bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparative Restriction Enzyme Analysis of Methylation (CREAM) Reveals Methylome Variability Within a ClonalIn VitroCannabis Population
Authors: Boissinot Justin , Adamek Kristian , Jones Andrew Maxwell Phineas , Normandeau Eric , Boyle Brian , Torkamaneh Davoud 
Published: August 2023

AbstractThe primary focus of medicinal cannabis research is to ensure the stability of cannabis lines for consistent administration of chemically consistent products to patients. In recent years, tissue culture has emerged as a valuable technique for genetic preservation and rapid production of cannabis clones. However, there is concern that the physical and chemical conditions of the growing media can induce somaclonal variation, potentially impacting the viability and uniformity of clones. To address this concern, we developed Comparative Restriction Enzyme Analysis of Methylation (CREAM), a novel method to assess DNA methylation patterns and used it to assess a population of 78 cannabis clones maintained in tissue culture. Through bioinformatics analysis of the methylome, we successfully detected 2,272 polymorphic methylated regions among the clones. Remarkably, our results demonstrated that DNA methylation patterns were preserved across subcultures within the clonal population, allowing us to distinguish between two subsets of clonal lines used in this study. These findings significantly contribute to our understanding of the epigenetic variability within clonal lines in medicinal cannabis produced through tissue culture techniques. This knowledge is crucial for understanding the effects of tissue culture on DNA methylation and ensuring the consistency and reliability of medicinal cannabis products with therapeutic properties. Additionally, the CREAM method is a fast and affordable technology to get a first glimpse at methylation in a biological system. It offers a valuable tool for studying epigenetic variation in other plant species, thereby facilitating broader applications in plant biotechnology and crop improvement.
Difference in Kernel Shape and Endocarp Anatomy Promote Dehiscence in Pistachio Endocarp
Authors: Zhang Shuxiao , Wang Minmin , Chernikova Alisa , Eagle Shaina , Bedell Kaleigh Marie , Nguyen Karen , Blanco-Ulate Barbara , Jernstedt Judy , Drakakaki Georgia 
Published: August 2023

AbstractA fully split shell in pistachio (Pistacia vera) is a trait that is preferred by consumers and is a criterion in evaluating the grade of the pistachio nut. However, while the expanding kernel has been hypothesized to provide the physical force needed for shell split, the mechanisms that control shell split remains unknown. Furthermore, it is intriguing how the shell, or endocarp, splits at the suture ridge when there is no clear dehiscence zone.The objectives of this study were to 1) identify traits associated with dehiscence in fruit in the high split rate cultivar Golden Hills when compared to the lower split rate cultivar Kerman and determine the anatomical features associated with endocarp dehiscence at the suture region, and 2) examine the effect of kernel shape on endocarp dehiscence.We determined that despite of the fact that the pistachio endocarp is primarily composed of a single type of polylobate schlerenchyma cell, specialization of cell shape and size at the suture site results in smaller, more flattened cells. We report that there is a furrowing of the shell at the dorsal and apical suture site, where dehiscence initiates. This furrowing is not observed at the ventral suture site or in the indehiscent fruit ofPistacia atlantica, a species that has been used as rootstock forP. vera. In addition, the size of the kernel in the sagittal axis (the width) is strongly associated with higher split rate. Based on our results, a tentative model emerges where, in the absence of specialized cell types, cell shape modification can create an anatomically distinct region that is mechanically weak in the endocarp for the initiation of dehiscence, while the force from the width of the kernel is necessary for the shell split rate difference as observed in cultivars.
Double mutation of open syntaxin and UNC-18 P334A leads to excitatory-inhibitory imbalance and impairs multiple aspects ofC. elegansbehavior
Authors: Huang Mengjia , Wang Ya , Chow Chun Hin , Stepien Karolina P. , Indrawinata Karen , Xu Junjie , Argiropoulos Peter , Xie Xiaoyu , Sugita Kyoko , Tien Chi-Wei , Lee Soomin , Monnier Philippe P. , Rizo Josep , Gao Shangbang , Sugita Shuzo 
Published: August 2023

AbstractSNARE and Sec/Munc18 proteins are essential in synaptic vesicle exocytosis. Open form t-SNARE syntaxin and UNC-18 P334A are well-studied exocytosis-enhancing mutants. Here we investigate the interrelationship between the two mutations by generating double mutants in various genetic backgrounds inC. elegans. While each single mutation rescued the motility ofCAPS/unc-31andsynaptotagmin/snt-1mutants significantly, double mutations unexpectedly worsened motility or lost their rescuing effects. Electrophysiological analyses revealed that simultaneous mutations of open syntaxin and gain-of-function P334A UNC-18 induces a strong imbalance of excitatory over inhibitory transmission. In liposome fusion assays performed with mammalian proteins, the enhancement of fusion caused by the two mutations individually was abolished when the two mutations were introduced simultaneously, consistent with what we observed inC. elegans. We conclude that open syntaxin and P334A UNC-18 do not have additive beneficial effects, and this extends toC. elegans’characteristics such as motility, growth, offspring bared, body size, and exocytosis, as well as liposome fusion in vitro. Our results also reveal unexpected differences between the regulation of exocytosis in excitatory versus inhibitory synapses.
Enriched atlas of lncRNA and protein-coding genes for the GRCg7b chicken assembly and its functional annotation across 47 tissues
Authors: Degalez Fabien , Charles Mathieu , Foissac Sylvain , Zhou Haijuan , Guan Dailu , Fang Lingzhao , Klopp Christophe , Allain Coralie , Lagoutte Laetitia , Lecerf Frédéric , Acloque Hervé , Giuffra Elisabetta , Pitel Frédérique , Lagarrigue Sandrine 
Published: August 2023

ABSTRACTGene atlases for livestock are steadily improving thanks to new genome assemblies and new expression data improving the gene annotation. However, gene content varies across databases due to differences in RNA sequencing data and bioinformatics pipelines, especially for long non-coding RNAs (lncRNAs) which have higher tissue and developmental specificity and are harder to consistently identify compared to protein coding genes (PCGs). As done previously in 2020 for chicken assemblies galgal5 and GRCg6a, we provide a new gene atlas, lncRNA-enriched, for the latest GRCg7b chicken assembly, integrating “NCBI RefSeq”, “EMBL-EBI Ensembl/GENCODE” reference annotations and other resources such as FAANG and NONCODE. As a result, the number of PCGs increases from 18,022 (RefSeq) and 17,007 (Ensembl) to 24,102, and that of lncRNAs from 5,789 (RefSeq) and 11,944 (Ensembl) to 44,428. Using 1,400 public RNA-seq transcriptome representing 47 tissues, we provided expression evidence for 35,257 (79%) lncRNAs and 22,468 (93%) PCGs, supporting the relevance of this atlas. Further characterization including tissue-specificity, sex-differential expression and gene configurations are provided. We also identifiend conserved miRNA-hosting genes with human counterparts, suggesting common function. The annotated atlas is available atwww.fragencode.org/lnchickenatlas.html.
LncRNA SNHG16 modulates paclitaxel resistance in breast cancer by regulating Let-7a-5p/DUSP7
Authors: Zhao Hui , Wang Lei , Feng Yueqing , Yang Junzheng 
Published: August 2023

AbstractObjectiveTo investigate the effect of long chain non coding RNA (lncRNA) small nucleolar RNA host gene 6 (SNHG16) on paclitaxel (PTX) resistance in breast cancer and to understand its underlying mechanism, to lay a foundation for decreasing the PTX resistance in breast cancer treatment and improving the therapeutic quality for breast cancer patients.MethodsPTX was used to induce the establishment of PTX resistant breast cancer cell lines; the control group (normal cultured MCF-7/PTX cells), si-NC group, si-SNHG16 group, si-SNHG16+anti miR-NC group, and si-SNHG16 +anti-Let-7a-5p group were set to compared the effect of SNHG16 on the PTX resistance in MCF-7 cells; MTT assay, Flow cytometry, and Transwell invasion assay were used to determine the PTX resistance, apoptosis, and invasion ability of MCF-7 cells in different groups, respectively; for further assess the effect of SNHG16 on the PTX resistance, nude mouse tumor transplantation experiment was used; and the potential mechanism of SNHG16 regulated the PTX resistance in MCF-7 cells was explored by double luciferase reporter gene detection method and gene silencing technology.ResultsThe expression of SNHG16 gene and DUSP7 protein in MCF-7 cell line was the highest, and the expression of Let-7a-5p was the lowest compared with the various breast cancer cell lines (human breast epithelial cell line MCF-10A and human breast cancer cell lines MDA-MB-468 and MDA-MB-453) (P<0.05); PTX could increase the expression of SNHG16 gene and DUSP7 protein, and reduce the expression of Let-7a-5pin MCF-7 cell line (P<0.05); the results of cell experiment and nude mouse transplantation tumor experiment demonstrated that inhibiting the expression of SNHG16 gene could reduce the invasive ability and promote cell apoptosis of MCF-7/PTX cells (P<0.05), and inhibit tumor growth and reduce the PTX resistance in breast cancer transplantation models; simultaneous inhibition of Let-7a-5p and SNHG16 had a weakening effect in MCF-7/PTX cells (P<0.05). Double luciferase reporter gene detection and gene silencing technology demonstrated that inhibiting SNHG16 gene expression could increase the expression of Let-7a-5p and decrease the expression of DUSP7 (P<0.05); inhibiting the Let-7a-5p gene could increase the expression of DUSP7.ConclusionsInhibiting SNHG16 gene could upregulate Let-7a-5p expression and downregulate the expression of DUSP7 to inhibit MCF-7 cell invasion, promote MCF-7 cell apoptosis, and reduce the PTX resistance in MCF-7 cells and nude mouse tumor transplantation models, those data demonstrated that SNHG16-Let-7a-5p-DUSP7 axis maybe a potential therapeutic strategy for decreasing the PTX resistance in breast cancer in the future.
Lineage-based scaling of germline intercellular bridges during oogenesis
Authors: Shaikh Umayr , Sherlock Kathleen , Wilson Julia , Gilliland William , Lewellyn Lindsay 
Published: August 2023

AbstractThe size of subcellular structures must be tightly controlled to maintain normal cell function. Despite its importance, few studies have determined how the size of organelles or other structures is maintained during development, when cells are growing, dividing, and rearranging. The developing egg chamber is a powerful model in which to study the relative growth rates of subcellular structures. The egg chamber contains a cluster of sixteen germline cells, which are connected through intercellular bridges called ring canals. As the egg chamber grows, the germline cells and the ring canals that connect them increase in size. Here, we demonstrate that ring canal size scaling is related to lineage; the largest, “first born” ring canals increase in size at a relatively slower rate than ring canals derived from subsequent mitotic divisions. This lineage-based scaling relationship is maintained even if directed transport is reduced, ring canal size is altered, or in egg chambers with twice as many germline cells. Analysis of lines that produce larger or smaller mature eggs reveals different strategies could be used to alter final egg size.Summary StatementUsing the fruit fly egg chamber as a model, this study demonstrates that the size and scaling of germline intercellular bridges vary based on lineage.
Dynamics of chromatin accessibility during human first-trimester neurodevelopment
Authors: Mannens Camiel C.A. , Hu Lijuan , Lönnerberg Peter , Schipper Marijn , Reagor Caleb , Li Xiaofei , He Xiaoling , Barker Roger A. , Sundström Erik , Posthuma Danielle , Linnarsson Sten 
Published: August 2023

AbstractThe human brain is capable of highly complex functions that develops through a tightly organized cascade of patterning events, expressed transcription factors and changes in chromatin accessibility. While extensive datasets exist describing gene expression across the developing brain with single-cell resolution, similar atlases of chromatin accessibility have been primarily focused on the forebrain. Here, we focus on the chromatin landscape and paired gene expression across the developing human brain to provide a comprehensive single cell atlas during the first trimester (6 - 13 post-conceptional weeks). We identified 135 clusters across half a million nuclei and using the multiomic measurements linked candidatecis-regulatory elements (cCREs) to gene expression. We found an increase in the number of accessible regions driven both by age and neuronal differentiation. Using a convolutional neural network we identified putative functional TF-binding sites in enhancers characterizing neuronal subtypes and we applied this model to cCREs upstream ofESRRBto elucidate its activation mechanism. Finally, by linking disease-associated SNPs to cCREs we validated putative pathogenic mechanisms in several diseases and identified midbrain-derived GABAergic neurons as being the most vulnerable to major depressive disorder related mutations. Together, our findings provide a higher degree of detail to some key gene regulatory mechanisms underlying the emergence of cell types during the first trimester. We anticipate this resource to be a valuable reference for future studies related to human neurodevelopment, such as identifying cell type specific enhancers that can be used for highly specific targeting inin vitromodels.
Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) LANA Prevents KSHV Episomes from Degradation
Authors: Nakajima Ken-ichi , Inagaki Tomoki , Espera Jonna Magdallene , Izumiya Yoshihiro 
Published: August 2023

ABSTRACTProtein knock-down with an inducible degradation system is a powerful tool to study proteins of interest in living cells. Here, we adopted the auxin-inducible degron (AID) approach to detail Kaposi’s Sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) function in latency maintenance and inducible viral lytic gene expression. We fused the mini-AID (mAID) tag at the LANA N-terminus with KSHV BAC16 recombination, and iSLK cells were stably infected with the recombinant KSHV encoding mAID-LANA. Incubation with 5-phenyl-indole-3-acetic acid (5-Ph-IAA), a derivative of natural auxin, rapidly degraded LANA within 1.5 h. In contrast to our hypothesis, depletion of LANA did not trigger lytic reactivation but rather decreased inducible lytic gene expression when we stimulated reactivation with a combination of ORF50 protein expression and sodium butyrate treatment. Decreased overall lytic gene induction seemed to associate with a rapid loss of KSHV genomes in the absence of LANA. The rapid loss of viral genomic DNA was blocked by treatment with lysosomal inhibitor chloroquine. Furthermore, siRNA-mediated knockdown of cellular innate immune proteins, cyclic AMP-GMP synthase (cGAS) and Stimulator of Interferon Genes (STING), and other autophagy-related genes rescued the degradation of viral genomic DNA upon LANA depletion. These results suggest that LANA is actively protecting viral genomic DNA from sensing by cGAS-STING signaling axis, and add novel insights into the role of LANA in latency maintenance.IMPORTANCEKSHV LANA plays a wide variety of roles in latency maintenance and lytic gene expression. We adopted the inducible protein knockdown approach and revealed that depletion of LANA induced rapid degradation of viral genomic DNA. The viral genome degradation was rescued by inhibition of the cellular innate immune pathway and autophagy. These observations suggest that LANA might play a role in hiding KSHV episome from cellular innate immune DNA sensors. Our study thus provides novel insights into the role of LANA in latency maintenance.
Pectin methylesterase activity is required for RALF1 peptide signalling output
Authors: Rößling Ann-Kathrin , Dünser Kai , Liu Chenlu , Lauw Susan , Rodriguez-Franco Marta , Kalmbach Lothar , Barbez Elke , Kleine-Vehn Jürgen 
Published: August 2023

AbstractThe extracellular matrix plays an integrative role in cellular responses in plants, but its contribution to the signalling of extracellular ligands largely remains to be explored. RAPID ALKALINIZATION FACTORs (RALFs) are extracellular peptide hormones that play pivotal roles in various physiological processes. Here, we address a crucial connection between the de-methylesterification machinery of the cell wall component pectin and RALF1 activity. Pectin is a polysaccharide, contributing to the structural integrity of the cell wall. Our data illustrate that the pharmacological and genetic interference with PECTIN METHYL ESTERASEs (PMEs) abolishes RALF1-induced root growth repression. Our data suggest that positively charged RALF1 peptides bind negatively charged, de-methylesterified pectin with high avidity. We illustrate that the RALF1 association with de-methylesterified pectin is required for its FERONIA-dependent perception, contributing to the control of the extracellular matrix and the regulation of plasma membrane dynamics. Notably, this mode of action is independent of the FER-dependent extracellular matrix sensing mechanism provided by FER interaction with the Leucine-Rich Repeat Extensin (LRX) proteins. We propose that the methylation status of pectin acts as a contextualizing signalling scaffold for RALF peptides, linking extracellular matrix dynamics to peptide hormone-mediated responses.
A noveladiposeloss-of-function mutant inDrosophila
Authors: Losurdo Nicole A. , Bibo Adriana , Bedke Jacob , Link Nichole 
Published: August 2023

ABSTRACTTo identify genes required for brain growth, we took an RNAi knockdown reverse genetic approach in Drosophila. One potential candidate isolated from this effort is the anti-lipogenic geneadipose(adp).Adphas an established role in the negative regulation of lipogenesis in the fat body of the fly and adipose tissue in mammals. While fat is key to proper development in general,adphas not been investigated during brain development. Here we found that RNAi knockdown ofadpin neuronal stem cells and neurons results in reduced brain lobe volume and sought to replicate this with a mutant fly. We generated a noveladpmutant that acts as a loss-of-function mutant based on buoyancy assay results. We found that despite a change in fat content in the body overall and an increase in the number of larger (>5μm) brain lipid droplets, there was no change in the brain lobe volume of mutant larvae. Overall, our work describes a noveladpmutant that can functionally replace the long-standingadp60mutant and shows that theadpgene has no obvious involvement in brain growth.
Knockout studies using CD34+ hematopoietic cells suggest that CD44 is a physiological human neutrophil E-selectin ligand
Authors: Zhu Yuqi , Neelamegham Sriram 
Published: August 2023

ABSTRACTThe recruitment of peripheral blood neutrophils at sites of inflammation involves a multistep cascade, starting with E- and P-selectin expressed on the inflamed vascular endothelium binding sialofucosylated glycans on leukocytes. As the glycoconjugate biosynthesis pathways in different cells are distinct, the precise carbohydrate ligands of selectins varies both across species, and between different immune cell populations in a given species. To study this aspect in human neutrophils, we developed a protocol to perform CRISPR/Cas9 gene-editing on CD34+ hHSCs (human hematopoietic stem/progenitor cells) as they are differentiated towards neutrophil lineage. This protocol initially uses a cocktail of SCF (stem-cell factor), IL-3 (interleukin-3) and FLT-3L (FMS-like tyrosine kinase 3 ligand) to expand the stem/progenitor cells followed by directed differentiation to neutrophils using G-CSF (granulocyte colony-stimulating factor). Microfluidics based assays were performed on a confocal microscope platform to characterize the rolling phenotype of each edited cell type in mixed populations. These studies demonstrated that CD44, but not CD43, is a major E-selectin ligand on human neutrophils. The loss of function results were validated by developing sialofucosylated recombinant CD44. This glycosylated protein supported both robust E-selectin binding in a cell-free assay, and it competitively blocked neutrophil adhesion to E-selectin on inflamed endothelial cells. Together, the study establishes important methods to study human neutrophil biology and determines that sialoflucosylated-CD44 is a physiological human E-selectin ligand.KEY POINTSA CRISPR-Cas9 based protocol was optimized to knockout genes in primary human neutrophils derived from CD34+ hHSCsUnder physiological fluid shear conditions, a sialofucosylated form of CD44 expressed on human neutrophils is a major E-selectin ligandVISUAL ABSTRACT
Characterization of pediatric porcine pulmonary valves as a model for tissue engineered heart valves
Authors: Nejad Shouka Parvin , Mirani Bahram , Simmons Craig A. 
Published: August 2023

AbstractHeart valve tissue engineering holds the potential to transform the surgical management of congenital heart defects affecting the pediatric pulmonary valve (PV) by offering a viable valve replacement with the compositional, architectural and mechanical properties required to functionin situ. While aiming to recapitulate the native valve, the minimum requirement for tissue engineered heart valves (TEHVs) has historically been adequate mechanical function at implantation. However, long-termin situfunctionality of TEHVs remains elusive, suggesting that a closer approximation of the native valve is required. The realization of biomimetic engineered pediatric PV is impeded by insufficient characterization of healthy pediatric tissue. In this study, we comprehensively characterized the planar biaxial tensile behaviour, extracellular matrix (ECM) composition and organization, and valvular interstitial cell (VIC) phenotypes of PVs from piglets to provide benchmarks for TEHVs. The piglet PV possessed an anisotropic and non-linear tension-strain profile from which material constants for a predictive constitutive model were derived. Further, the ECM of the pediatric PV possessed a trilayer organization populated by collagen, glycosaminoglycans, and elastin. Biochemical quantification of ECM proteins normalized to wet weight and DNA content of PV tissue revealed homogenous distribution of proteins across sampled regions of the leaflet. Finally, the predominant phenotype of VICs in the piglet PV was quiescent vimentin-expressing fibroblasts, with a small proportion of activated α-smooth muscle actin-expressing myofibroblasts residing primarily at the base of the leaflet. Overall, the properties characterized in this study can be used to inform TEHV design parameters towards generation of biomimetic pediatric PVs.Graphical Abstract