Computational Optogenetics: Empirically-Derived Voltage- and Light-Sensitive Channelrhodopsin-2 Model

Channelrhodospin-2 (ChR2), a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silicoprediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R) with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, and used these data to develop a model of ChR2 with empirically-derived voltage- and irradiance- dependence, where parameters were fine-tuned via simulated annealing optimization. This ChR2 model offers: 1) accurate inward rectification in the current-voltage response across irradiances; 2) empirically-derived voltage- and light-dependent kinetics (activation, deactivation and recovery from inactivation); and 3) accurate amplitude and morphology of the response across voltage and irradiance settings. Temperature-scaling factors (Q10) were derived and model kinetics was adjusted to physiological temperatures. Using optical action potential clamp, we experimentally validated model-predicted ChR2 behavior in guinea pig ventricular myocytes. The model was then incorporated in a variety of cardiac myocytes, including human ventricular, atrial and Purkinje cell models. We demonstrate the ability of ChR2 to trigger action potentials in human cardiomyocytes at relatively low light levels, as well as the differential response of these cells to light, with the Purkinje cells being most easily excitable and ventricular cells requiring the highest irradiance at all pulse durations. This new experimentally-validated ChR2 model will facilitate virtual experimentation in neural and cardiac optogenetics at the cell and organ level and provide guidance for the development of in vivo tools

T-Lymphoblastic Lymphoma Cells Express High Levels of BCL2, S1P1, and ICAM1, Leading to a Blockade of Tumor Cell Intravasation

The molecular events underlying the progression of T-lymphoblastic lymphoma (T-LBL) to acute T-lymphoblastic leukemia (T-ALL) remain elusive. In our zebrafish model, concomitant overexpression of bcl-2 with Myc accelerated T-LBL onset while inhibiting progression to T-ALL. The T-LBL cells failed to invade the vasculature and showed evidence of increased homotypic cell-cell adhesion and autophagy. Further analysis using clinical biopsy specimens revealed autophagy and increased levels of BCL2, S1P1, and ICAM1 in human T-LBL compared with T-ALL. Inhibition of S1P1 signaling in T-LBL cells led to decreased homotypic adhesion in vitro and increased tumor cell intravasation in vivo. Thus, blockade of intravasation and hematologic dissemination in T-LBL is due to elevated S1P1 signaling, increased expression of ICAM1, and augmented homotypic cell-cell adhesion.Stem Cell and Regenerative Biolog

ABCB1 (MDR1) polymorphisms and ovarian cancer progression and survival: A comprehensive analysis from the Ovarian Cancer Association Consortium and The Cancer Genome Atlas

<b>Objective</b> <i>ABCB1</i> encodes the multi-drug efflux pump P-glycoprotein (P-gp) and has been implicated in multi-drug resistance. We comprehensively evaluated this gene and flanking regions for an association with clinical outcome in epithelial ovarian cancer (EOC).<p></p> <b>Methods</b> The best candidates from fine-mapping analysis of 21 <i>ABCB1</i> SNPs tagging C1236T (rs1128503), G2677T/A (rs2032582), and C3435T (rs1045642) were analysed in 4616 European invasive EOC patients from thirteen Ovarian Cancer Association Consortium (OCAC) studies and The Cancer Genome Atlas (TCGA). Additionally we analysed 1,562 imputed SNPs around ABCB1 in patients receiving cytoreductive surgery and either ‘standard’ first-line paclitaxel–carboplatin chemotherapy (n = 1158) or any first-line chemotherapy regimen (n = 2867). We also evaluated ABCB1 expression in primary tumours from 143 EOC patients.<p></p> <b>Result</b> Fine-mapping revealed that rs1128503, rs2032582, and rs1045642 were the best candidates in optimally debulked patients. However, we observed no significant association between any SNP and either progression-free survival or overall survival in analysis of data from 14 studies. There was a marginal association between rs1128503 and overall survival in patients with nil residual disease (HR 0.88, 95% CI 0.77–1.01; p = 0.07). In contrast, <i>ABCB1</i> expression in the primary tumour may confer worse prognosis in patients with sub-optimally debulked tumours.<p></p> <b>Conclusion</b> Our study represents the largest analysis of <i>ABCB1</i> SNPs and EOC progression and survival to date, but has not identified additional signals, or validated reported associations with progression-free survival for rs1128503, rs2032582, and rs1045642. However, we cannot rule out the possibility of a subtle effect of rs1128503, or other SNPs linked to it, on overall survival.<p></p&gt

The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends

DNA damage is a common hazard that all cells have to combat. Saccharomyces cerevisiae HMO2 is a high mobility group protein (HMGB) that is a component of the chromatin-remodeling complex INO80, which is involved in double strand break (DSB) repair. We show here using DNA end-joining and exonuclease protection assays that HMO2 binds preferentially to DNA ends. While HMO2 binds DNA with both blunt and cohesive ends, the sequence of a single stranded overhang significantly affects binding, supporting the conclusion that HMO2 recognizes features at DNA ends. Analysis of the effect of duplex length on the ability of HMO2 to protect DNA from exonucleolytic cleavage suggests that more than one HMO2 must assemble at each DNA end. HMO2 binds supercoiled DNA with higher affinity than linear DNA and has a preference for DNA with lesions such as pairs of tandem mismatches; however, comparison of DNA constructs of increasing length suggests that HMO2 may not bind stably as a monomer to distorted DNA. The remarkable ability of HMO2 to protect DNA from exonucleolytic cleavage, combined with reports that HMO2 arrives early at DNA DSBs, suggests that HMO2 may play a role in DSB repair beyond INO80 recruitment

Literature Triage and Indexing in the Mouse Genome Informatics (MGI) Group

The Mouse Genome Informatics (MGI; "http://www.informatics.jax.org":http://www.informatics.jax.org) group is comprised of several collaborating projects including the Mouse Genome Database (MGD) Project, the Gene Expression Database (GXD) Project, the Mouse Tumor Biology (MTB) Database Project, and the Gene Ontology (GO) Project. Literature identification and collection is performed cooperatively amongst the groups.

In recent years many institutional libraries have transitioned from a focus largely on print holdings to one of electronic access to journals. This change has necessitated adaptation on the part of the MGI curatorial group. Whereas the majority of journals covered by the group used to be surveyed in paper form, those journals are now surveyed electronically. Approximately 160 journals have been identified as those most relevant to the various database groups. Each curator in the group has the responsibility of scanning several journals for articles relevant to any of the database projects. Articles chosen via this process are marked as to their potential significance for various projects. Each article is catalogued in a Master Bibliography section of the MGI database system and annotated to the database sections for which it has been identified as relevant. A secondary triage process allows curators from each group to scan the chosen articles and mark ones desired for their project if such annotation has been missed on the initial scan.

Once articles have been identified for each database project a variety of processes are implemented to further categorize and index data from those articles. For example, the Alleles and Phenotype section of the MGD database indexes each article marked for MGD and in this indexing process they identify each mouse gene and allele examined in the article. The GXD database indexing process has a different focus. In this case articles are indexed with regard to the stage of development used in the study as well as the assay technique used. In each case the indexing gives an overview of the data held in the article and assists in the more extensive curation performed in the following step of the curation process. Indexing also provides each group with valuable information used to prioritize and streamline the overall curation process.

The MGI projects are supported by NHGRI grants HG000330, HG00273, and HG003622, NICHD grant HD033745, and NCI grant CA089713

Li Wenliang, a face to the frontline healthcare worker? The first doctor to notify the emergence of the SARS-CoV-2 (COVID-19) outbreak

Dr Li Wenliang, who lost his life to the novel coronavirus, SARS-CoV-2, became the face of the threat of SARS-CoV-2 to frontline workers, the clinicians taking care of patients. Li, 34, was an ophthalmologist at Wuhan Central Hospital. On 30th December, 2019, when the Wuhan municipal health service sent out an alert, he reportedly warned a closed group of ex-medical school classmates on the WeChat social media site of “Seven cases of severe acute respiratory syndrome (SARS) like illness with links with the Huanan Seafood Wholesale Market” at his hospital. He was among eight people reprimanded by security officers for “spreading rumours”. In a tragic turn of events, he subsequently contracted SARS-CoV-2 and, after a period in intensive care, died on the morning of Friday 7th February, 2020 (South China Morning Post, 2020). This case is a stark reminder of the risks of emerging disease outbreaks for healthcare workers (HCWs). Dr Li Wenliang’s name is added to the long list of HCW that were at the forefront of outbreaks of SARS, Ebola, MERS and now SARS-CoV-2. It is important to recognise that it was the clinicians in Wuhan who sounded the alarm about the emergence of SARS-CoV-2 which was rapidly identified after these clinicians sent samples to a reference laboratory for next generation sequencing (NGS) (Zhou et al., 2020)

A Novel Labeling Approach Identifies Three Stability Levels of Acetylcholine Receptors in the Mouse Neuromuscular Junction In Vivo

The turnover of acetylcholine receptors at the neuromuscular junction is regulated in an activity-dependent manner. Upon denervation and under various other pathological conditions, receptor half-life is decreased., in our setup the same animals are used throughout the whole measurement period, thereby permitting a dramatic reduction of animal numbers at increased data quality. We identified three stability levels of acetylcholine receptors depending on the presence or absence of innervation: one pool of receptors with a long half-life of ∼13 days, a second with an intermediate half-life of ∼8 days, and a third with a short half-life of ∼1 day. Data were highly reproducible from animal to animal and followed simple exponential terms. The principal outcomes of these measurements were reproduced by an optical pulse-labeling assay introduced recently.A novel assay to determine kinetics of acetylcholine receptor turnover with small animal numbers is presented. Our data show that nerve activity acts on muscle acetylcholine receptor stability by at least two different means, one shifting receptor lifetime from short to intermediate and another, which further increases receptor stability to a long lifetime. We hypothesize on possible molecular mechanisms

PI3Ks Maintain the Structural Integrity of T-Tubules in Cardiac Myocytes

Phosphoinositide 3-kinases (PI3Ks) regulate numerous physiological processes including some aspects of cardiac function. Although regulation of cardiac contraction by individual PI3K isoforms has been studied, little is known about the cardiac consequences of downregulating multiple PI3Ks concurrently.Genetic ablation of both p110α and p110β in cardiac myocytes throughout development or in adult mice caused heart failure and death. Ventricular myocytes from double knockout animals showed transverse tubule (T-tubule) loss and disorganization, misalignment of L-type Ca(2+) channels in the T-tubules with ryanodine receptors in the sarcoplasmic reticulum, and reduced Ca(2+) transients and contractility. Junctophilin-2, which is thought to tether T-tubules to the sarcoplasmic reticulum, was mislocalized in the double PI3K-null myocytes without a change in expression level.PI3K p110α and p110β are required to maintain the organized network of T-tubules that is vital for efficient Ca(2+)-induced Ca(2+) release and ventricular contraction. PI3Ks maintain T-tubule organization by regulating junctophilin-2 localization. These results could have important medical implications because several PI3K inhibitors that target both isoforms are being used to treat cancer patients in clinical trials

Snowmass Theory Frontier: Effective Field Theory

We summarize recent progress in the development, application, and understanding of effective field theories and highlight promising directions for future research. This Report is prepared as the TF02 "Effective Field Theory" topical group summary for the Theory Frontier as part of the Snowmass 2021 process.Comment: 12 page