bluuebunny/crossref_metadata_2025 huggingface dataset:
Robust Binding Capability and Occasional Gene Loss of Telomere-Binding Proteins Underlying Telomere Evolution in Nematoda
Authors: Song Hobum , Kim Seonhong , Lim Daisy S. , Choi Hee-Jung , Lee Junho
Published: July 2024

Telomeres, the nucleoprotein complexes that protect the ends of linear chromosomes, are essential for maintaining the stability of eukaryotic genomes. As telomeres generally consist of repetitive DNA associated with specifically bound proteins, telomeric repeat motifs (TRMs) are thought to be difficult to evolve. However, a recent study identified nematodes with telomeric repeats distinct from the canonical TTAGGC motif. Here, we investigated how telomere repeats could have evolved despite the challenge posed by the specificity of telomere-binding proteins (TBPs) to the telomeric DNA in Nematoda. We performed a phylogenetic analysis and electrophoresis mobility shift assays to assess the binding affinities of two TBPs, which displayed different conservation patterns. Our results revealed that the well-conserved protein CEH-37 exhibits limited specificity, unable to distinguish telomeric repeats found in nematodes except for the TTAGGG motif, while the less conserved POT proteins displayed rigid specificity. These findings suggest that the emergence of novel telomeric repeat motifs correlated with the characteristics and evolutionary outcomes of TBPs in Nematoda. Our study not only revealed the dynamics of telomere evolution but also enhanced the understanding of the evolutionary relationship between proteins and DNAs.

Lay summary

Telomeres are chromosomal end structures composed of repetitive DNA where telomere-binding proteins (TBPs) specifically bind. This nucleoprotein complex is crucial for eukaryotes to maintain their genome integrity, making it difficult to evolve. However, a recent study identified nematodes with non- canonical telomeric repeat motifs (TRMs). Through an analysis of TBP conservation in Nematoda and assessments of their binding affinities, we observed that the binding capabilities of TBPs align with their conservation patterns. This correlation is also linked to the emergence of novel TRMs, explaining the evolution progress of TRMs in Nematoda. The evolutionary process discovered in this research is not limited to telomeres, providing further insights into evolutionary relationships between proteins and DNA.
Development of shuttle vector-based transformation systems forChlamydia pecorumandChlamydia caviae
Authors: Fässler Nadja , Biggel Michael , Jelocnik Martina , Borel Nicole , Marti Hanna
Published: July 2024

Chlamydia (C.) abortus,C. caviaeandC. pecorumare obligate intracellular, zoonotic pathogens, which have all been associated with community-acquired pneumonia in humans.C. abortusis the causative agent of enzootic ovine abortion in small ruminants and can lead to miscarriage in women.C. caviaecauses conjunctivitis in guinea pigs, whileC. pecorumis found in livestock, resulting in economic losses and contributing to the decline of the koala population in Australia. Studying the biology of these bacteria has been challenging due to a dearth of genetic tools. This study aimed to establish transformation systems forC. abortusandC. pecorumusing shuttle vectors and to expand upon already existing protocols forC. caviae. Shuttle vectors comprised the cryptic plasmid of the chlamydial species of interest, the pUC19 origin of replication (ori), a beta-lactamase (bla), and genes that mediate heterologous expression of fluorescent proteins (GFP, mNeonGreen, mScarlet). AC. suis-tailored transformation protocol and a previously established protocol forC. psittaci, C. trachomatisandC. pneumoniaewere applied. WhileC. pecorumandC. caviaetransformation experiments were successful, transformation ofC. abortusremained ineffective. Shuttle vectors yielded stable transformants over several passages in the presence and absence of selective antibiotics while the fluorescence intensity of GFP was superior compared to mNeonGreen. Finally, we co-cultured GFP- and mScarlet-expressingC. pecorumstrains demonstrating that both fluorophores can be detected in the same cell or even inclusion, possibly promoting homologous recombination. These findings open new avenues into our understanding of interstrain and interspecies co-infection dynamics bothin vitroandin vivo.
Material usage in commercial lateral flow assay kits
Authors: Wöhrle Marie-Louise , Street Alice , Kersaudy-Kerhoas Maïwenn
Published: July 2024

Objective

To assess material usage in commercial lateral flow assay kits and determine baselines for quantitative material requirements in target product profiles for new tests.

Methods

We collected and weighted the components of 21 different commercial COVID-19 lateral flow test kits, with emergency use authorisations from the UK MHRA, EU, the US FDA, and the WHO EUL. We took test kits apart manually, classified components and weighted them individually.

Findings

Large variations in the total average weights of COVID-19 LFA cassette test kits were observed from 13 g per test to 84 g. The average weight of standard LFA housing in the sample is 4.1 g per casing (range 2.47 g - 6.54 g) whilst the outer packaging makes up between 25% and over 88% of the whole kit and was found to be a large source of weight variations, followed by instructions and LFA housing types.

Conclusion

The contribution of LFA tests to global plastic pollution is set to grow year-on-year due to increasingly decentralised testing. Wide variation in the weight of components included in existing COVID-19 test kits suggests there is scope for manufacturers to reduce the amount of materials, including plastic, in test products. We propose that a quantitative baseline of material usage is introduced in future target product profiles for LFA format test kits. This would limit the number of products with a large volume of plastic from reaching the market and reduce the burden of plastic waste from diagnostic testing on local waste management systems.
Depletion of skin bacteria by topical antibiotic treatment accelerates onset of Zika virus disease in mice
Authors: Langendries Lana , Verwimp Sam , Jacobs Sofie , Alpizar Yeranddy Aguiar , Malengier-Devlies Bert , Abdelnabi Rana , Raes Jeroen , Callewaert Chris , Yshii Lidia , Delang Leen
Published: July 2024

Mosquito-borne flaviviruses have emerged as global health threats due to their rapid spread and high disease burden. The initial site of virus replication is the mosquito biting site in the host skin, yet the role of host skin bacteria remains unclear. Here, we observed accelerated progression of Zika virus (ZIKV)-induced disease in mice with depleted skin bacteria by topical antibiotic treatment prior to ZIKV infection. Increased viral loads in the blood, draining lymph node, spleen and brain of antibiotic-treated mice suggested faster ZIKV dissemination. Flow cytometry revealed increased frequencies of T cells in the lymph nodes, along with enhanced activation of T cells in the brain upon antibiotic treatment. The antibiotic-induced phenotype was rescued by restoring the skin bacteria prior to infection. Our findings demonstrate an increased susceptibility to ZIKV infection in mice with depleted skin microbiomes, suggesting a potential role of skin bacteria in modulating the infection outcome.
PRC1.6 localizes on chromatin with the human silencing hub (HUSH) complex for promoter-specific silencing
Authors: Rodríguez Tomás C. , Yurkovetskiy Leonid , Nagalekshmi Karthika , Lam Chin Hung Oscar , Jazbec Eva , Maitland Stacy A. , Wolfe Scot A. , Sontheimer Erik J. , Luban Jeremy
Published: July 2024

An obligate step in the life cycle of HIV-1 and other retroviruses is the establishment of the provirus in target cell chromosomes. Transcriptional regulation of proviruses is complex, and understanding the mechanisms underlying this regulation has ramifications for fundamental biology, human health, and gene therapy implementation. The three core components of the Human Silencing Hub (HUSH) complex, TASOR, MPHOSPH8 (MPP8), and PPHLN1 (Periphilin 1), were identified in forward genetic screens for host genes that repress provirus expression. Subsequent loss-of-function screens revealed accessory proteins that collaborate with the HUSH complex to silence proviruses in particular contexts. To identify proteins associated with a HUSH complex-repressed provirus in human cells, we developed a technique, Provirus Proximal Proteomics, based on proximity labeling with C-BERST (dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging). Our screen exploited a lentiviral reporter that is silenced by the HUSH complex in a manner that is independent of the integration site in chromatin. Our data reveal that proviruses silenced by the HUSH complex are associated with DNA repair, mRNA processing, and transcriptional silencing proteins, including L3MBTL2, a member of the non-canonical polycomb repressive complex 1.6 (PRC1.6). A forward genetic screen confirmed that PRC1.6 components L3MBTL2 and MGA contribute to HUSH complex-mediated silencing. PRC1.6 was then shown to silence HUSH-sensitive proviruses in a promoter-specific manner. Genome wide profiling showed striking colocalization of the PRC1.6 and HUSH complexes on chromatin, primarily at sites of active promoters. Finally, PRC1.6 binding at a subset of genes that are silenced by the HUSH complex was dependent on the core HUSH complex component MPP8. These studies offer new tools with great potential for studying the transcriptional regulation of proviruses and reveal crosstalk between the HUSH complex and PRC1.6.
Investigation of host plant contact withDiaphorina citri(Hemipreta: Psyllidae) by detectingD. citri-derived environmental DNA
Authors: Fujikawa T. , Fujiwara K. , Inoue H. , Hatomi H. , Hayashikawa S.
Published: July 2024

Citrus greening (Huanglongbing) disease has serious impacts on citrus production. Field monitoring of the Asian citrus psyllidDiaphorina citri(Hemiptera: Psyllidae), a vector of citrus greening disease, is essential to prevent the invasion and spread of this disease. This study reported a new method for determining the presence ofD. citriand traces of contact with host plants by collecting leaves of host plants from the survey area and detecting the environmental DNA (eDNA) derived fromD. citri. The results showed that the method can determine the presence ofD. citri.AsD. citri-derived eDNA is contained in DNA solutions of plants with a history of contact withD. citri, we succeeded in detecting not only genes conserved inD. citrimitochondria but also genes ofD. citrisymbiont organisms consisting ofWolbachia,Wolbachiaphage,CandidatusCarsonella sp., andCandidatusProfftella sp.Diaphorina citri-derived eDNA could be detected in host plants even after only 10 min of contact withD. citriand could still be detected 6 months after contact. This technology has the potential to traceD. citrifrom their traces without individual detection and is expected to greatly contribute to the early detection and invasion warning of citrus greening disease in the future.
A conformational fingerprint for amyloidogenic light chains
Authors: Paissoni Cristina , Puri Sarita , Broggini Luca , Sriramoju Manoj K. , Maritan Martina , Russo Rosaria , Speranzini Valentina , Ballabio Federico , Nuvolone Mario , Merlini Giampaolo , Palladini Giovanni , Hsu Shang-Te Danny , Ricagno Stefano , Camilloni Carlo
Published: July 2024

Immunoglobulin light chain amyloidosis (AL) and multiple myeloma (MM) both share the overproduction of a clonal light chain (LC). However, while LCs in MM remain soluble in circulation, AL LCs misfold into toxic soluble species and amyloid fibrils that accumulate in organs, leading to distinct clinical manifestations. The significant sequence variability of LCs has hindered understanding of the mechanisms driving LC aggregation. Nevertheless, emerging biochemical properties, including dimer stability, conformational dynamics, and proteolysis susceptibility, distinguish AL LCs from those in MM under native conditions. This study aimed to identify a conformational fingerprint distinguishing AL from MM LCs. Using small-angle X-ray scattering (SAXS) under native conditions, we analyzed four AL and two MM LCs. We observed that AL LCs exhibited a slightly larger radius of gyration and greater deviations from X-ray crystallography-determined or predicted structures, reflecting enhanced conformational dynamics. SAXS data, integrated with molecular dynamics (MD) simulations, revealed a conformational ensemble where LCs adopt multiple states, with variable and constant domains either bent or straight. AL LCs displayed a distinct, low-populated, straight conformation (termed H state), which maximized solvent accessibility at the interface between constant and variable domains. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experimentally validated this H state. These findings reconcile diverse experimental observations and provide a precise structural target for future drug design efforts.
In vitroone-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics system
Authors: Tanaka Ryota , Tamao Kenji , Ono Mana , Yamayoshi Seiya , Kawaoka Yoshihiro , Su’etsugu Masayuki , Noji Hiroyuki , Tabata Kazuhito V.
Published: July 2024

The reverse genetics system, which allows the generation of influenza viruses from plasmids encoding viral genome, is a powerful tool for basic research on viral infection mechanisms and application research such as vaccine development. However, conventional plasmid construction usingEscherichia coli(E. coli) cloning is time-consuming and has difficulties handling DNA encoding genes toxic for E. coli or highly repeated sequences. These limitations hamper rapid virus synthesis. In this study, we establish a very rapid in vitro one-pot plasmid construction (IVOC) based virus synthesis. This method dramatically reduced the time for genome plasmid construction, which was used for virus synthesis, from several days or more to about 8 hours. Moreover, infectious viruses could be synthesized with a similar yield to the conventionalE. colicloning-based method with high accuracy. The applicability of this method was also demonstrated by the generation of recombinant viruses carrying reporter genes from the IVOC products. This method is expected to potentially advance further understanding of influenza viruses and apply to other RNA viruses.
Larva in the loop, a closed loop machine interface system forDanio reriolarvae
Authors: Jutoy John , Jung Erica
Published: July 2024

The optokinetic response (OKR) in Zebrafish (Danio Rerio) had been characterized for its robust response to visual stimuli. Expanding on these works, we developed a novel closed loop control schema to drive a robot utilizing the OKR of Zebrafish larvae. Our system keeps the body of a larva constrained via a novel agarose mold holder that allows for eye movement and vision. The larva is then put under a microscope camera and processed through computer vision to track its eyes via ellipse fitting. Relative eye angle data is then parsed through an algorithm and used to send movement signals to a robot on a lined track. Simultaneously, the robot returns its relative position with respect to the line and converts that information into an OKR stimulation animation which is displayed on an LCD screen in the ventral plane of the larva, thus closing the loop. Through this work we show the capability of larvae OKR to keep a robot on a linear track after an initial oblique entrance to the line. This work displays the potential of our system and how it can pave the way to a Zebrafish Brain-Machine Interface.
The impact of ERUPRon mitochondrial integrity mediated by PDK4
Authors: Mallick Priyanka , Maity Sebabrata , Mondal Rupsha , Roy Trina , Chakrabarti Oishee , Chakrabarti Saikat
Published: July 2024

ER and mitochondrial stress are often interconnected and considered as major contributors to aging as well as neurodegeneration. Co-ordinated induction of ERUPRand mitoUPRhas been observed in diabetes and pulmonary disorders. However, in the context of aging and neurodegeneration, regulation of this intra-organellar crosstalk has remained relatively elusive. Here, we demonstrate that pyruvate dehydrogenase kinase 4 (PDK4), a mitochondrial protein accumulates at the ER-mitochondrial contact sites (MAMs) during ER stress. Classically, PDK4 is known to phosphorylate PDHA1 (pyruvate dehydrogenase E1 subunit alpha 1), and plays a significant role in regulating the oxidative phosphorylation driven ATP production. In this study, we propose a non-canonical function of PDK4; we show that it acts as a connecting link between ERUPRand mitoUPRwith significance in aging and neurodegeneration. Transcriptomics analyses show increased PDK4 levels upon drug induced ER stress. We detect elevated PDK4 levels in brain lysates of Alzheimer’s disease (AD) patients, as well as inin vivoandex vivoAD models. Additionally, exogenous expression of PDK4 was found to refine ER-mitochondrial communication, significantly altering mitochondrial morphology and function. Further, we also observe defective autophagic clearance of mitochondria under such conditions. It is prudent to suggest that elevated PDK4 levels could be one of the key factors connecting ERUPRwith mitoUPR, a phenotypic contributor in aging and at least some neurodegenerative diseases.
NanoTag - an IgG-free method for mapping DNA-protein interactions
Authors: Dimitriu Maria A. , Arzate-Mejía Rodrigo G. , Steg Leonard C. , Germain Pierre-Luc , Mansuy Isabelle M.
Published: July 2024

Genome-wide profiling of DNA-protein interactions in cells can provide important information about mechanisms of gene regulation. Most current methods for genome-wide profiling of DNA-bound proteins, such as ChIP-seq and CUT&Tag, use conventional IgG antibodies to bind target protein(s), which limits their applicability to targets for which high affinity and high specificity antibodies are available. Here we describe NanoTag, a novel method derived from CUT&Tag that is IgG-free and uses a nanobody to profile DNA-protein interactions. NanoTag is based on an anti-GFP nanobody-Tn5 transposase fusion that allows mapping GFP-tagged proteins associated with chromatin in a fast and cost-effective manner. We demonstrate the utility of NanoTag by profiling the histone mark H3K4me3 via its binding partner TATA box-binding protein-associated factor 3 (TAF3) and the transcription factors Nanog and CTCF in mouse embryonic stem cells expressing GFP-tagged targets. For the targets examined, NanoTag data shows high correlation to CUT&Tag data and displays a similarly high signal-to-noise ratio. Overall, NanoTag provides a flexible, IgG-free and cost-effective method to generate high resolution DNA-binding profiles in cells or tissues.
CUTS RNA Biosensor for the Real-Time Detection of TDP-43 Loss-of-Function
Authors: Xie Longxin , Merjane Jessica , Bergmann Cristian A , Xu Jiazhen , Hurtle Bryan , Donnelly Christopher J
Published: July 2024

Given the mounting evidence implicating TDP-43 dysfunction in several neurodegenerative diseases, there is a pressing need to establish accessible tools to sense and quantify TDP-43 loss-of-function (LOF). These tools are crucial for assessing potential disease contributors and exploring therapeutic candidates in TDP-43 proteinopathies. Here, we develop a sensitive and accurate real-time sensor for TDP-43 LOF: the CUTS (CFTR UNC13A TDP-43 Loss-of-Function) system. This system combines previously reported cryptic exons regulated by TDP-43 with a reporter, enabling the tracking of TDP-43 LOF through live microscopy and RNA/protein-based assays. We demonstrate CUTS’ effectiveness in detecting LOF caused by TDP-43 mislocalization and RNA binding dysfunction, and pathological aggregation. Our results highlight the sensitivity and accuracy of the CUTS system in detecting and quantifying TDP-43 LOF, opening avenues to explore unknown TDP-43 interactions that regulate its function. In addition, by replacing the fluorescent tag in the CUTS system with the coding sequence for TDP-43, we show significant recovery of its function under TDP-43 LOF conditions, highlighting CUTS’ potential for self-regulating gene therapy applications. In summary, CUTS represents a versatile platform for evaluating TDP-43 LOF in real-time and advancing gene-replacement therapies in neurodegenerative diseases associated with TDP-43 dysfunction.

Highlights

CUTS is a cryptic exon RNA biosensor enabling real-time detection of TDP-43 loss of splicing function.

CUTS exhibits a significant linear relationship with TDP-43 loss.

CUTS can deliver an autoregulated gene payload in response to TDP-43 loss-of-function.

TDP-43 homotypic phase transitions induce loss of splicing function via CUTS detection.
DeepOrientation: Deep Orientation Estimation of Macromolecules in Cryo-electron tomography
Authors: Hajarolasvadi Noushin , Phelippeau Harold , Martinez-Sanchez Antonio , Brandt Robert , Suau Pierre-Nicolas , Baum Daniel
Published: July 2024

Orientation estimation of macromolecules in cryo-electron tomography (cryo-ET) images is one of the fundamental steps in applying subtomogram averaging. The standard method in particle picking and orientation estimation is template matching (TM), which is computationally very expensive, with its performance depending linearly on the number of template orientations. In addition to conventional image processing methods like TM, the investigation of crowded cell environments using cryo-ET has also been attempted with deep learning (DL) methods. These attempts were restricted to macromolecule localization and identification while orientation estimation was not addressed due to a lack of a large enough dataset of ground truth annotations suitable for DL. To this end, we first generate a large-scale synthetic dataset of 450 tomograms containing almost 200K samples of two macromolecular structures using the PolNet simulator. Utilizing this synthetic dataset, we address the problem of particle orientation estimation as a regression problem by proposing a DL-based model based on multi-layer perceptron networks and a six-degree-of-freedom orientation representation. The iso-surface visualizations of the averaged subtomograms show that the predicted results by the network are overly similar to that of ground truth. Our work shows that orientation estimation of particles using DL methods is in principle possible provided that ground truth data is available. What remains to be solved is the gap between synthetic and experimental data. The source code is available athttps://github.com/noushinha/DeepOrientation.
The bat Influenza A virus subtype H18N11 induces nanoscale MHCII clustering upon host cell attachment
Authors: Osman Maria Kaukab , Robert Jonathan , Broich Lukas , Frank Dennis , Grosse Robert , Schwemmle Martin , Wrobel Antoni , Ciminski Kevin , Sieben Christian , Reuther Peter
Published: July 2024

Prior to the discovery of the bat influenza A virus (IAV) subtypes H17N10 and H18N11, it was believed that all IAVs bind via the viral hemagglutinin (HA) to sialic acid residues to mediate attachment and subsequent viral entry. Both HA subtypes, H17 and H18, however, do not bind to sialic acids but instead engage a proteinaceous receptor: major histocompatibility complex class II (MHCII). The mechanistic details of this hitherto unknown protein-mediated entry are not understood. Since conventional IAVs require attachment to clusters of sialylated glycans to overcome the low affinity of the HA-sialic acid interaction, we hypothesized that bat HA would likewise interact with multiple MHCII molecules. Here, we used photoactivated localization microscopy (PALM) on fixed and live cells expressing MHCII fused to appropriate fluorescent reporters. We show that bat IAV particles attach to pre-existing MHCII clusters present on susceptible cells and that the local HA-MHCII interaction results in an increased cluster size. To measure the impact of viral attachment on the dynamics of MHCII, we utilized an experimental setup designated “inverse infection” where intact viral particles were immobilized on coverslips before live MHCII-expressing cells were seeded on top. Recording the trajectories of single MHCII molecules, this approach revealed that the mobility of MHCII was indeed slowed down in viral proximity leading to a local enrichment of MHCII molecules beneath the viral particle. Taken together, these data suggest that attachment of viral particles leads to clustering of MHCII, a process similar to the MHCII dynamics observed during the formation of an immunological synapse.
Mechanisms of life cycle simplification in African trypanosomes
Authors: Oldrieve Guy R , Venter Frank , Cayla Mathieu , Verney Mylene , Hébert Laurent , Geerts Manon , Reet Nick Van , Matthews Keith R
Published: July 2024

African trypanosomes are important parasites in sub-Saharan Africa that undergo a quorum-sensing dependent development to morphologically stumpy forms in mammalian hosts to favour disease transmission by tsetse flies1. However, some trypanosome lineages have simplified their lifecycle by escaping dependence on tsetse allowing an expanded geographical range, with direct transmission between hosts achieved via biting flies and vampire bats (Trypanosoma brucei evansi, causing the disease ‘surra’) or through sexual transmission (Trypanosoma brucei equiperdum, causing ‘dourine’). Concomitantly, stumpy formation is reduced or lost, and the isolates are described as monomorphic, with infections spread widely in Africa, Asia, South America and parts of Europe. Here, using genomic analysis of distinct field isolates, we identified and functionally confirmed molecular changes that accompany the loss of the stumpy transmission stage in monomorphic clades. Further, using laboratory selection for resistance to the parasite’s quorum-sensing signal, we identified reversible steps in the initial development of monomorphism. This study identifies a trajectory of events that simplify the life cycle in emergent and established monomorphic trypanosomes, with impact on disease spread, vector control strategies, geographical range and virulence.
Cohesin-mediated 3D contacts tune enhancer-promoter regulation
Authors: Guckelberger Philine , Doughty Benjamin R. , Munson Glen , Rao Suhas S. P. , Tan Yingxuan , Cai Xiangmeng Shawn , Fulco Charles P. , Nasser Joseph , Mualim Kristy S. , Bergman Drew T. , Ray Judhajeet , Jagoda Evelyn , Munger Chad J. , Gschwind Andreas R. , Sheth Maya U. , Tan Anthony S. , Pulido Saul Godinez , Mitra Namita , Weisz David , Shamim Muhammad Saad , Durand Neva C. , Mahajan Ragini , Khan Ruqayya , Steinmetz Lars M. , Kanemaki Masato T. , Lander Eric S. , Meissner Alexander , Aiden Erez Lieberman , Engreitz Jesse M.
Published: July 2024

Enhancers are key drivers of gene regulation thought to act via 3D physical interactions with the promoters of their target genes. However, genome-wide depletions of architectural proteins such as cohesin result in only limited changes in gene expression, despite a loss of contact domains and loops. Consequently, the role of cohesin and 3D contacts in enhancer function remains debated. Here, we developed CRISPRi of regulatory elements upon degron operation (CRUDO), a novel approach to measure how changes in contact frequency impact enhancer effects on target genes by perturbing enhancers with CRISPRi and measuring gene expression in the presence or absence of cohesin. We systematically perturbed all 1,039 candidate enhancers near five cohesin-dependent genes and identified 34 enhancer-gene regulatory interactions. Of 26 regulatory interactions with sufficient statistical power to evaluate cohesin dependence, 18 show cohesin-dependent effects. A decrease in enhancer-promoter contact frequency upon removal of cohesin is frequently accompanied by a decrease in the regulatory effect of the enhancer on gene expression, consistent with a contact-based model for enhancer function. However, changes in contact frequency and regulatory effects on gene expression vary as a function of distance, with distal enhancers (e.g., >50Kb) experiencing much larger changes than proximal ones (e.g., <50Kb). Because most enhancers are located close to their target genes, these observations can explain how only a small subset of genes — those with strong distal enhancers — are sensitive to cohesin. Together, our results illuminate how 3D contacts, influenced by both cohesin and genomic distance, tune enhancer effects on gene expression.
Integrative LC-MS and GC-MS Metabolic Profiling Unveils Dynamic Changes during Barley Malting
Authors: Rani Heena , Whitcomb Sarah J
Published: July 2024

Malting, a crucial process for beer production, involves complex biochemical transformations affecting sensory attributes and product quality. Despite extensive research on storage carbohydrates and proteins involved in malting, a detailed understanding of metabolic alterations during this process remains elusive, limiting our ability to assess and enhance malt quality. Our study employed untargeted GC-MS and LC-MS metabolite profiling to elucidate these changes across six malting stages: dry seed, post-steeping (DOG0), germination (DOG1, DOG3, DOG5), and kilning. We identified a total of 4980 known metabolites, with approximately 82% exhibiting significant changes. Statistical analysis revealed stage-dependent metabolic shifts, with most significant shifts occurring from DOG1 to DOG3 and during kilning. Dynamic changes in various chemical classes and metabolic pathways provide insights into processes critical for malt quality and beer production. Additionally, metabolites associated with antimicrobial properties and stress responses were identified, underscoring the interplay between barley and microbial metabolic processes during malting.

Highlights

GC-MS and LC-MS profiling were performed to track metabolic changes during malting.

Identified 4980 known compounds belonging to 346 chemical classes during malting.

Many microbial metabolites demonstrated increased abundance in finished malt.

The most significant metabolic shifts occurred during early germination and kilning.
Glycolytic flux controls retinal progenitor cell differentiation via regulating Wnt signaling
Authors: Hanna Joseph , Touahri Yacine , Pak Alissa , Belfiore Lauren , van Oosten Edwin , David Luke Ajay , Han Sisu , Ilnytskyy Yaroslav , Kovalchuk Igor , Kurrasch Deborah , Okawa Satoshi , del Sol Antonio , Screaton Robert A , Aubert Isabelle , Schuurmans Carol
Published: July 2024

Metabolic pathways are remodeled in response to energy and other homeostatic demands and are dynamically regulated during embryonic development, suggesting a role in guiding cellular differentiation. Here, we show that glycolytic flux is required and sufficient to bias multipotent retinal progenitor cells (RPCs) to acquire a rod photoreceptor fate in the murine retina. In an RPC-specific conditional knock-out of Phosphatase and tensin homolog (Pten-cKO) and an RPC-specific conditional gain-of-function of dominant active PFKB3 (cytoPFKB3), glycolytic gene expression and activity are elevated, correlating with precocious rod photoreceptor differentiation and outer segment maturation. Conversely, glycolytic inhibition in retinal explants, achieved either with 2-deoxy-d-glucose, a competitive inhibitor of glucose metabolism, by lowering media pH, which disables PKM2, a rate-limiting enzyme, or by inhibiting lactate/H+ symporters, which lowers intracellular pH, suppresses RPC proliferation and photoreceptor differentiation. Mechanistically, we show that Wnt signaling, the top-upregulated pathway in Pten-cKO retinas, is a glycolysis-dependent pathway. Pharmacological and genetic perturbation of Wnt signaling using a Ctnnb1-cKO phenocopies glycolytic inhibition, suppressing RPC proliferation, photoreceptor differentiation and outer segment maturation. Thus, developmental rewiring of glycolytic flux modulates Wnt signaling to drive rod photoreceptor differentiation and maturation, an instructive role that may be exploited therapeutically for cell replacement strategies.
An energy coupling factor transporter ofStreptococcus sanguinisimpacts antibiotic susceptibility as well as metal and membrane homeostasis
Authors: Rudzite Marta , O’Toole G. A.
Published: July 2024

Streptococcus sanguinisis a prevalent member of human microbiome capable of acting as a causative agent of oral and respiratory infections.S. sanguiniscompetitive success within the infection niche is dependent on acquisition of metal ions and vitamins. Among the systems that bacteria use for micronutrient uptake is the energy coupling factor (ECF) transporter system EcfAAT. Here we describe physiological changes arising from EcfAAT transporter disruption. We found that EcfAAT contributes toS. sanguinisantibiotic sensitivity as well as metal and membrane homeostasis. Specifically, our work found that disruption of EcfAAT results in increased polymyxin susceptibility. We performed assessment of cell-associated metal content and found depletion of iron, magnesium, and manganese. Furthermore, membrane composition analysis revealed significant enrichment in unsaturated fatty acid species resulting in increased membrane fluidity. Our results demonstrate how disruption of a single EcfAAT transporter can have broad consequences on bacterial cell homeostasis. ECF transporters are of interest within the context of infection biology in bacterial species other than streptococci, hence work described here will further the understanding of how micronutrient uptake systems contribute to bacterial pathogenesis.

Importance

Proficiency in micronutrient uptake is key for pathogen success in bacteria-bacteria and bacteria-host interactions within the infection context. Micronutrient uptake mechanisms are of interest in furthering the understanding of bacterial physiology within infection niche and as targets for design of antimicrobials. Here we describe how a deletion of a nutrient uptake transporter inS. sanguinisalters bacterial sensitivity to antibiotics. We also show that a defect in this candidate nutrient uptake system has consequences on the intracellular metal content, and also results in changes in membrane fatty acid composition and fluidity. This study demonstrates how disruption of a single nutrient uptake system disrupts bacterial physiology resulting in increased antibiotic sensitivity.
Synergistic reinforcement learning by cooperation of the cerebellum and basal ganglia
Authors: Yoshida Tatsumi , Sugino Hikaru , Yamamoto Hinako , Tanno Sho , Tamura Mikihide , Igarashi Jun , Isomura Yoshikazu , Hira Riichiro
Published: July 2024

The cerebral cortex, cerebellum, and basal ganglia are crucial for flexible learning in mammals. Recent studies suggest that reinforcement learning might occur in both the basal ganglia and cerebellum, as cerebellar climbing fibers represent reward prediction error. If the same learning mechanism via reward prediction error occurs simultaneously, it remains unclear how these two regions co-function. Here, we recorded neuronal activity in the output of cerebellum and basal ganglia, the cerebellar nuclei (CN) and substantia nigra pars reticulata (SNr), respectively, from ChR2 transgenic rats with high-density Neuropixels probes while optogenetically stimulating the cerebral cortex. The temporal response patterns could be categorized into two classes in both CN and SNr. Among them, the fast excitatory response of the CN due to the input of mossy fibers and the inhibitory response of the SNr via the direct pathway were synchronized. To further investigate this significance, we constructed a spiking network simulation based on connectome data and a reservoir model that mimics the experimental results. In the reservoir model, plasticity of both cerebellum and basal ganglia was essential for successful learning. Learning was impeded when cerebellar or basal ganglia output timing deviated by 10 ms from real data. Notably, the addition of β-oscillation to the cerebral cortex enhanced learning. These results indicate that coordinated output of the cerebellum and basal ganglia, with input from the cerebral cortex in a narrow frequency band, facilitates brain-wide synergistic reinforcement learning.

Significance Statement

The cerebral cortex, cerebellum, and basal ganglia support learning. Recent research suggests that both the basal ganglia and cerebellum use a similar learning process called reinforcement learning, which involves predicting rewards. To understand how these brain regions work together, we recorded brain activity in rats while photo-stimulating the cerebral cortex. We found that two types of responses in the cerebellum and basal ganglia were synchronized, which might help activate the cerebral cortex. A computer model showed that precise timing of signals from both the cerebellum and basal ganglia is important for learning. This timing was important only when the cerebral cortex worked in a specific frequency range. Our findings suggest that coordinated brain activity enhances learning.
The neonatal Fc receptor and DPP4 are human astrovirus receptors
Authors: Ingle Harshad , Molleston Jerome M. , Hall Paige D. , Bui Duyen , Wang Leran , Foster Lynne , Antia Avan , Ding Siyuan , Lee Sanghyun , Fremont Daved H. , Baldridge Megan T.
Published: July 2024

Human astroviruses (HAstV) are major global causes of gastroenteritis, but little is known about host factors required for their cellular entry. Here, we utilized complementary CRISPR-Cas9-based knockout and activation screening approaches and identified neonatal Fc receptor (FcRn) and dipeptidyl-peptidase IV (DPP4) as entry factors for HAstV infection of human intestinal epithelial cells. Disruption of FcRn or DPP4 reduced HAstV infection in permissive cells and, reciprocally, overexpression of these factors in non-permissive cells was sufficient to promote infection. We observed direct binding between FcRn and HAstV virions as well as purified spike protein. Finally, inhibitors for DPP4 and FcRn currently in clinical use prevent HAstV infection in cell lines and primary human enteroids. Thus, our results reveal mechanisms of HAstV entry as well as druggable targets.

One-Sentence Summary

Targeting FcRn or DPP4 using available therapies effectively prevents human astrovirus infection in human enteroid cultures.
Identification of stable reference genes for quantitative real-time PCR in human fibroblasts from lymph nodes and synovium
Authors: Rasouli S. , van Ansenwoude C.M.J. , Semmelink J.F. , van Baarsen L.G.M. , de Jong T.A.
Published: July 2024

Real-time quantitative PCR (RT–qPCR) has emerged as an accurate and widely used technique for measuring gene expression levels. However, its reliability depends on the selection of appropriate reference genes to normalize for sample input. Accordingly, the identification of reference genes characterized by stable expression in cells and conditions of interest is essential for ensuring accurate expression values. To date, no study has specifically identified suitable reference genes for primary human cultured fibroblast-like synoviocytes (FLS) and lymph node stromal cells (LNSCs) within the context of rheumatoid arthritis (RA). These stromal cells play a critical role in the pathogenesis of disease. In this study, we evaluated the suitability of 15 candidate reference genes for normalizing transcript expression in FLS and LNSCs subjected to various in vitro stimuli. We included traditional reference genes often used for transcript normalization in fibroblasts as well as candidate genes identified as suitable reference genes via GeneVestigator analysis of publicly available transcriptomic data. RefFinder algorithms were used to identify the most stable reference genes for transcript normalization across the cell types and different experimental conditions. We determined that the optimal number of reference genes for every experimental condition tested was two;RPLP0andPOLR2Gexhibited the greatest stability across different experimental conditions for LNSCs. However, for FLS, we observed greater variability in the most stable reference genes across different experimental conditions. AlthoughPOLR2GandTBPemerged as the most stable reference genes under unstimulated conditions, our findings indicated that FLS require distinct reference genes for transcript normalization depending on the specific experimental conditions. Validation of the selected reference genes for normalizing the expression levels of metabolic genes in unstimulated FLS emphasized the importance of prior evaluation of potential reference genes, as arbitrary selection of reference genes could lead to data misinterpretation. This study constitutes the first systematic analysis for selecting optimal reference genes for transcript normalization in different types of human fibroblasts. Our findings emphasize the importance of proper selection of reference genes for each experimental condition separately when applying standard quantitative PCR technology for assessing gene expression levels.
Cryo-ET reveals thein situarchitecture of the polar tube invasion apparatus from microsporidian parasites
Authors: Usmani Mahrukh , Coudray Nicolas , Riggi Margot , Raghu Rishwanth , Ramchandani Harshita , Bobe Daija , Kopylov Mykhailo , Zhong Ellen D. , Iwasa Janet H. , Ekiert Damian C. , Bhabha Gira
Published: July 2024

Microsporidia are divergent fungal pathogens that employ a harpoon-like apparatus called the polar tube (PT) to invade host cells. The PT architecture and its association with neighboring organelles remain poorly understood. Here, we use cryo-electron tomography to investigate the structural cell biology of the PT in dormant spores from the human-infecting microsporidian species,Encephalitozoon intestinalis. Segmentation and subtomogram averaging of the PT reveal at least four layers: two protein-based layers surrounded by a membrane, and filled with a dense core. Regularly spaced protein filaments form the structural skeleton of the PT. Combining cryo-electron tomography with cellular modeling, we propose a model for the 3-dimensional organization of the polaroplast, an organelle that is continuous with the membrane layer that envelops the PT. Our results reveal the ultrastructure of the microsporidian invasion apparatusin situ, laying the foundation for understanding infection mechanisms.
Modified Chlorophyll Pigment at ChlD1Tunes Photosystem II Beyond the Red-Light Limit
Authors: Allgöwer Friederike , Sirohiwal Abhishek , Gamiz-Hernandez Ana P. , Pöverlein Maximilian C. , Fantuzzi Andrea , Rutherford A. William , Kaila Ville R. I.
Published: July 2024

Photosystem II (PSII) is powered by the light-capturing properties of chlorophyllapigments that define the spectral range of oxygenic photosynthesis. Some photosynthetic cyanobacteria can acclimate to growth in longer wavelength light by replacing five chlorophylls for long wavelength pigments in specific locations, including one in the reaction center (RC). However, the exact location and the nature of this long wavelength pigment still remain uncertain. Here we have addressed the color-tuning mechanism of the farred light PSII (FRL-PSII) by excited state calculations at both theab initiocorrelated (ADC2) and linear-response time-dependent density functional theory (LR-TDDFT) levels in combination with large-scale hybrid quantum/classical (QM/MM) simulations and atomistic molecular dynamics. We show that substitution of a single chlorophyll pigment (ChlD1) at the RC by chlorophylldleads to a spectral shift beyond the far-red light limit, as a result of the protein electrostatic, polarization and electronic coupling effects that reproduce key structural and spectroscopic observations. Pigment substitution at the ChlD1site further results in a low site energy within the RC that could function as a sink for the excitation energy and initiate the primary charge separation reaction, driving the water oxidation. Our findings provide a basis for understanding color-tuning mechanisms and bioenergetic principles of oxygenic photosynthesis at the far-red light limit.
IFRS-9, expected loan loss provisioning and bank liquidity creation: early evidence
Authors: Ghosh Saibal
Published: July 2024

Purpose

The relevance of both microeconomic and macroeconomic factors in driving banks’ liquidity creation within a cross-country setup has been addressed in prior research. The purpose of this study is to explore whether and how the recent International Financial Reporting Standards-9 (IFRS-9) accounting standards affect this relationship.

Design/methodology/approach

The author exploits the staggered implementation of IFRS-9 across countries and use a difference-in-differences framework to tease out the causal impact.

Findings

The findings indicate that the enactment of IFRS-9 leads to an increase in asset-side liquidity creation and a decline of broadly similar magnitude in liability-side liquidity creation. As a result, total liquidity creation remains unaltered. Disaggregatedly, the evidence shows that all the key channels on the asset and liability side are instrumental in explaining this behaviour.

Originality/value

The author views this as one of the early studies in a cross-country setup to explore the interlinkage between IFRS-9 and bank liquidity creation. Since liquidity creation measure provides a comprehensive metric of liquidity supplied by banks to the market, the study seeks to inform the policy debate on the role of these recently instituted accounting standards on bank liquidity behaviour.
Information behavior of weaver community in Varanasi: an analytical study
Authors: Ansari Mohd Shoaib , Tripathi Aditya
Published: July 2024

Purpose

This study aims to evaluate the information behavior of the Banarasi Sarees weaving community and propose an information-focused policy tailored to their needs.

Design/methodology/approach

This study was conducted in seven weaver-centered areas in Varanasi and was designed around semi-structured interviews to explore various information-related aspects among the weavers. It aims to focus on their knowledge of weaving, their information needs, seeking behavior, preferred communication channels, prioritization of information and methods for enhancing information-related situations.

Findings

It was found that a lack of information among weavers significantly impacts their livelihoods and businesses. They struggle to make product, pricing and marketing decisions without access to accurate, timely and relevant information. They cannot expand their businesses and invest in new equipment and materials. The weavers need a library with a specialized collection and a qualified librarian to guide them in using the information sources. They also need a dedicated information system and community information centers to provide better information services.

Originality/value

This study is a work whose expected results and findings would help policymakers adopt measures to encourage providing information services through public libraries and community information centers. The results of this study may be further used to frame information policy for weavers and other occupational communities.