Targeting lactate dehydrogenase B-dependent mitochondrial metabolism affects tumor initiating cells and inhibits tumorigenesis of non-small cell lung cancer by inducing mtDNA damage.

Once considered a waste product of anaerobic cellular metabolism, lactate has been identified as a critical regulator of tumorigenesis, maintenance, and progression. The putative primary function of lactate dehydrogenase B (LDHB) is to catalyze the conversion of lactate to pyruvate; however, its role in regulating metabolism during tumorigenesis is largely unknown. To determine whether LDHB plays a pivotal role in tumorigenesis, we performed 2D and 3D in vitro experiments, utilized a conventional xenograft tumor model, and developed a novel genetically engineered mouse model (GEMM) of non-small cell lung cancer (NSCLC), in which we combined an LDHB deletion allele with an inducible model of lung adenocarcinoma driven by the concomitant loss of p53 (also known as Trp53) and expression of oncogenic KRAS (G12D) (KP). Here, we show that epithelial-like, tumor-initiating NSCLC cells feature oxidative phosphorylation (OXPHOS) phenotype that is regulated by LDHB-mediated lactate metabolism. We show that silencing of LDHB induces persistent mitochondrial DNA damage, decreases mitochondrial respiratory complex activity and OXPHOS, resulting in reduced levels of mitochondria-dependent metabolites, e.g., TCA intermediates, amino acids, and nucleotides. Inhibition of LDHB dramatically reduced the survival of tumor-initiating cells and sphere formation in vitro, which can be partially restored by nucleotide supplementation. In addition, LDHB silencing reduced tumor initiation and growth of xenograft tumors. Furthermore, we report for the first time that homozygous deletion of LDHB significantly reduced lung tumorigenesis upon the concomitant loss of Tp53 and expression of oncogenic KRAS without considerably affecting the animal's health status, thereby identifying LDHB as a potential target for NSCLC therapy. In conclusion, our study shows for the first time that LDHB is essential for the maintenance of mitochondrial metabolism, especially nucleotide metabolism, demonstrating that LDHB is crucial for the survival and proliferation of NSCLC tumor-initiating cells and tumorigenesis.

Schedule-Dependent Treatment Increases Chemotherapy Efficacy in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare but aggressive thoracic malignancy with limited treatment options. One of the standard treatments for MPM is chemotherapy, which consists of concurrent treatment with pemetrexed and cisplatin. Pemetrexed limits tumor growth by inhibiting critical metabolic enzymes involved in nucleotide synthesis. Cisplatin causes direct DNA damage, such as intra-strand and inter-strand cross-links, which are repaired by the nucleotide excision repair pathway, which depends on relatively high nucleotide levels. We hypothesized that prolonged pretreatment with pemetrexed might deplete nucleotide pools, thereby sensitizing cancer cells to subsequent cisplatin treatment. The MPM cell lines ACC-MESO-1 and NCI-H28 were treated for 72 h with pemetrexed. Three treatment schedules were evaluated by initiating 24 h of cisplatin treatment at 0 h (concomitant), 24 h, and 48 h relative to pemetrexed treatment, resulting in either concomitant administration or pemetrexed pretreatment for 24 h or 48 h, respectively. Multicolor flow cytometry was performed to detect γH2AX (phosphorylation of histone H2AX), a surrogate marker for the activation of the DNA damage response pathway. DAPI staining of DNA was used to analyze cell cycle distribution. Forward and side scatter intensity was used to distinguish subpopulations based on cellular size and granularity, respectively. Our study revealed that prolonged pemetrexed pretreatment for 48 h prior to cisplatin significantly reduced long-term cell growth. Specifically, pretreatment for 48 h with pemetrexed induced a cell cycle arrest, mainly in the G2/M phase, accumulation of persistent DNA damage, and induction of a senescence phenotype. The present study demonstrates that optimizing the treatment schedule by pretreatment with pemetrexed increases the efficacy of the pemetrexed-cisplatin combination therapy in MPM. We show that the observed benefits are associated with the persistence of treatment-induced DNA damage. Our study suggests that an adjustment of the treatment schedule could improve the efficacy of the standard chemotherapy regimen for MPM and might improve patient outcomes.

Morphological factors associated with giant vacuoles with I-pores in Schlemm's canal endothelial cells of human eyes: A serial block-face scanning electron microscopy study.

Increased intraocular pressure (IOP) is the main risk factor for primary open-angle glaucoma and results from impaired drainage of aqueous humor (AH) through the trabecular outflow pathway. AH must pass the inner wall (IW) endothelium of Schlemm's canal (SC), which is a monolayer held together by tight junctions, to exit the eye. One route across the IW is through giant vacuoles (GVs) with their basal openings and intracellular pores (I-pores). AH drainage through the trabecular outflow pathway is segmental. Whether more GVs with both basal openings and I-pores are present in the active flow areas and factors that may influence formation of GVs with I-pores have not been fully elucidated due to limitations in imaging methods. In this study, we applied a relatively new technique, serial block-face scanning electron microscopy (SBF-SEM), to investigate morphological factors associated with GVs with I-pores in different flow areas. Two normal human donor eyes were perfused at 15 mmHg with fluorescent tracers to label the outflow pattern followed by perfusion-fixation. Six radial wedges of trabecular meshwork including SC (2 each from high-, low-, and non-flow areas) were imaged using SBF-SEM (total: 9802 images). Total GVs, I-pores, basal openings, and four types of GVs were identified. Percentages of GVs with I-pores and basal openings and number of I-pores/GV were determined. Overall, 14.4% (477/3302) of GVs had I-pores. Overall percentage of GVs with both I-pores and basal openings was higher in high- (15.7%), than low- (12.6%) or non-flow (7.3%) areas. Of GVs with I-pores, 83.2% had a single I-pore; 16.8% had multiple I-pores (range: 2-6). Additionally, 180 GVs (90 with I-pores and 90 without I-pores) were randomly selected, manually segmented, and three-dimensionally (3D) reconstructed to determine size, shape, and thickness of the cellular lining. Size of GVs (including median volume, surface area, and maximal cross-sectional area) with I-pores (n = 90) was significantly larger than GVs without I-pores (n = 90) using 3D-reconstructed GVs (P ≤ 0.01). Most I-pores (73.3%; 66/90) were located on or close to GV's maximal cross-sectional area with significant thinning of the cellular lining. Our results suggest that larger size and thinner cellular lining of GVs may contribute to formation of GVs with I-pores. More GVs with I-pores and basal openings were observed in high-flow areas, suggesting these GVs do provide a channel through which AH passes into SC and that increasing this type of GV may be a potential strategy to increase aqueous outflow for glaucoma treatment.

Cisplatin-resistant A549 non-small cell lung cancer cells can be identified by increased mitochondrial mass and are sensitive to pemetrexed treatment.

BACKGROUND: Cisplatin plus pemetrexed combination therapy is considered the standard treatment for patients with advanced, non-squamous, non-small-cell lung cancer (NSCLC). However, advanced NSCLC has a 5-year survival rate of below 10%, which is mainly due to therapy resistance. We previously showed that the NSCLC cell line A549 harbors different subpopulations including a mesenchymal-like subpopulation characterized by increased chemo- and radiotherapy resistance. Recently, therapy resistance in hematological and solid tumors has been associated with increased mitochondrial activity. Thus, the aim of this study was to investigate the role of the mitochondrial activity in NSCLC chemotherapy resistance. METHODS: Based on MitoTracker staining, subpopulations characterized by the highest 10% (Mito-High) or lowest 10% (Mito-Low) mitochondrial mass content were sorted by FACS (Fluorescence-Activated Cell Sorting) from paraclonal cultures of the NSCLC A549 cell line . Mitochondrial DNA copy numbers were quantified by real-time PCR whereas basal cellular respiration was measured by high-resolution respirometry. Cisplatin and pemetrexed response were quantified by proliferation and colony formation assay. RESULTS: Pemetrexed treatment of parental A549 cells increased mitochondrial mass over time. FACS-sorted paraclonal Mito-High cells featured increased mitochondrial mass and mitochondrial DNA copy number compared to the Mito-Low cells. Paraclonal Mito-High cells featured an increased proliferation rate and were significantly more resistant to cisplatin treatment than Mito-Low cells. Interestingly, cisplatin-resistant, paraclonal Mito-High cells were significantly more sensitive to pemetrexed treatment than Mito-Low cells. We provide a working model explaining the molecular mechanism underlying the increased cisplatin- and decreased pemetrexed resistance of a distinct subpopulation characterized by high mitochondrial mass. CONCLUSIONS: This study revealed that cisplatin resistant A549 lung cancer cells can be identified by their increased levels of mitochondrial mass. However, Mito-High cells feature an increased sensitivity to pemetrexed treatment. Thus, pemetrexed and cisplatin target reciprocal lung cancer subpopulations, which could explain the increased efficacy of the combination therapy in the clinical setting.

Heterologous expression and purification of a marine alginate lyase in Escherichia coli.

Alginate lyase digestion is an efficient way to degrade alginate into oligosaccharides, which are useful in various areas including chemistry, pharmacy and food industry. Here we determined the sequence of Vibrio sp. QY102 sourced alginate lyase, and set up its heterologous expression in E. coli. We improved its secretion efficiency by replacing the original secretive sequence by E. coli specific signal peptide ompA. We successfully purified the full-length protein in shake flask culture, however, degradation happened during fed batch cultivation. By domain and disorder examination, we found that the protein was completely functional by expressing the C terminal fragment alone. For the final strain we constructed (HMS-ompA-CF), the extracellular enzyme activity reached 375 U/ml in shake flask and 1789 U/ml in fed batch cultivation (5 L bioreactor). And the final protein yield reached 0.58 g/L in fed batch cultivation. We determined that the optimal pH and temperature for the shortened alginate lyase were 7.0 and 39 °C, respectively.

Glycosaminoglycans from a Sea Snake (Lapemis curtus): Extraction, Structural Characterization and Antioxidant Activity.

Sea snakes have wide application prospects in medicine, health food and other fields. Several novel polysaccharides were successfully obtained from the skin and the meat of a sea snake (Lapemis curtus). The structures of polysaccharides LSP3 and LMP3, which were extracted and purified from Lapemis curtus, were determined to be new and highly heterogenic glycosaminoglycans (GAGs) by means of FT-IR, ESI-MS/MS and NMR. LSP3 is a hybrid dermatan sulfate (DS) and composed of 48% 4-sulfated disaccharides (Di4S), 42% 6-sulfated disaccharides (Di6S) and 5% disulfated disaccharides (Di2,6S), while LMP3 is a hybrid chondroitin sulfate (CS) and composed of 70% Di4S, 20% Di6S, and 8% Di2,6S. More importantly, LSP3 and LMP3 showed a strong scavenging ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, iron (Fe2+) chelating activity and total antioxidant capacity in vitro, especially LSP3, with high contents of uronic acid and sulfate, which possessed a higher scavenging ability of DPPH radicals than other fractions. These data suggested that the sea snake polysaccharides could be promising candidates for natural antioxidant ingredients.

Green Synthesis of Gold Nanoparticles Using Carrageenan Oligosaccharide and Their In Vitro Antitumor Activity.

Gold nanoparticles (AuNPs) have been widely used in catalysis, photothermal therapy, and targeted drug delivery. Carrageenan oligosaccharide (CAO) derived from marine red algae was used as a reducing and capping agent to obtain AuNPs by an eco-friendly, efficient, and simple synthetic route for the first time. The synthetic conditions of AuNPs were optimized by response surface methodology (RSM), and the CAO-AuNPs obtained were demonstrated to be ellipsoidal, stable and crystalline by means of transmission electron microscopy (TEM), scanning electron microscopy (SEM) and X-ray diffraction (XRD). The CAO-AuNPs showed localized surface plasmon resonance (LSPR) oscillation at about 530 nm with a mean diameter of 35 ± 8 nm. The zeta potential of CAO-AuNPs was around -20 mV, which was related to the negatively charged CAO around AuNPs. The CAO-AuNPs exhibited significant cytotoxic activities to HCT-116 and MDA-MB-231 cells, which could be a promising nanomaterial for drug delivery.

Regulating unfolded protein response activator HAC1p for production of thermostable raw-starch hydrolyzing α-amylase in Pichia pastoris.

Unfolded protein response (UPR) usually happens when expressing heterologous proteins in high level, which may help cells to facilitate protein processing. Here, we evaluated the effects of the UPR activator HAC1p on a raw-starch hydrolyzing α-amylase (Gs4j-amyA), so as to improve heterologous production of the enzyme in Pichia pastoris. The gene (amyA) encoding Gs4j-amyA was first codon-optimized and expressed in P. pastoris under the control of the AOX1 promoter. A high gene dosage (12 copies) of amyA facilitated amylase expression which produced an enzyme activity of 305 U/ml. A spliced HAC1 encoding an UPR activator HAC1p was then co-expressed and the dosage effects of HAC1 on amylase expression was investigated. Six copies of HAC1 driven by AOX1 promoter produced a high amylase activity of 2200 U/ml, further increasing by 621%. However, excessive gene dosages driven by the same promoter led to a titration effect of its transcription factors and decreased the amount of amyA transcripts. Thus, constitutive expression of HAC1 by GAP promotor was further involved and Gs4j-amyA activity reached 3700 U/ml finally, which was further increased by 68.2%. Moreover, Gs4j-amyA was glycosylated in P. pastoris which generated higher enzyme activity than that in E. coli. Generally, regulating HAC1p expression by different strategies enhanced amylase production by 11.1 folds, indicating a reference for expression of other proteins in P. pastoris.

Effective pH pretreatment and cell disruption method for real-time intracellular enzyme activity assay of a marine fungus covered with pigments.

Filamentous fungi are capable producers of many bioactive compounds, and real-time intracellular enzyme activity assay is an essential guidance for their bioprocess developments. However, there are many difficulties in preparing homogenate for enzyme activity assay, such as disrupting fungal cell with complicated cellular structure and solid cell wall, removing abundant extracellular metabolites accumulating on mycelia, and so on. Halorosellinia sp. (No. 1403) was a marine-derived filamentous fungus producing a potential antitumor compound 1403C, and the deep red pigments (with main component of 1403C) covering on its mycelia showed strong absorption in a wide range, which critically affected the measurement of many enzyme activities. In this study, we developed an effective pH pretreatment and cell disruption method to prepare homogenate for enzyme activity assay. When mycelia were washed by the solution with pH 5.0 for 3 min, most pigments could be removed without severe loss on enzyme activities. Afterward, grinding with mini bead for 15 min with alternating cooling could effectively disrupt both cell wall and mitochondrial membrane. These methods have been successfully applied on real-time intracellular enzyme activity assay of Halorosellinia sp. (No. 1403) and can offer enlightenment for other filamentous fungi with similar problems.

Peroxisome-targeted and tandem repeat multimer expressions of human antimicrobial peptide LL37 in Pichia pastoris.

Although the human antimicrobial peptide LL37 has a broad spectrum of antimicrobial activities, it easily damages host cells following heterologous expressions. This study attempted two strategies to alleviate its damage to host cells when expressed in Pichia pastoris using the AOX1 promoter. Tandem repeat multimers of LL37 were first designed, and secretion expression strains GS115-9K-(DPLL37DP)n (n = 2, 4, 6 and 8) containing different copies of the LL37 gene were constructed. However, LL37 tandems still killed the cells after 96 hr of induction. Subsequently, peroxisome-targeted expression was performed by adding a peroxisomal targeting signal 1 (SKL) at the C-terminus of LL37. The LL37 expression strain GS115-3.5K-LL37-SKL showed no significant inhibition in the cells after induction. Antibacterial activity assays showed that the recombinant LL37 expressed in peroxisomes had good antimicrobial activities. Then, a strain GS115-3.5K-LL37-GFP-SKL producing LL37, green fluorescent protein, and SKL fusion proteins was constructed, and the fusion protein was confirmed to be targeting the peroxisomes. However, protein extraction analysis indicated that most of the fusion proteins were still located in the cell debris after cell disruption, and further studies are required to extract more proteins from the peroxisome membrane.

Stimulatory effect of ethanol on libertellenone H biosynthesis by Arctic fungus Eutypella sp. D-1.

Libertellenone H (1) was a promising antitumor diterpenoid isolated from Arctic fungus Eutypella sp. D-1, however, its production was very limited. In this study, we investigated the effects of ethanol on cell growth and libertellenone H production. The mycelium in ethanol-feeding cultures was fragmented and dispersed, and the titer of libertellenone H was remarkably increased to 4.88 mg l(-1) in an optimal feeding manner, which was 16.4-fold higher than the control group. To provide an insight into the cell response to ethanol, genes critical to the libertellenone H biosynthesis were successfully cloned and their transcription levels were determined. The results suggested that the gene transcription levels of 3-hydroxy-3-methyl glutaric acyl coenzyme A reductase and geranylgeranyl diphosphate synthase were up-regulated by ethanol stimulation. The results from this study were helpful for further understanding of the ethanol function on diterpenes biosynthesis as well as developing more effective strategies for over-production of these desired secondary metabolites.

[Automatic classification method of star spectra data based on manifold fuzzy twin support vector machine].

Support vector machine (SVM) with good leaning ability and generalization is widely used in the star spectra data classification. But when the scale of data becomes larger, the shortages of SVM appear: the calculation amount is quite large and the classification speed is too slow. In order to solve the above problems, twin support vector machine (TWSVM) was proposed by Jayadeva. The advantage of TSVM is that the time cost is reduced to 1/4 of that of SVM. While all the methods mentioned above only focus on the global characteristics and neglect the local characteristics. In view of this, an automatic classification method of star spectra data based on manifold fuzzy twin support vector machine (MF-TSVM) is proposed in this paper. In MF-TSVM, manifold-based discriminant analysis (MDA) is used to obtain the global and local characteristics of the input data and the fuzzy membership is introduced to reduce the influences of noise and singular data on the classification results. Comparative experiments with current classification methods, such as C-SVM and KNN, on the SDSS star spectra datasets verify the effectiveness of the proposed method.

[Effect of adjuvant therapy with Dingguier umbilical paste on intestinal flora of diarrhea infants and its action characteristics].

The applicator therapy is a unique method to treat infant diarrhea in traditional Chinese medicines and widely applied in clinical practice. Currently, many researchers have proved the rationality of the therapy based on the traditional Chinese medicine mechanism and on the data from clinical practice, but its action mechanism is uncertain at present. In this study, with the assistance of pediatric practitioners, the automated ribosomal intergenic-spacer analysis (ARISA) was adopted to study the effect of the adjuvant therapy with Dingguier umbilical paste on intestinal flora of diarrhea infants, in which Dingguier umbilical paste served as the adjuvant therapy in oral traditional Chinese medicines and fecal samples of infants with different diarrhea symptoms were collected and used as the study materials. The results showed that the adjuvant therapy had a significant effect on the shift of intestinal flora, which was associated with the decrease in the similarity difference to the normal control group and the increase in the number of operational taxonomic units (OTUs) shared with the normal control group. Additionally, adjuvant therapy with Dingguier umbilical paste also showed long action duration and increased OTUs number. These results indicated that Dingguier umbilical paste has the effect in restoring the micro-ecosystem of unbalanced intestinal bacteria. Intestinal flora may be one of major targets for the applicator therapy for the infant diarrhea, but not for the single oral traditional Chinese medicine for infant diarrhea.

Circulating tumor DNA- and cancer tissue-based next-generation sequencing reveals comparable consistency in targeted gene mutations for advanced or metastatic non-small cell lung cancer.

BACKGROUND: Molecular subtyping is an essential complementarity after pathological analyses for targeted therapy. This study aimed to investigate the consistency of next-generation sequencing (NGS) results between circulating tumor DNA (ctDNA)-based and tissue-based in non-small cell lung cancer (NSCLC) and identify the patient characteristics that favor ctDNA testing. METHODS: Patients who diagnosed with NSCLC and received both ctDNA- and cancer tissue-based NGS before surgery or systemic treatment in Lung Cancer Center, Sichuan University West China Hospital between December 2017 and August 2022 were enrolled. A 425-cancer panel with a HiSeq 4000 NGS platform was used for NGS. The unweighted Cohen's kappa coefficient was employed to discriminate the high-concordance group from the low-concordance group with a cutoff value of 0.6. Six machine learning models were used to identify patient characteristics that relate to high concordance between ctDNA-based and tissue-based NGS. RESULTS: A total of 85 patients were enrolled, of which 22.4% (19/85) had stage III disease and 56.5% had stage IV disease. Forty-four patients (51.8%) showed consistent gene mutation types between ctDNA-based and tissue-based NGS, while one patient (1.2%) tested negative in both approaches. Advanced diseases and metastases to other organs would be fit for the ctDNA-based NGS, and the generalized linear model showed that T stage, M stage, and tumor mutation burden were the critical discriminators to predict the consistency of results between ctDNA-based and tissue-based NGS. CONCLUSION: ctDNA-based NGS showed comparable detection performance in the targeted gene mutations compared with tissue-based NGS, and it could be considered in advanced or metastatic NSCLC.

Adaptive evolution of plasmid and chromosome contributes to the fitness of a blaNDM-bearing cointegrate plasmid in Escherichia coli.

Large cointegrate plasmids recruit genetic features of their parental plasmids and serve as important vectors in the spread of antibiotic resistance. They are now frequently found in clinical settings, raising the issue of how to limit their further transmission. Here, we conducted evolutionary research of a large blaNDM-positive cointegrate within Escherichia coli C600, and discovered that adaptive evolution of chromosome and plasmid jointly improved bacterial fitness, which was manifested as enhanced survival ability for in vivo and in vitro pairwise competition, biofilm formation, and gut colonization ability. From the plasmid aspect, large-scale DNA fragment loss is observed in an evolved clone. Although the evolved plasmid imposes a negligible fitness cost on host bacteria, its conjugation frequency is greatly reduced, and the deficiency of anti-SOS gene psiB is found responsible for the impaired horizontal transferability rather than the reduced fitness cost. These findings unveil an evolutionary strategy in which the plasmid horizontal transferability and fitness cost are balanced. From the chromosome perspective, all evolved clones exhibit parallel mutations in the transcriptional regulatory stringent starvation Protein A gene sspA. Through a sspA knockout mutant, transcriptome analysis, in vitro transcriptional activity assay, RT-qPCR, motility test, and scanning electron microscopy techniques, we demonstrated that the mutation in sspA reduces its transcriptional inhibitory capacity, thereby improving bacterial fitness, biofilm formation ability, and gut colonization ability by promoting bacterial flagella synthesis. These findings expand our knowledge of how cointegrate plasmids adapt to new bacterial hosts.

Chemotherapy increases CDA expression and sensitizes malignant pleural mesothelioma cells to capecitabine treatment.

The combination of cisplatin and pemetrexed remains the gold standard chemotherapy for malignant pleural mesothelioma (MPM), although resistance and poor response pose a significant challenge. Cytidine deaminase (CDA) is a key enzyme in the nucleotide salvage pathway and is involved in the adaptive stress response to chemotherapy. The cytidine analog capecitabine and its metabolite 5'-deoxy-5-fluorocytidine (5'-DFCR) are converted via CDA to 5-fluorouracil, which affects DNA and RNA metabolism. This study investigated a schedule-dependent treatment strategy, proposing that initial chemotherapy induces CDA expression, sensitizing cells to subsequent capecitabine treatment. Basal CDA protein expression was low in different mesothelioma cell lines but increased in the corresponding xenografts. Standard chemotherapy increased CDA protein levels in MPM cells in vitro and in vivo in a schedule-dependent manner. This was associated with epithelial-to-mesenchymal transition and with HIF-1alpha expression at the transcriptional level. In addition, pretreatment with cisplatin and pemetrexed in combination sensitized MPM xenografts to capecitabine. Analysis of a tissue microarray (TMA) consisting of samples from 98 human MPM patients revealed that most human MPM samples had negative CDA expression. While survival curves based on CDA expression in matched samples clearly separated, significance was not reached due to the limited sample size. In non-matched samples, CDA expression before but not after neoadjuvant therapy was significantly associated with worse overall survival. In conclusion, chemotherapy increases CDA expression in xenografts, which is consistent with our in vitro results in MPM and lung cancer. A subset of matched patient samples showed increased CDA expression after therapy, suggesting that a schedule-dependent treatment strategy based on chemotherapy and capecitabine may benefit a selected MPM patient population.

BAP1 Deficiency Inflames the Tumor Immune Microenvironment and Is a Candidate Biomarker for Immunotherapy Response in Malignant Pleural Mesothelioma.

Introduction: Malignant pleural mesothelioma (MPM) is a rare and universally lethal malignancy with limited treatment options. Immunotherapy with immune checkpoint inhibitors (ICIs) has recently been approved for unresectable MPM, but response to ICIs is heterogeneous, and reliable biomarkers for prospective selection of appropriate subpopulations likely to benefit from ICIs remain elusive. Methods: We performed multiscale integrative analyses of published primary tumor data set from The Cancer Genome Atlas (TCGA) and the French cohort E-MTAB-1719 to unravel the tumor immune microenvironment of MPM deficient in BAP1, one of the most frequently mutated tumor suppressor genes (TSGs) in the disease. The molecular profiling results were validated in independent cohorts of patients with MPM using immunohistochemistry and multiplex immunohistochemistry. Results: We revealed that BAP1 deficiency enriches immune-associated pathways in MPM, leading to increased mRNA signatures of interferon alfa/gamma response, activating dendritic cells, immune checkpoint receptors, and T-cell inflammation. This finding was confirmed in independent patient cohorts, where MPM tumors with low BAP1 levels are associated with an inflammatory tumor immune microenvironment characterized by increased exhausted precursor T-cells and macrophages but decreased myeloid-derived suppressor cells (MDSCs). In addition, BAP1low MPM cells are in close proximity to T cells and therefore can potentially be targeted with ICIs. Finally, we revealed that BAP1-proficient MPM is associated with a hyperactive mitogen-activated protein kinase (MAPK) pathway and may benefit from treatment with MEK inhibitors (MEKis). Conclusion: Our results suggest that BAP1 plays an immunomodulatory role in MPM and that BAP1-deficient MPM may benefit from immunotherapy, which merits further clinical investigation.

Analysis of postpartum hypertension in women with preeclampsia.

Postpartum hypertension including persistent and recurrent hypertension could significantly affect maternal morbidity in preeclampsia. Data on the postpartum management of women with preeclampsia is limited. The objective of this study was to investigate the details of women experiencing persistent postpartum hypertension (PerPPH) or developing recurrent postpartum hypertension (RecPPH) after birth and whether the treatment with anti-hypertensive drugs could shorten the hospital stay. We also compared the clinical parameters in women who developed RecPPH and who did not. Data on 188 preeclamptic women, including the severity or time of onset, duration of hospital admission postpartum, and blood pressure during the admission were collected and analyzed. Overall, 30% of preeclamptic women developed RecPPH on day 1, 13% on day 3, and 12% on day 5 after birth. Women with severe preeclampsia or early onset preeclampsia are more likely to develop RecPPH, compared to women with mild or late onset preeclampsia. The overall time in days before discharge was not different between women with normal blood pressure and women with abnormal blood pressure 1 h after birth, regardless of the severity or gestation of onset. However, women with severe or early onset preeclampsia stayed longer in the hospital, compared to women with mild or late onset preeclampsia. In addition, women with severe or early onset preeclampsia or early delivery increased risk of developing RecPPH. In conclusion, we demonstrate that RecPPH became apparent on day 1 after delivery, and hence close monitoring of blood pressure even if initially seemingly normal after birth is important.

The Associations Between Alanine Aminotransferase and Other Biochemical Parameters in Lean PCOS.

To explore the associations of alanine aminotransferase in lean women of polycystic ovary syndrome (PCOS) with other biochemical parameters and the potential risk factors. This is a retrospective cohort study with lean PCOS (n = 91) and healthy controls (n = 45); we reviewed the electrical records and databases of the PCOS patients in our infertility clinic between January 2019 and September 2021; independent t-test, linear correlation analysis, and multiple linear regression were used to explore the associations. Higher levels of luteinizing hormone, total testosterone, thyroid stimulating hormone, platelet count, lymphocyte count, homocysteine, alanine aminotransferase (ALT), and uric acid were identified in lean PCOS patients, while follicle-stimulating hormone level was lower in in lean PCOS as expected (P < 0.05). Of note, the linear correlation showed that BMI, total testosterone, white blood cell count, lymphocyte count, aspartate aminotransferase, and uric acid were positively associated with alanine aminotransferase (r = 0.232, 0.318, 0.218, 0.388, 0.602, 0.353 respectively, P < 0.05). After multiple linear regression was performed, total testosterone and aspartate aminotransferase were independently and positively correlated with alanine aminotransferase in lean PCOS (B = 0.251, 0.605 respectively, P < 0.05). Higher level of ALT was identified in the lean PCOS. BMI, white blood cell count, lymphocyte count, aspartate aminotransferase, uric acid, and total testosterone were positively correlated with ALT in lean PCOS. Total testosterone and aspartate aminotransferase were independently and positively associated with ALT in lean PCOS after multiple linear regression. There might exist a potential risk of afflicting liver impairment for the lean PCOS women in the earlier period. Early examination and intervention might be necessary to prevent or delay the progression of the liver disease as soon as the diagnosis of PCOS.

Dynamic evolution and transmission of a blaNDM-1-bearing fusion plasmid in a clinical Escherichia coli.

Plasmids are the main driving forces for the rapid dissemination of blaNDM-1. In recent years, blaNDM-1-carrying fusion plasmids have been frequently reported. However, the evolutionary patterns of blaNDM-1-carrying fusion plasmids remain largely unknown. Herein, we reported a blaNDM-1-bearing fusion plasmid pZX35-269k possessing IncFII and IncA/C2 replicons from clinical ST349 E. coli 13ZX35. The backbone of pZX35-269k was structurally unstable, which was manifested in different types of structural dissociation during conjugation and passage, thereby forming various daughter plasmids. Moreover, the same events were observed in the clinical setting as well. We found that pZX35-269k exhibited highly identical to two plasmids (pZX30-70k and pZX30-192k) in 13ZX30, both of which were isolated from the same hospital. Sequence analysis highlighted that two plasmids in 13ZX30 evolved from pZX35-269k through homologous recombination of a 4856-bp fragment. Collectively, this study confirmed the transmission and structural evolution of a blaNDM-1-bearing fusion plasmid in both laboratory and clinical settings, and provided clear evidence of plasmid spread and evolution in clinical settings. Such versatile plasmids may represent a potential risk for the public health.