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1.

Introduction

Swine dysentery caused by Brachyspira hyodysenteriae is a production limiting disease in pig farming. Currently antimicrobial therapy is the only treatment and control method available.

Objective

The aim of this study was to characterize the metabolic response of porcine colon explants to infection by B. hyodysenteriae.

Methods

Porcine colon explants exposed to B. hyodysenteriae were analyzed for histopathological, metabolic and pro-inflammatory gene expression changes.

Results

Significant epithelial necrosis, increased levels of l-citrulline and IL-1α were observed on explants infected with B. hyodysenteriae.

Conclusions

The spirochete induces necrosis in vitro likely through an inflammatory process mediated by IL-1α and NO.
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2.

Background

All Helicobacter pylori-infected patients are recommended for eradication with an appropriate regimen in each geographic area. The choice of the therapy is somewhat dependent on the antimicrobial susceptibility. The rate of clarithromycin resistance has been increasing and is associated with failure; thus, susceptibility testing is recommended before triple therapy with clarithromycin. However, antimicrobial susceptibility testing is not yet clinically available and an alternative newly developed acid inhibitor vonoprazan is used for triple therapy in Japan. The aim of this study was to determine whether vonoprazan-based triple therapy is plausible treatment in H. pylori eradication.

Methods

A retrospective observational study of H. pylori eradication was conducted in a single institute. The patients who requested antimicrobial susceptibility testing were treated with susceptibility-guided proton pump inhibitor-based triple therapy in International University of Health and Welfare Hospital from 2013 to 2016. Other patients were treated with empirical treatment with a proton pump inhibitor. From 2015 to 2016, vonoprazan-based triple treatment (vonoprazan, 20 mg; amoxicillin, 750 mg; and clarithromycin, 200 or 400 mg, b.i.d.) was conducted, and its effectiveness was compared with susceptibility-guided proton pump inhibitor-based triple therapy. We also investigated the improvement in eradication rate when antimicrobial susceptibility testing was performed, and compared the outcomes of vonoprazan-based and proton pump inhibitor-based empirical therapy.

Results

A total of 1355 patients who received first-line eradication treatment were enrolled in the present study. The eradication rates of the empirical proton pump inhibitor-based therapy and the vonoprazan-based therapy group in a per-protocol analysis were 86.3% (95% CI 83.8–88.8) and 97.4% (95% CI 95.7–99.1), respectively. In 212 patients who received antimicrobial susceptibility testing, the rate of clarithromycin resistant was 23.5% and the eradication rate in susceptibility-guided treatment was 95.7% (95% CI 92.9–98.4). The difference between susceptibility-guided and vonoprazan-based therapy was ??1.7% (95% CI ??4.9 to 1.5%), and the non-inferiority of vonoprazan-based triple therapy was confirmed.

Conclusions

Vonoprazan-based triple therapy was effective as susceptibility-guided triple therapy for H. pylori eradication. An empirical triple therapy with vonoprazan is preferable even in area with high rates of clarithromycin-resistance.Trial registration The study was retrospectively registered in University Hospital Medical Information Network (UMIN000032351)
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3.

Introduction

Hypoxia commonly occurs in cancers and is highly related with the occurrence, development and metastasis of cancer. Treatment of triple negative breast cancer remains challenge. Knowledge about the metabolic status of triple negative breast cancer cell lines in hypoxia is valuable for the understanding of molecular mechanisms of this tumor subtype to develop effective therapeutics.

Objectives

Comprehensively characterize the metabolic profiles of triple negative breast cancer cell line MDA-MB-231 in normoxia and hypoxia and the pathways involved in metabolic changes in hypoxia.

Methods

Differences in metabolic profiles affected pathways of MDA-MB-231 cells in normoxia and hypoxia were characterized using GC–MS based untargeted and stable isotope assisted metabolomic techniques.

Results

Thirty-three metabolites were significantly changed in hypoxia and nine pathways were involved. Hypoxia increased glycolysis, inhibited TCA cycle, pentose phosphate pathway and pyruvate carboxylation, while increased glutaminolysis in MDA-MB-231 cells.

Conclusion

The current results provide metabolic differences of MDA-MB-231 cells in normoxia and hypoxia conditions as well as the involved metabolic pathways, demonstrating the power of combined use of untargeted and stable isotope-assisted metabolomic methods in comprehensive metabolomic analysis.
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4.

Background

Silver nanoparticles (AgNPs) are potential antimicrobials agents, which can be considered as an alternative to antibiotics for the treatment of infections caused by multi-drug resistant bacteria. The antimicrobial effects of double and triple combinations of AgNPs, visible blue light, and the conventional antibiotics amoxicillin, azithromycin, clarithromycin, linezolid, and vancomycin, against ten clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) were investigated.

Methods

The antimicrobial activity of AgNPs, applied in combination with blue light, against selected isolates of MRSA was investigated at 1/2–1/128 of its minimal inhibitory concentration (MIC) in 24-well plates. The wells were exposed to blue light source at 460 nm and 250 mW for 1 h using a photon emitting diode. Samples were taken at different time intervals, and viable bacterial counts were determined. The double combinations of AgNPs and each of the antibiotics were assessed by the checkerboard method. The killing assay was used to test possible synergistic effects when blue light was further combined to AgNPs and each antibiotic at a time against selected isolates of MRSA.

Results

The bactericidal activity of AgNPs, at sub-MIC, and blue light was significantly (p < 0.001) enhanced when both agents were applied in combination compared to each agent alone. Similarly, synergistic interactions were observed when AgNPs were combined with amoxicillin, azithromycin, clarithromycin or linezolid in 30–40 % of the double combinations with no observed antagonistic interaction against the tested isolates. Combination of the AgNPs with vancomycin did not result in enhanced killing against all isolates tested. The antimicrobial activity against MRSA isolates was significantly enhanced in triple combinations of AgNPs, blue light and antibiotic, compared to treatments involving one or two agents. The bactericidal activities were highest when azithromycin or clarithromycin was included in the triple therapy compared to the other antibiotics tested.

Conclusions

A new strategy can be used to combat serious infections caused by MRSA by combining AgNPs, blue light, and antibiotics. This triple therapy may include antibiotics, which have been proven to be ineffective against MRSA. The suggested approach would be useful to face the fast-growing drug-resistance with the slow development of new antimicrobial agents, and to preserve last resort antibiotics such as vancomycin.
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5.

Introduction

Quantification of tetrahydrofolates (THFs), important metabolites in the Wood–Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen.

Objective

To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors.

Methods

Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC–MS/MS was used for quantification.

Results

Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP.

Conclusion

Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.
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6.

Background

The clinical outcomes and cost implications of a diagnostic shift from an EIA- to PCR-based assay for Clostridium difficile infection (CDI) have not been completely described in the literature.

Methods

The impact of the PCR-based assay on the incidence and duration of CDI therapy was compared to the EIA assay for patients with a negative CDI diagnostic result. Secondary clinical and economic outcomes were also evaluated. Independent predictors of receipt of antibiotic therapy were assessed via logistic regression.

Results

141 EIA and 140 PCR patients were included. Significantly more patients were started or continued on anti-CDI antibiotic therapy after a known negative assay result in the EIA group (26 patients vs. 8 patients, P = 0.002). Duration of antibiotic therapy after a known negative result was significantly shorter in the PCR group (1 vs. 4 days, P = 0.029) and a 23% reduction in the number of tests obtained per patient was observed (1.41 ± 0.86 vs. 1.82 ± 1.35, P = 0.007). The over fourfold difference in per-test cost of the EIA assay ($8.33 vs. $42.86, P < 0.0001) was offset by the overall medication costs required for the increased treatment in the EIA group ($546.60 vs. $188.96, P = 0.191). Utilization of the EIA-based CDI assay was associated with increased odds of CDI treatment after a negative test (aOR 4.71, 95% CI 1.93–11.46, P = 0.001).

Conclusion

The transition from an EIA to PCR-based assay for diagnosing CDI resulted in a significant decrease in the number of patients treated and the duration of treatment in response to a negative test result. This significant decrease in treatment resulted in decreased costs offsetting the utilization of a more expensive molecular test for patients with a negative CDI diagnostic result.
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7.

Background

Co-administration of anti-tuberculosis and antiretroviral therapy is often inevitable in high-burden countries where tuberculosis is the most common opportunistic infection associated with HIV/AIDS. Concurrent use of rifampicin and several antiretroviral drugs is complicated by pharmacokinetic drug-drug interaction.

Method

Pubmed and Google search following the key words tuberculosis, HIV, emtricitabine, tenofovir efavirenz, interaction were used to find relevant information on each drug of the fixed dose combination AtriplaR

Results

Information on generic name, trade name, pharmacokinetic parameter, metabolism and the pharmacokinetic interaction with Anti-TB drugs of emtricitabine, tenofovir, and efavirenz was obtained.

Conclusion

Fixed dose combination of emtricitabine/tenofovir/efavirenz (ATRIPLAR) which has been approved by Food and Drug Administration shows promising results as far as safety and efficacy is concerned in TB/HIV co-infection patients, hence can be considered effective and safe antiretroviral drug in TB/HIV management for adult and children above 3 years of age.
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8.

Introduction

Aqueous–methanol mixtures have successfully been applied to extract a broad range of metabolites from plant tissue. However, a certain amount of material remains insoluble.

Objectives

To enlarge the metabolic compendium, two ionic liquids were selected to extract the methanol insoluble part of trunk from Betula pendula.

Methods

The extracted compounds were analyzed by LC/MS and GC/MS.

Results

The results show that 1-butyl-3-methylimidazolium acetate (IL-Ac) predominantly resulted in fatty acids, whereas 1-ethyl-3-methylimidazolium tosylate (IL-Tos) mostly yielded phenolic structures. Interestingly, bark yielded more ionic liquid soluble metabolites compared to interior wood.

Conclusion

From this one can conclude that the application of ionic liquids may expand the metabolic snapshot.
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9.

Objective

To improve the diagnosis and treatment of Penicilliosis marneffei without human immunodeficiency virus infection.

Methods

Analyze and review the clinical features, diagnosis and treatment of six cases of P. marneffei without human immunodeficiency virus infection at The First Affiliated Hospital of Fujian Medical University.

Results

Two cases were diagnosed in the ENT Department, three cases in the respiratory department and one case in the dermatological department. Penicillium marneffei infection was confirmed by sputum culture, blood culture and tissue biopsy. After definite diagnosis, one refused further treatment, and others showed significant improvement.

Conclusion

Penicilliosis marneffei is insidious onset and easy to be escaped and misdiagnosed. To achieve early diagnosis and appropriate treatment, doubtful cases should be alerted for the diagnoses as P. marneffei.
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10.

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.
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11.

Background

Tuberculosis (TB) is a contagious infectious disease caused by Mycobacterium tuberculosis (Mtb). This disease with two million deaths per year has the highest mortality rate among bacterial infections. The only available vaccine against TB is BCG vaccine. BCG is an effective vaccine against TB in childhood, however, due to some limitations, has not proper efficiency in adults. Also, BCG cannot produce an adequately protective response against reactivation of latent infections.

Objective

In the present study we will review the most recent findings about contribution of HspX protein in the vaccines against tuberculosis.

Methods

Therefore, many attempts have been made to improve BCG or to find its replacement. Most of the subunit vaccines for TB in various phases of clinical trials were constructed as prophylactic vaccines using Mtb proteins expressed in the replicating stage. These vaccines might prevent active TB but not reactivation of latent tuberculosis infection (LTBI). A literature search was performed on various online databases (PubMed, Scopus, and Google Scholar) regarding the roles of HspX protein in tuberculosis vaccines.

Results

Ideal subunit post-exposure vaccines should target all forms of TB infection, including active symptomatic and dormant (latent) asymptomatic forms. Among these subunit vaccines, HspX is the most important latent phase antigen of M. tuberculosis with a strong immunological response. There are many studies that have evaluated the immunogenicity of this protein to improve TB vaccine.

Conclusion

According to the studies, HspX protein is a good candidate for development of subunit vaccines against TB infection.
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12.

Background

Outer membrane vesicles (OMVs) of Acinetobacter baumannii are cytotoxic and elicit a potent innate immune response. OMVs were first identified in A. baumannii DU202, an extensively drug-resistant clinical strain. Herein, we investigated protein components of A. baumannii DU202 OMVs following antibiotic treatment by proteogenomic analysis.

Methods

Purified OMVs from A. baumannii DU202 grown in different antibiotic culture conditions were screened for pathogenic and immunogenic effects, and subjected to quantitative proteomic analysis by one-dimensional electrophoresis and liquid chromatography combined with tandem mass spectrometry (1DE-LC-MS/MS). Protein components modulated by imipenem were identified and discussed.

Results

OMV secretion was increased >?twofold following imipenem treatment, and cytotoxicity toward A549 human lung carcinoma cells was elevated. A total of 277 proteins were identified as components of OMVs by imipenem treatment, among which β-lactamase OXA-23, various proteases, outer membrane proteins, β-barrel assembly machine proteins, peptidyl-prolyl cis–trans isomerases and inherent prophage head subunit proteins were significantly upregulated.

Conclusion

In vitro stress such as antibiotic treatment can modulate proteome components in A. baumannii OMVs and thereby influence pathogenicity.
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13.

Introduction

Mass spectrometry imaging (MSI) experiments result in complex multi-dimensional datasets, which require specialist data analysis tools.

Objectives

We have developed massPix—an R package for analysing and interpreting data from MSI of lipids in tissue.

Methods

massPix produces single ion images, performs multivariate statistics and provides putative lipid annotations based on accurate mass matching against generated lipid libraries.

Results

Classification of tissue regions with high spectral similarly can be carried out by principal components analysis (PCA) or k-means clustering.

Conclusion

massPix is an open-source tool for the analysis and statistical interpretation of MSI data, and is particularly useful for lipidomics applications.
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14.

Objective

To develop a reliable and easy to use expression system for antibiotic production improvement of Streptomyces.

Results

A two-compound T7 RNA polymerase-dependent gene expression system was developed to fulfill this demand. In this system, the T7 RNA polymerase coding sequence was optimized based on the codon usage of Streptomyces coelicolor. To evaluate the functionality of this system, we constructed an activator gene overexpression strain for enhancement of actinorhodin production. By overexpression of the positive regulator actII-ORF4 with this system, the maximum actinorhodin yield of engineered strain was 15-fold higher and the fermentation time was decreased by 48 h.

Conclusion

The modified two-compound T7 expression system improves both antibiotic production and accelerates the fermentation process in Streptomyces. This provides a general and useful strategy for strain improvement of important antibiotic producing Streptomyces strains.
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15.

Purpose

To evaluate the efficiency of corneal collagen cross-linking (CXL) in addition to topical voriconazole in cases with mycotic keratitis.

Design

Retrospective case series in a tertiary university hospital.

Participants

CXL was performed on 13 patients with mycotic keratitis who presented poor or no response to topical voriconazole treatment.

Methods

The clinical features, symptoms, treatment results and complications were recorded retrospectively. The corneal infection was graded according to the depth of infection into the stroma (from grade 1 to grade 3). The visual analogue scale was used to calculate the pain score before and 2 days after surgery.

Main Outcome Measures

Grade of the corneal infection.

Results

Mean age of 13 patients (6 female and 7 male) was 42.4 ± 17.7 years (20–74 years). Fungus was demonstrated in culture (eight patients) or cytological examination (five patients). Seven of the 13 patients (54%) were healed with topical voriconazole and CXL adjuvant treatment in 26 ± 10 days (15–40 days). The remaining six patients did not respond to CXL treatment; they initially presented with higher grade ulcers. Pre- and post-operative pain score values were 8 ± 0.8 and 3.5 ± 1, respectively (p < 0.05).

Conclusions

The current study suggests that adjunctive CXL treatment is effective in patients with small and superficial mycotic ulcers. These observations require further research by large randomized clinical trials.
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16.

Background

Botulism is a potentially fatal infection characterized by progressive muscle weakness, bulbar paralysis, constipation and other autonomic dysfunctions. A recent report suggested that cancer chemotherapy might increase the risk for the intestinal toxemia botulism in both adults and children.

Case presentation

We report a 5-year-old boy, who developed general muscle weakness, constipation, ptosis and mydriasis during the third induction therapy for relapsed acute myeloid leukemia. He had recent histories of multiple antibiotic therapy for bacteremia and intake of well water at home. Repeated bacterial cultures identified Clostridium botulinum producing botulinum neurotoxin A. Botulinum toxin A was isolated from his stools at 17, 21, and 23 days after the onset. Symptoms were self-limiting, and were fully recovered without anti-botulinum toxin globulin therapy.

Conclusion

This is the second report of a pediatric case with cancer chemotherapy-associated intestinal toxemia botulism. Our case provides further evidence that the immunocompromised status due to anti-cancer treatments increases the risk for the development of botulism at all ages in childhood.
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17.

Introduction

Several studies have observed serum lipid changes during malaria infection in humans. All of them were focused at analysis of lipoproteins, not specific lipid molecules. The aim of our study was to identify novel patterns of lipid species in malaria infected patients using lipidomics profiling, to enhance diagnosis of malaria and to evaluate biochemical pathways activated during parasite infection.

Methods

Using a multivariate characterization approach, 60 samples were representatively selected, 20 from each category (mild, severe and controls) of the 690 study participants between age of 0.5–6 years. Lipids from patient’s plasma were extracted with chloroform/methanol mixture and subjected to lipid profiling with application of the LCMS-QTOF method.

Results

We observed a structured plasma lipid response among the malaria-infected patients as compared to healthy controls, demonstrated by higher levels of a majority of plasma lipids with the exception of even-chain length lysophosphatidylcholines and triglycerides with lower mass and higher saturation of the fatty acid chains. An inverse lipid profile relationship was observed when plasma lipids were correlated to parasitaemia.

Conclusions

This study demonstrates how mapping the full physiological lipid response in plasma from malaria-infected individuals can be used to understand biochemical processes during infection. It also gives insights to how the levels of these molecules relate to acute immune responses.
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18.

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.
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19.

Introduction

In spite of advances in antibiotics, urinary tract infection (UTI) is still among the most common reasons for antibiotic medication worldwide. Persicaria capitata (Buch.-Ham. ex D. Don) H.Gross (P. capitata) is a herbal medicine used by the Miao people in China to treat UTI. However studies of its mechanism are challenging, owing to the complexity of P. capitata with multiple constituents acting on multiple metabolic pathways.

Objective

The objective of this study was to explore the working mechanism of P. capitata on urinary tract infection.

Methods

Relinqing® granule, which is solely made from aqueous extracts of the whole P. capitata plant, was used in this study. Urine metabolomics based on gas chromatography-mass spectroscopy was employed to assess the metabolic changes caused by administration of Relinqing® granule in a UTI mouse model. Female specific-pathogen-free Kunming mice were divided into control group (mock infection, saline treatment), model group (E.coli infection, saline treatment), Relinqing® group (E.coli infection, Relinqing® granule treatment), ciprofloxacin group (E.coli infection, ciprofloxacin treatment), and sham-Relinqing® group (no surgery, Relinqing® granule treatment).

Results

The results showed that after the treatments, urine levels of itaconic acid in Relinqing® group increased by 4.9 fold and 11.3 fold compared with model and ciprofloxacin groups respectively. Itaconic acid is an endogenous antibacterial metabolite produced by macrophages, which also functions as a checkpoint for metabolic reprogramming of macrophage.

Conclusion

Our findings suggest that this herbal medicine can cure urinary tract infection through modulation of immune system.
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20.

Introduction

The differences in fecal metabolome between ankylosing spondylitis (AS)/rheumatoid arthritis (RA) patients and healthy individuals could be the reason for an autoimmune disorder.

Objectives

The study explored the fecal metabolome difference between AS/RA patients and healthy controls to clarify human immune disturbance.

Methods

Fecal samples from 109 individuals (healthy controls 34, AS 40, and RA 35) were analyzed by 1H NMR spectroscopy. Data were analyzed with principal component analysis (PCA) and orthogonal projection to latent structure discriminant (OPLS-DA) analysis.

Results

Significant differences in the fecal metabolic profiles could distinguish AS/RA patients from healthy controls but could not distinguish between AS and RA patients. The significantly decreased metabolites in AS/RA patients were butyrate, propionate, methionine, and hypoxanthine. Significantly increased metabolites in AS/RA patients were taurine, methanol, fumarate, and tryptophan.

Conclusion

The metabolome variations in feces indicated AS and RA were two homologous diseases that could not be distinguished by 1H NMR metabolomics.
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