bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of salinity and water dilution on environmental DNA degradation in freshwater environments
Authors: Saito Tatsuya , Doi Hideyuki
Published: May 2021

Environmental DNA (eDNA) analysis methods have been developed to detect the distribution and abundance/biomass of organisms in various environments. eDNA generally degrades quickly, thus the study of eDNA degradation is critical for eDNA evaluation. However, there have only been a few studies of eDNA degradation experiments in which the salt concentration and water dilution were controlled. In this study, the effects of degradation were experimentally evaluated by controlling the salinity and water dilution of pond water. An experiment was conducted to evaluate the effects of salinity and dilution on eDNA detection with fragmental eDNA and free cell-derived eDNA using pond water, diluted pond water, and saline pond water. We quantified the eDNA copies of free cells, fragmental DNA, and the eDNA from Cyprinus carpio. In both the diluted and saline pond water, we found that the degradation rate of eDNA was much slower than that in pond water. Furthermore, the DNA concentration did not exponentially decrease in both the saline purified water and purified water samples. For the lower degradation rate in salt water, we interpreted that salts may affect DNA degradation factors such as microbe compositions and activities. The effect of salinity and dilution on eDNA detection provides fundamental information about the degradation process of eDNA, which is essential to understand the behavior of eDNA in natural environments.
Self-Supervised Pretraining for Transferable Quantitative Phase Image Cell Segmentation
Authors: Vicar Tomas , Chmelik Jiri , Jakubicek Roman , Chmelikova Larisa , Gumulec Jaromir , Balvan Jan , Provaznik Ivo , Kolar Radim
Published: May 2021

In this paper, U-Net-based method for robust adherent cell segmentation for quantitative phase microscopy image is designed and optimised. We designed and evaluated four specific post-processing pipelines. To increase the transferability to different cell types, non-deep learning transfer with adjustable parameters is used in the post-processing step. Additionally, we proposed a self-supervised pretraining technique using nonlabelled data, which is trained to reconstruct multiple image distortions and improved the segmentation performance by from 0.67 to 0.70 of Object-wise Intersection over Union. Moreover, we publish a new dataset of manually labelled images suitable for this task together with the unlabelled data for self-supervised pretraining.

Graphical Abstract

Highlights

Four strategies for instance cell segmentation with U-Net were compared.

Specialised post-processing pipelines with tunable/optimizable parameters were designed for each segmentation strategy.

Transferability to different cell types by optimisation of post-processing parameters was tested.

The proposed self-supervised pretraining method improved both segmentation performance and transferability to different cell types.

A new manually labelled quantitative phase imaging dataset for cell segmentation with unlabelled data for self-supervised pretraining was created.
Three classes of epigenomic regulators converge to hyperactivate the essential maternal gene deadhead within a heterochromatin mini-domain
Authors: Torres-Campana Daniela , Horard Béatrice , Denaud Sandrine , Benoit Gérard , Loppin Benjamin , Orsi Guillermo A.
Published: May 2021

The formation of a diploid zygote is a highly complex cellular process that is entirely controlled by maternal gene products stored in the egg cytoplasm. This highly specialized transcriptional program is tightly controlled at the chromatin level in the female germline. As an extreme case in point, the massive and specific ovarian expression of the essential thioredoxin Deadhead (DHD) is critically regulated in Drosophila by the histone demethylase Lid and its partner, the histone deacetylase complex scaffold Sin3A, via yet unknown mechanisms. Here, we identified the Brahma chromatin remodeler sub-unit Snr1 and the insulator component Mod(mdg4) as essential for dhd expression and investigated how these epigenomic effectors act with Lid and Sin3A to hyperactivate dhd. Using Cut&Run chromatin profiling with a dedicated data analysis procedure, we found that dhd is intriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3A, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, we show that these effectors activate dhd independently of H3K27me3/H3K9me3, and that these marks are not required to repress dhd. Together, our study demonstrates an atypical and critical role for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to trigger tissue-specific hyperactivation within a unique heterochromatin mini-domain.

AUTHOR SUMMARY

Gene expression is tightly regulated by conserved protein complexes that act at the chromatin level to allow or restrict transcription. Such epigenetic control of gene activity defines the identity of different cell types during development, as well as their response to environmental cues. Yet, how multiple chromatin factors converge to achieve precise gene regulation remains difficult to address, partly due to the lack of biological situations where these intricate relationships can be studied. In this paper, we have addressed this issue by dissecting the regulation of deadhead, an essential gene specifically and massively expressed in the Drosophila germline. Unexpectedly, we found that its hyperactivation occurs despite deadhead being embedded in an apparently unfavorable chromatin mini-domain, notably featuring repressive histone modifications. We further demonstrate that four chromatin effectors, Lid, Sin3A, Snr1 and Mod(mdg4), have distinct, atypical and essential roles to ensure deadhead expression within this chromatin environment. Together, our findings put into perspective our understanding on these regulatory factors by illustrating how they can exert a biologically essential function via non-canonical mechanisms.
Knockdown of a novel ATPase domain of capsid protein inhibits genome packaging in potato leaf roll virus
Authors: Kumar Jitesh , Kumar Ravi Ranjan , Das Dilip Kumar , Mohanty Auroshikha , Rajani Kumari , Kumari Namaste , Kumar Vinod , Kumar Sunil , Ranjan Tushar
Published: May 2021

Potato leaf roll virus (PLRV) uses powerful molecular machines to package its genome into a viral capsid employing ATP as fuel. Although, recent bioinformatics and structural studies have revealed detailed mechanism of DNA packaging, little is known about the mechanochemistry of genome packaging in small plant viruses such as PLRV. We have identified a novel P-loop-containing ATPase domain with two Walker A-like motifs, two arginine fingers, and two sensor motifs distributed throughout the polypeptide chain of PLRV capsid protein (CP). The composition and arrangement of the ATP binding and hydrolysis domain of PLRV CP is unique and rarely reported. The discovery of the system sheds new light on the mechanism of viral genome packaging, regulation of viral assembly process, and evolution of plant viruses. Here, we used the RNAi approach to suppress CP gene expression, which in turn prevented PLRV genome packaging and assembly in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with siRNA constructs against the ATPase domain of CP exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of CP gene expression is an efficient method for generating PLRV-resistant potato plants. Moreover, our findings provide a robust approach to generate PLRV-resistant potato plants, which can be further extended to other species. Finally, we propose a new mechanism of genome packaging and assembly in plant viruses.
Signatures of genetic variation in human microRNAs point to processes of positive selection related to population-specific disease risks
Authors: Villegas-Mirón Pablo , Gallego Alicia , Bertranpetit Jaume , Laayouni Hafid , Espinosa-Parrilla Yolanda
Published: May 2021

The occurrence of natural variation in human microRNAs has been the focus of numerous studies during the last twenty years. Most of them have been dedicated to study the role of specific mutations in diseases, like cancer, while a minor fraction seek to analyse the diversity profiles of microRNAs in the genomes of human populations. In the present study we analyse the latest human microRNA annotations in the light of the most updated catalog of genetic variation provided by the 1000 Genomes Project. We show by means of thein silicoanalysis of noncoding variation of microRNAs that the level of evolutionary constraint of these sequences is governed by the interplay of different factors, like their evolutionary age or the genomic location where they emerged. The role of mutations in the shaping of microRNA-driven regulatory interactions is emphasized with the acknowledgement that, while the whole microRNA sequence is highly conserved, the seed region shows a pattern of higher genetic diversity that appears to be caused by the dramatic frequency shifts of a fraction of human microRNAs. We highlight the participation of these microRNAs in population-specific processes by identifying that not only the seed, but also the loop, are particularly differentiated regions among human populations. The quantitative computational comparison of signatures of population differentiation showed that candidate microRNAs with the largest differences are enriched in variants implicated in gene expression levels (eQTLs), selective sweeps and pathological processes. We explore the implication of these evolutionary-driven microRNAs and their SNPs in human diseases, such as different types of cancer, and discuss their role in population-specific disease risk.
Cooperative multivalent receptor binding promotes exposure of the SARS-CoV-2 fusion machinery core
Authors: Pak Alexander J. , Yu Alvin , Ke Zunlong , Briggs John A. G. , Voth Gregory A.
Published: May 2021

The molecular events that permit the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to bind, fuse, and enter cells are important to understand for both fundamental and therapeutic reasons. Spike proteins consist of S1 and S2 domains, which recognize angiotensin-converting enzyme 2 (ACE2) receptors and contain the viral fusion machinery, respectively. Ostensibly, the binding of spike trimers to ACE2 receptors promotes the preparation of the fusion machinery by dissociation of the S1 domains. We report the development of bottom-up coarse-grained (CG) models validated with cryo-electron tomography (cryo-ET) data, and the use of CG molecular dynamics simulations to investigate the dynamical mechanisms involved in viral binding and exposure of the S2 trimeric core. We show that spike trimers cooperatively bind to multiple ACE2 dimers at virion-cell interfaces. The multivalent interaction cyclically and processively induces S1 dissociation, thereby exposing the S2 core containing the fusion machinery. Our simulations thus reveal an important concerted interaction between spike trimers and ACE2 dimers that primes the virus for membrane fusion and entry.
Dissection of multiple sclerosis genetics identifies B and CD4+ T cells as driver cell subsets
Authors: Guo Michael H. , Sama Prashanth , LaBarre Brenna A. , Lokhande Hrishikesh , Balibalos John , Chu Ci , Du Xiaomi , Kheradpour Pouya , Kim Charles C , Oniskey Taylor , Snyder Thomas , Soghoian Damien Z , Weiner Howard L. , Chitnis Tanuja , Patsopoulos Nikolaos A.
Published: May 2021

Multiple sclerosis (MS) is an autoimmune condition of the central nervous system with a well-characterized genetic background. Prior analyses of MS genetics have identified broad enrichments across peripheral immune cells, yet the driver immune subsets are unclear. We utilized chromatin accessibility data across hematopoietic cells to identify cell type-specific enrichments of MS genetic signals. We found that CD4 T and B cells were independently enriched for MS genetics and further refined the driver subsets to Th17 and memory B cells, respectively. We replicated our findings in data from untreated and treated MS patients and found that immunomodulatory treatments suppress chromatin accessibility at driver cell types. Integration of statistical fine-mapping and chromatin interactions nominated numerous putative causal genes, illustrating complex interplay between shared and cell-specific genes. Our study highlights how careful integration of genetics and epigenetics can provide fine-scale insights into causal cell types and nominate new genes and pathways for disease.
A UAV-based high-throughput phenotyping approach to assess time-series nitrogen responses and identify traits associated genetic components in maize
Authors: Rodene Eric , Xu Gen , Delen Semra Palali , Smith Christine , Ge Yufeng , Schnable James , Yang Jinliang
Published: May 2021

Advancements in the use of genome-wide markers have provided new opportunities for dissecting the genetic components that control phenotypic trait variation. However, cost-effectively characterizing agronomically important phenotypic traits on a large scale remains a bottleneck. Unmanned aerial vehicle (UAV)-based high-throughput phenotyping has recently become a prominent method, as it allows large numbers of plants to be analyzed in a time-series manner. In this experiment, 233 inbred lines from the maize diversity panel were grown in a replicated incomplete block under both nitrogen-limited conditions and following conventional agronomic practices. UAV images were collected during different plant developmental stages throughout the growing season. A pipeline for extracting plot-level images, filtering images to remove non-foliage elements, and calculating canopy coverage and greenness ratings based on vegetation indices (VIs) was developed. After applying the pipeline, about half a million plot-level image clips were obtained for 12 different time points. High correlations were detected between VIs and ground truth physiological and yield-related traits collected from the same plots, i.e., Vegetative Index (VEG) vs. leaf nitrogen levels (Pearson correlation coefficient, R = 0.73), Woebbecke index vs. leaf area (R = -0.52), and Visible Atmospherically Resistant Index (VARI) vs. 20 kernel weight – a yield component trait (R = 0.40). The genome-wide association study was performed using canopy coverage and each of the VIs at each date, resulting in N = 29 unique genomic regions associated with image extracted traits from three or more of the 12 total time points. A candidate gene Zm00001d031997, a maize homolog of the Arabidopsis HCF244 (high chlorophyll fluorescence 244), located underneath the leading SNPs of the canopy coverage associated signals that were repeatedly detected under both nitrogen conditions. The plot-level time-series phenotypic data and the trait-associated genes provide great opportunities to advance plant science and to facilitate plant breeding.
Towards a Mechanical Model for anisotropic Glioma Spread using Darcy’s law
Authors: Reséndiz Margarita
Published: May 2021

The growth of a tumor within a finite domain (skull) generates mechanical forces that alter the physical interactions among cells. The relationship between these forces and the tumor architecture remains an open problem subjected to extensive research. Recently, it has been determined that those regions of high mechanical compression can accelerate and intensify the invasive capacity of the malignant cells, forming an irregular tumor whose full extent and edges are difficult to identify.

In the present paper, we propose a one-dimensional mathematical model that describes the process of proliferation and diffusion of glioma cells taking into account the mechanical compression generated during its expansion. Supported on the mixture theory, we model the brain-tumor system as a multiphase mixture of cancer cells, healthy cells, biological fluids and extracellular matrix whose densities determine the mechanical loads generated during the volumetric growth. Our model provides a detailed understanding of the pressure distribution on the interface boundary between healthy and cancer cells. It validates the hypothesis that the conferred ability of cancer cells to proliferate depends strongly on the mechanical pressure sensed. Through the analysis of the mechanical pressure, we determine that the anisotropic loads promote cancer cells to grow preferentially in the directions of low mechanical compression.
Island-specific evolution of a sex-primed autosome in the planarianSchmidtea mediterranea
Authors: Guo Longhua , Bloom Joshua , Serrate Daniel Dols , David Eyal Ben , Schubert Olga T. , Kazuma Kaiya , Ho Katarina , Wei Yubao , Leighton Daniel , Boocock James , Vergara Tzitziki Lemus , Riutort Marta , Alvarado Alejandro Sánchez , Kruglyak Leonid
Published: May 2021

The sexual biotype of the planarianSchmidtea mediterraneais a hermaphrodite indigenous to Tunisia and several Mediterranean islands. Here, we isolated individual chromosomes and used sequencing, Hi-C and linkage mapping to assemble a chromosome-scale genome reference. The linkage map revealed an extremely low rate of recombination on chromosome 1. We confirmed suppression of recombination on chromosome 1 by quantifying recombination events in individual sperm and oocytes. We showed that the extensive heterozygous regions, previously designated as J and V haplotypes, comprise essentially all of chromosome 1. Genome sequencing of individuals isolated in the wild indicated that this heterozygosity has evolved specifically in populations from Sardinia and Corsica. We found that chromosome 1 acquired many genes that determine the development of female and male reproductive systems, along with haplotype-specific expression of such sex specific genes. These molecular signatures and restricted recombination in a hermaphrodite led us to propose that chromosome 1 is a sex-primed autosome, providing direct molecular evidence for the traditional model of how sex chromosomes may have evolved from autosomes.
A method for single molecule localization microscopy of tissues reveals non-random distribution of nuclear pores in Drosophila
Authors: Cheng Jinmei , Allgeyer Edward S. , Richens Jennifer H. , Džafić Edo , Palandri Amandine , Lewkow Bohdan , Sirinakis George , Johnston Daniel St
Published: May 2021

Single Molecule Localisation Microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues, because of high background from out of focus emitters. Here we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-PAINT routinely gives 30nm resolution or better at depths greater than 20 µm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to cultured cells. Lamin Dm0 shows a complementary localisation to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C distributes the nuclear pores more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Since nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate chromatin organisation locally and contribute to the disease phenotypes caused by human Lamin A/C laminopathies.
Species’ traits and exposure as a future lens for quantifying seabird bycatch vulnerability in global fisheries
Authors: Richards Cerren , Cooke Robert S. C. , Bowler Diana E. , Boerder Kristina , Bates Amanda E.
Published: May 2021

Fisheries bycatch, the incidental mortality of non-target species, is a global threat to seabirds and a major driver of their declines worldwide. Identifying the most vulnerable species is core to developing sustainable fisheries management strategies that aim to improve conservation outcomes. To advance this goal, we present a preliminary vulnerability framework that integrates dimensions of species’ exposure, sensitivity, and adaptive capacity to fisheries bycatch to classify species into five vulnerability classes. The framework combines species’ traits and distribution ranges for 341 seabirds, along with a spatially resolved fishing effort dataset. Overall, we find most species have high vulnerability scores for the sensitivity and adaptive capacity dimensions. By contrast, exposure is more variable across species, and thus the median scores calculated within seabird families is low. We further find 46 species have high exposure to fishing activities, but are not identified as vulnerable to bycatch, whilst 133 species have lower exposure, but are vulnerable to bycatch. Thus, the framework has been valuable for revealing patterns between and within the vulnerability dimensions. Still, further methodological development, additional traits, and greater availability of threat data are required to advance the framework and provide a new lens for quantifying seabird bycatch vulnerability that complements existing efforts, such as the International Union for Conservation of Nature (IUCN) Red List.
Entry Pathway for the Inverse Agonist Ligand in the G Protein-Coupled Receptor Rhodopsin
Authors: Tian He , Gunnison Kathryn M. , Kazmi Manija A. , Sakmar Thomas P. , Huber Thomas
Published: May 2021

While the number of high-resolution structures of ligand-bound G protein-coupled receptors (GPCRs) has been steadily climbing, ligand binding and unbinding pathways remain largely undefined. The visual photoreceptor rhodopsin (Rho) represents a curious case among GPCRs because its primary ligand 11-cis-retinal (11CR) is an inverse agonist, which partitions into the bilayer and is likely to enter its orthosteric binding pocket through an intermembranous pathway. Light activates Rho by converting 11CR to all-trans-retinal (ATR), which serves as an agonist ligand. The light-triggered switch from the inactive to the active conformation creates two openings in the transmembrane region, suggesting pathways for exit of ATR and subsequent entry of 11CR to regenerate Rho. However, stabilization of an active ligand-free opsin conformation has been found to inhibit 11CR binding. Here we address this paradox of opsin regeneration with 11CR. We used genetic code expansion to engineer Rho mutants that serve as fluorescence sensors for measuring 11CR binding kinetics and energetics. We found mutations that alter a channel between transmembrane helices 5 and 6 (TM5/6) dramatically affect 11CR binding kinetics, but not ATR release kinetics. Our data provide direct experimental evidence for 11CR entry between TM5/6 in Rho that involves dynamic allosteric control of the ligand entry channel. Our findings can be extended to other visual pigments and a wide range of GPCRs with hydrophobic ligands that are hypothesized to enter their binding pockets through transmembrane pores.
Gaseous Signaling Compounds (Hydrogen Sulfide and Nitric Oxide) and Their Relative Roles in Affecting Anaerobic HeLa 229 Cell Viability
Authors: Plotkin Balbina J. , Sigar Ira M. , Kaminski Amber
Published: May 2021

Metabolic pathways supporting long-term anaerobic cell viability have not been identified. The effect NO and H2S pathway effectors have on HeLa 229 cell viability was measured after 10 days anaerobic incubation. The addition of arginine or xanthine (NO pathway precursors) consistently increased HeLa cell viability by 13.1- and 4.4-fold, respectively. Allopurinol, a xanthine oxidase inhibitor, also increased viability, as compared to control levels. In contrast, inhibition of iNOS by 1400W increased cell viability by 79-fold. Regarding the H2S pathway, precursor cysteine enhanced viability by 9.8-fold with the greatest number of viable cells measured in response to the presence of a H2S donor (GYY4137), or an inhibitor of glutathione synthesis, propargylglycine (40- and 85-fold, respectively). These results demonstrate that the constitutive level of cell viability after extended (10 days) growth without oxygen can be modulated by affecting NO or H2S generating pathways.
Natural variation in a short region of the Acidovorax citrulli type III-secreted effector AopW1 is associated with differences in cytotoxicity
Authors: Jiménez-Guerrero Irene , Sonawane Monica , Eckshtain-Levi Noam , Da Silva Gustavo Mateus , Pérez-Montaño Francisco , Leibman-Markus Meirav , Noda-Garcia Lianet , Bar Maya , Burdman Saul
Published: May 2021

Bacterial fruit blotch (BFB) is a serious disease of melon and watermelon caused by the Gram-negative bacterium Acidovorax citrulli. The strains of the pathogen can be divided into two major genetic groups, I and II. While group I strains are strongly associated with melon, group II strains are more aggressive on watermelon. Like many pathogenic bacteria, A. citrulli secretes a variety of protein effectors to the host cell via the type III secretion system. In the present study, we characterized AopW1, an A. citrulli type III-secreted effector that shares similarity with the actin cytoskeletondisrupting effector HopW1 of Pseudomonas syringae and with effectors from other plant-pathogenic bacterial species. aopW1 is present in group I and II strains, encoding products of 485 amino acids. Although highly conserved in most of the sequence, AopW1 has a highly variable region (HVR) within amino acid positions 147 to 192, including 14 amino acid differences between groups. Here we show that group I AopW1 is more toxic to yeast and plant cells than group II AopW1, having a stronger actin filament disruption activity, and increased ability to reduce plant callose deposition. We demonstrate the role of some of these 14 amino acid positions in determining the phenotypic differences between the two versions of the effector. Moreover, cellular analyses revealed that in addition to the interaction with actin filaments, AopW1 is localized to the endoplasmic reticulum, chloroplasts, and early and recycling plant endosomes, with differences observed between the two AopW1 versions. Finally, we show that overexpression of the endosome-associated protein EHD1 that increases cellular recycling, attenuates the toxic effects exerted by AopW1 and increases defence responses. This study provides insights into the HopW1 family of bacterial effectors and their interactions with the plant cell and provides first evidence on the involvement of EHD1 in response to biotic stress.
Non-neutralizing antibodies targeting the immunogenic regions of HIV-1 envelope reduce mucosal infection and virus burden in humanized mice
Authors: Hioe Catarina E. , Li Guangming , Liu Xiaomei , Tsahouridis Ourania , He Xiuting , Funaki Masaya , Klingler Jéromine , Tang Alex F. , Feyznezhad Roya , Heindel Daniel W. , Wang Xiao-Hong , Spencer David A. , Hu Guangna , Satija Namita , Prévost Jérémie , Finzi Andrés , Hessell Ann , Wang Shixia , Lu Shan , Chen Benjamin K. , Zolla-Pazner Susan , Upadhyay Chitra , Alvarez Raymond , Su Lishan
Published: May 2021

Antibodies are principal immune components elicited by vaccines to induce protection from microbial pathogens. In the Thai RV144 HIV-1 vaccine trial, vaccine efficacy was 31% and the sole primary correlate of reduced risk was shown to be vigorous antibody response targeting the V1V2 region of HIV-1 envelope. Antibodies against V3 also were inversely correlated with infection risk in subsets of vaccinees. Antibodies recognizing these regions, however, do not exhibit potent neutralizing activity. Therefore, we examined the antiviral potential of poorly neutralizing monoclonal antibodies (mAbs) against immunodominant V1V2 and V3 sites by passive administration of human mAbs to humanized mice engrafted with CD34+ hematopoietic stem cells, followed by mucosal challenge with an HIV-1 infectious molecular clone (IMC) expressing the envelope of a tier 2 resistant HIV-1 strain. Treatment with anti-V1V2 mAb 2158 or anti-V3 mAb 2219 did not prevent infection, but both reduced the virus burden, and V3 mAb 2219 displayed a superior potency compared to V1V2 mAb 2158. While these mAbs had no or weak neutralizing activity and elicited undetectable levels of antibody-dependent cellular cytotoxicity (ADCC), V3 mAb 2219 displayed a greater capacity to bind virus- and cell-associated HIV-1 envelope and to mediate antibody-dependent cellular phagocytosis (ADCP) and C1q complement binding as compared to V1V2 mAb 2158. Mutations in the Fc region of 2219 abolished these effector activities and abrogated virus control in humanized mice. These results demonstrate the importance of Fc functions other than ADCC for antibodies without potent neutralizing activity.
Structure-function relationship for a divergent Atg8 protein required for a non-autophagic function in malaria parasites
Authors: Walczak Marta , Meister Thomas R. , Zhu Yili , Yeh Ellen
Published: May 2021

Atg8 family proteins are highly-conserved eukaryotic proteins with diverse autophagy and non-autophagic functions in eukaryotes. While the structural features required for conserved autophagy functions of Atg8 are well-established, little is known about the molecular changes that facilitated acquisition of divergent, non-autophagic functions of Atg8. The malaria parasite Plasmodium falciparum offers a unique opportunity to study non-autophagic functions of Atg8 family proteins because it encodes a single Atg8 homolog whose only essential function is in the inheritance of an unusual secondary plastid called the apicoplast. Here we used functional complementation to investigate the structure-function relationship for this divergent Atg8 protein. We showed that the LC3-interacting region (LIR) docking site (LDS), the major interaction interface of Atg8 protein family, is not sufficient for PfAtg8 apicoplast function. Other regions previously implicated in canonical Atg8 interactions, the ubiquitin-interacting motif (UIM) docking site (UDS) and the N-terminal helix are not required for PfAtg8 function. Finally, the unique Apicomplexan-specific loop previously implicated in interaction with membrane conjugation machinery in vitro, is not required in vivo neither for membrane conjugation nor for the effector function of PfAtg8. These results suggest that the effector function of PfAtg8 is mediated by structural features distinct from those previously identified for macroautophagy and selective autophagy functions.

Importance

The most extensively studied role of Atg8 proteins is in autophagy. However, it is clear that they have other non-autophagic functions critical to cell function and disease pathogenesis yet understudied compared to their canonical role in autophagy. Mammalian cells contain multiple Atg8 paralogs that have diverse, specialized functions. Gaining molecular insight into their non-autophagic functions is difficult because of redundancy between the homologs and their role in both autophagy and non-autophagic pathways. Malaria parasites such as Plasmodium falciparum are a unique system to study a novel, non-autophagic function of Atg8 separate from its role in autophagy: They have only one Atg8 protein whose only essential function is in the inheritance of the apicoplast, a unique secondary plastid organelle. Insights into the molecular basis of PfAtg8’s function in apicoplast biogenesis will have important implications for the evolution of diverse non-autophagic functions of the Atg8 protein family.
Cholesterol Oxidation Modulates the Formation of Liquid-Ordered Domains in Model Membranes
Authors: Smith Paul , Petrov Peter G. , Lorenz Christian D.
Published: May 2021

7-ketocholesterol (KChol) is one of the most cytotoxic oxysterols found in the plasma membrane, and increased levels of KChol are associated with numerous pathologies. It is thought to induce apoptosis via inactivation of the phosphatidylinositol 3-kinase/Akt signaling pathway — a pathway that depends on lipid-rafts as signaling platforms. By means of coarse-grained molecular dynamics simulations, we demonstrate that KChol disrupts the liquid-liquid phase separation seen in an equimolar mixture of (dipalmitoylphosphatidylcholine) DPPC, (dioleoylphosphatidylcholine) DOPC, and Cholesterol (Chol). This disruption occurs via two mechanisms: i) KChol adopts a wider range of orientations with the membrane, which disrupts the packing of neigh-boring lipids and ii) KChol has no preference for DPPC over DOPC, which is the main driving force for lateral demixing in DPPC/DOPC/Chol membranes. This provides a molecular description of the means by which KChol induces apoptosis, and illustrates that a single chemical substitution to cholesterol can have a profound impact on the lateral organization of lipid membranes.

Graphical TOC Entry
Distinct mechanoreceptor pezo-1 isoforms modulate food intake in the nematode Caenorhabditis elegans
Authors: Hughes K , Shah A , Bai X , Adams J , Bauer R , Jackson J , Harris E , Ficca A , Freebairn P , Mohammed S , Fernández EM , Bainbridge C , Brocco MA , Stein W , Vidal-Gadea AG
Published: May 2021

Two PIEZO mechanosensitive cation channels, PIEZO1 and PIEZO2, have been identified in mammals, where they are involved in numerous sensory processes. While structurally similar, PIEZO channels are expressed in distinct tissues and exhibit unique properties. How different PIEZOs transduce force, how their transduction mechanism varies, and how their unique properties match the functional needs of the tissues they are expressed in remain all-important unanswered questions. The nematode Caenorhabditis elegans has a single PIEZO ortholog (pezo-1) predicted to have twelve isoforms. These isoforms share many transmembrane domains but differ in those that distinguish PIEZO1 and PIEZO2 in mammals. We used transcriptional and translational reporters to show that putative promoter sequences immediately upstream of the start codon of long pezo-1 isoforms predominantly drive GFP expression in mesodermally derived tissues (such as muscle and glands). In contrast, sequences upstream of shorter pezo-1 isoforms resulted in GFP expression primarily in neurons. Putative promoters upstream of different isoforms drove GFP expression in different cells of the same organs of the digestive system. The observed unique pattern of complementary expression suggests that different isoforms could possess distinct functions within these organs. We used mutant analysis to show that pharyngeal muscles and glands require long pezo-1 isoforms to respond appropriately to the presence of food. The number of pezo-1 isoforms in C. elegans, their putative differential pattern of expression, and roles in experimentally tractable processes make this an attractive system to investigate the molecular basis for functional differences between members of the PIEZO family of mechanoreceptors.
CuAAC stabilization of an NMR mixed labeled dimer
Authors: Sapienza Paul J. , Currie Michelle M. , Li Kelin , Aubé Jeffrey , Lee Andrew L.
Published: May 2021

Homo dimers are the most abundant type of enzyme in cells and as such, they represent the archetypal system for studying the remarkable phenomenon of allostery. In these systems, in which the allosteric features are manifest by the effect of the first binding event on the similar event at the second site, the most informative state is the asymmetric single bound (lig1) form, yet it tends to be elusive thermodynamically. Here we take significant steps towards obtaining milligram quantities of pure lig1 of the allosteric homodimer, chorismate mutase, in the form of a mixed isotopically labeled dimer stabilized by Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) between the subunits. Below, we outline several critical steps required to generate high yields of both types of unnatural amino acid-containing proteins, and overcome multiple pitfalls intrinsic to CuAAC to obtain high yields of pure, fully intact, and active mixed labeled dimer. These data not only will make possible NMR-based investigations of allostery envisioned by us, but should also facilitate other structural applications where specific linkage of proteins is helpful.
CmP signaling network unveils novel biomarkers for triple negative breast cancer in African American women
Authors: Abou-Fadel Johnathan , Grajeda Brian , Jiang Xiaoting , Cailing-De La O Alyssa-Marie D. , Flores Esmeralda , Padarti Akhil , Bhalli Muaz , Le Alexander , Zhang Jun
Published: May 2021

Breast cancer is the most commonly diagnosed cancer worldwide and remains the second leading cause of cancer death. While breast cancer mortality has steadily declined over the past decades through medical advances, an alarming disparity in breast cancer mortality has emerged between African American women (AAW) and Caucasian American women (CAW); and new evidence suggests more aggressive behavior of triple-negative breast cancer (TNBC) in AAW may contribute to racial differences in tumor biology and mortality. Progesterone (PRG) is capable of exerting its cellular effects through either its classic, non-classic or combined responses through binding to either classic nuclear PRG receptors (nPRs) or non-classic membrane PRG receptors (mPRs), warranting both pathways an equally important status in PRG-mediated signaling. In our previous report, we demonstrated that the CCM signaling complex (CSC) consisting of CCM1, CCM2, and CCM3 proteins can couple both nPRs and mPRs signaling cascades to form a CSC-mPRs-PRG-nPRs (CmPn) signaling network in nPR positive(+) breast cancer cells. In this report, we furthered our research by establishing the CSC-mPRs-PRG (CmP) signaling network in nPR(-) breast cancer cells, demonstrating that a common core mechanism exists, regardless of nPR(+/-) cell type. This is the first report stating that inducible expression patterns exist between CCMs and major mPRs in TNBC cells. Furthermore, we firstly show mPRs in TNBC cells are localized in the nucleus and participate in nucleocytoplasmic shuttling in a coordinately synchronized fashion with CCM proteins under steroid actions, following the same cellular distribution as other well-defined steroid hormone receptors. Finally, for the first time, we deconvoluted the CmP signalosome by using multi-omics approaches, which helped us understand key factors within the CmP network, and identify 21 specific biomarkers with potential clinical applications associated with AAW-TNBC tumorigenesis. These novel biomarkers could have immediate clinical implications to dramatically improve health disparities among AAW-TNBCs.
Genomic Selection for End-Use Quality and Processing Traits in Soft White Winter Wheat Breeding Program with Machine and Deep Learning Models
Authors: Sandhu Karansher S. , Aoun Meriem , Morris Craig , Carter Arron H.
Published: May 2021

Breeding for grain yield, biotic and abiotic stress resistance, and end-use quality are important goals of wheat breeding programs. Screening for end-use quality traits is usually secondary to grain yield due to high labor needs, cost of testing, and large seed requirements for phenotyping. Hence, testing is delayed until later stages in the breeding program. Delayed phenotyping results in advancement of inferior end-use quality lines into the program. Genomic selection provides an alternative to predict performance using genome-wide markers. Due to large datasets in breeding programs, we explored the potential of the machine and deep learning models to predict fourteen end-use quality traits in a winter wheat breeding program. The population used consisted of 666 wheat genotypes screened for five years (2015-19) at two locations (Pullman and Lind, WA, USA). Nine different models, including two machine learning (random forest and support vector machine) and two deep learning models (convolutional neural network and multilayer perceptron), were explored for cross-validation, forward, and across locations predictions. The prediction accuracies for different traits varied from 0.45-0.81, 0.29-0.55, and 0.27-0.50 under cross-validation, forward, and across location predictions. In general, forward prediction accuracies kept increasing over time due to increments in training data size and was more evident for machine and deep learning models. Deep learning models performed superior over the traditional ridge regression best linear unbiased prediction (RRBLUP) and Bayesian models under all prediction scenarios. The high accuracy observed for end-use quality traits in this study support predicting them in early generations, leading to the advancement of superior genotypes to more extensive grain yield trailing. Furthermore, the superior performance of machine and deep learning models strengthen the idea to include them in large scale breeding programs for predicting complex traits.
GraFT: Graph Filtered Temporal Dictionary Learning for Functional Neural Imaging
Authors: Charles Adam S. , Cermak Nathan , Affan Rifqi , Scott Ben , Schiller Jackie , Mishne Gal
Published: May 2021

Optical imaging of calcium signals in the brain has enabled researchers to observe the activity of hundreds-to-thousands of individual neurons simultaneously. Current methods predominantly focus on matrix factorization and aim at detecting neurons in the imaged field-of-view, and then inferring the corresponding time-traces. The explicit locality constraints on the cell shapes additionally limits the applicability to optical imaging at different scales (i.e., dendritic or widefield data). Here we present a new method that frames the problem of isolating independent fluorescing components as a dictionary learning problem. Specifically, we focus on the time-traces, which are the main quantity used in scientific discovery, and learn the dictionary of time traces with the spatial maps acting as the presence coefficients encoding which pixels the time traces are active in. Furthermore, we present a novel graph filtering model which redefines connectivity between pixels in terms of their shared temporal activity, rather than spatial proximity. This model greatly eases the ability of our method to handle data with complex non-local spatial structure, such as dendritic imaging. We demonstrate important properties of our method, such as robustness to initialization, implicitly inferring number of neurons and simultaneously detecting different neuronal types, on both synthetic data and real data examples. Specifically, we demonstrate applications of our method to calcium imaging both at the dendritic, somatic, and widefield scales.
Identification of multiple TAR DNA Binding Protein retropseudogene lineages during the evolution of primates
Authors: Opazo Juan C. , Zavala Kattina , Vargas-Chacoff Luis , Morera Francisco J. , Mardones Gonzalo A.
Published: May 2021

The TAR DNA Binding Protein (TARDBP) gene has gained attention in biomedicine after the discovery of several pathogenic mutations. The lack of knowledge about its evolutionary history contrasts with a large number of studies in the biomedical area. This study aimed to investigate the retrotransposition evolutionary dynamics associated with this gene in primates. We identified retropseudogenes that originated in the ancestors of anthropoids, catarrhines, and lemuriformes, i.e. the strepsirrhine clade that inhabit Madagascar. We also found species-specific retropseudogenes in the philippine tarsier, Bolivian squirrel monkey, capuchin monkey and vervet. Although retropseudogenes are not able to produce a functional protein, we can not rule out that they may represent genetic material upon which evolution acts on, especially with regulatory functions.
Cryptic genetic variations of alanine:glyoxylate aminotransferase shape its fitness and dynamics
Authors: Dindo Mirco , Pascarelli Stefano , Chiasserini Davide , Grottelli Silvia , Costantini Claudio , Uechi Gen-Ichiro , Giardina Giorgio , Laurino Paola , Cellini Barbara
Published: May 2021

Genetic variations expand the conformational landscape of proteins and may underlie cryptic properties that promote environmental adaptability. However, they can also represent modifying factors for disease susceptibility, by changing frustrated regions that in turn affect protein overall intracellular fitness. In this dichotomy between conservation and innovation, understanding at structural level how genetic variations keep the balance to maintain protein fitness represents an unmet need.

Herein, we took advantage of known genetic variations of human alanine:glyoxylate aminotransferase (AGT1), which is present as a common major allelic form (AGT-Ma) and a minor polymorphic form (AGT-Mi) expressed in 20% of Caucasian population. By crystallographic studies and molecular dynamics simulations we showed that the polymorphic amino acid substitutions shape the conformational flexibility of AGT1 so that three surface regions that are structured in AGT-Ma become disordered in AGT-Mi, thanks to plasticity effects propagated from the mutation site(s) to the whole structure. In-depth biochemical characterisation of variants from a library encompassing the three regions correlate this plasticity to a fitness window between AGT-Ma and AGT-Mi, and suggest the existence of cryptic functions related to protein-protein interactions. These results establish that naturally-occurring genetic variations tip the balance between stability and frustration to expand the potential innovability of the protein.
Cellular heterogeneity in DNA alkylation repair as a trade-off between cell survival and genetic plasticity
Authors: Vincent Maxence S. , Uphoff Stephan
Published: May 2021

DNA repair mechanisms fulfil a dual role, as they are essential for cell survival and genome maintenance. Here, we studied how cells regulate the interplay between DNA repair and mutation. We focused on the Escherichia coli adaptive response that increases resistance to DNA alkylation damage. Combination of single-molecule imaging and microfluidic-based single-cell microscopy showed that noise in the gene activation timing of the master regulator Ada is accurately propagated to generate a distinct subpopulation of cells in which all proteins of the adaptive response are absent. Although lack of these proteins causes extreme sensitivity to alkylation stress, cellular heterogeneity in DNA alkylation repair provides a functional benefit by increasing the evolvability of the whole population. We demonstrated this by monitoring the dynamics of nascent mutations during alkylation stress as well as the frequency of fixed mutations that are generated by the distinct subpopulations of the adaptive response. This highlighted that evolvability is a trade-off between mutability and cell survival. Stochastic modulation of DNA repair capacity by the adaptive response solves this trade-off through the generation of a viable hypermutable subpopulation of cells that acts as a source of genetic diversity in a clonal population.