Supergene Research Articles

Mycobacterial FtsEX-RipC interaction is required for normal growth and cell morphology in rifampicin and low ionic strength conditions.

Mycobacterial cell growth and division are coordinated with regulated peptidoglycan hydrolysis. Understanding cell wall gene complexes that govern normal cell division and elongation will aid in the development of tools to disarm the ability of mycobacteria to survive immune-like and antibiotic stresses. We combined genetic analyses and scanning electron microscopy to analyze morphological changes of mycobacterial FtsEX and RipC mutants in stressful conditions. We demonstrate that FtsE, FtsX, FtsEX, and RipC are conditionally required for the survival of Mycobacterium smegmatis during rifampicin treatment and in low-salt conditions. Growth defects in these conditions were characterized by short and bulgy cells as well as elongated filamentous cells. We also show that the FtsEX-RipC interaction is essential for the survival of M. smegmatis in rifampicin. Our results suggest that FtsE, FtsX, and RipC are required for normal cell wall regulation and ultimately for survival in stressful conditions.

NADPH oxidase exerts a B cell-intrinsic contribution to lupus risk by modulating endosomal TLR signals.

Genome-wide association studies in systemic lupus erythematosus (SLE) have linked loss-of-function mutations in phagocytic NADPH oxidase complex (NOX2) genes, including NCF1 and NCF2, to disease pathogenesis. The prevailing model holds that reduced NOX2 activity promotes SLE via defective efferocytosis, the immunologically silent clearance of apoptotic cells. Here, we describe a parallel B cell-intrinsic mechanism contributing to breaks in tolerance. In keeping with an important role for B cell Toll-like receptor (TLR) pathways in lupus pathogenesis, NOX2-deficient B cells exhibit enhanced signaling downstream of endosomal TLRs, increased humoral responses to nucleic acid-containing antigens, and the propensity toward humoral autoimmunity. Mechanistically, TLR-dependent NOX2 activation promotes LC3-mediated maturation of TLR-containing endosomes, resulting in signal termination. CRISPR-mediated disruption of NCF1 confirmed a direct role for NOX2 in regulating endosomal TLR signaling in primary human B cells. Together, these data highlight a new B cell-specific mechanism contributing to autoimmune risk in NCF1 and NCF2 variant carriers.

Integrated Analysis of lncRNAs and mRNAs Reveals Complex Gene Network Mediated by lncRNAs and Regulatory Function of MuLRR-RLK-AS in Response to Phytoplasma Infection in Mulberry.

Phytoplasma disease is one of the most serious infectious diseases that affects the growth and development of mulberry. Long non-coding RNAs (lncRNAs) play an important role in plants' defense systems; however, the contribution of lncRNAs in the response to phytoplasma infection in mulberry is still largely unknown. Herein, strand-specific RNA sequencing was performed to profile the mRNAs and lncRNAs involved in the response to phytoplasma infection in mulberry, and a total of 4169 genes were found to be differentially expressed (DE) between healthy and phytoplasma-infected leaves. Moreover, 1794 lncRNAs were identified, of which 742 lncRNAs were DE between healthy and infected leaves. Target prediction showed that there were 68 and 44 DE lncRNAs which may function as cis and trans-regulators, targeting 54 and 44 DE genes, respectively. These DE target genes are associated with biological processes such as metabolism, signaling, development, transcriptional regulation, etc. In addition, it was found that the expression of the antisense lncRNA (MuLRR-RLK-AS) of the leucine-rich repeat receptor-like protein kinase gene (MuLRR-RLK) was decreased in the phytoplasma-infected leaves. Interestingly, it was found that overexpression of MuLRR-RLK-AS can inhibit the expression of MuLRR-RLK. Moreover, it was found that the expression levels of PTI-related and MAPK genes in the transgenic MuLRR-RLK Arabidopsis plants were significantly higher than those in the wild-type plants when inoculated with pathogens, and the transgenic plants were conferred with strong disease resistance. Our results demonstrate that MuLRR-RLK-AS, as a trans-regulatory factor, can inhibit the expression of the MuLRR-RLK gene and is a negative regulatory factor for mulberry resistance. The information provided is particularly useful for understanding the functions and mechanisms of lncRNAs in the response to phytoplasma infection in mulberry.

Abstract PR-011: The role of tumor microenvironment in metastasis and relapses of medulloblastoma using immunocompetent mouse models

Abstract Medulloblastoma (MB) is an embryonal malignant tumor that arises in the cerebellum of infants, children, and adolescents. Despite highly aggressive therapies, including surgery, radiation, and chemotherapy, metastasis remains the primary cause of death in these patients. The tumor microenvironment (TME) has an important role in metastasis and relapse in several cancer types, but it’s role in high-risk MBs (metastatic and relapse) has been understudied. Here, we investigated the process of relapse in an immunocompetent Shh MB mouse model based on somatic mutagenesis induced by Sleeping Beauty (SB)-mediated transposition on a genetically engineered strain carrying a loss-of-function Patched 1 gene (Ptch1) mutation. Lack of one functional copy of the Ptch1 gene predisposes this strain to develop MB. Somatic mutations induced by the transposon increase penetrance of tumor formation in the triple mutant mice carrying the SB transposon system and the Ptch1 mutation. We used this model to study the mechanisms of chemoresistance to cisplatin and cyclophosphamide, that constitute the backbone of the high-dose chemotherapy used to treat patients under the age of 3, who cannot receive craniospinal irradiation. By treating the SB mouse model with surgery followed by high-dose fractionated chemotherapy, we developed a model of MB chemoresistance which is relevant to the infant SHH-MB patient population. While chemotherapy improved mouse survival, only a small number of animals were cured, mirroring the outcome seen in infant patients with Shh MB. Deep sequencing and mapping of transposon-gDNA junctions, along with gene-centric common insertion site analysis (gCIS), revealed that Trp53 transposon insertions are found exclusively at relapse. In the metastatic compartment we observed convergence of gCISes on genes coding for epigenetic regulators, among them a prominent enrichment was found for PRC2 complex genes. RNA-seq analysis on the transcriptome of primary and recurrent tumors indicated that a majority of the differentially expressed genes are associated with inflammatory responses, IL6, and TNF-α/NF-κB signaling. Immunohistochemistry to visualize the expression levels of inflammatory markers: IL-6, CCL2, and F4-80 and spatial targeted multiplexed assays (Geomx) confirmed the increase of immune cells at progression. ChIP-qPCR on cytokine loci revealed binding of PRC2 and deposition of PRC2 mediated repressive histone mark H3K27me3 on the promoter of CCL2 highlighting LOF mutations in PRC2 components as potential epigenetic mechanisms for immune recruitment at progression. Inhibition of immune activation through pharmacological strategies might be a possible therapeutic option for infant SHH-MB. Citation Format: Niusha Khazaei, Ana Castillo Orozco, Wajih Jawhar, Geoffroy Danieau, Kiran Narta, Michael Taylor, Sorana Morrissy, Ana Stuecklin, Livia Garzia. The role of tumor microenvironment in metastasis and relapses of medulloblastoma using immunocompetent mouse models [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr PR-011.

Norflurazon causes cell death and inhibits implantation-related genes in porcine trophectoderm and uterine luminal epithelial cells

Norflurazon, an inhibitor of carotenoid synthesis, is a pre-emergent herbicide that prevents growth of weeds. The norflurazon is known to hamper embryo development in non-mammals. However, specific toxic effects of norflurazon on mammalian maternal and fetal cells have not been elucidated. Thus, the hypothesis of this study is that norflurazon may influence the toxic effects between maternal and fetal cells during early pregnancy in pigs. We aimed to examine the toxic effects of norflurazon in porcine trophectoderm (Tr) and uterine luminal epithelium (LE) cells. Norflurazon, administered at 0, 20, 50 or 100 μM for 48 h was used to determine its effects on cell proliferation and cell-cycle arrest. For both uterine LE and Tr cell lines, norflurazone caused mitochondrial dysfunction by inhibiting mitochondrial respiration and ATP production, and down-regulated expression of mRNAs of mitochondrial complex genes. Norflurazon increased cell death by increasing intracellular calcium and regulating PI3K and MAPK cell signaling pathways, as well as endoplasmic reticulum (ER) stress, ER-mitochondrial contact, and autophagy-related target proteins. Norflurazone also inhibited expression of genes required for implantation of blastocysts, including SMAD2, SMAD4, and SPP1. These findings indicate that norflurazon may induce implantation failure in pigs and other mammals through adverse effects on both Tr and uterine LE cells.

Adipokinetic hormone signaling regulates adult dehydration resistance in the migratory locust

Drought events have become more severe under climate change, and this can pose a major threat to the survival of various organisms. The molecular mechanisms involved in dehydration resistance are not well known. Here, adults of the migratory locust, Locusta migratoria, were subjected to food-mediated dehydration, and adipokinetic hormone (AKH) signaling was found to play a key role in regulating dehydration resistance. Specifically, dehydration shortened the lifespan, increased the body weight loss, and reduced the water loss rate in adult locusts. Global transcriptome profiles revealed variations in tissue-specific gene expression between dehydration-resistant locusts and normal locusts. Importantly, dehydration selection and exposure induced prominent expression of AKH genes in the retrocerebral complex of adult locusts. Furthermore, individual knockdown of AKH1, AKH2, or AKH receptor (AKHR) accelerated water loss and shortened the lifespan of adult locusts under dehydration conditions, and trehalose supplementation ameliorated the negative effects caused by interference with AKH or AKHR. These findings demonstrated that AKH/AKHR signaling-dependent trehalose metabolism plays a crucial role in regulating locust dehydration resistance and thus provide novel insights into the regulatory mechanism underlying drought resistance.

WITHDRAWN: Exploring the Interplay of m6A regulators in renal cell carcinoma: Molecular insights and clinical implications

BackgroundThe m6A modification of N6-methyladenosine (m6A) modification regulates gene expression and functioning by affecting RNA splicing, stability, translocation, and translation. ObjectiveThis study used comprehensive bioinformatics analysis to elucidate the mechanisms by which m6A regulators function in kidney renal clear cell carcinoma (KIRC) and their interrelationships. MethodsThe KIRC data extracted from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases was used in this study. LASSO Cox regression, protein–protein interaction analysis, univariate Cox regression, and a predictive risk model. Cox regression, Kaplan–Meier analysis, consensus clustering, CIBERSORT, gene set enrichment analysis (GSEA), ESTIMATE, immune cell infiltration, and prognostic factors were investigated in KIRC utilizing computational methods. ResultsThe results of integrated analysis showed that KIRC has unique and complex gene patterns associated with m6A and is linked to different gene patterns. These patterns included an 11.68-fold change in insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3), a 3.35-fold change in ELAV-like RNA binding protein 2 (ELAVL2), a 2.34-fold change in fat mass- and obesity-associated protein (FTO), a 2.52-fold change in ELAVL4, and a 2.15-fold change in ELAVL3. By contrast, there was downregulation of IGF2BP2, zinc finger CCCH-type containing 14 (ZC3H14), and alpha-ketoglutarate-dependent dioxygenase (ALKBH6), with fold changes of 0.44, 0.66, and 0.68, respectively. Minor modifications ranging from 1 to 1.28 were seen in other m6A regulators. ConclusionThis study showed that m6A alteration affects KIRC tumor immunity. Moreover, m6A regulators such as IGF2BP3 have therapeutic and diagnostic potential, which could improve patient outcomes by personalized immunotherapy.

Adult epithelioid glioblastoma exhibits an extremely poor prognosis and high frequency of SWI/SNF complex mutation: Insights from a retrospective study.

Epithelioid glioblastoma (eGBM) is a rare subtype of GBM. Given the update of the definition of GBM, the understanding of the molecular characteristics and prognosis of "true" adult eGBM remains limited. Herein, we retrospectively analyzed the clinicopathological data of 39 adult eGBM cases. Adult eGBM primarily affected females, with a male-to-female ratio of 1:2.3. The average age of diagnosis was 53 years, and the tumor affected the temporal lobe in 41% of cases (16/39, 41%). Microscopically, the tumors consisted mainly or entirely of epithelioid cells. Perivascular infiltration (10/39, 25.6%) and leptomeningeal dissemination (7/39, 17.9%) were not uncommon. BRAF V600E mutation was detected in 40.9% of cases (n = 9/22). Next-generation sequencing revealed that CDKN2A/B homogeneous deletion was the most frequently mutated gene (8/10, 80%), followed by TERT promoter mutation (7/10, 70%), Cyclin-dependent kinases 4 or 6 (CDK4/6) amplification (5/10, 50%) and BRAF V600E mutation (50%, 5/10). Notably, the incidence of ARID1B mutation in eGBM was 50% (5/10), representing the first report of such a mutation in this subtype of GBM. ARID1B was known to be a subunit of the SWI/SNF chromatin remodeler. Chromosome analysis showed a 7+/10- signature in 90% (9/10) cases. Adult eGBM carried a dismal prognosis compared to GBM with IDH and H3 wild-type (typical GBM) (OS: 13.89 vs 24.30 months; P = .003) and even typical GBM without MGMT promoter methylation (OS: 13.89 vs 22.08 months; P = .036). Based on these findings, it can be concluded that adult eGBM harbors a high frequency of the 7+/10- signature and alterations in the MAPK pathway, SWI/SNF complex and cyclin-related genes and portends an extremely poor prognosis.

Integrating network pharmacology and experimental models to identify notoginsenoside R1 ameliorates atherosclerosis by inhibiting macrophage NLRP3 inflammasome activation.

Atherosclerosis is a cardiovascular disease, accounting for the most common mortality cause worldwide. Notoginsenoside R1 (NGR1) is a characteristic saponin of Radix notoginseng that exhibits anti-inflammatory and antioxidant effects while modulating lipid metabolism. Evidence suggests that NGR1 exerts cardioprotective, neuroprotective, and anti-atherosclerosis effects. However, underlying NGR1 mechanisms alleviating atherosclerosis (AS) have not been examined. This study used a network pharmacology approach to construct the drug-target-disease correlation and protein-protein interaction (PPI) network of NGR1 and AS. Moreover, functional annotation and pathway enrichment analyses deciphered the critical biological processes and signaling pathways potentially regulated by NGR1. The protective effect of NGR1 against AS and the underlying mechanism(s) was assessed in an atherogenic apolipoprotein E-deficient (ApoE-/-) mice in vivo and an oxidized low-density lipoprotein (ox-LDL)-induced macrophage model in vitro. The network pharmacology and molecular docking analyses revealed that NGR1 protects against AS by targeting the NLRP3/caspase-1/IL-1β pathway. NGR1 reduced foam cell formation in ox-LDL-induced macrophages and decreased atherosclerotic lesion formation, serum lipid metabolism, and inflammatory cytokines in AS mice in vivo. Therefore, NGR1 downregulates the NLRP3 inflammasome complex gene expression of NLRP3, caspase-1, ASC, IL-1β, and IL-18, in vivo and in vitro.

Super enhancer related gene ANP32B promotes the proliferation of acute myeloid leukemia by enhancing MYC through histone acetylation

BackgroundAcute myeloid leukemia (AML) is a malignancy of the hematopoietic system, and childhood AML accounts for about 20% of pediatric leukemia. ANP32B, an important nuclear protein associated with proliferation, has been found to regulate hematopoiesis and CML leukemogenesis by inhibiting p53 activity. However, recent study suggests that ANP32B exerts a suppressive effect on B-cell acute lymphoblastic leukemia (ALL) in mice by activating PU.1. Nevertheless, the precise underlying mechanism of ANP32B in AML remains elusive.ResultsSuper enhancer related gene ANP32B was significantly upregulated in AML patients. The expression of ANP32B exhibited a negative correlation with overall survival. Knocking down ANP32B suppressed the proliferation of AML cell lines MV4-11 and Kasumi-1, along with downregulation of C-MYC expression. Additionally, it led to a significant decrease in H3K27ac levels in AML cell lines. In vivo experiments further demonstrated that ANP32B knockdown effectively inhibited tumor growth.ConclusionsANP32B plays a significant role in promoting tumor proliferation in AML. The downregulation of ANP32B induces cell cycle arrest and promotes apoptosis in AML cell lines. Mechanistic analysis suggests that ANP32B may epigenetically regulate the expression of MYC through histone H3K27 acetylation. ANP32B could serve as a prognostic biomarker and potential therapeutic target for AML patients.

Only rare classical MHC-I alleles are highly expressed in the European house sparrow.

The exceptional polymorphism observed within genes of the major histocompatibility complex (MHC), a core component of the vertebrate immune system, has long fascinated biologists. The highly polymorphic classical MHC class-I (MHC-I) genes are maintained by pathogen-mediated balancing selection (PMBS), as shown by many sites subject to positive selection, while the more monomorphic non-classical MHC-I genes show signatures of purifying selection. In line with PMBS, at any point in time, rare classical MHC alleles are more likely than common classical MHC alleles to confer a selective advantage in host-pathogen interactions. Combining genomic and expression data from the blood of wild house sparrows Passer domesticus, we found that only rare classical MHC-I alleles were highly expressed, while common classical MHC-I alleles were lowly expressed or not expressed. Moreover, highly expressed rare classical MHC-I alleles had more positively selected sites, indicating exposure to stronger PMBS, compared with lowly expressed classical alleles. As predicted, the level of expression was unrelated to allele frequency in the monomorphic non-classical MHC-I alleles. Going beyond previous studies, we offer a fine-scale view of selection on classical MHC-I genes in a wild population by revealing differences in the strength of PMBS according to allele frequency and expression level.

Fatty Acid Amide Hydrolase and Cannabinoid Receptor Type 1 Genes Regulation is Modulated by Social Isolation in Rats.

Social isolation is a state of lack of social connections, involving the modulation of different molecular signalling cascades and associated with high risk of mental health issues. To investigate if and how gene expression is modulated by social experience at the central level, we analyzed the effects of 5weeks of social isolation in rats focusing on endocannabinoid system genes transcription in key brain regions involved in emotional control. We observed selective reduction in mRNA levels for fatty acid amide hydrolase (Faah) and cannabinoid receptor type 1 (Cnr1) genes in the amygdala complex and of Cnr1 in the prefrontal cortex of socially isolated rats when compared to controls, and these changes appear to be partially driven by trimethylation of Lysine 27 and acetylation of Lysine 9 at Histone 3. The alterations of Cnr1 transcriptional regulation result also directly correlated with those of oxytocin receptor gene. We here suggest that to counteract the effects of SI, it is of relevance to restore the endocannabinoid system homeostasis via the use of environmental triggers able to revert those epigenetic mechanisms accounting for the alterations observed.

Donor KIR2DL1 Allelic Polymorphism Influences Posthematopoietic Progenitor Cell Transplantation Outcomes in the T Cell Depleted and Reduced Intensity Conditioning Setting

The majority of established KIR clinical assessment algorithms used for donor selection for hematopoietic progenitor cell transplantation (HPCT) evaluate gene content (presence/absence) of the KIR gene complex. In comparison, relatively little is known about the impact of KIR allelic polymorphism. By analyzing donors of T cell depleted (TcD) reduced intensity conditioning (RIC) HPCT, this study investigated the influence on post-transplant outcome of 2 polymorphic residues of the inhibitory KIR2DL1. The aim of this study was to expand upon existing research into the influence of KIR2DL1 allelic polymorphism upon post-transplant outcome. The effects of allele groups upon transplant outcomes were investigated within a patient cohort using a defined treatment protocol of RIC with TcD. Using phylogenetic data, KIR2DL1 allelic polymorphism was categorized into groups on the basis of variation within codons 114 and 245 (positive or negative for the following groups: KIR2DL1*002/001g, KIR2DL1*003, KIR2DL1*004g) and the identification of null alleles. The influence of these KIR2DL1 allele groups in hematopoietic progenitor cell transplantation (HPCT) donors was assessed in the post-transplant data of 86 acute myelogenous leukemia patients receiving RIC TcD HPCT at a single center. KIR2DL1 allele groups in the donor significantly impacted upon 5-year post-transplant outcomes in RIC TcD HPCT. Donor KIR2DL1*003 presented the greatest influence upon post-transplant outcomes, with KIR2DL1*003 positive donors severely reducing 5-year post-transplant overall survival (OS) compared to those receiving a transplant from a KIR2DL1*003 negative donor (KIR2DL1*003 pos versus neg: 27.0% versus 60.0%, P = .008, pc = 0.024) and disease-free survival (DFS) (KIR2DL1*003 pos versus neg: 23.5% versus 60.0%, P = .004, pc = 0.012), and increasing 5-year relapse incidence (KIR2DL1*003 pos versus neg: 63.9% versus 27.2%, P = .009, pc = 0.027). KIR2DL1*003 homozygous and KIR2DL1*003 heterozygous grafts did not present significantly different post-transplant outcomes. Donors possessing the KIR2DL1*002/001 allele group were found to significantly improve post-transplant outcomes, with donors positive for the KIR2DL1*004 allele group presenting a trend towards improvement. KIR2DL1*002/001 allele group (KIR2DL1*002/001g) positive donors improved 5-year OS (KIR2DL1*002/001g pos versus neg: 56.4% versus 27.2%, P = .009, pc = 0.024) and DFS (KIR2DL1*002/001g pos versus neg: 53.8% versus 25.5%, P = .018, pc = 0.036). KIR2DL1*004 allele group (KIR2DL1*004g) positive donors trended towards improving 5-year OS (KIR2DL1*004g pos versus neg: 53.3% versus 35.5%, P = .097, pc = 0.097) and DFS (KIR2DL1*004g pos versus neg: 50.0% versus 33.9%, P = .121, pc = 0.121), and reducing relapse incidence (KIR2DL1*004g pos versus neg: 33.1% versus 54.0%, P = .079, pc = 0.152). The presented findings suggest donor selection algorithms for TcD RIC HPCT should consider avoiding KIR2DL1*003 positive donors, where possible, and contributes to the mounting evidence that KIR assessment in donor selection algorithms should reflect the conditioning regime protocol used.

Prenatal diagnosis and clinical management of cardiac rhabdomyoma: a single-center study.

The study aims to assess the ultrasonic features of fetal cardiac rhabdomyoma (CR), track the perinatal outcome and postnatal disease progression, investigate the clinical utility of ultrasound, MRI and tuberous sclerosis complex (TSC) gene analysis in CR evaluation, and offer evidence for determing of fetal CR prognosis. We conducted a retrospective analysis of prenatal ultrasound-diagnosed fetal CR cases in our hospital from June 2011 to June 2022, tracked the perinatal outcomes, regularly followed live infants to analyze cardiac lesion changes and disease progression, and compared the sensitivities of ultrasound, MRI and their combination in the detecting of intracranial sclerosing nodules. Our study included 54 fetuses with CR: 32 pregnancies were terminated, 22 were delivered, 35 were diagnosed with TSC, 13 had simple CR without TSC, and in 6 cases, remained unclear whether TSC accompanied the CR due to insufficient evidence. 45 fetuses (83.3%) had multiple lesions, while 9 fetuses (16.7%) presented with a single lesion. Twelve cases had intracardiac complications, all associated with multiple lesions, and these cases exhibited larger maximum tumor diameters than the non-complicated group. Multiple intracardiac lesions were more prevalent in the TSC group than in the simple CR group. However, there was no significant difference in maximum tumor diameter between the two groups. Among 30 fetuses who underwent fetal brain MRI, 23 were eventually diagnosed with TSC, with 11 fetuses showing intracranial sclerosis nodules by ultrasound and 15 by MRI, and the diagnostic consistency was moderate (k = 0.60). Twenty-two fetuses were born and followed up for 6-36 months. CR lesions diminished or disappeared in 18 infants (81.8%), while they remained unchanged in 4 infants (18.2%). Ten out of 12 (83.3%) surviving children diagnosed with TSC developed epilepsy, and 7 (58.3%) had neurodevelopmental dysfunction. The majority of CR cases involve multiple lesions, which are a primary risk factor for TSC. Through prenatal ultrasound examination is crucial for assessing fetal CR prognosis. Although ultrasound combined with MRI can detect intracranial sclerosis nodules in TSC fetuses, its sensitivity is limited. TSC gene sequencing is an essential diagnostic method. Simple CR cases without TSC generally have a favorable prognosis.

Optical Mapping: Detecting Genomic Resistance Cassettes in MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant bacterium with a global presence in healthcare facilities as well as community settings. The resistance of MRSA to beta-lactam antibiotics can be attributed to a mobile genetic element called the staphylococcal cassette chromosome mec (SCCmec), ranging from 23 to 68 kilobase pairs in length. The mec gene complex contained in SCCmec allows MRSA to survive in the presence of penicillin and other beta-lactam antibiotics. We demonstrate that optical mapping (OM) is able to identify the bacterium as S. aureus, followed by an investigation of the presence of kilobase pair range SCCmec elements by examining the associated OM-generated barcode patterns. By employing OM as an alternative to traditional DNA sequencing, we showcase its potential for the detection of complex genetic elements such as SCCmec in MRSA. This approach holds promise for enhancing our understanding of antibiotic resistance mechanisms and facilitating the development of targeted interventions against MRSA infections.

Diversity of MHC class II DRB alleles in the Northern chamois genus Rupicapra

Abstract Major histocompatibility complex (MHC) genes are commonly used markers for monitoring adaptive genetic and evolutionary potential of species. In this study, we investigated genetic variation of the MHC class II DRB locus in the chamois genus Rupicapra by using next-generation sequencing. Sequencing of 102 samples led to the identification of 25 alleles, 11 of which are novel. The high ratio of the relative rates of nonsynonymous to synonymous mutations (dN/dS) suggests a signal of positive selection on this locus. We analyzed patterns of genetic variation within and among 2 subspecies of Northern Chamois and compared them to previously published studies using neutral markers to provide a basis for assessing the effects of demographic processes. Our analyses have shown that alleles are likely to be maintained by balancing selection in different populations with similar frequencies and that this mechanism also works in small, isolated populations that are strongly affected by genetic drift.

Enhanced Expression of Glycolytic Enzymes and Succinate Dehydrogenase Complex Flavoprotein Subunit A by Mesothelin Promotes Glycolysis and Mitochondrial Respiration in Myeloblasts of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy characterized by rapid growth and uncontrolled proliferation of undifferentiated myeloid cells. Metabolic reprogramming is commonly observed in the bone marrow of AML patients, as leukemia cells require increased ATP supply to support disease progression. In this study, we examined the potential role of mesothelin as a metabolic modulator in myeloid cells in AML. Mesothelin is a well-known marker of solid tumors that promotes cancer cell proliferation and survival. We initially analyzed alterations in mesothelin expression in the myeloblast subpopulations, defined as SSC-Alow/CD45dim, obtained from the bone marrow of AML patients using flow cytometry. Our results showed overexpression of mesothelin in 34.8% of AML patients. Subsequently, metabolic changes in leukemia cells were evaluated by comparing the oxygen consumption rates (OCR) of bone marrow samples derived from adult AML patients. Notably, a higher OCR was observed in the mesothelin-positive compared to the mesothelin-low and non-expressing groups. Treatment with recombinant human mesothelin protein enhanced OCR and increased the mRNA expression of glycolytic enzymes and mitochondrial complex II in KG1α AML cells. Notably, siRNA targeting mesothelin in KG1α cells led to the reduction of glycolysis-related gene expression but had no effect on the mitochondrial complex gene. The collective results demonstrate that mesothelin induces metabolic changes in leukemia cells, facilitating the acquisition of a rapid supply of ATP for proliferation in AML. Therefore, the targeting of mesothelin presents a potentially promising approach to mitigating the progression of AML through the inhibition of glycolysis and mitochondrial respiration in myeloid cells.

Meat and meat products as potential sources of emerging MDR Bacillus cereus: groEL gene sequencing, toxigenic and antimicrobial resistance

BackgroundBacillus cereus is implicated in severe foodborne infection in humans. This study intended to assess the occurrence, groEL gene sequencing, biofilm production, and resistance profiles of emerged multidrug resistant (MDR) B. cereus in meat and meat product samples. Moreover, this work highlights the virulence and toxigenic genes (hblABCD complex, nheABC complex, cytK, ces, and pc-plc) and antimicrobial resistance genes (bla1, tetA, bla2, tetB, and ermA).MethodsConsequently, 200 samples (sausage, minced meat, luncheon, beef meat, and liver; n = 40 for each) were indiscriminately collected from commercial supermarkets in Port Said Province, Egypt, from March to May 2021. Subsequently, food samples were bacteriologically examined. The obtained isolates were tested for groEL gene sequence analysis, antibiotic susceptibility, biofilm production, and PCR screening of toxigenic and resistance genes.ResultsThe overall prevalence of B. cereus among the inspected food samples was 21%, where the highest predominance was detected in minced meat (42.5%), followed by beef meat (30%). The phylogenetic analysis of the groEL gene exposed that the examined B. cereus strain disclosed a notable genetic identity with other strains from the USA and China. Moreover, the obtained B. cereus strains revealed β-hemolytic activity, and 88.1% of the recovered strains tested positive for biofilm production. PCR evidenced that the obtained B. cereus strains usually inherited the nhe complex genes (nheA and nheC: 100%, and nheB: 83.3%), followed by cytK (76.2%), hbl complex (hblC and hblD: 59.5%, hblB: 16.6%, and hblA: 11.9%), ces (54.7%), and pc-plc (30.9%) virulence genes. Likewise, 42.9% of the examined B. cereus strains were MDR to six antimicrobial classes and encoded bla1, bla2, ermA, and tetA genes.ConclusionIn summary, this study highlights the presence of MDR B. cereus in meat and meat products, posing a significant public health risk. The contamination by B. cereus is common in minced meat and beef meat. The molecular assay is a reliable fundamental tool for screening emerging MDR B. cereus strains in meat and meat products.

HCCDB v2.0: Decompose Expression Variations by Single-cell RNA-seq and Spatial Transcriptomics in HCC.

Large-scale transcriptomic data are crucial for understanding the molecular features of hepatocellular carcinoma (HCC). Integrated 15 transcriptomic datasets of HCC clinical samples, the first version of HCC database (HCCDB v1.0) was released in 2018. Through the meta-analysis of differentially expressed genes and prognosis-related genes across multiple datasets, it provides a systematic view of the altered biological processes and the inter-patient heterogeneities of HCC with high reproducibility and robustness. With four years having passed, the database now needs integration of recently published datasets. Furthermore, the latest single-cell and spatial transcriptomics have provided a great opportunity to decipher complex gene expression variations at the cellular level with spatial architecture. Here, we present HCCDB v2.0, an updated version that combines bulk, single-cell, and spatial transcriptomic data of HCC clinical samples. It dramatically expands the bulk sample size by adding 1656 new samples from 11 datasets to the existing 3917 samples, thereby enhancing the reliability of transcriptomic meta-analysis. A total of 182,832 cells and 69,352 spatial spots are added to the single-cell and spatial transcriptomics sections, respectively. A novel single-cell level and 2-dimension (sc-2D) metric is proposed as well to summarize cell type-specific and dysregulated gene expression patterns. Results are all graphically visualized in our online portal, allowing users to easily retrieve data through a user-friendly interface and navigate between different views. With extensive clinical phenotypes and transcriptomic data in the database, we show two applications for identifying prognosis-associated cells and tumor microenvironment. HCCDB v2.0 is available at http://lifeome.net/database/hccdb2.

ESCHERICHIA COLI phenotypic characteristics and antagonistic activity in opisthorchiasis invasion

Opisthorchis felineus invasion in human causes inflammatory and dyskinetic disorders of the gastrointestinal tract accompanied by altered phenotypic characteristics in colon microbiota. The aim of research — study an impact of the Escherichia coli isolate phenotypic characteristics on Klebsiella spp. bacteria, isolated from colonic contents of patients with diagnosed opisthorchiasis as well as E. coli antagonistic activity. Materials and methods. The phenotypic properties of 54 E. coli isolates and 8 genus Klebsiella isolates obtained from colonic contents of patients with diagnosed opisthorchiasis were assessed. Identification of isolates and analysis of proteomic profiles were performed using Maldi BioTyper 3.0 software. 204 co-cultivation datasets were analyzed investigating antagonistic activity of E. coli isolates with varying properties on Klebsiella spp. E. coli and Klebsiella spp. isolates were examined by whole genome sequencing. Results. E. coli bacteria with typical phenotypic characteristics showed significantly more prominent antagonistic activity against Klebsiella spp. A significantly higher level of antagonistic activity against K. oxytoca bacteria vs K. pneumoniae strains. The proteomic bacterial strain profiles were divided into clusters depending on the level of antagonistic activity. E. coli molecular serotyping for O- and H-antigens revealed the genes of enterotoxigenic, enteroinvasive and extraintestinal pathogens in 60.0% of cases. Strains with the highest antagonistic activity index, which are carriers of the genes typical to enterotoxigenic E. coli sequence serotypes O6:H1 and O6:H5, were identified. The genome of such strains consisted of the largest number of virulence gene complexes: adhesins, invasins, toxins, bacteriocins. Multilocus sequence typing and sequence serotyping of E. coli and K. pneumoniae strains established their heterogeneity; K. oxytoca isolates were identified as ST242 and ST176. All strains were characterized by homology of antibiotic resistance markers (oqxA, oqxB, fosA) and a variety of beta-lactam resistance gene variants. Conclusion. It was found that E. coli isolates with typical phenotypic characteristics and carriers of virulence gene complexes exhibited significantly more pronounced antagonistic activity against Klebsiella spp. isolated from colonic contents of patients with diagnosed opisthorchiasis.