Loss of OVOL2 in Triple-Negative Breast Cancer Promotes Fatty Acid Oxidation Fueling Stemness Characteristics.

Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer, has a poor prognosis and lacks effective treatment strategies. Here, the study discovered that TNBC shows a decreased expression of epithelial transcription factor ovo-like 2 (OVOL2). The loss of OVOL2 promotes fatty acid oxidation (FAO), providing additional energy and NADPH to sustain stemness characteristics, including sphere-forming capacity and tumor initiation. Mechanistically, OVOL2 not only suppressed STAT3 phosphorylation by directly inhibiting JAK transcription but also recruited histone deacetylase 1 (HDAC1) to STAT3, thereby reducing the transcriptional activation of downstream genes carnitine palmitoyltransferase1 (CPT1A and CPT1B). PyVT-Ovol2 knockout mice develop a higher number of primary breast tumors with accelerated growth and increased lung-metastases. Furthermore, treatment with FAO inhibitors effectively reduces stemness characteristics of tumor cells, breast tumor initiation, and metastasis, especially in OVOL2-deficient breast tumors. The findings suggest that targeting JAK/STAT3 pathway and FAO is a promising therapeutic strategy for OVOL2-deficient TNBC.

Rapid and sensitive detection of nucleic acids using an RAA-CRISPR/Cas12b one-pot detection assay (Rcod).

Rapid, sensitive and specific methods are crucial for nucleic acid detection. CRISPR/Cas12b has recently been widely used in nucleic acid detection. However, due to its thermophagic property, DNA isothermal recombinase-aided amplification (RAA) and subsequent CRISPR/Cas12b detection require two separate reactions, which is cumbersome and inconvenient and may cause aerosol pollution. In this study, we propose an RAA-CRISPR/Cas12b one-pot detection assay (Rcod) for Bordetella pertussis detection without additional amplification product transfer steps. The time from sample processing to response time was less than 30 min using nucleic acid extraction-free method, and the sensitivity reached 0.2 copies/µL. In this system, Alicyclobacillus acidoterrestris Cas12b protein (AacCas12b) exhibited strong and specific trans-cleavage activity at a constant temperature of 37 °C, while the cis-cleavage activity was weak. This characteristic reduces the interference of AacCas12b with nucleic acids in the system. Compared with real-time PCR, our Rcod system detected B. pertussis in 221 clinical samples with a sensitivity and specificity of 97.96 % and 99.19 %, respectively, with nucleic acid extraction-free method. The rapid, sensitive and specific Rcod system provides ideas for the establishment of CRISPR-based one-step nucleic acid detection and may aid the development of reliable point-of-care nucleic acid tests. IMPORTANCE: Pertussis is an acute respiratory infection caused by B. pertussis that is highly contagious and potentially fatal, and early diagnosis is essential for the treatment of whooping cough. In this study, we found that AacCas12b has high and strongly specific trans-cleavage activity at lower temperatures. A RAA-CRISPR/Cas12b one-step detection platform (Rcod) without interference with amplification was developed. In addition, the combination of Rcod and nucleic acid extraction-free method can quickly and accurately detect the qualitative detection of B. pertussis, and the detection results are visualized, which makes the pathogen nucleic acid detection and analysis process simpler, and provides a new method for the rapid clinical diagnosis of B. pertussis.

Pirin Inhibits FAS-Mediated Apoptosis to Support Colorectal Cancer Survival.

Resistance to immunotherapy in colorectal cancer (CRC) is associated with obstruction of FAS (Apo-1 or CD95)-dependent apoptosis, a hallmark of cancer. Here it is demonstrated that the upregulation of pirin (PIR) protein in colon cancers promotes tumorigenesis. Knockout or inhibition of PIR dramatically increases FAS expression, FAS-dependent apoptosis and attenuates colorectal tumor formation in mice. Specifically, NFκB2 is a direct transcriptional activator of FAS and robustly suppressed by PIR in dual mechanisms. One is the disruption of NFκB2 complex (p52-RELB) association with FAS promoter, the other is the inhibition of NIK-mediated NFκB2 activation and nuclear translocation, leading to the inability of active NFκB2 complex toward the transcription of FAS. Furthermore, PIR interacts with FAS and recruits it in cytosol, preventing its membrane translocation and assembling. Importantly, knockdown or knockout of PIR dramatically sensitizes cells to FAS mAb- or active CD8+ T cells-triggered cell death. Taken together, a PIR-NIK-NFκB2-FAS survival pathway is established, which plays a key role in supporting CRC survival.

TET2 is recruited by CREB to promote Cebpb, Cebpa, and Pparg transcription by facilitating hydroxymethylation during adipocyte differentiation.

Ten-eleven translocation proteins (TETs) are dioxygenases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an important epigenetic mark that regulates gene expression during development and differentiation. Here, we found that the TET2 expression was positively associated with adipogenesis. Further, in vitro and in vivo experiments showed that TET2 deficiency blocked adipogenesis by inhibiting the expression of the key transcription factors CCAAT/enhancer-binding protein beta (C/EBPß), C/EBPα and peroxisome proliferator-activated receptor gamma (PPARγ). In addition, TET2 promoted 5hmC on the CpG islands (CGIs) of Cebpb, Cebpa and Pparg at the initial time point of their transcription, which requires the cAMP-responsive element-binding protein (CREB). At last, specific knockout of Tet2 in preadipocytes enabled mice to resist obesity and attenuated the obesity-associated insulin resistance. Together, TET2 is recruited by CREB to promote the expression of Cebpb, Cebpa and Pparg via 5hmC during adipogenesis and may be a potential therapeutic target for obesity and insulin resistance.

An extraction-free method for rapid detection of CYP2C19 * 2/3/17 polymorphisms in one tube using melting curve analysis.

Drug-metabolizing enzymes play an important role in the metabolism of drugs in vivo. Their activity is an important factor affecting the rate of drug metabolism, which directly determines the intensity and persistence of drug action. Patients taking medication can be divided into different metabolic types through detection of CYP2C19 drug-metabolizing enzyme gene polymorphisms, which can then be used for medication guidance for clopidogrel. Here, we describe a detection method based on real-time polymerase chain reaction (PCR). This method uses multicolor melting curve analysis to accurately identify different mutation sites and genotypes of CYP2C19 * 2, CYP2C19 * 3, and CYP2C19 * 17. The detection limit of plasmid samples was 1 copies µL-1 ; that of genomic samples was 0.1 ng µL-1 . The system can detect nine types of CYP2C19 * 2/3/17 at three sites in one tube, quickly achieving detection within 1 h. Combined with the sample release agent, sample extraction was completed in 5 s, achieving rapid diagnosis without extraction for timely diagnosis and treatment. Furthermore, the system is not limited to blood samples and can also be applied to oropharyngeal and saliva samples, increasing sampling diversity and convenience. When using clinical blood samples (n = 93), the detection system we established was able to quickly and accurately identify different genotypes, and the accuracy and effectiveness of the detection were confirmed by Sanger sequencing. Due to its accuracy, rapidity, simple operation, and low cost, detection technology based on real-time polymerase amplification combined with melting curve analysis is expected to become a powerful tool for detecting and guiding clopidogrel use in countries with limited resources.

Acinetobacter baumannii adaptation to the host pH microenvironment is mediated by allelic variation in a single residue of BauA protein.

Acinetobacter baumannii has been listed as one of the most critical pathogens in nosocomial infections; however, the key genes and mechanisms to adapt to the host microenvironment lack in-depth understanding. In this study, a total of 76 isolates (from 8 to 12 isolates per patient, spanning 128 to 188 days) were longitudinally collected from eight patients to investigate the within-host evolution of A. baumannii. A total of 70 within-host mutations were identified, 80% of which were nonsynonymous, indicating the important role of positive selection. Several evolutionary strategies of A. baumannii to increase its potential to adapt to the host microenvironment were identified, including hypermutation and recombination. Six genes were mutated in isolates from two or more patients, including two TonB-dependent receptor genes (bauA and BJAB07104_RS00665). In particular, the siderophore receptor gene bauA was mutated in multiple isolates from four patients with three MLST types, and all mutations were at amino acid 391 in ligand-binding sites. With 391T or 391A, BauA was more strongly bound to siderophores, which promoted the iron-absorption activity of A. baumannii at acidic or neutral pH, respectively. Through the A/T mutation at site 391 of BauA, A. baumannii displayed two reversible phases to adapt to distinct pH microenvironments. In conclusion, we demonstrated the comprehensive within-host evolutionary dynamics of A. baumannii, and discovered a key mutation of BauA site 391 as a genetic switch to adapt to different pH values, which may represent a model in the pathogen evolutionary adaption of the host microenvironment.

Suppression of preadipocyte determination by SOX4 limits white adipocyte hyperplasia in obesity.

Preadipocyte determination expanding the pool of preadipocytes is a vital process in adipocyte hyperplasia, but the molecular mechanisms underlying this process are yet to be elucidated. Herein, SRY-related HMG box transcription factor 4 (SOX4) was identified as a critical target in response to BMP4- and TGFß-regulated preadipocyte determination. SOX4 deficiency is sufficient to promote preadipocyte determination in mesenchymal stem cells (MSCs) and acquisition of preadipocyte properties in nonadipogenic lineages, while its overexpression impairs the adipogenic capacity of preadipocytes and converts them into nonadipogenic lineages. Mechanism studies indicated that SOX4 activates and cooperates with LEF1 to retain the nuclear localization of ß-catenin, thus mediating the crosstalk between TGFß/BMP4 signaling pathway and Wnt signaling pathway to regulate the preadipocyte determination. In vivo studies demonstrated that SOX4 promotes the adipogenic-nonadipogenic conversion and suppresses the adipocyte hyperplasia. Together, our findings highlight the importance of SOX4 in regulating the adipocyte hyperplasia in obesity.

Fast and visual detection of nucleic acids using a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM.

CRISPR-Cas12a (Cpf1) is widely used for pathogen detection. However, most Cas12a nucleic acid detection methods are limited by a PAM sequence requirement. Moreover, preamplification and Cas12a cleavage are separate. Here, we developed a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM sequence with high sensitivity and specificity that offers one-tube, rapid, and visually observable detection of nucleic acids. In this system, Cas12a detection and RPA amplification are performed simultaneously, without separate preamplification and product transfer steps, and 0.2 copies/µL of DNA and 0.4 copies/µL of RNA can be detected. In the ORCD system, the activity of Cas12a is the key to the nucleic acid detection; specifically, reducing Cas12a activity increases the sensitivity of ORCD assay detection of the PAM target. Furthermore, by combining this detection technique with a nucleic acid extraction-free method, our ORCD system can be used to extract, amplify and detect samples within 30 min, as verified with tests of 82 Bordetella pertussis clinical samples with a sensitivity and specificity of 97.30% and 100% compared with PCR. We also tested 13 SARS-CoV-2 samples with RT-ORCD, and the results were consistent with RT-PCR.

TPX2 enhances the transcription factor activation of PXR and enhances the resistance of hepatocellular carcinoma cells to antitumor drugs.

The pregnane X receptor (PXR) is an important regulator of hepatocellular carcinoma cellular resistance to antitumor drugs. Activation of PXR was modulated by the co-regulators. The target protein for the Xenopus plus end-directed kinesin-like protein (Xklp2) known as TPX2 that was previously considered as a tubulin regulator, also functions as the regulator of some transcription factors and pro-oncogenes in human malignances. However, the actions of TPX2 on PXR and HCC cells are still unclear. In the present study, our results demonstrate that the high expression of endogenous mRNA level of TPX2 not only correlated with the poor prognosis of advanced HCC patients who received sorafenib treatment but also with expression of PXR's downstream genes, cyp3a4 and/or mdr-1. Results from luciferase and real-time polymerase chain reaction (qPCR) showed that TPX2 leads to enhancement of the transcription factor activation of PXR. Protein-protein interactions between PXR and TPX2 were identified using co-immunoprecipitation. Mechanically, overexpression of TPX2 led to enhancement of PXR recruitment to its downstream gene cyp3a4's promoter region (the PXRE region) or enhancer region (the XREM region). Treatment of HCC cells with paclitaxel, a microtubule promoter, led to enhancement of the effects of TPX2, whereas vincristine, a microtubule depolymerizing agent caused a decrease in TPX2-associated effects. TPX2 was found to cause acceleration of the metabolism or clearance of sorafenib, a typical tyrosine kinase inhibitor (TKI) in HCC cells and in turn led to the resistance to sorafenib by HCC cells. By establishing novel actions of TXP2 on PXR in HCC cells, the results indicate that TPX2 could be considered a promising therapeutic target to enhance HCC cells sensitivity to antitumor drugs.

SOX4 promotes beige adipocyte-mediated adaptive thermogenesis by facilitating PRDM16-PPARγ complex.

Brown and beige fat protect against cold environments and obesity by catabolizing stored energy to generate heat. This process is achieved by controlling thermogenesis-related gene expression and the development of brown/beige fat through the induction of transcription factors, most notably PPARγ. However, the cofactors that induce the expression of thermogenic genes with PPARγ are still not well understood. In this study, we explored the role of SOX4 in adaptive thermogenesis and its relationship with PPARγ. Methods: Whole transcriptome deep sequencing (RNA-seq) analysis of inguinal subcutaneous white adipose tissue (iWAT) after cold stimulation was performed to identify genes with differential expression in mice. Indirect calorimetry detected oxygen consumption rate and heat generation. mRNA levels were analyzed by qPCR assays. Proteins were detected by immunoblotting and immunofluorescence. Interaction of proteins was detected by endogenous and exogenous Co-IP. ChIP-qPCR, FAIRE assay and luciferase reporter assays were used to investigate transcriptional regulation. Results: SOX4 was identified as the main transcriptional effector of thermogenesis. Mice with either adipocyte-specific or UCP1+ cells deletion of SOX4 exhibited significant cold intolerance, decreased energy expenditure, and beige adipocyte formation, which was attributed to decreased thermogenic gene expression. In addition, these mice developed obesity on a high-fat diet, with severe hepatic steatosis, insulin resistance, and inflammation. At the cell level, loss of SOX4 from preadipocytes inhibited the development of beige adipocytes, and loss of SOX4 from mature beige adipocytes reduced the expression of thermogenesis-related genes and energy metabolism. Mechanistically, SOX4 stimulated the transcriptional activity of Ucp1 by binding to PPARγ and activating its transcriptional function. These actions of SOX4 were, at least partly, mediated by recruiting PRDM16 to PPARγ, thus forming a transcriptional complex to elevate the expression of thermogenic genes. Conclusion: SOX4, as a coactivator of PPARγ, drives the thermogenic gene expression program and thermogenesis of beige fat, promoting energy expenditure. It has important physiological significance in resisting cold and obesity.

Loss of Mst1/2 activity promotes non-mitotic hair cell generation in the neonatal organ of Corti.

Mammalian sensory hair cells (HCs) have limited capacity for regeneration, which leads to permanent hearing loss after HC death. Here, we used in vitro RNA-sequencing to show that the Hippo signaling pathway is involved in HC damage and self-repair processes. Turning off Hippo signaling through Mst1/2 inhibition or Yap overexpression induces YAP nuclear accumulation, especially in supporting cells, which induces supernumerary HC production and HC regeneration after injury. Mechanistically, these effects of Hippo signaling work synergistically with the Notch pathway. Importantly, the supernumerary HCs not only express HC markers, but also have cilia structures that are able to form neural connections to auditory regions in vivo. Taken together, regulating Hippo suggests new strategies for promoting cochlear supporting cell proliferation, HC regeneration, and reconnection with neurons in mammals.

MTBP enhances the activation of transcription factor ETS-1 and promotes the proliferation of hepatocellular carcinoma cells.

Increasing evidence indicates that the oncoprotein murine double minute (MDM2) binding protein (MTBP) can be considered a pro-oncogene of human malignancies; however, its function and mechanisms in hepatocellular carcinoma (HCC) are still not clear. In the present work, our results demonstrate that MTBP could function as a co-activator of transcription factor E26 transformation-specific sequence (ETS-1), which plays an important role in HCC cell proliferation and/or metastasis and promotes proliferation of HCC cells. Using luciferase and real-time polymerase chain reaction (qPCR) assays, MTBP was found to enhance the transcription factor activation of ETS-1. The results from chromatin co-immunoprecipitation showed that MTBP enhanced the recruitment of ETS-1 to its downstream gene's (mmp1's) promoter region with ETS-1 binding sites. In cellular and nude mice models, overexpression of MTBP was shown to promote the proliferation of MHCC97-L cells with low endogenous MTBP levels, whereas the knockdown of MTBP led to inhibition of the proliferation of MHCC97-H cells that possessed high endogenous levels of MTBP. The effect of MTBP on ETS-1 was confirmed in the clinical specimens; the expression of MTBP was positively correlated with the downstream genes of ETS-1, mmp3, mmp9, and uPA. Therefore, by establishing the role of MTBP as a novel co-activator of ETS-1, this work expands our knowledge of MTBP or ETS-1 and helps to provide new ideas concerning HCC-related research.

[Construction and application of lentivirus overexpression vector with two labeling genes fused with CopGFP and PuroR].

Objective: To construct the lentivirus overexpression vector with two label genes fused with CopGFP and PuroR and to detect the emission of green fluorescence as well as resistance to puromycin in liver cancer cells infected with lentivirus packaged with the above vector. Methods: Firstly, two fragments containing copGFP and PuroR coding sequences were amplified from pCDH-CMV-MCS-copGFP and pLKO.1 respectively; secondly, the two amplified regions were fused with each other by recombinant PCR; thirdly, the fusion DNA fragment was cut and inserted into pCDH-CMV-MCS-copGFP vector, which was linearized with the same restriction endonuclease as used to digest fusion DNA fragment: BamH Ⅰ and Sal Ⅰ. The fusion region in the constructed vector was confirmed by DNA sequencing. The checked vector was co-transfected with package assistant plasmids, namely PLP1, PLP2 and VSVG into in 293T cells and the culture supernatant was subjected to centrifuge and infect liver cancer MHCC97H cells, which were then used to detect their resistance to puromycin (infected cells were treated with 1 mg/ml puromycin for 7 days after infection) and to observe green fluorescence emission in microscope. To determine its efficiency in expressing foreign target protein, the Sp1 coding region was inserted into the MCS sites of the vector, and Sp1 mRNA and protein expression levels were compared with the vehicle vector by RT-qPCR and Western blot. Results: The lentivirus overexpression vector with two label genes fused with CopGFP and PuroR was successfully constructed, and the liver cancer cells infected with lentivirus packaged with the vector expressing two labeling genes fused with CopGFP and PuroRshowed both emission of green fluorescence and resistance to puromycin simultaneously, while cells containing with the vector inserted with Sp1 coding region improved Sp1 mRNA level with 3.3 fold and protein level with 2.2 fold higher in comparison with cells containing the vehicle vector (P＜0.01). Conclusion: The fused label genes consisting of copGFP and PuroR are correctly cloned into the lentivirus vector and confer cells with the ability to emission of green fluorescence and resistance to puromycin, besides, the vector may promote the expression of the target gene with long coding sequence.

A Single Nucleotide Mixture Enhances the Antitumor Activity of Molecular-Targeted Drugs Against Hepatocellular Carcinoma.

New strategies for molecular-targeted drug therapy for advanced hepatocellular carcinoma (HCC) ignore the contribution of the nutritional status of patients and nutritional support to improve physical status and immunity. We aimed to elucidate the role of a single nucleotide mixture (SNM) in the anti-tumor therapy of HCC, and to explore the importance of a SNM as adjuvant therapy for HCC. Compared with a lipid emulsion (commonly used nutritional supplement for HCC patients), the SNM could not induce metabolic abnormalities in HCC cells (Warburg effect), and did not affect expression of metabolic abnormality-related factors in HCC cells. The SNM could also attenuate the lymphocyte injury induced by antitumor drugs in vitro and in vivo, and promote the recruitment and survival of lymphocytes in HCC tissues. Using HCC models in SCID (server combined immune-deficiency) mice or BalB/c mice, the SNM had anti-tumor activity, and could significantly upregulate the antitumor activity of molecular-targeted drugs (tyrosine-kinase inhibitors [TKI] and immune-checkpoint inhibitors [ICI]) against HCC. We employed research models in vivo and in vitro to reveal the anti-tumor activity of the SNM on HCC. Our findings expand understanding of the SNM and contribute to HCC (especially nutritional support) therapy.

Genetic Characterization of Four Groups of Chromosome-Borne Accessory Genetic Elements Carrying Drug Resistance Genes in Providencia.

Purpose: The aim of this study was to gain a deeper genomics and bioinformatics understanding of diversification of accessory genetic elements (AGEs) in Providencia. Methods: Herein, the complete genome sequences of five Providencia isolates from China were determined, and seven AGEs were identified from the chromosomes. Detailed genetic dissection and sequence comparison were applied to these seven AGEs, together with additional 10 chromosomal ones from GenBank (nine of them came from Providencia). Results: These 17 AGEs were divided into four groups: Tn6512 and its six derivatives, Tn6872 and its two derivatives, Tn6875 and its one derivative, and Tn7 and its four derivatives. These AGEs display high-level diversification in modular structures that had complex mosaic natures, and particularly different multidrug resistance (MDR) regions were presented in these AGEs. At least 52 drug resistance genes, involved in resistance to 15 different categories of antimicrobials and heavy metal, were found in 15 of these 17 AGEs. Conclusion: Integration of these AGEs into the Providencia chromosomes would contribute to the accumulation and distribution of drug resistance genes and enhance the ability of Providencia isolates to survive under drug selection pressure.

Longitudinal and proteome-wide analyses of antibodies in COVID-19 patients reveal features of the humoral immune response to SARS-CoV-2.

Introduction: The SARS-CoV-2 pandemic has endangered global health, the world economy, and societal values. Despite intensive measures taken around the world, morbidity and mortality remain high as many countries face new waves of infection and the spread of new variants. Worryingly, more and more variants are now being identified, such as 501Y.V1 (B.1.1.7) in the UK, 501Y.V2 (B.1.351) in South Africa, 501Y.V3 in Manaus, Brazil, and B.1.617/B.1.618 in India, which could lead to a severe epidemic rebound. Moreover, some variants have a stronger immune escape ability. To control the new SARS-CoV-2 variant, we may need to develop and redesign new vaccines repeatedly. So it is important to investigate how our immune system combats and responds to SARS-CoV-2 infection to develop safe and effective medical interventions. Objectives: In this study, we performed a longitudinal and proteome-wide analysis of antibodies in the COVID-19 patients to revealed some immune processes of COVID-19 patients against SARS-CoV-2 and found some dominant epitopes of a potential vaccine. Methods: Microarray assay, Antibody depletion assays, Neutralization assay. Results: We profiled a B-cell linear epitope landscape of SARS-CoV-2 and identified the epitopes specifically recognized by either IgM, IgG, or IgA. We found that epitopes more frequently recognized by IgM are enriched in non-structural proteins. We further identified epitopes with different immune responses in severe and mild patients. Moreover, we identified 12 dominant epitopes eliciting antibodies in most COVID-19 patients and identified five key amino acids of epitopes. Furthermore, we found epitope S-82 and S-15 are perfect immunogenic peptides and should be considered in vaccine design. Conclusion: This data provide useful information and rich resources for improving our understanding of viral infection and developing a novel vaccine/neutralizing antibodies for the treatment of SARS-CoV-2.

An NF-κB/OVOL2 circuit regulates glucose import and cell survival in non-small cell lung cancer.

BACKGROUND: Tumor cells tend to utilize glycolysis rather than aerobic respiration even under aerobic conditions. OVOL2, an inhibitory C2H2 zinc finger transcription factor, is a potential tumor suppressor in cancers. However, the association between OVOL2 and tumor energy metabolism is unknown. METHODS: Western blotting was used to determine the expression of OVOL2 in different non-small cell lung cancer (NSCLC) cell lines and mouse models. The metabolic parameters in NSCLC cells following overexpression or knockdown OVOL2 were examined. To define the mechanism by which OVOL2 regulates aerobic glycolysis, interacting protein of OVOl2 and downstream molecular events were identified by luciferase assay and co-immunoprecipitation. We documented the regulatory mechanism in mouse xenograft models. Finally, clinical relevance of OVOL2, NF-κB signaling and GLUT1 was measured by immunostaining. RESULTS: OVOL2 is downregulated in NSCLC and overexpression of OVOL2 inhibits the survival of cancer cells. Moreover, OVOL2 directly binds to P65 and inhibits the recruitment of P300 but facilitates the binding of HDAC1 to P65, which in turn negatively regulates NF-κB signaling to suppress GLUT1 translocation and glucose import. In contrast, OVOL2 expression is negatively regulated by NF-κB signaling in NSCLC cells via the ubiquitin-proteasome pathway. Re-expression of OVOL2 significantly compromise NF-κB signaling-induced GLUT1 translocation, aerobic glycolysis in NSCLC cells and mouse models. Immunostaining revealed inverse correlations between the OVOL2 and phosphorylated P65 levels and between the OVOL2 and membrane GLUT1 levels, and a strong correlation between the phosphorylated P65 and membrane GLUT1 levels. CONCLUSIONS: These results suggest a regulatory circuit linking NF-κB and OVOL2, which highlights the role of NF-κB signaling and OVOL2 in the modulation of glucose metabolism in NSCLC. Video Abstract.

An automated nucleic acid detection platform using digital microfluidics with an optimized Cas12a system.

Outbreaks of both influenza virus and the novel coronavirus SARS-CoV-2 are serious threats to human health and life. It is very important to establish a rapid, accurate test with large-scale detection potential to prevent the further spread of the epidemic. An optimized RPA-Cas12a-based platform combined with digital microfluidics (DMF), the RCD platform, was established to achieve the automated, rapid detection of influenza viruses and SARS-CoV-2. The probe in the RPA-Cas12a system was optimized to produce maximal fluorescence to increase the amplification signal. The reaction droplets in the platform were all at the microliter level and the detection could be accomplished within 30 min due to the effective mixing of droplets by digital microfluidic technology. The whole process from amplification to recognition is completed in the chip, which reduces the risk of aerosol contamination. One chip can contain multiple detection reaction areas, offering the potential for customized detection. The RCD platform demonstrated a high level of sensitivity, specificity (no false positives or negatives), speed (≤30 min), automation and multiplexing. We also used the RCD platform to detect nucleic acids from influenza patients and COVID-19 patients. The results were consistent with the findings of qPCR. The RCD platform is a one-step, rapid, highly sensitive and specific method with the advantages of digital microfluidic technology, which circumvents the shortcomings of manual operation. The development of the RCD platform provides potential for the isothermal automatic detection of nucleic acids during epidemics. Electronic Supplementary Material: Supplementary material is available in the online version of this article at 10.1007/s11426-021-1169-1.

Clinical and genomic analysis of hypermucoviscous Klebsiella pneumoniae isolates: Identification of new hypermucoviscosity associated genes.

Introduction: Hypermucoviscous Klebsiella pneumoniae (HmKp) poses an emerging and highly pathogenic global health threat. This study aimed to investigate the clinical and genomic characteristics of HmKp isolates to better understand the virulence mechanisms of the hypermucoviscous (HMV) phenotype. Methods: From May 2018 to August 2021, 203 non-repeat K. pneumoniae isolates causing invasive infections were collected from a hospital in Beijing, China. Isolates were divided into HmKp (n=90, 44.3%) and non-HmKp (n=113, 55.7%) groups according to string test results. Results: Multivariate regression showed that diabetes mellitus (odds ratio [OR]=2.20, 95% confidence interval (CI): 1.20-4.05, p=0.010) and liver abscess (OR=2.93, CI 95%:1.29-7.03, p=0.012) were associated with HmKp infections. K. pneumoniae was highly diverse, comprising 87 sequence types (STs) and 54 serotypes. Among HmKp isolates, ST23 was the most frequent ST (25/90, 27.8%), and the most prevalent serotypes were KL2 (31/90, 34.4%) and KL1 (27/90, 30.0%). Thirteen virulence genes were located on the capsular polysaccharide synthesis region of KL1 strains. HmKp isolates were sensitive to multiple antibiotics but carried more SHV-type extended spectrum ß-lactamase (ESBL) resistance genes (p<0.05), suggesting that the emergence of ESBL-mediated multidrug resistance in HmKp should be monitored carefully during treatment. Phylogenetic analysis disclosed that HmKp isolates were highly diverse. Comparative genomic analysis confirmed that the HMV phenotype is a plasmid-encoded virulence factor. Seventeen HmKp genes were highly associated with HmKp, and included rmpAC, 7 iron-acquisition-related genes, and pagO, which may promote liver abscess formation. Discussion: This investigation provides insight into the mechanisms producing the HMV phenotype.

Longitudinal Metabolomics Reveals Ornithine Cycle Dysregulation Correlates With Inflammation and Coagulation in COVID-19 Severe Patients.

COVID-19 is a severe disease in humans, as highlighted by the current global pandemic. Several studies about the metabolome of COVID-19 patients have revealed metabolic disorders and some potential diagnostic markers during disease progression. However, the longitudinal changes of metabolomics in COVID-19 patients, especially their association with disease progression, are still unclear. Here, we systematically analyzed the dynamic changes of the serum metabolome of COVID-19 patients, demonstrating that most of the metabolites did not recover by 1-3 days before discharge. A prominent signature in COVID-19 patients comprised metabolites of amino acids, peptides, and analogs, involving nine essential amino acids, 10 dipeptides, and four N-acetylated amino acids. The levels of 12 metabolites in amino acid metabolism, especially three metabolites of the ornithine cycle, were significantly higher in severe patients than in mild ones, mainly on days 1-3 or 4-6 since onset. Integrating blood metabolomic, biochemical, and cytokine data, we uncovered a highly correlated network, including 6 cytokines, 13 biochemical parameters, and 49 metabolites. Significantly, five ornithine cycle-related metabolites (ornithine, N-acetylornithine, 3-amino-2-piperidone, aspartic acid, and asparagine) highly correlated with "cytokine storms" and coagulation index. We discovered that the ornithine cycle dysregulation significantly correlated with inflammation and coagulation in severe patients, which may be a potential mechanism of COVID-19 pathogenicity. Our study provided a valuable resource for detailed exploration of metabolic factors in COVID-19 patients, guiding metabolic recovery, understanding the pathogenic mechanisms, and creating drugs against SARS-CoV-2 infection.