bluuebunny/crossref_metadata_2025 huggingface dataset:
REverse transcriptase ACTivity (REACT) assay for point-of-care measurement of established and emerging antiretrovirals for HIV treatment and prevention
Authors: Brainerd Cara , Singh Maya A , Tatka John , Craig Cosette , Gilligan-Steinberg Shane , Panpradist Nuttada , Chang Megan M , Lutz Barry , Olanrewaju Ayokunle O.
Published: August 2024

Maintaining adequate levels of antiretroviral (ARV) medications is crucial for the efficacy of HIV treatment and prevention regimens. Monitoring ARV levels can predict or prevent adverse health outcomes like treatment failure or drug resistance. However, conventional ARV measurement using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is slow, expensive, and centralized delaying clinical and behavioral interventions. We previously developed a rapid enzymatic assay for measuring nucleotide reverse transcriptase inhibitors (NRTIs) — the backbone of HIV treatment and prevention regimens — based on the drugs' termination of DNA synthesis by HIV reverse transcriptase (RT) enzyme. Here we expand our work to include non-nucleoside reverse transcriptase inhibitors (NNRTIs) — an ARV class used in established and emerging HIV treatment and prevention regimens. We demonstrate that the REverse Transcriptase ACTivity (REACT) assay can detect NNRTIs including medications used in oral and long-acting/extended-release HIV treatment and prevention. We demonstrate that REACT can measure NNRTIs spiked in either buffer or diluted plasma, and that fluorescence can be measured on both a traditional plate reader and an inexpensive portable reader that can be deployed in point-of-care (POC) settings. REACT measured clinically relevant concentrations of five NNRTI spiked in aqueous buffer. REACT measurements showed excellent agreement between the plate reader and the portable reader, with a high correlation in both aqueous buffer (Pearson's r=0.9807, P < 0.0001) and diluted plasma (Pearson's r = 0.9681, P < 0.0001). REACT has the potential to provide rapid measurement of NNRTIs in POC settings and may help to improve HIV treatment and prevention outcomes.
Development of a preclinical screening platform for clinically relevant therapy of Dravet syndrome
Authors: Mensah Jeffrey A. , Thomson Kyle E. , Huff Jennifer L. , Freeman Tia , Reilly Christopher A. , Rower Joseph E. , Metcalf Cameron S. , Wilcox Karen S.
Published: August 2024

Background

Patients with drug-resistant epilepsy, including Dravet syndrome (DS), are frequently prescribed multiple antiseizure medications (ASMs). Nevertheless, people with DS often have inadequate seizure control, and there is an ongoing unmet clinical need to identify novel therapeutics. As a proof-of-principle study to further validate and characterize theScn1aA1783V/WTmouse model and identify a drug screening paradigm with face, construct, and predictive value, we assessed the efficacy of subchronic administration of stiripentol add-on to clobazam and valproic acid at clinically relevant doses using theScn1aA1783V/WTmouse model.

Methods

We evaluated the efficacy of STP add-on to CLB and VPA using hyperthermia-induced and video-EEG monitoring of spontaneous seizure tests following a 14-day treatment. VPA was delivered via osmotic minipump, while STP and CLB were administered via food pellets delivered through automatic feeders. Bioanalytical assays were performed to evaluate drug concentrations in plasma and brain using liquid chromatography-tandem mass spectrometry.

Results

STP, CLB, N-desmethylclobazam, and VPA all yielded plasma concentrations within the human therapeutic plasma concentrations range. STP added to CLB and VPA significantly elevated the seizing temperatures in the hyperthermia-induced seizure assay. CLB, VPA, and STP coadministration significantly reduced spontaneous seizure frequency compared to CLB and VPA combined.

Significance

This research lays the groundwork for exploring effective add-on compounds to CLB and VPA in treating DS. The study further highlights the utility of theScn1aA1783V/WTmice in discovering therapies for DS-associated pharmacoresistant seizures.

Key points

Integrating pharmacokinetic studies to guide the selection of doses in preclinical studies to achieve target concentrations comparable to the human therapeutic range is crucial in successfully translating animal drug development studies to clinical use.

STP add-on to CLB and VPA significantly reduced spontaneous seizure frequency inScn1aA1783V/WTmice.

A triple-drug polytherapy approach that mimics the clinical treatment paradigm will be an essential preclinical drug screening strategy for identifying novel investigational compounds for Dravet syndrome.
The brain simulates actions and their consequences during REM sleep
Authors: Senzai Yuta , Scanziani Massimo
Published: August 2024

Vivid dreams mostly occur during a phase of sleep called REM1–5. During REM sleep, the brain’s internal representation of direction keeps shifting like that of an awake animal moving through its environment6–8. What causes these shifts, given the immobility of the sleeping animal? Here we show that the superior colliculus of the mouse, a motor command center involved in orienting movements9–15, issues motor commands during REM sleep, e.g. turn left, that are similar to those issued in the awake behaving animal. Strikingly, these motor commands, despite not being executed, shift the internal representation of direction as if the animal had turned. Thus, during REM sleep, the brain simulates actions by issuing motor commands that, while not executed, have consequences as if they had been. This study suggests that the sleeping brain, while disengaged from the external world, uses its internal model of the world to simulate interactions with it.
A method to increase the imaging efficiency of tiling light sheet microscopy using scanning non-coaxial beam arrays synchronized with regional virtual confocal slits
Authors: Gao Liang
Published: August 2024

We present a novel method to improve the imaging efficiency of tiling light sheet microscopy. In the method, scanning non-coaxial beam arrays synchronized with regional virtual confocal slits are used to illuminate imaging plane. There are two advantages. One is the imaging efficiency increases proportional to the number of excitation beams within the non-coaxial beam array. The other is the width of the regional virtual confocal slits could be very wide without admitting off-focus fluorescence generated by the non-coaxial beam array, which makes the method easy to adopt and very robust in practice. We describe the method in detail, characterize the method via numerical simulations. The results suggest that the imaging efficiency and feasibility of the tiling light sheet microscopy could be improved significantly without affecting the 3D imaging ability by using the method. In additions, we propose several configurations to implement the method in practice.
FusionESP: Improved enzyme-substrate pair prediction by fusing protein and chemical knowledge
Authors: Du Zhenjiao , Fu Weimin , Guo Xiaolong , Caragea Doina , Li Yonghui
Published: August 2024

To reduce the cost of experimental characterization of the potential substrates for enzymes, machine learning prediction model offers an alternative solution. Pretrained language models, as powerful approaches for protein and molecule representation, have been employed in the development of enzyme-substrate prediction models, achieving promising performance. In addition to continuing improvements in language models, effectively fusing encoders to handle multimodal prediction tasks is critical for further enhancing model performance using available representation methods. Here, we present FusionESP, a multimodal architecture that integrates protein and chemistry language models with a newly designed contrastive learning strategy for predicting enzyme-substrate pairs. Our best model achieved state-of-the-art performance with an accuracy of 94.77% on independent test data and exhibited better generalization capacity while requiring fewer computational resources and training data, compared to previous studies of finetuned encoder or employing more encoders. It also confirmed our hypothesis that embeddings of positive pairs are closer to each other in high-dimension space, while negative pairs exhibit the opposite trend. The proposed architecture is expected to be further applied to enhance performance in additional multimodality prediction tasks in biology. A user-friendly web server of FusionESP is established and freely accessible athttps://rqkjkgpsyu.us-east-1.awsapprunner.com/.
A quantitative pipeline for whole-mount deep imaging and multiscale analysis of gastruloids
Authors: Gros Alice , Vanaret Jules , Dunsing-Eichenauer Valentin , Rostan Agathe , Roudot Philippe , Lenne Pierre-François , Guignard Léo , Tlili Sham
Published: August 2024

Whole-mount 3D imaging at the cellular scale is a powerful tool for exploring the complex processes of organoid morphogenesis, as it allows examining tissue architecture, cell types, and morphology simultaneously in 3D models. To address the challenges of whole-mount imaging in dense and diffusive tissues, we focused on gastruloids—3D embryonic organoids derived from mouse embryonic stem cells that replicate key aspects ofin vivoorganogenesis and present significant imaging challenges. We developed an integrated two-photon microscopy pipeline to image immunostained and cleared gastruloidsin toto, achieving detailed visualization at both cellular and whole-mount scale. Our pipeline, implemented in Python with a Napari interface, includes image preprocessing to correct optical artifacts and segment individual cell nuclei. This enables comprehensive quantitative analysis of gene expression patterns, cellular events, and morphologies across multiple spatial scales using an interactive post-processing pipeline. This approach not only advances our understanding of gastruloids development but also lays the groundwork for applying these methods to other organoid and tumoroid systems, enhancing our ability to study a wide range of 3D biological models.
Comparative Analysis of Polysaccharide and Cell Wall Structure inAspergillus nidulansandAspergillus fumigatusby Solid-State NMR
Authors: Gautam Isha , Yarava Jayasubba Reddy , Xu Yifan , Li Reina , Scott Faith J. , Mentink-Vigier Frederic , Momany Michelle , Latgé Jean-Paul , Wang Tuo
Published: August 2024

Invasive aspergillosis poses a significant threat to immunocompromised patients, leading to high mortality rates associated with these infections. Targeting the biosynthesis of cell wall carbohydrates is a promising strategy for antifungal drug development and will be advanced by a molecular-level understanding of the native structures of polysaccharides within their cellular context. Solid-state NMR spectroscopy has recently provided detailed insights into the cell wall organization ofAspergillus fumigatus, but genetic and biochemical evidence highlights species-specific differences amongAspergillusspecies. In this study, we employed a combination of13C,15N, and1H-detection solid-state NMR, supplemented by Dynamic Nuclear Polarization (DNP), to compare the structural organization of cell wall polymers and their assembly in the cell walls ofA. fumigatusandA. nidulans, both of which are key model organisms and human pathogens. The two species exhibited a similar rigid core architecture, consisting of chitin, α-glucan, and β-glucan, which contributed to comparable cell wall properties, including polymer dynamics, water retention, and supramolecular organization. However, differences were observed in the chitin, galactosaminogalactan, protein, and lipid content, as well as in the dynamics of galactomannan and the structure of the glucan matrix.
Mapping eye, arm, and reward information in frontal motor cortices using electrocorticography in non-human primates
Authors: Ouchi Tomohiro , Scholl Leo R. , Rajeswaran Pavithra , Canfield Ryan A. , Smith Lydia I. , Orsborn Amy L.
Published: August 2024

Goal-directed reaches give rise to dynamic neural activity across the brain as we move our eyes and arms, and process outcomes. High spatiotemporal resolution mapping of multiple cortical areas will improve our understanding of how these neural computations are spatially and temporally distributed across the brain. In this study, we used micro-electrocorticography (µECoG) recordings in two male monkeys performing visually guided reaches to map information related to eye movements, arm movements, and receiving rewards over a 1.37 cm2area of frontal motor cortices (primary motor cortex, premotor cortex, frontal eye field, and dorsolateral pre-frontal cortex). Time-frequency and decoding analyses revealed that eye and arm movement information shifts across brain regions during a reach, likely reflecting shifts from planning to execution. We then used phase-based analyses to reveal potential overlaps of eye and arm information. We found that arm movement decoding performance was impacted by task-irrelevant eye movements, consistent with the presence of intermixed eye and arm information across much of motor cortices. Phase-based analyses also identified reward-related activity primarily around the principal sulcus in the pre-frontal cortex as well as near the arcuate sulcus in the premotor cortex. Our results demonstrate µECoG’s strengths for functional mapping and provide further detail on the spatial distribution of eye, arm, and reward information processing distributed across frontal cortices during reaching. These insights advance our understanding of the overlapping neural computations underlying coordinated movements and reveal opportunities to leverage these signals to enhance future brain-computer interfaces.

Significance statement

Picking up your coffee mug requires coordinating movements of your eyes and hand and processing the outcomes of those movements. Mapping how neural activity relates to different functions helps us understand how the brain performs these computations. Many mapping techniques have limited spatial or temporal resolution, restricting our ability to dissect computations that overlap closely in space and time. We used micro-electrocorticography recordings to map neural activity across multiple cortical areas while monkeys made goal-directed reaches. These measurements revealed high spatial and temporal resolution maps of neural activity related to eye, arm, and reward information processing. These maps reveal overlapping neural computations underlying movement and open opportunities to use eye and reward information to improve therapies to restore motor function.
Early atrial remodelling drives arrhythmia in Fabry Disease
Authors: Roy Ashwin , O’Shea Christopher , Dasi I Martinez Albert , Patel Leena , Cumberland Max , Nieves Daniel , Canagarajah Hansel , Thompson Sophie , Azad Amar , Price Anna , Hall Caitlin , Alvior Amor Mia , Rath Phalguni , Davies Ben , Rodriguez Blanca , Holmes Andrew P , Pavlovic Davor , Townend Jonathan N , Geberhiwot Tarekegn , Gehmlich Katja , Steeds Richard P
Published: August 2024

Background

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by α-galactosidase A (α-Gal A) deficiency, resulting in multi-organ accumulation of sphingolipid, namely globotriaosylceramide (Gb3). This triggers ventricular myocardial hypertrophy, fibrosis, and inflammation, driving arrhythmia and sudden death, a common cause of FD mortality. Atrial fibrillation (AF) is common in FD, yet the cellular mechanisms accounting for this are unknown. To address this, we conducted electrocardiography (ECG) analysis from a large cohort of adults with FD at varying stages of cardiomyopathy. Cellular contractile and electrophysiological function were examined in an atrial FD model, developed using gene-edited atrial cardiomyocytes and imputed intoin-silicoatrial models to provide insight into arrhythmia mechanisms.

Methods

In 115 adults with FD, ECG P-wave characteristics were compared with non-FD controls. Induced pluripotent stem cells (iPSCs) were genome-edited using CRISPR-Cas9 to introduce theGLAp.N215Svariant and differentiated into atrial cardiomyocytes (iPSC-CMs). Contraction, calcium handling and electrophysiology experiments were conducted to explore proarrhythmic mechanisms. A bi-atrialin-silicomodel was developed with the cellular changes induced byGLAp.N215SiPSC-CMs.

Results

ECG analysis demonstrated P-wave duration and PQ interval shortening in FD adults before onset of cardiomyopathy on imaging and biochemical criteria. FD patients exhibited a higher incidence of premature atrial contractions and increased risk of developing AF. In our cellular model,GLAp.N215SiPSC-CMs were deficient in α-Gal A and exhibited Gb3 accumulation. AtrialGLAp.N215SiPSC-CMs demonstrated a more positive diastolic membrane potential, faster action potential upstroke velocity, greater burden of delayed afterdepolarizations, greater contraction force, slower beat rate and dysfunction in calcium handling compared to wildtype iPSC-CMs. Inputting these changes into thein-silicomodel resulted in similar P-wave morphology changes to those seen in early FD cardiomyopathy and increased the action potential duration (APD) restitution slope, causing APD alternans and inducing AF.

Conclusions

These findings enhance our understanding of atrial myopathy in FD by providing novel insights into underpinning mechanisms for atrial arrhythmia and a rationale for early P-wave changes. These may be targeted in future research to develop therapeutic strategies to reduce the arrhythmic burden in FD and other atrial cardiomyopathies.
Mitochondrial calcium uniporter regulates human fibroblast-like synoviocytes invasion via altering mitochondrial dynamics and dictates rheumatoid arthritis pathogenesis
Authors: Promila Lakra , Sarkar Kabita , Guleria Shivika , Rathore Manisha , Singh Nishakumari C , Khan Shazia , Tomar Manendra Singh , Ammanathan Veena , Barthwal Manoj Kumar , Kumaravelu Jagavelu , Shrivastava Ashutosh , Mitra Kalyan , Guha Rajdeep , Aggarwal Amita , Lahiri Amit
Published: August 2024

Rheumatoid arthritis (RA) is characterized by the aggressive migration and invasion of fibroblast-like synoviocytes (FLS) into cartilage and bone, a process that is significantly influenced by mitochondrial calcium uptake. This study highlights the critical role of mitochondrial calcium uniporter (MCU) in regulating FLS migration and mitochondrial dynamics, and its potential as a therapeutic target in RA. Notably, RA-FLS exhibited increased MCU expression and mitochondrial dysfunction compared to controls. Treatment with Ru360, a potent MCU inhibitor significantly reduced RA-FLS migration, calcium influx, and mitochondrial reactive oxygen species (ROS) levels, while restoring mitochondrial morphology and enhancing ATP production. Further analysis of MCU complex expression revealed elevated levels of MCU and other regulatory subunits (EMRE, MICU1, MICU2) in RA-FLS compared to controls, indicating mitochondrial dysfunction in RA. Mechanistically, MCU inhibition altered gene expression related to cytoskeletal dynamics, focal adhesion, and metabolic pathways. In human RA-FLS, MCU inhibition suppressed migration and invasion both in vitro and in vivo. The interaction between MCU and Miro1, essential for mitochondrial transport, was validated, and its disruption impaired FLS migration. Our findings highlight MCU as a promising therapeutic target to inhibit FLS migration and ameliorate RA progression.
Behavior- and circuit-specific cortico-striatal decoupling during the early development of Parkinson’s disease-like syndrome
Authors: Yao Xu-Ran , Liu Yang , Zheng Wei-Tong , He Kai-Wen
Published: August 2024

Despite that cortico-striatal decoupling has been widely reported in individuals diagnosed with Parkinson’s Disease (PD), its onset, circuit specificity and underlying mechanism remain largely unclear. To investigate these questions, dual fiber photometry is established for the first time to evaluate cortico-striatal coupling during varied motor behaviors, whose cell-type resolution was provided by the usage of Cre transgenic mouse lines. Contralateral turning, digging and licking show distinct coupling patterns, among which digging induces the strongest coupling. Striatal D1R-expressed medium spiny neurons (dMSNs) and D2R-expressed MSNs (iMSNs) similarly contribute to the cortical-striatal coupling during turning and licking but not digging, with much tighter coupling between the dMSNs and the M1 cortex. In PD-like mouse model induced via intra-striatal injection of synthetic mouse wildtype α-synuclein preformed fibril (PFF), digging-associated cortical-striatal decoupling emerges as early as 1-month post induction (Mpi), which becomes significant since 2 Mpi and correlates with later-onset behavioral deficit. Notably, impaired dMSNs but not iMSNs mediate this decoupling, which can be rescued by activation of D1 but not D2 receptor. Mechanistically, we found an inverted U-shape decline in striatal dopamine level along the disease development in PFF-injected mice. Supplement with L-DOPA alleviates the decoupling and motor deficit, suggesting that early dopamine deficiency directly contributes to the cortical-striatal decoupling and the associated motor deficit. These findings provide new insights into the temporal profile and mechanisms underlying the PD-associated cortico-striatal decoupling, which has been implicated as functional biomarker for early diagnosis of PD.
Chronic and Acute Mediators of Passive Viscoelasticity in Human Skeletal Muscle Fibers
Authors: Privett Grace E. , Ricci Austin W. , Needham Karen Wiedenfeld , Callahan Damien M.
Published: August 2024

Cellular viscoelastic modulus in skeletal muscle tissue responds dynamically to chronic stressors, such as age and exercise. Passive tissue mechanics may also be sensitive to acute stimuli such as mechanical loading and/or activation-induced muscle fatigue. These insights are largely derived from preclinical studies of age and acute muscle activation. Therefore, we sought to understand the relative responsiveness of muscle cellular passive mechanics to chronic (resistance training) and acute (muscle fatigue) stressors in healthy young males and females categorized as “resistance trained” or “untrained”. We measured passive mechanics to test the hypothesis that Young’s Modulus and stress would be greater in fibers from trained versus untrained participants and both would be reduced following fatigue. We further assessed the translation of these findings to composite tissue in a sub-set of volunteers where muscle tissue bundles, containing both fibers and extracellular matrix, were analyzed in addition to single fibers. We report a main effect of training such that cellular passive mechanical measures were increased in single fibers from trained versus untrained participants. We likewise report reductions in passive mechanical measures following fatiguing exercise. Surprisingly, both training and acute fatigue only impacted muscle fiber passive measures in males, whereas females showed a more variable response across conditions. Last, we provide preliminary evidence supporting the translation of per-individual cellular differences to the tissue level. Together, these data suggest males respond more dynamically to acute and chronic stressors of muscle tissue mechanics, potentially linking cellular response and sex-dependent differences in musculotendinous injury risk.

New and noteworthy

We report that passive stress and modulus in single muscle fibers was higher in resistance trained healthy adults and fatiguing exercise reduced passive stress and modulus. In each case, dynamic responsiveness of muscle fibers to chronic and acute stressors was observed consistently in males, whereas responses in females varied considerably. We provide further evidence that cellular mechanisms may contribute to multicellular muscle tissue samples, suggesting these findings have relevance to in vivo tissue mechanics.
Anomalies in regional and chronological distributions of Omicron BA.1.1 lineage in the United States
Authors: Kakeya Hideki
Published: August 2024

This study compares the collection dates and locations of the Omicron BA.1 lineage and other major SARS-CoV- 2 mutants registered in NCBI GenBank and provides a detailed analysis of the emergence patterns of pure reverse mutants, which contain only reverse mutations and no other mutations in the surface glycoprotein. The results indicate that Omicron BA.1.1 and its pure reverse mutants were widely distributed throughout the United States from the early days of their emergence, showing a statistically significant difference compared to other major variants, which spread from a small number of sources. The peak emergence of BA.1.1 and BA.1.1.18 pure reverse mutants occurred a few weeks before the peak of all collected samples, whereas the peak of pure reverse mutants in major BA.1 variants and BA.2 coincides with the overall sampling peak. Although the peaks of BA.1.x collections are not all synchronous, the peaks of pure reverse mutants in the BA.1 lineage completely overlap, with the number of such mutants declining abruptly after the peak. These regional and temporal anomalies in the Omicron BA.1 lineage, especially in the BA.1.1 lineage, are virtually impossible to explain by current theories of natural mutation and spread by human-to-human infection.
Optogenetic quantification of source sink relationship in intact hearts to explain cardiac arrhythmia initiation and protection
Authors: Langen Judith S. , Boyle Patrick M. , Malan Daniela , Sasse Philipp
Published: August 2024

Increased cardiac excitability and reduced electrical coupling promote cardiac arrhythmia and can be quantified by input resistance (Rm), pacing threshold (Ithr), and cardiac length constant (λ). However, measurement of these parameters in the heart was not feasible, because the required homogenous current injection cannot be performed with electrical stimulation. Here, we overcame this problem by optogenetic current injection into all illuminated cardiomyocytes of mouse hearts in different action potential phases. Precisely triggered and patterned illumination enabled measuring Rmand λ, which both were smallest at diastole and larger during plateau and repolarization. Pharmacological and depolarization-induced reduction of inward rectifying K+currents (IK1), gap junction block and cardiac infarction reduced Ithrshowing the importance of high IK1density and intact cardiomyocyte coupling for preventing arrhythmia initiation. Simulations in a calibrated cardiomyocyte model were used to classify pro- and anti-arrhythmic mechanisms based on their effects on Rmand Ithr. Finally, combining experiments with simulations allowed for quantification of IK1inward rectification in the intact heart, identifying strong rectification as a new pro-arrhythmic concept.
Genomic analyses identify 15 susceptibility loci and revealHDAC2,SOX2-OT, andIGF2BP2in a naturally-occurring canine model of gastric cancer
Authors: Cook Shawna R. , Hugen Sanne , Hayward Jessica J. , Famula Thomas R. , Belanger Janelle M. , McNiel Elizabeth , Fieten Hille , Oberbauer Anita M. , Leegwater Peter A.J. , Ostrander Elaine A. , Mandigers Paul J.J. , Evans Jacquelyn M.
Published: August 2024

Gastric cancer (GC) is the fifth most common human cancer worldwide, but the genetic etiology is largely unknown. We performed a Bayesian genome-wide association study and selection analyses in a naturally-occurring canine model of GC, the Belgian Tervuren and Sheepdog breeds, to elucidate underlying genetic risk factors. We identified 15 loci with over 90% predictive accuracy for the GC phenotype. Variant filtering revealed germline putative regulatory variants for theEPAS1(HIF2A) andPTENgenes and a coding variant inCD101. Although closely related to Tervuren and Sheepdogs, Belgian Malinois rarely develop GC. Across-breed analyses uncovered protective haplotypes under selection in Malinois atSOX2-OTandIGF2BP2. Among Tervuren and Sheepdogs,HDAC2putative regulatory variants were present at comparatively high frequency and were associated with GC. Here, we describe a complex genetic architecture governing GC in a dog model, including genes such asPDZRN3, that have not been associated with human GC.
A range of voltage-clamp protocol designs for rapid capture of hERG kinetics
Authors: Lei Chon Lok , Whittaker Dominic G , Windley Monique J , Perry Matthew D , Hill Adam P , Mirams Gary R
Published: August 2024

We provide details of a series of short voltage-clamp protocols designed for gathering a large amount of information on hERG (Kv11.1) ion channel gating. The protocols have a limited number of steps and consist only of steps and ramps, making them easy to implement on any patch clamp setup, including automated platforms. The primary objective is to assist with parameterisation, selection and refinement of mathematical models of hERG gating. We detail a series of manual and automated model-driven designs, together with an explanation of their rationale and design criteria. Although the protocols are intended to study hERG1a currents, the approaches could be easily extended and generalised to other ion channel currents.
Differences in energy storage in sympatric salmonid morphs with contrasting lifestyles
Authors: Esin Evgeny V. , N. Markevich Grigorii N. , Shulgina Elena V. , Baskakova Yulia A. , Atemov Roman V. , Shkil Fedor N.
Published: August 2024

Although physiology ensures homeostasis and fitness in a particular environment, and ecological shifts cannot be realized without physiological changes, metabolic transformations during animal adaptive radiations still remain unexplored. We present a study of energy reserve storage in the salmonid assemblage inhabiting a cold-water Lake Kronotskoe. This assemblage diversified from Salvelinus malma and includes eight distinct ecomorphs with contrasting lifestyles and trophic specializations. We hypothesized that ecomorphs differ in energy storage and expenditure, and that their metabolic phenotypes should be among the primary targets of natural selection. To test this hypothesis, we compared the stored amount and ratio of carbohydrates, lipids, circulating peptides that supply the citric acid cycle, as well as proxy indicators of metabolic rate, the blood levels of plasma proteins (including albumin) and hemoglobin. Among ecomorphs, numerous significant differences in physiological parameters were found, closely related to the composition of food, the depth of habitat, and determined by internal factors, probably genetics. Each ecomorph has a specific metabolic phenotype corresponding to its tropho-ecological specialization and lifestyle. Metabolically advanced predators accumulate lipids; littoral insectivorous morphs grow slower and accumulate glycogen; amphipod feeders do not accumulate spare substances; the deepwater consumer of silt benthos differs in the most divergent physiological characteristics. We assume a specific selection on endocrine regulators of energy metabolism during the adaptive radiation of the assemblage, among which the most plausible candidates are thyroid hormones and leptin.
Genome Instability Precedes Viral Integration in Human Papilloma Virus Transformed Tonsillar Keratinocytes
Authors: Chan Kimberly , Tseng Christopher , Malarachi Emily , Goldrich David , Schneper Lisa , Sheldon Kathryn , Alam Samina , Aliaga Cesar , Chatterjee Sreejata , El-Bayoumy Karam , Meyers Craig , Goldenberg David , Broach James R.
Published: August 2024

Approximately 70% of oropharyngeal squamous carcinomas (OPSCC) are associated with human papillomavirus (HPV). While HPV-positive (HPV+) OPSCC responds better to standard therapies and patients with HPV+ tumor generally have better outcomes than those with HPV-negative (HPV-) tumors, a subset of HPV+ patients do have poor outcomes. Our previous work suggested that tumors with integrated virus exhibit significantly greater genome wide genomic instability than those with only episomal viral genomes and patients with HPV+ OPSCC with episomal viral genomes had better outcomes. To explore the causal relation between viral integration and genomic instability, we have examined the time course of viral integration and genetic instability in tonsillar keratinocytes transformed with HPV16. HPV-infected human tonsil keratinocyte cell lines were continuously passaged and every fifth passage some cells were retained for genomic analysis. Whole genome sequencing and optical genomic mapping confirmed that virus integrated in five of six cell lines while remaining episomal in the sixth. In all lines genome instability occurred during early passages, but essentially ceased following viral integration but continued to occur later passages in the episomal line. To test tumorigenicity of the cell lines, cells were injected subcutaneously into the flanks of nude mice. A cell line with the integrated virus induced tumors following injection in the nude mouse while that with the episomal virus did not. We conclude that genomic instability is not the result of viral integration but likely promotes integration. Moreover, those transformants with episomal virus appear to be less tumorigenic than those with integrated virus.
S-RNase Evolution in Self-Incompatibility: Phylogenomic Insights into Synteny with S-Like Genes
Authors: Liu Yunxiao , Zhang Yangxin , Han Songxue , Guo Bocheng , Liang Jiakai , Yu Ze , Yang Fan , Sun Yaqiang , Xue Jiayu , Lin Zongcheng , Schranz M. Eric , Guan Changfei , Ma Fengwang , Zhao Tao
Published: August 2024

S-RNases are essential in the gametophytic self-incompatibility (GSI) system of many flowering plants, where they act as stylar-S determinants. Despite their significance, the syntenic genomic origin and evolutionary trajectory of S-RNase genes in eudicots have remained largely unclear. Here, we performed large-scale phylogenetic and microsynteny network analyses of RNase T2 genes across 130 angiosperm genomes, encompassing 35 orders and 56 families. Based on the connections observed in the synteny network, particularly the persisting synteny block in none-SI Cucurbitaceae species, we infer that well-characterized S-RNase genes (Class III-A RNase genes) were duplicated and evolved from Class I S-like RNase genes, possibly as a result of the gamma triplication event shared by core eudicots. Additionally, we identified frequent lineage-specific gene transpositions of S-RNase genes across diverse angiosperm lineages, including Rosaceae, Solanaceae, and Rutaceae families, accompanied by a significant increase in transposable element (TE) activity near these genes. Our findings delineate the precise genomic origin and evolutionary path of eudicot S-RNase genes, enhancing our understanding of the evolution of the S-RNase-based GSI system.
Comprehensive double-mutant analysis of theBacillus subtilisenvelope using double-CRISPRi
Authors: Koo Byoung-Mo , Todor Horia , Sun Jiawei , van Gestel Jordi , Hawkins John S. , Hearne Cameron C. , Banta Amy B. , Huang Kerwyn Casey , Peters Jason M. , Gross Carol A.
Published: August 2024

Understanding bacterial gene function remains a major biological challenge. Double-mutant genetic interaction (GI) analysis addresses this challenge by uncovering the functional partners of targeted genes, allowing us to associate genes of unknown function with novel pathways and unravel connections between well-studied pathways, but is difficult to implement at the genome-scale. Here, we develop and use double-CRISPRi to systematically quantify genetic interactions at scale in theBacillus subtilisenvelope, including essential genes. We discover > 1000 known and novel genetic interactions. Our analysis pipeline and experimental follow-ups reveal the distinct roles of paralogous genes such as themreBandmblactin homologs, and identify new genes involved in the well-studied process of cell division. Overall, our study provides valuable insights into gene function and demonstrates the utility of double-CRISPRi for high-throughput dissection of bacterial gene networks, providing a blueprint for future studies in diverse bacterial species.
The Changing Immune Landscape of Innate-like T Cells and Innate Cells Throughout Life
Authors: Taheri Marziyeh , Menne Christopher , Anderson Jeremy , Li Shuo , Berzins Stuart P , Licciardi Paul V , Ashhurst Thomas M , Jalali Sedigheh , Pellicci Daniel G
Published: August 2024

Spectral flow cytometry is an advanced immunological tool that enables comprehensive analysis of the immune system by simultaneously comparing innate and adaptive immune cells. Here, using a 40-colour antibody panel we advance our knowledge of innate and innate-like T cells by investigating chemokine receptors, activation and maturation markers not usually assessed on these populations and examine age-related effects to these immune cell subsets. We characterised phenotypic changes of peripheral blood mononuclear cells (PBMC) in three age groups: newborn (cord blood), adults aged 20-30 years, and adults aged 70-80 years, focusing on innate-like T cells and innate cells, including MAIT cells, NKT cells, γδ T cells, ILCs, and Natural Killer (NK) cells. We identify subsets of double-negative (DN) T cells (CD4-CD8-) and CD161+T cells that increase in an age-related manner and exhibit a phenotype similar to innate-like T cells, MAIT cells and γδ T cells. Innate-like T cell subsets express similar patterns of the chemokine receptors and maturation markers CCR4, CCR6, CD27, CD38, CD57 and CD45RA, and resemble memory subsets of conventional CD4+T cells and CD8+T cells. We could detect ILCs in all age ranges, although the frequency of ILC1, ILC2, and ILC3 subsets decreased with age. Notably, we identify the NK maturation marker, CD57, as a universal marker that defines ageing populations of innate and adaptive immune cells. This study enhances our understanding of the ontogeny of human immune cells, highlighting significant age-related changes in the frequency and phenotype of immune cells.
Development of the FitBack online platform: enhancing global child fitness assessment, health-related interpretation, and surveillance
Authors: Sorić Maroje , Morrison Shawnda A. , Ortega Francisco B. , Carraro Attilio , Csányi Tamás , Leskošek Bojan , Mäestu Jarek , Radisavljević-Janić Snežana , Sardinha Luís B. , Scheuer Claude , Starc Gregor , Tammelin Tuija H. , Jurak Gregor
Published: August 2024

Introduction

Physical fitness is an important indicator of health in children. Accurately monitoring child and adolescent physical fitness is critical when designing effective public health interventions and strategies. This article aims to provide an overview of the FitBack platform and the health-related criteria that support the reporting system.

Methods

Four methodological phases were used to create the FitBack platform. First, a review of existing European fitness databases was conducted to identify common fitness tests and methodologies within a European framework. Second, investigators collated data to create European-level fitness norms, health-related criteria, and health-oriented educational resources dedicated to improving child fitness levels. Third, guidance on how to create one’s own fitness monitoring and surveillance system was created based on good practice examples. The FitBack platform was then piloted and launched based on data received from stakeholders who responded to ≥2 rounds of consultation dialogues.

Results

The final version of online platform, with special focus on newly developed Fitback health-related fitness criteria, are communicated herein.

Discussion

The FitBack web platform is a first-step towards creating a standardised, person-centred, multi-national fitness surveillance system for children, bridging existing gaps between European political recommendations, individual member states, and harmonious cooperation between sectors. This article also provided the detailed age- and sex-specific health-related fitness cut-points that are used by the FitBack platform, and that could be useful for researchers, practitioners as well as fitness monitoring systems.
Linking Adiponectin Gene Variants (+45T>G and +276G>T) to Adipokine Levels and Metabolic Syndrome in a North Indian Adult Women
Authors: Gupta Abhishek , Singh Arun Kumar , Gupta Priyanka , Gupta Vani
Published: August 2024

Background

Adiponectin, an adipocyte-derived adipokine, is often downregulated in obesity-related disorders. This study aimed to explore the association between adiponectin gene variants (+45T>G, rs2241766, and +276G>T, rs1501299) and circulating adipokine levels as well as metabolic syndrome in North Indian adult women.

Methods

We genotyped single nucleotide polymorphisms (SNPs) in 541 adult women, comprising 269 with metabolic syndrome (MetS) according to NCEP-ATP III criteria and 272 without MetS (wMetS; control). We assessed circulating levels of adiponectin, leptin, lipid profile, glucose, insulin, and HOMA-IR.

Results

Significant differences (p<0.01) were observed in circulating adipokines (adiponectin and leptin), lipid profile, glucose, insulin, HOMA-IR, and waist-to-hip ratio (WHR) between wMetS and MetS women. The frequency of the combined mutant genotype (TG+GG) at +45T>G was significantly lower (p=0.017) in MetS women, while the mutant G allele was higher (p=0.008) compared to the wild type. For the +276G>T variant, the frequency of the mutant T allele was significantly lower (p=0.027) in MetS women compared to wMetS women. The mutant genotypes GG of +45T>G and TT of +276G>T were significantly associated with lower adiponectin levels, higher leptin levels, and increased HOMA-IR (all p<0.001) in MetS women.

Conclusions

The findings suggest that adiponectin gene variants (+45T>G and +276G>T), along with reduced adiponectin levels and elevated HOMA-IR, may contribute significantly to the development of metabolic syndrome.
Clinical validation of RNA sequencing for Mendelian disorder diagnostics
Authors: Zhao Sen , Macakova Kristina , Sinson Jefferson C. , Dai Hongzheng , Rosenfeld Jill , Zapata Gladys E. , Li Shenglan , Ward Patricia , Wang Christiana , Qu Chunjing , Maywald Becky , Network Undiagnosed Diseases , Lee Brendan , Eng Christine , Liu Pengfei
Published: August 2024

Despite rapid advancements in clinical sequencing, over half of diagnostic evaluations still lack definitive results. RNA-seq has shown promise in research settings for bridging this gap by providing essential functional data for accurate interpretation of diagnostic sequencing results. However, despite advanced research pipelines, clinical translation of diagnostic RNA-seq has not yet been realized. We have developed and validated a clinical diagnostic RNA-seq test in a CLIA laboratory for individuals with suspected genetic disorders who have existing or concurrent comprehensive DNA diagnostic testing. This diagnostic RNA-seq test processes patient RNA samples from fibroblasts or blood and derives clinical interpretations based on the analytical detection of outliers in gene expressions and splicing patterns. The clinical validation involves 150 samples, including benchmark, negative, and positive samples. We developed provisional expression and splicing benchmarks using short-read and long-read RNA-seq data from the HG002 lymphoblastoid sample produced by the Genome in a Bottle Consortium. Our validation data achieved analytical sensitivity and specificity higher than 99% against the benchmarks. For clinical validation, we first established reference ranges for each gene and junction based on expression distributions from our control data. We then evaluated the clinical performance of our outlier-based pipeline using positive samples with previously identified diagnostic findings from the Undiagnosed Diseases Network project. Our pipeline identified 19 of 20 positive findings in both fibroblast and blood samples and highlighted the limitations of the test. Our study provides a paradigm and necessary resources for independent laboratories to validate a clinical RNA-seq test.
DeepSME: De Novo Nanopore Basecalling of Motif-insensitive DNA Methylation and Alignment-free Digital Information Decryptions at Single-Molecule Level
Authors: Fan Qingyuan , Zhao Xuyang , Li Junyao , Liu Ronghui , Liu Ming , Long Yanping , Fu Yang , Feng Qishun , Zhai Jixian , Pan Qing , Li Yi
Published: August 2024

Biomolecular encryption employing chemical modifications enables secure approaches for information storage and communications. However, constructing high information density pathways for rapid synthesis and readout remains a challenge to guarantee confidentiality, integrity, and availability (CIA). Here we develop a nanopore sequencing based protocol, demonstrated by complete substitution using 5-hydroxymethylcytosine (5hmC) for individual nucleotide recognition rather than sequential interactions. Such motif-insensitive methylation at the single-molecule level does not naturally exist and results in severe ion current disruption and a 67.2%-100% readout failure, which ensure its ability on the encryption of the data encoded inside the DNA. We further propose and establish an alignment-free DeepSME basecaller, which is a deep learning-based platform independent on prior models and knowledges. DeepSME utilizes a three-stage training pipeline that initiates tolerable for 11.55% errors, expands its neighboring k-mer dictionary model size from 4^6 to 4^9, and mitigates the errors by only three microbial genomes, giving rise to 92% precision with 92% recall. Fully 5hmC encrypted digital information were deciphered by DeepSME within 16× coverage depth. The versatile and transparent DeepSME pipeline and its F1-score performance of 86.4% surpassing all the state-of-the-art basecallers, support its great potential for meeting the rapidly increasing CIA demands of DNA-based secure communications.
Sidelobe suppressed Bessel beams for one-photon light-sheet microscopy
Authors: Taneja Chetna , George Jerin Geogy , Corsetti Stella , Wijesinge Philip , Bruce Graham D. , Zwart Maarten F. , Bhattacharya Shanti , Dholakia Kishan
Published: August 2024

Bessel beams (BB) have found widespread adoption in various forms of light-sheet microscopy. However, for one-photon fluorescence, the transverse profile of the beam poses challenges due to the detrimental effect of the sidelobes. Here, we mitigate this issue by using a computer generated phase element for generating a sidelobe suppressed Bessel beam (SSBB). We then progress to perform a comparison of biological imaging using SSBB to standard BB in a light-sheet geometry. The SSBB peak intensity is more than an order of magnitude higher than the first sidelobe. In contrast to a standard BB light-sheet, SSBB does not need deconvolution and propagates to depths exceeding 400μm in phantom samples maintaining a transverse size of 5μm. Finally, we demonstrate the advantage of using a SSBB light-sheet for biological applications by imaging fixed early-stage zebrafish larvae. In comparison to the standard BB, we observe a two-fold increase in contrast-to-noise ratio (CNR) when imaging the labelled cellular eye structures and the notochords. Our results provide an effective approach to generating and using SSBB light-sheets to enhance contrast for one-photon light-sheet microscopy.