bluuebunny/crossref_metadata_2025 huggingface dataset:
Phylogenomics revealed migration routes and adaptive radiation timing of Holarctic malaria vectors of the Maculipennis group
Authors: Yurchenko Andrey A. , Naumenko Anastasia N. , Artemov Gleb N. , Karagodin Dmitry A. , Hodge James M. , Velichevskaya Alena I. , Kokhanenko Alina A. , Bondarenko Semen M. , Abai Mohammad R. , Kamali Maryam , Gordeev Mikhail I. , Moskaev Anton V. , Caputo Beniamino , Aghayan Sargis A. , Baricheva Elina M. , Stegniy Vladimir N. , Sharakhova Maria V. , Sharakhov Igor V.
Published: August 2022

Background

Understanding the evolutionary relationships between closely related taxa is important for mosquitoes that transmit human diseases. Six out of 41 dominant malaria vectors in the world belong to the Maculipennis group, which is subdivided into two North American subgroups (Freeborni and Quadrimaculatus), and one Eurasian (Maculipennis) subgroup. Although previous studies considered the Nearctic subgroups as ancestral, details about their relationship with the Palearctic subgroup, and their migration times and routes from North America to Eurasia remain controversial. The Eurasian speciesAn. beklemisheviis currently included in the North American Quadrimaculatus subgroup adding to the uncertainties in mosquito systematics.

Results

To reconstruct historic relationships between the North American and Eurasian mosquitoes, we conducted a phylogenomic analysis of 11 Palearctic and 2 Nearctic species based on 1271 orthologous genes using their transcriptomic or genomic sequences. The analysis indicated that the Palearctic speciesAn. beklemisheviclusters together with other Eurasian species and represents a basal lineage among them. Also,An. beklemisheviis related more closely toAn. freeborni,which inhabits the Western United States, rather than toAn. quadrimaculatus,a species from the Eastern United States. The time-calibrated tree suggests a migration of mosquitoes in the Maculipennis group from North America to Eurasia about 20-25 million years ago through the Bering Land Bridge. A Hybridcheck analysis demonstrated highly significant signatures of introgression events between allopatric speciesAn. labranchiaeandAn. beklemishevi. The analysis also identified ancestral introgression events betweenAn. sacharoviand its Nearctic relativeAn. freebornidespite their current geographic isolation.

Conclusions

Our phylogenomic analyses reveal migration routes and adaptive radiation timing of Holarctic malaria vectors and strongly support inclusion ofAn. beklemisheviinto the Maculipennis subgroup. The vectorial capacity and the ability to diapause during winter evolved multiple times in Maculipennis evolution. Detailed knowledge of the evolutionary history in the Maculipennis subgroup will help us better understand the current and future patterns of malaria transmission in Eurasia.
A comprehensive single cell data analysis of in lymphoblastoid cells reveals the role of Super-enhancers in maintaining EBV latency
Authors: Yan Bingyu , Wang Chong , Chakravorty Srishti , Zhang Zonghao , Kadadi Simran D. , Zhuang Yuxin , Sirit Isabella , Hu Yonghua , Jung Minwoo , Sahoo Subhransu , Wang Luopin , Shao Kunming , Anderson Nicole L. , Trujillo-Ochoa Jorge L. , Liu Xing , Olson Matthew R. , Afzali Behdad , Zhao Bo , Kazemian Majid
Published: August 2022

We probed the lifecycle of EBV on a cell-by-cell basis using single cell RNA sequencing (scRNA-seq) data from nine publicly available lymphoblastoid cell lines (LCL). While the majority of LCLs comprised cells containing EBV in the latent phase, two other clusters of cells were clearly evident and were distinguished by distinct expression of host and viral genes. Notably, both were high expressors of EBV LMP1/BNLF2 and BZLF1 compared to another cluster that expressed neither gene. The two novel clusters differed from each other in their expression of EBV lytic genes, including glycoprotein gene GP350. The first cluster, comprising GP350−LMP1hi cells, expressed high levels of HIF1A and was transcriptionally regulated by HIF1-α. Treatment of LCLs with Pevonedistat, a drug that enhances HIF1-α signaling, markedly induced this cluster. The second cluster, containing GP350+LMP1hi cells, expressed EBV lytic genes. Host genes that are controlled by super-enhancers (SEs), such as transcription factors MYC and IRF4, had the lowest expression in this cluster. Functionally, the expression of genes regulated by MYC and IRF4 in GP350+LMP1hi cells were lower compared to other cells. Indeed, induction of EBV lytic reactivation in EBV+ AKATA reduced the expression of these SE-regulated genes. Furthermore, CRISPR-mediated perturbation of the MYC or IRF4 SEs in LCLs induced the lytic EBV gene expression, suggesting that host SEs and/or SE target genes are required for maintenance of EBV latency. Collectively, our study revealed EBV associated heterogeneity among LCLs that may have functional consequence on host and viral biology.

Importance

Epstein-Barr virus (EBV) establishes a life-long latency program within host cells. As such, EBV immortalized lymphoblastoid cells (LCLs) often carry the latent EBV genome and only a small percentage of LCLs containing lytic EBV. However, the cellular programs that distinguish latent from lytic cells and the heterogeneity of cells in latent or lytic phases remains poorly explored. To explore these unknowns, we reanalyzed publicly available single cell RNA-seq data from nine LCLs. This approach permitted the simultaneous study of cells in both latent and lytic phases. We identified three cell populations with distinct lytic/latent activity and further characterized the transcriptomes of these cells. We also identified a new role of super-enhancers in regulating EBV lytic replication. Collectively, our studies revealed EBV associated heterogeneity among LCLs that contribute to EBV life cycle and biology.
Genetic differences between 129S substrains affect antiretroviral immune responses
Authors: Zhang Robert Z. , Mele Vincent , Robben Lia , Kane Melissa
Published: August 2022

Inbred mouse lines vary in their ability to mount protective antiretroviral immune responses, and even closely related strains can exhibit opposing phenotypes upon retroviral infection. Here, we found that 129S mice inherit a previously unknown mechanism for the production of anti-murine leukemia virus (MLV) antibodies and control of infection. The resistant phenotype is controlled by two dominant loci that are independent from known MLV-resistance genes. We also show that production of anti-MLV antibodies in 129S7, but not 129S1 mice is independent of interferon gamma (IFNγ) signaling. Thus, our data indicate that 129S mice inherit an unknown mechanism for control of MLV infection and demonstrate that there is genetic variability in 129S substrains that affects their ability to mount antiviral immune responses.

Importance

Understanding the genetic basis for production of protective antiviral immune responses is crucial for the development of novel vaccines and adjuvants. Additionally, characterizing the genetic and phenotypic variability in inbred mice has implications for the selection of strains for targeted mutagenesis, choice of controls, and for broader understanding of the requirements for protective immunity.
Resting State Functional Connectivity Demonstrates Increased Segregation in Bilateral Temporal Lobe Epilepsy
Authors: Lucas Alfredo , Cornblath Eli J. , Sinha Nishant , Hadar Peter , Caciagli Lorenzo , Keller Simon S. , Bonilha Leonardo , Shinohara Russell T. , Stein Joel M. , Das Sandhitsu , Gleichgerrcht Ezequiel , Davis Kathryn A.
Published: August 2022

Temporal lobe epilepsy (TLE) is the most common type of focal epilepsy. An increasingly identified subset of patients with TLE consists of those who show bilaterally independent temporal lobe involvement during seizures. Bilateral TLE (BiTLE) remains understudied, likely due to its complex underlying pathophysiology and heterogeneous clinical presentation.

In this study, using a multicenter resting state functional MRI (rs-fMRI) dataset, we constructed whole brain functional networks of 19 patients with BiTLE, and compared them to those of 75 patients with unilateral TLE (UTLE). We quantified resting-state, whole-brain topological properties using metrics derived from network theory, including clustering coefficient, global efficiency, participation coefficient, and modularity. For each metric, we computed an average across all brain regions, and iterated this process across network densities ranging from 0.10-0.50. Curves of network density versus each network metric were compared between groups. Finally, we derived a combined metric, which we term the “integration-segregation axis”, by combining whole brain average clustering coefficient and global efficiency curves and applying principal component analysis (PCA)-based dimensionality reduction.

Compared to UTLE, BiTLE had decreased global efficiency (p=0.026), increased whole brain average clustering coefficient (p=0.035), and decreased whole brain average participation coefficient across a range of network densities (p=0.001). Modularity maximization yielded a larger number of smaller communities in BiTLE than in UTLE (p=0.016). Differences in network properties separate BiTLE and UTLE along the integration-segregation axis: 68% of patients with BiTLE were identified within the high segregation region, while only 41% of the UTLE patients were identified in the same region (p=0.042). Along the integration-segregation axis, UTLE patients with poor surgical outcomes were more similar to BiTLE than those with good surgical outcomes (p=0.72).

Increased interictal whole brain network segregation, as measured by rs-fMRI, is specific to BiTLE, and may assist in non-invasively identifying this patient population prior to intracranial EEG or device implantation.
Longitudinal neonatal brain development and socio-demographic correlates of infant outcomes following preterm birth
Authors: Vanes Lucy , Fenn-Moltu Sunniva , Hadaya Laila , Fitzgibbon Sean , Cordero-Grande Lucilio , Price Anthony , Chew Andrew , Falconer Shona , Arichi Tomoki , Counsell Serena J. , Hajnal Joseph V. , Batalle Dafnis , Edwards A. David , Nosarti Chiara
Published: August 2022

Preterm birth results in premature exposure of the brain to the extrauterine environment during a critical period of neurodevelopment. Consequently, infants born preterm are at a heightened risk of adverse behavioural outcomes in later life. We characterise longitudinal development of neonatal regional brain volume and functional connectivity in the first weeks following preterm birth, sociodemographic factors, and their respective relationships to psychomotor outcomes and psychopathology in toddlerhood. We study 121 preterm infants preterm who underwent magnetic resonance imaging shortly after birth, at term-equivalent age, or both. Longitudinal regional brain volume and functional connectivity were modelled as a function of psychopathology and psychomotor outcomes at 18 months. Better psychomotor functioning in toddlerhood was associated with greater relative right cerebellar volume and a more rapid decrease over time of sensorimotor degree centrality in the neonatal period. In contrast, increased 18-month psychopathology was associated with a more rapid decrease in relative regional subcortical volume. Furthermore, while socio-economic deprivation was related to both psychopathology and psychomotor outcomes, cognitively stimulating parenting predicted psychopathology only. Our study highlights the importance of longitudinal imaging to better predict toddler outcomes following preterm birth, as well as disparate environmental influences on separable facets of behavioural development in this population.
Real-time analysis of the cancer genome and fragmentome from plasma and urine short and long cell-free DNA using Nanopore sequencing
Authors: van der Pol Ymke , Tantyo Normastuti Adhini , Evander Nils , Hentschel Anouk E. , Ramaker Jip , Bahce Idris , Nieuwenhuijzen Jakko A. , Steenbergen Renske D.M. , Pegtel D. Michiel , Moldovan Norbert , Mouliere Florent
Published: August 2022

Cell-free DNA (cfDNA) can be isolated from blood and/or urine of cancer patients and analyzed with sequencing. Unfortunately, most conventional short-read sequencing methods are technically challenging, labor intensive and time consuming, requiring several days but more typically weeks to obtain interpretable data which are limited by a bias for short cfDNA fragments.

Here, we demonstrate that with Oxford Nanopore Technologies sequencing we can achieve economical and ultra-fast delivery of clinical data from liquid biopsies.

Our ‘ITSFASTR’ approach is able to deliver copy number aberrations, and cfDNA fragmentation profiles in less than 24 hours from sample collection. The tumorderived cfDNA fraction calculated from lung cancer patient plasma and urine from bladder cancer patients was highly correlated (R=0.98) to the tumor fraction calculated from conventional short-read sequencing of the same samples. cfDNA size profile and fragmentation patterns in plasma and urine exhibited the typical cfDNA features yet with a significantly higher proportion of fragments that exceed 300bp, exhibiting similar tumor fraction than shorter size fragments. Notably, comprehensive fragment-end composition and nucleosome profiling near transcription start sites can be determined from the same data.

We propose that ITSFASTR is the first point-of-care solution for obtaining genomic and fragmentomic results from liquid biopsies.
Risk factors for COVID-19 hospitalization or death during the first Omicron surge in adults: a large population-based case-control study
Authors: Lo TKT , Usman Hussain , Sikdar Khokan C. , Strong David , James Samantha , Ross Jordan , Saxinger Lynora M.
Published: August 2022

Background

Description of risk factors of severe acute COVID-19 outcomes with the consideration of vaccination status in the era of the Omicron variant of concern are limited.

Objectives

To examine the association of age, sex, underlying medical conditions, and COVID-19 vaccination with hospitalization, intensive-care unit (ICU) admission, or death due to the disease, using data from a period when Omicron was the dominant strain.

Methods

A population-based case-control study based on administrative health data, that included confirmed COVID-19 patients during January (2022) in Alberta, Canada. Patients who were non-residents, without the provincial healthcare insurance coverage, or ≤18 years of age were excluded. Patients with any severe outcome were the cases; and those without any hospitalization, ICU admission, or death were controls. Adjusted odds ratios, of the explanatory factors of a severe outcome, were estimated using a logistic regression model.

Results

There were 90,989 COVID-19 patients included in the analysis; 2% had severe outcomes and 98% were included in the control group. Overall, more COVID patients were found in the younger age-groups (72.0% ≤49 years old), females (56.5%), with no underlying conditions (59.5%), and fully vaccinated patients (90.4%). However, the adjusted odds ratios were highest in the 70–79 age group (28.32; 95% CI 20.6–38.9) or among ≥80 years old (29.8; 21.6–41.0), males (1.4; 1.3–1.6); unvaccinated (16.1; 13.8–18.8), or patients with ≥3 underlying conditions (13.1; 10.9–15.8).

Conclusion

Higher risk of severe acute COVID-19 outcomes were associated with older age, the male sex, and increased number of underlying medical conditions. Unvaccination or undervaccination remained as the greatest modifiable risk factor in prevention of severe COVID outcomes. These findings help inform medical decisions and allocation of scarce healthcare resources.
Keeping up with the pathogens: Improved antimicrobial resistance detection and prediction fromPseudomonasaeruginosa genomes
Authors: Madden Danielle E. , Baird Timothy , Bell Scott C. , McCarthy Kate L. , Price Erin P. , Sarovich Derek S.
Published: August 2022

Background

Antimicrobial resistance (AMR) is an intensifying threat that requires urgent mitigation to avoid a post-antibiotic era. The ESKAPE pathogen,Pseudomonas aeruginosa, represents one of the greatest AMR concerns due to increasing multi- and pan-drug resistance rates. Shotgun sequencing is quickly gaining traction forin silicoAMR profiling due to its unambiguity and transferability; however, accurate and comprehensive AMR prediction fromP. aeruginosagenomes remains an unsolved problem.

Methods

We first curated the most comprehensive database yet of knownP. aeruginosaAMR variants. Next, we performed comparative genomics and microbial genome-wide association study analysis across a Global isolate Dataset (n=1877) with paired antimicrobial phenotype and genomic data to identify novel AMR variants. Finally, the performance of ourP. aeruginosaAMR database, implemented in our ARDaP software, was compared with three previously publishedin silicoAMR gene detection or phenotype prediction tools – abritAMR, AMRFinderPlus, ResFinder – across both the Global Dataset and an analysis-naïve Validation Dataset (n=102).

Results

Our AMR database comprises 3639 mobile AMR genes and 733 AMR-conferring chromosomal variants, including 75 chromosomal variants not previously reported, and 284 chromosomal variants that we show are unlikely to confer AMR. Our pipeline achieved a genotype-phenotype balanced accuracy (bACC) of 85% and 81% across 10 clinically relevant antibiotics when tested against the Global and Validation Datasets, respectively, vs. just 56% and 54% with abritAMR, 58% and 54% with AMRFinderPlus, and 60% and 53% with ResFinder.

Conclusions

Our ARDaP software and associated AMR variant database provides the most accurate tool yet for predicting AMR phenotypes inP. aeruginosa, far surpassing the performance of current tools. Implementation of our ARDaP-compatible database for routine AMR prediction fromP. aeruginosagenomes and metagenomes will improve AMR identification, addressing a critical facet in combatting this treatment-refractory pathogen. However, knowledge gaps remain in our understanding of theP. aeruginosaresistome, particularly the basis of colistin AMR.
Structural and Dynamic Basis of DNA Capture and Translocation by Mitochondrial Twinkle Helicase
Authors: Li Zhuo , Kaur Parminder , Lo Chen-Yu , Chopra Neil , Smith Jamie , Wang Hong , Gao Yang
Published: August 2022

Twinkle is the sole helicase responsible for mitochondrial DNA replication. Twinkle can selfload onto and unwind mitochondrial DNA. Nearly 60 mutations on Twinkle have been linked to human mitochondrial diseases. Using cryo-electron microscopy (cryo-EM) and High-speed atomic force microscope (HS-AFM), we obtained the atomic-resolution structure of a vertebrate Twinkle homolog with DNA and captured in real-time how Twinkle is self-loaded onto DNA. Our data highlight the essential role of the non-catalytic N-terminal domain of Twinkle. The N-terminal domain interacts with the helicase domain to stabilize Twinkle hexamers during translocation and the new domain-domain interface is a hotspot for disease-related mutations. The N-terminal domains can protrude approximately 5 nm to capture nearby DNA and initialize Twinkle loading onto DNA. Moreover, structural analysis and subunit doping experiments suggest that Twinkle hydrolyzes ATP stochastically, which explains the low efficiency of Twinkle DNA unwinding and implicates additional regulations of Twinkle during mitochondrial DNA replication.
Transcriptome and chromatin accessibility mapping reveals a type I Interferon response triggered by Mycobacterium tuberculosis infection
Authors: Madden Katrina , El Hamra Rayan , Berton Stefania , Alvarez Gonzalo G. , Blais Alexandre , Sun Jim
Published: August 2022

Tuberculosis, a deadly infectious lung disease caused by Mycobacterium tuberculosis (Mtb), remains the leading cause of bacterial disease-related deaths worldwide. The success of Mtb as a human pathogen depends on its ability to manipulate host immune response pathways, many of which are regulated by epigenetic mechanisms that control the accessibility of chromatin to the transcriptional machinery. Recent reports suggest that host phosphatases, such as PPM1A, may play a role in the regulation of chromatin accessibility during bacterial infections. However, changes in genome-wide chromatin accessibility during Mtb infection and whether PPM1A plays a role in this process remains unknown. Using combinatorial chromatin accessibility (ATAC-seq) and transcriptomics (RNA-seq) profiling of wild-type (WT), PPM1A knockout (ΔPPM1A) and PPM1A overexpressing (PPM1A+) macrophages, we demonstrate that Mtb infection induces global chromatin remodeling consistent with changes in gene expression signatures. The strongest concordant chromatin accessibility and gene expression signature triggered by Mtb infection was enriched for genes involved in the type I interferon (IFN) signaling pathways. Modulation of PPM1A expression results in altered chromatin accessibility signatures during Mtb infection that are reflected in the total number, chromosome location and directionality of change. Transcription factor motif analysis revealed an enrichment for transcription factors involved in the type I IFN pathway during Mtb infection, including IRF4, MEF2A, and JDP2. In contrast, both deletion and overexpression of PPM1A produced unique transcription factor enrichment signatures linked to the genomic regions with altered chromatin accessibility. Our study demonstrates that altered type I IFN responses in Mtb-infected macrophages occurs as a result of genome-wide changes in chromatin accessibility, and that PPM1A likely plays a role in a subset of these signatures.
Phosphorylation of the Smooth Muscle Master Splicing Regulator RBPMS Regulates its Splicing Activity
Authors: Barnhart Michael D. , Yang Yi , Nakagaki-Silva Erick E. , Hammond Thomas H. , Pizzinga Mariavittoria , Gooding Clare , Stott Katherine , Smith Christopher W.J.
Published: August 2022

We previously identified RBPMS as a master regulator of alternative splicing in differentiated smooth muscle cells (SMCs). RBPMS is transcriptionally downregulated during SMC dedifferentiation, but we hypothesized that RBPMS protein activity might be acutely downregulated by post-translational modifications. Publicly available phosphoproteomic datasets reveal that Thr113 and Thr118 immediately adjacent to the RRM domain are commonly both phosphorylated. An RBPMS T113/118 phosphomimetic T/E mutant showed decreased splicing regulatory activity both in transfected cells and in a cell-free in vitro assay, while a non-phosphorylatable T/A mutant retained full activity. Loss of splicing activity was associated with a modest reduction in RNA affinity but significantly reduced RNA binding in nuclear extract. A lower degree of oligomerization of the T/E mutant might cause lower avidity of multivalent RNA binding. However, NMR analysis also revealed that the T113/118E peptide acts as an RNA mimic which can loop back and antagonize RNA-binding by the RRM domain. Finally, we identified ERK2 as the most likely kinase responsible for phosphorylation at Thr113 and Thr118. Collectively, our data identify a potential mechanism for rapid modulation of the SMC splicing program in response to external signals during the vascular injury response and atherogenesis.
Designing a Cas9/gRNA-assisted quantitative Real-Time PCR (CARP) assay for identification of point mutations leading to rifampicin resistance in the human pathogen Mycobacterium tuberculosis
Authors: Augustin Linus , Agarwal Nisheeth
Published: August 2022

A simple, rapid and low-cost diagnostic test, which can detect both the drug-sensitive and the drug-resistant tuberculosis (TB) cases is the need of the hour. Here, we developed a Cas9/gRNA-assisted quantitative Real-Time PCR (qRT-PCR) (CARP) assay to detect single nucleotide mutations causing drug resistance in the TB pathogen, Mycobacterium tuberculosis (Mtb). Guide RNAs (gRNAs) were designed against S531 and H526 positions in the rifampicin (RIF)-resistance-determining region (RR-DR) of the Mtb rpoB gene that exhibit frequent mutations in the RR clinical isolates of Mtb. Conditions were optimised for in vitro Cas9 cleavage such that single nucleotide changes at these positions can be recognised by Cas9/gRNA complex with 100% specificity. Further estimation of Cas9/gRNA-based cleavage of target DNA by qRT-PCR led to rapid detection of drug-resistant sequences. The newly designed CARP assay holds a great deal of promise in the diagnosis and prognosis of patients suffering from TB, in a cost-effective manner.
Turnover of the extracellular polymeric matrix in an EBPR microbial community
Authors: Tomás-Martínez Sergio , Zwolsman Erwin J. , Merlier Franck , Pabst Martin , Lin Yuemei , van Loosdrecht Mark C.M. , Weissbrodt David G.
Published: August 2022

Polyphosphate accumulating organisms (PAOs) are responsible for enhanced biological phosphate removal (EBPR) from wastewater, where they grow embedded in a matrix of extracellular polymeric substances (EPS). EPS comprise a mixture of biopolymers like polysaccharides or (glyco)proteins. Despite previous studies, little is known about the dynamics of EPS in mixed cultures, and their production by PAOs and potential consumption by flanking microbes. EPS are biodegradable and have been suggested to be a substrate for other organisms in the community. Studying EPS turnover can help elucidate their biosynthesis and biodegradation cycles. We analyzed the turnover of proteins and polysaccharides in the EPS of an enrichment culture of PAOs relative to the turnover of internal proteins. An anaerobic-aerobic sequencing batch reactor (SBR) simulating EBPR conditions was operated to enrich for PAOs. After achieving a stable culture, carbon source was switched to uniformly 13C-labelled acetate. Samples were collected at the end of each aerobic phase. EPS were extracted by alkaline treatment. 13C enrichment in proteins and sugars (after hydrolysis of polysaccharides) in the extracted EPS were measured by mass spectrometry. The average turnover rate of sugars and proteins (0.167 and 0.192 d-1 respectively) was higher than the expected value based on the solid removal rate (0.132 d-1), and no significant difference was observed between intracellular and secreted proteins. This indicates that EPS from the PAO enriched community is not selectively degraded by flanking populations under stable EBPR process conditions. Instead, we observed general decay of biomass, which corresponds to a value of 0.048 d-1.

Graphical abstract

Key points

- Proteins showed a higher turnover rate than carbohydrates.

- Turnover of EPS was similar to the turnover of intracellular proteins.

- EPS is not preferentially consumed by flanking populations.
FUS binding to RNA prevents R-loops
Authors: Thompson Valery F. , Wieland Daniel R. , Mendoza-Leon Vivian , Janis Helen I. , Lay Michelle A. , Harrell Lucas M. , Schwartz Jacob C.
Published: August 2022

The protein FUS (FUSed in sarcoma) is a metazoan RNA-binding protein that influences RNA production by all three nuclear polymerases. FUS also binds nascent transcripts, RNA processing factors, RNA polymerases, and transcription machinery. We explored the role of FUS binding interactions for activity during transcription.In vitrorun-off transcription assays revealed FUS enhanced RNA produced by a non-eukaryote polymerase. Activity also reduced the formation of R-loops between RNA products and their DNA template. Analysis by domain mutation and deletion indicated RNA-binding was required for activity. We interpret that FUS binds and sequesters nascent transcripts to prevent R-loops forming with nearby DNA. DRIP-seq analysis showed that a knockdown of FUS increased R-loop enrichment near expressed genes. Prevention of R-loops by FUS binding to nascent transcripts has potential to affect transcription by any RNA polymerase, highlighting the broad impact FUS can have on RNA metabolism in cells and disease.
Cellular, Molecular, and Enzymatic Signatures of Thrombi are Vascular Bed-Dependent
Authors: Bender Matthew , Aggarwal Anu , Mix Doran , Godwin Matthew , Guntupalli Suman , Nanjundappa Aravinda , Tefera Leben , Hadadin Ihab , Bhandari Rohan , Tong Michael , Baldwin William M. , Fairchild Robert L , Gomes Marcelo , Campbell Joseph , Schumick David , Cameron Scott J.
Published: August 2022

Background

The contribution of arterial and venous thrombi to vascular remodeling is unclear. While catheter-extraction of thrombus in cerebrovascular accident (CVA) is time-sensitive, similar urgency is rare in managing venous thromboembolism (VTE).

Objectives

Our goal was to determine molecular cellular signatures of thrombus extracted by catheter from various vascular beds to gain insight into vascular remodeling.

Methods

Twenty-five patients underwent catheter-directed thrombectomy (CDT), 13 for acute CVA, 8 for pulmonary embolism (PE), and 4 for deep vein thrombosis (DVT). Protein and RNA extracted from thrombus was evaluated by immunoblotting and sequencing, respectively. Thrombus-derived enzymes for which substrate is present in the blood vessel wall were examined for enzymatic activity.

Results

Time from symptom onset to thrombus extraction was 7.7 ± 1.9 hours for CVA and 109 ± 55 hours for VTE. Protein concentration, white blood cell and red blood cell content were all greater in venous compared with arterial thrombus while platelet content was similar. Both venous and arterial thrombus contained multiple Matrix Metalloproteinase (MMP) isoforms. MMP9 specific activity was greater in venous than in arterial thrombus (57 ± 6 ng/mL.μg protein-1 vs. 24 ± 8 ng/mL.μg protein-1, P=0.0051).

Conclusions

Arterial and venous thrombus have dissimilar phenotypes, each with biologically-active enzymes known to remodel blood vessels, and enzymatic activity proportional to the white blood cell content which increases with thrombus age. These data suggest a mechanistically-important role for early CDT to avoid the consequences of irreversible vascular remodeling.

Condensed Abstract

Emergent extraction of acute thrombus from arterial vascular beds restores limb and end-organ perfusion and is widely-accepted to be the standard of care. Extraction of thrombus from venous vascular beds, however, is rarely considered urgent, even though many patients subsequently develop debilitating symptoms. By capitalizing on privileged thrombus extracted from multiple vascular beds, we gained mechanistic insight regarding the cellular composition and cell-derived enzymes secreted from thrombus that may remodel the vessel wall. This study shows thrombi are biologically-active entities, continuously recruiting circulating cells that secrete enzymes both proportional to thrombus age and the time of patient presentation.
Characterization of RNA Polymerase II Trigger Loop Mutations using Molecular Dynamics Simulations and Machine Learning
Authors: Dutagaci Bercem , Duan Bingbing , Qiu Chenxi , Kaplan Craig D. , Feig Michael
Published: August 2022

Catalysis and fidelity of multisubunit RNA polymerases rely on a highly conserved active site domain called the trigger loop (TL), which achieves roles in transcription through conformational changes and interaction with NTP substrates. The mutations of TL residues cause distinct effects on catalysis including hypo- and hyperactivity and altered fidelity. We applied molecular dynamics simulation (MD) and machine learning (ML) techniques to characterize TL mutations in the Saccharomyces cerevisiae RNA Polymerase II (Pol II) system. We did so to determine relationships between individual mutations and phenotypes and to associate phenotypes with MD simulated structural alterations. Using fitness values of mutants under various stress conditions, we modeled phenotypes along a spectrum of continual values. We found that ML could predict the phenotypes with 0.68 R2 correlation from amino acid sequences alone. It was more difficult to incorporate MD data to improve predictions from machine learning, presumably because MD data is too noisy and possibly incomplete to directly infer functional phenotypes. However, a variational auto-encoder model based on the MD data allowed the clustering of mutants with different phenotypes based on structural details. Overall, we found that lethal mutations tended to increase distances of TL residues to the NTP substrate, while viable loss-of-function (LOF) substitutions tended to confer an increase in distances between TL and bridge helix (BH). In contrast, GOF mutants generally have a disrupting effect on hydrophobic contacts among TL and nearby helices.

AUTHOR SUMMARY

RNA polymerase II (Pol II) synthesizes RNA with the help of an active site domain called trigger loop (TL). The mutations of TL cause changes in the activity of Pol II that could range from gain-of function (GOF) to loss-of-function (LOF) or lethal. This study provides a systematic characterization of the structural and functional outcomes of the TL mutations using molecular dynamics (MD) simulations and machine learning (ML). We obtained functional phenotypes of mutants by ML using the genetic fitness scores as the input. We revealed that mutant TL sequences could predict the functional outcomes at a relatively high correlation. Then, we performed MD simulations to relate the structural information to the phenotypes. The analysis of the MD data suggested that the lethal mutants had increased distances between the TL and the substrate, while a subset of LOF mutants showed increased distances between TL and another active site domain called bridge helix (BH). On the other hand, GOF mutants had effects on the hydrophobic interactions around the active site. Overall, this study enhances our understanding of the effects of TL mutations to the Pol II function.
Polynuclear ruthenium complexes are effective antibiotics against Pseudomonas aeruginosa
Authors: Weber Brent S. , Carfrae Lindsey A. , Woods Joshua J. , Klobucar Kristina , Bigham Nicholas P. , MacNair Craig R. , Raivio Tracy L. , Wilson Justin J. , Brown Eric D.
Published: August 2022

There is an urgent need to develop new antibiotics for the treatment of infections caused by drug-resistant Gram-negative bacteria. In particular, new and diverse chemical classes of antibiotics are needed, as most antibiotics in clinical development are derivatives of existing drugs. Despite a history of use as antimicrobials, metals and metal-based compounds have largely been overlooked as a source of new chemical matter for antibacterial drug discovery. In this work, we identify several ruthenium complexes, ruthenium red, Ru265, and Ru360’, that possess potent antibacterial activity against both laboratory and clinical isolates of Pseudomonas aeruginosa. Suppressors with increased resistance were sequenced and found to contain mutations in the mechanosensitive ion channel mscS-1 or the colRS two component system. The antibacterial activity of these compounds translated in vivo to Galleria mellonella larvae and mouse infection models. Finally, we identify strong synergy between these compounds and the antibiotic rifampicin, with a dose-sparing combination therapy showing efficacy in both infection models. Our findings provide clear evidence that these ruthenium complexes are effective antibacterial compounds against a critical priority pathogen and show promise for the development of future therapeutics.
Comparison of antibody responses to SARS-CoV-2 variants in Australian children
Authors: Toh Zheng Quan , Mazarakis Nadia , Nguyen Jill , Higgins Rachel A. , Anderson Jeremy , Ha Do Lien Anh , Burgner David P. , Curtis Nigel , Steer Andrew C. , Mulholland Kim , Crawford Nigel W. , Tosif Shidan , Licciardi Paul V
Published: August 2022

There is limited understanding of antibody responses in children across different SARS-CoV-2 variants. As part of an ongoing household cohort study, we assessed the antibody response among unvaccinated children infected with Wuhan, Delta or Omicron variants, as well as vaccinated children with breakthrough Omicron infection, using a SARS-CoV-2 S1-specific IgG assay and surrogate virus neutralisation test (sVNT). Most children infected with Delta (100%, 35/35) or Omicron (81.3%, 13/16) variants seroconverted by one month following infection. In contrast, 37.5% (21/56) children infected with Wuhan seroconverted, as previously reported. However, Omicron-infected children (GMC 46.4 BAU/ml; sVNT % inhibition: 16.3%) mounted a significantly lower antibody response than Delta (435.5 BAU/mL, sVNT=76.9%) or Wuhan (359.0 BAU/mL, sVNT=74.0%). Vaccinated children with breakthrough Omicron infection mounted the highest antibody response (2856 BAU/mL, sVNT=96.5%). Our findings suggest that despite a high seroconversion rate, Omicron infection in children results in lower antibody levels and function compared with Wuhan or Delta infection or with vaccinated children with breakthrough Omicron infection. Our data have important implications for public health measures and vaccination strategies to protect children.
Estimation of the Total Number of Infected Cases in the 5th Wave of COVID-19 in Hong Kong
Authors: Chow W.K. , Chow C.L. , Cheng C.H.
Published: August 2022

The total number of infected cases in a region in an epidemic is an important measure of the severity of the disease. With the increase in the number of infected people, the number of susceptible people will be reduced, and the recovery number is increased. The present study attempts to estimate the total number of infected cases in the 5th wave of COVID-19 in Hong Kong based on the daily additional cases supplied by the government based on two test schemes. Scheme 1 covers citizens suspected to be infected as referred by medical professionals, or requested or reported by citizens themselves, those returning from overseas, and those in close contact with the infected persons. Scheme 2 covers residents in buildings with a high concentration of virus in sewage. Polymerase chain reaction (PCR) test and then rapid antigen test (RAT) after 26 February 2022 were accepted by the Hong Kong Special Administrative Government in confirming infected cases. The number of infected cases in these two schemes were compared.

A prediction model on infection case was proposed based on the transient daily infection curves. The averaged recovery number was estimated by assuming a 10-day infection period, including an incubation period of 5 days, and another 5 days for recovery. The transient number of infected, susceptible, recovered people were then presented. An adjustment factor to extend the scenario to the whole population of 7 million in Hong Kong was estimated and applied to study infection number in Hong Kong. Further, it appears that the infection number at the later stage of the 5th wave is weak around end July 2022. However, the number stayed at a constant value in comparing with rapid rise at the early stage in February 2022, even though the gathering activities were kept normal.
The ZNF687 mutation of Paget’s disease associated with giant cell tumour causes severe bone remodelling alteration as a result of a deregulated osteoclast transcriptional program
Authors: Russo Sharon , di Carlo Federica Scotto , Fortunato Giorgio , Maurizi Antonio , Teti Anna , Licastro Danilo , Settembre Carmine , Gianfrancesco Fernando
Published: August 2022

Paget’s disease (PDB) is a late-onset bone remodelling disorder with a broad spectrum of symptoms and complications. One of the most aggressive forms is caused by the P937R mutation in theZNF687gene. Although the genetic involvement ofZNF687in PDB has been extensively studied, the molecular mechanisms underlying this association remains unclear. Here, we describe the firstZfp687knock-in mouse model and demonstrate that the mutation recapitulates the PDB phenotype, showing a severe bone remodelling alteration. Through micro-computed tomography analysis, we observed that 8-month-old mice showed a mainly osteolytic phase, with a significant decrease in the trabecular bone volume affecting the femurs and the vertebrae of both heterozygous and homozygous mutant mice. In contrast, osteoblast activity was deregulated, beginning to produce disorganised bone. Noteworthy, this phenotype became pervasive in 16-month-old mice, where osteoblast function overtook bone resorption as the predominant event, as highlighted by the presence of woven bone in histological analyses, consistent with the PDB phenotype. Furthermore, we detected osteophytes and intervertebral disc degeneration, outlining for the first time the link between osteoarthritis and PDB in a PDB mouse model. Finally, we generated CRISPR-Cas9-basedZfp687knock-out RAW 264.7 cells, and noted a remarkable impairment of osteoclast differentiation capacity, reinforcing the relevance of Zfp687 during this process. RNA-sequencing on wild type and KO clones identified a set of genes involved in osteoclastogenesis under the control of Zfp687, i.e.,Tspan7, Cpe, Vegfc, andGgt1. Thus, this study established an essential role of Zfp687 in the regulation of bone remodelling, and may offer the potential to therapeutically treat PDB.
Cell surface excess is essential for protrusions and motility in 3D matrix
Authors: Kapustina Maryna , Li Donna , Zhu James , Wall Brittany , Weinreb Violetta , Cheney Richard E.
Published: August 2022

To facilitate rapid changes in morphology without endangering cell integrity, each cell possesses a substantial amount of cell surface excess (CSE) that can be promptly deployed to cover cell extensions. CSE can be stored in different types of small surface projections such as filopodia, microvilli, and ridges, with rounded bleb-like projections being the most common and rapidly achieved form of storage. We demonstrate in this paper that cells migrating in 3D collagen use CSE to cover the developing protrusions. After retraction of a protrusion, the CSE this produces is stored over the cell body similar to the CSE produced by cell rounding. For the coordinated process of CSE storage and release, all cells should have specific mechanisms of regulation, and we hypothesize that microtubules (MT) play an important role in this mechanism. We show here that different effects of MT depolymerization on cell motility such as inhibiting mesenchymal motility and enhancing amoeboid, can be explained by the essential role of MT in CSE regulation and dynamics.
Metabolic adaptations underpin resistance to histone acetyltransferase inhibition
Authors: Bishop Timothy R. , Subramanian Chitra , Bilotta Eric M. , Garnar-Wortzel Leopold , Ramos Anissa R. , Zhang Yuxiang , Asiaban Joshua N. , Ott Christopher J. , Rock Charles O. , Erb Michael A.
Published: August 2022

Histone acetyltransferases (HAT) catalyze the acylation of lysine side chains and are implicated in diverse human cancers as both oncogenes and non-oncogene dependencies1. Acetyl-CoA-competitive HAT inhibitors have garnered attention as potential cancer therapeutics and the first clinical trial for this class is ongoing (NCT04606446). Despite broad enthusiasm for these targets, notably including CBP/p300 and KAT6A/B2–5, the potential mechanisms of therapeutic response and evolved drug resistance remain poorly understood. Using comparative transcriptional genomics, we found that the direct gene regulatory consequences of CBP/p300 HAT inhibition are indistinguishable in models of intrinsically hypersensitive and insensitive acute myeloid leukemia (AML). We therefore modelled acquired drug resistance using a forward genetic selection and identified dysregulation of coenzyme A (CoA) metabolism as a facile driver of resistance to HAT inhibitors. Specifically, drug resistance selected for mutations in PANK3, a pantothenate kinase that controls the rate limiting step in CoA biosynthesis6. These mutations prevent negative feedback inhibition, resulting in drastically elevated concentrations of intracellular acetyl-CoA, which directly outcompetes drug-target engagement. This not only impacts the activity of structurally diverse CBP/p300 HAT inhibitors, but also agents related to an investigational KAT6A/B inhibitor that is currently in Phase-1 clinical trials. We further validated these results using a genome-scale CRISPR/Cas9 loss-of-function genetic modifier screen, which identified additional gene-drug interactions between HAT inhibitors and the CoA biosynthetic pathway. Top hits from the screen included the phosphatase, PANK4, which negatively regulates CoA production and therefore suppresses sensitivity to HAT inhibition upon knockout7, as well as the pantothenate transporter, SLC5A68, which enhances sensitivity. Altogether, this work uncovers CoA plasticity as an unexpected but potentially class-wide liability of anti-cancer HAT inhibitors and will therefore buoy future efforts to optimize the efficacy of this new form of targeted therapy.
APE1 recruits ATRIP to ssDNA in an RPA-independent manner to promote the ATR DNA damage response
Authors: Lin Yunfeng , Li Jia , Zhao Haichao , McMahon Anne , Yan Shan
Published: August 2022

Cells have evolved the DNA damage response (DDR) pathways in response to DNA replication stress or DNA damage. In the ATR-Chk1 DDR pathway, it has been proposed that ATR is recruited to RPA-coated single-strand DNA (ssDNA) by direct ATRIP-RPA interaction. However, it remains elusive whether and how ATRIP is recruited to ssDNA in an RPA-independent manner. Here, we provide evidence that APE1 directly associates ssDNA to recruit ATRIP onto ssDNA in an RPA-independent fashion. The N-terminal motif within APE1 is required and sufficient for the APE1-ATRIP interaction in vitro and the distinct APE1-ATRIP interaction is required for ATRIP recruitment to ssDNA and the ATR-Chk1 DDR pathway activation in Xenopus egg extracts. In addition, APE1 directly associates with RPA70 and RPA32 via two distinct motifs. Taken together, our evidence identifies APE1 as a direct recruiter of ATRIP onto ssDNA independent of RPA in the activation of ATR DDR pathway.

APE1 associates with ssDNA and ATRIP directly via distinct motifs and thereby recruits ATRIP onto ssDNA independent of RPA to promote the ATR DDR.

APE1 interacts with RPA via distinct two motifs in vitro but such RPA-APE1 interaction is dispensable for ATRIP recruitment onto ssDNA.
Attachment/detachment of cortical myosin regulates cell junction exchange during cell rearrangement
Authors: Ikawa Keisuke , Ishihara Shuji , Tamori Yoichiro , Sugimura Kaoru
Published: August 2022

Epithelial cells remodel cell adhesion and change their neighbors to shape a tissue. This cell rearrangement proceeds in three steps: the shrinkage of a junction, exchange of junctions, and elongation of the newly generated junction. Herein, by combining live imaging and physical modeling, we showed that the formation of myosin-II (myo-II) cables around the cell vertices underlies the exchange of junctions. The local and transient detachment of myo-II from the cell cortex is coupled with the junction shrinkage and elongation via an interplay between the LIM domain-containing protein Jub and the tricellular junction protein M6. Furthermore, we developed a mechanical model based on the wetting theory and clarified the way by which the physical properties of myo-II cables are integrated with the junction geometry to induce the transition between the attached and detached states and support the unidirectionality of cell rearrangement. Collectively, the present study elucidates the orchestration of geometry, mechanics and signaling for exchanging junctions.