bluuebunny/crossref_metadata_2025 huggingface dataset:
Proximal binding pocket Arg717 substitutions in Escherichia coli AcrB cause clinically relevant divergencies in resistance profiles
Authors: Zwama Martijn , Nishino Kunihiko
Published: December 2021

Multidrug resistance (MDR) in bacteria can be caused by the over-expression of multidrug efflux pumps belonging to the Resistance-Nodulation-Division (RND) superfamily of proteins. These intrinsic or acquired pumps can export a wide range of antibiotics. Recently, amino acid substitutions within these pumps have been observed in resistant clinical strains. Among others, two of these worrying gain-of-function mutations are R717L and R717Q in the proximal binding pocket of efflux pump AcrB (AcrB-Sa) found in azithromycin-resistant Salmonella enterica spp. We investigated the ramifications of these (and other) mutations in phylogenetically closely related AcrB from Escherichia coli (AcrB-Ec). We found that AcrB-Ec harboring Arg717 substitutions were significantly more effective in exporting all tested macrolides, with an up to 8-fold increase in the minimum inhibitory concentration (MIC) values (from 16 to 128 μg/mL for azithromycin). Interestingly, gain-of-function was also seen for fluoroquinolones (2-fold higher MICs), while there was a consistent loss-of-function for the export of novobiocin and β-lactam cloxacillin (2-fold lower MICs), pointing to a protein adaptation, which simultaneously partly compromised the efflux ability of other compounds with different molecular properties. Disk diffusion susceptibility testing corroborated the findings, as the R717Q and R717L mutant strains had significantly smaller inhibition zones for macrolides and fluoroquinolones and a larger inhibition zone for novobiocin, compared to the wild type. The spread and independent emergences of these potent efflux pump mutations highlight the necessity of control of, and adjustments to, treatments with antibiotics and the need for novel antibiotics and efflux pump inhibitors.

Importance

The over-expression of multidrug efflux pumps is a major cause of multidrug-resistant bacteria. The emergence of gain-of-function mutations in these pumps is additionally worrying, as these render last-resort antibiotic treatment options ineffective. The Arg717-mutations in related Salmonella Typhi and Paratyphi A AcrB caused azithromycin-resistant strains, with resistance levels past breakpoints. This is worrying, as azithromycin is a last-resort antibiotic after fluoroquinolones to treat typhoid and paratyphoid fever. Our findings that the same substitutions in closely related Escherichia coli AcrB cause an increase in MICs for both fluoroquinolones and macrolides is worrying as it shows the effect of one amino acid substitution on a multitude of treatment options. Additionally, the finding that the substitutions negatively impact the export of other drugs, such as β-lactam cloxacillin, suggests that treatment with multiple antibiotics may mitigate resistance and improve treatment. Our findings also imply the urge to develop novel antibiotics and inhibitors.
Carbon dioxide, COVID-19 and the importance of restaurant ventilation: a case study from Spain approaching Christmas 2021
Authors: Moreno Teresa , Gibbons Wes
Published: December 2021

Restaurants present an especial challenge in the prevention of the spread of COVID-19 via exhalatory bioaerosols because customers are unprotected by facemasks while eating, so that ventilation protocols in such establishments become particularly important. However, despite the fact that this pandemic airborne disease has been with us for two full years, many restaurants are still not successfully prioritising air renovation as a key tool for reducing infection risk. We demonstrate this in the run-up to the 2021 Christmas celebrations by reporting on CO2 concentration data obtained from a hotel breakfast room and restaurants during the 5-day Spanish holiday period of 4th-8th December. In the case of the breakfast room, inadequate ventilation resulted in average CO2 levels ranging from 868 to 1237ppm on five consecutive days, with the highest levels coinciding with highest occupancy numbers. Inside the five restaurants, three of these were well ventilated, maintaining stable average CO2 concentrations below 700ppm. In contrast, two restaurants failed to keep average CO2 levels below 1000ppm, despite sporadic, but ineffective, attempts by one of them to ventilate the establishment. More effort needs to be made to foster in both restaurant managers and the general public an improved awareness of the value of CO2 concentrations as an infection risk proxy and the relevance of ventilation issues to the propagation of respiratory diseases.
Seroresponse to Inactivated and Recombinant Influenza Vaccines among Maintenance Hemodialysis Patients
Authors: Manley Harold J. , Lacson Eduardo K. , Aweh Gideon , Weiner Daniel E. , Miskulin Dana C. , Hsu Caroline M. , Kapoian Toros , Hayney Mary S. , Meyer Klemens B. , Johnson Doug S.
Published: December 2021

Rationale & Objectives

High dose influenza vaccine provides better protection against influenza infection in older adults than standard dose vaccine. We compared vaccine seroresponse among hemodialysis patients over 4 months after high dose inactivated (HD-IIV3), standard dose inactivated (SD-IIV4) or recombinant (RIV4) influenza vaccine.

Study Design

Prospective observational study.

Setting & Participants

Patients at four hemodialysis clinics who received influenza vaccination. Hemagglutination inhibition titers were measured at baseline and at 1, 2, 3 and 4 months following vaccination with HD-IIV3, SD-IIV4 or RIV4 influenza vaccine.

Outcome

The primary outcome was seroprotection rates at ≥1:40 and ≥1:160 (which correspond to antibody levels providing protection from infection in about 50% and 95% immunocompetent individuals, respectively) at 1, 3 and 4 months after vaccination.

Analytical approach

We determined GMT and seroprotection and seroconversion rates. Chi-square or Fisher exact tests were used for categorical data; continuous values were analyzed using Wilcoxon rank-sum tests. Generalized estimating equation was used to determine association between GMT and age.

Results

254 HD patients received HD-IIV3 (n=141), SD-IIV4 (n=36) or RIV4 (n=77) vaccine. A robust initial seroresponse to influenza A strains was observed after all 3 vaccines, with no difference in seroprotection rates at either the ≥1:40 or ≥1:160 titer at 1 and 2 months. Seroresponses to RIV4 and SD-IIV4 waned thereafter, such that by month 3 and 4, seroprotection by HD-IIV3 was significantly higher. Seroprotection rates were lower to the B strains across all three vaccines. Results trended similarly across patients aged below 65 years.

Limitations

Due to use of observational data, bias by unmeasured confounders may exist. Some of the subgroups by age were low in number.

Conclusions

Hemodialysis patients achieved high seroprotection rates after all 3 vaccines. The seroresponse waned more slowly with HD-IIV3 as compared to SD-IIV4 or RIV4 vaccine.
Rational Protein Engineering of Bacterial N-demethylases to Create Biocatalysts for the Production of Methylxanthines
Authors: Mills Shelby Brooks , Mock Meredith B. , Summers Ryan M.
Published: December 2021

Methylxanthines have a rich history as therapeutics and pharmaceuticals. However, natural dimethyl- and monomethylxanthines are difficult to produce synthetically, which has limited further exploration of these compounds in medicinal applications. A biosynthetic method for production of methylxanthines from whole cell biocatalysts is an attractive alternative. The bacterium Pseudomonas putida CBB5 contains a set of five enzymes, NdmABCDE, which are responsible for methylxanthine metabolism via N-demethylation to xanthine. The recent elucidation of the crystal structures of NdmA and NdmB, which remove the N1- and N3-methyl groups of caffeine, respectively, has opened new avenues to create biocatalysts for methylxanthine production. We have created a set of fifteen N-demethylase mutants and expressed them in E. coli BL21(DE3) as whole cell biocatalysts. The activity of each mutant was characterized for their affinity towards caffeine, theobromine, and theophylline. Two mutant enzymes in particular, labeled NdmA3 and NdmA4, both exhibited selectivity towards the N3-methyl group instead of the N1-methyl group. We also discovered that specific point mutations in NdmD resulted in the ability to tune the rate of the N-demethylase reaction. These new enzymes provide the capability of producing high-value methylxanthines, such as paraxanthine and 1-methylxanthine, through a biocatalytic route.
Ataxin-2, Twenty-four and Dicer-2 are components of a non-canonical cytoplasmic polyadenylation complex
Authors: Nadimpalli Hima Priyanka , Guitart Tanit , Coll Olga , Gebauer Fátima
Published: December 2021

Cytoplasmic polyadenylation is a mechanism to promote mRNA translation in a wide variety of biological contexts. A canonical complex centered around the conserved RNA-binding protein family CPEB has been shown to be responsible for this process. We have previously reported evidence for an alternative non-canonical, CPEB-independent complex inDrosophila, of which the RNA-interference factor Dicer-2 is a component. Here, we investigate Dicer-2 mRNA targets and protein co-factors in cytoplasmic polyadenylation. Using RIP-Seq analysis we identify hundreds of novel Dicer-2 target transcripts, ∼50% of which were previously found as targets of the cytoplasmic poly(A) polymerase Wispy, suggesting widespread roles of Dicer-2 in cytoplasmic polyadenylation. Large-scale immunoprecipitation revealed Ataxin-2 and Twenty-four among the high-confidence interactors of Dicer-2. Functional analysis indicate that both factors form an RNA-independent complex with Dicer-2, and are required for cytoplasmic polyadenylation of Dicer-2 targets. Our results reveal the composition of a novel cytoplasmic polyadenylation complex that operates duringDrosophilaearly embryogenesis.
Dissecting the stability determinants of a challenging de novo protein fold using massively parallel design and experimentation
Authors: Kim Tae-Eun , Tsuboyama Kotaro , Houliston Scott , Martell Cydney M. , Phoumyvong Claire M. , Lemak Alexander , Haddox Hugh K. , Arrowsmith Cheryl H. , Rocklin Gabriel J.
Published: December 2021

Designing entirely new protein structures remains challenging because we do not fully understand the biophysical determinants of folding stability. Yet some protein folds are easier to design than others. Previous work identified the 43-residue □ββ□ fold as especially challenging: the best designs had only a 2% success rate, compared to 39-87% success for other simple folds (1). This suggested the □ββ□ fold would be a useful model system for gaining a deeper understanding of folding stability determinants and for testing new protein design methods. Here, we designed over ten thousand new □ββ□ proteins and found over three thousand of them to fold into stable structures using a high-throughput protease-based assay. Nuclear magnetic resonance, hydrogen-deuterium exchange, circular dichroism, deep mutational scanning, and scrambled sequence control experiments indicated that our stable designs fold into their designed □ββ□ structures with exceptional stability for their small size. Our large dataset enabled us to quantify the influence of universal stability determinants including nonpolar burial, helix capping, and buried unsatisfied polar atoms, as well as stability determinants unique to the □ββ□ topology. Our work demonstrates how large-scale design and test cycles can solve challenging design problems while illuminating the biophysical determinants of folding.

Significance

Most computationally designed proteins fail to fold into their designed structures. This low success rate is a major obstacle to expanding the applications of protein design. In previous work, we discovered a small protein fold that was paradoxically challenging to design (only a 2% success rate) even though the fold itself is very simple. Here, we used a recently developed high-throughput approach to comprehensively examine the design rules for this simple fold. By designing over ten thousand proteins and experimentally measuring their folding stability, we discovered the key biophysical properties that determine the stability of these designs. Our results illustrate general lessons for protein design and also demonstrate how high-throughput stability studies can quantify the importance of different biophysical forces.
Physico-chemical principles of HDL-small RNA binding interactions
Authors: Michell Danielle L. , Allen Ryan M. , Cavnar Ashley B. , Contreras Danielle M. , Yu Minzhi , Semler Elizabeth M. , Ramirez Marisol A. , Zhu Wanying , May-Zhang Linda , Raby Chase A. , Castleberry Mark , Ifrim Anca , Carr John Jeffrey , Terry James G. , Schwendeman Anna , Davies Sean S. , Sheng Quanhu , Linton MacRae F. , Vickers Kasey C.
Published: December 2021

Extracellular small RNAs (sRNA) are abundant in many biofluids, but little is known about their mechanisms of transport and stability in RNase-rich environments. We previously reported that high-density lipoproteins (HDL) of mice were enriched with multiple classes of sRNA derived from the endogenous transcriptome, but also exogenous organisms. Here, we show that human HDL transports tRNA-derived sRNAs (tDRs) from host and non-host species which were found to be altered in human atherosclerosis. We hypothesized that HDL binds to tDRs through apolipoprotein A-I (apoA-I) and these interactions are conferred by RNA-specific features. We tested this using microscale thermophoresis and electrophoretic mobility shift assays and found that HDL bind tDRs and other single-stranded sRNAs with strong affinity, but not doublestranded RNA or DNA. Natural and synthetic RNA modifications influenced tDR binding to HDL. Reconstituted HDL bound tDRs only in the presence of apoA-I and purified apoA-I alone was sufficient for binding sRNA. Conversely, phosphatidylcholine vesicles did not bind tDRs. In summary, HDL preferentially binds to single-stranded sRNAs likely through non-ionic interactions with apoA-I. These studies highlight binding properties that likely enable extracellular RNA communication and provide a foundation for future studies to manipulate HDL-sRNA for therapeutic approaches to prevent or treat disease.
Tissue- and ethnicity-independent hypervariable DNA methylation states show evidence of establishment in the early human embryo
Authors: Derakhshan Maria , Kessler Noah J. , Ishida Miho , Demetriou Charalambos , Brucato Nicolas , Moore Gudrun E. , Fall Caroline H.D. , Chandak Giriraj R. , Ricaut Francois-Xavier , Prentice Andrew M. , Hellenthal Garrett , Silver Matt J.
Published: December 2021

We analysed DNA methylation data from 30 datasets comprising 3,474 individuals, 19 tissues and 8 ethnicities at CpGs covered by the Illumina450K array. We identified 4,143 hypervariable CpGs (“hvCpGs”) with methylation in the top 5% most variable sites across multiple tissues and ethnicities. hvCpG methylation was influenced but not determined by genetic variation, and was not linked to probe reliability, epigenetic drift, age, sex or cell heterogeneity effects. hvCpG methylation tended to covary across tissues derived from different germ-layers and hvCpGs were enriched for associations with periconceptional environment, proximity to ERV1 and ERVK retrovirus elements and parent-of-origin-specific methylation. They also showed distinctive methylation signatures in monozygotic twins. Together, these properties position hvCpGs as strong candidates for studying how stochastic and/or environmentally influenced DNA methylation states which are established in the early embryo and maintained stably thereafter can influence life-long health and disease.
An improved organ explant culture method reveals stem cell lineage dynamics in the adult Drosophila intestine
Authors: Marchetti Marco , Zhang Chenge , Edgar Bruce A.
Published: December 2021

In recent years, live-imaging techniques have been developed for the adult midgut of Drosophila melanogaster that allow temporal characterization of key processes involved in stem cell and tissue homeostasis. However, current organ culture techniques are limited to imaging sessions of ≤16 hours, an interval too short to track dynamic processes such as damage responses and regeneration, which can unfold over several days. Therefore, we developed a new organ explant culture protocol capable of sustaining midguts ex vivo for up to 3 days. This was made possible by the formulation of a culture medium specifically designed for adult Drosophila tissues with an increased Na+/K+ ratio and trehalose concentration, and by placing midguts at an air-liquid interface for enhanced oxygenation. We show that midgut progenitor cells can respond to gut epithelium damage ex vivo, proliferating and differentiating to replace lost cells, but are quiescent in healthy intestines. Using ex vivo gene induction to promote stem cell proliferation, we demonstrate that intestinal stem lineages can be traced through multiple cell divisions using live imaging. Both asymmetric and symmetric divisions can be identified in the reconstructed lineages. We find that daughter cells of asymmetric divisions remain in close proximity of each other, while the progeny of symmetric divisions actively move apart, with implications for cell differentiation and tissue organization. We show that the same culture set-up is useful for imaging adult renal tubules and ovaries for up to 72 hours. By enabling both long-term imaging and real-time ex vivo gene manipulation, our simple culture protocol provides a powerful tool for studies of epithelial biology and cell lineage behavior.
Histone demethylase UTX regulates glioblastoma progression through affecting periostin expression
Authors: Luan Yan , Liu Yingfei , Xue Jingwen , Wang Ke , Ma Kaige , Lu Haixia , Chen Xinlin , Liu Yong , Zhang Zhichao
Published: December 2021

The histone H3K27 demethylase UTX participates in regulating multiple cancer types. However, less is known about the UTX function in glioblastoma (GBM). This study aims to define the effect of UTX on GBM. GEPIA2 database analysis showed that UTX expression was significantly increased in GBM and inversely correlated with survival. Knockdown UTX inhibited GBM cell proliferation, migration, and invasion while promoting apoptosis. Moreover, knockdown UTX also hampered tumor growth in the heterotopic xenograft model. RNA-seq combined with qRT-PCR and ChIP-qPCR were used to identify the target genes. The results showed that the UTX-mediated genes were strongly associated with tumor progression and the extracellular environment. Protein-protein interaction analysis suggested that periostin (POSTN) interacted with most of the other UTX-mediated genes. POSTN supplement abolished the effect of UTX knockdown in GBM cells. Furthermore, silencing UTX exhibited similar antitumor effect in patient-derived glioblastoma stem-like cells, while UTX functions were partially restored after exposing POSTN. Our findings may reveal a new insight into the onset of gliomagenesis and progression, providing a promising therapeutic strategy for GBM treatment.

Bullet Points

UTX correlates with survival in glioblastoma. Silencing UTX decreased the levels of H3K27 methylation in the POSTN gene, thereby suppressing POSTN expression. UTX-mediated POSTN expression is crucial for glioblastoma cell growth and tumorigenesis.

UTX expression is increased in GBM and negatively correlated with survival.

UTX knockdown influences proliferation and apoptosis in both GBM cells and GSCs.

The antitumor effect of UTX knockdown is achieved by suppressing POSTN expression.
Intra-colony channel morphology in Escherichia coli biofilms is governed by nutrient availability and substrate stiffness
Authors: Bottura Beatrice , Rooney Liam M. , Hoskisson Paul A. , McConnell Gail
Published: December 2021

Nutrient-transporting channels have been recently discovered in mature Escherichia coli biofilms, however the relationship between intra-colony channel structure and the surrounding environmental conditions is poorly understood. Using a combination of fluorescence mesoscopy and a purpose-designed open-source quantitative image analysis pipeline, we show that growth substrate composition and nutrient availability have a profound effect on the morphology of intra-colony channels in mature E. coli biofilms. Under all nutrient conditions, intra-colony channel width was observed to increase non-linearly with radial distance from the centre of the biofilm. Notably, the channels were around 25% wider at the centre of glucose-limited biofilms compared to ammonium-limited biofilms. Channel density also differed in colonies grown on rich and minimal media, with the former creating a network of tightly packed channels and the latter leading to well-separated, wider channels with defined edges. Our approach paves the way for measurement of internal patterns in a wide range of biofilms, offering the potential for new insights into infection and pathogenicity.
Systematic analysis of low-affinity transcription factor binding site clusters in vitro and in vivo establishes their functional relevance
Authors: Shahein Amir , López-Malo Maria , Istomin Ivan , Olson Evan J. , Cheng Shiyu , Maerkl Sebastian J.
Published: December 2021

Transcription factor binding to a single binding site and its functional consequence in a promoter context are beginning to be relatively well understood. However, binding to clusters of sites has yet to be characterized in depth, and the functional relevance of binding site clusters remains uncertain. We employed a high-throughput biochemical method to characterize transcription factor binding to clusters varying across a range of affinities and configurations. We found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Furthermore, compared to an individual high-affinity binding site, small clusters with binding sites an order of magnitude lower in affinity give rise to higher mean occupancies at physiologically-relevant transcription factor concentrations in vitro. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters by inserting them into a synthetic minimal CYC1 and the native PHO5 S. cerevisiae promoter. In the minCYC1 promoter, clusters of low-affinity binding sites can generate transcriptional output comparable to a promoter containing three consensus binding sites. In the PHO5 promoter, replacing the native Pho4 binding sites with clusters of low-affinity binding sites recovered activation of these promoters as well. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters.
Translational readthrough as a potential therapeutic for AIPL1-associated Leber Congenital Amaurosis in a patient-derived iPSC-retinal organoid model
Authors: Leung Amy , Sacristan-Reviriego Almudena , Perdigão Pedro R. L. , Sai Hali , Georgiou Michalis , Kalitzeos Angelos , Carr Amanda-Jayne F. , Coffey Peter J. , Michaelides Michel , Bainbridge James , Cheetham Michael E. , van der Spuy Jacqueline
Published: December 2021

Leber Congenital Amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterised by severe sight impairment in infancy and rapidly progressive degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cGMP phosphodiesterase (PDE6) compared to control or CRISPR corrected organoids. Moreover, increased levels of cGMP were detected. The translational readthrough inducing drug (TRID) PTC124 was investigated as a potential therapeutic. LCA4 retinal organoids exhibited rescue of AIPL1 and PDE6; however, the level of full-length, functional AIPL1 induced through PTC124 treatment was insufficient to reduce cGMP levels and fully rescue the LCA4 phenotype. LCA4 retinal organoids are a valuable platform for the in vitro investigation of the molecular mechanisms that drive photoreceptor loss and for the evaluation of novel therapeutics.
Mathematical modeling suggests cooperation of plant-infecting viruses
Authors: Miller Joshua , Burch-Smith Tessa M. , Ganusov Vitaly V.
Published: December 2021

Viruses are major pathogens of agricultural crops. Viral infections often start after the virus enters the outer layer of a tissue or surface and many successful viruses, after local replication in the infected tissue, are able to spread systemically. Quantitative details of virus dynamics in plants, however, have been poorly understood, in part, because of the lack of experimental methods allowing to accurately measure the degree of infection in individual plant tissues. Recently, by using flow cytometry and two different flourescently-labeled strains of the Tobacco etch virus (TEV), Venus and BFP, kinetics of viral infection of individual cells in leaves of Nicotiana tabacum plants was followed over time [1]. A simple mathematical model, assuming that viral spread occurs from lower to upper leaves, was fitted to these data. While the the original model could accurately describe the kinetics of viral spread locally and systemically, we also found that many alternative versions of the model, for example, if viral spread starts at upper leaves and progresses to lower leaves or when virus dissemination is stopped due to an immune response, provided fits of the data with reasonable quality, and yet with different parameter estimates. These results strongly suggest that experimental measurements of the virus infection in individual leaves may not be sufficient to identify the pathways of viral dissemination between different leaves and reasons for viral control; we propose experiments that may allow discrimination between the alternatives. By analyzing the kinetics of coinfection of individual cells by Venus and BFP strains of TEV we found a strong deviation from the random infection model, suggesting cooperation between the two strains when infecting plant cells. Importantly, we showed that many mathematical models on the kinetics of coinfection of cells with two strains could not adequately describe the data, and the best fit model needed to assume i) different susceptibility of uninfected cells to infection by two viruses locally in the leaf vs. systemically from other leaves, and ii) decrease in the infection rate depending on the fraction of uninfected cells which could be due to a systemic immune response. Our results thus demonstrate the difficulty in reaching definite conclusions from extensive and yet limited experimental data and provide evidence of potential cooperation between different viral variants infecting individual cells in plants.
Synuclein alpha accumulation mediates podocyte injury in Fabry nephropathy
Authors: Abed A , Braun F , Sellung D , Woidy M , Eikrem O , Wanner N , von Cossel K , Muschol N , Gersting SW , Muntau AC , Kretz O , Najafian B , Tøndel C , Mauer M , Bork T , Grahammer F , Liang W , Eierhoff T , Römer W , Marti HP , Puelles VG , Schell C , Huber TB
Published: December 2021

Current therapies for Fabry disease are based on reversing intra-cellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosome dysfunction. However, their effect in the reversal of endorgan damage, like kidney injury and chronic kidney disease remains unclear. First, ultrastructural analysis of serial human kidney biopsies showed that longterm use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/CAS9-mediated α-Galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified alpha-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.
Antibiotic Resistance Genes and Taxa Analysis from Mat and Planktonic Microbiomes of Antarctic Perennial Ice-covered Lake Fryxell and Lake Bonney
Authors: Tallada Sheetal , Hall Grant , Barich Daniel , Morgan-Kiss Rachael M. , Slonczewski Joan L.
Published: December 2021

The perennial ice-covered lakes of the Antarctic McMurdo Dry Valleys harbor oligotrophic microbial communities that are separated geographically from other aquatic systems. Their microbiomes include planktonic microbes as well as lift-off mat communities that emerge from the ice. We used ShortBRED to quantify the antibiotic resistance genes (ARGs) from metagenomes of lift-off mats emerging from ice, and from filtered water samples of Lake Fryxell and Lake Bonney. The overall proportion of ARG hits was similar to that found in temperate-zone rural water bodies with moderate human inputs. Specific ARGs showed distinct distributions for the two lakes, and for mat versus planktonic sources. Metagenomic taxa distributions showed that mat phototrophs consisted mainly of cyanobacteria or betaproteobacteria, whereas the water column phototrophs were mainly protists. An enrichment culture of the betaproteobacterium Rhodoferax antarcticus from a Lake Fryxell mat sample showed an unusual mat-forming phenotype not previously reported for this species. Its genome showed no ARGs associated with betaproteobacteria, but had ARGs consistent with a minor Pseudomonas component. The Antarctic lake mats and water showed specific ARGs distinctive to the mat and water sources, but overall ARG levels were similar to those of temperate water bodies with moderate human inputs.
Neural defensive circuits underlie helping under threat in humans
Authors: Vieira Joana B. , Olsson Andreas
Published: December 2021

Empathy for others’ distress has long been considered the driving force of helping. However, when deciding to help others in danger, one must consider not only their distress, but also the risk to oneself. Whereas the role of self-defence in helping has been overlooked in human research, studies in other animals indicate defensive responses are necessary for the protection of conspecifics. In this pre-registered study (N=49), we demonstrate that human defensive neural circuits are implicated in helping others under threat. Participants underwent fMRI scanning while deciding whether to help another participant avoid aversive electrical shocks, at the risk of also being shocked. We found that higher engagement of neural circuits that coordinate fast escape from self-directed danger (including the insula, PAG and ACC) facilitated decisions to help others. Importantly, using Representational Similarity Analysis, we found that the strength with which the amygdala and insula uniquely represented the threat to oneself (and not the other’s distress) predicted helping. Our findings indicate that in humans, as other mammals, defensive mechanisms play a greater role in helping behaviour than previously understood.
The E3 ubiquitin ligase HECTD1 contributes to cell cycle progression through an effect on mitosis
Authors: Vaughan Natalie , Scholz Nico , Lindon Catherine , Licchesi Julien D. F.
Published: December 2021

Mechanistic studies of how protein ubiquitylation regulates the cell cycle, in particular during mitosis, has provided unique insights which have contributed to the emergence of the ‘Ubiquitin code’. In contrast to RING E3 ubiquitin ligases such as the APC/c ligase complex, the contribution of other E3 ligase families during cell cycle progression remains less well understood. Similarly, the contribution of ubiquitin chain types beyond homotypic K48 chains in S-phase or branched K11/K48 chains assembled by APC/c during mitosis, also remains to be fully determined. Our recent findings that HECTD1 ubiquitin ligase activity assembles branched K29/K48 ubiquitin linkages prompted us to evaluate its function during the cell cycle. We used transient knockdown and genetic knockout to show that HECTD1 depletion in HEK293T and HeLa cells decreases cell proliferation and we established that this is mediated through loss of its ubiquitin ligase activity. Interestingly, we found that HECTD1 depletion increases the proportion of cells with aligned chromosomes (Prometa/Metaphase). We confirmed this molecularly using phospho-Histone H3 (Ser28) as a marker of mitosis. Time-lapse microscopy of NEBD to anaphase onset established that HECTD1-depleted cells take on average longer to go through mitosis. To explore the mechanisms involved, we used proteomics to explore the endogenous HECTD1 interactome in mitosis and validated the Mitosis Checkpoint Complex protein BUB3 as a novel HECTD1 interactor. In line with this, we found that HECTD1 depletion reduces the activity of the Spindle Assembly Checkpoint. Overall, our data suggests a novel role for HECTD1 ubiquitin ligase activity in mitosis.
Functional organization of social perception in the human brain
Authors: Santavirta Severi , Karjalainen Tomi , Nazari-Farsani Sanaz , Hudson Matthew , Putkinen Vesa , Seppälä Kerttu , Sun Lihua , Glerean Enrico , Hirvonen Jussi , Karlsson Henry K. , Nummenmaa Lauri
Published: December 2021

Humans rapidly extract diverse and complex information from ongoing social interactions, but the perceptual and neural organization of the different aspects of social perception remains unresolved. We showed short film clips with rich social content to 97 healthy participants while their haemodynamic brain activity was measured with fMRI. The clips were annotated moment-to-moment for 112 social features. Cluster analysis revealed that 13 dimensions were sufficient for describing the social perceptual space. Regression analysis was used to map regional neural response profiles to different social features. Multivariate pattern analysis was then utilized to establish the spatial specificity of these responses. The results revealed a gradient in the processing of social information in the brain. Posterior temporal and occipital regions were broadly tuned to most social dimensions and the classifier revealed that these responses showed spatial specificity for social dimensions; in contrast Heschl gyri and parietal areas were also broadly associated with different social signals, yet the spatial patterns of responses did not differentiate social dimensions. Frontal and subcortical regions responded only to a limited number of social dimensions and the spatial response patterns did not differentiate social dimension. Altogether these results highlight the distributed nature of social processing in the brain.
Different temporal requirements for the LRR transmembrane receptors Tartan and Toll-2 in the formation of contractile interfaces at compartmental boundaries
Authors: Sharrock Thomas E. , Blanchard Guy B. , Evans Jenny , Sanson Bénédicte
Published: December 2021

Compartmental boundaries physically separate groups of epithelial cells, a property fundamental for the organization of the body plan in both insects and vertebrates. In many examples, this physical separation has been shown to be the consequence of a regulated increase in contractility of the actomyosin cortex at boundary cell-cell interfaces, a property important in developmental morphogenesis beyond compartmental boundary formation. In this study, we took an unbiased screening approach to identify cell surface receptors required for actomyosin enrichment and polarisation at parasegmental boundaries (PSBs) in early Drosophila embryos, leading us to uncover different temporal requirements for two LRR receptors, Tartan and Toll-2. First, we find that Tartan is required during germband extension for actomyosin enrichment at PSBs, confirming an earlier report. Next, by following in real time the dynamics of loss of boundary straightness in tartan mutant embryos compared to wildtype and ftz mutant embryos, we show that Tartan is not required beyond germband extension. At this stage, actomyosin enrichment at PSBs becomes regulated by Wingless signalling. We find that Wingless signalling regulates Toll-2 expression and we show that Toll-2 is required for planar polarization of actomyosin after the completion of germ-band extension. Thus the formation of contractile interfaces at PSBs depends on a dynamic set of LRR receptors cues. Our study also suggests that the number of receptor cues is small and that the receptors are interchangeable.
Overlapping stimulons arising in response to divergent stresses inEscherichia coli
Authors: Wang Huijing , McElfresh GW , Wijesuriya Nishantha , Podgorny Adam , Hecht Andrew D. , Ray J. Christian J.
Published: December 2021

Cellular responses to stress can cause a similar change in some facets of fitness even if the stresses are different. Lactose as a sole carbon source forEscherichia coliis an established example: too little causes starvation while excessive lactose import causes toxicity as a side-effect. In anE. colistrain that is robust to osmotic and ionic differences in growth media, B REL606, the rate of antibiotic-tolerant persister formation is elevated in both starvation-inducing and toxicity-inducing concentrations of lactose in comparison to less stressful intermediate concentrations. Such similarities between starvation and toxification raise the question of how much the global stress response stimulon differs between them. We hypothesized that a common stress response is conserved between the two conditions, but that a previously shown threshold driving growth rate heterogeneity in a lactose-toxifying medium would reveal that the growing fraction of cells in that medium to be missing key stress responses that curb growth. To test this, we performed RNA-seq in three representative conditions for differential expression analysis. In comparison to nominally unstressed cultures, both stress conditions showed global shifts in gene expression, with informative similarities and differences. Functional analysis of pathways, gene ontology terms, and clusters of orthogonal groups revealed signatures of overflow metabolism, membrane component shifts, and altered cytosolic and periplasmic contents in toxified cultures. Starving cultures showed an increased tendency toward stringent response-like regulatory signatures. Along with other emerging evidence, our results show multiple possible pathways to stress responses, persistence, and possibly other phenotypes. These results suggest a set of overlapping responses that drives emergence of stress-tolerant phenotypes in diverse conditions.
CD4 T cells are rapidly depleted from tuberculosis granulomas following acute SIV co-infection
Authors: Foreman Taylor W. , Nelson Christine E. , Kauffman Keith D. , Lora Nickiana E. , Vinhaes Caian L. , Dorosky Danielle E. , Sakai Shunsuke , Gomez Felipe , Fleegle Joel D. , Parham Melanie , Perera Shehan R. , Lindestam Arlehamn Cecilia S. , Sette Alessandro ,   Tuberculosis Imaging Program, Brenchley Jason M. , Queiroz Artur T.L. , Andrade Bruno B. , Kabat Juraj , Via Laura E. , Barber Daniel L.
Published: December 2021

The HIV-mediated decline in circulating CD4 T cells correlates with increased risk of active tuberculosis (TB)1–4. However, HIV/Mycobacterium tuberculosis (Mtb) co-infected individuals also have an increased incidence of TB prior to loss of CD4 T cells in blood3,5, raising the possibility that HIV co-infection leads to disruption of CD4 T cell responses at the site of lung infection before they are observed systemically. Here we used a rhesus macaque model of SIV/Mtb co-infection to study the early effects of acute SIV infection on CD4 T cells in pulmonary Mtb granulomas. Two weeks after SIV co-infection CD4 T cells were dramatically depleted from granulomas, before significant bacterial outgrowth, disease reactivation as measured by PET-CT imaging, or CD4 T cell loss in blood, airways, and lymph nodes. Mtb-specific CD4 T cells, CCR5-expressing, in granulomas were preferentially depleted by SIV infection. Moreover, CD4 T cells were preferentially depleted from the granuloma core and lymphocyte cuff relative to B cell-rich regions, and live imaging of granuloma explants showed that SIV co-infection reduced T cell motility. Thus, Mtb-specific CD4 T cells in pulmonary granulomas may be decimated before many patients even experience the first symptoms of acute HIV infection.
RNA viral communities are structured by host plant phylogeny in oak and conifer leaves
Authors: ter Horst Anneliek M. , Fudyma Jane D. , Bak Aurélie , Hwang Min Sook , Santos-Medellín Christian , Stevens Kristian A. , Rizzo David M. , Al Rwahnih Maher , Emerson Joanne B.
Published: December 2021

Wild plants can suffer devastating diseases, experience asymptomatic, persistent infections, and serve as reservoirs for viruses of agricultural crops, yet we have a limited understanding of the natural plant virosphere. To access representatives of locally and globally distinct wild plants and investigate their viral diversity, we extracted and sequenced dsRNA from leaves from 16 healthy oak and conifer trees in the UC Davis Arboretum (Davis, California). From de novo assemblies, we recovered 389 RNA-dependent RNA polymerase (RdRp) gene sequences from 384 putative viral species, and a further 580 putative viral contigs were identified with virus prediction software followed by manual confirmation of virus annotation. Based on similarity to known viruses, most recovered viruses were predicted to infect plants or fungi, with the highest diversity and abundance observed in the Totiviridae and Mitoviridae families. Phyllosphere viral community composition differed significantly by host plant phylogeny, suggesting the potential for host-specific viromes. The phyllosphere viral community of one oak tree differed substantially from other oak viral communities and contained a greater proportion of putative mycoviral sequences, potentially due to the tree’s more advanced senescence at the time of sampling. These results suggest that oaks and conifers harbor a vast diversity of viruses with as-yet unknown roles in plant health and phyllosphere microbial ecology.
A tale of two plasmids: contributions of plasmid associated phenotypes to epidemiological success among Shigella
Authors: De Silva P. Malaka , Stenhouse George E. , Blackwell Grace A. , Bengtsson Rebecca J. , Jenkins Claire , Hall James P.J. , Baker Kate S.
Published: December 2021

Dissemination of antimicrobial resistance (AMR) genes by horizontal gene transfer (HGT) mediated through plasmids is a major global concern. Genomic epidemiology studies have shown varying success of different AMR plasmids during outbreaks, but the underlying reasons for these differences are unclear. Here, we investigated two Shigella plasmids (pKSR100 and pAPR100) that circulated in the same transmission network but had starkly contrasting epidemiological outcomes to identify plasmid features that may have contributed to the differences. We used plasmid comparative genomics to reveal divergence between the two plasmids in genes encoding AMR, SOS response alleviation, and conjugation. Experimental analyses revealed that these genomic differences corresponded with reduced conjugation rates for the epidemiologically successful pKSR100, but more extensive AMR, reduced fitness costs, and a reduced SOS response in the presence of antimicrobials, compared with the less successful pAPR100. The discrepant phenotypes between the two plasmids are consistent with the hypothesis that plasmid associated phenotypes contribute to determining the epidemiological outcome of AMR HGT and suggest that phenotypes relevant in responding to antimicrobial pressure and fitness impact may be more important than those around conjugation in this setting. Plasmid phenotypes could thus be valuable tools in conjunction with genomic epidemiology for predicting AMR dissemination.
DrosophilaSPG12 ortholog, reticulon-like 1, governs presynaptic ER organization and Ca2+dynamics
Authors: Pérez-Moreno Juan José , Smith Rebecca C , Oliva Megan K , Gallo Filomena , Ojha Shainy , Müller Karin H. , O’Kane Cahir J
Published: December 2021

Neuronal endoplasmic reticulum (ER) appears continuous throughout the cell. Its shape and continuity are influenced by ER-shaping proteins, mutations in which can cause distal axon degeneration in Hereditary Spastic Paraplegia (HSP). We therefore asked how loss ofRtnl1, aDrosophilaortholog of the human HSP geneRTN2(SPG12), which encodes an ER-shaping protein, affects ER organization and the function of presynaptic terminals. Loss of Rtnl1 depleted ER membrane markers atDrosophilapresynaptic motor terminals, and appeared to deplete narrow tubular ER while leaving cisternae largely unaffected, thus suggesting little change in resting Ca2+storage capacity. Nevertheless, these changes were accompanied by major reductions in activity-evoked Ca2+fluxes in the cytosol, ER lumen, and mitochondria, as well as reduced evoked and spontaneous neurotransmission. We found that reduced STIM-mediated ER-plasma membrane contacts underlie presynaptic Ca2+defects inRtnl1mutants. Our results show the importance of ER architecture in presynaptic physiology and function which are therefore potential factors in the pathology of HSP.
Music-selective cortex is sensitive to structure in both pitch and time
Authors: Boebinger Dana L. , Norman-Haignere Sam V. , McDermott Josh H. , Kanwisher Nancy G.
Published: December 2021

Converging evidence suggests that neural populations within human non-primary auditory cortex respond selectively to music. These neural populations respond strongly to a wide range of music stimuli, and weakly to other natural sounds and to synthetic control stimuli matched to music in many acoustic properties, suggesting that they are driven by high-level musical features. What are these features? Here we used fMRI to test the extent to which musical structure in pitch and time contribute to music-selective neural responses. We used voxel decomposition to derive music-selective response components in each of 15 participants individually, and then measured the response of these components to synthetic music clips in which we selectively disrupted musical structure by scrambling either the note pitches and/or onset times. Both types of scrambling produced lower responses compared to when melodic or rhythmic structure was intact. This effect was much stronger in the music-selective component than in the other response components, even those with substantial spatial overlap with the music component. We further found no evidence for any cortical regions sensitive to pitch but not time structure, or vice versa. Our results suggest that the processing of melody and rhythm are intertwined within auditory cortex.
Cryo-EM structure of the human CST•Polα/Primase complex in a recruitment state
Authors: Cai Sarah W. , Zinder John C. , Svetlov Vladimir , Bush Martin W. , Nudler Evgeny , Walz Thomas , de Lange Titia
Published: December 2021

The CST•Polα/Primase complex is essential for telomere overhang maintenance and additionally functions to counteract resection at double-strand breaks. We report a 4.6-Å resolution cryo-EM structure of CST•Polα/Primase, captured prior to catalysis in a recruitment state, which provides insights into the architecture and stoichiometry of the fill-in machinery. Our model informs on human disease mutations that cause Coats plus syndrome.