bluuebunny/crossref_metadata_2025 huggingface dataset:
Helper NLRs Nrc2 and Nrc3 act co-dependently with Prf/Pto and activate MAPK signaling to induce immunity in tomato
Authors: Zhang Ning , Gan Joyce , Carneal Lauren , González-Tobón Juliana , Filiatrault Melanie , Martin Gregory B.
Published: August 2023

Plant intracellular immune receptors, primarily nucleotide-binding, leucine-rich repeat proteins (NLRs), can detect virulence proteins (effectors) from pathogens and activate NLR-triggered immunity (NTI). Recently, ‘sensor’ NLRs have been reported to function with ‘helper’ NLRs to activate immune responses. We investigated the role of two helper NLRs, Nrc2 (NLR required for cell death 2) and Nrc3, on immunity in tomato to the bacterial pathogenPseudomonas syringaepv.tomato(Pst) mediated by the sensor NLR Prf and the Pto kinase. Loss-of-function mutations in bothNrc2andNrc3completely compromised Prf/Pto-mediated NTI toPstcontaining the cognate effectors AvrPto and AvrPtoB. Annrc3mutant showed intermediate susceptibility between wild-type tomato plants and aPrfmutant, while annrc2mutant developed only mild disease symptoms. These observations indicate that Nrc2 and Nrc3 act additively to contribute to Prf/Pto-mediated immunity. We also examined at what point Nrc2 and Nrc3 act in the Prf/Pto-mediated immune response. In thenrc2/3mutant, programmed cell death (PCD) normally induced by constitutively-active variants of AvrPtoB, Pto or Prf was abolished, but that induced by M3Kα or Mkk2 was not. PCD induced by a constitutively active variant of Nrc3 was also abolished in aNicotiana benthamianaline with reduced expression ofPrf. MAPK activation triggered by expression of AvrPto in the wild-type Pto-expressing tomato plants was completely abolished in thenrc2/3 mutant. These results indicate that Nrc2 and Nrc3 act in concert with Prf/Pto and upstream of MAPK signaling. Nrc2 and Nrc3 were not required for the HR triggered by Ptr1, another sensor NLR mediatingPstresistance, although these helper NLRs do appear to be involved in resistance to certainPstrace 1 strains.
Irreversible PDMS bonding using flame activation of adhesives for fabrication of microfluidic and organ-on-chip devices
Authors: Singer Ryan , Hirota Jeremy A. , Dabaghi Mohammadhossein
Published: August 2023

Polydimethylsiloxane (PDMS) is widely used for microfluidics fabrication in many disciplines due to its ease of use in soft lithography and its ability to bond liquid-tight seals. A variety of PDMS-PDMS bonding methods exist, but each may have limitations for some applications. For example, chemical bonding via oxygen plasma treatment is reliable but requires expensive equipment and specialized training. Here we present a rapid, low-cost, and accessible method for irreversible PDMS bonding in which flame treatment activates PDMS and Nitto 5302A adhesive surfaces. Using this technique, PDMS microchannels can be fabricated with a bonding integrity of up to 325 kPa burst pressure. This technique is suitable for fabricating organ-on-chip devices for cell line and primary cell culture, supporting cell viability and establishment of key cellular features in apical and basal compartments, while maintaining bonding integrity.
Ehrlichia chaffeensisco-opts phagocytic hemocytes for systemic dissemination in the Lone Star tick,Amblyomma americanum
Authors: Adegoke Abdulsalam , Hanson Julia , Smith Ryan , Karim Shahid
Published: August 2023

Hematophagous arthropods can acquire and transmit several pathogens of medical importance. In ticks, the innate immune system is crucial in the outcome between vector-pathogen interaction and overall vector competence. However, the specific immune response(s) elicited by the immune cells known as hemocytes remains largely undefined inEhrlichiachaffeensisand its competent tick vector,Amblyomma americanum. Here, we show that granulocytes, professional phagocytic cells, are integral in eliciting immune responses against commensal and pathogen infection. The chemical depletion of granulocytes led to decreased phagocytic efficiency of tissues-associated hemocytes. We demonstrateE. chaffeensiscan infect circulating hemocytes, and both cell-free plasma and hemocytes fromE. chaffeensis-infected ticks can establishEhrlichiainfection in recipient ticks. Lastly, we provide evidence to show granulocytes play a dual role inE. chaffeensisinfection. Depleting granulocytic hemocytes increasedEhrlichiaload in the salivary gland and midgut tissues. In contrast, granulocyte depletion led to a reduced systemic load ofEhrlichia. This study has identified multiple roles for granulocytic hemocytes in the control and systemic dissemination ofE. chaffeensisinfection.
Trabeculations of the porcine and human cardiac ventricles are different in number but similar in total volume
Authors: Jensen Bjarke , Salvatori Daniela , Schouten Jacobine , Meijborg Veronique M. F. , Lauridsen Henrik , Agger Peter
Published: August 2023

An intricate meshwork of trabeculations lines the luminal side of cardiac ventricles. Compaction, a developmental process, is thought to reduce trabeculations by adding them to the neighboring compact wall which is then enlarged. When pig, a plausible cardiac donor for xenotransplantation, is compared to human, the ventricular walls appear to have fewer trabeculations. We hypothesized the trabecular volume is proportionally smaller in pig than in human. Macroscopically, we observed in sixteen pig hearts that the ventricular walls harbor few but large trabeculations. Close inspection revealed a high number of tiny trabeculations, a few hundred, within the recesses of the large trabeculations. While tiny, these were still larger than embryonic trabeculations and even when considering their number, the total tally of trabeculations in pig was much fewer than in human. Volumetrics based on high-resolution MRI of additional six pig hearts compared to six human hearts, revealed the left ventricles were not significantly differently trabeculated (21.5 versus 22.8%, respectively), and the porcine right ventricles were only slightly less trabeculated (42.1 versus 49.3%, respectively). We then analyzed volumetrically ten pig embryonic hearts from gestational day 14 to 35. The trabecular and compact layer always grew, as did the intertrabecular recesses, in contrast to what compaction predicts. The proportions of the trabecular and compact layers changed substantially, nonetheless, due to differences in their growth rate rather than compaction. In conclusion, processes that affect the trabecular morphology do not necessarily affect the proportion of trabecular-to-compact myocardium and they are then distinct from compaction.
Long noncoding RNAs and mRNAs profiling in ovary during laying and broodiness in Taihe Black-Bone Silky Fowls (Gallus gallus domesticus Brisson)
Authors: Tan Yuting , Huang Yunyan , Xu Chunhui , Huang Xuan , Yin Zhaozheng
Published: August 2023

Broodiness is one of the important factors affecting poultry egg production, with the black-bone Silky (Gallus gallus domesticus Brisson) exhibiting strong broodiness behavior. Changes in ovarian signaling can offer insights into the mechanisms influencing broodiness. However, research comparing the characteristics of long non- coding RNAs (lncRNAs) in the ovaries during the broody chickens (BC) and high egg- laying chickens (GC) groups of poultry is still limited. In this study, we employed RNA-seq to analyze the ovarian transcriptomes (lncRNAs and mRNAs) of eight brooding and high egg-laying Taihe Black-Bone Silky Fowls (TBsf). We identified a total of 16,444 mRNAs and 18,756 lncRNAs, among which 349 mRNAs and 651 lncRNAs (P < 0.05) showed significantly different expression (DE) between the BC and GC groups. Additionally, we identified the cis-regulated and trans-regulated target genes of differentially abundant lncRNA transcripts and constructed an lncRNA- mRNA trans-regulated interaction network associated with ovarian follicle development. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analyses revealed that DE mRNAs and target genes of DE lncRNAs were associated with neuroactive ligand-receptor interaction, CCR6 chemokine receptor binding, G-protein coupled receptor binding, Cytokine-cytokine receptor interaction, and ECM-receptor interaction pathways. Our study provides a catalogue of lncRNAs and mRNAs related to ovarian development and constructs a predicted interaction network of differentially abundant lncRNAs and Differentially Expressed Genes (DEGs) in TBsf, thus enhancing our understanding of the interactions between lncRNAs and genes regulating brooding behavior.

Author summary

In this study, we performed transcriptome sequencing analysis of the ovaries from BC and GC groups in TBsf. A total of 349 significantly differentially expressed mRNAs and 651 significantly differentially expressed lncRNAs were detected. Furthermore, we identified 10 key genes that may affect reproductive performance in chickens: STC1, MMP13, IL8, AT2, FSHB, RARB, THOC7, FGF12, EPHA1, and NPY5R. Based on GO and KEGG enrichment analyses, we preliminarily explored five pathways that may influence egg production performance: neuroactive ligand-receptor interaction, CCR6 chemokine receptor binding, G-protein coupled receptor binding, cytokine-cytokine receptor interaction and ECM-receptor interaction. These genes and pathways may play crucial roles in improving broodiness behavior. This study establishes a foundation for subsequent screening of candidate functional genes and functional validation of important production traits in TBsf, providing a theoretical basis for further molecular breeding and enhancing egg-laying performance in TBsf.
Bioengineering of a human innervated cardiac muscle model
Authors: Schneider Lennart Valentin , Guobin Bao , Methi Aditi , Jensen Ole , Schmoll Kea Aline , Setya Michael Gani , Sakib Sadman , Fahud Aminath Luveysa , Brockmöller Jürgen , Fischer André , Liaw Norman Y. , Zimmermann Wolfram-Hubertus , Zafeiriou Maria-Patapia
Published: August 2023

Cardiac autonomic neurons control cardiac contractility. Dysregulation of the autonomic nervous system can lead to sympathetic overdrive resulting in heart failure and an increased incidence of fatal arrhythmias. Here, we introduce innervated engineered human myocardium (iEHM), a novel model of neuro-cardiac junctions, constructed by fusion of a bioengineered neural organoid (BENO) patterned to autonomic nervous system and engineered human myocardium (EHM). Projections of sympathetic neurons into engineered human myocardium formed presynaptic terminals in close proximity to cardiomyocytes and an extensive vascular network co-developing in the tissues. Contractile responses to optogenetic stimulation of the accordingly engineered neuronal component demonstrated functionality of neuro-cardiac junctions in iEHM. This model will serve as a human surrogate system to delineate neuron and cardiac cell contribution to brain and heart diseases and is an important step towards engineering a human brain to heart axis in a dish.
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

The 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

A 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

SNARE 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

Gene 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

Objective

To 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.

Methods

PTX 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.

Results

The 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.

Conclusions

Inhibiting 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

The 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 Statement

Using 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

The 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

Protein 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.

IMPORTANCE

KSHV 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

The 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

To 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

The 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 POINTS

A CRISPR-Cas9 based protocol was optimized to knockout genes in primary human neutrophils derived from CD34+ hHSCs

Under physiological fluid shear conditions, a sialofucosylated form of CD44 expressed on human neutrophils is a major E-selectin ligand

VISUAL 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

Heart 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
Copy number of the eukaryotic rRNA gene can be counted comprehensively
Authors: Yabuki Akinori , Hoshino Tatsuhiko , Nakamura Tamiko , Mizuno Keiko
Published: August 2023

Gene sequence has been widely used in molecular ecology. For instance, the ribosomal RNA (rRNA) gene has been widely used as a biological marker to understand microbial communities. The variety of the detected rRNA gene sequences reflects the diversity of the microorganisms existing in the analyzed sample. Their biomass also can be estimated by applying quantitative sequencing with the information of rRNA gene copy numbers in genomes, however, the information on rRNA gene copy number is still limited. Especially, the copy number in microbial eukaryotes is much less understood than that of prokaryotes, possibly because of the large and complex structure of eukaryotic genomes. In this communication, we report an alternative approach that is more appropriate from the existing method of quantitative sequencing and demonstrate that the copy number of eukaryotic rRNA can be measured efficiently and comprehensively. By applying this approach widely, information on the eukaryotic rRNA copy number can be determined, and their community structures can be depicted and compared more efficiently.
A conserved transcription factor controls gluconeogenesis via distinct targets in hypersaline-adapted archaea with diverse metabolic capabilities
Authors: Hackley Rylee K. , Vreugdenhil-Hayslette Angie , Darnell Cynthia L. , Schmid Amy K.
Published: August 2023

Timely regulation of carbon metabolic pathways is essential for cellular processes and to prevent futile cycling of intracellular metabolites. InHalobacterium salinarum, a hypersaline adapted archaeon, a sugar-sensing TrmB family protein controls gluconeogenesis and other biosynthetic pathways. Notably,Hbt. salinarumdoes not utilize carbohydrates for energy, uncommon among Haloarchaea. We characterized a TrmB-family transcriptional regulator in a saccharolytic generalist,Haloarcula hispanica, to investigate whether the targets and function of TrmB, or its regulon, is conserved in related species with distinct metabolic capabilities. InHar. hispanica, TrmB binds to 15 sites across the genome and induces the expression of genes primarily involved in gluconeogenesis and tryptophan biosynthesis. An important regulatory control point inHbt. salinarum, activation ofppsAand repression ofpykA, is absent inHar. hispanica. Contrary to its role inHbt. salinarumand saccharolytic hyperthermophiles, TrmB does not act as a global regulator: it does not directly repress the expression of glycolytic enzymes, peripheral pathways such as cofactor biosynthesis, or catabolism of other carbon sources inHar. hispanica. Cumulatively, these findings suggest re-wiring of the TrmB regulon alongside metabolic network evolution in Haloarchaea.
Neuroendocrine control of synaptic transmission by PHAC-1 inC. elegans
Authors: Stratigi Aikaterini , Soler-García Miguel , Krout Mia , Shukla Shikha , Bono Mario De , Richmond Janet E. , Laurent Patrick
Published: August 2023

A dynamic interplay between synaptic and neuromodulatory signalling guarantees flexible but robust neuronal circuits. Presynaptic modulation plays a crucial role in controlling the excitatory-inhibitory balance within networks. Here, we designed a genetic screen to identify genes involved in the neuromodulation of theC. elegansneuromuscular junctions (NMJ) and identified the orthologs of the Protein Phosphatase 1 regulatory subunit PHACTR1 (phac-1)and the presynaptic phosphoproteins Synapsin (snn-1). Fivede novovariants of human PHACTR1 are associated with severe early-onset epilepsies (DEE70). To understand the impact of these variants, we introduced the DEE70 mutations intophac-1. These mutations resulted in the formation of a constitutively active PP1-PHAC-1 holoenzyme that disrupts cholinergic signalling at the NMJ. By using quantitative fluorescence imaging, electron microscopy and electrophysiology, we found that the constitutive holoenzyme alters the synaptic vesicle cycle, reduces the synaptic vesicle reserve pool, and increases neuropeptide release by dense-core vesicles. Notably, while SNN-1 phosphoregulation contributes to NMJ signalling, genetic interactions suggest that SNN-1 is not the main effector of PP1-PHAC-1 holoenzyme signalling. Collectively, our results confirm the pathogenicity of DEE70 variants, clarify their dominant-positive effects, and provide evidence of a presynaptic mode of action for DEE70.
The Role of Decompressive Craniectomy following Microsurgical Repair of a Ruptured Aneurysm: Analysis of a South Australian Cerebrovascular Registry
Authors: O’Donohoe Tom J , Ovenden Christopher , Bouras George , Chidambaram Seevakan , Plummer Stephanie , Davidson Andrew S , Kleinig Timothy , Abou-Hamden Amal
Published: August 2023

Background

Decompressive craniectomy (DC) remains a controversial intervention for established or anticipated intracranial hypertension among patients with aneurysmal subarachnoid haemorrhage (aSAH).

Methods

We identified aSAH patients who underwent DC following microsurgical aneurysm repair from a prospectively maintained cerebrovascular registry and compared their outcomes with a propensity-matched cohort who did not. Logistic regression was used to identify predictors of undergoing decompressive surgery and post-operative outcome. The outcomes of interest were inpatient mortality, unfavourable outcome at first and final follow-up, NIS-Subarachnoid Hemorrhage Outcome Measure and modified Rankin Score (mRS) at first and final follow-up.

Results

A total of 246 consecutive patients with aSAH underwent microsurgical clipping of the culprit aneurysm between 01/09/2011 and 20/07/2020. Of these, 46 patients underwent DC and were included in the final analysis. Unsurprisingly, patients treated with DC had a greater chance of unfavourable outcome (p<0.001) and higher median mRS (p<0.001) compared with those who did not at final follow-up. Despite this, almost two-thirds (64.1%) of patients undergoing a DC had a favourable outcome at this time-point. When compared with a propensity-matched cohort who did not, patients treated with DC fared worse at all endpoints. Multivariable logistic regression revealed that the presence of intracerebral haemorrhage (ICH) and increased pre-operative mid-line shift were predictive of undergoing DC, and WFNS grade ≥ 4 and a delayed ischaemic neurological deficit requiring endovascular angioplasty were associated with an unfavourable outcome.

Conclusions

Our data suggest that DC can be performed with acceptable rates of morbidity and mortality, particularly among patients who present with lower grade aSAH. Further research is required to determine the superiority, or otherwise, of DC compared with structured medical management of intracranial hypertension in this context, and to identify predictors of requiring decompressive surgery and patient outcome.
An amortized approach to non-linear mixed-effects modeling based on neural posterior estimation
Authors: Arruda Jonas , Schälte Yannik , Peiter Clemens , Teplytska Olga , Jaehde Ulrich , Hasenauer Jan
Published: August 2023

Non-linear mixed-effects models are a powerful tool for studying heterogeneous populations in various fields, including biology, medicine, economics, and engineering. Here, the aim is to find a distribution over the parameters that describe the whole population using a model that can generate simulations for an individual of that population. However, fitting these distributions to data is computationally challenging if the description of individuals is complex and the population is large. To address this issue, we propose a novel machine learning-based approach: We exploit neural density estimation based on conditional normalizing flows to approximate individual-specific posterior distributions in an amortized fashion, thereby allowing for efficient inference of population parameters. Applying this approach to problems from cell biology and pharmacology, we demonstrate its unseen flexibility and scalability to large data sets compared to established methods.
Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO2
Authors: Brotherton Deborah H. , Nijjar Sarbjit , Savva Christos G. , Dale Nicholas , Cameron Alexander D.
Published: August 2023

Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO2. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E mutation pushes a conformational equilibrium towards the channel having a constricted pore entrance, similar to effects seen on raising the partial pressure of CO2. In previous structures of connexins, the cytoplasmic loop, important in regulation and where K125 is located, is disordered. Through further cryo-EM studies we trap distinct states of Cx26 and observe density for the cytoplasmic loop. The interplay between the position of this loop, the conformations of the transmembrane helices and the position of the N-terminal helix, which controls the aperture to the pore, provides a mechanism for regulation.
Multi-scale characterisation of homologous recombination deficiency in breast cancer
Authors: Jacobson Daniel H. , Pan Shi , Fisher Jasmin , Secrier Maria
Published: August 2023

Background

Homologous recombination is a robust, broadly error-free mechanism of double strand break repair, and deficiencies cause reliance on alternative repair processes, leading to sensitivity to PARP inhibitors. Patients displaying homologous recombination deficiency can be identified using ‘mutational signatures’. However, these patterns are difficult to reliably infer from exome sequencing. Additionally, as mutational signatures are a historical record of mutagenic processes, this limits their utility in describing the current status of a tumour.

Results

Here, we apply two methods for classifying homologous recombination deficiency in breast cancer to explore the features and heterogeneity associated with this phenotype. Firstly, we apply a likelihood-based method which leverages small insertions and deletions to improve classification of homologous recombination deficiency in exome sequenced breast cancers. We find that whilst BRCA+ homologous recombination deficient samples display strong similarities to those harbouring BRCA defects, they deviate in microenvironmental features such as hypoxic signalling. Secondly, using this classification we develop a 228-gene transcriptional signature which simultaneously characterises homologous recombination deficiency and BRCA1/2-defect status, and is linked with PARP inhibitor response. Finally, we apply this signature to single-cell RNA-sequenced breast cancer cohorts to study homologous recombination deficiency at single cell resolution and demonstrate that these cells present a distinct milieu of interactions with immune cells in their microenvironment compared to their HR proficient counterparts, which could inform checkpoint blockade for HRD tumours.

Conclusions

We apply multi-scale approaches to characterise homologous recombination deficiency in breast cancer through the development of mutational and transcriptional signatures. We show that indels, even at low levels, can improve homologous recombination deficiency classification. Additionally, we demonstrate the heterogeneity of homologous recombination deficiency, especially in relation to BRCA status, and show that indications of this feature can be captured at a single-cell level, enabling further investigations into interactions between DNA repair deficient cells and their tumour microenvironment.
Navigating multiple accountabilities through managers’ boundary work in professional service firms
Authors: Azambuja Ricardo , Baudot Lisa , Malsch Bertrand
Published: August 2023

Purpose

This study explores the professional work of managers in professional service firms (PSFs) by focusing on the relational position of managers as they interface between diverse groups of actors and navigate a multiplicity of accountabilities.

Design/methodology/approach

Drawing on an ethnographic study of managers in a Brazilian PSF, this study demonstrates through observations, interviews and shadowing how PSF managers address multiple accountabilities in the conduct of professional work.

Findings

To navigate multiple accountabilities, PSF managers perform several forms of boundary work, which the authors conceptualize as “revamping” and “remolding” operations and “translating” and “transforming” understandings. The form of boundary work performed depends on the configuration of two elements of accountability demands: knowledge orientation and extent of exposure. Although analytically distinct, these elements intertwine, suggesting the need for a dynamic perspective on accountability. The analysis shows that professional work extends beyond fixed or passive views of hierarchical and intrinsic accountability to emphasize that managers exude accountability of an adaptive nature.

Research limitations/implications

This study illustrates and theorizes the role of boundary work in PSFs, develops its link to accountability, and identifies its scholarly and practical affordances and limitations for understanding managers’ professional work when navigating multiple accountabilities.

Originality/value

The findings reveal perspectives and behaviors of managers embedded in PSFs, illustrating their unstructured and agentic experiences of accountability and boundary work in practice. The insights can be extended to other contexts where managers face multiple accountabilities in conducting professional work.