bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct lipid interactions control SARS-CoV-2 M protein conformational dynamics and virus assembly
Authors: Dutta Mandira , Dolan Kimberly A. , Amiar Souad , Bass Elijah J. , Sultana Rokaia , Voth Gregory A. , Brohawn Stephen G. , Stahelin Robert V.
Published: November 2024

M is the most abundant structural membrane protein in coronaviruses and is essential for the formation of infectious virus particles. SARS-CoV-2 M adopts two conformations, Mshortand Mlong, and regulated transition between states is hypothesized to coordinate viral assembly and budding. However, the factors that regulate M conformation and roles for each state are unknown. Here, we discover a direct M-sphingolipid interaction that controls M conformational dynamics and virus assembly. We show M binds Golgi-enriched anionic lipids including ceramide-1-phosphate (C1P). Molecular dynamics simulations show C1P interaction promotes a long to short transition and energetically stabilizes Mshort. Cryo-EM structures show C1P specifically binds Mshortat a conserved site bridging transmembrane and cytoplasmic regions. Disrupting Mshort-C1P interaction alters M subcellular localization, reduces interaction with Spike and E, and impairs subsequent virus-like particle cell entry. Together, these results show endogenous signaling lipids regulate M structure and support a model in which Mshortis stabilized in the early endomembrane system to organize other structural proteins prior to viral budding.
Increasing brain half-life of antibodies by additional binding to myelin oligodendrocyte glycoprotein, a CNS specific protein
Authors: Cuypers Marie-Lynn , Jaspers Tom , Clerckx Jarne , Leekens Simon , Cawthorne Christopher , Bormans Guy , Cleeren Frederik , Geukens Nick , Strooper Bart De , Dewilde Maarten
Published: November 2024

Background

Therapeutic antibodies for the treatment of neurological disease show great potential, but their applications are rather limited due to limited brain exposure. The most well-studied approach to enhance brain influx of protein therapeutics, is receptor-mediated transcytosis (RMT) by targeting nutrient receptors to shuttle protein therapeutics over the blood-brain barrier (BBB) along with their endogenous cargos. While higher brain exposure is achieved with RMT, the timeframe is short due to rather fast brain clearance. Therefore, we aim to increase the brain half-life of antibodies by binding to myelin oligodendrocyte glycoprotein (MOG), a CNS specific protein.

Methods

Alpaca immunization with mouse/human MOG, and subsequent phage selections and screenings for MOG binding VHHs were performed to find mouse/human cross-reactive VHHs. Their ability to increase the brain half-life of antibodies was evaluated by coupling two different MOG VHHs (low/high affinity) in a mono- and bivalent format to an anti-β-secretase 1 or anti-SarsCov2 antibody fused to an anti-transferrin receptor (TfR) VHH for active transport over the BBB. Brain pharmacokinetics and pharmacodynamics, CNS and peripheral biodistribution, and brain toxicity were evaluated after intravenous administration to balb/c mice.

Results

Additional binding to MOG increases the Cmaxand brain half-life of antibodies that are actively shuttled over the BBB. biMOGLow:monoTfR:SarsCov antibodies could be detected in brain 49 days after a single intravenous injection, which is a major improvement compared to monoTfR:SarsCov antibodies which cannot be detected in brain anymore one week post treatment. Additional MOG binding of antibodies does not affect peripheral biodistribution but alters brain distribution to white matter localization and less neuronal internalization.

Conclusions

We have discovered mouse/human/cyno cross-reactive anti-MOG VHHs which have the ability to drastically increase brain exposure of antibodies. Combining MOG and TfR binding leads to distinct PK, biodistribution, and brain exposure, differentiating it from the highly investigated TfR- shuttling. It is the first time such long brain antibody exposure is demonstrated after one single dose.

This new approach of adding a binding moiety for brain specific targets to RMT shuttling antibodies is a huge advancement for the field and paves the way for further research into brain half-life extension.
Air-Liquid Interface Model for Influenza Aerosol Exposure In Vitro
Authors: Seibert Brittany , Caceres C. Joaquin , Gay L. Claire , Shetty Nishit , Faccin Flavio Cargnin , Carnaccini Silvia , Walters Matthew , Marr Linsey C. , Lowen Anice C. , Rajao Daniela S. , Perez Daniel R.
Published: November 2024

Airborne transmission is an essential mode of infection and spread of influenza viruses among humans. However, most studies use liquid inoculum for virus infection. To better replicate natural airborne infectionsin vitro, we generated a calm-aerosol settling chamber system designed to examine the aerosol infectivity of influenza viruses in different cell types. Aerosol inoculation was characterized for multiple influenza A virus (FLUAV) subtypes, including a pandemic 2009 H1N1, a seasonal swine H3N2, and an avian H9N2 using this exposure system. While each FLUAV strain displayed high infectivity within MDCK cells via liquid inoculation, differences in infectivity were observed during airborne inoculation. This was further observed in recently developed immortalized differentiated human airway epithelial cells (BCi-NS1.1) cultured in an air-liquid interface. The airborne infectious dose 50 for each virus was based on the exposure dose per well. Our findings indicate that this system has the potential to enhance our understanding of the factors influencing influenza transmission via the airborne route. This could be invaluable for conducting risk assessments, potentially reducing the reliance on extensive and costlyin vivoanimal studies.

Importance

This study presents a significant advancement in influenza research by developing a novel in vitro system to assess aerosol infectivity, a crucial aspect of influenza transmission. The system’s ability to differentiate between mammalian-adapted and avian-adapted influenza viruses based on their aerosol infectivity offers a valuable tool for pre-screening the pandemic potential of different strains. This could potentially streamline the risk assessment process and inform public health preparedness strategies. Moreover, the system’s capacity to examine aerosol infectivity in human airway epithelial cells provides a more relevant model for studying virus-host interactions in natural airborne infections. Overall, this study provides an accessible platform for investigating aerosol infectivity, which could significantly contribute to our understanding of influenza transmission and pandemic preparedness.
Is Probiotic Supplementation During Pregnancy Safe for Healthy Populations? A Systematic Review of Preclinical Studies
Authors: Coca Mario R. , Perez-Fernandez Cristian , Ruiz-Sobremazas Diego , Morales-Navas Miguel , Colomina Teresa , López-Granero Caridad , Sanchez-Santed Fernando
Published: November 2024

Gut-brain axis and probiotics supplementation during gestational period have gained an especial attention in the field of neurodevelopmental disorders. However, the influence of prenatal probiotics in physiology and neurodevelopment of healthy organisms is widely unexplored and probiotics are commercially such as dietetic supplementations without the necessity of ensuring safety in healthy organisms, as is required for drugs. The aim is to present and discuss the various biochemical, developmental and behavioral effects that prenatal probiotics have on the offspring of healthy organisms in rodent models and assess their security during gestation. We included a total of 15 preclinical studies discarding all those that did not have an appropriate control for the probiotic-treated group. Results suggest that probiotics, particularlyLactobacillusstrains, influenced anxiety-like behaviors in mice and GABAergic system maturation, showing an apparent biphasic effect, reducing GABA functioning in early life but increasing them in later stages; and both prenatalLactobacillusandBifidobacterium, influenced microbiota and general development of healthy organisms. However, the significant variations in methodologies used across studies complicate the ability to elucidate specific conclusions related to bacterial strains or doses. Further research is imperative to ensure the safety and regulation of probiotic interventions, particularly during the gestational period.
A neural network model that generates salt concentration memory-dependent chemotaxis inCaenorhabditis elegans
Authors: Hironaka Masakatsu , Sumi Tomonari
Published: November 2024

A neuroanatomical minimal network model was revisited to elucidate the mechanism of salt concentration memory-dependent chemotaxis observed inCaenorhabditis elegans. C. elegansmemorizes the salt concentration during cultivation, manifesting a pronounced taste preference for this concentration. The right-side head sensory neuron, designated ASER, exhibits a response to a decrease in salt concentration. The basal level of glutamate transmission from ASER has been demonstrated to transiently increase and decrease when the current environmental salt concentrations are lower and higher, respectively, than that during previous cultivation. Given the sensitivity of excitatory/inhibitory glutamate receptors expressed on the postsynaptic AIY interneurons, it can be anticipated that the ASER-AIY synaptic transmission will undergo a reversal due to alterations in the basal glutamate release. The neural network model, derived with the hypothesis, reproduced the salt concentration memory-dependent preference behavior and revealed the modular neural circuit function downstream of ASE that is responsible for salt klinotaxis.
Time course of Quadriceps thickness changes over six months post-anterior cruciate ligament reconstruction: unveiling critical impairments in vastii muscles
Authors: De la Fuente Carlos , Silvestre Rony , Martinez-Valdes Eduardo , Yañez Roberto , Abat Ferran , Neira Alejandro , de Andrade André Gustavo , Carpes Felipe P
Published: November 2024

The mechanisms behind the persistent Quadriceps weakness observed after an Anterior Cruciate Ligament reconstruction (ACLr) remain debatable. We described the time course of changes in Quadriceps thickness, strength, and thigh circumference over six months following an ACLr. We also studied the causal relation between Quadriceps thickness, total thickness and strength. Quadriceps thicknesses, Quadriceps strength, and thigh circumference were measured preoperative, 3, and 6 months post-ACLr surgery in 103 patients (77 men and 26 women). Limbs and time were compared with repeated-measures ANOVA and causal relations through mediation analysis (α = 5%). From 0 to 3 months post-surgery, Quadriceps strength (p<0.05), and VI (p<0.05) and VL (p<0.001) thicknesses was reduced. From 3 to 6 months, Quadriceps strength (p<0.05), total Quadriceps thickness (p<0.001), and VI (p<0.001) and VL (p<0.05) thicknesses increased. VM and RF thicknesses increase to 3 months after ACLr (p<0.05). Quadriceps strength, circumference at 5 cm, VI, VL, VM, and total Quadriceps thickness remain lower than the contralateral limb 6 months following an ACLr (p<0.05). The VI thickness determine indirectly (p<0.001) through the total Quadriceps thickness the Quadriceps strength pattern, while the VL thickness directly determined it (p=0.034). Quadriceps thickness adapts heterogeneously, with VI and VL atrophying for up to three months, explaining the post-ACLr quadriceps weakness. By 6 months, VI does not recover its thickness proportion, and VI and VL do not return to pre-surgery conditions. Both vastii muscles primarily determine Quadriceps strength changes, eliciting the VI thickness as a key biomarker for Quadriceps weakness following ACLr.
Recommended Interventions for Enhanced Recovery After Cesarean Delivery in the United States
Authors: Sultan Pervez , Zakowski M , Joudi Kareem , Singh DJ , Gregory Kimberly D. , Lyell D , Carvalho Brendan ,   Study investigators
Published: November 2024

Background

Existing enhanced recovery after cesarean delivery (ERAC) professional society recommendations lack intersociety collaboration, multidisciplinary expert input and involvement of patient stakeholders. This initiative aimed to develop a multidisciplinary set of ERAC interventions endorsed by relevant US professional societies and patient representatives.

Methods

This American Society of Anesthesiologists (ASA) and American College of Obstetricians and Gynecologists (ACOG) led initiative received IRB approval from Stanford University. In total 19 stakeholders were invited to participate in this Delphi study, including 13 representatives from 8 US professional societies (American Society of Anesthesiologists (ASA - 1 representative), Society for Obstetric Anesthesia and Perinatology (SOAP - 2), American College of Obstetricians and Gynecologists (ACOG - 4), Society for Maternal Fetal Medicine (SMFM - 1), Society of OBGYN Hospitalists (SOGH - 1), Enhanced Recovery After Surgery Society (ERAS Society - 1), Association of Women’s Health Obstetric and Neonatal Nurses ((AWHONN -2), American Association of Nurse Anesthesiology (AANA - 1), a physical therapist, a lactation expert and 4 patient representatives from diverse backgrounds. A three-round modified Delphi approach was conducted (2 rounds of electronic questionnaires and a 3rdround of e-discussion), to produce the final set of ERAC recommendations. An initial list of 70 interventions was compiled based on a previously published systematic review of ERAC studies and professional society guidelines/recommendations (ERAS Society, SOAP, and Healthcare Canada). Consensus was obtained for the final list of interventions, with strong consensus defined as ≥70% agreement and weak consensus as 50–69% agreement.

Results

All professional societies that were approached provided content-expert stakeholders to participate, and all stakeholders completed all rounds of the Delphi process. A final set of 32 interventions (6 preoperative, 13 intraoperative and 13 postoperative) achieved strong consensus.

Conclusions

This US intersociety and multidisciplinary Delphi study resulted in a list of ERAC interventions, which should be considered when implementing ERAC within US labor and delivery units.
A Randomized Clinical Trial Comparing Visual Inspection with Acetic Acid (VIA) to Pocket Colposcopy for the Triage of HPV+ women living with HIV in Kisumu, Kenya
Authors: Dotson Mary E. , Steinberg Eliza , Santos Maria Olivia , Ambaka Jeniffer , Huchko Megan , Ramanujam Nimmi
Published: November 2024

Objective

The World Health Organization recommends a “screen, triage, treat” approach for cervical cancer screening for Women Living with HIV (WLWH) in resource-limited settings, with Human Papillomavirus (HPV) testing preferred for screening. We assessed the use of the Pocket colposcope as an adjunct tool to Visual Assessment with Acetic Acid (VIA) for the triage of HPV+ WLWH.

Methods

We carried out a randomized clinical trial across six clinics in Kisumu, Kenya between November 2022 and April 2023 (NCT04998318). WLWH who screened positive with self-collected HPV were randomized to either the VIA or Pocket arm. Exam positivity was determined by presence or absence of aceto-white epithelium (AWE). Directed biopsies were performed on AWE; if negative, two random biopsies were taken. Pathology was used to determine diagnostic accuracy. Providers and participants took brief surveys after each exam.

Findings

The rate of a positive exams was 17.3% for VIA compared to 14.3% for the Pocket. The overall rate of CIN2/3 was 15.4%, with 12.2% in the VIA Arm and is 18.8% in the Pocket Arm. Pocket and VIA performed comparably on all sensitivity, specificity and negative predictive value (NPV). For Pocket compared to VIA, Sensitivity was 26.3% vs 25.0%; specificity was 88.9% vs 84.0%; and NPV was 82.9% vs 87.1%. However, the positive predictive value (PPV) of the Pocket colposcope arm was almost a factor of two higher than that of the VIA arm (Pocket arm PPV was 375 is and that of the VIA arm was 20.6%). The Pocket Colposcope was acceptable to providers and patients for clinic-based triage of HPV positivity.

Conclusion

Provider assessment with the Pocket colposcope detected significantly more treatable disease, thereby reducing the need for overtreatment. This study indicates that the Pocket colposcope is a feasible, lower cost colposcopic device, which could facilitate biopsy-confirmation of disease, increase provider training, patient education and facilitate remote diagnosis.
Potential causal links between genetic variants inSAMM50,SUGP1,MAU2, andGATAD2Aand liver fat in individuals with normal weight
Authors: Piras Ignazio S. , Don Janith , Schork Nicholas J. , DiStefano Johanna K.
Published: November 2024

Background & Aims

Metabolic-associated steatotic liver disease (MASLD) affects individuals across a range of body weights, but little is known about the genetic determinants of this condition in individuals with normal BMI. This study aimed to identify genetic variants contributing to hepatic fat levels in normal-weight individuals.

Methods

We conducted a genome-wide association study (GWAS) using magnetic resonance imaging-proton density fat fraction (MRI-PDFF) data from 10,918 normal-weight participants (BMI < 25 kg/m²) of European ancestry from the UK Biobank. We employed a case-control (CC) design to assess hepatic steatosis and a quantitative trait (QT) design for liver fat content. Fine mapping was conducted to identify potential causal variants, while Multi-marker Analysis of GenoMic Annotation (MAGMA) and transcriptome-wide association studies (TWAS) were performed to assess gene-level associations and impute liver-specific gene expression.

Results

We identified 241 (CC-GWAS) and 418 (QT-GWAS) significant variants located predominantly in genes on chromosomes 19 and 22, includingPNPLA3,TM6SF2, andSAMM50. Fine mapping revealed three potential causal variants inSUGP1,GATAD2A, andMAU2. MAGMA analysis identified eight genes in CC-GWAS and 19 in QT-GWAS, including the novel association withRFXANK. TWAS detectedMBOAT7andSAMM50, withSAMM50being further implicated as a potential causal gene through TWAS fine mapping.

Conclusions

This study, only the second to identify associations at the genome-wide level, represents a significant step forward in understanding the genetic basis of hepatic steatosis in normal-weight individuals, identifying both novel and established genetic variants. These findings provide insights into the pathophysiology of MASLD in this understudied population, emphasizing the importance of genetic predisposition beyond obesity-related mechanisms, and provide potential targets for personalized treatment and prevention strategies.

Impact and implications

This study provides relevant insights into the genetic risk factors of MASLD in normal-weight individuals, a population often overlooked in liver disease research. By identifying both novel and established genetic variants, this work highlights the genetic predisposition to hepatic steatosis independent of obesity-related factors. The findings offer potential targets for early detection and therapeutic intervention in normal-weight individuals, which could be applied by physicians, patients, and policymakers to inform precision medicine approaches and preventive strategies. The study provides valuable insights based on a population of European ancestry, and future research can build upon these findings to explore their applicability to patients from diverse ethnic groups.
Perceived barriers and facilitators to implementing preventive therapy for tuberculosis in Malaysian prisons: Perspectives from prison personnel
Authors: Chen Phoebe , Altice Frederick , Ghoshal Divya , Ahmad Ahsan , Long Jiang , Saifi Rumana , Koh Hui Moon , Nagadevi Naga , Baharudin Mohd Norbayusri , Min Fui Esther Wong , Kamarulzaman Adeeba , Shenoi Sheela
Published: November 2024

Tuberculosis (TB) is a major health threat worldwide. TB is concentrated in prisons, where TB preventive therapy (TPT) implementation has been limited. Prisons generally, and especially in Malaysia, are overcrowded, minimally resourced, and have high prevalence of latent TB.

As prison personnel are largely responsible for TPT implementation, their perspectives were sought to identify barriers and potential solutions for implementing TPT in Malaysia’s largest prison. A focus group using nominal group technique (NGT) was conducted with 9 prison officers with experience related to TPT to generate a list and rank-order the top TPT barriers. The process was repeated to assess facilitators.

The top three barriers to implementing TPT were: 1) disruptions involved in prison activities (e.g., intra- and extra-prison transfers; 8 votes); 2) insufficient workforce (5 votes), and unanticipated inter-prison transfers (4 votes). Potential solutions included implementing care coordination protocols between officers and medical staff (8 votes), placing a medical hold until TPT completion (5 votes), and improving medication delivery logistics (4 votes). All prison officers supported a short-course TPT regimen administered once-weekly for 12 weeks (i.e. rifapentine and isoniazid (3HP)), over a daily TPT regimen for 6 months (i.e. isoniazid (26H)), due to less logistics and staff time required.

From the perspective of prison officer stakeholders, logistical constraints commonly observed in prisons were perceived to impact the implementation of TPT, yet they prioritized pragmatic solutions that align with known implementation strategies to employ to optimize TPT delivery, including developing stakeholder relationships between medical and prison personnel and changing infrastructure to address frequent transfers within and between prisons and the community.
Impact of implementation of rapid syndromic molecular diagnostics on self-reported clinical and public health practice: a qualitative study in small island health services
Authors: Pae Riinu , Millest Adam , Tirion Anna , Dryden Matthew , Lee John E. , Wight Natalie , Fraser Graham , Toplass Janice , Weston Dale
Published: November 2024

Objective

This evaluation aimed to assess the impact of implementing Biofire© filmarray rapid syndromic molecular diagnostics systems in UK Overseas Territories, which are small jurisdictions with historically limited microbiological diagnostic capacity. The diagnostic systems were installed to improve patient management and public health response.

Methods

We conducted a qualitative evaluation. Data were gathered through semi-structured interviews and a focus group with clinicians, laboratory staff, and Chief Medical Officers.

Results

The interviewees’ reported substantial improvements in diagnostic capabilities. Interviewees shared that implementation reduced test turnaround times to 1-24 hours compared to several days to weeks pre-implementation, enabling faster and more accurate clinical management and improving clinician and patient satisfaction. Reduced reliance on off-island reference laboratories and patient medical evacuations was reported, contributing to potential cost savings and increased health system resilience. Respondents found rapid diagnostics to be useful in the context of communicable disease outbreaks. However, high test cartridge costs, supply and logistics issues, and lack of or low utilisation of protocols were noted challenges.

Conclusion

The implementation of this rapid automated syndromic molecular diagnostics technology markedly enhanced diagnostic capacity in territories included in this evaluation, particularly for respiratory, bloodstream, and gastrointestinal infections. This advancement accelerated diagnosis, was seen to improve patient management and antimicrobial stewardship. Despite these benefits, challenges remain. Further research is needed to assess the long-term impact on clinical practice, health outcomes, and cost-effectiveness, particularly in the unique contexts of small islands.
How to minimize mechanical power during controlled mechanical ventilation
Authors: Fabry Ben
Published: November 2024

High intrapulmonary pressures, large tidal volumes, and elevated respiratory rates during controlled mechanical ventilation can lead to barotrauma, volutrauma, and atelectrauma. Mechanical power - defined as the product of the pressure-volume integral and respiratory rate - consolidates these three risk factors into a single, intuitive parameter. Several studies have demonstrated that higher mechanical power correlates with an increased risk of lung injury and mortality, prompting the suggestion that mechanical power should be minimized. However, under the constraint of maintaining a fixed alveolar minute ventilation and positive end-expiratory pressure (PEEP), it remains unclear how to adjust respiratory rate and tidal volume to minimize mechanical power. This study provides an analytical solution to this optimization problem. Accordingly, only the elastic component of mechanical power should be targeted for minimization. Regardless of lung elastance or resistance, or the mode and settings of the ventilator, the elastic power is minimized at a tidal volume equal to twice the anatomic dead space, or approximately 4.4 ml/kg of body weight.
Integrating Common and Rare Variants Improves Polygenic Risk Prediction Across Diverse Populations
Authors: Williams Jacob , Chen Tony , Hua Xing , Wong Wendy , Yu Kai , Kraft Peter , Li Xihao , Zhang Haoyu
Published: November 2024

Polygenic risk scores (PRS) predict complex traits by aggregating genetic effects across the genome, yet most models focus on common variants, overlooking rare variants that may contribute to hidden heritability. We developed RICE, a new PRS framework integrating both common and rare variants to improve genetic risk prediction across diverse ancestries. RICE constructs separate PRSs: for common variants, it integrates methods using ensemble learning; for rare variants, it uses gene-level testing with functional annotations and penalized regression. We evaluated RICE using simulated datasets and sequencing data from UK Biobank and All of Us, involving up to 740 million genetic variants from 361,939 individuals across diverse ancestries and 11 complex traits. In real data analysis, RICE improved predictive accuracy by an average of 25.7% compared to leading common variant PRS methods. Our findings demonstrate that incorporating rare variants significantly enhances PRS, providing a more accurate and inclusive approach to genetic risk prediction.
Early transcriptional changes in neutrophil-mediated processes following recanalization after ischemic stroke
Authors: Bui Truong An , Ma Yonglie , Jickling Glen C , Winship Ian R
Published: November 2024

Background

Ischemic stroke is a leading cause of mortality and long-term disability globally. Current recanalization therapies, including mechanical thrombectomy and thrombolysis, aim to restore blood flow in acute ischemic stroke patients. However, many patients with successful recanalization still experience poor clinical outcomes. This phenomenon, where the restoration of blood flow does not translate into functional recovery, is known as futile recanalization.

Methods

Using a murine middle cerebral occlusion (MCAO) model that mimics a large vessel occlusion with recanalization, a comprehensive microarray analysis of gene expression from blood samples collected during MCAO and after recanalization (N=44) was performed.

Results

Il1r2, Cd55, Mmp8, Cd14,andCd69were key regulatory genes expressed early after MCAO and recanalization. A comparison with gene expression data from human stroke patients and rat MCAO model (GSE16561 and GSE21136) revealedVcanas a differentially expressed gene conserved across species, marking it as a novel indicator of recanalization that was detected as early as 3 hours post-recanalization (4 hours post-MCAO) in mice and 24 hours after recanalization rats (MCAO-thrombectomy) and humans (rtPA-thrombolysis). Leukocyte and neutrophil activation pathways were significantly enriched in the mouse data and human samples from GSE16561, with greater upregulation in female subjects. The analysis identified key miRNAs regulating gene expression in response to recanalization, and NFE4 and MTF1 as crucial transcription factors regulating these processes. Based on these data, a coregulatory network underlying neutrophil activity was constructed, highlighting its central role in early recanalization responses, especially in females.

Conclusions

This study identified new genomic markers in blood associated with recanalization, and identified critical age- and sex-specific factors. By mapping a coregulatory network of interacting genes and neutrophil-related pathways, the data provides insights to inform future research and develop targeted therapies. Such therapies can improve recanalization efficacy or modulate leukocytes to reduce futile recanalization, ultimately enhancing clinical outcomes for ischemic stroke patients.

Graphical Abstract
Distant Ribose 2’-O-Methylation of 23S rRNA Helix 69 Pre-Orders the Capreomycin Drug Binding Pocket at the Ribosome Subunit Interface
Authors: Nandi Suparno , Dey Debayan , Srinivas Pooja , Dunham Christine M. , Conn Graeme L.
Published: November 2024

Loss of ribosomal RNA (rRNA) modifications incorporated by the intrinsic methyltransferase TlyA results in reduced sensitivity to tuberactinomycin antibiotics such as capreomycin. However, the mechanism by which rRNA methylation alters drug binding, particularly at the distant but functionally more important site in 23S rRNA Helix 69 (H69), is currently unknown. We determined high-resolution cryo-electron microscopy structures of theMycolicibacterium smegmatis70S ribosome with or without the two ribose 2’-O-methyl modifications incorporated by TlyA. In the unmodified ribosome, the tip of H69 adopts a more compact conformation, positioning two key nucleotides (A2137 and C2138) such that interactions with capreomycin would be lost and the binding pocket partially occluded. In contrast, methylation of 23S rRNA nucleotide C2144 results in conformational changes that propagate from the site of modification to the H69 tip, resulting in its movement away from h44, a more favorable positioning of C2138 and adoption of a more open conformation to enable capreomycin binding. Methylation of h44 also results in structural rearrangements at the H69-h44 interface that further support antibiotic binding. These structures thus reveal the effect and regulation of distant rRNA methylation on ribosome-targeting antibiotic binding.
A Proximity MAP of RAB GTPases
Authors: St-Laurent Véronique Gaudeault , Marchand Benoit , Larcher Raphaëlle , Nassari Sonya , Bourassa Francis , Moreau Mathilde , Jean Dominique , Boisvert François-Michel , Brunet Marie A. , Jean Steve
Published: November 2024

RAB GTPases are the most abundant family of small GTPases and regulate multiple aspects of membrane trafficking events, from cargo sorting to vesicle budding, transport, docking, and fusion. To regulate these processes, RABs are tightly regulated by guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs). Activated RABs recruit effector proteins that regulate trafficking. Identifying RAB-associated proteins has proven to be difficult because their association with interacting proteins is often transient. Recent advances in proximity labeling approaches that allow for the covalent labeling of neighbors of proteins of interest now permit the cataloging of proteins in the vicinity of RAB GTPases. Here, we report APEX2 proximity labeling of 23 human RABs and their neighboring proteomes. We have used bioinformatic analyses to map specific proximal proteins for an extensive array of RAB GTPases, and RAB localization can be inferred from their adjacent proteins. Focusing on specific examples, we identified a physical interaction between RAB25 and DENND6A, which affects cell migration. We also show functional relationships between RAB14 and the EARP complex, or between RAB14 and SHIP164 and its close ortholog UHRF1BP1. Our dataset provides an extensive resource to the community and helps define novel functional connections between RAB GTPases and their neighboring proteins.
R-Loop control and mitochondria genome stability require the 5’-3’ exonuclease/flap endonuclease OEX1
Authors: Schatz-Daas Déborah , Le Blevenec Anaïs , Moratti Fabio G. , Chung Kin Pan , Mercier Pierre , Iqbal Rana Khalid , Vallet Elody , Dietrich André , Bock Ralph , Weber-Lotfi Frédérique , Gualberto José M.
Published: November 2024

Maintenance of the plant organelle genomes involves factors mostly inherited from their bacterial symbiotic ancestors. In bacteria, a major player in genome maintenance is DNA Polymerase I (Pol I), which provides a 5’-3’-exonuclease/flap-endonuclease activity required for multiple replication and repair functions. In plant organelles, DNA polymerases POL1A and POL1B are evolutionarily derived from DNA Pol I but lack this domain. In Arabidopsis, OEX1 and OEX2 (Organellar Exonucleases 1 and 2) represent this missing domain and are targeted to mitochondria and chloroplasts, respectively. Anoex1mutant allele shows developmental and fertility defects that correlate with the differential segregation of mitochondrial DNA (mtDNA) subgenomes generated by recombination, suggesting that OEX1 processes replication and recombination intermediates whose accumulation results in genome instability. Alternative splicing generates two OEX1 isoforms that can differentially interact with POL1A and POL1B and variably affect mtDNA repair.

Recombinant OEX1 has 5’-3’-exonuclease and flap endonuclease activities, the latter being a key function in replication and repair. Furthermore, OEX1 has high affinity for RNA:DNA hybrids, rapidly degrading RNA in Okazaki-like structures and R-loops. Consistent with a role in suppressing R-loops,oex1plants accumulate R-loops in highly transcribed mtDNA regions. Taken together, our results show that OEX1 plays multiple important roles in the processes required to maintain mtDNA stability.
Characterizing the effect of impeller design in plant cell fermentations using CFD modeling
Authors: Manickavasagam Vidya Muthulakshmi , Anupindi Kameswararao , Bhatt Nirav , Srivastava Smita
Published: November 2024

Cultivation of plant cell cultures in conventional bioreactors designed for microbial cells often results in the decrease of biomass productivity as compared to that in shake flasks, presumably due to the imbalance between the mass transfer requirements and compromise with cell viability. Hit and trial methods are usually adapted to achieve high biomass productivity in the bioreactor. In this study, a rational approach has been developed to choose a suitable impeller forViola odoratacell suspension culture using computational fluid dynamics (CFD). A two-phase CFD model was employed to characterize the non-Newtonian fluid dynamics in a stirred tank reactor using different impeller designs, a setric, Rushton and marine impeller. The simulations were performed adopting Euler-Euler approach for the two-phase flow and dispersedκ− ε turbulence model. The numerical model was validated with good agreement with experimental determination of volumetric mass transfer coefficient. The impact of impeller design was then investigated on critical process parameters like mixing, oxygen mass transfer and shear. The developed CFD model demonstrated that setric impeller is a suitable choice forV. odoratacell cultivation offering low-shear environment at equivalent velocity magnitudes at reactor bottom with higher cell-lift capabilities which is preferable in high cell-density plant cell cultivations.
Essential role for HSP40 in asexual replication and thermotolerance of malaria parasites
Authors: Roper Brianne , Kannan Deepika , Mathews Emily S. , John Audrey R. Odom
Published: November 2024

Plasmodium falciparum,the parasite responsible for nearly all cases of severe malaria, must survive challenging environments to persist in its human host. Symptomatic malaria is characterized by periodic fevers corresponding to the 48-hour asexual reproduction ofP. falciparumin red blood cells. As a result,P. falciparumhas evolved a diverse collection of heat shock proteins to mitigate the stresses induced by temperature shifts. Among the assortment of heat shock proteins inP. falciparum, there is only one predicted canonical cytosolic J domain protein, HSP40 (PF3D7_1437900). Here, we generate a HSP40 tunable knockdown strain ofP. falciparumto investigate the biological function of HSP40 during the intraerythrocytic lifecycle. We determine that HSP40 is required for malaria parasite asexual replication and survival of febrile temperatures. Previous reports have connected proteotoxic and thermal stress responses in malaria parasites. However, we find HSP40 has a specific role in heat shock survival and does not mitigate the proteotoxic stresses induced by artemisinin or proteosome inhibition. Following HSP40 knockdown, malaria parasites have a cell cycle progression defect and reduced nuclear replication. Untargeted proteomics reveal HSP40 depletion leads to a multifaceted downregulation of DNA replication and repair pathways. Additionally, we find HSP40 knockdown sensitizes parasites to DNA replication inhibition. Overall, these studies define the specialized role of the J domain protein HSP40 in malaria parasites during the blood stages of infection.

Author Summary

Malaria parasites have evolved to survive and cause disease in humans even when they are challenged by the temperature shifts of fever. We are interested in uncovering how malaria parasites persist despite exposure to febrile temperatures. We know the parasite has a diverse collection of heat shock proteins that are important for proper folding of many proteins within cells during stress conditions. In this study, we define the role of one specific heat shock protein, HSP40, by generating a strain of the human malaria parasiteP. falciparumwhere we control the expression of HSP40 during the red blood cell stages of infection. We find that HSP40 is required for malaria parasites to replicate in red blood cells. We demonstrate HSP40 expression to be vital for malaria parasite survival of febrile temperature stress. Additionally, we determine that without HSP40, DNA replication and repair is disrupted. Our work has uncovered essential parasite biology that may be exploited for the development of new antimalarials.
A green lifetime biosensor for calcium that remains bright over its full dynamic range
Authors: van der Linden Franka H. , Thornquist Stephen C. , Beek Rick M. ter , Huijts Jelle Y. , Hink Mark A. , Gadella Theodorus W.J. , Maimon Gaby , Goedhart Joachim
Published: November 2024

Fluorescent biosensors toggle between two states and for the vast majority of biosensors one state is bright and the other state is dim. As a consequence, there is a substantial difference in the signal-to-noise ratio (SNR) for the two states. The dim state has a low signal-to-noise ratio, which is problematic when precise, quantitative measurements are needed. During the engineering of a red-shifted variant of an mTurquoise-based calcium sensor, we serendipitously generated a green-emitting sensor that shows high brightness in both the calcium-bound and -unbound state, while still showing a calcium dependent lifetime change of >1 nanosecond. This sensor, named G-Ca-FLITS, is comparable in brightness to the bright state of GCaMP3 and jGCaMP7c in mammalian cells. The calcium induced loss in fluorescence intensity is only around 30% and therefore we observe little variation in the SNR when calcium levels change. G-Ca-FLITS shows negligible sensitivity to pH in the physiological range, like its turquoise parent. Using fluorescence lifetime imaging (FLIM), we measured the calcium concentration with G-Ca-FLITS in various organelles and observed in HeLa cells transient and spatially heterogeneous calcium elevations in mitochondria. Finally, we evaluated the use of G-Ca-FLITS and its turquoise predecessor for two-photon FLIM inDrosophilabrains.

Abstract Figure
CD3, CD4, and CD8 staining, but not PD-L1, are positively correlated with overall survival in Asian breast cancer
Authors: Pan Jia-Wern , Makmur Haslina , Tai Ju-Yi , Ng Pei-Sze , Leong Herman Xin Yang , Hasan Siti Norhidayu , Yip Cheng-Har , Rajadurai Pathmanathan , Teo Soo-Hwang
Published: November 2024

Given the fundamental importance of T lymphocytes in the effectiveness of cancer immunotherapy, and potentially in the response to chemotherapy and radiotherapy, immune cell markers have been evaluated as predictive and prognostic biomarkers, with conflicting results in the different subtypes of breast cancer and in different studies. In this study, we report the association of T lymphocyte markers with overall survival in a cross-sectional cohort of Asian breast cancer patients. Formalin-fixed, paraffin-embedded tumour samples from 576 breast cancer patients from a Malaysian hospital were stained with CD3, CD4, CD8 and PD-L1 antibodies, and area-based positive staining was quantified from digitized whole slide images. At a median follow up of 68 months, we report that CD3, CD4 and CD8 scores, but not PDL1 scores, were associated with overall survival, with the strongest associations seen in triple-negative breast cancer (TNBC). CD3, CD4, and CD8 scores were also compared to genomic and transcriptomic data from previous studies, and found to have a negative association with copy number aberrations, but no association with tumour mutational burden or neoantigen load. Taken together, T lymphocyte markers may be associated with good prognosis in Asian breast cancer patients.
Dual CRISPR-Interference Strategy for Targeting Synthetic Lethal Interactions between Non-Coding RNAs in Cancer Cells
Authors: Schloßhauer Jeffrey L. , Shamloo Sama , Schamrin Katja , Imig Jochen
Published: November 2024

Long non-coding RNAs (lncRNAs) represent a vast and functionally diverse class of RNA molecules, with over 100,000 predicted in the human genome. Although lncRNAs are less conserved across species compared to protein-coding genes, they play critical roles in gene regulation, chromatin interactions, and cancer progression. Their involvement in cancer make them promising therapeutic targets. CRISPR interference (CRISPRi), utilizing catalytically inactive Cas9 fused with a transcriptional repressor such as KRAB-MeCP2, offers a precise method for targeting nuclear lncRNAs and assessing their functions. This study introduces a dual CRISPRi system using orthogonal CRISPRi technologies fromStaphylococcus aureusandStreptococcus pyogenesdCas9-KRAB, optimized for combinatorial targeting of lncRNAs in human melanoma cells. The protocol facilitates combinatorial gene knockdown or synthetic lethal screening of lncRNA pairs, providing a novel tool for cancer research. By exploring synthetic lethality between lncRNAs, this approach can help identify lncRNA interactions critical for cancer cell survival, offering new therapeutic strategies. The dual system’s functionality is demonstrated, highlighting its potential in identifying critical cancer-specific lncRNA interactions.

This study presents a dual CRISPRi system targeting long non-coding RNAs in melanoma cells. It enables combinatorial gene knockdown and synthetic lethal screening, identifying cancerspecific lncRNA interactions for potential therapeutic strategies.
Predicting Tumor Mutational Burden from UHF-Dielectrophoresis Crossover Frequency
Authors: Daverat Héloïse , Blasco Nina , Robert Sandrine , Pothier Arnaud , Rovini Amandine , Boutaud Marie , Jemfer Charlotte , Dalmay Claire , Lalloué Fabrice , Durand Karine , Naves Thomas
Published: November 2024

Tumor Mutational Burden (TMB) has emerged as a crucial biomarker to guide patient eligibility for immunotherapy. However, whole exome sequencing, the gold-standard method for TMB measurement, remains limited in accessibility due to its high costs, operational complexity, and lengthy processing times. To address these limitations, we investigated whether Ultra-High-Frequency (UHF) technology could serve as a novel approach to assess TMB by analyzing the crossover frequencies or electromagnetic signature (EMS) of cancer cells on a lab-on-a-chip biosensor, integrating microfluidics and dielectrophoresis.

In a panel of 12 cancer cell lines with varying TMB levels, we observed that EMS showed an upward shift correlating with higher TMB, particularly in solid tumor cell lines. This finding suggests a potential relationship between TMB and EMS. To further explore this hypothesis, we artificially increased mutation levels by treating cells with the highly mutagenic compound N-ethyl-N-nitrosourea (ENU). Results showed that EMS captured significant TMB variations in ENU-treated cells with enhanced proliferative capacity compared to their parental counterparts. These results underscore the importance of matched control samples for reliable EMS measurements. Altogether, our findings highlight the potential of EMS to detect TMB variations associated with proliferative activity, a key hallmark of cancer cells, thereby enabling a more precise stratification of cancer cells.

Highlights

We propose a new biosensor to improve patient stratification for ICI eligibility

High frequency fields and dielectric spectroscopy can estimate TMB in cancer cells

Significant changes in UHF-DEP signatures correlate with varying TMB levels

UHF-DEP provides a cheap, rapid and label-free method to predict ICI response

Offers an innovative and complementary marker to conventional diagnostic approaches

Graphical abstract
Chemogenetic manipulation of the ventral pallidum-nucleus accumbens shell pathway modulates sucrose consumption independent of motivation in female rats
Authors: Peroutka Markie , Rivero Covelo Ignacio
Published: November 2024

Here we investigated the role of the ventral pallidum (VP) to nucleus accumbens shell (AcbSh) pathway in mediating sucrose consumption and motivation for food in female rats. Using chemogenetic techniques, excitatory and inhibitory designer receptors were expressed in VP neurons projecting to the AcbSh. Behavioral assays assessed both lever pressing under a progressive ratio schedule and free sucrose consumption following activation or inhibition of this pathway. Results showed that inhibition of VP-AcbSh projections increased sucrose intake, while excitation reduced it, without affecting motivation to work for food. These findings indicate a specific influence of the VP-AcbSh pathway on sucrose consumption independent of food-seeking motivation. The study highlights the importance of differentiating VP efferents in understanding distinct roles in feeding behaviors and suggests that the VP-AcbSh pathway modulates consumption based on food type and potentially other variables.
Vcfexpress: flexible, rapid user-expressions to filter and format VCFs
Authors: Pedersen Brent S. , Quinlan Aaron R.
Published: November 2024

Motivation

Variant Call Format (VCF) files are the standard output format for various software tools that identify genetic variation from DNA sequencing experiments. Downstream analyses require the ability to query, filter, and modify them simply and efficiently. Several tools are available to perform these operations from the command line, including BCFTools, vembrane, slivar, and others.

Results

Here, we introduce vcfexpress, a new, high-performance toolset for the analysis of VCF files, written in the Rust programming language. It is nearly as fast as BCFTools, but adds functionality to execute user expressions in the lua programming language for precise filtering and reporting of variants from a VCF or BCF file. We demonstrate performance and flexibility by comparing vcfexpress to other tools using the vembrane benchmark.

Availability

vcfexpress is available under the MIT license athttps://github.com/brentp/vcfexpress

Contact

bpederse@gmail.com,aquinlan@genetics.utah.edu

Supplementary information

Supplementary data are available atBioinformaticsonline.