bluuebunny/crossref_metadata_2025 huggingface dataset:
Lifespan differences in visual short-term memory load-modulated functional connectivity
Authors: Lugtmeijer Selma , Geerligs Linda , Tsvetanov Kamen A. , Mitchell Daniel J. , Cam-CAN  , Campbell Karen L.
Published: September 2022

Working memory is critical to higher-order executive processes and declines throughout the adult lifespan. However, our understanding of the neural mechanisms underlying this decline is limited. Recent work suggests that functional connectivity between frontal control and posterior visual regions may be critical, but examinations of age differences therein have been limited to a small set of brain regions and extreme group designs (i.e., comparing young and older adults). In this study, we build on previous research by using a lifespan cohort and a whole-brain approach to investigate working memory load-modulated functional connectivity in relation to age and performance. The article reports on analysis of the Cambridge Centre for Ageing and Neuroscience (Cam-CAN) data. Participants from a population-based lifespan cohort (N=111, age 23-86) performed a visual short-term memory task during functional magnetic resonance imaging. Visual short-term memory was measured with a delayed recall task for visual motion with three different loads. Whole-brain load-modulated connectivity was estimated using psychophysiological interactions in a hundred regions of interest, sorted into seven networks (Schaefer et al., 2018, Yeo et al., 2011). Results showed that load-modulated functional connectivity was strongest within the dorsal attention network followed by the visual network during encoding and maintenance. With increasing age, load-modulated functional connectivity strength decreased throughout the cortex. Within the dorsal attention network, increased load-modulated connectivity strength was related to better task performance in an age-invariant way. Our results demonstrate the widespread negative impact of age on the modulation of functional connectivity by working memory load. Older adults might already be close to ceiling in terms of their resources at the lowest load and therefore less able to further increase connectivity with increasing task demands.

Highlights

We examine visual short-term memory load-related functional connectivity

And how age and performance affect modulation of connectivity by memory load

Modulation of connectivity is strongest in dorsal attention and visual networks

Load-modulated connectivity strength decreases with increasing age

Load-modulated connectivity in the dorsal attention network relates to performance
Gain-of-function dynamin-2 mutations linked to centronuclear myopathy impair Ca2+-induced exocytosis in human myoblasts
Authors: Bayonés Lucas , Guerra-Fernández María José , Hinostroza Fernando , Báez-Matus Ximena , Vásquez-Navarrete Jacqueline , Gallo Luciana I. , Parra Sergio , Martínez Agustín D. , González-Jamett Arlek , Marengo Fernando D. , Cárdenas Ana M.
Published: September 2022

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as reporter of the GLUT4 vesicle-trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 μM ionomycin, indicating that in addition to impair exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.
Associations of Minor Histocompatibility Antigens with Clinical Outcomes Following Allogeneic Hematopoietic Cell Transplantation
Authors: Jadi Othmane , Tang Hancong , Olsen Kelly , Vensko Steven , Zhu Qianqian , Wang Yiwen , Haiman Christopher A , Pooler Loreall , Sheng Xin , Brock Guy , Webb Amy , Pasquini Marcelo C. , McCarthy Philip L , Spellman Stephen R. , Hahn Theresa , Vincent Benjamin , Armistead Paul , Sucheston-Campbell Lara E.
Published: September 2022

The role of minor histocompatibility antigens (mHAs) in mediating graft versus leukemia (GvL) and graft versus host disease (GvHD) following allogeneic hematopoietic cell transplantation (alloHCT) is recognized but not well-characterized. By implementing improved methods for mHA prediction in two large patient cohorts, this study aimed to comprehensively explore the role of mHAs in alloHCT by analyzing whether (1) the number of predicted mHAs, or (2) individual mHAs are associated with clinical outcomes using multi-variate survival models corrected for multiple testing. Cox proportional hazard results showed that patients with a class I mHA count greater than the population median had an increased hazard of GvHD mortality (HR=1.39, 95%CI 1.01, 1.77, P=0.046). Competing risk analyses identified the class I mHAs DLRCKYISL (gene GSTP), WEHGPTSLL (CRISPLD2) and STSPTTNVL (SERPINF2) were associated with increased GVHD death (HR=2.84, 95%CI 1.52, 5.31, P=0.01), decreased leukemia-free survival (LFS) (HR=1.94,95%CI 1.27, 2.95, P=0.044), and increased disease-related mortality (DRM) (HR=2.32, 95%CI 1.5, 3.6, P=0.008), respectively. One class II mHA YQEIAAIPSAGRERQ (TACC2) was associated with increased risk of treatment-related mortality (TRM) (HR=3.05, 95%CI 1.75, 5.31, P=0.02). WEHGPTSLL and STSPTTNVL were present in conjunction within HLA haplotype B*40:01-C*03:04 and showed a positive dose-response relationship with increased all-cause mortality and DRM and decreased LFS, indicating these two mHAs contribute to risk of mortality in an additive manner. Our study reports the first large scale investigation of the associations of predicted class I and class II mHA peptides with clinical outcomes following alloHCT.

Graphical Abstract
Membrane Retrieval after Immediately Releasable Pool (IRP) Exocytosis is produced by Dynamin-Dependent and Dynamin-Independent Mechanisms
Authors: Bayonés Lucas , Montenegro Mauricio , Moya-Díaz José , Alfonso-Bueno Samuel , Gallo Luciana I. , Marengo Fernando D.
Published: September 2022

The importance of the immediately releasable pool (IRP) of vesicles was proposed to reside in the maintenance of chromaffin cell secretion during the firing of action potentials at basal physiological frequencies. To accomplish this duty, IRP should be replenished as a function of time. We have previously reported that an action potential-like stimulus (APls) triggers the release of ∽50% IRP, followed by a fast dynamin-dependent endocytosis and an associated rapid replenishment process. In this work we investigated the endocytosis and IRP replenishment produced after the exocytosis of variable IRP fractions in mice primary chromaffin cell cultures. Exocytosis and endocytosis were estimated by membrane capacitance measurements obtained in patch-clamped cells. In addition to the dynamin-dependent fast endocytosis activated after the application of APls or 5 ms squared depolarizations, we found that depolarizations lasting 25-50 ms, which release >80% of IRP, are related with a fast dynamin-independent, Ca2+- and protein kinase C (PKC)-dependent endocytosis (time constant < 1 s). PKC inhibitors, such as staurosporine, bisindolylmaleimide XI and prolonged treatments with high concentrations of phorbol esters, reduced and decelerated this endocytosis. Additionally, we found that the inhibition of PKC also abolished a slow component of replenishment (time constant ∽8 s) observed after total IRP exocytosis. Therefore, our results suggest that PKC contributes to the coordination of membrane retrieval and vesicle replenishment mechanisms that occur after the complete exocytosis of IRP.
Artificial Intelligence based Liver Portal Tract Region Identification and Quantification with Transplant Biopsy Whole-Slide Images
Authors: Yu Hanyi , Sharifai Nima , Jiang Kun , Wang Fusheng , Teodoro George , Farris Alton B. , Kong Jun
Published: September 2022

Liver fibrosis staging is clinically important for liver disease progression prediction. As the portal tract fibrotic quantity and size in a liver biopsy correlate with the fibrosis stage, an accurate analysis of portal tract regions is clinically critical. Manual annotations of portal tract regions, however, are time-consuming and subject to large inter- and intra-observer variability. To address such a challenge, we develop a Multiple Up-sampling and Spatial Attention guided UNet model (MUSA-UNet) to segment liver portal tract regions in whole-slide images of liver tissue slides. To enhance the segmentation performance, we propose to use depth-wise separable convolution, the spatial attention mechanism, the residual connection, and multiple up-sampling paths in the developed model. This study includes 53 histopathology whole slide images from patients who received liver transplantation. In total, 6,012 patches derived from 30 images are used for our deep learning model training and validation. The remaining 23 whole slide images are utilized for the model testing. The average liver portal tract segmentation performance of the developed MUSA-UNet is 0.94 (Precision), 0.85 (Recall), 0.89 (F1 Score), 0.89 (Accuracy), 0.80 (Jaccard Index), and 0.91 (Fowlkes–Mallows Index), respectively. The clinical Scheuer fibrosis stage presents a strong correlation with the resulting average portal tract fibrotic area (R=0.681, p<0.001) and portal tract percentage (R=0.335, p=0.02) computed from the MUSA-UNet segmentation results. In conclusion, our developed deep learning model MUSA-UNet can accurately segment portal tract regions from whole-slide images of liver tissue biopsies, presenting its promising potential to assist liver disease diagnosis in a computational manner.
Biologic that disrupts PDE11A4 homodimerization in hippocampus CA1 reverses age-related proteinopathies in PDE11A4 and cognitive decline of social memories
Authors: Pilarzyk Katy , Capell Will , Rips-Goodwin Audrey , Porcher Latarsha , Kelly Michy P.
Published: September 2022

Age-related proteinopathies in phosphodiesterase 11A (PDE11A), an enzyme that degrades 3’,5’-cAMP/cGMP and is enriched in the ventral hippocampal formation (VHIPP), drive age-related cognitive decline (ARCD) of social memories. In the VHIPP, age-related increases in PDE11A4 occur specifically within the membrane compartment and ectopically accumulate in filamentous structures termed ghost axons. Previousin vitrostudies show that disrupting PDE11 homodimerization by expressing an isolated PDE11A-GAFB domain, which acts as a “negative sink” for monomers, selectively degrades membrane-associated PDE11A4 and prevents the punctate accumulation of PDE11A4. Therefore, we determined if disrupting PDE11A4 homodimerizationin vivovia the expression of an isolated PDE11A4-GAFB domain would be sufficient to reverse 1) age-related accumulations of PDE11A4 in VHIPP ghost axons and 2) ARCD of social memories. Indeed,in vivolentiviral expression of the isolated PDE11A4-GAFB domain in hippocampal CA1 reversed the age-related accumulation of PDE11A4 in ghost axons, reversed ACRD of social transmission of food preference memory (STFP), and improved remote long-term memory for social odor recognition (SOR) without affecting memory for non-social odor recognition.In vitrostudies suggest that disrupting homodimerization of PDE11A4 does not directly alter the catalytic activity of the enzyme but may reverse age-related decreases in cGMP by dispersing the accumulation of the enzyme independently of other intramolecular mechanisms previously established to disperse PDE11A4 (e.g., phosphorylation of PDE11A4 at serine 162). Altogether, these data suggest that a biologic designed to disrupt PDE11A4 homodimerization may serve to ameliorate age-related deficits in hippocampal cyclic nucleotide signaling and subsequent ARCD of remote social memory.
In Totoimaging of early Enteric Nervous System Development reveals that gut colonization is tied to proliferation downstream of Ret
Authors: Baker Phillip A. , Ibarra-García-Padilla Rodrigo , Venkatesh Akshaya , Singleton Eileen W. , Uribe Rosa. A.
Published: September 2022

The enteric nervous system (ENS) is a vast intrinsic network of neurons and glia within the gastrointestinal tract and is largely derived from enteric neural crest cells (ENCCs) that emigrate into the gut during vertebrate embryonic development. Study of ENCC migration dynamics and their genetic regulators provides great insights into fundamentals of collective cell migration and nervous system formation, and are a pertinent subject for study due to their relevance to the human congenital disease, Hirschsprung disease (HSCR). For the first time, we performedin totogut imaging and single-cell generation tracing of ENCC migration in WT and a novelretheterozygous background zebrafish (retwmr1/+) to gain insight into ENCC dynamicsin vivo. We observed thatretwmr1/+zebrafish produced fewer ENCCs while localized along the gut, which failed to reach the hindgut, resulting in HSCR-like phenotypes. Specifically, we observed a proliferation dependent migration mechanism, where cell divisions were associated with inter-cell distances and migration speed. Lastly, we detected a premature neuronal differentiation gene expression signature inretwmr1/+ENCCs, collectively suggesting that Ret signaling may function to regulate maintenance of a stem-state in ENCCs.
openNAU: An open-source platform for normalizing, analyzing, and visualizing untargeted metabolomics data
Authors: Sun Qingrong , Xu Qingqing , Wang Majie , Wang Yongcheng , Zhang Dandan , Lai Maode
Published: September 2022

Motivation

As an important part of metabolomics analysis, untargeted metabolomics has become a powerful tool in the study of tumor mechanisms and the discovery of metabolic markers with high-throughput spectrometric data which also brings great challenges to data analysis from the extraction of raw data to the identification of differential metabolites. To date, a large number of analytical tools and processes have been developed and constructed to serve untargeted metabolomics research. The different selection of analytical tools and parameter settings lead to varied results of untargeted metabolomics data. Our goal is to establish an easily operated platform and obtain a repeatable analysis result.

Results

We used the R language basic environment to construct the preprocessing system of the original data and the LAMP (Linux + Apache + MySQL + PHP) architecture to build a cloud mass spectrum data analysis system. An open-source analysis software for untargeted metabolomics data (openNAU) was constructed. It includes the extraction of raw mass data and quality control for the identification of differential metabolic ion peaks. A reference metabolomics database based on public databases was also constructed. Finally, a complete analysis system platform for untargeted metabolomics was established. This platform provides a complete template interface for the addition and updating of the analysis process, so we can finish complex analyses of untargeted metabolomics with simple human-computer interactions.

Availability and Implementation

The source code can be downloaded from https://github.com/zjuRong/openNAU.

Contact

sunqingrong@zju.edu.cn
OPUS-Fold3: a gradient-based protein all-atom folding and docking framework on TensorFlow
Authors: Xu Gang , Luo Zhenwei , Zhou Ruhong , Wang Qinghua , Ma Jianpeng
Published: September 2022

For refining and designing protein structures, it is essential to have an efficient protein folding and docking framework that generates a protein 3D structure based on given constraints. In this study, we introduce OPUS-Fold3 as a gradient-based, all-atom protein folding and docking framework, which accurately generates 3D protein structures in compliance with specified constraints, such as a potential function as long as it can be expressed as a function of positions of heavy atoms. Our tests show that, for example, OPUS-Fold3 achieves performance comparable to pyRosetta in backbone folding, and significantly better in side-chain modeling. Developed using Python and TensorFlow 2.4, OPUS-Fold3 is user-friendly for any source-code level modifications and can be seamlessly combined with other deep learning models, thus facilitating collaboration between the biology and AI communities. The source code of OPUS-Fold3 can be downloaded fromhttp://github.com/OPUS-MaLab/opus_fold3. It is freely available for academic usage.
A TetR-like transcriptional regulator in Stenotrophomonas maltophilia involved in fatty acid metabolism is controlled by quorum sensing signals
Authors: Coves Xavier , Huedo Pol , Bravo Marc , Conchillo-Solé Òscar , Gómez Andromeda-Celeste , Esteve-Codina Anna , Dabad Marc , Gut Marta , Daura Xavier , Yero Daniel , Gibert Isidre
Published: September 2022

Stenotrophomonas maltophilia is an environmental bacterium and it is also an emerging opportunistic multidrug-resistant pathogen. It uses the endogenous DSF quorum sensing (QS) system to coordinate population behaviors and to regulate virulence processes but can also respond to exogenous AHL signals produced by neighboring bacteria. Whole-transcriptome sequencing analyses were performed for S. maltophilia K279a in the exponential and stationary phases as well as in exponential cultures after treatment with exogenous DSF or AHLs. The results revealed that at the beginning of the stationary phase 1673 genes are differentially expressed. COG analysis showed that most of these genes were enriched for energetic metabolism processes and regulation of gene expression. After adding DSF or AHLs, 28 or 82 genes were found deregulated, respectively, 22 of which upregulated by both autoinducers. Interestingly, among these later genes, 14 were also upregulated in the stationary phase. Gene functions regulated by all conditions include lipid and amino acid metabolism, stress response and signal transduction, nitrogen and iron metabolism, and adaptation to microoxic conditions. Among the common top upregulated QS core genes, a putative TetR-like regulator (Smlt2053) was selected for functional characterization. This regulator has been shown to control a narrow regulon, including its own operon. It was found to sense long-chain fatty acids, including the QS signal DSF, and regulate a β-oxidation catabolic pathway. Overall, our findings provide clues on the role that the QS could have in S. maltophilia in the transition from the exponential to the stationary phase and bacterial fitness under high-density growth.

IMPORTANCE

The quorum sensing system in Stenotrophomonas maltophilia, in addition to coordinating the bacterial population, controls virulence-associated phenotypes, such as biofilm formation, motility, protease production, and antibiotic resistance mechanisms. Biofilm formation is frequently associated with the persistence and chronic nature of nosocomial infections. In addition, biofilms exhibit high resistance to antibiotics, making treatment of these infections extremely difficult. The importance of studying the metabolic and regulatory systems controlled by quorum sensing autoinducers will make it possible to discover new targets to control pathogenicity mechanisms in S. maltophilia.
Record-matching of STR profiles with fragmentary genomic SNP data
Authors: Kim Jaehee , Rosenberg Noah A.
Published: September 2022

In many forensic settings, identity of a DNA sample is sought from poor-quality DNA, for which the typical STR loci tabulated in forensic databases are not possible to reliably genotype. Genome-wide SNPs, however, can potentially be genotyped from such samples via next-generation sequencing, so that queries can in principle compare SNP genotypes from DNA samples of interest to STR genotype profiles that represent proposed matches. We use genetic record-matching to evaluate the possibility of testing SNP profiles obtained from poor-quality DNA samples to identify exact and relatedness matches to STR profiles. Using 2,504 whole-genome sequences, we show that in some settings, similar match accuracies to those seen with full coverage of the genome are obtained by genetic record-matching for SNP data that represent 5-10% genomic coverage. Thus, if even a fraction of random genomic SNPs can be genotyped by next-generation sequencing, then the potential may exist to test the resulting genotype profiles for matches to profiles consisting exclusively of nonoverlapping STR loci. The result has implications in relation to criminal justice, mass disasters, missing-person cases, studies of ancient DNA, and genomic privacy.
Dectin-1 Stimulation Promotes a Distinct Inflammatory Signature in the Setting of HIV-infection and Aging
Authors: Kumar Archit , Wang Jiawei , Zhou Haowen , Radcliffe Chris , Wyk Brent Vander , Allore Heather G. , Tsang Sui , Barakat Lydia , Mohanty Subhasis , Zhao Hongyu , Shaw Albert C. , Zapata Heidi J.
Published: September 2022

Dectin-1 is an innate immune receptor that recognizes and binds β-1,3/1,6 glucans on fungi. We evaluated Dectin-1 function in myeloid cells in a cohort of HIV-positive and HIV-negative young and older adults. Stimulation of monocytes with β-D-glucans induced a pro-inflammatory phenotype in monocytes of HIV-infected individuals that was characterized by increased levels of IL-12, TNF-α, and IL-6, with some age-associated cytokine increases also noted. Dendritic cells showed a striking HIV-associated increase in IFN-α production. These increases in cytokine production paralleled increases in Dectin-1 surface expression in both monocytes and dendritic cells that were noted with both HIV and aging. Differential gene expression analysis showed that HIV-positive older adults had a distinct gene signature compared to other cohorts characterized by a robust TNF-α and coagulation response (increased at baseline), a persistent IFN-α and IFN-γ response, and an activated dendritic cell signature/M1 macrophage signature upon Dectin-1 stimulation. Dectin-1 stimulation induced a strong upregulation of MTORC1 signaling in all cohorts, although increased in the HIV-Older cohort (stimulation and baseline). Overall, our study demonstrates that the HIV Aging population has a distinct immune signature in response to Dectin-1 stimulation. This signature may contribute to the pro-inflammatory environment that is associated with HIV and Aging.
Alpha kinase 3 signaling at the M-band maintains sarcomere integrity and proteostasis in striated muscle
Authors: McNamara James W. , Parker Benjamin L. , Voges Holly K. , Mehdiabadi Neda R. , Bolk Francesca , Chung Jin D. , Charitakis Natalie , Molendijk Jeffrey , Lal Sean , Ramialison Mirana , Karavendzas Kathy , Pointer Hayley L. , Syrris Petros , Lopes Luis R. , Elliott Perry M. , Lynch Gordon S. , Mills Richard J. , Hudson James E. , Watt Kevin I. , Porrello Enzo R. , Elliott David A
Published: September 2022

Pathogenic variants in alpha kinase 3 (ALPK3) cause cardiomyopathy and musculoskeletal disease. How ALPK3 mutations result in disease remains unclear because little is known about this atypical kinase. Using a suite of engineered human pluripotent stem cells (hPSCs) we show that ALPK3 localizes to the M-Band of the sarcomere. ALPK3 deficiency disrupted sarcomeric organization and calcium kinetics in hPSC-derived cardiomyocytes and reduced force generation in cardiac organoids. Phosphoproteomic profiling identified ALPK3-dependant phospho-peptides that were enriched for sarcomeric components of the M-band and the ubiquitin-binding protein SQSTM1. Analysis of the ALPK3 interactome confirmed binding to M-band proteins including SQSTM1. Importantly, in hPSC-derived cardiomyocytes modeling ALPK3 deficiency and cardiomyopathic ALPK3 mutations, sarcomeric organization and M-band localization ofSQSTM1 were abnormal. These data suggest ALPK3 has an integral role in maintaining sarcomere integrity and proteostasis in striated muscle. We propose this mechanism may underly disease pathogenesis in patients with ALPK3 variants.
ANKRD26 is a new regulator of type I cytokine receptor signaling in normal and pathological hematopoiesis
Authors: Basso-Valentina Francesca , Donada Alessandro , Manchev Vladimir T , Lisetto Manuel , Balayn Nathalie , Martin Jean Edouard , Muller Delphine , Marin Oyarzun Cecilia Paola , Duparc Hélène , Arkoun Brahim , Cumin Alessandro , Faivre Lionel , Droin Nathalie , Biunno Ida , Balduini Alessandra , Debili Najet , Antony-Debré Iléana , Marty Caroline , Vainchenker William , Plo Isabelle , Favier Remi , Raslova Hana
Published: September 2022

Sustained ANKRD26 expression associated with germline ANKRD26 mutations causes Thrombocytopenia 2 (THC2), an inherited platelet disorder associated with leukemia predisposition. Some of those patients present also erythrocytosis and/or leukocytosis. Using multiple human-relevant in vitro models (cell lines, primary patient cells and patient-derived iPSCs) we demonstrate for the first time that ANKRD26 is expressed during the early steps of erythroid, megakaryocyte and granulocyte differentiation, and is necessary for progenitor proliferation. As differentiation progresses, ANKRD26 expression is progressively silenced, to complete the cellular maturation of the three myeloid lineages. In primary cells, abnormal ANKRD26 expression in committed progenitors directly impacts the proliferation/differentiation balance for these three cell types. We show that ANKRD26 interacts with and crucially modulates the activity of MPL, EPOR and G-CSFR, three homodimeric type I cytokine receptors that regulate blood cell production. Higher than normal levels of ANKRD26 prevent the receptor internalization, which leads to increased signaling and cytokine hypersensitivity. Altogether these findings show that ANKRD26 overexpression or the absence of its silencing during differentiation are responsible for myeloid blood cell abnormalities in THC2 patients.
Wide Window Acquisition and AI-based data analysis to reach deep proteome coverage for a wide sample range, including single cell proteomic inputs
Authors: Mayer Rupert L. , Matzinger Manuel , Schmücker Anna , Stejskal Karel , Krššáková Gabriela , Berger Frédéric , Mechtler Karl
Published: September 2022

A comprehensive proteome map is essential to elucidate molecular pathways and protein functions. Although great improvements in sample preparation, instrumentation and data analysis already yielded impressive results, current studies suffer from a limited proteomic depth and dynamic range therefore lacking low abundant or highly hydrophobic proteins. Here, we combine and benchmark advanced micro pillar array columns (µPAC™) operated at nanoflow with Wide Window Acquisition (WWA) and the AI-based CHIMERYS™ search engine for data analysis to maximize chromatographic separation power, sensitivity and proteome coverage.

Our data shows that µPAC™ columns clearly outperform classical packed bed columns boosting peptide IDs by up to 140%. Already at classical narrow isolation widths CHIMERYS™ boosted ID rates by a factor of 2.6 compared to the conventional search engine MS Amanda 2.0. By combining CHIMERYS™ with WWA, even a 4.6-fold increase in ID rates could be achieved.

Using our optimized workflow, we were further able to identify more than 10,000 proteins from a single 2 h gradient shotgun analysis. We further investigated the applicability of WWA for single cell inputs and found that the choice of the optimal isolation window width depends on sample input and complexity. Using a short 5.5 cm column and very high flow rates during loading and column equilibration we improved sample throughput to ∼100 samples per day while maintaining high protein ID numbers. We believe that this is especially important for the single cell field where throughput is one of the most limiting factors.

Finally, we applied our optimized workflow on immunoprecipitations of Smarca5/SNF2H and found 32 additional interaction partners compared to the original workflow utilizing a packed bed column. These additional interaction partners include previously described interaction partners of Smarca5 like Baz2b as well as undescribed interactors including Arid1a, which is also involved in chromatin remodeling and has been described as key player in neurodevelopmental and malignant disorders.
Ventral temporal and posteromedial sulcal morphology in autism spectrum disorder
Authors: Benitez Javier Ramos , Kannan Sandhya , Hastings William L. , Parker Benjamin J. , Willbrand Ethan H. , Weiner Kevin S.
Published: September 2022

Two recent parallel research tracks link tertiary sulcal morphology—sulci that emerge last in gestation and continue to develop after birth—with functional features of the cerebral cortex and cognition, respectively. The first track identified a relationship between the mid-fusiform sulcus (MFS) in ventral temporal cortex (VTC) and cognition in individuals with Autism Spectrum Disorder (ASD). The second track identified a new tertiary sulcus, the inframarginal sulcus (IFRMS), that serves as a tripartite landmark within the posteromedial cortex (PMC). As VTC and PMC are structurally and functionally different in individuals with ASD compared to neurotypical controls (NTs), here, we integrated these two tracks with a twofold approach. First, we tested if there are morphological differences in VTC and PMC sulci between 50 NTs and 50 individuals with ASD. Second, we tested if tertiary sulcal morphology was linked to cognition in ASD individuals. Our twofold approach replicates and extends recent findings in five ways. First, in terms of replication, the standard deviation (STD) of MFS cortical thickness (CT) was increased in ASDs compared to NTs. Second, MFS length was shorter in ASDs compared to NTs. Third, the CT STD effect extended to other VTC and PMC sulci. Fourth, a subset of VTC and PMC morphological features were correlated between regions in ASD. Fifth, IFRMS depth was negatively associated with ADOS-GS score. These results empirically support a relationship between later-developing, tertiary sulci and ASD, providing a novel framework to study the relationship between brain structure and cognition in additional neurodevelopmental disorders in future studies.

Lay Summary

We observed that some, but not all, morphological features of later-developing tertiary indentations (sulci) in the cerebral cortex differed significantly between neurotypical controls and individuals with autism spectrum disorder (ASD). In ASD, a subset of sulcal morphological features also correlated between brain areas and one feature reflected differences in cognition. Thus, studying these structures provides insight into how individual variability in structure is related to individual variability in cognition in ASD.
Pro-inflammatory chemokines CCL5, CXCL12, and CX3CL1 bind to and activate platelet integrin αIIbβ3 in an allosteric manner
Authors: Takada Yoko K , Fujita Masaaki , Takada Yoshikazu
Published: September 2022

Previous studies showed that pro-inflammatory chemokines CX3CL1 and CXCL12 bound to the allosteric binding site (site 2) of integrins and allosterically activated integrins, in addition to the classical ligand-binding site (site 1). We showed that CCL5 also bound to site 2, in addition to site 1, and activated soluble integrin αvβ3. Platelet integrin αIIbβ3, a fibrinogen receptor, is critical for hemostasis and thrombus formation and activation of αIIbβ3 is a key event for thrombus formation. Activation of αIIbβ3 is known to be mediated exclusively by inside-out signaling. We studied if αIIbβ3 can be allosterically activated. We discovered that CCL5, CXCL12, and CX3CL1 are new ligands for αIIbβ3. Notably they enhanced the binding of monovalent ligand to soluble αIIbβ3 in 1 mM Ca2+by binding to site 2. They activated cell-surface αIIbβ3 on CHO cells quickly (half maximal response <1 min) and at low concentrations (1-10 ng/ml) compared to soluble αIIbβ3, probably because chemokines bind to cell surface proteoglycans. Notably, activation of αIIbβ3 by the chemokines was several times more potent than 1 mM Mn2+. Since CCL5 and CXCL12 are stored in platelet granules and rapidly transported to the surface upon platelet activation, we hypothesized that they are released from the granules and allosterically activate αIIbβ3 by binding to site 2.

Transmembrane CX3CL1 on activated endothelial cells likely mediates platelet-endothelial interaction by binding to and activating αIIbβ3. Also, over-produced chemokines during inflammation may trigger αIIbβ3 activation, which is a possible missing link between inflammation and thrombosis.
Transcriptional changes in human palate and skin healing
Authors: Leonardo Trevor R , Chen Lin , Schrementi Megan E , Shi Junhe , Marucha Phillip T , Glass Kimberly , DiPietro Luisa A
Published: September 2022

Most human tissue injuries lead to the formation of a fibrous scar and result in the loss of functional tissue. One adult tissue that exhibits a more regenerative response to injury with minimal scarring is the oral mucosa. We generated a microarray gene expression dataset to examine the response to injury from human palate and skin excisional biopsies spanning the first seven days after wounding. Differential expression analyses were performed in each tissue to identify genes overexpressed or underexpressed over time compared to baseline unwounded tissue. To attribute biological processes of interest to these gene expression changes, gene set enrichment analysis was used to identify core gene sets that are enriched over the time-course of the wound healing process with respect to unwounded tissue. This analysis identified gene sets uniquely enriched in either palate or skin wounds and gene sets that are enriched in both tissues in at least one time point after injury. Finally, a cell-deconvolution analysis was performed to better understand the cell type distribution in these tissues and how it changes over the time course of wound healing. This work provides a source of human wound gene expression data that includes two tissue types with distinct regenerative and scarring phenotypes.
COVID-19-associated AKI in hospitalized US patients: incidence, temporal trends, geographical distribution, risk factors and mortality
Authors:   Masthead, Yoo Yun Jae , Wilkins Kenneth J. , Alakwaa Fadhl , Liu Feifan , Torre-Healy Luke A. , Krichevsky Spencer , Hong Stephanie S. , Sakhuja Ankit , Potu Chetan K. , Saltz Joel H. , Saran Rajiv , Zhu Richard L. , Setoguchi Soko , Kane-Gill Sandra L. , Mallipattu Sandeep K. , He Yongqun , Ellison David H. , Byrd James Brian , Parikh Chirag R. , Moffitt Richard A. , Koraishy Farrukh M.
Published: September 2022

Background

Acute kidney injury (AKI) is associated with mortality in patients hospitalized with COVID-19, however, its incidence, geographic distribution, and temporal trends since the start of the pandemic are understudied.

Methods

Electronic health record data were obtained from 53 health systems in the United States (US) in the National COVID Cohort Collaborative (N3C). We selected hospitalized adults diagnosed with COVID-19 between March 6th, 2020, and January 6th, 2022. AKI was determined with serum creatinine (SCr) and diagnosis codes. Time were divided into 16-weeks (P1-6) periods and geographical regions into Northeast, Midwest, South, and West. Multivariable models were used to analyze the risk factors for AKI or mortality.

Results

Out of a total cohort of 306,061, 126,478 (41.0 %) patients had AKI. Among these, 17.9% lacked a diagnosis code but had AKI based on the change in SCr. Similar to patients coded for AKI, these patients had higher mortality compared to those without AKI. The incidence of AKI was highest in P1 (49.3%), reduced in P2 (40.6%), and relatively stable thereafter. Compared to the Midwest, the Northeast, South, and West had higher adjusted AKI incidence in P1, subsequently, the South and West regions continued to have the highest relative incidence. In multivariable models, AKI defined by either SCr or diagnostic code, and the severity of AKI was associated with mortality.

Conclusions

Uncoded cases of COVID-19-associated AKI are common and associated with mortality. The incidence and distribution of COVID-19-associated AKI have changed since the first wave of the pandemic in the US.
Sodium content of menu items in New York City chain restaurants following enforcement of the sodium warning icon rule, 2015-2017
Authors: Sisti Julia S. , Prasad Divya , Niederman Sarah , Mezzacca Tamar Adjoian , Anekwe Amaka V. , Clapp Jenifer , Farley Shannon M.
Published: September 2022

In 2016, New York City (NYC) began enforcing a sodium warning regulation at chain restaurants, requiring placement of an icon next to any menu item containing ≥2,300 mg sodium. As menu labeling may improve menu nutritional composition, we investigated whether sodium content of menu items changed following enforcement of the sodium warning icon. All menu offerings at 10 quick-service (QSR) and 3 full-service (FSR) chain restaurants were photographed in 2015 (baseline) and 2017 (follow-up) and matched to nutritional information from restaurant websites; items were categorized as being available at both baseline and follow-up, or at only one timepoint. Linear and logistic regression models, respectively, assessed changes in calculated mean sodium-per-serving and the odds of an item containing ≥2,300 mg sodium. At baseline, mean per-serving sodium content was 2,160 mg at FSR and 1,070 mg at QSR, and 40.6% of FSR items and 7.2% of QSR items contained ≥2,300 mg sodium per serving. Sodium content did not differ when comparing all items offered at follow-up to all offered at baseline (21 mg, 95% CI: -60,101), or when comparing new versus discontinued items (17 mg, 95% CI: -154, 187). At follow-up, there was a non-significant increase in the overall likelihood of items requiring a warning icon (OR=1.32, 95% CI: 0.97,1.79). When comparing new versus discontinued items, there was a twofold increase in the odds of requiring a warning icon (OR=2.08, 95% CI: 1.02,4.24). Our findings both highlight high sodium content of menu items at popular chain restaurants and underscore difficulties in motivating restaurants to reduce sodium levels.
Organoid-based single-cell spatiotemporal gene expression landscape of human embryonic development and hematopoiesis
Authors: Chao Yiming , Xiang Yang , Xiao Jiashun , Zhang Shihui , Zheng Weizhong , Wan Xiaomeng , Li Zhuoxuan , Gao Mingze , Wang Gefei , Chen Zhilin , Ebrahimkhani Mo , Yang Can , Wu Angela Ruohao , Liu Pentao , Huang Yuanhua , Sugimura Ryohichi
Published: September 2022

Single-cell level characterization of embryonic development is a major benchmark of human developmental biology. Spatiotemporal analysis of stem-cell-derived embryos offers conceptual and technical advances in the field. Here, we defined the single-cell spatiotemporal gene expression landscape of human embryonic development with stem-cell-derived organoids. We established the human embryonic organoid (HEMO) from expanded potential stem cells and achieved both embryonic and extraembryonic tissues in the same organoid. Time-series single-cell RNA sequencing paired with single-cell resolution spatial revealed human embryonic development signatures such as extraembryonic placenta, yolk sac hematopoiesis neural crest, blood vessels, and cardiac mesoderm. Hematopoietic tissues eventually predominated HEMO with erythropoiesis, mekagaryopiesis, and myelopoiesis. Cell-cell communication network analysis demonstrated that trophoblast-like tissues supplied WNT signaling in neural crest cells to facilitate maturation and migration. Single-cell resolution spatial transcriptomics defined the yolk sac erythro-megakaryopoietic niche. Vitronectin-integrin signaling, a major contributor to megakaryocyte maturation, was predominant in the yolk sac niche in HEMO and to human fetal samples. Overall, our study advances the spatiotemporal analysis of human embryonic development in stem-cell-derived organoids.

Highlights

Modeling human embryonic development from stem cells

Used of both 10X Chromium and 10X Visium to define the gene expression landscape of embryonic development and hematopoiesis

WNT signaling as a regulator of neural crest maturation and EMT

VTN-ITGA2B as the main contributor to Mk maturation within the yolk sac erythro-megakaryopoietic niche

Graphical abstract
Structural Basis for pre-tRNA Recognition and Processing by the Human tRNA Splicing Endonuclease Complex
Authors: Hayne Cassandra K. , Butay Kevin John U. , Stewart Zachary D. , Krahn Juno M. , Perera Lalith , Williams Jason G. , Petrovitch Robert M. , Deterding Leesa J. , Matera A. Gregory , Borgnia Mario J. , Stanley Robin E.
Published: September 2022

Across all walks of life, certain transfer RNA (tRNA) transcripts contain introns. Pre-tRNAs with introns require splicing to form the mature anticodon stem loop (ASL). In eukaryotes, tRNA splicing is initiated by the heterotetrameric tRNA splicing endonuclease (TSEN) complex. All TSEN subunits are essential and mutations within the complex are associated with a family of neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). The pathogenesis of PCH is poorly understood. Moreover, a lack of structures for any eukaryotic TSEN complex has hindered our understanding of tRNA recognition and processing. Here, we report Cryo-Electron Microscopy (cryo-EM) structures of the human TSEN•pre-tRNA complex, trapped in the pre-cleavage state, at near atomic resolution. These structures reveal the overall architecture of the complex, along with extensive tRNA binding interfaces within the complex. Although it shares structural homology with archaeal TSENs, the human TSEN complex contains additional features important for recognizing the acceptor stem and D-arm of the pre-tRNA. Our findings also establish the TSEN54 subunit as more than a simple molecular ruler; it functions as a pivotal scaffold for the pre-tRNA and the two endonuclease subunits, TSEN2 and TSEN34. Finally, the human TSEN structures enable detailed visualization of the molecular environments of PCH-causing missense mutations, providing crucial insight into the mechanism of eukaryotic pre-tRNA splicing and neurodevelopmental disease.
FDA-Approved Drug Screening in Patient-Derived Organoids Demonstrates Potential of Drug Repurposing for Rare Cystic Fibrosis Genotypes
Authors: de Poel E. , Spelier S. , Hagemeijer M.C. , van Mourik P. , Suen S.W.F. , Vonk A.M. , Brunsveld J.E. , Ithakisiou G. N. , Kruisselbrink E. , Oppelaar H. , Berkers G. , de Winter-de Groot K.M. , Heida-Michel S. , Jans S.R. , van Panhuis H. , Bakker M. , van der Meer R. , Roukema J. , Dompeling E. , Weersink E.J.M. , Koppelman G.H. , Blaazer A.R. , Muijlwijk-Koezen J.E. , van der Ent C.K. , Beekman J.M.
Published: September 2022

Background

Preclinical cell-based assays that recapitulate human disease play an important role in drug repurposing. We previously developed a functional forskolin induced swelling (FIS) assay using patient-derived intestinal organoids (PDIOs), allowing functional characterization of CFTR, the gene mutated in people with cystic fibrosis (pwCF). CFTR function-increasing pharmacotherapies have revolutionized treatment for approximately 85% of people with CF, but a large unmet need remains to identify new treatments for all pwCF.

Methods

We used 76 non-homozygous F508del-CFTR PDIOs to test the efficacy of 1400 FDA-approved drugs on improving CFTR function, as measured in FIS assays.

Results

Based on the results of a secondary validation screen, we investigated CFTR elevating function of PDE4 inhibitors and currently existing CFTR modulators in further detail. We show that PDE4 inhibitors are potent CFTR function inducers in PDIOs and that CFTR modulator treatment rescues of CF genotypes that are currently not eligible for this therapy.

Conclusions

This study exemplifies the feasibility of high-throughput compound screening using PDIOs and we show the potential of repurposing drugs for pwCF that are currently not eligible for therapies.

One-sentence Summary

We screened 1400 FDA-approved drugs in CF patient-derived intestinal organoids using the previously established functional FIS assay, and show the potential of repurposing PDE4 inhibitors and CFTR modulators for rare CF genotypes.
A cryogenic, coincident fluorescence, electron and ion beam microscope
Authors: Boltje Daan B. , Hoogenboom Jacob P. , Jakobi Arjen J. , Jensen Grant J. , Jonker Caspar T.H. , Kaag Max J. , Koster Abraham J. , Last Mart G.F. , de Agrela Pinto Cecilia , Plitzko Jürgen M. , Raunser Stefan , Tacke Sebastian , Wang Zhexin , van der Wee Ernest B. , Wepf Roger , den Hoedt Sander
Published: September 2022

Cryogenic electron tomography (cryo-ET) combined with sub-tomogram averaging, allows in-situ visualisation and structure determination of macromolecular complexes at sub-nanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in-situ fluoresence microscopy-guided FIB fabrication of a frozen-hydrated lamella to solve this problem: we built a coincident 3-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing focused ion-beam scanning electron microscope (FIB-SEM). We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope (TEM) tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.
PERK Kinase Activity is Regulated by Copper Binding-A New Regulatory Paradigm for Modulation of ER Stress Tolerance
Authors: Newton Sarah E. Bond , Beratan Noah R. , Akay-Espinoza Cagla , Bond Margaret K. , Shi Xinglong , Perhacs Julia , Gidalevitz Tali , Brady Donita C. , Jordan-Sciutto Kelly L.
Published: September 2022

PERK is a kinase that, in response to ER stress, mediates duplicitous homeostatic and pro-apoptotic signaling. Thus, intricate regulation is required for physiologic function. Attempts to modulate PERK activity have shown that the determinants of adaptive vs. mal-adaptive signaling remain ambiguous. Here, with purified protein, we provide direct evidence that PERK binds copper, identify the residues that coordinate binding, and demonstrate that binding is necessary for kinase activity. Furthermore cellular PERK activity can be modulated via copper availability, and this regulatory relationship can be manipulated to dictate ER stress tolerance. Critically, these phenomenon translate to phenotypesin vivo, asC. elegansharboring a “copper-binding mutant” of PERK exhibit enhanced ER-stress sensitivity. The copper-PERK paradigm adds onto a new class of dynamic copper-binding enzymes and implicates that copper homeostasis, as a regulator of PERK, may constitute a previously unknown variable to resolve long-standing ambiguity in endeavors to therapeutically target PERK.