bluuebunny/crossref_metadata_2025 huggingface dataset:
AlphaBridge: tools for the analysis of predicted macromolecular complexes
Authors: Álvarez-Salmoral Daniel , Borza Razvan , Xie Ren , Joosten Robbie P. , Hekkelman Maarten L. , Perrakis Anastassis
Published: October 2024

Artificial intelligence (AI)-powered protein structure prediction methods have revolutionised how life scientists explore macromolecular function. Using AI to predict the structure of macromolecular complexes, is gaining attention for modelling known interactions and evaluating the likelihood of proteins forming multimers or interacting with other proteins or nucleic acids. There is a growing need for tools to efficiently evaluate these predicted models. We introduce new tools that use AlphaFold3’s “predicted local-distance difference test” (pLDDT), “predicted aligned error” (PAE), and “predicted distance error” (PDE) matrices in a graph-based community clustering approach to label sequence motifs involved in binary interactions. The resulting “interaction image” is processed through a multidimensional image algorithm to cluster interacting sequence motifs in three-dimensional binary interfaces. This method allows us to present the interaction information between multiple proteins and nucleic acids back to two-dimensional space using chord diagrams. These “AlphaBridge” diagrams summarise predicted interfaces and intermolecular interactions, including prediction confidence and sequence conservation scores. They are valuable for efficient screening of predictions, ranking, and scoring the confidence of predicted interactions, prior to more detailed (and resource intensive) analysis.
Generative Models Validation via Manifold Recapitulation Analysis
Authors: Lazzaro Nicolo’ , Leonardi Gianluca , Marchesi Raffaele , Datres Massimiliano , Saiani Anna , Tessadori Jacopo , Granados Alejandro , Henriksson Johan , Chierici Marco , Jurman Giuseppe , Tebaldi Toma , Sales Gabriele
Published: October 2024

Single-cell transcriptomics increasingly relies on nonlinear models to harness the dimensionality and growing volume of data. However, most model validation focuses on local manifold fidelity (e.g., Mean Squared Error and other data likelihood metrics), with little attention to the global manifold topology these models should ideally be learning. To address this limitation, we have implemented a robust scoring pipeline aimed at validating a model’s ability to reproduce the entire reference manifold. The Python library Cytobench demonstrates this approach, along with Jupyter Notebooks and an example dataset to help users get started with the workflow. Manifold recapitulation analysis can be used to develop and assess models intended to learn the full network of cellular dynamics, as well as to validate their performance on external datasets.

Availability

A Python library implementing the scoring pipeline has been made available via pip and can be inspected atGitHubalongside some Jupyter Notebooks demonstrating its application.

Contact

nlazzaro@fbk.euortoma.tebaldi@unitn.it
Malat1regulates female Th2 cell cytokine expression through controlling early differentiation and response to IL2
Authors: Gwynne Mags , West Katie A. , van Dongen Stijn , Kourtzelis Ioannis , Coverley Dawn , Teichmann Sarah A. , James Kylie R. , Hewitson James P. , Lagos Dimitris
Published: October 2024

Beyond sex hormone and sex chromosome-driven mechanisms, little is known about cell intrinsic mediators of immune sexual dimorphism. We show thatMalat1is required for appropriate cytokine expression in female but not male Th2 cells.Malat1deficiency impairsin vitroTh2 differentiation of naïve CD4+T cells from female mice, characterised by transcriptome-wide effects and suppression of cytokine expression, particularly IL10. Mechanistically, naïve CD4+T cells fromMalat1−/−female mice demonstrate altered early activation kinetics, followed by impairment of the early differentiation gene expression programme, including up-regulation of an interferon responsive gene module. This is followed by suppression of IL2Rα and IL2Rγ expression and IL2-mediated differentiation. Male CD4+T cell activation and Th2 differentiation are not sensitive toMalat1loss due to stronger early activation, higher interferon responsive gene expression during early differentiation, maintenance of IL2Rα expression independently ofMalat1, and lower sensitivity to exogenous IL2 during late differentiation.In vivofemale, but not male,Malat1−/−mice demonstrate altered Th2 differentiation characterised by a reduction in IL10+Th2 cells in both lung and spleen following priming and challenge withSchistosoma mansonieggs, a model of lung type 2 inflammation. These findings revealMalat1as a determinant of immune sexual dimorphism.
Identifying simultaneous inhibition of riboflavin metabolism and MEK as a novel therapeutic strategy for pancreatic cancer
Authors: Ooshio Takako , Satoh Yusuke , Jiang Hui , Liu Tongwei , Fujii Kiyonaga , Ishikawa Takamasa , Igarashi Kaori , Saitoh Kaori , Kato Keiko , Mashima Tetsuo , Jin Shigeki , Matoba Kotaro , Mack Matthias , Osawa Tsuyoshi , Seimiya Hiroyuki , Soga Tomoyoshi , Sonoshita Masahiro
Published: October 2024

Pancreatic ductal adenocarcinoma (PDAC) remains a formidable challenge with a survival rate of approximately 10%, largely due to the lack of diagnostic and therapeutic options. To identify novel therapeutic targets for PDAC, we previously developed aDrosophila melanogastermodel, the ’4-hit’ fly, which mimics the alterations inKRAS,TP53,CDKN2A, andSMAD4genes observed in PDAC. Through whole-body genetic screening using the4-hitmodel, we identified riboflavin (RF) metabolism as a novel potential therapeutic target for PDAC. Additionally, transcript levels of four out of five genes that are involved in RF metabolism were elevated in human PDAC compared to non-tumor tissues. Roseoflavin (RoF), an antimetabolite of RF, resulted in a variety range of metabolic changes, decreased mitochondrial respiratory chain activity, and suppressed proliferation in cultured PDAC cells. Furthermore, the combination of RoF with the MEK inhibitor drug trametinib significantly suppressed the growth of human PDAC xenografts compared to either treatment alone. These findings underscore that dual targeting of RF metabolism and MEK represents a novel therapeutic strategy for PDAC.
Long-term warming inverts the relationship between ecosystem function and microbial resource acquisition
Authors: Shinfuku Melissa S. , DeAngelis Kristen M.
Published: October 2024

Soil microbial traits drive ecosystem functions, which can explain the positive correlation between microbial functional diversity and ecosystem function. However, microbial adaptation to climate change related warming stress can shift microbial traits with direct implications for soil carbon cycling. Here, we investigated how long-term warming affects the relationship between microbial trait diversity and ecosystem function. Soils were sampled after 24 years of +5°C warming alongside unheated control soils from the Harvard Forest Long-Term Ecological Research site. Ecosystem function was estimated from six different enzyme activities and microbial biomass. Functional diversity was calculated from metatranscriptomics sequencing, where reads were assigned to yield, acquisition, or stress trait categories. We found that in organic horizon soils, warming decreased the richness of acquisition-related traits. In the mineral soils, we observed that heated soils exhibited a negative relationship with the richness of acquisition-related traits. These results suggest that microbial communities exposed to long-term warming are shifting away from a resource acquisition life history strategy.
3D Multiplexed Immunohistochemistry Using Opal-TSA Amplification for Enhanced Imaging of Pulmonary TB Lesions
Authors: Lata Suruchi , Yabaji Shivraj M. , O’Connell Aoife K , Gertje Hans P , Crossland Nicholas A , Kramnik Igor
Published: October 2024

Mycobacterium tuberculosishijacks the host immune system and persists for several years before the onset of active disease. Spatial characterization of epithelial compartments, immune cell populations and bacteria simultaneously within tissue specimens provide significant information about host pathogen interactions. Here, we present a protocol to detect multiple protein markers using Opal™-TSA conjugated fluorescent dyes in free floating 10% neutral buffered formalin fixed thick tissue sections (50-100 μm), with minimal additional tissue processing not requiring specialized equipment. Use of thick sections provides more information as compared to classic thin microtomy sections (3-10 μm), including the capacity for Z stacking and three-dimensional image rendering. Importantly, reduced tissue processing of samples with this method preserves endogenous fluorescent reporter signal. Use of Opal™-TSA conjugated fluorescent dyes enhances the sensitivity of low expressing proteins and supports the use of primary antibodies raised in the same species.

Before you begin

This protocol describes the specific use of Mtb infected mouse lungs, but is applicable to any tissue type and species of origin.

Institutional permissions

Obtain institutional permission to perform animal studies and collect tissues under an approved Institutional Animal Care and Use Committee (IACUC) or Institutional Review Board protocol. Our protocol was approved by Boston University’s Institutional Animal Care and Use Committee (IACUC protocol number PROTO201800218).

Mice

B6J.C3-Sst1C3HeB/FejKrmn and B6. Sst1S, ifnb-YFP mice were developed in our laboratory (available from MMRRC stock # 043908-UNC).

Highlights

Compatible with Opal™-TSA conjugated fluorescent dyes resulting in enhanced signal sensitivity with low background noise

Reduced tissue processing preserves endogenous fluorescent reporter signals

Compatible with primary antibodies raised in the same species

Doesn’t require specialized automated instruments

Graphical Abstract
Vibrio mimicuscarrying the Type III Secretion System 2 (T3SS2) and TDH toxin ofVibrio parahaemolyticusin an integrative conjugative element context
Authors: Morgado Sergio Mascarenhas , da Fonseca Erica Lourenço , Paulo Vicente Ana Carolina
Published: October 2024

Objectives

Identify possible virulence factors in the genomes ofVibrio mimicusthat may be determinants of diarrheal cases/outbreaks.

Methods

AllV. mimicusgenomes from Genbank were retrieved and the virulome was searched with Abricate using the VFDB database. Genomic islands (GIs) and integrative conjugative elements (ICEs) were identified using IslandViewer and ICEberg, respectively.

Results

Five ctx-negativeV. mimicusgenomes carrying the Type III Secretion System 2 (T3SS2) and the TDH/TRH toxin fromVibrio parahaemolyticuswere identified. The T3SS-positive genomes presented the phylotypes T3SS2α and T3SS2β and formed two clusters and one singleton throughout theV. mimicusphylogeny. Genomes carrying T3SS2α and T3SS2β were associated with thetdhandtrhgenes, respectively. Furthermore, genomic analyses characterized an integrative conjugative element (ICE) with a size of ∼150 kb carrying bothV. parahaemolyticusvirulence determinants.

Conclusions

In addition to looking for cholera toxin genes inV. mimicuscases/outbreaks, it is important to considerV. parahaemolyticustoxins such as TDH and TRH. Furthermore, an ICE was identified and characterized inV. mimicus, being associated with T3SS2 and the TDH toxin, which is worrying, as it can disseminate these virulence factors among Vibrio spp.
African origin haplotype protective for Alzheimer’s disease inAPOEε4 carriers: exploring potential mechanisms
Authors: Bertholim-Nasciben Luciana , Nuytemans Karen , Van Booven Derek , Rajabli Farid , Moura Sofia , Ramirez Aura M. , Dykxhoorn Derek M. , Wang Liyong , Scott William K. , Davis David A. , Vontell Regina T. , McInerney Katalina F. , Cuccaro Michael L. , Byrd Goldie S. , Haines Jonathan L. , Gearing Marla , Adams Larry D. , Pericak-Vance Margaret A. ,   ADSP, Young Juan I. , Griswold Anthony J. , Vance Jeffery M.
Published: October 2024

APOEε4 is the strongest genetic risk factor for Alzheimer’s disease (AD) with approximately 50% of AD patients carrying at least oneAPOEε4 allele. Our group identified a protective interaction betweenAPOEε4 with the African-specific A allele of rs10423769, which reduces the AD risk effect ofAPOEε4 homozygotes by approximately 75%. The protective variant lies 2Mb fromAPOEin a region of segmental duplications (SD) of chromosome 19 containing a cluster of pregnancy specific beta-1 glycoprotein genes (PSGs) and a long non-coding RNA. Using both short and long read sequencing, we demonstrate that rs10423769_A allele lies within a unique single haplotype inside this region of segmental duplication. We identified the protective haplotype in all African ancestry populations studied, including both West and East Africans, suggesting the variant has an old origin. Long-read sequencing identified both structural and DNA methylation differences between the protective rs10423769_A allele and non-protective haplotypes. An expanded variable number tandem repeat (VNTR) containing multiple MEF2 family transcription factor binding motifs was found associated with the protective haplotype (p-value = 2.9e-10). These findings provide novel insights into the mechanisms of this African-origin protective variant for AD inAPOEε4 carriers and supports the importance of including all ancestries in AD research.
Lipid hydrogen isotope compositions primarily reflect growth water in the model archaeonSulfolobus acidocaldarius
Authors: Harris Carolynn M , Kopf Sebastian , Rhim Jeemin Hannah , Cobban Alec , Elling Felix J , Feng Xiahong , McFarlin Jamie , Weber Yuki , Zhang Yujiao , Zhou Alice , Batther Harpreet , Pearson Ann , Leavitt William D.
Published: October 2024

The stable hydrogen isotope composition (δ2H) of lipid biomarkers can track environmental processes and remain stable over geologically relevant time scales, enabling studies of past climate, hydrology, and ecology. Most research has focused on lipids from the domain Eukarya (e.g., plant waxes, long-chain alkanes), and the potential of prokaryotic lipid biomarkers from the domain Archaea to offer unique insights into environments not captured by eukaryotic lipids remains unclear. Here, we investigate the H-isotope composition of biphytanes in Sulfolobus acidocaldarius, a model thermoacidophile and obligate heterotroph. We conducted a series of experiments that varied temperature, pH, shaking rate, electron acceptor availability, or electron donor flux. From these experiments, we quantified the lipid/water H-isotope fractionation (2ϵL/W) values for core biphytane chains derived from tetraether lipids. The2ϵL/Wvalues are consistently negative (-230 to -180 ‰) and are relatively invariant across all experiments despite the 20-fold change in doubling times and 2-fold change in lipid cyclization. The magnitude and relative invariance of2ϵL/Wvalues are consistent with studies on other heterotrophic archaea and suggests archaeal lipids may be faithful recorders of the δ2H composition of growth water. Our study highlights the potential of archaeal lipid2ϵL/Was a hydrological proxy, offering new insights into environments where traditional proxies, such as plant-derived lipids, are not available, including extreme environments and extraterrestrial settings.
A sequence-specific RNA acetylation catalyst
Authors: Thalalla Gamage Supuni , Howpay Manage Shereen , Sas-Chen Aldema , Nir Ronit , Burkhart Brett W. , Jhulki Isita , Link Courtney N. , Penikalapati Manini S. , Jones Jane E. , Iyer Lakshminarayan M. , Aravind L. , Santangelo Thomas J. , Schwartz Schraga , Meier Jordan L.
Published: October 2024

N4-acetylcytidine (ac4C) is a ubiquitous RNA modification incorporated by cytidine acetyltransferase enzymes. Here, we report the biochemical characterization ofThermococcus kodakarensisNat10 (TkNat10), an RNA acetyltransferase involved in archaeal thermotolerance. We demonstrate that TkNat10’s catalytic activity is critical forT. kodakarensisfitness at elevated temperatures. Unlike eukaryotic homologs, TkNat10 exhibits robust stand-alone activity, modifying diverse RNA substrates in a temperature, ATP, and acetyl-CoA-dependent manner. Transcriptome-wide analysis reveals TkNat10 preferentially modifies unstructured RNAs containing a 5’-CCG-3’ consensus sequence. Using a high-throughput mutagenesis approach, we define sequence and structural determinants of TkNat10 substrate recognition. We find TkNat10 can be engineered to use non-native acyl-CoA donors, providing insight into its cofactor specificity. Finally, we demonstrate TkNat10’s utility for site-specific acetylation of RNA oligonucleotides, enabling analysis of ac4C-dependent RNA-protein interactions. Our findings establish a framework for understanding archaeal RNA acetylation and a new tool for studying the functional consequences of ac4C in diverse RNA contexts.
Crosstalk between three CRISPR-Cas types enables primed type VI-A adaptation inListeria seeligeri
Authors: Margolis Shally R. , Meeske Alexander J.
Published: October 2024

CRISPR-Cas systems confer adaptive immunity to their prokaryotic hosts through the process of adaptation, where sequences are captured from foreign nucleic acids and integrated as spacers in the CRISPR array, and thereby enable crRNA-guided interference against new threats. While the Cas1-2 integrase is critical for adaptation, it is absent from many CRISPR-Cas loci, rendering the mechanism of spacer acquisition unclear for these systems. Here we show that the RNA-targeting type VI-A CRISPR system ofListeria seeligeriacquires spacers from DNA substrates through the action of a promiscuous Cas1-2 integrase encoded by a co-occurring type II-C system, in a transcription-independent manner. We further demonstrate that the type II-C integration complex is strongly stimulated by preexisting spacers in a third CRISPR system (type I-B) which imperfectly match phage targets and prime type VI-A adaptation. Altogether, our results reveal an unprecedented degree of communication among CRISPR-Cas loci encoded by a single organism.
Stoichiometry of HIV-1 Envelope Glycoprotein Protomers with Changes That Stabilize or Destabilize the Pretriggered Conformation
Authors: Zhang Zhiqing , Anang Saumya , Wang Qian , Nguyen Hanh T. , Chen Hung-Ching , Chiu Ta-Jung , Yang Derek , Smith Amos B. , Sodroski Joseph G.
Published: October 2024

During human immunodeficiency virus (HIV-1) entry into host cells, binding to the receptors, CD4 and CCR5/CXCR4, triggers conformational changes in the metastable envelope glycoprotein (Env) trimer ((gp120-gp41)3). CD4 binding induces Env to make transitions from its pretriggered conformation (PTC) to more “open” conformations that are sensitive to inhibition by antibodies, CD4-mimetic compounds (CD4mcs) and exposure to cold. Changes in functional membrane Envs have been identified that either stabilize or destabilize the PTC. Here, we investigate the stoichiometric requirements for the PTC-stabilizing and -destabilizing changes in the Env protomers. To this end, we generated viruses bearing Envs with mixed protomers exhibiting different degrees of PTC stability and determined the sensitivity of the viruses to cold (0°C) and, in some cases, to a CD4mc. The number of stabilized Env protomers required to achieve stabilization of the PTC was inversely related to the degree of PTC stabilization that resulted from the introduced Env change. For strongly stabilizing Env changes, modification of a single protomer was sufficient to achieve PTC stabilization; given adequate stability, the modified protomer can apparently constrain the conformation of the other two protomers to maintain the PTC. Weakly stabilizing Env changes needed to be present in all three protomers to achieve efficient stabilization of the PTC. In many cases, the PTC was disrupted when destabilizing changes were present in only a single protomer. These complementary results suggest that conformational symmetry among the protomers of the functional Env trimer is conducive to the integrity of the PTC.

IMPORTANCE

The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer consists of three protomers. In response to receptor binding, the flexible Env changes its conformation to mediate virus entry into host cells. The shape-shifting ability of Env also contributes to HIV-1’s capacity to evade the host immune system. The pretriggered (State-1) conformation (PTC) of Env is an important target for virus entry inhibitors and host antibodies, but is unstable and therefore incompletely characterized. Changes in Env amino acids that either stabilize or destabilize the PTC have been identified. Here, we define how many Env protomers need to be modified by these changes to achieve stabilization or destabilization of the PTC. These results can guide the placement of changes in the HIV-1 Env protomers to control the movement of the Env trimer from the PTC, allowing better characterization of this elusive conformation and testing of its utility in vaccines.
Regulation of Crassulacean Acid Metabolism at the protein level in the CAM plant Kalanchoë laxiflora
Authors: Schiller Katharina , Janshoff Saskia , Zenker Sanja , Viehöver Prisca , Hartwell James , Eirich Jürgen , Finkemeier Iris , Bräutigam Andrea
Published: October 2024

Crassulacean acid metabolism (CAM) is an adaptation to environments where water availability is seasonal or extremely low. It serves to ensure survival and/ or maintain productivity in these adverse environments. CAM has repeatedly evolved although it requires a large and complex set of enzymes and transporters and regulatory processes to control metabolite flux and pools. To test potential regulatory levels, we analyze the CAM plant Kalanchoë laxiflora embedded in the context of available CAM genome and transcriptome sequences. We show that CAM associated transcripts and proteins do not show a binary on/off pattern between day and night in K. laxiflora. Instead, we observe that many CAM plants display shared amino acid changes compared to C3 plants especially in starch metabolism. Phosphoproteomics identifies phosphoproteome changes in K. laxiflora between day and night. Taken together, the analyses demonstrate the CAM photosynthesis is regulated at the levels of transcripts and proteins.
The life history traits of phages in a cocktail determine coinfection dynamics and efficacy
Authors: Khan Ruqaiyah , Chaudhari Harshali V. , Inamdar Mandar M. , Kondabagil Kiran
Published: October 2024

Phage cocktails are preferred over single phages for efficacious and broader-spectrum therapy. An ideal phage cocktail should have a minimum number of phages with efficient infection kinetics and delay the emergence of resistance in bacterial populations. This study examined population dynamics of the common host and combinations of two phages (N4, KKE5P, and Ec_YwIITB1) through experimental and modeling approaches to gaining insights into how phage life history traits influence the outcome of infection in the short-term of approximately an infection cycle, and whether it is informative for developing efficacious cocktails. We tested the killing efficacy of a cocktail containing two divergent phages (N4 and Ec_YwIITB1) with similar adsorption rates but differed in their latency period. Because of the shorter latency period, phage N4 dominated under all conditions tested. The cocktail essentially behaves as a single phage. When two phages (N4 and KKE5P), with similar adsorption rates and latency periods but targeted different host receptors were used, it not only resulted in the efficient replication of both phages but also improved the suppression of the emergence of resistance when compared to the N4 and Ec_YwIITB1 combination, thus behaving like an ideal cocktail. The ODE-based mathematical model demonstrated predictive capabilities consistent with experimental observations and offered insights into infection dynamics. The model may aid in phage selection and optimizing cocktail formulation based on phage life history traits. This study highlights the need for thorough characterization of phage growth parameters and informed combinations of phages, as random combinations could lead to undesirable outcomes.
Cortical layer 6b mediates state-dependent changes in brain activity and effects of orexin on waking and sleep
Authors: Meijer Elise J. , Mueller Marissa , Krone Lukas B. , Yamagata Tomoko , Hoerder-Suabedissen Anna , Wilcox Sian , Alfonsa Hannah , Chakrabarty Atreyi , Guidi Luiz , Oliver Peter , Vyazovskiy Vladyslav V , Molnar Zoltan
Published: October 2024

One of the most distinctive features of the mammalian cerebral cortex is its laminar structure. Of all cortical layers, layer 6b (L6b) is by far the least-studied, despite exhibiting direct sensitivity to orexin and having widespread connectivity, suggesting an important role in regulating cortical oscillations and brain state. We performed chronic electroencephalogram (EEG) recordings in mice in which a subset of L6b neurons was conditionally silenced, during undisturbed conditions, after sleep deprivation (SD), and after intracerebroventricular (ICV) administration of orexin. While the total amount of waking and sleep or the response to SD were not altered, L6b-'silenced' mice showed a slowing of theta-frequency (6-9 Hz) during wake and REM sleep, and a marked reduction of total EEG power, especially in NREM sleep. The infusion of orexin A increased wakefulness in both genotypes, but the effect was more pronounced in L6b-silenced mice, while the increase in theta-activity by orexin B was attenuated in L6b silenced animals. In summary, we show the role of cortical L6b in state-dependent brain oscillations and global vigilance state control, which could be mediated by orexinergic neurotransmission. Our findings provide new insights in the understanding of abnormal regulation of arousal states in neurodevelopmental and anxiety disorders.
A near telomere-to-telomere phased reference assembly for the male mountain gorilla
Authors: Nelson David R. , Muvunyi Richard , Hazzouri Khaled M. , Tumushime Jean-Claude , Nzayisenga Gaspard , Julius Nziza , Meert Wim , Karim Latifa , Coppieters Wouter , Munson Katherine M. , Yoo DongAhn , Eichler Evan E. , Salehi-Ashtiani Kourosh , Twizere Jean-Claude
Published: October 2024

The endangered mountain gorilla, Gorilla beringei beringei, faces numerous threats to its survival, highlighting the urgent need for genomic resources to aid conservation efforts. Here, we present a near telomere-to-telomere, haplotype-phased reference genome assembly for a male mountain gorilla generated using PacBio HiFi (26.77× ave. coverage) and Oxford Nanopore Technologies (52.87× ave. coverage) data. The resulting non-scaffolded assembly exhibits exceptional contiguity, with contig N50 of ∼95 Mbp for the combined pseudohaplotype (3,540,458,497 bp), 56.5 Mbp (3.1 Gbp) and 51.0 Mbp (3.2 Gbp) for each haplotype, an average QV of 65.15 (error rate = 3.1 × 10-7), and a BUSCO score of 98.4%. These represent substantial improvements over most other available primate genomes. This first high-quality reference genome of the mountain gorilla provides an invaluable resource for future studies on gorilla evolution, adaptation, and conservation, ultimately contributing to the long-term survival of this iconic species.
A randomized controlled trial of the effectiveness of a community-based rabies vaccination strategy
Authors: Lankester Felix , Lugelo Ahmed , Changalucha Joel , Anderson Danni , Duamor Christian Tetteh , Czupryna Anna , Lushasi Kennedy , Ferguson Elaine , Swai Emmanuel S. , Nonga Hezron , Sambo Maganga , Cleaveland Sarah , Wyke Sally , Johnson Paul C. D. , Hampson Katie
Published: October 2024

Background

Approximately 60,000 people die from dog-mediated rabies annually. Low and heterogeneous coverage reduces the effectiveness of dog vaccination campaigns that can eliminate rabies. Campaigns typically involve teams travelling annually to villages to deliver cold chain stored vaccines from centralized headquarters. Thermotolerant vaccines enable novel decentralized delivery of locally-stored vaccines by communities throughout the year. We compared the effectiveness of annual team-based versus continuous community-based dog vaccination strategies.

Methods

We conducted a cluster randomized controlled trial across Mara region, Tanzania. Trial clusters were administrative wards (112, on average four villages each). For the team-based arm vaccinators hosted annual static-point clinics, whilst for the community-based arm, a ward-based animal health officer with a village community leader managed vaccinations using vaccines stored within the ward. We measured vaccination coverage, the primary outcome, twice annually per cluster (month 1 and 11) through household surveys over three years (November 2020 to October 2023) and examined spatial and temporal coverage variations as secondary outcomes.

Findings

Community-based delivery achieved significantly higher coverage (49-62%) than team-based delivery (22-46%), and consistently exceeded the critical threshold for herd immunity (40%), Odds ratio (OR): 1.48-3.49. The lower less uniform coverage achieved through team-based delivery had a higher monthly probability of falling below the critical threshold (0.6, 95% CI: 0.38-0.81) vs 0.18 (95% CI: 0.04-0.40). Greater declines in coverage over the year were recorded in the team-based arm compared to the community-based

Conclusion

Community-based mass dog vaccination achieves higher more consistent coverage than team-based delivery across settings typical of many sub-Saharan African countries. This approach could play an important role in national rabies elimination programmes aiming to end human rabies deaths by 2030 as part of the global ‘zero by 30’ strategy.

Funding

Department of Health and Human Services of the National Institutes of Health (R01AI141712), Wellcome Trust (207569/Z/17/Z, 224520/Z/21/Z) and MSD Animal Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Small-Molecule Modulators of Lipid Raft Stability and Protein-Raft Partitioning
Authors: Stefanski Katherine M. , Huang Hui , Luu Dustin D. , Hutchison James M. , Saksena Nilabh , Fisch Alexander J. , Hasaka Thomas P. , Bauer Joshua A. , Kenworthy Anne K. , Van Horn Wade D. , Sanders Charles R.
Published: October 2024

Development of an understanding of membrane nanodomains colloquially known as “lipid rafts” has been hindered by a lack of pharmacological tools to manipulate rafts and protein affinity for rafts. We screened 24,000 small molecules for modulators of the affinity of peripheral myelin protein 22 (PMP22) for rafts in giant plasma membrane vesicles (GPMVs). Hits were counter-screened against another raft protein, MAL, and tested for impact on raft, leading to two classes of compounds. Class I molecules altered the raft affinity of PMP22 and MAL and also reduced raft formation in a protein-dependent manner. Class II molecules modulated raft formation in a protein-independent manner. This suggests independent forces work collectively to stabilize lipid rafts. Both classes of compounds altered membrane fluidity in cells and modulated TRPM8 channel function. These compounds provide new tools for probing lipid raft function in cells and for furthering our understanding of raft biophysics.

Teaser

Compounds have been discovered that modulate the affinity of membrane proteins for lipid rafts as well as raft formation.
The effects ofCDC45mutations on DNA replication and genome stability
Authors: Denkiewicz-Kruk Milena , Chaudhry Deepali , Krasilia Alina , Jedrychowska Malgorzata , Fijalkowska Iwona J. , Dmowski Michal
Published: October 2024

Cdc45 is a non-catalytic subunit of the CMG helicase complex and is recruited to the autonomously replicating sequence at the onset of DNA replication. Cdc45 protein is required for the initiation of the DNA replication process as well as for the nascent DNA strand synthesis. It interacts with Mcm2 and Psf1 elements of CMG helicase, as well as with Sld3, and initiation factor, and Pol2, the catalytic subunit of DNA polymerase epsilon (Pol ε). In this study, we analyzed the effects of amino acid substitutions in Cdc45 regions involved in the interaction of this protein with Mcm2-7 (Cdc45-1), Psf1 (Cdc45-26), and Sld3 (Cdc45-25, Cdc45-35). We found that mutations inCDC45result in defective DNA replication. At the restrictive temperature, mutant cells are unable to initiate DNA replication, while in permissive conditions, they demonstrate DNA synthesis delay. Moreover, we observed increased mutation rates, mainly dependent on DNA polymerase zeta (Pol ζ), as well as increased incidence of replication errors. These findings contribute to our understanding of Cdc45’s function in eukaryotic cells. Changes in the cell functioning observed in this study, related to the defect in Cdc45 function, may be helpful in understanding some diseases associated withCDC45.
TIPP3 and TIPP3-fast: Improved Abundance Profiling in Metagenomics
Authors: Shen Chengze , Wedell Eleanor , Pop Mihai , Warnow Tandy
Published: October 2024

We present TIPP3 and TIPP3-fast, new tools for abundance profiling in metagenomic datasets. Like its predecessor, TIPP2, the TIPP3 pipeline uses a maximum likelihood approach to place reads into labeled taxonomies using marker genes, but it achieves superior accuracy to TIPP2 by enabling the use of much larger taxonomies through improved algorithmic techniques. We show that TIPP3 outperforms leading methods for abundance profiling in two important contexts: when reads come from genomes not already in a public database (i.e., novel genomes) and when reads contain sequencing errors. We also show that TIPP3-fast has slightly lower accuracy than TIPP3, but is still more accurate than other leading methods and uses a small fraction of TIPP3’s runtime. Additionally, we highlight the potential benefits of restricting abundance profiling methods to those reads that map to marker genes (i.e., using a filtered marker-gene based analysis), which we show typically improves accuracy. TIPP3 is freely available athttps://github.com/c5shen/TIPP3.

Author summary

TIPP3 is a new marker gene-based abundance profiling tool that builds on TIPP and TIPP2 with significant enhancements. TIPP3 supports larger reference packages (∼ 55,000 sequences per marker gene) and achieves higher accuracy in abundance profiling, especially with challenging input reads containing sequencing errors or novel genomes. TIPP3 outperforms TIPP2 and other leading methods in profiling accuracy, and its fast version TIPP3-fast is competitive in runtime with the competing methods while being more accurate under challenging conditions. TIPP3 is open-source and available athttps://github.com/c5shen/TIPP3.
Machine Learning Approach to Single Cell Transcriptomic Analysis of Sjogren’s Disease Reveals Altered Activation States of B and T Lymphocytes
Authors: McDermott Maxwell , Li Wenyi , Wang Yin-Hu , Lacruz Rodrigo , Nadorp Bettina , Feske Stefan
Published: October 2024

Sjogren’s Disease (SjD) is an autoimmune disorder characterized by salivary and lacrimal gland dysfunction and immune cell infiltration leading to gland inflammation and destruction. Although SjD is a common disease, its pathogenesis is not fully understood. In this study, we conducted a single-cell transcriptome analysis of peripheral blood mononuclear cells (PBMC) from patients with SjD and symptomatic non-SjD controls to identify cell types and functional changes involved in SjD pathogenesis. All PBMC populations showed marked differences in gene expression between SjD patients and controls, particularly an increase in interferon (IFN) signaling gene signatures. T and B cells of SjD patients displayed a depletion of ribosomal gene expression and pathways linked to protein translation. SjD patients had increased frequencies of naive B cells, which featured a unique gene expression profile (GEP) distinct from controls and had hallmarks of B cell hyperactivation. Non-negative matrix factorization (NMF) also identified several non-overlapping GEPs in CD4+and CD8+T cells with differential usage in SjD patients and controls. Of these, only theTh1 activationGEP was enriched in T cells of SjD patients whereas the other two GEPs were depleted in T cells, emphasizing the important role of Th1 cells in SjD. Our study provides evidence for aberrant and unique gene expression patterns in both B and T lymphocytes of SjD patients that point to their altered activation states and may provide new insights into the pathogenesis of SjD.
Highly porous granular hydrogels reinforced by electrospun hydrogel fibers for long-term stability
Authors: Whitewolf Jack , Riley Lindsay , Brady Brooke , Grewal Greg , Segura Tatiana , Highley Chris
Published: October 2024

Granular hydrogels (GHs) formed from hydrogel microparticles (HMPs) possess inherent, microscale porosity. In GHs including microporous annealed particle (MAP) hydrogels as well as unannealed GHs, increases in pore size and total porosity correlate with enhanced cellular infiltration and growth. However, increased porosity or pore size are typically achieved by decreasing HMP density, reducing interparticle contacts and weakening hydrogel structure. Here, to achieve a highly porous GH without compromising stability, we developed an approach using sacrificial HMPs to achieve high, mesoscale porosity and high-aspect ratio hydrogel fibers to reinforce the scaffold. The use of sacrificial HMPs circumvented the need to decrease packing density, creating GHs with up to 60% porosity. The hydrogel fibers span particles in the porous space, stabilizing the porous structure by enhancing interactions among GH components. Upon 5-10% v/v fiber incorporation, highly porous GHs retained their structure over 28 days ofin vivoculture, compared to 4 days in GHs without fibers. These scaffolds supported and promoted endothelial cell growth as porosity increased. Overall, this study presents a stable, highly porous granular scaffold system with high processability and cytocompatibility.
Effects of organic and inorganic contaminants and their mixtures on metabolic health and gene expression in developmentally exposed zebrafish
Authors: Bérubé Roxanne , LeFauve Matthew K. , Khalaf Aicha , Aminioroomi Darya , Kassotis Christopher D.
Published: October 2024

Organic and inorganic chemicals co-occur in household dust, and these chemicals have been determined to have endocrine and metabolic disrupting effects. While there is increasing study of chemical mixtures, the effects of complex mixtures mimicking household dust and other environmental matrices have not been well studied and their potential metabolism disrupting effects are thus poorly understood. Previous research has demonstrated high potency adipogenic effects of residential household dust extracts usingin vitroadipogenesis assays. More recent research simplified this to a mixture relevant to household dust and comprised of common co-occurring organic and inorganic contaminants, finding that these complex combinations often exhibited additive or even synergistic effects in cell models. This study aimed to translate our previous in vitro observation to an in vivo model, the developing zebrafish, to evaluate the metabolic effects of early exposure to organic and inorganic chemicals, individually and in mixtures. Zebrafish embryos were exposed from 1 day post fertilization (dpf) to 6 dpf, then metabolic energy expenditure, swimming behavior and gene expression were measured. Globally, we observed that most mixtures did not reflect the effects of individual chemicals; the BFR mixture produced a less potent effect when compared to the individual chemicals, while the PFAS and the inorganic mixtures seemed to have a more potent effect than the individual chemicals. Finally, the environmental mixture, mimicking household dust proportions, was less potent than the inorganic chemical mix alone. Additional work is necessary to better understand the mixture effect of inorganic and organic chemicals combined.
Fragment-based development of small molecule inhibitors targetingMycobacterium tuberculosischolesterol metabolism
Authors: Kavanagh Madeline E. , McLean Kirsty J. , Gilbert Sophie H. , Amadi Cecilia , Snee Matthew , Tunnicliffe Richard B. , Arora Kriti , Boshoff Helena I. , Fanourakis Alexander , Rebello-Lopez Maria Jose , Ortega-Muro Fatima , Levy Colin W. , Munro Andrew W. , Leys David , Abell Chris , Coyne Anthony G.
Published: October 2024

Mycobacterium tuberculosis(Mtb) is the world’s most deadly infectious pathogen and new drugs are urgently required to combat the emergence of multi-(MDR) and extensively-(XDR) drug resistant strains. The bacterium specifically upregulates sterol uptake pathways in infected macrophages and the metabolism of host-derived cholesterol is essential forMtb’slong-term survivalin vivo.Here, we report the development of antitubercular small molecules that inhibit theMtbcholesterol oxidases CYP125 and CYP142, which catalyze the initial step of cholesterol metabolism. An efficient biophysical fragment screen was used to characterize the structure-activity relationships of CYP125 and CYP142, and identify a non-azole small molecule1athat can bind to the heme cofactor of both enzymes. A structure-guided fragment-linking strategy was used to optimize the binding affinity of1a, yielding a potent dual CYP125/142 inhibitor5m(KDCYP125/CYP142 = 0.04/0.16 µM). Compound5mpotently inhibits the catalytic activity of CYP125 and CYP142in vitro(KIvalues < 0.1 µM), and rapidly depletesMtbintracellular ATP (IC50= 0.15 µM). The compound has antimicrobial activity against both drug susceptible and MDRMtb (MIC99values 0.4 - 1.5 µM)in extracellular assays, and inhibits the growth ofMtbin human macrophages (MIC = 1.7 µM) with good selectivity over mammalian cytotoxicity (LD50≥ 50 µM). The combination of small molecule inhibitors and structural data reported here provide useful tools to study the role of cholesterol metabolism inMtband are a promising step towards novel antibiotics targeting bioenergetic pathways, which could be used to help combat MDR-TB.
Application of2H2O to quantify red blood cell protein synthesis rates in young trained and untrained males and females
Authors: Kontro Hilkka , McGlory Chris , MacInnis Martin J
Published: October 2024

Aim

Compare the fractional synthetic rate (FSR) of hemoglobin in trained and untrained humans (Hb FSR).

Methods

We employed deuterated water (2H2O) to measure Hb FSR in young males (n=10) and females (n=10) who were aerobically trained (n=5 per sex) and untrained (n=5 per sex). Overall, participants had a meanof 49.8 [SD: 10.9] mL/kg/min, hemoglobin mass of 775 [180] g, and red blood cell volume of 2370 [550] mL. After an initial loading dose, participants ingested2H2O daily for 28 days to maintain a stable2H body water enrichment (∼0.9 atom percent deuterium (APD)), as measured in saliva samples collected every 2-3 days.2H-enriched alanine was measured in RBC protein using gas chromatography combustion isotope ratio mass spectrometry.

Results

The increase in APD for Hb protein was nonlinear for the first two weeks, but stabilized from day 14 to day 28, with mean APD reaching 0.033 [0.005] % on day 28. Hb FSR calculated over this 2-week period was 0.84 [0.15] %/day, which equated to an Hb absolute synthetic rate of 6.5 [2.2] g/day and a lifespan of 126 [30] days. Hb FSR was not different trained (0.83 [0.19] %/day) and untrained (0.86 [0.24] %/day, P=0.81) individuals or between males (0.80 [0.25] %/day) and females (0.88 [0.17] %/day), P=0.24).

Conclusion

Habitual endurance training does not appear to affect Hb FSR, but the use of2H2O to measure Hb FSR in humans has potential applications to many experimental and clinical scenarios.
Monitoring the Coating of Single DNA Origami Nanostructures with a Molecular Fluorescence Lifetime Sensor
Authors: Scheckenbach Michael , Brüggenthies Gereon Andreas , Schröder Tim , Betuker Karina , Wassermann Lea , Tinnefeld Philip , Heuer-Jungemann Amelie , Glembockyte Viktorija
Published: October 2024

The high functionality of DNA nanostructures makes them a promising tool for biomedical applications, their intrinsic instability under application-relevant conditions, still remains challenging. Protective coating of DNA nanostructures with materials like silica or cationic polymers has evolved as a simple, yet powerful strategy to improve their stability even under extreme conditions. While over time, various materials and protocols have been developed, the characterization and quality assessment of the coating is either time consuming, highly invasive or lacks detailed insights on single nanostructures. Here, we introduce a cyanine dye based molecular sensor designed to non-invasively probe the coating of DNA origami by either a cationic polymer or by silica, in real-time and on a single nanostructure level. The cyanine dye reports changes in its local environment upon coating via increased fluorescence lifetime induced by steric restriction and water exclusion. Exploiting the addressability of DNA origami, the molecular sensor can be placed at selected positions to probe the coating layer with nanometer precision. We demonstrate the reversibility of the sensor and use it to study the stability of the different coatings in degrading conditions. To showcase the potential for correlative studies, we combine the molecular fluorescence lifetime sensor with DNA PAINT super-resolution imaging to investigate coating and structural integrity as well as preserved addressability of DNA nanostructures. The reported sensor presents a valuable tool to probe the coating of DNA nanodevices in complex biochemical environments in real-time and at the single nanosensor level and aids the development of novel stabilization strategies.
Territorial behavior as a route of social microbial transmission in an asocial mammal
Authors: Petrullo Lauren , Webber Quinn , Raulo Aura , Boutin Stan , Lane Jeffrey E. , McAdam Andrew G. , Dantzer Ben
Published: October 2024

Microbial transmission is a major benefit of sociality, facilitated by affiliative behaviors such as grooming and communal nesting in group-living animals. The spread of microbial symbionts through these pathways, and their incorporation into host microbiomes, can enhance host health and fitness by contributing to pathogen protection and metabolic flexibility. Are pathways that facilitate microbial transfer across hosts also present in animals that do not form social groups because territoriality limits social interactions and prevents group formation? Here, we addressed this question by combining longitudinal sampling of individual gut microbial communities, demographic data, and dynamic behavioral and spatial measures of territoriality from a non-social, highly territorial small mammal: wild North American red squirrels (Tamiasciurus hudsonicus).As squirrel densities increased, individual gut microbial communities became richer and more phylogenetically diverse, while among-individual differences in composition decreased. This pattern was characterized primarily by increases in obligately anaerobic and non-sporulating taxa with little to no tolerance for oxygen-rich environments, suggesting social rather than environmental routes of transmission. Moreover, territorial intrusions—in which conspecifics were found on within an individual’s territorial space—increased gut microbial diversity among individuals defending larger territorial spaces. Using an intrusion-based social network analysis, we found that that pairs with stronger social association (via intrusions) exhibited higher gut microbial similarity. Taken together, our findings provide some of the first evidence for social microbial transmission in a non-social species, and suggest that increased density and territorial behavior can diversify and homogenize host gut microbial communities despite social isolation.