bluuebunny/crossref_metadata_2025 huggingface dataset:
RTICBM-74 is a Brain-Penetrant CB1Receptor Allosteric Modulator that Reduces Alcohol Intake in Rats
Authors: Lovelock Dennis , Nguyen Thuy , Van Voorhies Kalynn , Zhang Yanan , Besheer Joyce
Published: September 2021

The endocannabinoid system is implicated in the neuronal mechanisms of alcohol use disorder (AUD), with the cannabinoid receptor subtype 1 (CB1) representing a promising target for AUD therapeutic interventions. We have previously shown negative allosteric modulators (NAMs) of the CB1receptor attenuated the reinstatement of other drugs of abuse including cocaine and methamphetamine in rats; however, their effects on alcohol-related behaviors have not been investigated. Here, we tested the pharmacokinetic properties of one such CB1NAM, RTICBM-74, and its effects on alcohol self-administration in rats. RTICBM-74 showed low aqueous solubility and high protein binding but had excellent half-life and low clearance against rat liver microsomes and hepatocytes, and excellent brain penetrance in rats. RTICBM-74 pretreatment specifically reduced alcohol intake across a range of doses in male or female Wistar or Long-Evans rats that were trained to self-administer alcohol. These effects were similar to the CB1antagonist/inverse agonist rimonabant which was tested as a positive control. Importantly, RTICBM-74 was effective at reducing alcohol intake at doses that did not affect locomotion or sucrose self-administration. Our findings suggest that CB1NAMs such as RTICBM-74 have promising therapeutic potential in treatment of AUD.

SIGNIFICANCE STATEMENT

The present work shows that a metabolically stable and brain penetrant CB1receptor negative allosteric modulator (NAM) reduces alcohol self-administration in rats without affecting locomotion or sucrose self-administration, suggesting potential therapeutic relevance for the treatment of AUD.
Single-Molecule Localization Microscopy of 3D Orientation and Anisotropic Wobble using a Polarized Vortex Point Spread Function
Authors: Ding Tianben , Lew Matthew D.
Published: September 2021

Within condensed matter, single fluorophores are sensitive probes of their chemical environments, but it is difficult to use their limited photon budget to image precisely their positions, 3D orientations, and rotational diffusion simultaneously. We demonstrate the polarized vortex point spread function (PSF) for measuring these parameters, including characterizing the anisotropy of a molecule’s wobble, simultaneously from a single image. Even when imaging dim emitters (∼500 photons detected), the polarized vortex PSF is able to obtain 12 nm localization precision, 4-8° orientation precision, and 26° wobble precision. We use the vortex PSF to measure the emission anisotropy of fluorescent beads, the wobble dynamics of Nile red (NR) within supported lipid bilayers, and the distinct orientation signatures of NR in contact with amyloid-beta fibrils, oligomers, and tangles. The unparalleled sensitivity of the vortex PSF transforms single-molecule microscopes into nanoscale orientation imaging spectrometers, where the orientations and wobbles of individual probes reveal structures and organization of soft matter that are nearly impossible to perceive using molecular positions alone.
A root for massive crown-of-thorns starfish outbreaks in the Pacific Ocean
Authors: Yasuda Nina , Inoue June , Hall Michael R. , Nair Manoj R. , Adjeroud Mehdi , Fortes Miguel D. , Nishida Mutsumi , Tuivavalagi Nat , Ravago-Gotanco Rachel , Forsman Zac H. , Hassan Soliman Taha Basheir , Koyanagi Ryo , Hisada Kanako , Motti Cherie A. , Satoh Noriyuki
Published: September 2021

Recurring outbreaks of crown-of-thorns starfish (COTS) severely damage healthy corals in the Western Pacific Ocean. To determine the source of outbreaking COTS larvae and their dispersal routes across the Western Pacific, complete mitochondrial genomes were sequenced from 243 individuals collected in 11 reef regions. Our results indicate that Pacific COTS comprise two major clades, an East-Central Pacific clade (ECP-C) and a Pan-Pacific clade (PP-C). The ECP-C consists of COTS from French Polynesia (FP), Fiji, Vanuatu and the Great Barrier Reef (GBR), and does not appear prone to outbreaks. In contrast, the PP-C, which repeatedly spawns outbreaks, is a large clade comprising COTS from FP, Fiji, Vanuatu, GBR, Papua New Guinea, Vietnam, the Philippines, Japan, Micronesia, and the Marshall Islands. Given the nature of Pacific Ocean currents, the vast area encompassing FP, Fiji, Vanuatu, and the GBR likely supplies larvae for repeated outbreaks, exacerbated by anthropogenic environmental changes, such as eutrophication.
Life-history genotype explains variation in migration activity in Atlantic salmon (Salmo salar)
Authors: Niemelä Petri T. , Klemme Ines , Karvonen Anssi , Hyvärinen Pekka , Debes Paul V. , Erkinaro Jaakko , Sinclair-Waters Marion , Pritchard Victoria L. , Härkönen Laura , Primmer Craig R.
Published: September 2021

One of the most important life-history continuums is the fast–slow axis, where “fast” individuals mature earlier than “slow” individuals. “Fast” individuals are predicted to be more active than “slow” individuals; high activity is required to maintain a fast life-history strategy. Recent meta-analyses revealed mixed evidence for such integration. Here, we test whether known life-history genotypes differ in activity expression by using Atlantic salmon (Salmo salar) as a model. In salmon, variation in Vgll3, a transcription co-factor, explains ∼40% of variation in maturation timing. We predicted that the allele related to early maturation (vgll3*E) would be associated with increased activity. We used an automated surveillance system to follow ∼1900 juveniles including both migrants and non-migrants (i.e. smolt and parr fish, respectively) in semi-natural conditions over 31 days (∼580 000 activity measurements). Against our prediction,vgll3did not explain variation in activity in pooled migrant and non-migrant data. However, in migrants,vgll3explained variation in activity according to our prediction in a sex-dependent manner. Specifically, in females thevgll3*E allele was related to increasing activity, whereas in males thevgll3*L allele (later maturation allele) was related to increasing activity. These sex-dependent effects might be a mechanism maintaining within-population genetic life-history variation.
Weak selection and the separation of eco-evo time scales using perturbation analysis
Authors: Gerlee Philip
Published: September 2021

We show that under the assumption of weak frequency-dependent selection a wide class of population dynamical models can be analysed using perturbation theory. The inner solution corresponds to the ecological dynamics, where to zeroth order, the genotype frequencies remain constant. The outer solution provides the evolutionary dynamics and corresponds, to zeroth order, to a generalisation of the replicator equation. We apply this method to a model of public goods dynamics and construct, using matched asymptotic expansions, a composite solution valid for all times. We also analyse a Lotka-Volterra model of predator competition and show that to zeroth order the fraction of wild-type predators follows a replicator equation with a constant selection coefficient given by the predator death rate. For both models we investigate how the error between approximate solutions and the solution to the full model depend on the order of the approximation, and show using numerical comparison, for k = 1 and 2, that the error scales according to εk+1, where ε is the strength of selection and k is the order of the approximation.
The individuality of shape asymmetries of the human cerebral cortex
Authors: Chen Yu-Chi , Arnatkeviciute Aurina , McTavish Eugene , Pang James C. , Chopra Sidhant , Suo Chao , Fornito Alex , Aquino Kevin M. ,   Alzheimer’s Disease Neuroimaging Initiative
Published: September 2021

Asymmetries of the cerebral cortex are found across diverse phyla and are particularly pronounced in humans, with important implications for brain function and disease. However, many prior studies have confounded asymmetries due to size with those due to shape. Here, we introduce a novel approach to characterize asymmetries of the whole cortical shape, independent of size, across different spatial frequencies using magnetic resonance imaging data in three independent datasets. We find that cortical shape asymmetry is highly individualized and robust, akin to a cortical fingerprint, and identifies individuals more accurately than size-based descriptors, such as cortical thickness and surface area, or measures of inter-regional functional coupling of brain activity. Individual identifiability is optimal at coarse spatial scales (∼37 mm wavelength), and shape asymmetries show scale-specific associations with sex and cognition, but not handedness. While unihemispheric cortical shape shows significant heritability at coarse scales (∼65 mm wavelength), shape asymmetries are determined primarily by subject-specific environmental effects. Thus, coarse-scale shape asymmetries are highly personalized, sexually dimorphic, linked to individual differences in cognition, and are primarily driven by stochastic environmental influences.
The role of superstition of cognitive control during neurofeedback training
Authors: Grössinger Doris , Fischmeister Florian Ph.S , Witte Matthias , Koschutnig Karl , Ninaus Manuel , Neuper Christa , Kober Silvia Erika , Wood Guilherme
Published: September 2021

Background

Real-time fMRI neurofeedback is growing in reputation as a means to alter brain activity patterns and alleviate psychiatric symptoms. Activity in ventral striatum structures is considered an index of training efficacy. fMRI response in these brain regions indicates neurofeedback-driven associative learning. Here we investigated the impact of mere superstition of control as observed during neurofeedback training on patterns of fMRI activation.

Methods

We examined the brain activations of a large sample of young participants (n = 97, 50 female, age range 18-54yrs) in a simple fMRI task. Participants saw a display similar to that typically used for real-time fMRI. They were instructed to watch the bars’ movements or to control them with their own brain activity. Bar movements were not connected with brain activity of participants in any way and perceptions of control were superstitious. After the pretended control condition, they rated how well they were able to control the bars’ movements.

Results

Strong activation in the basal ganglia and ventral striatum as well as in large portions of the anterior insula, supplementary motor area, and the middle frontal gyrus due to the superstition of brain control.

Conclusions

The superstition of control over one’s own brain activity in a pretended neurofeedback training session activates the same neural networks as neurofeedback-driven learning. Therefore, activity in the basal ganglia and ventral striatum cannot be taken as evidence for neurofeedback-driven associative learning unless its effects are proven to supersede those elicited by appropriate sham conditions.
An improved culture protocol for the differentiation and survival of human promyelocytic leukaemia PLB-985 cell-line into mature neutrophil-like granulocytes
Authors: Shaayau Shehu , Cross Andrew L. , Wright Helen L. , Edwards Steven W.
Published: September 2021

Circulating blood neutrophils are short-lived, lack proliferation capacity and cannot be transfectedin vitroto express exogenous genes or proteins. These properties have made theex vivogenetic manipulation of neutrophils challenging and hindered biochemical and molecular studies investigating the function of specific genes and proteins. Improved methodology for differentiating cell lines into mature neutrophil-like phenotypes, with similar morphological and functional properties to blood neutrophils would, therefore, be an important tool to probe the molecular properties of mature cells. The PLB-985 cell line was cultured in RPMI-1640 medium supplemented foetal calf serum (FCS) and penicillin/streptomycin. For induction of differentiation into neutrophil-like cells, the medium was supplemented with sodium pyruvate,N, N-dimethylformamide (DMF) and all-transretinoic acid (ATRA), FCS and penicillin/streptomycin. The cytokines G-CSF and GM-CSF were used to enhance differentiation, prolong viability and delay the progression of the differentiated cells into apoptosis. The modified culture protocol and conditions induced PLB-985 cells to differentiate into mature, neutrophil-like granulocytes that resembled the morphology of mature blood neutrophils as evident by acquisition of a multi-lobed nucleus and granulated cytoplasm. These modified culture conditions resulted in enhanced differentiation into neutrophil-like cells and the apoptosis of these differentiated cells was delayed by supplementation with cytokines. This experimental system should be useful for studies probing the function of specific genes and proteins in human neutrophils.
A data compendium of Mycobacterium tuberculosis antibiotic resistance
Authors: Brankin Alice , Malone Kerri M ,   The CRyPTIC Consortium
Published: September 2021

The Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) presents here a compendium of 15,211 Mycobacterium tuberculosis global clinical isolates, all of which have undergone whole genome sequencing (WGS) and have had their minimum inhibitory concentrations to 13 antitubercular drugs measured in a single assay. It is the largest matched phenotypic and genotypic dataset for M. tuberculosis to date. Here, we provide a summary detailing the breadth of data collected, along with a description of how the isolates were collected and uniformly processed in CRyPTIC partner laboratories across 23 countries. The compendium contains 6,814 isolates resistant to at least one drug, including 2,129 samples that fully satisfy the clinical definitions of rifampicin resistant (RR), multi-drug resistant (MDR), pre-extensively drug resistant (pre-XDR) or extensively drug resistant (XDR). Accurate prediction of resistance status (sensitive/resistant) to eight antitubercular drugs by using a genetic mutation catalogue is presented along with the presence of suspected resistance-conferring mutations for isolates resistant to the newly introduced drugs bedaquiline, clofazimine, delamanid and linezolid. Finally, a case study of rifampicin mono-resistance demonstrates how this compendium could be used to advance our genetic understanding of rare resistance phenotypes. The compendium is fully open-source and it is hoped that the dataset will facilitate and inspire future research for years to come.
Reward enhances memory via age-varying online and offline neural mechanisms across development
Authors: Cohen Alexandra O. , Glover Morgan M. , Shen Xinxu , Phaneuf Camille V. , Avallone Kristen N. , Davachi Lila , Hartley Catherine A.
Published: September 2021

Reward motivation enhances memory through interactions between mesolimbic, hippocampal, and cortical systems — both during and after encoding. Developmental changes in these distributed neural circuits may lead to age-related differences in reward-motivated memory and the underlying neural mechanisms. Converging evidence from cross-species studies suggests that subcortical dopamine signaling is increased during adolescence, which may lead to stronger memory representations of rewarding, relative to mundane, events and changes in the contributions of underlying subcortical and cortical brain mechanisms across age. Here, we used fMRI to examine how reward motivation influences the “online” encoding and “offline” post-encoding brain mechanisms that support long-term associative memory from childhood to adulthood in human participants of both sexes. We found that reward motivation led to both age-invariant enhancements and nonlinear age-related differences in associative memory after 24 hours. Furthermore, reward-related memory benefits were linked to age-varying neural mechanisms. During encoding, interactions between the prefrontal cortex and ventral tegmental area (VTA) were associated with better high-reward memory to a greater degree with increasing age. Pre- to post-encoding changes in functional connectivity between the anterior hippocampus and VTA were also associated with better high-reward memory, but more so at younger ages. Our findings suggest that there may be developmental differences in the contributions of offline subcortical and online cortical brain mechanisms supporting reward-motivated memory.

Significance Statement

A substantial body of research has examined the neural mechanisms through which reward influences memory formation in adults. However, despite extensive evidence that both reward processing and associative memory undergo dynamic change across development, few studies have examined age-related changes in these processes. We found both age-invariant and nonlinear age-related differences in reward-motivated memory. Moreover, our findings point to developmental differences in the processes through which reward modulates the prioritization of information in long-term memory – with greater early reliance on offline subcortical consolidation mechanisms and increased contribution of systems-level online encoding circuitry with increasing age. These results highlight dynamic developmental changes in the cognitive and neural mechanisms through which motivationally salient information is prioritized in memory from childhood to adulthood.
The creation of ghost forests is driven by physical, ecological, and disturbance factors but remains a rare phenomenon
Authors: Field Christopher R.
Published: September 2021

Marine transgression and the landward migration of coastlines in response to sea-level rise will determine the future of coastal ecosystems and species worldwide, yet no complete model of these processes exist. Ghost forests, areas where coastal forest has been killed by saltwater inundation, are an attention-grabbing early indicator of marine transgression. Research on marine transgression to date has largely been limited to place-based studies that, due to logistical constraints and foci of expertise, investigate subsets of the potential drivers of marine transgression. Here I take advantage of new open source datasets to provide the first systematic analysis of marine transgression across large scales, focusing on the creation of ghost forests across a region of ecological and scientific importance, the northeast U.S. My analysis provides the first synthesis of the physical, ecological, and disturbance factors that influence marine transgression. It also provides crucial regional context for more intensive studies that have a limited geographic scope. I found that ghost forests are a rare phenomenon in the landscape context: 95% of recent forest loss is concentrated in less than 3.86% of marshes, and between 2000 and 2018, only 0.88% of the entire forested area of the transgression zone experienced loss. As a result of this rarity, regional variation in marine transgression is largely driven by opportunities for rare events, which are more numerous when suitable conditions, such as shallow slopes, cover extensive areas. Quantifying recent trends in the rate of forest loss found little evidence of acceleration, with evidence instead suggesting that fewer ghost forests are being created. I also found that physical, ecological, and disturbance factors, including hurricane impacts, were all important for understanding recent trends in forest loss, suggesting that an interdisciplinary approach is warranted for future analyses and modeling of marine transgression. Such interdisciplinary research is urgently needed, as the current rate of marine transgression points to the likelihood of near-term losses of coastal wetlands, with dire implications for the species that depend on them.
Analysis of Multisensory-Motor Integration in Olfactory Navigation of Silkmoth, Bombyx mori, using Virtual Reality System
Authors: Yamada Mayu , Ohashi Hirono , Hosoda Koh , Kurabayashi Daisuke , Shigaki Shunsuke
Published: September 2021

Most animals survive and thrive due to navigation behavior to reach their destinations. In order to navigate, it is important for animals to integrate information obtained from multisensory inputs and use that information to modulate their behavior. In this study, by using a virtual reality (VR) system for an insect, we investigated how an adult silkmoth integrates visual and wind direction information during female search behavior (olfactory behavior). According to the behavioral experiments using the VR system, the silkmoth had the highest navigation success rate when odor, vision, and wind information were correctly provided. However, we found that the success rate of the search significantly reduced if wind direction information was provided that was incorrect from the direction actually detected. This indicates that it is important to acquire not only odor information, but also wind direction information correctly. In other words, Behavior was modulated by the degree of co-incidence between the direction of arrival of the odor and the direction of arrival of the wind, and posture control (angular velocity control) was modulated by visual information. We mathematically modeled the modulation of behavior using multisensory information and evaluated it by simulation. As a result, the mathematical model not only succeeded in reproducing the actual female search behavior of the silkmoth, but can also improve search success relative to the conventional odor source search algorithm.
Aberrant epigenetic and transcriptional events associated with breast cancer risk
Authors: Marino Natascia , German Rana , Podicheti Ram , Rush Douglas B. , Rockey Pam , Huang Jie , Sandusky George E. , Temm Constance J. , Althouse Sandra K. , Nephew Kenneth P. , Nakshatri Harikrishna , Liu Jun , Vode Ashley , Cao Sha , Storniolo Anna Maria
Published: September 2021

Background

Genome-wide association studies have identified several breast cancer susceptibility loci. However, biomarkers for risk assessment are still missing. Here, we investigated cancer-related molecular changes detected in tissues from women at high risk for breast cancer prior to disease manifestation. Disease-free breast tissue cores donated by healthy women (N=146, median age=39 years) were processed for both methylome (MethylCap) and transcriptome (Illumina’s HiSeq4000) sequencing. Analysis of tissue microarray and primary breast epithelial cells was used to confirm gene expression dysregulation.

Results

Transcriptomic analysis identified 69 differentially expressed genes between women at either high and those at average risk of breast cancer (Tyrer-Cuzick model) at FDR<0.05 and fold change≥2. The majority of the identified genes were involved in DNA damage checkpoint, cell cycle, and cell adhesion. Two genes, FAM83A and NEK2, were overexpressed in tissue sections (FDR<0.01) and primary epithelial cells (p<0.05) from high-risk breasts. Moreover, 1698 DNA methylation aberrations were identified in high-risk breast tissues (FDR<0.05), partially overlapped with cancer-related signatures and correlated with transcriptional changes (p<0.05, r≤0.5). Finally, among the participants, 35 women donated breast biopsies at two time points, and age-related molecular alterations enhanced in high-risk subjects were identified.

Conclusions

Normal breast tissue from women at high risk of breast cancer bears molecular aberrations that may contribute to breast cancer susceptibility. This study is the first molecular characterization of the true normal breast tissues and provides an opportunity to investigate molecular markers of breast cancer risk, which may lead to new preventive approaches.
Sensitivity of discrete symmetry metrics: implications for metric choice
Authors: Hill Allen D , Nantel Julie
Published: September 2021

Gait asymmetry is present in several pathological populations, including those with Parkinson’s disease, Huntington’s disease, and stroke survivors. Previous studies suggest that commonly used discrete symmetry metrics, which compare single bilateral variables, may not be equally sensitive to underlying effects of asymmetry, and the use of a metric with low sensitivity could result in unnecessarily low statistical power. The purpose of this study was to provide a comprehensive assessment of the sensitivity of commonly used discrete symmetry metrics to better inform design of future studies. Monte Carlo simulations were used to estimate the statistical power of each symmetry metric at a range of asymmetry magnitudes, group/condition variabilities, and sample sizes. Power was estimated by repeated comparison of simulated symmetric and asymmetric data with a paired t-test, where the proportion of significant results is equivalent to the power. Simulation results confirmed that not all common discrete symmetry metrics are equally sensitive to reference effects of asymmetry. Multiple symmetry metrics exhibit equivalent sensitivities, but the most sensitive discrete symmetry metric in all cases is a bilateral difference (e.g. left - right). A ratio (e.g. left/right) has poor sensitivity when group/condition variability is not small, but a log-transformation produces increased sensitivity. Additionally, two metrics which included an absolute value in their definitions showed increased sensitivity when the absolute value was removed. Future studies should consider metric sensitivity when designing analyses to reduce the possibility of underpowered research.

Summary statement

Statistical power is an important factor in study design. Our results show that not all discrete symmetry metrics have similar or sufficient sensitivity to detect effects of asymmetry.
Naïve andin vitro-activated primary mouse CD8+T cells retainin vivoimmune responsiveness after electroporation-based CRISPR/Cas9 genetic engineering
Authors: Pfenninger Petra , Yerly Laura , Abe Jun
Published: September 2021

CRISPR/Cas9 technology has revolutionized genetic engineering of primary cells. Although its use is gaining momentum in studies on CD8+T cell biology, it remains elusive to what extent CRISPR/Cas9 affectsin vivofunction of CD8+T cells. Here, we optimized nucleofection-based CRISPR/Cas9 genetic engineering of naïve andin vitro-activated primary mouse CD8+T cells and tested theirin vivoimmune responses. Nucleofection of naïve CD8+T cells preserved theirin vivoantiviral immune responsiveness to an extent that is indistinguishable from non-nucleofected cells, whereasin vitroactivation of CD8+T cells prior to nucleofection led to slightly impaired expansion/survival. Of note, different target proteins displayed distinct decay rates after gene editing. This is in stark contrast to a comparable period of time required to complete gene inactivation. Thus, for optimal experimental design, it is crucial to determine the kinetics of the loss of target gene product to adapt incubation period after gene editing. In sum, nucleofection-based CRISPR/Cas9 genome editing achieves efficient and rapid generation of mutant CD8+T cells without imposing detrimental constraints on theirin vivofunctions.
Examining Batch Effect in Histopathology as a Distributionally Robust Optimization Problem
Authors: Hari Surya Narayanan , Nyman Jackson , Mehta Nicita , Elmarakeby Haitham , Jiang Bowen , Dietlein Felix , Rosenthal Jacob , Sengupta Eshna , Chowdhury Alexander , Umeton Renato , Van Allen Eliezer M.
Published: September 2021

Computer vision (CV) approaches applied to digital pathology have informed biological discovery and development of tools to help inform clinical decision-making. However, batch effects in the images have the potential to introduce spurious confounders and represent a major challenge to effective analysis and interpretation of these data. Standard methods to circumvent learning such confounders include (i) application of image augmentation techniques and (ii) examination of the learning process by evaluating through external validation (e.g., unseen data coming from a comparable dataset collected at another hospital). Here, we show that the source site of a histopathology slide can be learned from the image using CV algorithms in spite of image augmentation, and we explore these source site predictions using interpretability tools. A CV model trained using Empirical Risk Minimization (ERM) risks learning this source-site signal as a spurious correlate in the weak-label regime, which we abate by using a training method with abstention. We find that a patch based classifier trained using abstention outperformed a model trained using ERM by 9.9, 10 and 19.4% F1 in the binary classification tasks of identifying tumor versus normal tissue in lung adenocarcinoma, Gleason score in prostate adenocarcinoma, and tumor tissue grade in clear cell renal cell carcinoma, respectively, at the expense of up to 80% coverage (defined as the percent of tiles not abstained on by the model). Further, by examining the areas abstained by the model, we find that the model trained using abstention is more robust to heterogeneity, artifacts and spurious correlates in the tissue. Thus, a method trained with abstention may offer novel insights into relevant areas of the tissue contributing to a particular phenotype. Together, we suggest using data augmentation methods that help mitigate a digital pathology model’s reliance on potentially spurious visual features, as well as selecting models that can identify features truly relevant for translational discovery and clinical decision support.
Integrating Transcriptomics, Genomics, and Imaging in Alzheimer’s Disease: A Federated Model
Authors: Wu Jianfeng , Chen Yanxi , Wang Panwen , Caselli Richard J , Thompson Paul M , Wang Junwen , Wang Yalin ,   for the Alzheimer’s Disease Neuroimaging Initiative
Published: September 2021

Alzheimer’s disease (AD) affects more than 1 in 9 people age 65 and older and becomes an urgent public health concern as the global population ages. In clinical practice, structural magnetic resonance imaging (sMRI) is the most accessible and widely used diagnostic imaging modality. Additionally, genome-wide association studies (GWAS) and transcriptomics – the study of gene expression – also play an important role in understanding AD etiology and progression. Sophisticated imaging genetics systems have been developed to discover genetic factors that consistently affect brain function and structure. However, most studies to date focused on the relationships between brain sMRI and GWAS or brain sMRI and transcriptomics. To our knowledge, few methods have been developed to discover and infer multimodal relationships among sMRI, GWAS, and transcriptomics. To address this, we propose a novel federated model, Genotype-Expression-Imaging Data Integration (GEIDI), to identify genetic and transcriptomic influences on brain sMRI measures. The relationships between brain imaging measures and gene expression are allowed to depend on a person’s genotype at the single-nucleotide polymorphism (SNP) level, making the inferences adaptive and personalized. We performed extensive experiments on publicly available Alzheimer’s Disease Neuroimaging Initiative (ADNI) dataset. Experimental results demonstrated our proposed method outperformed state-of-the-art expression quantitative trait loci (eQTL) methods for detecting genetic and transcriptomic factors related to AD and has stable performance when data are integrated from multiple sites. Our GEIDI approach may offer novel insights into the relationship among image biomarkers, genotypes, and gene expression and help discover novel genetic targets for potential AD drug treatments.
A genetic trap in yeast for inhibitors of SARS-CoV-2 main protease
Authors: Alalam Hanna , Sigurdardóttir Sunniva , Bourgard Catarina , Tiukova Ievgeniia , King Ross D. , Grøtli Morten , Sunnerhagen Per
Published: September 2021

The ongoing COVID-19 pandemic urges searches for antiviral agents that can block infection or ameliorate its symptoms. Using dissimilar search strategies for new antivirals will improve our overall chances of finding effective treatments. Here, we have established an experimental platform for screening of small molecule inhibitors of SARS-CoV-2 main protease in Saccharomyces cerevisiae cells, genetically engineered to enhance cellular uptake of small molecules in the environment. The system consists of a fusion of the E. coli toxin MazF and its antitoxin MazE, with insertion of a protease cleavage site in the linker peptide connecting the MazE and MazF moieties. Expression of the viral protease confers cleavage of the MazEF fusion, releasing the MazF toxin from its antitoxin, resulting in growth inhibition. In the presence of a small molecule inhibiting the protease, cleavage is blocked and the MazF toxin remains inhibited, promoting growth. The system thus allows positive selection for inhibitors. The engineered yeast strain is tagged with a fluorescent marker protein, allowing precise monitoring of its growth in the presence or absence of inhibitor. We detect an established main protease inhibitor down to 10 μM by a robust growth increase. The system is suitable for robotized large-scale screens. It allows in vivo evaluation of drug candidates, and is rapidly adaptable for new variants of the protease with deviant site specificities.

IMPORTANCE

The COVID-19 pandemic may continue several years before vaccination campaigns can put an end it globally. Thus, the need for discovery of new antiviral drug candidates will remain. We have engineered a system in yeast cells for detection of small molecule inhibitors of one attractive drug target of SARS-CoV-2, its main protease which is required for viral replication. To detect inhibitors in live cells brings the advantage that only compounds capable of entering the cell and remain stable there, will score in the system. Moreover, by its design in yeast, the system is rapidly adaptable for tuning of detection level, eventual modification of protease cleavage site in case of future mutant variants of the SARS-CoV-2 main protease, or even for other proteases.
Little hope for the polyploid endemic Pyrenean Larkspur (Delphinium montanum): evidences from population genomics and Ecological Niche Modelling
Authors: Salvado Pascaline , Aymerich Boixader Pere , Parera Josep , Bonfill Albert Vila , Martin Maria , Quélennec Céline , Lewin Jean-Marc , Delorme-Hinoux Valérie , Bertrand Joris A. M.
Published: September 2021

Species endemic to restricted geographical ranges represent a particular conservation issue, be it for their heritage interest. In a context of global change, this is particularly the case for plants which belong to high-mountain ecosystems and, because of their ecological requirements, are doomed to survive or disappear on their ‘sky islands’. The Pyrenean Larkspur (Delphinium montanum, Ranunculaceae) is endemic to the Eastern part of the Pyrenees (France and Spain). It is now only observable at a dozen of localities and some populations show signs of decline, such as a recurrent lack of flowering. Implementing population genomic approach (e.g. RAD-seq like) is particularly useful to understand genomic patterns of diversity and differentiation in order to provide recommendations in term of conservation. However, it remains challenging for species such asD. montanumthat are autotetraploid with a large genome size (1C-value > 10 pg) as most methods currently available were developed for diploid species. A Bayesian framework able to call genotypes with uncertainty allowed us to assess genetic diversity and population structure in this system. Our results show evidence for inbreeding (meanGIS= 0.361) within all the populations and substantial population structure (meanGST= 0.403) at the metapopulation level. In addition to a lack of connectivity between populations, spatial projections of Ecological Niche Modelling analyses under different climatic scenarios predict a dramatic decrease of suitable habitat forD. montanumin the future. Based on these results, we discuss the relevance and feasibility of different conservation measures.
Levels of Notch-regulated transcription are modulated by tissue movements at gastrulation
Authors: Falo-Sanjuan Julia , Bray Sarah J.
Published: September 2021

Cells sense and integrate external information from diverse sources that include mechanical cues. Shaping of tissues during development may thus require coordination between mechanical forces from morphogenesis and cell-cell signalling to confer appropriate changes in gene expression. By live-imaging Notch-induced transcription in real time we have discovered that morphogenetic movements duringDrosophilagastrulation bring about an increase in activity-levels of a Notch responsive enhancer. Mutations that disrupt the timing of gastrulation resulted in concomitant delays in transcription up-regulation that correlated with the start of mesoderm invagination. As a similar gastrulation-induced effect was detected when transcription was elicited by the intracellular domain NICD, it cannot be attributed to forces exerted on Notch receptor activation. A Notch independentvndenhancer also exhibited a modest gastrulation-induced activity increase in the same stripe of cells. Together, these observations argue that gastrulation-associated forces act on the nucleus to modulate transcription levels. This regulation was uncoupled when the complex linking the nucleoskeleton and cytoskeleton (LINC) was disrupted, indicating a likely conduit. We propose that the coupling between tissue level mechanics, arising from gastrulation, and enhancer activity represents a general mechanism for ensuring correct tissue specification during development and that Notch dependent enhancers are highly sensitive to this regulation.
CRAM 3.1: Advances in the CRAM File Format
Authors: Bonfield James K
Published: September 2021

Motivation

CRAM has established itself as a high compression alternative to the BAM file format for DNA sequencing data. We describe updates to further improve this on modern sequencing instruments.

Results

With Illumina data CRAM 3.1 is 7 to 15% smaller than the equivalent CRAM 3.0 file, and 50 to 70% smaller than the corresponding BAM file. Long-read technology shows more modest compression due to the presence of high-entropy signals.

Availability

The CRAM 3.0 specification is freely available from https://samtools.github.io/hts-specs/CRAMv3.pdf. The CRAM 3.1 improvements are available from https://github.com/samtools/hts-specs/pull/433, with OpenSource implementations in HTSlib and HTScodecs.

Contact

jkb@sanger.ac.uk

Supplementary information

Supplementary data are available online
Decoding the Mechanism of Specific RNA Targeting by Ribosomal Methytransferases
Authors: Singh Juhi , Raina Rahul , Vinothkumar Kutti R. , Anand Ruchi
Published: September 2021

Methylation of specific nucleotides is integral for ribosomal biogenesis and serves as a common way to confer antibiotic resistance by pathogenic bacteria. Here, by determining the high-resolution structure of 30S-KsgA by cryo-EM, a state was captured, where KsgA juxtaposes between helices h44 and h45, separating them, thereby enabling remodeling of the surrounded rRNA and allowing the cognate site to enter the methylation pocket. With the structure as a guide, factors that direct the enzyme to its cognate site with high fidelity were unearthed by creating several mutant versions of the ribosomes, where interacting bases in the catalytic helix h45 and surrounding helices h44, h24, and h27 were mutated and evaluated for their methylation efficiency. The biochemical studies delineated specificity hotspots that enable KsgA to achieve an induced fit. This study enables the identification of distal exclusive allosteric pocket and other divergent structural elements in each rMTase, which can be exploited to develop strategies to reverse methylation, mediated drug resistance.
Fast and reliable sgRNA efficiency testing using HIReff
Authors: Marks David , Anstatt Jannis , Bachmann Lisa , Villarejo Lucia Gallego , Geidies Alexander , Müller Thorsten
Published: September 2021

CRISPR/Cas9 is the method of choice for gene editing like the endogenous knock-in of sequences in order to investigate protein function, abundance or intracellular localization. One of the crucial steps in the preparation of CRISPR/Cas9-mediated knock-ins is the design of sgRNAs, which need to be tested carefully in order to minimize off-target binding and reach highest cleavage efficiency. Usually, sgRNA is evaluated via mismatch cleavage assays, like Surveyor or T7 endonuclease 1 assay. We demonstrate that these methods are often highly cost- and time-intensive with a low sensitivity and high fail rate. As an alternative, we present a new HITI-based sgRNA efficiency (HIReff) test to precisely evaluate sgRNA efficiency. HIReff is based on a sophisticated integration vector with on-site generation of a linear donor fragment that allows a comparably easy read-out via fluorescence signal and integrates several internal controls. Next to a quantifiable sgRNA assessment, HIReff provides additional information on the ‘gene/protein to be studied’ abundance, subcellular localization and promoter activity and allows derivation of fluorescence protein labeled clonal lines. We highlight benefits of HIReff in comparison to commonly used enzyme-based assays and demonstrate improved practicability and high sensitivity, while being less time-, labor- and costintensive at the same time. Our results suggest HIReff as a fast and easy-to-use alternative for sgRNA efficiency testing.
Source-to-Target Automatic Rotating Estimation (STARE) – a publicly-available, blood-free quantification approach for PET tracers with irreversible kinetics: Theoretical framework and validation for [18F]FDG
Authors: Bartlett Elizabeth A , Ogden R Todd , Mann J John , Zanderigo Francesca
Published: September 2021

Introduction

Full quantification of positron emission tomography (PET) data requires an input function. This generally means arterial blood sampling, which is invasive, labor-intensive and burdensome. There is no current, standardized method to fully quantify PET radiotracers with irreversible kinetics in the absence of blood data. Here, we present Source-to-Target Automatic Rotating Estimation (STARE), a novel, data-driven approach to quantify the net influx rate (Ki) of irreversible PET radiotracers, that requires only individual-level PET data and no blood data. We validate STARE with [18F]FDG PET and assess its performance using simulations.

Methods

STARE builds upon a source-to-target tissue model, where the tracer time activity curves (TACs) in multiple “target” regions are expressed at once as a function of a “source” region, based on the two-tissue irreversible compartment model, and separates target region Ki from source Ki by fitting the source-to-target model across all target regions simultaneously. To ensure identifiability, data-driven, subject-specific anchoring is used in the STARE minimization, which takes advantage of the PET signal in a vasculature cluster in the FOV that is automatically extracted and partial volume-corrected. To avoid the need for any a priori determination of a single source region, each of the considered regions acts in turn as the source, and a final Ki is estimated in each region by averaging the estimates obtained in each source rotation.

Results

In a large dataset of [18F]FDG human scans (N=69), STARE Ki estimates were in good agreement with corresponding arterial blood-based estimates (regression slope=0.88, r=0.80), and were precisely estimated, as assessed by comparing STARE Ki estimates across several runs of the algorithm (coefficient of variation across runs=6.74 ± 2.48%). In simulations, STARE Ki estimates were largely robust to factors that influence the individualized anchoring used within its algorithm.

Conclusion

Through simulations and application to [18F]FDG PET data, feasibility is demonstrated for STARE blood-free, data-driven quantification of Ki. Future work will include applying STARE to PET data obtained with a portable PET camera and to other irreversible radiotracers.
Phylodynamic Pattern of Genetic Clusters, Paradigm Shift on Spatio-temporal Distribution of Clades, and Impact of Spike Glycoprotein Mutations of SARS-CoV-2 Isolates from India
Authors: Subramanian Siva , Kitambi Satish
Published: September 2021

Background: The COVID-19 pandemic is associated with high morbidity and mortality, with the emergence of numerous variants. The dynamics of SARS-CoV-2 with respect to clade distribution is uneven, unpredictable and fast changing. Aims: Retrieving the complete genomes of SARS-CoV-2 from India and subjecting them to analysis on phylogenetic clade diversity, Spike (S) protein mutations and their functional consequences such as immune escape features and impact on infectivity. Methods: Whole genome of SARS-CoV-2 isolates (n=4,326) deposited from India during the period from January 2020 to December 2020 is retrieved from GISAID and various analyses performed using in silico tools. Results: Notable clade dynamicity is observed indicating the emergence of diverse SARS-CoV-2 variants across the country. GR clade is predominant over the other clades and the distribution pattern of clades is uneven. D614G is the commonest and predominant mutation found among the S-protein followed by L54F. Mutation score prediction analyses reveal that there are several mutations in S-protein including the RBD and NTD regions that can influence the virulence of virus. Besides, mutations having immune escape features as well as impacting the immunogenicity and virulence through changes in the glycosylation patterns are identified. Conclusions: The study has revealed emergence of variants with shifting of clade dynamics within a year in India. It is shown uneven distribution of clades across the nation requiring timely deposition of SARS-CoV-2 sequences. Functional evaluation of mutations in S-protein reveals their significance in virulence, immune escape features and disease severity besides impacting therapeutics and prophylaxis.
Synaptotagmin-1-dependent phasic axonal dopamine release is dispensable for basic motor behaviors in mice
Authors: Delignat-Lavaud Benoît , Kano Jana , Ducrot Charles , Massé Ian , Mukherjee Sriparna , Giguère Nicolas , Moquin Luc , Lévesque Catherine , Burke Samuel , Denis Raphaëlle , Bourque Marie-Josée , Tchung Alex , Neto Pedro Rosa- , Lévesque Daniel , Beaumont Louis De , Trudeau Louis-Éric
Published: September 2021

In Parkinson’s disease (PD), motor dysfunctions only become apparent after extensive loss of DA innervation. This resilience has been hypothesized to be due to the ability of many motor behaviors to be sustained through a diffuse basal tone of DA; but experimental evidence for this is limited. Here we show that conditional deletion of the calcium sensor synaptotagmin-1 (Syt1) in DA neurons (Syt1 cKODAmice) abrogates most activity-dependent axonal DA release in the striatum and mesencephalon, leaving somatodendritic (STD) DA release intact. Strikingly, Syt1 cKODAmice showed intact performance in multiple unconditioned DA-dependent motor tasks and even in a task evaluating conditioned motivation for food. Considering that basal extracellular DA levels in the striatum were unchanged, our findings suggest that activity-dependent DA release is dispensable for such tasks and that they can be sustained by a basal tone of extracellular DA. Taken together, our findings reveal the striking resilience of DA-dependent motor functions in the context of a near-abolition of phasic DA release, shedding new light on why extensive loss of DA innervation is required to reveal motor dysfunctions in PD.