Expression, purification and refolding of pro-MMP-2 from inclusion bodies of E. coli.

MMP-2 has been reported as the most validated target for cancer progression and deserves further investigation. However, due to the lack of methods for obtaining large amounts of highly purified and bioactive MMP-2, identifying specific substrates and developing specific inhibitors of MMP-2 remains extremely difficult. In this study, the DNA fragment coding for pro-MMP-2 was inserted into plasmid pET28a in an oriented manner, and the resulting recombinant protein was effectively expressed and led to accumulation as inclusion bodies in E. coli. This protein was easy to purify to near homogeneity by the combination of common inclusion bodies purification procedure and cold ethanol fractionation. Then, our results of gelatin zymography and fluorometric assay revealed that pro-MMP-2 at least partially restored its natural structure and enzymatic activity after renaturation. We obtained approximately 11 mg refolded pro-MMP-2 protein from 1 L LB broth, which was higher than other strategies previously reported. In conclusion, a simple and cost-effective procedure for obtaining high amounts of functional MMP-2 was developed, which would contribute to the progress of studies on the gamut of biological action of this important proteinase. Furthermore, our protocol should be appropriate for the expression, purification, and refolding of other bacterial toxic proteins.

Feasibility and efficacy of modified fixed citrate concentration protocol using only commercial preparations in critically ill patients: a prospective cohort study with a historical control group.

BACKGROUND: The cumbersome program and the shortage of commercial solution hindered the regular application of regional citrate anticoagulation (RCA). It is urgent to simplify the protocol using only commercial preparations. The aim of this study was to explore the feasibility and efficacy of the modified protocol for continuous veno-venous hemofiltration (CVVH) in unselected critically ill patients. METHODS: A prospective cohort study was conducted in 66 patients who received a new protocol combining fixed citrate concentration with modified algorithm for supplements (i.e., fixed protocol), and compared the efficacy, safety and convenience for this group to a historical control group with a traditional protocol (n = 64), where citrate was titrated according to the circuit ionized calcium concentration (i.e., titrated protocol). The convenience was defined as the demand for monitoring test and dose adjustment of any supplement. RESULTS: The filter lifespan was 63.2 ± 16.1 h in the fixed group and 51.9 ± 17.7 h in the titrated group, respectively. Kaplan-Meier survival analysis demonstrated longer circuit lifetime for fixed group (log-rank, p = 0.026). The incidence of circuit clotting was lower in the fixed protocol (15.2% vs. 29.7% in the titrated protocol, p = 0.047). Moreover, compared with the titrated group, patients with fixed protocol had less demand for monitoring test and dose adjustment of any supplement (the number of times per person per day) (3.3 [IQR 2.3-4.5] vs. 5.7 [IQR 3.3-6.9], p = 0.001 and 1.9 [IQR 0.5-2.7] vs. 6.3 [IQR 4.2-7.9], p < 0.001; respectively). No new onset bleeding complications occurred in all patients. The overall incidence of suspected citrate accumulation was 4.6% and there was no difference between the two groups (p = 0.969), yet a lower rate of metabolic alkalosis was found in the fixed group (3.0% vs. 14.1%, p = 0.024). CONCLUSIONS: Our modified fixed citrate concentration protocol is feasible, safe and effective to enhance the circuit lifespan and the convenience of implementation while maintaining a similar safety when compared to the traditional protocol. Using only commercial preparations may be helpful for widespread application of RCA. TRIAL REGISTRATION: Clinicaltrials.gov. NCT02663960 . Registered 26 January 2016.

Stress ulcer prophylaxis in intensive care unit patients receiving enteral nutrition: a systematic review and meta-analysis.

BACKGROUND: Pharmacologic stress ulcer prophylaxis (SUP) is recommended in critically ill patients with high risk of stress-related gastrointestinal (GI) bleeding. However, as to patients receiving enteral feeding, the preventive effect of SUP is not well-known. Therefore, we performed a meta-analysis of randomized controlled trials (RCTs) to evaluate the effect of pharmacologic SUP in enterally fed patients on stress-related GI bleeding and other clinical outcomes. METHODS: We searched PubMed, Embase, and the Cochrane database from inception through 30 Sep 2017. Eligible trials were RCTs comparing pharmacologic SUP to either placebo or no prophylaxis in enterally fed patients in the ICU. Results were expressed as risk ratio (RR) and mean difference (MD) with accompanying 95% confidence interval (CI). Heterogeneity, subgroup analysis, sensitivity analysis and publication bias were explored. RESULTS: Seven studies (n = 889 patients) were included. There was no statistically significant difference in GI bleeding (RR 0.80; 95% CI, 0.49 to 1.31, p = 0.37) between groups. This finding was confirmed by further subgroup analyses and sensitivity analysis. In addition, SUP had no effect on overall mortality (RR 1.21; 95% CI, 0.94 to 1.56, p = 0.14), Clostridium difficile infection (RR 0.89; 95% CI, 0.25 to 3.19, p = 0.86), length of stay in the ICU (MD 0.04 days; 95% CI, -0.79 to 0.87, p = 0.92), duration of mechanical ventilation (MD -0.38 days; 95% CI, -1.48 to 0.72, p = 0.50), but was associated with an increased risk of hospital-acquired pneumonia (RR 1.53; 95% CI, 1.04 to 2.27; p = 0.03). CONCLUSIONS: Our results suggested that in patients receiving enteral feeding, pharmacologic SUP is not beneficial and combined interventions may even increase the risk of nosocomial pneumonia.

Metagenomic next-generation sequencing: A promising tool for diagnosis and treatment of suspected pneumonia in rheumatic patients with acute respiratory failure: Retrospective cohort study.

Background: The effectiveness of metagenomic next-generation sequencing (mNGS) in respiratory pathogen detection and clinical decision-making in critically rheumatic patients remains largely unexplored. Methods: A single-center retrospective study of 58 rheumatic patients who were admitted to ICU due to suspected pneumonia with acute respiratory failure if they underwent both bronchoalveolar lavage fluid specimen mNGS and combined microbiological tests (CMTs) was conducted to compare their diagnostic performance, using clinical composite diagnosis as the gold standard. Treatment modifications based on mNGS results were also reviewed. Results: Forty-three patients were diagnosed with microbiologically confirmed pneumonia and 15 were considered as a non-infectious disease. mNGS outperformed CMTs in the accurate diagnosis of infectious and non-infectious lung infiltration (98.1% [57/58] vs. 87.9% [51/58], P = 0.031). A total of 94 causative pathogens were defined by the gold standard and 27 patients had polymicrobial pneumonia. The sensitivity of pathogen detection and complete concordance with the gold standard by mNGS exceeded those by CMTs (92.6% [87/94] vs. 76.6% [72/94], P < 0.001 and 72.1% [31/43] vs. 51.2% [22/43], P = 0.004, respectively). Moreover, 22 pathogens were detected only by mNGS and confirmed by orthogonal test. Accordingly, the etiological diagnosis changed in 19 cases, and the empirical treatment improved in 14 cases, including 8 cases of rescue treatment and 11 of antibiotics de-escalation. At the pathogen-type level, both methods were comparable for bacteria, but mNGS was advantageous to identify viruses (accuracy: 100% vs. 81%, P = 0.004). For Pneumocystis jirovecii detection, mNGS improved the sensitivity compared with Gomori's methenamine silver stain (91.7% vs. 4.2%, P < 0.001) and was higher than polymerase chain reaction (79.2%), but the difference was not significant (P = 0.289). In terms of Aspergillus, the better sensitivity with a combination of culture and galactomannan test than that with mNGS was found (100% vs. 66.7%, P = 0.033). Conclusions: mNGS has an excellent accuracy in etiological diagnosis and pathogen detection of suspected pneumonia in critically rheumatic patients, which has potential significance for clinical decision-making. Its superiority to different types of pathogens depends on the comprehensiveness of CMTs.

Metagenomic next-generation sequencing for the diagnosis of suspected pneumonia in immunocompromised patients.

OBJECTIVES: To evaluate the potential of metagenomic next-generation sequencing (mNGS), compared with that of comprehensive conventional microbiological tests (CMTs), of bronchoalveolar lavage fluid (BALF) as a front-line diagnostic for immunocompromised patients with suspected pneumonia. METHODS: Sixty critically ill immunocompromised patients undergoing both mNGS of BALF and CMTs for suspected pneumonia were retrospectively analysed. The diagnostic performance was compared between mNGS and CMTs, using the composite diagnosis as the reference standard. RESULTS: Forty-nine patients were diagnosed with microbiologically confirmed pneumonia, with 55% having polymicrobial infections. There was no significant difference in the overall diagnostic accuracy between mNGS and CMTs (61.7% vs 76.7%; P = 0.11). mNGS and CMTs had comparable diagnostic accuracy for bacterial and viral infections. Although mNGS identified more viral pneumonia, it had a much lower diagnostic accuracy for fungal infections (76.7% vs 99.2%; P < 0.001), mainly due to the low sensitivity for invasive pulmonary aspergillosis (45.5% vs 100%; P < 0.001). CONCLUSION: The overall diagnostic performance of BALF mNGS as a first-line diagnostic was similar to that of comprehensive CMTs, except in the case of a lack of consideration of potential pathogens or limited CMTs. The combination of mNGS and CMTs may be the best diagnostic strategy.

Etiologies and outcomes of rheumatology patients with acute respiratory failure requiring intensive care: a single-center medical records review study of 259 patients.

OBJECTIVE: The etiologies of acute respiratory failure in patients with systemic rheumatic diseases (SRDs) requiring intensive care remain unknown. This study was undertaken to investigate the etiologies and outcomes. METHODS: A medical records review study was performed of 259 adult SRDs patients with respiratory failure admitted to medical ICU across a 5-year period. The etiologies were classified as infection, SRD exacerbation, and undetermined. The factors associated with ICU mortality were identified with multivariate logistic regression analysis. RESULTS: The etiologies of respiratory failure included infection (n = 209, 80.7%), SRD exacerbation (n = 71, 27.4%), and undetermined (n = 21, 8.1%). The most common pathogen was Pneumocystis jirovecii (39.8%), followed by Aspergillus spp. (33.2%), and cytomegalovirus (23.2%). The ICU mortality rate was 59.8%. A high acute physiology and chronic health evaluation II score (OR 1.118, 95% CI 1.054 to 1.186, p < 0.001), a PaO2/FiO2 ratio < 100 mmHg (OR 3.918, 95% CI 2.199 to 6.892, p < 0.001), and a diagnosis of dermatomyositis/polymyositis (OR 4.898, 95% CI 1.949 to 12.309, p = 0.001), vasculitis (OR 3.007, 95% CI 1.237 to 7.309, p = 0.015), and Pneumocystis pneumonia (OR 2.345, 95% CI 1.168 to 4.705, p = 0.016) were associated with increased mortality. CONCLUSIONS: Opportunistic infections and SRD exacerbation were the most common etiologies of acute respiratory failure in patients with SRDs requiring ICU admission, with high ICU mortality. Development of a standard protocol for differential diagnosis in this population might help initiate definitive therapy and improve clinical outcome. Key Points • Infections, especially with opportunistic infections, were the leading cause of acute respiratory failure in critically ill rheumatology patients, with high mortality. • Severity of illness, certain types of rheumatic diseases, and opportunistic fungal infections were associated with increased mortality. • Using a comprehensive diagnostic workup might help to confirm the infective etiology and improve outcome.