Engineering of Corynebacterium glutamicum for the secretory production of recombinant proteins via Tat-dependent pathway

Corynebacterium glutamicum, which has been an industrial producer of various L-amino acids, nucleic acids, and vitamins, is now also regarded as a potential host for the secretory production of recombinant proteins since it exhibits numerous ideal features for protein secretion: (i) it has a single cellular membrane as a gram-positive bacterium, which allows proteins to be easily secreted into the extracellular medium. (ii) C. glutamicum secretes only a few endogenous proteins into the culture medium, which allows the simpler purification of target proteins in downstream process. (iii), secreted proteins from C. glutamicum can be kept stable because extracellular protease activity is rarely detectable. To harness its potential as an industrial platform for recombinant protein production, the development of an efficient secretion system is necessary. To achieve this goal first, we engineered several genetic parts in C. glutamicum: (i) synthetic promoters, (ii) plasmid copy number, (iii) signal peptides, (iv) co-expression of secretion machinery proteins. Using the engineered host-vector systems, gram-scale production of recombinant proteins could be achieved in fed-batch cultivation

Longitudinal changes in health-related quality of life according to clinical course among patients with non-tuberculous mycobacterial pulmonary disease: a prospective cohort study

Improvement in health-related quality of life (HRQL) has been suggested as an alternative treatment goal of non-tuberculous mycobacterial pulmonary disease (NTM-PD). This study was performed to elucidate the longitudinal changes in HRQL using St. Georges Respiratory Questionnaire (SGRQ) among patients with NTM-PD according to their clinical course. Patients with NTM-PD who participated in Seoul National University Hospitals prospective NTM cohort were screened. Participants for whom the SGRQ score was estimated with the one-year interval for ≥ three times were included. The longitudinal trends of the SGRQ score were assessed. The impact of the clinical course on the change in the SGRQ score was elucidated using multilevel mixed-effects linear regression with a repeated-measures model. In total, 114 patients were analyzed. During the median 5-year observation period, 53 patients started anti-mycobacterial treatment and 61 patients were observed without treatment. Among the treated patients, 24 (45.2%) achieved microbiological cure. Patients who required treatment eventually had worsening SGRQ scores with time compared with patients who could be observed without treatment (P < 0.001). In cured patients, the SGRQ score decreased from 33.9 at baseline to 20.8 at 1 year post-treatment (P < 0.001), 21.3 at 2 years (P < 0.001), and 17.6 at 3 years (P < 0.001). The SGRQ scores also decreased for 2 years of treatment in patients with NTM-PD that could not be cured, although this decrease did not last for 3 years of treatment. Worsening HRQL scores were associated with the initiation of treatment and, in turn, treatment improved HRQL scores of patients with NTM-PD. This study was registered to the ClinicalTrials.gov (Identifier: NCT01616745 / registration date: June 12, 2012). The protocol was retrospectively registered

Nontuberculous mycobacterial pulmonary disease diagnosed by two methods: a prospective cohort study

Background Microbiological criteria for diagnosing nontuberculous mycobacterial pulmonary disease (NTM-PD) include positive culture results from at least two separately expectorated sputum specimens or one bronchial washing or lavage. However, the clinical similarities and differences between patients diagnosed by these two methods remain unclear. We compared clinical features and prognoses of patients with NTM-PD diagnosed from both specimen types. Methods We analysed data from patients who participated in the Seoul National University Hospital NTM-PD cohort (ClinicalTrials.gov identifier: NCT01616745). Baseline demographics, symptoms, radiographic findings, disease progression, and treatment responses were summarized and compared between patients diagnosed from sputum specimens and patients diagnosed from bronchoscopic specimens. Results Three hundred forty-seven patients were included in the analyses. Of these, 279 (80.4%) were diagnosed from two separately expectorated sputum specimens, and 68 (19.6%) were diagnosed from bronchoscopic specimens. Patients diagnosed from sputum specimens had more frequent and severe cough, sputum, postnasal drip, and high St. Georges Respiratory Questionnaire scores. However, the extent and severity of the radiographic lesions, disease progression, and treatment responses were similar for both groups. Further analysis based on the following three groups (sputum culture positive, sputum culture negative/bronchoscopy, and scanty sputum/bronchoscopy groups) suggested that the scanty sputum/bronchoscopy group appeared to have the worst prognosis in terms of both time to progression and time to culture conversion. Conclusions Although some symptoms and quality of life were worse in patients with NTM-PD diagnosed from sputum specimens, their prognoses were similar to those of patients diagnosed by bronchoscopic specimen. We recommend bronchoscopic sampling for patients in whom NTM-PD is suspected clinically or radiographically, especially those who have no or scanty sputum.This work was supported by grant from the Seoul National University College of Medicine Research Fund, year 2017 (800–20170102). The funder did not have any role in the stud

Kidney Transplantation from a Donor Following Cardiac Death Supported with Extracorporeal Membrane Oxygenation

To expand the donor pool, organ donation after cardiac death (DCD) has emerged. However, kidneys from DCD donors have a period of long warm ischemia between cardiac arrest and the harvesting of the organs. Recently, we used extracorporeal membrane oxygenation (ECMO) to minimize ischemic injury during 'no touch' periods in a Maastricht category II DCD donor and performed two successful kidney transplantations. The kidneys were procured from a 49-yr-old male donor. The warm ischemia time was 31 min, and the time of maintained circulation using ECMO was 7 hr 55 min. The cold ischemia time was 9 hr 15 min. The kidneys were transplanted into two recipients and functioned immediately after reperfusion. The grafts showed excellent function at one and three months post-transplantation; serum creatinine (SCr) levels were 1.0 mg/dL and 0.8 mg/dL and the estimated glomerular filtration rates (eGFR) were 63 mL/min/1.73 m2 and 78 mL/min/1.73 m2 in the first recipient, and SCr levels were 1.1 mg/dL and 1.0 mg/dL and eGFR were 56 mL/min/1.73 m2 and 64 mL/min/1.73 m2 in the second recipient. In conclusion, it is suggested that kidney transplantation from a category II DCD donor assisted by ECMO is a reasonable modality for expanding donor pool

Anti-Inflammatory Potential of Carpomitra costata

Marine algae have valuable health and dietary benefits. The present study aimed to investigate whether an ethanol extract of Carpomitra costata (CCE) could inhibit the inflammatory response to LPS. CCE attenuated the production of proinflammatory mediators, such as prostaglandin E2 (PGE2) and nitric oxide (NO), by inhibiting inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-induced RAW264.7 macrophages. CCE also inhibited the expression of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6. CCE suppressed the LPS-induced DNA-binding activity of (NF-κB) and activator protein-1 (AP-1). In addition, CCE attenuated the LPS-stimulated phosphorylation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK) and phosphatidylinositol 3′-kinase/Akt (PI3K/Akt). Functional aspects of the JNK and Akt signaling pathways were analyzed using specific inhibitors, which attenuated the LPS-induced production of proinflammatory cytokines, and NO and PGE2 expression by suppressing AP-1 and NF-κB activity. In particular, the AP-1 signaling pathway is not involved in the production of inflammatory cytokines, such as IL-6, TNF-α, and IL-1β. These results suggested that CCE might exert its anti-inflammatory action by downregulating transcriptional factors (NF-κB and AP-1) through JNK and Akt signaling pathways. The current study suggested that CCE might be a valuable candidate for the treatment of inflammatory disorders

Impact of Caveolin-1 Expression on the Prognosis of Transitional Cell Carcinoma of the Upper Urinary Tract

This study aimed to investigate the relationship of caveolin-1 expression with prognosis in patients with transitional cell carcinoma of the upper urinary tract (TCC-UUT). Formalin-fixed, paraffin-embedded tissue sections of TCC-UUT from 98 patients, who had undergone radical nephroureterectomy, were stained immunohistochemically using antibodies against caveolin-1. The expression pattern of caveolin-1 was compared with the clinicopathological variables. The caveolin-1 expression was significantly correlated with T stage (p<0.001) and grade (p=0.036). The survival rate of patients with caveolin-1 positive tumors was significantly lower than that of patients with caveolin-1 negative tumors (p<0.0001). The univariate analyses identified T stage, grade, and caveolin-1 expression as significant prognostic factors for cancer-specific survival, whereas the multivariate analyses indicated that T stage and caveolin-1 expression were independent prognostic factors. These results show that the increased expression of caveolin-1 is associated with tumor progression and poor prognosis in TCC-UUT, suggesting that caveolin-1 may play an important role in the progression of TCC-UUT

Cotransplanted Bone Marrow Derived Mesenchymal Stem Cells (MSC) Enhanced Engraftment of Hematopoietic Stem Cells in a MSC-dose Dependent Manner in NOD/SCID Mice

Transplantation of marrow-derived mesenchymal stem cells (MSCs), expanded by culture in addition to whole bone marrow, has been shown to enhance engraftment of human hematopoietic stem cells (HSCs). Our hypothesis was that there might be an optimum ratio range that could enhance engraftment. We examined the percent donor chimerism according to the ratio of HSCs to MSCs in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We tested a series of ratios of co-transplanted CD34+-selected bone marrow cells, and marrow-derived MSCs into sublethally irradiated NOD/SCID mice. In all experiments, 1×105 bone marrow derived human CD34+ cells were administered to each mouse and human MSCs from different donors were infused concomitantly. We repeated the procedure three times and evaluated engraftment with flow cytometry four weeks after each transplantation. Serial ratios of HSCs to MSCs were 1:0, 1:1, 1:2 and 1:4, in the first experiment, 1:0, 1:1, 1:2, 1:4 and 1:8 in the second and 1:0, 1:1, 1:4, 1:8 and 1:16 in the third. Cotransplantation of HSCs and MSCs enhanced engraftment as the dose of MSCs increased. Our results suggest that the optimal ratio of HSCs and MSCs for cotransplantation might be in the range of 1:8-1:16; whereas, an excessive dose of MSCs might decrease engraftment efficiency