Reverse Transcription-polymerase Chain Reaction Experiments Research Articles

Overexpression of β-lactamase genes (blaKPC, blaSHV) and novel CirA deficiencies contribute to decreased cefiderocol susceptibility in carbapenem-resistant Klebsiella pneumoniae before its approval in China.

Cefiderocol (FDC) is an effective antibiotic that is used to treat severe infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). The mechanisms underlying FDC resistance and molecular epidemiology in China remain unclear. We collected 477 non-duplicate CRKP clinical isolates in central China and characterized their susceptibility to FDC, virulence genes, and sequence typing. The overall FDC susceptibility rate of CRKP was 99.2% in central China, which was higher than that in North America and Europe (96.1%), with MIC50/90 values of 1/2 mg/L. The decrease in FDC susceptibility in central China was concentrated in the ST11 CRKP-carrying virulence plasmids. Whole-genome sequencing (WGS) and quantitative reverse transcription PCR (qRT-PCR) experiments showed that serine β-lactamases, especially highly expressed KPC and SHV, substantially decreased FDC susceptibility in four FDC non-susceptible isolates (two resistant and two intermediate isolates). Notably, different CirA deficiencies, p.E450GfsTer16 and p.E133Ter, were found in both of the resistant isolates. In contrast, global WGS data indicate that the resistance mechanisms in North America and Europe were primarily associated with NDM and KPC variants, predominantly found in ST307 and ST147. Overall, FDC exhibits excellent activity against CRKP in central China, with resistance mechanisms primarily related to high KPC and SHV expression, along with deficiencies in CirA, frequently observed in ST11. This is remarkably different from the situation in North America and Europe and will directly impact the choice of clinical interventions. Additionally, the surveillance of FDC resistance in China is imperative.

Shikonin induces ferroptosis in osteosarcomas through the mitochondrial ROS-regulated HIF-1α/HO-1 axis

BackgroundThe most common malignant bone tumour is osteosarcoma, which has an unsatisfactory prognosis and unsatisfactory treatment. Ferroptosis shows promise as an effective OS therapy. A substance extracted from the Lithospermum erythrorhizon, Shikonin (SHK), inhibits a number of tumours, including ovarian, gastric, and lung cancers. However, whether SHK induces OS ferroptosis and its mechanisms are not clear. PurposeOur study is aimed at investigating whether SHK causes ferroptosis and elucidating its molecular mechanism. MethodsCell counting Kit-8, cell cycle and cell apoptosis assay were utilised to assess therapeutic effect of SHK against OS. Normal cells, including H9C2, AML-12 and HK-2, haematoxylin and eosin staining and mice serum biomarker tests were used to assess SHK biosafety. Malondialdehyde (MDA) levels, the glutathione (GSH)/oxidized glutathione (GSSG) ratio, reactive oxygen species (ROS), lipid peroxide (LPO), and intracellular Fe2+ detection, quantitative reverse transcription PCR (qRT-PCR), Western blotting (WB) and rescue experiments were employed to confirm whether SHK induced ferroptosis in OS cells. Molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay were used to evaluate the direct binding between SHK and hypoxia-inducible factor-1α (HIF-1α). Protein stability and degradation analysis, small RNA interference, flow cytometry, qRT-PCR, and WB were used to investigate the molecular mechanism of ferroptosis. In vivo xenografts of nude mouse was used to study the anti-OS effect of SHK. ResultsSHK significantly reduced OS cell viability and induced apoptosis and G2/M arrest. SHK increased intracellular levels of MDA, ROS, LPO, and Fe2+ while simultaneously reducing the GSH/GSSG ratio and GPX4 and SLC7A11 expression. CETSA and DARTS results demonstrated that SHK did not bind targetly to HIF-1α. Instead, mitochondrial ROS (MitoROS) promoted HIF-1α expression, resulting in HO-1 overexpression, excess Fe2+ production, ROS accumulation and GPX depletion, and ferroptosis. Furthermore, inhibition of MitoROS using Mito-TEMPO downregulated HIF-1α/HO-1 axis and mitigated the SHK-induced ferroptosis. In vivo, SHK effectively suppressed OS growth with favourable biosafety, confirming the molecular mechanism underlying SHK-induced ferroptosis in OS. ConclusionWe observe that HIF-1α/HO-1 axis is the crucial factor in SHK-induced OS ferroptosis. Importantly, we demonstrate that HIF-1α is indirectly regulated by MitoROS rather than SHK bound directly to HIF-1α. Our research suggest that SHK is a potential drug candidate for OS treatment and may help in identifying novel therapeutic targets for OS.

Surface exclusion of IncC conjugative plasmids and their relatives.

The phenomenon of exclusion allows conjugative plasmids to selectively impede the entry of identical or related elements into their host cell to prevent the resulting instability. Entry exclusion blocks DNA translocation into the recipient cell, whereas surface exclusion destabilizes the mating pair. IncC conjugative plasmids largely contribute to the dissemination of antibiotic-resistance genes in Gammaproteobacteria. IncC plasmids are known to exert exclusion against their relatives, including IncC and IncA plasmids, yet the entry exclusion factor eexC alone does not account for the totality of the exclusion phenotype. In this study, a transposon-directed insertion sequencing approach identified sfx as necessary and sufficient for the remaining exclusion phenotype. Sfx is an exclusion factor unrelated to the ones described to date. A cell fractionation assay localized Sfx in the outer membrane. Reverse transcription PCR and beta-galactosidase experiments showed that sfx is expressed constitutively at a higher level than eexC. A search in Gammaproteobacteria genomes identified Sfx homologs encoded by IncC, IncA and related, untyped conjugative plasmids and an uncharacterized family of integrative and mobilizable elements that likely rely on IncC plasmids for their mobility. Mating assays demonstrated that sfx is not required in the donor for exclusion, ruling out Sfx as the exclusion target. Instead, complementation assays revealed that the putative adhesin TraN in the donor mediates the specificity of surface exclusion. Mating assays with TraN homologs from related untyped plasmids from Aeromonas spp. and Photobacterium damselae identified two surface exclusion groups, with each Sfx being specific of TraN homologs from the same group. Together, these results allow us to better understand the apparent incompatibility between IncA and IncC plasmids and to propose a mechanistic model for surface exclusion mediated by Sfx in IncC plasmids and related elements, with implications for the rampant dissemination of antibiotic resistance.

Transcriptome Study of rd1Mouse Brain and Association with Parkinson's Disease.

Degeneration of the retina is intrinsically associated with the pathogenesis and progression of neurodegenerative diseases. However, the cellular and molecular mechanisms underlying the association between neurodegeneration and retinal degeneration are still under exploration due to the complexity of the connectivity network of the nervous system. In this study, RNA-seq data from the brains of model retinitis pigmentosa (RP) mice and previously studied Parkinson's disease (PD) mice were analyzed to explore the commonalities between retinal degenerative and neurodegenerative diseases. Differentially expressed genes in RP were compared with neurodegenerative disease-related genes and intersecting genes were identified, including Cnr1 and Septin14. These genes were verified by quantitative real-time reverse transcription PCR and Western blotting experiments. The key proteins CNR1 and SEPTIN14 were found to be potential cotherapeutic targets for retinal degeneration and neurodegenerative disease. In conclusion, understanding the commonalities between retinal degenerative diseases and neurodegenerative processes in the brain will not only facilitate the interpretation of the underlying pathomechanisms but also contribute to early diagnosis and the development of new therapeutic strategies.

Genome-wide analysis of the GRAS gene family in white clover (Trifolium repens L.) provides insight into its critical role in response to cold stress

The GRAS transcription factor (TF) is a plant-specific regulator that plays a critical role in plant growth, development and response to various abiotic stresses. While the functions of many GRAS TFs have been extensively studied in numerous model plants with fully sequenced genomes, the GRAS TFs in white clover (Trifolium repens L.) remain elusive despite the sequencing of its genome. To bridge this knowledge gap, homologs of GRAS TFs from Arabidopsis were employed and subjected to a genome-wide blast against white clover proteins. This analysis identified and characterized 102 putative GRAS genes, designated as TrGRAS, based on the presence of GRAS domains in positive blast hits. Concurrently, an exploration of their gene structures, classification, evolutionary relationships and conservative motifs provided insights into the distinctive features of TrGRAS in white clover. Furthermore, gene duplication analysis revealed an expansion of TrGRAS genes in the PAT1 and LISCL subfamilies, indicating functional diversity within the white clover genome. Lastly, RNA-seq and quantitative reverse transcription-PCR (qRT-PCR) experiments confirmed their higher expression during the early or intermittent responses to cold stress, suggesting their potential and crucial roles in determining the cold tolerances of white clover. The analysis of TrGRAS genes, coupled with expression studies, contributes to the functional characterization of TrGRAS genes in white clover. This knowledge is pivotal for informing genetic improvement strategies in future breeding efforts aimed at enhancing cold tolerance in white clover.

Medicinal cannabis oil improves anxiety-like and depressive-like behaviors in CCS mice via the BDNF/TRPC6 signaling pathway

BackgroundPost-traumatic stress disorder (PTSD) refers to a chronic impairing psychiatric disorder occurring after exposure to the severe traumatic event. Studies have demonstrated that medicinal cannabis oil plays an important role in neuroprotection, but the mechanism by which it exerts anti-PTSD effects remains unclear. MethodsThe chronic complex stress (CCS) simulating the conditions of long voyage stress for 4 weeks was used to establish the PTSD mice model. After that, behavioral tests were used to evaluate PTSD-like behaviors in mice. Mouse brain tissue index was detected and hematoxylin-eosin staining was used to assess pathological changes in the hippocampus. The indicators of cell apoptosis and the BDNF/TRPC6 signaling activation in the mice hippocampus were detected by western blotting or real-time quantitative reverse transcription PCR experiments. ResultsWe established the PTSD mice model induced by CCS, which exhibited significant PTSD-like phenotypes, including increased anxiety-like and depression-like behaviors. Medicinal cannabis oil treatment significantly ameliorated PTSD-like behaviors and improved brain histomorphological abnormalities in CCS mice. Mechanistically, medicinal cannabis oil reduced CCS-induced cell apoptosis and enhanced the activation of BDNF/TRPC6 signaling pathway. ConclusionsWe constructed a PTSD model with CCS and medicinal cannabis oil that significantly improved anxiety-like and depressive-like behaviors in CCS mice, which may play an anti-PTSD role by stimulating the BDNF/TRPC6 signaling pathway.

Genome assembly and transcriptomic analysis to elucidate the ability of Nasonovia ribisnigri to break host plant resistance.

Aphid genomic resources enable the study of complex life history traits and provide information on vector biology, host adaption and speciation. The currant-lettuce aphid (Nasonovia ribisnigri (Hemiptera: Aphididae) (Mosley)) is a cosmopolitan pest of outdoor lettuce (Lactuca sativa (Asterales: Asteraceae) (Linnaeus)). Until recently, the use of resistant cultivars was an effective method for managing N. ribisnigri. A resistant cultivar containing a single gene (Nr-locus), introduced in the 1980s, conferred complete resistance to feeding. Overreliance of this Nr-locus in lettuce resulted in N. ribisnigri's ability to break resistance mechanism, with first reports during 2003. Our work attempts to understand which candidate gene(s) are associated with this resistance-breaking mechanism. We present two de novo draft assembles for N. ribisnigri genomes, corresponding to both avirulent (Nr-locus susceptible) and virulent (Nr-locus resistant) biotypes. Changes in gene expression of the two N. ribisnigri biotypes were investigated using transcriptomic analyses of RNA-sequencing (RNA-seq) data to understand the potential mechanisms of resistance to the Nr-locus in lettuce. The draft genome assemblies were 94.2% and 91.4% complete for the avirulent and virulent biotypes, respectively. Out of the 18,872 differentially expressed genes, a single gene/locus was identified in N. ribisnigri that was shared between two resistant-breaking biotypes. This locus was further explored and validated in Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) experiments and has predicted localisations in both the cytoplasm and nucleus. This is the first study to provide evidence that a single gene/locus is likely responsible for the ability of N. ribisnigri to overcome the Nr-locus resistance in the lettuce host.

Identification of transcriptionally active transposons in Barley

BackgroundThe genomes of many major crops including barley (Hordeum vulgare) consist of numerous transposons. Despite their important roles in crop genome evolution and morphological variations, most of these elements are silent or truncated and unable to be mobile in host genomes. Thus far, only a very limited number of active transposons were identified in plants.ResultsWe analyzed the barley full-length cDNA (FLcDNA) sequences and detected 71 unique FLcDNAs exhibiting significant sequence similarity to the extant transposase proteins. These FLcDNAs were then used to search against the genome of a malting barley cultivar ‘Morex’, seven new intact transposons were identified. Sequence alignments indicated that six intact transposons contained the entire FLcDNAs whereas another one served as 3’ untranslated region (3’ UTR) of a barley gene. Our reverse transcription-PCR (RT-PCR) experiment further confirmed the expression of these six transposons and revealed their differential expression. We conducted genome-wide transposon comparisons and detected polymorphisms of three transposon families between the genomes of ‘Morex’ and other three genotypes including the wild barley (Hordeum spontaneum, B1K-04-12) and two cultivated barley varieties, ‘Golden Promise’ and ‘Lasa Goumang’. Lastly, we screened the transcripts of all annotated barley genes and found that some transposons may serve as the coding regions (CDSs) or UTRs of barley genes.ConclusionWe identified six newly expressed transposons in the barley genome and revealed the recent mobility of three transposon families. Our efforts provide a valuable resource for understanding the effects of transposons on barley genome evolution and for developing novel molecular tools for barley genetic improvement and other research.

Role for ubiquitin-specific protease 7 (USP7) in the treatment and the immune response to hepatocellular carcinoma: potential mechanisms.

Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that can affect or regulate a variety of cellular activities. The purpose of this study was to investigate therapeutic and immunologic effects of USP7 in hepatocellular carcinoma (HCC), and as well to evaluate potential mechanisms of action. USP7-related gene expression and clinical data were obtained from The Cancer Genome Atlas (TCGA) dataset, International Cancer Genome Consortium (ICGC) dataset, and Gene Expression Omnibus (GEO) dataset. Pathways associated with USP7 were determined by gene set enrichment analysis (GSEA). The relationships among USP7, immunity, and drug therapy were also investigated and potential mechanisms of action were explored. TCGA database results demonstrated USP7 mRNA expression levels to be upregulated in HCC tissues. Results were validated with UALCAN, ICGC, and GSE10143 datasets, as well as immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) experiments and were consistent with TCGA database findings (all P<0.05). GSEA analysis demonstrated increased USP7 levels to be associated with CHEMOKINE, Janus kinase/signal transducer and activator of transcription (JAK-STAT), mitogen-activated protein kinase (MAPK), P53, vascular endothelial growth factor (VEGF), and wingless (WNT) signaling pathways. Based on immune correlation analysis, USP7 was dramatically associated with immune cells and immune checkpoint molecules. In terms of drug therapy, USP7 expression levels were significantly related to HCC sensitivity to ciclosporin, talazoparib, dabrafenib, trametinib, paclitaxel, sorafenib, bortezomib, sunitinib, and crizotinib. Based on these results, we mechanistically propose an association between USP7 and these four drug targets: B-Raf proto-oncogene serine/threonine protein kinase (BRAF), mitogen-activated extracellular signal-regulated kinase (MEK), DNA topoisomerase I (TOPOI), and poly ADP-ribose polymerase (PARP). USP7 plays a therapeutic and immunological role in HCC. The four drug targets BRAF, MEK, TOPOI, and PARP are implicated in the USP7 mechanism of action.

GAD1 contributes to the progression and drug resistance in castration resistant prostate cancer

BackgroundProstate cancer is currently the second most lethal malignancy in men worldwide due to metastasis and invasion in advanced stages. Studies have revealed that androgen deprivation therapy can induce stable remission in patients with advanced prostate cancer, although most patients will develop castration-resistant prostate cancer (CRPC) in 1–2 years. Docetaxel and enzalutamide improve survival in patients with CRPC, although only for a short time, eventually patients develop primary or secondary resistance, causing disease progression or biochemical relapse.MethodsThe gene expression profiles of docetaxel-sensitive or -resistant prostate cancer cell lines, namely GSE33455, GSE36135, GSE78201, GSE104935, and GSE143408, were sequentially analyzed for differentially expressed genes and progress-free interval significance. Subsequently, the overall survival significance and clinic-pathological features were analyzed by the R package. The implications of hub genes mutations, methylation in prostate cancer and the relationship with the tumor immune cell infiltration microenvironment were assessed with the help of cBioPortal, UALCAN and TISIDB web resources. Finally, effects of the hub genes on the progression and drug resistance in prostate cancer were explored using reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, cell phenotype, and drug sensitivity.ResultGlutamate decarboxylase 1 (GAD1) was tentatively identified by bioinformatic analysis as an hub gene for the development of drug resistance, including docetaxel and enzalutamide, in prostate cancer. Additionally, GAD1 expression, mutation and methylation were significantly correlated with the clinicopathological features and the tumor immune microenvironment. RT-PCR, immunohistochemistry, cell phenotype and drug sensitivity experiments further demonstrated that GAD1 promoted prostate cancer progression and decreased the therapeutic effect of docetaxel or enzalutamide.ConclusionThis research confirmed that GAD1 was a hub gene in the progression and development of drug resistance in prostate cancer. This helped to explain prostate cancer drug resistance and provides new immune-related therapeutic targets and biomarkers for it.

Molecular characterization and functional analysis of a mollusk Rel homologous gene

Members of the nuclear factor-kappa B (NF-κB) family are crucial regulators of physiological processes such as apoptosis, inflammation, and the immune response, acting as vital transcription factors to perform their function. In this study, we identified a NF-κB homologous gene (CfRel1) in Zhikong scallops. The 3006-bp-long open reading frame encodes 1001 amino acids. The N-terminus of the CfRel1 protein harbors a conserved Rel homology domain (RHD) that contains a DNA-binding domain and a dimerization domain. According to the multiple sequence alignment results, both the DNA-binding and dimerization domains are highly conserved. Phylogenetic analysis indicated that CfRel1 is closely related to both the Dorsal protein of Pinctada fucata and the Rel2 protein of Crassostrea gigas. CfRel1 mRNA was expressed in all tissues tested in the quantitative reverse transcription PCR experiments, with hepatopancreatic tissue expressing the highest levels. Furthermore, after stimulation with lipopolysaccharide, peptidoglycan, or polyinosinic:polycytidylic acid, the mRNA expression level of CfRel1 was markedly increased. The co-immunoprecipitation test results showed that CfRel1 interacted with scallop IκB protein through its RHD DNA-binding domain, suggesting that IκB may regulate the activity of Rel1 by binding to this domain. Dual-luciferase reporter gene assays revealed that CfRel1 overexpression in HEK293T cells activated the activator protein 1 (AP-1), NF-κB, interferon (IFN)α, IFNβ, and IFNγ reporter genes, indicating the diverse functions of the protein. In summary, CfRel1 is capable of responding to attacks from pathogen-associated molecular patterns, participating in immune signaling, and activating NF-κB and IFN reporter genes. Our findings contribute to the advancement of invertebrate innate immunity theory, enrich the theory of comparative immunology, and serve as a reference for the future screening of disease-resistant strains in scallops.

BIIB021, an orally available and small-molecule inhibitor of HSP90, activates intrinsic apoptotic pathway in human cervical adenocarcinoma cell line (HeLa).

Heat shock protein 90 (HSP90) is a highly conserved ATP-dependent chaperone protein that plays a vital role in tumorigenesis. This study aims to investigate the apoptosis inducer role of BIIB021 (orally available HSP90 inhibitors) compound via inhibition of HSP90 activity in the human cervical cancer cell line (HeLa). The anticancer potential of BIIB021 was determined by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)] cell proliferation assay against the human cervical cancer cell line (HeLa). ATPase and luciferase aggregation assays were carried out to detect the HSP90 inhibitor potential of BIIB021. To determine the antiproliferative mechanism of the BIIB021, the expression level of the pro-apoptotic and antiapoptotic markers was determined by reverse transcription polymerase chain reaction (RT-PCR) and ELISA experiments. BIIB021 exhibited a cytotoxic effect on HeLa cell proliferation and the inhibitory concentration (IC)50 dose of BIIB021 was found to be 14.79 nM at 48 h. BIIB021 decreased the ATP hydrolysis rate of HSP90 and blocked the refolding of the desaturated luciferase in the presence of ATP. To understand the antiproliferative mechanism of the BIIB021 in HeLa cells, the mRNA and protein expression levels of the apoptotic markers [BCL-2 associated X (BAX), B-cell lymphoma 2 (BCL-2), cytochrome-c (CYT-c), and caspase-3 (CAS-3)] were determined by RT-PCR and ELISA experiments. The results obtained indicated that BIIB021 decreased BCL-2 levels and increased BAX, CYT-c, and CAS-3 levels in human cervical cancer cells. These results confirmed that BIIB021 inhibited the chaperone activity of HSP90, resulting in anti-proliferating effects in cervical cancer cells via the induction of the intrinsic apoptotic pathways.

Gelatin methacryloyl and Laponite bioink for 3D bioprinted organotypic tumor modeling

Three-dimensional (3D) in vitro tumor models that can capture the pathophysiology of human tumors are essential for cancer biology and drug development. However, simulating the tumor microenvironment is still challenging because it consists of a heterogeneous mixture of various cellular components and biological factors. In this regard, current extracellular matrix (ECM)-mimicking hydrogels used in tumor tissue engineering lack physical interactions that can keep biological factors released by encapsulated cells within the hydrogel and improve paracrine interactions. Here, we developed a nanoengineered ion-covalent cross-linkable bioink to construct 3D bioprinted organotypic tumor models. The bioink was designed to implement the tumor ECM by creating an interpenetrating network composed of gelatin methacryloyl (GelMA), a light cross-linkable polymer, and synthetic nanosilicate (Laponite) that exhibits a unique ionic charge to improve retention of biological factors released by the encapsulated cells and assist in paracrine signals. The physical properties related to printability were evaluated to analyze the effect of Laponite hydrogel on bioink. Low GelMA (5%) with high Laponite (2.5%–3.5%) composite hydrogels and high GelMA (10%) with low Laponite (1.0%–2.0%) composite hydrogels showed acceptable mechanical properties for 3D printing. However, a low GelMA composite hydrogel with a high Laponite content could not provide acceptable cell viability. Fluorescent cell labeling studies showed that as the proportion of Laponite increased, the cells became more aggregated to form larger 3D tumor structures. Reverse transcription-polymerase chain reaction (RT-qPCR) and western blot experiments showed that an increase in the Laponite ratio induces upregulation of growth factor and tissue remodeling-related genes and proteins in tumor cells. In contrast, cell cycle and proliferation-related genes were downregulated. On the other hand, concerning fibroblasts, the increase in the Laponite ratio indicated an overall upregulation of the mesenchymal phenotype-related genes and proteins. Our study may provide a rationale for using Laponite-based hydrogels in 3D cancer modeling.

Penumbra-targeted CircOGDH siRNA-loaded nanoparticles alleviate neuronal apoptosis in focal brain ischaemia

BackgroundNanoparticles (NPs) are a class of substances that can be loaded with therapeutic agents delivered to specific areas. In our earlier research, we identified a neuron-derived circular RNA (circRNA), circular...

#6771 IDENTIFICATION OF KEY GENES IN DIABETIC NEPHROPATHY BASED ON LIPID METABOLISM

Abstract Background and Aims Diabetic nephropathy (DN), which is one of the most common systemic microvascular complications of diabetes mellitus, is extremely harmful to the patients’ health. There were some studies had shown that the disturbance of lipid metabolism was connected with the progression of DN. Therefore, the purpose of our study was to find the lipid metabolism-related hub genes in DN and provide a better reference for the diagnosis of DN. Method The Gene Expression Omnibus (GEO) database was used to download the gene expression profile data of DN and healthy samples (GSE142153), and we obtained the lipid metabolism-related genes from the Molecular Signatures Database (MSigDB). Differentially expressed genes (DEGs) between DN and healthy samples were analyzed and the weighted gene co-expression network analysis (WGCNA) was performed to examine the connection between genes and clinical traits and screen the key module genes in DN. Next, we utilized the Venn Diagram R package to identify the lipid metabolism-related genes in DN, and the Protein-Protein Interaction (PPI) of these genes was constructed. Then we carried out the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Moreover, the hub genes were identified using two machine learning algorithms, and the Gene Set Enrichment Analysis (GSEA) was used to analyze the functions of the hub genes. Furthermore, the immune infiltration discrepancies between DN and healthy samples and the correlation between the immune cells and hub genes were estimated. Finally, quantitative reverse transcription-PCR (qRT-PCR) experiment verified the expression of key genes. Results A total of 1445 DEGs were found in DN samples compared to healthy samples, and 694 DN-related genes in yellow and turquoise modules were identified by WGCNA. Next, we used the Venn Diagram R package to further identify 17 genes that were related to lipid metabolism and constructed a PPI network. Then GO analysis revealed that these 17 genes were significantly correlated with ‘phospholipid biosynthetic process’ and ‘cholesterol biosynthetic process’, while the KEGG analysis showed these lipid metabolism-related genes were enriched in ‘glycerophospholipid metabolism’ and ‘fatty acid degradation’. Moreover, SAMD8 and CYP51A1 were identified through the intersections of two machine learning algorithms. The results of GSEA analysis revealed that the ‘mitochondrial matrix’ and ‘GTPase activity’ were the significantly enriched GO terms in SAMD8 and CYP51A1, and the KEGG pathways of them were mainly concentrated in ‘pathways of neurodegeneration - multiple diseases’. Immune infiltration analysis suggested that there were 9 immune cells expressed differently in DN and healthy samples, and both SAMD8 and CYP51A1 were significantly correlated with activated B cell and effector memory CD8 T cell. Finally, qRT-PCR confirmed the expression of SAMD8 and CYP51A1 in DN was high. Conclusion In summary, the lipid metabolism-related genes SAMD8 and CYP51A1 may play key roles in DN.

ZDHHC15 promotes glioma malignancy and acts as a novel prognostic biomarker for patients with glioma

BackgroundGlioma is the most common and aggressive tumor in the adult brain. Recent studies have indicated that Zinc finger DHHC-type palmitoyltransferases (ZDHHCs) play vital roles in regulating the progression of glioma. ZDHHC15, a member of the ZDHHCs family, participates in various physiological activities in the brain. However, the biological functions and related mechanisms of ZDHHC15 in glioma remain poorly understood.MethodsData from multiple glioma-associated datasets were used to investigate the expression profiles and potential biological functions of ZDHHC15 in glioma. Expression of ZDHHC15 and its association with clinicopathological characteristics in glioma were validated by quantitative reverse transcription PCR (RT-qPCR) and immunohistochemical experiments. GO enrichment analysis, KEGG analysis, GSEA analysis, CCK-8, EdU, transwell, and western blotting assays were performed to confirm the functions and mechanism of ZDHHC15 in glioma. Moreover, we performed Kaplan-Meier analysis and Cox progression analysis to explore the prognostic significance of ZDHHC15 in glioma patients.ResultsZDHHC15 expression was significantly up-regulated in glioma and positively associated with malignant phenotypes. Results from the GO and KEGG enrichment analysis revealed that ZDHHC15 was involved in regulating cell cycle and migration. Knockdown of ZDHHC15 inhibited glioma cell proliferation and migration, while overexpression of ZDHHC15 presented opposite effects on glioma cells. Besides, results from GSEA analysis suggested that ZDHHC15 was enriched in STAT3 signaling pathway. Knockdown or overexpression of ZDHHC15 indeed affected the activation of STAT3 signaling pathway. Additionally, we identified ZDHHC15 as an independent prognostic biomarker in glioma, and higher expression of ZDHHC15 predicted a poorer prognosis in glioma patients.ConclusionOur findings suggest that ZDHHC15 promotes glioma malignancy and can serve as a novel prognostic biomarker for glioma patients. Targeting ZDHHC15 may be a promising therapeutic strategy for glioma.

Abstract 3103: Dual targeting MYC G-quadruplex and topoisomerase I by indenoisoquinolines for cancer therapy

Abstract MYC is one of the most important oncogenes and is overexpressed in a number of human cancers. The G-quadruplex formed in the MYC oncogene promoter (MycG4) is a transcriptional silencer and amenable to small molecule targeting. G-quadruplexes are noncanonical four-stranded DNA or RNA secondary structures that have emerged as novel therapeutic targets. Indenoisoquinolines are human topoisomerase I inhibitors with improved physicochemical and biological properties as compared to the clinically used camptothecin anticancer drugs. Three indenoisoquinolines, indotecan (LMP400), indimitecan (LMP776), and LMP744, have entered phase I clinical trials in adults with relapsed solid tumors and lymphomas. However, some indenoisoquinolines showed potent anticancer activity without being strong topoisomerase I inhibitors, suggesting additional mechanisms of action. Herein, we demonstrate that a large number of anticancer indenoisoquinolines strongly bind and stabilize MycG4 in vitro, using fluorescence, nuclear magnetic resonance (NMR), and circular dichroism (CD) spectroscopy, as well as gel electromobility shift assay (EMSA). In addition, MycG4-interactive indenoisoquinolines lower MYC mRNA and protein levels in vivo, as shown by western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) experiments. Structure-activity-relationships of MycG4 recognition by indenoisoquinolines were investigated to understand structural requirements for binding. Systemic analysis of indenoisoquinoline derivatives led to the discovery of a synergistic effect of MYC inhibition and topoisomerase I inhibition on anticancer activity. Therefore, these results indicate that targeting the MYC promoter G-quadruplex to downregulate MYC is a new mechanism of action for anticancer indenoisoquinolines. Moreover, our data reveals dual targeting of MycG4 and topoisomerase I as a novel strategy for cancer therapy. Citation Format: Danzhou Yang, Mark Cushman, Kai-Bo Wang, Mohamed S. Elsayed, Guanhui Wu, Adam J. Buric, Mercedes DeMoss. Dual targeting MYC G-quadruplex and topoisomerase I by indenoisoquinolines for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3103.

Ginsenoside Rh2 suppresses colon cancer growth by targeting the miR-150-3p/SRCIN1/Wnt axis

Ginsenoside Rh2, which is extracted from ginseng, exerts antitumor activity. Recent studies suggest that Rh2 may suppress the growth of colon cancer (CC) in vitro. However, the underlying mechanism remains unclear. In this study, we identified the relative levels of miR-150-3p in CC tissues and cells by a comprehensive strategy of data mining, computational biology, and real-time reverse transcription PCR (qRT-PCR) experiments. The regulatory effects of miR-150-3p/SRCIN1 on the proliferative and invasive abilities of CC cells are evaluated by CCK-8, EdU, wound healing, and transwell assays. Cell cycle- and apoptosis-related protein levels are assessed by western blot analysis. An in vivo tumor formation assay was conducted to explore the effects of miR-150-3p on tumor growth. Furthermore, bioinformatics and dual luciferase reporter assays are applied to determine the functional binding of miRNA to mRNA of the target gene. Finally, the relationship between Rh2 and miR-150-3p was further verified in SW620 and HCT-116 cells. miR-150-3p is downregulated in CC tissues and cell lines. Functional assays indicate that the upregulation of miR-150-3p inhibits tumor growth both in vivo and in vitro. In addition, SRCIN1 is upregulated in CC and predicts a poor prognosis, and it is the direct target for miR-150-3p. Moreover, the miR-150-3p mimic decreases Topflash/Fopflash-dependent luciferase activity, resulting in the inhibition of Wnt pathway activity. Rh2 can suppress the growth of CC by increasing miR-150-3p expression. Rh2 alleviates the accelerating effect on Wnt pathway activity, cell proliferation/migration, and colony formation caused by miR-150-3p inhibition. Rh2 inhibits the miR-150-3p/SRCIN1/Wnt axis to suppress colon cancer growth.

Regulation of Multidrug Efflux Pumps by TetR Family Transcriptional Repressor Negatively Affects Secondary Metabolism in Streptomyces coelicolor A3(2).

Streptomyces spp. are well-known producers of bioactive secondary metabolites (SMs) that serve as pharmaceutical agents. In addition to their ability to produce SMs, Streptomyces spp. have evolved diverse membrane transport systems to protect cells against antibiotics produced by itself or other microorganisms. We previously screened mutants of Streptomyces coelicolor that show a phenotype of reduced undecylprodigiosin (RED) production in a combined-culture with Tsukamurella pulmonis. Here, we identified a point mutation, which reduced RED production, by performing genome resequencing and genetic complementation. We found that inactivation of the sco1718 gene encoding the TetR family transcriptional regulator (TFR) produced a deficient phenotype for several SMs in Streptomyces coelicolor A3(2). In the genome of S. coelicolor A3(2), two other sets of TFR and two-component ATP-binding cassette (ABC) transporter genes (sco4358-4360 and sco5384-5382) were found which had similar effects on the phenotype for both secondary metabolism and antibiotic resistance. An electrophoretic mobility shift assay and quantitative reverse transcription-PCR experiments demonstrated that TFRs repressed the expression of each adjacent two-component ABC transporter genes by binding to the operator sequence. Notably, the Δsco1718 mutant showed increased resistance to several antibiotics of other actinomycete origin. Our results imply the switching of cell metabolism to direct offense (antibiotic production) or defense (efflux pump activation) using costly and limited quantities of cell energy sources (e.g., ATP) in the soil ecosystem. IMPORTANCE The bacterial metabolic potential to synthesize diverse secondary metabolites in the environment has been revealed by recent (meta)genomics of both unculturable and culturable bacteria. These studies imply that bacteria are continuously exposed to harmful chemical compounds in the environment. Streptomyces spp. contain antibiotic efflux pumps and SM biosynthetic gene clusters. However, the mechanism by which soil bacteria, including Streptomyces, survive against toxic compounds in the environment remains unclear. Here, we identified three sets of TFR-ABC transporter genes in Streptomyces coelicolor A3(2). We found that each TFR controlled the expression of respective ABC transporter, and the expression of all ABC transporters negatively impacted SM production and increased antibiotic resistance. Notably, bioinformatic analysis indicated that these TFR-ABC transporter gene sets are highly conserved and widely distributed in the genome of Streptomyces species, indicating the importance of systematic regulation that directs antibiotic production and xenobiotic excretion.

Investigating Genetic Characteristics of Chinese Holstein Cow’s Milk Somatic Cell Score by Genetic Parameter Estimation and Genome-Wide Association

The quality and safety of milk is challenged by cow mastitis, and the value of somatic cell score (SCS) in milk is closely related to the occurrence of mastitis. This study aimed to analyze the genetic characteristics of SCS across the first three parities in Chinese Holstein cattle, as well as to investigate potential candidate genes and biological processes that may play a potential role in the progress of cow mastitis. In this respect, we evaluated genetic parameters and conducted a genome-wide association study based on the test-day records of SCS for Chinese Holstein cows; we also validated key candidate genes using a quantitative reverse transcription PCR (RT-qPCR) experiment in primary bovine mammary epithelial cells (bMECs). The heritability of the SCS 305-day performance in milk varied between 0.07 and 0.24, and decreased with increasing parity. As the time interval grew larger, the genetic and permanent environmental correlations with the number of days in milk (DIM) weakened. Six significant single-nucleotide polymorphisms (SNPs) were identified in the association analysis, one of which was located within the exonic region of CD44. This exon-associated SNP may modify the activity of the protein encoded by the CD44. A total of 32 genes within the two hundred kilobase (kb) range of significant SNPs were detected, and these genes were markedly enriched in eight Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and 22 biological processes, mainly participating in the progress of transmembrane transport, inflammatory factor regulation, cellular responses, the Toll-like receptor signaling pathway, and the MAPK signaling pathway. Nine genes, including the PKD2, KCNAB1, SLC35A4, SPP1, IBSP, CD14, CD44, MAPK10, and ABCG2 genes, were selected as candidate genes that could have critical functions in cow mastitis. These findings can serve as a foundation for molecular breeding and as valuable data for reducing the incidence of mastitis of Chinese Holstein cattle at the molecular level.