bluuebunny/crossref_metadata_2025 huggingface dataset:
Systematic functional screening of chromatin factors identifies strong lineage and disease dependencies in normal and malignant haematopoiesis
Authors: Lara-Astiaso D , Goñi-Salaverri A , Mendieta-Esteban J , Narayan N , Del Valle C , Gross T , Giotopoulos G , Navarro-Alonso M , Zazpe J , Marchese F , Torrea N , Calvo IA , Lopez C , Alignani D , Lopez A , Saez B , Taylor-King J. P , Prosper F , Fortelny N , Huntly B. J. P
Published: August 2022

Interactions between transcription factors (TF) and chromatin factors (CF) regulate gene expression programmes to determine cellular fate. However, unlike for TF, the exact role of CF in this process is poorly understood. Using haematopoiesis as a model system and utilising novel functional CRISPR screens ex vivo and in vivo, coupled with Perturb-Seq, CF binding and genome-wide chromatin accessibility in primary murine cells, we assess the role of 550 chromatin factors in lineage choice in normal haematopoiesis and the maintenance of acute myeloid leukaemia (AML). These studies demonstrate marked specificity for a large number of CFs in lineage determination, highlighting functional diversity within specific families of chromatin regulators, including MLL-H3K4-methyltransferases and different BAF-complexes, that regulate disparate lineage decisions across haematopoiesis. Conversely, we demonstrate that unrelated Repressive complexes function similarly to restrain excessive myeloid differentiation and protect lineage diversity. We identify interactions between CF and TF that, at least in part, explain the regulatory function of CF and link Brd9-loss to a premalignant state. Utilising similar experiments in a relevant murine AML model, we demonstrate opposing effects for CF in normal haematopoiesis and AML, where MLL-H3K4-methyltransferases, c-BAF-remodelers and Repressive complexes prevent differentiation and maintain leukaemic fitness. We show that this alteration relates to differential utilisation of TF by CF complexes between normal and malignant haematopoiesis, highlighting corrupted TF-CF interactions as potential novel avenues for therapeutic intervention in AML. Together, this study provides novel insights on the functional diversity of chromatin factors in governing cell-fate.
A Dual Role for LAR-RPTP in Regulating Long-distance Transport and Synaptic Retention of AMPARs, Essential for Long Term Associative Memory
Authors: Pierce D.M. , Lenninger Z. , Doser R.L. , Knight K.M. , Stetak A. , Hoerndli F.J.
Published: August 2022

The AMPA subtype of ionotropic glutamate receptors (AMPARs) plays an essential role in excitatory synaptic transmission, learning, and memory. The majority of AMPARs are made in the cell body and are transported by molecular motors to synapses. Maintaining the proper number of synaptic receptors requires coordinated regulation of receptor production, export from the soma and delivery at synapses. This major logistical process is essential for circuit function and behavior. Although recent studies have shown that long-distance synaptic transport is regulated by neuronal activity, little is known about the mechanisms that coordinate somatic export or synaptic delivery and removal. Here we show that loss of the PTP-3A isoform of the receptor tyrosine phosphatase PTP-3 (the C. elegans homologue of vertebrate LAR-RPTP) leads to a ∼60% decrease in AMPAR transport; this affects synaptic delivery of AMPARs and synaptic functions necessary for long-term associative olfactory memory in C. elegans. Interestingly, while complete loss of PTP-3A leads to defects in transport and local synaptic trafficking of AMPARs, loss of only PTP-3 phosphatase function affects local synaptic recycling and retention of AMPARs. Finally, we show that the N-terminus of PTP-3A regulates transport, whereas the C-terminal regulates synaptic retention of AMPARs. Altogether, our results suggest a model in which the two domains of PTP-3/LAR-RPTPs have specific, complementary roles in coordinating somatic export and local retention of AMPARs essential for long-term associative memory.
Measurement error associated with gait cycle selection in treadmill running at various speeds
Authors: Fox Aaron S. , Bonacci Jason , Warmenhoven John , Keast Meghan F.
Published: August 2022

A common approach in biomechanical analysis of running technique is to average data from several gait cycles to compute a ‘representative mean.’ However, the impact of the quantity and selection of gait cycles on biomechanical measures is not well understood. We examined the effects of gait cycle selection on kinematic data by: (i) comparing representative means calculated from varying numbers of gait cycles to ‘global’ means from the entire capture period; and (ii) comparing representative means from varying numbers of gait cycles sampled from different parts of the capture period. We used a public dataset (n = 28) of lower limb kinematics captured during a 30-second period of treadmill running at three speeds (2.5m · s-1, 3.5m · s-1 and 4.5m · s-1). ‘Ground truth’ values were determined by averaging data across all collected strides and compared to representative means calculated from random samples (1,000 samples) of n (range = 5—30) consecutive gait cycles. We also compared representative means calculated from n (range = 5—15) consecutive gait cycles randomly sampled (1,000 samples) from within the same data capture period. The mean, variance and range of the absolute error of the representative mean compared to the ‘ground truth’ mean progressively reduced across all speeds as the number of gait cycles used increased. Similar magnitudes of ‘error’ were observed between the 2.5m · s-1 and 3.5m · s-1 speeds at comparable gait cycle numbers — where the maximum errors were < 1.5 degrees even with a small number of gait cycles (i.e. 5-10). At the 4.5m · s-1 speed, maximum errors typically exceeded 2-4 degrees when a lower number of gait cycles were used. Subsequently, a higher number of gait cycles (i.e. 25-30) was required to achieve low errors (i.e. 1-2 degrees) at the 4.5m · s-1 speed. The mean, variance and range of absolute error of representative means calculated from different parts of the capture period was consistent irrespective of the number of gait cycles used. The error between representative means was low (i.e. <1.5 degrees) and consistent across the different number of gait cycles at the 2.5m · s-1 and 3.5m · s-1 speeds, and consistent but larger (i.e. up to 2-4 degrees) at the 4.5m · s-1 speed. Our findings suggest that selecting as many gait cycles as possible from a treadmill running bout will minimise potential ‘error.’ Analysing a small sample (i.e. 5-10 cycles) will typically result in minimal ‘error’ (i.e. < 2 degrees), particularly at lower speeds (i.e. 2.5m · s-1 and 3.5m · s-1). Researchers and clinicians should consider the balance between practicalities of collecting and analysing a smaller number of gait cycles against the potential ‘error’ when determining their methodological approach. Irrespective of the number of gait cycles used, we recommend that the potential ‘error’ introduced by the choice of gait cycle number be considered when interpreting the magnitude of effects in treadmill-based running studies.
A microglia-CD4+ T cell partnership generates protective anti-tumor immunity to glioblastoma
Authors: Chen Dan , Varanasi Siva Karthik , Hara Toshiro , Traina Kacie , McDonald Bryan , Farsakoglu Yagmur , Clanton Josh , Xu Shihao , Mann Thomas H. , Du Victor , Chung H. Kay , Xu Ziyan , Tripple Victoria , Casillas Eduardo , Ma Shixin , O’Connor Carolyn , Yang Qiyuan , Zheng Ye , Hunter Tony , Lemke Greg , Kaech Susan M.
Published: August 2022

The limited efficacy of immunotherapies against glioblastoma illustrates the urgent need to better understand the interactions between the central nervous system and the immune system. Here, we showed that a protective response to αCTLA-4 therapy depended on a mutualistic relationship between microglia and CD4+ T cells. Suppression of gliomas by CD4+ T cells did not require tumor-intrinsic MHC-II expression, but rather was dependent on the selective expression of MHC-II and antigen presentation by local microglia that in turn, sustained CD4+ T cell tumoricidal effector functions. CD4+ T cell secretion of IFNγ made the glioma cells vulnerable to enhanced tumor surveillance and phagocytosis by microglia via the AXL/MER tyrosine kinase receptors that were necessary for tumor suppression. This work illustrates a novel partnership between CD4+ T cells and microglia that unleashes the tumoricidal properties of microglia that can be harnessed to improve immunotherapies for glioblastoma.
Dendritic cell-Natural Killer cell Crosstalk Modulates T cell activation in Response to Influenza A Viral Infection
Authors: Harvey Abigail G. , Graves Athens M. , Uppalapati Chandana K. , Matthews Saoirse M. , Rosenberg Stephanie , Fagerlie Madison H. , Guinan Jack , Lopez Brina , Kronstad Lisa M.
Published: August 2022

Influenza viruses lead to substantial morbidity and mortality including ~3-5 million cases of severe illness and ~290,000-650,000 deaths annually. One of the major hurdles regarding influenza vaccine efficacy is generating a durable, robust cellular immune response. Appropriate stimulation of the innate immune system is key to generating cellular immunity. Crosstalk between innate dendritic cells (DC) and natural killer (NK) cells plays a key role in activating virus-specific T cells, yet the mechanisms used by influenza A viruses (IAV) to govern this process remain incompletely understood. Here, we used anex vivoautologous human primary immune cell culture system to evaluate the impact of genetically distinct IAV strains on DC-NK cell crosstalk and subsequent T cell activation. We report that the addition of NK cells to cultures containing both DCs and naïve T cells led to an increase in the frequency of CD69+and CD25+T cells and elevated levels of IFN-γ, TNF, and IL-10. However, upon IAV infection of DCs, the addition of NK cells to cultures no longer increased the frequency of CD25+T cells nor elevated IFN-γ, TNF, and IL-10 cytokine levels. Investigation of the impact of IAV infection on DC-NK crosstalk revealed that exposure of DCs to influenza virus in co-culture led to an increased frequency of HLA-DR+and a decreased frequency of CD83+and CD86+cells–molecules involved in stimulating T cell activation. An expansion of an HLA-DR+NK cell subset was observed following culture with influenza-infected DCs in a contact-dependent and cytokine independent-manner. Overall, our results indicate a role for DC-NK cell crosstalk in T cell priming in the context of influenza infection, informing the immunological mechanisms that could be manipulated for the next generation influenza vaccine or immunotherapeutic.
Somatic and terminal CB1 receptors are differentially coupled to voltage-gated sodium channels in neocortical neurons
Authors: Steiger Luke , Tsintsadze Timur , Mattheisen Glynis B. , Smith Stephen M.
Published: August 2022

Endogenous cannabinoid signaling is vital for important brain functions and can be modified pharmacologically to treat pain, epilepsy, and posttraumatic stress disorder. Endocannabinoid mediated changes to excitability are predominantly attributed to 2-arachidonoylglycerol at synapses. Here we identify a pathway in the neocortex by which anandamide, the other major endocannabinoid, powerfully inhibits sodium conductances in the soma resulting in a loss of neuronal excitability. This pathway is mediated by the cannabinoid receptor, and its activation results in a decrease of recurrent action potential generation. The synthetic cannabinoid, WIN 55,212-2, also inhibits VGSC currents indicating this pathway is positioned to mediate the actions of exogenous cannabinoids.

Highlights

Anandamide (AEA), a major endocannabinoid, indirectly inhibits VGSC currents in neocortical neurons.

This prevalent signaling pathway involves AEA activation of CB1 and other G-protein-coupled receptors localized to the intracellular compartment of neurons.

CB1 activation by AEA reduces VGSC availability at the soma but not at the axonal compartment suggesting tighter functional coupling between VGSCs and CB1 at the cell body.

Cannabinoid action on somatic CB1 inhibits VGSCs with high efficacy, providing a parallel pathway outside of nerve terminals, by which these ligands reduce neuronal excitability in the neocortex.
Engineering precise adenine base editor with infinitesimal rates of bystander mutations and off-target editing
Authors: Chen Liang , Zhang Shun , Xue Niannian , Hong Mengjia , Zhang Xiaohui , Zhang Dan , Yang Jing , Bai Sijia , Huang Yifan , Meng Haowei , Wu Hao , Luan Changming , Zhu Biyun , Ru Gaomeng , Liu Meizhen , Liu Mingyao , Cheng Yiyun , Yi Chengqi , Song Gaojie , Wang Liren , Li Dali
Published: August 2022

Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise the concerns of safety issues. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1-2nt with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision than ABE8e) and was further confirmed through a library of guide RNA-target sequence pairs. Due to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic SNVs when fused to Cas9 variants with expanded PAM compatibility.
Evolution of constitutive bacteriocin production and release
Authors: Bhattacharya Amrita , Lively Curtis M
Published: August 2022

The production of the bacterial anti-competitor toxins, called bacteriocins, is widely described as spite, because the production of the toxins is costly and often requires cell death for release. Given these costs, it reasonable to think that bacteriocin production should be induced by the presence of unrelated competitor strains. Nonetheless, recent evidence in the insect-pathogenic bacteriumXenorhabdus koppenhoeffrishowed that bacteriocin production occurs regardless of the presence of unrelated competitors. Could the constitutive production of bacteriocins be favored by natural selection? Here we use a mathematical model to examine this question, both within and between subpopulations. The model assumes that bacteriocin production is constitutive and costly, and that toxin release only occurs during natural cell death. We found that constitutive bacteriocin producers can outcompete non-producer sensitive strains within populations, and that it can spread in a metapopulation provided there is local competition for patches. Hence, the evolution of bacteriocin production does not require the detection of competitors or “suicidal” release of toxins.
Elements of divergence in germline determination in closely related species
Authors: Morita Shumpei , Oulhen Nathalie , Foster Stephany , Wessel Gary M.
Published: August 2022

Evolutionary transitions enable the wide diversity in life histories of plants and animals. This is particularly germane in the development of the germ line in which fitness is a direct readout of evolutionary change. Here, we focused on the gem line of two distinct sea urchin species who shared a common ancestor 50 million years ago. Even though they both rely on inherited mechanisms to specify their germ line, the integration of stage-matched single cell RNA-seq (scRNA-seq) datasets from these two sea urchins revealed a variety of differences in gene expression, including a broader expression of the germ line factor Nanos2 in Lytechinus variegatus (Lv) compared to Strongylocentrotus purpuratus (Sp). In Sp, Nanos2 mRNA expression is highly restricted to the primordial germ cells (PGCs) by a lability element in its 3’UTR. This element is lacking in the mRNA of Lv Nanos2, explaining how this mRNA more broadly accumulates in the Lv embryos. We discovered that the Lv Nanos2 3’UTR instead leads to a germline specific translation of the protein. The results emphasize that regulatory mechanisms resulting in germline diversity rely less on transcriptional regulation and more on post-transcriptional and post-translational restrictions of key gene products, such as Nanos2.

Highlights

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The first integration of scRNA-seq datasets comparing two echinoderm species.

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We find Nanos2 positive cells in the embryonic soma of Lytechinus variegatus, an unusual occurrence, but not in Strongylocentrous purpuratus.

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We discovered that this somatic Nanos2 mRNA is lacking an important regulatory element (GNARLE) in its 3’UTR

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Instead, in Lv, the 3’UTR of Nanos2 leads to its specific translation in the germ cells.
Dancing with the ions: symport and antiport mechanisms of human glutamate transporters
Authors: Qiu Biao , Boudker Olga
Published: August 2022

Excitatory amino acid transporters (EAATs) pump glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton and counter-transporting a potassium ion via an elevator mechanism. Despite the availability of structures, the symport and antiport mechanisms remain unclear. We report high-resolution Cryo-EM structures of human EAAT3 bound to the neurotransmitter glutamate with symported ions, potassium ions, sodium ions alone, or in the absence of ligands. We show that an evolutionarily conserved occluded translocation intermediate has a dramatically higher affinity for the neurotransmitter and the counter-transported potassium ion than outward- or inward-facing transporters and plays a crucial role in ion coupling. We propose a comprehensive ion coupling mechanism involving a choreographed interplay between bound solutes, conformations of conserved amino acid motifs, and movements of the gating hairpin and the substrate-binding domain.
Changes in behavioral priority influence the accessibility but not the quality of working memory content
Authors: Ester Edward F. , Pytel Paige
Published: August 2022

Evolving behavioral goals require the existence of selection mechanisms that prioritize task-relevant working memory (WM) content for action. Selecting an item stored in WM is known to blunt and/or reverse information loss in stimulus-specific representations of that item reconstructed from human brain activity, but extant studies have focused on all-or-none circumstances that allow or disallow an agent to select one of several items stored in WM. Conversely, behavioral studies suggest that humans can flexibly assign different levels of priority to different items stored in WM, but how doing so influences neural representations of WM content is unclear. One possibility is that assigning different levels of priority to items in WM influences the quality of those representations, resulting in more robust neural representations of high- vs. low-priority WM content. A second – and non-exclusive – possibility is that asymmetries in behavioral priority influence how rapidly neural representations of high- vs. low-priority WM content can be selected and reported. We tested these possibilities in two experiments by decoding high- and low-priority WM content from EEG recordings obtained while human volunteers performed a retrospectively cued WM task. Probabilistic changes in the behavioral relevance of a remembered item had no effect on our ability to decode it from EEG signals; instead, these changes influenced the latency at which above-chance decoding performance was reached. Thus, our results indicate that probabilistic changes in the behavioral relevance of WM content influence the ease with which memories can be accessed and retrieved independently of their strength.
Genetic architecture and evolution of color variation in American black bears
Authors: Puckett Emily E. , Davis Isis S. , Harper Dawn C. , Wakamatsu Kazumasa , Battu Gopal , Belant Jerrold L. , Beyer Dean E. , Carpenter Colin , Crupi Anthony P. , Davidson Maria , DePerno Christopher S. , Forman Nicholas , Fowler Nicholas L. , Garshelis David L. , Gould Nicholas , Gunther Kerry , Haroldson Mark , Ito Shosuke , Kocka David , Lackey Carl , Leahy Ryan , Lee-Roney Caitlin , Lewis Tania , Lutto Ashley , McGowan Kelly , Olfenbuttel Colleen , Orlando Mike , Platt Alexander , Pollard Matthew D. , Ramaker Megan , Reich Heather , Sajecki Jaime L. , Sell Stephanie K. , Strules Jennifer , Thompson Seth , van Manen Frank , Whitman Craig , Williamson Ryan , Winslow Frederic , Kaelin Christopher B. , Marks Michael S. , Barsh Gregory S.
Published: August 2022

Color variation is a frequent evolutionary substrate for camouflage in small mammals but the underlying genetics and evolutionary forces that drive color variation in natural populations of large mammals are mostly unexplained. The American black bear, Ursus americanus, exhibits a range of colors including the cinnamon morph which has a similar color to the brown bear, U. arctos, and is found at high frequency in the American southwest. Reflectance and chemical melanin measurements showed little distinction between U. arctos and cinnamon U. americanus individuals. We used a genome-wide association for hair color as a quantitative trait in 151 U. americanus individuals and identified a single major locus (P < 10−13). Additional genomic and functional studies identified a missense alteration (R153C) in Tyrosinase-related protein 1 (TYRP1) that impaired protein localization and decreased pigment production. Population genetic analyses and demographic modeling indicated that the R153C variant arose 9.36kya in a southwestern population where it likely provided a selective advantage, spreading both northwards and eastwards by gene flow. A different TYRP1 allele, R114C, contributes to the characteristic brown color of U. arctos, but is not fixed across the range.

HIGHLIGHTS

The cinnamon morph of American black bears and brown bears have different missense mutations in TYRP1 that account for their similar coloration

TYRP1 variants in American black bears and brown bears are loss-of-function alleles associated with impaired protein localization to melanosomes

In American black bears, the variant causing the cinnamon morph arose 9,360 years ago in the western lineage where it provides an adaptive advantage, and has spread northwards and eastwards by migration
A comprehensive model of Drosophila epithelium reveals the role of embryo geometry and cell topology in mechanical responses
Authors: Cheikh Mohamad Ibrahim , Tchoufag Joel , Osterfield Miriam , Dean Kevin , Bhaduri Swayamdipta , Chuzhong Zhang , Mandadapu Kranthi Kiran , Doubrovinski Konstantin
Published: August 2022

In order to understand morphogenesis, it is necessary to know the material properties or forces shaping the living tissue. In spite of this need, very fewin vivomeasurements are currently available. Here, using the early Drosophila embryo as a model, we describe a novel cantilever-based technique which allows for the simultaneous quantification of applied force and tissue displacement in a living embryo. By analyzing data from a series of experiments in which embryonic epithelium is subjected to developmentally relevant perturbations, we conclude that the response to applied force is adiabatic and is dominated by elastic forces and geometric constraints, or system size effects. Crucially, computational modeling of the experimental data indicated that the apical surface of the epithelium must be softer than the basal surface, a result which we confirmed experimentally. Further, we used the combination of experimental data and comprehensive computational model to estimate the elastic modulus of the apical surface and set a lower bound on the elastic modulus of the basal surface. More generally, our investigations revealed important general features that we believe should be more widely addressed when quantitatively modeling tissue mechanics in any system. Specifically, different compartments of the same cell can have very different mechanical properties; when they do, they can contribute differently to different mechanical stimuli and cannot be merely averaged together. Additionally, tissue geometry can play a substantial role in mechanical response, and cannot be neglected.
Direct conversion of somatic cells into ‘insulin-producing-cells’ by user-defined multiplex-epigenetic-engineering vector (MEEV-β)
Authors: Naqvi Raza Ali , Naqvi Afsar R , Priyadarshini Medha
Published: August 2022

We demonstrate here a single-step and user-friendly approach to generate insulin producing cells by gRNA driven specific-activation of PDX1, NKX6.1, MAFA, Insulin and Glut2 genes in somatic cells via multiplex-epigenetic-engineering-vector (MEEV-β) containing dCas9.P300core developed by us. Sorted Glut2+ cells could secrete insulin in response to glucose challenge and showed expression of β-cell specific transcription factors: NKX2.2, and aforementioned genes. Expression of Cav1.3, GSK3β,, KJNC11, and SLC30A8 genes substantiated the functional insulin secreting machinery genes in these Glut2+ cells. Also, absence of ARX and GCG expression in these cells highlighted the specificity of the conversion.
mtFociCounter for automated single-cell mitochondrial nucleoid quantification and reproducible foci analysis
Authors: Rey Timo , Tábara Luis Carlos , Prudent Julien , Minczuk Michal
Published: August 2022

Mitochondrial DNA (mtDNA) encodes the core subunits for OXPHOS and is essential in eukaryotes. mtDNA is packed into distinct foci (nucleoids) inside mitochondria, and the number of mtDNA differs between cell-types, and is affected in several human diseases. Today, common protocols estimate per-cell mtDNA-molecule numbers by sequencing or qPCR from bulk samples. However, this does not allow insight into cell-to-cell heterogeneity and can mask phenotypical sub-populations. Here, we presentmtFociCounter, a single-cell image-analysis tool for reproducible quantification of nucleoids and other foci.mtFociCounteris a light-weight, open-source freeware and overcomes current limitations to reproducible single-cell analysis of mitochondrial foci. We demonstrate its use by analysing 2165 single fibroblasts, and observe a large cell-to-cell heterogeneity in nucleoid numbers. In addition,mtFociCounterquantifies mitochondrial content and our results show good correlation (R=0.90) between nucleoid number and mitochondrial area, and we find nucleoid density is less variable than nucleoid numbers in wild-type cells. Finally, we demonstratemtFociCounterreadily detects differences in foci numbers upon sample-treatment, and applies to superresolution microscopy. Together, we presentmtFociCounteras a solution to reproducibly quantify cellular foci in single cells and our results highlight the importance of accounting for cell-to-cell variance and mitochondrial context in mitochondrial nucleoid analysis.
Viruses Diversity and Interactions with Hosts in Deep-Sea Hydrothermal Vents
Authors: Cheng Ruolin , Li Xiaofeng , Jiang Lijing , Gong Linfeng , Geslin Claire , Shao Zongze
Published: August 2022

Background

Deep-sea harbor enormous viruses, yet their diversity and interactions with hosts in hydrothermal ecosystem are largely unknown. Here, we analyzed the viral composition, distribution, host preference and metabolic potential in different inhabits of global hydrothermal vents.

Results

From 34 samples of eight vent sites, a total of 4,662 viral populations were recovered from the metagenome assemblies, encompassing diverse phylogenetic groups and defining many novel lineages. Apart for the abundant unclassified viruses, tailed phages are most predominant across the global hydrothermal vents, while single-stranded DNA viruses including Microviridae and small eukaryotic viruses also constitute a significant part of the viromes. These viral populations were grouped into 1,138 genus-level clusters by protein-sharing network analysis. More than half were exclusively of hydrothermal origin, reflecting the remarkable novelty of hydrothermal viruses. Among the typical niches, vent plumes own the largest number of viral clusters compared to diffuse flows and sediments. Moreover, merely 11% of the viral populations can be linked to specific hosts, which are the key microbial taxa of hydrothermal habitats, such as Gammaproteobacteria and Campylobacteraeota. Intriguingly, vent viromes shared some metabolic features in common that they encode auxiliary genes extensively involved in metabolisms of carbohydrate, amino acid, cofactors and vitamins. Specifically in plume viruses, various auxiliary genes related with the methane, nitrogen and sulfur metabolisms were observed, indicating their contribution to host’s energy conservation. Moreover, the prevalence of sulfur relay pathway genes notified the significant role of vent virus in stabilizing tRNA structure, which promotes host’s adaptation to the steep environmental gradients.

Conclusions

The deep-sea hydrothermal systems reserve an untapped viral diversity with novelty. They may affect both vent prokaryotic and eukaryotic communities, and modulate host metabolisms related to vent adaptability. More explorations are needed to depict global vent virus diversity and their role in the unique ecosystem.
Conserved and divergent peptide substrate binding properties of bacterial Hsp70
Authors: Sun Yiyue , Xu Hongke , Li Jiong , Zhu Hanmo , Ma Hongwei , Ma Yiming , Yang Jiao
Published: August 2022

The 70-kDa heat shock protein (Hsp70) interacts with the polypeptide segments of abundant native proteins to fulfill various cellular activities in both stress and normal conditions. However, a non-native linear polypeptide NR (NRLLLTG) is widely used for the study of Hsp70 substrate binding properties, which is too simple to reflect the complex status of Hsp70 substrates in living organisms. To further broaden our knowledge in this area, we screened 2645 polypeptides derived from 78 biologically relevant proteins and identified eight native peptide substrates (named VP1∼VP8) for bacterial Hsp70 DnaK. Consistent with previous findings, the amino acid distribution in VP1∼VP8 were enriched in aliphatic and basic residues, and most of their residues were buried in folded proteins as well. Besides, the substrate binding properties for seven polypeptides were largely the same as observed in NR, suggesting their conserved binding mode to DnaK. However, VP5, which contains more percentage of positively charged residues, demonstrates divergent substrate binding properties during in-vitro biochemical studies. Moreover, VP5 efficiently inhibits the refolding activity of DnaK and bacterial viability, implying its potential to be a good lead peptide for antibacterial drug development.
The ChlorON Series: Turn-on Fluorescent Protein Sensors for Imaging Labile Chloride in Living Cells
Authors: Tutol Jasmine N. , Ong Whitney S. Y. , Phelps Shelby M. , Peng Weicheng , Goenawan Helen , Dodani Sheel C.
Published: August 2022

Beyond its role as the queen electrolyte, chloride can also serve as an allosteric regulator or even a signaling ion. To illuminate this essential anion across such a spectrum of biological processes, researchers have relied on fluorescence imaging with genetically encoded sensors. In large part, these have been derived from the green fluorescent protein found in the jellyfishAequorea victoria. However, a standalone sensor with a turn-on intensiometric response at physiological pH has yet to be reported. Here, we address this technology gap by building on our discovery of mNeonGreen (mNG) derived from lanYFP found in the cephalochordateBranchiostoma lanceolatum. Targeted engineering of two non-coordinating residues in the chloride binding pocket of mNG unlocks the ChlorON series.In vitrospectroscopy reveals that the binding of chloride tunes the chromophore environment to give rise to the turn-on response. We further showcase how this unique sensing mechanism can be exploited for directly imaging labile chloride in living cells with spatial and temporal resolution, accelerating the path forward for fundamental and translational aspects of chloride biology.

Abstract Figure
Degradation pathways for organic matter of terrestrial origin are widespread and expressed in Arctic Ocean microbiomes
Authors: Grevesse Thomas , Guéguen Céline , Onana Vera E. , Walsh David A.
Published: August 2022

Background

The Arctic Ocean receives massive freshwater input and a correspondingly large amount of humic-rich organic matter of terrestrial origin. Global warming, permafrost melt, and a changing hydrological cycle will contribute to an intensification of terrestrial organic matter release to the Arctic Ocean. Although considered recalcitrant to degradation due to complex aromatic structures, humic substances can serve as substrate for microbial growth in terrestrial environments. However, the capacity of marine microbiomes to process aromatic-rich humic substances, and how this processing may contribute to carbon and nutrient cycling in a changing Arctic Ocean, is relatively unexplored. Here, we used a combination of metagenomics and metatranscriptomics to assess the prevalence and diversity of metabolic pathways and bacterial taxa involved in aromatic compound degradation in the salinity-stratified summer waters of the Canada Basin in the western Arctic Ocean.

Results

Community-scale meta-omics profiling revealed that 22 complete pathways for processing aromatic compounds were present and expressed in the Canada Basin, including those for aromatic ring fission and upstream funnelling pathways to access diverse aromatic compounds of terrestrial origin. A phylogenetically diverse set of functional marker genes and transcripts were associated with fluorescent dissolved organic matter, a component of which is of terrestrial origin. Pathways were common throughout global ocean microbiomes, but were more abundant in the Canada Basin. Genome-resolved analyses identified 12 clades ofAlphaproteobacteria, includingRhodospirillales, as central contributors to aromatic compound processing. These genomes were mostly restricted in their biogeographical distribution to the Arctic Ocean, and were enriched in aromatic compound processing genes compared to their closest relatives from other oceans.

Conclusion

Overall, the detection of a phylogenetically diverse set of genes and transcripts implicated in aromatic compound processing supports the view that Arctic Ocean microbiomes have the capacity to metabolize humic substances of terrestrial origin. In addition, the demonstration that bacterial genomes replete with aromatic compound degradation genes exhibit a limited distribution outside of the Arctic Ocean suggests that processing humic substances is an adaptive trait of the Arctic Ocean microbiome. Future increases in terrestrial organic matter input to the Arctic Ocean may increase the prominence of aromatic compound processing bacteria and their contribution to Arctic carbon and nutrient cycles.
Enhanced production of recombinant HALT-1 pore-forming toxin using two-step chromatographic procedure
Authors: Yap Wei Yuen , Loo Lok Wenn , Sha Hong Xi , Hwang Jung Shan
Published: August 2022

Hydra actinoporin-like toxin-1 (HALT-1) has been isolated from Hydra magnipapillata and is highly cytolytic against various human cells including erythrocyte. Previously, recombinant HALT-1 (rHALT-1) was expressed in Escherichia coli and purified by the nickel affinity chromatography. In this study, we improved the purification of rHALT-1 by two-step purifications. Bacterial cell lysate containing rHALT-1 was subjected to the sulphopropyl (SP) cation exchange chromatography with different buffers, pHs, and NaCl concentrations. The results indicated that both phosphate and acetate buffers facilitated the strong binding of rHALT-1 to SP resins, and the buffers containing 150 mM and 200 mM NaCl, respectively, removed protein impurities but retain most rHALT-1 in the column. When combining the nickel affinity chromatography and the SP cation exchange chromatography, the purity of rHALT-1 was highly enhanced. In subsequent cytotoxicity assays, 50% of cells could be lysed at ∼18 and ∼22 μg/ml of rHALT-1 purified with phosphate and acetate buffers, respectively.

HALT-1 is a soluble α-pore-forming toxin of 18.38 kDa.

rHALT-1 was purified by nickel affinity chromatography followed by SP cation exchange chromatography.

The cytotoxicity of purified rHALT-1 using 2-step purifications via either phosphate or acetate buffer was comparable to those previously reported.

Graphical abstract

Specifications table
lncRNAHOTAIRM1Coordinates with RNA Processing Factors in DNA Damage Repair
Authors: Chuang Tzu-Wei , Wu Pei-Yu , Chang Yao-Ming , Tarn Woan-Yuh
Published: August 2022

The eukaryotic RNA processing factor Y14 participates in double-strand break (DSB) repair via its RNA-dependent interaction with the non-homologous end-joining (NHEJ) complex. We identified the long non-coding RNAHOTAIRM1as a candidate that mediates this interaction.HOTAIRM1localized to DNA damage sites induced by ionizing radiation. Depletion ofHOTAIRM1delayed the recruitment of DNA damage response and repair factors to DNA lesions and reduced DNA repair efficiency. Identification of theHOTAIRM1interactome revealed a large set of RNA processing factors including mRNA surveillance factors. The surveillance factors Upf1 and SMG6 localized to DNA damage sites in aHOTAIRM1-dependent manner. Depletion of Upf1 or SMG6 increased the level of DSB-induced non-coding transcripts at damaged sites, indicating a pivotal role for Upf1/SMG6-mediated RNA degradation in DNA repair. We conclude thatHOTAIRM1serves as an assembly scaffold for both DNA repair and RNA processing factors that act in concert to repair DSBs.
Structural basis and dynamics of Chikungunya alphavirus RNA capping by the nsP1 capping pores
Authors: Jones Rhian , Homs Michael , Rabat Nadia , Zamarreño Noelia , Arranz Rocio , Reguera Juan
Published: August 2022

Alphaviruses are emerging positive stranded RNA virus which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The non-structural protein 1 (nsP1) is responsible for RNA capping and the gating of replication organelles by assembling into monotopic membrane-associated dodecameric pores (Jones R. et al. Nature 2021). The capping path is unique for Alphavirus; beginning with the N7 methylation of a GTP molecule, followed by the covalent linkage of a m7GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA (Ahola T. et al. PNAS 1995). Here we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and SAM, how it reaches a metastable post-methylation state with SAH and m7GTP in the active site, the subsequent covalent transfer of m7GMP to nsP1 and post-reaction conformational changes triggering the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of intermediates of the reaction. Our data identify the molecular determinants allowing each pathway transition, provide explanation for the need for the SAM methyl donor all along the pathway and new clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together our results set new ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals.

Significance statement

Here we present biochemical and structural characterization of the capping pathway carried out by the Chikungunya virus nsP1 capping pores. We provide five Cryo-EM structures representative of the different steps of the reaction. These structures reveal the molecular determinants and dynamics associated with the alphavirus capping process. In addition, we biochemically show the RNA capping specificity and the reversibility of the reaction which allow nsP1 to cap and decap RNAs and to release intermediates of the reaction. These data provide a new biochemical clues on the enzymatic activity of nsP1 capping pores and a new structural landscape that will be instrumental for the design of effective antivirals targeting the viral RNA capping for blocking the infection.
Atomistic simulations of the E. coli ribosome provide selection criteria for translationally active substrates
Authors: Watson Zoe L. , Knudson Isaac , Ward Fred R. , Miller Scott J. , Cate Jamie H. D. , Schepartz Alanna , Abramyan Ara M.
Published: August 2022

As genetic code expansion advances beyond L-α-amino acids to backbone modifications and new polymerization chemistries, the field faces an increasingly broad challenge to discover what the ribosome can accommodate. Although the E. coli ribosome tolerates non-L-α-amino acids in vitro, few structural insights are available, and the boundary conditions for efficient bond formation are unknown. We describe a 2.1 Å cryo-EM structure of the E. coli ribosome containing well-resolved α-amino acid monomers coupled with a computational approach for which energy surface minima produced by metadynamics trend in agreement with established incorporation efficiencies. Reactive monomers across diverse structural classes favor a conformational space characterized by an A-site nucleophile to P-site carbonyl distance of < 4 Å and a Bürgi-Dunitz angle of 90-110°. Monomers whose free energy minima fall outside these regions do not react. Application of this model should accelerate the in vivo and in vitro ribosomal synthesis and application of sequence-defined, non-peptide heterooligomers.
Propagation of viral genomes by replicating ammonia-oxidising archaea during soil nitrification
Authors: Lee Sungeun , Sieradzki Ella T. , Nicol Graeme W. , Hazard Christina
Published: August 2022

Ammonia-oxidising archaea (AOA) are a ubiquitous component of microbial communities and dominate the first stage of nitrification in some soils. While we are beginning to understand soil virus dynamics, we have no knowledge of the composition or activity of those infecting nitrifiers or their potential to influence processes. This study aimed to characterise viruses having infected autotrophic AOA in two nitrifying soils of contrasting pH by following transfer of assimilated CO2-derived13C from host to virus via DNA stable-isotope probing and metagenomic analysis. Incorporation of13C into low GC mol% AOA and virus genomes increased DNA buoyant density in CsCl gradients but resulted in co-migration with dominant non-enriched high GC mol% genomes, reducing sequencing depth and contig assembly. We therefore developed a hybrid approach where AOA and virus genomes were assembled from low buoyant density DNA with subsequent mapping of13C isotopically enriched high buoyant density DNA reads to identify activity of AOA. Metagenome-assembled genomes were different between the two soils and represented a broad diversity of active populations. Sixty-four AOA-infecting viral operational taxonomic units (vOTUs) were identified with no clear relatedness to previously characterised prokaryote viruses. These vOTUs were also distinct between soils, with 42% enriched in13C derived from hosts. The majority were predicted as capable of lysogeny and auxiliary metabolic genes included an AOA-specific multicopper oxidase suggesting infection may augment copper uptake essential for central metabolic functioning. These findings indicate virus infection of AOA may be a frequent process during nitrification with potential to influence host physiology and activity.
Phylogenetic Association and Genetic Factors in Cold Stress Tolerance in Campylobacter jejuni
Authors: Hur Jeong In , Kim Jinshil , Ryu Sangryeol , Jeon Byeonghwa
Published: August 2022

Campylobacter jejuni is a major foodborne pathogen transmitted to humans primarily via contaminated poultry meat. Since poultry meat is generally processed, distributed, and stored in the cold chain, the survival of C. jejuni at refrigeration temperatures crucially affects human exposure to C. jejuni. Here, we investigated genetic factors associated with cold stress tolerance in C. jejuni. Seventy-nine C. jejuni strains isolated from retail raw chicken exhibited different survival levels at 4°C for 21 days. Multilocus sequence typing (MLST) clonal complex (CC)-21 and CC-443 were dominant among cold stress-tolerant strains, whereas CC-45 was common among cold stress-sensitive strains. Genome-wide average nucleotide identity (ANI) analysis identified a phylogenetic cluster associated with cold stress tolerance. Moreover, a pan-genome analysis revealed 58 genes distinctively present in the cold stress-tolerant phylogenetic cluster. Among the 58 genes, cfrA, encoding the ferric enterobactin receptor involved in ion transport and metabolism, was selected for further analysis. Remarkably, the viability of a ΔcfrA mutant at 4°C was significantly decreased, while the levels of total reactive oxygen species and intracellular iron exceeded those in the wild type. Additionally, a knockout mutation of cfrA also significantly decreased the viability of three cold stress-tolerant isolates at 4°C, confirming the role of cfrA in cold stress tolerance. The results of this study demonstrate that unique phylogenetic clusters of C. jejuni associated with cold stress tolerance exist and that cfrA is a genetic factor contributing to cold stress tolerance in C. jejuni.

IMPORTANCE

The tolerance of foodborne pathogens to environmental stresses significantly affects food safety. Several studies have demonstrated that C. jejuni survives extended exposure to low temperatures, but the mechanisms of cold stress tolerance are not fully understood. Here, we demonstrate that C. jejuni strains in certain phylogenetic groups exhibit increased tolerance to cold stress. Notably, cfrA is present in the phylogenetic cluster associated with cold stress tolerance and plays a role in C. jejuni survival at low temperatures by alleviating oxidative stress. This is the first study to discover phylogenetic associations involving cold stress tolerance and identify genetic elements conferring cold stress tolerance in C. jejuni.
The high-resolution in vivo measurement of replication fork velocity and pausing by lag-time analysis
Authors: Huang Dean , Johnson Anna E. , Sim Brandon S. , Lo Teresa , Merrikh Houra , Wiggins Paul A.
Published: August 2022

An important step towards understanding the mechanistic basis of the central dogma is the quantitative characterization of the dynamics of nucleic-acid-bound molecular motors in the context of the living cell, where a crowded cytoplasm as well as competing and potentially antagonistic processes may significantly affect their rapidity and reliability. To capture these dynamics, we develop a novel method, lag-time analysis, for measuring in vivo dynamics. The approach uses exponential growth as the stopwatch to resolve dynamics in an asynchronous culture and therefore circumvents the difficulties and potential artifacts associated with synchronization or fluorescent labeling. Although lag-time analysis has the potential to be widely applicable to the quantitative analysis of in vivo dynamics, we focus on an important application: characterizing replication dynamics. To benchmark the approach, we analyze replication dynamics in three different species and a collection of mutants. We provide the first quantitative locus-specific measurements of fork velocity, in units of kb per second, as well as replisome-pause durations, some with the precision of seconds. The measured fork velocity is observed to be both locus and time dependent, even in wild-type cells. In addition to quantitatively characterizing known phenomena, we detect brief, locus-specific pauses at rDNA in wild-type cells for the first time. We also observe temporal fork velocity oscillations in three highly-divergent bacterial species. Lag-time analysis not only has great potential to offer new insights into replication, as demonstrated in the paper, but also has potential to provide quantitative insights into other important processes.