bluuebunny/crossref_metadata_2025 huggingface dataset:
Human NMDAR autoantibodies disrupt excitatory-inhibitory balance leading to hippocampal network hypersynchrony
Authors: Ceanga Mihai , Rahmati Vahid , Haselmann Holger , Schmidl Lars , Hunter Daniel , Kreye Jakob , Prüss Harald , Groc Laurent , Hallermann Stefan , Dalmau Josep , Ori Alessandro , Heckmann Manfred , Geis Christian
Published: March 2022

Specific autoantibodies against the NMDA-receptor (NMDAR) GluN1 subunit cause severe and debilitating NMDAR-encephalitis. Autoantibodies induce prototypic disease symptoms resembling schizophrenia, including psychosis and cognitive dysfunction. Using a mouse passive transfer model applying human monoclonal anti-GluN1-autoantibodies, we observed CA1 pyramidal neuron hypoexcitability, reduced AMPA-receptor (AMPAR) signaling, and faster synaptic inhibition resulting in disrupted excitatory-inhibitory balance. Functional alterations were supported by widespread remodeling of the hippocampal proteome, including changes in glutamatergic and GABAergic neurotransmission. At the network level, anti-GluN1-autoantibodies amplified gamma oscillations and disrupted theta-gamma coupling. A data-informed network model revealed that lower AMPAR strength and faster GABAA-receptor current kinetics chiefly account for these abnormal oscillations. As predicted by our model and evidenced experimentally, positive allosteric modulation of AMPARs alleviated aberrant gamma activity and thus reinforced the causative effects of the excitatory-inhibitory imbalance. Collectively, NMDAR-hypofunction-induced aberrant synaptic, cellular, and network dynamics provide new mechanistic insights into disease symptoms in NMDAR-encephalitis and schizophrenia.
Physiological alterations in microglial morphology associate with the sleep-wake cycle in a brain region-specific manner
Authors: Steffens Sarah Katharina , Stenberg Tarja Helena , Wigren Henna-Kaisa
Published: March 2022

Long-term total sleep deprivation induces changes in cortical - and hippocampal microglial morphology that closely resemble the microglial response to the gram-negative bacterial cell wall component lipopolysaccharide (LPS). A recent study found evidence that microglia could modify vigilance-states/sleep, but only few studies investigated microglial throughout the diurnal behavioral inactivity/activity pattern or the naturally occurring sleep-wake cycle, and those who have, only concentrated on the cortical or hippocampal microglia. As microglia demonstrate regional heterogeneity, we compared microglial diurnal morphological alterations in the somatosensory cortex (SC) and dorsal hippocampus (HC) to the basal forebrain (BF), which is a subcortical brain area involved in the regulation of vigilance states.

We collected mouse brain samples every 3h throughout the 24h light-dark-cycle and applied a 3D reconstruction method for the acquired confocal microscopy images for each brain area separately. While microglial regional heterogeneity was evident, stimulation of microglia with LPS caused comparable microglial responses in all brain areas. When comparing microglial features between the 12h light- and dark periods, regional heterogeneity re-appeared. As most of the morphological alterations occurred during the light period-the habitual sleeping period of the mice, we performed polysomnography to study the possible interaction of microglial morphology and sleep. We found that cortical-, but not HC- or BF microglial territory and volume negatively correlated with sleep slow wave activity (SWA), an electroencephalic feature of non-REM sleep (NREMS). Since microglia are sensitive to neuronal activity, we propose that the regional differences reflect vigilance-state specific neuronal activity patterns.

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Main Points

Microglia show morphological differences between the somatosensory cortex (SC), hippocampus (HC) and basal forebrain (BF) under physiological conditions.

Cortical-, but not HC- or BF microglial cell volume negatively correlates with non-REM sleep slow wave activity.
Blinding efficacy and adverse events following repeated transcranial alternating current, direct current, and random noise stimulation
Authors: Sheffield James G. , Ramerpresad Sumientra , Brem Anna-Katharine , Mansfield Karen , Orhan Umut , Dillard Michael , McKanna James , Plessow Franziska , Thompson Todd , Santarnecchi Emiliano , Pascual-Leone Alvaro , Pavel Misha , Mathan Santosh , Kadosh Roi Cohen
Published: March 2022

As transcranial electrical stimulation (tES) protocols advance, assumptions underlying the technique need to be retested to ensure they still hold. Whilst the safety of stimulation has been demonstrated mainly for a small number of sessions, and small sample size, adverse events (AEs) following multiple sessions remain largely untested. Similarly, whilst blinding procedures are typically assumed to be effective, the effect of multiple stimulation sessions on the efficacy of blinding procedures also remains under question. This is especially relevant in multisite projects where small unintentional variations in protocol could lead to inter-site difference. We report AE and blinding data from 1,019 participants who received up to 11 semi-consecutive sessions of active or sham transcranial alternating current stimulation (tACS), direct current stimulation (tDCS), and random noise stimulation (tRNS), at 4 sites in the UK and US. We found that AEs were often best predicted by factors other than tES, such as testing site or session number. Results from the blinding analysis suggested that blinding was less effective for tDCS and tACS than tRNS. The occurrence of AEs did not appear to be linked to tES despite the use of smaller electrodes or repeated delivery. However, blinding efficacy was impacted in tES conditions with higher cutaneous sensation, highlighting a need for alternative stimulation blinding protocols. This may be increasingly necessary in studies wishing to deliver stimulation with higher intensities.
A mass spectrometry-based method for the determination ofin vivobiodistribution of tumor targeting small molecule-metal conjugates
Authors: Gilardoni Ettore , Zana Aureliano , Galbiati Andrea , Sturm Theo , Millul Jacopo , Cazzamalli Samuele , Neri Dario , Stucchi Riccardo
Published: March 2022

Nuclear medicine plays a key role in modern diagnosis and cancer therapy. The development of tumor targeting radionuclide conjugates (also named Small Molecule-Radio Conjugates - SMRCs) represents a significant improvement over the clinical use of metabolic radiotracers (e.g., [18F]-Fluorodeoxyglucose) for imaging and over the application of biocidal external beam radiations for therapy. During the discovery of SMRCs, molecular candidates must be carefully evaluated typically by performing biodistribution assays in preclinical tumor models. Quantification methodologies based on radioactive counts are typically demanding due to safety concerns, availability of radioactive material, and infrastructures. In this article, we report the development of a mass spectrometry (MS)-based method for the detection and quantification of small molecule-metal conjugates (SMMCs) as cold surrogates of SMRCs. We applied this methodology for the evaluation of the biodistribution of a particular class of tumor-targeting drug candidates based onnatLu,natGa,natF and directed against Fibroblast Activation Protein (FAP). The reliability of the LC-MS analysis was validated by direct comparison of MSbased and radioactivity-based biodistribution data. Results show that MS biodistribution of stable isotope metal conjugates is an orthogonal tool for the preclinical characterization of different classes of radiopharmaceuticals.
Flipped elevational pattern of pollination mode in tropical vs. temperate Americas
Authors: Dellinger Agnes S. , Hamilton Ashley M. , Wessinger Carolyn A. , Smith Stacey
Published: March 2022

Aim

Abiotic factors, such as temperature and precipitation, vary markedly along elevational gradients, and can in turn, shape key biotic interactions, such as herbivory and pollination. Despite the well-known effects of climatic conditions on pollinator activity and efficiency, we know little about the role of climate in pollinator shifts in animal-pollinated plants at broad geographic scales. Here we investigate patterns of altitudinal turnover in pollination mode across the Americas, with a focus on the most common pollinators (bees and hummingbirds). Specifically, we test Cruden’s classic hypothesis that plants are likely to shift to bird pollination at high elevations because endothermic pollinators are more reliable in cold and rainy conditions.

Location

Americas

Time period

Current

Major taxa studied

2232 plant taxa from 26 clades

Methods

We collated information on pollination mode (1262 insect-pollinated, 970 vertebrate-pollinated) for the study taxa from the literature, and used GBIF occurrence data to estimate median distributions and bioclimatic attributes of each species. We used (phylogenetic) GLMMs to test for associations between pollination mode and ecogeographic variables.

Results

To our surprise, we found flipped elevational patterns of insect- and vertebrate-pollination strategies across latitudes, with vertebrate pollination dominating at high elevations in the tropics, but not in temperate zones. We term this pattern the ‘Tropical flip’. We recovered a strong association of vertebrate-pollinated plants with moist, forested habitats across latitudes, while insect-pollinated plants were often found in cool and dry or warm and moist conditions.

Main conclusions

Altitudinal gradients in temperature may not serve as a universal explanation for shifts among endothermic insect and ectothermic vertebrate pollination. Instead, strong abiotic niche differentiation among insect- and vertebrate-pollinated plants, along with competition for pollination niche space, has likely shaped the ‘tropical flip’.
Systematic Characterization of Double Emulsion Droplets for Biological Applications
Authors: Calhoun Suzanne G. K. , Brower Kara K , Suja Vineeth Chandran , Kim Gaeun , Wang Ningning , McCully Alexandra L. , Kusumaatmaja Halim , Fuller Gerald G. , Fordyce Polly M.
Published: March 2022

Double emulsion droplets (DEs) are water/oil/water droplets that can be sorted via Fluorescence-Activated Cell Sorting (FACS), allowing for new opportunities in high-throughput cellular analysis, enzymatic screening, and synthetic biology. These applications require stable, uniform droplets with predictable microreactor volumes. However, predicting DE droplet size, shell thickness, and stability as a function of flow rate has remained challenging for monodisperse single core droplets and those containing biologically-relevant buffers, which influence bulk and interfacial properties. As a result, developing novel DE-based bioassays has typically required extensive initial optimization of flow rates to find conditions that produce stable droplets of the desired size and shell thickness. To address this challenge, we conducted systematic size parameterization quantifying how differences in flow rates and buffer properties (viscosity and interfacial tension at water/oil interfaces) alter droplet size and stability, across 6 inner aqueous buffers used across applications such as cellular lysis, microbial growth, and drug delivery, quantifying the size and shell thickness of >22,000 droplets overall. We restricted our study to stable single core droplets generated in a 2-step dripping-dripping formation regime in a straightforward PDMS device. Using data from 138 unique conditions (flow rates and buffer composition), we also demonstrated that a recent physically-derived size law of Wang et al1 can accurately predict double emulsion shell thickness for >95% of observations. Finally, we validated the utility of this size law by using it to accurately predict droplet sizes for a novel bioassay that requires encapsulating growth media for bacteria in droplets. This work has the potential to enable new screening-based biological applications by simplifying novel DE bioassay development.
Crippled Coronavirus: 5’-PolyU targeted Oligo prevents development of infectious Virions
Authors: Ullah Hemayet , Averick Saadyah , Tang Qiyi
Published: March 2022

Current RNA viral pandemic of COVID-19 has been worsened by rapidly spreading viral variants. To inhibit mutation-based development of new escape variants, elements that are indispensable for the virus may be targeted. The 5’-polyU tract of the antigenome offers one such target. Host cells do not harbor 5’-polyU tracts on any of their transcripts, making the tract an attractive virus-specific target. We hypothesize that inhibiting the 5’-polyU by complementary oligonucleotide can limit the use of the tract as template for virus to generate 3’ polyA tails of RNA viruses. Here, we used a frameshift-inducing DNA oligonucleotide with 3’ polyAs to target the 5’-polyU tract of mouse coronavirus (MHV-A59). Results from assays for double stranded RNA (dsRNA) synthesis, infectivity of released virions, and syncytium formation indicate that the oligonucleotide treatment prevented generation of infectious virions. Our results show a unique mode of action of the designed 3’-polyA oligonucleotide against mouse coronavirus which leaves host cells unaffected. This strategy can be adopted for the development of novel classes of oligonucleotide-based drugs that inhibit the production of infectious RNA viruses, including the coronaviruses. Since the 5’-polyU tract is conserved and is essential for variants of coronaviruses, this strategy can potentially address coronavirus variant emergence as well.
Meta-analysis and experimental re-evaluation of the Boyle van ‘t Hoff relation with osmoregulation modelled by linear elastic principles and ion-osmolyte leakage
Authors: Olver Dominic J , Azam Iqra , Benson James D
Published: March 2022

In this study we challenge the paradigm of using the Boyle van ’t Hoff (BvH) relation to relate cell size as a linear function of inverse extracellular osmotic pressure for short time periods (~5 to 30 mins). We present alternative models that account for mechanical resistance (turgor model) and ion-osmolyte leakage (leak model), which is not accounted for by the BvH relation. To test the BvH relation and the alternative models, we conducted a meta-analysis of published BvH datasets, as well as new experiments using a HepG2 cell line. Our meta-analysis showed that the BvH relation may be assumed of the hypertonic region but cannot be assumed a priori over the hyper- and hypotonic region. Both alternative models perform better than the BvH relation but are nearly indistinguishable when plotted. The return to isotonic conditions plot indicated neither alternative model accurate predicts return volumes for HepG2 cells. However, a combined turgor-leak model accurately predicts both the BvH plot and the return to isotonic conditions plot. Moreover, this turgor-leak model provides a facile method to estimate the membrane-cortex Young’s modulus and the cell membrane permeability to intracellular ions/osmolytes during periods of osmotic challenge, and predicts a novel passive method of volume regulation without the need for ion pumps.
Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors
Authors: Moon Patrick , Boike Lydia , Dovala Dustin , Henning Nathaniel J. , Knapp Mark , Spradlin Jessica N. , Ward Carl C. , Wolleb Helene , Zammit Charlotte M. , Fuller Daniel , Blake Gabrielle , Murphy Jason P. , Wang Feng , Lu Yipin , Moquin Stephanie A. , Tandeske Laura , Hesse Matthew J. , McKenna Jeffrey M. , Tallarico John A. , Schirle Markus , Toste F. Dean , Nomura Daniel K.
Published: March 2022

While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly “druggable” or relatively easy-to-drug target is the coronavirus Main Protease (3CLpro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.
ALS-linked KIF5A ΔExon27 mutant causes neuronal toxicity through gain of function
Authors: Pant Devesh C. , Parameswaran Janani , Rao Lu , Shi Liang , Chilukuri Ganesh , McEachin Zachary T. , Glass Jonathan , Bassell Gary J. , Gennerich Arne , Jiang Jie
Published: March 2022

Mutations in the human kinesin family member 5A (KIF5A) gene were recently identified as a genetic cause of amyotrophic lateral sclerosis (ALS). Several KIF5A ALS variants cause exon 27 skipping and produce motor proteins with an altered C-terminal tail (referred to as ΔExon27). However, the underlying pathogenic mechanism is still unknown. In this study, we performed a comprehensive analysis of ΔExon27 at the single-molecule, cellular, and organism levels. Our results show that ΔExon27 is prone to form cytoplasmic aggregates and is neurotoxic. The mutation relieves motor autoinhibition and increases motor self-association, leading to drastically enhanced processivity on microtubules. Finally, ectopic expression of ΔExon27 in Drosophila melanogaster causes wing defects, motor impairment, paralysis and premature death. Our results suggest gain of function as an underlying disease mechanism in KIF5A-associated ALS.
Characterization of Hydrophobic Interactions of SARS-CoV-2 and MERS-CoV Spike Protein Fusion Peptides Using Single Molecule Force Measurements
Authors: Qiu Cindy , Whittaker Gary R. , Gellman Samuel H. , Daniel Susan , Abbott Nicholas L.
Published: March 2022

We address the challenge of understanding how hydrophobic interactions are encoded by fusion peptide sequences within coronavirus (CoV) spike proteins. Within the fusion peptides of SARS-CoV-2 and MERS-CoV, a largely conserved peptide sequence called FP1 (SFIEDLLFNK and SAIEDLLFDK in SARS-2 and MERS, respectively) has been proposed to play a key role in encoding hydrophobic interactions that drive viral-host cell membrane fusion. While a non-polar triad (LLF) is common to both FP1 sequences, and thought to dominate the encoding of hydrophobic interactions, FP1 from SARS and MERS differ in two residues (Phe 2 versus Ala 2 and Asn 9 versus Asp 9, respectively). Here we explore if single molecule force measurements can quantify hydrophobic interactions encoded by FP1 sequences, and then ask if sequence variations between FP1 from SARS and MERS lead to significant differences in hydrophobic interactions. We find that both SARS-2 and MERS wild-type FP1 generate measurable hydrophobic interactions at the single molecule level, but that SARS-2 FP1 encodes a substantially stronger hydrophobic interaction than its MERS counterpart (1.91 ± 0.03 nN versus 0.68 ± 0.03 nN, respectively). By performing force measurements with FP1 sequences with single amino acid substitutions, we determine that a single residue mutation (Phe 2 versus Ala 2) causes the almost threefold difference in the hydrophobic interaction strength generated by the FP1 of SARS-2 versus MERS, despite the presence of LLF in both sequences. Infrared spectroscopy and circular dichroism measurements support the proposal that the outsized influence of Phe 2 versus Ala 2 on the hydrophobic interaction arises from variation in the secondary structure adopted by FP1. Overall, these insights reveal how single residue diversity in viral fusion peptides, including FP1 of SARS-CoV-2 and MERS-CoV, can lead to substantial changes in intermolecular interactions proposed to play a key role in viral fusion, and hint at strategies for regulating hydrophobic interactions of peptides in a range of contexts.

SIGNIFICANCE

Fusion of coronaviruses (CoVs) and host cells is mediated by the insertion of the fusion peptide (FP) of the viral spike protein into the host cell membrane. Hydrophobic interactions between FPs with their host cell membranes regulate the viral membrane fusion process and are key to determining infection ability. However, it is not fully understood how the amino acid sequences in FPs mediate hydrophobic interactions. We use single-molecule force measurements to characterize hydrophobic interactions of FPs from SARS-CoV-2 and MERS-CoV. Our findings provide insight into the mechanisms by which the amino acid composition of FPs encodes hydrophobic interactions and their implications for fusion activity critical to the spread of infection.
Behavioral differences among domestic cats in the response to cat-attracting plants and their volatile compounds reveal a potential distinct mechanism of action for actinidine
Authors: Bol Sebastiaan , Scaffidi Adrian , Bunnik Evelien M. , Flematti Gavin R.
Published: March 2022

It has been known for centuries that cats respond euphorically to Nepeta cataria (catnip). Recently, we have shown that Lonicera tatarica (Tatarian honeysuckle), Actinidia polygama (silver vine) and Valeriana officinalis (valerian) can also elicit this “catnip response”. The aim of this study was to learn if the behavior seen in response to these plants is similar to the response to catnip. Furthermore, we studied if these responses are fixed or if there are differences between cats. While nepetalactone was identified decades ago as the molecule responsible for the “catnip response”, we know that this volatile is found almost exclusively in catnip. Therefore, we also aimed to identify other compounds in these alternative plants that can elicit the blissful behavior in cats. Bioassays with 6 cats were performed in a stress-free environment, where 6 plants and 13 single compounds were each tested for at least 100 and 17 hours, respectively. All responses were video recorded and BORIS software was used to analyze the cats’ behavior. Both response duration and behavior differed significantly between the cats. While individual cats had preferences for particular plants, the behavior of individual cats was consistent among all plants. About half a dozen lactones similar in structure to nepetalactone were able to elicit the “catnip response”, as were the structurally more distinct molecules actinidine and dihydroactinidiolide. Most cats did not respond to actinidine, whereas those who did, responded longer to this volatile than any of the other secondary plant metabolites, and different behavior was observed. Interestingly, dihydroactinidiolide was also found in excretions and secretions of the red fox, making this the first report of a compound produced by a mammal, that can elicit the “catnip response”. A range of different cat-attracting compounds was detected by chemical analysis of plant materials but differences in cat behavior could not be directly related to differences in chemical composition of the plants. Together with among other results of habituation / dishabituation experiments, this indicates that additional cat-attracting compounds may be present in the plant materials that remain to be discovered. Collectively, these findings suggest that both the personality of the cat and genetic variation in the genes encoding olfactory receptors may play a role in how cats respond to cat-attracting plants. Furthermore, the data suggest a potential distinct mechanism of action for actinidine.
Lack of robust evidence for aWolbachiainfection inAnopheles gambiaefrom Burkina Faso
Authors: Sawadogo Simon P. , Kabore Didier A. , Tibiri Ezechiel B. , Hughes Angela , Gnankine Olivier , Quek Shannon , Diabaté Abdoulaye , Ranson Hilary , Hughes Grant L. , Dabiré Roch K.
Published: March 2022

The endosymbiontWolbachiacan have major effects on the reproductive fitness, and vectorial capacity of host insects and may provide new avenues to control mosquito borne pathogens.Anopheles gambiaes.l is the major vector of malaria in Africa but the use ofWolbachiain this species has been limited by challenges in establishing stable transinfected lines and uncertainty around native infections. High frequencies of infection ofWolbachiahave been previously reported inAn. gambiaecollected from the Valle du Kou region of Burkina Faso in 2011 and 2014. Here we re-evaluated the occurrence ofWolbachiain natural samples, collected from Valle du Kou over a 12-year time span, and in addition, expanded sampling to other sites in Burkina Faso. Our results showed that, in contrast to earlier reports,Wolbachiais present at an extremely low prevalence in natural population ofAn. gambiae. From 5,341 samples analysed only 29 were positive forWolbachiaby nested PCR representing 0.54% of prevalence. No positive samples were found with regular PCR. Phylogenetic analysis of 16S rRNA gene amplicons clustered across supergroup B, with some having similarity to sequences previously found inAnophelesfrom Burkina Faso. However, we cannot discount the possibility that the amplicon positive samples we detected were due to environmental contamination or were false positives. Regardless, the lack of a prominent native infection inAn. gambiaes.l. is encouraging for applications utilisingWolbachiatraninsfected mosquitoes for malaria control.
Cell size asymmetries in the sea star embryo
Authors: Barone Vanessa , Byrne Maria , Lyons Deirdre C.
Published: March 2022

Cell size asymmetries are often linked to cell fate decisions, due to cell volumes and cell fate determinants being unequally partitioned during asymmetric cell divisions. A clear example is found in the sea urchin embryo, where a characteristic and obvious unequal 4th cleavage generates micromeres, which are necessary for mesendoderm cell fate specification. Unlike sea urchin development, sea star development is generally thought to have only equal cleavage. However, subtle cell size asymmetries can be observed in sea star embryos; whether those cell size asymmetries are consistently produced during sea star development and if they are involved in cell fate decisions remains unknown. Using confocal live imaging of early embryos we quantified cell size asymmetries in 16-cell stage embryos of two sea star species, Patiria miniata and Patiriella regularis. Using photoconversion to perform lineage tracing, we find that the position of the smallest cells of P. miniata embryos is biased toward anterior ventral tissues. However, both blastomere dissociation and mechanical removal of one small cell do not prevent dorsoventral (DV) axis formation, suggesting that embryos compensate for the loss of those cells and asymmetric partitioning of maternal determinants is not strictly necessary for DV patterning. Finally, we show that manipulating cell size to introduce artificial cell size asymmetries is not sufficient to direct the positioning of the future DV axis in P. miniata embryos. Our results show that although cell size asymmetries are consistently produced during sea star early cleavage and may be predictive of the DV axis, they are not necessary to instruct DV axis formation.
Predicted binding interface between coronavirus nsp3 and nsp4
Authors: Hensel Zach
Published: March 2022

Double membrane vesicles (DMVs) in coronavirus-infected cells feature pores that span both membranes. DMV pores were observed to have six-fold symmetry and include the nsp3 protein. Co-expression of SARS-CoV nsp3 and nsp4 induces DMV formation, and elements of nsp3 and nsp4 have been identified that are essential for membrane disruption. I describe a predicted luminal binding interface between nsp3 and nsp4 that is membrane-associated, conserved in SARS-CoV-2 during the COVID-19 pandemic and in diverse coronaviruses, and stable in molecular dynamics simulation. Combined with structure predictions for the full-length nsp4 monomer and cryo-EM data, this suggests a DMV pore model in which nsp4 spans both membranes with nsp3 and nsp4 inserted into the same bilayer. This approach may be able to identify additional protein-protein interactions between coronavirus proteins.
Chromosomal integration of blaCTX-M genes in diverse Escherichia coli isolates recovered from river water in Japan
Authors: Gomi Ryota , Yamamoto Masaki , Tanaka Michio , Matsumura Yasufumi
Published: March 2022

Occurrence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBLEC) in environmental waters is of great concern. However, unlike clinical ESBLEC, their genetic characteristics, in particular the genetic contexts of ESBL genes, are not well understood. In this study, we sequenced and analyzed the genomes of CTX-M-producing E. coli isolates recovered from river water to fully characterize the genetic contexts of blaCTX-M genes. Among the 14 isolates with completed genomes, blaCTX-M genes were detected on the chromosome in nine isolates. All but one chromosomal blaCTX-M genes were associated with ISEcp1 and were carried on different transposition units ranging in size from 2,855 bp to 11,093 bp; the exception, blaCTX-M-2, was associated with ISCR1. The remaining five isolates carried blaCTX-M genes on epidemic IncI1 plasmids of different sequence types (STs) (ST3, ST16, ST113, and ST167) (n = 4) or on an IncB/O/K/Z plasmid (n = 1). This study revealed that environmental E. coli carry blaCTX-M genes in diverse genetic contexts. Apparent high prevalence of chromosomal blaCTX-M potentially indicates that some E. coli can stably maintain blaCTX-M genes in environmental waters, though further studies are needed to confirm this.
Mutation Rate Evolution Drives Immune Escape In Mismatch Repair-Deficient Cancer
Authors: Kayhanian Hamzeh , Barmpoutis Panagiotis , Lakatos Eszter , Cross William , Caravagna Giulio , Zapata Luis , Litchfield Kevin , Steele Christopher , Waddingham William , Patel Dominic , Milite Salvatore , Jin Chen , Baker Ann-Marie , Ross Christopher , Alexander Daniel , Khan Khurum , Hochhauser Daniel , Novelli Marco , Werner Benjamin , Guppy Naomi , Linares Josep ,   Genomics England Research Consortium, Ligtenberg Marjolijn J.L. , Nagtegaal Iris D. , Sottoriva Andrea , Graham Trevor , Pillay Nischalan , Rodriguez-Justo Manuel , Shiu Kai-Keen , Jansen Marnix
Published: March 2022

Mutation rate optimisation drives evolution and immune evasion of bacteria and lentiviral strains, including HIV. Whether evolving cancer lineages similarly adapt mutation rates to increase tumour cell fitness is unknown. Here, by mapping the clonal topography of mismatch repair-deficient (MMRd) colorectal cancer, we show that genomic MMRd mutability co-evolves with neoantigen selection to drive intratumour diversification and immune escape. Mechanistically, we find that microsatellite instability modulates subclonal DNA repair by toggling two hypermutable mononucleotide homopolymer runs in the mismatch repair genes MSH6 and MSH3 (C8 and A8, respectively) through stochastic frameshift switching. Spontaneous mutation and reversion at these evolvability switches modulates subclonal mutation rate, mutation bias, and clonal HLA diversity during MMRd cancer evolution. Combined experimental and simulation studies demonstrate that subclonal immune selection favours incremental MMR mutations. MMRd cancers thus fuel intratumour heterogeneity by adapting subclonal mutation rate and mutation bias to immune selection, revealing a conserved co-evolutionary arms race between neoantigen selection and adaptive genomic mutability. Our work reveals layers of mutational complexity and microsatellite biology in MMRd cancer evolution previously hidden in bulk analyses.
Ultrastructure Expansion Microscopy (U-ExM) in Trypanosoma cruzi: localization of tubulin isoforms and isotypes
Authors: Alonso Victoria Lucia
Published: March 2022

Ultrastructure Expansion Microscopy (U-ExM) is a recently developed technique that enables the increase of the spatial resolution within a cell or a tissue for microscopic imaging by physically expanding the sample. For the first time, I report a detailed protocol validating the use of U-ExM in Trypanosoma cruzi and quantifying the expansion factors of different subcellular compartments. I was able to determine the localization patterns of different tubulin isoforms, such as α-tubulin and β-tubulin. Also, I immunolocalized acetylated and tyrosinated α-tubulin isotypes in epimastigotes and use mitochondrial cell-permeable dyes to identify this organelle. Finally, U-ExM was also performed in trypomastigotes and amastigotes validating this technique in all life cycle stages of T. cruzi.
Transcriptional circuitry of NKX2-1 and SOX1 defines an unrecognized lineage subtype of small cell lung cancer
Authors: Kong Ranran , Patel Ayushi S. , Sato Takashi , Yoo Seungyeul , Bao Li , Sinha Abhilasha , Jiang Feng , Tian Yang , Fridrikh Maya , Liu Shuhui , Feng Jie , He Xijing , Jiang Jiantao , Ma Yuefeng , Grullon Karina , Yang Dawei , Powell Charles A. , Beasley Mary Beth , Zhu Jun , Snyder Eric L. , Li Shaomin , Watanabe Hideo
Published: March 2022

Rationale

The current molecular classification of small cell lung cancer (SCLC) based on expression of four lineage transcription factors still leaves its major subtype SCLC-A as a heterogeneous group, necessitating more precise characterization of lineage subclasses.

Objectives

To refine the current SCLC classification with epigenomic profiles and to identify features of the re-defined SCLC subtypes.

Methods

We performed unsupervised clustering of epigenomic profiles on 25 SCLC cell lines. Functional significance was evaluated by cell growth, apoptosis and xenograft using CRISPR-Cas9-mediated deletion. The specific cistromic profiles by ChIP-seq and its functional transcriptional partners using co-immunoprecipitation followed by mass spectrometry were determined. Rb1fl/flTrp53fl/fl and Rb1fl/flNkx2-1fl/fl mouse models were engineered to explore the function of Nkx2-1 in tumor initiation and differentiation. H3K27ac profiles were analyzed to reveal 6 human SCLC specimen and 20 mice tumors epigenomic landscapes.

Measurements and Main Results

We identified an epigenomic subclusters of the major SCLC-A subtype, named SCLC-Aα and SCLC-Aσ. SCLC-Aα was characterized by the presence of a super-enhancer at the NKX2-1 locus, which was observed in human SCLC specimens and a murine SCLC model. We found NKX2-1, a dual lung and neural lineage factor, is uniquely relevant in SCLC-Aα. We further found maintenance of this neural identity in SCLC-Aα is mediated by collaborative transcriptional activity with another neuronal transcriptional factor SOX1.

Conclusions

We comprehensively describe an additional epigenomic heterogeneity of the major SCLC-A subtype, and define SCLC-Aα subtype by the core regulatory circuitry representing NKX2-1 and SOX1 super-enhancers and their functional collaborations to maintain neuronal linage state.
Sensory Tetanization to Induce LTP-Like Plasticity: A Review and Reassessment of the Approach
Authors: Dias James W. , McClaskey Carolyn M. , Rumschlag Jeffrey A. , Harris Kelly C.
Published: March 2022

Great interest has been given to developing non-invasive approaches for studying cortical plasticity in humans. High frequency presentation of auditory and visual stimuli, or sensory tetanization, can induce long-term-potentiation-like (LTP-like) changes in cortical activity. However, contrasting effects across studies suggest that sensory tetanization may be unreliable. We review these contrasting effects, conduct our own study of auditory and visual tetanization, and perform meta-analyses to determine the average effect of sensory tetanization across studies. We measured auditory-evoked amplitude changes in a group of younger (18-29 years of age) and older (55-83 years of age) adults following tetanization to 1 kHz and 4 kHz tone bursts and following a slow-presentation control. We also measured visual-evoked amplitude changes following tetanization to horizontal and vertical sign gradients. Auditory and visual response amplitudes decreased following tetanization, consistent with some studies but contrasting with others finding amplitude increases (i.e., LTP-like changes). Older adults exhibited more modest auditory-evoked amplitude decreases, but visual-evoked amplitude decreases like those of younger adults. Changes in response amplitude were not specific to tetanized stimuli. Importantly, slow presentation of auditory tone-bursts produced response amplitude changes approximating those observed following tetanization in younger adults. Meta-analyses of visual and auditory tetanization studies found that the overall effect of sensory tetanization was not significant across studies or study sites. The results suggest that sensory tetanization may not produce reliable changes in cortical responses and more work is needed to determine the validity of sensory tetanization as a method for inducing human cortical plasticity in vivo.
LanA (Language Atlas): A probabilistic atlas for the language network based on fMRI data from >800 individuals
Authors: Lipkin Benjamin , Tuckute Greta , Affourtit Josef , Small Hannah , Mineroff Zachary , Kean Hope , Jouravlev Olessia , Rakocevic Lara , Pritchett Brianna , Siegelman Matthew , Hoeflin Caitlyn , Pongos Alvincé , Blank Idan A. , Struhl Melissa Kline , Ivanova Anna , Shannon Steven , Sathe Aalok , Hoffmann Malte , Nieto-Castañón Alfonso , Fedorenko Evelina
Published: March 2022

Two analytic traditions characterize fMRI language research. One relies on averaging activations voxel-wise across individuals. This approach has limitations: because of inter-individual variability in the locations of language areas, a location in a common brain space cannot be meaningfully linked to function. An alternative approach relies on identifying language areas in each individual using a functional ‘localizer’. Because of its greater sensitivity, functional resolution, and interpretability, functional localization is gaining popularity, but it is not always feasible, and cannot be applied retroactively to past studies. We provide a solution for bridging these currently disjoint approaches in the form of aprobabilistic functional atlascreated from fMRI data for an extensively validated language localizer in 806 individuals. This atlas enables estimating the probability that any given location in a common brain space belongs to the language network, and thus can help interpret group-level peaks and meta-analyses of such peaks, and lesion locations in patient investigations. More meaningful comparisons of findings across studies should increase robustness and replicability in language research.
SRSF10 is essential for progenitor spermatogonia expansion by regulating alternative splicing
Authors: Liu Wenbo , Lu Xukun , Zhao Zheng-Hui , Li Qian-Nan , Xue Yue , Gao Zheng , Sun Si-Min , Lei Wen-Long , Li Lei , An Geng , Liu Hanyan , Han Zhiming , Ouyang Ying-Chun , Hou Yi , Wang Zhen-Bo , Sun Qing-Yuan , Liu Jianqiao
Published: March 2022

Alternative splicing expands the transcriptome and proteome complexity and plays essential roles in tissue development and human diseases. However, how alternative splicing regulates spermatogenesis remains largely unknown. Here, using germ cell-specific knockout mouse model, we demonstrated that the splicing factor Srsf10 is essential for spermatogenesis and male fertility. Depletion of Srsf10 in germ cells had little effect on the formation of SSCs but impeded the expansion of progenitor spermatogonia, leading to the failure of spermatogonia differentiation and meiosis initiation. This was further evidenced by the decreased expression of progenitor cell markers in bulk RNA-seq, and much less progenitor and differentiating spermatogonia in single-cell RNA-seq data. Furthermore, the expression of genes involved in cell cycle was abnormal in all subtypes of spermatogonia identified in single-cell RNA-seq data. Notably, using isolated spermatogonia, we found that Srsf10 depletion disturbed the alternative splicing of hundreds of genes, which were preferentially associated with cell cycle, mitotic cell cycle checkpoint and germ cell development, including Dazl, Kit, Ret, Sycp1, Nasp and Bora. These data suggest that SRSF10 is critical for the expansion of progenitor spermatogonia by regulating alternative splicing, expanding our understanding of the mechanism underlying spermatogenesis.
Taste quality and hunger interactions in a feeding sensorimotor circuit
Authors: Shiu Philip K. , Sterne Gabriella R. , Engert Stefanie , Dickson Barry J. , Scott Kristin
Published: March 2022

Taste detection and hunger state dynamically regulate the decision to initiate feeding. To study how context-appropriate feeding decisions are generated, we combined synaptic resolution circuit reconstruction with targeted genetic access to specific neurons to elucidate a gustatory sensorimotor circuit for feeding initiation in adult Drosophila melanogaster. This circuit connects gustatory sensory neurons to proboscis motor neurons through three intermediate layers. Most neurons in this pathway are necessary and sufficient for proboscis extension, a feeding initiation behavior, and respond selectively to sugar taste detection. Pathway activity is amplified by hunger signals that act at select second-order neurons to promote feeding initiation in food-deprived animals. In contrast, the feeding initiation circuit is inhibited by a bitter taste pathway that impinges on premotor neurons, illuminating a local motif that weighs sugar and bitter taste detection to adjust behavioral outcome. Together, these studies reveal central mechanisms for the integration of external taste detection and internal nutritive state to flexibly execute a critical feeding decision.
POSITIVE RATE DEPENDENT ACTION POTENTIAL PROLONGATION BY MODULATING POTASSIUM ION CHANNELS
Authors: Cabo Candido
Published: March 2022

Pharmacological agents that prolong action potential duration (APD) to a larger extent at slow rates than at the fast excitation rates typical of ventricular tachycardia exhibit reverse rate dependence. Reverse rate dependence has been linked to the lack of efficacy of class III agents at preventing arrhythmias. In this report we show that, in computer models of the ventricular action potential, APD prolongation by accelerating phase 2 repolarization (by increasing IKs) and decelerating phase 3 repolarization (by blocking IKr and IK1) results in a robust positive rate dependence (i.e., larger APD prolongation at fast rates than at slow rates). In contrast, APD prolongation by blocking a specific potassium channel type results in reverse rate dependence or a marginal positive rate dependence. Interventions that result in a strong positive rate dependence tend to decrease the repolarization reserve because they require substantial IK1 block. However, limiting IK1 block to ∼50% results in a strong positive rate dependence with moderate decrease in repolarization reserve. In conclusion, the use of a combination of IKs activators and IKr and IK1 blockers could result in APD prolongation that potentially maximizes anti-arrhythmic effects (by maximizing APD prolongation at fast excitation rates) and minimizes pro-arrhythmic effects (by minimizing APD prolongation at slow excitation rates).
Structural maturation of SYCP1-mediated meiotic chromosome synapsis through conformational remodelling by molecular adapter SYCE3
Authors: Crichton James H. , Dunce James M. , Dunne Orla M. , Salmon Lucy J. , Devenney Paul S. , Lawson Jennifer , Adams Ian R. , Davies Owen R.
Published: March 2022

In meiosis, a supramolecular protein structure, the synaptonemal complex (SC), assembles between homologous chromosomes to facilitate their recombination. Mammalian SC formation is thought to involve hierarchical zipper-like assembly of an SYCP1 protein lattice that recruits stabilising central element (CE) proteins as it extends. Here, we combine biochemical approaches with separation-of-function mutagenesis in mice to uncover that, rather than stabilising the SYCP1 lattice, the CE protein SYCE3 actively remodels this structure during synapsis. We find that SYCP1 tetramers undergo conformational change into 2:1 heterotrimers upon SYCE3-binding, removing their assembly interfaces and disrupting the SYCP1 lattice. SYCE3 then establishes a new lattice by its self-assembly mimicking the role of the disrupted interface in tethering together SYCP1 dimers. SYCE3 also interacts with CE complexes SYCE1-SIX6OS1 and SYCE2-TEX12, providing a mechanism for their recruitment. Thus, SYCE3 remodels the SYCP1 lattice into a CE-binding integrated SYCP1-SYCE3 lattice to achieve long-range synapsis by a mature SC.
Identifying the C. elegans vulval transcriptome
Authors: Zhang Qi , Hrach Heather , Mangone Marco , Reiner David J.
Published: March 2022

Development of the C. elegans vulva is a classic model of organogenesis. This system, which starts with six equipotent cells, encompasses diverse types of developmental event, including developmental competence, multiple signaling events to control precise and faithful patterning of three cell fates, execution and proliferation of specific cell lineages, and a series of sophisticated morphogenetic events. Early events have been subjected to extensive mutational and genetic investigations and later events to cell biological analyses. We infer the existence of dramatically changing profiles of gene expression that accompanies the observed changes in development. Yet except from serendipitous discovery of several transcription factors expressed in dynamic patterns in vulval lineages, our knowledge of the transcriptomic landscape during vulval development is minimal. This study describes the composition of a vulva-specific transcriptome. We used tissue specific harvesting of mRNAs via immunoprecipitation of epitope-tagged poly(A) binding protein, PAB-1, heterologously expressed by a promoter known to express GFP in vulval cells throughout their development. The identified transcriptome was small but tightly interconnected. From this data set we identified several genes with identified functions in development of the vulva and validated more with promoter-GFP reporters of expression. For one target, lag-1, promoter-GFP expression was limited but fluorescent tag of the endogenous protein revealed extensive expression. Thus, we have identified a transcriptome of the C. elegans as a launching pad for exploration of functions of these genes in organogenesis.