Effectiveness and safety of regimens containing linezolid for treatment of Mycobacterium abscessus pulmonary Disease.

OBJECTIVE: To evaluate the effectiveness and safety of linezolid-containing regimens for treatment of M. abscessus pulmonary disease. METHODS: The records of 336 patients with M. abscessus pulmonary disease who were admitted to Shanghai Pulmonary Hospital from January 2018 to December 2020 were retrospectively analyzed. A total of 164 patients received a linezolid-containing regimen and 172 controls did not. The effectiveness, safety, antibiotic susceptibility profiles, outcomes, culture conversion, cavity closure, and adverse reactions were compared in these two groups. RESULTS: The two groups had similar treatment success (56.1% vs. 48.8%; P > 0.05), but treatment duration was shorter in the linezolid group (16.0 months [inter-quartile ranges, IQR: 15.0-17.0] vs. 18.0 months [IQR: 16.0-18.0]; P < 0.01). The rates of sputum culture conversion were similar (53.7% vs. 46.5%, P > 0.05), but time to conversion was shorter in the linezolid group (3.5 months [IQR: 2.5-4.4] vs. 5.5 months [IQR: 4.0-6.8]; P < 0.01). The linezolid group had a higher rate of cavity closure (55.2% vs. 28.6%, P < 0.05) and a shorter time to cavity closure (3.5 months [IQR: 2.5-4.4] vs. 5.5 months [IQR: 4.0-6.8]; P < 0.01). Anemia and peripheral neuropathy were more common in the linezolid group (17.7% vs. 1.7%, P < 0.01; 12.8% vs. 0.6%, P < 0.01). CONCLUSIONS: The linezolid and control groups had similar treatment success rates. The linezolid group had a shorter treatment duration, shorter time to sputum culture conversion, and higher rate and shorter time to lung cavity closure. More patients receiving linezolid developed anemia and peripheral neuropathy.

EBUS-GS with the GeneXpert MTB/RIF assay for diagnosis of Mycobacterium tuberculosis infection of isolated pulmonary nodules.

OBJECTIVE: Investigate the use of endobronchial ultrasonography with a guide sheath (EBUS-GS) combined with Gene Xpert MTB/RIF (Xpert) for diagnosis of Mycobacterium tuberculosis (MTB) infection in isolated pulmonary nodules. METHODS: Patients who had isolated pulmonary nodules and unknown diagnoses at our institution from October 2020 to December 2021 were prospectively examined using EBUS-GS and Xpert. The diagnostic values of using EBUS-GS or bronchoalveolar lavage fluid (BALF) with acid-fast staining, MGIT 960 culture, pathological examination, and Xpert for isolated pulmonary nodules caused by MTB infection were compared using receiver operating characteristic (ROC) analysis. RESULTS: There were 135 patients, 64 with isolated pulmonary tuberculomas and 71 with non-tuberculous lesions. The sensitivity of EBUS-GS with Xpert was significantly higher than BALF with Xpert (57.81% vs. 34.78%, P = 0.017). Use of EBUS-GS with Xpert and MGIT 960 culture further increased the sensitivity to 62.50% (95%CI 50.64-74.36) and increased the specificity to 100%. The AUC values of BALF with MGIT 960 culture was 0.663(95%CI 0.543-0.783) and BALF with Xpert was 0.674 (95%CI 0.556-0.792). The AUC values of EBUS-GS with MGIT 960 culture was 0.680 (95%CI 0.554-0.743), with pathological examination was 0.713 (95%CI 0.573-0.760), and with Xpert was 0.789 (95%CI 0.655-0.829). CONCLUSION: Use of EBUS-GS with Xpert had high sensitivity and specificity in the diagnosis of isolated pulmonary tuberculoma. This method has significant potential for use in clinical practice.

Detection of Hepatitis C virus RNA using a novel hybridization chain reaction method that competitively dampens cascade amplification.

The hybridization chain reaction (HCR) is widely used for biosensing. However, HCR does not provide the required sensitivity. In this study, we reported a method to improve the sensitivity of HCR by dampening the cascade amplification. First, we designed a biosensor based on HCR, and an initiator DNA was used to trigger the cascade amplification. Optimization of the reaction was then performed, and the results showed that the limit of detection (LOD) for the initiator DNA was about 2.5 nM. Second, we designed a series of inhibitory DNAs to dampen the HCR cascade amplification, and DNA dampeners (50 nM) were applied in the presence of the DNA initiator (50 nM). One of the DNA dampeners (D5) showed the best inhibitory efficiency of greater than 80%. This was further applied at concentrations ranging from 0 nM to 10 nM to prohibit the HCR amplification caused by a 2.5 nM initiator DNA (the limit of detection for this initiator DNA). The results showed that 0.156 nM of D5 could significantly inhibit the signal amplification (p<0.05). Additionally, the limit of detection for the dampener D5 was 16 times lower than that for the initiator DNA. Based on this detection method, we achieved a detection limit as low as 0.625 nM for HCV-RNAs. In summary, we developed a novel method with improved sensitivity to detect the target designed to prohibit the HCR cascade. Overall, this method could be used to qualitatively detect the presence of single-stranded DNA/RNA.

Detrimental Role of PDZ-RhoGEF in Pathological Cardiac Hypertrophy.

BACKGROUND: Postsynaptic density 95/disk-large/ZO-1 Rho guanine nucleotide exchange factor (PDZ-RhoGEF, PRG) functions as a RhoGEF for activated Gα13 and transmits activation signals to downstream signaling pathways in various pathological processes. Although the prohypertrophic effect of activated Gα13 (guanine nucleotide binding protein alpha 13; a heterotrimeric G protein) is well-established, the role of PDZ-RhoGEF in pathological cardiac hypertrophy is still obscure. METHODS: Genetically engineered mice and neonatal rat ventricular myocytes were generated to investigate the function of PRG in pathological myocardial hypertrophy. The prohypertrophic stimuli-induced alternations in the morphology and intracellular signaling were measured in myocardium and neonatal rat ventricular myocytes. Furthermore, multiple molecular methodologies were used to identify the precise molecular mechanisms underlying PDZ-RhoGEF function. RESULTS: Increased PDZ-RhoGEF expression was documented in both hypertrophied hearts and neonatal rat ventricular myocytes. Upon prohypertrophic stimuli, the PDZ-RhoGEF-deficient hearts displayed alleviated cardiomyocyte enlargement and attenuated collagen deposition with improved cardiac function, whereas the adverse hypertrophic responses in hearts and neonatal rat ventricular myocytes were markedly exaggerated by PDZ-RhoGEF overexpression. Mechanistically, RhoA (ras homolog family member A)-dependent signaling pathways may function as the downstream effectors of PDZ-RhoGEF in hypertrophic remodeling, as confirmed by rescue experiments using a RhoA inhibitor and dominant-negative RhoA. Furthermore, PDZ-RhoGEF is associated with activated Gα13 and contributes to Gα13-mediated activation of RhoA-dependent signaling. CONCLUSIONS: Our data provide the first evidence that PDZ-RhoGEF promotes pathological cardiac hypertrophy by linking activated Gα13 to RhoA-dependent signaling pathways. Therefore, PDZ-RhoGEF has the potential to be a diagnostic marker or therapeutic target for pathological cardiac hypertrophy.

Detection of Ureaplasma urealyticum by Catalytic Hairpin Assembly Combined with a Lateral Flow Immunoassay Strip.

Ureaplasma urealyticum is a common genital mycoplasma in men and women, which can cause reproductive tract infection and infertility, and is also related to adverse pregnancy outcomes and neonatal diseases. Pathogen culture and polymerase chain reaction (PCR) are the main methods for the diagnosis of U. urealyticum. However, pathogen culture takes too long, and PCR requires professional personnel and sophisticated instruments. Here, we report a simple, convenient, sensitive, and specific detection method, which combines catalytic hairpin assembly with a lateral flow immunoassay strip. Only a water bath and a fluorescence reader are needed to detect the results in 30 min. We can realize the point-of-care testing of U. urealyticum by this method. To verify this method, we selected 10 clinical samples for testing, and the test results were exactly the same as the clinical report.

Association of RAGE gene Gly82Ser polymorphism with coronary artery disease and ischemic stroke: A systematic review and meta-analysis.

BACKGROUND: The receptor for advanced glycosylation end products (RAGE) has been widely linked to diabetic atherosclerosis, but its effects on coronary artery disease (CAD) and ischemic stroke (IS) remain controversial. The Gly82Ser polymorphism is located in the ligand-binding V domain of RAGE, suggesting a possible influence of this variant on RAGE function. The aim of the present study is to clarify the association between the RAGE Gly82Ser polymorphism and susceptibility to CAD and IS. METHODS: Eligible studies were identified through a comprehensive literature search. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to evaluate the association of Gly82Ser polymorphism with CAD and IS risk. Fixed- or random-effects model was used depending on the heterogeneity between studies. A funnel plot and Egger linear regression test were applied to assess publication bias. We also performed subgroup analyses to investigate potential sources of heterogeneity. RESULTS: A total of 16 eligible articles containing 18 studies were analyzed. The pooled analysis indicated that the Gly82Ser polymorphism significantly increased CAD risk in recessive and homozygous genetic models (SS vs GS + GG: OR = 1.34, 95% CI = 1.09-1.64; SS vs GG: OR = 1.38, 95% CI = 1.12-1.71). A significant association between the Gly82Ser polymorphism and IS risk was observed in all tested models except the heterozygous genetic model (GS + SS vs GG: OR = 1.20, 95% CI = 1.04-1.38; SS vs GS + GG: OR = 2.20, 95% CI = 1.74-2.78; SS vs GG: OR = 2.23, 95% CI = 1.72-2.91; S vs G: OR = 1.32, 95% CI = 1.05-1.65). Subgroup analysis suggested an association between CAD and IS risk and the Gly82Ser polymorphism in the Chinese population, but not in the non-Chinese population. CONCLUSIONS: The current meta-analysis suggests that the RAGE Gly82Ser polymorphism is associated with an increased risk of CAD and IS, especially in the Chinese population. However, better-designed studies with larger sample sizes are needed to validate the results.

Bioscreening and expression of a camel anti-CTGF VHH nanobody and its renaturation by a novel dialysis-dilution method.

The variable regions of the camel heavy chain antibody, also known as nanobody is the smallest antibody with antigen-binding efficiency. CTGF is considered important during extracellular matrix deposition which was involved in the pathogenesis of fibrosis related diseases. There are several anti-CTGF-C nanobody drugs under developing in pharmacy. In this study, we described the screening of a novel anti-CTGF-C nanobody from the peripheral blood of immunized camel by phage display. The screened nanobody was further expressed and purified from E. coli cells. A sophisticated dialysis-dilution method was designed for the in vitro refolding of the nanobody. The results showed that the expressed nanobody was consisted of 135 amino acid and mainly expressed as inclusion body in E. coli cells. The dialysis-dilution method was very effective and the recovery rate of the renaturation was more than 80 %. The ELISA result suggested the nanobody had been well refolded showing a superior CTGF binding activity to the commercial mouse anti-CTGF-C mAb. In conclusion, the anti-CTGF-C nonobody had been successfully screened by phage display. The dialysis-dilution refolding method was very effective and the recovery rate reached over 80 %.

A Novel Pentapeptide Targeting Integrin ß3-Subunit Inhibits Platelet Aggregation and Its Application in Rat for Thrombosis Prevention.

BACKGROUND: Antiplatelet therapy plays a pivotal role in the prevention and treatment of thrombotic diseases. We reported the screening of P1C as a novel integrin-binding peptide from the C-terminal of connective tissue growth factor. Primary study indicated that P1C has potential against platelet aggregation. OBJECTIVES: We aimed to find the shortest active unit from the P1C fragments and explore its in vivo and in vitro activities. METHODS: A series of truncated P1C fragments was prepared and screened for antiplatelet activity. The most active fragment was evaluated using coagulation assays. Flow cytometry and confocal microscopy were used to determine the interaction between the peptide and the integrin. The in vivo potential was further explored using two types of rat models. RESULTS: From a series of truncated P1C forms, a so-called P1Cm peptide of 5-amino acids, namely, IRTPK was screened out as the shortest active unit with superior activity. Coagulation experiments and an in vivo toxicity assay demonstrated that P1Cm is safe in vivo and inhibits ADP- and TH-induced human platelet aggregation in vitro in a concentration-dependent manner. Furthermore, it has limited effect on the coagulation parameters. Flow cytometry and confocal microscopy experiments consistently indicated that the peptide specifically binds the ß3-subunit of integrin on platelets. Further experiments using rat models of artery-vein shunt and carotid arterial thrombosis illustrated that P1Cm can effectively prevent thrombosis formation. CONCLUSION: P1Cm may be a new, promising antithrombotic alternative to currently available antiplatelet treatments.

A Hybrid Peptide PTS that Facilitates Transmembrane Delivery and Its Application for the Rapid In vivo Imaging via Near-Infrared Fluorescence Imaging.

BACKGROUND AND PURPOSE: Intravital imaging provides invaluable readouts for clinical diagnoses and therapies and shows great potential in the design of individualized drug dosage regimes. Ts is a mammalian free cell membrane-penetrating peptide. This study aimed to introduce a novel approach to the design of a cancer-selective peptide on the basis of a membrane-penetrating peptide and to explore its potential as a carrier of medical substances. EXPERIMENTAL APPROACH: Ts was linked with a αvß3-binding peptide P1c to create a hybrid referred to as PTS. The hybrid was labeled with an FITC or Cy5.5 as an imaging indicator to evaluate its in vitro and in vivo bioactivity. KEY RESULTS: Hemolysis tests proved that in comparison with Ts, PTS caused similar or even less leakage of human erythrocytes at concentrations of up to 1 mmol/L. Flow cytometry assay and confocal microscopy demonstrated the following. (1) P1c alone could target and mostly halt at the cancer cell membrane. (2) Ts alone could not bind to the membrane sufficiently. (3) P1c greatly enhanced the binding affinity of PTS with MDA-MB-231 breast cancer cells that upregulated αvß3. (4) Ts conferred PTS with the ability to traverse a cell membrane and thus facilitate the transmembrane delivery of imaging probes. In vivo near-infrared fluorescence (NIRF) imaging demonstrated that the imaging probes were rapidly concentrated in a MDA-MB-231 tumor tissue within 1 h after intravenous injection. CONCLUSIONS AND IMPLICATIONS: PTS exhibited the capability of targeting specific tumors and greatly facilitating the transmembrane delivery of imaging probes.

Computed tomography image analysis before and after treatment of anti-neutrophil cytoplasmic antibody-associated pulmonary interstitial fibrosis in 8 patients.

PURPOSE: The purpose of this study was to observe the treatment response of anti-neutrophil cytoplasmic antibody (ANCA)-associated pulmonary interstitial fibrosis in 8 patients before and after glucocorticoid or immunosuppressive therapy. METHODS: The clinical features and computed tomography imaging findings of the 8 patients in our hospital from October 2011 to October 2013, were retrospectively analyzed. FINDINGS: Mean age of the 8 patients was 72.6 (range 60-80) years. Five patients exhibited cough, sputum, and chest tightness, including 2 patients with fever. One patient developed hemoptysis, 1 patient exhibited abnormal urinalysis and developed renal insufficiency, and 1 patient developed limb pain. Two patients exhibited high urine erythrocytes and 2 patients had renal dysfunction and urinary abnormalities. One of the latter patients, upon renal biopsy, had focal proliferative necrotizing glomerulonephritis (consistent with vasculitis damage) with stage II to III mild nephropathy. Seven cases were anti-myeloperoxidase-ANCA, and 1 case was anti-proteinase 3-ANCA. All 8 cases exhibited streaks and grid shadows in chest imaging; 2 cases exhibited limited ground-glass patches; 1 case displayed multiple large patches of exudative shadows, indicating diffuse alveolar hemorrhage; 2 cases exhibited obvious honeycomb manifestations; and 1 case exhibited significant traction bronchiectasis. The ground-glass opacities disappeared after corticosteroid or immunosuppressive therapy; however, for streaks and grid shadows, no significant changes in the images were observed after treatment from 2 weeks to 10 months. IMPLICATIONS: ANCA-associated pulmonary interstitial fibrosis most often in elderly patients with many complications. In these patients ground-glass opacities in computed tomography images, corticosteroid or immunosuppressant therapy may be effective. Clinicians should consider the poor efficacy and side effects of these therapies in the fibrosis stage of the disease.

Multiplex real-time PCR for RRM1, XRCC1, TUBB3 and TS mRNA for prediction of response of non-small cell lung cancer to chemoradiotherapy.

BACKGROUND: This study was aimed to establish a novel method to simultaneously detect expression of four genes, ribonucleotide reductase subunit M1(RRM1), X-ray repair cross-complementing gene 1 (XRCC1), thymidylate synthase (TS) and class III ß-tubulin (TUBB3), and to assess their application in the clinic for prediction of response of non-small cell lung cancer (NSCLC) to chemoradiotherapy. MATERIALS AND METHODS: We have designed four gene molecular beacon (MB) probes for multiplex quantitative real-time polymerase chain reactions to examine RRM1, XRCC1, TUBB3 and TS mRNA expression in paraffin-embedded specimens from 50 patients with advanced or metastatic carcinomas. Twenty one NSCLC patients receiving cisplatin- based first-line treatment were analyzed. RESULTS: These molecular beacon probes could specially bind to their target genes in homogeneous solutions. Patients with low RRM1 and XRCC1 mRNA levels were found to have apparently higher response rates to chemoradiotherapy compared with those with high levels of RRM1 and XRCC1 expression (p<0.05). The TS gene expression level was not significantly associated with chemotherapy response (p>0.05). CONCLUSIONS: A method of simultaneously detecting four molecular markers was successfully established and applied for evaluation of chemoradiotherapy response. It may be a useful tool in personalized cancer therapy.