bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of high temperature and heavy precipitation on drinking water quality and child hand contamination levels in rural kenya
Authors: Powers Julie E. , Mureithi Maryanne , Mboya John , Campolo Jake , Swarthout Jenna M. , Pajka Joseph , Null Clair , Pickering Amy J.
Published: October 2022

Climate change may impact human health through the influence of weather on environmental transmission of diarrhea. Previous studies have found that high temperatures and heavy precipitation are associated with increased diarrhea prevalence, but the underlying causal mechanisms are not clear. We linked measurements of Escherichia coli in source water (n=1,673), stored drinking water (n=8,924), and hand rinses from children <2 years old (n=2,660) with publicly available gridded temperature and precipitation data (at ≤0.2 degree spatial resolution and daily temporal resolution) by the GPS coordinates and date of sample collection. Measurements were collected over a 3-year period across a 2500 km2 area in rural Kenya. In drinking water sources, high 7-day temperature was associated with a 0.16 increase in log10E. coli levels (p<0.001), while heavy 7-day total precipitation was associated with a 0.29 increase in log10E. coli levels (p<0.001). In household stored drinking water, heavy 7-day precipitation was associated with a 0.079 increase in log10E. coli levels (p=0.042). On child hands, high 7-day temperature was associated with a 0.39 decrease in log10E. coli levels (p<0.001). Our findings provide insight on how climate change could impact environmental transmission of bacterial pathogens in Kenya, and suggest water treatment could be a mitigation strategy.
Mechanoresponse of epithelial monolayers to in-plane and out-of-plane curvatures imposed by 3D microwells
Authors: Luciano Marine , Versaevel Marie , Vercruysse Eléonore , Gabriele Sylvain
Published: October 2022

The organization of epithelial tissues with precise spatial definition is essential to various biological processes and to generate curved epithelial structures. However, the regulation of the architecture and dynamics of collective epithelial assemblies by the matrix curvature remains understudied. Here, we photopolymerize microwells of various diameters in hydrogels to form curved epithelial structures such as breast epithelial lobules, and study how in-plane and out-of-plane curvatures modulate the mechanoresponse of epithelial tissues. In-plane curvature governed by the microwell radius drives the centripetal orientation of cells and nuclei close to the edge of the microwell, resulting from contractile forces exerted by a supracellular actomyosin purse-string. Convex out-of-plane curvature imposed at the microwell entrance leads to a vertical orientation of the nuclei towards the microwell axis. We demonstrated that increasing the out-of-plane curvature leads to more flatten and elongated nuclear morphologies with high levels of compacted chromatin. Epithelial cells exhibit higher directionality and speed around the microwell edge, demonstrating that the out-of-plane curvature significantly enhances the cellular trafficking. These findings demonstrate the importance of in-plane and out-of-plane curvatures in epithelial organization and how both can be leveraged to facilitate the engineering of curved structures to study curvature-dependent mechanotransduction pathways.
Structure of mycobacterial respiratory Complex I
Authors: Liang Yingke , Plourde Alicia , Bueler Stephanie A. , Liu Jun , Brzezinski Peter , Vahidi Siavash , Rubinstein John L.
Published: October 2022

Oxidative phosphorylation, the combined activity of the electron transport chain (ETC) and adenosine triphosphate synthase, has emerged as a valuable target for the treatment of infection by Mycobacterium tuberculosis and other mycobacteria. The mycobacterial ETC is highly branched with multiple dehydrogenases transferring electrons to a membrane-bound pool of menaquinone and multiple oxidases transferring electrons from the pool. The proton-pumping type I nicotinamide adenine dinucleotide (NADH) dehydrogenase (Complex I) is found at low abundance in the plasma membranes of mycobacteria in typical in vitro culture conditions and is often considered dispensable. We found that growth of Mycobacterium smegmatis in carbon-limited conditions greatly increased the abundance of Complex I and allowed isolation of a rotenone-sensitive preparation of the enzyme. Determination of the structure of the complex by cryoEM revealed the “orphan” two-component response regulator protein MSMEG_2064 as a subunit of the assembly. MSMEG_2064 in the complex occupies a site similar to the proposed redox sensing subunit NDUFA9 in eukaryotic Complex I. An apparent purine nucleoside triphosphate within the NuoG subunit resembles the GTP-derived molybdenum cofactor in homologous formate dehydrogenase enzymes. The membrane region of the complex binds acyl phosphatidylinositol dimannoside, a characteristic three-tailed lipid from the mycobacterial membrane. The structure also shows menaquinone, which is preferentially used over ubiquinone by gram-positive bacteria, in two different positions along the quinone channel and suggests that menaquinone interacts more extensively than ubiquinone with a key catalytic histidine residue in the enzyme.
Microbial mat compositions and localization patterns explain the virulence of black band disease in corals
Authors: Wada Naohisa , Iguchi Akira , Urabe Yuta , Yoshioka Yuki , Abe Natsumi , Takase Kazuki , Hayashi Shuji , Kawanabe Sakiko , Sato Yui , Tang Sen-Lin , Mano Nobuhiro
Published: October 2022

Black band disease (BBD) in corals is characterized by a distinctive, band-like microbial mat, which spreads across the tissues and often kills infected colonies. The microbial mat is dominated by cyanobacteria but also commonly contains sulfide-oxidizing bacteria (SOB), sulfate-reducing bacteria (SRB), and other microbes. The migration rate in BBD varies across different environmental conditions including temperature, light, and pH. However, whether variations in the migration rates reflect differences in the microbial consortium within the BBD mat remains unknown. Here, we show that the micro-scale surface structure, bacterial composition, and spatial distribution differed across BBD lesions with different migration rates. The migration rate was positively correlated with the relative abundance of potential SOBs belonging toArcobacteraceaelocalized in the middle layer within the mat and negatively correlated with the relative abundance of other potential SOBs belonging toRhodobacteraceae. Our study highlights the microbial composition in BBD as an important determinant of virulence.
Structural transition of the ground-state structure to steady-state structures by sequential binding of ATP to V/A-ATPase
Authors: Nakanishi Atsuko , Kishikawa Jun-ichi , Mitsuoka Kaoru , Yokoyama Ken
Published: October 2022

V/A-ATPase is a rotary ATPase that shares a common rotary catalytic mechanism with FoF1 ATP synthase. Structural images of V/A-ATPase obtained by single-particle cryo-electron microscopy (cryo-EM) during ATP hydrolysis identified several intermediates, revealing the rotary mechanism under steady-state conditions. Here, we identified the cryo-EM structures of V/A-ATPase corresponding to short-lived initial intermediates during the activation of the ground state structure by time-resolving snapshot analysis. These intermediate structures provide insights into how the ground-state structure changes to the active, steady state through the sequential binding of ATP to its three catalytic sites. All the intermediate structures of V/A-ATPase adopt the same asymmetric structure, whereas the three catalytic dimers adopt different conformations. This is significantly different from the initial activation process of FoF1, where the overall structure of the F1 domain changes during the transition from a pseudo-symmetric to a canonical asymmetric structure. Our findings will enhance the future prospects for the initial activation processes of the enzymes with dynamical information, which contains unknown intermediate structures in their functional pathway.
Host Phylogeny Shapes Viral Transmission Networks in an Island Ecosystem
Authors: French Rebecca K. , Anderson Sandra H. , Cain Kristal E. , Greene Terry C. , Minor Maria , Miskelly Colin M. , Montoya Jose M. , Muller Chris G. , Taylor Michael W. ,   Kākāpō Recovery Team, Holmes Edward C.
Published: October 2022

Viral transmission between host species underpins disease emergence. Both host phylogenetic relatedness and aspects of their ecology, such as species interactions and predator-prey relationships, may govern cross-species virus transmission and zoonotic risk, although their relative impact is unknown. By characterising the virome of a relatively isolated island ecological community linked through a food web we show that phylogenetic barriers result in distantly related host species sharing fewer viruses. Host ecology had a much smaller influence on overall virome composition. Network analysis revealed that hosts with a high diversity of viruses were more likely to gain new viruses, and that generalist viruses were more likely to infect new hosts. Such a highly connected ecological community heightens the risk of disease emergence, particularly among closely related species.

One-Sentence Summary

Sequencing of an entire island virome reveals that closely related hosts have highly connected virus communities, increasing emergence risk.
DNA-measuring Wadjet SMC ATPases restrict smaller circular plasmids by DNA cleavage
Authors: Liu Hon Wing , Roisné-Hamelin Florian , Beckert Bertrand , Myasnikov Alexander , Li Yan , Gruber Stephan
Published: October 2022

Structural maintenance of chromosomes (SMC) complexes fold DNA by loop extrusion to support chromosome segregation, genome maintenance, and gene expression. Wadjet systems (JetABCD/MksBEFG/EptABCD) are derivative SMC complexes with proposed roles in bacterial immunity against selfish DNA elements. Here, we show that JetABCD systems restrict extrachromosomal circular DNA with an upper size limit of about 100 kb, while a linear plasmid evades restriction. Recombinant preparations of a JetABCD complex cleave circular DNA regardless of its helical topology but not linear DNA; cleavage occurs at random positions and depends on ATP as well as the SMC ATPase. We solve a structure of the JetABCD core by cryo-EM revealing an alternative dimer-of-dimers configuration. The two SMC DNA motor units face in opposite orientations—rather than the same as observed with MukBEF—possibly representing a restriction-specific state of JetABCD. We hypothesize that JetABCD is a DNA-shape-specific endonuclease and present a model for the activation of DNA cleavage exclusively when extrusion of an entire plasmid has been completed by a single JetABCD complex. The self-stalling of two opposing DNA motor units may serve as signal to trigger DNA cleavage. Complete extrusion by a single complex cannot be achieved on the much larger chromosome, explaining how self-DNA may evade cleavage.
Context matters: environmental microbiota of ice cream processing facilities affects the inhibitory performance of two lactic acid bacteria against Listeria monocytogenes
Authors: Rolon M. Laura , Chandross-Cohen Tyler , Kaylegian Kerry E. , Roberts Robert F , Kovac Jasna
Published: October 2022

Pathogenic L. monocytogenes may inhabit dairy processing environments, increasing the risk for cross-contamination of foods. Using biocontrol microorganisms that inhibit or outcompete L. monocytogenes to complement sanitation of dairy processing facilities may enhance the control of L. monocytogenes. However, it remains unknown whether the resident microbiota of dairy processing facilities affects the antilisterial activity of biocontrol strains. Here, two lactic acid bacteria (LAB) strains (Enterococcus PS01155 and PS01156) were tested for their biocontrol potential in the context of microbiomes collected from three ice cream processing facilities (A, B, and C). Antilisterial ability was assessed by co-culturing LABs with 8-L. monocytogenes strains in the presence of microbiota for 3 days at 15°C, followed by quantification of the most probable number of attached L. monocytogenes. L. monocytogenes concentration increased by 0.38±0.77 log10 MPN/sample in treatments containing microbiota from facility A, while it decreased by 0.99±1.13 and 2.54±0.84 log10 MPN/sample in treatments with microbiota from facilities B and C, respectively. The attachment of LAB to an abiotic surface was assessed by co-culturing LABs in with the microbiomes at 15°C for 3 days, followed by characterization of attached microbiota composition using amplicon sequencing. All samples containing microbiomes from facilities A and B had high relative abundance of Pseudomonas, while samples with facility C microbiome had high relative abundance of Enterococcus. Overall, we show that microbiota composition of ice cream processing facilities affected the antilisterial ability of LABs.

IMPORTANCE

Antilisterial lactic acid bacteria strains had been proposed as biological pathogen control agents for application in food processing environments. However, the effect of resident food processing environment microbiota on the performance on antilisterial lactic acid bacteria strains is poorly understood. Our study shows that the composition of the microbiota collected from ice cream processing facilities’ environmental surfaces can affect the attachment and inhibitory effect of lactic acid bacteria strains against L. monocytogenes. Further studies are therefore needed to evaluate whether individual microbial taxa affect antilisterial properties of lactic acid bacteria strains and to characterize the underlying mechanisms.
Kinetics of naturally induced binding and neutralizing anti-SARS-CoV-2 antibody levels and potencies among Kenyan patients with diverse grades of COVID-19 severity
Authors: Kimotho John , Sein Yiakon , Sayed Shahin , Shah Reena , Mwai Kennedy , Saleh Mansoor , Wanjiku Perpetual , Mwacharo Jedidah , Nyagwange James , Karanja Henry , Kutima Bernadette , Gitonga John , Mugo Daisy , Karanu Ann , Moranga Linda , Oluoch Vivian , Shah Jasmit , Mutiso Julius , Mburu Alfred , Nneka Zaitun , Betti Peter , Mutinda Wanzila Usyu , Abdi Abdirahman Issak , Bejon Philip , Ochola-Oyier Lynette Isabella , Warimwe George M. , Nduati Eunice , Ndungu Francis M.
Published: October 2022

Background

Given the low levels of COVID-19 vaccine coverage in Sub-Saharan Africa, despite high levels of natural SARS-CoV-2 exposures, strategies for extending the breadth and longevity of naturally acquired immunity are warranted. Designing such strategies will require a good understanding of natural immunity.

Methods

We used ELISA to measure whole-spike IgG and spike-receptor binding domain (RBD) total immunoglobulins (Igs) on 585 plasma samples collected longitudinally over five successive time points within six months of COVID-19 diagnosis in 309 COVID-19 patients. We measured antibody neutralizing potency against the wild-type (Wuhan) SARS-CoV-2 pseudo-virus in a subset of 51 patients over three successive time points. Binding and neutralizing antibody levels and potencies were then tested for correlations with COVID-19 severities, graded according to the National Institute of Health (NIH), USA criteria.

Results

Rates of sero-conversion increased from Day 0 (day of PCR testing) to Day 180 (six months) (63.6% to 100 %) and (69.3 % to 97%) for anti-spike IgG and anti-spike-RBD binding Igs, respectively. Levels of these binding antibodies peaked at Day 28 (P<0.0001) and were subsequently maintained for six months without significant decay (p>0.99). Similarly, antibody neutralizing potencies peaked at Day 28 (p<0.0001) but had decreased by three-folds, six months after COVID-19 diagnosis (p<0.0001). Binding antibodies levels were highly correlated with neutralizing antibody potencies at all the time points analyzed (r>0.6, P<0.0001). Levels and potencies of binding and neutralizing antibodies increased with disease severity.

Conclusion

Most COVID-19 patients from Sub-Saharan Africa generate SARS-CoV-2 specific binding antibodies that remain stable during the first six months of infection. Although antibody binding levels and neutralizing potencies were directly correlated, the respective neutralizing antibodies decayed three-fold by the sixth month of COVID-19 diagnosis suggesting that they are short-lived, consistent with what has been observed elsewhere. Thus, just like for other populations, regular vaccination boosters will be required to broaden and sustain the high levels of predominantly naturally acquired anti-SARS-CoV-2 neutralizing antibodies.
Estimating the potential impact and diagnostic requirements for SARS-CoV-2 test-and-treat programs
Authors: Han Alvin X. , Hannay Emma , Carmona Sergio , Rodriguez Bill , Nichols Brooke E. , Russell Colin A.
Published: October 2022

Oral antivirals have the potential to reduce the public health burden of COVID-19. However, now that we have exited the emergency phase of the COVID-19 pandemic, declining SARS-CoV-2 clinical testing rates (average testing rates = ≪10 tests/100,000 people/day in low- and-middle income countries; <100 tests/100,000 people/day in high-income countries; September 2023) make the development of effective test-and-treat programs challenging. We used an agent-based model to investigate how testing rates and strategies affect the use and effectiveness of oral antiviral test-to-treat programs in four country archetypes of different income levels and demographies. We find that in the post-emergency phase of the pandemic, in countries where low testing rates are driven by limited testing capacity, significant population-level impact of test-and-treat programs can only be achieved by both increasing testing rates and prioritizing individuals with greater risk of severe disease. However, for all countries, significant reductions in severe cases with antivirals are only possible if testing rates were substantially increased with high willingness of people to seek testing. Comparing the potential population-level reductions in severe disease outcomes of test-to-treat programs and vaccination shows that test-and-treat strategies are likely substantially more resource intensive requiring very high levels of testing (>>100 tests/100,000 people/day) and antiviral use suggesting that vaccination should be a higher priority.
Non-Small Cell Lung Cancer Patients Treated with Anti-PD1 Immunotherapy Show Distinct Microbial Signatures and Metabolic Pathways According to Clinical Outcomes
Authors: Dora David , Ligeti Balazs , Kovacs Tamas , Revisnyei Peter , Galffy Gabriella , Dulka Edit , Krizsán Dániel , Kalcsevszki Regina , Megyesfalvi Zsolt , Dome Balazs , Weiss Glenn J. , Lohinai Zoltan
Published: October 2022

Background

Clinical outcomes of immune checkpoint inhibitor (ICI)-treated non-small cell lung cancer (NSCLC) patients might be associated with the gut metagenome.

Methods

Sixty-two Caucasian advanced-stage NSCLC patients treated with anti-PD1 immunotherapy were included. Gut bacterial signatures were evaluated by metagenomic sequencing and correlated with progression-free survival (PFS), PD-L1 expression defined by immunohistochemistry, and other clinicopathological parameters. The predictive role of PFS-related key bacteria was confirmed with multivariate statistical models and validated on an additional patient cohort (n=60). The functional microbial signature of distinct patient groups were determined by analyzing metabolic pathways. A random forest (RF) machine learning algorithm was used to assess the predictive power of taxonomic and metabolic microbial signatures.

Results

Alpha-diversity showed no significant difference in any comparison. However, there was a significant difference in beta-diversity between patients with long- (>6 months) vs. short (≤6 months) PFS and between chemotherapy (CHT)-treated vs. CHT-naive cases. Short PFS was associated with increased abundance of Firmicutes (F) and Actinobacteria phyla, whereas elevated abundance of Euryarcheota was specific for low-PD-L1 expression. F/Bacteroides (F/B) ratio was significantly increased in patients with short PFS. Multivariate analysis revealed an association between Alistipes shahii, Alistipes finegoldii, Barnesiella visceriola, and long PFS. In contrast, Streptococcus salivarius, Streptococcus vestibularis, and Bifidobacterium breve were associated with short PFS. Taxonomic profiles performed superiorly in predicting PFS (AUC=0.74), while metabolic pathways including Amino Acid Synthesis and Fermentation were better predictors of PD-L1 expression (AUC=0.87).

Conclusion

The specific metagenomic features of the gut microbiome including bacterial taxonomy and metabolic pathways are suggestive of ICI efficacy and PD-L1 expression in NSCLC patients.
Integrative genetic analysis identifies FLVCR1 as an essential component of choline transport in mammals
Authors: Kenny Timothy C. , Khan Artem , Son Yeeun , Yue Lishu , Heissel Søren , Gamazon Eric R. , Alwaseem Hanan , Hite Richard , Birsoy Kıvanç
Published: October 2022

Genome-wide association studies (GWAS) of serum metabolites have the potential to uncover genes that influence human metabolism. Here, we combined an integrative genetic analysis associating serum metabolites to membrane transporters with a coessentiality map of metabolic genes. This analysis revealed a connection between feline leukemia virus subgroup C cellular receptor 1 (FLVCR1) - a plasma membrane protein - and phosphocholine, a downstream metabolite of choline metabolism. Loss of FLVCR1 in human cells and in mice strongly impairs choline metabolism due to a block in choline import. Consistently, CRISPR-based genetic screens identified several components of the membrane phospholipid machinery as synthetic lethal with FLVCR1 loss. Finally, cells lacking FLVCR1 exhibit mitochondrial defects and upregulate the integrated stress response (ISR) through heme regulated inhibitors kinase (HRI). Altogether, these findings identify FLVCR1 as a universal mediator of choline transport in mammals and provide a platform to discover substrates for unknown metabolite transporters.
Three components of human brain gene expression reflect normative developmental programmes with specific links to neurodevelopmental disorders
Authors: Dear Richard , Wagstyl Konrad , Seidlitz Jakob , Markello Ross D. , Arnatkevičiūtė Aurina , Anderson Kevin M. , Bethlehem Richard A.I. ,   Lifespan Brain Chart Consortium, Raznahan Armin , Bullmore Edward T. , Vértes Petra E.
Published: October 2022

Human brain organisation emerges from the coordinated transcription of thousands of genes, and the first principal component (C1) of spatial whole genome expression was shown to reflect cortical hierarchy. Here, optimised processing of the Allen Human Brain Atlas revealed two new components of brain transcription, C2 and C3, which were distinctively enriched for neuronal, metabolic and immune processes, cell-types and cytoarchitecture, and genetic variants associated with intelligence. Using additional datasets (PsychENCODE, Allen Cell Atlas, and BrainSpan), we found that C1-C3 represent generalisable transcriptional programmes that are coordinated within cells, and differentially phased during foetal and postnatal development. Autism spectrum disorder and schizophrenia were specifically associated with C1/C2 and C3, respectively, across neuroimaging, gene expression, and genome-wide association studies. Evidence converged especially in support of C3 as a normative transcriptional programme for adolescent brain development, which can lead to atypical supragranular brain connectivity in people at high genetic risk for schizophrenia.
Whole-mount smFISH allows combining RNA and protein quantification at cellular and subcellular resolution
Authors: Zhao Lihua , Fonseca Alejandro , Meschichi Anis , Sicard Adrien , Rosa Stefanie
Published: October 2022

Multicellular organisms result from complex developmental processes largely orchestrated through the quantitative spatiotemporal regulation of gene expression. Yet, obtaining absolute counts of mRNAs at a 3-dimensional resolution remains challenging, especially in plants, due to high levels of tissue autofluorescence that prevent the detection of diffraction-limited fluorescent spots. In situ hybridization methods based on amplification cycles have recently emerged, but they are laborious and often lead to quantification biases. In this article, we present a simple method based on single molecule RNA fluorescence in situ hybridization (smFISH) to visualize and count the number of mRNA molecules in several intact plant tissues. In addition, with the use of fluorescent protein reporters, our method also enables simultaneous detection of mRNA and protein quantity, as well as subcellular distribution, in single cells. With this method, research in plants can now fully explore the benefits of the quantitative analysis of transcription and protein levels at cellular and subcellular resolution in plant tissues.
Typhoid toxin hijacks Wnt5a to potentiate TGFβ-mediated senescence and Salmonella infections
Authors: ElGhazaly Mohamed , Collins Mark O , Ibler Angela EM , Humphreys Daniel
Published: October 2022

Damage to our genome causes acute senescence in mammalian cells, which undergo growth arrest and release a secretome that elicits cell cycle arrest in bystander cells through the senescence-associated secretory phenotype (SASP). Thus, acute senescence is a powerful tumour suppressor. Salmonella enterica hijacks senescence through its typhoid toxin, which usurps unidentified factors in the stress secretome of senescent cells to mediate intracellular infections. Here, transcriptomics of toxin-induced senescent cells (txSCs) and proteomics of their secretome identified secreted ligands that activate the TGFβ pathway through SMAD transcription factors. The ligand Wnt5a established a self-amplifying positive feedback loop driving TGFβ signalling, which enforced autocrine senescence in txSCs and paracrine senescence in naive bystander cells by activation of DDRs. Wnt5a and GDF15 increased host cell susceptibility to infection. The study reveals how an innate defence against cancer is co-opted by a bacterial pathogen to cause widespread damage and mediate infections.
eCRUIS captures RNA-protein interaction in vitro and in vivo
Authors: Zhang Ziheng , Zhang Yuanbing , Liu Ji-Long
Published: October 2022

As an information bridge between DNA and protein, RNA regulates cellular processes and gene expression in a variety of ways. From synthesis to degradation, RNA is associated with a series of RNA-binding proteins. Therefore, it is very important to develop innovative methods to study the interaction between RNA and protein. Previously, we developed an RNA-centric method, called CRISPR-based RNA-United Interacting System (CRUIS), to capture RNA-protein interaction in cells. On this basis, here we develop an enhanced CRUIS (eCRUIS) by combining the power of dCas13d and the engineered promiscuous ligase TurboID. The new version allows us to label RNA-binding proteins on the target RNA within 30 minutes, which may be used in vivo. By introducing bait-assay with exogenous RNA, we confirm that eCRUIS can effectively label RNA-binding proteins on bait RNA in a short time. eCRUIS provides a wider range of in vitro and in vivo applications.
High-resolution structure of a microtubule-like tube composed of FtsZ–monobody complexes
Authors: Fujita Junso , Amesaka Hiroshi , Yoshizawa Takuya , Kuroda Natsuko , Kamimura Natsuki , Hara Mizuho , Inoue Tsuyoshi , Namba Keiichi , Tanaka Shun-ichi , Matsumura Hiroyoshi
Published: October 2022

FtsZ, a bacterial tubulin homologue, forms protofilaments and the Z-ring, which acts as a scaffold for accessory proteins during cell division. Although various studies have revealed its molecular mechanisms, the lack of high-resolution solution structures has hindered the understanding of the detailed mechanisms. Here, we developed a monobody (Mb) that binds FtsZs from Escherichia coli and Klebsiella pneumoniae (KpFtsZ) without affecting their GTPase activities. When expressed in E. coli cells, the Mb did not inhibit Z-ring formation but did inhibit cell division. The crystal structures of the KpFtsZ–Mb complexes revealed the epitope, and the cryoEM structure at 2.67 Å resolution showed a double helical tube consisting of two KpFtsZ protofilaments stabilized by the Mb filling interfilament gaps. Our structural analyses highlight the similarity between the microtubule and the FtsZ tube and the importance of the plasticity of FtsZ protofilaments.
Heterogeneous and Surface-Catalyzed Amyloid Aggregation Monitored by Spatially Resolved Fluorescence and Single Molecule Microscopy
Authors: Zhou Xin , Sinkjær Anders Wilgaard , Zhang Min , Pinholt Henrik Dahl , Mørck Nielsen Hanne , Hatzakis Nikos S. , van de Weert Marco , Foderà Vito
Published: October 2022

Amyloid aggregation is associated with many diseases and may also occur in therapeutic protein formulations. Addition of co-solutes is a key strategy to modulate the stability of proteins in pharmaceutical formulations and select inhibitors for drug design in the context of diseases. However, the heterogeneous nature of this multi-component system in terms of structures and mechanisms poses a number of challenges for the analysis of the chemical reaction. Combining a spatially resolved fluorescence approach with single molecule microscopy and machine learning approaches, we disentangle the different contributions from multiple species within a single aggregation experiment. Moreover, we link the presence of interfaces to the degree of heterogeneity of the aggregation kinetics and retrieve the rate constants and underlying mechanisms for single aggregation events, providing a general tool for a comprehensive analysis of self-assembly reactions.

Table of Contents
STAT5b is a key effector of NRG-1/ERBB4-mediated cardiomyocyte growth
Authors: Vaparanta Katri , Jokilammi Anne , Paatero Ilkka , Merilahti Johannes A. , Heliste Juho , Hemanthakumar Karthik Amudhala , Kivelä Riikka , Alitalo Kari , Taimen Pekka , Elenius Klaus
Published: October 2022

The growth factor neuregulin-1 (NRG-1) regulates hypertrophic and hyperplastic myocardial growth and is currently under clinical investigation as a treatment for heart failure. We have previously demonstrated that an isoform of the NRG-1 receptor ERBB4 (ERBB4 JM-b) expressed in cardiomyocytes selectively regulates the activation of STAT5b. To explore the role of STAT5b in NRG-1/EBBB4 mediated cardiomyocyte growth, severalin vitroandin vivomodels were utilized. The downregulation of NRG-1/ERBB4 signaling consistently reduced STAT5b activation and transcription of STAT5b target genesIgf1, MycandCdkn1ain murinein vitroandin vivomodels of myocardial growth.Stat5bknock-down in primary cardiomyocytes ablated NRG-1-induced cardiomyocyte hypertrophy. Stat5b was activated during NRG-1-induced hyperplastic myocardial growth and chemical inhibition of the Nrg-1/Erbb4 pathway led to the loss of myocardial growth and Stat5 activation in zebrafish embryos. Moreover, CRISPR/Cas9-mediated knock-down ofstat5bin zebrafish embryos resulted in reduced myocardial growth and heart failure as indicated by reduced ventricular ejection fraction. Dynamin-2 was discovered to control the cell surface localization of ERBB4 and the chemical inhibition of dynamin-2 downregulated NRG-1/ERBB4/STAT5b signaling in models of hypertrophic and hyperplastic myocardial growth. Finally, the activation of the NRG-1/ERBB4/STAT5b signaling pathway was explored in clinical samples representing pathological cardiac hypertrophy. The NRG-1/ERBB4/STAT5b signaling pathway was differentially regulated both at the mRNA and protein levels in the myocardium of patients with pathological cardiac hypertrophy as compared to myocardium of control subjects. These results establish the role for STAT5b, and dynamin-2 in NRG-1/ERBB4-mediated myocardial growth.
Assessing the influence of distinct IVF culture media on human pre-implantation development using single-embryo transcriptomics
Authors: Ducreux Bastien , Barberet Julie , Guilleman Magali , Pérez-Palacios Raquel , Teissandier Aurélie , Bourc’his Déborah , Fauque Patricia
Published: October 2022

The use of Assisted Reproductive Technologies (ART) is consistently rising across the world. However, making an informed choice on which embryo culture medium should be preferred to ensure satisfactory pregnancy rates and the health of future children critically lacks scientific background. Particularly, embryos within their first days of development are highly sensitive to their micro-environment. Here, we aimed to determine the impact of culture media composition on gene expression of human preimplantation embryos. By employing single-embryo RNA-sequencing after 2 or 5 days of post-fertilization culture in different commercially available media, we revealed medium-specific differences in gene expression changes. In particular, we found that culture medium composition can affect the dynamics of expression of developmentally relevant genes at day-2 but the differences were mitigated at the blastocyst stage. This study highlights the ability of embryos conceived in suboptimal in vitro culture media to recover proper transcriptome competency.
Functional genomics of cattle through integration of multi-omics data
Authors: Beiki Hamid , Murdoch Brenda M. , Park Carissa A. , Kern Chandlar , Kontechy Denise , Becker Gabrielle , Rincon Gonzalo , Jiang Honglin , Zhou Huaijun , Thorne Jacob , Koltes James E. , Michal Jennifer J. , Davenport Kimberly , Rijnkels Monique , Ross Pablo J. , Hu Rui , Corum Sarah , McKay Stephanie , Smith Timothy P.L. , Liu Wansheng , Ma Wenzhi , Zhang Xiaohui , Xu Xiaoqing , Han Xuelei , Jiang Zhihua , Hu Zhi-Liang , Reecy James M.
Published: October 2022

Functional annotation of the bovine genome was performed by characterizing the spectrum of RNA transcription using a multi-omics approach, combining long- and short-read transcript sequencing and orthogonal data to identify promoters and enhancers and to determine boundaries of open chromatin. A total number of 171,985 unique transcripts (50% protein-coding) representing 35,150 unique genes (64% protein-coding) were identified across tissues. Among them, 159,033 transcripts (92% of the total) were structurally validated by independent datasets such as PacBio Iso-seq, ONT-seq,de novoassembled transcripts from RNA-seq, or Ensembl and NCBI gene sets. In addition, all transcripts were supported by extensive independent data from different technologies such as WTTS-seq, RAMPAGE, ChIP-seq, and ATAC-seq. A large proportion of identified transcripts (69%) were novel, of which 87% were produced by known genes and 13% by novel genes. A median of two 5’ untranslated regions was detected per gene, an increase from Ensembl and NCBI annotations (single). Around 50% of protein-coding genes in each tissue were bifunctional and transcribed both coding and noncoding isoforms. Furthermore, we identified 3,744 genes that functioned as non-coding genes in fetal tissues, but as protein coding genes in adult tissues. Our new bovine genome annotation extended more than 11,000 known gene borders compared to Ensembl or NCBI annotations. The resulting bovine transcriptome was integrated with publicly available QTL data to study tissue-tissue interconnection involved in different traits and construct the first bovine trait similarity network. These validated results show significant improvement over current bovine genome annotations.
A Theory for High-Throughput Genetic Interaction Screening
Authors: McCarthy Madeline E. , Dodd William B. , Lu Xiaoming , Patel Nishi D. , Haskell Charlotte V. , Sanabria Hugo , Blenner Mark A. , Birtwistle Marc R.
Published: October 2022

Systematic, genome-scale genetic screens have been instrumental for elucidating genotype-phenotype relationships, but approaches for probing genetic interactions have been limited to at most ∼100 pre-selected gene combinations in mammalian cells. Here, we introduce a theory for high-throughput genetic interaction screens. The theory extends our recently developed Multiplexing using Spectral Imaging and Combinatorics (MuSIC) approach to propose ∼105 spectrally unique, genetically-encoded MuSIC barcodes from 18 currently available fluorescent proteins. Simulation studies based on constraints imposed by spectral flow cytometry equipment suggest that genetic interaction screens at the human genome-scale may be possible if MuSIC barcodes can be paired to guide RNAs. While experimental testing of this theory awaits, it offers transformative potential for genetic perturbation technology and knowledge of genetic function. More broadly, the availability of a genome-scale spectral barcode library for non-destructive identification of single-cells could find more widespread applications such as traditional genetic screening and high-dimensional lineage tracing.
Imaging of lactate metabolism in retinal Müller cells with a FRET nanosensor
Authors: Calbiague García Víctor , Chen Yiyi , Cádiz Bárbara , Paquet-Durand François , Schmachtenberg Oliver
Published: October 2022

Müller cells, the glial cells of the retina, provide metabolic support for photoreceptors and inner retinal neurons, and have been proposed as source of the significant lactate production of this tissue. To better understand the role of lactate in retinal metabolism, we expressed a lactate and a glucose nanosensor in organotypic mouse retinal explants cultured for 14 days, and used FRET imaging in acute vibratome sections of the explants to study metabolite flux in real time. Pharmacological manipulation with specific monocarboxylate transporter (MCT) inhibitors and immunohistochemistry revealed the functional expression of MCT2 and MCT4 in Müller cells. The introduction of nanosensors to measure key metabolites at the cellular level may contribute to a better understanding of heretofore poorly understood issues in retinal metabolism.
Cytoskeletal association of ATP citrate lyase controls the mechanodynamics of macropinocytosis
Authors: Puccini Joseph , Bar-Sagi Dafna
Published: October 2022

Macropinocytosis is an actin-dependent mode of non-selective endocytosis that mediates the uptake of extracellular fluid-phase cargoes. It is now well-recognized that tumor cells exploit macropinocytosis to internalize macromolecules that can be catabolized and used to support cell growth and proliferation under nutrient limiting conditions. Therefore, the identification of molecular mechanisms that control macropinocytosis is fundamental to the understanding of the metabolic adaptive landscape of tumor cells. Here we report that the acetyl-CoA producing enzyme, ATP citrate lyase (ACLY), is a key regulator of macropinocytosis and describe a heretofore unappreciated association of ACLY with the actin cytoskeleton. The cytoskeletal tethering of ACLY is required for the spatially-defined acetylation of heterodimeric actin capping protein, which we identify as an essential mediator of the actin remodeling events that drive membrane ruffling and macropinocytosis. Furthermore, we identify a requirement for mitochondrial-derived citrate, an ACLY substrate, for macropinocytosis, and show that mitochondria traffic to cell periphery regions juxtaposed to plasma membrane ruffles. Collectively these findings establish a new mode of metabolite compartmentalization that supports the spatiotemporal modulation of membrane-cytoskeletal interactions required for macropinocytosis by coupling regional acetyl-CoA availability with dynamic protein acetylation.

Significance

The scavenging of extracellular macromolecules via macropinocytosis is a key adaptive mechanism that supports the metabolic fitness of cancer cells. Although the importance of macropinocytosis for sustaining tumor cell growth under nutrient limiting conditions is well documented, less is known about the molecular mechanisms by which macropinocytosis is regulated. This study describes a previously uncharacterized dependence of macropinocytosis on the compartmentalized generation of acetyl-CoA through the association of ACLY with the actin cytoskeleton. This metabolic channeling process establishes a new mechanistic framework for understanding actin remodeling events that drive macropinocytosis.
Opposing transcription factors MYCL and HEY1 mediate the Notch-dependent airway stem cell fate decision
Authors: Byrnes Lauren E. , Deleon Rachel , Reiter Jeremy F. , Choksi Semil P.
Published: October 2022

Tissue function depends on the relative proportions of multiple cell types. In the airway, basal stem cells differentiate into both multiciliated and secretory cells, which together protect the lungs from inhaled pathogens and particulates. To define how airway stem cells differentiate, we mapped differentiation trajectories using single-cell mRNA sequencing (scRNA-seq) and identified a transitional intermediate cell state in between basal stem cells and differentiated cells. These intermediate cells induce different gene expression programs that precede differentiation into either multiciliated or secretory cells. For example, we found that within the intermediate cell population, multiciliated cell precursors express Mycl, encoding a MYC-family transcription factor, and secretory cell precursors express Hey1, encoding a transcriptional repressor. We also found that Notch signaling acts on intermediate cells to repress Mycl and induce Hey1. We further show MYCL expression is sufficient to drive multiciliated cell fate, whereas HEY1 expression is sufficient to repress multiciliated cell fate. Using CUT&RUN, we made the surprising observation that MYCL and HEY1 bind to many of the same regulatory elements near genes encoding early regulators of multiciliated cell differentiation. We conclude that intermediate cells receiving Notch signals induce HEY1 to repress the multiciliated cell fate and become secretory cells, while intermediate cells not receiving Notch signals induce MYCL to promote the multiciliated cell fate. These experiments reveal that during airway stem cell differentiation Notch signaling balances the production of two different cell types by regulating the functions of two opposing transcription factors, MYCL and HEY1.
Characterizing Aerosol Generating Procedures with Background Oriented Schlieren
Authors: Howard N. Scott , Alrefaie Abdulaziz , Mejia Nicholas A. , Ugbeye Tosan , Schmidt Bryan E.
Published: October 2022

The potential for characterizing aerosol generating procedures (AGPs) using background oriented schlieren (BOS) flow visualization was investigated in two clinical situations. A human-scale BOS system was used on a manikin simulating jet ventilation and extubation. A novel approach to representation of the BOS images using line integral convolution allows direct evaluation of both magnitude and direction of the refractive index gradient field. Plumes issuing from the manikin’s mouth were clearly visualized and characterized in both experiments, and it is recommended that BOS be adapted into a clinical tool for risk evaluation in clinical environments.
Integrative dissection of gene regulatory elements at base resolution
Authors: Chen Zeyu , Javed Nauman , Moore Molly , Wu Jingyi , Vinyard Michael , Pinello Luca , Najm Fadi J. , Bernstein Bradley E.
Published: October 2022

Although vast numbers of putative gene regulatory elements have been cataloged, the sequence motifs and individual bases that underlie their functions remain largely unknown. Here we combine epigenetic perturbations, base editing, and deep learning models to dissect regulatory sequences within the exemplar immune locus encoding CD69. Focusing on a differentially accessible and acetylated upstream enhancer, we find that the complementary strategies converge on a ∼170 base interval as critical for CD69 induction in stimulated Jurkat T cells. We pinpoint individual cytosine to thymine base edits that markedly reduce element accessibility and acetylation, with corresponding reduction of CD69 expression. The most potent base edits may be explained by their effect on binding competition between the transcriptional activator GATA3 and the repressor BHLHE40. Systematic analysis of GATA and bHLH/Ebox motifs suggests that interplay between these factors plays a general role in rapid T cell transcriptional responses. Our study provides a framework for parsing gene regulatory elements in their endogenous chromatin contexts and identifying operative artificial variants.

Highlights

Base editing screens and deep learning pinpoint sequences and single bases affecting immune gene expression

An artificial C-to-T variant in a regulatory element suppresses CD69 expression by altering the balance of transcription factor binding

Competition between GATA3 and BHLHE40 regulates inducible immune genes and T cell states
Rabbit Development as a Model for Single Cell Comparative Genomics
Authors: Ton Mai-Linh N. , Keitley Daniel , Theeuwes Bart , Guibentif Carolina , Ahnfelt-Rønne Jonas , Andreassen Thomas Kjærgaard , Calero-Nieto Fernando J. , Imaz-Rosshandler Ivan , Pijuan-Sala Blanca , Nichols Jennifer , Benito-Gutiérrez Èlia , Marioni John C. , Göttgens Berthold
Published: October 2022

Biomedical research relies heavily on the use of model organisms to gain insight into human health and development. Traditionally, the mouse has been the favored vertebrate model, due to its experimental and genetic tractability. Non-rodent embryological studies however highlight that many aspects of early mouse development, including the egg-cylinder topology of the embryo and its method of implantation, diverge from other mammals, thus complicating inferences about human development. In this study, we constructed a morphological and molecular atlas of rabbit development, which like the human embryo, develops as a flat-bilaminar disc. We report transcriptional and chromatin accessibility profiles of almost 180,000 single cells and high-resolution histology sections from embryos spanning gastrulation, implantation, amniogenesis, and early organogenesis. Using a novel computational pipeline, we compare the transcriptional landscape of rabbit and mouse at the scale of the entire organism, revealing that extra-embryonic tissues, as well as gut and PGC cell types, are highly divergent between species. Focusing on these extra-embryonic tissues, which are highly accessible in the rabbit, we characterize the gene regulatory programs underlying trophoblast differentiation and identify novel signaling interactions involving the yolk sac mesothelium during hematopoiesis. Finally, we demonstrate how the combination of both rabbit and mouse atlases can be leveraged to extract new biological insights from sparse macaque and human data. The datasets and analysis pipelines reported here set a framework for a broader cross-species approach to decipher early mammalian development, and are readily adaptable to deploy single cell comparative genomics more broadly across biomedical research.
Is the Stalk of the SARS-CoV-2 Spike Protein Druggable?
Authors: Pipitò Ludovico , Reynolds Christopher A. , Deganutti Giuseppe
Published: October 2022

The SARS-CoV-2 virus spike protein (SP) is the vector of the virus infectivity. The high propensity to mutate in key regions responsible for the recognition of the human angiotensinconverting enzyme 2 (hACE2) or the antibodies produced by the immune system following infection or vaccination makes subunit 1 of the SP a difficult to target and, to date, efforts have not delivered any ACE2 binding inhibitor yet. The inherent flexibility of the stalk region, within subunit S2, is key to SARS-CoV-2 high infectivity because it facilitates the receptor binding domain encounter with ACE2. Thus, it could be a valuable therapeutic target. By employing a fragment-based strategy, we computationally studied the druggability of the conserved part of the SP stalk by means of an integrated approach that combines molecular docking with high-throughput molecular dynamics simulations. Our results suggest that the druggability of the stalk is challenging and provide the structural basis for such difficulty.