bluuebunny/crossref_metadata_2025 huggingface dataset:
Self-Directed Home-Based Dim-Light Melatonin Onset Collection: The Circadia Pilot Study
Authors: Bormes Gregory , Love Jessica , Oluwaseun Akeju , Cherry Jakob , Kunorozva Lovemore , Qadri Salim , Rahman Shadab A. , Westover Brandon , Winkelman John , Lane Jacqueline M.
Published: May 2023

Study Objectives

To test the feasibility of a novel at-home salivary Dim Light Melatonin Onset (DLMO) assessment protocol to measure the endogenous circadian phase of 10 individuals (1 Advanced Sleep-Wake Phase Disorder patient (ASWPD), 4 Delayed Sleep-Wake Phase Disorder patients (DSWPD), and 5 controls).

Methods

The study involved 10 participants (sex at birth: females = 9; male= 1), who ranged between 27 to 63 years old, with an average age of 38 years old. Our study population consisted of 7 individuals who identified as white and 3 who identified as Asian. Our participants were diverse in gender identity (woman = 7, male = 1, transgender = 1, nonbinary = 1, none = 1).The study tracked the sleep and activity patterns of 10 individuals over a 5-6 week period using self-reported online sleep diaries and objective actigraphy data. Participants completed two self-directed DLMO assessments, approximately one week apart, adhering to objective compliance measures. Participants completed the study entirely remotely: they completed all sleep diaries and other evaluations online and were mailed a kit with all materials needed to perform the actigraphy and at-home sample collections.

Results

Salivary DLMO times were calculated for 8/10 participants using the Hockeystick method. DLMO times were on average 3 hours and 18 minutes earlier than self-reported sleep onset times (DSPD: 12:04 AM, controls: 9:55 PM.) Among the 6 participants for whom we calculated two separate DLMO times, DLMOs 1 and 2 were 96% correlated (p<0.0005.)

Conclusions

Our results indicate that self-directed, at-home DLMO assessments are feasible and accurate. The current protocol may serve as a framework to reliably assess circadian phase in both clinical and general populations.
Complexes of vertebrate TMC1/2 and CIB2/3 proteins form hair-cell mechanotransduction cation channels
Authors: Giese Arnaud P. J. , Weng Wei-Hsiang , Kindt Katie S. , Chang Hui Ho Vanessa , Montgomery Jonathan S. , Ratzan Evan M. , Beirl Alisha J. , Rivera Roberto Aponte , Lotthammer Jeffrey M. , Walujkar Sanket , Foster Mark P. , Zobeiri Omid A. , Holt Jeffrey R. , Riazuddin Saima , Cullen Kathleen E. , Sotomayor Marcos , Ahmed Zubair M.
Published: May 2023

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechano-electrical transduction (MET) apparatus. These interactions have been proposed to be functionally relevant across mechanosensory organs and vertebrate species. Here we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.
Stroke Hospitalization Administration & Monitoring: Routine Or Covid-19 Care (SHAMROCC)
Authors: Langlois-Thérien Timothé , Shamy Michel , Dewar Brian , Ramsay Tim , Lun Ronda , Blacquière Dylan , Fahed Robert , Dowlatshahi Dar , Stotts Grant , Ducroux Célina
Published: May 2023

BACKGROUND

Monitoring stroke patients in critical-care units for 24 hours after thrombolysis or endovascular thrombectomy is considered standard of care but is not evidence-based. Due to the Covid-19 pandemic, our center modified its protocol in April 2021 with 24-hour critical-care monitoring no longer being guaranteed for stroke patients. We aim to compare the incidence and timing of complications over the first 24 hours post-reperfusion therapies and their association to hospital unit in 2019, 2020 and 2021.

METHODS

We conducted a single-center retrospective cohort study. We analyzed data from stroke patients treated with thrombolysis and/or endovascular thrombectomy at our center in 2019 (pre-Covid-19, standard of care), 2020 (during Covid-19, standard of care) and 2021 (during Covid-19, new protocol). Data extracted included demographics, the nature and timing of complications within the first 24 hours, and the unit at the time of any complication. Major complications included neurologic deterioration, symptomatic intracranial hemorrhage, recurrent stroke, myocardial infarction, systemic bleeding, rapid assessment of critical events call, and death.

RESULTS

Three hundred forty-nine patients were included in our study: 78 patients in 2019, 115 patients in 2020, and 156 patients in 2021. In 2021, 32% of patients experienced at least one complication within the first 24 hours compared to 34% in 2020 and 27% in 2019. In 2021, 33% of patients admitted to critical-care units had a complication compared to 31% in 2020 and 26% in 2019. In 2021, 70% of complications had occurred by hour eight compared to 49% in 2020 and 29% in 2019. Patients with low NIHSS score at presentation and treated only with tPA had significantly less complications.

CONCLUSIONS

Despite the change of protocol in April 2021, the incidence and timing of complications did not significantly worsen compared to prior years and were not associated with hospital location. Further research is required to evaluate the necessity of critical care monitoring for 24 hours in this population, specifically patients with low NIHSS score at presentation and treated only with tPA.
Engineering a highly durable adeno-associated virus receptor for analytical applications
Authors: Yoshida Kouhei , Tsunekawa Yuji , Kurihara Kento , Watanabe Kazuya , Makino Yuriko , Tanaka Toru , Ide Teruhiko , Okada Takashi
Published: May 2023

Adeno-associated virus (AAV) is a major viral vector used in gene therapy. There are multiple AAV serotypes, and many engineered AAV serotypes with modified capsids altering tissue tropism are enthusiastically being developed. The universal AAV receptor (AAVR) is an essential receptor for multiple AAV infections. Since most AAV serotypes used in gene therapy infect cells via interaction with AAVR, the quantification of the vector-binding ability of AAV to AAVR could be an important quality check for therapeutic AAV vectors. To enable a steady evaluation of the AAV-AAVR interaction, we created an engineered AAVR through mutagenesis. Engineered AAVR showed high durability against acid while retaining its AAV-binding activity and an affinity chromatography column with engineered AAVR was also developed. This column enabled repeated binding and acid dissociation measurements of AAVR with various AAV serotypes. Our data showed that the binding affinities of AAV to AAVR were diverse among serotypes, providing insight into the relationship with the infection efficiency of AAV vectors. Thus, this affinity column can be used in process development for quality checks, quantitating capsid titers, and affinity purification of AAV vectors. Furthermore, this column may work for a useful tool of novel AAV vector capsid engineering.
Genetically Encodablein situGelation Redox-Responsive Collagen-Like Protein Hydrogel for Accelerating Diabetic Wound Healing
Authors: Jia Shuang , Wang Jie , Wang Xiaojie , Liu Xing , Li Shubin , Li Yimiao , Li Jiaqi , Wang Jieqi , Man Shad , Guo Zhao , Sun Yinan , Jia Zhenzhen , Wang Liyao , Li Xinyu
Published: May 2023

Genetically encoded collagen-like protein-based hydrogels have demonstrated remarkable efficacy in promoting the healing process in diabetic patients. However, the current methods for preparing these hydrogels pose significant challenges due to harsh reaction conditions and the reliance on chemical crosslinkers. In this study, we present a genetically encoded approach that allows for the creation of protein hydrogels without the need for chemical additives. Our design involves the genetic encoding of paired-cysteine residues at the C- and N-terminals of a meticulously engineered collagen-like recombination protein. The protein-based hydrogel undergoes a gel-sol transition in response to redox stimulation, achieving a sol-gel transition. We provide evidence that the co-incubation of the protein hydrogel with 3T3 cells not only enhances cell viability but also promotes cell migration. Moreover, the application of the protein hydrogel significantly accelerates the healing of diabetic wounds by upregulating the expression of COL-1α and CK-14, while simultaneously reducing oxidant stress in the wound microenvironment. Our study highlights a straightforward and chemical-free strategy for the preparation of redox-responsive protein hydrogels. Importantly, our findings underscore the potential of this hydrogel system for effectively treating diabetic wounds, offering a promising avenue for future therapeutic applications.
Telomerase inhibition by MST-312 sensitises breast cancer cells to anti-cancer properties of Plumbagin
Authors: Sameni Safoura , Viswanathan Ramya , Quan Gavin Yong-NG , Martinez-Lopezm Wilner , Hande Prakash
Published: May 2023

Breast cancer is the most common cause of malignancy and the second most common cause of cancer death in women. This heterogeneous disease is currently broadly classified as ER, PG positive luminal tumours, HER2 amplified tumours and triple-negative breast cancers (TNBC). Natural plant derived compounds are proven to be promising anti-cancer chemotherapeutics agents with minimal cytotoxic effects on healthy cells. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) is a phytochemical derived from the roots ofPlumbago zeylanicaand it is known to possess anti-cancer properties similar to other compounds of naphthoquinones. In about 90 % of cancer cells, the telomerase enzyme activity is revived to add telomeric repeats to evade apoptosis. In this study, a combinatorial approach of combining anti-cancer compound Plumbagin to induce genotoxicity and a potent telomerase inhibitor, MST-312 (synthetic derivative of tea-catechins) was used to determine the synthetic lethality in breast cancer cells such as MDA-MB-231 (TNBC) and MCF-7 (lumina) cells. MDA-MB-231 cells were responsive to combination treatment to both short-term (48 hours) and long-term treatment (14 days) in a synergistic manner, whereas in MCF-7, the combination treatment was more effective in the long-term regimen. Furthermore, the cytotoxic effects of the Plumbagin and MST-312 combination treatment were not recoverable after the short-term treatment. In conclusion, combination treatment of MST-312 and Plumbagin is proven to be more effective than single Plumbagin compound treatment, in inducing DNA damage and telomere dysfunction leading to greater genome instability, cell cycle arrest and eventually cell death in cancer cells.
Structural basis of substrate progression through the chaperonin cycle
Authors: Gardner Scott , Darrow Michele C. , Lukyanova Natasha , Thalassinos Konstantinos , Saibil Helen R.
Published: May 2023

The bacterial chaperonin GroEL-GroES promotes protein folding through ATP-regulated cycles of substrate protein binding, encapsulation, and release. Here, we have used cryoEM to determine structures of GroEL, GroEL-ADP·BeF3, and GroEL-ADP·AlF3-GroES all complexed with the model substrate Rubisco. Our structures provide a series of snapshots that show how the conformation and interactions of non-native Rubisco change as it proceeds through the GroEL-GroES reaction cycle. We observe specific charged and hydrophobic GroEL residues forming strong initial contacts with non-native Rubisco. Binding of ATP or ADP·BeF3to GroEL-Rubisco results in the formation of an intermediate GroEL complex displaying striking asymmetry in the ATP/ADP·BeF3-bound ring. In this ring, four GroEL subunits bind Rubisco and the other three are in the GroES-accepting conformation, explaining how GroEL can recruit GroES without releasing bound substrate. Our cryoEM structures of stalled GroEL-ADP·AlF3-Rubisco-GroES complexes show Rubisco folding intermediates interacting with GroEL-GroES via different sets of residues.
Improving Multichannel Raw Electroencephalography-based Diagnosis of Major Depressive Disorder by Pretraining Deep Learning Models with Single Channel Sleep Stage Data*
Authors: Ellis Charles A. , Sattiraju Abhinav , Miller Robyn L. , Calhoun Vince D.
Published: May 2023

As the field of deep learning has grown in recent years, its application to the domain of raw resting-state electroencephalography (EEG) has also increased. Relative to traditional machine learning methods or deep learning methods applied to extracted features, there are fewer methods for developing deep learning models on small raw EEG datasets. One potential approach for enhancing deep learning performance in this case is the use of transfer learning. In this study, we propose a novel EEG transfer learning approach wherein we first train a model on a large publicly available sleep stage classification dataset. We then use the learned representations to develop a classifier for automated major depressive disorder diagnosis with raw multichannel EEG. We find that our approach improves model performance, and we further examine how transfer learning affected the representations learned by the model through a pair of explainability analyses. Our proposed approach represents a significant step forward for the domain raw resting-state EEG classification. Furthermore, it has the potential to expand the use of deep learning methods across more raw EEG datasets and lead to the development of more reliable EEG classifiers.

Clinical Relevance

The proposed approach takes the field of deep learning in EEG a step closer to the robustness needed for clinical implementation.
Large Scale Foundation Model on Single-cell Transcriptomics
Authors: Hao Minsheng , Gong Jing , Zeng Xin , Liu Chiming , Guo Yucheng , Cheng Xingyi , Wang Taifeng , Ma Jianzhu , Song Le , Zhang Xuegong
Published: May 2023

Large-scale pretrained models have become foundation models leading to breakthroughs in natural language processing and related fields. Developing foundation models in life science for deciphering the “languages” of cells and facilitating biomedical research is promising yet challenging. We developed a large-scale pretrained model scFoundation with 100M parameters for this purpose. scFoundation was trained on over 50 million human single-cell transcriptomics data, which contain high-throughput observations on the complex molecular features in all known types of cells. scFoundation is currently the largest model in terms of the size of trainable parameters, dimensionality of genes and the number of cells used in the pre-training. Experiments showed that scFoundation can serve as a foundation model for single-cell transcriptomics and achieve state-of-the-art performances in a diverse array of downstream tasks, such as gene expression enhancement, tissue drug response prediction, single-cell drug response classification, and single-cell perturbation prediction.
Common evolutionary origins of the bacterial glycyl tRNA synthetase and alanyl tRNA synthetase
Authors: Alvarez-Carreño Claudia , Arciniega Marcelino , Ribas de Pouplana Lluis , Petrov Anton S. , Hernández-González Adriana , Valencia-Sánchez Marco Igor , Williams Loren Dean , Torres-Larios Alfredo
Published: May 2023

Aminoacyl-tRNA synthetases (aaRSs) establish the genetic code. Each aaRS covalently links a given canonical amino acid to a cognate set of tRNA isoacceptors. Glycyl tRNA aminoacylation is unusual in that it is catalyzed by different aaRSs in different lineages of the Tree of Life. We have investigated the phylogenetic distribution and evolutionary history of bacterial glycyl tRNA synthetase (bacGlyRS). This enzyme is found in early diverging bacterial phyla such as Firmicutes, Acidobacteria, and Proteobacteria, but not in archaea or eukarya. We observe relationships between each of six domains of bacGlyRS and six domains of four different RNA-modifying proteins. Component domains of bacGlyRS show common ancestry with i) the catalytic domain of class II tRNA synthetases; ii) the HD domain of the bacterial RNase Y; iii) the body and tail domains of the archaeal CCA-adding enzyme; iv) the anti-codon binding domain of the arginyl tRNA synthetase; and v) a previously unrecognized domain that we call ATL (Ancient tRNA latch). The ATL domain is found only in bacGlyRS and in the universal alanyl tRNA synthetase (uniAlaRS). Further, the catalytic domain of bacGlyRS is more closely related to the catalytic domain of uniAlaRS than to any other aminoacyl tRNA synthetase. The combined data suggest that the ATL and catalytic domains of these two enzymes are ancestral to bacGlyRS and uniAlaRS, which emerged from common protein ancestors by bricolage, stepwise accumulation of protein domains, before the last universal common ancestor of life.
Arpin deficiency increases actomyosin contractility and vascular permeability
Authors: Montoya-García Armando , Guerrero-Fonseca Idaira M. , Chánez-Paredes Sandra D. , Hernández-Almaraz Karina B. , León-Vega Iliana I. , Silva-Olivares Angélica , Bentazos Abigail , Mondragón-Castelán Mónica , Mondragón-Flores Ricardo , Salinas-Lara Citlaltepetl , Vargas-Robles Hilda , Schnoor Michael
Published: May 2023

Arpin was discovered as an inhibitor of the Arp2/3 complex localized at the lamellipodial tip of fibroblasts, where it regulated migration steering. Recently, we showed that arpin stabilizes the epithelial barrier in an Arp2/3-dependent manner. However, expression and functions of arpin in endothelial cells (EC) have not yet been described. Arpin mRNA and protein are expressed in EC and downregulated by pro-inflammatory cytokines. Arpin depletion in HUVEC causes the formation of actomyosin stress fibers leading to increased permeability in an Arp2/3-independent manner. Instead, inhibitors of ROCK1 and ZIPK, kinases involved in the generation of stress fibers, normalize the loss-of-arpin effects on actin filaments and permeability. Arpin-deficient mice are viable but show a characteristic vascular phenotype in the lung including edema, microhemorrhage and vascular congestion, increased F-actin levels and vascular permeability. Our data show that, apart from being an Arp2/3 inhibitor, arpin is also a regulator of actomyosin contractility and endothelial barrier integrity.

The expression and functions of arpin in endothelial cells are unknown. We show that arpin controls actomyosin contractility and endothelial barrier integrity in an Arp2/3-independent manner via ROCK1/ZIPK. Arpin-deficient mice are viable, but also show increased basal and induced vascular permeability.
A dual hit of α-synuclein internalization and immune challenge leads to the formation and maintenance of Lewy body-like inclusions in human dopaminergic neurons
Authors: Bayati Armin , Ayoubi Riham , Aguila Adriana , Zorca Cornelia E. , Han Chanshuai , Banks Emily , Nguyen-Renou Emmanuelle , Luo Wen , Shlaifer Irina , Del Cid-Pellitero Esther , Yaqubi Moein , Fon Edward A. , Stratton Jo Anne , Durcan Thomas M. , Nahirney Patrick C. , McPherson Peter S.
Published: May 2023

Lewy bodies (LBs), rich in α-synuclein, are a hallmark of Parkinson’s disease (PD). Understanding their biogenesis is likely to provide insight into the pathophysiology of PD, yet a cellular model for LB formation remains elusive. The realization that the immune challenge is a trigger for neurodegenerative diseases has been a breakthrough in the understanding of PD. Here, iPSC-derived human dopaminergic (DA) neurons from multiple healthy donors were found to form LB-like inclusions following treatment with α- synuclein preformed fibrils, but only when coupled to an immune challenge (interferon-gamma or interleukin-1 beta) or when co-cultured with activated microglia. Human cortical neurons derived from the same iPSC lines did not form LB-like inclusions. Exposure to interferon-gamma impairs autophagy in a lysosomal-specific mannerin vitro,similar to the disruption of proteostasis pathways that contribute to PD. We find that lysosomal membrane proteins LAMP1 and LAMP2 and transcription factors regulating lysosomal biogenesis and function are downregulated in DA but not cortical neurons. Finally, due to the excellent sample preservation afforded by cells compared to post-mortem PD brain tissue, we conclude that the LB-like inclusions in DA neurons are membrane-bound, suggesting they are not limited to the cytoplasmic compartment.

In Brief

Bayati et al. identify that iPSC-derived dopaminergic neurons undergoing a dual hit treatment of exogenous α-synuclein fibrils and proinflammatory cytokines form Lewy body-like inclusions. The dual hit treatment also led to the downregulation of lysosomal proteins. Characterization of inclusions revealed that inclusions were membrane-bound and LC3B-positive, suggesting they are dysfunctional autophagosomes.

Highlights

α-synuclein preformed fibril administration coupled with Interferon-gamma exposure leads dopaminergic neurons to form Lewy body-like inclusions

Inclusions are filamentous, membranous, and filled with aberrant organelles

Impaired autophagic flux and downregulation of TFEB, NRF2, LAMP1, and LAMP2 correlated with inclusion formation

Activation of NRF2 through the treatment of neurons with the antioxidant perillaldehyde, prevents inclusion formation
A comprehensive workflow for target adaptive sampling long-read sequencing applied to hereditary cancer patient genomes
Authors: Nakamura Wataru , Hirata Makoto , Oda Satoyo , Chiba Kenichi , Okada Ai , Mateos Raúl Nicolás , Sugawa Masahiro , Iida Naoko , Ushiama Mineko , Tanabe Noriko , Sakamoto Hiromi , Kawai Yosuke , Tokunaga Katsushi ,   NCBN Controls WGS Consortium, Tsujimoto Shinichi , Shiba Norio , Ito Shuichi , Yoshida Teruhiko , Shiraishi Yuichi
Published: June 2023

Innovations in sequencing technology have led to the discovery of novel mutations that cause inherited diseases. However, many patients with suspected genetic diseases remain undiagnosed. Long-read sequencing technologies are expected to significantly improve the diagnostic rate by overcoming the limitations of short-read sequencing. In addition, Oxford Nanopore Technologies (ONT) offers a computationally-driven target enrichment technology, adaptive sampling, which enables intensive analysis of targeted gene regions at low cost. In this study, we developed an efficient computational workflow for target adaptive sampling long-read sequencing (TAS-LRS) and evaluated it through application to 33 genomes collected from suspected hereditary cancer patients. Our workflow can identify single nucleotide variants with nearly the same accuracy as the short-read platform and elucidate complex forms of structural variations. We also newly identified SVAs affecting theAPCgene in two patients with familial adenomatous polyposis, as well as their sites of origin. In addition, we demonstrated that off-target reads from adaptive sampling, which are typically discarded, can be effectively used to accurately genotype common SNPs across the entire genome, enabling the calculation of a polygenic risk score. Furthermore, we identified allele-specificMLH1promoter hypermethylation in a Lynch syndrome patient. In summary, our workflow with TAS-LRS can simultaneously capture monogenic risk variants including complex structural variations, polygenic background as well as epigenetic alterations, and will be an efficient platform for genetic disease research and diagnosis.
Reducing the equity gap in under-5 mortality through an innovative community health program in Ethiopia: An implementation research study
Authors: Drown Laura , Amberbir Alemayehu , Teklu Alula M. , Zelalem Meseret , Tariku Abreham , Tadesse Yared , Gebeyehu Solomon , Semu Yirdachew , Ntawukuriryayo Jovial Thomas , VanderZanden Amelia , Binagwaho Agnes , Hirschhorn Lisa R.
Published: May 2023

Background

The Ethiopian government implemented a national community health program, the Health Extension Program (HEP), to provide community-based health services to address persisting access-related barriers to care using health extension workers (HEWs). We used implementation research to understand how Ethiopia leveraged the HEP to widely implement evidence-based interventions (EBIs) known to reduce under-5 mortality (U5M) and address health inequities.

Methods

This study was part of a six-country case study series using implementation research to understand how countries implemented EBIs between 2000-2015. Our mixed-methods research was informed by a hybrid implementation science framework using desk review of published and gray literature, analysis of existing data sources, and 11 key informant interviews. We used implementation of pneumococcal conjugate vaccine (PCV-10) and integrated community case management (iCCM) to illustrate Ethiopia’s ability to rapidly integrate interventions into existing systems at a national level through leveraging the HEP and other implementation strategies and contextual factors which influenced implementation outcomes.

Results

Ethiopia implemented numerous EBIs known to address leading causes of U5M, leveraging the HEP as a platform for delivery to successfully introduce and scale new EBIs nationally. By 2014/15, estimated coverage of three doses of PCV-10 was at 76%, with high acceptability (nearly 100%) of vaccines in the community. Between 2000 and 2015, we found evidence of improved care-seeking; coverage of oral rehydration solution for treatment of diarrhea, a service included in iCCM, doubled over this period.

HEWs made health services more accessible to rural and pastoralist communities, which account for over 80% of the population, with previously low access, a contextual factor that had been a barrier to high coverage of interventions.

Conclusions

Leveraging the HEP as a platform for service delivery allowed Ethiopia to successfully introduce and scale existing and new EBIs nationally, improving feasibility and reach of introduction and scale-up of interventions. Additional efforts are required to reduce the equity gap in coverage of EBIs including PCV-10 and iCCM among pastoralist and rural communities. As other countries continue to work towards reducing U5M, Ethiopia’s experience provides important lessons in effectively delivering key EBIs in the presence of challenging contextual factors.
Analytical validation of HepatoPredict kit to assess hepatocellular carcinoma prognosis prior to a liver transplantation
Authors: Gonçalves-Reis Maria , Proença Daniela , Frazão Laura P. , Neto João L. , Silva Sílvia , Pinto-Marques Hugo , Pereira-Lea José B. , Cardoso Joana
Published: June 2023

Background

The best curative treatment for hepatocellular carcinoma (HCC) is liver transplant (LT), but the limited number of organs available for LT dictates strict eligibility criteria. Despite this patient selection stringency, current criteria often fail in pinpointing patients at risk of HCC relapse and in identifying good prognosis patients that could benefit from a LT. HepatoPredict kit was developed and clinically validated to forecast the benefit of LT in patients diagnosed with HCC. By combining clinical variables and a gene expression signature in an ensemble of machine learning algorithms, HepatoPredict stratifies HCC patients according to their risk of relapse after LT.

Methods

Aiming at the characterization of the analytical performance of HepatoPredict kit in terms of sensitivity, specificity and robustness, several variables were tested which included reproducibility between operators and between RNA extractions and RT-qPCR runs, interference of input RNA levels or varying reagent levels. The described methodologies, included in the HepatoPredict kit, were tested according to analytical validation criteria of multi-target genomic assays described in guidelines such as ISO201395-2019, MIQE, CLSI-MM16, CLSI-MM17, and CLSI-EP17-A. Furthermore, a new retrained version of the HepatoPredict algorithms is also presented and tested.

Results

The results of the analytical performance demonstrated that the HepatoPredict kit performed within the required levels of robustness (p> 0.05), analytical specificity (inclusivity ≥ 95 %), and sensitivity (LoB, LoD, linear range, and amplification efficiency between 90 – 110 %). The introduced operator, equipment, input RNA and reagents into the assay had no significant impact on HepatoPredict classifier results. As demonstrated in a previous clinical validation, a new retrained version of the HepatoPredict algorithm still outperformed current clinical criteria, in the accurate identification of HCC patients that more likely will benefit from a LT.

Conclusions

Despite the variations in the molecular and clinical variables, the prognostic information obtained with HepatoPredict kit and does not change and can accurately identify HCC patients more likely to benefit from a LT. HepatoPredict performance robustness also validates its easy integration into standard diagnostic laboratories.
STAREG: an empirical Bayesian approach to detect replicable spatially variable genes in spatial transcriptomic studies
Authors: Li Yan , Zhou Xiang , Chen Rui , Zhang Xianyang , Cao Hongyuan
Published: May 2023

Identifying replicable genes that display spatial expression patterns from different yet related spatially resolved transcriptomic studies provides stronger scientific evidence and more powerful inference. We present an empirical Bayesian method, STAREG, for identifying replicable spatially variable genes in data generated from various spatially resolved transcriptomic techniques. STAREG models the joint distribution ofp-values from different studies with a mixture model and accounts for the heterogeneity of different studies. It provides effective control of the false discovery rate and has higher power by borrowing information across genes and different studies. Moreover, it provides different rankings of important spatially variable genes. With the EM algorithm in combination with pool-adjacent-violator-algorithm (PAVA), STAREG is scalable to datasets with tens of thousands of genes measured on tens of thousands of spatial spots without any tuning parameters. Analyzing three pairs of spatially resolved transcriptomic datasets using STAREG, we show that it makes biological discoveries that otherwise cannot be obtained by using existing methods.
Non-invasive vagus nerve stimulation decreases vagally mediated heart rate variability
Authors: Kaduk Kristin , Petrella Alessandro , Müller Sophie J. , Koenig Julian , Kroemer Nils B.
Published: May 2023

The vagus nerve plays a critical role in balancing the body’s physiological functions, including the cardiovascular system. Measures of heart rate (HR) and its variability (HRV) may provide non-invasive proxies of vagal activity in humans, but transcutaneous auricular vagus nerve stimulation (taVNS) has produced mixed effects so far—limited by a lack of studies stimulating the right branch. Here, we used a randomized cross-over design to study the effects of taVNS on HR and HRV. To estimate how the side of the stimulation (left vs. right ear) affects cardiovascular function, we recorded an electrocardiogram in four sessions per person (factors: Stimulation × Side). To evaluate potential interactions with physiological states, we investigated three phases per session: baseline, during stimulation (taVNS vs. sham), and after consuming a milkshake (∼400 kcal) with concurrent stimulation. First, we found moderate evidence against an effect of taVNS on HR (BF10=0.21). Second, taVNS decreased HRV (multivariatep=.004) independent of physiological state with strong evidence for RMSSD (BF10=15.11) and HF-HRV (BF10=11.80). Third, taVNS-induced changes were comparable across sides and more strongly correlated (vs. sham), indicating similar cardiovascular effects independent of the stimulation side. We conclude that taVNS reduces HRV without altering HR, contradicting the common assumption that increased HRV indexes a heightened vagal tone. Instead, our results support a putative role of vagal afferent activation in arousal. Crucially, modulatory effects on the cardiovascular system can be safely elicited by taVNS on both sides, opening new options for treatment.

Graphical Abstract

Created withBioRender.com
New strategy for bioplastic and exopolysaccharides production: Enrichment of field microbiomes with cyanobacteria
Authors: Altamira-Algarra Beatriz , Rueda Estel , Lage Artai , San León David , Martínez-Blanch Juan F. , Nogales Juan , García Joan , Gonzalez-Flo Eva
Published: May 2023

Seven photosynthethic microbiomes were collected from field environmental samples to test their potential in polyhydroxybutirate (PHB) and exopolysaccharides (EPS) production, two alternatives to chemical-based polymers. Microscope observations together with microbial sequence analysis revealed the microbiome enrichment in cyanobacteria after culture growth under phosphorus limitation. PHB and EPS production were studied under three culture factors (phototrophy, mixotrophy and heterotrophy) by evaluating and optimizing the effect of three parameters (organic and inorganic carbon and days under light:dark cycles) by Box-Behnken design. Results showed that optimal conditions for both biopolymers synthesis were microbiome-dependent; however, the addition of organic carbon boosted PHB production in all the tested microbiomes, producing up to 14%dcwPHB with the addition of 1.2 g acetate·L-1and seven days under light:dark photoperiods. The highest EPS production was 59 mg·L-1with the addition of 1.2 g acetate·L-1and four days under light:dark photoperiods. The methodology used in this article is suitable for enriching microbiomes in cyanobacteria, and for testing the best conditions for bioproducts synthesis for further scale up.
Multisensory integration enhances audiovisual responses in the Mauthner cell
Authors: Otero-Coronel Santiago , Preuss Thomas , Medan Violeta
Published: May 2023

Multisensory integration combines information from multiple sensory modalities to create a coherent perception of the world. In contexts where sensory information is limited or equivocal, it also allows animals to integrate individually ambiguous stimuli into a clearer or more accurate percept and, thus, react with a more adaptive behavioral response. Although responses to multisensory stimuli have been described at the neuronal and behavioral levels, a causal or direct link between these two is still missing. In this study, we studied the integration of audiovisual inputs in the Mauthner cell, a command neuron necessary and sufficient to trigger a stereotypical escape response in fish. We performed intracellular recordings in adult goldfish while presenting a diverse range of stimuli to determine which stimulus properties affect their integration. Our results show that stimulus modality, intensity, temporal structure, and interstimulus delay affect input summation. Mechanistically, we found that the distinct decay dynamics of feedforward inhibition triggered by auditory and visual stimuli can account for certain aspects of input integration. Altogether, this is a rare example of the characterization of multisensory integration in a cell with clear behavioral relevance, providing both phenomenological and mechanistic insights into how multisensory integration depends on stimulus properties.
Melanin found in wheat spike husks
Authors: Bazhenov Mikhail S. , Litvinov Dmitry Y. , Divashuk Mikhail G.
Published: May 2023

Melanin is the dark polymer pigment found in all kingdoms of life. Plant melanin, formed through the oxidation and polymerization of phenolic compounds, does not contain nitrogen, however it possesses similar properties with melanin of animal, fungal or bacterial origin. Melanin in plants is usually found in seed coats or fruit pericarp and is ascribed mechanical barrier or some other protective functions. Wild and formerly locally cultivated wheat species, like Persian wheat (Triticum carthlicumNevski) frequently exhibit black color of spike husks and awns. The pigment causing it and the biological purpose of this coloration was not clarified before. In this paper using standard chemical extraction procedures for anthocyanins and melanin, ultraviolet-visible-near-infrared spectroscopy and hyperspectral imaging, we prove that the black color of Persian wheat spikes is caused by melanin but not anthocyanins. Also, we show that the dark pigment in husks and awns is located in epiderma and subepidermal sclerenchyma cells, that implies melanin potentially to have mechanical-enhancing and protection function. Other possible functions of melanin in cereals are discussed.
The CDK12 inhibitor SR-4835 functions as a molecular glue that promotes cyclin K degradation in melanoma
Authors: Houles Thibault , Boucher Jonathan , Lavoie Geneviève , MacLeod Graham , Lin Sichun , Angers Stephane , Roux Philippe P.
Published: May 2023

CDK12 is a transcriptional cyclin-dependent kinase (CDK) that interacts with cyclin K to regulate different aspects of gene expression. The CDK12-cyclin K complex phosphorylates several substrates, including RNA polymerase II (Pol II), and thereby regulates transcription elongation, RNA splicing, as well as cleavage and polyadenylation. Because of its implication in cancer, including breast cancer and melanoma, multiple pharmacological inhibitors of CDK12 have been identified to date, including THZ531 and SR-4835. While both CDK12 inhibitors affect Poll II phosphorylation, we found that SR-4835 uniquely promotes cyclin K degradation via the proteasome. Using loss-of-function genetic screening, we found that SR-4835 cytotoxicity depends on a functional CUL4-RBX1-DDB1 ubiquitin ligase complex. Consistent with this, we show that DDB1 is required for cyclin K degradation, and that SR-4835 promotes DDB1 interaction with the CDK12-cyclin K complex. Docking studies and structure-activity relationship analyses of SR-4835 revealed the importance of the benzimidazole side-chain in molecular glue activity. Together, our results indicate that SR-4835 acts as a molecular glue that recruits the CDK12-cyclin K complex to the CUL4-RBX1-DDB1 ubiquitin ligase complex to target cyclin K for degradation.
Cell specification and functional interactions in the pig blastocyst inferred from single cell transcriptomics
Authors: Dufour Adrien , Kurilo Cyril , Stöckl Jan B. , Laloë Denis , Bailly Yoann , Manceau Patrick , Martins Frédéric , Turhan Ali G. , Ferchaud Stéphane , Pain Bertrand , Fröhlich Thomas , Foissac Sylvain , Artus Jérôme , Acloque Hervé
Published: May 2023

The early embryonic development of the pig comprises a longin uteropre- and peri-implantation development, which dramatically differs from mouse and human. During this pro-tracted peri-implantation period, an intimate dialogue between the embryo and the uterus is established through a complex series of paracrine signals. It leads to concomitant drastic changes in the embryonic morphology and uterine receptivity to implantation. From day 7 after fertilization, the spherical blastocyst elongates as ovoid, tubular and filamentous wich transforms from a 0.5-1 mm diameter sphere to a 1000 mm long filamentous blastocyst at day 16. At the same time, the inner cell mass moves up to the trophoblast as the Rauber’s layer disappears and evolves as an embryonic disc that is directly exposed to molecules present in the uterine fluids. These drastic changes occurring before implantation are driven and coordinated by interactions between embryonic cells and maternal tissues.

To better understand the biology of pig embryos before implantation, we generated a large dataset of single-cell RNAseq at different embryonic stages (early and hatched blastocyst, spheroid and ovoid conceptus) and proteomic datasets from corresponding uterine fluids. These data were cleaned, filtered and represent a total of 34,888 cells. We first characterised the embryonic and extra-embryonic cell populations and their evolution, and identified population-specific markers of the three main populations (epiblast, trophectoderm and hypoblast). Our analysis also confirmed known functions and predicted new biological functions associated with these cell populations.We then inferred gene regulatory networks working on modules of gene regulation (regulon) and selected those that were specifically active in each embryonic population. We confirmed the relevance of the identified regulons by a meta-analysis of two other scRNAseq datasets (porcine and human preimplantation embryos). We then linked these regulons to signalling pathways and biological processes. Our results confirm the molecular specificity and functionality of the three main cell populations and identify novel stage-specific subpopulations. In particular, we discovered two previously unknown subpopulations of the trophectoderm, one characterised by the expression of LRP2, which could represent a subpopulation of progenitor cells, and the other, expressing many pro-apoptotic markers, which could correspond to the cells of the Rauber’s layer. We also provide new insights into the biology of these populations, their reciprocal functional interactions and the molecular dialogue established with the maternal uterine environment.
Genetically engineering endothelial niche in human kidney organoids enables multilineage maturation, vascularization and de novo cell types
Authors: Maggiore Joseph C. , LeGraw Ryan , Przepiorski Aneta , Velazquez Jeremy , Chaney Christopher , Streeter Evan , Silva-Barbosa Anne , Franks Jonathan , Hislop Joshua , Hill Alex , Wu Haojia , Pfister Katherine , Howden Sara E. , Watkins Simon C. , Little Melissa , Humphreys Benjamin D. , Watson Alan , Stolz Donna B. , Kiani Samira , Davidson Alan J. , Carroll Thomas J. , Cleaver Ondine , Sims-Lucas Sunder , Ebrahimkhani Mo R. , Hukriede Neil A.
Published: May 2023

Vascularization plays a critical role in organ maturation and cell type development. Drug discovery, organ mimicry, and ultimately transplantation in a clinical setting thereby hinges on achieving robust vascularization ofin vitroengineered organs. Here, focusing on human kidney organoids, we overcome this hurdle by combining an inducibleETS translocation variant 2(ETV2) human induced pluripotent stem cell (iPSC) line, which directs endothelial fate, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive vascularization by endothelial cells with an identity most closely related to endogenous kidney endothelia. Vascularized organoids also show increased maturation of nephron structures including more mature podocytes with improved marker expression, foot process interdigitation, an associated fenestrated endothelium, and the presence of renin+cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Furthermore, this approach is orthogonal to native tissue differentiation paths, hence readily adaptable to other organoid systems and thus has the potential for a broad impact on basic and translational organoid studies.

Translational Statement

Developing therapies for patients with kidney diseases relies on a morphologically and physiologically representativein vitromodel. Human kidney organoids are an attractive model to recapitulate kidney physiology, however, they are limited by the absence of a vascular network and mature cell populations. In this work, we have generated a genetically inducible endothelial niche that, when combined with an established kidney organoid protocol, induces the maturation of a robust endothelial cell network, induces a more mature podocyte population, and induces the emergence a functional renin population. This advance significantly increases the clinical relevance of human kidney organoids for etiological studies of kidney disease and future regenerative medicine strategies.

Graphical Abstract

Genetically engineered endothelial niche induces mature cell populations in human kidney organoids
A GPCR screening in human keratinocytes identifies that the metabolite receptor HCAR3 controls epithelial proliferation, migration, and cellular respiration
Authors: Pilar Pedro M. , Lund Katherine , Kang Sun Woo Sophie , Chen Ting , Stuelten Christina H. , Porat-Shliom Natalie , Iglesias-Bartolome Ramiro
Published: May 2023

Epithelial cells in the skin and other tissues rely on signals from their environment to maintain homeostasis and respond to injury, and G protein-coupled receptors (GPCRs) play a critical role in this communication. A better understanding of the GPCRs expressed in epithelial cells will contribute to understanding the relationship between cells and their niche and could lead to developing new therapies to modulate cell fate. This study used human primary keratinocytes as a model to investigate the specific GPCRs regulating epithelial cell proliferation and differentiation. We identified three key receptors, hydroxycarboxylic acid-receptor 3 (HCAR3), leukotriene B4-receptor 1 (LTB4R), and G Protein-Coupled Receptor 137 (GPR137) and found that knockdown of these receptors led to changes in numerous gene networks that are important for maintaining cell identity and promoting proliferation while inhibiting differentiation. Our study also revealed that the metabolite receptor HCAR3 regulates keratinocyte migration and cellular metabolism. Knockdown of HCAR3 led to reduced keratinocyte migration and respiration, which could be attributed to altered metabolite use and aberrant mitochondrial morphology caused by the absence of the receptor. This study contributes to understanding the complex interplay between GPCR signaling and epithelial cell fate decisions.
The role of subicular VIP-expressing interneurons on seizure dynamics in the intrahippocampal kainic acid model of temporal lobe epilepsy
Authors: Rahimi Sadegh , Salami Pariya , Matulewicz Pawel , Schmuck Armin , Bukovac Anneliese , Ramos-Prats Arnau , Tasan Ramon Osman , Drexel Meinrad
Published: May 2023

The subiculum, a key output region of the hippocampus, is increasingly recognized as playing a crucial role in seizure initiation and spread. The subiculum consists of glutamatergic pyramidal cells, which show alterations in intrinsic excitability in the course of epilepsy, and multiple types of GABAergic interneurons, which exhibit varying characteristics in epilepsy. In this study, we aimed to assess the role of the vasoactive intestinal peptide interneurons (VIP-INs) of the ventral subiculum in the pathophysiology of temporal lobe epilepsy. We observed that an anatomically restricted inhibition of VIP-INs of the ventral subiculum was sufficient to reduce seizures in the intrahippocampal kainic acid model of epilepsy, changing the circadian rhythm of seizures, emphasizing the critical role of this small cell population in modulating TLE. As we expected, permanent unilateral or bilateral silencing of VIP-INs of the ventral subiculum in non-epileptic animals did not induce seizures or epileptiform activity. Interestingly, transient activation of VIP-INs of the ventral subiculum was enough to increase the frequency of seizures in the acute seizure model. Our results offer new perspectives on the crucial involvement of VIP-INs of the ventral subiculum in the pathophysiology of TLE. Given the observed predominant disinhibitory role of the VIP-INs input in subicular microcircuits, modifications of this input could be considered in the development of therapeutic strategies to improve seizure control.