bluuebunny/crossref_metadata_2025 huggingface dataset:
An overview of next generation sequencing strategies and genomics tools used for tuberculosis research
Authors: Deb Sushanta , Basu Jhinuk , Choudhary Megha
Published: July 2024

Tuberculosis (TB) is a grave public health concern and is considered the foremost contributor to human mortality resulting from infectious disease. Due to the stringent clonality and extremely restricted genomic diversity, conventional methods prove inefficient for in-depth exploration of minor genomic variations and the evolutionary dynamics operating in Mycobacterium tuberculosis (M.tb) populations. Until now, the majority of reviews have primarily focused on delineating the application of whole-genome sequencing (WGS) in predicting antibiotic resistant genes, surveillance of drug resistance strains, and M.tb lineage classifications. Despite the growing use of next generation sequencing (NGS) and WGS analysis in TB research, there are limited studies that provide a comprehensive summary of there role in studying macroevolution, minor genetic variations, assessing mixed TB infections, and tracking transmission networks at an individual level. This highlights the need for systematic effort to fully explore the potential of WGS and its associated tools in advancing our understanding of TB epidemiology and disease transmission. We delve into the recent bioinformatics pipelines and NGS strategies that leverage various genetic features and simultaneous exploration of host-pathogen protein expression profile to decipher the genetic heterogeneity and host-pathogen interaction dynamics of the M.tb infections. This review highlights the potential benefits and limitations of NGS and bioinformatics tools and discusses their role in TB detection and epidemiology. Overall, this review could be a valuable resource for researchers and clinicians interested in NGS-based approaches in TB research.
Top-down and bottom-up microbiome engineering approaches to enable biomanufacturing from waste biomass
Authors: Lyu Xuejiao , Nuhu Mujaheed , Candry Pieter , Wolfanger Jenna , Betenbaugh Michael , Saldivar Alexis , Zuniga Cristal , Wang Ying , Shrestha Shilva
Published: July 2024

Growing environmental concerns and the need to adopt a circular economy have highlighted the importance of waste valorization for resource recovery. Microbial consortia-enabled biotechnologies have made significant developments in the biomanufacturing of valuable resources from waste biomass that serve as suitable alternatives to petrochemical-derived products. These microbial consortia-based processes are designed following a top-down or bottom-up engineering approach. The top-down approach is a classical method that uses environmental variables to selectively steer an existing microbial consortium to achieve a target function. While high-throughput sequencing has enabled microbial community characterization, the major challenge is to disentangle complex microbial interactions and manipulate the structure and function accordingly. The bottom-up approach uses prior knowledge of the metabolic pathway and possible interactions among consortium partners to design and engineer synthetic microbial consortia. This strategy offers some control over the composition and function of the consortium for targeted bioprocesses, but challenges remain in optimal assembly methods and long-term stability. In this review, we present the recent advancements, challenges, and opportunities for further improvement using top-down and bottom-up approaches for microbiome engineering. As the bottom-up approach is relatively a new concept for waste valorization, this review explores the assembly and design of synthetic microbial consortia, ecological engineering principles to optimize microbial consortia, and metabolic engineering approaches for efficient conversion. Integration of top-down and bottom-up approaches along with developments in metabolic modeling to predict and optimize consortia function are also highlighted.

One-Sentence Summary

This review highlights the microbial consortia-driven waste valorization for biomanufacturing through top-down and bottom-up design approaches and describes strategies, tools, and unexplored opportunities to optimize the design and stability of such consortia.
Predictors of operative ischemic cerebrovascular complications in skull base tumor resections: Experience in low-resource setting
Authors: Shiferaw Mestet Yibeltal , Baleh Abat Sahlu , Gizaw Abel , Teklemariam Tsegazeab Laeke , Aklilu Abenezer Tirsit , Awedew Atalel Fentahun , Anley Denekew Tenaw , Mekuria Bereket Hailu , Yesuf Ermias Fikiru , Yigzaw Mengistu Ayele , Molla Henok Teshome , Adise Mldie Alemu , Awano Mekides Musie , Teym Abraham
Published: July 2024

Background

Ischemic cerebrovascular complications following skull base tumor resections remain a significant factor impacting both short-term and long-term patient outcomes. This study aims to improve risk stratification, surgical decision-making, and postoperative care protocols.

Methods

A retrospective cohort study on predictors of ischemic cerebrovascular complications among patients who underwent skull base tumor resection was conducted at 2 high-volume neurosurgical centers in Ethiopia from 2018 to 2023. Binary logistic analysis was performed to see the association of each predictor variable.

Results

The study included 266 patients, with 65.5% being female. The median age and tumor size were 37 (± IQR = 17) years and 4.9 cm (± IQR 1.5), respectively. Ischemic cerebrovascular complications occurred in 19.9% of patients. Middle cranial fossa tumors and tumors spanning both anterior and middle cranial fossa (AOR = 6.75, 95% CI: 1.66–27.54, P < .008), grades 3–5 vascular encasement (AOR = 5.04, 95% CI: 1.79–14.12, P < .002), near-total resection and gross total resection (AOR = 2.89, 95% CI: 1.01–8.24, P < .048), and difficult hemostasis (AOR = 9.37, 95% CI: 3.19–27.52, P < .000) were significantly associated with iatrogenic vascular injury. Subarachnoid hemorrhage had a statistically significant association with vasospasm (AOR = 12.27, 95% CI: 1.99–75.37, P = .007).

Conclusions

Surgery-related ischemic cerebrovascular complications are common. Thorough perioperative risk stratification and proactive treatment planning are crucial to mitigate vascular insults associated with it. In low-resource settings, neurosurgical services are provided without advanced instruments, leading to more complications. Therefore, it is important to focus on improving neurosurgical setup to enhance patient outcomes.
Understanding the interfacial water structure in electrocatalysis
Authors: Shi Guoshuai , Lu Tingyu , Zhang Liming
Published: July 2024

The structure of interfacial water molecules plays a crucial role in modulating the electrochemical surface kinetics. This article provides an in-depth understanding of the water molecule structure inside the double layer and its main influencing factors at the molecular scale.
Cavitating pulmonary lesion secondary to mycobacterium szulgai infection—case report of a rare pathogenic organism
Authors: Sohail Aroon , Hare James , Ibrahim Abdalla
Published: July 2024

Mycobacterium szulgai (MS) is a species of non-tuberculous mycobacterium (NTM), which very rarely is identified as the causative pathogen of pulmonary infections. Due to its rarity, there are limitations in the existing literature regarding the diagnosis, investigation and treatment of MS pulmonary infection. Our case report provides further information regarding the clinical, microbiological and radiological findings associated with MS pulmonary infection with suggestions provided on its long term management.
An Update in Claviceps paspali Disease: A Comprehensive Analysis on Field and Greenhouse Paspalum spp. Infection
Authors: Oberti Hector , Reyno Rafael , Do Canto Javier , Castro Lucia , Murchio Sara , Rossi Carlos , Ayala Walter , Dalla-Rizza Marco
Published: July 2024

Paspalum L. is a genus of the Poaceae family, with many species serving as well-adapted forage plants in subtropical climates and continuous grazing systems. However, Claviceps paspali, an ascomycete of the order Hypocreales, represents a major threat to the Paspalum species. This fungus induces ergot disease, characterized by the replacement of infected flower seeds with sclerotia, which adversely affects seed production and animal health through mycotoxin production. Although ergot disease is reported in many countries, no totally resistant Paspalum cultivars have been reported for commercial use. This study comparatively evaluated disease development in six Paspalum species under greenhouse conditions with three specific isolates of C. paspali, along with field trials. In addition, field isolates of C. paspali were analyzed for phenotypic characteristics and phylogenetic relationships. Greenhouse evaluation revealed variable susceptibility among Paspalum species, with P. malacophyllum, P. notatum, and P. umbrosum being the most resistant. In field trials P. dilatatum showed the highest severity index, and P. umbrosum and P. malacophyllum were less affected. The phenotypic characterization of 22 C. paspali isolates showed variability in pigmentation and growth rates, with potato dextrose agar being the culture medium in which the highest growth rate was observed. Phylogenetic analysis of the isolates identified two well-supported lineages between C. paspali species. This research reports an ergot resistance gradient among Paspalum species, identifying genuine sources of resistance. In addition, virulent isolates of C. paspali potentially useful for rapid screening of accessions or crosses in Paspalum breeding programs were identified.

[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
Horizontal and Vertical Distribution of Clarireedia spp. in Asymptomatic and Symptomatic Creeping Bentgrass Cultivars
Authors: Groben Glen , Schaefer Brian , Clarke Bruce B. , Murphy James A. , Purdon Patrick , Koch Paul , Zhang Ning
Published: July 2024

Dollar spot is an important disease of both cool- and warm-season turfgrasses caused by six fungal species in the genus Clarireedia, yet the ecology and epidemiology of these pathogens remains poorly understood. The goal of this study was to determine the distribution of Clarireedia in asymptomatic and symptomatic creeping bentgrass (Agrostis stolonifera) in the field using a previously developed quantitative PCR assay. To determine the horizontal distribution of the pathogen, the abundance of Clarireedia spp. was measured in leaf and crown tissue from 90, 1-cm-diameter cores spaced 10 cm apart in May 2019 and 2020 (asymptomatic tissue) and August 2019 and July 2020 (symptomatic tissue). Thirty-seven to 69% of cores sampled from asymptomatic turfgrass and 77 to 95% of cores taken from symptomatic turfgrass yielded positive detections for Clarireedia. Spatial analysis indicated that Clarireedia was randomly distributed in the field in both asymptomatic and symptomatic turfgrass. To assess the vertical distribution of the pathogen, the abundance of Clarireedia was measured in the foliar, crown, and thatch layers of 39, 1-cm-diameter × 2.5-cm-deep cores of creeping bentgrass maintained at fairway height (9.5 mm) during 2019 and 2020. Clarireedia was most abundant in foliar tissue, followed by crown tissue and thatch (lowest abundance) throughout the 2-year study. Both studies provide evidence that Clarireedia is widely distributed in turfgrass swards prior to symptom development and can persist within turfgrass as an endophyte. These findings will improve our understanding of Clarireedia epidemiology and may lead to more sustainable dollar spot management.
Ginkgetin improved experimental colitis by inhibiting intestinal epithelial cell apoptosis through EGFR/PI3K/AKT signaling
Authors: Geng Zhijun , Zuo Lugen , Li Jing , Yin Lixia , Yang Jingjing , Duan Ting , Wang Lian , Zhang Xiaofeng , Song Xue , Wang Yueyue , Hu Jianguo
Published: July 2024

Excessive apoptosis of intestinal epithelial cells leads to intestinal barrier dysfunction, which is not only one of the pathological features of inflammatory bowel disease (IBD) but also a therapeutic target. A natural plant extract, Ginkgetin (GK), has been reported to have anti‐apoptotic activity, but its role in IBD is unknown. This study aimed to explore whether GK has anti‐colitis effects and related mechanisms. An experimental colitis model induced by dextran sulfate sodium (DSS) was established, and GK was found to relieve colitis in DSS‐induced mice as evidenced by improvements in weight loss, colon shortening, Disease Activity Index (DAI), macroscopic and tissue scores, and proinflammatory mediators. In addition, in DSS mice and TNF‐α‐induced colonic organoids, GK protected the intestinal barrier and inhibited intestinal epithelial cell apoptosis, by improving permeability and inhibiting the number of apoptotic cells and the expression of key apoptotic regulators (cleaved caspase 3, Bax and Bcl‐2). The underlying mechanism of GK's protective effect was explored by bioinformatics, rescue experiments and molecular docking, and it was found that GK might directly target and activate EGFR, thereby interfering with PI3K/AKT signaling to inhibit apoptosis of intestinal epithelial cells in vivo and in vitro. In conclusion, GK inhibited intestinal epithelial apoptosis in mice with experimental colitis, at least in part, by activating EGFR and interfering with PI3K/AKT activation, explaining the underlying mechanism for ameliorating colitis, which may provide new options for the treatment of IBD.
Identification of differential m6A RNA methylomes and ALKBH5 as a potential prevention target in the developmental neurotoxicity induced by multiple sevoflurane exposures
Authors: Meng Xiaowen , Wang Yichan , Zhao Weiming , Chen Ying , Li Wenting , Peng Ke , Xu Hanbing , Yang Yufan , Shan Xisheng , Huo Wenwen , Liu Huayue , Ji Fuhai
Published: July 2024

Sevoflurane, as a commonly used inhaled anesthetic for pediatric patients, has been reported that multiple sevoflurane exposures are associated with a greater risk of developing neurocognitive disorder. N6‐Methyladenosine (m6A), as the most common mRNA modification in eukaryotes, has emerged as a crucial regulator of brain function in processes involving synaptic plasticity, learning and memory, and neurodevelopment. Nevertheless, the relevance of m6A RNA methylation in the multiple sevoflurane exposure‐induced developmental neurotoxicity remains mostly elusive. Herein, we evaluated the genome‐wide m6A RNA modification and gene expression in hippocampus of mice that received with multiple sevoflurane exposures using m6A‐sequencing (m6A‐seq) and RNA‐sequencing (RNA‐seq). We discovered 19 genes with differences in the m6A methylated modification and differential expression in the hippocampus. Among these genes, we determined that a total of nine differential expressed genes may be closely associated with the occurrence of developmental neurotoxicity induced by multiple sevoflurane exposures. We further found that the alkB homolog 5 (ALKBH5), but not methyltransferase‐like 3 (METTL3) and Wilms tumor 1‐associated protein (WTAP), were increased in the hippocampus of mice that received with multiple sevoflurane exposures. And the IOX1, as an inhibitor of ALKBH5, significantly improved the learning and memory defects and reduced neuronal damage in the hippocampus of mice induced by multiple sevoflurane exposures. The current study revealed the role of m6A methylated modification and m6A‐related regulators in sevoflurane‐induced cognitive impairment, which might provide a novel insight into identifying biomarkers and therapeutic strategies for inhaled anesthetic‐induced developmental neurotoxicity.
Ultraslow serotonin oscillations in the hippocampus delineate substates across NREM and waking
Authors: Cooper Claire , Parthier Daniel , Sibille Jérémie , Tukker John , Tritsch Nicolas X. , Schmitz Dietmar
Published: July 2024

Beyond the vast array of functional roles attributed to serotonin (5-HT) in the brain, changes in 5-HT levels have been shown to accompany changes in behavioral states, including WAKE, NREM and REM sleep. Whether 5-HT dynamics at shorter time scales can be seen to delineate substates within these larger brain states remains an open question. Here, we performed simultaneous recordings of extracellular 5-HT using a recently-developed G Protein–Coupled Receptor-Activation–Based 5-HT sensor (GRAB5-HT3.0) and local field potential (LFP) in the hippocampal CA1, which revealed the presence of prominent ultraslow (<0.05 Hz) 5-HT oscillations both during NREM and WAKE states. Interestingly, the phase of these ultraslow 5-HT oscillations was found to distinguish substates both within and across larger behavioral states. Hippocampal ripples occurred preferentially on the falling phase of ultraslow 5-HT oscillations during both NREM and WAKE, with higher power ripples concentrating near the peak specifically during NREM. By contrast, hippocampal-cortical coherence was strongest and microarousals and EMG peaks were most prevalent during the rising phase in both wake and NREM. Overall, ultraslow 5-HT oscillations delineate substates within the larger behavioral states of NREM and WAKE, thus potentially temporally segregating internal memory consolidation processes from arousal-related functions.
Influence of asymmetric microchannels in the structure and function of engineered neuronal circuits
Authors: Mateus JC , Melo P , Aroso M , Charlot B , Aguiar P
Published: July 2024

Understanding the intricate structure-function relationships of neuronal circuits is crucial for unraveling how the brain sustains efficient information transfer. In specific brain regions, like the hippocampus, neurons are organized in layers and form unidirectional connectivity, which is thought to help ensure controlled signal flow and information processing. In recent years, researchers have tried emulating these structural principles by providing cultured neurons with asymmetric environmental cues, namely microfluidics’ microchannels that promote directed axonal growth. Even though a few reports have claimed achieving unidirectional connectivity ofin vitroneuronal circuits, given the lack of functional characterization, whether this structural connectivity correlates with functional connectivity remains unknown.

We have replicated and tested the performance of asymmetric microchannel designs previously reported in the literature to be successful in the promotion of directed axonal growth, as well as other custom variations. A new variation of “Arrowhead”, termed “Rams”, was the best-performing motif with a ∼76% probability per microchannel of allowing strictly unidirectional connections at 14 days in vitro. Importantly, we assessed the functional implications of these different asymmetric microchannel designs. For this purpose, we combined custom microfluidics with microelectrode array (MEA) technology to record the electrophysiological activity of two segregated populations of hippocampal neurons (“Source” and “Target”). This functional characterization revealed that up to ∼94% of the spiking activity recorded along microchannels with the “Rams” motif propagates towards the “Target” population. Moreover, our results indicate that these engineered circuits also tended to exhibit network-level synchronizations with defined directionality.

Overall, this characterization of the structure-function relationships promoted by asymmetric microchannels has the potential to provide insights into how neuronal circuits use specific network architectures for effective computations. Moreover, the here-developed devices and approaches may be used in a wide range of applications, such as disease modeling or preclinical drug screening.
Are cell length and volume interchangeable in cell cycle analysis?
Authors: Kar Prathitha , Amir Ariel
Published: July 2024

Cell length has been used as a proxy for cell size in cell cycle modeling studies. A previous study, however, brought into question the validity of this assumption, noting that correlations between cell lengths can be different from those involving cell volume if cell width fluctuations are taken into account. If cell volume is regulated, data analysis involving cell lengths will lead to an incorrect inference of the cell size control mechanism. We used conditional correlation of length variables conditioned upon radius variables to elucidate the underlying volume control mechanism. Using the conditional correlation on previous mother machine datasets measuring lengths at birth and division, and the cell radius for multiple cells, we find that the cell volume control strategy is consistent with an adder model. Further, using the conditional correlation, we conclude that measurement noise constitutes a significant portion of the radius variability in the experimental datasets. To conclude, cell length and cell volume can often be used interchangeably owing to small cell width fluctuations.
Genomic insights into red squirrels in Scotland reveals loss of heterozygosity associated with extreme founder effects
Authors: Marr Melissa M. , Humble Emily , Lurz Peter W. W. , Wilson Liam A. , Milne Elspeth , Beckmann Katie M. , Schoenebeck Jeffrey , Fung Uva-Yu-Yan , Kitchener Andrew C. , Kortland Kenny , Edwards Colin , Ogden Rob
Published: July 2024

Remnant populations of endangered species often have complex demographic histories associated with human impact. This can present challenges for conservation as the genetic status of these populations are often a-typical of natural populations, and may require bespoke management. The Eurasian red squirrel,Sciurus vulgaris (L., 1758), is endangered in the UK. Scotland represents a key stronghold, but Scottish populations have been subjected to intense anthropogenic influence, including wide-spread extirpations, reintroductions and competition from an invasive species. This study examined the genetic legacy of these events through whole genome resequencing of 106 red squirrels. Using SNP and genotype likelihood datasets, previously undetected population structure and patterns of gene-flow were uncovered. One off-shore island, three mainland Scottish populations, and a key east-coast migration corridor were observed. An abrupt historical population bottleneck related to extreme founder effects has led to a severe and prolonged depression in genome-wide heterozygosity, which is amongst the lowest reported for any species. Current designated red squirrel conservation stronghold locations do not encompass all existing diversity. These findings highlight the genetic legacies of past anthropogenic influence on long-term diversity in endangered taxa. Continuing management interventions and regular genetic monitoring are recommended to safeguard and improve future diversity.
STRPsearch: fast detection of structured tandem repeat proteins
Authors: Mozaffari Soroush , Arrías Paula Nazarena , Clementel Damiano , Piovesan Damiano , Ferrari Carlo , Tosatto Silvio C. E. , Monzon Alexander Miguel
Published: July 2024

Motivation

State-of-the-art prediction methods are generating millions of publicly available protein structures. Structured Tandem Repeats Proteins (STRPs) constitute a subclass of tandem repeats characterized by repetitive structural motifs. STRPs exhibit distinct propensities for secondary structure and form regular tertiary structures, often comprising large molecular assemblies. They can perform important and diverse biological functions due to their highly degenerated sequences, which maintain a similar structure while displaying a variable number of repeat units. This suggests a disconnection between structural size and protein function. However, automatic detection of STRPs remains challenging with current state-of-the-art tools due to their lack of accuracy and long execution times, hindering their application on large datasets. In most cases, manual curation is the most accurate method for detecting and classifying them, making it impossible to inspect millions of structures.

Results

We present STRPsearch, a novel computational tool for rapid identification, classification, and mapping of STRPs. Leveraging the manually curated entries in RepeatsDB as the known conformational space of the STRPs, STRPsearch utilizes the latest advancements in structural alignment techniques for a fast and accurate detection of repeated structural motifs in protein structures, followed by an innovative approach to map units and insertions through the generation of TM-score graphs. STRPsearch can serve researchers in structural bioinformatics and protein science as an efficient and practical tool for analysis and detection of STRPs.

Availability and implementation

STRPsearch is coded in Python, all the scripts and the associated documentation are available athttps://github.com/BioComputingUP/STRPsearch.

Contact

alexander.monzon@unipd.it,silvio.tosatto@unipd.it

Supplementary information

Supplementary data are available..
Search for candidate substances from the spiny red gurnardChelidonichthys spinosus, as novel food additives that color cultivated meat red
Authors: Shimada Eriko , Tsuruwaka Yusuke
Published: July 2024

The spiny red gurnardChelidonichthys spinosusis a fish with a unique evolution of brightly colored fins consisting of various colors. In Japan, the fish is also used as an ingredient for fish paste products such as fish cake, because of its small edible portion in the processed food industry.

We previously reported on the preparation of “sashimi” in the field of cellular agriculture. In this study, we attempted to explore for cell types suitable for “surimi” or fish paste products and to establish a culture method. In particular, we focused on the development of raw materials for cultivated meat containing pigment cells from edible fish, the spiny red gurnard. As a result, migratory cells were succeeded to be observed from a portion of the fish fin by the migration method. Three types of cells were found in the migrating cells: epithelial-like, fibroblast-like, and those cells containing pigment. Five or more passages of culture were possible, suggesting that the cells could be used as one of the raw materials for cultivated meat surimi in the future. The pigmented cells in particular are expected to be a naturally occurring material that can be used to color surimi.
Transposable element accumulation drives genome size increase inHylesia metabus(Lepidoptera: Saturniidae), an urticating moth species from South America
Authors: Perrier Charles , Allio Rémi , Legeai Fabrice , Gautier Mathieu , Bénéluz Frédéric , Marande William , Theron Anthony , Rodde Nathalie , Herrera Melfran , Saune Laure , Parrinello Hugues , Mcclure Melanie , Arias Mónica
Published: July 2024

We present the first nuclear genome assembly and a complete mitogenome forHylesia metabus(Arthropoda; Insecta; Lepidoptera; Saturniidae). The assembled nuclear genome sequence is 1,271 Mb long, which is among the 10 largest lepidopteran genome assemblies published to date. It is scaffolded in 31 pseudo chromosomes, has a BUSCO score of 99.5%, and has a highly conserved synteny compared to phylogenetically close species. Repetitive elements make up 67% of the nuclear genome and are mainly located in intergenic regions, among which LINEs were predominant, with CR1-Zenon being the most abundant. Phylogenetic and comparative analyses ofH. metabusassembly and 17 additional Saturniidae and Sphingidae assemblies suggested that an accumulation of repetitive elements likely led to the increased size ofH. metabus’genome. Gene annotation using Helixer identified 26,122 transcripts. The Z scaffold was identified using both a synteny analysis and variations of coverage for two resequenced male and femaleH. metabus. TheH. metabusnuclear genome and mitogenome assemblies can be found and browsed on the BIPAA website and constitute useful resources for future population and comparative genomics studies.
How to choose the optimal RNA-Seq library characteristics for alternative splicing analysis
Authors: Ladwig Annika , Klostermann Melina , Zarnack Kathi
Published: July 2024

Alternative splicing (AS) is a key layer of regulation in eukaryotic gene expression that is investigated in all areas of life sciences. Differences in AS between conditions can be quantified from transcriptome-wide short-read RNA sequencing (RNA-Seq) data with designated computational tools. However, not all short-read RNA-Seq data are equally suited for AS analysis. Here, we perform an exemplary AS analysis to showcase the impact of the RNA-Seq library characteristics on the obtained results. Using three standard ENCODE datasets with widespread AS changes, we modulate read length, read depth and the number of replicates and compare their influence on the detection, quantification and classification of AS events with the state-of-the-art AS algorithm MAJIQ. We find that longer reads and a higher read depth are the most effective measures to improve the sensitivity and precision of the analysis. From our results, we provide a recommendation on how to best choose the short-read RNA-Seq library specifications for an AS analysis.
Developing a Soft Micropatterned Substrate to Enhance Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs)
Authors: Maaref Yasaman , Jannati Shayan , Akbari Mohsen , Chiao Mu , Tibbits Glen F
Published: July 2024

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer numerous advantages as a biological model, yet their inherent immaturity compared to adult cardiomyocytes poses significant limitations. This study addresses hiPSC-CM immaturity by introducing a novel physiologically relevant micropatterned substrate for long-term culture and maturation. A novel microfabrication technique combining laser etching and casting creates a micropatterned polydimethylsiloxane (PDMS) substrate with varying stiffness, from 2 to 50 kPa, mimicking healthy and fibrotic cardiac tissue, respectively. Platinum electrodes integrated into the cell culture chamber enabled pacing of cells at various frequencies. Subsequently, cells were transferred to the incubator for time-course analysis, ensuring contamination-free conditions. Cell contractility, cytosolic Ca2+transient, sarcomere orientation, distribution, and nucleus aspect ratio are analyzed in a 2D hiPSC-CM monolayer up to 90 days post-replating in relation to substrate micropattern dimensions. Culturing hiPSC-CMs for three weeks on a micropatterned PDMS substrate (2.5-5 µm deep, 20 µm center-to-center spacing of grooves, 2-5 kPa stiffness) emerges as optimal for cardiomyocyte alignment, nucleus aspect ratio, contractility, and cytosolic Ca2+transient. The study provides significant insights into substrate stiffness effects on hiPSC-CM contractility and Ca2+transient at immature and mature states. Maximum contractility and fastest Ca2+transient kinetics occur in mature hiPSC-CMs cultured for two to four weeks, with the optimum at three weeks, on a soft micropatterned PDMS substrate. This new substrate offers a promising platform for disease modeling and therapeutic interventions.
Kidney mRNA-protein expression correlation: What can we learn from the Human Protein Atlas?
Authors: Acoba Dianne , Reznichenko Anna
Published: July 2024

The Human Protein Atlas (HPA), with more than 10 million immunohistochemical images showing tissue- and cell-specific protein expression levels and subcellular localization information, is widely used in kidney research. The HPA contains comprehensive data on multi-tissue transcript and protein abundance, allowing for comparisons across tissues. However, while visual and intuitive to interpret, immunohistochemistry is limited by its semi-quantitative nature. This can lead to mismatches in protein expression measurements across different platforms. We performed a comparison of the HPA’s kidney-specific RNA sequencing and immunohistochemistry data to determine if the mRNA and protein abundance levels are concordant. Our study demonstrates that there is a discordance between mRNA and protein expression in the kidney based on HPA data. Using an external validatory mass spectrometry dataset, we show that more than 500 proteins undetected by immunohistochemistry are robustly measured by mass spectrometry. The HPA transcriptome data, on the other hand, exhibit similar transcript detection levels as other kidney RNA-seq datasets. Such discordance in mRNA-protein expression could be due to both biological and technical reasons, such as transcriptional dynamics, translation rates, protein half-lives, and measurement errors. This is further complicated by the heterogeneity of the kidney tissue itself, which can increase the discordance if the cell populations or tissue compartment samples do not match. As such, shedding light on the mRNA-protein relationship of the kidney-specific HPA data can provide more context to our scientific inferences when we discuss renal gene and protein quantification.
An INS-1 β-cell proteome highlights the role of fatty acid biosynthesis in glucose-stimulated insulin secretion
Authors: Stremmel Nina , Lemke Oliver , Textoris-Taube Kathrin , Ludwig Daniela , Mülleder Michael , Muenzner Julia , Ralser Markus
Published: July 2024

Pancreatic beta cells secrete insulin as a response to rising glucose level, a process known as glucose-stimulated insulin secretion (GSIS). In this study, we used liquid chromatography tandem mass spectrometry and data-independent acquisition to acquire proteomes of rat pancreatic INS-1 832/13 beta cells that were short-term stimulated with glucose concentrations ranging from 0 to 20 mM, quantifying the behavior of 3703 proteins across 11 concentrations. Ensemble clustering of proteome profiles revealed unique response patterns of proteins expressed by INS-1 cells. 237 proteins, amongst them proteins associated with vesicular SNARE interactions, protein export, and pancreatic secretion showed an increase in abundance upon glucose stimulation, whilst the majority of proteins, including those associated with metabolic pathways such as glycolysis, the TCA cycle and the respiratory chain, did not respond to rising glucose concentrations. Interestingly, we observe that enzymes participating in fatty acid metabolism, responded distinctly, showing a “switch-on” response upon release of glucose starvation with no further changes in abundance upon increasing glucose levels. We speculate that increased activity of fatty acid metabolic activity might either be part of GSIS by replenishing membrane lipids required for vesicle-mediated exocytosis and/or by providing an electron sink to compensate for the increase in glucose catabolism.

Significance of the Study

We used high-throughput proteomics to capture comprehensive proteome changes 30 minutes post stimulation in the INS-1 832/13 beta cell line. Our study provides insights into the metabolic regulation of glucose-stimulated insulin secretion in pancreatic beta cells, specifically highlighting the early role of fatty acid biosynthesis. These findings suggest a necessary shift in focus from electrochemical to metabolic mechanisms in understanding GSIS, paving the way for future research. As the first to document proteome alterations in the initial phase of GSIS, our study furthermore documents the extent of protein abundance variability when obtaining data after short stimulation times, and therefore highlights the necessity of well-controlled study design and biological replicates. The recorded data set complements existing metabolomic and transcriptomic studies, providing a valuable resource for subsequent investigations.
Prophage-encodedHm-oscargene recapitulatesWolbachia-induced male killing in the tea tortrix mothHomona magnanima
Authors: Arai Hiroshi , Katsuma Susumu , Matsuda-Imai Noriko , Lin Shiou-Ruei , Inoue Maki N , Kageyama Daisuke
Published: July 2024

Wolbachia are maternally transmitted bacterial symbionts that are ubiquitous among arthropods. They can hijack host reproduction in various ways, including male killing (MK), where the sons of infected mothers are killed during development. The recent discovery of MK-associated Wolbachia genes, i.e., oscar in Ostrinia moths and wmk in Drosophila flies, stimulates our interest in the diversity and commonality of MK mechanisms, which remain largely unclear. We recently discovered that a Wolbachia symbiont of the moth Homona magnanima carries an MK-associated prophage region encoding homologs of oscar (Hm-oscar) and wmk (wmk-1-4). Here, we investigated the effects of these genes in the native host. Upon transient overexpression, Hm-oscar, but not wmk, induced male lethality in H. magnanima, in contrast to our observations in Drosophila, where the wmk homologs, but not Hm-oscar, killed the males. Hm-oscar disrupted sex determination in male embryos by inducing a female-type doublesex splicing and impaired dosage compensation, recapitulating the Wolbachia phenotype. Cell-based transfection assays confirmed that Hm-oscar suppressed the function of masculinizer, the primary male sex determinant involved in lepidopteran dosage compensation. Our study highlights the conserved roles of oscar homologs in Wolbachia-induced lepidopteran MK and argues that Wolbachia have evolved multiple MK mechanisms in insects.
Synchronous theta networks characterize successful memory retrieval
Authors: Rao Aditya M. , DeHaan Riley D. , Kahana Michael J.
Published: July 2024

Memory retrieval activates regions across the brain, including not only the hippocampus and medial temporal lobe (MTL), but also frontal, parietal, and lateral temporal cortical regions. What remains unclear, however, is how these regions communicate to organize retrieval-specific process-ing. Here, we elucidate the role of theta (3–8 Hz) synchronization, broadly implicated in memory function, during the spontaneous retrieval of episodic memories. Analyzing a dataset of 382 neurosurgical patients (213 male, 168 female, 1 unknown) implanted with intracranial electrodes who completed a free recall task, we find that synchronous networks of theta phase synchrony span the brain in the moments before spontaneous recall, in comparison to periods of deliberation and incorrect recalls. Hubs of the retrieval network, which systematically synchronize with other regions, appear throughout the prefrontal cortex and lateral and medial temporal lobes, as well as other areas. Theta synchrony increases appear more prominently for slow (3 Hz) theta than for fast (8 Hz) theta in the recall–deliberation contrast, but not in the encoding or recall–intrusion contrast, and theta power and synchrony positively correlate throughout the theta band. These results implicate diffuse brain-wide synchronization of theta rhythms, especially slow theta, in episodic memory retrieval.

Significance Statement

Analyzing intracranial recordings from 382 subjects who completed an episodic free recall experi-ment, we study the brain-wide theta synchrony effects of memory retrieval. The literature has not previously described the whole-brain regional distribution of these effects nor studied them with respect to intrusions. We show that a whole-brain theta synchrony effect marks the recall accuracy contrast, that distributed synchronous hubs constitute a whole-brain retrieval network, and that theta synchrony in the successful encoding, successful retrieval, and recall accuracy contrasts corre-lates positively with theta power increases at a region. These findings advance our understanding of the role and localization of theta synchrony effects during human memory retrieval.
METTL3 shapes m6A epitranscriptomic landscape for successful human placentation
Authors: Kumar Ram Parikshan , Kumar Rajnish , Ganguly Avishek , Ghosh Ananya , Ray Soma , Islam Md. Rashedul , Saha Abhik , Roy Namrata , Dasgupta Purbasa , Knowles Taylor , Niloy Asef Jawad , Marsh Courtney , Paul Soumen
Published: July 2024

Methyltransferase-like 3 (METTL3), the catalytic enzyme of methyltransferase complex for m6A methylation of RNA, is essential for mammalian development. However, the importance of METTL3 in human placentation remains largely unexplored. Here, we show that a fine balance of METTL3 function in trophoblast cells is essential for successful human placentation. Both loss-of and gain-in METTL3 functions are associated with adverse human pregnancies. A subset of recurrent pregnancy losses and preterm pregnancies are often associated with loss of METTL3 expression in trophoblast progenitors. In contrast, METTL3 is induced in pregnancies associated with fetal growth restriction (FGR). Our loss of function analyses showed that METTL3 is essential for the maintenance of human TSC self-renewal and their differentiation to extravillous trophoblast cells (EVTs). In contrast, loss of METTL3 in human TSCs promotes syncytiotrophoblast (STB) development. Global analyses of RNA m6A modification and METTL3-RNA interaction in human TSCs showed that METTL3 regulates m6A modifications on the mRNA molecules of critical trophoblast regulators, includingGATA2, GATA3, TEAD1, TEAD4, WWTR1, YAP1, TFAP2CandASCL2, and loss of METTL3 leads to depletion of mRNA molecules of these critical regulators. Importantly, conditional deletion ofMettl3in trophoblast progenitors of an early post-implantation mouse embryo also leads to arrested self-renewal. Hence, our findings indicate that METLL3 is a conserved epitranscriptomic governor in trophoblast progenitors and ensures successful placentation by regulating their self-renewal and dictating their differentiation fate.
REAL-TIME VISUALIZATION OF SPLICEOSOME ASSEMBLY REVEALS BASIC PRINCIPLES OF SPLICE SITE SELECTION
Authors: Donovan Benjamin T , Wang Bixuan , Garcia Gloria R , Mount Stephen M , Larson Daniel R
Published: July 2024

The spliceosome is a megadalton protein-RNA complex which removes introns from pre-mRNA, yet the dynamic early assembly steps have not been structurally resolved. Specifically, how the spliceosome selects the correct 3’ splice site (3’SS) amongst highly similar non-functional sites is not known. Here, we develop a kinetic model of splice site selection based on single-molecule U2AF heterodimer imagingin vitroandin vivo. The model successfully predicts alternative splicing patterns and indicates that 3’SS selection occurs while U2AF is in complex with the spliceosome, not during initial binding. This finding indicates the spliceosome operates in a ‘partial’ kinetic proofreading regime, catalyzed in part by the helicase DDX42, which increases selectivity to the underlying U2AF binding site while still allowing for efficient forward progression.

ONE-SENTENCE SUMMARY

We apply a kinetic proofreading model to elucidate how transient U2AF binding leads to high fidelity splice site selection.
Experimental reproduction of viral replication and disease in dairy calves and lactating cows inoculated with highly pathogenic avian influenza H5N1 clade 2.3.4.4b
Authors: Baker Amy L. , Arruda Bailey , Palmer Mitchell V. , Boggiatto Paola , Sarlo Davila Kaitlyn , Buckley Alexandra , Ciacci Zanella Giovana , Snyder Celeste A. , Anderson Tavis K. , Hutter Carl , Nguyen Thao-Quyen , Markin Alexey , Lantz Kristina , Posey Erin A. , Torchetti Mia Kim , Robbe-Austerman Suelee , Magstadt Drew R. , Gorden Patrick J.
Published: July 2024

Highly pathogenic avian influenza (HPAI) H5N1 of the hemagglutinin clade 2.3.4.4b was detected in the United States in late 2021 and continues to circulate in all four North American flyways to date. In addition to impacting poultry, these HPAI viruses caused mortality events in wild bird species and wild mammals. Transmission in multiple host species raises the concern for mammalian adaptation. On March 25, 2024, HPAI H5N1 clade 2.3.4.4b was confirmed in a dairy cow in Texas in response to a multi-state investigation into milk production losses. Over one hundred positive herds were rapidly identified in Texas and eleven other U.S. states. The case description included reduced feed intake and rumen motility in lactating cows, decreased milk production, and thick yellow milk. The diagnostic investigation revealed detections of viral RNA in milk and mammary tissue with alveolar epithelial degeneration and necrosis, and positive immunoreactivity of glandular epithelium by immunohistochemistry. A single transmission event, likely from avian species to dairy cattle, followed by limited local transmission preceded the onward lateral transmission of H5N1 clade 2.3.4.4b genotype B3.13. We sought to experimentally reproduce infection with genotype B3.13 in Holstein yearling heifers and lactating cows. The heifers were inoculated by an aerosol respiratory route and the cows by an intramammary route. Clinical disease was mild in the heifers, but infection was confirmed by virus detection, lesions, and seroconversion. Clinical disease in lactating cows included decreased rumen motility, changes to milk appearance, and production losses consistent with field reports of viral mastitis. Infection was confirmed by high levels of viral RNA detected in milk, virus isolation, lesions in mammary tissue, and seroconversion. This study provides the foundation to investigate additional routes of infection, transmission, and intervention strategies.
Scalable Antigen-Antibody Binding Affinity Landscape: A Case Study with ENHERTU
Authors: Li Wei
Published: July 2024

Optimization of binding affinities for antibody-drug conjugates (ADCs) is inextricably linked to their therapeutic efficacy and specificity, where the majority of ADCs are engineered to achieve equilibrium dissociation constants (Kdvalues) in the range of 10−9to 10−10M. Yet, there is a paucity of published data delineating the optimal binding affinity or its range that ensures improved therapeutic outcomes for ADCs. This study addresses this issue by integrating structural biophysics within a scalable in silico workflow to generate antigen-antibody binding affinity landscapes, with a focus on Trastuzumab, a monoclonal antibody employed in the treatment of HER2-positive breast cancer. By leveraging high-throughput computational techniques, including homology structural modeling and structural biophysics-based Kdcalculations, this research puts forward a set of high-accuracy structural and intermolecular binding affinity data for Her2-Trastuzumab-Pertuzumab (PDB entry 6OGE). Beyond the design of Her2-targeting ADCs with enhanced efficacy and specificity, this scalable antigen-antibody binding affinity landscape also offers a technically feasible workflow for the high-throughput generation of synthetic structural and biophysical data with reasonable accuracy. Overall, in combination with artificial intelligence (e.g., deep learning) algorithms, this synthetic data approach aims to catalyze a paradigm shift in the discovery and design of antibodies and ADCs with improved efficacy and specificity.

SIGNIFICANCE

With Trastuzumab as an example, this study presents a scalable computational biophysical generation of antigen-antibody binding affinity landscapes, serving two purposes: design of Her2-targeting ADCs with enhanced efficacy and specificity and continued accumulation of synthetic structural biophysics data for the development of useful AI-based drug discovery and design model in future. This scalable approach is broadly applicable to databases such as Protein Data Bank.