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

High triglyceride/HDL cholesterol ratio is associated with silent brain infarcts in a healthy population

Background Triglycerides (TG)/high-density lipoprotein (HDL) cholesterol ratio is a marker of small/dense low-density lipoprotein particles, which are closely associated with various metabolic and vascular diseases. However, the role of TG/HDL cholesterol ratio in cerebrovascular diseases has not been well studied. In this study, we evaluated the relationship between TG/HDL cholesterol ratio and the presence of silent brain infarct (SBI) in a neurologically healthy population. Methods We retrospectively evaluated consecutive participants in health check-ups between January 2006 and December 2013. SBI was defined as an asymptomatic, well-defined lesion with a diameter of ≥3 mm on T1- or T2-weighted images. TG/HDL cholesterol ratio was calculated after dividing absolute TG levels by absolute HDL cholesterol levels. Results Of 3172 healthy participants, 263 (8.3%) had SBI lesions. In multivariate analysis, TG/HDL cholesterol ratio was independently associated with SBI (adjusted odds ratio [aOR] = 1.16, 95% confidence interval [CI] = 1.00 to 1.34, P = 0.047). This association was prominent in males (aOR = 1.23, 95% CI = 1.03 to 1.48, P = 0.021), but not in females. In the analyses of the relationships between lipid parameters and SBI lesion burden, TG/HDL cholesterol ratio was positively correlated, and total cholesterol/TG ratio was negatively correlated with SBI lesion burden, in dose-response manners (P for trend = 0.015 and 0.002, respectively). Conclusions The TG/HDL cholesterol ratio was positively associated with the prevalence of SBI in a neurologically healthy population

High triglyceride-glucose index is associated with subclinical cerebral small vessel disease in a healthy population: a cross-sectional study

The triglyceride-glucose (TyG) index is a marker of insulin resistance (IR) and has been associated with various metabolic syndromes, cardiovascular diseases, and cerebrovascular diseases. However, limited information is available regarding its association with subclinical cerebral small vessel disease (cSVD). In this study, we evaluated the relationship between the TyG index and cSVD, including silent brain infarcts (SBIs) and white matter hyperintensity (WMH). We assessed health check-up participants aged 40–79years from 2006 to 2013. The TyG index was calculated using the log scale of fasting triglyceride (mg/dL) × fasting glucose (mg/dL)/2. The Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) was also calculated. This was compared with two insulin surrogates and cSVD as another IR indicator and compared the association between two insulin surrogates and cSVD. SBI was measured for both prevalence and burden. The WMH volume was quantitatively rated using a computer-assisted semi-automated technique. Results A total of 2615 participants were evaluated (median age: 56years, male sex: 53%). In the multivariable logistic regression analysis, the TyG index was seen to be associated with SBI prevalence (adjusted odds ratio: 1.39; 95% confidence interval [CI] = 1.06–1.81). Further quantitative analyses showed a positive dose–response relationship between the TyG index and SBI burden (P for trend = 0.006). In multivariable linear regression analysis, the TyG index was also found to be related to the volume of WMH (β = 0.084; 95% CI = 0.013 to 0.154). Additionally, the TyG index showed a similar or slightly stronger association with the prevalence of SBI and the volume of WMH than did HOMA-IR. A high TyG index was associated with a higher prevalence and burden of cSVD in a neurologically healthy population. This marker of IR could be a convenient and useful predictor of cSVD

3D garment digitisation for virtual wardrobe using a commodity depth sensor

5-Aminovaleric acid (5AVA) is an important five-carbon platform chemical that can be used for the synthesis of polymers and other chemicals of industrial interest. Enzymatic conversion of L-lysine to 5AVA has been achieved by employing lysine 2-monooxygenase encoded by the davB gene and 5-aminovaleramidase encoded by the davA gene. Additionally, a recombinant Escherichia coli strain expressing the davB and davA genes has been developed for bioconversion of L-lysine to 5AVA. To use glucose and xylose derived from lignocellulosic biomass as substrates, rather than L-lysine as a substrate, we previously examined direct fermentative production of 5AVA from glucose by metabolically engineered E. coli strains. However, the yield and productivity of 5AVA achieved by recombinant E. coli strains remain very low. Thus, Corynebacterium glutamicum, a highly efficient L-lysine producing microorganism, should be useful in the development of direct fermentative production of 5AVA using L-lysine as a precursor for 5AVA. Here, we report the development of metabolically engineered C. glutamicum strains for enhanced fermentative production of 5AVA from glucose.Various expression vectors containing different promoters and origins of replication were examined for optimal expression of Pseudomonas putida davB and davA genes encoding lysine 2-monooxygenase and delta-aminovaleramidase, respectively. Among them, expression of the C. glutamicum codon-optimized davA gene fused with His-Tag at its N-Terminal and the davB gene as an operon under a strong synthetic H promoter (plasmid p36davAB3) in C. glutamicum enabled the most efficient production of 5AVA. Flask culture and fed-batch culture of this strain produced 6.9 and 19.7\ua0g/L (together with 11.9\ua0g/L glutaric acid as major byproduct) of 5AVA, respectively. Homology modeling suggested that endogenous gamma-aminobutyrate aminotransferase encoded by the gabT gene might be responsible for the conversion of 5AVA to glutaric acid in recombinant C. glutamicum. Fed-batch culture of a C. glutamicum gabT mutant-harboring p36davAB3 produced 33.1\ua0g/L 5AVA with much reduced (2.0\ua0g/L) production of glutaric acid.Corynebacterium glutamicum was successfully engineered to produce 5AVA from glucose by optimizing the expression of two key enzymes, lysine 2-monooxygenase and delta-aminovaleramidase. In addition, production of glutaric acid, a major byproduct, was significantly reduced by employing C. glutamicum gabT mutant as a host strain. The metabolically engineered C. glutamicum strains developed in this study should be useful for enhanced fermentative production of the novel C5 platform chemical 5AVA from renewable resources

Idiopathic retroperitoneal fibrosis associated with Hashimoto's thyroiditis in a patient with a single functioning kidney

AbstractRetroperitoneal fibrosis (RPF) is a rare disease characterized by the presence of fibroinflammatory tissue around the abdominal aorta and ureteral entrapment in most cases. Idiopathic RPF is frequently reported in association with autoimmune diseases; however, there have been few reports of idiopathic RPF associated with Hashimoto's thyroiditis. Here, we report a case of idiopathic RPF with Hashimoto's thyroiditis in a patient with a single functioning kidney, which was successfully treated by corticosteroid therapy and transient intraureteral stent insertion with a double-J catheter

Mind bomb 1 in the lymphopoietic niches is essential for T and marginal zone B cell development

Notch signaling regulates lineage decisions at multiple stages of lymphocyte development, and Notch activation requires the endocytosis of Notch ligands in the signal-sending cells. Four E3 ubiquitin ligases, Mind bomb (Mib) 1, Mib2, Neuralized (Neur) 1, and Neur2, regulate the Notch ligands to activate Notch signaling, but their roles in lymphocyte development have not been defined. We show that Mib1 regulates T and marginal zone B (MZB) cell development in the lymphopoietic niches. Inactivation of the Mib1 gene, but not the other E3 ligases, Mib2, Neur1, and Neur2, abrogated T and MZB cell development. Reciprocal bone marrow (BM) transplantation experiments revealed that Mib1 in the thymic and splenic niches is essential for T and MZB cell development. Interestingly, when BM cells from transgenic Notch reporter mice were transplanted into Mib1-null mice, the Notch signaling was abolished in the double-negative thymocytes. In addition, the endocytosis of Dll1 was impaired in the Mib1-null microenvironment. Moreover, the block in T cell development and the failure of Dll1 endocytosis were also observed in coculture system by Mib1 knockdown. Our study reveals that Mib1 is the essential E3 ligase in T and MZB cell development, through the regulation of Notch ligands in the thymic and splenic microenvironments

Cost-effectiveness of using amyloid positron emission tomography in individuals with mild cognitive impairment

Background Amyloid positron emission tomography (PET) makes it possible to diagnose Alzheimers disease (AD) in its prodromal phase including mild cognitive impairment (MCI). This study evaluated the cost-effectiveness of including amyloid-PET for assessing individuals with MCI. Methods The target population was 60-year-old patients who were diagnosed with MCI. We constructed a Markov model for the natural history of AD with the amyloid positivity (AP). Because amyloid-PET can detect the AP MCI state, AD detection can be made faster by reducing the follow-up interval for a high-risk group. The health outcomes were evaluated in quality-adjusted life years (QALYs) and the final results of cost-effectiveness analysis were presented in the form of the Incremental Cost-Effectiveness Ratio (ICER). To handle parameter uncertainties, one-way sensitivity analyses for various variables were performed. Results Our model showed that amyloid-PET increased QALYs by 0.003 in individuals with MCI. The estimated additional costs for adopting amyloid-PET amounted to a total of 1250 USD per patient when compared with the cost when amyloid-PET is not adopted. The ICER was 3,71,545 USD per QALY. According to the sensitivity analyses, treatment effect of Donepezil and virtual intervention effect in MCI state were the most influential factors. Conclusions In our model, using amyloid-PET at the MCI stage was not cost-effective. Future advances in management of cognitive impairment would enhance QALYs, and consequently improve cost-effectiveness.This project was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HC15C1509 to JW Han, JH Park, TJ Lee, and HG Jeong). Additional funding came from the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea [HI09C1379 (A092077) to KW Kim]

Predicting the Interactome of Xanthomonas oryzae pathovar oryzae for target selection and DB service

<p>Abstract</p> <p>Background</p> <p>Protein-protein interactions (PPIs) play key roles in various cellular functions. In addition, some critical inter-species interactions such as host-pathogen interactions and pathogenicity occur through PPIs. Phytopathogenic bacteria infect hosts through attachment to host tissue, enzyme secretion, exopolysaccharides production, toxins release, iron acquisition, and effector proteins secretion. Many such mechanisms involve some kind of protein-protein interaction in hosts. Our first aim was to predict the whole protein interaction pairs (interactome) of <it>Xanthomonas oryzae </it>pathovar oryzae (Xoo) that is an important pathogenic bacterium that causes bacterial blight (BB) in rice. We developed a detection protocol to find possibly interacting proteins in its host using whole genome PPI prediction algorithms. The second aim was to build a DB server and a bioinformatic procedure for finding target proteins in Xoo for developing pesticides that block host-pathogen protein interactions within critical biochemical pathways.</p> <p>Description</p> <p>A PPI network in Xoo proteome was predicted by bioinformatics algorithms: PSIMAP, PEIMAP, and iPfam. We present the resultant species specific interaction network and host-pathogen interaction, XooNET. It is a comprehensive predicted initial PPI data for Xoo. XooNET can be used by experimentalists to pick up protein targets for blocking pathological interactions. XooNET uses most of the major types of PPI algorithms. They are: 1) Protein Structural Interactome MAP (PSIMAP), a method using structural domain of SCOP, 2) Protein Experimental Interactome MAP (PEIMAP), a common method using public resources of experimental protein interaction information such as HPRD, BIND, DIP, MINT, IntAct, and BioGrid, and 3) Domain-domain interactions, a method using Pfam domains such as iPfam. Additionally, XooNET provides information on network properties of the Xoo interactome.</p> <p>Conclusion</p> <p>XooNET is an open and free public database server for protein interaction information for Xoo. It contains 4,538 proteins and 26,932 possible interactions consisting of 18,503 (PSIMAP), 3,118 (PEIMAP), and 8,938 (iPfam) pairs. In addition, XooNET provides 3,407 possible interaction pairs between two sets of proteins; 141 Xoo proteins that are predicted as membrane proteins and rice proteomes. The resultant interacting partners of a query protein can be easily retrieved by users as well as the interaction networks in graphical web interfaces. XooNET is freely available from <url>http://bioportal.kobic.kr/XooNET/</url>.</p

Prognostic factors in children with extracranial germ cell tumors treated with cisplatin-based chemotherapy

PurposeTo evaluate the outcomes and prognostic factors in children with extracranial germ cell tumors (GCTs) treated at a single institution.MethodsSixty-six children diagnosed with extracranial GCTs between 1996 and 2012 were included in the study. Primary treatment was surgical excision, followed by six cycles of cisplatin-based chemotherapy. The survival rates were compared according to the International Germ Cell Cancer Cooperative Group classification used for GCTs in adults to validate the classification guidelines for GCTs in children.ResultsThe median patient age was 4.4 years. In 34 patients (51.5%), the primary tumor site was the gonad. Extragonadal GCTs were detected in 32 patients. The 5-year overall survival and event-free survival (EFS) were 92.0%±3.5% and 90.4%±3.7%, respectively. In univariate analysis, tumor histology, metastasis, and elevated alpha-fetoprotein were not prognostic factors in children with extracranial GCTs. However, EFS was poorer in patients with mediastinal disease (n=12, 66.7%±13.6 %) than in those with nonmediastinal disease (n=54, 96.0%±2.8%) (P=0.001). The 5-year EFS was lower in patients older than 10 years, (n=21, 80.0%±8.9%) compared with those younger than 10 years (n=45, 95.2%±3.3%) (P=0.04). Multivariate analysis identified the mediastinal tumor site as the only independent prognostic factor.ConclusionThe prognosis of children with extracranial GCTs was favorable. However, nongerminomatous mediastinal tumors were associated with poor survival in children. Further research is needed to improve the prognosis of children with malignant mediastinal GCTs