bluuebunny/crossref_metadata_2025 huggingface dataset:
IQMMA: an efficient MS1 intensity extraction using multiple feature detection algorithms for DDA proteomics
Authors: Postoenko Valeriy I. , Garibova Leyla A. , Levitsky Lev I. , Bubis Julia A. , Gorshkov Mikhail V. , Ivanov Mark V.
Published: February 2023

One of the key steps in data dependent acquisition (DDA) proteomics is detection of peptide isotopic clusters, also called «features», in MS1 spectra and matching them to MS/MS-based peptide identifications. A number of peptide feature detection tools became available in recent years each relying on its own matching algorithm. Here, we provide an integrated solution, Intensity-based Quantitative Mix and Match Approach (IQMMA), which integrates a number of peptide feature detection algorithms and returns the most probable intensity values for the MS/MS-based identifications. IQMMA was tested using available proteomic data acquired for both well-characterized (ground truth) and real-world biological samples, including a mix ofE. Colidigest spiked at different concentrations into the HeLa digest used as a background and a set of glioblastoma cell lines. Three open-source feature detection algorithms were integrated: Dinosaur, biosaur2 and OpenMS FeatureFinder. Neither of them was found optimal when applied individually to all the datasets employed in this work, however, their combined use in IQMMA improved efficiency of subsequent protein quantitation. The software implementing IQMMA is freely available athttps://github.com/PostoenkoVI/IQMMAunder Apache 2.0 license.
Virus-host dynamics in archaeal groundwater biofilms and the associated bacterial community composition
Authors: Turzynski Victoria , Griesdorn Lea , Moraru Cristina , Soares André , Simon Sophie A. , Stach Tom L. , Rahlff Janina , Esser Sarah P. , Probst Alexander J.
Published: February 2023

Lytic viruses can be prevalent in deep groundwater, yet their spatial and temporal distribution in such an ecosystem remains unexplored. Here, we tackle this gap of knowledge by studying viral infections in individual, archaea-dominated biofilm flocks sampled from deep anoxic groundwater over a period of three years. Using virusFISH whose detection efficiency for individual viral particles was 15%, we show a significant and steady increase of virus infections between the years 2019 and 2022. Based on various fluorescence micrographs of individual biofilm flocks, we determined different stages of viral infections in biofilms for single sampling events, demonstrating the progression of infection of biofilms in deep groundwater. Biofilms associated with many host cells undergoing lysis showed a substantial accumulation of filamentous microbes around infected cells probably feeding off host cell debris. Using 16S rRNA gene sequencing across ten individual biofilm flocks from one sampling event, we determined that the associated bacterial community remains relatively constant and was dominated byDesulfobacterota. Given the stability of the virus-host interaction in these deep groundwater samples, we postulate that the virus-host dynamics ofCa. Altiarchaeum hamiconexum and its abundant virus Altivir_l_MSI described herein represent a suitable model system for studying deep biosphere virus-host interactions in future research endeavors.
Widespread reductions in body size are paired with stable assemblage biomass
Authors: Martins Inês S. , Schrodt Franziska , Blowes Shane A. , Bates Amanda E. , Bjorkman Anne D. , Brambilla Viviana , Carvajal-Quintero Juan , Chow Cher F. Y. , Daskalova Gergana N. , Edwards Kyle , Eisenhauer Nico , Field Richard , Fontrodona-Eslava Ada , Henn Jonathan J , van Klink Roel , Madin Joshua S. , Magurran Anne E. , McWilliam Michael , Moyes Faye , Pugh Brittany , Sagouis Alban , Trindade-Santos Isaac , McGill Brian , Chase Jonathan M. , Dornelas Maria
Published: February 2023

Biotic responses to global change include directional shifts in organismal traits. Body size, an integrative trait that determines demographic rates and ecosystem functions, is often thought to be shrinking in the Anthropocene. Here, we assess the prevalence of body size change in six taxon groups across 5,032 assemblage time-series spanning 1960-2020. Using the Price equation to partition this change into within-species body size versus compositional changes, we detect prevailing decreases in body size through time. Change in assemblage composition contributes more to body size changes than within-species trends, but both components show substantial variation in magnitude and direction. The biomass of assemblages remains remarkably stable as decreases in body size trade-off with increases in abundance.

One-Sentence Summary

Variable within-species and compositional trends combine into shrinking body size, abundance increases and stable biomass.
RAB7 deficiency impairs pulmonary artery endothelial function and promotes pulmonary hypertension
Authors: Piper Bryce , Bogamuwa Srimathi , Hossain Tanvir , Farkas Daniela , Rosas Lorena , Green Adam , Newcomb Geoffrey , Sun Nuo , Horowitz Jeffrey C. , Bhagwani Aneel R , Yang Hu , Kudryashova Tatiana V. , Rojas Mauricio , Mora Ana L. , Yan Pearlly , Mallampalli Rama K. , Goncharova Elena A. , Eckmann David M. , Farkas Laszlo
Published: February 2023

Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with limited treatment options. Endothelial dysfunction plays a central role in development and progression of PAH, yet the underlying mechanisms are incompletely understood. The endosome-lysosome system is important to maintain cellular health and the small GTPase RAB7 regulates many functions of this system. Here, we explored the role of RAB7 in endothelial cell (EC) function and lung vascular homeostasis. We found reduced expression of RAB7 in ECs from PAH patients. Endothelial haploinsufficiency of RAB7 caused spontaneous PH in mice. Silencing of RAB7 in ECs induced broad changes in gene expression revealed via RNA sequencing and RAB7 silenced ECs showed impaired angiogenesis, expansion of a senescent cell fraction, combined with impaired endolysosomal trafficking and degradation, which suggests inhibition of autophagy at the pre-degradation level. Further, mitochondrial membrane potential and oxidative phosphorylation were decreased, and glycolysis was enhanced. Treatment with the RAB7 activator ML-098 reduced established PH in chronic hypoxia/SU5416 rats. In conclusion, we demonstrate here for the first time the fundamental impairment of EC function by loss of RAB7 that leads to PH and show RAB7 activation as a potential therapeutic strategy in a preclinical model of PH.
Structure-informed Language Models Are Protein Designers
Authors: Zheng Zaixiang , Deng Yifan , Xue Dongyu , Zhou Yi , Ye Fei , Gu Quanquan
Published: February 2023

This paper demonstrates that language models are strong structure-based protein designers. We present LM-Design, a generic approach to reprogramming sequence-based protein language models (pLMs), that have learned massive sequential evolutionary knowledge from the universe of natural protein sequences, to acquire an immediate capability to design preferable protein sequences for given folds. We conduct astructural surgeryonpLMs, where a lightweight structural adapter is implanted intopLMs and endows it with structural awareness. During inference, iterative refinement is performed to effectively optimize the generated protein sequences. Experiments show that LM-Designimproves the state-of-the-art results by a large margin, leading to 4% to 12% accuracy gains in sequence recovery (e.g., 55.65%/56.63% on CATH 4.2/4.3 single-chain benchmarks, and>60% when designing protein complexes). We provide extensive and in-depth analyses, which verify that LM-Designcan (1) indeed leverage both structural and sequential knowledge to accurately handle structurally non-deterministic regions, (2) benefit from scaling data and model size, and (3) generalize to other proteins (e.g., antibodies andde novoproteins).
BCR-ABL promotes hematopoietic stem and progenitor cell formation in embryonic stem cells
Authors: Artus Jérôme , Zenych Alina , Simanic Isidora , Desterke Christophe , Clay Denis , Saïm Sonia , Ijjeh Yousef , de Souza Lucas Eduardo Botelho , Coignard Sabrina , Bennaceur-Griscelli Annelise , Turhan Ali G. , Foudi Adlen
Published: February 2023

Generating Hematopoietic Stem Cells (HSCs) from Pluripotent Stem Cells (PSCs) has been a long-lasting quest in the field of hematopoiesis. Previous studies suggested that enforced expression of BCR-ABL, the unique oncogenic driver of Chronic Myelogeneous Leukemia (CML), in Embryonic Stem Cells (ESCs)-derived hematopoietic cells is sufficient to confer long-termin vivorepopulating potential. To precisely uncover the molecular events regulated by the Tyrosine-kinase activity of BCR-ABL1 (p210) during the course of hematopoietic differentiation, we engineered a Tet-ON inducible system to modulate its expression in murine ESC. We showed in unique site-directed knock-in ESC model, thatBCR-ABLexpression tightly regulated by doxycycline (dox) controls the formation and the maintenance of immature hematopoietic progenitors. Interestingly, these progenitors can be expandedin vitrofor several passages in the presence of dox. Our analysis of cell surface markers and transcriptome compared to wild-type fetal and adult HSCs unraveled a similar molecular signature. LTC-IC assay confirmed their self-renewal capacities albeit with a differentiation bias towards erythroid and myeloid cells. Collectively, our novel Tet-ON system represents a uniquein vitromodel to shed lights on ESC-derived hematopoiesis, CML initiation and maintenance.

KEY POINTS

We report a unique BCR-ABL-induced-embryonic stem cell -derived hematopoiesis model in murine embryonic stem cells

This BCR-ABL-induced self-renewal and differentiation model can be of major interest to uncover molecular events required for ESC-derived hematopoiesis
Label-free tumor cells classification using deep learning and high-content imaging
Authors: Piansaddhayanon Chawan , Koracharkornradt Chonnuttida , Laosaengpha Napat , Tao Qingyi , Ingrungruanglert Praewphan , Israsena Nipan , Chuangsuwanich Ekapol , Sriswasdi Sira
Published: February 2023

Many studies have shown that cellular morphology can be used to distinguish spiked-in tumor cells in blood sample background. However, most validation experiments included only homogeneous cell lines and inadequately captured the broad morphological heterogeneity of cancer cells. Further-more, normal, non-blood cells could be erroneously classified as cancer because their morphology differ from blood cells. Here, we constructed a database of microscopic images of organoid-derived cancer and normal cell with diverse morphology and developed a proof-of-concept deep learning model that can distinguish cancer cells from normal cells within an unlabeled microscopy image. In total, more than 75,000 organoid-drived cells from 3 cholangiocarcinoma patients were collected. The model achieved an area under the receiver operating characteristics curve (AUROC) of 0.78 and can generalize to cell images from an unseen patient. These resources serve as a foundation for an automated, robust platform for circulating tumor cell detection.
Low dimensionality of phenotypic space as an emergent property of coordinated teams in biological regulatory networks
Authors: Hari Kishore , Harlapur Pradyumna , Saxena Aashna , Haldar Kushal , Girish Aishwarya , Malpani Tanisha , Levine Herbert , Jolly Mohit Kumar
Published: February 2023

Biological networks driving cell-fate decisions involve complex interactions, but they often give rise to only a few phenotypes, thus exhibiting low-dimensional dynamics. The network design principles that govern such cell-fate canalization remain unclear. Here, we investigate networks across diverse biological contexts– Epithelial-Mesenchymal Transition, Small Cell Lung Cancer, and Gonadal cell-fate determination – to reveal that the presence of two mutually antagonistic, well-coordinated teams of nodes leads to low-dimensional phenotypic space such that the first principal component (PC1) axis can capture most of the variance. Further analysis of artificial team-based networks and random counterparts of biological networks reveals that the principal component decomposition is determined by the team strength within these networks, demonstrating how the underlying network structure governs PC1 variance. The presence of low dimensionality in corresponding transcriptomic data confirms the applicability of our observations. We propose that team-based topology in biological networks are critical for generating a cell-fate canalization landscape.
Common synaptic inputs and persistent inward currents of vastus lateralis motor units are reduced in older age
Authors: Guo Yuxiao , Jones Eleanor J. , Škarabot Jakob , Inns Thomas B. , Phillips Bethan E. , Atherton Philip J. , Piasecki Mathew
Published: February 2023

Although muscle atrophy may partially account for age-related strength decline, it is further influenced by alterations of neural input to muscle. Persistent inward currents and the level of common synaptic inputs to motoneurons influence neuromuscular function. However, these have not yet been described in aged human quadriceps.

High density surface electromyography (HDsEMG) signals were collected from the vastus lateralis of 15 young (mean±SD, 23 ± 5 y) and 15 older (67 ± 9 y) men during submaximal sustained and 20-s ramped contractions. HDsEMG signals were decomposed to identify individual motor unit discharges, from which delta F and intramuscular coherence were estimated.

Older participants produced significantly lower knee extensor torque (p<0.001) and poorer force tracking ability (p<0.001) than young. Older participants also had lower delta F (p=0.001) and coherence estimates in the alpha frequency band (p<0.001) during ramp contractions when compared to young.

Persistent inward currents and common synaptic inputs are lower in the vastus lateralis of older males when compared to young. These data highlight altered neural input to the clinically and functionally important quadriceps, further underpinning age-related loss of function which may occur independently of the loss of muscle mass.

Key Points

The age-related loss of muscle mass is exceeded by the loss of function, which is influenced by structural and functional alterations of the nervous system.

Motoneuronal persistent inward currents and common synaptic inputs play an important role in the activation of motor units and subsequent force generation and control ability.

Here we show reduced estimates of persistent inward currents and lower common synaptic inputs to older vastus lateralis, potentially contributing to observed lower strength and poorer force tracking.

These findings highlight decrements of the aged human motor system, accompanied by muscle atrophy in functionally relevant muscle groups, which should be considered in the application of interventions targeting aged human muscle.
Towards Universal Cell Embeddings: Integrating Single-cell RNA-seq Datasets across Species with SATURN
Authors: Rosen Yanay , Brbić Maria , Roohani Yusuf , Swanson Kyle , Li Ziang , Leskovec Jure
Published: February 2023

Analysis of single-cell datasets generated from diverse organisms offers unprecedented opportunities to unravel fundamental evolutionary processes of conservation and diversification of cell types. However, inter-species genomic differences limit the joint analysis of cross-species datasets to homologous genes. Here, we present SATURN, a deep learning method for learning universal cell embeddings that encodes genes’ biological properties using protein language models. By coupling protein embeddings from language models with RNA expression, SATURN integrates datasets profiled from different species regardless of their genomic similarity. SATURN has a unique ability to detect functionally related genes co-expressed across species, redefining differential expression for cross-species analysis. We apply SATURN to three species whole-organism atlases and frog and zebrafish embryogenesis datasets. We show that cell embeddings learnt in SATURN can be effectively used to transfer annotations across species and identify both homologous and species-specific cell types, even across evolutionarily remote species. Finally, we use SATURN to reannotate the five species Cell Atlas of Human Trabecular Meshwork and Aqueous Outflow Structures and find evidence of potentially divergent functions between glaucoma associated genes in humans and other species.
UVB modifies skin immune-stroma cross-talk and promotes effector T cell recruitment during crypticLeishmania donovaniinfection
Authors: de Oca Marcela Montes , Dey Shoumit , Van Bocxlaer Katrien , Ashwin Helen , Brown Najmeeyah , Myburgh Elmarie , Dey Nidhi S , Rani Gulab Fatima , Muscutt Edward , Osman Mohamed , Perez-Mazliah Damian , James Sally , Gilbert Lesley , Chatterjee Mitali , Kaye Paul M
Published: February 2023

Many parasites of significant public health importance assume skin residency without causing overt pathlogy. How immune and stromal cells respond to such “cryptic” infections and how exposure to UVB alters such responses in poorly understood. We combined scRNA-seq, spatial transcriptomics and inferential network analysis to address these questions in a model of cryptic skin infection byLeishmania donovani. In infected C57BL/6 mice, p-selectin and CXCL12 interactions dominate intercellular communication between leucocytes, fibroblast and endothelial cells, but effector T cell function remains muted. Following UVB exposure, increased numbers of IFNγ+CD4+Th1 cells and NK cells enter the skin, communicating with stromal cells via CCL5-CCR5 and LFA-1-ICAM1/2. However, spatial mapping indicated that Th1 cells and macrophages occupied distinct niches after UVB exposure, likely limiting effector function. Our data provide the first holistic view of the immune landscape during crypticL. donovaniinfection and demonstrate how UVB exposure fundamentally reshapes this response.

GRAPHICAL ABSTRACT
The organotypic culture of mouse seminiferous tubules as a reliable methodology for the study of meiosisin vitro
Authors: López-Jiménez P , Berenguer I , Pérez-Moreno I , de Aledo J.G. , Parra MT , Page J , Gómez R
Published: February 2023

Male mouse meiosis has been traditionally studied using descriptive methods like histological sections, spreading or squashing techniques, which allow the observation of fixed meiocytes in either wildtype or genetically modified mice. For these studies, the sacrifice of the males and the extraction of the testicles are required to obtain the material of study. Other functionalin vivostudies include the administration of intravenous or intraperitoneal drugs, or the exposure to mutagenic agents or generators of DNA damage, in order to study their impact on meiosis progression. However, in these studies, the exposure times or drug concentration are important limitations to consider when acknowledging animal welfare. Recently, several approaches have been proposed to offer alternative methodologies that allow thein vitrostudy of spermatocytes with a considerable reduction in the use of animals. Here we revisit and validate an optimal technique of organotypic culture of fragments of seminiferous tubules for meiotic studies. This technique is a trustable methodology to develop functional studies that preserve the histological configuration of the seminiferous tubule, aim homogeneity of the procedures (the use of the same animal for different study conditions), and allow procedures that would compromise the animal welfare. Therefore, this methodology is highly recommendable for the study of meiosis and spermatogenesis, while it supports the principle of 3R′s for animal research.
Mathematically mapping the network of cells in the tumor microenvironment
Authors: van Santvoort Mike , Lapuente-Santana Óscar , Finotello Francesca , van der Hoorn Pim , Eduati Federica
Published: February 2023

Cell-cell interaction networks are pivotal in cancer development and treatment response. These networks can be inferred from data; however, this process often combines data from multiple patients, and/or creates networks on a cell-types level. It creates a good average overview of cell-cell interaction networks but fails to capture patient heterogeneity and/or masks potentially relevant local network structures. We propose a mathematical model based on random graphs (called RaCInG) to alleviate these issues using prior knowledge on potential cellular interactions and patient’s bulk RNA-seq data. We have applied RaCInG to extract 444 network features related to the tumor microenvironment, unveiled associations with immune response and subtypes, and identified cancer-type specific differences in inter-cellular signaling. Additionally, we have used RaCInG to explain how immune phenotypes regulated by context-specific intercellular communication affect immunotherapy response. RaCInG is a modular pipeline, and we envision its application for cell-cell interaction reconstruction in different contexts.
Genome sequencing, assembly and annotation of the carob tree, Ceratonia siliqua(Eudicots:Fabaceae)
Authors: Bibi Androniki C. , Ioannidis Panagiotis , Bazakos Christos , Kalantidis Kriton
Published: February 2023

The carob tree,Ceratonia siliqua, is an evergreen tree that belongs to the family of Fabaceae. It has been cultivated for thousands of years in Mediterranean countries and thus, has a considerable impact on the nutrition of humans as well as other animals of this region. Despite its importance, genomic resources are still scarce for this plant species. To fill this gap, we undertook the sequencing, assembly and annotation of the carob tree genome, which resulted in the first, nearly chromosome-level assembly for this plant species. The total assembly size is 492 Mbp which is close to the previously estimated genome size. The assembly N50 is 34.99 Mbp, thus showing a high contiguity, which is also evident from the fact that >98% of the assembly is contained in as few as 17 very large contigs. Moreover, both the genome sequence as well as the predicted gene set contain more than 96% of conserved, full-length BUSCOs. Finally, a comparative orthology analysis with 10 other plant species showed that the vast majority of the predicted carob tree genes have orthologs in the other plants and only a small fraction of them appears to be species-specific. Additional analyses are in progress for a more detailed study of the agronomic traits of this important legume.
Theta and alpha EEG oscillations reflect sleep need — except during the wake maintenance zone
Authors: Snipes Sophia , Meier Elias , Meissner Sarah , Landolt Hans-Peter , Huber Reto
Published: February 2023

Increasing time spent awake results in accumulated sleep need, a process known as sleep homeostasis. Sleep homeostasis combines with a 24 h circadian rhythm to determine when and for how long we sleep. Both sleep homeostasis and the circadian rhythm substantially affect spectral power of the wake electroencephalogram (EEG), but not in ways predicted by current models. Specifically, these models hypothesize that time spent awake increases neuronal synaptic strength, which increases synchronization and should therefore increase oscillatory activity. However, the dominant wake EEG oscillations, measured as theta (4-8 Hz) and alpha power (8-12 Hz), do not follow the predicted buildup in homeostatic sleep pressure with time awake. This is due to a limitation of spectral power analysis, which does not distinguish between changes in the amplitude of oscillations from changes in the quantity of oscillations present in the signal. We wished to determine whether the amplitudes of EEG oscillations would specifically reflect homeostatic sleep pressure, independently from changes in quantity. We collected data from 18 young healthy adults during a 4-h sleep / 24-h extended wake paradigm. We indeed found that theta and alpha oscillation amplitudes reflect homeostatic sleep pressure, increasing along a saturating exponential function with time awake. Instead, theta quantities increased linearly with time awake, and alpha quantities decreased. Notably, theta and alpha amplitudes temporarily decreased during the wake maintenance zone (WMZ), a 3-4 h time window just before bedtime when it is difficult to fall asleep. Using pupillometry, we also found that mean pupil diameter increased during this window, while variance decreased. These results suggest that the WMZ is dependent on an alerting signal from the ascending arousal system. The WMZ therefore counteracts the observed build-up in homeostatic sleep pressure reflected in EEG amplitudes by temporarily desynchronizing cortical activity.
The fitness effects of codon composition of the horizontally transferred antibiotic resistance genes intensify at sub-lethal antibiotic levels
Authors: Shaferman Michael , Gensel Melis , Alon Noga , Alasad Khawla , Rotblat Barak , Serohijos Adrian W.R. , Alfonta Lital , Bershtein Shimon
Published: February 2023

The rampant variability in codon bias existing between bacterial genomes is expected to interfere with horizontal gene transfer (HGT), a phenomenon that drives bacterial adaptation. However, delineating the constraints imposed by codon bias on functional integration of the transferred genes is complicated by multiple genomic and functional barriers controlling HGT, and by the dependence of the evolutionary outcomes of HGT on the host’s environment. Here, we designed an experimental system in which codon composition of the transferred genes is the only variable triggering fitness change of the host. We replacedE. coli’s chromosomalfolAgene encoding dihydrofolate reductase, an essential enzyme that constitutes a target for trimethoprim, with combinatorial libraries of synonymous codons offolAgenes from trimethoprim-sensitiveListeria grayiand trimethoprim-resistantNeisseria sicca.The resulting populations underwent selection at a range of trimethoprim concentrations, and the ensuing changes in variant frequencies were used to infer the fitness effects of the individual combinations of codons. We found that when HGT causes overstabilization of the 5’-end mRNA, the fitness contribution of mRNA folding stability dominates over that of codon optimality. The 5’-end overstabilization can also lead to mRNA accumulation outside of the polysome, thus preventing the decay of the foreign transcripts despite the codon composition-driven reduction in translation efficiency. Importantly, the fitness effects of mRNA stability or codon optimality become apparent only at sub-lethal levels of trimethoprim individually tailored for each library, emphasizing the central role of the host’s environment in shaping the codon bias compatibility of horizontally transferred genes.
Deformed wingsis an Atg8a-interacting protein that negatively regulates autophagy
Authors: Kołodziej Marta , Tsapras Panagiotis , Konstantinou Aristi , Cameron Alexander D. , Promponas Vasilis , Nezis Ioannis P.
Published: February 2023

LC3 (microtubule-associated protein 1 light chain 3, called Atg8 in yeast andDrosophila), is one of the most well-studied autophagy-related proteins. LC3 controls selectivity of autophagic degradation by interacting with LIR (LC3-interacting region) motifs also known as AIM (Atg8-interacting motifs) on selective autophagy receptors that carry cargo for degradation. Non-degradative roles of LIR motif-dependent interactions of LC3 are poorly understood. Here, we used yeast-two hybrid screening and Alpha Fold structural predictions, and we identified transcription factor Dwg (Deformed wings) as an Atg8a-interacting protein inDrosophila. Dwg-Atg8a interaction is LIR-motif dependent. We further show that Dwg is a negative regulator of autophagy. Our results provide novel insights on non-degradative roles of LIR motif dependent interactions of Atg8a and the transcriptional regulation of autophagy inDrosophila.
Chemo-sEVs release in cisplatin-resistance ovarian cancer cells are regulated by the lysosomal function
Authors: Cerda-Troncoso Cristóbal , Grünenwald Felipe , Arias-Muñoz Eloísa , Cavieres Viviana A. , Caceres-Verschae Albano , Hernández Sergio , Gaete-Ramírez Belén , Álvarez-Astudillo Francisca , Acuña Rodrigo A , Ostrowski Matias , Burgos Patricia V. , Varas-Godoy Manuel
Published: February 2023

Ovarian cancer (OvCa) is an aggressive disease usually treated with cisplatin (CDDP)-based therapy. However, among the different types of cancers treated with CDDP, OvCa commonly develops chemoresistance to this treatment. The small extracellular vesicles (sEVs) play a central role in chemoresistance. In response to chemotherapy, resistant cells secrete sEVs named chemo-sEVs characterized by specific cargo landscape content involved in the transfer of chemoresistance to recipient cells. sEVs encompass a variety of vesicle types, including exosomes, and are formed as intraluminal vesicles (ILVs) within multivesicular endosomes (MVEs). MVEs follow at least two trafficking pathways regulated by RAB GTPase family members; 1) a secretory pathway where MVEs fuse with the plasma membrane (PM) for sEVs secretion, where RAB27A is the most studied; 2) a degradative pathway where MVEs fuse with lysosomes, an event controlled by RAB7. There is growing evidence suggesting that a loss of lysosomal function can increase sEVs secretion; however, whether sEVs secretion and the transfer of CDDP chemoresistance in OvCa is the result of a fine regulation between these two MVEs trafficking pathways is unknown. In this work, we study the status of these two pathways, between CDDP-sensitive (A2780) and CDDP-resistant (A2780cis) OvCa cells. We found A2780cis cells have an increased number of MVEs and ILVs structures, together with higher levels of ESCRTs machinery components and RAB27A, compared to A2780 cells. Moreover, CDDP promotes the secretion of chemo-sEVs in A2780cis cells. Interestingly, chemo-sEVs contain a high number of proteins related to DNA damage response. In addition, we determine A2780cis cells have a poor lysosomal function with reduced levels of RAB7. Surprisingly, silencing of RAB27A in A2780cis cells was found to be sufficient to restore lysosomal function and levels of RAB7 in A2780cis cells, switching into an A2780-like cellular phenotype. Next, we found rapamycin, a potent enhancer of lysosomal function, reduced the secretion of chemo-sEVs. Taken together, these results indicate that the secretion of chemo-sEVs in OvCa cells is determined by the balance between secretory MVEs and MVEs that are destined for lysosomal degradation. Thus, our results suggest that adjusting this balance between these two MVEs trafficking pathways could be a promising strategy for overcoming CDDP chemoresistance in OvCa.

Graphical Abstract
A Soluble Platelet-Derived Growth Factor Receptor-β Originates via Pre-mRNA Splicing in the Healthy Brain and is Differentially Regulated during Hypoxia and Aging
Authors: Payne Laura Beth , Abdelazim Hanaa , Hoque Maruf , Barnes Audra , Mironovova Zuzana , Willi Caroline E. , Darden Jordan , Jenkins-Houk Clifton , Sedovy Meghan W. , Johnstone Scott R. , Chappell John C.
Published: February 2023

ABSTRACT/SUMMARY

The platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFRβ) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases (RTKs) like PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFRβ (sPDGFRβ) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFRβ variants, and specifically during tissue homeostasis. Here, we found sPDGFRβ protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFRβ isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFRβ by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRβ transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRβ protein was detected throughout the brain parenchyma in distinct regions such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFRβ variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRβ variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRβ likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFRβ in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion – critical processes underlying neuronal health and function, and in turn memory and cognition.
Structural basis for inhibition of mycobacterial ATP synthase by squaramides and second generation diarylquinolines
Authors: Courbon Gautier M. , Palme Paul R. , Mann Lea , Richter Adrian , Imming Peter , Rubinstein John L.
Published: February 2023

Mycobacteria, such asMycobacterium tuberculosis, depend on the activity of adenosine triphosphate (ATP) synthase for growth. The diarylquinoline bedaquiline (BDQ), a mycobacterial ATP synthase inhibitor, is an important medication for treatment of drug-resistant tuberculosis but suffers from off-target effects and is susceptible to resistance mutations. Consequently, both new and improved mycobacterial ATP synthase inhibitors are needed. We used electron cryomicroscopy and biochemical assays to study the interaction ofMycobacterium smegmatisATP synthase with the second generation diarylquinoline TBAJ-876 and the squaramide inhibitor SQ31f. The aryl groups of TBAJ-876 improve binding compared to BDQ, while SQ31f, which blocks ATP synthesis ~10 times more potently than ATP hydrolysis, binds a previously unknown site in the enzyme’s proton-conducting channel. Remarkably, BDQ, TBAJ-876, and SQ31f all induce similar conformational changes in ATP synthase, suggesting the resulting conformation is particularly suited for drug binding. Further, high concentrations of the diarylquinolines uncouple the transmembrane proton motive force while for SQ31f they do not, which may explain why high concentrations of diarylquinolines have been reported to kill mycobacteria while SQ31f has not.
Using single-cell RNA sequencing to generate cell-type-specific split-GAL4 reagents throughout development
Authors: Chen Yu-Chieh David , Chen Yen-Chung , Rajesh Raghuvanshi , Shoji Nathalie , Jacy Maisha , Lacin Haluk , Erclik Ted , Desplan Claude
Published: February 2023

Cell-type-specific tools facilitate the identification and functional characterization of distinct cell types, which underly the complexity of neuronal circuits. A large collection of existing genetic tools in Drosophila relies on enhancer activity to label different subsets of cells. These enhancer-based GAL4 lines often fail to show a predicable expression pattern to reflect the expression of nearby gene(s), partly due to an incomplete capture of the full gene regulatory elements. While genetic intersectional technique such as the split-GAL4 system further improve cell-type-specificity, it requires significant time and resource to generate and screen through combinations of enhancer expression patterns. In addition, since existing enhancer-based split-GAL4 lines that show cell-type-specific labeling in adult are not necessarily active nor specific in early development, there is a relative lack of tools for the study of neural development. Here, we use an existing single-cell RNA sequencing (scRNAseq) dataset to select gene pairs and provide an efficient pipeline to generate cell-type-specific split-GAL4 lines based on the native genetic regulatory elements. These gene-specific split-GAL4 lines can be generated from a large collection of coding intronic MiMIC/CRIMIC lines either by embryo injection orin vivocassette swapping crosses and/or CRISPR knock-in at the N or C terminal of the gene. We use the developing Drosophila visual system as a model to demonstrate the high prediction power of scRNAseq-guided gene specific split-GAL4 lines in targeting known cell types. The toolkit allows efficient cluster annotation in scRNAseq datasets but also the identification of novel cell types. Lastly, the gene-specific split-GAL4 lines are broadly applicable to Drosophila tissues. Our work opens new avenues for generating cell-type-specific tools for the targeted manipulation of distinct cell types throughout development and represents a valuable resource to the fly research community.

Significance Statement

Understanding the functional role of individual cell types in the nervous systems has remained a major challenge for neuroscience researchers, partly due to incomplete identification and characterization of underlying cell types. To study the development of individual cell types and their functional roles in health and disease, experimental access to a specific cell type is often a prerequisite. Here, we establish an experimental pipeline to generate gene-specific split-GAL4 guided by single-cell RNA sequencing datasets. These lines show high accuracy for labeling targeted cell types from early developmental stages to adulthood and can be applied to any tissues in Drosophila. The collection of gene-speicifc-split-GAL4 will provide a valuable resource to the entire fly research community.
Role of the small protein Mco6 in the mitochondrial sorting and assembly machinery
Authors: Busto Jon V. , Mathar Hannah , Steiert Conny , Schneider Eva F. , Straub Sebastian P. , Ellenrieder Lars , Song Jiyao , Stiller Sebastian B. , Lübbert Philipp , Guiard Bernard , den Brave Fabian , Schulte Uwe , Fakler Bernd , Becker Thomas , Wiedemann Nils
Published: February 2023

The majority of mitochondrial precursor proteins are imported through the Tom40 β-barrel channel of the translocase of the outer membrane (TOM). The sorting and assembly machinery (SAM) is essential for β-barrel membrane protein insertion into the outer membrane and thus required for the assembly of the TOM complex. Here we demonstrate that the a-helical outer membrane protein Mco6 forms a complex with the mitochondrial distribution and morphology protein Mdm10 as part of the SAM machinery. Moreover, Mco6 also interacts with the subunit Mim1 of the mitochondrial import complex (MIM), which is itself required for the biogenesis of a-helical outer membrane proteins.MCO6andMDM10display a negative genetic interaction and aMCO6-MDM10yeast double mutant contains reduced levels of TOM complex. Cells lacking Mco6 affect the levels of Mdm10 and MIM-subunits associated with assembly defects of the TOM complex. Thus, this work reveals a role of the SAMMco6complex for the biogenesis of the mitochondrial outer membrane.
Archaic Introgression Shaped Human Circadian Traits
Authors: Velazquez-Arcelay Keila , Colbran Laura L. , McArthur Evonne , Brand Colin , Rinker David , Siemann Justin , McMahon Douglas , Capra John A.
Published: February 2023

Introduction

When the ancestors of modern Eurasians migrated out of Africa and interbred with Eurasian archaic hominins, namely Neanderthals and Denisovans, DNA of archaic ancestry integrated into the genomes of anatomically modern humans. This process potentially accelerated adaptation to Eurasian environmental factors, including reduced ultra-violet radiation and increased variation in seasonal dynamics. However, whether these groups differed substantially in circadian biology, and whether archaic introgression adaptively contributed to human chronotypes remains unknown.

Results

Here we traced the evolution of chronotype based on genomes from archaic hominins and present-day humans. First, we inferred differences in circadian gene sequences, splicing, and regulation between archaic hominins and modern humans. We identified 28 circadian genes containing variants with potential to alter splicing in archaics (e.g.,CLOCK,PER2,RORB,RORC), and 16 circadian genes likely divergently regulated between present-day humans and archaic hominins, includingRORA. These differences suggest the potential for introgression to modify circadian gene expression. Testing this hypothesis, we found that introgressed variants are enriched among eQTLs for circadian genes. Supporting the functional relevance of these regulatory effects, we found that many introgressed alleles have associations with chronotype. Strikingly, the strongest introgressed effects on chronotype increase morningness, consistent with adaptations to high latitude in other species. Finally, we identified several circadian loci with evidence of adaptive introgression or latitudinal clines in allele frequency.

Conclusions

These findings identify differences in circadian gene regulation between modern humans and archaic hominins and support the contribution of introgression via coordinated effects on variation in human chronotype.

SIGNIFICANCE STATEMENT

Interbreeding between humans and Neanderthals created the potential for adaptive introgression as humans moved into environments that had been populated by Neanderthals for hundreds of thousands of years. Here we discover lineage-specific genetic differences in circadian genes and their regulatory elements between humans and Neanderthals. We show that introgressed alleles are enriched for effects on circadian gene regulation, consistently increase propensity for morningness in Europeans, and show evidence of adaptive introgression or associations between latitude and frequency. These results expand our understanding of how the genomes of humans and our closest relatives responded to environments with different light/dark cycles, and demonstrate a coordinated contribution of admixture to human chronotype in a direction that is consistent with adaptation to higher latitudes.
A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes
Authors: Ellis Nicole A. , Myers Kevin S. , Tung Jessica , Ward Anne Davidson , Johnston Kathryn , Bonnington Katherine E. , Donohue Timothy J. , Machner Matthias P.
Published: February 2023

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed amultiplex, randomizedCRISPR interferencesequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44Legionella pneumophilavirulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged withL. pneumophilabearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes.lpg2888andlpg3000were particularly fascinating for their apparent redundant functions duringL. pneumophilahuman macrophage infection, whilelpg3000alone was essential forL. pneumophilavirulence in the amoeban hostAcanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.
MICA: A multi-omics method to predict gene regulatory networks in early human embryos
Authors: Alanis-Lobato Gregorio , Bartlett Thomas E. , Huang Qiulin , Simon Claire , McCarthy Afshan , Elder Kay , Snell Phil , Christie Leila , Niakan Kathy K.
Published: February 2023

Recent advances in single-cell-omics have been transformative to characterise cell types in challenging to study biological contexts. It is technically difficult to infer transcription factor-gene regulatory interactions from these datasets, especially in contexts with limited single-cell sample number such as the early human embryo. Here we systematically assessed the application of four different linear or non-linear gene regulatory network (GRN) prediction strategies to single-cell simulated and human embryo transcriptome datasets. We also compared how gene expression normalisation methods impact on regulatory network predictions, finding that TPM (transcripts per million reads) outperformed alternative methods. We identified more reproducible GRN inferences using a non-linear method based on mutual information (MI) applied to single-cell transcriptome datasets refined with chromatin accessibility (CA) (called MICA), compared to alternative network prediction methods tested. MICA captures complex non-monotonic dependencies and feedback loops. Using MICA, we generated the first GRN inferences in early human development. MICA predicted co-localisation of the AP-1 transcription factor subunit proto-oncogene JUND and the TFAP2C transcription factor AP-2ψ in human preimplantation embryos. These interactions were supported by previous findings in other developmental and stem cell contexts. Overall, our comparative analysis of gene regulatory network prediction methods defines a pipeline that can be applied to single-cell multi-omics datasets in especially challenging contexts to infer interactions between transcription factor expression and target gene regulation.
The cerebellum promotes sequential foraging strategies and contributes to the directional modulation of hippocampal place cells
Authors: Zhang Lu , Fournier Julien , Fallahnezhad Mehdi , Paradis Anne-Lise , Rochefort Christelle , Rondi-Reig Laure
Published: February 2023

The cerebellum contributes to goal-directed navigation abilities and place coding in the hippocampus. Here we investigated its contribution to foraging strategies. We recorded hippocampal neurons in mice with impaired PKC-dependent cerebellar functions (L7-PKCI) and in their littermate controls while they performed a task where they were rewarded for visiting a subset of hidden locations. We found that L7-PKCI and control mice developed different foraging strategies: while control mice repeated spatial sequences to maximize their rewards, L7-PKCI mice persisted to use a random foraging strategy. Sequential foraging was associated with more place cells exhibiting theta-phase precession and theta rate modulation. Recording in the dark showed that PKC-dependent cerebellar functions controlled how self-motion cues contribute to the selectivity of place cells for both position and direction. Thus, the cerebellum contributes to the development of optimal sequential paths during foraging, possibly by controlling how self-motion and theta signals contribute to place cells coding.