Negative Regulatory Gene Research Articles

Genetic Factors and MicroRNAs in the Development of Gallbladder Cancer: The Prospective Clinical Targets.

Gallbladder cancer (GBC) is an uncommon condition in which malignant (cancer) cells are detected in gallbladder tissue. Cancer is often triggered when normal cells turn malignant and begin to spread. Cancer can also be caused by genetic anomalies that result in uncontrolled cell proliferation and tumor development. MicroRNAs (also known as miRNAs or miRs) are a group of small, endogenous, non-coding RNAs of 19-23 nucleotides in length, which play a key role in post-transcriptional gene regulation. These miRNAs serve as negative gene regulators by supervising target genes and regulating biological processes, including cell proliferation, migration, invasion, and apoptosis. Cancer development and progression relate to aberrant miRNA expression. This review demonstrated the implication of various genetic factors and microRNAs in developing and regulating GBC. This suggests the potential of genes and RNAs as the diagnostic, prognostic, and therapeutic targets in gallbladder cancer.

Abstract 1420: Mechanism of NPRL2 gene therapy induced antitumor immunity in KRAS/STK11mt aPD1 resistant metastatic NSCLC

Abstract NPRL2/TUSC4 is a tumor suppressor gene whose expression is reduced in many cancers including NSCLC. Restoration of NPRL2 induces cell cycle arrest and apoptosis. We investigated the antitumor immune responses to NPRL2 gene therapy in aPD1 resistant KRAS/STK11mt NSCLC in a humanized mouse model. Humanized mice were generated by transplanting human cord blood-derived CD34 stem cells into NSG mice. Mice harboring > 25% human CD45 cells were considered humanized. Lung metastases were developed in humanized mice by injecting KRAS/STK11mt/aPD1R A549 cells, which were treated with i.v. injection of NPRL2 nanoparticles (DOTAP-NPRL2) with or without pembrolizumab (aPD1). NPRL2 treatment reduced lung metastases, whereas pembrolizumab was ineffective. The antitumor effect was greater in humanized than non-humanized mice suggesting the role of immune cells. The antitumor effect was associated with increased infiltration of human cytotoxic immune cells and decreased numbers of Treg in tumors. NPRL2+ pembrolizumab was not synergistic in this resistant model but was synergistic in KRASwt/aPD1S H1299 tumors. Cytotoxic immune cells in tumors were associated with the antitumor effect. Consistent with A549, NPRL2 showed a significantly strong antitumor effect on other KRASmt/aPD1R syngeneic LLC2 tumors whereas aPD1 was not effective. The antitumor effect of NPRL2 was correlated with HLA-DR+ DC, CD11c DC, TILs and NK cells in TME. The antitumor effect of NPRL2 was abolished upon in-vivo depletion of CD4, CD8 T, and MΦ in the LLC2 tumor model. However, no effect was found on in-vivo depletion of NK cells. Nanostring analysis on lung metastasis resulted in a distinct pattern of human gene expression by NPRL2. T cell functional genes, including IFNγ, CD8b, CD7, TNFSF18, ITGA1, GATA3 and TBX21 were significantly increased. Conversely, the negative regulatory genes, including FOXP3, TGFB1, TGFB2, and IL-10RA were inhibited. NPRL2 also downregulated T cell co-inhibitory molecules, including CTLA4, ICOS, LAG3, PDCD1, CD274, IDO1, PDCD1LG2, CD47, and KLRB1. Stably expressing NPRL2 clones were established, and tumors in humanized mice with these clones exhibited significantly slower growth compared to controls. TME analysis showed an upregulation of human CD45, CD3, CD8 T, HLA-DR+ DC and a downregulation of Tregs, CD3+PD1+T, MDSC, and CD163+ TAM in tumors expressing NPRL2. The stable cells showed a substantial increase in both colony formation inhibition and apoptosis. Stable clones showed heightened sensitivity to carboplatin in colony formation, apoptosis, and PARP cleavage assays. Stable expression of NPRL2 resulted in the downregulation of both AKT-mTOR and MAPK pathways by inhibition of pAKT, pmTOR, pPRAS40, p4E-BP1, pS6, and pERK1/2. Taken together, NPRL2 gene therapy induces antitumor activity on KRAS/STK11mt/aPD1R tumors through DC-mediated antigen presentation and cytotoxic immune cell activation Citation Format: Ismail M. Meraz, Mourad Majidi, Renduo Song, Feng Meng, Lihui Gao, Qi Wang, Jing Wang, Elizabeth Shpall, Jack A. Roth. Mechanism of NPRL2 gene therapy induced antitumor immunity in KRAS/STK11mt aPD1 resistant metastatic NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1420.

Discovering therapeutic possibilities for polycystic ovary syndrome by targeting XIST and its associated ceRNA network through the analysis of transcriptome data

Long non-coding RNA (lncRNA) regulates many physiological processes by acting as competitive endogenous RNA (ceRNA). The dysregulation of lncRNA X-inactive specific transcript (XIST) has been shown in various human disorders. However, its role in the pathogenesis of polycystic ovary syndrome (PCOS) is yet to be explored. This study aimed to explore the underlying mechanism of XIST in the pathogenesis of PCOS, specifically through dataset functional analysis. GEO PCOS datasets including RNA-seq, microarray, and miRNA-seq in granulosa cells (GCs) and blood, were examined and comprehensively analyzed. Enrichment analysis, ROC curve constructions, lncRNA-miRNA-mRNA interaction network analyses, and qRT-PCR validation were performed followed by a series of drug signature screenings. Our results revealed significant dysregulation in the expression of 1131 mRNAs, 30 miRNAs, and XIST in GCs of PCOS patients compared to healthy individuals. Of the120 XIST-correlated upregulated genes, 25 were enriched in inflammation-related pathways. Additionally, 5 miRNAs were identified as negative regulators of XIST-correlated genes. Accordingly, a ceRNA network containing XIST-miRNAs-mRNAs interactions was constructed. Furthermore, 6 genes, including AQP9, ETS2, PLAU, PLEK, SOCS3, and TNFRSF1B served as both GCs and blood-based biomarkers. By analyzing the number of interactions among XIST, miRNAs, and mRNAs, we pinpointed ETS2 as the pivotal gene within the ceRNA network. Our findings reveal a novel XIST- hsa-miR-146a-5p, hsa-miR-144-3p, and hsa-miR-1271-5p-ETS2 axis that comprehensively elucidates the XIST-associated mechanism underlying PCOS onset. qRT-PCR analysis further confirmed the, overexpression of both XIST and ETS2 . Furthermore, our results demonstrated that XIST and ETS2 were correlated with some assisted reproductive technologies outcomes. Finally, we identified two novel compounds including, methotrexate/folate and threonine using drug–gene interaction databases for PCOS management. These findings provide novel insights into the molecular etiology, diagnosis, and potential therapeutic interventions for PCOS.

Ursodeoxycholic acid ameliorates erectile dysfunction and corporal fibrosis in diabetic rats by inhibiting the TGF-β1/Smad2 pathway.

Corporal tissue fibrosis is critical in diabetes-associated erectile dysfunction. Transforming growth factor-β1/Small mothers against decapentaplegic-2 (TGF-β1/Smad2) contributes to the induction of fibrosis in corporal tissue. Smad7 is accepted as a general negative regulator of Smad signaling, although its role in corporal fibrosis is unknown. Ursodeoxycholic acid (UDCA) is a hydrophilic bile acid used for biliary and liver related disorders and has antifibrotic effects in the liver. This study investigated the effects of UDCA on diabetic erectile dysfunction. Forty-eight male Spraque Dawley rats were divided into six groups: nondiabetic (n = 6), nondiabetic+20 mg/kg UDCA (n = 6), nondiabetic+80 mg/kg UDCA (n = 6), diabetic (n = 10), diabetic+20 mg/kg UDCA (n = 10), diabetic+80 mg/kg UDCA (n = 10). Diabetes was induced by intraperitoneal injection of 60 mg/kg Streptozocin. UDCA (20 and 80 mg/kg/day) or saline was subsequently administered via oral gavage for 56 days. Erectile function was evaluated as measurement of maximum intracavernosal pressure (m-ICP)/mean arterial pressure (MAP) and total ICP/MAP. Corporal tissues were evaluated by Western blotting and Masson's trichrome staining. Electrical stimulation-induced m-ICP/MAP responses were higher in UDCA-treated diabetic rats compared to untreated diabetic rats, respectively (20 mg/kg; 4 V: 0.77 ± 0.11 vs 0.45 ± 0.09, p = 0.0001 and 80 mg/kg; 4 V: 0.78 ± 0.11 vs 0.45 ± 0.09, p = 0.0001) UDCA prevented the increase in phospho-Smad2 and fibronectin protein expressions in diabetic corporal tissue both at 20 mg/kg (p = 0.0002, p = 0.002 respectively) and 80 mg/kg doses (p < 0.0001 for both). Smad7 protein expressions were significantly increased in the UDCA-treated diabetic groups compared to the untreated diabetic group (20 mg/kg: p = 0.0079; 80 mg/kg: p = 0.004). Furthermore, UDCA significantly prevented diabetes-induced increase in collagen (20 mg/kg: p = 0.0172; 80 mg/kg: p = 0.0003) and smooth muscle loss (20 mg/kg: p = 0.044; 80 mg/kg: p = 0.039). In conclusion, UDCA has a potential protective effect on erectile function in diabetic rats by altering fibrotic pathways via inhibition of TGF-β1/Smad2 and activation of Smad7.

Convergent and divergent genes expression profiles associated with brain-wide functional connectome dysfunction in deficit and non-deficit schizophrenia.

Deficit schizophrenia (DS) is a subtype of schizophrenia characterized by the primary and persistent negative symptoms. Previous studies have identified differences in brain functions between DS and non-deficit schizophrenia (NDS) patients. However, the genetic regulation features underlying these abnormal changes are still unknown. This study aimed to detect the altered patterns of functional connectivity (FC) in DS and NDS and investigate the gene expression profiles underlying these abnormal FC. The study recruited 82 DS patients, 96 NDS patients, and 124 healthy controls (CN). Voxel-based unbiased brain-wide association study was performed to reveal altered patterns of FC in DS and NDS patients. Machine learning techniques were used to access the utility of altered FC for diseases diagnosis. Weighted gene co-expression network analysis (WGCNA) was employed to explore the associations between altered FC and gene expression of 6 donated brains. Enrichment analysis was conducted to identify the genetic profiles, and the spatio-temporal expression patterns of the key genes were further explored. Comparing to CN, 23 and 20 brain regions with altered FC were identified in DS and NDS patients. The altered FC among these regions showed significant correlations with the SDS scores and exhibited high efficiency in disease classification. WGCNA revealed associations between DS/NDS-related gene expression and altered FC. Additionally, 22 overlapped genes, including 12 positive regulation genes and 10 negative regulation genes, were found between NDS and DS. Enrichment analyses demonstrated relationships between identified genes and significant pathways related to cellular response, neuro regulation, receptor binding, and channel activity. Spatial and temporal gene expression profiles of SCN1B showed the lowest expression at the initiation of embryonic development, while DPYSL3 exhibited rapid increased in the fetal. The present study revealed different altered patterns of FC in DS and NDS patients and highlighted the potential value of FC in disease classification. The associations between gene expression and neuroimaging provided insights into specific and common genetic regulation underlying these brain functional changes in DS and NDS, suggesting a potential genetic-imaging pathogenesis of schizophrenia.

CRISPR/Cas9-based genome editing and functional analysis of SlHyPRP1 and SlDEA1 genes of Solanum lycopersicum L. in imparting genetic tolerance to multiple stress factors.

CRISPR/Cas is a breakthrough genome editing system because of its precision, target specificity, and efficiency. As a speed breeding system, it is more robust than the conventional breeding and biotechnological approaches for qualitative and quantitative trait improvement. Tomato (Solanum lycopersicum L.) is an economically important crop, but its yield and productivity have been severely impacted due to different abiotic and biotic stresses. The recently identified SlHyPRP1 and SlDEA1 are two potential negative regulatory genes in response to different abiotic (drought and salinity) and biotic stress (bacterial leaf spot and bacterial wilt) conditions in S. lycopersicum L. The present study aimed to evaluate the drought, salinity, bacterial leaf spot, and bacterial wilt tolerance response in S. lycopersicum L. crop through CRISPR/Cas9 genome editing of SlHyPRP1 and SlDEA1 and their functional analysis. The transient single- and dual-gene SlHyPRP1 and SlDEA1 CRISPR-edited plants were phenotypically better responsive to multiple stress factors taken under the study. The CRISPR-edited SlHyPRP1 and SlDEA1 plants showed a higher level of chlorophyll and proline content compared to wild-type (WT) plants under abiotic stress conditions. Reactive oxygen species accumulation and the cell death count per total area of leaves and roots under biotic stress were less in CRISPR-edited SlHyPRP1 and SlDEA1 plants compared to WT plants. The study reveals that the combined loss-of-function of SlHyPRP1 along with SlDEA1 is essential for imparting significant multi-stress tolerance (drought, salinity, bacterial leaf spot, and bacterial wilt) in S. lycopersicum L. The main feature of the study is the detailed genetic characterization of SlDEA1, a poorly studied 8CM family gene in multi-stress tolerance, through the CRISPR/Cas9 gene editing system. The study revealed the key negative regulatory role of SlDEA1 that function together as an anchor gene with SlHyPRP1 in imparting multi-stress tolerance in S. lycopersicum L. It was interesting that the present study also showed that transient CRISPR/Cas9 editing events of SlHyPRP1 and SlDEA1 genes were successfully replicated in stably generated parent-genome-edited line (GEd0) and genome-edited first-generation lines (GEd1) of S. lycopersicum L. With these upshots, the study's key findings demonstrate outstanding value in developing sustainable multi-stress tolerance in S. lycopersicum L. and other crops to cope with climate change.

Targeting transcription factor pu.1 for improving neurologic outcomes after spinal cord injury.

After spinal cord injury (SCI), lipid metabolism dysregulation at the lesion site exacerbates secondary damage. The transcription factor pu.1 has been implicated as a negative regulator of multiple lipid metabolism-related genes and pathways. However, its role in post-SCI lipid metabolism remains unclear. We employed a mouse model of complete T10 crush SCI. Non-targeted metabolomics and bioinformatics analysis were utilized to investigate lipid metabolism at the lesion site after SCI. Polarized light imaging was used to evaluate the presence of cholesterol crystals. DB1976, a specific inhibitor of pu.1, was administered to examine its impact on local lipid metabolism after SCI. Immunofluorescence staining was performed to assess pu.1 expression and distribution, and to evaluate lipid droplet formation, astrocytic/fibrotic scar development, inflammatory cell infiltration, and tight junctions within the vasculature. Non-targeted metabolomics and bioinformatics analyses revealed significant alterations in lipid metabolism components after SCI. Moreover, immunofluorescence staining and polarized light imaging demonstrated substantial BODIPY+ lipid droplet accumulation and persistent cholesterol crystal formation at the lesion site after SCI. Increased pu.1 expression was predominantly observed within macrophages/microglia at the lesion site after SCI. DB1976 treatment significantly mitigated lipid droplet accumulation and cholesterol crystal formation, reduced CD68+ macrophage/microglial infiltration, and attenuated fibrotic scar formation. Moreover, DB1976 treatment promoted the expression of claudin-5 and zonula occludens-1 between vascular endothelial cells and enhanced GFAP+ glial connectivity after SCI. Our study reveals a significant correlation between lipid metabolism disturbance post-SCI and transcription factor pu.1 upregulation, specifically in macrophages/microglia at the lesion site. Thus, targeted pu.1 modulation has the potential to yield promising results by substantially diminishing the deposition of lipid metabolism byproducts at the lesion site and fostering a milieu conducive to SCI repair.

Regulation of daptomycin biosynthesis in Streptomyces roseosporus: new insights from genomic analysis and synthetic biology to accelerate lipopeptide discovery and commercial production.

Covering 2005-2024Daptomycin is a clinically important antibiotic that treats Gram-positive infections of skin and skin structure, bacteremia, and right-sided endocarditis, including those caused by methicillin-resistant Staphylococcus aureus (MRSA). Daptomycin is now generic, and many companies are involved in manufacturing and commercializing this life-saving medicine. There has been much recent interest in improving the daptomycin fermentation of Streptomyces roseosporus by mutagenesis, metabolic engineering, and synthetic biology methods. The genome sequences of two strains discovered and developed at Eli Lilly and Company, a wild-type low-producer and a high-producer induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis, are available for comparitive studies. DNA sequence analysis of the daptomycin biosynthetic gene clusters (BGCs) from these strains indicates that the high producer has two mutations in a large promoter region that drives the transcription of a giant multicistronic mRNA that includes all nine genes involved in daptomycin biosynthesis. The locations of translational start and stop codons strongly suggest that all nine genes are translationally coupled by overlapping stop and start codons or by 70S ribosome scanning. This report also reviews recent studies on this promoter region that have identified at least ten positive or negative regulatory genes suitable to manipulate by metabolic engineering, synthetic biology and focused mutagenesis for strain improvement. Improvements in daptomycin production will also enable high-level production of novel lipopeptide antibiotics identified by genome mining and combinatorial biosynthesis, and accelerate clinical and commercial development of superior lipopeptide antibiotics.

Diurnal switches in diazotrophic lifestyle increase nitrogen contribution to cereals

Uncoupling of biological nitrogen fixation from ammonia assimilation is a prerequisite step for engineering ammonia excretion and improvement of plant-associative nitrogen fixation. In this study, we have identified an amino acid substitution in glutamine synthetase, which provides temperature sensitive biosynthesis of glutamine, the intracellular metabolic signal of the nitrogen status. As a consequence, negative feedback regulation of genes and enzymes subject to nitrogen regulation, including nitrogenase is thermally controlled, enabling ammonia excretion in engineered Escherichia coli and the plant-associated diazotroph Klebsiella oxytoca at 23 °C, but not at 30 °C. We demonstrate that this temperature profile can be exploited to provide diurnal oscillation of ammonia excretion when variant bacteria are used to inoculate cereal crops. We provide evidence that diurnal temperature variation improves nitrogen donation to the plant because the inoculant bacteria have the ability to recover and proliferate at higher temperatures during the daytime.

Neo-functionalization in Saccharomyces cerevisiae: a novel Nrg1-Rtg3 chimeric transcriptional modulator is essential to maintain mitochondrial DNA integrity.

In Saccharomyces cerevisiae, the transcriptional repressor Nrg1 (Negative Regulator of Glucose-repressed genes) and the β-Zip transcription factor Rtg3 (ReTroGrade regulation) mediate glucose repression and signalling from the mitochondria to the nucleus, respectively. Here, we show a novel function of these two proteins, in which alanine promotes the formation of a chimeric Nrg1/Rtg3 regulator that represses the ALT2 gene (encoding an alanine transaminase paralog of unknown function). An NRG1/NRG2 paralogous pair, resulting from a post-wide genome small-scale duplication event, is present in the Saccharomyces genus. Neo-functionalization of only one paralog resulted in the ability of Nrg1 to interact with Rtg3. Both nrg1Δ and rtg3Δ single mutant strains were unable to use ethanol and showed a typical petite (small) phenotype on glucose. Neither of the wild-type genes complemented the petite phenotype, suggesting irreversible mitochondrial DNA damage in these mutants. Neither nrg1Δ nor rtg3Δ mutant strains expressed genes encoded by any of the five polycistronic units transcribed from mitochondrial DNA in S. cerevisiae. This, and the direct measurement of the mitochondrial DNA gene complement, confirmed that irreversible damage of the mitochondrial DNA occurred in both mutant strains, which is consistent with the essential role of the chimeric Nrg1/Rtg3 regulator in mitochondrial DNA maintenance.

Genome-wide association study and high-quality gene mining related to soybean protein and fat

BackgroundSoybean is one of the most important oil crops in the world, and its protein and fat are the primary sources of edible oil and vegetable protein. The effective components in soybean protein and fat have positive effects on improving human immunity, anti-tumor, and regulating blood lipids and metabolism. Therefore, increasing the contents of protein and fat in soybeans is essential for improving the quality of soybeans.ResultsThis study selected 292 soybean lines from different regions as experimental materials, based on SLAF-seq sequencing technology, and performed genome-wide association study (GWAS) on the phenotype data from 2019–2021 Planted at the experimental base of Jilin Agricultural University, such as the contents of protein and fat of soybeans. Through the GLM model and MLM model, four SNP sites (Gm09_39012959, Gm12_35492373, Gm16_9297124, and Gm20_24678362) that were significantly related to soybean fat content were associated for three consecutive years, and two SNP sites (Gm09_39012959 and Gm20_24678362) that were significantly related to soybean protein content were associated. By the annotation and enrichment of genes within the 100 Kb region of SNP loci flanking, two genes (Glyma.09G158100 and Glyma.09G158200) related to soybean protein synthesis and one gene (Glyma.12G180200) related to lipid metabolism were selected. By the preliminary verification of expression levels of genes with qPCR, it is found that during the periods of R6 and R7 of the accumulation of soybean protein and fat, Glyma.09G158100 and Glyma.09G158200 are positive regulatory genes that promote protein synthesis and accumulation, while Glyma.12G180200 is the negative regulatory gene that inhibits fat accumulation.ConclusionsThese results lay the basis for further verifying the gene function and studying the molecular mechanisms regulating the accumulation of protein and fat in soybean seeds.

Assessment of cytisine as an insecticide candidate for Hyphantria cunea management: Toxicological, biochemical, and control potential insights

In the present study, the toxicological effects of cytisine on the H. cunea larvae were investigated, and the potential of cytisine as a botanical insecticide through field simulation experiments was evaluated. The results showed that cytisine treatment (0.25–2.5%) exerted significant biotoxic effects on the H. cunea larvae, including diminished weight, disruption of both positive (HcCKS1, HcPLK, HcCCNA) and negative (HcGADD and HcCDKN) regulatory genes associated with larval growth, increased mortality, and heightened oxidative damage (H2O2 and MDA). Cytisine treatment significantly reduced glucose content and inhibited the expression of key rate-limiting enzyme genes (HcPFK, HcPK, HcHK1, HcCS, and HcIDH2) within glycolysis and the tricarboxylic acid cycle pathways. Under cytisine treatment, detoxification enzyme activities (CarE and GST) and expression of detoxification genes (HcCarE1, HcCarE2, HcCarE3, HcGST1, and HcGST3) were inhibited in H. cunea larvae. An increased contents of SOD, CAT, ASA and T-AOC, as well as expression of antioxidant enzyme genes HcSOD1 and HcCAT2, was found in cytisine-treated H. cunea larvae. Simultaneously, this is accompanied by a significant reduction in the expression of four antioxidant enzyme genes (e.g., HcPOD1 and HcPOD2). In the field experiment, a cytisine aqueous solution (25 g/L) with pre-sprayed and directly sprayed ways demonstrated potent insecticidal activity against H. cunea larvae, achieving a mortality rate of 53.75% and 100% at 24 h, respectively. Taken together, cytisine has significantly weight inhibition and lethal toxicity on the H. cunea larvae, and can be developed as a botanical insecticide for H. cunea control.

Wheat powdery mildew resistance: from gene identification to immunity deployment.

Powdery mildew is one of the most devastating diseases on wheat and is caused by the obligate biotrophic phytopathogen Blumeria graminis f. sp. tritici (Bgt). Due to the complexity of the large genome of wheat and its close relatives, the identification of powdery mildew resistance genes had been hampered for a long time until recent progress in large-scale sequencing, genomics, and rapid gene isolation techniques. Here, we describe and summarize the current advances in wheat powdery mildew resistance, emphasizing the most recent discoveries about the identification of genes conferring powdery mildew resistance and the similarity, diversity and molecular function of those genes. Multilayered resistance to powdery mildew in wheat could be used for counteracting Bgt, including durable, broad spectrum but partial resistance, as well as race-specific and mostly complete resistance mediated by nucleotide-binding and leucine rich repeat domain (NLR) proteins. In addition to the above mentioned layers, manipulation of susceptibility (S) and negative regulator genes may represent another layer that can be used for durable and broad-spectrum resistance in wheat. We propose that it is promising to develop effective and durable strategies to combat powdery mildew in wheat by simultaneous deployment of multilayered immunity.

The enigmatical manipulators in the capsule synthesis of Pasteurella multocida: Iron acquisition proteins

Aim: Spontaneous capsular loss or capsular changes in Pasteurella multocida can result from repeated laboratory passages, positive or negative regulatory genes, or an unknown gene. This study, it was aimed to compare the properties of phenotypic, genotypic, and biotypic of each non-typical, and typical Pasteu-rella multocida strain, to determine the dominant genes on capsule synthesis. Materials and Methods: Fifty-six strains, which capsular type was determined, and thirty -six, which capsular type was not determined, were used in this study. Multiple linear regression analysis was used based on the presence of dominant genes (serogroup, serotype, toxin, adhesin, iron acquisition, and protectin) in the isolates. Results: When colony morphologies of strains were evaluated of these strains by culture methods, mucoid colony formation was commonly detected in typical strains (87.5%), in contrast to non-typical strains (27.7%). In typical strains, the highest percentages of ptfA, ompA, and tadD genes were 78.57%, 75%, and 69.64%, respectively. In non-typical strains, the highest rates of ompA, ptfA, and tadD genes were 61.1%, 52.78%, and 52.78%, respectively. According to multiple linear regression analysis results, the together hgbA with hgbB genes caused the increase of capsule synthesis in these strains. The presence of the ompA gene in these strains was secondly a induction on these strains. Other genes were not effective in capsule synthesis in these strains. Conclusion: It was determined that the most significant effect in the forming of non-typical P. multocida strains was related to not enough HgbA and HgbB genes. It was supposed that P. multocida may not be heavily encapsulated under iron-restricted conditions. Consequently, P. multocida may change its capsule or lose its capsule related to iron acquisition proteins.

Identifying Treatment Resistance Related Pathways by Analyzing Serum Extracellular Vesicles of Patients With Resistant Versus Regressed Retinoblastoma.

To identify the genes and pathways responsible for treatment resistance (TR) in retinoblastoma (RB) by analyzing serum small extracellular vesicles (sEVs) of patients with TR active RB (TR-RB) and completely regressed RB (CR-RB). Serum-derived sEVs were characterized by transmission electron microscopy and nanoparticle tracking analysis. sEV transcriptome profiles of two TR-RB and one CR-RB with good response (>20 years tumor free) were compared to their age-matched controls (n = 3). Gene expression data were analyzed by the R Bioconductor package. The CD9 protein and mRNA expression of CD9, CD63, and CD81 were studied in five RB tumors and two control retinae by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. The isolated serum sEVs were round shaped and within the expected size (30-150 nm), and they had zeta potentials ranging from -10.8 to 15.9 mV. The mean ± SD concentrations of sEVs for two adults and four children were 1.1 × 1012 ± 0.1 and 5.8 × 1011 ± 1.7 particles/mL. Based on log2 fold change of ±2 and P < 0.05 criteria, there were 492 dysregulated genes in TR-RB and 184 in CR-RB. KAT2B, VWA1, CX3CL1, MLYCD, NR2F2, USP46-AS1, miR6724-4, and LINC01257 genes were specifically dysregulated in TR-RB. Negative regulation of apoptotic signaling, cell growth, and proton transport genes were greater than fivefold expressed only in TR-RB. CD9, CD63, and CD81 mRNA levels were high in RB tumors versus control retina, with increased and variable CD9 immunoreactivity in the invasive areas of the tumor. Serum sEVs could serve as a potential liquid biopsy source for understanding TR mechanisms in RB.

A New Insight into 6-Pentyl-2H-pyran-2-one against Peronophythora litchii via TOR Pathway.

The litchi downy blight disease of litchi caused by Peronophythora litchii accounts for severe losses in the field and during storage. While ample quantitative studies have shown that 6-pentyl-2H-pyran-2-one (6PP) possesses antifungal activities against multiple plant pathogenic fungi, the regulatory mechanisms of 6PP-mediated inhibition of fungal pathogenesis and growth are still unknown. Here, we investigated the potential molecular targets of 6PP in the phytopathogenic oomycetes P. litchii through integrated deployment of RNA-sequencing, functional genetics, and biochemical techniques to investigate the regulatory effects of 6PP against P. litchii. Previously we demonstrated that 6PP exerted significant oomyticidal activities. Also, comparative transcriptomic evaluation of P. litchii strains treated with 6PP Revealed significant up-regulations in the expression profile of TOR pathway-related genes, including PlCytochrome C and the transcription factors PlYY1. We also noticed that 6PP treatment down-regulated putative negative regulatory genes of the TOR pathway, including PlSpm1 and PlrhoH12 in P. litchii. Protein-ligand binding analyses revealed stable affinities between PlYY1, PlCytochrome C, PlSpm1, PlrhoH12 proteins, and the 6PP ligand. Phenotypic characterization of PlYY1 targeted gene deletion strains generated in this study using CRISPR/Cas9 and homologous recombination strategies significantly reduced the vegetative growth, sporangium, encystment, zoospore release, and pathogenicity of P. litchii. These findings suggest that 6PP-mediated activation of PlYY1 expression positively regulates TOR-related responses and significantly influences vegetative growth and the virulence of P. litchii. The current investigations revealed novel targets for 6PP and underscored the potential of deploying 6PP in developing management strategies for controlling the litchi downy blight pathogen.

Systematic profiling of subtelomeric silencing factors in budding yeast.

Subtelomeric gene silencing is the negative transcriptional regulation of genes located close to telomeres. This phenomenon occurs in a variety of eukaryotes with salient physiological implications, such as cell adherence, virulence, immune-system escape, and aging. The process has been widely studied in the budding yeast Saccharomyces cerevisiae, where genes involved in this process have been identified mostly on a gene-by-gene basis. Here, we introduce a quantitative approach to study gene silencing, that couples the classical URA3 reporter with GFP monitoring, amenable to high-throughput flow cytometry analysis. This dual silencing reporter was integrated into several subtelomeric loci in the genome, where it showed a gradual range of silencing effects. By crossing strains with this dual reporter at the COS12 and YFR057W subtelomeric query loci with gene-deletion mutants, we carried out a large-scale forward screen for potential silencing factors. The approach was replicable and allowed accurate detection of expression changes. Results of our comprehensive screen suggest that the main players influencing subtelomeric silencing were previously known, but additional potential factors underlying chromatin conformation are involved. We validate and report the novel silencing factor LGE1, a protein with unknown molecular function required for histone H2B ubiquitination. Our strategy can be readily combined with other reporters and gene perturbation collections, making it a versatile tool to study gene silencing at a genome-wide scale.

Pathogenic characterization of Klebsiella pneumoniae resistant to carbapenems and polymyxin

Objective: Analysis and investigation of pathogenic characteristics of polymyxin-and carbapenem-resistant Klebsiella pneumoniae (PR-CRKP). Methods: A total of 23 PR-CRKP strains isolated from clinical specimens from the General Hospital of Southern Theater Command from March 2019 to July 2021 were retrospectively collected, Whole-genome sequencing was performed on 23 PR-CRKP strains, resistance genes were identified by comparison of the CARD and the ResFinder database, high-resolution typing of PR-CRKP strains was analyzed by core genomic multilocus sequencing (cgMLST) and single nucleotide polymorphism (SNP); polymyxin resistance genes were determined by PCR and sequencing. Results: All PR-CRKP strains were KPC-2 producing ST11 types. cgMLST results showed that the evolutionary distance between the PR-CRKP strains and Klebsiella pneumoniae in mainland China was 66.44 on average, which is more closely related than foreign strains; the 23 PR-CRKP strains were divided into 3 main subclusters based on SNP phylogenetic trees, with some aggregation among Clade 2-1 in the isolation department and date. The two-component negative regulatory gene mgrB has seven mutation types including point mutations, different insertion fragments and different insertion positions. Conclusion: The close affinity of PR-CRKP strains indicate the possibility of nosocomial clonal transmission and the need to strengthen surveillance of PR-CRKP strains to prevent epidemic transmission of PR-CRKP.

The Potential Mechanism of Cannabidiol (CBD) Treatment of Epilepsy in Pentetrazol (PTZ) Kindling Mice Uncovered by Multi-Omics Analysis

Cannabidiol (CBD) is the main active ingredient in the cannabis plant used for treating epilepsy and related diseases. However, how CBD ameliorates epilepsy and its effect on the hippocampus remains unknown. Herein, we evaluated how CBD ameliorates seizure degree in pentylenetetrazol (PTZ) induced epilepsy mice after being exposed to CBD (10 mg/kg p.o). In addition, transcriptome and metabolomic analysis were performed on the hippocampus. Our results suggested that CBD could alleviate PTZ-induced seizure, of which the NPTX2, Gprc5c, Lipg, and Stc2 genes were significantly down-regulated in mice after being exposed to PTZ. Transcriptome analysis showed 97 differently expressed genes (CBD) and the PTZ groups. Metabonomic analysis revealed that compared with the PTZ group, 41 up-regulated and 67 down-regulated metabolites were identified in the hippocampus of epileptic mice exposed to CBD. The correlation analysis for transcriptome and metabolome showed that (±) 15-HETE and carnitine C6:0 were at the core of the network and were involved in the positive or negative regulation of the related genes after being treated with CBD. In conclusion, CBD ameliorates epilepsy by acting on the metabolism, calcium signaling pathway, and tuberculosis pathways in the hippocampus. Our study provided a practical basis for the therapeutic potential of treating epilepsy using CBD.

RRS1 shapes robust root system to enhance drought resistance in rice.

A strong root system facilitates the absorption of water and nutrients from the soil, to improve the growth of crops. However, to date, there are still very few root development regulatory genes that can be used in crop breeding for agriculture. In this study, we cloned a negative regulator gene of root development, Robust Root System 1 (RRS1), which encodes an R2R3-type MYB family transcription factor. RRS1 knockout plants showed enhanced root growth, including longer root length, longer lateral root length, and larger lateral root density. RRS1 represses root development by directly activating the expression of OsIAA3 which is involved in the auxin signaling pathway. A natural variation in the coding region of RRS1 changes the transcriptional activity of its protein. RRS1T allele, originating from wild rice, possibly increases root length by means of weakening regulation of OsIAA3. Knockout of RRS1 enhances drought resistance by promoting water absorption and improving water use efficiency. This study provides a new gene resource for improving root systems and cultivating drought-resistant rice varieties with important values in agricultural applications.