bluuebunny/crossref_metadata_2025_split 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

AbstractTelomeres, 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 summaryTelomeres 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

AbstractChlamydia (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

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

SummaryMosquito-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

ABSTRACTAn 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

AbstractCitrus 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

AbstractImmunoglobulin 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

AbstractThe 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

AbstractThe 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

AbstractER 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

AbstractGenome-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

AbstractGiven 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.HighlightsCUTS 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

AbstractOrientation 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.