bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant dimeric PDZ protein inhibitors for long-term relief of chronic pain by AAV therapeutics
Authors: Noes-Holt Gith , Jensen Kathrine L. , Richner Mette , Comaposada-Baro Raquel , Sivertsen Line , Jager Sara E. , Jiménez-Fernández Lucía , Andersen Rita C. , Lilja Jamila H. , Larsen Andreas H. , Kowenicki Marco B. K. , Boesgaard Sofie P. , Houser Grace A. , Christensen Nikolaj R. , Tappe-Theodor Anke , Vægter Christian B. , Kuner Rohini , Madsen Kenneth L. , Sørensen Andreas T.
Published: August 2024

The inadequate state of current pain treatments, the chronic nature of particularly neuropathic pain, and the high impact on quality of life render chronic pain conditions relevant for gene therapy. Here, we describe the development and application of self-assembling dimeric peptide inhibitors of the pain-associated scaffolding protein PICK1 (protein interacting with C-kinase 1) delivered by adeno-associated viral (AAV) vectors. In mice, these peptides prevented mechanical allodynia in inflammatory and neuropathic pain models and reversed neuropathic pain in advanced stages up to one year. Pain relief was obtained by targeting several relays along the somatosensory pain pathways unaccompanied by overt adverse side effects, while selective transduction of peripheral neurons was sufficient for providing full pain relief. We further confirmed PICK1 expression and peptide target engagement in mice and human donor tissue, and we conclude that AAV therapeutics, based on recombinant PICK1 inhibitors, represent a potential clinically meaningful strategy for persistent neuropathic pain conditions.

One Sentence Summary

Alleviating neuropathic pain by PICK1-directed gene therapy.
A Novel Methodology to Recalibrate Pathogenic Range of SCA36 Repeat Expansions for PGT-M
Authors: Liu Fulin , Huang Wen , Liao Ling , Yang Jiyun
Published: August 2024

Background

Spinocerebellar ataxia-36 (SCA36) is an inherited neurodegenerative disorder caused by the heterozygous expansion of an intronic GGCCTG hexanucleotide repeat in the NOP56 gene on chromosome 20p13. Unaffected individuals typically carry 3 to 14 repeats, whereas affected individuals carry 650 to 2,500. However, based on a single study, this pathogenic range was conservatively established, limiting its extended clinical applicability such as preimplantation genetic testing (PGT). In this study, we propose a novel methodology to recalibrate the pathogenic range of SCA36 repeat expansion.

Methods

We conducted a comprehensive literature review and collected examination data from 2012 onward. We used the gamma distribution to describe the data distribution and applied Bayesian methods to update the prior distribution with data from recent publications. Based on the recalibrated distribution, the 95% confidence interval (CI) was used to determine the new lower boundary of the pathogenic range. A pedigree was collected to validate the proposal with long-read sequencing (LRS) applied to detect the high GC content and long length of repeat expansions.

Results

Our results, based on 2 studies, indicate that the data distribution is well-described by gamma distribution. The prior, likelihood and posterior distributions within the 95% CI for the integrated research of SCA36 pathogenic repeat expansions were [446, +∞), [124, +∞), and [484, +∞), respectively. These recalibrated pathogenic ranges were validated by an authentic case: a proband diagnosed with SCA36 carrying 418 repeats and her daughter with 499 repeats, under the detection of LRS.

Conclusions

Therefore, we proposed a novel methodology that integrates updated data, 95% CI using Bayesian methods and LRS for accurate detection of repeat expansions of dynamic mutations to present an up-to-date pathogenic range of SCA36, as well as other similar diseases.
Unsupervised Dimensionality Reduction Techniques for the Assessment of ASD Biomarkers
Authors: Jacokes Zachary , Adoremos Ian , Hussain Arham Rameez , Newman Benjamin T. , Pelphrey Kevin A. , Van Horn John Darrell ,   the ACE GENDAAR Consortium
Published: August 2024

Autism Spectrum Disorder (ASD) encompasses a range of developmental disabilities marked by differences in social functioning, cognition, and behavior. Both genetic and environmental factors are known to contribute to ASD, yet the exact etiological factors remain unclear. Developing integrative models to explore the effects of gene expression on behavioral and cognitive traits attributed to ASD can uncover environmental and genetic interactions. A notable aspect of ASD research is the sex-wise diagnostic disparity: males are diagnosed more frequently than females, which suggests potential sex-specific biological influences. Investigating neuronal microstructure, particularly axonal conduction velocity offers insights into the neural basis of ASD. Developing robust models that evaluate the vast multidimensional datasets generated from genetic and microstructural processing poses significant challenges. Traditional feature selection techniques have limitations; thus, this research aims to integrate principal component analysis (PCA) with supervised machine learning algorithms to navigate the complex data space. By leveraging various neuroimaging techniques and transcriptomics data analysis methods, this methodology seeks to contextualize the complex genetic and phenotypic heterogeneity linked to sex differences in ASD and pave the way for tailored interventions.
Continuous bedside neuroimaging using high-density diffuse optical tomography in a pediatric patient on extracorporeal support
Authors: McMorrow Sophia R. , Park Sung Min , George Tessa G. , Sobolewski Chloe M. , Yang Dalin , King Kelsey T. , Kenley Jeanette , Smyser Christopher D. , Culver Joseph P. , Guilliams Kristin P. , Said Ahmed S. , Eggebrecht Adam T.
Published: August 2024

Background

Extracorporeal membrane oxygenation (ECMO) provides life support for severe, reversible cardiac or respiratory failure, yet is associated with significant neurological risks including stroke. Currently available neuroimaging methods have limited sensitivity or specificity to detect early brain injury, have little real-time ability to assess interventions, and/or pose additional risks. Here, we present a case study of high-density diffuse optical tomography (HD-DOT) for bedside neuroimaging in pediatric ECMO.

Methods

A young infant supported on ECMO following cardiac surgery underwent continuous HD-DOT imaging over a two-hour duration that included baseline support and a clamp trial to test the ability to separate from ECMO. After stringent data quality assessments, we estimated cortical parcel-based brain functional connectivity (FC), evaluated spatial correlations between neighboring temporal epochs throughout the recording to evaluate test-retest reliability of brain FC, and calculated paired t-tests between the brain-wide set of test-retest values to test for significant changes in brain FC.

Results

High-fidelity bedside HD-DOT data were acquired without disruptions to patient care. During the baseline period, we observed strong test-retest with consistent bilateral FC patterns. Significant disruptions in cortical FC reflected concurrent changes in cerebral blood flow during the clamp trial and persisted after ECMO resumed.

Conclusions

Our results demonstrate the feasibility of continuous bedside HD-DOT neuroimaging in pediatric ECMO. HD-DOT can potentially provide clinically relevant information on cortical FC during ECMO support.
Automated Coronary Artery Calcium Scoring Using Convolutional Neural Networks: Enhancing Cardiovascular Risk Assessment in Chest CT Scans
Authors: Mansoor Masab A. , Grindem David J.
Published: August 2024

Background

Coronary artery calcium (CAC) scoring is valuable for cardiovascular risk assessment but often time-consuming and subject to variability. This study aimed to develop and validate a convolutional neural network (CNN) model for automated CAC scoring in chest CT scans, potentially enhancing efficiency and accuracy.

Methods

We utilized 10,000 chest CT scans from a public dataset, split into training (n=7,000), validation (n=1,500), and testing (n=1,500) sets. A 3D CNN model based on ResNet-50 was developed and trained for CAC detection and quantification. Performance was evaluated on the test set and compared to manual scoring by three experienced radiologists.

Results

The CNN model achieved 93.7% accuracy in detecting CAC, with 87.4% sensitivity and 92.1% specificity for identifying clinically significant CAC (Agatston score >100) in the test set (n=1,500). The model showed strong correlation with manual CAC scores (r=0.89, p<0.001). Automated scoring reduced processing time by 78% compared to manual techniques, averaging 18.3 seconds per scan. The model demonstrated consistent performance across diverse patient demographics and CT types. In a subset of patients with follow-up data (n=500), the model’s risk stratification was comparable to the Framingham Risk Score in predicting cardiovascular events (AUC 0.76 vs 0.74, p=0.09).

Conclusions

The CNN-based automated CAC scoring system demonstrated high accuracy and efficiency, potentially enabling more widespread cardiovascular risk assessment in routine chest CT scans. Future research should focus on prospective validation and investigation of long-term patient outcomes when integrating this technology into clinical practice.
Surgical window of opportunity trial reveals mechanisms of response and resistance to navtemadlin (KRT-232) in patients with recurrent glioblastoma
Authors: Rendo Veronica , Lee Eudocia Q. , Bossi Connor , Khuu Nicholas , Rudek Michelle A. , Pal Sangita , Reynolds Abigail R. N. , Fassinou Auriole C.R. , Ayoub Georges , Lapinskas Emily , Pisano William , Jeang John , Stopka Sylwia A. , Regan Michael S. , Spetz Johan , Desai Arati , Lieberman Frank , Fisher Joy D. , Pelton Kristine , Huang Raymond Y. , Nabors Louis B. , Holdhoff Matthias , Danda Neeraja , Strowd Roy , Desideri Serena , Walbert Tobias , Ye Xiaobu , Agar Nathalie Y. R. , Grossman Stuart A. , Alexander Brian M. , Wen Patrick Y. , Ligon Keith L. , Beroukhim Rameen
Published: August 2024

We investigated the effectiveness of navtemadlin (KRT-232) in treating recurrent glioblastoma. A surgical window-of-opportunity trial (NCT03107780) was conducted on 21 patients to determine achievable drug concentrations within tumor tissue and examine mechanisms of response and resistance. Both 120 mg and 240 mg daily dosing achieved a pharmacodynamic impact. Sequencing of three recurrent tumors revealed an absence ofTP53-inactivating mutations, indicating alternative mechanisms of resistance. In patient-derived GBM models, the lower range of clinically achieved navtemadlin concentrations induced partial tumor cell death as monotherapy. However, combining navtemadlin with temozolomide increased apoptotic rates while sparing normal bone marrow cells in vitro, which in return underwent reversible growth arrest. These results indicate that clinically achievable doses of navtemadlin generate significant pharmacodynamic effects and suggest that combined treatment with standard-of-care DNA damaging chemotherapy is a route to durable survival benefits.

Statement of significance

Tissue sampling during this clinical trial allowed us to assess mechanisms of response and resistance associated with navtemadlin treatment in recurrent GBM. We report that clinically achievable doses of navtemadlin induce pharmacodynamic effects in tumor tissue, and suggest combinations with standard-of-care chemotherapy for durable clinical benefit.
Pemivibart is less active against recent SARS-CoV-2 JN.1 sublineages
Authors: Wang Qian , Guo Yicheng , Ho Jerren , Ho David D.
Published: August 2024

Protection from COVID-19 vaccination is suboptimal in many immunocompromised individuals. In March 2024, the Food and Drug Administration issued an Emergency Use Authorization for pemivibart (Permagard/VYD222), an engineered human monoclonal antibody, for pre-exposure prophylaxis in this vulnerable population. However, SARS-CoV-2 has since evolved extensively, resulting in multiple Omicron JN.1 sublineages. We therefore evaluated the in vitro neutralizing activity of pemivibart against the prevalent forms of JN.1, including KP.2, KP.3, KP.2.3, LB.1, and, importantly, KP.3.1.1, which is now expanding most rapidly. A panel of VSV-based pseudoviruses representing major JN.1 sublineages was generated to assess their susceptibility to pemivibart neutralization in vitro. Structural analyses were then conducted to understand the impact of specific spike mutations on the virus-neutralization results. Pemivibart neutralized both JN.1 and KP.2 in vitro with comparable activity, whereas its potency was decreased slightly against LB.1, KP.2.3, and KP.3 but substantially against KP.3.1.1. Critically, the 50% inhibitory concentration of pemivibart against KP.3.1.1 was ∼6 µg/mL, or ∼32.7 fold higher than that of JN.1 in our study. Structural analyses suggest that Q493E and the S31-deletion mutations in viral spike contribute to the antibody evasion, with the latter having a more pronounced effect. Our findings show that pemivibart has lost substantial neutralizing activity in vitro against KP.3.1.1, the most rapidly expanding lineage of SARS-CoV-2 today. Close monitoring of its clinical efficacy is therefore warranted. These results also highlight the imperative to expand our arsenal of preventive agents to protect millions of immunocompromised individuals who could not respond robustly to COVID-19 vaccines.
Mechanism that regulates the mutual inhibition controls functional switch of UNC-13
Authors: Liu Haowen , Li Lei , Wang Jiafan , Hu Jiayi , Xia Jingyao , Ma Cong , Kang Lijun , Hu Zhitao
Published: August 2024

Munc13 plays a crucial role in short-term synaptic plasticity by regulating synaptic vesicle (SV) exocytosis and neurotransmitter release at the presynaptic terminals. However, the intricate mechanisms governing these processes have remained elusive due to the presence of multiple functional domains within Munc13, each playing distinct roles in neurotransmitter release. Here we report a coordinated mechanism in theC. elegansMunc13 homolog UNC-13 that controls the functional switch of UNC-13 during synaptic transmission. Mutations disrupting the interactions of C1 and C2B with diacylglycerol (DAG) and phosphatidylinositol 4,5-bisphosphate (PIP2) on the plasma membrane induced the gain-of-function state of UNC-13L, the long UNC-13 isoform, resulting in enhanced SV release. Concurrent mutations in both domains counteracted this enhancement, highlighting the functional interdependence of C1 and C2B. Intriguingly, the individual C1 and C2B domains exhibited significantly stronger facilitation of SV release compared to the presence of both domains, supporting a mutual inhibition of C1 and C2B under basal conditions. Moreover, the N-terminal C2A and X domains exhibited opposite regulation on the functional switch of UNC-13L. Furthermore, we identified the polybasic motif in the C2B domain that facilitates SV release. Finally, we found that disruption of C1 and C2B membrane interaction in UNC-13S, the short isoform, leads to functional switch between gain-of-function and loss-of-function. Collectively, our findings provide a novel mechanism for SV exocytosis wherein UNC-13 undergoes functional switches through the coordination of its major domains, thereby regulating synaptic transmission and short-term synaptic plasticity.
Inference technique for the synaptic conductances in rhythmically active networks and application to respiratory central pattern generation circuits
Authors: Molkov Yaroslav I. , Borgmann Anke , Koizumi Hidehiko , Hama Noriyuki , Zhang Ruli , Smith Jeffrey C.
Published: August 2024

Unraveling synaptic interactions between excitatory and inhibitory interneurons within rhythmic neural circuits, such as central pattern generation (CPG) circuits for rhythmic motor behaviors, is critical for deciphering circuit interactions and functional architecture, which is a major problem for understanding how neural circuits operate. Here we present a general method for extracting and separating patterns of inhibitory and excitatory synaptic conductances at high temporal resolution from single neuronal intracellular recordings in rhythmically active networks. These post-synaptic conductances reflect the combined synaptic inputs from the key interacting neuronal populations and can reveal the functional connectome of the active circuits. To illustrate the applicability of our analytic technique, we employ our method to infer the synaptic conductance profiles in identified rhythmically active interneurons within key microcircuits of the mammalian (mature rat) brainstem respiratory CPG and provide a perspective on how our approach can resolve the functional interactions and circuit organization of these interneuron populations. We demonstrate the versatility of our approach, which can be applied to any other rhythmic circuits where conditions allow for neuronal intracellular recordings.
Estimating the burden of mpox among MSM in South Africa
Authors: McCabe Ruth , Johnson Leigh F. , Whittles Lilith
Published: August 2024

Despite seeing few cases during the 2022-23 mpox global outbreak, recent reports of 22 cases among men-who-have-sex-with-men (MSM) in South Africa, including three deaths, have raised concerns about underreported community transmission. We used a Monte Carlo simulation model to estimate the true epidemic size, considering increased severity of mpox among MSM living with advanced HIV (MSMLAHIV), documented overrepresentation of people living with HIV (PLHIV) among mpox cases, and HIV prevalence in South Africa. We estimate there have been between 220-450 cases among MSM LHIV in South Africa, implying a total of 290-560 cases among all MSM. We provide an upper bound of 750-1,600 cases as a sensitivity analysis where the prevalence of HIV among mpox patients is the same as population prevalence among MSM in South Africa. Estimates in both scenarios suggest a substantial number of undetected cases, with case ascertainment rates estimated between 1% and 8%. Our findings underscore the need for enhanced surveillance, targeted public health interventions, and awareness campaigns to mitigate the outbreak’s impact at a population-level.
Preoperative Exposure to Fine Particulate Matter and Risk of Postoperative Complications: A Single Center Observational Cohort Bayesian Analysis
Authors: Pearson John F. , Jacobson Cameron K. , Riss Calvin S. , Strickland Matthew J. , Lee Longyin , Wan Neng , Benney Tabitha M. , Pace Nathan L. , Goodrich Ben K. , Gabry Jonah S. , Pham John V. , Kartchner Cade K. , Wood Jake S. , Andreae Michael H.
Published: August 2024

Background

While exposure to fine particulate matter air pollution (PM2.5) is known to cause adverse health effects, its impact on postoperative outcomes in US adults remains understudied. Perioperative exposure to PM2.5may induce inflammation that interacts insidiously with the surgical stress response, leading to higher postoperative complications.

Methods

We conducted a single center, retrospective cohort study using data from 49,615 surgical patients living along Utah’s Wasatch Front and who underwent elective surgical procedures at a single academic medical center from 2016-2018. Patients’ addresses were geocoded and linked to daily Census-tract level PM2.5estimates. We hypothesized that elevated PM2.5concentrations in the week prior to surgery would be associated with an increase in a bundle of major postoperative complications. A hierarchical Bayesians regression model was fit adjusting for age, sex, season, neighborhood disadvantage, and the Elixhauser index of comorbidities.

Results

Postoperative complications increased in a dose-dependent manner with higher concentrations of PM2.5exposure, with a relative increase of 8% in the odds of complications (OR=1.082) for every 10ug/m3increase in the highest single-day 24-hr PM2.5exposure during the 7 days prior to surgery. For a 30 fold increase in PM2.5(1 ug/m3to 30ug/m3) the odds of complication rose to over 27% (95%CI: 4%-55%). The association persisted after controlling for comorbidities and confounders; our inferences were robust to modeling choices and sensitivity analysis.

Conclusions

In this large Utah cohort, exposure to elevated PM2.5concentrations in the week before surgery was associated with a dose-dependent increase in postoperative complications, suggesting a potential impact of air pollution on surgical outcomes. These findings merit replication in larger datasets to identify populations at risk and define the interaction and impact of different pollutants. PM2.5exposure is a potential perioperative risk factor and, given the unmitigated air pollution in urban areas, a global health concern.
Characterising grey-white matter relationships in recent-onset psychosis and its association with cognitive function
Authors: Saito Yoshito , Pantelis Christos , Cropley Vanessa , Laskaris Liliana , Wannan Cassandra M. J. , Syeda Warda T.
Published: August 2024

Individuals with recent-onset psychosis (ROP) present widespread grey matter (GM) reductions and white matter (WM) abnormalities. However, relationships between GM and WM changes and their association with cognitive impairment, a key symptom of ROP, are unclear. Using a multiblock partial least squares correlation (MB-PLS-C) analysis, we examined multivariate GM-WM relationships and their association with cognitive abilities in ROP. We used T1 and diffusion-weighted images from 71 non-affective ROP individuals (age 22.1±3.2) and 71 matched controls. We performed MB-PLS-C between GM thickness and WM fractional anisotropy (FA) and between GM surface area and WM FA to identify multivariate GM-WM patterns and analysed correlations between these patterns and cognitive abilities. MB-PLS-C identified a ‘GM thickness’-‘WM FA’ pattern representing group differences, explaining 12.38% of the variance and associated with frontal and temporal GM regions and seven WM tracts, including the corticospinal tract. MB-PLS-C also identified a ‘GM surface area’-‘WM FA’ pattern showing group differences, explaining 18.92% and related with cingulate, frontal, temporal, and parietal GM regions and 15 WM tracts, including the inferior cerebellar peduncle. The ‘GM thickness’-‘WM FA’ pattern describing group differences was significantly correlated with processing speed in ROP. There was no association between cognition and the ‘GM surface area’-‘WM FA’ pattern. MB-PLS-C identified differential whole-brain GM-WM relationships, indicating a potential signature of brain alterations in ROP. Our findings of a relationship between cognitive function and GM-WM patterns for GM thickness rather than for surface area have implications for our understanding of brain-behaviour relationships neurodevelopmentally in psychosis.
Opto-CLIP reveals dynamic FMRP regulation of mRNAs upon CA1 neuronal activation
Authors: Singer Ruth A. , Rajchin Veronika , Park Kwanghoon , Heintz Nathaniel , Darnell Robert B.
Published: August 2024

Neuronal diversity and function are intricately linked to the dynamic regulation of RNA metabolism. Electrophysiologic studies of synaptic plasticity, models for learning and memory, are disrupted in Fragile X Syndrome (FXS). FXS is characterized by the loss of FMRP, an RNA-binding protein (RBP) known to suppress translation of specific neuronal RNAs. Synaptic plasticity in CA1 excitatory hippocampal neurons is protein-synthesis dependent, suggesting a role for FMRP in FXS-related synaptic deficits. To explore this model, we developed Opto-CLIP, integrating optogenetics with cell-type specific FMRP-CLIP and RiboTag in CA1 neurons, allowing investigation of activity-induced FMRP regulation. We tracked changes in FMRP binding and ribosome-associated RNA profiles 30 minutes after neuronal activation. Our findings reveal distinct temporal dynamics for FMRP transcript regulation in the cell body versus the synapse. In the cell body, FMRP binding to transcripts encoding nuclear functions is relieved, potentially allowing rapid transcriptional responses to neuronal activation. At the synapse, FMRP binding to transcripts encoding synaptic targets was relatively stable, with variability in translational control across target categories. These results offer fresh insights into the dynamic regulation of RNA by FMRP in response to neuronal activation and provide a foundation for future research into the mechanisms of RBP-mediated synaptic plasticity.
One-Pot Time-Induced Proteome Integral Solubility Alteration (OPTI-PISA) assay for automated and sensitive drug target identification
Authors: Meng Zhaowei , Saei Amir Ata , Lyu Hezheng , Gaetani Massimiliano , Zubarev Roman A.
Published: August 2024

Proteome Integral Solubility Alteration (PISA) assay is widely used for identifying drug targets, but it is labour-intensive and time-consuming, and requires a substantial amount of biological sample. Aiming at enabling automation and greatly reducing the sample amount, we developed One-Pot Time-Induced (OPTI)-PISA. Here we demonstrate OPTI-PISA performance on identifying targets of multiple drugs in cell lysate and scaling down the sample amount to sub-microgram levels, making PISA method suitable for NanoProteomics.
Increased autonomous bioluminescence emission from mammalian cells by enhanced cofactor synthesis
Authors: Brinker Theresa , Gregor Carola
Published: August 2024

The bacterial bioluminescence system has been successfully implemented in mammalian cell lines, enabling substrate-free luminescence imaging of living cells. One of the major limitations of the system is its comparatively low brightness. To improve light emission, we aimed at increasing the cellular production of FMNH2and NADPH which serve as cosubstrates in the bacterial bioluminescence reaction. We coexpressed different proteins involved in the synthesis of these two cofactors together with the proteins of the bacterial bioluminescence system in different mammalian cell lines. Combined expression of a riboflavin kinase (RFK) and a constitutively active Akt2 variant (Akt2CA) that participate in the cellular production of FMN and NADP+, respectively, increased bioluminescence emission up to 2.4–fold. The improved brightness allows autonomous bioluminescence imaging of mammalian cells at a higher signal-to-noise ratio and enhanced spatiotemporal resolution.
A Chemoenzymatic Strategy for Site-Specific Glyco-Tagging of Native Proteins for the Development of Biologicals
Authors: Gimeno Ana , Ehlers Anna M. , Delgado Sandra , Langenbach Jan-Willem H. , van den Bos Leendert J. , Kruijtzer John A.W. , Guigas Bruno G.A. , Boons Geert-Jan
Published: August 2024

Glycosylation is an attractive approach to enhance biological properties of pharmaceutical proteins, however, precise installation of glycans for structure-function studies remains challenging. Here, we describe a chemoenzymatic methodology for glyco-tagging of proteins by peptidoligase catalyzed modification of theN-terminus of a protein with a synthetic glycopeptide ester having anN-acetyl-glucosamine (GlcNAc) moiety to generate aN-GlcNAc modified protein. The GlcNAc moiety can be elaborated into complex glycans bytrans-glycosylation using a well-defined sugar oxazolines and mutant forms of endo β-N-acetylglucosaminidases (ENGases). The glyco-tagging methodology makes it possible to modifyon-demandtherapeutic proteins, including proteins heterologously expressed inE. coli,with diverse glycan structures. As a proof of principle, theN-terminus of interleukin (IL)-18 and interferon (IFN)α-2a was modified by a glycopeptide harboring a complexN-glycan without compromising biological potencies. The glyco-tagging methodology was also used to prepare several glycosylated insulin variants that exhibit reduced oligomerization, aggregation and fibrillization yet maintained cell signaling properties, which is attractive for the development of insulins with improved shelf-lives. It was found that by employing different peptidoligases, it is possible to modify either the A or both chains of human insulin.
Examining the Biophysical Properties of the Inner Membrane of Gram-Negative ESKAPE Pathogens
Authors: Gazerani Golbarg , Piercey Lesley R. , Reema Syeda , Wilson Katie A.
Published: August 2024

The World Health Organization has identified multidrug-resistant bacteria as a serious global health threat. Gram-negative bacteria are particularly prone to antibiotic resistance, and their high rate of antibiotic resistance has been suggested to be related to the complex structure of their cell membrane. The outer membrane of Gram-negative bacteria contains lipopolysaccharides that protect the bacteria against threats such as antibiotics, while the inner membrane houses 20-30% of the bacterial cellular proteins. Given the cell membrane’s critical role in bacterial survival, antibiotics targeting the cell membrane have been proposed to combat bacterial infections. However, a deeper understanding of the biophysical properties of the bacterial cell membrane is crucial for developing effective and specific antibiotics. In this study, Martini coarse-grain molecular dynamics simulations were used to investigate the interplay between membrane composition and biophysical properties of the inner membrane across four pathogenic bacterial species:Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae, andEscherichia coli. The simulations indicate the impact of species-specific membrane composition on dictating the overall membrane properties. Specifically, cardiolipin concentration in the inner membrane is a key factor influencing the membrane features. Model membranes with varying concentrations of bacterial lipids (phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin) further support the significant role of cardiolipin in determining the membrane biophysical properties. The bacterial inner membrane models developed in this work pave the way for future simulations of bacterial membrane proteins and for simulations investigating novel strategies aimed at disrupting the bacterial membrane to treat antibiotic-resistant infections.
The pro-tumoral and anti-tumoral roles of EphA4 on T regulatory cells and tumor associated macrophages during HNSCC tumor progression
Authors: Corbo Sophia , Nguyen Diemmy , Bhatia Shilpa , Darragh Laurel B. , Abdelazeem Khalid N.M. , Court Benjamin Van , Olimpo Nicholas A. , Gadwa Jacob , Yu Justin , Hodgson Chloe , Samedi Von , Garcia Enrique Sanz , Siu Lillian , Saviola Anthony , Heasley Lynn E. , Knitz Michael W. , Pasquale Elena B. , Karam Sana D.
Published: August 2024

Head and Neck Squamous Cell Carcinoma (HNSCC) is a deadly cancer with poor response to targeted therapy, largely driven by an immunosuppressive tumor microenvironment (TME). Here we examine the immune-modulatory role of the receptor tyrosine kinase EphA4 in HNSCC progression. Within the TME, EphA4 is primarily expressed on regulatory T cells (Tregs) and macrophages. In contrast ephrinB2, an activating ligand of EphA4, is expressed in tumor blood vessels. Using genetically engineered mouse models, we show that EphA4 expressed in Tregs promotes tumor growth, whereas EphA4 expressed in monocytes inhibits tumor growth. In contrast, ephrinB2 knockout in blood vessels reduces both intratumoral Tregs and macrophages. A novel specific EphA4 inhibitor, APY-d3-PEG4, reverses the accelerated tumor growth we had previously reported with EphB4 cancer cell knockout. EphA4 knockout in macrophages not only enhanced their differentiation into M2 macrophage but also increased Treg suppressive activity. APY-d3-PEG4 reversed the accelerated growth seen in the EphA4 knockout of monocytes but conferred no additional benefit when EphA4 was knocked out on Tregs. Underscoring an EphA4-mediated interplay between Tregs and macrophages, we found that knockout of EphA4 in Tregs not only decreases their activation but also reduces tumor infiltration of pro-tumoral M2 macrophages. These data identify Tregs as a primary target of APY-d3-PEG4 and suggest a role for Tregs in regulating macrophage conversion. These data also support the possible anti-cancer therapeutic value of bispecific peptides or antibodies capable of promoting EphA4 blockade in Tregs but not macrophages.

Significance

EphA4 in regulatory T cells has a pro-tumoral effect while EphA4 in macrophages plays an anti-tumoral role underscoring the necessity of developing biologically rational therapeutics.
One step 4x and 12x 3D-ExM: robust super-resolution microscopy in cell biology
Authors: Norman Roshan X , Chen Yu-Chia , Recchia Emma E , Loi Jonathan , Rosemarie Quincy , Lesko Sydney L , Patel Smit , Sherer Nathan , Takaku Motoki , Burkard Mark E , Suzuki Aussie
Published: August 2024

Super-resolution microscopy has become an indispensable tool across diverse research fields, offering unprecedented insights into biological architectures with nanometer scale resolution. Compared to traditional nanometer-scale imaging methods such as electron microscopy, super-resolution microscopy offers several advantages, including the simultaneous labeling of multiple target biomolecules with high specificity and simpler sample preparation, making it accessible to most researchers. In this study, we introduce two optimized methods of super-resolution imaging: 4-fold and 12-fold 3D-isotropic and preserved Expansion Microscopy (4x and 12x 3D-ExM). 3D-ExM is a straightforward expansion microscopy method featuring a single-step process, providing robust and reproducible 3D isotropic expansion for both 2D and 3D cell culture models. With standard confocal microscopy, 12x 3D-ExM achieves a lateral resolution of under 30 nm, enabling the visualization of nanoscale structures, including chromosomes, kinetochores, nuclear pore complexes, and Epstein-Barr virus particles. These results demonstrate that 3D-ExM provides cost-effective and user-friendly super-resolution microscopy, making it highly suitable for a wide range of cell biology research, including studies on cellular and chromatin architectures.
The conserved ATPase PCH-2 controls the number and distribution of crossovers by antagonizing their formation inC. elegans
Authors: Patel Bhumil , Grobler Maryke , Herrera Alberto , Logari Elias , Ortiz Valery , Bhalla Needhi
Published: August 2024

Meiotic crossover recombination is essential for both accurate chromosome segregation and the generation of new haplotypes for natural selection to act upon. This requirement is known as crossover assurance and is one example of crossover control. While the conserved role of the ATPase, PCH-2, during meiotic prophase has been enigmatic, a universal phenotype whenpch-2or its orthologs are mutated is a change in the number and distribution of meiotic crossovers. Here, we show that PCH-2 controls the number and distribution of crossovers by antagonizing their formation. This antagonism produces different effects at different stages of meiotic prophase: early in meiotic prophase, PCH-2 prevents double strand breaks from becoming crossover-eligible intermediates, limiting crossover formation at sites of initial double strand break formation and homolog interactions. Later in meiotic prophase, PCH-2 winnows the number of crossover-eligible intermediates, contributing to the designation of crossovers and ultimately, crossover assurance. We also demonstrate that PCH-2 accomplishes this regulation through the meiotic HORMAD, HIM-3. Our data strongly support a model in which PCH-2’s conserved role is to remodel meiotic HORMADs throughout meiotic prophase to destabilize crossover-eligible precursors, coordinate meiotic recombination with synapsis, and contribute to the progressive implementation of meiotic recombination, guaranteeing crossover control.
Discovery and description of gammanonin: a widely distributed natural product from Gammaproteobacteria
Authors: Costa Jonas H , Adams Eva E , Johnston Chad W
Published: August 2024

Antibiotics are essential for modern medicine, but their use drives the evolution of antimicrobial resistance (AMR) that limits the long-term efficacy of any one drug. To keep pace with AMR and preserve our ability to treat bacterial infections, it is essential that we identify antibiotics with new structures and targets that are not affected by clinical resistance. Historically, most developmental candidates for antibiotics have come from microbial natural products, as they feature chemical structures and biological activities that have been honed over millions of years of evolution. Unfortunately, as classical bioactivity screens for natural product discovery are blind to the pharmacological properties of their hits, they often identify molecules with functional groups that limit their utility as drugs. One prominent example is actinonin, an inhibitor of bacterial peptide deformylase (PDF) whose activity is dependent on a hydroxamate moiety associated with toxicityin vivo. The abundance of bacterial genomes now presents an opportunity for target-based natural product discovery, where biosynthetic pathways can be mined for molecules that possess desired activities but lack toxic moieties. Here, we use bioinformatics to lead a chemotype-sensitive, target-based search for natural product inhibitors of bacterial PDF that lacks the conserved and problematic metal chelating group. We describe the discovery, heterologous expression, biosynthesis, total synthesis, and activity of the molecule gammanonin: an apparent actinonin homologue from Gammaproteobacteria. Moving forward, we hope this chemotype and target-driven methodology will help to expedite the discovery of new leads for antibiotic development.
PMS2 has both pro-mutagenic and anti-mutagenic effects on repeat instability in the Repeat Expansion Diseases
Authors: Walker Alexandra , Jimenez Diego Antonio , Usdin Karen , Zhao Xiaonan
Published: August 2024

Genome Wide Association studies (GWAS) have implicated PMS2 as a modifier of somatic expansion in Huntington’s disease (HD), one of >45 known Repeat Expansion Diseases (REDs). PMS2 is a subunit of the MutLα complex, a major component of the mismatch repair (MMR) system, a repair pathway that is involved in the generation of expansions in many different REDs. However, while MLH3, a subunit of a second MutL complex, MutLγ, is required for all expansions, PMS2 has been shown to protect against expansion in some model systems but to drive expansion in others. To better understand PMS2’s behavior, we have compared the effect of the loss of PMS2 in different tissues of an HD mouse model (CAG/CTG repeats) and a mouse model for the Fragile X-related disorders (FXDs), disorders that result from a CGG/CCG repeat expansion. Mice heterozygous forPms2show increased expansions in most expansion-prone tissues in both disease models. However, inPms2null mice expansions of both repeats increased in some tissues but decreased in others. Thus, the previously reported differences in the effects of PMS2 in different model systems do not reflect fundamentally different roles played by PMS2 in different REDs, but rather the paradoxical effects of PMS2 in different cellular contexts. These findings have important implications not only for the mechanism of expansion and the development of therapeutic approaches to reduce the pathology generated by repeat expansion, but also for our understanding of normal MMR.
Human-specific features of cerebellar astrocytes and Purkinje cells: an anatomical comparison with mice and macaques
Authors: Hercher Christa , Ellerbeck Kristin , Toutée Louise , Ye Xinyu , Mpai Refilwe , Davoli Maria Antonietta , Watt Alanna J. , Murai Keith K. , Turecki Gustavo , Mechawar Naguib
Published: August 2024

Little is known about the morphological diversity and distribution of cerebellar astrocytes in the human brain and how or if these features differ from those of cerebellar astrocytes in species used to model human illnesses. To address this, we performed a comparative post-mortem examination of cerebellar astrocytes and Purkinje cells (PCs) in healthy humans, macaques, and mice using microscopy-based techniques. Visualizing with canonical astrocyte markers glial fibrillary acidic protein (GFAP) and aldehyde dehydrogenase-1 family member L1 (ALDH1L1), we mapped astrocytes within a complete cerebellar hemisphere. Astrocytes were observed to be differentially distributed across the cerebellar layers, displayed overall increases in area coverages with evolution, and showed features uniquely hominoid. Stereological quantifications in 3 functionally distinct cerebellar lobules demonstrated opposing trends for the canonical astrocyte markers across species with ALDH1L1+ astrocytes increasing with evolution and GFAP+ astrocytes decreasing. PC analyses revealed that while humans have the lowest PC densities, their cell body sizes were the largest with more ALDH1L1 immunoreactive astrocytes surrounding. Notably, the cognitive lobule crus I displayed the highest ratio of Bergmann glia to PC in all species. These findings align with the growing literature for astrocyte and PC heterogeneity and suggest cerebellar astrocyte and PC divergence both within and across species, possibly indicative of a role for these cells in higher-order cerebellar processing.
Cryo-ET of actin cytoskeleton and membrane structure in lamellipodia formation using optogenetics
Authors: Inaba Hironori , Imasaki Tsuyoshi , Aoyama Kazuhiro , Yoshihara Shogo , Takazaki Hiroko , Kato Takayuki , Goto Hidemasa , Mitsuoka Kaoru , Nitta Ryo , Nakata Takao
Published: August 2024

Lamellipodia are sheet-like protrusions essential for migration and endocytosis, yet the ultrastructure of the actin cytoskeleton during lamellipodia formation remains underexplored. Here, we combined the optogenetic tool PA-Rac1 with cryo-ET to enable ultrastructural analysis of newly formed lamellipodia. We successfully visualized lamellipodia at various extension stages, representing phases of their formation. In minor extensions, several unbundled actin filaments formed “Minor protrusions” at the leading edge. For moderately extended lamellipodia, cross-linked actin filaments formed small filopodia-like structures, termed “mini filopodia.” In fully extended lamellipodia, filopodia matured at multiple points, and cross-linked actin filaments running nearly parallel to the leading edge increased throughout the lamellipodia. These observations suggest that actin polymerization begins in specific plasma membrane regions, forming mini filopodia that either mature into full filopodia or detach from the leading edge to form parallel filaments. This actin turnover likely drives lamellipodial protrusion, providing new insights into actin dynamics and cell migration.
MIRO1 controls energy production and proliferation of smooth muscle cells
Authors: Qian Lan , Koval Olha M. , Endoni Benney T. , Juhr Denise , Stein Colleen S. , Allamargot Chantal , Lin Li-Hsien , Guo Deng-Fu , Rahmouni Kamal , Boudreau Ryan L. , Streeter Jennifer , Thiel William H. , Grumbach Isabella M.
Published: August 2024

Background

The outer mitochondrial Rho GTPase 1, MIRO1, mediates mitochondrial motility within cells, but implications for vascular smooth muscle cell (VSMC) physiology and its roles invascular diseases, such as neointima formation following vascular injury are widely unknown.

Methods

An in vivo model of selective Miro1 deletion in VSMCs was generated, and the animals were subjected to carotid artery ligation. The molecular mechanisms relevant to VSMC proliferation were then explored in explanted VSMCs by imaging mitochondrial positioning and cristae structure and assessing the effects on ATP production, metabolic function and interactions with components of the electron transport chain (ETC).

Results

MIRO1 was robustly expressed in VSMCs within human atherosclerotic plaques and promoted VSMC proliferation and neointima formation in mice by blocking cell-cycle progression at G1/S, mitochondrial positioning, and PDGF-induced ATP production and respiration; overexpression of a MIRO1 mutant lacking the EF hands that are required for mitochondrial mobility did not fully rescue these effects. At the ultrastructural level, Miro1 deletion distorted the mitochondrial cristae and reduced the formation of super complexes and the activity of ETC complex I.

Conclusions

Mitochondrial motility is essential for VSMC proliferation and relies on MIRO1. The EF-hands of MIRO1 regulate the intracellular positioning of mitochondria. Additionally, the absence of MIRO1 leads to distorted mitochondrial cristae and reduced ATP generation. Our findings demonstrate that motility is linked to mitochondrial ATP production. We elucidated two unrecognized mechanisms through which MIRO1 influences cell proliferation by modulating mitochondria: first, by managing mitochondrial placement via Ca2+-dependent EF hands, and second, by affecting cristae structure and ATP synthesis.
ANO1 expression is associated with male survival in lung squamous cell carcinoma
Authors: Afolabi Oluwadamilare I.
Published: August 2024

Lung cancer is the leading cause of cancer deaths, with lung squamous cell carcinoma (LUSC) accounting for a substantial proportion of cases. LUSC exhibits significant variability in patient outcomes, influenced by clinicopathological factors such as stage, age, and sex, with men exhibiting higher rates of incidence and poorer outcomes compared to women. Therefore, prognosis modeling and customized approaches to LUSC therapy require the characterization of the biological mechanisms that differentiate tumors and patient outcomes across sexes. Using data from The Cancer Genome Atlas (TCGA), this study characterized gene expression patterns that distinguish male and female LUSC. Specifically, differential expression, survival, and Cox regression analyses assessed the prognostic value of ANO1 (Anoctamin 1) expression and methylation in LUSC. Analyses uncovered a significant overexpression of ANO1 in a subset of male LUSC tumors compared to a much lower expression in normal lung and female LUSC tumors. High ANO1 expression was associated with poor survival outcomes in male subjects. High ANO1 gene body methylation mirrored gene expression and was similarly associated with poor survival outcomes. ANO1’s prognostic value remained significant in a multivariate Cox regression analysis, establishing it as an independent prognostic biomarker. ANO1’s marked sex-specific differences in expression and prognostic value indicate its role in the sex disparity of LUSC survival, highlighting its potential as a biomarker and a target for sex-specific customized therapies.