bluuebunny/crossref_metadata_2025 huggingface dataset:
A rapid visualization method for detecting rotavirus A by combining nuclear acid sequence-based amplification with the CRISPR-Cas12a assay
Authors: Chen Yue , Wu Junhua , Gao E-bin , Lu Yanbo , Qiu Haiyan
Published: October 2024

Introduction. Rotavirus A is the most common pathogen causing diarrhoea in children less than 5 years, leading to severe complications such as dehydration, electrolyte imbalances, acidosis, myocarditis, convulsions, pneumonia, and other life-threatening conditions.

Gap statement. There is an urgent need for a rapid and efficient nucleic acid detection strategy to enable early diagnosis and treatment, preventing rotavirus transmission and associated complications.

Aim. This article aimed to develop a nuclear acid sequence-based amplification (NASBA)-Cas12a system for detecting rotavirus A using fluorescence intensity or lateral flow strips.

Methodology. The NASBA technology was combined with the clustered regularly interspaced short palindromic repeats-Cas12a system to establish a NASBA-Cas12a system for detecting rotavirus A.

Results. The NASBA-Cas12a system could detect rotavirus A at 37 ℃ within 70 min and had no cross-reactivity with other viruses, achieving a limit of detection of 1.2 copies μl–1. This system demonstrated a sensitivity of 100%, specificity of 90%, positive predictive value of 97.22% and negative predictive value of 100%. The kappa value was 0.933, indicating that the NASBA-Cas12a system was highly consistent with reverse transcription-PCR.

Conclusion. The NASBA-Cas12a system exhibited high sensitivity and specificity for detecting rotavirus A, showing great potential for clinical application.
Nicotine promotes pathogenic bacterial growth and biofilm formation in peri-implant
Authors: Hu Rong , Qian Huifen , Wang Xiangyun , Peng Bei , Huang Dahai
Published: October 2024

Introduction. Peri-implantitis is a plaque-associated disease that leads to implant loss and arises from bacterial biofilms on the surface of the implant. Smoking is a risk factor for peri-implantitis and impedes treatment effectiveness. Additionally, aryl hydrocarbon receptor (AHR), IL−6, and IL-22 levels are related to peri-implantitis.

Aim. We aimed to investigate the effects of nicotine on inflammatory response, bacterial growth and biofilm formation.

Hypothesis/Gap Statement. We hypothesized that nicotine promoted pathogenic bacterial growth and biofilm formation, thereby aggravating inflammation.

Methodology. The expression of AHR, IL-6 and IL-22 was measured in peri-implant sulci fluid using quantitative PCR and Western blot analyses. The cementum was incubated with bacterial suspension including Porphyromonas gingivalis, Streptococcus sanguinis and Fusobacterium nucleatum and treated with 100, 200, 250 and 300 µg ml−1 nicotine, and then, the absorbance and number of colony-forming units were detected. Biofilm formation was evaluated using the tissue culture plate method and safranin O staining. Carbohydrates and proteins were measured by the phenol–sulfuric acid method and the bicinchoninic acid method, respectively.

Results. The results indicated that smoking increased the levels of AHR, IL-6 and IL-22. Functionally, nicotine promoted the growth of P. gingivalis, S. sanguinis and F. nucleatum. Additionally, it promoted the biofilm formation of these bacteria and increased the contents of carbohydrates and proteins.

Conclusion. Nicotine promoted bacterial growth and biofilm build-up, suggesting that smoking may aggravate the progression of peri-implantitis.
Pattern and Risk Factors for Nocturnal Enuresis among Children in Aseer Region, Southwestern Saudi Arabia: A cross-sectional study
Authors: Alqahtani Youssef Ali , Shati Ayed A. , Alhanshani Ahmad A , Al-Garni Abdulaziz M. , Mahmood Syed Esam
Published: October 2024

Background

Nocturnal enuresis is defined as persistent bed-wetting at night beyond the age of five year old. To assess the prevalence and risk factors for nocturnal enuresis in children of the Aseer region.

Materials and methods

A descriptive cross-sectional survey via an online questionnaire was conducted targeting all accessible populations who are involved in childcare (5 to 18 years of age) in the Aseer region of Saudi Arabia. The questionnaire was circulated online using social media platforms by the research team. The questionnaire included items to address sociodemographic data of the child and the family, their medical history, and bed-wetting-related information.

Results

The study included 466 children, of which 145 (31.1%) complained of recurrent bedwetting. More than half of the children who experienced nocturnal enuresis were males (58.6%), and 31.7% were aged between 7-8 years, with a mean age of 5.9 years. A comparable proportion of fathers (59.3%) and mothers (57.9%) of the sampled children were university graduates. A total of 104 (71.7%) children started bed-wetting at 5 years of age, and 19.3% experienced bed-wetting from 6-7 years. Also, 126 (90%) children reported bed-wetting during sleep. Attention-deficit hyperactivity disorder, dark phobia, family troubles, and exposure to bullying were the most frequently reported risk factors.

Conclusion

Nearly 1 out of 3 children experienced bed-wetting, which is relatively common in boys than girls. Early toilet training and a supportive parental attitude toward bed-wetting is essential to improve the child’s quality of life.
Menstrual hygiene management improvement in selected communities using nurturing care group approach
Authors: Dwumfour-Asare Bismark , Appiah-Effah Eugene , Tidwell James Ben , Nyarko Kwabena Biritwum
Published: October 2024

Improved menstrual hygiene management (MHM) is key to socioeconomic empowerment of women through improved well-being. Safe MHM is challenging in developing countries especially in rural and low-income settings. The study assessed the influence of World Vision’s piloted project of Nurturing Care Group approach on MHM in rural communities of Ghana. Four (4) communities each from Savelugu Municipal and Sekyere East District Assemblies were selected for a cross-sectional qualitative survey using 16 focus group discussions. Data was analysed using inductive mixed method with content analysis. Findings from 162 study participants showed satisfactory MHM awareness and practices among young and old females; MHM awareness and education sources included relatives, friends, NGOs, public and social institutions (e.g., health centres, churches, schools etc); intervention improved MHM understanding, behaviour and personal hygiene practices. Some MHM challenges that persisted included non-availability and prohibitive high cost of MHM materials, burden of managing menstrual pains, inadequate knowledge in handling menstrual intricacies, menstrual taboos, and stigma. Menstrual stigma was comparatively low among intervention communities than controls, but there was no difference regarding menstrual taboos across all communities largely due to religious and superstitious beliefs. Championing local production and use of reusable menstrual pads from fabrics was considered an innovation to ease sanitary pad cost burden and non-availability of menstrual pads. MHM was positively influenced through improved understanding of personal hygiene practices, and reduced stigmatization among others. Subsequent interventions would require adequate scope and resources to work with stakeholders to facilitate change processes with persistent MHM challenge like taboos.
A spatially-resolved blueprint of the developing human lung reveals a WNT-driven niche for basal stem cells
Authors: Hsu Peggy P. , Conchola Ansley S. , Frum Tristan , Dong Xiangning , Tudrick Lila , Ponnusamy Varun , Downey Michael S. , Wu Manqi , Yang Mengkun , Lee Yusoo , Niestroy Emma , Tsai Yu-Hwai , Wu Angeline , Glass Ian A. , Merajver Sofia D. , Spence Jason R.
Published: October 2024

Organs are composed of diverse cell types that change across space and time during development. To interrogate this diversity, we micro-dissected developing human lungs along the proximal-distal axis during the late pseudoglandular stage and generated an integrated analysis of single-nucleus sequencing and spatial transcriptomics, creating a cellularly-resolved atlas of the lung. These rich datasets revealed positional niches and cellular heterogeneity along the proximal-distal axis, including the identification of a unique population of TP63+basal cells, the primary stem cell of the airway, marked by expression ofLGR5andLGR6. Analysis of theLGR5+basal cell niche and functional experiments with primary organoid models suggest a tonic level of WNT pathway activity inLGR5+basal cells that is potentiated by mesenchyme-derived R-SPONDIN. We found that basal cell self-renewal is enhanced by WNT activity, suggesting that the WNT pathway plays a previously unappreciated but critical role in airway stem cell maintenance during human development. These results enhance our fundamental understanding of the positional and cellular heterogeneity in the developing human lungs and begin to reveal unique niches that maintain homeostasis throughout the lung.
The PHD3-FOXO3 axis modulates the interferon type I response in microglia aggravating Alzheimer’s disease progression
Authors: Sanchez-Garcia Manuel A. , Lara-Ureña Nieves , March-Diaz Rosana , Ortega-de San Luis Clara , Quiñones-Cañete Silvia , Barba-Reyes Juan M. , Cabello-Rivera Daniel , Muñoz-Cabello Ana M. , Mora-Romero Bella , Romero-Molina Carmen , Heras-Garvin Antonio , Navarro Victoria , Lopez-Barneo Jose , Vizuete Marisa , Vitorica Javier , Muñoz-Manchado Ana B. , Cokman Matthew , Rosales-Nieves Alicia E. , Pascual Alberto
Published: October 2024

Microglia respond to Alzheimer’s disease (AD) with a variety of transcriptional responses. However, the regulation of specific transcriptional signatures and the contribution of each individual response to disease progression is only starting to be characterized. We have previously shown that hypoxiaviahypoxia inducible factor 1 (HIF1) is a strong regulator of Aß plaque-associated microglia (AßAM). Here, we characterize the role of HIF1-mediated transcription ofEgln3, encoding for PHD3, in AßAM. We show that oligomeric Aß treatment (oAß)in vitroinduces the expression ofHif1aandEgln3in microglia, which correlates with the transcriptional activation of genes involved in the interferon type I signature (IFNS) in a PHD3-dependent manner. Mechanistically, we demonstrate FOXO3 to be an important repressor of IFNS in microglia, whose abundance decreases upon Aß presence, and, correspondingly, both in human single-nucleus (sn) and mouse AßAM transcriptomics, FOXO3 DNA binding sites define the IFNS. FOXO3 repression of the IFNS is dependent on PHD3, with our results suggesting a physical interaction between both proteinsin vitro.In vivo, loss of PHD3 correlate with abrogation of the IFNS and activation of the disease-associated microglia signature (DAM) in AßAM. Transcriptional changes in microglia associate with increased microglia proximity to Aß plaques, augmented phagocytosis of Aß by microglia, reduced parenchymal levels of Aß, and an increase in small-sized plaques. PHD3 deficiency also reduced the Aß plaque-associated neuropathology and rescued behavioural deficits of an AD mouse model. Finally, we also demonstrate that microglial PHD3 overexpression during development in the absence of Aß pathology is sufficient to induce the IFNS and to behavioural alterations. Altogether, our data strongly indicate that the PHD3-FOXO3 axis controls the microglial IFNS in a cell autonomous manner, contributing to the progression of AD.
Proteolysis-targeting vaccines (PROTAVs) for robust combination immunotherapy of melanoma
Authors: Wang Qiyan , Su Ting , Cheng Furong , Zhou Shurong , Liu Xiang , Wang Mi , Xu You , Tang Ri , Liao Shimiao , Dailey Jordan , Xiao Guolan , Yang Chunpeng , Wen Hanning , Zheng Weijia , Wen Bo , Tyc Katarzyna M , Liu Jinze , Sun Duxin , Wang Shaomeng , Zhu Guizhi
Published: October 2024

Protein/peptide subunit vaccines are promising to promote the tumor therapeutic efficacy of immune checkpoint blockade (ICB). However, current protein/peptide vaccines elicit limited antitumor T cell responses, leading to suboptimal therapeutic efficacy. Here, we present proteolysis-targeting vaccines (PROTAVs) that facilitate antigen proteolytic processing and cross-presentation to potentiate T cell responses for robust ICB combination immunotherapy of melanoma. PROTAVs are modular conjugates of protein/peptide antigens, E3 ligase-binding ligands, and linkers. In antigen-presenting cells (APCs), PROTAVs bind to E3 ligases to rapidly ubiquitinate PROTAV antigens, facilitating antigen proteolytic processing by proteasome, and thereby promoting antigen cross-presentation to T cells and potentiating CD8+T cell responses. We developed a melanoma PROTAV using a tandem peptide of trivalent melanoma-associated antigens. Co-delivered by lipid nanoparticles (LNPs) with bivalent immunostimulant adjuvants, this PROTAV promotes the quantity and quality of melanoma-specific CD8+T cells in mice. Further, combining PROTAV and ICB ameliorates the immunosuppressive melanoma microenvironment. As a result, PROTAV and ICB combination enhances melanoma complete regression rates and eradicated 100% largeBrafV600Emelanoma without recurrence in syngeneic mice. PROTAVs hold the potential for robust tumor combination immunotherapy.
Regulatory T cells crosstalk with tumor and endothelium through lymphotoxin signaling
Authors: Piao Wenji , Wu Long , Xiong Yanbao , Zapas Gregory C. , Paluskievicz Christina M. , Oakes Robert S. , Pettit Sarah M. , Schmitz Jessica , Ivanyi Philipp , Shetty Amol C. , Song Yang , Kong Dejun , Sleeth Margaret , Hippen Keli L. , Lee Young , Li Lushen , Shirkey Marina W. , Kensiski Allison , Alvi Aamna , Ho Kevin , Saxena Vikas , Bräsen Jan H. , Jewell Christopher M. , Blazar Bruce R. , Abdi Reza , Bromberg Jonathan S.
Published: October 2024

Regulatory T cells (Tregs) are suppressors of anti-tumor immunity that exert multifaceted functions by signaling surrounding cells. We revealed Tregs use their high-level surface lymphotoxin (LT)α1β2 to preferentially stimulate LTβ receptor (LTβR) nonclassical NFκB signaling on both tumor and lymphatic endothelial cells (LECs) to accelerate tumor growth and metastasis. Selectively targeting LTβR nonclassical NFκB pathways on both tumors and LECs cocultured with Tregs, inhibited tumor growth and migration in vitro. Further, we identified protumorigenic chemokines and interferon-stimulated response genes selectively driven by LTβR nonclassical NFκB in melanoma cells. Endothelial specific genes related to oncogenic process such as SOX18 and FLRT2 were identified to be driven under LTβR nonclassical NFκB in LECs. Leveragingin vivoTreg LTα1β2 interactions with LTβR on tumor and LECs, transfer of WT but not LTα-deficient Tregs promoted transplanted WT B16F10 growth and tumor cell-derived CXCL1 and CXCL10 secretion in LTβR-deficient host mice, and increased endothelial specific genes related to tumor angiogenesis and lymphangiogenesis, in WT mice bearing LTβR-depleted melanoma. Selectively blocking LTβR nonclassical NFκB pathways remarkably suppressed tumor growth and lymphatic metastasis by reducing tumor cell and LEC-derived CXCL1 and CXCL10 production, restricting Treg and myeloid-derived suppressor cell (MDSC) recruitment to tumor. It also retained intratumoral effector T cells, especially IFNγ+CD8 T cells by restraining Treg facilitated lymphatic vessel permeability. Our data revealed that Treg LTα1β2 promotes LTβR nonclassical NFκB signaling in tumor cells and LECs providing a rational strategy to modulate Treg-mediated protumorigenic molecules to prevent tumor growth and metastasis.
New Statistical Metric for Robust Target Detection in Cryo-EM Using 2DTM
Authors: Zhang Kexin , Cossio Pilar , Rangan Aaditya , Lucas Bronwyn , Grigorieff Nikolaus
Published: October 2024

2D template matching (2DTM) can be used to detect molecules and their assemblies in cellular cryo-EM images with high positional and orientational accuracy. While 2DTM successfully detects spherical targets such as large ribosomal subunits, challenges remain in detecting smaller and more aspherical targets in various environments. In this work, a novel 2DTM metric, referred to as the 2DTM p-value, is developed to extend the 2DTM framework to more complex applications. The 2DTM p-value combines information from two previously used 2DTM metrics, namely the 2DTM signal-to-noise ratio (SNR) and z-score, which are derived from the cross-correlation coefficient between the target and the template. The 2DTM p-value demonstrates robust detection accuracies under various imaging and sample conditions and outperforms the 2DTM SNR and z-score alone. Specifically, the 2DTM p-value improves the detection of aspherical targets such as a modified artificial tubulin patch particle (500 kDa) and a much smaller clathrin monomer (193 kDa) in simulated data. It also accurately recovers mature 60S ribosomes in yeast lamellae samples, even under conditions of increased Gaussian noise. The new metric will enable the detection of a wider variety of targets in both purified and cellular samples through 2DTM.
Host Genome Drives the Diversity, Richness, and Beneficial Microbes in the Shrimp Microbiota: A Hologenome Perspective
Authors: Cornejo-Granados Fernanda , Gallardo-Becerra Luigui , Romero-Hidalgo Sandra , Lopez-Zavala Alonso A. , Sotelo-Mundo Rogerio R. , Cota-Huizar Andres , Ochoa-Leyva Adrian
Published: October 2024

Despite the growing understanding of the factors influencing the shrimp microbiome, the impact of host genetics on modulating the intestinal microbiota still needs to be studied. Moreover, the existing studies were typically conducted using animal models and under laboratory conditions. Here, we investigated the effect of two shrimp genetics, fast-growth (Gen1) and disease-resistant (Gen2), on shaping the microbiota of the hepatopancreas and the intestine in open-pond aquaculture farming conditions. First, we identified significant genomic differences between the two genetic lines using genome-wide SNP analysis. Then, the microbiota analysis confirmed that organs had a more substantial impact, explaining 33.9% of the variability, followed by the genetic line, which explained 17.3%. The microbiota of the hepatopancreas was more significantly affected by the genetic line than the intestine. Gen1 exhibited higher richness, diversity, niche breadth, and abundance of beneficial microbes than Gen2, suggesting that Gen1 had a more generalist microbiota. By comparing the microbiota of another set of healthy and diseased shrimps, we confirmed that a higher presence of beneficial microbes was linked to a healthy shrimp status. Additionally, we genotyped and determined the microbiota of wild-type shrimps, proving that they differ from those observed in both genetic lines. Interestingly, ponds with Gen1 had better productivity than Gen2, suggesting a potential link between higher productivity and the microbiota selected by Gen1. Our study highlights the importance of the holobiome perspective in breeding and management programs. It suggests that a specific genetic line and its associated microbiota could be used to select the larvae to be cultivated, improving shrimp aquaculture production.
A multi-step completion process model of cell plasticity
Authors: Chen Chen M. , Yu Rosemary
Published: October 2024

Plasticity is the potential for cells or cell populations to change their phenotypes and behaviours in response to internal or external cues. A common way to study a plasticity programme is to track the underlying molecular changes over time, by collecting omics time-series data. However, there remains a lack of mathematical models to elucidate and predict molecular behaviours in a plasticity programme using omics time-series data. Here we report a new mathematical framework that models cell plasticity as a multi-step completion process, where the system moves from the initial state along a path guided by multiple intermediate attractors until the final state (i.e. a new homeostasis) is reached. In benchmarking tests, we show that our developed Control Point (CP) model fits omics time-series data and identifies attractor states that are well-aligned with prior knowledge. Importantly, we show that the CP model can make non-trivial predictions such as the molecular outcomes of blocking a plasticity programme from reaching completion, in a quantitative and time-resolved manner.

Significance statement

Cell plasticity is fundamental to a wide range of complex biological processes, for example in embryonic development, immunology, regenerative medicine, aging, cancer, and others. We developed a new mathematical model that leverages omics time-series data to understand and make accurate predictions about cell plasticity. This can lead to new discoveries and open up new avenues of research in medicine and bioengineering.
Potent and durable gene modulation in heart and muscle with chemically defined siRNAs
Authors: Fakih Hassan H. , Lochmann Clemens , Gagnon Rosemary , Summers Ashley , Caiazzi Jillian , Buchwald Julianna E. , Tang Qi , Maru Bruktawit , Hildebrand Samuel R. , UI Abideen Mohammad Zain , Furgal Raymond C. , Gross Katherine Y. , Yang Yen , Cooper David , Monopoli Kathryn R. , Echeverria Dimas , Shim JaeHyuck , Yamada Ken , Alterman Julia F. , Khvorova Anastasia
Published: October 2024

Small interfering RNA (siRNAs) hold immense promise for treating cardiac and muscular diseases, but robust and scalable delivery to these tissues remains a challenge. Recent advances in delivery strategies to muscle include conjugation of biologics (antibody/antibody fragments, peptides), which are currently in clinical development. However, the manufacturing of biologic-siRNA conjugates is a challenging and complex process. By contrast, lipophilic siRNAs are readily chemically synthesized at scale and support sufficient cardiac and skeletal muscle delivery. In this work, we refine siRNA design elements to enhance potency and durability and support clinically relevant silencing in muscle. Applying this strategy for siRNAs targeting myostatin (MSTN), a key target in muscle-wasting conditions, we show that a single subcutaneous dose in mice achieved robust and durable silencing (∼80% inhibition up to 6 weeks, ∼30% at 14 weeks). Biweekly dosing resulted in >95% reduction of circulatingMSTNfor half a year, with no observed systemic or target-related toxicity.MSTNinhibition resulted in muscle growth and increased lean muscle mass, correlating with improved grip strength. Interestingly, the functional impact on muscle growth and strength significantly outlasts the target silencing, suggesting extended pharmacological effects. Systemic administration was equally efficacious in all muscle groups tested, including skeletal muscle, heart, tongue and diaphragm. The informational nature of the muscle-active chemically defined siRNA scaffold was confirmed by demonstrating muscle and heart efficacy with three additional targets. Our findings pave the way for potent and long-lasting gene modulation in muscle using chemically defined, lipophilic siRNAs, offering a new avenue for treating muscular diseases.
Atomic-Level Investigation ofKCNJ2Mutations Associated with Ventricular Arrhythmic Syndrome Phenotypes
Authors: Munawar Saba , Anderson Corey L , Reilly Louise , Woltz Ryan , Kayani Yusra Sajid , Chiamvimonvat Nipavan , Eckhardt Lee L.
Published: October 2024

KCNJ2encodes the inward rectifying potassium channel (Kir2.1) that createsIK1which maintains the cardiac resting membrane potential and regulates excitability. Mutations inKCNJ2have been linked to several clinical phenotypes associated with ventricular arrhythmia and sudden death including Andersen-Tawil syndrome (ATS) related to loss of function mutations and Short QT Syndrome 3 related to gain of function mutations. Detailed structural-functional relationships to explain the arrhythmia phenotypes are understudied and limit the capacity to provide precision medicine. Here, we combine in-depth and complementary computational molecular modeling techniques with functional analysis from three patients with ATS that harborKCNJ2mutations R67Q, R218L, and G300D. Whole-cell patch-clamp experiments revealed loss of function in homomeric mutant channels. Full-length Kir2.1 models were developed for structure-based investigation, and mutations were introduced in both open and closed conformations. Site-directed mutagenesis identified altered interaction profiles contributing to structural perturbations. Molecular dynamics simulations assessed the impact of each mutation on overall channel conformation and stability. Principal component analysis and normal mode analysis revealed mutation-specific structural perturbations. These findings extend beyond previous studies, offering atomic-level characterization of mutation-specific perturbations. Our multifaceted approach provides first atomic-level insights into the molecular mechanisms underlying ATS, paving the way for targeted therapeutic strategies.

Study Highlights

1) Clinical mutation analysis confirmed loss-of-function.

2) Mutation analysis revealed that these clinical mutations dramatically alter the interaction pattern of the mutated residue and subsequently disturbs channel stability.

3) The molecular dynamics based RMSD and RMSF evaluations show that the open conformation state of the channel is more stable comparative to the closed state however, the mutations impact channel conformations regardless of conductance state.

4) The PCA (principal component analysis) and PCA based NM (normal mode) analysis revealed that these clinicalKCNJ2mutations caused significant conformational changes, even distant from the specific residue.

4) This study is the first extensivein silicoand experimental analysis ofKCNJ2clinical mutations that start from the arrhythmia phenotype and lead to an in-depth atomic-level investigation. These newly resolved features pave the way towards a better understanding of the molecular disease mechanism and new therapeutic strategies.
EDTA tubes are suitable for insulin and C-peptide measurement in resource-limited settings and can be stored at room temperature for up to 24 hours
Authors: Mubiru Nathan , Mukasa Rogers , Sekitoleko Isaac , Balungi Priscilla A , Kakumba Ronald M. , Ongaria Terry , Nkabura Hubert , Nyirenda Moffat , Niwaha Anxious J , Nakanga Wisdom P
Published: October 2024

Introduction

Insulin and C-peptide assessment are important in characterization and management of diabetes. However, their adoption and increased clinical use in low resource settings (LRSs) is partly hindered by logistical factors including supplies required for pre-analytical sample handling and limited infrastructure. We aimed to determine the effects of altered sample processing conditions on stability of insulin and C-peptide at the pre-analytical stage.

Methods

We investigated the stability of C-peptide and insulin in serum and plasma collected, preservative type, time to centrifugation, storage conditions and duration of storage on the stability of C-peptide and insulin over 24 hours.

Results

Both C-peptide and insulin levels remained stable above 90% from baseline p=1.000 & p=0.776 over 24 hours for samples stored in K2EDTA tubes, whether at room temperature or in a cooler box, both as centrifuged and uncentrifuged whole blood. In contrast, samples collected in plain serum tubes kept at room temperature and uncentrifuged C-peptide and insulin levels decreased significantly to 51%, p=0.006 and 62%, p=0. 083 respectively, similarly insulin levels for centrifuged samples declined to 64%, p=0.083 All iced and centrifuged serum samples remained above 90% of baseline concentration.

Conclusions

In resource limited settings where insulin and c-peptide tests are limited to central laboratories and highly dependent on sample referral systems, these tests can be reliably measured without the need for immediate centrifugation or processing from samples collected in whole blood K2EDTA tubes uncentrifuged kept at room temperature and processed within 24hours.

Message

K2EDTA tubes can be used for sample collection in resource limited setting kept at room temperature for up to 24hrs for insulin and c-peptide assays.

K2EDTA plasma is more stable than serum for insulin and c-peptide measurement and should be used in resource limited settings
Virtual epilepsy patient cohort: generation and evaluation
Authors: Dollomaja Borana , Wang Huifang E. , Guye Maxime , Makhalova Julia , Bartolomei Fabrice , Jirsa Viktor K.
Published: October 2024

Epilepsy is a prevalent brain disorder, characterized by sudden, abnormal brain activity, making it difficult to live with. One-third of people with epilepsy do not respond to anti-epileptic drugs. Drug-resistant epilepsy is treated with brain surgery. Successful surgical treatment relies on identifying brain regions responsible for seizure onset, known as epileptogenic zones (EZ). Despite various methods for EZ estimation, evaluating their efficacy remains challenging due to a lack of ground truth for empirical data. To address this, we generated and evaluated a cohort of 30 virtual epilepsy patients, using patient-specific anatomical and functional data from 30 real drug-resistant epilepsy patients. This personalized modelling, based on the patient’s brain data, is called a virtual brain twin. For each virtual patient, we provided data that included anatomically parcellated brain regions, structural connectivity, reconstructed intracranial electrodes, simulated brain activity at both the brain region and electrode levels, and key parameters of the virtual brain twin. These key parameters, which include the EZ hypothesis, serve as the ground-truth for simulated brain activity. For each virtual brain twin, we generated synthetic spontaneous seizures, stimulation-induced seizures and interictal activity. We systematically evaluated these simulated brain signals by quantitatively comparing them against their corresponding empirical intracranial recordings. Simulated signals based on patient-specific EZ captured spatio-temporal seizure generation and propagation. Through in-silico exploration of stimulation parameters, we also demonstrated the role of patient-specific stimulation location and amplitude in reproducing empirically stimulated seizures. The virtual epileptic cohort is openly available, and can be directly used to systematically evaluate methods for the estimation of EZ or source localization using ground truth EZ parameters and source signals.
Appraisal of Gene Expression-Based Classifiers for Neuropsychiatric Disorders: A Meta-Regression
Authors: Razavi Ali , Arensman Brittany , Barnett Eric J , Lee Leo A , Faraone Stephen V , Glatt Stephen J , Hess Jonathan L
Published: October 2024

A substantial body of research examines the potential of gene-expression-based biomarkers for diagnosing and selecting treatments for neuropsychiatric disorders, yet no clear consensus has been reached regarding the influence of controllable factors such as study design and model selection on the performance of gene-expression-based classifiers. To investigate study characteristics and methodologies that influence the accuracy of studies using transcriptomics to classify neuropsychiatric disorders, we conducted a literature review and meta-regression of relevant studies. We extracted several characteristics from each study, including the number of samples in a training dataset, approach for model validation, and classification model. Using univariate and multi-variate mixed-effect meta-regression analyses, we estimated the association between these study characteristics and reported classification accuracies. Machine Learning (ML) models accounted for 55% of all models, Deep Learning (DL) models accounted for 20% and variations of Logistic Regression models making up the remaining 25%. Support vector machine(SVM) was the most common model type (17%).The use of withheld test samples (56%) was the most frequent approach for validating performance of classification models. We found significant associations between reported accuracies and study-rated bias risk, model type, class ratio, and validation approach. Overall, this review provides helpful insights into study characteristics that significantly influence classification accuracies and emphasizes the importance of prudent methodologies for training and evaluating classification models to mitigate biased accuracy estimates.
Fate plasticity of interneuron specification
Authors: Mostajo-Radji Mohammed A. , Leon Walter R. Mancia , Breevoort Arnar , Gonzalez-Ferrer Jesus , Schweiger Hunter E. , Lehrer Julian , Zhou Li , Schmitz Matthew T. , Perez Yonatan , Mukhtar Tanzila , Chu Julia , Andrews Madeline G. , Sullivan Frederika N. , Tejera Dario , Choy Eric C. , Paredes Mercedes F. , Teodorescu Mircea , Kriegstein Arnold R. , Alvarez-Buylla Arturo , Pollen Alex A.
Published: October 2024

The generation of neuronal subtypes in the mammalian central nervous system is driven by competing genetic programs. The medial ganglionic eminence (MGE) gives rise to two major cortical interneuron (cIN) populations, marked by Somatostatin (Sst) and Parvalbumin (Pvalb), which develop on different timelines. The extent to which external signals influence these identities remains poorly understood. Pvalb-positive cINs are particularly important for regulating cortical circuits through strong perisomatic inhibition, yet they have been difficult to modelin vitro. Here we investigated the role of the environment in shaping and maintaining Pvalb cINs. We grafted mouse MGE progenitors into a variety of 2D and 3D co-culture models, including mouse and human cortical, MGE, and thalamic systems with dissociated cells, organoids, organotypic cultures, and conditioned media. Across models, we observed distinct proportions of Sst- and Pvalb-positive cIN descendants. Strikingly, grafting MGE progenitors into 3D human, but not mouse, corticogenesis models led to efficient, non-autonomous differentiation of Pvalb-positive cINs. This differentiation was characterized by upregulation of Pvalb maturation markers, downregulation of Sst-specific markers, and the formation of perineuronal nets. Furthermore, lineage-traced postmitotic Sst-positive cINs, when grafted onto human cortical models, also upregulated Pvalb expression. These results reveal an unexpected level of fate plasticity in MGE-derived cINs, demonstrating that their identities can be dynamically shaped by the surrounding environment.
Systematic empirical evaluation of individual base editing targets: validating therapeutic targets inUSH2Aand comparison of methods
Authors: Tachida Yuki , Manian Kannan V. , Butcher Rossano , Levy Jonathan M. , Pendse Nachiket , Hennessey Erin , Liu David R. , Pierce Eric A. , Liu Qin , Comander Jason
Published: October 2024

Base editing shows promise for the correction of human mutations at a higher efficiency than other repair methods and is especially attractive for mutations in large genes that are not amenable to gene augmentation therapy. Here, we demonstrate a comprehensive workflow for in vitro screening of potential therapeutic base editing targets for theUSH2Agene and empirically validate the efficiency of adenine and cytosine base editor/guide combinations for correcting 35USH2Amutations. Editing efficiency and bystander edits are compared between different target templates (plasmids versus transgenes) and assays (Next generation sequencing versus Sanger), as well as comparisons between unbiased empirical results and computational predictions. Based on these observations, practical assay recommendations are discussed. Finally, a humanized knock-in mouse model was created with the best-performing target, the nonsense mutation c.11864G>A p.(Trp3955*). Split-intein AAV9 delivery of editing reagents resulted in the restoration of USH2A protein and a correction rate of 65 ± 3% at the mutant base pair and of 52 ± 3% excluding bystander amino acid changes. This efficiency compares favorably to a prior genome editing strategy tested in the retina that completed a clinical trial and demonstrates the effectiveness of this overall strategy to identify and test base editing reagents with the potential for human therapeutic applications.
Illuminating root-soil mechanics
Authors: Le Houx James , Fletcher Daniel McKay , Leonardi Alberto , Williams Katherine A. , Walker Nancy , Alvarez-Borges Fernando , Afsar Dizaj Ebrahim , Murthy Madhu , Smith Ronan , Perera Liam , Aslani Navid , James Andrew , Ahmed Sharif , Roose Tiina , Ruiz Siul
Published: October 2024

Soil compaction and escalating global drought increase soil strength and stiffness. It remains unclear which plant root biomechanical mechanisms/traits enable growth in these harsh conditions. Here, we combine synchrotron X-ray computed tomography with spatially resolved X-ray diffraction to characterize the biomechanics of a replica root-soil system. We map the strain field around the root tip, finding strong agreement with finite element simulations, thereby demonstrating a promising new in-vivo measurement protocol.
A comprehensive multi-omics signature of doxorubicin-induced cellular senescence in the postmenopausal human ovary.
Authors: Devrukhkar Pooja R , Watson Mark , Soygur Bikem , Wu Fei , Bons Joanna , Anvari Hannah , Atazhanova Tomiris , Martin Nicolas , Tran Tommy , Schneider Kevin , Rose Jacob P , Shanes Elisheva , Pavone Mary Ellen , Campisi Judith , Furman David , Melov Simon , Schilling Birgit , Duncan Francesca E
Published: October 2024

A major aging hallmark is the accumulation of cellular senescence burden. Over time senescent cells contribute to tissue deterioration through chronic inflammation and fibrosis driven by the Senescence-Associated Secretory Phenotype (SASP). The human ovary is one of the first organs to age, and prominent age-related fibroinflammation within the ovarian microenvironment is consistent with the presence of senescent cells, but these cells have not been characterized in the human ovary. We thus established a doxorubicin-induced model of cellular senescence to establish a senotype (gene/protein signature of a senescence cell state) for ovarian senescent cells. Explants of human postmenopausal ovarian cortex and medulla were treated with doxorubicin for 24 hours followed by culture for up to 10 days in a doxorubicin-free medium. Tissue viability was confirmed by histology, lack of apoptosis, and continued glucose consumption by explants. Single nuclei sequencing and proteomics revealed an unbiased signature of ovarian senescence. We identified distinct senescence profiles for the cortex and medulla, driven predominantly by epithelial and stromal cells. Proteomics uncovered subregional differences in addition to 120 proteins common to the cortex and medulla SASP. Integration of transcriptomic and proteomic analyses revealed 26 shared markers, defining a senotype of doxorubicin-induced senescence unique to the postmenopausal ovary. A subset of these proteins: Lumican, SOD2, MYH9, and Periostin were mapped onto native tissue to reveal compartment-specific localization. This senotype will help determine the role of cellular senescence in ovarian aging, inform biomarker development to identify, and therapeutic applications to slow or reverse ovarian aging, senescence, and cancer.
A novel behavioral apparatus for spontaneous exploration and operant conditioning of social information under spatial conditions in rats
Authors: Wise Taylor B. , Templer Victoria L. , Burwell Rebecca D.
Published: October 2024

Background

Evidence from the fields of evolutionary biology and neuroscience supports the theory that spatial cognition and social cognition share neural mechanisms. Rodent models are widely used to study either spatial or social cognition, but few studies have explored the interactions between the spatial and social cognitive domains due to the lack of appropriate paradigms.

New method

Our study introduces the Vertical Maze (VM), a novel behavioral apparatus designed to measure multiple aspects of spatial and social cognition. The VM features a standard 3-chamber maze positioned above multilevel columns allowing for the presentation of conspecifics at varying spatial distances and familiarity levels. This arrangement enables conspecifics to serve as discriminative stimuli for both social and spatial spontaneous and goal-oriented assessments. The three-dimensional design of the VM allows rats to use multisensory cues to judge distance, direction, and social identity of conspecifics.

Results

In the present study, we found that rats can 1) discriminate the spatial distance of conspecifics located below them in an operant conditioning task, and 2) discriminate social novelty when conspecifics are presented below at the near, middle, and far distances in a spontaneous exploration task. Critically, it was necessary for rats to explore all levels of the maze to perform these discriminations.

Comparison with existing methods

This new method advances the field by permitting the presentation of social information (conspecifics) at different spatial distances. The use of conspecifics to serve as stimuli for both social and spatial discriminations allows more direct comparison of behavioral measures across these information domains. Importantly, the presentation of conspecifics as stimuli below the 3-chamber level of the maze engages auditory, visual, and olfactory systems, encouraging a robust multisensory representation of conspecifics presented at a distance.

Conclusions

Our results confirm that the VM is an effective tool for studying both spatial and social cognition, facilitating the development of novel automated tasks in these areas. This new method opens new avenues for investigating the neural and cognitive foundations of spatial and social behavior, as well as for exploring the possibility of shared mechanisms across these cognitive domains.
Glycerol improves the viability of a cryopreserved choanoflagellate
Authors: Chandra Sheel , Rutaganira Florentine U.
Published: October 2024

The colonial choanoflagellateSalpingoeca rosettais a tractable model system for studying the origins of multicellularity, but long-term storage strategies for this species have not been tested. In this study, we probed each stage of cryopreservation (freeze-down, long-term storage, recovery) to identify the optimal protocol for recovery ofS. rosettaand co-cultured bacterial cells. Dimethyl sulfoxide (Me2SO; commonly referred to as DMSO), the current cryoprotective agent (CPA) standard, proved to be worse than glycerol at comparable concentrations. Samples treated with either CPA at 5% showed the poorest recovery. Our results identified 15% glycerol as the most effective CPA for bothS. rosettaandEchinicola pacifica. We also determined that ultra-low temperature freezers can be sufficient for short-term storage. We propose 15% glycerol and liquid phase nitrogen as the standard cryopreservation protocol forS. rosettacultures and as a starting point for testing long-term storage strategies for other choanoflagellates and heterotrophic protists.
Cross-task implications: How hippocampal event boundary responses predict unrelated memory performance
Authors: van Dijk Daphne , Collin Silvy H.P.
Published: October 2024

Hippocampal responses at event boundaries have been shown to predict memory performance for these events. However, are these hippocampal event boundary responses specific to memory for those particular events, or can they also have predictive power across various memory tasks? We used data from the Cam-CAN project (fMRI data from continuous movie viewing and memory results from an unrelated Famous Faces Task, N = 630) to determine whether hippocampal responses at event boundaries during the continuous movie viewing were indicative of memory performance in the unrelated Famous Faces task using various machine learning algorithms. The results showed that memory performance in the Famous Faces Task could be predicted based on participants’ hippocampal event boundary responses in another task, which suggests that the hippocampal event boundary responses are indicative for general memory performance. This might indicate importance of these hippocampal event boundary responses in terms of general information processing of the human brain.
The challenge of chromatin model comparison and validation - a project from the first international 4D Nucleome Hackathon
Authors: Kubica Jędrzej , Korsak Sevastianos , Clerkin Ariana Brenner , Kouřil David , Schirman Dvir , Yadavalli Anurupa Devi , Banecki Krzysztof , Kadlof Michał , Busby Ben , Plewczynski Dariusz
Published: October 2024

The computational modeling of chromatin structure is highly complex and challenging due to the hierarchical organization of chromatin, which reflects its diverse biophysical principles, as well as inherent dynamism, which underlies its complexity. The variety of methods for chromatin structure modeling, which are based on different approaches, assumptions and scales of modeling, suggests that there is a necessity for a comprehensive benchmark. This inspired us to conduct a project at the NIH-funded 4D Nucleome Hackathon on March 18-21, 2024 at The University of Washington in Seattle, USA. The hackathon provided an amazing opportunity to gather an international, multi-institutional and unbiased group of experts to discuss, understand and undertake the challenges of chromatin model comparison and validation. These challenges seem straightforward in theory, however in practice, they are challenging and ambiguous. To address them, we developed a bioinformatics workflow for chromatin model comparison and validation, in which we use distance matrices to represent chromatin models, and we calculate Spearman correlation coefficients between pairs of matrices to estimate correlations between models, as well as between models and experimental data. During the 4-day hackathon, we tested our workflow on several distinct software packages for chromatin structure modeling and we discovered several challenges that include: 1) different aspects of chromatin biophysics and scales complicate model comparisons, 2) expertise in biology, bioinformatics, and physics is necessary to conduct a comprehensive research on chromatin structure, 3) bioinformatic software, which is often developed in academic settings, is characterized by insufficient support and documentation. Therefore, our work constitutes a way to advance the modeling of the 3D organization of the human genome, while emphasizing the importance of establishing guidelines for software development and standardization.
A genetic framework for RNAi inheritance inCaenorhabditis elegans
Authors: Schreier Jan , Kielisch Fridolin , Ketting René F.
Published: October 2024

Gene regulation by RNA interference (RNAi) is a conserved process driven by double-stranded RNA (dsRNA). It responds to exogenous cues and drives endogenous gene regulation. InCaenorhabditis elegans, RNAi can be inherited from parents to offspring. While a number of factors have been implicated in this inheritance process, we do not understand how and when they function. Using a new inheritance assay, we establish a hierarchy amongst previously identified inheritance factors. The nuclear argonaute protein HRDE-1 was required for RNAi establishment in parents and offspring, but not for the inheritance process. In contrast, the cytoplasmic argonaute protein WAGO-3 was the only factor essential for inheritance, via sperm and oocyte, while not affecting establishment in either parent or offspring. We propose a cycle between nuclear and cytoplasmic argonaute proteins, where nuclear activity drives most of the silencing and cytoplasmic activity ensures inheritance. Finally, we implicate the RNA helicase ZNFX-1 as a factor that controls the entry of exogenous versus endogenous small RNAs into this cycle, ensuring a proper balance between gene silencing and activity.