Multiomics analysis of metabolic heterogeneity in cervical cancer cell lines with or without HPV.

Metabolomics analysis revealed the metabolic heterogeneity of cervical cancer (CC) cell lines C33A and CaSki, and their molecular mechanisms were explored. Using the modified Bligh-Dyer method, the endogenous metabolites of C33A and CaSki cells were divided into polar and nonpolar fractions. The metabolites were analysed by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Then, the differential metabolites were screened by combining multivariate statistical analysis and volcano maps, and functional enrichment and pathway analysis of the differential metabolites were performed. Finally, association analysis was carried out in combination with transcriptomics, and the important differential metabolisms were experimentally verified by real-time PCR (RT-qPCR) and oil red staining. The results showed that between the C33A and CaSki cell lines, there were significant differences in amino acids, nucleotides and lipids, such as in threonine, arachidonic acid and hypoxanthine, in the metabolic pathways. These compounds could be used as markers of differences in cellular metabolism. The heterogeneity of lipid metabolism accounted for 87.8%, among which C33A cells exhibited higher contents of fatty acid polar derivatives, while CaSki cells showed higher contents of free fatty acids and glycerides. Based on correlation analysis of the above metabolic differences in HPV pathways as well as lipid metabolism-related genes, p53 and the genes involved in lipid metabolism pathways, such as Peroxisome Proliferator Activated Receptor Gamma(PPARG) and stearoyl-CoA desaturase (SCD), are relevant to the metabolic heterogeneity of the cells. The differential expression of some genes was validated by RT-qPCR. CaSki cells showed significantly higher glyceride levels than that of C33A cells, as verified by oil red O staining and glyceride assays. The above results showed that the metabolomic differences between C33A and CaSki cells were relatively obvious, especially in lipid metabolism, which might be related to the decreased expression of PPARG and p53 caused by HPV E6. Further studies on the molecular mechanism of lipid metabolism heterogeneity in cervical cancer cell lines with or without HPV could provide a new reference for the development of CC and individualized treatments of tumour patients.

The Role of Hypoxia and Cancer Stem Cells in Development of Glioblastoma.

Glioblastoma multiform (GBM) is recognized as the most malignant brain tumor with a high level of hypoxia, containing a small population of glioblastoma stem like cells (GSCs). These GSCs have the capacity of self-renewal, proliferation, invasion and recapitulating the parent tumor, and are major causes of radio-and chemoresistance of GBM. Upregulated expression of hypoxia inducible factors (HIFs) in hypoxia fundamentally contributes to maintenance and progression of GSCs. Therefore, we thoroughly reviewed the currently acknowledged roles of hypoxia-associated GSCs in development of GBM. In detail, we recapitulated general features of GBM, especially GSC-related features, and delineated essential responses resulted from interactions between GSC and hypoxia, including hypoxia-induced signatures, genes and pathways, and hypoxia-regulated metabolic alterations. Five hypothesized GSC niches are discussed and integrated into one comprehensive concept: hypoxic peri-arteriolar niche of GSCs. Autophagy, another protective mechanism against chemotherapy, is also closely related to hypoxia and is a potential therapeutic target for GBM. In addition, potential causes of therapeutic resistance (chemo-, radio-, surgical-, immuno-), and chemotherapeutic agents which can improve the therapeutic effects of chemo-, radio-, or immunotherapy are introduced and discussed. At last, as a potential approach to reverse the hypoxic microenvironment in GBM, hyperbaric oxygen therapy (HBOT) might be an adjuvant therapy to chemo-and radiotherapy after surgery. In conclusion, we focus on demonstrating the important role of hypoxia on development of GBM, especially by affecting the function of GSCs. Important advantages have been made to understand the complicated responses induced by hypoxia in GBM. Further exploration of targeting hypoxia and GSCs can help to develop novel therapeutic strategies to improve the survival of GBM patients.

CEBPD is a master transcriptional factor for hypoxia regulated proteins in glioblastoma and augments hypoxia induced invasion through extracellular matrix-integrin mediated EGFR/PI3K pathway.

Hypoxia contributes to the initiation and progression of glioblastoma by regulating a cohort of genes called hypoxia-regulated genes (HRGs) which form a complex molecular interacting network (HRG-MINW). Transcription factors (TFs) often play central roles for MINW. The key TFs for hypoxia induced reactions were explored using proteomic analysis to identify a set of hypoxia-regulated proteins (HRPs) in GBM cells. Next, systematic TF analysis identified CEBPD as a top TF that regulates the greatest number of HRPs and HRGs. Clinical sample and public database analysis revealed that CEBPD is significantly up-regulated in GBM, high levels of CEBPD predict poor prognosis. In addition, CEBPD is highly expressed in hypoxic condition both in GBM tissue and cell lines. For molecular mechanisms, HIF1α and HIF2α can activate the CEBPD promotor. In vitro and in vivo experiments demonstrated that CEBPD knockdown impaired the invasion and growth capacity of GBM cells, especially in hypoxia condition. Next, proteomic analysis identified that CEBPD target proteins are mainly involved in the EGFR/PI3K pathway and extracellular matrix (ECM) functions. WB assays revealed that CEBPD significantly positively regulated EGFR/PI3K pathway. Chromatin immunoprecipitation (ChIP) qPCR/Seq analysis and Luciferase reporter assay demonstrated that CEBPD binds and activates the promotor of a key ECM protein FN1 (fibronectin). In addition, the interactions of FN1 and its integrin receptors are necessary for CEBPD-induced EGFR/PI3K activation by promoting EGFR phosphorylation. Furthermore, GBM sample analysis in the database corroborated that CEBPD is positively correlated with the pathway activities of EGFR/PI3K and HIF1α, especially in highly hypoxic samples. At last, HRPs are also enriched in ECM proteins, indicating that ECM activities are important components of hypoxia induced responses in GBM. In conclusion, CEPBD plays important regulatory roles in the GBM HRG-MINW as a key TF, which activates the EGFR/PI3K pathway through ECM, especially FN1, mediated EGFR phosphorylation.

Polyphosphate kinase 1 is involved in formation, the morphology and ultramicrostructure of biofilm of Mycobacterium smegmatis and its survivability in macrophage.

The most unique characteristic of Mycobacterium tuberculosis is persistence in the human host, and the biofilm formation is related to the persistance. Polyphosphate (polyP) kinase 1 (PPK1) is conserved in Mycobacteria and is responsible for polyP synthesis. polyP is a chain molecule linked by high-energy phosphate bonds, which is considered to play a very important role in bacterial persistence. However, the relationship of PPK1 and mycobacterial biofilm formation is still adequately unclear. In current study, ppk1-deficient mutant (MT), ppk1-complemented (CT) and wild-type strains of M. smegmatis mc2 155 were used to investigate the formation, morphology and ultramicrostructure of the biofilm and to analyze the lipid levels and susceptibility to vancomycin antibiotic. And then WT, MT and CT strains were used to infect macrophages and to analyze the expression levels of various inflammatory factors, respectively. We found that PPK1 was required for M. smegmatis polyP production in vivo and polyP deficiency not only attenuated the biofilm formation, but also altered the phenotype and ultramicrostructure of the biofilm and reduced the cell lipid composition (except for C16.1 and C17.1, most of the fatty acid components from C8-C24). Moreover, the ppk1-deficient mutant was also significantly more sensitive to vancomycin which targets the cell wall, and its ability to survive in macrophages was decreased, which was related to the change of the expression level of inflammatory factors in macrophage. This study demonstrates that the PPK1 can affect the biofilm structure through affecting the content of short chain fatty acid and promote intracellular survival of M. smegmatis by altering the expression of inflammatory factors. These findings establish a basis for investigating the role of PPK1 in the persistence of M. tuberculosis, and provide clues for treating latent infection of M. tuberculosis with PPK1 as a potential drug target.

Supramolecular coordination complexes (SCCs) with aggregation-induced emission for in vitro photodynamic therapy.

Photodynamic therapy (PDT) has attracted a great deal of attention in the field of cancer treatment due to its non-invasive nature, high repeatability and minimal side effects. Due to the dual effect of organic small molecule donors and platinum receptors, supramolecular coordination complexes (SCCs) exhibit stronger reactive oxygen species (ROS) production capacity and have become a promising class of photosensitizers (PSs). Herein, we report a rhomboid SCC MD-CN based on the D-A structure with aggregation-induced emission (AIE). The results show that the as-prepared nanoparticles (NPs) had excellent photosensitization efficiency and good biocompatibility. Most importantly, they exhibited potential killing effects on cancer cells in vitro under light irradiation.

NIR-II phototherapy agents with aggregation-induced emission characteristics for tumor imaging and therapy.

As one of the major public health concerns, malignant tumors threaten people's lives. With the increasing demand for early accurate diagnosis and the safe treatment of tumors, non-invasive optical imaging (including fluorescence imaging and photoacoustic imaging) and phototherapy (including photothermal therapy and photodynamic therapy) have received much attention. In particular, light in the near-infrared second region (NIR-II) has been attracting research interest, owing to its deep penetration, minimal tissue autofluorescence, and decreased tissue absorption and scattering. Among all biological materials, organic nanomaterials with aggregation-induced emission (AIE) properties have attracted significant attention, owing to various incomparable advantages, such as high brightness, good photostability, tunable photophysical properties, and good biosafety. To modulate the working optical region of AIE molecules to the NIR-II region, many researchers have tried a variety of methods in recent years, and the focus of this review is to summarize the three most common methods from the perspective of molecular design strategies. In addition, this article briefly reviews the recent five-year progress of NIR-II AIE luminophores in tumor imaging and phototherapy applications. The research status is also summarized and prospected, with the hope of contributing to further research.

[Pathogenesis of Secondary Pulmonary Tuberculosis and Role of Cord Factor in Secondary Infection].

The primary and secondary tuberculosis features two completely different pathogenesis.At present,the pathogenesis of primary tuberculosis has been clear,whereas that of secondary tuberculosis remains unclear.In order to decipher the mechanism of secondary infection of Mycobacterium tuberculosis and provide insights into vaccine research and drug development,this paper reviews the problems of the widely accepted mechanism of secondary infection,the new findings of the research on the mechanism,as well as the role of cord factors.

A Novel Prognostic Model and Practical Nomogram for Predicting the Outcomes of Colorectal Cancer: Based on Tumor Biomarkers and Log Odds of Positive Lymph Node Scheme.

BACKGROUND: Emerging evidence shows that serum tumor biomarkers (TBs) and log odds of positive lymph node scheme (LODDS) are closely associated with the prognosis of colorectal cancer (CRC) patients. The aim of our study is to validate the predictive value of TBs and LODDS clinically and to develop a robust prognostic model to predict the overall survival (OS) of patients with CRC. METHODS: CRC patients who underwent radical resection and with no preoperative chemotherapy were enrolled in the study. The eligible population were randomized into training (70%) and test (30%) cohorts for the comprehensive evaluation of the prognostic model. Clinical implications of serum biomarkers and LODDS were identified by univariate and multivariate Cox proportion regression analysis. The predictive ability and discriminative performance were evaluated by Kaplan-Meier (K-M) curves and receiver operating characteristic (ROC) curves. Clinical applicability of the prognostic model was assessed by decision curve analysis (DCA), and the corresponding nomogram was constructed based on the above factors. RESULTS: A total of 1,202 eligible CRC patients were incorporated into our study. Multivariable COX analysis demonstrated that CA199 (HR = 1.304), CA125 (HR = 1.429), CEA (HR = 1.307), and LODDS (HR = 1.488) were independent risk factors for OS (all P < 0.0001). K-M curves showed that the high-risk group possessed a shorter OS than the low-risk counterparts. The area under curves (AUCs) of the model for 1-, 3- and 5-year OS were 86.04, 78.70, and 76.66% respectively for the train cohort (80.35, 77.59, and 74.26% for test cohort). Logistic DCA and survival DCA confirmed that the prognostic model displayed more clinical benefits than the conventional AJCC 8th TNM stage and CEA model. The nomograms were built accordingly, and the calibration plot for the probability of survival at 3- or 5-years after surgery showed an optimal agreement between prediction and actual observation. CONCLUSIONS: Preoperative serum TBs and LODDS have significant clinical implications for CRC patients. A novel prognostic model incorporating common TBs (CA199, CA125, and CEA) and LODDS displayed better predictive performance than both single factor and the TNM classification. A novel nomogram incorporating TBs and LODDS could individually predict OS in patients with CRC.

Preferences for Artificial Intelligence Clinicians Before and During the COVID-19 Pandemic: Discrete Choice Experiment and Propensity Score Matching Study.

BACKGROUND: Artificial intelligence (AI) methods can potentially be used to relieve the pressure that the COVID-19 pandemic has exerted on public health. In cases of medical resource shortages caused by the pandemic, changes in people's preferences for AI clinicians and traditional clinicians are worth exploring. OBJECTIVE: We aimed to quantify and compare people's preferences for AI clinicians and traditional clinicians before and during the COVID-19 pandemic, and to assess whether people's preferences were affected by the pressure of pandemic. METHODS: We used the propensity score matching method to match two different groups of respondents with similar demographic characteristics. Respondents were recruited in 2017 and 2020. A total of 2048 respondents (2017: n=1520; 2020: n=528) completed the questionnaire and were included in the analysis. Multinomial logit models and latent class models were used to assess people's preferences for different diagnosis methods. RESULTS: In total, 84.7% (1115/1317) of respondents in the 2017 group and 91.3% (482/528) of respondents in the 2020 group were confident that AI diagnosis methods would outperform human clinician diagnosis methods in the future. Both groups of matched respondents believed that the most important attribute of diagnosis was accuracy, and they preferred to receive combined diagnoses from both AI and human clinicians (2017: odds ratio [OR] 1.645, 95% CI 1.535-1.763; P<.001; 2020: OR 1.513, 95% CI 1.413-1.621; P<.001; reference: clinician diagnoses). The latent class model identified three classes with different attribute priorities. In class 1, preferences for combined diagnoses and accuracy remained constant in 2017 and 2020, and high accuracy (eg, 100% accuracy in 2017: OR 1.357, 95% CI 1.164-1.581) was preferred. In class 2, the matched data from 2017 were similar to those from 2020; combined diagnoses from both AI and human clinicians (2017: OR 1.204, 95% CI 1.039-1.394; P=.011; 2020: OR 2.009, 95% CI 1.826-2.211; P<.001; reference: clinician diagnoses) and an outpatient waiting time of 20 minutes (2017: OR 1.349, 95% CI 1.065-1.708; P<.001; 2020: OR 1.488, 95% CI 1.287-1.721; P<.001; reference: 0 minutes) were consistently preferred. In class 3, the respondents in the 2017 and 2020 groups preferred different diagnosis methods; respondents in the 2017 group preferred clinician diagnoses, whereas respondents in the 2020 group preferred AI diagnoses. In the latent class, which was stratified according to sex, all male and female respondents in the 2017 and 2020 groups believed that accuracy was the most important attribute of diagnosis. CONCLUSIONS: Individuals' preferences for receiving clinical diagnoses from AI and human clinicians were generally unaffected by the pandemic. Respondents believed that accuracy and expense were the most important attributes of diagnosis. These findings can be used to guide policies that are relevant to the development of AI-based health care.

N-myc downstream-regulated gene 2 promotes the protein stability of estrogen receptor beta via inhibition of ubiquitin-protein ligase E3A to suppress colorectal cancer.

BACKGROUND: N-myc downstream-regulated gene 2 (NDRG2) and estrogen receptor beta (ERß) both play key roles in cellular differentiation in colorectal cancer (CRC). Previous studies have demonstrated that ERß co-locates with and directly transactivates NDRG2. However, the effect of NDRG2 on ERß and its underlying mechanism remain largely unknown. Our aim of the study is to explore the effect of NDRG2 on ERß and their contributions to progression of CRC. METHODS: The Cancer Genome Atlas (TCGA) database was first utilized to validate the clinical significance of ERß and NDRG2 in CRC. MTT and scratch migration assays were carried out to verify the role of ERß and NDRG2 in CRC cells. Western blotting and polymerase chain reaction were performed to analyze the effect of NDRG2 on ERß, and an immunoprecipitation assay was conducted to explore the protein-protein interaction. RESULTS: ERß and NDRG2 were both found to be significantly down-regulated in tumor tissues from the TCGA-CRC database. NDRG2 was also observed to enhance the protein stability of ERß while could not change messenger RNA (mRNA) level of ESR2 (encoding ERß). A positive relationship was found to exist between the two proteins in CRC cells, with NDRG2 prolonging the half-life of ERß and improving its nuclear translocation. Through detecting expression of ERß downstream genes (such as TP53 and JNK) and performing related function experiment, we demonstrated that NDRG2 could promote transcriptional activation of ERß target genes and enhance the function of tumor suppressors when the ERß agonist diarylpropionitrile (DPN). The immunoprecipitation assay showed that NDRG2 could affect the complex components of ubiquitin-protein ligase E3A (UBE3A, known as E6AP) and ERß, reducing the ubiquitin-mediated proteasome degradation of ERß. CONCLUSIONS: In the current study, we found that NDRG2 could bind with UBE3A to hinder the binding of UBE3A with ERß. Moreover, a positive feedback loop was discovered between NDRG2 and ERß, which provides a novel insight and therapeutic target for CRC.

Essential oil from Fructus Alpinia zerumbet (fruit of Alpinia zerumbet (Pers.) Burtt.et Smith) protected against aortic endothelial cell injury and inflammation in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Fructus Alpinia zerumbet (FAZ), a dry and ripe fruit of Alpinia zerumbet (Pers.) Burtt. et Smith, is widely used as a spice to treat cardiovascular diseases in clinic as a miao folk medicine in Guizhou Province of China. Essential oil extracted from FAZ (EOFAZ) is the key bioactive ingredients. AIM OF THE STUDY: This study aimed to examine the effects and mechanisms of EOFAZ on lipopolysaccharide (LPS)-induced endothelial cell injury, inflammation and apoptosis in vitro and in vivo. MATERIALS AND METHODS: For the in vitro study, LPS-treated human aortic endothelial cells were used to perform PCR, western blot analysis and immunofluorescence. For the in vivo study, male mouse were divided into four groups, vehicle control group and LPS group received 0.5% Tween-80 in saline; and two EOFAZ groups receive different dose of EOFAZ (90 mg kg -1·day-1, 180 mg kg -1·day-1) respectively. Each group was fed for 7 days by intragastrical administration at daily base. Then, except vehicle control group received saline, mice in other three groups were administered with LPS (1 mg kg -1, dissolved in saline) by intraperitoneal injection. 24 h later, Aorta tissue was collected and frozen immediately in liquid N2, stored at -80 °C for western blot analysis. RESULTS: We found that EOFAZ completely prevented LPS-induced HAEC activation and inflammation in vitro and in vivo, as assessed by expression of endothelial adhesion molecules, ICAM-1 and VCAM-1. Similarly, EOFAZ significantly blunted LPS-induced endothelial injury, as tested by MTT assay, LDH release and caspase-3 activation. We further demonstrated that TLR4-dependent NF-κB signaling may be involved in the process. CONCLUSION: EOFAZ protected against LPS-induced endothelial cell injury and inflammation likely via inhibition of TLR4-dependent NF-κB signaling.

Inhibiting Jumoji domain containing protein 3 (JMJD3) prevent neuronal apoptosis from stroke.

Control of p53 by histone methylation is closely related in the neuronal apoptosis following ischemic stroke. In mammalian cells, demethylation of methylated lysine residue of histones is catalyzed by Jumonji domain-containing proteins (JMJD) family. Among them, JMJD3 is reported to be a hypoxic target gene and expressed in all cell types of brain including neurons. However, the role of JMJD3 on process of neuronal apoptosis after ischemic stroke is still largely unknown. PCR, immunostaining and Western blotting results indicated that JMJD3 expression was upregulated in cultured neurons upon oxygen-glucose deprivation (OGD) injury. Jmjd3-/- neurons exhibited inhibited cell apoptosis and tolerance to the OGD injury. Chromatin immunoprecipitation and promoter reporter assays showed that the underlying mechanism was through transcriptional activation of p53, thus altering the downstream Bax and Caspase-3 genes. Silencing Jmjd3 improved neurological deficit and reduced infarct volume following ischemic injury in vivo. The present study suggested that JMJD3 was a critical promoter of neuronal apoptosis by regulating the expression of Bax and Caspase-3, and inhibition of JMJD3 might provide a new therapeutic intervention for treating cerebral ischemia.

[Polyphosphate and its physiological function in Mycobacteria - A review].

Tuberculosis is still a global infectious disease. New drugs to shorten the course of treatment and new vaccines are the key points to control tuberculosis. The physiological study of Mycobacteria will contribute to the above-mentioned purposes. Polyphosphate plays an important role in the stress adaptation in bacteria. And there are two classes of enzymes:polyphosphate kinase and exopolyphosphatase involved in the polyphosphate metabolism to control the dynamic equilibrium of polyphosphate level in Mycobacteria. Present paper summarized the progress in metabolism and physiological roles of polyphosphate in Mycobacteria, to provide useful information for studying the physiological function of polyphosphate in Mycobacteria.

Polyphosphate kinase of Lysinibacillus sphaericus and its effects on accumulation of polyphosphate and bacterial growth.

Lysinibacillus sphaericus produces parasporal crystal proteins (Bin toxins) that are specifically toxic to Culex and Anopheles mosquito larvae. It has long persistence in mosquito breeding sites due to the recycle of spores in mosquito cadaver and certain environments. This bacterium is unable to metabolize sugar (except N-acetylglucosamine) but can use proteinaceous materials. Previous studies have shown that inorganic polyphosphate (polyP) is essential for cell growth and responses to nutritional stringencies and environmental stresses and branched-chain amino acids (BCAAs) are useful intracellular signals for bacterial adaption to nutritional environments. However, relatively little is known about the role of polyP and the regulation link between polyP and BCAAs in nutritional limitation adaptation in a bacterium incapable of polysaccharide utilization. Here, a primary enzyme involved in polyP biosynthesis, polyphosphate kinase (PPK), was identified from L. sphaericus, and the polyP accumulation in different nutritional media was investigated. Furthermore, a ppk null mutant and the ppk-complemented strain were constructed and the effect of deletion on cell growth, polyP accumulation and expression of BCAAs biosynthetic genes (ilvBHC) was surveyed. The result showed that deletion of ppk resulted in a deficiency in polyP accumulation rather than cell growth in Luria-Bertani (LB) medium. Further studies demonstrated that the cell growth was evidently retarded and the ilvBHC expression was significantly reduced when ppk mutant was transferred from LB to limited-amino acid (LA) medium and that these phenotypes were significantly restored in the ppk-complemented strain. These results suggested that PPK plays a role in polyP accumulation and expression of genes involved in BCAAs biosynthesis, providing a hint for further understanding of the role of PPK in nutritional limitation in L. sphaericus.

A novel transcriptional activator, tubX, is required for the stability of Bacillus sphaericus mosquitocidal plasmid pBsph.

Stable maintenance of the low-copy-number plasmid pBsph in Bacillus sphaericus requires a partitioning (par) system that consists of a filament-forming protein, B. sphaericus TubZ (TubZ-Bs); a centromere-binding protein, TubR-Bs; and a centromere-like DNA site, tubC, composed of three blocks (I, II, and III) of 12-bp degenerate repeats. Previous studies have shown that mini-pBsph replicons encoding the TubZ system are segregationally highly unstable, whereas the native pBsph is stably maintained. However, the mechanism underlying the stability discrepancy between pBsph and its minireplicon is poorly understood. Here orf187 (encoding TubX), a gene downstream of tubZ-Bs, was found to play a role in plasmid stabilization. Null mutation or overexpression of tubX resulted in a defect in pBsph stability and a significant decrease in the level of tubRZ-Bs expression, and the TubX-null phenotype was suppressed by ectopic expression of a wild-type copy of tubX and additional tubRZ-Bs. An electrophoresis mobility shift assay (EMSA) and a DNase I footprinting assay revealed that the TubX protein bound directly to five 8-bp degenerate repeats located in the par promoter region and that TubX competed with TubR-Bs for binding to the par promoter. Further studies demonstrated that TubX significantly stimulated the transcription of the par operon in the absence of tubR-Bs, and a higher level of gene activation was observed when tubR-Bs was present. These results suggested that TubX positively regulates tubRZ-Bs transcription by interfering with TubR-Bs-mediated repression and binding directly to the tubRZ-Bs promoter region.

Collagen-like glycoprotein BclS is involved in the formation of filamentous structures of the Lysinibacillus sphaericus exosporium.

Lysinibacillus sphaericus produces mosquitocidal binary toxins (Bin toxins) deposited within a balloon-like exosporium during sporulation. Unlike Bacillus cereus group strains, the exosporium of L. sphaericus is usually devoid of the hair-like nap, an external filamentous structure formed by a collagen-like protein, BclA. In this study, a new collagen-like exosporium protein encoded by Bsph_0411 (BclS) from L. sphaericus C3-41 was characterized. Thin-section electron microscopy revealed that deletion of bclS resulted in the loss of the filamentous structures that attach to the exosporium basal layer and spread through the interspace of spores. In vivo visualization of BclS-green fluorescent protein (GFP)/mCherry fusion proteins revealed a dynamic pattern of fluorescence that encased the spore from the mother cell-distal (MCD) pole of the forespore, and the BclS-GFP fusions were found to be located in the interspace of the spore, as confirmed by three-dimensional (3D) superresolution fluorescence microscopy. Further studies demonstrated that the bclS mutant spores were more sensitive to wet-heat treatment and germinated at a lower rate than wild-type spores and that these phenotypes were significantly restored in the bclS-complemented strain. These results suggested novel roles of collagen-like protein in exosporium assembly and spore germination, providing a hint for a further understanding of the genetic basis of the high level of persistence of Bin toxins in nature.

A new tubRZ operon involved in the maintenance of the Bacillus sphaericus mosquitocidal plasmid pBsph.

pBsph is a mosquitocidal plasmid first identified from Bacillus sphaericus, encoding binary toxins (Bin toxins) that are highly toxic to mosquito larvae. This plasmid plays an important role in the maintenance and evolution of the bin genes in B. sphaericus. However, little is known about its replication and partitioning. Here, we identified a 2.4 kb minimal replicon of pBsph plasmid that contained an operon encoding TubR-Bs and TubZ-Bs, each of which was shown to be required for plasmid replication. TubR-Bs was shown to be a transcriptional repressor of tubRZ-Bs genes and could bind cooperatively to the replication origin of eleven 12 bp degenerate repeats in three blocks, and this binding was essential for plasmid replication. TubZ-Bs exhibited GTPase activities and interacted with TubR-Bs : DNA complex to form a ternary nucleoprotein apparatus. Electron and fluorescence microscopy revealed that TubZ-Bs assembled filaments both in vitro and in vivo, and two point mutations in TubZ-Bs (T114A and Y260A) that severely impaired the GTPase and polymerization activities were found to be defective for plasmid maintenance. Further investigation demonstrated that overproduction of TubZ-Bs-GFP or its mutant forms significantly reduced the stability of pBsph. Taken together, these results suggested that TubR-Bs and TubZ-Bs are involved in the replication and probably in the partitioning of pBsph plasmid, increasing our understanding of the genetic particularity of TubZ systems.

Mycobacterium sulfur metabolism and implications for novel drug targets.

New antibiotic targets are urgently needed to tackle the multidrug resistant and latent Mycobacterium tuberculosis, the causative agent of the most formidable infectious disease tuberculosis. Sulfur metabolism is essential for the survival and virulence of many pathogens including M. tuberculosis. The absence of most genes involved in microbial sulfur metabolism in human beings suggests abundant novel potential antibiotic targets in pathogen sulfur metabolism. In this article, a comparative genomic landscape of Mycobacterium sulfur metabolism, such as the uptake, activation, and reduction of sulfate and allied enzymes, the biosynthesis pathway of some sulfated metabolites, and the enzymes involved in these pathways were presented. Novel clues for antibiotic targets are put forward.

[Regulatory mechanism underlying pathogen biofilm formation and potential drug targets].

Bacterial communities usually develop biofilms abound in nature niche. The development of biofilm is a highly dynamic and complex process coordinated by multiple mechanisms, of which two-component system and quorum sensing are two well-defined systems. Biofilm is involved in the virulence of many pathogens. Therefore, targeting the key factors involved in the biofilm formation represents a novel and promising avenue for developing better antibiotics.