Identification of the Effects of Chondroitin Sulfate on Inhibiting CDKs in Colorectal Cancer Based on Bioinformatic Analysis and Experimental Validation.

With a high occurrence rate and high mortality, the treatment of colorectal cancer (CRC) is increasingly attracting the attention of scholars. Hub genes that determine the phenotypes of CRC become essential for targeted therapy. In the present study, the importance of cyclin-dependent kinases (CDKs) on the occurrence of CRC was identified by data mining of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The results showed that the gene expression levels of CDK1, CDK4, and CDK6 were obviously changed in different stages of CRC. Among the CDKs, CDK4 was suggested as an independent risk factor for CRC based on Cox analysis. Furthermore, chondroitin sulfate (CS), a kind of dietary supplement to treat osteoarthritis, was predicted to treat CRC based on its chemical structure and GEO datasets. Cell assay experiments with the human CRC cell line HCT-116 also verified this prediction. CS inhibited the gene and protein expression levels of CDKs and increased the ratios of apoptotic or dead HCT-116 cells by regulating mitogen-activated protein (MAP) kinase pathways. Our data highlight the essential roles of CDKs in CRC carcinogenesis and the effects of CS on treating CRC, both of which will contribute to the future CRC treatment.

Semi-empirical model to estimate ideal conditions for the growth of large protein crystals.

A large high-quality crystal is required to specify the positions of H atoms in neutron structural analysis. Consequently, several methods have been proposed for obtaining such large crystals, and theoretical considerations for growing them have been presented. However, further investigation is required to obtain a numerical model that can provide quantitative experimental conditions for obtaining a single large crystal. In the case of protein crystallization experiments, the amount of sample is often limited. Therefore, it is more realistic to make a rough estimation from a small number of experiments. This paper proposes a method of estimating the optimum experimental conditions for the growth of large protein crystals by performing a small number of experiments using a micro-batch method and reporting a numerical model based on nucleation theory and a linear approximation of the crystal-growth rate. Specifically, micro-batch experiments are performed to provide the empirical parameters for the model and to help to estimate the conditions for the growth of a crystal of a predetermined size using a certain sample concentration and volume. This method is offered as a step on the path towards efficiently and rationally producing large crystals that can be subjected to neutron diffraction without depending on luck or on performing many experiments. It is expected to contribute to drug design and the elucidation of protein molecular functions and mechanisms by obtaining positional information on H atoms in the protein molecule, which is an advantage of neutron diffraction.

Association Between Autistic Symptoms and Self-Stigma in Patients with Schizophrenia Spectrum Disorders.

PURPOSE: Self-stigma negatively influences self-esteem, quality of life, self-efficacy, treatment adherence, and recovery in psychiatric patients. By revealing personality traits that influence self-stigma, we can gain useful knowledge for the management of self-stigma. A previous meta-analysis indicated that patients with schizophrenia have higher scores on the Autism-Spectrum Quotient (AQ) than healthy controls. However, the relationship between autistic symptoms and self-stigma in patients with schizophrenia spectrum disorders remains unclear. Therefore, the present study aimed to reveal the association between autistic symptoms and self-stigma in patients with schizophrenia spectrum disorders. PATIENTS AND METHODS: We recruited 127 patients with schizophrenia spectrum disorders (schizophrenia, schizoaffective disorder, and delusional disorder). We assessed participants' self-stigma and autistic symptoms using the Internalized Stigma for Mental Illness (ISMI) scale and the Autism-Spectrum Quotient (AQ), respectively. The differences in the scores of ISMI and AQ according to patient characteristics were investigated. Multiple regression analysis controlling for age and gender was performed to determine the relationship between the total scores on the AQ and IMSI scale. RESULTS: Female patients showed a higher level of self-stigma than males. Unmarried patients showed a significantly higher score on the AQ than married patients. Multiple regression analysis adjusted for age and gender indicated that the total score on AQ might be a predictor of the overall rating on ISMI in patients with schizophrenia spectrum disorders. CONCLUSION: This study is the first to reveal the association between autistic symptoms and self-stigma in patients with schizophrenia spectrum disorders. Our results highlight the importance of considering autistic symptoms in the assessment and management of self-stigma in patients with schizophrenia spectrum disorders.

Improved preparation of group-specific component (Gc) protein to derive macrophage activating factor.

Cancer immunotherapy has recently attracted attention as an approach for cancer treatment through the activation of the immune system. Group-specific component (Gc) protein is a precursor for macrophage activating factor (GcMAF), which has a promising immunomodulatory effect on the suppression of tumor growth and angiogenesis. In this study, we successfully purified Gc protein from human serum using anion-exchange chromatography combined with affinity chromatography using a 25-OH-D3-immobilized column. The purity of Gc protein reached 95.0% after anion-exchange chromatography. The known allelic variants of Gc protein are classified into three subtypes-Gc1F, Gc1S and Gc2. The fragment sequence of residues 412-424 determined according to their MS/MS spectra is available to evaluate the subtypes of Gc protein. The data showed that the Gc protein purified in this study consisted of the Gc1F and Gc2 subtypes. Our method improved the purity of Gc protein, which was not affected by the treatment to convert it into GcMAF using ß-galactosidase- or neuraminidase-immobilized resin, and will be useful for biological studies and/or advanced clinical uses of GcMAF, such as cancer immunotherapy.

Orally administered heat-killed Lactobacillus paracasei MCC1849 enhances antigen-specific IgA secretion and induces follicular helper T cells in mice.

Antigen-specific immunoglobulin (Ig) A plays a major role in host defense against infections in gut mucosal tissue. Follicular helper T (Tfh) cells are located in germinal centers and promote IgA production via interactions with germinal center B cells. Several studies have demonstrated that some lactic acid bacteria (LAB) strains activate the host's acquired immune system, inducing IgA secretion in the intestine. However, the precise molecular mechanisms underlying the effects of LAB on IgA production and Tfh cells are not fully resolved. Lactobacillus paracasei MCC1849 is a probiotic strain isolated from the intestine of a healthy adult. In this study, we investigated the effects of orally administered heat-killed MCC1849 on IgA production in the intestine and on Tfh cell induction in vivo. We found that orally administered MCC1849 induced antigen-specific IgA production in the small intestine, serum and lungs. We also observed that MCC1849 increased the proportion of IgA+ B cells and Tfh cells in Peyer's patches (PPs). In addition, MCC1849 increased the gene expression of IL-12p40, IL-10, IL-21, STAT4 and Bcl-6 associated with Tfh cell differentiation. These results suggest that orally administered MCC1849 enhances antigen-specific IgA production and likely affects Tfh cell differentiation in PPs.

Electrodiagnostic Characterization of Hereditary Neuropathy With Liability to Pressure Palsies.

OBJECTIVES: The study objective was electrodiagnostic characterization of a large cohort of patients with genetically confirmed hereditary neuropathy with liability to pressure palsies (HNPP). METHODS: A retrospective review was conducted on all patients with HNPP seen at the neuromuscular clinic (London, Canada) from 1977 to 2015. Clinical data obtained included patient characteristics, examination findings, and nerve conduction study results. RESULTS: A total of 46 patients were analyzed. The mean age and median disease duration were 42.6 and 5.0 years, respectively. Most patients had abnormalities on sural nerve conduction studies. The most common focal neuropathies at compressive sites were ulnar nerve at the elbow (85.7%), distal median nerve at the wrist (84.4%), and fibular nerve at the fibular head (36.7%). Distal median neuropathy was associated with a mean terminal latency of 6.64 milliseconds. CONCLUSIONS: The presence of polyneuropathy, median terminal motor latency prolongation, and multiple compressive neuropathies are the most common findings associated with HNPP.

Lifestyle Modifications Versus Antihypertensive Medications in Reducing Cardiovascular Events in an Aging Society: A Success Rate-oriented Simulation.

Objective It is difficult to compare directly the practical effects of lifestyle modifications and antihypertensive medications on reducing cardiovascular disease (CVD). The purpose of this study was to compare the hypothetical potential of lifestyle modifications with that of antihypertensive medications in reducing CVD in an aging society using a success rate-oriented simulation. Methods We constructed a simulation model for virtual Japanese subpopulations according to sex and age at 10-year intervals from 40 years of age as an example of an aging society. The fractional incidence rate of CVD was calculated as the product of the incidence rate at each systolic blood pressure (SBP) level and the proportion of the SBP frequency distribution in the fractional subpopulations of each SBP. The total incidence rate was calculated by the definite integral of the fractional incidence rate at each SBP level in the sex- and age-specific subpopulations. Results If we consider the effects of lifestyle modifications on metabolic factors and transfer them onto SBP, the reductions in the total incidence rate of CVD were competitive between lifestyle modifications and antihypertensive medications in realistic scenarios. In middle-aged women, the preventive effects of both approaches were limited due to a low incidence rate. In middle-aged men and extremely elderly subjects whose adherence to antihypertensive medications is predicted to be low, lifestyle modifications could be an alternative choice. Conclusion The success rate-oriented simulation suggests that the effectiveness of lifestyle modifications or antihypertensive medications in preventing cardiovascular events largely depends on the baseline incidence rate and sex- and age-specific behavioral factors.

Age-related changes in gut microbiota composition from newborn to centenarian: a cross-sectional study.

BACKGROUND: It has been reported that the composition of human gut microbiota changes with age; however, few studies have used molecular techniques to investigate the long-term, sequential changes in gut microbiota composition. In this study, we investigated the sequential changes in gut microbiota composition in newborn to centenarian Japanese subjects. RESULTS: Fecal samples from 367 healthy Japanese subjects between the ages of 0 and 104 years were analyzed by high-throughput sequencing of amplicons derived from the V3-V4 region of the 16S rRNA gene. Analysis based on bacterial co-abundance groups (CAGs) defined by Kendall correlations between genera revealed that certain transition types of microbiota were enriched in infants, adults, elderly individuals and both infant and elderly subjects. More positive correlations between the relative abundances of genera were observed in the elderly-associated CAGs compared with the infant- and adult-associated CAGs. Hierarchical Ward's linkage clustering based on the abundance of genera indicated five clusters, with median (interquartile range) ages of 3 (0-35), 33 (24-45), 42 (32-62), 77 (36-84) and 94 (86-98) years. Subjects were predominantly clustered with their matched age; however, some of them fell into mismatched age clusters. Furthermore, clustering based on the proportion of transporters predicted by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) showed that subjects were divided into two age-related groups, the adult-enriched and infant/elderly-enriched clusters. Notably, all the drug transporters based on Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology groups were found in the infant/elderly-enriched cluster. CONCLUSION: Our results indicate some patterns and transition points in the compositional changes in gut microbiota with age. In addition, the transporter property prediction results suggest that nutrients in the gut might play an important role in changing the gut microbiota composition with age.

Plastid Proteomic Analysis in Tomato Fruit Development.

To better understand the mechanism of plastid differentiation from chloroplast to chromoplast, we examined proteome and plastid changes over four distinct developmental stages of 'Micro-Tom' fruit. Additionally, to discover more about the relationship between fruit color and plastid differentiation, we also analyzed and compared 'Micro-Tom' results with those from two other varieties, 'Black' and 'White Beauty'. We confirmed that proteins related to photosynthesis remain through the orange maturity stage of 'Micro-Tom', and also learned that thylakoids no longer exist at this stage. These results suggest that at a minimum there are changes in plastid morphology occurring before all related proteins change. We also compared 'Micro-Tom' fruits with 'Black' and 'White Beauty' using two-dimensional gel electrophoresis. We found a decrease of CHRC (plastid-lipid-associated protein) and HrBP1 (harpin binding protein-1) in the 'Black' and 'White Beauty' varieties. CHRC is involved in carotenoid accumulation and stabilization. HrBP1 in Arabidopsis has a sequence similar to proteins in the PAP/fibrillin family. These proteins have characteristics and functions similar to lipocalin, an example of which is the transport of hydrophobic molecules. We detected spots of TIL (temperature-induced lipocalin) in 2D-PAGE results, however the number of spots and their isoelectric points differed between 'Micro-Tom' and 'Black'/'White Beauty'. Lipocalin has various functions including those related to environmental stress response, apoptosis induction, membrane formation and fixation, regulation of immune response, cell growth, and metabolism adjustment. Lipocalin related proteins such as TIL and HrBP1 could be related to the accumulation of carotenoids, fruit color and the differentiation of chromoplast.

Lifestyle modifications supported by regional health nurses lowered insulin resistance, oxidative stress and central blood pressure in subjects with metabolic syndrome.

BACKGROUND: This study was attempted to investigate whether lifestyle modifications supported by regional health nurses should improve cardio-metabolic factors--including adipocytokines, oxidative stress, and arterial stiffness--in subjects with metabolic syndrome. METHODS: Thirty-six subjects with metabolic syndrome were enrolled, 28 of whom completed the 6-month lifestyle modifications (male:female=19:9). Blood and urine test results were examined in relation to metabolic factors before and after 6-month nutritional and physical activity modifications. In addition, oral glucose tolerance tests were performed and arterial stiffness was measured by brachial-ankle pulse wave velocity and radial augmentation index before and after them. RESULTS: Six-month lifestyle modifications significantly reduced body weight, homeostasis model assessment index, and low-density lipoprotein cholesterol (LDL-C). They significantly attenuated oxidative stress measured by the urinary 8-hydroxy-2-deoxyguanosine/creatinine ratio. They also lowered brachial and central systolic blood pressure. They tended to decrease waist circumferences and the levels of C-reactive protein. However they did not significantly change the levels of adipocytokines, including tumour necrosis factor, soluble tumour necrosis factor receptors, and interleukin 6, or arterial stiffness measured by brachial-ankle pulse wave velocity and radial augmentation index. CONCLUSIONS: Six-month lifestyle modifications supported by regional health nurses lowered body weight, insulin resistance, LDL-C, oxidative stress, and peripheral and central blood pressure in subjects with metabolic syndrome.

Changes in nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis among healthy children attending a day-care centre before and after official financial support for the 7-valent pneumococcal conjugate vaccine and H. influenzae type b vaccine in Japan.

The 7-valent pneumococcal conjugate vaccine (PCV7) and Haemophilus influenzae type b (Hib) vaccine reduce nasopharyngeal carriage of vaccine-type bacteria, which may in turn influence the presence of other nasopharyngeal bacterial pathogens. To investigate this possibility, nasopharyngeal carriage of potential pathogens was examined before and after official financial support was provided to offer the PCV7 and Hib vaccines in healthy children attending a day care centre in Japan during 2011-2012. Despite a virtual disappearance of PCV7 serotypes over time, the overall pneumococcal carriage rate remained unchanged. Although others have reported an increase in PCV13 serotypes following PCV7 vaccination, only non-PCV13 serotypes were observed to have increased in this study. The majority of H. influenzae isolates were non-typeable and Hib was not found. Our data identified an unexpected pattern of pneumococcal serotype replacement following PCV7. Continuous monitoring of pneumococcal carriage is important for decisions regarding the future of national vaccination policy in Japan.

JAXA protein crystallization in space: ongoing improvements for growing high-quality crystals.

The Japan Aerospace Exploration Agency (JAXA) started a high-quality protein crystal growth project, now called JAXA PCG, on the International Space Station (ISS) in 2002. Using the counter-diffusion technique, 14 sessions of experiments have been performed as of 2012 with 580 proteins crystallized in total. Over the course of these experiments, a user-friendly interface framework for high accessibility has been constructed and crystallization techniques improved; devices to maximize the use of the microgravity environment have been designed, resulting in some high-resolution crystal growth. If crystallization conditions were carefully fixed in ground-based experiments, high-quality protein crystals grew in microgravity in many experiments on the ISS, especially when a highly homogeneous protein sample and a viscous crystallization solution were employed. In this article, the current status of JAXA PCG is discussed, and a rational approach to high-quality protein crystal growth in microgravity based on numerical analyses is explained.

Numerical model of protein crystal growth in a diffusive field such as the microgravity environment.

It is said that the microgravity environment positively affects the quality of protein crystal growth. The formation of a protein depletion zone and an impurity depletion zone due to the suppression of convection flow were thought to be the major reasons. In microgravity, the incorporation of molecules into a crystal largely depends on diffusive transport, so the incorporated molecules will be allocated in an orderly manner and the impurity uptake will be suppressed, resulting in highly ordered crystals. Previously, these effects were numerically studied in a steady state using a simplified model and it was determined that the combination of the diffusion coefficient of the protein molecule (D) and the kinetic constant for the protein molecule (ß) could be used as an index of the extent of these depletion zones. In this report, numerical analysis of these depletion zones around a growing crystal in a non-steady (i.e. transient) state is introduced, suggesting that this model may be used for the quantitative analysis of these depletion zones in the microgravity environment.

Ectopic localization of auxin and cytokinin in tobacco seedlings by the plant-oncogenic AK-6b gene of Agrobacterium tumefaciens AKE10.

The oncogenic 6b gene of Agrobacterium tumefaciens induces a number of morphological and metabolic alterations in plants. Although molecular functions associated with the 6b genes have been proposed, including auxin transport, sugar transport, transcriptional regulation, and miRNA metabolism, so far an unequivocal conclusion has not been obtained. We investigated the association between auxin accumulation and tumor development of the tobacco seedlings expressing the AK-6b gene under the control of the dexamethasone-inducible promoter. Indole-3-acetic acid (IAA) localization was examined by immunochemical staining with monoclonal antibody against IAA and by histochemical analysis using the IAA-specific induced construct, DR5::GUS (ß-glucuronidase). Both procedures indicated that IAA preferentially accumulated in the tumorous protrusions as well as in newly developing vascular bundles in the tumors. Furthermore, true leaves also showed abaxial IAA localization, leading to altered leaves in which the adaxial and abaxial identities were no longer evident. Co-localization of cytokinin and auxin in the abaxial tumors was verified by immunochemical staining with an antibody against cytokinin. Treatment of AK-6b-seedlings with N-1-naphthylphthalamic acid, an inhibitor of polar auxin transport, promoted the morphological severity of phenotypes, whereas 1-naphthoxyacetic acid, a specific auxin influx carrier inhibitor, induced tumor regression on cotyledons and new tumorous proliferations on hypocotyls. Prominent accumulation of both auxin and cytokinin was observed in both regressed and newly developing tumors. We suggest from these results that modulation of auxin/cytokinin localization as a result of AK-6b gene expression is responsible for the tumorous proliferation.

Pure hemi-chorea resulting from an acute phase of contralateral thalamic lacunar infarction: a case report.

BACKGROUND: Thalamic lesions give rise to a variety of clinical syndromes such as pure sensory stroke, ataxic hemiparesis, and rarely involuntary movements including chorea. Generally and classically, lacunar infarction in the subthalamic nucleus has been regarded as the lesion mainly responsible for hemi-chorea and hemi-ballismus, on the basis of previous anatomical studies. CASE PRESENTATION: This report describes the case of an 81-year-old man who developed sudden-onset pure hemi-chorea in the right limbs resulting from an acute phase of left thalamic lacunar infarction detected on a diffusion-weighted image (DWI) in an MRI study. The patient had no other neurological symptoms such as ataxic hemiparesis and sensory disturbance. A single-photon emission computed tomography (SPECT) study using the (99m)Tc-ECD Patlak plot method demonstrated significant perfusional asymmetry between the right and left thalami (p = 0.0035), consistent with the left thalamic lesion on DWI. CONCLUSION: It is speculated that this perfusional asymmetry, in particular the hypoperfusion in the left thalamus, detected by SPECT might play the most important role in the contralateral pure hemi-chorea as a rare neurological manifestation in this case.

High-Quality Protein Crystal Growth of Mouse Lipocalin-Type Prostaglandin D Synthase in Microgravity.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH(2) to PGD(2) and is involved in the regulation of pain and of nonrapid eye movement sleep and the differentiation of male genital organs and adipocytes, etc. L-PGDS is secreted into various body fluids and binds various lipophilic compounds with high affinities, acting also as an extracellular transporter. Mouse L-PGDS with a C65A mutation was previously crystallized with citrate or malonate as a precipitant, and the X-ray crystallographic structure was determined at 2.0 Å resolution. To obtain high-quality crystals, we tried, unsuccessfully, to crystallize the C65A mutant in microgravity under the same conditions used in the previous study. After further purifying the protein and changing the precipitant to polyethylene glycol (PEG) 8000, high-quality crystals were grown in microgravity. The precipitant solution was 40% (w/v) PEG 8000, 100 mM sodium chloride, and 100 mM HEPES-NaOH (pH 7.0). Crystals grew on board the International Space Station for 11 weeks in 2007, yielding single crystals of the wild-type L-PGDS and the C65A mutant, both of which diffracted at around 1.0 Å resolution. The crystal quality was markedly improved through the use of a high-viscosity precipitant solution in microgravity, in combination with the use of a highly purified protein.

The ß-catenin HMP-2 functions downstream of Src in parallel with the Wnt pathway in early embryogenesis of C. elegans.

The Wnt and Src pathways are widely used signal transduction pathways in development. ß-catenin is utilized in both pathways, as a signal transducer and a component of the cadherin cell adhesion complex, respectively. A C. elegans ß-catenin HMP-2 is involved in cell adhesion, but its signaling role has been unknown. Here, we report that in early embryogenesis HMP-2 acts as a signaling molecule in the Src signal. During early embryogenesis in C. elegans, the Wnt and Src pathways are redundantly involved in endoderm induction at the four-cell stage and spindle orientation in an ABar blastomere. RNAi experiments demonstrated that HMP-2 functions in the Src pathway, but in parallel with the Wnt pathway in these processes. HMP-2 localized at the cell boundaries and nuclei, and its localization at cell boundaries was negatively regulated by SRC-1. In addition, HMP-2 was Tyr-phosphorylated in a SRC-1-dependent manner in vivo. Taken together, we propose that HMP-2 functions downstream of the Src signaling pathway and contribute to endoderm induction and ABar spindle orientation, in parallel with the Wnt signaling pathway.

Optimization of salt concentration in PEG-based crystallization solutions.

Although polyethylene glycol (PEG) is the most widely used precipitant in protein crystallization, the concentration of co-existing salt in the solution has not been well discussed. To determine the optimum salt concentration range, several kinds of protein were crystallized in a 30% PEG 4000 solution at various NaCl concentrations with various pH levels. It was found that, if crystallization occurred, the lowest effective salt concentration depended on the pH of the protein solution and the pI of the protein molecule; that is, higher salt concentrations were required for crystal growth if the difference between pH and pI was increasing. The linear relationship between the charge density of the protein and the ionic strength of the crystallization solution was further verified. These results suggested that the lowest effective concentration of salt in a crystallization solution can be predicted before performing a crystallization experiment. Our results can be a tip for tuning crystallization conditions by the vapor-diffusion method.

Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment.

Human hematopoietic prostaglandin synthase, one of the better therapeutic target enzymes for allergy and inflammation, was crystallized with 22 inhibitors and in three inhibitor-free conditions in microgravity. Most of the space-grown crystals showed better X-ray diffraction patterns than the terrestrially grown ones, indicating the advantage of a microgravity environment on protein crystallization, especially in the case of this protein.

Antiobesity effects of Bifidobacterium breve strain B-3 supplementation in a mouse model with high-fat diet-induced obesity.

The aim of the present study was to evaluate the anti-obesity activity of a probiotic bifidobacterial strain in a mouse model with obesity induced by a high-fat diet. The mice were fed a high-fat diet supplemented with Bifidobacterium breve B-3 at 10(8) or 10(9) CFU/d for 8 weeks. B. breve B-3 supplementation dose-dependently suppressed the accumulation of body weight and epididymal fat, and improved the serum levels of total cholesterol, fasting glucose and insulin. The bifidobacterial counts in the caecal contents and feces were significantly increased with the B. breve B-3 administration. The expression of genes related to fat metabolism and insulin sensitivity in the gut and epididymal fat tissue was up-regulated by this administration. These results suggest that the use of B. breve B-3 would be effective in reducing the risk of obesity.