lab (https://www.claridgechang.net/) is seeking a motivated and skilled Research Assistant. The successful candidate will work on an innovative project focused on developing and implementing cell-specific pharmacology methods in Drosophila melanogaster for neuroscience research, to understand the effects of targeted drug delivery on specific neural cell types.

Key Responsibilities:

• Develop and apply cell-targeted techniques for restricted drug delivery in Drosophila to better understand neural mechanisms underlying various brain disorders.
• Perform and support research-project tasks, including molecular cloning, fly genetics, cell culture, electrophysiology, and imaging, under the supervision of the Principal Investigator or their designate.
• Plan, organize, and conduct research studies within the project scope.
• Accurately monitor, document, and report experimental results and research findings.
• Assist in drafting research papers and review articles.
• Provide guidance and support to other researchers, undergraduate and graduate students.
• Maintain laboratory safety protocols, manage laboratory inventory, order and prepare common reagents.
• Assist with procurement, general lab maintenance, housekeeping, and fly stock maintenance.

Job Requirements:

• Degree in Biological Sciences or related fields with a keen interest in neuroscience.
• Strong laboratory skills in Drosophila experiments, molecular biology, cell culture, and confocal microscopy.
• Familiarity with programmatic data analysis using Python, R, or similar methods.
• Prior experience in scientific sourcing and procurement.
• Excellent written and oral communication skills.
• Proficiency in microscopy techniques (e.g., laser confocal microscopy) and image analysis tools (e.g., ImageJ, Zeiss LSM Browser).
• Self-motivated, able to work independently, prioritize tasks, and collaborate in a diverse research environment.

By joining our team, you will contribute to cutting-edge research that advances our understanding of cell-targeted pharmacology in neuroscience. Apply now by submitting your resume, highlighting your relevant experience and skills.

Please email [email protected] with your CV. Please use code ACC-RA-DART-202307 in the subject line. We regret that only shortlisted candidates will be notified.

The Signature Research Programme in Neuroscience and Behavioural Disorders (NBD) is focused on understanding the structure and function of the nervous system, and the neural mechanisms underlying human neurological, psychiatric and ophthalmological disorders.

Controlling neuronal activity with light is an important method in neuroscience, The Ministory of Education has funded a five-year project to study and use optogenetic tools. This exciting project aims to develop new optogenetic tools to inhibit neuronal activity. The Claridge-Chang Lab at Duke-NUS Medical School (https://www.claridgechang.net/) is seeking to appoint a Research Assistant, ideally with a strong background in neuroscience, to join this project. We would be especially interested in scientists with experience in molecular cloning, electrophysiology, cell culture, fly genetics, and/or imaging.  

The selected candidate will perform a variety of research activities which include planning, organising and conducting research studies within the overall scope of a research project under the supervision of the Principal Investigator or his/her designate, including but not limited to the following: -

• Perform tasks and support all aspects of the research project.
• Conduct accurate monitoring, documenting and reporting of experimental results and/or research findings.
• Assist in writing the draft of research papers and review articles.
• Assist to provide guidance and support to other researchers, as well as undergraduate and graduate students.
• Assist with housekeeping of the laboratory area, general maintenance tasks and animal stock maintenance.
• Maintain safety protocols, order/prepare common reagents and maintain laboratory inventory.
• Perform other related duties incidental to the work described therein.

Job Requirements

• Degree in Biological Sciences or other related fields with a keen interest in neuroscience.
• Possess laboratory experimental skills including the relevant skill sets to perform the following techniques - cell culture, electrophysiology, transfection, molecular biology (sub-cloning methods including restriction digests, PCR and gel electrophoresis), Drosophila experiments, imaging.
• Possess skills in microscopy such as laser confocal microscopy, image analysis (ImageJ, Zeiss LSM Browser, PhotoShop, Metamorph and/or Matlab).
• Have skills in bioinformatics data analysis such as Python, R or similar analysis methods.
• Prior experience in scientific sourcing & procurement.
• Proven knowledge, skills and abilities with excellent written and oral communication skills.
• A self-motivated individual who is able to work independently, prioritise, multi-task and work collaboratively in a research environment of diverse workforce.

 
Please email [email protected] with your CV.  We regret that only shortlisted candidates will be notified.

We are looking to recruit a Data Scientist.

Job Description
The Signature Research Programme in Neuroscience and Behavioural Disorders (NBD) is focused on understanding the structure and function of the nervous system, and the neural mechanisms underlying human neurological, psychiatric and ophthalmological disorders.
The Claridge-Chang Laboratory, Duke-NUS Medical School is looking for a data scientist to help transform the way scientists analyze data. The position is funded for two years. Recently, the laboratory has led the way in introducing a new way of data analysis and estimation statistics. It has achieved this progress with a suite of software, Data Analysis with Bootstrapped Estimation (DABEST), available as libraries in R, Python, and a web application. These software tools have wide usage, representing an important shift in statistical culture in biomedical research.

• https://github.com/ACCLAB/DABEST-python
• https://github.com/ACCLAB/dabestr
• https://www.estimationstats.com/#/

The existing software covers five of the most widely used analysis models, suitable for a variety of small data sets and common experimental designs. The laboratory has won a competitive grant and the candidate will work on DABEST for a wider set of data types, including visualizing large data with estimation graphics. This project will be in collaboration with Assoc Prof. Hyungwon Choi, NUS Medicine.  The candidate will benefit from the dynamic environment of two laboratories with world-class expertise in experimental design, advanced statistical analysis of high-dimensional data, and data visualization.
The role will involve both software development for new modules of estimation statistics, and application of estimation methods to exciting research questions, encouraging a productive dialogue between analysis methods development and application. The selected candidate will perform a variety of research activities, including but not limited to the following:

• Develop the DABEST visualization package for new categories of estimation data analysis.
• Develop an empirical Bayes method for analysis of high-dimensional analysis and visualisation with estimation.
• Document the code and write introductory materials to introduce the new methods.
• Record instructional videos introducing the methods and describe the new methods to a broad audience.
• Coordinate the estimationstats.com web application.
• Analyse and process data from neuroscience and proteomic experiments, using estimation and other statistical methods.
• Contribute to project management, presentations and publications of research work
• Provide guidance to junior researchers as well as undergraduate and graduate students.
  

Job Requirements

• PhD or Master’s Degree in Statistics, Data Science, Bioinformatics, Computational Biology or related areas with demonstrated expertise in statistical computing and data visualisation.
• Experience in programming and software development, as well as machine learning would be advantageous.
• Experience with statistical modelling and analysis of large-scale data.
• Has good programming skills in R and Python.
• Demonstrate good knowledge, skills and expertise, with potential to achieve research and methods-development excellence.
• Able to effectively multi-task and perform well under pressure, both independently and as part of a team.
• Able to prioritise and work collaboratively in a diverse workforce.

ApplyPlease email a CV and cover letter to claridge-chang.adam<at>duke-nus.edu.sg.

This article was originally published in the Straits Times 10 February 2022

On my way to work, I often walk past a smoking area. While second-hand smoke can harm passersby, cigarette smoke can at least be seen and smelled so the risk to others is apparent. Like tobacco smoke, the Covid-19 virus is also airborne: it is exhaled, floats around and can fill a room. But unlike smoke,  its airborne particles are odourless and invisible.

The transmission mode of Covid-19 has been controversial. Some people still believe that the main routes of transmission are through touching contaminated objects that can carry infection such as doorknobs, or through coming into contact with mouth droplets.
However, a large number of studies have now demonstrated that the major route of transmission is via tiny airborne particles called aerosols.

So the announcement a week ago by Singapore’s Building and Construction Authority (BCA) that it is launching a major study into the use of natural ventilation to reduce transmission inside buildings is welcome and timely (The Straits Times, Jan 30, ‘Study to look at reducing disease spread in Singapore buildings’).

As the planned study suggests, a simple, effective approach to deal with airborne transmission will be to open buildings to allow more outdoor air.
Innovating modern versions of traditional fan-based cooling will make indoor spaces comfortable, while facilitating the elimination of contaminated air. In the short term, ventilation can be improved by opening windows, and holding gatherings outdoors whenever possible.

However, not every indoor space can be opened up, and air conditioning will remain a reality. How can that issue be addressed? And how did it take so long to move away from the focus on droplets, and recognise Covid’s airborne transmission?

Droplets v aerosols
The critical difference between droplets and aerosols is that droplets are so large that they fall to the ground within a few metres in seconds, while aerosol particles are small enough to float in the air for minutes or even hours.

In March 2021, a group of scientists led by Trisha Greenhalgh, professor of Primary Care Health Sciences at Oxford University, published a short letter listing 10 points of evidence supporting the Covid virus’s airborne transmission. One devastating observation was the frequency of long-range infections.

One such outbreak occurred in a restaurant in Guangdong. At three adjacent tables, nine people were infected by the index patient. Security video confirmed that the index patient never had close contact with the other tables - so droplet transmission was ruled out. All three tables were cooled by the same air conditioner. Some of the infected people were right next to the airconditioner’s blower, and so were seemingly “upwind” of the index patient.

A team of scientists, headed by Min Kang at the Guangdong Provincial Centre for Disease Control and Prevention, used smoke to track airflow. They discovered that the air was recirculating, and that the “upwind” infected people were effectively 10 metres downwind of the index patient.

Long-distance outbreaks are common - hence the word “super-spreader”.
Early in the pandemic, on February 11 2020, the World Health Organisation’s director-general, Tedros Ghebreyesus, got it right: he announced  the virus’s airborne transmission.

He said: “This is airborne. Corona is airborne.” But in the following month, WHO officials backtracked. Dr Tedros himself wrote “ ... actually it’s not airborne”.

But throughout the first year of the pandemic, evidence for airborne transmission accumulated and scientists increased the pressure, culminating in Professor Greenhalgh’s letter. On April 30, 2021, rather quietly, WHO updated its website to acknowledge that aerosol transmission plays a role. Even today WHO appears to minimise airborne transmission.

WHO’s stance can influence national policies. Currently, the Singapore Ministry of Health’s (MOH) main website, under the heading “How does Covid-19 spread?” starts off by echoing WHO’s old droplet claims.

It adds later on: “While there have been limited reports of airborne transmission outside of health care settings internationally, its role and extent are under further study. MOH will continue to monitor the evidence as it emerges.”

Nevertheless, since 2020, MOH, together with the National Environmental Agency, and the Building and Construction Authority (BCA), has been advising ways to improve ventilation to fight Sars2 (as the Covid-19 virus is known). And last year, the agencies advised that Sars2 “can also be spread through virus aerosols in the air under certain settings, such as enclosed environments which are poorly ventilated”, along with updated advice to building owners and facility managers on how to improve indoor air quality. Indeed, the adoption of some of these airborne-oriented measures might have already helped to reduce transmission.

What can and needs to be done
And barely a week ago came the BCA’s announcement looking into natural ventilation.
Natural ventilation is an excellent solution for many settings, however not all indoor spaces can be opened up in this way, and air conditioning will continue to play a role. So we also need to invest in better air monitoring, filtration, and purification to keep our indoor air clean.

Carbon dioxide monitors can measure how much air in a space has already been breathed. Particulate monitors can measure the extent of particle and aerosol contamination.

Air purifiers with HEPA-type (High Efficiency Particulate Air) filters clear aerosols effectively. As an interim solution, affordable do-it-yourself purifiers can be constructed and installed within a day, before buildings’ air-handling systems are upgraded. If HEPA-type filters had been used in the Guangdong restaurant’s air conditioner, that outbreak might have been prevented.

Ultimately, we need new building codes and designs that will make indoor air as safe and healthy as outside air. 

David Fisman, professor of Epidemiology at the University of Toronto and co-author of the Greenhalgh letter jokes, “Air: it’s the new poop.” People in wealthy countries don’t tolerate faeces in their water supply because it causes potentially fatal diseases. So why should they tolerate workplaces, schools, or restaurants with potentially deadly exhaled air?

Or an even closer analogy: I’m old enough to remember when indoor smoking was allowed, and a bar could contain so much exhaled smoke it would be hard to see across the room. Now it seems absurd that we would allow public spaces with high levels of visible carcinogenic aerosols.

One day, we will think the same way about invisible virus aerosols. To control the coming waves of  Covid variants - and the next pandemic - let’s hope that day comes soon.

-Adam Claridge-Chang

By Adam Claridge-Chang.
A version of this opinion piece was first published by the South China Morning Post on 14 October 2021.

Singapore’s vaccination rate is one of the highest in the world, above 80 per cent. Encouraged by this, the city-state has tried to reopen and renormalize. However, the recent COVID-19 surge has grown faster than expected, hospitals are feeling the impact, restrictions were reintroduced, and mobility remains well below pre-pandemic levels.

Here and abroad, there is a vigorous debate between two groups on how to ‘exit’ from the pandemic.

On one side are the ‘eliminationists’. They point to China which advocates that the safest way to defend against COVID-19 is to eliminate it. Lockdowns and quarantines, they argue, are a price worth paying to keep everyone safe. They highlight that eliminationist economies have overall grown faster than mitigation economies. Eliminationists note the potential threat of novel variants and that long COVID is incompletely understood.

On the other side are those advocating to re-open society. They argue that, since vaccines protect the vast majority from severe disease, allowing Delta to spread is acceptable and even desirable for post-infection immunity. The re-openers point to well-vaccinated countries in Europe that lifted regulations without major spikes in deaths. Re-openers say that ongoing travel and social restrictions will do needless damage to society, mental health, and the economy. 

Both camps make strong cases. But what if we could do both? What if we could strongly suppress COVID-19 while also re-normalising society? Some believe this can be done with at-home testing.

In the pandemic’s first days, the WHO urged the world to “test, test, test”. By identifying and isolating cases, they said, the chains of transmission could be broken. Polymerase chain reaction (PCR) testing—a powerful way to detect viral genes (RNA)—was widely implemented. 

Later, another kind of test became available: the antigen rapid test (ART). ART kits are like a pregnancy test for COVID: they require no special electronics or skills, and can be used by anyone. For the past year, Dr Michael Mina, an assistant professor of epidemiology at the Harvard T.H. Chan School of Public Health in the US, has been advocating that the widespread use of ARTs could force COVID-19 transmission into collapse. He bases this on ART’s three critical advantages.

First, ARTs can reliably detect if someone is in the roughly weeklong infectious phase, when they secrete millions of viruses. This high-virus phase corresponds to the interval when ARTs are highly sensitive, making them an almost ideal tool for someone to know when they are contagious. Critics of ARTs say that PCR is much more sensitive, but this can actually be a liability when it detects a residual amount of RNA during a waning, non-infectious case. Data from a study by the UK Covid-19 Lateral Flow Oversight Team indicates that ART sensitivity for contagious cases, even when asymptomatic, is typically around 97 per cent. This means that, with rapid testing, positive individuals can reliably know which few days they need to stay home, while everyone else testing negative can confidently get on with their lives. 

Second, ARTs are very fast. The Delta variant becomes infectious 1–2 days earlier than last year’s strains, effectively outrunning PCR tests, which can take days to give a result. An ART can let someone know if they are contagious in just 20 minutes, beating Delta’s pace.

Third, ART technology lends itself to mass production at low cost (although in many places this has not yet been fully realized). ARTs will be most effective when testing is affordable for everyone. Transmission will be more effectively slowed as ARTs are used more widely. Therefore, better control will be achieved when ARTs are low cost or, ideally, free.

Indeed, some European nations have made low-cost ARTs integral to their re-opening strategy. In Germany, the federal regulator has authorized dozens of different ART brands, driving prices under €2.00. In the UK, households can order a free pack of seven tests every day, or collect free test packs from local collection points. 

However, places like the United States and Singapore have been slower to increase ART availability. The US has stringently regulated ARTs as medical devices, resulting in only a handful of approved home-test vendors, high prices, and shortages. President Biden is moving to fix this, invoking the US Defense Production Act to help increase production and directly buying millions of tests.

In Singapore, one especially clever application is using ARTs in coordination with automated contact tracing: close contacts are given free kits and expected to test daily for a week in case they become positive. However, approvals of home-use ARTs have been relatively slow—just a few brands approved at the time of this writing. Prices at the major retailers remain high, around S$10 per test. In Germany, an ART costs roughly the same as a ride on Singapore’s Mass Rapid Transit (MRT). Broadening vendor approvals should lower prices, so that everyone can afford to test regularly. A survey in the US showed that, if available for U$1.00 (S$1.35), as many as 79% would use ARTs regularly. Low cost would also make it easier for governments and employers to distribute kits freely to citizens and staff.

Whatever we do now is preparation for the next variant wave or future pandemics. Self-testing is already as easy as other hygiene routines like tooth-brushing. We know it can be made as cheap as a daily commute. When that happens, we will find out whether we can crush the pandemic with a new kind of ‘MRT’: mass rapid testing.