bluuebunny/crossref_metadata_2025 huggingface dataset:
New-to-nature PHA synthase design using deep learning
Authors: Tenkanen Tuula , Ylinen Anna , Jouhten Paula , Penttilä Merja , Castillo Sandra
Published: October 2024

Polyhydroxyalkanaoate (PHA) synthases are a group of complex, dimeric enzymes which catalyse polymerization of Rhydroxyacids into PHAs. PHA properties depend on their monomer composition but enzymes found in nature have narrow specificities to certain R-hydroxyacids. In this study, a conditional variational autoencoder was used for the first time to design new-to-nature PHA synthases. The model was trained with natural protein sequences obtained from Uniprot and was used for the creation of approximately 10 000 new PHA synthase enzymes. Out of these, 16 sequences were selected forin vivovalidation. The selection criteria included the presence of conserved residues such as catalytic amino acids and amino acids in the dimer interface and structural features like the number ofα-helixes in the N-terminal part of the enzyme. Two of the new-to-nature PHA synthases that had substantial numbers of amino acid substitutions (87 and 98) with respect to the most similar native enzymes were confirmed active and produced poly(hydroxybutyrate) (PHB) when expressed in yeastS. cerevisiae. PHA including PHB have high potential as biodegradable and biocompatible materials. Ultimately the model-designed new-to-nature PHA synthases, could expand the PHA material properties to suit new application areas.
An ancient competition for the conserved branchpoint sequence influences physiological and evolutionary outcomes in splicing
Authors: Pereira de Castro Karen Larissa , Abril Jose M. , Liao Kuo-Chieh , Hao Haiping , Donohue John Paul , Russell William K. , Fagg W. Samuel
Published: October 2024

Recognition of the intron branchpoint during spliceosome assembly is a multistep process that defines both mRNA structure and amount. A branchpoint sequence motif UACUAAC is variably conserved in eukaryotic genomes, but in some organisms more than one protein can recognize it. Here we show that SF1 and Quaking (QKI) compete for a subset of intron branchpoints with the sequence ACUAA. SF1 activates exon inclusion through this sequence, but QKI represses the inclusion of alternatively spliced exons with this intron branchpoint sequence. Using mutant reporters derived from a natural intron with two branchpoint-like sequences, we find that when either branchpoint sequence is mutated, the other is used as a branchpoint, but when both are present, neither is used due to high affinity binding and strong splicing repression by QKI. QKI occupancy at the dual branchpoint site directly prevents SF1 binding and subsequent recruitment of spliceosome-associated factors. Finally, the ectopic expression of QKI in budding yeast (which lacksQKI) is lethal, due at least in part to widespread splicing repression. In conclusion, QKI can function as a splicing repressor by directly competing with SF1/BBP for a subset of branchpoint sequences that closely mirror its high affinity binding site. This suggests thatQKIand degenerate branchpoint sequences may have co-evolved as a means through which specific gene expression patterns could be maintained in QKI-expressing or non-expressing cells in metazoans, plants, and animals.
BEREN: A bioinformatic tool for recovering Giant viruses, Polinton-like Viruses, and Virophages in metagenomic data
Authors: Minch Benjamin , Moniruzzaman Mohammad
Published: October 2024

Viruses in the kingdomBamfordvirae, specifically giant viruses (NCLDVs) in the phylumNucleocytoviricotaand smaller members in thePreplasmiviricotaphylum, are widespread and important groups of viruses that infect eukaryotes. While viruses in this kingdom such as giant viruses, polinton-like viruses, and virophages have gained large interest from researchers in recent years, there is still a lack of streamlined tools for the recovery of their genomes from metagenomic datasets. Here, we present BEREN, a comprehensive bioinformatic tool to unlock the diversity of these viruses in metagenomes through five modules for NCLDV genome, contig, and marker gene recovery, metabolic protein annotation, andPreplasmiviricotagenome identification and annotation. BEREN’s performance was benchmarked against other mainstream virus recovery tools using a mock metagenome, demonstrating superior recovery rates of NCLDV contigs andPreplasmiviricotagenomes. Applied to a real-world dataset from the Baltic Sea, BEREN identified diverseBamfordviraemembers, giving insight into viral interactions and metabolic functions in this region. Overall, BEREN offers a user-friendly, transparent bioinformatic solution for studying the ecological and functional roles of these eukaryotic viruses, facilitating broader access to their metagenomic analysis.
Template-Independent Enzymatic RNA Synthesis
Authors: Karalkar Nilesh B , Kent Tatiana , Tredinnick Taylor , Betancurt-Anzola Leonardo , Delarue Marc , Pomerantz Richard , Benner Steven A
Published: October 2024

A route to prepare ribonucleoside triphosphates featuring a 3’-aminoxy (3’-O-NH2) removable blocking group is reported here. We then show that versions of two DNA polymerases, human DNA polymerase theta (Polθ) and mimiviral PrimPol, accept these triphosphates as substrates to add single nucleotides to an RNA primer under engineered conditions. Cleaving the O-N bond in the 3’-O-NH2group within the extended primer regenerates the 3’-OH group, facilitating subsequent polymerase cycles that add a second, selected, nucleotide. These enzymes and triphosphates together enable template-independent enzymatic RNA synthesis (TIERS) exploiting a cyclic reversible termination framework. The study shows that this process is ready for instrument adaptation by using it to add three ribonucleotides in three cycles using an engineered Polθ. This work creates a new way to synthesize RNA with a de novo defined sequence, without requiring the protecting groups, hazardous solvents, and sensitive reagents that bedevil phosphoramidite-based RNA synthesis.
Aβ*56 is present in brain extracts enriched for extracellular proteins
Authors: Liu Peng , Lapcinski Ian P. , Ashe Karen H.
Published: October 2024

Amyloid-β (Aβ) oligomers are believed to be important in the pathogenesis of Alzheimer’s disease (AD). Aβ*56 is an Aβ oligomer that has been reported in several lines of transgenic mice modeling AD, including Tg2576, hAPP-J20, 3xTgAD, 5xFAD, and APPTTA. In Tg2576 mice, Aβ*56 appears several months before neuritic plaques and impairs memory when injected into the brains of healthy rodents. Aβ*56 has several distinctive biochemical features: 1) it is soluble in aqueous buffers, 2) it is stable in the ionic detergent sodium dodecyl sulfate (SDS), 3) it has an apparent mass of 56 kDa whether measured by denaturing SDS polyacrylamide gel electrophoresis or non-denaturing size-exclusion chromatography, 4) it binds to A11 conformational antibodies that recognize non-fibrillar oligomeric assemblies, and 5) it contains canonical Aβ(1-40) and/or Aβ(1-42). Here, we show its presence in Tg2576 brain extracts enriched for extracellular proteins and conclude that Aβ*56 is present in the extracellular space.
Cryo-EM of native membranes reveals an intimate connection between the Krebs cycle and respiration in mycobacteria
Authors: Di Trani Justin M. , Yu Jiacheng , Courbon Gautier M. , Lobez Rodriguez Ana Paula , Cheung Chen-Yi , Liang Yingke , Coupland Claire E. , Bueler Stephanie A. , Cook Gregory M. , Brzezinski Peter , Rubinstein John L.
Published: October 2024

Imaging of endogenous protein complexes in their native membranes can reveal protein-protein interactions that are lost upon detergent solubilization. To investigate interactions in the mycobacterial oxidative phosphorylation machinery, we prepared inverted membrane vesicles fromMycobacterium smegmatisand enriched for vesicles containing complexes of interest by affinity chromatography. Electron cryomicroscopy (cryo-EM) of these vesicles revealed that malate-quinone oxidoreductase (Mqo), an enzyme from the Krebs cycle, physically associates with the electron transport chain Complex III2IV2(CIII2CIV2) supercomplex. Analysis of the Mqo:CIII2CIV2interaction shows that CIII2CIV2is necessary for malate-driven, but not NADH- driven, electron transport chain activity and oxygen consumption. Further, the association of Mqo with CIII2CIV2enables electron transfer from malate to CIII2CIV2with millisecond kinetics. Together, these findings indicate a connection between the Krebs cycle and respiration that directs electrons along a single branch of the mycobacterial electron transport chain.
Directed assembly of single-stranded DNA fragments for data storage via enzyme-free catalytic splint ligation
Authors: Mendonsa Gemma , Chari Sriram , Bao Mengdi , Herdendorf Brett , Reddy Anil
Published: October 2024

Oligonucleotides or gene fragments can be ligated in a specified order to create longer DNA assemblies. We present a method where DNA symbols, or oligos designed to encode information for data storage, are joined to linker sequences at either end. These linkers dictate the assembly order of the symbols; the order of the symbols can be changed by changing the sequences of the linkers attached to them. Utilizing a ligating DNAzyme as a catalytic splint, we achieve room-temperature, enzyme-free assembly, offering a cost-effective alternative to traditional enzyme-based ligation methods. We demonstrate this technique by assembling three different five-symbol constructs, with the order of the symbols determined by their linking ends. This linker directed assembly technique allows data-encoding symbols to be assembled in any desired order. Furthermore, the DNAzyme-based assembly method is versatile and can be applied to various DNA assembly applications, particularly where cost-effectiveness and efficient room-temperature ligation are required.
Tunable, self-contained gene dosage control via proteolytic cleavage of CRISPR-Cas systems
Authors: Katz Noa , An Connie , Lee Yu-Ju , Tycko Josh , Zhang Meng , Kang Jeewoo , Bintu Lacramioara , Bassik Michael C , Huang Wei-Hsiang , Gao Xiaojing J
Published: October 2024

Gene therapy holds great therapeutic potential. Yet, controlling cargo expression in single cells is limited due to the variability of delivery methods. We implement an incoherent feedforward loop based on proteolytic cleavage of CRISPR-Cas activation or inhibition systems to reduce gene expression variability against the variability of vector delivery. We demonstrate dosage control for activation and inhibition, post-delivery tuning, and RNA-based delivery, for a genome-integrated marker. We then target theRAI1gene, the haploinsufficiency and triplosensitivity of which cause two autism-related syndromes, Smith-Magenis-Syndrome (SMS) and Potocki-Lupski-Syndrome, respectively. We demonstrate dosage control for RAI1 activation in HEK293s, Neuro-2As, and mouse cortical neurons via AAVs and lentiviruses. Finally, we activate the intactRAI1copy in SMS patient-derived cells to an estimated two-copy healthy range, avoiding the harmful three-copy regime. Our circuit paves the way for viable therapy in dosage-sensitive disorders, creating precise and tunable gene regulation systems for basic and translational research.
Activation and modulation of the host response to DNA damage by an integrative and conjugative element
Authors: McKeithen-Mead Saria , Anderson Mary E. , García-Heredia Alam , Grossman Alan D.
Published: October 2024

Mobile genetic elements help drive horizontal gene transfer and bacterial evolution. Conjugative elements and temperate bacteriophages can be stably maintained in host cells. They can alter host physiology and regulatory responses and typically carry genes that are beneficial to their hosts. We found that ICEBs1, an integrative and conjugative element ofBacillus subtilis, inhibits the host response to DNA damage (the SOS response). Activation of ICEBs1before DNA damage reduced host cell lysis that was caused by SOS-mediated activation of two resident prophages. Further, activation of ICEBs1itself activated the SOS response in a subpopulation of cells, and this activation was attenuated by the functions of the ICEBs1genesydcTandyddA(nowramTandramA, for RecA modulator). Double mutant analyses indicated that RamA functions to inhibit and RamT functions to both inhibit and activate the SOS response. Both RamT and RamA caused a reduction in RecA filaments, one of the early steps in activation of the SOS response. We suspect that there are several different mechanisms by which mobile genetic elements that generate ssDNA during their lifecycle inhibit the host SOS response and RecA function, as RamT and RamA differ from the known SOS inhibitors encoded by conjugative elements.
Infectivity and persistence of influenza viruses in raw milk
Authors: Zulli Alessandro , Zhang Mengyang , Jong Sehee , Blish Catherine , Boehm Alexandria B.
Published: October 2024

Influenza A viruses present a significant public health risk, with recent outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in dairy cattle raising concerns about potential transmission through raw milk consumption. This study investigated the persistence of influenza A virus PR8 (IAV PR8) in raw cow milk at 4 °C. We found that IAV PR8 remained infectious in raw milk for up to 5 days, with a decay rate constant of −2.05 day−1. In contrast, viral RNA remained detectable and stable for at least 57 days, with no significant degradation. Pasteurization (63°C for 30 minutes) significantly reduced detectable viral RNA concentrations, but reduction was less than 1 log. These findings highlight the potential risk of zoonotic virus transmission through raw milk consumption and underscore the importance of milk pasteurization. The prolonged persistence of viral RNA in both raw and pasteurized milk has implications for food safety assessments and environmental monitoring, particularly in the context of environmental surveillance for influenza viruses.

Synopsis

Influenza A RNA is persistent in milk, even after pasteurization, and it remains infectious for 5 days in refrigerated milk.
A novelex vivomodel of muscle contraction to measure muscle endocrine and paracrine signalling
Authors: Dabadie Carole , Belaidi Élise , Moretta Antonio , Lemarié Lucas , Allard Bruno , Kneppers Anita , Mounier Rémi
Published: October 2024

Skeletal muscle is a highly organized tissue that possesses the ability to contract and that exerts metabolic, endocrine and paracrine functions. Previous studies explored these functions throughin vitromodels, computational analyses, and endpoint measurements. To include the cellular complexity that modulates muscle endocrine and paracrine functions, and to allow us to study the kinetics of released and/or depleted factors, we developed a novel setup forex vivocontraction of whole skeletal muscle.Ex vivocontraction of theExtensor Digitorum Longus(EDL) induced by either electrical field stimulation or direct nerve stimulation resulted in a reduction of relative maximal force and a depletion of muscle glycogen content. Differential proteomics confirmed that proteins upregulated upon electrical pulse stimulation (EPS) were enriched for biological processes and molecular functions associated to cytoskeletal organization and muscle contraction. Finally, EPS induced the release of lactate, and IL6 secretion was induced after cessation of EPS. Taken together, we have developed and validated a new model ofex vivocontraction that includes the full cellular complexity of the muscle and mimics thein vivophysiological response to contraction. Importantly, this model supports for the first time to study the kinetics of the endocrine and paracrine response of the muscle to contraction, and to investigate its effects on various cell-typesin vitro.
Chai-1: Decoding the molecular interactions of life
Authors:   Chai Discovery, Boitreaud Jacques , Dent Jack , McPartlon Matthew , Meier Joshua , Reis Vinicius , Rogozhnikov Alex , Wu Kevin
Published: October 2024

We introduce Chai-1, a multi-modal foundation model for molecular structure prediction that performs at the state-of-the-art across a variety of tasks relevant to drug discovery. Chai-1 can optionally be prompted with experimental restraints (e.g. derived from wet-lab data) which boosts performance by double-digit percentage points. Chai-1 can also be run in single-sequence mode with-out MSAs while preserving most of its performance. We release Chai-1 model weights and inference code as a Python package for non-commercial use and via a web interface where it can be used for free including for commercial drug discovery purposes.
Polyserine peptides are toxic and exacerbate tau pathology in mice
Authors: Van Alstyne Meaghan , Nguyen Vanessa L. , Hoeffer Charles A. , Parker Roy
Published: October 2024

Polyserine domains mediate the association of nuclear RNA binding proteins with cytoplasmic tau aggregates that occurs across tauopathy models and patient samples. In cell lines, polyserine peptides co-localize with and promote formation of tau aggregates suggesting the cytoplasmic mislocalization of polyserine-containing proteins might contribute to human disease. Moreover, polyserine can be produced by repeat associated non-AUG translation in CAG repeat expansion diseases. However, whether polyserine expressed in a mammalian brain is toxic and/or can exacerbate tau pathology is unknown. Here, we used AAV9-mediated delivery to express a 42-repeat polyserine protein in wild-type and tau transgenic mouse models. We observe that polyserine expression has toxic effects in wild-type animals indicated by reduced weight, behavioral abnormalities and a striking loss of Purkinje cells. Moreover, in the presence of a pathogenic variant of human tau, polyserine exacerbates disease markers such as phosphorylated and insoluble tau levels and the seeding capacity of brain extracts. These findings demonstrate that polyserine domains can promote tau-mediated pathology in a mouse model and are consistent with the hypothesis that cytoplasmic mislocalization of polyserine containing proteins might contribute to the progression of human tauopathies.
Pairwise interactions, feedback rule changes, and deliberative decisions underlie honeybee inflight group coordination
Authors: Islam Md. Saiful , Faruque Imraan
Published: October 2024

Systematic descriptions of the underlying interaction rules that insects use to support group and swarm flight has the potential to contribute to mathematics, biology, and robotics, including aerial swarming under sensory and computational limitations. This study analyzes 1,000 trajectories of flying honeybees in crowded conditions approaching a moving stimulus and finds how during this stimulus, honeybees coordinate flight through pairwise interactions involving a novel three-zone decision-making process. The experimental setup consists of 3-D position reconstructions via a high speed camera system recording honeybee foragers returning to a hive entrance actuated to move robotically. The analysis consists of neighborhood identification through three methods (cross-correlation, distance threshold, and average distance threshold), which reveals the dominant interaction is pairwise. The individual leader-follower pair interactions are then tested against three regulation candidates: optic flow, relative velocity, and optical expansion rate, based on minimizing root mean square error. The results show that each follower demonstrates a three stage process involving a feedback rule change, linked by an intermediate observation/decision phase. During the initial “lock” phase, an insect maintains a consistent optical expansion rate until inter-agent distance closes to 10 cm. The regulation candidates then undergo large variations during a relatively long observation/decision zone, with 1.04 seconds being the average time in the decision zone. 79% of the paired insect entries into the decision zone result in subsequent re-engagement to track the same initial leader, while 21% result in disengagement from the group behavior. Visual regulation candidate comparison in the third stage indicates that upon re-engagement, the follower relative velocity is regulated to provide consistent velocity matching between agents. The third stage’s velocity tracking is consistent with a closed-loop feedback proportional-integral (PI) controller regulating velocity tracking error. Across the insect population studied, the proportional gain remained showed minimal variability over individuals, a derivative gain was considered and found negligible, and the integral gain varied by individual. Collectively, these findings underscore the existence of an alternative swarm architecture, highlighting individual decision-making capabilities, feedback regulation target changes, and the presence of reactive, deliberative, and moderate (PI control) timescale interaction rules contained within aerial groups.
SMYD5 is a ribosomal methyltransferase which trimethylates RPL40 lysine 22 through recognition of a KXY motif
Authors: Hamey Joshua J. , Shah Manan , Wade John D. , Bartolec Tara K. , Wettenhall Richard E. H. , Quinlan Kate G.R. , Williamson Nicholas A. , Wilkins Marc R.
Published: October 2024

The eukaryotic ribosome is highly modified by protein methylation, yet many of the responsible methyltransferases remain unknown. Here we have identified SMYD5 as a ribosomal protein methyltransferase that catalyses trimethylation of RPL40/eL40 at lysine 22. Through a systematic mass spectrometry-based approach, we show that the human ribosome has 12 primary sites of protein methylation, including at RPL40 K22. Throughin vitromethylation of synthetic RPL40 using fractionated lysate, we then identify SMYD5 as a candidate RPL40 K22 methyltransferase. We show that recombinant SMYD5 has robust activity towards RPL40 K22in vitro, and that active site mutations ablate this activity. Knockouts ofSMYD5in K562 cells show a complete loss of RPL40 K22 methylation and decrease polysome levels. By systematic analysis of its recognition motif, we show that SMYD5 requires a tyrosine in the +2 position, and thereby is incapable of methylating its previously reported histone substrates.
PROPERMAB: an integrative framework forin silicoprediction of antibody developability using machine learning
Authors: Li Bian , Luo Shukun , Wang Wenhua , Xu Jiahui , Liu Dingjiang , Shameem Mohammed , Mattila John , Franklin Matthew , Hawkins Peter G. , Atwal Gurinder S.
Published: October 2024

Selection of lead therapeutic molecules is often driven predominantly by pharmacological efficacy and safety. Candidate developability, such as biophysical properties that affect the formulation of the molecule into a product, is usually evaluated only toward the end of the drug development pipeline. The ability to evaluate developability properties early in the process of antibody therapeutic development could accelerate the timeline from discovery to clinic and save considerable resources.In silicopredictive approaches, such as machine learning models, which map molecules to predictions of developability properties could offer a cost-effective and high-throughput alternative to experiments for antibody developability assessment. We developed a computational framework, PROPERMAB, for large-scale and efficientin silicoprediction of developability properties for monoclonal antibodies, using custom molecular features and machine learning modeling. We demonstrate the power of PROPERMABby using it to develop models to predict antibody hydrophobic interaction chromatography retention time and high-concentration viscosity. We further show that structure-derived features can be rapidly and accurately predicted directly from sequences by pre-training simple models for molecular features, thus providing the ability to scale these approaches to repertoire-scale sequence datasets.
Mapping synaptic ensembles throughin vitrofunctional cell assemblies
Authors: Zaccaria Clara , Malkoç Asiye , Auslender Ilya , Heydari Yasaman , Canossa Marco , Vignoli Beatrice , Pavesi Lorenzo
Published: October 2024

In memory circuits, synaptic engrams consist of ensembles of strengthened synapses that form between engram neurons encoding distinct memory traces. As proposed in Hebb’s seminal postulate, these potentiated synapses enable the functional cell assembly essential for neuronal wiring. While existing technologies can track potentiated synapses in behaving animals, understanding thein vivospatial-temporal organization of synaptic engrams remains challenging. We developed anin vitrohybrid system that integrates digital light processing with optogenetics and used optical stimulation to synchronize the firing patterns of two-neuron modules within a network, supporting Hebb’s postulate experimentally. After illumination, we observed that synapses predominantly strengthen on dendrites between the two illuminated neurons, reflecting neuron responses to the wiring activity. This observation provides a strong conceptual foundation for our methodology. Our setup also allows for direct illumination and strengthening of synaptic ensembles, granting spatial-temporal control over the synaptic networks. Building on Hebb’s theoretical framework, our system offers a solid experimental approach to test the fundamental principles underlying synaptic engrams.
Step-by-step methodology for building a capsule to sample small intestine content
Authors: Ollagnier Catherine , Tretola Marco , García Viñado Inés , Morel Benoit
Published: October 2024

Background

Research on the intestinal microbiome has been hindered by limited access to intestinal content. Recently, a few capsule prototypes have demonstrated their potential for sampling intestinal material while using the natural pathway. However, access to these capsules is restricted because most of them are not yet commercially available. Pigs offer significant potential to inform human research due to the many physiological similarities between the two species. The unique features of pig anatomy have made it difficult to conduct research using swallowable devices. This article provides a detailed account of the manufacturing process and composition of a capsule, along with all the necessary steps for successfully sampling small intestine content in pigs.

Results

The capsule moves passively through the digestive tract, relying solely on intestinal peristalsis for propulsion. Engineered to open when it encounters a pH level greater than 6, the upper part dissolves, allowing intestinal fluids to enter the inner chamber. This triggers a plunger to expand, drawing luminal content into the storage chamber. Once the plunger mechanism is fully extended the capsule is automatically sealed. The capsule has a size of a 0 hard capsule and consists of two main components: a dissolvable exterior with an enteric coating and a 3D-printed bottom part. The printing files of the 3D-printed bottom part are provided for replication. In vitro testing shows that the capsule can withstand two hours in an acidic medium and successfully samples within an hour of being transferred to a neutral medium. When tested in vivo in pigs, the capsule successfully collected intestinal content from the upper and middle sections of the small intestine.

Conclusions

This article provides essential details for the rapid development of a cost-effective tool that has been already validated for non-invasive sampling of small intestine content in pigs. By providing access to the exact production steps and printing files, this article empowers others to innovate and expand upon this foundational work. This open-source approach opens up new avenues for intestinal research, making it more accessible and adaptable for a wide range of studies in both animal and human models.
Systematic Mining of gut microbiota biomarkers for IBD
Authors: Zhang Yuchen , Lai Wenkai , Wang Meiling , Lai Shirong , Liu Qing , Luo Qi , Dou Lijun , Yang Fenglong
Published: October 2024

Background

Inflammatory bowel disease (IBD), encompassing ulcerative colitis (UC) and Crohn’s disease (CD), represents a chronic inflammatory condition with an incompletely understood etiology. Emerging evidence suggests that alterations in gut microbiota composition play a pivotal role in disease development. Here, we leveraged gut fecal metagenomic data (3044 samples: 2248 IBD and 796 healthy) from publicly available sources to explore microbiome biomarkers related to IBD to provide new ideas for clinical treatment.

Results

Our analyses revealed marked disparities in microbial species abundance and composition between IBD and healthy samples in both male and female subjects from the United States, whereas such distinctions were absent in subjects from Spain. Hierarchical and systematic investigations uncovered microbial phyla, such as Verrucomicrobia and Firmicutes, associated with IBD in US cohorts. Furthermore, we identified 127 highly correlated pathways with these differential microbes, covering functions such as peptidoglycan biosynthesis III, dTDP-L-rhamnose biosynthesis I, starch degradation, and glucose-1-phosphate degradation. Gut microbial metabolites were predicted based on metagenomic data using the MelonnPan workflow and 16 metabolites with significant differences were identified that collectively contribute to energy metabolism, digestion, skin integrity and general bodily function.

Conclusions

This study identified differences in microbial species and metabolic pathways related to Inflammatory Bowel Disease (IBD) through hierarchical and systematic analysis, which can aid in the clinical diagnosis of IBD. Pathways such as Peptidoglycan Biosynthesis III and dTDP-L-Rhamnose Biosynthesis I are associated with the generation of bacterial cell walls, and disruptions in these pathways affect bacterial activity, leading to an imbalance in the host’s gut microbiota. Based on the hypotheses regarding the pathogenesis of IBD derived from the above mining results, it provides a theoretical basis for selecting precise treatment options.
Predicting treatment-free remission outcomes in Chronic Myeloid Leukemia patients using an integrated model of tumor-immune dynamics
Authors: Fassoni Artur C. , Yong Agnes , Clark Richard E. , Roeder Ingo , Glauche Ingmar
Published: October 2024

The interactions between tumor and the immune system are main factors in determining cancer treatment outcomes. In Chronic Myeloid Leukemia (CML), considerable evidence shows that the dynamics between residual leukemia and the patient’s immune system can result in either sustained disease control, leading to treatment-free remission (TFR), or disease recurrence. The question remains how to integrate mechanistic and data-driven models to support prediction of treatment outcomes. Starting from classical ecological modeling concepts, which allow to explicitly account for immune interactions at the cellular level, we incorporate time-course data on natural killer (NK) cell number, function, and their tumor-induced suppression into our general model of CML treatment. We identify relevant time scales governing treatment and immune response, enabling refined model calibration using tumor and NK cell time courses from different datasets. While the model successfully describes patient-specific response dynamics, critical parameters for predicting treatment outcome remain uncertain. However, by explicitly incorporating tumor load changes in response to TKI dose alterations, these parameters can be estimated and used to derive model predictions for treatment cessation. Further exploring dynamic changes in the number of functional immune cells, we suggest specific measurement strategies of immune effector cell populations to enhance prediction accuracy for CML recurrence following treatment cessation. The generalizability and flexibility of our approach represent a significant step towards quantitative, personalized medicine that integrates tumor-immune dynamics to guide clinical decisions and optimize dynamic cancer therapies.
Structural and functional evidence supports re-defining mouse higher order visual areas into a single area V2
Authors: Rowley Declan P. , Sedigh-Sarvestani Madineh
Published: October 2024

The mouse has become one of the main organisms for studies of the visual system. As a result, there is increased effort to understand universal principles of visual processing by comparing the mouse visual system to that of other species. In primates and other well-studied species including cats and tree shrews, the visual cortex is parcellated into an area V1 and several higher order areas defined by structural and functional differences, and a near complete map of the visual field. In mice, the visual cortex beyond V1 is parcellated into several higher order areas, with less notable structural and functional differences, partial coverage of the visual field, and areal boundaries defined by reversals in progression of the visual field. Notably, recent work in tree shrews and primates has shown that reversals in progression of the visual field can be a hallmark of complex retinotopic mapping within a single visual area. This, and other lines of evidence discussed here, provides a compelling case that the apparent existence of multiple higher order visual areas in the mouse is related to the false assumption of simple retinotopy. Specifically, we use simulations to show that complex retinotopy within a single visual area can recapitulate the appearance of multiple areal borders beyond mouse V1. In addition, we show that many reported differences in functional properties between higher order visual areas can be better explained by retinotopic differences rather than areal identity. Our proposal to reclassify some of the higher order visual areas in the mouse into a single area V2 is not mere semantics because areal definitions influence experimental design and data analysis. Furthermore, such a reclassification would produce a common set of rules for defining areal boundaries among mammals and would bring the mouse visual system into agreement with evolutionary evidence for a single area V2 in related lineages.
CREB3gain of function variants protect against ALS
Authors: Megat Salim , Marques Christine , Hernan Godoy Marina , Sellier Chantal , Stuart-Lopez Geoffrey , Dirrig-Grosch Sylvie , Gorin Charlotte , Brunet Aurore , Fischer Mathieu , Keime Céline , Kessler Pascal , Mendoza-Parra Marco Antonio , Scholz Sonja , Ferrucci Luigi , Ludolph Albert , Traynor Bryan , Chio Adriano , Dupuis Luc , Rouaux Caroline
Published: October 2024

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly evolving neurodegenerative disease that arises from the loss of glutamatergic corticospinal neurons (CSN) and cholinergic motoneurons (MN). The disease is mostly sporadic, but genetics is expected to highly contribute to disease onset and progression. Genome wide association studies identified a few genetic disease modifiers, mostly associated with a negative outcome, and demonstrated that ALS is primarily a disease of excitatory glutamatergic neurons. Here, we reasoned that at least a subpart of genetic disease modifiers may directly modulate the molecular pathways selectively activated in vulnerable neurons as the disease progresses, and concentrated on CSN for their selective vulnerability and glutamatergic identity. We implemented comparative cross-species transcriptomics using snRNAseq data from postmortem motor cortex of ALS patients and controls, and longitudinal RNAseq data from anatomically defined CSN purified from theSod1G86Rmouse model of ALS. We report that disease vulnerable neuronal populations undergo ER stress and altered mRNA translation, and identify the transcription factor CREB3 and its regulatory network as a resilience marker of neuronal dysfunction in ALS. Using genetic and epidemiologic analyses we further identify the rare variant CREB3R119G(rs11538707) as a new disease modifier in ALS. Through gain of function, CREB3R119Gdecreases both the risk of developing ALS and the progression rate of ALS patients. This study reveals novel genetic variants that protect against ALS and highlights the benefice of combining transcriptomics and genetics to identify new disease modifiers and therapeutic targets.
Circulating microRNAs as biomarkers of chronic kidney disease and its association with renal and cardiovascular outcomes in non-dialysis patients
Authors: Ramil-Gómez Olalla , Cerqueira Ana , Moura Sara Reis , Carvalho Eduarda , Paulo Núria , Brandão Isabel , Santos Janete , Quaranta Patricia , Sousa Maria João , Pinho Ana , Diniz Hugo , Almeida Maria Inês , Pestana Manuel , Alencastre Inês Soares
Published: October 2024

Chronic kidney disease (CKD) affects over 10% of the population worldwide and entails a significant risk for cardiovascular disease (CVD), leading to a 500-fold increase in cardiovascular mortality in advanced stages. Still, the pathophysiological mechanisms underlying kidney-heart intercommunication remain largely unknown. In recent years, microRNAs (miRNAs) emerged as important key regulators of gene expression that may serve as biomarkers for several diseases, however, their role in kidney-heart crosstalk in CKD remains underexplored. In this study, we evaluated the expression of a miRNA panel in plasma samples from non-dialysis CKD patients and explored their association with main comorbidities and significant outcomes in a 5-year follow-up. Results show that miR-30c-5p and miR-132-3p were downregulated in CKD patients presenting a significant power to discriminate the disease state. Importantly, only miR-30c-5p was downregulated in early glomerular filtration rate (GFR) categories, being able to discriminate these CKD patients from non-diseased individuals. Concerning cardiovascular outcomes, miR-199a-5p was found to be associated with an increased frequency of CVD. When analyzing the major disease outcomes in a 5-year time, miR-199a-5p upregulation at baseline was associated with increased mortality, while miR-324-3p was downregulated in patients who progressed to more advanced stages of the disease. These findings highlight the involvement of novel circulating miRNAs in CKD onset and progression, and identify, for the first time, the enrollment of miR-199a-5p in kidney-heart pathophysiological crosstalk, paving the way for the establishment of new biomarkers and therapeutic targets for CKD and its outcomes.
HighALPS: Ultra-High-Throughput Marker-Gene Amplicon Library Preparation and Sequencing on the Illumina NextSeq and NovaSeq Platforms
Authors: Flörl Lena , Cabrera Paula Momo , Moccia Maria Domenica , Plüss Serafina , Bokulich Nicholas A.
Published: October 2024

Microbiome research using amplicon sequencing of microbial marker genes has surged over the past decade, propelled by protocols for highly multiplexed sequencing with barcoded primer constructs. Newer Illumina platforms like the NovaSeq and NextSeq series significantly outperform older sequencers in terms of reads, output, and runtime. However these platforms are more prone to index-hopping, which limits the application of protocols designed for older platforms such as the Earth Microbiome Project (EMP) protocols, hence there is a need to adapt these established protocols. Here, we present an ultra-High-throughputAmpliconLibraryPreparation andSequencing protocol (HighALPS) incorporating the capabilities of these newer sequencing platforms, designed for both 16S rRNA gene and fungal internal transcribed spacer (ITS) domain sequencing. Our results demonstrate good run performance across different sequencing platforms and flow cells, with successful sequencing of mock communities, validating the protocol’s effectiveness. The new HighALPS library preparation method offers a robust, cost effective, and ultra-high-throughput solution for microbiome research, compatible with the latest sequencing technologies. This protocol allows multiplexing thousands of samples in a single run at a read depth of tens of millions of sequences per sample.
A collection of split-Gal4 drivers targeting conserved signaling ligands inDrosophila
Authors: Ewen-Campen Ben , Joshi Neha , Hermon Ashley Suraj , Thakkar Tanuj , Zirin Jonathan , Perrimon Norbert
Published: October 2024

Communication between cells in metazoan organisms is mediated by a remarkably small number of highly conserved signaling pathways. Given the relatively small number of signaling pathways, the existence of multiple related ligands for many of these pathways is thought to represent a key evolutionary innovation for encoding complexity into cell-cell signaling. Relatedly, crosstalk and other interactions between pathways is another critical feature which allows a modest number pathways to ultimately generate an enormously diverse range of outcomes. It would thus be useful to have genetic tools to identify and manipulate not only those cells which express a given signaling ligand, but also those cells that specifically co-express pairs of signaling ligands. Here, we present a collection of split-Gal4 knock-in lines targeting many of the ligands for highly conserved signaling pathways inDrosophila(Notch, Hedgehog, FGF, EGF, TGFβ, JAK/STAT, JNK, and PVR). We demonstrate that these lines faithfully recapitulate the endogenous expression pattern of their targets, and that they can be used to specifically identify the cells and tissues that co-express pairs of signaling ligands. As a proof of principle, we demonstrate that the 4th chromosome TGFβligandsmyoglianinandmaverickare broadly co-expressed in muscles and other tissues of both larva and adults, and that the JAK/STAT ligandsupd2andupd3are partially co-expressed from cells of the midgut following gut damage. Together with our previously collection of split-Gal4 lines targeting the seven Wnt ligands, this resource allowsDrosophilaresearchers to identify and genetically manipulate cells that specifically express pairs of conserved ligands from nearly all the major intercellular signaling pathways.
Escherichia coligrowth under low pH, low temperature, and high osmotic stress conditions requires thewzxEflippase for the enterobacterial common antigen intermediate
Authors: Yamaguchi Saki , Ishikawa Kazuya , Furuta Kazuyuki , Kaito Chikara
Published: October 2024

Colanic acid and enterobacterial common antigen (ECA) are cell surface polysaccharides that are produced by manyE. coliisolates. Colanic acid is induced under low pH, low temperature, and hyperosmotic conditions and is important inE. coliresistance to these stresses; however, the role of the ECA is unclear. In this study, we observed that knockout of the flippasewzxE, which converts ECA intermediates from the cytoplasmic side of the inner membrane to the periplasmic side, resulted in low pH sensitivity inE. coli. ThewzxE-knockout mutant showed reduced growth potential and viable counts in the extracts of several vegetables (cherry tomatoes, carrots, celery, lettuce, and spinach), which are known to be low pH environments. A double knockout strain ofwzxEandwecF, which encodes an enzyme that synthesizes an ECA intermediate, did not show sensitivity to low pH, nor did a double knockout mutant ofwzxEandwcaJ, which encodes a colanic acid synthase. ThewzxE-knockout mutant was sensitive to low temperature or hyperosmotic conditions, which induced colanic acid synthesis, and these sensitivities were abolished by the additional knockout ofwcaJ. These results suggest that ECA intermediates causeE. colisusceptibility to low pH, low temperature, and high osmotic pressure in a colanic acid-dependent manner, and thatwzxEsuppresses this negative effect.

Importance

Polysaccharides covering bacterial cell surfaces, such as colanic acid, confer resistance to various stresses, such as low pH. However, the role of enterobacterial common antigens, carbohydrate antigens that are conserved throughout enterobacteria, in stress resistance is unclear. Our results suggest that lipid III enterobacterial common antigen, a substrate of flippase, causes sensitivity ofEscherichia colito low pH, low temperature, and high osmolarity in dependence on colanic acid synthesis, whilewzxEinhibits this negative effect. ThewzxE-knockout mutant was sensitive to crude vegetable extracts, suggesting that the creation of WzxE inhibitors could lead to new food poisoning prevention agents.
Sex-specific effects of intensity and dose of physical activity on BOLD-fMRI cerebrovascular reactivity and cerebral pulsatility
Authors: Potvin-Jutras Zacharie , Intzandt Brittany , Mohammadi Hanieh , Liu Peiying , Chen Jean J. , Gauthier Claudine J.
Published: October 2024

Cerebrovascular reactivity (CVR) and cerebral pulsatility (CP) are important indicators of cerebrovascular health and have been shown to be associated with physical activity (PA). Sex differences have been shown to influence the impact of PA on cerebrovascular health. However, the sex-specific effects of PA on CP and CVR, particularly in relation to intensity and dosage of PA, remains unknown. Thus, this cross-sectional study aimed to evaluate the sex-specific effects of different intensities and doses of PA on CVR and CP. The Human Connectome - Aging dataset was used, including 626 participants (350 females, 276 males) aged 36-85 (mean age: 58.8 ± 14.1 years). Females were stratified into premenopausal and postmenopausal groups to assess the potential influence of menopausal status. Novel tools based solely on resting state fMRI data were used to estimate both CVR and CP. The International Physical Activity Questionnaire was used to quantify weekly self-reported PA as metabolic equivalent of task. Results indicated that both sexes and menopausal subgroups revealed negative linear relationships between relative CVR and PA. Furthermore, females presented a unique non-linear relationship between relative CVR and total PA in the cerebral cortex. In females, there were also relationships with total and walking PA in occipital and cingulate regions. In males, we observed relationships between total or vigorous PA and CVR in parietal and cingulate regions. Sex-specific effects were also observed with CP, whereby females benefited across a greater number of regions and intensities than males, especially in the postmenopause group. Overall, males and females appear to benefit from different amounts and intensities of PA, with menopause status significantly influencing the effect of PA on cerebrovascular outcomes, underscoring the need for sex-specific recommendations in promoting cerebrovascular health.
RPS19 and RPL5, the most commonly mutated genes in Diamond Blackfan anemia, impact DNA double-strand break repair
Authors: DeCleene Nicholas F. , Asik Elif , Sanchez Anthony , Williams Christopher L. , Kabotyanski Elena B. , Zhao Na , CHATTERJEE NIMRAT , Miller Kyle , Wang Yu Hsiu , Bertuch Alison A
Published: October 2024

Diamond Blackfan anemia (DBA) is caused by germline heterozygous loss-of-function pathogenic variants (PVs) in ribosomal protein (RP) genes, most commonlyRPS19andRPL5. In addition to red cell aplasia, individuals with DBA are at increased risk of various cancers. Importantly, the mechanism(s) underlying cancer predisposition are poorly understood. We found that DBA patient-derived lymphoblastoid cells had persistent γ-H2AX foci following ionizing radiation (IR) treatment, suggesting DNA double-strand break (DSB) repair defects. RPS19- and RPL5-knocked down (KD) CD34+ cells had delayed repair of IR-induced DSBs, further implicating these RPs in DSB repair. Assessing the impact of RPS19- and RPL5-KD on specific DSB repair pathways, we found RPS19-KD decreased the efficiency of pathways requiring extensive end-resection, whereas RPL5-KD increased end-joining pathways. Additionally, RAD51 was reduced in RPS19- and RPL5-KD and RPS19- and RPL5-mutated DBA cells, whereas RPS19-deficient cells also had a reduction in PARP1 and BRCA2 proteins. RPS19-KD cells had an increase in nuclear RPA2 and a decrease in nuclear RAD51 foci post-IR, reflective of alterations in early, critical steps of homologous recombination. Notably, RPS19 and RPL5 interacted with poly(ADP)-ribose chains noncovalently, were recruited to DSBs in a poly(ADP)-ribose polymerase activity-dependent manner, and interacted with Ku70 and histone H2A. RPL5's recruitment, but not RPS19's, also required p53, suggesting that RPS19 and RPL5 directly participate in DSB repair via different pathways. We propose that defective DSB repair arising from haploinsufficiency of these RPs may underline the cancer predisposition in DBA.