Metabolic Analysis of Intracellular Pathogenic Bacteria Using NMR.

Pathogen proliferation and virulence depend on available nutrients, and these vary when the pathogen moves from outside of the host cell (extracellular) to the inside of the host cell (intracellular). Nuclear Magnetic Resonance (NMR) is a versatile analytical method, which lends itself for metabolic studies. In this chapter, we describe how 1H NMR can be combined with a cellular infection model to study the metabolic crosstalk between a bacterial pathogen and its host both in the extracellular and intracellular compartments. Central carbon metabolism is highlighted by using glucose labeled with the stable isotope 13C.

Visualizing Metabolism in Biotechnologically Important Yeasts with dDNP NMR Reveals Evolutionary Strategies and Glycolytic Logic.

Saccharomyces cerevisiae has long been a pillar of biotechnological production and basic research. More recently, strides to exploit the functional repertoire of nonconventional yeasts for biotechnological production have been made. Genomes and genetic tools for these yeasts are not always available, and yeast genomics alone may be insufficient to determine the functional features in yeast metabolism. Hence, functional assays of metabolism, ideally in the living cell, are best suited to characterize the cellular biochemistry of such yeasts. Advanced in cell NMR methods can allow the direct observation of carbohydrate influx into central metabolism on a seconds time scale: dDNP NMR spectroscopy temporarily enhances the nuclear spin polarization of substrates by more than 4 orders of magnitude prior to functional assays probing central metabolism. We use various dDNP enhanced carbohydrates for in-cell NMR to compare the metabolism of S. cerevisiae and nonconventional yeasts, with an emphasis on the wine yeast Hanseniaspora uvarum. In-cell observations indicated more rapid exhaustion of free cytosolic NAD+ in H. uvarum and alternative routes for pyruvate conversion, in particular, rapid amination to alanine. In-cell observations indicated that S. cerevisiae outcompetes other biotechnologically relevant yeasts by rapid ethanol formation due to the efficient adaptation of cofactor pools and the removal of competing reactions from the cytosol. By contrast, other yeasts were better poised to use redox neutral processes that avoided CO2-emission. Beyond visualizing the different cellular strategies for arriving at redox neutral end points, in-cell dDNP NMR probing showed that glycolytic logic is more conserved: nontoxic precursors of cellular building blocks formed high-population intermediates in the influx of glucose into the central metabolism of eight different biotechnologically important yeasts. Unsupervised clustering validated that the observation of rapid intracellular chemistry is a viable means to functionally classify biotechnologically important organisms.

Working Regime Criteria for Microscale Electrohydrodynamic Conduction Pumps.

We investigated the microscale electrohydrodynamic (EHD) conduction pumps in a wide range of working regimes, from the saturation regime to the ohmic regime. We showed that the existing macro- and microscale theoretical models could not accurately predict the electric force of microscale EHD conduction pumps, especially for the cases of a strong diffusion effect. We clarified that the failure is caused by a rough estimate of the heterocharge layer thickness. We revised the expression of heterocharge layer thickness by considering the diffusion effect and developed a new theoretical model for the microscale EHD conduction pumps based on the revised expression of heterocharge layer thickness. The results showed that our model can accurately predict the dimensionless electric force of the microscale EHD conduction pumps even for the cases of a strong diffusion effect. Furthermore, we developed a working regime map of microscale EHD conduction pumps and found that the microscale EHD conduction pumps more easily fall into the saturation regime compared with the macroscale EHD conduction pumps due to the enhanced diffusion effect; in other words, the microscale EHD conduction pumps have a wider saturation regime. We showed that the conduction number C0 could not distinguish the working regime of the microscale EHD conduction pumps because it does not take the diffusion effect into account. By employing the revised expression of heterocharge layer thickness, we proposed a new dimensionless number, C0D to distinguish the working regimes of microscale EHD conduction pumps.

Rapid probing of glucose influx into cancer cell metabolism: using adjuvant and a pH-dependent collection of central metabolites to improve in-cell D-DNP NMR.

Changes to metabolism are a hallmark of many diseases. Disease metabolism under physiological conditions can be probed in real time with in-cell NMR assays. Here, we pursued a systematic approach towards improved in-cell NMR assays. Unambiguous identifications of metabolites and of intracellular pH are afforded by a comprehensive, downloadable collection of spectral data for central carbon metabolites in the physiological pH range (4.0-8.0). Chemical shifts of glycolytic intermediates provide unique pH dependent patterns akin to a barcode. Using hyperpolarized 13C1 enriched glucose as the probe molecule of central metabolism in cancer, we find that early glycolytic intermediates are detectable in PC-3 prostate cancer cell lines, concurrently yielding intracellular pH. Using non-enriched and non-enhanced pyruvate as an adjuvant, reactions of the pentose phosphate pathway become additionally detectable, without significant changes to the barriers in upper glycolysis and to intracellular pH. The scope of tracers for in-cell observations can thus be improved by the presence of adjuvants, showing that a recently proposed effect of pyruvate in the tumor environment is paralleled by a rerouting of cancer cell metabolism towards producing building blocks for proliferation. Overall, the combined use of reference data for compound identification, site specific labelling for reducing overlap, and use of adjuvant afford increasingly detailed insight into disease metabolism.

Formate Metabolism in Shigella flexneri and Its Effect on HeLa Cells at Different Stages during the Infectious Process.

Shigellosis caused by Shigella is one of the most important foodborne illnesses in global health, but little is known about the metabolic cross talk between this bacterial pathogen and its host cells during the different stages of the infection process. A detailed understanding of the metabolism can potentially lead to new drug targets remedying the pressing problem of antibiotic resistance. Here, we use stable isotope-resolved metabolomics as an unbiased and fast method to investigate how Shigella metabolizes 13C-glucose in three different environments: inside the host cells, adhering to the host cells, and alone in suspension. We find that especially formate metabolism by bacteria is sensitive to these different environments. The role of formate in pathogen metabolism is sparsely described in the literature compared to the roles of acetate and butyrate. However, its metabolic pathway is regarded as a potential drug target due to its production in microorganisms and its absence in humans. Our study provides new knowledge about the regulatory effect of formate. Bacterial metabolism of formate is pH dependent when studied alone in culture medium, whereas this effect is less pronounced when the bacteria adhere to the host cells. Once the bacteria are inside the host cells, we find that formate accumulation is reduced. Formate also affects the host cells resulting in a reduced infection rate. This was correlated to an increased immune response. Thus, intriguingly formate plays a double role in pathogenesis by increasing the virulence of Shigella and at the same time stimulating the immune response of the host. IMPORTANCE Bacterial infection is a pressing societal concern due to development of resistance toward known antibiotics. Central carbon metabolism has been suggested as a potential new target for drug development, but metabolic changes upon infection remain incompletely understood. Here, we used a cellular infection model to study how the bacterial pathogen Shigella adapts its metabolism depending on the environment starting from the extracellular medium until Shigella successfully invaded and proliferated inside host cells. The mixed-acid fermentation of Shigella was the major metabolic pathway during the infectious process, and the glucose-derived metabolite formate surprisingly played a divergent role in the pathogen and in the host cell. Our data show reduced infection rate when both host cells and bacteria were treated with formate, which correlated with an upregulated immune response in the host cells. The formate metabolism in Shigella thus potentially provides a route toward alternative treatment strategies for Shigella prevention.

A magnetic field controlled memristor towards the design of an implantable detector.

Memristors, which combine the behaviors of memory and resistive switching (RS), have a wide application prospect in information processing and artificial neural networks. The RS memory behaviors of memristors are primarily determined by the functional layer materials, device structure, and working conditions. Herein, a CuMnO2 nanomaterial with the manganese copper ore structure was prepared on a Ti substrate by hydrothermal method, and a memristor with the Ag/CuMnO2/Ti sandwich structure was developed. The RS memory behavior of the as-prepared memristor can be regulated through a low magnetic field (MF), and thus the resistance value of device shows a multi-level resistance states. Compared with other regulation factors, the MF can remotely adjust and control the RS characteristics of memristor, which is a non-invasive and non-destructive regulatory means. The MF regulated memristor can not only be used for multi-level high-density information storage, but also it can protect the health of special populations by identifying the MF intensity of the surrounding environment. When the device is operated in an MF environment, the change of resistance value of the device in both high resistance state (HRS) and low resistance state (LRS) is mainly attributed to the influence of Loren magnetic force on conductive ions.

Memristor-based neural networks: a bridge from device to artificial intelligence.

Since the beginning of the 21st century, there is no doubt that the importance of artificial intelligence has been highlighted in many fields, among which the memristor-based artificial neural network technology is expected to break through the limitation of von Neumann so as to realize the replication of the human brain by enabling strong parallel computing ability and efficient data processing and become an important way towards the next generation of artificial intelligence. A new type of nanodevice, namely memristor, which is based on the variability of its resistance value, not only has very important applications in nonvolatile information storage, but also presents obsessive progressiveness in highly integrated circuits, making it one of the most promising circuit components in the post-Moore era. In particular, memristors can effectively simulate neural synapses and build neural networks; thus, they can be applied for the preparation of various artificial intelligence systems. This study reviews the research progress of memristors in artificial neural networks in detail and highlights the structural advantages and frontier applications of neural networks based on memristors. Finally, some urgent problems and challenges in current research are summarized and corresponding solutions and future development trends are put forward.

In-Depth Physical Mechanism Analysis and Wearable Applications of HfOx-Based Flexible Memristors.

Since memristors as an emerging nonlinear electronic component have been considered the most promising candidate for integrating nonvolatile memory and advanced computing technology, the in-depth reveal of the memristive mechanism and the realization of hardware fabrication have facilitated their wide applications in next-generation artificial intelligence. Flexible memristors have shown great promising prospects in wearable electronics and artificial electronic skin (e-skin), but in-depth research on the physical mechanism is still lacking. Here, a flexible memristive device with a Ag/HfOx/Ti/PET crossbar structure was fabricated, and a remarkable analog switching characteristic similar to synaptic behavior was observed. Through detailed data fitting and in-depth physical mechanism analysis, it is confirmed that the analog switching characteristics of the device are mainly caused by carrier tunneling. Furthermore, the memristive properties of the Ag/HfOx/Ag/PET device can be attributed to the conductive filaments formed by the redox reaction of the active metal Ag. Finally, the interfacial barrier is extracted by the Arrhenius diagram and the energy band diagram, which is drawn to clearly demonstrate the conduction mechanism of charge trapping in the device. Therefore, the HfOx-based flexible memristor with analog switching behavior and stable memory performance lays the foundation for cutting-edge applications in wearable electronics and smart e-skin.

Prediction of distant metastasis and survival prediction of gastric cancer patients with metastasis to the liver, lung, bone, and brain: research based on the SEER database.

BACKGROUND: Gastric cancer (GC) is a globally important disease. It is the 5th most common malignancy and the 4th most common cause of death from cancer in the world. Patients with GC are often at an advanced stage when they are first diagnosed, and their overall prognosis is poor due to locally advanced and distant metastasis. This study sought to establish a predictive model of GC distant metastasis and survival that can be used to guide individualized treatment. METHODS: Patients diagnosed with GC from the Surveillance, Epidemiology, and End Results database were enrolled in the study. Univariate and multivariate logistic regression analyses were used to identify risk and prognostic factors for GC patients with distant metastasis. The factors were then used to construct nomograms to predict the probability of distant metastasis and the survival time of GC patients. Receiver operating characteristic (ROC) curve and decision curve analyses were used to verify the prediction ability of the nomograms. RESULTS: We established a comprehensive nomogram to predict the survival time of GC patients and 4 nomograms to predict distant metastasis. Nomograms could help oncologists to formulate treatment strategies and provide hospice care under an overall management model. CONCLUSIONS: Establishing a prediction model for distant metastasis and the survival of GC patients is of great clinical significance. The prediction of distant metastasis could help clinicians to make individualized assessments of patients and formulate individualized examination measures. Survival prediction models could help oncologists to formulate good treatment strategies and provide hospice care.

Biomechanical feasibility of semi-rigid stabilization and semi-rigid lumbar interbody fusion: a finite element study.

BACKGROUND: Semi-rigid lumbar fusion offers a compromise between pedicle screw-based rigid fixation and non-instrumented lumbar fusion. However, the use of semi-rigid interspinous stabilization (SIS) with interspinous spacer and ligamentoplasty and semi-rigid posterior instrumentation (SPI) to assist interbody cage as fusion constructs remained controversial. The purpose of this study is to investigate the biomechanical properties of semi-rigidly stabilized lumbar fusion using SIS or SPI and their effect on adjacent levels using finite element (FE) method. METHOD: Eight FE models were constructed to simulate the lumbosacral spine. In the non-fusion constructs, semi-rigid stabilization with (i) semi-rigid interspinous spacer and artificial ligaments (PD-SIS), and (ii) PI with semi-rigid rods were simulated (PD + SPI). For fusion constructs, the spinal models were implanted with (iii) PEEK cage only (Cage), (iv) PEEK cage and SIS (Cage+SIS), (v) PEEK cage and SPI (Cage+SPI), (vi) PEEK cage and rigid PI (Cage+PI). RESULT: The comparison of flexion-extension range of motion (ROM) in the operated level showed the difference between Cage+SIS, Cage+SPI, and Cage+PI was less than 0.05 degree. In axial rotation, ROM of Cage+SIS were greater than Cage+PI by 0.81 degree. In the infrajacent level, while Cage+PI increased the ROM by 24.1, 27,7, 25.9, and 10.3% and Cage+SPI increased the ROM by 26.1, 30.0, 27.1, and 10.8% in flexion, extension, lateral bending and axial rotation respectively, Cage+SIS only increased the ROM by 3.6, 2.8, and 11.2% in flexion, extension, and lateral bending and reduced the ROM by 1.5% in axial rotation. The comparison of the von Mises stress showed that SIS reduced the adjacent IVD stress by 9.0%. The simulation of the strain energy showed a difference between constructs less than 7.9%, but all constructs increased the strain energy in the infradjacent level. CONCLUSION: FE simulation showed semi-rigid fusion constructs including Cage+SIS and Cage+SPI can provide sufficient stabilization and flexion-extension ROM reduction at the fusion level. In addition, SIS-assisted fusion resulted in less hypermobility and less von Mises stress in the adjacent levels. However, SIS-assisted fusion had a disadvantage of less ROM reduction in lateral bending and axial rotation. Further clinical studies are warranted to investigate the clinical efficacy and safety of semi-rigid fusions.

Acupuncture for Quality of Life in Gastric Cancer Patients Undergoing Adjuvant Chemotherapy.

CONTEXT: Patients with gastric cancer experience health-related quality of life (HRQOL) decline during adjuvant chemotherapy following gastrectomy. OBJECTIVES: This pilot study aimed to evaluate the preliminary effect and feasibility of electro-acupuncture (EA) for HRQOL and symptom burden in these patients. METHODS: In this open-label, multicenter, parallel controlled trial, gastric cancer patients who planned to receive adjuvant chemotherapy were randomly assigned to receive high-dose EA (seven times each chemotherapy cycle for three cycles), low-dose EA (three times each chemotherapy cycle), or usual care only. The acupoints prescription consisted of bilateral ST36, PC6, SP4, and DU20, EX-HN3, and selected Back-shu points. Patients completed the Functional Assessment of Cancer Therapy-Gastric (FACT-Ga) weekly, and the Edmonton Symptom Assessment System (ESAS). The primary outcome was the difference among the groups on the gastric cancer subscale (GaCS) of the FACT-Ga. RESULTS: Of the 66 randomized patients, 58 were analyzed according to intention-to-treat principle, and 45 were in the per-protocol set (PPS). The average scores in PPS of GaCS were 52.12±9.71, 51.85±12.36, and 45.37±8.61 in high-dose EA, low-dose EA, and control groups, respectively. EA was significantly associated with improved average GaCS scores when compared with control group (51.98±10.91 vs. 45.37±8.61, P = 0.039). EA treatment also produced ESAS relief at the end of intervention (14.36 ± 12.28 vs. 23.91 ± 15.52, P = 0.027). Participants in EA groups had fewer grade ≥3 leukopenia (0% vs. 15.79%, P = 0.031) and neutropenia (2.56% vs. 26.31%, P = 0.012). CONCLUSION: EA showed promising effects in improving HRQOL, controlling symptom burden, and reducing toxicity during adjuvant chemotherapy in gastric cancer patients. Future adequately powered trials are feasible and needed to confirm the specific effect of EA.

Universal germline testing among patients with colorectal cancer: clinical actionability and optimised panel.

PURPOSE: Universal germline testing in patients with colorectal cancer (CRC) with a multigene panel can detect various hereditary cancer syndromes. This study was performed to understand how to choose a testing panel and whether the result would affect clinical management. METHODS: We prospectively enrolled 486 eligible patients with CRC, including all patients with CRC diagnosed under age 70 years and patients with CRC diagnosed over 70 years with hereditary risk features between November 2017 and January 2018. All participants received germline testing for various hereditary cancer syndromes. RESULTS: The prevalence of germline pathogenic variants (PVs) in cancer susceptibility genes was 7.8% (38/486), including 25 PVs in genes with high-risk CRC susceptibility (the minimal testing set) and 13 PVs in genes with moderate-risk CRC susceptibility or increased cancer risk other than CRC (the additional testing set). All the clinically relevant PVs were found in patients diagnosed under age 70 years. Among them, 11 patients would not have been diagnosed if testing reserved to present guidelines. Most (36/38) of the patients with PVs benefited from enhanced surveillance and tailored treatment. PVs in genes from the minimal testing set were found in all age groups, while patients carried PVs in genes from the additional testing set were older than 40 years. CONCLUSION: Universal germline testing for cancer susceptibility genes should be recommended among all patients with CRC diagnosed under age 70 years. A broad panel including genes from the additional testing set might be considered for patients with CRC older than 40 years to clarify inheritance risks. TRIAL REGISTRATION NUMBER: NCT03365986.

Genotypic Diversity of Ciprofloxacin Nonsusceptibility and Its Relationship with Minimum Inhibitory Concentrations in Nontyphoidal Salmonella Clinical Isolates in Taiwan.

This study analyzed the genetic diversity of ciprofloxacin (CIP) nonsusceptibility and the relationship between two major mechanisms and minimum inhibitory concentrations (MICs) of CIP in nontyphoidal Salmonella (NTS). Chromosomal mutations in quinolone resistance-determining regions (QRDRs) and plasmid-mediated quinolone resistance (PMQR) genes were searched from ResFinder, ARG-ANNOT, and PubMed for designing the sequencing regions in gyrA, gyrB, parC, and parE, and the 13 polymerase chain reactions for PMQR genes. We found that QRDR mutations were detected in gyrA (82.1%), parC (59.0%), and parE (20.5%) but not in gyrB among the 39 isolates. Five of the 13 PMQR genes were identified, including oqxA (28.2%), oqxB (28.2%), qnrS (18.0%), aac(6')-Ib-cr (10.3%), and qnrB (5.1%), which correlated with the MICs of CIP within 0.25-2 µg/mL, and it was found that oxqAB contributed more than qnr genes to increase the MICs. All the isolates contained either QRDR mutations (53.8%), PMQR genes (15.4%), or both (30.8%). QRDR mutations (84.6%) were more commonly detected than PMQR genes (46.2%). QRDR mutation numbers were significantly associated with MICs (p < 0.001). Double mutations in gyrA and parC determined high CIP resistance (MICs ≥ 4 µg/mL). PMQR genes contributed to intermediate to low CIP resistance (MICs 0.25-2 µg/mL), thus providing insights into mechanisms underlying CIP resistance.

Efficacy and safety of weekly rifapentine and isoniazid for tuberculosis prevention in Chinese silicosis patients: a randomized controlled trial.

OBJECTIVE: The aim was to evaluate the efficacy, safety and completion rate of 3-month, once-weekly rifapentine and isoniazid for tuberculosis (TB) prevention among Chinese silicosis patients. METHODS: Male silicosis patients without human immunodeficiency virus infection, aged 18 years to 65 years, with or without latent TB infection, were randomized 1:1 to receive rifapentine/isoniazid under direct observation (3RPT/INH group) or were untreated (observation group). Active TB incidence was compared between the two groups with 37 months of follow-up. Safety profile and complete rates were evaluated. RESULTS: A total of 1227 adults with silicosis were screened; 513 eligible participants were enrolled and assigned to 3RPT/INH (n = 254) vs. observation (n = 259). Twenty-eight participants were diagnosed with active TB, and 9 and 19 in the 3RPT/INH group and observation groups, respectively. In the intention-to-treat analysis, the cumulative active TB rate was 3.5% (9/254) in the 3RPT/INH group and 7.3% (19/259) in the observation group (log rank p 0.055). On per protocol analysis, the cumulative active TB rates were 0.7% (1/139) and 7.3% (19/259), respectively (log rank p 0.01). Owing to an unexpected high frequency of adverse events (70.4%) and Grade 3 or 4 AEs (7.9%), the completion rate of the 3RPT/INH regimen was 54.7% (139/254). Twenty-six (10.8%) participants had flu-like systemic drug reactions; five (2.1%) experienced hepatotoxicity. DISCUSSION: Weekly rifapentine/isoniazid prophylaxis prevented active TB among Chinese people with silicosis when taken, irrespective of LTBI screening; efficacy was reduced by lack of compliance. The regimen must be used with caution because of the high rates of adverse effects. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov number: NCT02430259.

The establishment of a hub differential gene model to predict prognosis in stage II and III right- and left-sided colon cancer patients.

BACKGROUND: It is now recognized that the symptoms of colon cancer differ according to whether the tumor is located on the left or right side of the patient. The results of the present study point to the differences in the tissue and embryonic origins of left- and right-sided colon cancer that cause the variations in molecular typing. The research purpose of this study is to establish a core differential gene scoring model and proved its effect. METHODS: We downloaded transcriptome data and clinical information from The Cancer Genome Atlas (TCGA). A total of 243 patients in stages II and III were grouped according to the colon cancer site. Then we screened for differential transcriptome products. The corresponding differential gene were performing a corresponding protein interaction analysis. We used 12 algorithms in Cytoscape to calculate the hub genes and a total of 37 hub genes were obtained finally. We extracted the first principal component value (PC1) of the hub genes to evaluate the effectiveness of screening. Cox regression analysis was performed for the differential genes. Finally, we performed a prognostic analysis on right-sided colon cancer patients using the BST2 gene, PC1 and relevant clinical information. RESULTS: After screening for differentially expressed genes, 37 hub genes were obtained with appropriate algorithms. PC1 showed differences in hub genes between left- and right-sided colon cancer patients. BST2 and 31 other genes were identified as significant by Cox regression analysis and were significantly mutated in patients with right-sided colon cancer. Finally, we selected the BST2 gene and relevant clinical information as the prognostic factors to build a scoring model. The prediction effect of the model was satisfied. CONCLUSIONS: We constructed a prognostic model based on BST2, PC1, and other relevant clinical information and proved its good effect.

Low prevalence of mismatch repair deficiency in Chinese colorectal cancers: a multicenter study.

BACKGROUND: Although universal testing for mismatch repair deficiency (dMMR) has been recommended to all colorectal cancer (CRC) patients, related evidence for the Chinese population is lacking. Here, we investigated the prevalence and clinicopathological features of dMMR patients in a large Chinese CRC cohort. METHODS: We included 7,373 CRC patients treated at four Chinese medical centers between August 2010 and September 2016. Patients' baseline characteristics and pathological features were recorded. The clinicopathological features were compared between patients with MLH1/PMS2 deficiency (dMLH1/PMS2) and MSH2/MSH6 deficiency (dMSH2/MSH6). RESULTS: Among the investigated patients, 654 (8.9%) were identified with dMMR CRCs and, of them, 401 (61.3%) were males, with a median age of 55 years (range, 22-87 years); 355 (54.3%) had stage II CRC based on American Joint Committee on Cancer 8th edition. The prevalence of the dMLH1/PMS2 group and the dMSH2/MSH6 group were 51.5% (337/654) and 25.1% (164/654), respectively. Compared with dMSH2/MSH6 patients, those with dMLH1/PMS2 were older (57 vs 52 years, P < 0.001), more likely to be female (45.7% vs 31.5%, P = 0.004), prone to having tumors located in the right-hand side of the colon (59.0% vs 47.6%, P = 0.015), and less likely to have a family history of tumors (29.7% vs 43.3%, P = 0.003). CONCLUSIONS: The prevalence of dMMR in Chinese CRC patients was low, especially in the dMLH1/PMS2 group. The clinicopathological features were different between dMMR subgroups.

Role of wzxE in Salmonella Typhimurium lipopolysaccharide biosynthesis and interleukin-8 secretion regulation in human intestinal epithelial cells.

In Salmonella Typhimurium (S. Typhimurium), lipopolysaccharide (LPS) anchored on the bacterial outer membrane is a major immune stimulus that can broadly activate immune cells and induce innate immune responses. wzxE is involved in bacterial LPS biosynthesis but has rarely been reported in Salmonella; wzxE encodes a flipase that can flip the precursor of LPS across the membrane into the periplasm space. Our preliminary data showed that the wzxE transposon mutant of S. Typhimurium could not significantly adhere to and invade into HEp-2 cells, but the mechanism remains unknown. In this study, we infected human LS174T, Caco-2, HeLa, and THP-1 cells with the wild-type S. Typhimurium strain SL1344, its wzxE mutant, and its complemented strain. wzxE depletion significantly attenuated bacterial adhesion and internalization in the four cell types. In addition, the postinfectious production of interleukin-8 (IL-8) was significantly decreased in the Caco-2 cells infected with the wzxE mutant. Bacterial LPS stained with polymyxin B probe also exhibited a reduced signal in the wzxE mutant. The silver staining of purified LPS demonstrated a significant reduction of the O-antigen (OAg) chain in the wzxE mutant. To confirm the role of OAg in the wzxE mutant during infection, we treated the HT-29 cells with the S. Typhimurium strain SL1344, its wzxE mutant, and their purified LPS, which revealed significantly decreased IL-8 secretion in the HT-29 cells treated with purified LPS from the wzxE mutant and with the wzxE mutant. In conclusion, wzxE mediates LPS biosynthesis and plays a major role in bacterial pathogenesis by regulating OAg flipping.

PD-1 blockade in neoadjuvant setting of DNA mismatch repair-deficient/microsatellite instability-high colorectal cancer.

Background: Although PD-1 blockade has significantly improved the survival of metastatic colorectal cancer with DNA Mismatch Repair-Deficient/Microsatellite Instability-High (MSI-H), the data on neoadjuvant setting is limited. Methods: In this retrospective study, we enrolled eight patients with advanced MSI-H colorectal cancer from three hospitals. Four patients are locally advanced and four are metastatic. All the patients received at least two doses of PD-1 antibody with or without chemotherapy as neoadjuvant therapy. The aim of the present study was to evaluate the short-term efficacy and toxicities of this strategy. Results: All the enrolled eight patients had a major response in imaging and/or pathological evaluation. Five of the seven resected patients were evaluated as pathological complete response. One patient without surgery has a clinical complete response (cCR) tumor response. Conclusions: Neoadjuvant PD-1 blockade induced tumor regression with a major clinical and pathological response in advanced dMMR/MSI-H colorectal cancer. Further studies are required to evaluate the long-term effect of this strategy.

speG Is Required for Intracellular Replication of Salmonella in Various Human Cells and Affects Its Polyamine Metabolism and Global Transcriptomes.

The speG gene has been reported to regulate polyamine metabolism in Escherichia coli and Shigella, but its role in Salmonella remains unknown. Our preliminary studies have revealed that speG widely affects the transcriptomes of infected in vitro M and Caco-2 cells and that it is required for the intracellular replication of Salmonella enterica serovar Typhimurium (S. Typhimurium) in HeLa cells. In this study, we demonstrated that speG plays a time-dependent and cell type-independent role in the intracellular replication of S. Typhimurium. Moreover, high-performance liquid chromatography (HPLC) of four major polyamines demonstrated putrescine, spermine, and cadaverine as the leading polyamines in S. Typhimurium. The deletion of speG significantly increased the levels of the three polyamines in intracellular S. Typhimurium, suggesting the inhibitory effect of speG on the biosynthesis of these polyamines. The deletion of speG was associated with elevated levels of these polyamines in the attenuated intracellular replication of S. Typhimurium in host cells. This result was subsequently validated by the dose-dependent suppression of intracellular proliferation after the addition of the polyamines. Furthermore, our RNA transcriptome analysis of S. Typhimurium SL1344 and its speG mutant outside and inside Caco-2 cells revealed that speG regulates the genes associated with flagellar biosynthesis, fimbrial expression, and functions of types III and I secretion systems. speG also affects the expression of genes that have been rarely reported to correlate with polyamine metabolism in Salmonella, including those associated with the periplasmic nitrate reductase system, glucarate metabolism, the phosphotransferase system, cytochromes, and the succinate reductase complex in S. Typhimurium in the mid-log growth phase, as well as those in the ilv-leu and histidine biosynthesis operons of intracellular S. Typhimurium after invasion in Caco-2 cells. In the present study, we characterized the phenotypes and transcriptome effects of speG in S. Typhimurium and reviewed the relevant literature to facilitate a more comprehensive understanding of the potential role of speG in the polyamine metabolism and virulence regulation of Salmonella.

Safety of intraoperative chemotherapy with 5-FU for colorectal cancer patients receiving curative resection: a randomized, multicenter, prospective, phase III IOCCRC trial (IOCCRC).

PURPOSE: The safety and efficacy of intraoperative chemotherapy in colorectal cancer have not yet been extensively investigated. This randomized control trial was designed to compare the safety and efficacy of intraoperative chemotherapy in combination with surgical resection to those of traditional surgical resection alone. METHODS: From January 2011 to January 2016, 696 colorectal cancer patients were enrolled in this study: 341 patients were randomly assigned to the intraoperative chemotherapy, which consist of portal vein chemotherapy, intraluminal chemotherapy and intraperitoneal chemotherapy, plus surgery group, whereas 344 patients were randomized to the control group to undergo surgery alone. Eleven patients withdrew consent. RESULTS: Intraoperative chemotherapy did not increase the rate of surgical complications, and no severe chemotherapy-associated side effects were observed. Four patients in each of the intraoperative chemotherapy and the control groups experienced anastomotic leakage and underwent a second operation (1.2 vs. 1.2%, P = 0.99). There were no deaths within 90 days after surgery in the chemotherapy group, whereas one patient died in the control group. Intraoperative chemotherapy did not decrease the rate of patients who received postoperative chemotherapy between the intraoperative group and control group (29.3 vs. 30.2%, P = 0.795). CONCLUSIONS: Intraoperative chemotherapy can be safely performed during colorectal surgery; however, follow-up is necessary for a better assessment of its efficacy. ClinicalTrial.gov Register Number: NCT01465451.