GlnR activated transcription of nitrogen metabolic pathway genes facilitates biofilm formation by mycobacterium abscessus.

OBJECTIVES: Nitrogen is indispensable for the synthesis of biomacromolecules. The correlation between nitrogen metabolism and Mycobacterium abscessus (M. abscessus) biofilm formation is unclear. This study constructed global nitrogen regulator gene GlnR (Mab_0744) knockout (ΔglnR) and complementation (ΔglnR::glnR) M. abscessus strains. METHODS: Global nitrogen regulator gene glnR (Mab_0744) knockout (ΔglnR) and complementation (ΔglnR::glnR) M. abscessus strains were constructed. Sauton's medium was used to culture M. abscessus pellicle biofilm. To test the antibiotic susceptibility of pellicle biofilm, clarithromycin, amikacin, cefoxitin or imipenem was added to the medium under biofilms after 14 days of incubation. RT-qPCR and ChIP-qPCR were performed to analyse the transcriptional regulatory function of GlnR. RESULTS: GlnR knockout decreased the growth rate of planktonic cells, reduced biofilm mass and wrinkle formation, and diminished the resistance of biofilms to antibiotics. However, the susceptibility of planktonic cells to antibiotics was not changed by glnR knockout. The growth rate of planktonic ΔglnR cells was accelerated by adding nitrogen sources to the medium; the addition of glutamine or sodium glutamate rescued ΔglnR biofilm morphology and resistance to amikacin, cefoxitin, clarithromycin and imipenem. GlnR bound the promoter region and activated the transcription of eight nitrogen metabolic pathway genes (i.e. glnA, amt, ansP, nirB, nirD, glnD, glnK and narK3), which are closely related to glutamine/glutamate biosynthesis and, thus, regulate biofilm formation. CONCLUSION: This study provides insights into the mechanisms of M. abscessus biofilm formation and its resistance to antibiotics.

Impaired ESX-3 Induces Bedaquiline Persistence in Mycobacterium abscessus Growing Under Iron-Limited Conditions.

ESX-3 is a secretion pathway which is essential for mycobactin-mediated iron acquisition under iron-limited conditions. Although present in all Mycobacterium sp., ESX-3 remains to be elucidated in Mycobacterium abscessus. In the study reported here, impaired ESX-3 seriously restricts the growth of M. abscesses under iron-limited conditions; growth is salvaged by functional ESX-3 or iron supplementation. Notably, impaired ESX-3 does not kill M. abscesses when environmental iron is insufficient but induces persistence to bedaquiline, a diarylquinoline class antibiotic used to treat multidrug-resistant mycobacteria. One potential mechanism contributing to persistence is the iron deficiency due to impaired ESX-3 suppressing succinate dehydrogenase activity, which dysregulates the tricarboxylic acid cycle and inactivates bedaquiline. Experiments conducted here also demonstrate that the regulator, MtrA, can bind ESX-3 and promote the survival of M. abscessus. As such, this study suggests that a novel pathway involving MtrA, ESX-3, iron metabolism, and the TCA cycle contributes to bedaquiline persistence in M. abscesses growing under iron-limited conditions.

sRNA21, a novel small RNA, protects Mycobacterium abscessus against oxidative stress.

BACKGROUND: During infection, Mycobacterium abscessus encounters numerous environmental changes and adapts to them using a variety of complex mechanisms. Non-coding small RNAs (sRNAs) have been shown in other bacteria to be involved in post-transcriptional regulatory pathways, including environmental stress adaptation. However, the potential role of sRNAs in the resistance to oxidative stress in M. abscessus was not clearly described. METHODS: In the present study, we analyzed putative sRNAs identified by RNA-sequencing (RNA-seq) experiments in M. abscessus ATCC_19977 under oxidative stress, and the transcription profiles of sRNAs with differential expression were verified by quantitative reverse transcription-PCR (qRT-PCR). Six sRNA overexpression strains were constructed, and the differences in growth curves between these strains and the control strain were verified. An upregulated sRNA under oxidative stress was selected and named sRNA21. The survival ability of the sRNA21 overexpression strain was assessed, and computer-based approaches were used to predict the targets and pathways regulated by sRNA21. The total ATP production and NAD+ /NADH ratio of the sRNA21 overexpression strain were measured. The expression level of antioxidase-related genes and the activity of antioxidase were tested to confirm the interaction of sRNA21 with the predicted target genes in silico. RESULTS: In total, 14 putative sRNAs were identified under oxidative stress, and the qRT-PCR analysis of six sRNAs showed comparable results to RNA-seq assays. Overexpression of sRNA21 in M. abscessus increased cell growth rate and intracellular ATP level before and after peroxide exposure. The expression of genes encoding alkyl hydroperoxidase and superoxide dismutase was significantly increased, and superoxide dismutase activity was enhanced in the sRNA21 overexpression strain. Meanwhile, after sRNA21 overexpression, the intracellular NAD+ /NADH ratio decreased, indicating changes in redox homeostasis. CONCLUSIONS: Our findings show that sRNA21 is an oxidative stress-induced sRNA that increases M. abscessus survival and promotes the expression of antioxidant enzymes under oxidative stress. These findings may provide new insights into the adaptive transcriptional response of M. abscessus to oxidative stress.

Antibacterial peptide RP557 increases the antibiotic sensitivity of Mycobacterium abscessus by inhibiting biofilm formation.

Biofilm formation is an important factor for Mycobacterium abscessus to resist harsh environment and produce drug resistance. The anti-biofilm activity of a newly designed antibacterial peptide, RP557, was investigated. The effect of RP557 alone or in combination with several clinically effective antibiotics, including clarithromycin, amikacin, cefoxitin and imipenem, on M. abscessus growth in biofilms was determined. Microstructural changes in biofilms after RP557 treatment were observed by scanning electron microscope. The effect of RP557 on the viability of bacteria was determined by Syto9/PI staining and fluorescence microscopy. Finally, the potential mechanism of RP557 action on biofilm development was explored by transcriptome analysis. M. abscessus growing in biofilms showed increased resistance to antimicrobial drugs. RP557 alone exhibited only moderate anti-M. abscessus activity in vitro, but significantly increased the antibiotic sensitivity of M. abscessus in biofilms. The inhibitory effect of RP557 on biofilm formation was visualized by the scanning electron microscope; fluorescence staining demonstrated increased bacterial death in response to RP557 treatment. Furthermore, comparative analysis of transcriptomic data suggested RP557 may inhibit biofilm formation by down-regulating nitrogen and fatty acid metabolism, as well as peptidoglycan biosynthesis. As such, RP557 is a potential candidate to include in novel strategies to treat M. abscessus infections.

Improvement of sample discrimination using laser-induced breakdown spectroscopy with multiple-setting spectra.

Laser-induced breakdown spectroscopy (LIBS) is a promising multi-elemental analysis technique and has the advantages of rapidness and minimal sample preparation. In traditional LIBS measurement, sample spectra are generally collected based on a single set of fixed experimental parameters, such as laser energy and delay time. When samples have the same main components and similar component concentrations, the difference in their spectral intensities becomes less obvious. This can lower the sensitivity of LIBS measurement and pose a threat to the accuracy and robustness of LIBS qualitative analysis. In this work, we propose a new method to increase the spectral difference between similar samples, namely multiple-setting spectra. For each sample, it adopts different sets of experimental parameters and obtains a group of spectra to increase the fingerprint spectral information. The effectiveness of the proposed method is theoretically verified and then tested on 11 similar coal samples. Specifically, the sample spectra were collected with different laser energy and delay time, and processed by principal component analysis (PCA) and Davies-Bouldin index (DBI). The results show that the use of multiple-settings spectra can significantly improve the sample discrimination accuracy from 81.8% to 96.4%. In addition, the proposed method can maintain the efficiency and cost of LIBS measurement.

A Novel Oxazolidinone, Contezolid (MRX-I), Expresses Anti-Mycobacterium abscessus Activity In Vitro.

An evaluation of the anti-Mycobacterium abscessus activity expressed by a novel oxazolidinone, contezolid (MRX-I), toward 12 reference strains and 194 clinical isolates was conducted. Contezolid was active against M. abscessus in vitro, with effects comparable to the anti-M. abscessus effects of linezolid both extracellularly and intracellularly. Contezolid did not antagonize the most frequently used anti-M. abscessus drugs, and preexposure to contezolid did not induce drug resistance. These results provide a novel approach to treating M. abscessus infections.

MAB_2355c Confers Macrolide Resistance in Mycobacterium abscessus by Ribosome Protection.

Macrolide resistance is always a concern when treating Mycobacterium abscessus infections. MAB_2355c was identified previously as a possible new factor that confers the intrinsic resistance of 194 clinical M. abscessus isolates to clarithromycin. Herein, the potential mechanism by which MAB_2355c exerts macrolide resistance was explored by bioinformatics analysis, MAB_2355c cloning and protein purification, ATP hydrolysis assay, gene knockout and complementation, antibiotic sensitivity, and transcription-translation assays. MAB_2355c is a putative ATP-binding cassette F (ABC-F) family protein. Purified MAB_2355c protein exhibits ATP hydrolysis activity, which can be inhibited by ribosome-targeting antibiotics. MAB_2355c mRNA expression is upregulated more significantly after exposure to macrolides than after exposure to other ribosome-targeting antibiotics. MAB_2355c deleted strains showed increased sensitivity to macrolides, which was reduced by MAB_2355c complementation. Finally, MAB_2355c rescued the transcription and translation activities affected by macrolides in vitro. These findings suggest that MAB_2355c confers the resistance of M. abscessus to macrolides by ribosome protection, thus complementing other known resistance mechanisms.

Sitafloxacin Expresses Potent Anti-Mycobacterium abscessus Activity.

Therapeutic options for treating Mycobacterium abscessus infections are extremely limited; quinolones are important. The in vitro anti-M. abscessus activities of nine quinolones, emphasizing sitafloxacin, were investigated. Antimicrobial susceptibility testing was performed on 10 non-tuberculous mycobacterium reference strains and 194 clinical, M. abscessus isolates. The activity of sitafloxacin against intracellular M. abscessus residing within macrophages was also evaluated. A checkerboard assay was conducted to determine synergy between sitafloxacin and 10 clinically important antibiotics. Among the nine quinolones tested, sitafloxacin exhibited the greatest anti-M. abscessus activity with MIC50 and MIC90 of 1 and 2 mg/L, respectively. Sitafloxacin exerted a bacteriostatic effect on M. abscessus and inhibited the intracellular growth of M. abscessus at concentrations equivalent to clarithromycin. No antagonism between sitafloxacin and 10 clinically important anti-M. abscessus antibiotics was evident. In summary, sitafloxacin exhibited a significant advantage relative to other quinolones in inhibiting the growth of M. abscessus in vitro, suggesting the potential inclusion of sitafloxacin in new strategies to treat M. abscessus infections.

Selective Photocatalytic Reduction of CO2 to CH4 Modulated by Chloride Modification on Bi2WO6 Nanosheets.

Solar-driven photocatalytic CO2 reduction into CH4 with H2O is considered to be a promising way to alleviate the energy crisis and greenhouse effect. However, current CO2 photoreduction technologies tend to overlook the role of photooxidation half reaction as well as the effect of the protons produced by water oxidation on CH4 generation, resulting in low CO2 conversion efficiency and poor CH4 selectivity. In the present study, a series of chloride-modified Bi2WO6 nanosheets were constructed in view of chloride-assisted photocatalytic water oxidation. The results show that the CH4 yield of the synthesized sample can be enhanced up to about 10 times compared to that with no Cl- modification. Besides, the selectivity of CH4 can be regulated by the loading amount of chloride, varying from 51.29% for Bi2WO6 to 94.98% for the maximum. The increase of product yield is attributed to chloride modification, which not only changed the morphology of the catalyst, but also modified the pathway of water oxidation. Further studies on intermediate products and the density functional theory calculation confirm that the Cl- ions on Bi2WO6 nanosheets not only promote H2O oxidation, but also lower the energy barrier for intermediate *CHO generation, thus facilitating CH4 production. The results gained herein may provide some illuminating insights into the design of a highly selective photocatalyst for efficient CO2 reduction.

Hint1 Overexpression Inhibits the Cell Cycle and Induces Cell Apoptosis in Human Osteosarcoma Cells.

BACKGROUND: New evidence suggests that histidine triad nucleotide-binding protein 1 (Hint1) exerts a tumor suppressor effect in various human tumors, such as colorectal cancer and gastric cancer. However, it has not been reported whether Hint1 is involved in the occurrence and development of osteosarcoma (OS). MATERIALS AND METHODS: The present study investigated the role of Hint1 in human OS cells by using cell lines, including 143B, U2OS, KHOS-240S, Saos-2 and MG-63. Cell proliferation and apoptosis were detected by flow cytometry. RESULTS: The present result revealed that Hint1 is downregulated in these cell lines. The overexpression of Hint1 by adenovirus transfection in 143B and MG63 cell lines suppressed the proliferation and cell cycle, and increased the cell apoptosis. Mechanically, it was found that Hint1 downregulated the cyclin D1 expression via FOXO1 inhibition. Furthermore, FOXO1 overexpression in the 143B and MG63 cell lines significantly blurred the effects of Hint1 on cellular proliferation and apoptosis. CONCLUSION: The present study indicates that Hint1 inhibits the development of OS by regulating FoxO1-cyclin D1, suggesting that Hint1 may be a new method for the treatment of OS.

Efficacy and safety of osimertinib in treating EGFR-mutated advanced NSCLC: A meta-analysis.

Osimertinib is the only Food and Drug Administration-approved third-generation epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitor (TKI). A meta-analysis was performed to aggregate the mixed results of published clinical trials to assess the efficacy and safety of osimertinib. A systematic search of the PubMed, Web of Science, and Cochrane Library electronic databases was performed to identify eligible literature. The primary endpoints were overall response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and adverse events (AEs). A total of 3,086 advanced nonsmall cell lung cancer (NSCLC) patients from 11 studies have been identified. The aggregate efficacy parameters for treatment-naïve patients with EGFR-TKI-sensitizing mutations are as follows: ORR 79% (95% CI 75-84%), DCR 97% (95% CI 95-99%), 6-month PFS 83% (95% CI 80-87%), and 12-month PFS 64% (95% CI 59-69%). The aggregate efficacy parameters for advanced NSCLC harboring T790M mutations after earlier-generation EGFR-TKI therapy are as follows: ORR 58% (95% CI 46-71%), DCR 80% (95% CI 63-98%), 6-month PFS 63% (95% CI 58-69%), and 12-month PFS 32% (95% CI 17-47%). EGFR-TKI-naïve patients with EGFR-positive mutations tend to have longer median PFS than EGFR-TKI-pretreated counterparts (19.17 vs. 10.58 months). The most common AEs were diarrhea and rash, of which the pooled incidences were 44 and 42%, respectively. Generally, osimertinib is a favorable treatment option for previously treated T790M mutation-positive advanced NSCLC as well as a preferable therapy for untreated EGFR mutation-positive advanced NSCLC. Additionally, osimertinib is well tolerated by most patients.

The efficacy and safety of ALK inhibitors in the treatment of ALK-positive non-small cell lung cancer: A network meta-analysis.

PURPOSE: The current study was carried out to compare the effectiveness and safety of different ALK inhibitors in treating ALK+ NSCLC. METHODS: Progression-free survival (PFS), disease control rate (DCR), overall response rate (ORR), and intracranial ORR and DCR have been aggregated to appraise the effectiveness of each ALKi. The discontinuation rate due to adverse events (AEs) was pooled to evaluate their safety. Bayesian network meta-analyses were used to compare the ORR, DCR, PFS, and discontinuation rate of patients treated with alectinib, ceritinib, crizotinib, and chemotherapy. RESULTS: Compared with chemotherapy, ALK inhibitors significantly prolonged PFS [hazard ratio (HR) and 95% confidence interval (CI): alectinib, 0.50 (0.43-0.58); ceritinib, 0.75 (0.69-0.83); crizotinib, 0.71 (0.66-0.76)]. The ORRs were significantly higher for ALK inhibitors than for chemotherapy [odds ratio (OR) and corresponding 95% CI: alectinib, 11.69 (4.29-36.56); ceritinib, 7.85 (3.44-19.27); crizotinib, 6.04 (3.33-11.71)]. The discontinuation rates were lower for ALK inhibitors than for chemotherapy [OR and corresponding 95% CI: alectinib, 0.42 (0.12-1.36); ceritinib, 0.52 (0.20-1.35); crizotinib, 0.70 (0.30-1.62)]. CONCLUSIONS: ALK+ NSCLC patients treated with ALKi tend to have longer PFS than those treated with chemotherapy. ALKi-naïve patients tended to response better than their ALKi-pretreated counterparts. Alectinib appeared to be preferable for treating brain metastases due to its high intracranial efficacy. Patients treated with alectinib or ceritinib tended to have higher ORR and DCR than patients with similar baselines treated with crizotinib or chemotherapy. No significant differences in discontinuation rate were found for alectinib, ceritinib, crizotinib, and chemotherapy.

Pharmacological mechanism of roflumilast in the treatment of asthma-COPD overlap.

Asthma-COPD overlap (ACO) is a type of incomplete obstructive airway disease that has a high incidence and mortality. Nevertheless, there is currently no clear definition of ACO and no effective intervention. The newly discovered phosphodiesterase-4 inhibitor, roflumilast, has shown initial efficacy for treating asthma, COPD, and ACO. The mechanism of roflumilast, however, remains unclear, and there has been no interpretation through systematic review to date. The determination of a definite mechanism of roflumilast will guide the clinician's decisions regarding medication use, standardized diagnosis, and treatment guidelines. For this reason, we have systematically reviewed the therapeutic mechanism of roflumilast for ACO and provided reference for the clinical application of roflumilast in ACO.

The efficacy and safety of alectinib in the treatment of ALK+ NSCLC: a systematic review and meta-analysis.

BACKGROUND: Alectinib is a second-generation anaplastic lymphoma kinase (ALK) inhibitor approved by the US Food and Drug Administration to treat crizotinib-refractory non-small cell lung cancer. We performed this meta-analysis to synthesize the results of different clinical trials to evaluate the efficacy and safety of alectinib. METHODS: A search of 3 databases, including PubMed, Web of Science, and the Cochrane Library, was performed from the inception of each database through September 5, 2017. We have pooled the overall response rate (ORR), disease control rate, progression-free survival, and intracranial ORR to evaluate the efficacy of alectinib. Discontinuation rate, rate of dose reduction or interruption due to adverse events as well as the incidence of several adverse events were aggregated to evaluate its safety. RESULTS: A total of 8 studies with 626 patients have been included in our study. The pooled efficacy parameters are as follows: ORR 70% (95% CI: 57% to 82%), disease control rate 88% (95% CI: 82% to 94%), progression-free survival 9.36 months (95% CI: 7.38% to 11.34%), and intracranial ORR 52% (95% CI: 45% to 59%). ALK inhibitor-naïve patients tend to have better responses than crizotinib-pretreated patients. The aggregate discontinuation rate is 7% (95% CI: 4% to 10%), and the pooled rate of dose reduction or interruption is 33% (95% CI: 24% to 42%). The incidences of most adverse events were relatively low, while the incidences of 2 frequently reported adverse events, myalgia (18%) and anemia (25%), were even higher than with the first-generation ALK inhibitor crizotinib. CONCLUSION: Generally, alectinib is a drug with preferable efficacy and tolerable adverse effects, and it is suitable for the treatment of intracranial metastases.

Prognostic role of the neutrophil-to-lymphocyte ratio in pancreatic cancer: A meta-analysis containing 8252 patients.

Several studies were carried out to explore the prognostic role of neutrophil-to-lymphocyte ratio (NLR) in pancreatic cancer, however, with contradictory results. The objectives of this study were to summarize the prognostic value of NLR in pancreatic cancer. Embase, PubMed and Cochrane Library were comprehensively retrieved. All the cohort studies focusing on the prognostic value of NLR in pancreatic cancer were eligible. 37 papers containing 43 cohort studies with pancreatic cancer were finally included into this study. The results presented that patients with low NLR might have longer OS when compared to the patients with high NLR (HR = 1.81, 95%CI = 1.59-2.05, P < 0.00001; I2 = 82%). Similar results were detected in the subgroup analyses of OS, which was based on the analysis model, ethnicity, treatment, sample size and cut-off value. In additions, low NLR was significantly associated with longer DFS when compared to high NLR in pancreatic cancer (HR = 1.66, 95%CI = 1.17-2.35, P = 0.005; I2 = 67%). Moreover, patients with low NLR had significantly smaller tumor size (P = 0.0007), better differentiation (P = 0.003), earlier stage (P = 0.02) and low CA-199 level (P = 0.007). In conclusion, it was revealed that low NLR was a favorable predictor of OS and DFS in patients with pancreatic cancer, and NLR is a promising prognostic biomarker for pancreatic cancer.

Glia maturation factor-ß: a potential therapeutic target in neurodegeneration and neuroinflammation.

Glia maturation factor-ß (GMFB) is considered to be a growth and differentiation factor for both glia and neurons. GMFB has been found to be upregulated in several neuroinflammation and neurodegeneration conditions. It may function by mediating apoptosis and by modulating the expression of superoxide dismutase, granulocyte-macrophage colony-stimulating factor, and neurotrophin. In this review, we mainly discussed the role of GMFB in several neuroinflammatory and neurodegenerative diseases. On review of the literature, we propose that GMFB may be a promising therapeutic target for neuroinflammatory and neurodegenerative diseases.

The applications of liquid biopsy in resistance surveillance of anaplastic lymphoma kinase inhibitor.

With the clinical promotion of precision medicine and individualized medical care, molecular targeted medicine has been used to treat non-small cell lung cancer (NSCLC) patients and proved to be significantly effective. Anaplastic lymphoma kinase (ALK) inhibitor is one of the most important specific therapeutic agents for patients with ALK-positive NSCLC. It can extend the survival of patients. However, resistance to the ALK inhibitor inevitably develops in the application process. So, the real-time resistance surveillance is particularly important, and liquid biopsy is one of the most potential inspection methods. Circulating tumor cells, circulating free tumor DNA and exosome in body fluid are used as the main detection biomarkers to reflect the occurrence of resistance in real time through sequencing or counting and then to guide the follow-up treatment.