The Role of nmcR, ampR, and ampD in the Regulation of the Class A Carbapenemase NmcA in Enterobacter ludwigii

Various carbapenemases have been identified in the Enterobacteriaceae. However, the induction and corresponding regulator genes of carbapenemase NmcA has rarely been detected in the Enterobacter cloacae complex (ECC). The NmcA-positive isolate ECC NR1491 was first detected in Japan in 2013. It was characterized and its induction system elucidated by evaluating its associated regulator genes nmcR, ampD, and ampR. The isolate was highly resistant to all β-lactams except for third generation cephalosporins (3GC). Whole-genome analysis revealed that blaNmcA was located on a novel 29-kb putatively mobile element called EludIMEX-1 inserted into the chromosome. The inducibility of β-lactamase activity by various agents was evaluated. Cefoxitin was confirmed as a strong concentration-independent β-lactamase inducer. In contrast, carbapenems induced β-lactamase in a concentration-dependent manner. All selected 3GC-mutants harboring substitutions on ampD (as ampR and nmcR were unchanged) were highly resistant to 3GC. The ampD mutant strain NR3901 presented with a 700 × increase in β-lactamase activity with or without induction. Similar upregulation was also observed for ampC and nmcA. NR1491 (pKU412) was obtained by transforming the ampR mutant (135Asn) clone plasmid whose expression increased by ∼100×. Like NR3901, it was highly resistant to 3GC. Overexpression of ampC, rather than nmcA, may have accounted for the higher MIC in NR1491. The ampR mutant repressed nmcA despite induction and it remains unclear how it stimulates nmcA transcription via induction. Future experiments should analyze the roles of nmcR mutant strains.

“Endogenous Melatonin Concentration Along with the Expression of Mitochondrial Regulator Genes is Elevated by the Serum Shock Process in the U87-MG Cell Line”

According to the World Health Organization, glioblastoma, also known as the fourth grade in the development of astrocytoma, is a glial tumor limited to the central nervous system with a strong ability to invade the brain parenchyma. Melatonin can be generated outside of the pineal gland tissue, according to new research. Melatonin is produced by mitochondria independently but in concert with cell demands, and it plays an important function in cell cycle and metabolism regulation. As a result, we set out to investigate the association between cell metabolism and the serum shock-induced increase in endogenous melatonin, as well as the percentage of cell proliferation.Background: Melatonin can be produced in the mitochondria organelle of glioblastoma cells without the involvement of the pineal gland, according to new research. Regarding the physiological function of melatonin secreted by the pineal gland in the regulation of rhythmicity, the goal of this study was to see if the glioblastoma cell's melatonin production ability could be influenced using a typical serum shock technique established for cellular rhythm regulator.Material and methods: First, U87-MG glioblastoma cells were cultured in a DMEM medium containing 10% FBS and then cells were treated with a standard serum shock process (no FBS, 8h). The concentration of melatonin was measured using ELISA method in supernatant and cell extracts of Shock and control groups. The cell proliferation was measured by using BrdU staining and flow cytometry assessment. The gene expression levels of some mitochondria or circadian related genes including TFAM, BMAL1, PPARGC1A(PGC1-α), and DNM1L(DRP1) were measured, using qRT-PCR method.Results: In comparison to the control group, serum shock treated U87-MG glioblastoma cells had higher concentrations of cellular and released endogenous melatonin (two times). At the mRNA level, we discovered considerable upregulation of mitochondrial or circadian regulator genes (TFAM, BMAL1, PPARGC1A, and DNM1L); in the shock group compared to the control group (P <0.0002). Furthermore, although the percentage of proliferative cells (Brdu positive) was higher in the shock group, it was not statistically significant.Conclusion: The serum shock procedure has a significant impact on the U87-MG cell line's cellular activity. In terms of the study's findings, it's worth noting that an increase in endogenous melatonin concentration influences several signaling pathways within the U87-MG cell line, as seen by the increased expression of candidate genes.In light of the findings of this study, it's worth noting that further research into the role of endogenous melatonin and its effects on cancer cells is critical, and that comparing the results of normal and cancer cells can reveal the hotspots of the signaling pathways involved, which could facilitate in better understanding the biology of glioblastoma.

Integrative analysis of CRISPR screening data uncovers new opportunities for optimizing cancer immunotherapy

Background In recent years, the application of functional genetic immuno-oncology screens has showcased the striking ability to identify potential regulators engaged in tumor-immune interactions. Although these screens have yielded substantial data, few studies have attempted to systematically aggregate and analyze them. Methods In this study, a comprehensive data collection of tumor immunity-associated functional screens was performed. Large-scale genomic data sets were exploited to conduct integrative analyses. Results We identified 105 regulator genes that could mediate resistance or sensitivity to immune cell-induced tumor elimination. Further analysis identified MON2 as a novel immune-oncology target with considerable therapeutic potential. In addition, based on the 105 genes, a signature named CTIS (CRISPR screening-based tumor-intrinsic immune score) for predicting response to immune checkpoint blockade (ICB) and several immunomodulatory agents with the potential to augment the efficacy of ICB were also determined. Conclusion Overall, our findings provide insights into immune oncology and open up novel opportunities for improving the efficacy of current immunotherapy agents.

Expression of pluripotency-related genes in human glioblastoma

Background Cancer is a group of heterogeneous diseases characterized by several disruptions of the genetic and epigenetic components of cell biology. Some types of cancer have been shown to be constituted by a mosaic of cells with variable differentiation states, with more aggressive tumors being more undifferentiated. In most cases, undifferentiated tumor cells express associated embryonic markers such as the OCT4, NANOG, SOX2 and CARM1 genes. The ectopic or reminiscent expression of some master regulator genes of pluripotency has been indicated as the cause of the poorly differentiated state of tumors, and based on the evidence of some reports, can be used as a possible therapeutic target. Considering this information, a more detailed investigation of the expression of pluripotency-associated genes is necessary to evaluate the roles of these genes in the etiology of some tumors and their use targets of therapy. Methods The expression of four pluripotency-related genes was investigated (OCT4, NANOG, SOX2 and CARM1) in the most malignant primary human brain tumor, glioblastoma (GBM). Results and Conclusion The results demonstrated a signature of OCT4/SOX2/CARM1 genes and a significant increase of CARM1 expression in GBM cases.

Hippo Pathway Effector Tead1 Induces Cardiac Fibroblast to Cardiomyocyte Reprogramming

Background The conversion of fibroblasts into induced cardiomyocytes may regenerate myocardial tissue from cardiac scar through in situ cell transdifferentiation. The efficiency transdifferentiation is low, especially for human cells. We explored the leveraging of Hippo pathway intermediates to enhance induced cardiomyocyte generation. Methods and Results We screened Hippo effectors Yap (yes‐associated protein), Taz (transcriptional activator binding domain), and Tead1 (TEA domain transcription factor 1; Td) for their reprogramming efficacy with cardio‐differentiating factors Gata4, Mef2C, and Tbx5 (GMT). Td induced nearly 3‐fold increased expression of cardiomyocyte marker cTnT (cardiac troponin T) by mouse embryonic and adult rat fibroblasts versus GMT administration alone ( P <0.0001), while Yap and Taz failed to enhance cTnT expression. Serial substitution demonstrated that Td replacement of TBX5 induced the greatest cTnT expression enhancement and sarcomere organization in rat fibroblasts treated with all GMT substitutions (GMTd versus GMT: 17±1.2% versus 5.4±0.3%, P <0.0001). Cell contractility (beating) was seen in 6% of GMTd‐treated cells by 4 weeks after treatment, whereas no beating GMT‐treated cells were observed. Human cardiac fibroblasts likewise demonstrated increased cTnT expression with GMTd versus GMT treatment (7.5±0.3% versus 3.0±0.3%, P <0.01). Mechanistically, GMTd administration increased expression of the trimethylated lysine 4 of histone 3 (H3K4me3) mark at the promoter regions of cardio‐differentiation genes and mitochondrial biogenesis regulator genes in rat and human fibroblast, compared with GMT. Conclusions These data suggest that the Hippo pathway intermediate Tead1 is an important regulator of cardiac reprogramming that increases the efficiency of maturate induced cardiomyocytes generation and may be a vital component of human cardiodifferentiation strategies.

Empowering Engineered Muscle in Biohybrid Pump by Extending Connexin 43 Duration with Reduced Graphene Oxides

Engineered skeletal muscle act as therapeutics invaluable to treat injured or diseased muscle and a living material essential to assemble biological machinery. For normal development, skeletal myoblasts should express connexin 43, one of the gap junction proteins that promote myoblast fusion and myogenesis, during the early differentiation stage. However, myoblasts cultured in vitro often down-regulate connexin 43 before differentiation, limiting myogenesis and muscle contraction. This study demonstrates that tethering myoblasts with reduced graphene oxide (rGO) slows connexin 43 regression during early differentiation and increases myogenic mRNA synthesis. The whole RNA sequencing also confirms that the rGO on cells increases regulator genes for myogenesis, including troponin, while decreasing negative regulator genes. The resulting myotubes generated a three-fold larger contraction force than the rGO-free myotubes. Accordingly, a valveless biohybrid pump assembled with the rGO-tethered muscle increased the fluid velocity and flow rate considerably. The results of this study would provide an important foundation for developing physiologically relevant muscle and powering up biomachines that will be used for various bioscience studies and unexplored applications.

Dynamic genome plasticity during unisexual reproduction in the human fungal pathogen Cryptococcus deneoformans

Genome copy number variation occurs during each mitotic and meiotic cycle and it is crucial for organisms to maintain their natural ploidy. Defects in ploidy transitions can lead to chromosome instability, which is a hallmark of cancer. Ploidy in the haploid human fungal pathogen Cryptococcus neoformans is exquisitely orchestrated and ranges from haploid to polyploid during sexual development and under various environmental and host conditions. However, the mechanisms controlling these ploidy transitions are largely unknown. During C. deneoformans (formerly C. neoformans var. neoformans, serotype D) unisexual reproduction, ploidy increases prior to the onset of meiosis, can be independent from cell-cell fusion and nuclear fusion, and likely occurs through an endoreplication pathway. To elucidate the molecular mechanisms underlying this ploidy transition, we identified twenty cell cycle-regulating genes encoding cyclins, cyclin-dependent kinases (CDK), and CDK regulators. We characterized four cyclin genes and two CDK regulator genes that were differentially expressed during unisexual reproduction and contributed to diploidization. To detect ploidy transition events, we generated a ploidy reporter, called NURAT, which can detect copy number increases via double selection for nourseothricin-resistant, uracil-prototrophic cells. Utilizing this ploidy reporter, we showed that ploidy transition from haploid to diploid can be detected during the early phases of unisexual reproduction. Interestingly, selection for the NURAT reporter revealed several instances of segmental aneuploidy of multiple chromosomes, which conferred azole resistance in some isolates. These findings provide further evidence of ploidy plasticity in fungi with significant biological and public health implications.

Pan-Cancer Analyses of Pyroptosis With Functional Implications in Prognosis and Immunotherapy in Cancer

Background Programmed cell death is an active and orderly form of cell death regulated by intracellular genes, which plays an important role in the normal occurrence and development of the immune system, and pyroptosis has been found to be involved in the tumorigenesis and development. However, compressive analysis and biological regulation about pyroptosis genes are lack in cancers. Methods Using the data from the The Cancer Genome Atlas, we established a score level model to quantify the pyroptosis level of cancer. Multi-omics bioinformatical analyses was performed to detect pyroptosis-related molecular features and effect of pyroptosis on immunotherapy in cancer. Results In the present study, we performed a comprehensive analysis of pyroptosis and its regulator genes in cancers. Most pyroptosis genes were aberrantly expressed among different cancer types, which is contributed by the CAN frequency and differences of DNA methylation level in cancer. We established the modeling of the pyroptosis level and found that pyroptosis showed dual roles across cancers, while the pyroptosis levels were different in multiple and be significantly associated with clinical prognosis. The dual role of pyroptosis also affect the effects of immunotherapy in several cancers. Multiple pyroptosis genes showed close connections with drug sensitivity across cancers, and may be considered as therapy targets in cancer. Conclusions Our comprehensive analyses provide new insight into the functions of pyroptosis in the initiation, development, and progression and treatment across cancers, suggesting corresponding prognostic and therapeutic utility.

The over-expression of phasin and regulator genes promoting the synthesis of polyhydroxybutyrate in Cupriavidus necator H16 under non-stress conditions

Cupriavidus necator H16 is an ideal strain for polyhydroxybutyrate (PHB) production from CO 2 . Low-oxygen-stress can induce PHB synthesis in C. necator H16 while reducing bacterial growth under chemoautotrophic culture. The optimum growth and PHB synthesis of C. necator H16 cannot be achieved simultaneously, which restricts PHB production. The present study was initiated to address the issue through comparative transcriptome and gene function analysis. Firstly, the comparative transcriptome of C. necator H16 chemoautotrophically cultured under low-oxygen-stress and non-stress conditions was studied. Three types of transcription different genes were discovered: PHB enzymatic synthesis, PHB granulation, and regulators. Under low-oxygen-stress condition, acetoacetyl-CoA reductase gene phaB2 , PHB synthase gene phaC2 , phasins genes phaP1 and phaP2 , regulators genes uspA and rpoN were up-regulated 3.0, 2.5, 1.8, 2.7, 3.5, 1.6 folds, respectively. Secondly, the functions of up-regulated genes and their applications in PHB synthesis were further studied. It was found that the over-expression of phaP1 , phaP2 , uspA , and rpoN can induce PHB synthesis under non-stress condition, while phaB2 and phaC2 have no significant effect. Under the optimum condition, PHB percentage content in C. necator H16 was respectively increased by 37.2%, 28.4%, 15.8%, and 41.0% with the over-expression of phaP1 , phaP2 , uspA , and rpoN , and the corresponding PHB production increased by 49.8%, 42.9%, 47.0%, and 77.5% under non-stress chemoautotrophic conditions. Similar promotion by phaP1 , phaP2 , uspA , and rpoN was observed in heterotrophically cultured C. necator H16. The PHB percentage content and PHB production were respectively increased by 54.4% and 103.1% with the over-expression of rpoN under non-stress heterotrophic conditions. Importance Microbial fixation of CO 2 is an effective way to reduce greenhouse gases. Some microbes such as C. necator H16 usually accumulate PHB when they grow under stress. Low-oxygen-stress can induce PHB synthesis when C. necator H16 is autotrophically cultured with CO 2 , H 2 , and O 2 , while under stress, growth is restricted and total PHB yield is reduced. Achieving the optimal bacterial growth and PHB synthesis at the same time is an ideal condition for transforming CO 2 into PHB by C. necator H16. The present study was initiated to clarify the molecular basis of low-oxygen-stress promoting PHB accumulation and to realize the optimal PHB production by C. necator H16. Genes up-regulated under non-stress conditions were identified through comparative transcriptome analysis and over-expression of phasin and regulator genes were demonstrated to promote PHB synthesis in C. necator H16.

Heterogeneity of acetylcholine receptor autoantibody-mediated complement activity in patients with myasthenia gravis.

Background: Autoantibodies targeting the acetylcholine receptor (AChR) in the serum of myasthenia gravis (MG) patients are broadly polyclonal and heterogeneous in their pathogenic capacity. Specifically, AChR autoantibody-mediated pathology occurs through three mechanisms that include complement-directed tissue damage, blocking of the acetylcholine binding site on the AChR, and modulation (internalization) of the AChR. Clinical assays used for diagnosis and prognosis measure only AChR autoantibody binding and they provide weak association with disease burden, thereby limiting understanding of mechanistic heterogeneity, and monitoring therapeutic response. Objective: To develop an in-vitro cell-based assay that measures AChR autoantibody-mediated complement membrane attack complex (MAC) formation. Methods: A HEK293T cell line, which is commonly used for live cell-based AChR autoantibody binding assays, was modified such that the expression of the complement regulator genes (CD46, CD55 and CD59) were disrupted using CRISPR/Cas9 genome editing. This modified cell line was used to measure serum AChR autoantibody-mediated complement MAC formation via flow cytometry. Results: AChR autoantibody-mediated MAC formation required the use of a modified HEK293T cell line in which the surface expression of three complement regulator genes was absent. Serum samples (n=155) from 97 clinically confirmed AChR patients were tested along with 32 healthy donor (HD) samples; the MG cohort included a wide range of disease burden and AChR autoantibody titer. AChR autoantibodies were detected in 139 of the 155 (89.7%) AChR patient samples via a live cell-based assay. Of the 139 AChR positive samples, autoantibody-mediated MAC formation was detected in 83 (59.7%), while no autoantibodies or MAC formation was detected in samples from the HD group. Autoantibody-mediated MAC formation positively associated with autoantibody binding in most MG patient samples. However, a subset displayed a disassociation between binding and MAC formation. Conclusions: We demonstrate the development of a novel assay for evaluating AChR autoantibody-mediated complement activity. It is anticipated that this assay will afford a deeper understanding of the heterogeneous disease pathology and allow for the identification of MG patients who may benefit from complement inhibitor therapy.