Effects of erythromycin on biofilm formation and resistance mutation of Escherichia coli on pristine and UV-aged polystyrene microplastics.

Microplastics (MPs) and antibiotics co-occur widely in the environment and pose combined risk to microbial communities. The present study investigated the effects of erythromycin on biofilm formation and resistance mutation of a model bacterium, E. coli, on the surface of pristine and UV-aged polystyrene (PS) MPs sized 1-2 mm. The properties of UV-aged PS were significantly altered compared to pristine PS, with notable increases in specific surface area, carbonyl index, hydrophilicity, and hydroxyl radical content. Importantly, the adsorption capacity of UV-aged PS towards erythromycin was approximately 8-fold higher than that of pristine PS. Biofilms colonizing on UV-aged PS had a greater cell count (5.6 × 108 CFU mg-1) and a higher frequency of resistance mutation (1.0 × 10-7) than those on pristine PS (1.4 × 108 CFU mg-1 and 1.4 × 10-8, respectively). Moreover, erythromycin at 0.1 and 1.0 mg L-1 significantly (p < 0.05) promoted the formation and resistance mutation of biofilm on both pristine and UV-aged PS. DNA sequencing results confirmed that the biofilm resistance was attributed to point mutations in rpoB segment of the bacterial genome. qPCR results demonstrated that both UV aging and erythromycin repressed the expression levels of a global regulator rpoS in biofilm bacteria, as well as two DNA mismatch repair genes mutS and uvrD, which was likely to contribute to increased resistance mutation frequency.

A degradome-related signature for predicting the prognosis and immunotherapy benefit in stomach adenocarcinoma based on machine learning procedure.

Stomach adenocarcinoma (STAD) is one of the subtype of gastric cancer with high invasiveness, extreme heterogeneity, high morbidity, and high mortality. The degradome is the most abundant class of cellular enzymes that play an essential role in regulating cellular activity and carcinogenesis. An integrative machine learning procedure including 10 methods was performed to develop a prognostic degradome-based prognostic signature (DPS) in TCGA, GSE15459, GSE26253, and GSE62254 datasets. Investigations of the DPS concerning immune infiltration, immunotherapy benefits, and drug priority were orchestrated. The DPS developed by Enet [alpha = 0.3] method was regarded as the optimal prognostic model. The DPS had a stable and powerful performance in predicting the clinical outcome of STAD and served as an independent risk factor in training and testing cohorts. The C-index of DPS was higher than that of age, sex, and clinical stage. STAD patients with low DPS scores had a higher abundance of B cells, CD8+ T cells, higher cytolytic scores, and T cell co-stimulation scores. Moreover, low DPS score indicated a lower tumor immune dysfunction and exclusion score, lower T cell dysfunction and exclusion score, higher PD1&CTLA4 immunophenoscore, and higher tumor mutation burden score in STAD, demonstrating a better immunotherapy response. STAD patients with a high DPS score had a lower IC50 value of common chemotherapy and targeted therapy regimens (Cisplatin, Docetaxel, Gefitinib, etc). Our study developed an optimal DPS for STAD. The DPS could predict the prognosis, risk stratification and guide treatment for STAD patients.

A prognostic nomogram for recurrence survival in post-surgical patients with varicose veins of the lower extremities.

Varicose veins of the lower extremities (VVLEs) are prevalent globally. This study aims to identify prognostic factors and develop a prediction model for recurrence survival (RS) in VVLEs patients after surgery. A retrospective analysis of VVLEs patients from the Third Hospital of Nanchang was conducted between April 2017 and March 2022. A LASSO (Least Absolute Shrinkage and Selection Operator) regression model pinpointed significant recurrence predictors, culminating in a prognostic nomogram. The model's performance was evaluated by C-index, receiver operating characteristic (ROC) curves, calibration plots, and decision curve analysis (DCA). The LASSO regression identified seven predictors for the nomogram predicting 1-, 2-, and 5-year RS. These predictors were age, body mass index (BMI), hypertension, diabetes, the Clinical Etiological Anatomical Pathophysiological (CEAP) grade, iliac vein compression syndrome (IVCS), and postoperative compression stocking duration (PCSD). The nomogram's C-index was 0.716, with AUCs (Area Under the Curve scores) of 0.705, 0.725, and 0.758 for 1-, 2-, and 5-year RS, respectively. Calibration and decision curve analyses validated the model's predictive accuracy and clinical utility. Kaplan-Meier analysis distinguished between low and high-risk groups with significant prognostic differences (P < 0.05). This study has successfully developed and validated a nomogram for predicting RS in patients with VVLEs after surgery, enhancing personalized care and informing clinical decision-making.

Global transmission of broad-host-range plasmids derived from the human gut microbiome.

Broad-host-range (BHR) plasmids in human gut bacteria are of considerable interest for their ability to mediate horizontal gene transfer (HGT) across large phylogenetic distance. However, the human gut plasmids, especially the BHR plasmids, remain largely unknown. Here, we identified the plasmids in the draft genomes of gut bacterial isolates from Chinese and American donors, resulting in 5372 plasmid-like clusters (PLCs), of which, 820 PLCs (comPLCs) were estimated with > 60% completeness genomes and only 155 (18.9%) were classified to known replicon types (n = 37). We observed that 175 comPLCs had a broad host range across distinct bacterial genera, of which, 71 were detected in at least two human populations of Chinese, American, Spanish, and Danish, and 13 were highly prevalent (>10%) in at least one human population. Haplotype analyses of two widespread PLCs demonstrated their spreading and evolutionary trajectory, suggesting frequent and recent exchanges of the BHR plasmids in environments. In conclusion, we obtained a large collection of plasmid sequences in human gut bacteria and demonstrated that a subset of the BHR plasmids can be transmitted globally, thus facilitating extensive HGT (e.g. antibiotic resistance genes) events. This study highlights the potential implications of the plasmids for global human health.

Characterization of a novel genus of jumbo phages and their application in wastewater treatment.

Phages widely exist in numerous environments from wastewater to deep ocean, representing a huge virus diversity, yet remain poorly characterized. Among them, jumbo phages are of particular interests due to their large genome (>200 kb) and unusual biology. To date, only six strains of jumbo phages infecting Klebsiella pneumoniae have been described. Here, we report the isolation and characterization of two jumbo phages from hospital wastewater representing the sixth genus: φKp5130 and φKp9438. Both phages showed lytic activity against broad range of clinical antibiotic-resistant K. pneumoniae strains and distinct physiology including long latent period, small burst size, and high resistance to thermal and pH stress. The treatment of sewage water with the phages cocktail resulted in dramatic decline in K. pneumoniae population. Overall, this study provides detailed molecular and genomics characterization of two novel jumbo phages, expands viral diversity, and provides novel candidate phages to facilitate environmental wastewater treatment.

Multi-scale feature pyramid fusion network for medical image segmentation.

PURPOSE: Medical image segmentation is the most widely used technique in diagnostic and clinical research. However, accurate segmentation of target organs from blurred border regions and low-contrast adjacent organs in Computed tomography (CT) imaging is crucial for clinical diagnosis and treatment. METHODS: In this article, we propose a Multi-Scale Feature Pyramid Fusion Network (MS-Net) based on the codec structure formed by the combination of Multi-Scale Attention Module (MSAM) and Stacked Feature Pyramid Module (SFPM). Among them, MSAM is used to skip connections, which aims to extract different levels of context details by dynamically adjusting the receptive fields under different network depths; the SFPM including multi-scale strategies and multi-layer Feature Perception Module (FPM) is nested in the network at the deepest point, which aims to better focus the network's attention on the target organ by adaptively increasing the weight of the features of interest. RESULTS: Experiments demonstrate that the proposed MS-Net significantly improved the Dice score from 91.74% to 94.54% on CHAOS, from 97.59% to 98.59% on Lung, and from 82.55% to 86.06% on ISIC 2018, compared with U-Net. Additionally, comparisons with other six state-of-the-art codec structures also show the presented network has great advantages on evaluation indicators such as Miou, Dice, ACC and AUC. CONCLUSION: The experimental results show that both the MSAM and SFPM techniques proposed in this paper can assist the network to improve the segmentation effect, so that the proposed MS-Net method achieves better results in the CHAOS, Lung and ISIC 2018 segmentation tasks.

Characterization of the evolution trajectory and immune profiling of new histologic patterns in lung adenocarcinoma.

In lung adenocarcinoma (LUAD), the appearance of morphologically diverse tumor regions, termed histological patterns, is closely associated with disease progression and lymph node metastasis. However, the molecular characteristics of the histological patterns in LUAD and the underlying molecular evolutionary mechanisms between the histological patterns in primary tumors and lymph node metastases are poorly understood. Here, we re-analyzed the large TCGA-LUAD dataset and depicted a comprehensive profiling of the genome and transcriptome across the histological patterns in LUAD. Tumor phylogenetic trajectory analysis suggested that the complex glands is more apt to metastasize to the lymph node. Further deconvolution of the tumor microenvironment demonstrated that the complex glands had a higher infiltration of cancer-associated fibroblasts (CAFs). Single-cell transcriptome profiling of complex glands pattern identified a novel CAF subtype co-expressing fibroblast activation protein-alpha (FAP) and stimulator of interferon genes (STING). Moreover, our data demonstrated that FAP is an important downstream effector of STING in CAFs. In summary, our results provide the basis for the development of innovative therapeutic guidelines and intervention strategies for LUAD patients.

Comparative genomic analysis of five freshwater cyanophages and reference-guided metagenomic data mining.

BACKGROUND: As important producers using photosynthesis on Earth, cyanobacteria contribute to the oxygenation of atmosphere and the primary production of biosphere. However, due to the eutrophication of urban waterbodies and global warming, uncontrollable growth of cyanobacteria usually leads to the seasonal outbreak of cyanobacterial blooms. Cyanophages, a group of viruses that specifically infect and lyse cyanobacteria, are considered as potential environment-friendly agents to control the harmful blooms. Compared to the marine counterparts, only a few freshwater cyanophages have been isolated and genome sequenced to date, largely limiting their characterizations and applications. RESULTS: Here, we isolated five freshwater cyanophages varying in tail morphology, termed Pam1~Pam5, all of which infect the cyanobacterium Pseudanabaena mucicola Chao 1806 that was isolated from the bloom-suffering Lake Chaohu in Anhui, China. The whole-genome sequencing showed that cyanophages Pam1~Pam5 all contain a dsDNA genome, varying in size from 36 to 142 Kb. Phylogenetic analyses suggested that Pam1~Pam5 possess different DNA packaging mechanisms and are evolutionarily distinct from each other. Notably, Pam1 and Pam5 have lysogeny-associated gene clusters, whereas Pam2 possesses 9 punctuated DNA segments identical to the CRISPR spacers in the host genome. Metagenomic data-based calculation of the relative abundance of Pam1~Pam5 at the Nanfei estuary towards the Lake Chaohu revealed that the short-tailed Pam1 and Pam5 account for the majority of the five cyanophages. Moreover, comparative analyses of the reference genomes of Pam1~Pam5 and previously reported cyanophages enabled us to identify three circular and seven linear contigs of virtual freshwater cyanophages from the metagenomic data of the Lake Chaohu. CONCLUSIONS: We propose a high-throughput strategy to systematically identify cyanophages based on the currently available metagenomic data and the very limited reference genomes of experimentally isolated cyanophages. This strategy could be applied to mine the complete or partial genomes of unculturable bacteriophages and viruses. Transformation of the synthesized whole genomes of these virtual phages/viruses to proper hosts will enable the rescue of bona fide viral particles and eventually enrich the library of microorganisms that exist on Earth. Video abstract.

Role of environmental stresses in elevating resistance mutations in bacteria: Phenomena and mechanisms.

Mutations are an important origin of antibiotic resistance in bacteria. While there is increasing evidence showing promoted resistance mutations by environmental stresses, no retrospective research has yet been conducted on this phenomenon and its mechanisms. Herein, we summarized the phenomena of stress-elevated resistance mutations in bacteria, generalized the regulatory mechanisms and discussed the environmental and human health implications. It is shown that both chemical pollutants, such as antibiotics and other pharmaceuticals, biocides, metals, nanoparticles and disinfection byproducts, and non-chemical stressors, such as ultraviolet radiation, electrical stimulation and starvation, are capable of elevating resistance mutations in bacteria. Notably, resistance mutations are more likely to occur under sublethal or subinhibitory levels of these stresses, suggesting a considerable environmental concern. Further, mechanisms for stress-induced mutations are summarized in several points, namely oxidative stress, SOS response, DNA replication and repair systems, RpoS regulon and biofilm formation, all of which are readily provoked by common environmental stresses. Given bacteria in the environment are confronted with a variety of unfavorable conditions, we propose that the stress-elevated resistance mutations are a universal phenomenon in the environment and represent a nonnegligible risk factor for ecosystems and human health. The present review identifies a need for taking into account the pollutants' ability to elevate resistance mutations when assessing their environmental and human health risks and highlights the necessity of including resistance mutations as a target to prevent antibiotic resistance evolution.

Harnessing stepping-stone hosts to engineer, select, and reboot synthetic bacteriophages in one pot.

Advances in synthetic genomics have led to a great demand for genetic manipulation. Trimming any process to simplify and accelerate streamlining of genetic code into life holds great promise for synthesizing and studying organisms. Here, we develop a simple but powerful stepping-stone strategy to promote genome refactoring of viruses in one pot, validated by successful cross-genus and cross-order rebooting of 90 phages infecting 4 orders of popular pathogens. Genomic sequencing suggests that rebooting outcome is associated with gene number and DNA polymerase availability within phage genomes. We integrate recombineering, screening, and rebooting processes in one pot and demonstrate genome assembly and genome editing of phages by stepping-stone hosts in an efficient and economic manner. Under this framework, in vitro assembly, yeast-based assembly, or genetic manipulation of native hosts are not required. As additional stepping-stone hosts are being developed, this framework will open doors for synthetic phages targeting more pathogens and commensals.

Large-Scale Genomic Epidemiology of Klebsiella pneumoniae Identified Clone Divergence with Hypervirulent Plus Antimicrobial-Resistant Characteristics Causing Within-Ward Strain Transmissions.

Global dissemination of K. pneumoniae clones poses health hazards to the public. Genomic epidemiology studies with comprehensive data set further revealed clone divergence, showing a high complexity in evolution. Moreover, clones carrying both acquired virulent and antimicrobial-resistant genes emerged and might replace the carbapenem-resistant clones. Co-occurrence of virulence and resistance is emerging. An unbiased collection of 3,061 clinical K. pneumoniae isolates (January 5, 2013 to July 24, 2018) underwent whole-genome sequencing. Pairwise core-genome single-nucleotide polymorphism (cgSNP) distances identified clone divergence and transmission events. A sum of 2,193 nonduplicated genomes clustered into four phenotypically indistinguishable species complexes. 93% (n = 2,035) were KpI with its largest clonal group (CG) being CG11 (n = 406). Three hundred ninety-three were ST11 and three hundred seventy-four carried blaKPC-2. Noticeably, CG11 is divided into two main subclones based on the capsule synthesis K loci (KL). CG11-KL64 showed a clear hypervirulent plus antimicrobial-resistant (hv+AMR) characteristic. Besides, the phylogenetic structure revealed the clone divergence of CG25, and this is the first report with sufficient CG25 genomes to identify the divergence. The outcomes of the hv+AMR CG25 cluster 1 affected patients were poorer (P < 0.05). Moreover, two episodes of strain transmissions were associated with CG25 cluster 1. Other transmissions were associated with ST20 and ST307. Genomic epidemiology identified clone divergence of CG11 and CG25. The hv+AMR subclones pose greater threats on a global scale. Nosocomial transmissions of the high-risk clones raised our concerns about the evolution and transmission of emerging clones among newborns and critically ill patients. IMPORTANCE The convergence of AMR and acquired virulence posing higher risks to the public is a focusing point. With sufficient genomes and genotypes, we successfully identify the convergence in two subclones, the previously reported CG11-KL64, and the newly reported CG25 cluster 1. The novel finding of the CG25 divergence was not only revealed by the phylogenetic tree but also confirmed by the clinical outcome data and the accessory genome patterns. Moreover, the transmission subclones circulated in two clinically important wards highlights the deficiency of infection control program using conventional methods. Without the assistance of whole-genome sequencing, the transmissions of high-risk clones could not be identified.

Population Bottlenecks and Intra-host Evolution During Human-to-Human Transmission of SARS-CoV-2.

The emergence of the novel human coronavirus, SARS-CoV-2, causes a global COVID-19 (coronavirus disease 2019) pandemic. Here, we have characterized and compared viral populations of SARS-CoV-2 among COVID-19 patients within and across households. Our work showed an active viral replication activity in the human respiratory tract and the co-existence of genetically distinct viruses within the same host. The inter-host comparison among viral populations further revealed a narrow transmission bottleneck between patients from the same households, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions.

Global Landscape of Clostridioides Difficile Phylogeography, Antibiotic Susceptibility, and Toxin Polymorphisms by Post-Hoc Whole-Genome Sequencing from the MODIFY I/II Studies.

INTRODUCTION: Clostridioides (Clostridium) difficile infection, the leading cause of healthcare-associated diarrhea, represents a significant burden on global healthcare systems. Despite being a global issue, information on C. difficile from a global perspective is lacking. The aim of this study is to model the global phylogeography of clinical C. difficile. METHODS: Using samples collected from the MODIFY I and II studies (NCT01241552, NCT01513239), we performed whole-genome sequencing of 1501 clinical isolates including 37 novel sequence types (STs), representing the largest worldwide collection to date. RESULTS: Our data showed ribotypes, multi-locus sequence typing clades, and whole-genome phylogeny were in good accordance. The clinical C. difficile genome was found to be more conserved than previously reported (61% core genes), and modest recombination rates of 1.4-5.0 were observed across clades. We observed a significant continent distribution preference among five C. difficile clades (Benjamini-Hochberg corrected Fisher's exact test P < 0.01); moreover, weak association between geographic and genetic distance among ribotypes suggested sources beyond healthcare-related transmission. Markedly different trends of antibiotic susceptibility among lineages and regions were identified, and three novel mutations (in pyridoxamine 5'-phosphate oxidase family protein: Tyr130Ser, Tyr130Cys, and a promoter SNP) associated with metronidazole-reduced susceptibility were discovered on a nim-related gene and its promotor by genome-wide association study. Toxin gene polymorphisms were shown to vary within and between prevalent ribotypes, and novel severe mutations were found on the tcdC toxin regulator protein. CONCLUSION: Our systematic characterization of a global set of clinical trial C. difficile isolates from infected individuals demonstrated the complexity of the genetic makeup of this pathogenic organism. The geographic variability of clades, variability in toxin genes, and mutations associated with antibiotic susceptibility indicate a highly complex interaction of C. difficile between host and environment. This dataset will provide a useful resource for validation of findings and future research of C. difficile.

Intra-host variation and evolutionary dynamics of SARS-CoV-2 populations in COVID-19 patients.

BACKGROUND: Since early February 2021, the causative agent of COVID-19, SARS-CoV-2, has infected over 104 million people with more than 2 million deaths according to official reports. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown. METHODS: Using high-throughput sequencing of metatranscriptomic and hybrid captured libraries, we characterized consensus genomes and intra-host single nucleotide variations (iSNVs) of serial samples collected from eight patients with COVID-19. The distribution of iSNVs along the SARS-CoV-2 genome was analyzed and co-occurring iSNVs among COVID-19 patients were identified. We also compared the evolutionary dynamics of SARS-CoV-2 population in the respiratory tract (RT) and gastrointestinal tract (GIT). RESULTS: The 32 consensus genomes revealed the co-existence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in a single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparing allele frequencies of the iSNVs revealed a clear genetic differentiation between intra-host populations from the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events during intra-host migrations. Compared to RT populations, the GIT populations showed a better maintenance and rapid development of viral genetic diversity following the suspected intra-host bottlenecks. CONCLUSIONS: Our findings here illustrate the intra-host bottlenecks and evolutionary dynamics of SARS-CoV-2 in different anatomic sites and may provide new insights to understand the virus-host interactions of coronaviruses and other RNA viruses.

Multiple approaches for massively parallel sequencing of SARS-CoV-2 genomes directly from clinical samples.

BACKGROUND: COVID-19 (coronavirus disease 2019) has caused a major epidemic worldwide; however, much is yet to be known about the epidemiology and evolution of the virus partly due to the scarcity of full-length SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genomes reported. One reason is that the challenges underneath sequencing SARS-CoV-2 directly from clinical samples have not been completely tackled, i.e., sequencing samples with low viral load often results in insufficient viral reads for analyses. METHODS: We applied a novel multiplex PCR amplicon (amplicon)-based and hybrid capture (capture)-based sequencing, as well as ultra-high-throughput metatranscriptomic (meta) sequencing in retrieving complete genomes, inter-individual and intra-individual variations of SARS-CoV-2 from serials dilutions of a cultured isolate, and eight clinical samples covering a range of sample types and viral loads. We also examined and compared the sensitivity, accuracy, and other characteristics of these approaches in a comprehensive manner. RESULTS: We demonstrated that both amplicon and capture methods efficiently enriched SARS-CoV-2 content from clinical samples, while the enrichment efficiency of amplicon outran that of capture in more challenging samples. We found that capture was not as accurate as meta and amplicon in identifying between-sample variations, whereas amplicon method was not as accurate as the other two in investigating within-sample variations, suggesting amplicon sequencing was not suitable for studying virus-host interactions and viral transmission that heavily rely on intra-host dynamics. We illustrated that meta uncovered rich genetic information in the clinical samples besides SARS-CoV-2, providing references for clinical diagnostics and therapeutics. Taken all factors above and cost-effectiveness into consideration, we proposed guidance for how to choose sequencing strategy for SARS-CoV-2 under different situations. CONCLUSIONS: This is, to the best of our knowledge, the first work systematically investigating inter- and intra-individual variations of SARS-CoV-2 using amplicon- and capture-based whole-genome sequencing, as well as the first comparative study among multiple approaches. Our work offers practical solutions for genome sequencing and analyses of SARS-CoV-2 and other emerging viruses.

Prophage Hunter: an integrative hunting tool for active prophages.

Identifying active prophages is critical for studying coevolution of phage and bacteria, investigating phage physiology and biochemistry, and engineering designer phages for diverse applications. We present Prophage Hunter, a tool aimed at hunting for active prophages from whole genome assembly of bacteria. Combining sequence similarity-based matching and genetic features-based machine learning classification, we developed a novel scoring system that exhibits higher accuracy than current tools in predicting active prophages on the validation datasets. The option of skipping similarity matching is also available so that there's higher chance for novel phages to be discovered. Prophage Hunter provides a one-stop web service to extract prophage genomes from bacterial genomes, evaluate the activity of the prophages, identify phylogenetically related phages, and annotate the function of phage proteins. Prophage Hunter is freely available at https://pro-hunter.bgi.com/.

1,520 reference genomes from cultivated human gut bacteria enable functional microbiome analyses.

Reference genomes are essential for metagenomic analyses and functional characterization of the human gut microbiota. We present the Culturable Genome Reference (CGR), a collection of 1,520 nonredundant, high-quality draft genomes generated from >6,000 bacteria cultivated from fecal samples of healthy humans. Of the 1,520 genomes, which were chosen to cover all major bacterial phyla and genera in the human gut, 264 are not represented in existing reference genome catalogs. We show that this increase in the number of reference bacterial genomes improves the rate of mapping metagenomic sequencing reads from 50% to >70%, enabling higher-resolution descriptions of the human gut microbiome. We use the CGR genomes to annotate functions of 338 bacterial species, showing the utility of this resource for functional studies. We also carry out a pan-genome analysis of 38 important human gut species, which reveals the diversity and specificity of functional enrichment between their core and dispensable genomes.

Study of the Expression of Bacterial Multidrug Efflux Pumps in Anaerobic Conditions.

Bacterial multidrug efflux pumps belong to a class of membrane transporter proteins that dedicate to the extrusion of a diverse range of substances out of cells including all classes of currently available antibiotics. They constitute an important mechanism of bacterial antibiotic and multidrug resistance. Since many ecological niches of bacteria and the infection foci in animal host display low oxygen tension under which condition bacterial pathogens undergo fundamental changes on their metabolic modes, it is necessary to study the expression profiles of drug efflux pumps under these physiologically and clinically relevant conditions. In this chapter, we first introduce procedures to culture bacteria under anaerobic conditions, which is achieved using screw-capped Pyrex culture tubes without agitation. We then introduce ß-galactosidase activity assay using promoter-lacZ (encoding the ß-galactosidase enzyme) fusion to measure the expression of efflux pumps at transcriptional level, and Western blot using chromosomal FLAG-tagged construct to examine the expression of these proteins at translational level. Applications of these gene expression studies to reveal the regulatory mechanisms of efflux genes expression as well as their physiological functions are also discussed.

[One case of gastric duplication cyst withectopic pancreas in adults].

Gastric duplication cyst is a very rare gastrointestinal tract malformation that accounts for 2%-4% of alimentary tract duplications. It is a diagnostic dilemma for doctors because its clinical and radiological manifest is usually nonspecific. At the present stage, it can only rely on surgery. We should pay attention to ectopic pancreas resection and ligation of pancreatic duct during operation. There was one case of gastric duplication cyst with ectopic pancreas in adults from the Second Affiliated Hospital of Nanchang University.

[Laparoscopic-assisted resection for colorectal cancer without incision at abdomen versus traditional laparoscopic resection: A Meta-analysis].

OBJECTIVE: To evaluate the safety, feasibility and effectiveness of laparoscopic-assisted resection of colorectal cancer without incision at abdomen vs the traditional laparoscopic resection.
 Methods: We retrieved literature published from August, 2005 to August, 2015 to compare laparoscopic-assisted resection for colorectal cancer without incision at abdomen with the traditional laparoscopic resection. The clinical indicators were extracted from literature met inclusion criteria. The RevMan 5.3 software with a Meta-analysis was used.
 Results: Seven literature with a total of 621 patients, including 262 in laparoscopic-assisted resection of colorectal cancer without auxiliary incision at abdomen group (NOSE group) and 359 in conventional laparoscopic colorectal resection group (LAP group), were enrolled. The Meta-analysis showed that the total complication rate in the NOSE group was significantly less than that in the LAP group (OR=0.31, 95% CI 0.18 to 0.53, P<0.05). Complications of incision in the NOSE group were less than those in the LAP group (OR= 0.15, 95% CI 0.05 to 0.40, P=0.0002). Postoperative bleeding (OR=1.52, 95% CI 0.38 to 6.18, P=0.55), intestinal obstruction (OR= 0.30, 95% CI 0.09 to 0.98, P=0.05), anastomotic complications (OR=0.92, 95% CI 0.28 to 3.07, P=0.89), and other related complications (OR=0.63, 95% CI 0.23 to 1.66, P=0.35) showed no significant difference between the 2 groups (P>0.05). Hospitalization (MD=-0.66, 95% CI -1.33 to 0.01, P=0.05), duration of surgery (MD=14.78, 95% CI -1.75 to 31.31, P=0.08), bleeding amount (MD=-12.81, 95% CI -40.36 to 14.74, P=0.36), the tumor size (SMD=-0.40, 95% CI -0.87 to 0.08, P=0.10), the number of lymph node dissection (MD=-0.49, 95% CI 1.80 to 0.82, P=0.46), and the recurrence of 2-year follow-up (OR=1.15, 95% CI 0.38 to 3.50, P=0.81) were not statistically significant between the 2 groups. Time of gas passage (SMD=-0.62, 95% CI -0.82 to -0.42, P<0.001) and time of regular diet after surgery (SMD=-0.60, 95% CI -1.15 to 0.05, P=0.03) in the NOSE group were earlier than those in the LAP group. The postoperative pain score (MD=-1.49, 95% CI -1.97 to -1.01, P<0.001) in the NOSE group was significantly lower than that in the LAP group. Cosmetic surgery in the NOSE group had a higher index (MD=1.37, 95% CI 0.59 to 2.14, P=0.0005) compared with that in the LAP group.
 Conclusion: Laparoscopic-assisted resection for colorectal cancer without auxiliary incision at abdomen can obviously reduce the incidence of incision complications, and the patients can recover early and incision is showed more cosmetic. The method is safe, feasible, and effective.