bluuebunny/crossref_metadata_2025 huggingface dataset:
Probabilistic mixture models improve calibration of panel-derived tumor mutational burden in the context of both tumor-normal and tumor-only sequencing
Authors: Anaya Jordan , Sidhom John-William , Cummings Craig A. , Baras Alexander S. ,   AACR Project GENIE Consortium
Published: May 2022

Background

Tumor mutational burden (TMB) has been investigated as a biomarker for immune checkpoint blockade (ICB) therapy. Increasingly, TMB is being estimated with gene panel-based assays (as opposed to full exome sequencing) and different gene panels cover overlapping but distinct genomic coordinates, making comparisons across panels difficult. Previous studies have suggested that standardization and calibration to exome-derived TMB be done for each panel to ensure comparability. With TMB cutoffs being developed from panel-based assays, there is a need to understand how to properly estimate exomic TMB values from different panel-based assays. Design: Our approach to calibration of panel-derived TMB to exomic TMB proposes the use of probabilistic mixture models that allow for nonlinear relationships along with heteroscedastic error. We examined various inputs including nonsynonymous, synonymous, and hotspot counts along with genetic ancestry. Using the TCGA cohort we generated a tumor-only version of the panel-restricted data by reintroducing private germline variants. Results: We were able to model more accurately the distribution of both tumor-normal and tumor-only data using the proposed probabilistic mixture models as compared to linear regression. Applying a model trained on tumor-normal data to tumor-only input results in biased TMB predictions. Including synonymous mutations resulted in better regression metrics across both data types, but ultimately a model able to dynamically weight the various input mutation types exhibited optimal performance. Including genetic ancestry improved model performance only in the context of tumor-only data, wherein private germline variants are observed.

Significance

A probabilistic mixture model better models the nonlinearity and heteroscedasticity of the data as compared to linear regression. Tumor-only panel data is needed to properly calibrate tumor-only panels to exomic TMB. Leveraging the uncertainty of point estimates from these models better informs cohort stratification in terms of TMB.
Immune activation state modulates the retrieval of infant engrams
Authors: Power Sarah D. , Stewart Erika , Zielke Louisa G. , Byrne Eric Patrick , Ortega-de San Luis Clara , Lynch Lydia , Ryan Tomás J.
Published: May 2022

Infantile amnesia is possibly the most ubiquitous form of memory loss in mammals. Despite its widespread relevance, little is known about the biological conditions for infantile amnesia to occur and its effect on the engram cells that encode a memory. We investigated how memories are stored in the brain throughout development by integrating engram labeling technology with mouse models of infantile amnesia. Here, we discovered a phenomenon in which male offspring in maternal immune activation models of autism spectrum disorder do not experience infantile amnesia. We rescued the same apparently forgotten infantile memories in mice by optogenetically reactivating dentate gyrus engram cells labeled during complex infant development experiences. Further, we were able to permanently reinstate lost infantile memories by artificially updating the memory engram, demonstrating that infantile amnesia is a reversible process. Our findings suggest that immune activation during development modulates innate, and reversible, forgetting switches that determine whether infantile amnesia will occur.
The Devil Is In The Details: Landscape Features Are Insufficient To Explain Patterns Of Non-Native Fishes Distribution In North Patagonian Streams
Authors: Lallement Mailén Elizabeth , Rechencq Magalí , Zattara Eduardo Enrique
Published: May 2022

Ecological communities are structured by combinations of factors known as habitat templates. These templates work as a filter allowing only species with particular traits or phenotypes to establish and persist excluding all others. Defining which habitat variables and spatial scales drive the assembly of freshwater communities is key to effective and efficient management of fluvial ecosystems. We took advantage of the relatively recent and well-studied history of salmonid introductions in Patagonia to evaluate if non-native species show different patterns of association with abiotic factors depending on the spatial scale of the environmental filter. We used a hierarchical approach to characterized environmental variables at the basin and reach scales to assess their influence on the presence, abundance and structure of the salmonid assemblages in breeding streams. We saw no evidence that presence/absence patterns of salmonid distribution were driven by landscape variables, except for those basins with physical environmental barriers to colonization. However, we did find evidence for relative abundances being influenced by climatic and geomorphological variables (e.g., precipitation and relief). Our results do not support a scenario in which any of the salmonid species modulates the distribution of the other species, suggesting that interference has played only a minor role in determining current fish distribution in fluvial systems of the region. Instead, current patterns of presence and abundance of salmonids are best explained as the product of environmental filters. Our findings contribute to our understanding of the ecology of individual species and provide insight into the mechanisms structuring fish assemblages in Southern Hemisphere’s lotic systems.
Wild-type MECP2 expression coincides with age-dependent sensory phenotypes in a female mouse model for Rett syndrome
Authors: Mykins Michael , Layo-Carris Dana , Dunn Logan Reid , Skinner David Wilson , McBryar Alexandra Hart , Perez Sarah , Shultz Trinity Rose , Willems Andrew , Lau Billy You Bun , Hong Tian , Krishnan Keerthi
Published: May 2022

Rett syndrome is characterized by an early period of typical development and then, regression of learned motor and speech skills in girls. Loss of MECP2 protein is thought to cause Rett syndrome phenotypes. The specific underlying mechanisms from typical developmental trajectory to regression features throughout life are unclear. Lack of established timelines to study the molecular, cellular, and behavioral features of regression in female mouse models is a major contributing factor. Due to random X-chromosome inactivation, female patients with Rett syndrome and female mouse models for Rett syndrome (Mecp2Heterozygous, Het) express a functional copy of wild-type MECP2 protein in approximately half of all cells. As MECP2 expression is regulated during early postnatal development and experience, we characterized the expression of wild-type MECP2 in the primary somatosensory cortex of female Het mice. Here, we report increased MECP2 levels in non-parvalbumin-positive neurons of 6-week-old adolescent Het relative to age-matched wild-type controls, while also displaying similar levels of perineuronal net expression, mild tactile sensory perception deficits, and efficient pup retrieval behavior. In contrast, 12-week-old adult Het express MECP2 at levels similar to age-matched wild-type mice, show increased perineuronal net expression in the cortex, and display significant tactile sensory perception deficits. Thus, we have identified a set of behavioral metrics and the cellular substrates to study regression during a specific time in the female Het mouse model, which coincide with changes in wild-type MECP2 expression. We speculate that the precocious increase in MECP2 expression within specific cell types of adolescent Het may provide compensatory benefits at the behavioral level, while the inability to further increase MECP2 levels leads to regressive behavioral phenotypes over time.
First-in-human evaluation of cutaneous innate and adaptive immunomodulation by mosquito bites
Authors: Guerrero David , My Vo Hoa Thi , Lon Chanthap , Bohl Jennifer A. , Nhik Sreynik , Chea Sophana , Man Somnang , Sreng Sokunthea , Pacheco Andrea R. , Ly Sokna , Sath Rathanak , Lay Sokchea , Missé Dorothee , Huy Rekol , Leang Rithea , Kry Hok , Valenzuela Jesus G. , Oliveira Fabiano , Cantaert Tineke , Manning Jessica E.
Published: May 2022

Mosquito-borne viruses are a growing global threat. Initial viral inoculation occurs in the skin via the mosquito ‘bite’, eliciting immune responses that shape the establishment of infection and pathogenesis. We aimed to cutaneous innate and adaptive immune responses to mosquito bites in individuals from endemic areas. In this single-arm, cross-sectional interventional study, we enrolled 30 healthy adult participants aged 18 to 45 years of age from Cambodia between October 2020 and January 2021. We performed 3-mm skin biopsies at baseline as well as 30 minutes, 4 hours, and 48 hours after a controlled feeding by uninfectedAedes aegyptimosquitos. The primary endpoints were measurement of changes in early and late innate responses in bitten vs unbitten skin by gene expression profiling, immunophenotyping, and cytokine profiling. Results revealed induction of neutrophil degranulation and recruitment of skin-resident dendritic cells and M2-macrophages in ‘bitten’ skin. As the immune reaction progressed over time, T cell priming and regulatory pathways were upregulated along with a shift to a Th2-driven response and CD8+ T cell activation. In accordance, participants’ bitten skin cells produced less pro-inflammatory cytokines when stimulated byAe. aegyptisalivary gland extract. No unexpected adverse events occurred, and one patient was lost to follow-up at Day 14. These results identify key immune genes, cell types, and pathways in the human response to mosquito bites that can be leveraged to develop novel therapeutics and vector-targeted vaccine candidates to arboviral diseases.

Graphical Abstract

Panel shows evolution of the human skin response toAedes aegyptibites over time.
Spatial patterns of evolutionary diversity in Cactaceae show low ecological representation within protected areas
Authors: Amaral Danilo Trabuco , Bonatelli Isabel A. S. , Romeiro-Brito Monique , Moraes Evandro Marsola , Franco Fernando Faria
Published: May 2022

Mapping biodiversity patterns across taxa and environments is crucial to address the evolutionary and ecological dimensions of species distribution, suggesting areas of particular importance for conservation purposes. Within Cactaceae, spatial diversity patterns are poorly explored, as well as the abiotic factors that may predict these patterns. We gathered geographic and genetic data from 922 cactus species, which are tightly associated with drylands, to evaluate diversity patterns, such as phylogenetic diversity and endemism, paleo-, neo-, and superendemism, and the environmental predictor variables of such patterns in a global analysis. Hotspot areas of cacti diversity are scattered along the Neotropical and Nearctic regions, mainly in the desertic portion of Mesoamerica, Caribbean Island, and the dry diagonal of South America. The geomorphological features of these regions may create a complexity of areas that work as locally buffered zones over time, which triggers local events of diversification and speciation. Desert and dryland/dry forest areas comprise paleo- and superendemism and may act as both museums and cradles of species, displaying great importance for conservation. Past climates, topography, soil features, and solar irradiance seem to be the main predictors of distinct endemism types. The hotspot areas that encompass a major part of the endemism cells are outside or poorly covered by formal protection units. The current legally protected areas are not able to conserve cactus evolutionary history. Given the rapid anthropogenic disturbance, efforts must be reinforced to monitor biodiversity and the environment and to define/plan current and new protected areas.
Functional metagenomic screening in microfluidic droplets identifies a β-glucuronidase in an unprecedented sequence neighbourhood
Authors: Neun Stefanie , Brear Paul , Campbell Eleanor , Tryfona Theodora , Omari Kamel El , Wagner Armin , Dupree Paul , Hyvönen Marko , Hollfelder Florian
Published: May 2022

The abundance of recorded protein sequence data stands in contrast to the small number of experimentally verified functional annotation. Here we screened a million-membered metagenomic library at ultrahigh throughput in microfluidic droplets for β-glucuronidase activity. We identified SN243, a genuine β-glucuronidase with little homology to previously studied enzymes of this type, as a glycoside hydrolase (GH) 3 family member. This GH family had no recorded evidence of β-glucuronidases at the outset of this study, showing that a functional metagenomic approach can shed light on assignments that are currently ‘unpredictable’ by bioinformatics. Kinetic analyses of SN243 characterised it as a promiscuous catalyst and structural analysis suggests regions of divergence from homologous GH3 members creating a wide-open active site. With a screening throughput of >107 library members per day, picolitre volume microfluidic droplets enable functional assignments that complement current enzyme database dictionaries and provide bridgeheads for the annotation of unexplored sequence space.
Effects of axon branching and asymmetry between the branches on transport, mean age, and age density distributions of mitochondria in neurons: a computational study
Authors: Kuznetsov Ivan A. , Kuznetsov Andrey V.
Published: May 2022

We report a computational study of mitochondria transport in a branched axon with two branches of different sizes. For comparison, we also investigate mitochondria transport in an axon with symmetric branches and in a straight (unbranched) axon. The interest in understanding mitochondria transport in branched axons is motivated by the large size of arbors of dopaminergic neurons, which die in Parkinson’s disease. Since the failure of energy supply of multiple demand sites located in various axonal branches may be a possible reason for the death of these neurons, we were interested in investigating how branching affects mitochondria transport. Besides investigating mitochondria fluxes between the demand sites and mitochondria concentrations, we also studied how the mean age of mitochondria and mitochondria age densities depend on the distance from the soma. We established that if the axon splits into two branches of unequal length, the mean ages of mitochondria and age density distributions in the demand sites are affected by how the mitochondria flux splits at the branching junction (what portion of mitochondria enter the shorter branch and what portion enter the longer branch). However, if the axon splits into two branches of equal length, the mean ages and age densities of mitochondria are independent of how the mitochondria flux splits at the branching junction. This even holds for the case when all mitochondria enter one branch, which is equivalent to a straight axon. Because the mitochondrial membrane potential (which many researchers view as a proxy for mitochondrial health) decreases with mitochondria age, the independence of mitochondria age on whether the axon is symmetrically branched or straight (providing the two axons are of the same length), and on how the mitochondria flux splits at the branching junction, may explain how dopaminergic neurons can sustain very large arbors and still maintain mitochondrial health across branch extremities.
A Feed-forward Pathway Drives LRRK2 kinase Membrane Recruitment and Activation
Authors: Vides Edmundo G. , Adhikari Ayan , Chiang Claire Y. , Lis Pawel , Purlyte Elena , Limouse Charles , Shumate Justin L. , Spínola-Lasso Elena , Dhekne Herschel S. , Alessi Dario R. , Pfeffer Suzanne R.
Published: May 2022

Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson’s disease and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this domain, termed “Site #1”, can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed “Site #2”, that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity.
Neural Implementation of Precise Temporal Patterns in Motor Cortex
Authors: Ning Yuxiao , Zheng Tianyu , Wan Guihua , Han Jiawei , Liu Tengjun , Zhang Shaomin
Published: May 2022

One of the most concerned problems in neuroscience is how neurons communicate and convey information through spikes. There is abundant evidence in sensory systems to support the use of precise timing of spikes to encode information. However, it remains unknown whether precise temporal patterns could be generated to drive output in the primary motor cortex (M1), a brain area containing ample recurrent connections that may destroy temporal fidelity. Here, we used a novel brain-machine interface that mapped the temporal order and precision of motor cortex activity to the auditory cursor and reward to guide the generation of precise temporal patterns in M1. During the course of learning, rats performed the “temporal neuroprosthetics” in a goal-directed manner with increasing proficiency. Precisely timed spiking activity in M1 was volitionally and robustly produced under this “temporal neuroprosthetics”, demonstrating the feasibility of M1 implementing temporal codes. Population analysis showed that the local network was coordinated in a fine time scale as the overall excitation heightened. Furthermore, we found that the directed connection between neurons assigned to directly control the output (“direct neurons”) was strengthened throughout learning, as well as connections in the subnetwork that contains direct neurons. Network models revealed that excitatory gain and strengthening of subnetwork connectivity transitioned neural states to a more synchronous regime, which improved the sensitivity for coincidence detection and, thus, the precision of spike patterns. Therefore, our results suggested the recurrent connections facilitate the implementation of precise temporal patterns instead of impairing them, which provided new perspectives on the fine-timescale activity and dynamics of M1.
Molecular docking, simulation and binding free energy analysis of small molecules as PfHT1 inhibitors
Authors: Owoloye Afolabi J. , Ligali Funmilayo C. , Enejoh Ojochenemi A. , Musa Adesola Z. , Aina Oluwagbemiga , Idowu Emmanuel T. , Oyebola Kolapo M.
Published: May 2022

Malaria chemotherapy has been plagued by parasite resistance. Novel drugs must be continually explored for malaria treatment. Plasmodium falciparum requires host glucose for survival and proliferation. Protein involved in hexose permeation, P. falciparum hexose transporter 1 (PfHT1) is a potential drug target. We performed high throughput virtual screening of 21,352 small-molecule compounds against PfHT1. The stability of the lead compound complexes was evaluated via molecular dynamics (MD) simulation for 100 nanoseconds. We also investigated the pharmacodynamic, pharmacokinetic and physiological characteristics of the compounds in accordance with Lipinksi rules for drug-likeness to bind and inhibit PfHT1. Molecular docking and free binding energy analyses were carried out using Molecular Mechanics with Generalised Born and Surface Area (MMGBSA) solvation to determine the selectivity of the hit compounds for PfHT1 over the human glucose transporter (hGLUT1) orthologue. Five important compounds were identified: Hyperoside (CID5281643); avicularin (CID5490064); sylibin (CID5213); harpagoside (CID5481542) and quercetagetin (CID5281680). The compounds formed intermolecular interaction with the binding pocket of the target via conserved amino acid residues (Val314, Gly183, Thr49, Asn52, Gly183, Ser315, Ser317, and Asn48). The MMGBSA analysis of the complexes yielded high free binding energies. Four (CID5281643, CID5490064, CID5213, and CID5481542) of the identified compounds were found to be stable within the PfHT1 binding pocket throughout the 100 nanoseconds simulation run time. The four compounds demonstrated higher affinity for PfHT1 than the human major glucose transporter (hGLUT1). This investigation demonstrates the inhibition potential of sylibin, hyperoside, harpagoside, and avicularin against PfHT1 receptor. Robust preclinical investigations are required to validate the chemotherapeutic properties of the identified compounds.
Lysosomal alterations and decreased electrophysiological activity in CLN3 disease (966 bp deletion, E295K) patient-derived cortical neurons
Authors: Chear Sueanne , Perry Sharn , Wilson Richard , Bindoff Aidan , Talbot Jana , Ware Tyson L , Grubman Alexandra , Vickers James C , Pébay Alice , Ruddle Jonathan B , King Anna E , Hewitt Alex W , Cook Anthony L
Published: May 2022

CLN3 disease is a lysosomal storage disorder associated with fatal neurodegeneration that is caused by mutations in CLN3. Most individuals with CLN3 disease carry at least one allele with a 966 bp deletion in CLN3 which results in the deletion of exons 7 and 8. There is a need for more physiologically relevant human cell-based CLN3 disease models to better understand the cellular changes during the disease process. Using CRISPR/Cas9, we corrected the 966 bp deletion mutation in human induced pluripotent stem cells (iPSCs) of a compound heterozygous patient (CLN3 Δ 966 bp and E295K). The isogenic deletion-corrected and unedited CLN3 patient iPSCs were used for disease modeling. iPSC-derived neurons carrying this particular CLN3 mutation (CLN3 neurons) had lower functional activity as recorded using microelectrode arrays for most of the culture period. Proteomics analysis showed downregulation of proteins related to axon guidance and endocytosis at day in vitro (DIV) 14 and 42 in CLN3 neurons. This was accompanied by an increase in lysosomal-related proteins in CLN3 neurons. Western blot analysis revealed hyperglycosylation of the lysosomal marker, Lysosome Associated Membrane Protein 1 (LAMP1) in CLN3 neurons at DIV 14, 28 and 42, which was not apparent in control neurons. Ultrastructural analysis of CLN3 neurons showed numerous membrane-bound vacuoles containing diverse types of storage material, ranging from curvilinear deposits, multilamellar structures to osmiophilic deposits. Our findings suggest alterations in lysosomal function and neurodevelopment involving axon guidance and synaptic transmission in CLN3-deficient neuronal derivatives, which could be potential targets for therapy.
Association between zinc deficiency and aorta stiffness in non-diabetic hemodialysis patients
Authors: Ishioka Kunihiro , Hidaka Sumi , Fujiwara Naoki , Yamano Mizuki , Mochida Yasuhiro , Oka Machiko , Maesato Kyoko , Moriya Hidekazu , Ohtake Takayasu , Kobayashi Shuzo
Published: May 2022

Objectives

Zinc deficiency (Zn < 60 μg/dL) is known to play an important role for vascular calcification. However, little data is available regarding the association between zinc deficiency and aorta stiffness in dialysis patients. Thus, we studied the relationship between zinc deficiency and aorta stiffness in non-diabetic hemodialysis(HD) patients.

Methods

Of 150 patients receiving maintenance HD at our hospital, we included 79 non-diabetic HD patients (age: 70±11 years, 49 men) after excluding 71 diabetic HD patients. Zinc deficiency was defined as Zn <60 μg/dL during pre-HD blood sampling. The association between zinc deficiency and aorta stiffness was analyzed. Aorta stiffness was evaluated as brachial-ankle pulse wave velocity (baPWV). Other surrogate markers for cardiovascular complications were also measured.

Results

The zinc deficiency group (ZD group) included 45 patients (57.0%). Compared to the zinc non-deficiency group (ZND group), patients with ZD group were significantly older, higher levels of CRP and hypoalbuminemia. Moreover, they had significantly higher levels of baPWV, and lower levels of ankle-brachial pressure index (ABI) (p<0.05). After adjusting for hypoalubuminuria, and CRP, multivariate analysis showed that age and zinc level were independent predictors of baPWV.

Conclusion

The study suggested that zinc deficiency may be an independent risk factor for aorta stiffness, even after adjusting for malnutrition and inflammation.
APOE Expression and Secretion are Modulated by Mitochondrial Dysfunction
Authors: Wynne Meghan E. , Ogunbona Oluwaseun , Lane Alicia R. , Gokhale Avanti , Zlatic Stephanie , Xu Chongchong , Wen Zhexing , Duong Duc , Rayaprolu Sruti , Ivanova Anna , Ortlund Eric A. , Dammer Eric B. , Seyfried Nicholas T. , Roberts Blaine R. , Crocker Amanda , Shanbhag Vinit , Petris Michael , Senoo Nanami , Kandasamy Selvaraju , Claypool Steven M. , Barrientos Antoni , Wingo Aliza P. , Wingo Thomas S. , Rangaraju Srikant , Levey Allan , Werner Erica , Faundez Victor
Published: May 2022

Mitochondria influence cellular function through both cell-autonomous and non-cell autonomous mechanisms, such as production of paracrine and endocrine factors. Here, we demonstrate that mitochondrial regulation of the secretome is more extensive than previously appreciated, as both genetic and pharmacological disruption of the electron transport chain caused upregulation of the Alzheimer’s disease risk factor apolipoprotein E (APOE) and other secretome components. This upregulation of secretory proteins was of a similar extent as modifications to the mitochondrial annotated proteome. Indirect disruption of the electron transport chain by gene editing of SLC25A mitochondrial membrane transporters as well as direct genetic and pharmacological disruption of either complexes I, III, or the copper-containing complex IV of the electron transport chain, elicited upregulation of APOE transcript, protein, and secretion, up to 49-fold. These APOE phenotypes were robustly expressed in diverse cell types and iPSC-derived human astrocytes as part of an inflammatory gene expression program. Moreover, age- and genotype-dependent decline in brain levels of respiratory complex I preceded an increase in APOE in the 5xFAD mouse model. We propose that mitochondria act as novel upstream regulators of APOE-dependent cellular processes in health and disease.
Challenges and lessons learned from the rapid operationalization of a prospective cohort to study the natural history and neurodevelopmental outcomes of postnatal Zika virus infection among infants and children in rural Guatemala
Authors: Paniagua-Avila Alejandra , Olson Daniel , Connery Amy , Calvimontes D. Mirella , Bolanos Guillermo A. , Lamb Molly M. , Bauer Desiree , Ralda Aida , Rojop Neudy , Barrios Eduardo , Chacon Andrea , Gomez Melissa , Arroyave Paola , Hernandez Sara , Martinez Maria Alejandra , Bunge-Montes Saskia , Colbert Alison , Arias Kareen , Brazeale Garret , Holliday Andrea , Tomashek Kay M. , El Sahly Hana M. , Keitel Wendy , Munoz Flor M. , Asturias Edwin J.
Published: May 2022

During the course of the 2015-2017 outbreak of Zika virus (ZIKV) infection in the Americas, the emerging virus was recognized as a congenital infection that could damage the developing brain. As the Latin American ZIKV outbreak advanced, the scientific and public health community questioned if this newly recognized neurotropic flavivirus could affect the developing brain of infants and young children infected after birth. We report here the design, methods and rapid operationalization of a prospective natural history cohort study aimed at evaluating the potential neurological and neurodevelopmental effects of postnatal Zika virus infection in infants and young children, which had become epidemic in Central America. This study enrolled a cohort of 500 mothers and their infants, along with nearly 400 children 1.5-3.5 years of age who were born during the initial phase of the ZIKV epidemic in a rural area of Guatemala. Our solutions and lessons learned while tackling real-life challenges may serve as a guide to other researchers carrying out studies of emerging infectious diseases of public health priority in resource-constrained settings.
N-ACT: An Interpretable Deep Learning Model for Automatic Cell Type and Salient Gene Identification
Authors: Heydari A. Ali , Davalos Oscar A. , Hoyer Katrina K. , Sindi Suzanne S.
Published: May 2022

Single-cell RNA sequencing (scRNAseq) is rapidly advancing our understanding of cellular composition within complex tissues and organisms. A major limitation in most scRNAseq analysis pipelines is the reliance on manual annotations to determine cell identities, which are time consuming, subjective, and require expertise. Given the surge in cell sequencing, supervised methods–especially deep learning models–have been developed for automatic cell type identification (ACTI), which achieve high accuracy and scalability. However, all existing deep learning frameworks for ACTI lack interpretability and are used as “black-box” models. We present N-ACT (Neural-Attention for Cell Type identification): the first-of-its-kind interpretable deep neural network for ACTI utilizing neural attention to detect salient genes for use in cell-types identification. We compare N-ACT to conventional annotation methods on two previously manually annotated data sets, demonstrating that N-ACT accurately identifies marker genes and cell types in an unsupervised manner, while performing comparably on multiple data sets to current state-of-the-art model in traditional supervised ACTI.
Modelling spatially autocorrelated detection probabilities in spatial capture-recapture using random effects
Authors: Dey Soumen , Moqanaki Ehsan M. , Milleret Cyril , Dupont Pierre , Tourani Mahdieh , Bischof Richard
Published: May 2022

Spatial capture-recapture (SCR) models are now widely used for estimating density from repeated individual spatial encounters. SCR accounts for the inherent spatial autocorrelation in individual detections by modelling detection probabilities as a function of distance between the detectors and individual activity centres. However, additional spatial heterogeneity in detection probability may still creep in due to environmental or sampling characteristics. if unaccounted for, such variation can lead to pronounced bias in population size estimates.

Using simulations, we describe and test three Bayesian SCR models that use generalized linear mixed models (GLMM) to account for latent heterogeneity in baseline detection probability across detectors using: independent random effects (RE), spatially autocorrelated random effects (SARE), and a twogroup finite mixture model (FM).

Overall, SARE provided the least biased population size estimates (median RB: -9 – 6%). When spatial autocorrelation was high, SARE also performed best at predicting the spatial pattern of heterogeneity in detection probability. At intermediate levels of autocorrelation, spatially-explicit estimates of detection probability obtained with FM where more accurate than those generated by SARE and RE. In cases where the number of detections per detector is realistically low (at most 1), all GLMMs considered here may require dimension reduction of the random effects by pooling baseline detection probability parameters across neighboring detectors (“aggregation”) to avoid over-parameterization.

The added complexity and computational overhead associated with SCR-GLMMs may only be justified in extreme cases of spatial heterogeneity. However, even in less extreme cases, detecting and estimating spatially heterogeneous detection probability may assist in planning or adjusting monitoring schemes.
Proximity labelling identifies pro-migratory endocytic recycling cargo and machinery of the Rab4 and Rab11 families
Authors: Wilson Beverley , Gemperle Jakub , Lawless Craig , Flett Chloe , Hartshorn Matthew , Hinde Eleanor , Harrison Thomas , Chastney Megan , Taylor Sarah , Allen Jennifer , Caswell Patrick
Published: May 2022

Endocytic recycling controls the return of internalised cargos to the plasma membrane to coordinate their positioning, availability and downstream signalling. The Rab4 and Rab11 small GTPase families regulate distinct recycling routes, broadly classified as fast recycling from early endosomes (Rab4) and slow recycling from perinuclear recycling endosomes (Rab11), and both routes handle a broad range of overlapping cargos to regulate cell behaviour. We adopted a proximity labelling approach, BioID, to identify and compare the protein complexes recruited by Rab4a, Rab11a and Rab25 (a Rab11 family member implicated in cancer aggressiveness), revealing statistically robust protein-protein interaction networks of both new and well characterised cargos and trafficking machinery in migratory cancer cells. Gene ontological analysis of these interconnected networks revealed that these endocytic recycling pathways are intrinsically connected to cell motility and cell adhesion. Using a knock sideways relocalisation approach we were further able to confirm novel links between Rab11/25 and the ESCPE-1 and retromer multiprotein sorting complexes and identify new endocytic recycling machinery associated with Rab4, Rab11 and Rab25 that regulates cancer cell migration in 3D-matrix.
LAWS: Local Alignment for Water Sites — tracking ordered water in simulations
Authors: Klyshko Eugene , Kim Justin Sung-Ho , Rauscher Sarah
Published: May 2022

Protein solvation plays a crucial role in protein function; accurate modeling of protein-water interactions is important for understanding the molecular basis of protein function. In addition to protein structure, x-ray crystallography provides positions of crystallographic water sites (CWS) coordinated by the protein, which can serve as a strong benchmark for modeling accuracy. Such comparison requires special method-ological considerations that take into account the dynamic nature of proteins. However, existing methods for analyzing CWS in MD simulations rely on global alignment of the protein onto the crystal structure, which introduces substantial errors in the case of significant structural deviations. Here, we propose a method called local alignment for water sites (LAWS), which is based on multilateration — an algorithm widely used in GPS tracking. LAWS considers the contacts formed by CWS and protein atoms in the crystal structure and uses these interaction distances to track the CWS in a simulation. LAWS provides a framework to quantify how perturbations of the local protein environment affect the preservation of crystallographic waters in simulations. We applied our method to a 1 µs simulation of a protein crystal and demonstrate that 76% of CWS are preserved. Compared to existing methods, LAWS identifies more high-confidence (low B-factor) CWS, which are characterized by more prominent water density peaks and a less-perturbed protein environment.
A critical role for spike synchrony in determining steep 1/f slopes in the setting of bursting EEG patterns
Authors: Yi Justin D. , Akbari Yama
Published: May 2022

EEG signals are commonly analyzed in the frequency-domain. Analysis of the power spectrum commonly demonstrates a reproducible fall-off of high frequencies, sometimes referred to as the “1/f slope”. The 1/f slope comes about from a linear fit of the power spectral density in log-log form and takes typical values between (minus) 0–4. This slope has been recently hypothesized to correlate with the ratio of excitatory to inhibitory synaptic weights. However, we demonstrate models of excitatory-inhibitory (E/I) balance only explain small values of 1/f slope (< 2). We seek to construct a model to explain large slopes (> 2), which have been reported under conditions of anesthesia. Using simulations of clustered spike pulses, we find that pulse widths on the order of 1-10 ms result in a transition to steep 1/f slopes between 3–4. This trend also holds when measuring the 1/f slope from a spiking recurrent network model in a synchronous regime. We conclude that steep 1/f slopes result from synchronous spiking in bulk microcircuit activity. This suggests interpretations of steep 1/f slopes should consider spike synchrony in addition to other factors like E/I balance.
Antisense pairing and SNORD13 structure guide RNA cytidine acetylation
Authors: Thalalla Gamage Supuni , Bortolin-Cavaillé Marie-Line , Link Courtney , Bryson Keri , Sas-Chen Aldema , Schwartz Schraga , Cavaillé Jérôme , Meier Jordan L.
Published: May 2022

N4-acetylcytidine (ac4C) is an RNA nucleobase found in all domains of life. Establishment of ac4C in helix 45 (h45) of human 18S ribosomal RNA (rRNA) requires the combined activity of the acetyltransferase NAT10 and the box C/D snoRNA SNORD13. However, the molecular mechanisms governing RNA-guided nucleobase acetylation in humans remain unexplored. Here we report two assays that enable the study of SNORD13-dependent RNA acetylation in human cells. First, we demonstrate that ectopic expression of SNORD13 rescues h45 in a SNORD13 knockout cell line. Next, we show mutant snoRNAs can be used in combination with nucleotide resolution ac4C sequencing to define structure and sequence elements critical for SNORD13 function. Finally, we develop a second method that reports on the substrate specificity of endogenous NAT10-SNORD13 via mutational analysis of an ectopically-expressed pre-rRNA substrate. By combining mutational analysis of these reconstituted systems with nucleotide resolution ac4C sequencing, our studies reveal plasticity in the molecular determinants underlying RNA-guided cytidine acetylation that is distinct from deposition of other well-studied rRNA modifications (e.g. pseudouridine). Overall, our studies provide a new approach to reconstitute RNA-guided cytidine acetylation in human cells as well as nucleotide resolution insights into the mechanisms governing this process.

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Arabidopsis cell suspension culture that lacks circadian rhythms can be recovered by constitutive ELF3 expression
Authors: Laosuntisuk Kanjana , Desai Jigar S. , Doherty Colleen J.
Published: May 2022

Callus and cell suspension culture techniques are valuable tools in plant biotechnology and are widely used in fundamental and applied research. For studies in callus and cell suspension cultures to be relevant, it is essential to know if the underlying biochemistry is similar to intact plants. This study examined the expression of core circadian genes in Arabidopsis callus from the cell suspension named AT2 and found that the circadian rhythms were impaired. The circadian waveforms are similar to intact plants in the light/dark cycles, but the circadian expression in the AT2 callus stopped in the free-running, constant light conditions. Temperature cycles could drive the rhythmic expression in constant conditions, but there were novel peaks at the point of temperature transitions unique to each clock gene. We found that callus freshly induced from seedlings had normal oscillations, like intact plants, suggesting that the loss of the circadian oscillation in the AT2 callus was specific to this callus. We determined that neither the media composition nor the source of the AT2 callus caused this disruption. We observed thatELF3expression was not differentially expressed between dawn and dusk in both entrained, light-dark cycles and constant light conditions. Overexpression ofELF3in the AT2 callus partially recovers the circadian oscillation in the AT2 callus. This work shows that while callus and cell suspension cultures can be valuable tools for investigating plant responses, careful evaluation of their phenotype is important. Moreover, the altered circadian rhythms under constant light and temperature cycles in the AT2 callus could be useful backgrounds to understand the connections driving circadian oscillators and light and temperature sensing at the cellular level.
Antibiotic acyldepsipeptides stimulate theStreptomycesClp-ATPase/ClpP complex for accelerated proteolysis
Authors: Reinhardt Laura , Thomy Dhana , Lakemeyer Markus , Ortega Joaquin , Sieber Stephan A. , Sass Peter , Brötz-Oesterhelt Heike
Published: May 2022

Clp proteases consist of a proteolytic, tetradecameric core ClpP and AAA+ Clp-ATPases. Streptomycetes, producers of a plethora of secondary metabolites, encode up to five different ClpP homologs and the composition of their unusually complex Clp protease machinery has remained unsolved. Here, we report on the composition of the house-keeping Clp protease inStreptomyces, consisting of a hetero-tetradecameric core built of ClpP1, ClpP2 and the cognate Clp-ATPases ClpX, ClpC1 or ClpC2, all interacting with ClpP2 only. ADEP antibiotics dysregulate the Clp protease for unregulated proteolysis. We observed that ADEP bindsStreptomycesClpP1, but not ClpP2, thereby not only triggering the degradation of non-native protein substrates but also accelerating Clp-ATPase-dependent proteolysis. The explanation is the concomitant binding of ADEP and Clp-ATPases to opposite sides of the ClpP1P2 barrel, hence revealing a third, so far unknown mechanism of ADEP action, i.e., the accelerated proteolysis of native protein substrates by the Clp protease.

Significance

Clp proteases are antibiotic and anti-cancer drug targets. Composed of the proteolytic core ClpP and a regulatory Clp-ATPase, the protease machinery is important for protein homeostasis and regulatory proteolysis. The acyldepsipeptide antibiotic ADEP targets ClpP and has shown promise for treating multi-resistant and persistent bacterial infections. The molecular mechanism of ADEP is multi-layered. Here, we present a new way how ADEP can deregulate the Clp protease system. Clp-ATPases and ADEP bind to opposite sides ofStreptomycesClpP, accelerating the degradation of natural Clp protease substrates. We also demonstrate the composition of the majorStreptomycesClp protease complex, a heteromeric ClpP1P2 core with the Clp-ATPases ClpX, ClpC1 or ClpC2 exclusively bound to ClpP2, and the killing mechanism of ADEP inStreptomyces.
Efficient parameter calibration and real-time simulation of large scale spiking neural networks with GeNN and NEST
Authors: Schmitt Felix Johannes , Rostami Vahid , Nawrot Martin Paul
Published: May 2022

Spiking neural networks (SNN) represent the state-of-the-art approach to the biologically realistic modeling of nervous system function. The systematic calibration for multiple free model parameters is necessary to achieve robust network function and demands high computing power and large memory resources. Special requirements arise from closed-loop model simulation in virtual environments, and from real-time simulation in robotic application. Here, we compare two complementary approaches to efficient large scale and realtime SNN simulation. The widely used NEural Simulation Tool (NEST) parallelizes simulation across multiple CPU cores. The GPU-enhanced Neural Network (GeNN) simulator uses the highly parallel GPU-based architecture to gain simulation speed. We quantify fixed and variable simulation costs on single machines with different hardware configurations. As benchmark model we use a spiking cortical attractor network with a topology of densely connected excitatory and inhibitory neuron clusters with homogeneous or distributed synaptic time constants and in comparison to the random balanced network. We show that simulation time scales linearly with the simulated biological model time and, for large networks, approximately linearly with the model size as dominated by the number of synaptic connections. Additional fixed costs with GeNN are almost independent of model size, while fixed costs with NEST increase linearly with model size. We demonstrate how GeNN can be used for simulating networks with up to 3.5 · 106neurons (> 3 · 1012synapses) on a high-end GPU, and up to 250, 000 neurons (25 ·109synapses) on a low-cost GPU. Real-time simulation was achieved for networks with 100, 000 neurons. Network calibration and parameter grid search can be efficiently achieved using batch processing. We discuss the advantages and disadvantages of both approaches for different use cases.
Analysis of the contribution of intrinsic disorder in shaping potyvirus genetic diversity
Authors: Lafforgue Guillaume , Michon Thierry , Charon Justine
Published: May 2022

Intrinsically disordered regions (IDRs) are abundant in the proteome of RNA viruses. The multifunctional properties of these regions are widely documented and their structural flexibility is associated with low constraint in the amino acid positions. Therefore, from an evolutionary stand point, these regions could have a greater mutational permissiveness than highly structured regions (ORs for Ordered Regions). They could thus provide a potential adaptive reservoir. To address this hypothesis, we compared the mutational robustness of IDRs and ORs in the genome of potyviruses, a major genus of plant viruses. For this purpose, a simulation model (DOI: 10.5281/zenodo.6396239) was built and used to distinguish a possible selection phenomenon in the biological data sets from randomly generated mutations. We analyzed several short-term experimental evolution datasets. An analysis was also performed on the natural diversity of three different species of potyviruses reflecting the long-term evolution. We observed that the mutational robustness of IDRs is significantly higher than that of ORs. Moreover, the substitutions in the ORs are very constrained by the conservation of the physico-chemical properties of the amino acids. This feature is not found in the IDRs where the substitutions tend to be more random. This reflects the weak structural constraints in these regions, in which an amino acid polymorphism is naturally conserved in the course of evolution, potyvirus IDRs and ODRs follow different evolutive paths with respect to their mutational robustness. These results force to consider the hypothesis that during selection, adaptive solutions could emerge from the amino acid polymorphism carried by IDRs.