bluuebunny/crossref_metadata_2025 huggingface dataset:
How do gepotidacin and zolifodacin stabilize DNA-cleavage complexes with bacterial type IIA topoisomerases? 2. A Single Moving Metal Mechanism
Authors: Nicholls Robert A. , Morgan Harry , Warren Anna J. , Ward Simon E. , Long Fei , Murshudov Garib N. , Sutormin Dimitry , Bax Benjamin D.
Published: October 2024

DNA gyrase is a type IIA topoisomerase that can create temporary double-stranded DNA breaks to regulate DNA topology and an archetypical target of antibiotics. The widely used quinolone class of drugs use a water-metal ion bridge in interacting with the GyrA subunit of DNA gyrase. Zoliflodacin sits in the same pocket as quinolones but interacts with the GyrB subunit and also stabilizes lethal double-stranded DNA-breaks. Gepotidacin had been observed to sit on the twofold axis of the complex, midway between the two four base-pair separated DNA-cleavage sites and has been observed to stabilize singe-stranded DNA-breaks. Here we use information from three crystal structures of complexes ofStaphlococcus aureusDNA gyrase (one with a precursor of gepotidacin and one with the progenitor of zoliflodacin) to propose a simple single moving metal-ion catalyzed DNA-cleavage mechanism. Our model explains why the catalytic tyrosine is in the tyrosinate (negatively charged) form for DNA-cleavage. Movement of a single catalytic metal-ion (Mg2+or Mn2+) guides water mediated protonation and cleavage of the scissile phosphate which is then accepted by the catalytic tyrosinate. Type IIA topoisomerases need to be able to rapidly cut the DNA when it becomes positively supercoiled (in front of replication forks and transcription bubbles) and we propose that the original purpose of the small Greek Key domain, common to all type IIA topoisomerases, was to allow access of the catalytic metal to the DNA-cleavage site. Although the proposed mechanism is consistent with published data, it is not proven and other mechanisms have been proposed. Finally how such mechanisms can be experimentally distinguished is considered.
Investigating the genetic architecture of biotic stress response in stone fruit tree orchards under natural infections with a multi-environment GWAS approach
Authors: Serrie Marie , Segura Vincent , Blanc Alain , Brun Laurent , Dlalah Naïma , Gilles Frédéric , Heurtevin Laure , Le-Pans Mathilde , Signoret Véronique , Viret Sabrina , Audergon Jean-Marc , Quilot Bénédicte , Roth Morgane
Published: October 2024

The mapping and introduction of sustainable plant immunity to pests and diseases in fruit tree is still a major challenge in modern breeding. This study aims at deciphering the genetic architecture underlying resistance or tolerance across environments for major pests and diseases in peach (P. persica) and apricot (P. armeniaca). We set up a multi-environment trial (MET) approach by studying two core collections of 206 peach and 150 apricot accessions deployed under low phytosanitary conditions in respectively three and two environmentally contrasted locations in South-East of France. To capture the complex dynamics of pest and disease spread in naturally infected orchards, visual scoring of symptoms was repeated within and between 3 years, for five and two pests and diseases respectively for peach and apricot, resulting in the maximum of damage score and the AUDPC. These traits were used as phenotypic inputs in our genome-wide association studies (GWAS) strategy, and leading to the identification of: i) non-additive genotype–phenotype associations, ii) environment-shared QTLs iii) environment-specific QTLs, and iv) interactive QTLs which changes in direction (‘antagonist’) or intensity (‘differential’) according to the environment. By conducting GWAS with multiple methods, we successfully identified a total of 60 high confidence QTLs, leading to the identification of 87 candidate genes, the majority belonging to the Leucine-rich repeat containing receptors (LRR-CRs) family gene. Finally, we provided a comparative analysis of our results on peach and apricot, two closely related species. The present results contribute to the development of genomics-assisted breeding to improve biotic resilience inPrunusvarieties.
Estimating the rate of sexual reproduction and the inbreeding rate inLeishmania
Authors: Nicoll Andrew G. , Kilford Hannah , Grace Cooper Alastair , Reis-Cunha João Luís , Jeffares Daniel C. , Constable George W. A.
Published: October 2024

Many eukaryotic species undergo sexual reproduction facultatively, either in response to stress or in particular environments. For the remainder of their life cycle they reproduce by mitosis. This facultative sex may be rare, which alters features of their evolutionary dynamics. Both the frequency of sex and the degree of inbreeding have been challenging to estimate from genome-scale polymorphism data. Here, we describe a method to estimate both these parameters based on the Moran model of heterozygous sites. We apply this method to population genomic data fromLeishmaniaparasitic protozoans from theLeishmania donovanispecies complex, showing that these parasites undergo sexual reproduction approximately once per 10,000 generations and that populations vary considerably in the extent of their inbreeding.
Carafe enables high qualityin silicospectral library generation for data-independent acquisition proteomics
Authors: Wen Bo , Hsu Chris , Zeng Wen-Feng , Riffle Michael , Chang Alexis , Mudge Miranda , Nunn Brook , Berg Matthew D. , Villén Judit , MacCoss Michael J. , Noble William S.
Published: October 2024

Data-independent acquisition (DIA)-based mass spectrometry is becoming an increasingly popular mass spectrometry acquisition strategy for carrying out quantitative proteomics experiments. Most of the popular DIA search engines make use ofin silicogenerated spectral libraries. However, the generation of high-quality spectral libraries for DIA data analysis remains a challenge, particularly because most such libraries are generated directly from data-dependent acquisition (DDA) data or are fromin silicoprediction using models trained on DDA data. In this study, we developed Carafe, a tool that generates high-quality experiment-specificin silicospectral libraries by training deep learning models directly on DIA data. We demonstrate the performance of Carafe on a wide range of DIA datasets, where we observe improved fragment ion intensity prediction and peptide detection relative to existing pretrained DDA models.
Repeated presentation of visual threats drives innate fear habituation and is modulated by environmental and physiological factors
Authors: Carroll Jordan N. , Myers Brent , Vaaga Christopher E.
Published: October 2024

To survive predation, animals must be able to detect and appropriately respond to predator threats in their environment. Such defensive behaviors are thought to utilize hard-wired neural circuits for threat detection, sensorimotor integration, and execution of ethologically relevant behaviors. Despite being hard-wired, defensive behaviors (i.e. fear responses) are not fixed, but rather show remarkable flexibility, suggesting that extrinsic factors such as threat history, environmental contexts, and physiological state may alter innate defensive behavioral responses. The goal of the present study was to examine how extrinsic and intrinsic factors influence innate defensive behaviors in response to visual threats. In the absence of a protective shelter, our results indicate that mice showed robust freezing behavior following both looming (proximal) and sweeping (distal) threats, with increased behavioral vigor in response to looming stimuli, which represent a higher threat imminence. Repeated presentation of looming or sweeping stimuli at short inter-trial intervals resulted in robust habituation of freezing, which was accelerated at longer inter-trial intervals, regardless of contextual cues. Finally, physiological factors such as acute stress further disrupted innate freezing habituation, resulting in a delayed habituation phenotype, consistent with a heightened fear state. Together, our results indicate that extrinsic factors such as threat history, environmental familiarity, and physiological stressors have robust and diverse effects on defensive behaviors, highlighting the behavioral flexibility in how mice respond to predator threats.
FBXW7 regulates MYRF levels to control myelin capacity and homeostasis in the adult CNS
Authors: Collins Hannah Y. , Doan Ryan A. , Li Jiaxing , Early Jason E. , Madden Megan E. , Simkins Tyrell , Lyons David A. , Monk Kelly R. , Emery Ben
Published: October 2024

Myelin, along with the oligodendrocytes (OLs) that produce it, is essential for proper central nervous system (CNS) function in vertebrates. Although the accurate targeting of myelin to axons and its maintenance are critical for CNS performance, the molecular pathways that regulate these processes remain poorly understood. Through a combination of zebrafish genetics, mouse models, and primary OL cultures, we found FBXW7, a recognition subunit of an E3 ubiquitin ligase complex, is a regulator of adult myelination in the CNS. Loss ofFbxw7in myelinating OLs resulted in increased myelin sheath lengths with no change in myelin thickness. As the animals aged, they developed progressive abnormalities including myelin outfolds, disrupted paranodal organization, and ectopic ensheathment of neuronal cell bodies with myelin. Through biochemical studies we found that FBXW7 directly binds and degrades the N-terminal of Myelin Regulatory Factor (N-MYRF), to control the balance between oligodendrocyte myelin growth and homeostasis.
Epigenetic derepression of H3K9me3 mitigates Alzheimer-related pathology and improves cognition via immunomodulation and Vgf induction
Authors: Fuchs Dieu-Trang , Vit Jean-Philippe , Rentsendorj Altan , Sheyn Julia , Shi Haoshen , Koronyo Yosef , Chepurna Oksana , Davis Miyah R. , Wilson Jered W. , Schneider Lon S. , Graham Stuart L. , Gupta Vivek K. , Fahnestock Margaret , Sun Tao , Kleinman Michael T. , Horne David , Mirzaei Mehdi , Black Keith L. , Koronyo-Hamaoui Maya
Published: October 2024

We investigated the role of histone 3 lysine 9 trimethylation (H3K9me3), an epigenetic mechanism involved in the repression of synaptic plasticity and memory-related genes, within aging and Alzheimer’s disease (AD). Our study reveals that elevated cortical H3K9me3 strongly correlates with cognitive dysfunction in individuals with mild cognitive impairment (MCI) and AD. In old (18 months) and younger (14 months) APPSWE/PS1ΔE9 and 3xTg AD mouse models, inhibiting SUV39H1 methyltransferase with ETP69, substantially reduces cerebral H3K9me3 levels and attenuates amyloid-β burden, tau pathology, and gliosis. Administration of ETP69 further promotes dendritic spine formation, leading to rapid and sustained improvements in cognitive function. Proteomics analysis indicates that a significant proportion of dysregulated proteins in the brains of AD-model mice are reversed by ETP69. These proteins are enriched for synaptic plasticity and learning-related pathways. ETP69 exerts its effects through multiple neuroprotective mechanisms, including regulation of neuroinflammation, induction of both blood and cerebral-infiltrating monocytes involved in cerebral Aβ clearance. Moreover, ETP69 activates brain-derived neurotrophic factor (Bdnf) network, and particularly its downstream effector neurosecretory protein Vgf. These findings support the pharmacological inhibition of H3K9me3-mediated gene silencing to reverse AD-related pathology and cognitive decline.
a2iHelper: a Python toolkit for a differential editing site analysis of RNA-Seq data
Authors: Ribas Guilherme T. , Guizelini Dieval , Riella Leonardo V. , Riella Cristian V.
Published: October 2024

Background

A-to-I RNA editing, mediated byADARenzymes, plays a crucial role in cancer and autoimmune disorders but lacks standardized tools for differential analysis. After reviewing 55 studies, it highlights significant methodological heterogeneity, hindering result comparability and reproducibility. To address this, we developed a2iHelper, a Python package that streamlines RNA editing analysis by filtering noise, performing statistical analyses, and generating visualizations. a2iHelper integrates seamlessly with Python machine learning tools, aiming to standardize and simplify RNA editing research.

Results

We applied our methods to analyze A-to-I editing in a public dataset comparing wild-type andADARknockout. The source code is open, freely available on GitHub, and organized in a well-documented Python package. Using Snakemake for preprocessing, we conducted differential editing analysis on the top 104 most edited genes. The results include p-values from statistical tests, Odds ratios for Manhattan plots, and correlations between editing frequencies and gene expression, visualized in various plots.

Conclusions

We developed a2iHelper, a Python-based package for analyzing and visualizing A-to-I RNA editing data. It allows researchers with minimal programming experience to perform organized, reproducible editing analyses. Novice users can easily detect editing sites, filter noise, and generate figures, while advanced users can integrate functionalities into their workflows. a2iHelper runs on personal computers without needing High-Performance Computing resources.
From Simulations to Inference: Using Machine Learning to Tune Patient-Specific Finite-Element Models of the Middle Ear Towards Objective Diagnosis
Authors: Motallebzadeh Hamid , Deistler Michael , Schönleitner Florian M. , Macke Jakob H. , Puria Sunil
Published: October 2024

Computational models, particularly finite-element (FE) models, are essential for interpreting experimental data and predicting system behavior, especially when direct measurements are limited. A major challenge in tuning these models is the large number of parameters involved. Traditional methods, such as one-by-one sensitivity analyses, are time-consuming, subjective, and often return only a single set of parameter values, focusing on reproducing averaged data rather than capturing the full variability of experimental measurements.

In this study, we applied simulation-based inference (SBI) using neural posterior estimation (NPE) to tune an FE model of the human middle ear. The training dataset consisted of 10,000 FE simulations of stapes velocity, ear-canal (EC) input impedance, and absorbance, paired with seven FE parameter values randomly sampled within plausible ranges. The neural network learned the association between parameters and simulation outcomes, returning the probability distribution of parameter values that can reproduce experimental data.

Our approach successfully identified parameter sets that reproduced three experimental datasets simultaneously. By accounting for experimental noise and variability during training, the method provided a probability distribution of parameters, representing all valid combinations that could fit the data, rather than tuning to averaged values. The network demonstrated robustness to noise and exhibited an efficient learning curve due to the large training dataset.

SBI offers an objective alternative to laborious sensitivity analyses, providing probability distributions for each parameter and uncovering interactions between them. This method can be applied to any biological FE model, and we demonstrated its effectiveness using a middle-ear model. Importantly, it holds promise for objective differential diagnosis of conductive hearing loss by providing insight into the mechanical properties of the middle ear.
Virion-associated influenza hemagglutinin clusters upon sialic acid binding visualized by cryo-electron tomography
Authors: Huang Qiuyu J. , Kim Ryan , Song Kangkang , Grigorieff Nikolaus , Munro James B. , Schiffer Celia A. , Somasundaran Mohan
Published: October 2024

Influenza viruses are enveloped, negative sense single-stranded RNA viruses covered in a dense layer of glycoproteins. Hemagglutinin (HA) accounts for 80-90% of influenza glycoprotein and plays a role in host cell binding and membrane fusion. While previous studies have characterized structures of receptor-free and receptor-bound HA in vitro, the effect of receptor binding on HA organization and structure on virions remains unknown. Here, we used cryo-electron tomography (cryoET) to visualize influenza virions bound to a sialic acid receptor mimic. Overall, receptor binding did not result in significant changes in viral morphology; however, we observed rearrangements of HA trimer organization and orientation. Compared to the even inter-glycoprotein spacing of unliganded HA trimers, receptor binding promotes HA trimer clustering and formation of a triplet of trimers. Subtomogram averaging and refinement yielded 8-10 Å reconstructions that allowed us to visualize specific contacts between HAs from neighboring trimers and identify molecular features that mediate clustering. Taken together, we present new structural evidence that receptor binding triggers clustering of HA trimers, revealing an additional layer of HA dynamics and plasticity.
A resource for extracellular vesicles from activated CD4 T cells that relay pro-inflammatory signals
Authors: Jainarayanan Ashwin K. , Mahla Ranjeet Singh , Capera Jesusa , Elanchezhian Mirudula , Gupta Dhanu , Anand Nithishwer Mouroug , Thomas Tom , Beckers Daniel , Leithner Alexander , Achar Sooraj R , Valvo Salvatore , Conceição Mariana , Kurz Elke , Devaprasad Abhinandan , Amin Sakina , Berridge Georgina , Hester Svenja , Fischer Roman , Céspedes Pablo F. , Dustin Lynn B. , Wood Matthew J. A. , Dustin Michael L.
Published: October 2024

CD4 T helper cells (TH cells) play a vital role in coordinating and amplifying the immune response to specific pathogens. They constitutively produce different kinds of extracellular vesicles (EVs), which mediate cell-to cell communication and play diverse roles in immune regulation and inflammatory processes. Here we provide a resource documenting the composition of activated TH cell EVs and demonstrating their ability to instigate pro-inflammatory response in antigen-presenting cells (APCs). EVs were characterized by lipidomics, proteomics, and NanoFCM. The activated TH cells derived EVs (act-EVs) were found to be enriched in TH cell-specific proteins, transmembrane and cytosolic EV marker proteins, and HLA proteins relative to resting CD4 T cells EVs (rest-EVs). The pro-inflammatory effect of act-EVs vs rest-EVs on donor matched APCs were characterized by chemokine and cytokine profiling and flow cytometry analysis. There was no discernible contrast in endotoxin levels between act-EVs and rest-EVs. Functional distinctions were seen to arise from variations in the content and composition of these EVs. Moreover, we validated our findings with an in-vivo investigation in mice, demonstrating the recruitment of monocytes, dendritic cells (DCs), neutrophils, and NK cells in the spleen, accompanied by the release of pro-inflammatory cytokines in the serum after administering act-EVs. In summary, this study sheds light on the role of TH cell released EVs in modulating the immune response during pro-inflammatory responses and this resource provides a foundation for development of novel therapeutics on EV based scaffolds.
Widespread Prevalence of CD19 Exon 5-6 Skipping In Indian Pediatric B-cell Acute Lymphoblastic Leukemia Patients
Authors: Srivastava Devesh , Gupta Anurag , Verma Nishant , Misra Ashish
Published: October 2024

B-cell acute lymphoblastic leukemia (B-ALL) is characterized by the malignant burgeoning of abnormal B-cell lymphoblasts. In recent years, the use of CART therapy which targets CD19 antigen present on the surface of B-cells, has gained significant attention as a treatment option against aggressive and refractory forms of B-ALL. However, the loss of CD19 antigen on B-cell surface due to aberrant splicing under therapy pressure has been suggested as one of the main factors for the emerging CART therapy resistance. Herein, using RT-PCR based splice assays we examined CD19 splicing patterns in 43 primary pediatric B-ALL patient samples spread across various subtypes. We observed that CD19 isoform lacking exon 5-6 exists in ∼ 55% of pediatric patients at the initial diagnosis stage itself. Using in-silico analysis, we identified RNA binding proteins, RC3H1 and MBNL1, as potential regulators of exon 5-6 skipping. Furthermore, qRT-PCR analysis in patient samples revealed that RC3H1 and MBNL1 are significantly upregulated in samples exhibiting exon 5-6 skipping. Taken together, we for the first time report the existence of aberrantly splicedCD19isoform lacking exon 5-6 in primary pediatric patients, and this occurrence could potentially result from RC3H1 and MBNL1 dysregulation.
Acceptability of the routine use of pulse oximeters into the Integrated Management of Childhood Illness guidelines at primary health centers in West Africa: a mixed-methods study.
Authors: Louart Sarah , HEDIBLE Gildas Boris , Balde Habibata , Coulibaly Abdourahmane , Dagobi Abdoua Elhadji , Kadio Kadidiatou , Neboua Desire , Zair Zineb , LEROY Valeriane , Ridde Valery ,   Aire Research Study Group
Published: October 2024

Introduction: To better identify illnesses with severe hypoxemia in children aged under-5 years, the AIRE project has implemented the routine use of a Pulse Oximeter (PO) into the Integrated Management of Childhood Illness (IMCI) guidelines at primary health centers (PHC) in Burkina Faso, Guinea, Mali, and Niger. We aimed to measure and understand the acceptability among healthcare worker.s (HCW) and children.s families (CF) of using PO in these contexts. Methods: Based on an original conceptual framework, we conducted a convergent mixed methods study to assess HCW.s and CF.s acceptability. We used quantitative questionnaires based on the Likert scale to conduct four repeated cross-sectional studies among all HCW on duty in the 202 PHC involved in the AIRE project before the intervention (before and after PO use training), six months after the introduction of the PO, and two months after project completion. We also conducted semi-structured interviews with HCW (n=102) and CF (n=59). Results: From March 2021 to December 2022, 486, 537, 538, and 476 HCW completed the four acceptability surveys. Overall, 31% of HCW had mitigated feelings about PO use before the training, 46% found it rather acceptable and 23% strongly acceptable. At the end of the project, it was respectively 15%, 34%, and 51%. PO training was consistently associated with greater HCW acceptability. HCW found many advantages in using PO, such as improved care for children, more accurate diagnosis, and a boost in their confidence in childcare management. Nevertheless, perceived increased workload and consultation time, as well as difficulties in referring children to hospital remain challenging. CF did not necessarily understand the new device's purpose, but their opinions of the technology were generally positive. Conclusion: The PO use, integrated into IMCI consultations, seems to be accepted by HCW and CF, although sustainable challenges remain. Keywords: Acceptability, Innovation, West Africa, Children under five, Pulse oximeters
Cardiopulmonary Exercise Tests in People with Chronic Stroke: Interpretation and Clinical Application
Authors: McCartney Kiersten M. , Edwards David , Pohlig Ryan , Boyne Pierce , Wright Tamara , Wright Henry , Overstreet Matthew , Reisman Darcy S.
Published: October 2024

Objective

To understand in people with stroke: 1) reasons for cardiopulmonary exercise test termination, 2) how frequently secondary criteria indicating a maximal test are met, and 3) how additional clinical measures provide context to interpreting test data.

Design

A secondary analysis from the Promoting Recovery Optimization of Walking Activity in Stroke (NCT02835313) clinical trial.

Setting

Four outpatient rehabilitation clinics.

Participants

People with chronic stroke able to walk without assistance of another person.

Intervention

Participants (n= 250) were randomized into a high-intensity treadmill or a step activity monitoring group. Cardiopulmonary exercise tests were conducted pre- (n= 247) and post-intervention (n= 185).

Main Measures

The primary measure was reason for cardiopulmonary exercise test termination. Secondary measures included: oxygen consumption, ventilatory threshold, peak heart rate, respiratory exchange ratio, six-minute walk test, and fastest walking speed. General Linear Mixed Methods were used to determine the fixed main effects of group and time, and their interaction for outcome variables.

Results

There were 4 categories of test termination. Only 1.9% of tests achieved the threshold to confirm a maximal aerobic effort. Despite one group receiving a high-intensity walking intervention, there were no between group differences in oxygen consumption or ventilatory threshold. There were significant differences between groups in measures of walking capacity.

Conclusions

Analyses indicate few with chronic stroke demonstrate a maximal aerobic effort on a cardiopulmonary exercise test. If the cardiorespiratory system is not thoroughly taxed during these tests in people with chronic stroke, interpreting results as their cardiorespiratory fitness should be done cautiously.

Clinical Messages

People with stroke rarely (< 2%) attain an aerobic maximum on cardiopulmonary exercise tests.

People with chronic stroke, frequently stop these tests due to biomechanical reasons which could reflect neuromuscular impairments, not cardiorespiratory impairments.

Interpretation of test results as a measure of solely cardiorespiratory fitness or to prescribe exercise should be done cautiously in people with chronic stroke.
Voluntary Exercise Attenuates Tumor Growth in a Preclinical Model of Castration-Resistant Prostate Cancer
Authors: Berger Nicolas , Kugler Benjamin , Han Dong , Li Muqing , Nguyen Paul , Anderson Meaghan , Zhang Songqi , Cai Changmeng , Zou Kai
Published: October 2024

Purpose

To examine the effects of voluntary exercise training on tumor growth and explore the underlying intratumoral molecular pathways and processes responsible for the beneficial effects of VWR on tumor initiation and progression in a mouse model of Castration-Resistant Prostate Cancer (CRPC).

Methods

Male immunodeficient mice (SCID) were castrated and subcutaneously inoculated with human CWR-22RV1 cancer cells to construct CRPC xenograft model before randomly assigned to either voluntary wheel running (VWR) or sedentary (SED) group (n=6/group). After three weeks, tumor tissues were collected. Tumor size was measured and calculated. mRNA expression of markers of DNA replication, Androgen Receptor (AR) signaling, and mitochondrial dynamics was determined by RT-PCR. Protein expression of mitochondrial content and dynamics was determined by western blotting. Finally, RNA-sequencing analysis was performed in the tumor tissues.

Results

Voluntary wheel running resulted in smaller tumor volume at the initial stage and attenuated tumor progression throughout the time course (P < 0.05). The reduction of tumor volume in VWR group was coincided with lower mRNA expression of DNA replication markers (MCM2,MCM6, andMCM7), AR signaling (ELOVL5andFKBP5) and regulatory proteins of mitochondrial fission (Drp1 and Fis1) and fusion (MFN1 and OPA1) when compared to the SED group (P<0.05). More importantly, RNA sequencing data further revealed that pathways related to pathways related to angiogenesis, extracellular matrix formation and endothelial cell proliferation were downregulated.

Conclusions

Three weeks of VWR was effective in delaying tumor initiation and progression, which coincided with reduced transcription of DNA replication, AR signaling targets and mitochondrial dynamics. We further identified reduced molecular pathways/processes related to angiogenesis that may be responsible for the delayed tumor initiation and progression by VWR.
Experiments and modelling of pulmonary surfactant disruption by aerosolised compounds
Authors: Barlow Hugh , Sengupta Sreyoshee , Baltazar Maria Teresa , Sørli Jorid B.
Published: October 2024

Within the deep lung, pulmonary surfactant coats the air-liquid interface at the surface of the alveoli. This complex mixture of amphiphilic molecules and proteins modifies the surface tension and mechanical properties of this interface to assist with breathing. In this study, we examine the effects on pulmonary surfactant function by two industrially used compounds composing surfactants and polymers. Using an experimental method previously developed to imitate the in vivo exposure in the alveoli[1], we quantify the change in the dilational rheology of the pulmonary surfactant due to the introduction of two widely used chemicals; Benzalkonium Chloride (BAC) and Polyhexamethylene Biguanide (PHMB). We observe that these chemicals alter the dilational rheology of the surfactant monolayer. Using a mechanistic theory, we are able to semi-quantitatively model the changes induced by the introduction of these compounds to the pulmonary surfactant.
Critical-Size Defect Tibialis Anterior (TA) Muscle Regeneration using Ex-Vivo Mice Hindlimbs Culturing under Dynamic Mechanical Loading
Authors: Jacho Diego , Huynh James , Crowe Emily , Rabino Agustin , Yildirim Mine , Czernik Piotr J. , Lecka-Czernik Beata , Garcia-Mata Rafael , Yildirim-Ayan Eda
Published: October 2024

In this study, we introduced an innovative computer-controlled ex vivo mice hindlimb culturing platform operating under dynamic loading, coupled with injectable cell-laden nanofibrous matrix (PNCOL), to investigate tissue response and therapeutic outcomes in critical size defect tibialis anterior (TA) muscle regeneration. The combination of mechanical stimulation and cell therapy offers a distinctive opportunity to delve into the regenerative rehabilitation field and create sustainable solutions in musculoskeletal (MSK) tissue regeneration. The application of mechanical loading on the whole mice hindlimbs increased total bone area and marrow area suggesting an increase in periosteal bone formation and resorption on the endosteal surface. Viability assessments confirmed the sustained culturing of the samples throughout the study. Then, the effect of mechanical loading and PNCOL injection on muscle regeneration at the TA defect site was evaluated. Histological analyses revealed enhanced muscle regeneration in PNCOL-treated hindlimbs. Structural analysis of the defect area through scanning electron microscopy (SEM) showed regeneration of ECM fibers at the defect site in PNCOL-treated groups. An analysis of cytokine levels in conditioned media at the end experiment showed changes in the number of proteins with the role in wound healing, muscle regeneration WNT, and IGF-1 signaling suggesting an anabolic effect of mechanical stimulation on muscle and bone. Similarly, gene expression analysis showed a significant upregulation of PAX7, Mrf4, MYF5, and TGFβ1 mRNA levels, indicating enhanced muscle regeneration after coupled mechanical loading and PNCOL treatments. Lastly, immunostaining showed an increase in tissue regeneration and anti-inflammatory response (CD206) in PNCOL-treated groups. Overall, the ex vivo hindlimb organ culturing platform– maintained tissue functions under mechanical loading, while PNCOL treatment promoted muscle tissue regeneration and reduced inflammation. These findings demonstrated the potential of multidimensional approaches for enhancing therapeutic outcomes in MSK disorders. In addition, this study aligns with the growing emphasis on minimizing the number of animals used in research and developing a robust sense of responsible animal experimentation through introducing dynamic ex-vivo muscle organ culturing platform.
Male and female gametophytes meet after winter: Testing the overwintering hypothesis for delayed fertilization in Japanese stone oak
Authors: Shagawa Takenori , Ogawa Kota , Kanaoka Masahiro M , Satake Akiko
Published: October 2024

Premise

Fertilization delays of nearly a year have been observed in many acorn-producing Fagaceae trees. Despite this long-standing recognition, the underlying mechanisms and adaptive significance of delayed fertilization remain largely unexplored. It has been hypothesized that these prolonged delays may serve as an adaptive strategy, allowing ovules to be fertilized and seeds to develop during more favorable seasons, thereby avoiding the adverse conditions of winter. However, empirical evidence to support this hypothesis is still lacking.

Methods

To test the overwintering hypothesis, we observed the seasonal progression of pollen tube growth and ovule development inLithocarpus edulis, a species exhibiting two flowering seasons, spring and autumn. We conducted monthly observations of pollen tube growth and ovule development in both spring and autumn flower samples. We employed microtome techniques and scanning confocal microscopy to closely examine the seasonal progression of pollen tube growth and ovule development.

Results

We found that pollen tubes were arrested at the style-joining site, and ovules remained immature in both spring and autumn samples prior to winter. Following winter, pollen tube regrowth and ovule maturation were synchronized in the subsequent spring in both spring and autumn samples.

Conclusions

Our results support the overwintering hypothesis, suggesting that delayed fertilization may serve as an adaptive strategy to ensure fertilization in spring, overcoming the reproductive challenges posed by cold winter conditions. Our study introduces a new avenue for studying the temporal organization of plant reproductive phenology, specifically the alignment of flowering, pollination, fertilization, and fruiting in seasonal environments.
Mapping DHPS Evolvability: Identification of Novel Evolutionarily Critical Sub-Structure through Evolutionary and Structural Analyses of DHPS
Authors: Sanyal Dwipanjan , Shivram A. , Banerjee Suharto , Uversky Vladimir N. , Chivukula Aneesh , Chattopadhyay Krishnananda , Chowdhury Sourav
Published: October 2024

Protein evolution shapes pathogen adaptation-landscape, particularly in developing drug resistance. The rapid evolution of target proteins under antibiotic pressure leads to escape mutations leading to the problem of antibiotic resistance. A deep understanding of the evolutionary dynamics of antibiotic target proteins presents a plausible intervention strategy for disrupting the evolutionary trajectory of resistance. Mutations in Dihydropteroate synthase (DHPS), an essential folate pathway protein and a key target for sulfonamide antibiotics, result in reduced antibiotic binding, leading to resistance. Deploying an array of statistical analyses on the DHPS sequence-space and integrating those with deep mutational analysis and structure-based network-topology models we identified critical DHPS-subsequences. Our analysis of the frustration landscape of DHPS predicts how conformational and mutational changes shift the energy distributions within the DHPS substructures. Combining dimensionality reduction and optimality analysis we identified a substructure critical to DHPS evolvability, and computed its druggablity. Our integrated evolution and structure-informed framework identified a DHPS-substructure with significant evolutionary and structural impact. Targeting this region could constrain DHPS evolvability and disrupt the resistome, presenting a new avenue for antibiotic development and contributing to the broader effort to address the problem of antibiotic resistance.
Long-read Spatial Transcriptomic Profiling of Patient-derived ccRCC Tumoroids Reveals Heterogeneity in Isoform and Gene Expression
Authors: Elshani Mustafa , Abdullah Hazem , Zhang Ying , Laird Alexander , Mullen Peter , Harrison David J.
Published: October 2024

Clear cell renal cell carcinoma (ccRCC) is the most prevalent type of kidney cancer characterized by its diverse tumor composition featuring various subclonal populations that hinder effective treatment responses. Tumoroids present an avenue for modelling this diversity and replicating the intricate tumor heterogeneity. Spatial transcriptomics preserves the spatial context of gene expression enabling us to study distinct tumor areas and the influence on overall diversity.

Our spatial transcriptomics analysis uncovered tumor clusters with distinct genetic profiles that showcase various functional areas in depth and offer valuable understandings into the diversity of ccRCC types. Some of these tumor clusters exhibited activity in genes responsible for protein catabolism and reduced abundance of genes related to mitochondrial respiration processes. We also show isoform expression within tumoroids, in particular glutaminase (GLS) especially with the prevalence of the highly metabolically active GAC isoform that is expressed in regions where mitochondrial gene abundance is lower; whereas the KGA isoform displayed a more focal expression pattern. Combining long-read spatial transcriptomics with organoid models presents a novel strategy for unravelling gene and transcript level complexity.
CXCL10/CXCR3 Signaling Induces Neural Senescence and Cognitive Impairments
Authors: Patel Monika , Kumar Sakesh , Singh Aditya , Yadav Prem N
Published: October 2024

Chemokine receptors belong to the G-protein-coupled receptors family and multiple lines of emerging evidence suggest that several chemokines are elevated in aging associated with central nervous system disorders. Increased level of CXCL10 in central nervous system is reported in several neurodegenerative diseases, including Multiple sclerosis, Alzheimer’s disease, and Virus-associated dementia. We also observed significantly increased expression of CXCL10 and CXCR3 in the prefrontal cortex and hippocampus of aged C57BL/6J mice (12- and 18-month-old mice). This leads us to hypothesize that CXCL10, being a component of SASPs, may aggravate/perpetuate the brain aging process and, finally, neurodegenerative diseases. To test this hypothesis, we administered CXCL10 (intracerebroventricular: ICV, 0.5 pg/ hrs, 28 days) in 8-month-old C57BL/6J mice. We observed increased expression of senescent marker proteins p16INK4a, p21Cip1, p53 and decreased expression of pRB in the prefrontal cortex, which was blocked by CXCR3-specific antagonist AMG487. Furthermore, chronic infusion of CXCL10 induced learning and memory deficits in Y-maze, social recognition, contextual freeze tests and c-FOS expression in the prefrontal cortex. To further determine the specificity of CXCL10/CXCR3 signaling, we treated the primary cortical neuron (Daysin vitro: DIV 7-8) with CXCL10 and found increased senescence in CXCR3 dependent fashion. Using RFP-EGFP-LC3-tagged transgenic mice, we also showed CXCL10/CXCR3 signaling attenuates autophagic flux in primary cortical neurons. Lastly, using a c-FOS-iRFP reporter, we observed that increased CXCL10/CXCR3 signaling impairs glutamatergic signaling in primary cortical neurons. These results support the hypothesis that increased CXCL10/CXCR3 facilitates brain aging and could be targeted for the management of aging-associated CNS disorders.
The cGAS-STING pathway is anin vivomodifier of genomic instability syndromes
Authors: Bergman Marva , Goshtchevsky Uri , Atlan Tehila , Astre Gwendoline , Halabi Ryan , El Hosniyah , Moses Eitan , Lemus Aaron J.J. , Benayoun Bérénice A. , Tzfati Yehuda , Ben-Ami Ido , Harel Itamar
Published: October 2024

Mutations in genes involved in DNA damage repair (DDR) often lead to premature aging syndromes. While recent evidence suggests that inflammation, alongside mutation accumulation and cell death, may drive disease phenotypes, its precise contribution toin vivopathophysiology remains unclear. Here, by modeling Ataxia Telangiectasia (A-T) and Bloom Syndrome in the African turquoise killifish (N. furzeri), we replicate key phenotypes of DDR syndromes, including infertility, cytoplasmic DNA fragments, and reduced lifespan. The link between DDR defects and inflammation is attributed to the activation of the cGAS-STING pathway and interferon signaling by cytoplasmic DNA. Accordingly, mutating cGAS partially rescues germline defects and senescence in A-T fish. Double mutants also display reversal of telomere abnormalities and suppression of transposable elements, underscoring cGAS’s non-canonical role as a DDR inhibitor. Our findings emphasize the role of interferon signaling in A-T pathology and identify the cGAS-STING pathway as a potential therapeutic target for genomic instability syndromes.
The Src family kinase inhibitor drug Dasatinib and glucocorticoids display synergistic activity against tongue squamous cell carcinoma and reduce MET kinase activity
Authors: Hmedat Ali N.A. , Doondeea Jessica , Ebner Daniel , Feller Stephan M. , Lewitzky Marc
Published: October 2024

Background

Tongue squamous cell carcinoma (TSCC) is an aggressive cancer associated with a poor prognosis and limited treatment options, necessitating new drug targets to improve therapeutic outcomes. Our current work focuses on protein tyrosine kinases as well-known targets for successful cancer therapies.

Methods

Western blot analysis of tyrosine phosphorylation patterns in 34 TSCC lines identified Src family kinases (SFKs) as the main contributors. Inhibition of SFKs with PP2 and Dasatinib led to profound biological effects. A high-throughput screen with 1600 FDA-approved drugs was performed with three TSCC lines to discover drugs that act synergistically with Dasatinib against TSCC cell viability. Glucocorticoids emerged as potential candidates and were further investigated in 2D culture and by 3D soft agar colony formation. Dexamethasone was chosen as the major tool for our analyses. Since Dasatinib and glucocorticoids are known for their pleiotropic actions on cells, we analyzed effects on cell cycle, senescence, autophagy and cell signaling.

Results

A panel of 34 TSCC lines showed a surprisingly homogenous pTyr-protein pattern and a prominent 130 kDa pTyr-protein. Inhibition of SFK activity greatly reduced overall pTyr-protein levels and p130Cas tyrosine phosphorylation. It also impaired TSCC viability in 2D cell culture and 3D soft agar colony formation. A high-throughput drug combination screen with Dasatinib identified glucocorticoids as promising candidates for synergistic activity. Dasatinib and Dexamethasone combination treatment showed strong synergistic effects on Src and p130Cas phosphorylation and led to reduced p130Cas expression. Dexamethasone also suppressed phosphorylation of the MET kinase and its key substrate Gab1. On the cellular level, Dasatinib combination with glucocorticoids led to G1 cell cycle arrest, increased senescence and enhanced autophagy. This was also reflected by effects on cell cycle regulatory proteins, including CDKs and cyclins.

Conclusion

This work is the first to show a strong synergistic activity of Dasatinib in combination with clinically used glucocorticoids in solid tumors. Furthermore, the tyrosine kinase MET and its effector protein Gab1 are newly identified glucocorticoid targets. Given the extensive research on MET as a drug target in various cancers, our findings have the potential to advance future cancer treatments.
Lung epithelial progenitor-mediated release of TGF-β regulates induction and persistence of lung CD8+TRMcells following mucosal BCG vaccination
Authors: Blake Judith A. , Seifert Julia , Ruscher Roland , Giacomin Paul R. , Doolan Denise L. , Kupz Andreas
Published: October 2024

A principal reason for the high global morbidity and mortality of tuberculosis (TB) is the lack of efficacy of the only licensed TB vaccine, Bacillus Calmette-Guérin (BCG), as parenteral BCG does not induce local pulmonary immune memory. Animal studies have shown that mucosal BCG vaccination provides superior protection against TB due to generation of lung resident memory T cells (TRM). Here, we demonstrated that following mucosal vaccination with the genetically modified virulent BCG strain, BCG::RD1, distal airway epithelial progenitors were mobilized to assist with restoration of alveolar epithelium. By way of their migration-mediated activation of latent TGF-β, lung CD8+TRMdifferentiation was induced. Mucosal vaccinations using nonvirulent strains of BCG in which airway epithelial progenitors were not mobilized, as well as genetic inhibition of migration-mediated activation of TGF-β, resulted in significantly lower numbers of lung CD8+TRM. In addition, we discovered CD8+cells with ex-lung and stem-like TRMphenotypes that persisted in the lung-draining mediastinal lymph nodes for up to four months following mucosal BCG vaccination. These results link airway epithelial progenitor-mediated repair of injured lung tissue with induction of resident T cell memory and delineate why persistence of TRMin the lung is short-lived. These findings may explain why mucosal vaccination with virulent BCG strains is more protective against TB and thus have notable implications for future TB vaccine development.

One Sentence Summary

Following lung damage due to inhalation of virulent BCG, distal airway epithelial progenitor cells interact with lung CD8+T cells to induce their differentiation into resident memory T cells via migration-mediated activation of TGF-β.
A First-in-Class Dual Degrader of Bcl-2/Bcl-xL Reverses HIV Latency and Eliminates Ex Vivo Reservoirs
Authors: Chang Lin-Chun , Yin Michael T , Laird Gregory M. , Ritter Kristen D. , Shah Jayesh , Debnath Asim K.
Published: October 2024

The persistence of latent HIV-1 proviruses in CD4+ T cells is a major obstacle to curing HIV. The “shock and kill” strategy involves reversing latency with latency-reversing agents (LRAs) and selectively inducing cell death in infected cells. However, current LRAs have shown limited efficacy in eliminating the ex vivo HIV reservoir. We repurposed PZ703b, a pro-apoptotic protein degrader initially developed for anti-leukemia therapy, to target HIV eradication. PZ703b induced degradation of Bcl-2 and Bcl-xL, activating the non-canonical NF-kB pathway and caspases cascade, resulting in latency reversal and selective apoptosis of infected cells. Treatment of ex vivo CD4+ T cells from ART-suppressed HIV-1 patients achieved a ∼50% reduction in the replication-competent reservoir. Our study provides proof-of-concept for using protein degraders to reverse HIV latency and induce cell death, highlighting PZ703b’s potential in HIV cure strategies. This approach may pave the way for novel therapeutic interventions aimed at eliminating the HIV-inducible reservoir.
Blood cultures contain populations of genetically diverseCandida albicansstrains that may differ in echinocandin tolerance and virulence
Authors: Fleres Giuseppe , Cheng Shaoji , Badrane Hassan , Dupont Christopher L. , Espinoza Josh L. , Abbey Darren , Driscoll Eileen , Newbrough Anthony , Hao Binghua , Mansour Akila , Nguyen M. Hong , Clancy Cornelius J.
Published: October 2024

It is unknown whether within-patientCandida albicansdiversity is common during bloodstream infections (BSIs). We determined whole genome sequences of 10C. albicansstrains from blood cultures (BCs) in each of 4 patients. BCs in 3 patients contained mixed populations of strains that differed by large-scale genetic variants, including chromosome (Chr) 5 or 7 aneuploidy (n=2) and Chr1 loss of heterozygosity (n=1). Chr7 trisomy (Tri7) strains from patient MN were attenuated for hyphal and biofilm formation in vitro compared to euploid strains, due at least in part toNRG1over-expression. Nevertheless, representative Tri7 strain M1 underwent filamentation during disseminated candidiasis (DC) in mice. M1 was more fit than euploid strain M2 during DC and mouse gastrointestinal colonization, and in blood ex vivo. M1 and M2 exhibited identical echinocandin minimum inhibitory concentrations, but M2 was more tolerant to micafungin in vitro. Furthermore, M2 was more competitive with M1 in mouse kidneys following micafungin treatment than it was in absence of micafungin. Tri7 strains represented 74% of patient MN’s baseline BC population, but after 1d and 3d of echinocandin treatment, euploid strains were 93% and 98% of the BC population, respectively. Findings suggest that echinocandin tolerant, euploid strains were a subpopulation to more virulent Tri7 strains at baseline and then were selected upon echinocandin exposure. In conclusion, BCs in at least some patients are comprised of diverseC. albicanspopulations not recognized by the clinical lab, rather than single strains. Clinical relevance ofC. albicansdiversity and antifungal tolerance merits further investigation.