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Leuconostoccitreum, a hetero-fermentative type of lactic acid bacteria, is a crucial probiotic candidate because of its ability to promote human health. However, inefficient gene manipulation tools limit its utilization in bioindustries. We report, for the first time, the development of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference (CRISPRi) system for engineering L. citreum. For reliable expression, the expression system of synthetic single guide RNA (sgRNA) and the deactivated Cas9 of Streptococcus pyogenes (SpdCas9) were constructed in a bicistronic design (BCD) platform using a high-copy-number plasmid. The expression of SpdCas9 and sgRNA was optimized by examining the combination of two synthetic promoters and Shine&ndash, Dalgarno sequences, the strong expression of sgRNA and the weak expression of SpdCas9 exhibited the most significant downregulation (20-fold decrease) of the target gene (sfGFP), without cell growth retardation caused by SpdCas9 overexpression. The feasibility of the optimized CRISPRi system was demonstrated by modulating the biosynthesis of riboflavin. Using the CRISPRi system, the expression of ribF and folE genes was downregulated (3.3-fold and 5.6-fold decreases, respectively), thereby improving riboflavin production. In addition, the co-expression of the rib operon was introduced and the production of riboflavin was further increased up to 1.7 mg/L, which was 1.53 times higher than that of the wild-type strain.

The lactic acid bacteria (LAB) Leuconostoc citreum are non-sporulating hetero-fermentative bacteria that play an important role in the fermented food industry. In this study, for the enhanced and reliable production of recombinant proteins in L. citreum, we developed a bicistronic design (BCD) expression system which includes a short leader peptide (1st cistron) followed by target genes (2nd cistron) under the control of a single promoter. Using superfolder green fluorescent protein (sfGFP) as a reporter, the functionality of BCD in L. citreum was verified. Further, to improve the expression in BCD, we tried to engineer a Shine-Dalgarno sequence (SD2) for the 2nd cistron and a promoter by FACS screening of random libraries, and both strong SD2 (eSD2) and promoter (P710V4) were successfully isolated. The usefulness of the engineered BCD with P710V4 and eSD2 was further validated using three model proteins—glutathione-s-transferase, human growth hormone, and α-amylase. All examined proteins were successfully produced with levels highly increased compared with those in the original BCD as well as the monocistronic design (MCD) expression system.

Objectives: The purpose of this study was to evaluate the effects of Polyporus Herbal-acupuncture(PO-HA) at KI10 (Umgok) on nephritis induced by lipopolysaccharide(LPS) in rats. Methods: Rats were allocated into normal, control, and 2 experimental groups. The rats in the control group were intra-peritoneally injected with LPS for nephritis induction. The rats in the groups of experiment 1 and experiment 2 were treated with Saline injection, and PO-HA, respectively at KI10 three times for a week and then intra-peritoneally injected with LPS. To evaluate the effects of PO-HA at KI10, WBC count in blood, creatine, tumor necrosis factor-alpha (TNF-α), cytokine-induced neutrophil chemoattractant 1(CINC-1) in serum, urinary volume, creatinine, total protein in urine, myeloperoxidase(MPO) in kidney were measured. Results: PO-HA at KI10 significantly suppressed the increase of WBC in blood, TNF-α , CINC-1 in serum, MPO in kidney of LPS-stimulated rats. PO-HA at KI10 significantly suppressed the increase creatinine, total protein in urine of LPS-stimulated rats. Conclusions: According to these results, it is postulated that PO-HA at KI10 has an anti-inflammatory and renal-protective effects on LPS-induced nephritis in rats. Therefore, it is suggested that PO-HA at KI10 may be an useful therapeutics for nephritis in clinical field after further researches.

Objectives : The purpose of this study is to report the clinical effects of Korean medical treatment with Hominis Placenta Pharmacopunture(HPP) on cold hypersensitivity of hands. Methods : A 46-year-old female patient whose chief complaint was cold hypersensitivity of hands was treated with Placenta Pharmacopunture at LI4 and LI10, ten times for a month. Acupuncture and herbal medicine were added depending on the symptoms. Digital Infrared Thermal Imaging(D.I.T.I) was taken and Visual Analog Scale(VAS) was examined before and after each treatment. Results : After the treatments, the symptoms were improved. Conclusions : DITI and VAS showed that the patient`s cold hypersensitivity of hands was improved after Korean medical treatment with Hominis Placenta Pharmacopuncture.

Cardiovascular complications have been major concerns in the treatment of diabetes, and up to 80% of all deaths in diabetic patients are linked to cardiovascular problems. Impaired angiogenesis is one of the most serious symptoms associated with diabetes, resulting in delayed wound healing and lower limb amputation. Endothelial progenitor cells (EPCs), a subpopulation of adult stem cells, are recruited from bone marrow to the injured vessel to promote endothelial regeneration and neovascularization, playing an important role in angiogenesis. Interestingly, several clinical studies have showed that the number of recruited EPCs is reduced and their function is decreased under diabetic conditions, implying that diabetic EPC dysfunction may contribute to defective angiogenesis and resultant cardiovascular complications in diabetes. To recover the functional abilities of diabetic EPCs and to address possible application of EPC cell therapy to diabetic patients, some studies provided explanations for diabetic EPC dysfunction including increased oxidative stress, involvement of the inflammatory response, alteration in the nitric oxide pathway and reduced signals for EPC recruitment. This review discusses clinical evidence of impairment of EPC functions under diabetic conditions and the suggested mechanisms for diabetic EPC dysfunction.

Background The NSCLC patients who experienced good clinical responses to an EGFR-TKI will inevitably develop acquired resistance. A great deal of research is being carried out to discover the molecular mechanisms underlying this resistance. In comparison, few studies have been conducted to find out about the clinical characteristics of acquired resistance in the patients who had responded to an EGFR-TKI. Herein we investigated clinical characteristics of NSCLC patients who experienced acquired resistance during gefitinib therapy. Patients and methods We reviewed NSCLC patients who showed a clinical benefit from initial gefitinib therapy. All clinical data were obtained from 11 centers of Korean Molecular Lung Cancer Group (KMLCG). The clinical manifestations of acquired resistance, time to progression (TTP), and post-progression survival (PPS) after gefitinib failure were analyzed retrospectively. Results A total of 417 patients were recruited. Median TTP was 10.2 months (95% CI, 9.5–10.9). TTP showed a significant longer duration in female, non-smoker, and patients with adenocarcinoma. At the time of acquired resistance, 63.3% of the patients showed symptomatic deterioration. Sites of disease progression were as follows: primary lung lesion in 58.4%, previous metastasis in 38.3%, and new metastasis in 54.2%. Patients with EGFR wild type showed a tendency of higher frequency in symptomatic deterioration and newly development of CNS metastasis compared with patients with EGFR mutation. There was a significant difference in newly development of lung metastasis between patients with exon 19 deletion and those with L858R mutation (41.4% vs. 6.3%, p =0.02). PPS was 8.9 months (95% CI, 7.4–10.4). Smoking history, PS, new CNS lesion and subsequent chemotherapy were independent factors for PPS. Conclusion This study suggests that clinical manifestations of acquired resistance may be different according to EGFR mutation status and EGFR mutation genotype. In addition, subsequent chemotherapy confers clinical benefit in terms of PPS in NSCLC patients who experienced acquired resistance after gefitinib therapy.

The C5aR antagonist (C5aRA) 1 , which blocks the interaction of C5a anaphylatoxin and its receptor C5aR, is one of the most potent therapeutic agents for the treatment of various autoimmune diseases and acute inflammatory conditions. Here we developed an efficient C5aRA production system using Escherichia coli . To produce functional C5aRA, which contains three disulfide bonds, we used E. coli Origami (DE3), which possessed an oxidative cytoplasm, as the production host. To improve solubility and ease in purification, we examined the effectiveness of three different fusion partners, including N utilization substrate A (NusA), maltose-binding protein (MBP), and thioredoxin A (TrxA), as well as three different culture temperatures (i.e., 25, 30, and 37 °C). Among the three fusion partners, MBP exhibited the highest solubility in the fusion protein at all tested temperatures. However, the highest biological activity against C5aR was observed with the NusA fusion. For large-scale production, batch fermentation was also performed using a NusA-fused C5aRA production system by using a lab-scale bioreactor. After a 12-h cultivation, approximately 496 mg/L of NusA-fused C5aRA could be produced.

Human kringle domains (KDs) are ubiquitously expressed binding modulators that fold into seven flexible loops and it has been previously demonstrated that KDs can be engineered toward target-specific binding proteins as a non-antibody protein scaffold. Here, we report a method for efficient expression of a KD derivative (KD548)-a promising anti-cancer agent-by high-cell-density culture of Escherichia coli at a preparative scale production. The correct folding of KD548 requires three disulfide bonds. Nevertheless, cytoplasmic expression of KD548 in E. coli led to good yields of highly soluble proteins with high activity. For efficient expression, four sets of expression systems consisting of different promoters (lac or T7) and fusion tags (His or FLAG) were examined. Of these, the expression system using a combination of the T7 promoter with the FLAG tag resulted in the highest production in shake flask cultivation as well as in high-cell-density cultivation performed in a 6.6-L jar bioreactor. When protein expression was induced at high-cell density (optical density [OD] = 100) and when complex feeding solutions were supplemented, cell density (maximum OD = 184) and production yield (approximately 5.4 g/L) were significantly enhanced to values that were much higher than those found previously with Pichia cultivation (8 mg/L).

Undifferentiated sarcoma is an uncommon primary malignant tumor of the liver typically occurring in older children. It is also referred to as malignant mesenchymoma, fibromyxosarcoma, or mesenchymal sarcoma. We experienced a case of undifferentiated sarcoma in 72-year-old male. Contrast enhanced liver CT scan revealed a 3.4 cm heterogeneously enhancing, ill-defined, and low attenuated mass in the left liver and subtle intrahepatic duct dilatation. And, in tubogram, there were segmental stenosis and occlusion from the hilum to the proximal common bile duct. We did ultrasonography guided liver biopsy. The pathologic finding revealed infiltrative growth of atypical cells with rhabdoid features. Some atypical cells showed clear cytoplasm, but no organoid pattern was identified. The stroma around atypical cells was filled with eosinophilic hyaline material. These tumor cells were positive for vimentin only, and the tumor was consistent with undifferentiated sarcoma of the liver.

BACKGROUND Pulmonary lymphangioleiomyomatosis (LAM) is a rare disease occurring in women of reproductive age and leading to progressive respiratory failure in spite of treatment. In Korea the first case was reported in 1984 and by 1997 a total of 23 cases had been reported. The clinical findings of these Korean cases are reviewed. METHODS The details of 10 cases of LAM on file at Seoul National University Hospital were reviewed together with those of 13 cases previously reported from other Korean institutes. Two, including the only one to be reported in a man, were excluded after reviewing the clinical, radiological, and pathological findings, leaving a total of 21 cases in the present study. RESULTS All 21 patients were women and in all cases the disease was proven pathologically. The mean (SD) age at onset of symptoms was 32 (8.6) years. The most common symptoms were dyspnoea and pneumothorax which were seen in 19 (90%) and 13 (76%) patients, respectively. Pulmonary function tests showed decreased transfer factor (Tlco) (100%) and airflow limitation (67%). All the cases had characteristic cysts on high resolution computed tomographic (HRCT) scanning. The overall severity score based on HRCT scans correlated with the percentage predicted Tlco/Va (p = 0.03) and FEV 1 /FVC (p = 0.02). The patients were all treated with medroxyprogesterone and/or tamoxifen. Follow up was possible in 10 cases. Two of these patients appeared to stabilise with no appreciable change clinically or in lung function on medroxyprogesterone and/or tamoxifen, but the remaining patients all deteriorated with two dying of respiratory insufficiency and one of infection following lung transplantation. CONCLUSIONS As in other countries, in Korea LAM occurs exclusively in women and progresses despite hormonal treatment.