Gene Symbol Research Articles

Transcriptionally induced nucleoid-associated protein-like ccr1 in combined-culture serves as a global effector of Streptomyces secondary metabolism.

Combined-cultures involving mycolic acid-containing bacteria (MACB) can stimulate secondary metabolite (SM) production in actinomycetes. In a prior investigation, we screened Streptomyces coelicolor JCM4020 mutants with diminished production of SMs, specifically undecylprodigiosin (RED), which was enhanced by introducing the MACB Tsukamurella pulmonis TP-B0596. In this study, we conducted mutational analysis that pinpointed the sco1842 gene, which we assigned the gene name ccr1 (combined-culture related regulatory protein no. 1), as a crucial factor in the deficient phenotype observed in the production of various major SMs in S. coelicolor A3(2). Notably, the Ccr1 (SCO1842) homolog was found to be highly conserved throughout the Streptomyces genome. Although Ccr1 lacked conserved motifs, in-depth examination revealed the presence of a helix-turn-helix (HTH) motif in the N-terminal region and a helicase C-terminal domain (HCTD) motif in the C-terminal region in some of its homologs. Ccr1 was predicted to be a nucleoid-associated protein (NAP), and its impact on gene transcription was validated by RNA-seq analysis that revealed genome-wide variations. Furthermore, RT-qPCR demonstrated that ccr1 was transcriptionally activated in combined-culture with T. pulmonis, which indicated that Ccr1 is involved in the response to bacterial interaction. We then investigated Streptomyces nigrescens HEK616 in combined-culture, and the knockout mutant of the ccr1 homolog displayed reduced production of streptoaminals and 5aTHQs. This finding reveals that the Ccr1 homolog in Streptomyces species is associated with SM production. Our study elucidates the existence of a new family of NAP-like proteins that evolved in Streptomyces species and play a pivotal role in SM production.

PCMT1 confirmed as a pan-cancer immune biomarker and a contributor to breast cancer metastasis.

Protein L-isoaspartyl (D-aspartyl) methyltransferase (PIMT, gene name PCMT1) is an enzyme that repairs proteins with altered aspartate residues by methylation, restoring their normal structure and function. This study conducted a comprehensive analysis of PCMT1 in pan-cancer. The Cancer Genome Atlas, Human Protein Atlas website, and the Genotype-Tissue Expression were utilized in analysis of PCMT1 expression. We examined the association between PCMT1 expression and various factors, including gene modifications, DNA methylation, immune cell infiltration, immunological checkpoints, drug susceptibility, tumor mutation burden (TMB), and microsatellite instability (MSI). Enrichment analyses determined the potential biological roles and pathways involving PCMT1. Our focus then shifted to the role of PCMT1 in breast invasive carcinoma (BRCA). We found that PCMT1 expression was aberrant in many tumors and significantly influenced the prognosis across several cancer types. Gene alterations in PCMT1 predominantly involved deep deletions and amplifications. A negative correlation was observed between DNA methylation and PCMT1 expression across all studied cancer types except thyroid carcinoma PCMT1 exhibited positive correlations with common lymphoid progenitor and CD4(+) T helper 2 cells, whereas it was inversely correlated with central and effector memory T cells, memory CD8(+) T cells, and CD4(+) T helper 1 cells. In many cancer types, PCMT1 expression closely correlated with immunological checkpoint inhibitors, TMB, and MSI. It was also significantly linked to pathways involved in epithelial-mesenchymal transition (EMT), highlighting its role in cancer metastasis. PCMT1 emerged as a significant predictor of breast cancer progression. In vitro experiments demonstrated that reducing PCMT1 expression decreased BRCA cell migration and invasiveness. Additionally, animal studies confirmed that inhibition of PCMT1 slowed tumor growth.

The Functional Diversity of the High-Affinity Nitrate Transporter Gene Family in Hexaploid Wheat: Insights from Distinct Expression Profiles.

High-affinity nitrate transporters (NRT) are key components for nitrogen (N) acquisition and distribution within plants. However, insights on these transporters in wheat are scarce. This study presents a comprehensive analysis of the NRT2 and NRT3 gene families, where the aim is to shed light on their functionality and to evaluate their responses to N availability. A total of 53 NRT2s and 11 NRT3s were identified in the bread wheat genome, and these were grouped into different clades and homoeologous subgroups. The transcriptional dynamics of the identified NRT2 and NRT3 genes, in response to N starvation and nitrate resupply, were examined by RT-qPCR in the roots and shoots of hydroponically grown wheat plants through a time course experiment. Additionally, the spatial expression patterns of these genes were explored within the plant. The NRT2s of clade 1, TaNRT2.1-2.6, showed a root-specific expression and significant upregulation in response to N starvation, thus emphasizing a role in N acquisition. However, most of the clade 2 NRT2s displayed reduced expression under N-starved conditions. Nitrate resupply after N starvation revealed rapid responsiveness in TaNRT2.1-2.6, while clade 2 genes exhibited gradual induction, primarily in the roots. TaNRT2.18 was highly expressed in above-ground tissues and exhibited distinct nitrate-related response patterns for roots and shoots. The TaNRT3 gene expression closely paralleled the profiles of TaNRT2.1-2.6 in response to nitrate induction. These findings enhance the understanding of NRT2 and NRT3 involvement in nitrogen uptake and utilization, and they could have practical implications for improving nitrogen use efficiency. The study also recommends a standardized nomenclature for wheat NRT2 genes, thereby addressing prior naming inconsistencies.

Neuroprotective effects of nanogold-based Ayurveda medicine Suvarna Bhasma against rotenone-induced Parkinson's-like model

BackgroundNeurodegenerative diseases have been one of the major concerns for human health. Genetic and environmental factors are believed to be responsible for neuronal diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease. It is difficult to restore normal nervous function after neurodegeneration; hence, prevention could be the best strategy against these diseases. Ayurved medicines such as Suvarna Bhasma (SB) have enormous potential to treat these neurological diseases. AimThe aim of this study is to examine the protective effect of SB against rotenone-induced Parkinson's-like model in zebrafish. Materials and methodsIn this study, we induced Parkinson's-like disease model in zebrafish by inducing it with rotenone (7 μg/L). We examined the behavioural, proteomics and dopamine alterations of rotenone induced zebrafish of SB pre-treated group as compared to the control group. ResultsThe behavioural experiments showed that due to rotenone exposure, Parkinson's-like behavioural abnormality was induced in zebrafish. However, because of SB treatment, this behavioural abnormality was reduced. The proteomics study of zebrafish brains clearly showed that the SB-treated group was not significantly affected due to rotenone exposure. However, in the SB non-treated group, expression of nine proteins that are linked to Parkinson's disease (gene name: sncgb, ywhae1, ywhah, uchl1, ywhaba, psma6a, ywhabl, ywhaqb, and ywhabb) were differentially expressed after rotenone exposure. Finally, prevention of dopamine alteration in SB-treated fish brains confirmed the protective action of SB against rotenone-induced Parkinson's-like model in zebrafish. ConclusionsThis study finds that Suvarna Bhasma has neuroprotective effects against Parkinson's-like disease model.

Microglial-specific knockdown of iron import gene, Slc11a2, blunts LPS-induced neuroinflammatory responses in a sex-specific manner

Neuroinflammation and microglial iron load are significant hallmarks found in several neurodegenerative diseases. In in vitro systems, microglia preferentially upregulate the iron importer, divalent metal transporter 1 (DMT1, gene name Slc11a2) in response to inflammatory stimuli, and it has been shown that iron can augment cellular inflammation, suggesting a feed-forward loop between mechanisms involved in iron import and inflammatory signaling. However, it is not understood how microglial iron import mechanisms contribute to inflammation in vivo, or whether altering a microglial iron-related gene affects the inflammatory response. These studies aimed to determine the effect of knocking down microglial iron import gene Slc11a2 on the inflammatory response in vivo. We generated a novel model of tamoxifen-inducible, microglial-specific Slc11a2 knockdown using Cx3cr1Cre-ERT2 mice. Transgenic male and female mice were administered intraperitoneal saline or lipopolysaccharide (LPS) and assessed for sickness behavior post-injection. Plasma cytokines and microglial bulk RNA sequencing (RNASeq) analyses were performed at 4 h post-LPS, and microglia were collected for gene expression analysis after 24 h. A subset of mice was assessed in a behavioral test battery following LPS-induced sickness recovery. Control male, but not female, mice significantly upregulated microglial Slc11a2 at 4 and 24 h following LPS. In Slc11a2 knockdown mice, we observed an improvement in the acute behavioral sickness response post-LPS in male, but not female, animals. Microglia from male, but not female, knockdown animals exhibited a significant decrease in LPS-provoked pro-inflammatory cytokine expression after 24 h. RNASeq data from male knockdown microglia 4 h post-LPS revealed a robust downregulation in inflammatory genes including Il6, Tnfα, and Il1β, and an increase in anti-inflammatory and homeostatic markers (e.g., Tgfbr1, Cx3cr1, and Trem2). This corresponded with a profound decrease in plasma pro-inflammatory cytokines 4 h post-LPS. At 4 h, male knockdown microglia also upregulated expression of markers of iron export, iron recycling, and iron homeostasis and decreased iron storage and import genes, along with pro-oxidant markers such as Cybb, Nos2, and Hif1α. Overall, this work elucidates how manipulating a specific gene involved in iron import in microglia alters acute inflammatory signaling and overall cell activation state in male mice. These data highlight a sex-specific link between a microglial iron import gene and the pro-inflammatory response to LPS in vivo, providing further insight into the mechanisms driving neuroinflammatory disease.

Deciphering the intricate hierarchical gene regulatory network: unraveling multi-level regulation and modifications driving secondary cell wall formation.

Wood quality is predominantly determined by the amount and the composition of secondary cell walls (SCWs). Consequently, unraveling the molecular regulatory mechanisms governing SCW formation is of paramount importance for genetic engineering aimed at enhancing wood properties. Although SCW formation is known to be governed by a hierarchical gene regulatory network (HGRN), our understanding of how a HGRN operates and regulates the formation of heterogeneous SCWs for plant development and adaption to ever-changing environment remains limited. In this review, we examined the HGRNs governing SCW formation and highlighted the significant key differences between herbaceous Arabidopsis and woody plant poplar. We clarified many confusions in existing literatures regarding the HGRNs and their orthologous gene names and functions. Additionally, we revealed many network motifs including feed-forward loops, feed-back loops, and negative and positive autoregulation in the HGRNs. We also conducted a thorough review of post-transcriptional and post-translational aspects, protein-protein interactions, and epigenetic modifications of the HGRNs. Furthermore, we summarized how the HGRNs respond to environmental factors and cues, influencing SCW biosynthesis through regulatory cascades, including many regulatory chains, wiring regulations, and network motifs. Finally, we highlighted the future research directions for gaining a further understanding of molecular regulatory mechanisms underlying SCW formation.

RNA Sequencing Analysis of Changes in Pancreatic RNA in Tree Shrews with Traumatic Systemic Inflammatory Response Syndrome after Treatment with Umbilical Cord Mesenchymal Stem Cells

Background: Systemic inflammatory response syndrome (SIRS) is a systemic nonspecific inflammatory response to severe injury caused by infectious or noninfectious factors. Methods: The impact method was used to make unilateral femoral comminuted fractures in tree shrews and lipopolysaccharide was injected intravenously to create a traumatic systemic inflammatory response syndrome model. The treatment group was infused with tree shrew umbilical cord mesenchymal stem cells via the tail vein. Result: After 10 days of treatment, 3 tree shrews were killed in each of the control group, the model group and the treatment group and pancreatic tissue was taken for RNA sequencing analysis. The results showed statistically significant changes in some genes. The two genes with the most significantly changed expression levels were ENSTBEG00000013408 (gene name TMEM211) and ENSTBEG00000006837 (gene name C1QL3). Their expression levels were increased in the model group and decreased after treatment. The changes in inflammatory factors indicate that the model group has an obvious inflammatory response and the treatment of umbilical cord mesenchymal stem cells has the effect of reducing inflammatory factors, which further proves the anti-inflammatory effect of umbilical cord mesenchymal stem cells. There was also an obvious change in the expression of the ENSTBEG00000021330 gene (gene name ATP8), which decreased in the model group and increased after treatment, indicating that ATP8 is a gene that contributes to the recovery of systemic inflammatory response syndrome. In the future, we may use this gene to treat systemic inflammatory response syndrome.

Discordance between different bioinformatic methods for identifying resistance genes from short-read genomic data, with a focus on Escherichia coli.

Several bioinformatics genotyping algorithms are now commonly used to characterize antimicrobial resistance (AMR) gene profiles in whole-genome sequencing (WGS) data, with a view to understanding AMR epidemiology and developing resistance prediction workflows using WGS in clinical settings. Accurately evaluating AMR in Enterobacterales, particularly Escherichia coli, is of major importance, because this is a common pathogen. However, robust comparisons of different genotyping approaches on relevant simulated and large real-life WGS datasets are lacking. Here, we used both simulated datasets and a large set of real E. coli WGS data (n=1818 isolates) to systematically investigate genotyping methods in greater detail. Simulated constructs and real sequences were processed using four different bioinformatic programs (ABRicate, ARIBA, KmerResistance and SRST2, run with the ResFinder database) and their outputs compared. For simulation tests where 3079 AMR gene variants were inserted into random sequence constructs, KmerResistance was correct for 3076 (99.9 %) simulations, ABRicate for 3054 (99.2 %), ARIBA for 2783 (90.4 %) and SRST2 for 2108 (68.5 %). For simulation tests where two closely related gene variants were inserted into random sequence constructs, KmerResistance identified the correct alleles in 35 338/46 318 (76.3 %) simulations, ABRicate identified them in 11 842/46 318 (25.6 %) simulations, ARIBA identified them in 1679/46 318 (3.6 %) simulations and SRST2 identified them in 2000/46 318 (4.3 %) simulations. In real data, across all methods, 1392/1818 (76 %) isolates had discrepant allele calls for at least 1 gene. In addition to highlighting areas for improvement in challenging scenarios, (e.g. identification of AMR genes at <10× coverage, identifying multiple closely related AMR genes present in the same sample), our evaluations identified some more systematic errors that could be readily soluble, such as repeated misclassification (i.e. naming) of genes as shorter variants of the same gene present within the reference resistance gene database. Such naming errors accounted for at least 2530/4321 (59 %) of the discrepancies seen in real data. Moreover, many of the remaining discrepancies were likely 'artefactual', with reporting of cut-off differences accounting for at least 1430/4321 (33 %) discrepants. Whilst we found that comparing outputs generated by running multiple algorithms on the same dataset could identify and resolve these algorithmic artefacts, the results of our evaluations emphasize the need for developing new and more robust genotyping algorithms to further improve accuracy and performance.

Uncovering the Hidden World of Aqueous Humor Proteins for Discovery of Biomarkers for Marfan Syndrome.

Ectopia lentis is a hallmark of Marfan syndrome (MFS), a genetic connective tissue disorder affecting 1/5000 to 1/10 000 individuals worldwide. Early detection in ophthalmology clinics and timely intervention of cardiovascular complications can be lifesaving. In this study, a modified proteomics workflow with liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based data-independent acquisition (DIA) and field asymmetric ion mobility spectrometry (FAIMS) to profile the proteomes of aqueous humor (AH) and lens tissue from MFS children with ectopia lentis is utilized. Over 2300 and 2938 comparable proteins are identified in AH and the lens capsule, respectively. Functional enrichment analyses uncovered dysregulation of complement and coagulation-related pathways, collagen binding, and cell adhesion in MFS. Through weighted correlation network analysis (WGCNA) and machine learning, distinct modules associated with clinical traits are constructed and a unique biomarker panel (Q14376, Q99972, P02760, Q07507; gene names: GALE, MYOC, AMBP, DPT) is defined. These biomarkers are further validated using advanced parallel reaction monitoring (PRM) in an independent patient cohort. The results provide novel insights into the proteome characterization of ectopia lentis and offer a promising approach for developing a valuable biomarker panel to aid in the early diagnosis of Marfan syndrome via AH proteome.

The alleles bc-ud and bc-ur (previously bc-4 gene), representing coding mutations within Vps4 AAA+ ATPase ESCRT protein, interact with other genes to condition resistance to BCMV and BCMNV in common bean.

Bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) have a damaging impact on global common bean (Phaseolus vulgaris L.) cultivation, causing potential yield losses of over 80%. The primary strategy for controlling these viruses is through host plant resistance. This research aimed to identify and validate structural variations for the bc-ud gene as revealed by long-read sequencing, develop an efficient DNA marker to assist selection of bc-ud in snap and dry beans, and examine the interactions between the bc-ud allele and other BCMV resistance genes. A gene (Phvul.005G125100) model on chromosome Pv05, encoding a vacuolar protein-sorting 4 (Vps4) AAA+ ATPase endosomal sorting complexes required for transport (ESCRT) protein, was identified as the best candidate gene for bc-ud. An 84-bp repetitive insertion variant within the gene, exhibited 100% co-segregation with the bc-ud resistance allele across 264 common bean accessions. The 84-bp repetitive insertion was labeled with an indel marker IND_05_36225873 which was useful for tracking the bc-ud allele across diverse germplasm. A different single nucleotide polymorphism variant within the same candidate gene was associated with the bc-4 gene. Segregation in F2 populations confirmed bc-ud and bc-4 were alleles, so bc-4 was renamed bc-ur to fit gene nomenclature guidelines. The interactions of bc-ud and bc-ur with other resistance genes, such as bc-1 (receptor-like kinase on Pv03) and bc-2 (Vps4 AAA+ ATPase ESCRT protein on Pv11), validated gene combinations in the differential "host groups" effective against specific BCMV/BCMNV "pathogroups." These findings increase our understanding of the Bc-u locus, and enhance our ability to develop more resilient bean varieties through marker-assisted selection, reducing the impact of BCMV and BCMNV.

A proteomic approach to identifying spermatozoa proteins in Indonesian native Madura bulls.

Proteins assist sperm mature, transit the female reproductive tract, and recognise sperm oocytes. Indigenous Indonesian bulls, Madura bulls, have not been studied for reproductive proteomics. As local Indonesian beef livestock, Madura cattle assist in achieving food security; hence, their number must be improved. Thus, the identification of molecular proteomics-based bull fertility biomarkers is needed. This study aimed to characterise the sperm fertility function of the superior Madura bull (Bos indicus × Bos Javanicus) spermatozoa proteome. Frozen semen from eight Madura superior bulls (Bos indicus × Bos javanicus) aged 4-8 years was obtained from the artificial insemination centre (AIC) in Singosari and Lembang. Madura superior bulls are those that have passed the bull breeding soundness evaluation. Frozen sperm were thawed and centrifuged at 3000 × g for 30 min. Proteins in sperm were characterised through proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting gene symbols for each protein were then subjected to bioinformatics tools, including UniProt, DAVID, and STRING databases. Regarding sperm fertility, the analysis revealed that 15 proteins were identified in the sperm of Madura bulls. Amongst the identified proteins, the superior Madura bull sperm contained several motilities, energy-related proteins, and chaperone proteins. A substantial portion of characterised proteins are linked to metabolic pathways and the tricarboxylic acid (TCA) cycle, contributing to sperm energy production. In conclusion, the first in-depth proteome identification of sperm related to sperm quality and bull fertility of a unique indigenous Madura breed of Indonesia was performed using the LC-MS/MS proteomic method. These findings may serve as a reference point for further studies related to the functions of bovine sperm and biomarkers of fertility and sperm quality.

Blood‐derived extracellular vesicles phosphoproteome as a resource of biomarker candidates for Alzheimer’s disease

AbstractBackgroundProtein phosphorylation plays a crucial role in cell communication, signaling and by extension is relevant to Alzheimer´s disease (AD) pathogenesis. Extracellular vesicles (EVs) are recognized as important mediators in intercellular communication and can be isolated from peripheral biofluids, where the content reflects the cellular origin. EVs’ role in AD pathophysiology and their value as biomarker resources is being unraveled. However, little is known regarding EVs’ phosphoproteome in AD and it has still to be determined if blood‐derived EVs are suitable for phosphoproteome profiling. Hence, this work aimed to characterize plasma‐derived EVs (pdEVs) and plasma supernatant EVs free (plasma‐depleted EVs) phosphoproteomes, to potential unravel disease mechanisms and/or new biomarker candidates for AD.MethodA pool of human plasma was used for pdEVs isolation using a precipitation‐based methodology. Both pdEVs and plasma‐depleted EVs samples were analyzed using an antibody‐based phosphospecific microarray. Characterization of the phosphoproteomes obtained was achieved by Gene Ontology enrichment analysis and the most relevant Reactome pathways associated, using ClueGo and CluePedia plugins from Cytoscape v3.9.1. In addition, protein‐protein interaction network of the pdEVs phosphoproteome was retrieved from STRING database and analyzed using Cytoscape “Network Analyzer”, employing the betweenness centrality option to highlight central nodes. Further, the pdEVs phosphoproteome (corresponding gene names) was overlapped with a list of genes associated with AD, collated from DisGenet database.ResultsMore than 70 proteins were enriched ≥40% in bdEVs when compared to plasma‐depleted. Top biological processes were mainly related to cell proliferation/division, apoptosis and phosphorylation. Reactome pathways analysis revealed terms associated to toll like receptor cascade and MAPK signaling. In the network constructed for the pdEVs enriched proteins, PTEN appeared as a central node. Additionally, overlap with AD gene list from DisGenet revealed that 60% of the pdEVs phosphoproteome is AD‐related. Among these, several kinases already described in senile plaques and/or neurofibrillary tangles were identified.ConclusionIn sum, bdEVs phosphoproteome is enriched in AD‐related targets, exhibiting potential as a biomarker candidate resource for AD diagnosis.This work was funded by the Alzheimer’s Association under Grant 2019‐AARG‐644347, supported by iBiMED‐UIDB/04501/2020,iBiMED‐UIDB/04501/2020,ADPro‐SAICT‐45‐2021‐02 and TSM individual PhD grant SFRH/BD/145979/2019. Health Ethics Committee ARS Centro(No.012804‐04.04.2012) and CNPD(No.369/2012).

Progressive dysregulation of circular RNAs in the hippocampus of a transgenic mouse model of amyloidosis

AbstractBackgroundCircular RNAs (circRNAs) are a class of RNAs that act as transcriptional regulators. They have a circular structure and are produced from back‐splicing events, which makes them more stable than linear RNAs. In Alzheimer’s disease (AD), the role of circRNAs remains elusive. Here, we aimed to evaluate whether the transcription of circRNAs is altered in the hippocampus, a region highly vulnerable to AD, of a transgenic amyloid mouse model.MethodWe selected hippocampal RNA‐sequencing or microarray datasets of circRNAs of the APPswe/PSEN1dE9, a widely used transgenic amyloid mouse model, from the NCBI databases. CircRNAs were detected with the CIRI2 algorithm by using the gene symbol of the parent transcript and the transcript ID. We identified the differently expressed circRNAs between APPswe/PSEN1dE9 and age‐matched wild‐types using the DESeq2 method (p‐value &lt; 0.01). The packages UpSetR and VennDiagram were used to uncover the circRNAs commonly altered between the studies. All analyses were performed in R.ResultFrom 12 databases, three had hippocampal circRNA data of 4, 6, and 8‐month‐old APPswe/PSEN1dE9 mice (GSE166393; GSE158995; and PRJNA712946, n = 3 per group). Amyloid‐beta (Aβ) starts to deposit at six months in the APPswe/PSEN1dE9 hippocampus. The number of dysregulated circRNAs increased with age: 41 (4‐month‐old), 82 (6‐month‐old), and 425 (8‐month‐old) (Figure 1). Interestingly, only one circRNA was shared between the three studies at all time points, originating from the CCDC141 gene. Additionally, 15 circRNAs were shared in at least two ages, such as CD2AP, FMN1, HOMER1, IL1RAP, NDST3, and TET1 (Please, see the full list of intersections in Figure 2). By dividing groups into pre‐plaque (4‐month‐old) and amyloid (6 and 8‐month‐old) phases, we found that four altered circRNAs were shared in both stages, 37 exclusively in pre‐amyloid stage and 490 in amyloid stage (Figure 3).ConclusionOur results demonstrate an increasing dysregulation in circRNAs profiles in the hippocampus of the APP/PS1 mice. Considering the role of circRNAs as transcriptional regulators, one could argue that these progressive changes may impact gene expression in AD‐related brain regions.

Abstract C009: Biomarker analyses for predicting the benefit from immune checkpoint inhibitors in EGFR-mutated non-small cell lung cancer

Abstract Background: Although immune checkpoint inhibitors (ICIs) are effective for non-small cell lung cancer (NSCLC), epidermal growth factor receptor (EGFR)-mutated NSCLC patients are unlikely to benefit from ICIs treatment. It has been reported, however, a small subset of patients harboring EGFR-mutations respond to the treatment, although no biomarkers have been identified. In this study, we retrospectively explored predictive biomarkers to identify the responders by addressing tumor mutational status and tumor microenvironment (TME). Methods: NSCLC patients harboring EGFR-sensitizing mutations who were treated with EGFR-TKIs followed by ICIs alone or ICIs combined with chemotherapy between October 1, 2014 and September 31, 2019 were enrolled into the study. Next-generation sequencing (NGS) was performed on Ion S5 system using DNAs extracted from tissue samples at diagnosis for evaluating genetic mutations and tumor mutational burden (TMB) using Oncomine Tumor Mutation Load Assay. We also evaluated the expression of PD-L1, CD8 and FoxP3 on tumor tissues by immunohistochemical staining. Gene enrichment analysis was performed using gene symbol of somatic mutations by KOBAS in database of KEGG pathway. Mutational signature was identified by SigProfilerAssignment with reference mutational signatures of COSMIC v3.3 using exported vcf file from Ion torrent FileExporter plugin. Fisher's exact test was performed by using EZR software version 1.61. Results: Thirty-four patients were enrolled and the tissue DNA for NGS was successfully collected from 27 patients. Patient demographics were as follows: male/female, 17/17; nonsmoker/smoker, 17/17; 32/2 adenocarcinoma/non-adenocarcinoma, 32/2; EGFR mutation in exon19del/L858R/other, 21/11/2; overall response (ORR) to the past EGFR-TKI treatment CR-PR/SD/PD, 27/2/5; type of ICI pembrolizumab/nivolumab/atezolizumab/other, 17/11/5/1; response to ICI treatment CR-PR/SD/PD, 10/1/22. There was no correlation between the duration of initial EGFR-TKI treatment and the duration of ICI therapy. Patients who had immune-related adverse event (irAE) had significantly longer progression free survival (PFS) with ICIs than those without irAE (p = 0.049). Significantly more CR and PR were observed in patients who do not harbor TP53 mutations (p = 0.033). No correlation was found between TMB and the response to ICI treatment. With regard to PD-L1 expression, tissue tumor proportion score or combined positive score did not correlate with the response to ICI treatment. Patients with high expression level of CD8 showed significantly longer PFS and overall survival (p = 0.039 and p = 0.006). Gene enrichment analysis in CR and PR group indicated that most enriched KEGG pathway was platinum drug resistance and one CR case had a mutational signature of SBS31, relevant to platinum drug treatment, suggesting the involvement of platinum drug treatment in the response to ICI treatment. Conclusions: TME defined by CD8 and TP53 mutational status may have predictive impact on ICIs treatment in EGFR-mutated NSCLC. Citation Format: Mitsuo Osuga, Koichi Azuma, Shoichi Itoh, Yoshitaka Kawa, Daijiro Harada, Yukihiro Toi, Kenta Murotani, Akito Hata, Toshiyuki Kozuki, Shunichi Sugawara, Miyako Satouchi, Nobuyuki Yamamoto, Yasuhiro Koh. Biomarker analyses for predicting the benefit from immune checkpoint inhibitors in EGFR-mutated non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C009.

A genome-wide and candidate gene association study of preterm birth in Korean pregnant women.

Preterm birth (PTB) refers to delivery before 37 weeks of gestation. Premature neonates exhibit higher neonatal morbidity and mortality rates than term neonates; therefore, it is crucial to predict and prevent PTB. Advancements enable the prediction and prevention of PTB using genetic approaches, especially by investigating its correlation with single nucleotide polymorphisms (SNPs). We aimed to identify impactive and relevant SNPs for the prediction of PTB via whole-genome sequencing analyses of the blood of 31 pregnant women with PTB (n = 13) and term birth (n = 18) who visited the Ewha Womans University Mokdong Hospital from November 1, 2018 to February 29, 2020. A genome-wide association study was performed using PLINK 1.9 software and 256 SNPs were selected and traced through protein-protein interactions. Moreover, a validation study by genotyping was performed on 60 other participants (preterm birth, n = 30; term birth, n = 30) for 25 SNPs related to ion channel binding and receptor complex pathways. Odds ratios were calculated using additive, dominant, and recessive genetic models. The risk of PTB in women with the AG allele of rs2485579 (gene name: RYR2) was significantly 4.82-fold increase, and the risk of PTB in women with the AG allele of rs7903957 (gene name: TBX5) was significantly 0.25-fold reduce. Our results suggest that rs2485579 (in RYR2) can be a genetic marker of PTB, which is considered through the association with abnormal cytoplasmic Ca2+ concentration and dysfunctional uterine contraction due to differences of RYR2 in the sarcoplasmic reticulum.

Discovery of a Novel, First-in-Class Bfl-1 BH3 Mimetic with Pro-Apoptotic Activity

Background: Intrinsic apoptosis is regulated by the B Cell lymphoma 2 (BCL-2) protein family. Anti-apoptotic family members, such as BCL-2, MCL-1 and BFL-1, sequester their pro-apoptotic counterparts through a highly conserved BH3 binding groove. Disruption of this balance leads to mitochondrial pore formation and subsequent cell death. To evade apoptosis, tumors often upregulate the expression of one or more anti-apoptotic family members and as such they have become attractive targets for anti-cancer drug development. BFL-1 (‘Bcl-2 related gene expressed in fetal liver’, gene name BCL2A1) is a lesser-known anti-apoptotic family member, physiologically mainly expressed in the hematopoietic system. BCL2A1 overexpression has been reported in many types of B cell lymphoma. Diffuse Large B Cell Lymphoma (DLBCL) patient sample analysis showed that BCL2A1 expression levels are second only to MCL1 within the anti-apoptotic family. Expression of BFL-1 was shown to be upregulated in MYC+/BCL2+ double hit lymphoma cell lines treated with the BCL-2 inhibitor, Venetoclax, in vivo. Interestingly, BFL-1 positive lymphomas are less sensitive to inhibition of BCL-2 and MCL-1. Hence, targeting BFL-1 is a rational and attractive strategy in DLBCL to improve clinical outcome. Methods: Using hit identification screens followed by iterative structure-activity relationship (SAR) analyses and structure-based design, compound A, a first-in-class, selective BFL-1 BH3 mimetic, was identified. Compound A was evaluated using biochemical, cellular in vitro and in vivo tumor efficacy models. Results: Compound A is a highly potent and selective compound in biochemical assays. In BFL-1 sensitive lymphoma cell lines characterized by high BFL-1 protein expression, compound A was shown to displace BAK and BIM from BFL-1, leading to downstream cleavage of caspase 3 and cell death. Interestingly, these cellular models were resistant to Venetoclax. Compound A exhibited tumor growth inhibition in an engineered BFL-1 overexpressing, MCL-1 KO human multiple myeloma xenograft model. However, in the high BFL-1 expressing SU-DHL-2 DLBCL human xenograft model, compound A did not induce significant PD (cleaved caspase 3) and efficacy in vivo. Conclusion: First-in-class selective BFL-1 BH3 mimetics are shown to specifically inhibit cell growth of lymphoma cell lines in vitro and can induce efficacy in vivo in a BFL-1 overexpressing model. Further studies may be useful to determine whether compound A elicits anti-tumor efficacy in lymphoma alone or in combination with other anti-apoptotic agents.

ICBcomb: a comprehensive expression database for immune checkpoint blockade combination therapy.

The success of immune checkpoint blockade (ICB) promotes the immunotherapy to be a new pillar in cancer treatment. However, the low response rate of the ICB therapy limits its application. To increase the response rate and enhance efficacy, the ICB combination therapy has emerged and its clinical trials are increasing. Nevertheless, the gene expression profile and its pattern of ICB combination were not comprehensively studied, which limits the understanding of the ICB combination therapy and the identification of new drugs. Here, we constructed ICBcomb (http://bioinfo.life.hust.edu.cn/ICBcomb/), a comprehensive database, by analyzing the human and mouse expression data of the ICB combination therapy and comparing them between groups treated with ICB, other drugs or their combinations. ICBcomb contains 1399 samples across 29 cancer types involving 52 drugs. It provides a user-friendly web interface for demonstrating the results of the available comparisons in the ICB combination therapy datasets with five functional modules: [1, 2] the 'Dataset/Disease' modules for browsing the expression, enrichment and comparison results in each dataset or disease; [3] the 'Gene' module for inputting a gene symbol and displaying its expression and comparison results across datasets/diseases; [4] the 'Gene Set' module for GSVA/GSEA enrichment analysis on the built-in gene sets and the user-input gene sets in different comparisons; [5] the 'Immune Cell' module for immune cell infiltration comparison between different groups by immune cell abundance analysis. The ICBcomb database provides the first resource for gene expression profile and comparison in ICB combination therapy, which may provide clues for discovering the mechanism of effective combination strategies and new combinatory drugs.

IDPpub: Illuminating the Dark Phosphoproteome Through PubMed Mining

Global phosphoproteomics experiments quantify tens of thousands of phosphorylation sites. However, data interpretation is hampered by our limited knowledge on functions, biological contexts, or precipitating enzymes of the phosphosites. This study establishes a repository of phosphosites with associated evidence in biomedical abstracts, using deep learning-based natural language processing techniques. Our model for illuminating the dark phosphoproteome through PubMed mining (IDPpub) was generated by fine-tuning BioBERT, a deep learning tool for biomedical text mining. Trained using sentences containing protein substrates and phosphorylation site positions from 3000 abstracts, the IDPpub model was then used to extract phosphorylation sites from all MEDLINE abstracts. The extracted proteins were normalized to gene symbols using the National Center for Biotechnology Information gene query, and sites were mapped to human UniProt sequences using ProtMapper and mouse UniProt sequences by direct match. Precision and recall were calculated using 150 curated abstracts, and utility was assessed by analyzing the CPTAC (Clinical Proteomics Tumor Analysis Consortium) pan-cancer phosphoproteomics datasets and the PhosphoSitePlus database. Using 10-fold cross validation, pairs of correct substrates and phosphosite positions were extracted with an average precision of 0.93 and recall of 0.94. After entity normalization and site mapping to human reference sequences, an independent validation achieved a precision of 0.91 and recall of 0.77. The IDPpub repository contains 18,458 unique human phosphorylation sites with evidence sentences from 58,227 abstracts and 5918 mouse sites in 14,610 abstracts. This included evidence sentences for 1803 sites identified in CPTAC studies that are not covered by manually curated functional information in PhosphoSitePlus. Evaluation results demonstrate the potential of IDPpub as an effective biomedical text mining tool for collecting phosphosites. Moreover, the repository (http://idppub.ptmax.org), which can be automatically updated, can serve as a powerful complement to existing resources.

TNF-α stimulated exosome derived from fibroblast-like synoviocytes isolated from rheumatoid arthritis patients promotes HUVEC migration, invasion and angiogenesis by targeting the miR-200a-3p/KLF6/VEGFA axis

The pathogenesis of rheumatoid arthritis (RA) is heavily impacted by the inflammation and activation of fibroblast-like synoviocytes (FLS). The objective of this investigation is to clarify the involvement of exosomes derived from FLS stimulated by tumour necrosis factor α (TNF-α) in angiogenesis and the underlying mechanisms. FLS cells were obtained from synovial fluid of RA patients and exosomes were obtained from FLS cell supernatant with TNF-α stimulation by ultracentrifugation. Exosomes were subsequently analysed using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. The functional effects of exosomes with TNF-α stimulation on human umbilical vein endothelial cells (HUVEC) migration, invasion, and angiogenesis was evaluated using wound scratch healing test, transwell invasion assay, and tube formation assay. DNA nanoball-seq (DNBSEQ) sequencing platform was utilised to analysis different expression miRNA from exosomes, miRNA and mRNA from HUVEC. The expression level of miR-200a-3p was determined through quantitative real-time polymerase chain reaction (qRT-PCR). The quantification of KLF6 and VEGFA expression levels were performed by qRT-PCR and western blot analysis. The validation of the association between miR-200a-3p and KLF6 was established through a fluorescence enzyme reporting assay. In comparison to exosome induced by PBS, exosome induced by TNF-α exhibited a substantial exacerbation of invasion, migration, and angiogenesis in HUVEC. 4 miRNAs in exosomes and HUVEC cells, namely miR-1246, miR-200a-3p, miR-30a-3p, and miR-99b-3p was obtained. MiR-200a-3p maintained high consistency with the sequencing results. We obtained 5 gene symbols, and KLF6 was chose for further investigation. The expression of miR-200a-3p in exosomes induced by TNF-α and in HUVEC treated with these exosomes demonstrated a significantly increase. Additionally, HUVEC cells displayed a notable decrease in KLF6 expression and a significant elevation in VEGFA expression. This was further confirmed by the fluorescence enzyme report assay, which provided evidence of the direct targeting of KLF6 by miR-200a-3p. Exosomes induced by TNF-α have the ability to enhance the migration, invasion, and angiogenesis of HUVEC cells via the miR-200a-3p/KLF6/VEGFA axis.

Immunoglobulin-like receptors in chickens: identification, functional characterization, and renaming to cluster homolog of immunoglobulin-like receptors

The cluster homolog of immunoglobulin-like receptors (CHIRs), previously known as the "chicken homolog of immunogloublin-like receptors," represents is a large group of transmembrane glycoproteins that direct the immune response. However, the full repertoire of putatively activating, inhibitory, or dual function CHIRA, CHIRB, and CHIRAB on chickens' immune responses is poorly understood. Herein, the study objective was to determine the genes encoding CHIR proteins and predict their function by searching canonical protein structure. A bioinformatics pipeline based on previous work was employed to search for the CHIRs from the newly updated broiler and layer genomes. The categorization into CHIRA, CHIRB, and CHIRAB types was assigned through motif searches, multiple sequence alignment, and phylogeny. In total, 150 protein-encoding genes on Chromosome 31 were identified as CHIRs. Gene members of each functional group (CHIRA, CHIRB, CHIRAB) were classified in accordance with previously recognized proteins. The genes were renamed to "cluster homolog of immunoglobulin-like receptors" (CHIRs) to allow for the naming of orthologous genes in other avian species. Additionally, expression analysis of the classified CHIRs across various reinforces their importance as immune regulators and activation in inflammatory tissues. Furthermore, over 1,000 diverse and rare CHIRs variants associated with differential Marek's disease response (P < 0.05) emphasize the impact of CHIRs on shaping avian immune responses in diverse contexts. The practical applications of these findings encompass advancing immunology, improving poultry health management, optimizing breeding programs for disease resistance, and enhancing overall animal health through a deeper understanding of the roles and functions of CHIRA, CHIRB, and CHIRAB types in avian immune responses.