Anti-Cancer Roles of Probiotic-Derived P8 Protein in Colorectal Cancer Cell Line DLD-1.

A novel probiotics-derived protein, P8, suppresses the growth of colorectal cancer (CRC). P8 can penetrate the cell membrane via endocytosis and cause cell cycle arrest in DLD-1 cells through down-regulation of CDK1/Cyclin B1. However, neither the protein involved in the endocytosis of P8 nor the cell cycle arrest targets of P8 are known. We identified two P8-interacting target proteins [importin subunit alpha-4 (KPNA3) and glycogen synthase kinase-3 beta (GSK3ß)] using P8 as a bait in pull-down assays of DLD-1 cell lysates. Endocytosed P8 in the cytosol was found to bind specifically to GSK3ß, preventing its inactivation by protein kinases AKT/CK1ε/PKA. The subsequent activation of GSK3ß led to strong phosphorylation (S33,37/T41) of ß-catenin, resulting in its subsequent degradation. P8 in the cytosol was also found to be translocated into the nucleus by KPNA3 and importin. In the nucleus, after its release, P8 binds directly to the intron regions of the GSK3ß gene, leading to dysregulation of GSK3ß transcription. GSK3ß is a key protein kinase in Wnt signaling, which controls cell proliferation during CRC development. P8 can result in a cell cycle arrest morphology in CRC cells, even when they are in the Wnt ON signaling state.

Toxicological Evaluation of a Probiotic-Based Delivery System for P8 Protein as an Anti-Colorectal Cancer Drug.

PURPOSE: This study aimed to toxicological evaluate a probiotics-based delivery system for p8 protein as an anti-colorectal cancer drug. INTRODUCTION: Lactic acid bacteria (LAB) have been widely ingested for many years and are regarded as very safe. Recently, a Pediococcus pentosaceus SL4 (PP) strain that secretes the probiotic-derived anti-cancer protein P8 (PP-P8) has been developed as an anti-colorectal cancer (CRC) biologic by Cell Biotech. We initially identified a Lactobacillus rhamnosus (LR)-derived anti-cancer protein, P8, that suppresses CRC growth. We also showed that P8 penetrates specifically into CRC cells (DLD-1 cells) through endocytosis. We then confirmed the efficacy of PP-P8, showing that oral administration of this agent significantly decreased tumor mass (~42%) relative to controls in a mouse CRC xenograft model. In terms of molecular mechanism, PP-P8 induces cell-cycle arrest in G2 phase through down-regulation of Cyclin B1 and Cdk1. In this study, we performed in vivo toxicology profiling to obtain evidence that PP-P8 is safe, with the goal of receiving approval for an investigational new drug application (IND). METHODS: Based on gene therapy guidelines of the Ministry of Food and Drug Safety (MFDS) of Korea, the potential undesirable effects of PP-P8 had to be investigated in intact small rodent or marmoset models prior to first-in-human (FIH) administration. The estimated doses of PP-P8 for FIH are 1.0×1010 - 1.0×1011 CFU/person (60 kg). Therefore, to perform toxicological investigations in non-clinical animal models, we orally administered PP-P8 at doses of 3.375 × 1011, 6.75 × 1011, and 13.5×1011 CFU/kg/day; thus the maximum dose was 800-8000-fold higher than the estimated dose for FIH. RESULTS: In our animal models, we observed no adverse effects of PP-P8 on clinicopathologic findings, relative organ weight, or tissue pathology. In addition, we observed no inflammation or ulceration during pathological necropsy. CONCLUSION: These non-clinical toxicology studies could be used to furnish valuable data for the safety certification of PP-P8.

Obstetrical, neonatal, and long-term outcomes of children conceived from in vitro matured oocytes.

OBJECTIVE: To investigate the obstetrical, neonatal, and long-term outcomes of in vitro maturation (IVM) compared with conventional in vitro fertilization (IVF) in women with polycystic ovarian syndrome (PCOS). DESIGN: Matched retrospective case-control study. SETTING: University fertility clinic. PATIENT(S): One hundred eighty-four patients undergoing IVM were compared with 366 patients undergoing conventional IVF. All had PCOS and were matched for patient age, gestational age at birth, and the number of fetuses. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Obstetrics, neonatal outcomes, and childhood medical problems and development. RESULT(S): Women's mean age at oocytes retrieval was 32.6 ± 2.9 years. Children's mean age was 7.5 ± 2.3 years. There were no differences in the frequency of obstetrical and neonatal outcomes between the two groups. No difference was found in birth weights between the two groups. The incidence of congenital anomalies was similar between the groups (4.3% in IVM group vs. 4.1% in IVF group). No significant difference was observed between the two groups in the frequency and duration of hospitalization during childhood. Growth developmental status of both groups was within normal range. CONCLUSION(S): In a matched setting between IVM and IVF babies born from women with PCOS, no significant increased risk associated with IVM was been identified after a mean follow-up of 7.5 years.

Effects of vitamin D deficiency and daily calcium intake on bone mineral density and osteoporosis in Korean postmenopausal woman.

OBJECTIVE: We evaluated the combined effects of vitamin D and daily calcium intake on bone mineral density (BMD) and osteoporosis in Korean postmenopausal women. METHODS: This study is a cross-sectional study consisting of 1,921 Korean postmenopausal women aged 45 to 70 years without thyroid dysfunction, from the 2008-2011 Korean National Health and Nutrition Examination Survey. Participants were classified into six groups according to serum 25-hydroxyvitamin D (25(OH)D) levels and daily calcium intake. BMD was measured using dual-energy X-ray absorptiometry at femur and at lumbar spine, and the serum vitamin D levels were measured by radioimmunoassay. RESULTS: The BMD divided according to serum 25(OH)D and daily calcium intakes were not statistically different among the groups. However, when both daily calcium intake and serum 25(OH)D were not sufficient, risk of osteopenia and osteoporosis showed significant increase in both femur neck and lumbar spine (odds ratio [OR] 2.242, P=0.006; OR 3.044, P=0.001; respectively). Although daily calcium intake was sufficient, risks of osteopenia and osteoporosis significantly increased in lumbar spine group if serum 25(OH)D is <20 ng/mL (OR 2.993, P=0.006). CONCLUSION: The combined effects of insufficient daily calcium intake and vitamin D deficiency may cause low BMD and increase in prevalence of osteopenia and osteoporosis in Korean postmenopausal women aged 45 to 70 years.

Obesity and hepatocellular carcinoma in patients receiving entecavir for chronic hepatitis B.

BACKGROUND/AIMS: This study aimed to clarify the effect of obesity on the development of hepatocellular carcinoma (HCC) in chronic hepatitis B (CHB) patients receiving antiviral treatment. METHODS: This study applied a retrospective analysis to a historical cohort in Bundang Jesaeng Hospital. In total, 102 CHB patients were treated with entecavir as an initial treatment for CHB and checked for obesity using a body composition analyzer. Hepatic steatosis was measured semiquantitatively using Hamaguchi's scoring system in ultrasonography. Risk factors for the development of HCC were analyzed, including obesity-related factors (body mass index [BMI], waist circumference [WC], waist-to-hip ratio [WHR], visceral fat area [VFA], and hepatic steatosis). RESULTS: The median follow-up duration of the patients was 45.2 months (interquartile range: 36.0-58.3 months). The cumulative incidence rates of HCC at 1 year, 3 years, and 5 years were 0%, 5.3%, and 9.0%, respectively. Univariable analysis revealed that the risk factors for HCC development were a platelet count of <120,000 /mm2 (hazard ratio [HR]=5.21, P=0.031), HBeAg negativity (HR=5.61, P=0.039), and liver cirrhosis (HR=10.26, P=0.031). Multivariable analysis showed that the significant risk factor for HCC development was liver cirrhosis (HR=9.07, P=0.042). However, none of the obesity-related risk factors were significantly associated with HCC: BMI ≥25 kg/m2 (HR=0.90, P=0.894), WC ≥90 cm (HR=1.10, P=0.912), WHR ≥0.9 (HR=1.94, P=0.386), VFA ≥100 cm2 (HR=1.69, P=0.495), and hepatic steatosis (HR=0.57, P=0.602). CONCLUSION: HCC development is associated with liver cirrhosis but not obesity-related factors in CHB patients receiving entecavir.

Serum Thyroid Stimulating Hormone Levels Are Associated with the Presence of Coronary Atherosclerosis in Healthy Postmenopausal Women.

OBJECTIVES: Menopause is a natural aging process causing estrogen deficiency, accelerating atherogenic processes including dyslipidemia. Prevalence of thyroid dysfunction is also high in postmenopausal women, and it is known to elevate the risk of cardiovascular disease (CVD). Therefore, we are to study on the associations in between serum thyroid stimulating hormone (TSH) and prevalence of CVD in postmenopausal women who have normal thyroid function. METHODS: We performed a retrospective review of 247 Korean postmenopausal women who visited the health promotion center from January, 2007 to December, 2009. Postmenopausal women with normal serum TSH were included in the study. Coronary atherosclerosis was assessed by 64-row multidetector computed tomography. RESULTS: In multiple linear regression analysis, serum TSH was associated with serum triglyceride (TG) (ß = 0.146, P = 0.023). In multiple logistic regression analysis, increasing age and serum TSH were associated with an increased risk of coronary atherosclerosis in euthyroid postmenopausal women (odds ratio [OR] = 1.107 [1.024-1.197], P = 0.011 and OR = 1.303 [1.024-1.658], P = 0.031, respectively). CONCLUSIONS: It revealed that significant predictor of serum TSH was serum TG, and increasing age and TSH were found to have associations with an increased risk of coronary atherosclerosis in euthyroid postmenopausal women. Screening and assessing risks for CVD in healthy postmenopausal women would be helpful before atherosclerosis develops.

Enhanced Raman spectrum of lawsone on Ag surface: vibrational analyses, frequency shifts, and molecular geometry.

The surface-enhanced Raman spectrum of lawsone, a well known natural dye, has been investigated. Activation with KNO(3) or Na(2)SO(4) solution was necessary to enhance the Raman signal, whereas addition of NaCl solution depletes the effects. In the enhanced Raman spectrum, the strong double-bond stretching bands are most distinctive and show large red shifts from those in the infrared and FT-Raman spectra. The observed strong double-bond stretching bands reflect lawsone coordinated perpendicular to the Ag surface. DFT computations have been carried out for the plausible configurations of lawsone coordinated to an adatom on the Ag surface. Lawsone coordinated to an Ag(+) adatom with H(+) released best reproduces the observed vibrational characteristics.

Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide.

TLR2 in association with TLR1 or TLR6 plays an important role in the innate immune response by recognizing microbial lipoproteins and lipopeptides. Here we present the crystal structures of the human TLR1-TLR2-lipopeptide complex and of the mouse TLR2-lipopeptide complex. Binding of the tri-acylated lipopeptide, Pam(3)CSK(4), induced the formation of an "m" shaped heterodimer of the TLR1 and TLR2 ectodomains whereas binding of the di-acylated lipopeptide, Pam(2)CSK(4), did not. The three lipid chains of Pam(3)CSK(4) mediate the heterodimerization of the receptor; the two ester-bound lipid chains are inserted into a pocket in TLR2, while the amide-bound lipid chain is inserted into a hydrophobic channel in TLR1. An extensive hydrogen-bonding network, as well as hydrophobic interactions, between TLR1 and TLR2 further stabilize the heterodimer. We propose that formation of the TLR1-TLR2 heterodimer brings the intracellular TIR domains close to each other to promote dimerization and initiate signaling.

Structural diversity of the hagfish variable lymphocyte receptors.

Variable lymphocyte receptors (VLRs) are recently discovered leucine-rich repeat (LRR) family proteins that mediate adaptive immune responses in jawless fish. Phylogenetically it is the oldest adaptive immune receptor and the first one with a non-immunoglobulin fold. We present the crystal structures of one VLR-A and two VLR-B clones from the inshore hagfish. The hagfish VLRs have the characteristic horseshoe-shaped structure of LRR family proteins. The backbone structures of their LRR modules are highly homologous, and the sequence variation is concentrated on the concave surface of the protein. The conservation of key residues suggests that our structures are likely to represent the LRR structures of the entire repertoire of jawless fish VLRs. The analysis of sequence variability, prediction of protein interaction surfaces, amino acid composition analysis, and structural comparison with other LRR proteins suggest that the hypervariable concave surface is the most probable antigen binding site of the VLR.