bluuebunny/crossref_metadata_2025 huggingface dataset:
Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons
Authors: Eguchi Kohgaku , Shigemoto Ryuichi
Published: July 2022

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an essential role in neuronal activities through interaction with various proteins involved in signaling at membranes. However, the distribution pattern of PI(4,5)P2 and the co-clustering with these proteins on the neuronal cell membranes remain elusive at the electron microscopic level. In this study, we quantitatively investigated the nanoscale distribution of PI(4,5)P2 on the neuronal cell membranes by SDS-digested freeze-fracture replica labeling with cryo-fixed mouse cerebellum. We demonstrate that PI(4,5)P2 makes clusters with a mean size of ∼ 1,000 nm2 and these clusters show preferential accumulation in specific membrane compartments depending on cell types. Purkinje cell (PC) spines and granule cell (GC) presynaptic active zones are particularly rich in the PI(4,5)P2 clusters. Furthermore, these clusters are extensively associated with clusters of CaV2.1 and GIRK3 throughout different membrane compartments of PCs, GCs, and molecular layer interneurons. In contrast, these clusters showed compartment-specific association with mGluR1α in PC spines. These results suggest that visualization of the nanoscale PI(4,5)P2 distribution may provide insight into the physiological functions of PI(4,5)P2 in neurons.
Breakdown of clonal cooperative architecture in multispecies biofilms and the spatial ecology of predation
Authors: Wucher Benjamin R. , Winans James B. , Elsayed Mennat , Kadouri Daniel E. , Nadell Carey D.
Published: July 2022

Adherence to surfaces and secretion of extracellular matrix, or biofilm formation, is common in the microbial world, but we often do not know how interaction at the cellular spatial scale translates to higher-order biofilm community ecology. Here we explore an especially understudied element of biofilm ecology, namely predation by the bacteriumBdellovibrio bacteriovorus. This predator can kill and consume many different Gram-negative bacteria, includingVibrio choleraeandEscherichia coli.V. choleraecan protect itself from predation within highly packed biofilm structures that it creates, whereasE. colibiofilms are highly susceptible toB. bacteriovorus. Here we explore how predator-prey dynamics change whenV. choleraeandE. coliare growing in biofilms together. We find that in dual species prey biofilms,E. colisurvival underB. bacteriovoruspredation increases, whereasV. choleraesurvival decreases.E. colibenefits from predator protection when it becomes embedded within expanding groups of highly packedV. cholerae. But we also find that the ordered, highly packed, and clonal biofilm structure ofV. choleraecan be disrupted ifV. choleraecells are directly adjacent toE. colicells at the start of biofilm growth. When this occurs, the two species become entangled, and the resulting disordered cell groups do not block predator entry. Because biofilm cell group structure depends on initial cell distributions at the start of prey biofilm growth, the colonization dynamics have a dramatic impact on the eventual multispecies biofilm architecture, which in turn determines to what extent both species survive exposure toB. bacteriovorus.

Significance Statement

Bacteria live in multispecies, spatially structured communities ubiquitously in the natural world. These communities, or biofilms, have a strong impact on microbial ecology, but we often do not know how cellular scale interactions determine overall biofilm structure and community dynamics. Here we explore this problem in the context of predator-prey interaction, with two prey species –Vibrio choleraeandEscherichia coli– being attacked by the bacterial predatorBdellovibrio bacteriovorus. We find that whenV. choleraeandE. coligrow together in biofilms, the architectures that they both produce change in ways that cannot be predicted from looking at each prey species alone, and that these changes in cell group structure impact the community dynamics of predator-prey interaction in biofilms.
Compartmentalization of specialized metabolites across vegetative and reproductive tissues in two sympatricPsychotria(Rubiaceae)
Authors: Schneider Gerald F. , Carlson Cole A. , Jos Elsa M. , Beckman Noelle G.
Published: July 2022

Premise of the study

The specialized metabolites of plants are recognized as key chemical traits in mediating the ecology and evolution of sundry plant-biotic interactions, from pollination to seed predation. Intra- and interspecific patterns of specialized metabolite diversity have been studied extensively in leaves, but the diverse biotic interactions which contribute to specialized metabolite diversity encompass all plant organs. Focusing on two species ofPsychotriashrubs, we investigate and compare patterns of specialized metabolite diversity in leaves and fruit with respect to each organ’s diversity of biotic interactions.

Methods

To evaluate associations between biotic interaction diversity and specialized metabolite diversity, we combine UPLC-MS metabolomic analysis of foliar and fruit specialized metabolites with existing surveys of leaf- and fruit-centered biotic interactions. We compare patterns of specialized metabolite richness and variance among vegetative and reproductive tissues, among plants, and between species.

Key results

In our study system, leaves are involved in a greater number of host-specific biotic interactions than fruit, while fruit-centric interactions are more ecologically diverse. This was reflected in specialized metabolite richness – leaves contained more than fruit, while each contained over 200 organ-specific specialized metabolites. Within each species, leaf- and fruit specialized metabolite composition varied independently of one another across plants. Specialized metabolite composition exhibited stronger contrasts between organs than between species.

Conclusions

As ecologically disparate plant organs with organ-specific specialized metabolite traits, leaves and fruit can each contribute to the tremendous overall diversity of plant specialized metabolites.
Posttranslational modifications of the DUX4 protein impact toxic function
Authors: Knox Renatta N. , Eidahl Jocelyn O. , Wallace Lindsay , Choudury Sarah , Rashnonejad Afrooz , Saad Nizar Y. , Hoover Michael E. , Zhang Liwen , Branson Owen E. , Freitas Michael A. , Harper Scott Q.
Published: July 2022

Objective

Facioscapulohumeral muscular dystrophy (FSHD) is caused by abnormal de-repression of the transcription factor DUX4, which is toxic to muscle in vitro and in vivo. While the transcriptional targets of DUX4 are known, the regulation of DUX4 protein and the molecular consequences of this regulation are unclear. Here, we used in vitro models of FSHD to identify and characterize DUX4 posttranslational modifications (PTMs) and their impact on the toxic function of DUX4.

Methods

DUX4 protein was immunoprecipitated and mass spectrometry performed to identify PTMs. We then extensively characterized DUX4 PTMs and potential enzyme modifiers using mutagenesis, proteomics and biochemical assays in human cell lines and human myoblast cell lines.

Results

Our in vitro screen of DUX4 PTM mutants identified arginine methyl-null and serine/threonine phosphomimetic mutants that protected cells against DUX4-mediated toxicity and reduced the ability of DUX4 to transactivate downstream gene targets, including FSHD biomarkers. Using additional proteomics and biochemical approaches, we identified protein kinase A (PKA) and a protein arginine methyltransferase (PRMT1) as components of the DUX4 complex. Importantly, over-expression of PRKACA, a catalytic subunit of the PKA holoenzyme, mitigated DUX4 toxicity, while pharmacologic inhibition of PRMT1 protected human myoblasts from DUX4-mediated apoptosis.

Interpretation

These results demonstrate that DUX4 is regulated by PTMs and that DUX4 PTMs, or associated modifying enzymes, may be druggable targets for FSHD therapy.
EpiCompare: R package for the comparison and quality control of epigenomic peak files
Authors: Choi Sera , Schilder Brian M. , Abbasova Leyla , Murphy Alan E. , Skene Nathan G.
Published: July 2022

EpiCompare combines a variety of downstream analysis tools to compare, quality control and benchmark different epigenomic datasets. The package requires minimal input from users, can be run with just one line of code and provides all results of the analysis in a single interactive HTML report. EpiCompare thus enables downstream analysis of multiple epigenomic datasets in a simple, effective and user-friendly manner.

Availability and Implementation

EpiCompare is available on Bioconductor (≥ v3.15):

https://bioconductor.org/packages/release/bioc/html/EpiCompare.html

All source code is publically available via GitHub:

https://github.com/neurogenomics/EpiCompare

Documentation website

https://neurogenomics.github.io/EpiCompare

EpiCompare DockerHub repository:

https://hub.docker.com/repository/docker/neurogenomicslab/epicompare
Resurrection genomics provides molecular and phenotypic evidence of rapid adaptation to salinization in a keystone aquatic species
Authors: Wersebe Matthew J. , Weider Lawrence J.
Published: July 2022

Ecologists and evolutionary biologists are increasingly cognizant of rapid adaptation in wild populations. Rapid adaptation to anthropogenic environmental change is critical for maintaining biodiversity and ecosystems services into the future. Anthropogenic salinization of freshwater ecosystems is quickly emerging as a primary threat, which is well documented in the northern temperate ecoregion. Specifically, many northern temperate lakes have undergone extensive salinization because of urbanization and the associated increase in impervious surfaces causing runoff, and the extensive use of road deicing salts (e.g., NaCl). It remains unclear if increasing salinization will lead to extirpation of species from these systems. Using a “resurrection genomics” approach, we investigated whether the keystone aquatic herbivore,Daphnia pulicaria, has evolved increased salinity tolerance in a severely salinized lake located in Minnesota, USA. Whole genome resequencing of 54Daphniaclones from the lake and hatched from resting eggs that represent a 25-year temporal contrast demonstrates that many regions of the genome containing genes related to osmoregulation are under selection in the study population. Tolerance assays of clones revealed that the most recent clones are more tolerant to salinity than older clones; this pattern is concomitant with the temporal pattern of stabilizing salinity in this lake. Together, our results demonstrate that keystone species such asDaphniacan rapidly adapt to increasing freshwater salinization. Further, our results indicate that rapid adaptation to salinity may allow lakeDaphniapopulations to persist in the face of anthropogenic salinization maintaining the food webs and ecosystem services they support despite global environmental change.

Significance Statement

Rapid adaptation to human-induced environmental change is critical for preserving biodiversity and ecosystem services into the future. A key question is whether populations of keystone species can rapidly adapt to maintain the ecosystems they support. We investigated rapid adaptation to anthropogenic salinization inDaphnia pulicaria, a keystone aquatic herbivore in lake ecosystems. By resuscitating decades-old resting eggs, we investigate genomic changes across an approximately 25-year temporal contrast from a severely salinized lake. We report that the genes showing signatures of natural selection throughout the genome are related to osmoregulation and ion regulation. Phenotyping clones for salinity tolerance revealed evidence that genetic changes may underlie rapid evolution. We provide molecular genomic and phenotypic evidence for rapid adaptation to salinity inD. pulicaria.
Circulating extracellular vesicles in lung cancer patients are not enriched in tumor-derived DNA fragments as revealed by whole genome sequencing
Authors: Moldovan Norbert , Verkuijlen Sandra , van der Pol Ymke , Bosch Leontien , van Weering Jan R.T. , Bahce Idris , Pegtel D. Michiel , Mouliere Florent
Published: July 2022

Liquid biopsies contain multiple analytes that can be mined to improve the detection and management of cancer. Beyond cell-free DNA (cfDNA), mutations have been detected in DNA associated with extracellular vesicles (EV-DNA). The genome-wide composition and structure of EV-DNA are poorly characterized, and it remains undecided whether circulating EVs are enriched in tumor signal compared to unfractionated cfDNA.

Here, using whole genome sequencing from selected lung cancer patients with a high cfDNA tumor content (>5%), we determined that the tumor fraction and heterogeneity are comparable between DNA associated with EVs and matched plasma cfDNA. DNA in EV fractions, obtained with standardized size-exclusion chromatography, are comprised of short ∼150-180 bp fragments and long >1000 bp fragments that are poor in tumor signal. Other fractions only exhibit short fragments with similar tumor DNA content. The composition in bases at the end of EV-DNA fragments, as well as their fragmentation patterns are similar to plasma cfDNA. Mitochondrial DNA is relatively enriched in EV fractions.

Our results highlight that cfDNA in plasma is of dual nature, either bound to proteins (including the nucleosome) but also associated to EV. cfDNA associated to small EV (including exosomes) is however not preferentially enriched in tumor signal.
Strong effects of temperature, population and age-at-maturity genotype on maturation probability for Atlantic salmon in a common garden setting
Authors: Åsheim Eirik R. , Debes Paul V , House Andrew , Niemelä Petri T. , Siren Jukka P. , Erkinaro Jaakko , Primmer Craig R
Published: July 2022

Age at maturity is a key life history trait and involves a trade-off between survival risk and reproductive investment, has close connections to fitness, and is an important factor for population structures. Temperature can have a dramatic influence on life history in ectotherms, but this influence may differ between populations. While an increasing number of studies have examined population-dependent reactions with temperature, few have investigated this in the context of maturation timing.

Atlantic salmon is a highly relevant study species for improving understanding of this topic as it displays considerable variation in life-history strategies, including maturation timing. Additionally, a large amount of this variation in maturation timing has been associated with a genomic region including the strong candidate genevgll3, but the effect of this gene in the context of different environments and populations has not been studied.

Using a large-scale common-garden experiment, we find strong effects of temperature, population, andvgll3genotype on maturation in 2-year-old male Atlantic salmon. Observed maturation probability was 4.8 times higher in individuals reared at a mean temperature of 8.6°C compared to 6.9°C. This temperature effect was population-specific and was higher in the southern population compared to the northern population, potentially due to a higher intrinsic growth in the southern population as well as growth-temperature interaction.

The early-maturationvgll3*E associated with a significantly higher maturation probability, but there was novgll3-interaction with temperature or population.

Both body condition and body mass associated strongly with maturation; the body-condition association was stronger in fish carrying thevgll3*E allele, and the body mass association was only present in the warm treatment.

Our findings demonstrate that the relative effect ofvgll3on maturation timing is similar for two populations and two thermal environments and gives new perspectives on the relative effect ofvgll3compared to such influences. Additionally, we show that populations can vary in their response to temperature change in terms of maturation timing, and that high intrinsic growth could potentially be associated with higher thermal sensitivity for life history variation.
The serine/threonine kinase Back seat driver prevents cell fusion to maintain cell identity
Authors: Yang Shuo , Johnson Aaron N.
Published: July 2022

Cell fate specification is essential for every major event of embryogenesis, and subsequent cell maturation ensures individual cell types acquire specialized functions. The mechanisms that regulate cell fate specification have been studied exhaustively, and each technological advance in developmental biology ushers in a new era of studies aimed at uncovering the most fundamental processes by which cells acquire unique identities. What is less appreciated is that mechanisms are in place to ensure cell identity is maintained throughout the life of the organism. The body wall musculature in the Drosophila embryo is a well-established model to study cell fate specification, as each hemisegment in the embryo generates and maintains thirty muscles with distinct identities. Here we show the serine/threonine kinase Back seat driver (Bsd), which regulates muscle morphogenesis, also maintains cell identity. Once specified, the thirty body wall muscles fuse with mononucleate muscle precursors that lack a specific identity to form multinucleate striated muscles. Importantly, body wall muscles do not fuse with each other and thereby maintain distinct identities. We show that Bsd prevents inappropriate fusion among the thirty body wall muscles. Thus, the regulation of cell fusion is one mechanism that maintains cell identity.
Synergism of interferon-beta with antiviral drugs against SARS-CoV-2 variants
Authors: Bojkova Denisa , Stack Richard , Rothenburger Tamara , Kandler Joshua D , Ciesek Sandra , Wass Mark N. , Michaelis Martin , Cinatl Jindrich
Published: July 2022

Omicron BA.1 variant isolates were previously shown to replicate less effectively in interferon-competent cells and to be more sensitive to interferon treatment than a Delta isolate. Here, an Omicron BA.2 isolate displayed intermediate replication patterns in interferon-competent Caco-2-F03 cells when compared to BA.1 and Delta isolates. Moreover, BA.2 was less sensitive than BA.1 and similarly sensitive as Delta to betaferon treatment. Delta and BA.1 displayed similar sensitivity to the approved anti-SARS-CoV-2 drugs remdesivir, nirmatrelvir, EIDD-1931 (the active metabolite of molnupiravir) and the protease inhibitor aprotinin, whereas BA.2 was less sensitive than Delta and BA.1 to EIDD-1931, nirmatrelvir and aprotinin. Nirmatrelvir, EIDD-1931, and aprotinin (but not remdesivir) exerted synergistic antiviral activity in combination with betaferon, with some differences in the extent of synergism detected between the different SARS-CoV-2 variants. In conclusion, even closely related SARS-CoV-2 (sub)variants can differ in their biology and in their response to antiviral treatments. Betaferon combinations with nirmatrelvir and, in particular, with EIDD-1931 and aprotinin displayed high levels of synergism, which makes them strong candidates for clinical testing. Notably, effective antiviral combination therapies are desirable, as a higher efficacy is expected to reduce resistance formation.
Npl3 functions in mRNP assembly by recruitment of mRNP components to the transcription site and their transfer onto the mRNA
Authors: Keil Philipp , Wulf Alexander , Kachariya Nitin , Reuscher Samira , Hühn Kristin , Silbern Ivan , Altmüller Janine , Stehle Ralf , Zarnack Kathi , Sattler Michael , Urlaub Henning , Sträßer Katja
Published: July 2022

RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large messenger ribonucleoprotein particle (mRNP) assemblies, these proteins often function in. Using UV-induced RNA-protein crosslinking and subsequent mass spectrometric analysis, we first identified more than 100in vivoRNA crosslinks in 16 nuclear mRNP components inS. cerevisiae. For functional analysis, we chose Npl3, for which we determined crosslinks in its two RNA recognition motifs (RRM) and in the flexible linker region connecting the two. Using NMR and structural analyses, we show that both RRM domains and the linker uniquely contribute to RNA recognition. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains of Npl3. Notably, thenpl3-Linkermutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of further mRNP components into nuclear mRNPs, establishing a function of Npl3 in nuclear mRNP assembly. Taken together, we determined the specific function of the RNA-binding activity of the nuclear mRNP component Npl3, an approach that can be applied to many RBPs in any RNA metabolic process.
RNA structure landscape ofS. cerevisiaeintrons
Authors: Rangan Ramya , Huang Rui , Hunter Oarteze , Pham Phillip , Ares Manuel , Das Rhiju
Published: July 2022

Pre-mRNA secondary structures are hypothesized to play widespread roles in regulating RNA processing pathways, but these structures have been difficult to visualizein vivo. Here, we characterizeS. cerevisiaepre-mRNA structures through transcriptome-wide dimethyl sulfate (DMS) probing, enriching for low-abundance pre-mRNA through splicing inhibition. We cross-validate structures found from phylogenetic and mutational studies and identify new structures within the majority of probed introns (102 of 161). We find widespread formation of “zipper stems” between the 5’ splice site and branch point, “downstream stems” between the branch point and the 3’ splice site, and previously uncharacterized long stems that distinguish pre-mRNA from spliced mRNA. Multi-dimensional chemical mapping reveals examples where intron structures can formin vitrowithout the presence of binding partners, and structure ensemble prediction suggests that such structures appear in introns across theSaccharomycesgenus. We develop a high-throughput functional assay to characterize variants of RNA structure (VARS-seq) and we apply the method on 135 sets of stems across 7 introns, identifying structured elements that alter retained intron levels at a distance from canonical splice sites. This transcriptome-wide inference of intron RNA structures suggests new ideas and model systems for understanding how pre-mRNA folding influences gene expression.
Longitudinal in vivo monitoring of axonal integrity after brain injury
Authors: Chornyy Sergiy , Borovicka Julie A. , Patel Davina , Shin Min-Kyoo , Vázquez-Rosa Edwin , Miller Emiko , Wilson Brigid , Pieper Andrew A. , Dana Hod
Published: July 2022

Traumatic brain injury-induced axonal degeneration leads to acute and chronic neuropsychiatric impairment, neuronal death, and accelerated neurodegenerative diseases of aging, including Alzheimer’s and Parkinson’s diseases. Thus, there is much interest in developing treatments that protect axons after injury. For this endeavor, extended comprehensive evaluation of axonal integrity in experimental systems is required to evaluate the efficacy of putative interventions in preclinical models. However, traditional histological tissue proccessing techniques are logistically prohibitive for assessments of long-term pathology. Here, we report a new method of longitudinally monitoring the functional activity of thalamocortical axons before and after injury in vivo in the same animal over an extended period of time. Specifically, we expressed an axonal-targeting genetically-encoded calcium indicator in the mouse dorsolateral geniculate nucleus and then recorded axonal activity patterns in the visual cortex in response to visual stimulation. We demonstrate the utility of this method for assessing in vivo aberrant axonal activity patterns after traumatic brain injury, as well as for evaluating the therapuetic efficacy of the neuroprotective P7C3-A20 pharmacologic agent in vivo. We found that P7C3-A20 treatment minimized most, but not all, of the pathological changes in axonal activity patterns after traumatic brain injury.
Non-trivial dynamics in a model of glial membrane voltage driven by open potassium pores
Authors: Janjic Predrag , Solev Dimitar , Kocarev Ljupco
Published: July 2022

Despite the molecular evidence that the nearly linear steady-state current-voltage relationship in mammalian astrocytes reflects a total current resulting from more than one differently regulated K+ conductances, detailed ODE models of membrane voltage Vm are still lacking. Repeated experimental results of deregulated expressions of major K+ channels in glia, Kir4.1, in models of neurodegenerative disease, as well as their altered rectification when assembling heteromeric Kir4.1/Kir5.1 channels have motivated us to attempt a detailed model incorporating the weaker potassium K2P-TREK1 current, in addition to Kir4.1, and study the stability of the resting state Vr. The main question is whether with a deregulated Kir conductivity the nominal resting state Vr remains stable, and the cell retains a trivial, potassium electrode behavior with Vm following EK. The minimal 2-dimensional model near Vr showed that certain alterations of Kir4.1 current may result in multistability of Vm if the typically observed K+ currents - Kir, K2P, and non-specific potassium leak are present. More specifically, a decrease or loss of outward Kir4.1 conductance (turning the channels into inwardly rectifying) introduces instability of Vr, near EK. That happens through robustly observed fold bifurcation giving birth to a second, much more depolarized stable resting state Vdr > −10 mV. Realistic time series were used to perturb the membrane model, from recordings of glial Vm during electrographic seizures. Simulations of the perturbed system by constant currents through gap-junctions and transient seizure-like discharges as local field potentials led to depolarization of the astrocyte and switching of Vm between the two stable states, in a downstate – upstate manner. If the prolonged depolarizations near Vdr prove experimentally plausible, such catastrophic instability would impact all aspects of the glial function, from metabolic support to membrane transport and practically all neuromodulatory roles assigned to glia.

Statement of Significance

The almost linear current-voltage relationship of most glial membranes results from multiple non-linear potassium leaky-pore, or background conductances. The corresponding channel types develop and deregulate independently, some of them asymmetrically – deregulate differently in different Vm ranges. Effect of those deregulations on whole-cell voltage responses has not been treated. We developed a minimal ODE model of voltage dynamics incorporating detailed models of the different potassium currents based on electrophysiological recordings. Parametrically inducing some of the reported deregulations of Kir current in glia resulted in instability of the nominal resting membrane potential and appearence of a second much more depolarized resting state. If prolonged glial depolarizations prove plausible such bistability would change the present beliefs about glial Vm dynamics.
α-syn overexpression, NRF2 suppression, and enhanced ferroptosis create a vicious cycle of neuronal loss in Parkinson’s disease
Authors: Anandhan A , Chen W , Nguyen N , Madhavan L , Dodson M , Zhang DD
Published: July 2022

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, affecting millions each year. Most PD cases (∼90%) are sporadic, resulting from the age-dependent accumulation of pathogenic effects. One key pathological hallmark of PD progression is the accumulation of alpha-synuclein (α-syn), which has been shown to negatively affect neuronal function and viability. Here, using 3- and 6-month-old Nrf2+/+ and Nrf2-/- mice overexpressing human α-syn (PD model), we show that loss of NRF2 increases markers of ferroptosis across PD-relevant brain regions. Increased ferroptosis was associated with an age- and genotype-dependent increase in α-syn pathology and behavioral deficits. Finally, we demonstrate that α-syn overexpression sensitizes neuronal cells and ex vivo brain slices to ferroptosis induction, which may be due to α-syn suppression of NRF2 at the protein level. Altogether, these results indicate that NRF2 is a critical anti-ferroptotic mediator of neuronal survival, and that the vicious cycle of α-syn overexpression and NRF2 suppression, leading to enhanced neuronal ferroptotic cell death, could represent a targetable and currently untapped means of preventing PD onset and progression.
GARP complex controls Golgi physiology by stabilizing COPI machinery and Golgi v-SNAREs
Authors: Khakurel Amrita , Kudlyk Tetyana , Pokrovskaya Irina , D’Souza Zinia , Lupashin Vladimir V.
Published: July 2022

GARP is an evolutionary conserved heterotetrameric protein complex that is thought to tether endosome-derived vesicles and promotes their fusion in thetrans-Golgi network. We have previously discovered the GARP’s role in maintaining Golgi glycosylation machinery. To further investigate the importance of the GARP complex for Golgi physiology, we employed Airyscan superresolution and electron microscopy, as well as the unbiased quantitative proteomic analysis of Golgi in RPE1 cells. Bothcisandtrans-Golgi compartments were significantly enlarged in GARP deficient cells with pronounced alterations of TGN morphology. In GARP-KO cells, proteomic analysis revealed a depletion of a subset of Golgi resident proteins, including Ca2+binding proteins, glycosylation enzymes, and v-SNAREs. We validated proteomics studies and discovered that two Golgi-resident proteins SDF4 and ATP2C1, related to Golgi calcium homeostasis, as well as intra-Golgi v-SNAREs GOSR1 and BET1L, are significantly depleted in GARP-KO cells. To test if SNARE depletion is responsible for the Golgi defects in GARP deficient cells, we created and analyzed GOSR1 and BET1L KO cell lines. Since GARP-KO was more deleterious to the Golgi physiology than SNARE-KOs, we have investigated other components of intra-Golgi vesicular trafficking, particularly COPI vesicular coat and its accessory proteins. We found that COPI is partially relocalized to the ERGIC compartment in GARP-KO cells. Moreover, COPI accessory proteins GOLPH3, ARFGAP1, GBF1 were displaced from the membrane and BIG1 was relocated to endolysosomal compartment in GARP-KO cells. We propose that the dysregulation of COPI machinery along with degradation of intra-Golgi v-SNAREs and alteration of Golgi Ca2+homeostasis are the major driving factors for the instability of Golgi resident proteins and glycosylation defects in GARP deficient cells.
Displacement experiments provide evidence for path integration in Drosophila
Authors: Titova Anna V. , Kau Benedikt E. , Tibor Shir , Mach Jana , Vo-Doan T. Thang , Wittlinger Matthias , Straw Andrew D.
Published: July 2022

Like many other animals, insects are capable of returning to previously visited locations using path integration. Recently, Drosophila has been added to the list of insects thought capable of this navigational memory. Existing experimental evidence, however, has a potential confound. Here we show that pheromones deposited at the site of reward might enable flies to find previously rewarding locations even without memory. Thus, we designed an experiment to determine if flies can use path integration memory despite potential pheromonal cues by displacing the flies shortly after an optogenetic reward. We found that rewarded flies returned to the location predicted by a memory-based model. Several analyses are consistent with path integration as the mechanism by which flies returned to the reward. We conclude that while pheromones may often be important in fly navigation and must be carefully controlled in future experiments, Drosophila may indeed be capable of performing path integration.
The chromatin landscape of the euryarchaeonHaloferax volcanii
Authors: Marinov Georgi K. , Bagdatli S. Tansu , Wu Tong , He Chuan , Kundaje Anshul , Greenleaf William J.
Published: July 2022

Archaea, together with Bacteria, represent the two main divisions of life on Earth, with many of the defining characteristics of the more complex eukaryotes tracing their origin to evolutionary innovations first made in their archaeal ancestors. One of the most notable such features is nucleosomal chromatin, although archaeal histones and chromatin differ significantly from those of eukaryotes. Despite increased interest in archaeal histones in recent years, the properties of archaeal chromatin have been little studied using genomic tools. Here, we adapt the ATAC-seq assay to archaea and use it to map the accessible landscape of the genome of the euryarchaeoteHaloferax volcanii. We integrate the resulting datasets with genome-wide maps of active transcription and single-stranded DNA (ssDNA) and find that whileH. volcaniipromoters exist in a preferentially accessible state, unlike most eukaryotes, modulation of transcriptional activity is not associated with changes in promoter accessibility. Applying orthogonal single-molecule footprinting methods, we quantify the absolute levels of physical protection ofH. volcanii, and find thatHaloferaxchromatin is similarly or only slightly more accessible, in aggregate, than that of eukaryotes. We also evaluate the degree of coordination of transcription within archaeal operons and make the unexpected observation that some CRISPR arrays are associated with highly prevalent ssDNA structures. These results provide a foundation for the future functional studies of archaeal chromatin.
Benchmarking Differential Abundance Analysis Methods for Correlated Microbiome Sequencing Data
Authors: Yang Lu , Chen Jun
Published: July 2022

Differential abundance analysis (DAA) is one central statistical task in microbiome data analysis. A robust and powerful DAA tool can help identify highly confident microbial candidates for further biological validation. Current microbiome studies frequently generate correlated samples from different microbiome sampling schemes such as spatial and temporal sampling. In the past decade, a number of DAA tools for correlated microbiome data (DAA-c) have been proposed. Disturbingly, different DAA-c tools could sometimes produce quite discordant results. To recommend the best practice to the field, we performed the first comprehensive evaluation of existing DAA-c tools using real data-based simulations. Overall, the linear model-based methods LinDA, MaAsLin2, and LDM are more robust than methods based on generalized linear models. The LinDA method is the only method that maintains reasonable performance in the presence of strong compositional effects.
Cortico-thalamo-cortical interactions modulate electrically evoked EEG responses in mice
Authors: Claar Leslie D. , Rembado Irene , Kuyat Jacqulyn R. , Russo Simone , Marks Lydia C. , Olsen Shawn R. , Koch Christof
Published: July 2022

Perturbational complexity analysis predicts the presence of consciousness in volunteers and patients by stimulating the brain with brief pulses, recording electroencephalographic (EEG) responses, and computing their spatiotemporal complexity. We examined the underlying neural circuits in mice by directly stimulating cortex while recording with EEG and Neuropixels probes during wakefulness and isoflurane anesthesia. When mice are awake, stimulation of deep cortical layers reliably evokes locally a brief pulse of excitation, followed by a bi-phasic sequence of 120 ms profoundoffperiod and areboundexcitation. A similar pattern, partially attributed to burst spiking, is seen in thalamic nuclei, and is associated with a pronounced late component in the evoked EEG. We infer that cortico-thalamo-cortical interactions drive the long-lasting evoked EEG signals elicited by deep cortical stimulation during the awake state. The cortical and thalamic off period and rebound excitation, and the late component in the EEG, are reduced during running and absent during anesthesia.
Regulatory roles of three-dimensional structures of chromatin domains
Authors: Li Kelly Yichen , Cao Qin , Wang Huating , Leung Danny , Yip Kevin Y.
Published: July 2022

1

Transcriptional enhancers usually, but not always, regulate genes within the same topologically associating domain (TAD). We hypothesize that this incomplete insulation is due to three-dimensional structures of corresponding chromatin domains in individual cells: Whereas enhancers and genes buried inside the “core” of a domain interact mostly with other regions in the same domain, those on the “surface” can more easily interact with the outside. Here we show that a simple measure, the intra-TAD ratio, can quantify the “coreness” of a region with respect to single-cell domains it belongs. We show that domain surfaces are permissive for high gene expression, and cell type-specific active cis-regulatory elements (CREs), active histone marks, and transcription factor binding sites are enriched on domain surfaces, most strongly in chromatin subcompartments typically considered inactive. These findings suggest a “domain surface CRE” model of gene regulation. We also find that disease-associated non-coding variants are enriched on domain surfaces.
Temporal regulation of TAK1 to counteract muscular dystrophy
Authors: Roy Anirban , Koike Tatiana E. , Joshi Aniket S. , da Silva Meiricris Tomaz , Mathukumalli Kavya , Wu Mingfu , Kumar Ashok
Published: July 2022

Muscular dystrophy is a group of genetic neuromuscular disorders that involves severe muscle wasting. Transforming growth factor β-activated kinase 1 (TAK1) is an important signaling protein that regulates cell survival, growth, and inflammation. TAK1 has been recently found to promote myofiber growth in skeletal muscle of adult mice. However, the role of TAK1 in muscle disorders remains poorly understood. In the present study, we have investigated how TAK1 affects progression of dystrophic phenotype in the mdx mouse model of Duchnne muscular dystrophy (DMD). TAK1 is highly activated during peak necrotic phase in mdx mice. Targeted inducible inactivation of TAK1 inhibits muscle injury, necroptosis, and accumulation of macrophages in dystrophic muscle of mdx mice. Additionally, targeted inactivation of TAK1 leads to the activation of autophagy and Notch and Wnt signaling in the dystrophic muscle. However, inactivation of TAK1 significantly reduces myofiber size and muscle contractile function in both young and adult mdx mice. Forced activation of TAK1 in skeletal muscle after peak necrotic phase induces myofiber growth and improves muscle histopathology in mdx mice. Our results suggest that targeted activation of TAK1 can ameliorate disease progression and improve muscle growth in DMD.

One Sentence Summary

Our results demonstrate that duly regulation of TAK1 activity ameliorates dystrophic phenotype in a mouse model of Duchnne Muscular Dystrophy.
hnRNPA1/2 homologhrpa-1coordinates with miRNAs to regulate gene expression duringC. elegansdevelopment
Authors: Hebbar Shilpa , Panzade Ganesh , Zinovyeva Anna
Published: July 2022

microRNAs (miRNAs) are small non-coding RNAs that play crucial roles in development and in disease. miRNAs associate with Argonaute proteins to form miRNA Induced Silencing Complexes (miRISCs), which post-transcriptionally repress gene expression. miRNA-mediated gene repression itself is subject to regulation by factors that can affect miRNA biogenesis or function. We previously identified HRPA-1, an hnRNPA/B homolog, as a putative physical interactor of miRNAs. Here, we report characterizations of both physical and genetic interactions between HRPA-1 and miRISC components. We confirmed HRPA-1 precipitation in let-7 and miR-58 pulldowns and detected an interaction between HRPA-1 and Argonaute. Deletion ofhrpa-1in amir-48 mir-241(nDf51)background enhanced themir-48 mir-241developmental defects, suggesting thathrpa-1may be important forlet-7family miRNA activity. Similarly, loss ofhrpa-1strongly enhanced developmental defects associated with two other miRNA mutants,lsy-6(ot150)andlet-7(n2853). Depletion of HRPA-1 modestly disrupted miRNA levels and affected global gene expression profiles. We identified a potential target ofhrpa-1, R06C1.4, whose knockdown partially recapitulates thehrpa-1(-)effects on miRNA mutant phenotypes. Overall, we demonstratehrpa-1andR06C1.4roles inC. elegansdevelopmental timing regulation and propose models describing possible coordinating modes of gene regulation by HRPA-1 and miRNAs.
p53 restoration in small cell lung cancer identifies a latent Cyclophilin-dependent necrosis mechanism
Authors: Acosta Jonuelle , Li Qinglan , Freeburg Nelson F. , Murali Nivitha , Grothusen Grant P. , Cicchini Michelle , Mai Hung , Gladstein Amy C. , Adler Keren M. , Doerig Katherine R. , Li Jinyang , Ruiz-Torres Miguel , Manning Kimberly L. , Stanger Ben Z. , Busino Luca , Wan Liling , Feldser David M.
Published: July 2022

The p53 tumor suppressor regulates multiple context-dependent tumor suppressive programs. Although p53 is mutated in ∼90% of small cell lung cancer (SCLC) tumors, how p53 mediates tumor suppression in this context is unknown. Here, using a mouse model of SCLC in which endogenous p53 expression can be conditionally and temporally regulated, we show that SCLC tumors maintain a requirement for p53 inactivation. However, we identified tumor subtype heterogeneity between SCLC tumors such that p53 reactivation induces a canonical senescence response in a subset of tumors, while, in others, p53 induces a non-apoptotic form of cell death that culminates in necrosis. We pinpointed the cyclophilin family of peptidyl prolyl cis-trans isomerases as critical determinants of a p53-induced transcriptional program that is specific to SCLC tumors and cell lines that are poised to undergo p53-mediated necrosis. Importantly, inhibition of cyclophilin isomerase activity suppresses SCLC subtype-specific p53-mediated death by limiting p53 transcriptional output without impacting chromatin binding. Our study demonstrates that intertumoral heterogeneity in SCLC can influence the biological response to p53 restoration, describes a novel mechanism of p53-regulated necrotic cell death, and uncovers new targets for the treatment of this most-recalcitrant tumor type.
Automated prediction of site and sequence of protein modification with ATRP initiators
Authors: Patel Arth , Smith Paige N. , Russell Alan J. , Carmali Sheiliza
Published: July 2022

One of the most straightforward and commonly used chemical modifications of proteins is to react surface amino groups (lysine residues) with activated esters. This chemistry has been used to generate protein-polymer conjugates, many of which are now approved therapeutics. Similar conjugates have also been generated by reacting activated ester atom transfer polymerization initiators with lysine residues to create biomacromolecular initiators for polymerization reactions. The reaction between activated esters and lysine amino groups is rapid and has been consistently described in almost every publication on the topic as a “random reaction”. A random reaction implies that every accessible lysine amino group on a protein molecule is equally reactive, and as a result, that the reaction is indiscriminate. Nonetheless, the literature contradicts itself by also suggesting that some lysine amino groups are more reactive than others (as a function of pKa, surface accessibility, temperature, and local environment). If the latter assumption is correct, then the outcome of these reactions cannot be random at all, and we should be able to predict the outcome from the structure of the protein. Predicting the non-random outcome of a reaction between surface lysines and reactive esters could transform the speed at which active bioconjugates can be developed and engineered. Herein, we describe a robust integrated tool that predicts the activated ester reactivity of every lysine in a protein, thereby allowing us to calculate the non-random sequence of reaction as a function of reaction conditions. Specifically, we have predicted the intrinsic reactivity of each lysine in multiple proteins with a bromine-functionalised N-hydroxysuccinimide initiator molecule. We have also shown that the model applied to PEGylation. The rules-based analysis has been coupled together in a single Python program that can bypass tedious trial and error experiments usually needed in protein-polymer conjugate design and synthesis.
EvoBind: in silico directed evolution of peptide binders with AlphaFold
Authors: Bryant Patrick , Elofsson Arne
Published: July 2022

Currently, there is no accurate method to computationally design peptide binders towards a specific protein interface using only a target structure. Experimental methods such as phage display can produce strong binders, but it is impossible to know where these bind without solving the structures. Using AlphaFold2 (AF) and other AI methods to distinguish true binders has proven highly successful but relies on the availability of binding scaffolds. Here, we develop EvoBind, an in silico directed-evolution platform based on AF that designs peptide binders towards an interface using only sequence information. We show that AF can distinguish between native and mutated peptide binders using the plDDT score and find that AF adapts the receptor interface structure to the binders during optimisation. We analyse previously designed minibinder proteins and show that AF can distinguish designed binders from non-binders. We compare ELISA ratios of different peptide binders and find the affinity can not be distinguished among binders, possibly due to varying binding sites and low AF confidence. We test the recovery of binding motifs and find that up to 75% of motifs are recovered. In principle, EvoBind can be used to design binders towards any interface conditioned on if AF can predict these. We expect that EvoBind will aid experimentalists substantially, providing a starting point for further laboratory analysis and optimisation. We hope that the use of AI-based methods will come to make binder design significantly cheaper and more accurate in tackling unmet clinical needs. EvoBind is freely available at: https://colab.research.google.com/github/patrickbryant1/EvoBind/blob/master/EvoBind.ipynb
Inositol acylation of phosphatidylinositol mannosides: a rapid mass response to membrane fluidization in mycobacteria
Authors: Nguyen Peter P. , Kado Takehiro , Prithviraj Malavika , Siegrist M. Sloan , Morita Yasu S.
Published: July 2022

Mycobacteria share an unusually complex, multilayered cell envelope, which contributes to adaptation to changing environments. The plasma membrane is the deepest layer of the cell envelope and acts as the final permeability barrier against outside molecules. There is an obvious need to maintain the plasma membrane integrity, but the adaptive responses of plasma membrane to stress exposure remain poorly understood. Using chemical treatment and heat stress to fluidize the membrane, we show here that phosphatidylinositol (PI)-anchored plasma membrane glycolipids known as PI mannosides (PIMs) rapidly remodel their structures upon membrane fluidization in Mycobacterium smegmatis. Without membrane stress, PIMs are predominantly in a tri-acylated form: two acyl chains of PI moiety plus one acyl chain modified at one of the mannose residues. Upon membrane fluidization, the fourth fatty acid is added to the inositol moiety of PIMs, making them tetra-acylated variants. PIM inositol acylation is a rapid response independent of de novo protein synthesis, representing one of the fastest mass conversions of lipid molecules found in nature. Strikingly, we found that M. smegmatis is more resistant to the bactericidal effect of a cationic detergent after benzyl alcohol preexposure. We further demonstrate that fluidization-induced PIM inositol acylation is conserved in pathogens such as Mycobacterium tuberculosis and Mycobacterium abscessus. Our results demonstrate that mycobacteria possess a mechanism to sense plasma membrane fluidity change. We suggest that inositol acylation of PIMs is a novel membrane stress response that enables mycobacterial cells to resist membrane fluidization.