bluuebunny/crossref_metadata_2025 huggingface dataset:
Extracellular ISG15 triggers ISGylation via a type-I interferon independent non-canonical mechanism to regulate host response during virus infection
Authors: Miller Lindsay Grace , Chiok Kim , Mariasoosai Charles , Mohanty Indira , Pandit Sudiksha , Deol Pallavi , Mehari Liyon , Teng Michael N. , Haas Arthur L. , Natesan Senthil , Miura Tanya A. , Bose Santanu
Published: July 2024

Type-I interferons (IFN) induce cellular proteins with antiviral activity. One such protein is Interferon Stimulated Gene 15 (ISG15). ISG15 is conjugated to proteins during ISGylation to confer antiviral activity and regulate cellular activities associated with inflammatory and neurodegenerative diseases and cancer. Apart from ISGylation, unconjugated free ISG15 is also released from cells during various conditions, including virus infection. The role of extracellular ISG15 during virus infection was unknown. We show that extracellular ISG15 triggers ISGylation and acts as a soluble antiviral factor to restrict virus infection via an IFN-independent mechanism. Specifically, extracellular ISG15 acts post-translationally to markedly enhance the stability of basal intracellular ISG15 protein levels to support ISGylation. Furthermore, extracellular ISG15 interacts with cell surface integrin (α5β1 integrins) molecules via its RGD-like motif to activate the integrin-FAK (Focal Adhesion Kinase) pathway resulting in IFN-independent ISGylation. Thus, our studies have identified extracellular ISG15 protein as a new soluble antiviral factor that confers IFN-independent non-canonical ISGylation via the integrin-FAK pathway by post-translational stabilization of intracellular ISG15 protein.
Generation of an inflammatory niche in an injectable hydrogel depot through recruitment of key immune cells improves efficacy of mRNA vaccines
Authors: Meany Emily L. , Klich John H. , Jons Carolyn K. , Mao Tianyang , Chaudhary Namit , Utz Ashley , Baillet Julie , Song Ye E. , Saouaf Olivia M. , Ou Ben S. , Williams Shoshana C. , Eckman Noah , Irvine Darrell J. , Appel Eric
Published: July 2024

Messenger RNA (mRNA) delivered in lipid nanoparticles (LNPs) rose to the forefront of vaccine candidates during the COVID-19 pandemic due in part to scalability, adaptability, and potency. Yet there remain critical areas for improvements of these vaccines in durability and breadth of humoral responses. In this work, we explore a modular strategy to target mRNA/LNPs to antigen presenting cells with an injectable polymer-nanoparticle (PNP) hydrogel depot technology which recruits key immune cells and forms an immunological niche in vivo. We characterize this niche on a single cell level and find it is highly tunable through incorporation of adjuvants like MPLAs and 3M-052. Delivering commercially available SARS-CoV-2 mRNA vaccines in PNP hydrogels improves the durability and quality of germinal center reactions, and the magnitude, breadth, and durability of humoral responses. The tunable immune niche formed within PNP hydrogels effectively skews immune responses based on encapsulated adjuvants, creating opportunities to precisely modulate mRNA/LNP vaccines for various indications from infectious diseases to cancers.
Cnidaria XIAP activates caspase-mediated cell death
Authors: Chen Yuan , Wu Meng , Yuan Zihao , Wang Qingyue , Xu Hang , Sun Li
Published: July 2024

In vertebrate, X-linked inhibitor of apoptosis (XIAP) is a potent inhibitor of apoptosis. XIAP inhibits apoptosis by interacting with proapoptotic caspases via the baculovirus IAP repeat (BIR) domains and mediating caspase ubiquitination via the really interesting new gene (RING) domain and ubiquitin-associated (UBA) domain. In invertebrate, mostly arthropods, XIAP is also known as an apoptosis inhibitor. To date, no study on basal metazoan XIAP has been documented. In the present work, we examined the biological activity of XIAP from jellyfish Aurelia coerulea (AcXIAP) and other non-bilaterians. AcXIAP possesses three BIRs and one RING domain but lacks the UBA domain. AcXIAP augmented the apoptosis-inducing activity of all of the four A. coerulea caspases, of both the initiator and the effector clades, identified in this study. AcXIAP activated caspase via one of the BIRs, which bound and stabilized the caspase, and the RING domain, which mediated ubiquitination on the p20 subunit of the caspase in a lysine-independent manner. Similar caspase-activating properties were also observed in the XIAP of hydra, coral, and sponge. In hydra, XIAP knockdown markedly decreased cell death induced by an apoptosis inducer. Together these results revealed the unconventional function and working mechanism of XIAP in Cnidaria, and shed new light into the functional and structural evolution of XIAP.
Osmotic Stress Influences Microtubule Drug Response Via WNK1 Kinase Signaling
Authors: Monfort-Vengut Ana , Sanz-Gómez Natalia , Ballesteros-Sánchez Sandra , Ortigosa Beatriz , Cambón Aitana , Ramos Maria , Lorenzo Ángela Montes-San , Rosa-Rosa Juan Manuel , Martínez-López Joaquín , Sánchez-Prieto Ricardo , Sotillo Rocío , de Cárcer Guillermo
Published: July 2024

Ion homeostasis is critical for numerous cellular processes, and disturbances in ionic balance underlie diverse pathological conditions, including cancer progression. Targeting ion homeostasis is even considered as a strategy to treat cancer. However, very little is known about how ion homeostasis may influence anticancer drug response. In a genome-wide CRISPR-Cas9 resistance drug screen, we identified and validated the master osmostress regulator WNK1 kinase as a modulator of the response to the mitotic drug rigosertib. Osmotic stress and WNK1 inactivation lead to an altered response not only to rigosertib treatment but also to other microtubule-related drugs, minimizing the prototypical mitotic arrest produced by these drugs. This effect is due to an alteration in microtubule stability and polymerization dynamics, likely maintained by fluctuations in intracellular molecular crowding upon WNK1 inactivation. This promotes resistance to microtubule depolymerizing drugs, and increased sensitivity to microtubule stabilizing drugs. In summary, our data proposes WNK1 osmoregulation activity as a biomarker for microtubule-associated chemotherapy response.
Chronic wounds and adaptivePseudomonas aeruginosa: A phenotypic and genotypic characterization
Authors: Buchholtz Kasandra , Jønsson Rie , Marvig Rasmus L. , Mojsoska Biljana , Krogfelt Karen Angeliki
Published: July 2024

Phenotypic and genetic diversity is found in varying prevalence in clinical populations where beneficial adaptations enable the bacteria to avoid recognition and eradication by the host immune system. This study aimed to investigate the presence ofPseudomonas aeruginosain chronic venous leg ulcers wounds over an 8-week time course. This was performed using genomic and phenotypic approaches to understand the survival and persistence ofPseudomonasstrains. The findings of this study show that the two patients were colonized with a recurringP. aeruginosagenotype with only minor phenotypic differences and few SNP differences, suggesting that thePseudomonasisolates present in the wound can survive and proliferate in the host’s hostile environment. The results provided from this study will allow us to understand clinically important traits inP. aeruginosaand provide insight into the dynamics and adaptations (or the lack thereof) in chronic wounds.

Highlights

Each patient is colonised overtime with a unique MLST

The total number of detected SNPs was 18 and 20 within each patient, respectively

One MLST belonged to the sequence type ST132, often observed in clinical isolates, whereas the other belonged to ST3244, previously seen inenvironmental isolates
microGWAS: a computational pipeline to perform large scale bacterial genome-wide association studies
Authors: Burgaya Judit , Damaris Bamu F. , Fiebig Jenny , Galardini Marco
Published: July 2024

Identifying genetic variants associated with bacterial phenotypes, such as virulence, host preference, and antimicrobial resistance, has great potential for a better understanding of the mechanisms involved in these traits. The availability of large collections of bacterial genomes has made genome-wide association studies (GWAS) a common approach for this purpose. The need to employ multiple software tools for data pre- and post-processing limits the application of these methods by experienced bioinformaticians. To address this issue, we have developed a pipeline to perform bacterial GWAS from a set of assemblies and annotations, with multiple phenotypes as targets. The associations are run using five sets of genetic variants: unitigs, gene presence/absence, rare variants (i.e. gene burden test), gene cluster specific k-mers, and all unitigs jointly. All variants passing the association threshold are further annotated to identify overrepresented biological processes and pathways. The results can be further augmented by generating a phylogenetic tree and by predicting the presence of antimicrobial resistance and virulence associated genes. We tested the microGWAS pipeline on a previously reported dataset onE. colivirulence, successfully identifying the causal variants, and providing further interpretation on the association results. The microGWAS pipeline integrates the state-of-the-art tools to perform bacterial GWAS into a single, user-friendly, and reproducible pipeline, allowing for the democratization of these analyses. The pipeline can be accessed, together with its documentation, at:https://github.com/microbial-pangenomes-lab/microGWAS.
Targeting NOTCH2-JAG1 juxtacrine signaling reverses macrophage-mediated tumor resistance to taxol
Authors: Yu Fazhi , Zhou Qin , Zhou Tong , Xie Yijia , Zhang Peng , He Wei , Yu Weiqiang , Cheng Aoxing , Liu Hanyuan , Wu Qingfa , Ma Xiaopeng , Guo Jing , Zhou Ying , Shi Jue , Yang Zhenye
Published: July 2024

Taxanes are widely used in chemotherapy, but primary and acquired resistance limit the clinical efficacy. Studies have shown tumor interaction with macrophages in the tumor microenvironment (TME) plays a significant role in taxane resistance, yet therapeutic strategies that directly deplete or repolarize macrophages are challenging and with considerable risk of side effects. Here we uncovered that tumor-macrophage interaction can be selectively targeted by inhibiting post-mitotic NOTCH2-JAG1 juxtacrine signaling in the TME, which strongly sensitizes paclitaxel response. Using translatome profiling, we found significant NOTCH2 upregulation during paclitaxel-induced prolonged mitosis. NOTCH2 was subsequently activated in the post-mitotic G1 phase by JAG1 expressed on the neighboring macrophages and tumor cells, which promoted tumor cell survival and upregulated cytokines that recruited JAG1-expressing macrophages, thus generating a positive feedback loop that further enhanced the pro-tumor NOTCH2 activity. By targeting this NOTCH2-JAG1 axis using NOTCH2 shRNA or a pan-NOTCH inhibitor, macrophage recruitment and paclitaxel resistance were significantly attenuated in multiple mouse tumor models of ovarian cancer. Clinical samples from paired primary and recurrent ovarian cancer patients also showed significant correlation of higher NOTCH2 expression with worse prognosis. Our results thus point to combining NOTCH2 inhibitor with taxane as an effective therapeutic strategy to selectively disrupt tumor-macrophage interaction in the TME and overcome macrophage-mediated taxane resistance in NOTCH2-positive tumors.
Short-Term Warming Induces Cyanobacterial Blooms and Antibiotic Resistance in a Freshwater Lake as Revealed by Metagenomics Analysis
Authors: Manna Bharat , Jay Emma , Zhang Wensi , Zhou Xueyang , Lyu Boyu , Thomas Gevargis Muramthookil , Singhal Naresh
Published: July 2024

Climate change poses significant risks to freshwater ecosystems, potentially exacerbating harmful cyanobacterial blooms and antibiotic resistance. We investigated these dual threats in Cosseys Reservoir, New Zealand, by simulating short-term warming scenarios and assessing the role of oxidative stress. Microcosms were subjected to Base (22°C), Normal (24°C), and projected Future (27°C) temperatures, with additional treatments including reactive oxygen species (ROS). Metagenomic analysis revealed substantial restructuring of microbial communities under warming conditions. Cyanobacterial abundance increased from 6.11% (initial) to 20.53% at 24°C and 10.66% at 27°C. Notably, ROS addition mimicked the effects of temperature increase on cyanobacterial proliferation. Toxin-producing families, includingMicrocystaceaeandNostocaceae, proliferated significantly. The microcystin synthesis gene (mcy) showed a strong positive association (R² = 0.88) with cyanobacterial abundance. Moreover, cyanobacteria exhibited enhanced nutrient acquisition (pstSgene, R² = 0.69) and upregulated nitrogen metabolism pathways under warming conditions. Concurrently, we observed a marked increase in antibiotic resistance gene (ARG) abundance with rising temperatures. The relative abundance of multidrug resistance genes was consistently high across all conditions (50.82 % of total ARGs). ROS stress further intensified ARG proliferation, particularly for efflux pump genes (e.g.,acrB,adeJ,ceoB,emrB,MexK,muxB). Co-association network analysis identified key antibiotic-resistant bacteria (e.g.,Streptococcus pneumoniae,Acinetobacter baylyi) and ARGs (e.g.,acrB,MexK,rpoB2,bacA) central to resistance dissemination under warming conditions. This study demonstrates that even modest temperature increases (2-5°C) can promote both cyanobacterial blooms and antibiotic resistance in freshwater ecosystems over short time scales. The synergistic effects of temperature and oxidative stress underscore the complex challenges posed by climate change to water quality and public health.

Highlights

Short-term warming promotes toxic cyanobacteria and antibiotic resistance in freshwater.

Harmful cyanobacteria and their metabolic potential increase under warming conditions.

Reactive oxygen species mimic and exacerbate temperature effects.

Oxidative stress response genes strongly correlate with ARG abundance.

Co-association networks identify key host pathogen central to resistance spread.
Reproducibly oriented cell divisions pattern the first flat body structures to set up dorsoventrality and de novo meristem formation inMarchantia polymorpha
Authors: Wallner Eva-Sophie , Dolan Liam
Published: July 2024

Land plant bodies develop from stem cells located in meristems. However, we know little about how meristems initiate from non-meristematic cells. The haploid body of bryophytes develops from unicellular spores in isolation from the parental plant, which allows all stages of development to be observed. We discovered that the Marchantia spore undergoes a series of precisely oriented cell divisions to generate a flat prothallus on which a meristem later develops de novo. The young sporeling comprises an early cell mass. One cell of the early cell mass elongates and undergoes a formative division that produces the prothalloblast, which initiates prothallus formation. A symmetric division of the prothalloblast followed by two transverse divisions generate a four-celled plate that expands into a flat disc through oblique divisions in three of the four plate cell-derived quadrants. One quadrant gives rise to a flat flabellum. A notch with a meristem and apical stem cell develops at the margin of the flabellum. The transcription factor MpC3HDZ is a marker of the first flat prothallus structure and polarises to the dorsal tissues of flabella and meristems. Mpc3hdzmutants are defective in setting up dorsoventrality and thallus body flatness. We report how a regular set of cell divisions forms the prothallus – the first dorsoventral structure – and how cells on the margin of the prothallus develop a dorsoventralised meristem de novo.
Oncogenic NOVA1 expression dysregulates alternative splicing in breast cancer
Authors: Moakley Daniel F , Zhang Chaolin
Published: July 2024

Neuro-Oncological Ventral Antigen 1 (NOVA1) is best known for its role in mediating an alternative splicing (AS) program in neurons, yet was first discovered as an antigen expressed in breast tumors, causing rare autoimmune reactions and paraneoplastic neurological disorders (PNDs). The PND model suggests a plausible role of the tumor antigen expression in tumor suppression, whereas it has emerged that NOVA may function as an oncogene in a variety of cancers. In addition, whether NOVA mediates AS in breast cancer remains unanswered. Here we examine the AS profiles of breast invasive carcinoma (BRCA) tumor samples and demonstrate that ectopicNOVA1expression led to the activation of neuron-like splicing patterns in many genes, including exons targeted by NOVA in the brain. The splicing dysregulation is especially prevalent in cell periphery and cytoskeleton genes related to cell-cell communication, actin-based movement, and neuronal functions. We find that NOVA1-mediated AS is most prominent in Luminal A tumors and highNOVA1expression in this subtype is associated with poorer prognosis. Our results suggest that ectopic NOVA1 in tumors has regulatory activity affecting pathways with high relevance to tumor progression and that this might be a more general mechanism for PND antigens.
TgATG9 is required for autophagosome biogenesis and maintenance of chronic infection inToxoplasma gondii
Authors: Thaprawat Pariyamon , Zhang Zhihai , Rentchler Eric C. , Wang Fengrong , Chalasani Shreya , Giuliano Christopher J. , Lourido Sebastian , Di Cristina Manlio , Klionsky Daniel J. , Carruthers Vern B.
Published: July 2024

Toxoplasma gondiiis a ubiquitous protozoan parasite that can reside long-term within hosts as intracellular tissue cysts comprised of chronic stage bradyzoites. To perturb chronic infection requires a better understanding of the cellular processes that mediate parasite persistence. Macroautophagy/autophagy is a catabolic and homeostatic pathway that is required forT. gondiichronic infection, although the molecular details of this process remain poorly understood. A key step in autophagy is the initial formation of the phagophore that sequesters cytoplasmic components and matures into a double-membraned autophagosome for delivery of the cargo to a cell’s digestive organelle for degradative recycling. WhileT. gondiiappears to have a reduced repertoire of autophagy proteins, it possesses a putative phospholipid scramblase, TgATG9. Through structural modeling and complementation assays, we show herein that TgATG9 can partially rescue bulk autophagy inatg9Δyeast. We demonstrated the importance of TgATG9 for proper autophagosome dynamics at the subcellular level using three-dimensional live cell lattice light sheet microscopy. Conditional knockdown of TgATG9 inT. gondiiafter bradyzoite differentiation resulted in markedly reduced parasite viability. Together, our findings provide insights into the molecular dynamics of autophagosome biogenesis within an early-branching eukaryote and pinpoint the indispensable role of autophagy in maintainingT. gondiichronic infection.
Structural Basis for the Activation of Proteinase-Activated Receptors (PARs) by Endogenous Ligands
Authors: Lyu Zongyang , Lyu Xiaoxuan , Xia Guliang , Carney Daniel , Alves Vinicius M. , Falk Mathew , Arora Nidhi , Zou Hua , McGrath Aaron , Kang Yanyong
Published: July 2024

The proteinase-activated receptor (PAR) subfamily of G protein-coupled receptors (GPCRs) include four members, PAR1-PAR4, that play critical roles in hemostasis, thrombosis, embryonic development, wound healing, inflammation, and cancer progression. The PARs share a unique activation mechanism driven by proteinase cleavage at a specific site within the extracellular amino-terminus, exposing a ‘tethered ligand’ that self-activates the receptor. Subsequent activation allows PAR family members to initiate complex intracellular signaling networks via traditional G protein-mediated pathways and beta-arrestin signaling and, in this way, the PARs link extracellular protease signaling molecules to cellular functions. Despite a primary reliance on biochemical studies for understanding tethered ligand recognition, direct structural visualization of these ligand-receptor complexes has been elusive. Here, we present structural snapshots of activated PAR1 and PAR2 bound to their endogenous tethered ligands, revealing, for the first time, shallow and constricted orthosteric binding pockets and highlighting critical residues involved in ligand recognition and receptor activation. Surprisingly, comparisons with antagonist-bound structures show minimal conformational changes in the TM6 helix, a typical signature of GPCR activation, with large movements of TM7 observed upon activation. These insights lead to the identification of a common mechanism for PAR1 and PAR2 activation and provide a structural template for designing novel antagonists targeting the orthosteric binding site, potentially opening new avenues for therapeutic interventions.
Differential circulating proteomic responses associated with ancestry during severe COVID-19 infection
Authors: Zheng Thomas M , Ilboudo Yann , Lu Tianyuan , Butler-Laporte Guillaume , Nakanishi Tomoko , Morrison David , Adra Darin , Cuddeback Lena , Brent Richards J.
Published: July 2024

Background

COVID-19 led to a disruption in nearly all aspects of society, yet these impacts were not the same across populations. During the pandemic, it became apparent that ancestry was associated with COVID-19 severity and morbidity, such that individuals of African descent tended to have worse outcomes than other populations. One factor that may influence COVID-19 outcomes is the circulating proteomic response to infection. This study examines how different ancestries had differential circulating protein levels in response to severe COVID-19 infection.

Methods

4,979 circulating proteins from 1,272 samples were measured using the SomaScan platform. We used a linear mixed model to assess the ancestry-specific association between the level of each protein and severe COVID-19 illness, accounting for sex, age, and days since symptom onset. We then compared each ancestry-specific effect size of severe COVID-19 illness on protein level to one another in a pairwise manner to generate Z-scores. These Z-scores were then converted into p-values and corrected for multiple comparisons using a Benjamini-Hochberg false discovery rate of 5%.

Results

Comparing ancestries, we found that 62% of the tested proteins are associated with severe COVID-19 in European-ancestry individuals, compared to controls. We found that 45% and 22% of the tested proteins were different between COVID-19 infected and control individuals in people of African and East Asian ancestry, respectively. There was a strong correlation in effect size between ancestries. We found that individuals of European and African ancestry had the most similar response with a Pearson correlation of 0.868, 95% CI [0.861, 0.875] while European and East Asian ancestries had a Pearson correlation of 0.645, 95% CI [0.628, 0.661] and, East Asian and African ancestries had a Pearson correlation of 0.709, 95% CI [0.695, 0.722]. However, we found 39 unique proteins that responded differently (FDR < 0.05) between the three ancestries.

Conclusions

Examining 4,979 protein levels in 1,272 samples, we identified that the majority of measured proteins had similar responses to infection across individuals of European, African and East Asian ancestry. However, there were 39 proteins that may have a differential response to infection, when stratified by ancestry. These proteins could be investigated to assess whether they explain the differences in observed severity of COVID-19 between ancestral populations.
Performance of LFSPROTP53germline carrier risk predictions compared to standard genetic counseling practice on prospectively collected probands
Authors: Corredor Jessica L. , Dodd-Eaton Elissa B. , Woodman-Ross Jacynda , Woodson Ashley , Nguyen Nam H. , Peng Gang , Green Sierra , Gutierrez Angelica M. , Arun Banu K. , Wang Wenyi
Published: July 2024

Purpose

Current clinical guidelines for genetic testing for Li-Fraumeni Syndrome (LFS) have many limitations, primarily the criteria don’t consider detailed personal and family history information and may miss many individuals with LFS. A personalized risk assessment tool, LFSPRO, was created to estimate a proband’s risk for LFS based on personal and family history information. The purpose of this study is to compare LFSPRO to existing clinical criteria to determine if LFSPRO can outperform these tools. Additionally, we gauged genetic counselors’ (GCs) experience using LFSPRO for their patients.

Methods

Between December 2021 and March 2024, GCs identified patients concerning for LFS based on the patients’ personal and family history information. This information was entered into LFSPRO to predict the risk to have a pathogenic/pathogenic (LP/P) germlineTP53variant. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) was compared between LFSPRO and Chompret criteria. Select GCs were asked to fill out surveys regarding their experience using LFSPRO following their genetic counseling appointments.

Results

LFSPRO’s sensitivity and specificity were 0.529 and 0.781 compared to Chompret’s respective 0.235 and 0.677. Additionally, LFSPRO had a positive predictive value (PPV) of 0.30 compared to Chompret’s 0.114. LFSPRO’s risk prediction was concordant with genetic testing results in 75% of probands. Eighty-one percent of GC surveys reported LFSPRO being concordant with the GC’s expectations and 75% would feel comfortable sharing the results with patients.

Conclusion

LFSPRO showed improved sensitivity and specificity compared to Chompret criteria and GCs report a positive experience with LFSPRO. LFSPRO can be used to increase access to genetic testing for patients at risk for LFS and could help healthcare providers give more direct risk assessments regarding LFS testing and management for patients.
Unveiling Chemotherapy’s Impact on Lung Cancer through Single-Cell Transcriptomics
Authors: Sayad Saed , Hiatt Mark , Mustafa Hazem
Published: July 2024

Background

Lung adenocarcinoma (LUAD) is the most common subtype of non-small cell lung cancer (NSCLC) and frequently affects non-smokers, especially women. It is characterized by a complex genetic profile and interactions with its microenvironment, which contribute to its aggressive and adaptable nature. Early symptoms are often subtle, leading to late diagnoses. Treatment approaches have advanced with targeted therapies and immunotherapy supplementing traditional chemotherapy and radiation. Despite these advancements, the prognosis remains variable, highlighting the need for continued research into new treatment strategies to improve outcomes.

Method

In this study, we employed Single-cell RNA Sequencing (scRNA-seq) to comprehensively analyze the impact of chemotherapy on lung adenocarcinoma at the individual cell level. By comparing before and after treatment samples, we assessed the differential expression of genes and pathways, revealing insights into how different cell types within the tumour respond to chemotherapy. This approach enabled us to pinpoint specific mechanisms of drug resistance and highlight potential therapeutic targets for overcoming these challenges.

Results

Our analysis uncovered substantial changes in gene expression between primary tumour cells and metastatic cells following chemotherapy. Notably, we observed that 45 pathways were shared between the top 50 upregulated pathways in the primary tumour and the top 50 downregulated pathways in the metastatic tumour post-chemotherapy. Conversely, there was no overlap between the top 50 downregulated pathways in the primary tumour and the top 50 upregulated pathways in the metastatic tumour after chemotherapy. This suggests that chemotherapy effectively downregulated the major upregulated pathways but did not upregulate the key downregulated pathways in metastatic tumours.

Conclusions

Integrating single-cell transcriptomics into LUAD research offers detailed insights into the tumour’s response to chemotherapy and its interaction with the immune system. This approach enhances our understanding of LUAD and aids in developing targeted and effective treatments. Based on our analysis, we hypothesize that combining chemotherapy with drugs designed to upregulate the downregulated pathways in primary tumour cells could significantly enhance treatment efficacy and improve patient outcomes.
Extracting the Sample Size From Randomized Controlled Trials in Explainable Fashion Using Natural Language Processing
Authors: Windisch Paul , Dennstädt Fabio , Koechli Carole , Förster Robert , Schröder Christina , Aebersold Daniel M. , Zwahlen Daniel R.
Published: July 2024

Background

Extracting the sample size from randomized controlled trials (RCTs) remains a challenge to developing better search functionalities or automating systematic reviews. Most current approaches rely on the sample size being explicitly mentioned in the abstract.

Methods

847 RCTs from high-impact medical journals were tagged with six different entities that could indicate the sample size. A named entity recognition (NER) model was trained to extract the entities and then deployed on a test set of 150 RCTs. The entities’ performance in predicting the actual number of trial participants who were randomized was assessed and possible combinations of the entities were evaluated to create predictive models.

Results

The most accurate model could make predictions for 64.7% of trials in the test set, and the resulting predictions were within 10% of the ground truth in 96.9% of cases. A less strict model could make a prediction for 96.0% of trials, and its predictions were within 10% of the ground truth in 88.2% of cases.

Conclusion

Training a named entity recognition model to predict the sample size from randomized controlled trials is feasible, not only if the sample size is explicitly mentioned but also if the sample size can be calculated, e.g., by adding up the number of patients in each arm.
Genetic co-occurrence networks identify polymorphisms within ontologies highly associated with preeclampsia
Authors: Obersterescu Andreea , Cox Brian J.
Published: July 2024

Polygenic diseases require the co-occurrence of multiple risk variants to initiate a pathology. An example is preeclampsia, a hypertensive disease of pregnancy with no known cure or therapy other than the often-preterm delivery of the neonate and placenta. Preeclampsia is challenging to predict due to symptomatic and outcome heterogeneity. Transcriptomic and genetic analysis suggests that this is a multi-syndromic and multigenic disease. Previous research applications of traditional GWAS methods to preeclampsia identified only a few alleles with marginal differences between cases and controls. We seek to identify genetic networks related to the pathophysiology of preeclampsia as potential avenues of therapeutic investigation and early genetic testing. We created a novel systems biology-based method that identifies networks of co-occurring SNPs associated with a trait or disease. The co-occurring pairs are assembled into higher-order associations using network graphs. We validated our method using simulation modelling and tested it against maternal genetic data of a previously assessed preeclampsia cohort. The genetic co-occurrence network identified SNPs in or near genes with ontological enrichment for VEGF, immunological and hormonal pathways, among others with known physiological disruption in preeclampsia. Our findings suggests that preeclampsia is caused by relatively common alleles (<5%) that accumulate in unfavorable combinations.
NETosis is an important component of chronic inflammation in patients with heart failure
Authors: Kostin Sawa , Richter Manfred , Krizanic Florian , Sasko Benjamin , Kelesidis Theodoros , Pagonas Nikolaos
Published: July 2024

Background and aim

We have previously demonstrated that heart failure (HF) is characterized by low-grade myocardial inflammation. However, the role of neutrophils (N), neutrophil extracellular traps (NETs) and neutrophil cell death by NETosis in the myocardium of patients with HF remains largely unknown. The present study investigated the number of neutrophils (N) and their proportion undergoing NETosis and developing NETs in HF.

Methods

We used quantitative confocal microscopy and NETosis markers in the left ventricular biopsies obtained from 5 control and from patients with HF due to dilated (DCM, n=7), inflammatory (infCMP, n=7) and ischemic cardiomyopathy (ICM, n=7). We used immunolabeling for CD45, CD66b and CD11b for (N) and citrullinated histone3 (citH3), peptidylarginine deiminase-4 (PAD-4), neutrophil elastase (NE) and myeoloperoxidase (MPO) for NETosis. These proteins were also investigated by quantitative fluorescence intensity analysis, Western blot and quantitative polymerase chain reaction (qPCR).

Results

Compared to control, the number of N was increased 3-4 times in HF. We found that using a single marker for NETosemarkers, 43.2% of N in DCM, 46.7% in ICM and 57.3% in infCMP experienced NETosis. The use of double labeling (NE with CitH3) showed that 55.6% of N developed NETosis in DCM, 57.9% in ICM and 79.4% in infCMP. The difference between the N who underwent NETosis in infCMP and those in DCM was statistically different (p<0.01). The proportion of N who developed NETosis or formed NETs in control tissue was less than 5% and differed significantly from that in HF patients, regardless of etiology (p<0.01). These results were confirmed by quantitative fluorescence analysis, Western blot and qPCR.

Conclusions

This is the first study to show the occurrence of NETosis in human heartsin situindicating that NETosis is an important component of low-grade myocardial inflammation in HF.

What is new?

Low-grade myocardial inflammation is a typical feature of heart failure and neutrophil cell death (NETosis) is an important component of this pathological process.

What are the clinical implications?

Preventing excessive neutrophil activation and inhibiting the major components involved in the NETosis program (neutrophil elastase, myeloperoxidase and peptidylarginine deiminase-4) are perspective targets for the treatment of HF.
Triton-X 100-treated virus-based ELLA demonstrates discordant antigenic evolution of influenza B virus haemagglutinin and neuraminidase
Authors: Do Thi H. T. , Wille Michelle , Wheatley Adam K. , Koutsakos Marios
Published: July 2024

Neuraminidase (NA)-specific antibodies have been associated with protection against influenza and thus NA is considered a promising target for next-generation vaccines against influenza A (IAV) and B viruses (IBV). NA inhibition (NI) by antibodies is typically assessed using an enzyme-linked lectin assay (ELLA). However, ELLA can be confounded by anti- hemagglutinin (anti-HA) antibodies that block NA by steric hindrance (termed HA interference). While strategies have been employed to overcome HA interference for IAV, similar approaches have not been assessed for IBV. We found HA interference is common in ELLA using IBV, rendering the technique unreliable. Anti-HA antibodies were not completely depleted from sera by HA-expressing cell lines and this approach was of limited utility. In contrast, we find that treatment of virions with Triton-X 100, but not Tween-20 or ether, efficiently separates the HA and NA components and overcomes interference caused by anti-HA antibodies. We also characterise a panel of recombinant IBV NA proteins that further validated the results from Triton-X 100-treated virus-based ELLA. Using these reagents and assays we demonstrate discordant antigenic evolution between IBV NA and HA over the last 80 years. This optimized ELLA protocol will facilitate further in-depth serological surveys of IBV immunity as well as antigenic characterisation of the IBV NA on a larger scale.

Importance

Influenza B viruses contribute to annual epidemics and may cause severe disease, especially in children. Consequently, several approaches are being explored to improve vaccine efficacy, including the addition of neuraminidase. Antigen selection and assessment of serological responses will require a reliable serological assay to specifically quantify Neuraminidase inhibition. While such assays have been assessed for influenza A viruses, this has not been done of influenza B viruses. Our study identifies a readily applicable strategy to measure inhibitory activity of neuraminidase-specific antibodies against influenza B virus without interference from anti-hemagglutinin antibodies. This will aid broader serological assessment of influenza B virus-specific antibodies and antigenic characterisation of the influenza B virus neuraminidase.
Environmental controls on crenarchaeol distributions in hydrothermal springs
Authors: Calhoun Amanda N. , Blewett Jerome , Colman Daniel R. , Amenabar Maximiliano J. , Harris Carolynn M. , Boyd Eric S. , Pearson Ann , Leavitt William D.
Published: July 2024

Abstract

Thermophilic archaea synthesize cellular membranes composed primarily of isoprenoid glycerol dibiphytanyl glycerol tetraethers (iGDGTs). Cells can adjust the structure of iGDGTs by increasing the number of cyclopentyl rings to enhance lipid packing, thereby decreasing membrane permeability and fluidity to maintain cellular function at high temperature and/or acidic pH. Certain archaea synthesize an iGDGT, crenarchaeol, with four cyclopentyl rings and a unique cyclohexyl ring, the function of which is unknown. Structural modeling suggests the cyclohexyl ring may increase membrane fluidity, potentially representing an adaptation that allowed thermophiles to radiate into cooler environments. To begin to investigate this hypothesis, iGDGT abundance was quantified in forty-one hydrothermal springs in Yellowstone National Park (YNP), USA, and contextualized within a compilation of global hydrothermal spring iGDGTs with pH values of 1.1 to 10.1 and temperatures of 16.3 to 95 °C. pH most strongly correlated with both crenarchaeol abundance and the number of cyclopentyl rings per iGDGT. Crenarchaeol abundance exhibited a nonlinear relationship with both pH and temperature, with highest abundances at pH 7.4 and 46 °C, then decreasing in abundance above and below these values. These observations support the hypothesis that crenarchaeol’s cyclohexyl ring facilitated the adaptation of thermophilic and acidophilic archaea to lower temperature and higher pH niches, enabling their radiation into the marine realm.

Importance

Archaea change the composition of their membrane lipids to alter the fluidity of their membranes to protect cellular functions from environmental stressors. Some archaea produce a lipid, crenarchaeol, with a unique six-membered ring, the effect of which on archaeal membrane dynamics remains unknown. In this study, we identify pH as the most important geochemical variable for archaeal membrane response in Yellowstone National Park hot springs. In addition, the lipid distributions we find support the hypothesis that crenarchaeol facilitated the archaeal evolutionary transition from hot and acidic to cool and neutral waters. We contextualize these findings in a literature compilation that spans the globe.
N-Glycosylation of MRS2 balances aerobic and anaerobic energy production by reducing rapid mitochondrial Mg2+influx in conditions of high glucose or impaired respiratory chain function
Authors: Peng Min , Mathew Neal D. , Anderson Vernon E. , Falk Marni J. , Nakamaru-Ogiso Eiko
Published: July 2024

N-linked glycoproteins function in numerous biological processes, modulating enzyme activities as well as protein folding, stability, oligomerization, and trafficking. WhileN-glycosylation of mitochondrial proteins has been detected by untargeted MS-analyses, the physiological existence and roles of mitochondrial proteinN-linked glycosylation remain under debate. Here, we report that MRS2, a mitochondrial inner membrane protein that functions as the high flux magnesium transporter, isN-glycosylated to various extents depending on cellular bioenergetic status. BothN-glycosylated and unglycosylated isoforms were consistently detected in mitochondria isolated from mouse liver, rat and mouse liver fibroblast cells (BRL 3A and AFT024, respectively) as well as human skin fibroblast cells. Immunoblotting of MRS2 showed it was bound to, and required stringent elution conditions to remove from, lectin affinity columns with covalently bound concanavalin A orLens culinarisagglutinin. Following peptide:N-glycosidase F (PNGase F) digestion of the stringently eluted proteins, the higher MrMRS2 bands gel-shifted to lower Mrand loss of lectin affinity was seen. BRL 3A cells treated with two differentN-linked glycosylation inhibitors, tunicamycin or 6-diazo-5-oxo-L-norleucine, resulted in decreased intensity or loss of the higher MrMRS2 isoform. To investigate the possible functional role of MRS2N-glycosylation, we measured rapid Mg2+influx capacity in intact mitochondria isolated from BRL 3A cells in control media or following treatment with tunicamycin or 6-diazo-5-oxo-L-norleucine. Interestingly, rapid Mg2+influx capacity increased in mitochondria isolated from BRL 3A cells treated with eitherN-glycosylation inhibitor. Forcing reliance on mitochondrial respiration by treatment with either galactose media or the glycolytic inhibitor 2-deoxyglucose or by minimizing glucose concentration similarly reduced theN-glycosylated isoform of MRS2, with a correlated concomitant increase in rapid Mg2+influx capacity. Conversely, inhibiting mitochondrial energy production in BRL 3A cells with either rotenone or oligomycin resulted in an increased fraction ofN-glycosylated MRS2, with decreased rapid Mg2+influx capacity. Collectively, these data provide strong evidence that MRS2N-glycosylation is directly involved in the regulation of mitochondrial matrix Mg2+, dynamically communicating relative cellular nutrient status and bioenergetic capacity by serving as a physiologic brake on the influx of mitochondrial matrix Mg2+under conditions of glucose excess or mitochondrial bioenergetic impairment.
Comprehensive deletion scan of anti-CRISPR AcrIIA4 reveals essential and dispensable domains for Cas9 inhibition
Authors: Iturralde Annette B , Weller Cory A , Sadhu Meru J
Published: July 2024

Delineating a protein’s essential and dispensable domains provides critical insight into how it carries out its function. Here, we developed a high-throughput method to synthesize and test the functionality of all possible in-frame and continuous deletions in a gene of interest, enabling rapid and unbiased determination of protein domain importance. Our approach generates precise deletions using a CRISPR library framework that is free from constraints of gRNA target site availability and efficacy. We applied our method to AcrIIA4, a phage-encoded anti-CRISPR protein that robustly inhibits SpCas9. Extensive structural characterization has shown that AcrIIA4 physically occupies the DNA-binding interfaces of several SpCas9 domains; nonetheless, the importance of each AcrIIA4 interaction for SpCas9 inhibition is unknown. We used our approach to determine the essential and dispensable regions of AcrIIA4. Surprisingly, not all contacts with SpCas9 were required, and in particular, we found that the AcrIIA4 loop that inserts into SpCas9’s RuvC catalytic domain can be deleted. Our results show that AcrIIA4 inhibits SpCas9 primarily by blocking PAM binding, and that its interaction with the SpCas9 catalytic domain is inessential.
A magnesium transporter CorA ofMycobacterium smegmatisenhances the tolerance of structurally unrelated antibiotics in the host cells
Authors: Chatterjee Debasmita , Daya Manasi A R , Rastogi Sumit Kumar , Panda Aditya Prasad , Biju Bayomi , Bhattacharyya Debleena , Ghosh Anindya Sundar
Published: July 2024

Ion transporters or channels are involved in maintaining metal homeostasis in bacterial cells by aiding the movement of metal ions across the cell, which might also facilitate the export of antimicrobials. Ubiquitous magnesium transporter, CorA ofMycobacterium smegmatisis well known for its role in maintaining magnesium homeostasis. However, little is known about its involvement in exerting antimicrobial resistance. Here, with the help of molecular genetics,in vivoandin silicostudies we tried to envisage the role of CorA ofM. smegmatisin antimicrobial resistance ofM. smegmatisandE. coli. Expression ofcorAinM. smegmatisandE. coliincreased the tolerance of the host cells towards various structurally unrelated antibiotics and anti-tubercular drugs. In addition, a significantly lower accumulation of norfloxacin and ofloxacin by the host cells expressingcorAfurther indicated its role in enhancing the efflux pump activity. Moreover, the presence of a sub-inhibitory concentration of Mg2+resulted in increased low-level tolerance towards the tested drugs. Furthermore, CorA enhanced the biofilm-forming ability of cells expressing it. Overall, we speculate that magnesium transporter CorA facilitates multi-drug efflux activity of the host cells where Mg2+might act as a facilitator in the process.

IMPORTANCE

Magnesium acts as a co-factor for various biochemical and physiological reactions, such as protein synthesis, cell membrane integrity, nucleic acid synthesis, etc. Metal transporters maintain metal homeostasis by regulating the uptake, efflux, or transportation of metals in certain necessary cellular compartments. In bacteria, magnesium ion (Mg2+) is mainly supplied by the CorA protein which is a ubiquitous family of transport proteins and extensively studied inE. coliandSalmonella sp. However, little is known about the functional relationship of metal transporters ofMycobacterium spwith extrusion of antibiotics, and their involvement in stress tolerance. Here, we report CorA (MSMEG_5056), a magnesium transporter ofMycobacterium smegmatisin influencing the extrusion of multiple structurally unrelated classes of drugs and enhancing the biofilm formation ofE. coliandMycobacterium smegmatis.

Abstract Figure

Graphical Abstract:

Hypothetical model of Antibiotic export by CorA.

Antibiotics bind to the closed state of the protein (left). During the transition to the open state (right), the to-and-fro motion between multiple open states drives the efflux of the antibiotic while facilitating the import of Mg2+. The colors of the models correspond to the chain ID. The bottom views of both the closed and open states are shown in the rectangular box, with the color indicated by their respective chain IDs.
Acute BRCAness Induction and AR Signaling Blockage through CDK12/7/9 Degradation Enhances PARP Inhibitor Sensitivity in Prostate Cancer
Authors: Gui Fu , Jiang Baishan , Jiang Jie , He Zhixiang , Tsujino Takuya , Takai Tomoaki , Arai Seiji , Pana Celine , Köllermann Jens , Bradshaw Gary Andrew , Eisert Robyn , Kalocsay Marian , Fassl Anne , Balk Steven P , Kibel Adam S. , Jia Li
Published: July 2024

Current treatments for advanced prostate cancer (PCa) primarily target the androgen receptor (AR) pathway. However, the emergence of castration-resistant prostate cancer (CRPC) and resistance to AR pathway inhibitors (APSIs) remains ongoing challenges. Here, we present BSJ-5-63, a novel proteolysis-targeting chimera (PROTAC) targeting cyclin-dependent kinases (CDKs) CDK12, CDK7, and CDK9, offering a multi-pronged approach to CRPC therapy. BSJ-5-63 degrades CDK12, diminishing BRCA1 and BRCA2 expression and inducing a sustained “BRCAness” state. This sensitizes cancer cells to PARP inhibitors (PARPis) regardless of their homologous recombination repair (HRR) status. Furthermore, CDK7 and CDK9 degradation attenuates AR signaling, enhancing its therapeutic efficacy. Preclinical studies, including bothin vitroandin vivoCRPC models, demonstrate that BSJ-5-63 exerts potent anti-tumor activity in both AR-positive and AR-negative setting. This study introduces BSJ-5-63 as a promising therapeutic agent that addresses both DNA repair and AR signaling mechanisms, with potential benefits for a board patient population.