Key factors for the survival of Lactiplantibacillus plantarum IDCC 3501 in manufacturing and storage.

Functional microbiome development has steadily increased; with this, the viability of microbial strains must be maintained not only after the manufacturing process but also at the time of consumption. Survival is threatened by various unavoidable factors during freeze-drying and shelf storage. Here, the aim was to optimize the manufacturing process of the functional strain Lactiplantibacillus plantarum IDCC 3501 after freeze-drying and storage. Explosive growth was achieved using a medium composition with two nitrogen sources and a mineral, and growth was drastically improved by neutralizing the medium pH during the culture of L. plantarum IDCC 3501. Culture optimization involved a smaller cell size, leading to less intracellular free water. Moreover, when maltodextrin (MD) powder was directly added to the harvested cells, some intracellular free water was extracted from the bacterial cells, resulting in a dramatic increase in the viability of L. plantarum IDCC 3501 after freeze-drying and subsequent storage. Furthermore, MD enhanced survival in a dose-dependent manner. Bacterial survival was correlated with lysozyme tolerance; therefore, the positive result might have been caused by the osmotic dehydration of intracellular free water, which would potentially damage the bacterial cells via ice crystallization and/or a phase transition during freeze-drying. These critical factors of L. plantarum IDCC 3501 processing provide perspectives on survival issues for manufacturing microbiome strains. KEY POINTS: • Culture conditions for probiotic bacteria were optimized for high growth yield. • Osmotic dehydration improved bacterial survival after manufacturing and shelf storage. • Reduction in intracellular free water content is crucial for intact survival.

Inhibitory effects of Bacillus velezensis ID-A01 supernatant against Streptococcus mutans.

BACKGROUND: Dental caries is a chronic oral disease caused by microbial infections, which result in erosion of the dental enamel and cause irreversible damage. Therefore, proper disease management techniques and the creation of an environment that prevents intraoral growth and biofilm formation of Streptococcus mutans in the early stages, are crucial to prevent the potential progression of dental plaque to disease. Here, we aimed to investigate antimicrobial and antibiofilm effects of the Bacillus velezensis ID-A01 supernatant (ID23029) against S. mutans, and its inhibitory effects on acidogenesis. RESULTS: A killing kinetics assay showed a peak lethality percentage of 94.5% after 6 h of exposure to ID23029. In sucrose-exposed conditions, ID23029 inhibited lactic acid formation, preventing the pH from falling below the threshold for enamel demineralization, and inhibited up to 96.6% of biofilm formation. This effect was maintained in the presence of lysozyme. Furthermore, ID23029 retained up to 92% lethality, even at an intraoral concentration at which lysozyme is ineffective against S. mutans. CONCLUSIONS: This study demonstrates the potential of the B. velezensis ID-A01 supernatant for the prevention and treatment of dental caries. Its eventual use in dental practice is encouraged, although further studies are required to confirm its beneficial effects.

Lifetime and diffusion distance of singlet oxygen in air under everyday atmospheric conditions.

Singlet oxygen is a toxic chemical but powerful oxidant, exploited in many chemical and biological applications. However, the lifetime of singlet oxygen in air under atmospheric conditions is yet to be known. This has limited safe usage of singlet oxygen in air, despite being a strong antimicrobial agent with the unique property of relaxing to breathable oxygen after serving its purpose. Here, we solve this long-standing problem by combining experimental and theoretical research efforts; we generate singlet oxygen using a photosensitizer at a local source and monitor the time-dependent extent of singlet oxygen reaction with probe molecules at a detector, precisely controlling the detector distance from the source. To explain our experimental results, we employ a theoretical model that fully accounts for singlet oxygen diffusion, radiative and nonradiative relaxations, and the bimolecular reaction with probe molecules at the detector. For all cases investigated, our model, with only two adjustable parameters, provides an excellent quantitative explanation of the experiment. From this analysis, we extract the lifetime of singlet oxygen in the air to be 2.80 s at 23 °C under 1 atm, during which time singlet oxygen diffuses about 0.992 cm. The correctness of this estimation is confirmed by a simple mean-first-passage time analysis of the maximum distance singlet oxygen can reach from the source. We also confirm the sterilization effects of singlet oxygen for distances up to 0.6-0.8 cm, depending on the bacteria strain in question, between the bacteria and the singlet oxygen source.

Effects of ID-CBT5101 in Preventing and Alleviating Osteoarthritis Symptoms in a Monosodium Iodoacetate-Induced Rat Model.

Osteoarthritis is a disease that affects the articular cartilage and osseous tissue, and can be worsened by aging, overweight status, and post-traumatic arthritis. The present study aimed to evaluate the effect of ID-CBT5101 (tyndallized Clostridium butyricum) on bone metabolism and the inflammatory response in a monosodium iodoacetate-induced rat model of osteoarthritis. ID-CBT5101 was administered orally at doses of 108 or 1010 CFU/day for 2 weeks before direct injection of monosodium iodoacetate (3 mg/50 µl of 0.9% saline) into the intra-articular space of the rats' right knees. The rats subsequently received the same doses of oral ID-CBT5101 for another 4 weeks. We evaluated the treatment effects based on serum biomarkers, mRNA expression, morphological and histopathological analyses of the knee joints, and weight-bearing distribution analysis. Compared with those in control rats, the ID-CBT5101 treatments significantly reduced the serum concentration of inflammation and bone metabolism markers (i.e., COX-2, IL-6, LTB4, and COMP), and significantly increased the concentration of IFN-γ and glycosaminoglycans. In addition, the ID-CBT5101 treatments inhibited the mRNA expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases (i.e., MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, and TIMP-2). Furthermore, the ID-CBT5101 treatments effectively preserved the knee cartilage and synovial membrane, and significantly decreased the amount of fibrous tissue. Moreover, compared with that of the negative control group, the ID-CBT5101 treatments increased the weight-bearing distribution by ≥20%. The results indicate that ID-CBT5101 prevented and alleviated osteoarthritis symptoms. Thus, ID-CBT5101 may be a novel therapeutic option for the management of osteoarthritis.

Stimuli-Responsive, Shape-Transforming Nanostructured Particles.

Development of particles that change shape in response to external stimuli has been a long-thought goal for producing bioinspired, smart materials. Herein, the temperature-driven transformation of the shape and morphology of polymer particles composed of polystyrene-b-poly(4-vinylpyridine) (PS-b-P4VP) block copolymers (BCPs) and temperature-responsive poly(N-isopropylacrylamide) (PNIPAM) surfactants is reported. PNIPAM acts as a temperature-responsive surfactant with two important roles. First, PNIPAM stabilizes oil-in-water droplets as a P4VP-selective surfactant, creating a nearly neutral interface between the PS and P4VP domains together with cetyltrimethylammonium bromide, a PS-selective surfactant, to form anisotropic PS-b-P4VP particles (i.e., convex lenses and ellipsoids). More importantly, the temperature-directed positioning of PNIPAM depending on its solubility determines the overall particle shape. Ellipsoidal particles are produced above the critical temperature, whereas convex lens-shaped particles are obtained below the critical temperature. Interestingly, given that the temperature at which particle shape change occurs depends solely on the lower critical solution temperature (LCST) of the polymer surfactants, facile tuning of the transition temperature is realized by employing other PNIPAM derivatives with different LCSTs. Furthermore, reversible transformations between different shapes of PS-b-P4VP particles are successfully demonstrated using a solvent-adsorption annealing with chloroform, suggesting great promise of these particles for sensing, smart coating, and drug delivery applications.

Analysis of Stage-Specific Gene Expression Profiles in the Uterine Endometrium during Pregnancy in Pigs.

The uterine endometrium plays a critical role in regulating the estrous cycle and the establishment and maintenance of pregnancy in mammalian species. Many studies have investigated the expression and function of genes in the uterine endometrium, but the global expression pattern of genes and relationships among genes differentially expressed in the uterine endometrium during gestation in pigs remain unclear. Thus, this study investigated global gene expression profiles using microarray in pigs. Diverse transcriptome analyses including clustering, network, and differentially expressed gene (DEG) analyses were performed to detect endometrial gene expression changes during the different gestation stages. In total, 6,991 genes were found to be differentially expressed by comparing genes expressed on day (D) 12 of pregnancy with those on D15, D30, D60, D90 and D114 of pregnancy, and clustering analysis of detected DEGs distinguished 8 clusters. Furthermore, several pregnancy-related hub genes such as ALPPL2, RANBP17, NF1B, SPP1, and CST6 were discovered through network analysis. Finally, detected hub genes were technically validated by quantitative RT-PCR. These results suggest the complex network characteristics involved in uterine endometrial gene expression during pregnancy and indicate that diverse patterns of stage-specific gene expression and network connections may play a critical role in endometrial remodeling and in placental and fetal development to establish and maintenance of pregnancy in pigs.

Does diabetes mellitus influence standardized uptake values of fluorodeoxyglucose positron emission tomography in colorectal cancer?

BACKGROUND/AIMS: Hyperglycemia is associated with decreased 2-(18)[F]fluoro-2-deoxy-D-glucose (FDG) uptake by tumors assessed by positron emission tomography (PET). In this retrospective study we investigated a comparison of standardized uptake values (SUVs) in patients with primary colorectal cancers who either had diabetes mellitus (DM) or were otherwise healthy. METHODS: The medical records of 397 patients who were diagnosed with colorectal cancer and underwent PET-CT between January 2006 and December 2012 were analyzed. Eighty patients with DM and 317 patients without DM were included. Clinical characteristics were reviewed and maximal standardized uptake values (SUVmax) were calculated in the primary colorectal lesions. RESULTS: There was no significant difference between tumor SUVmax in DM patients (10.60±5.78) and those without DM (10.92±5.44). In addition, no significant difference was detected between tumor SUVmax in DM patients with glycated hemoglobin (HbA1c) levels <8% (10.34±5.17) and those with HbA1c levels ≥8% (10.61±7.27). The maximum size of the primary colorectal tumor was associated with SUVmax in a linear regression analysis. CONCLUSION: The results of this study showed that DM did not influence FDG uptake values in colorectal cancer patients regardless of glucose levels.

Psychosocial stress in nurses with shift work schedule is associated with functional gastrointestinal disorders.

BACKGROUND/AIMS: The aim of this study was to investigate the role of psychosocial problems and their associations with rotating shift work in the development of functional gastrointestinal disorders. METHODS: In this cross-sectional observation study, survey was administered to nurses and nurse assistants in a referral hospital. In addi-tion to demographic questions, subjects were asked to complete the Rome III Questionnaire, Pittsburgh Sleep Quality Index and Rome III Psychosocial Alarm Questionnaire. RESULTS: Responses from 301 subjects were assessed. The overall prevalence of irritable bowel syndrome (IBS) and functional dyspepsia (FD) were 15.0% and 19.6%, respectively. Psychosocial alarms were prevalent in the nursing personnel (74.8% with alarm pres-ence and 23.3% with serious condition) and were more frequent among rotating shift workers (84.7% vs. 74.5% for alarm presence and 28.1% vs. 13.3% for serious condition). The prevalence of both IBS and FD significantly increased with psychoso-cial risk. An independent risk factor for IBS was serious psychosocial alarm (adjusted odds ratio [aOR], 10.75; 95% confidence interval (CI), 1.30-88.99; P = 0.028). Serious psychosocial alarm was an independent risk factor for FD (aOR, 7.84; 95% CI, 1.98-31.02; P = 0.003). Marriage (aOR 0.30; 95% CI, 0.09-0.93; P = 0.037) was associated with the decreased risk of FD. CONCLUSIONS: The high prevalence of psychosocial stress among nurses who work rotating shifts is associated with the development of func-tional gastrointestinal disorders.(J Neurogastroenterol Motil 2014;20:516-522).

A new statistical approach to predict bacteremia using electronic medical records.

BACKGROUND: Previous attempts to predict bacteremia have focused on selecting significant variables. However, these approaches have had limitations such as poor reproducibility in prediction accuracy and inconsistency in predictor selection. Here we propose a Bayesian approach to predict bacteremia based on the statistical distributions of clinical variables of previous patients, which has recently become possible through the adoption of electronic medical records. METHODS: In a derivation cohort, Bayesian prediction models were derived and their discriminative performance was compared with previous models under varying combinations of predictors. Then the Bayesian models were prospectively tested in a validation cohort. According to Bayesian probabilities of bacteremia, patients in both cohorts were grouped into bacteremia risk groups. RESULTS: Using the same prediction variables, the Bayesian predictions were more accurate than conventional rule-based predictions. Moreover, their better discriminative performance remained consistent despite variations in clinical variables. The receiver operating characteristic (ROC) area of the Bayesian model with 20 predictors was 0.70 ± 0.007 in the derivation cohort and 0.70 ± 0.018 in the validation cohort. The prevalence of bacteremia in groups I, II, and VI (grouped according to probability ratio) were 1.9%, 3.4%, and 20.0% in the derivation cohort, and 0.4%, 3.2%, and 18.4% in the validation cohort, respectively. The overall prevalence of bacteremia was 6.9% in both cohorts. CONCLUSIONS: In the present study, the Bayesian prediction model showed stable performance in predicting bacteremia and identifying risk groups, as the previous models did. The clinical significance of the Bayesian approach is expected to be demonstrated through a multicenter trial.

Analysis of the lysophosphatidic acid-generating enzyme ENPP2 in the uterus during pregnancy in pigs.

Lysophosphatidic acid (LPA), a simple phospholipid, plays a critical role in the establishment of pregnancy in pigs. LPA production is mediated by the action of ENPP2, a secreted lysophospholipase D (lysoPLD) that converts lysophosphatidylcholine to LPA. However, the mechanism that regulates LPA production by ENPP2 in the porcine uterus is not well understood. In this study, we evaluated ENPP2 expression during the estrous cycle and pregnancy in the uterine endometrium and in early stage conceptuses. We also evaluated lysoPLD activity in the uterine lumen. ENPP2 transcripts and proteins were detected in the uterine endometrium at all stages of the estrous cycle and pregnancy, with higher levels on Day (D) 12 and D15 of the estrous cycle and pregnancy. ENPP2 expression was localized mainly in luminal and glandular epithelial cells in the endometrium and was also detected in conceptuses on D12 of pregnancy. Secreted ENPP2 protein was detected in fluid flushing samples from the uterine lumen on D12 of the estrous cycle and pregnancy, with higher levels on D12 of pregnancy. LysoPLD activity was detected in uterine flushings on D12 of the estrous cycle and pregnancy, with higher levels on D12 of pregnancy. This study showed that uterine endometrium and conceptuses produce ENPP2 and secreted it into the uterine lumen where it has lysoPLD activity. These results suggest that ENPP2 may play an important role in the establishment of pregnancy in pigs by regulating LPA production at the maternal-conceptus interface.

Mesenchymal stem cells transfer mitochondria to the cells with virtually no mitochondrial function but not with pathogenic mtDNA mutations.

It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether the reported intercellular mitochondrial transfer could be replicated in different types of cells or under different experimental conditions, and tried to elucidate possible mechanism. Using biochemical selection methods, we found exponentially growing cells in restrictive media (uridine(-) and bromodeoxyuridine [BrdU](+)) during the coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mitochondrial DNA (mtDNA) identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B ρ(0) cells under uridine-free condition, induced increased chemotaxis in MSC, which was also supported by transcriptome analysis. Cytochalasin B, an inhibitor of chemotaxis and cytoskeletal assembly, blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion). Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential cell therapy-based mitochondrial restoration or mitochondrial gene therapy for human diseases caused by mitochondrial dysfunction.

Swine leukocyte antigen-DQ expression and its regulation by interferon-gamma at the maternal-fetal interface in pigs.

Successful pregnancy requires an appropriate intrauterine immune response to the conceptus, which is a semiallograft within the uterus. We reported that swine leukocyte antigen-DQA (SLA-DQA), a major histocompatibility complex (MHC) class II gene, is expressed in the uterine endometrium at the time of conceptus implantation in pigs. Because MHC molecules play critical roles in the immune system, SLA-DQ was hypothesized to be involved in immune regulation during pregnancy. Therefore, we examined expression of SLA-DQ in uterine endometrial tissues obtained during the estrous cycle and pregnancy. SLA-DQA and SLA-DQB mRNAs were detected as 1.3-kb and 1.2-kb bands, respectively. Real-time RT-PCR analysis indicated that SLA-DQA and SLA-DQB mRNA expression was affected by day and pregnancy status, with the highest expression on Day 15 of pregnancy. SLA-DQ was localized primarily to subepithelial stromal cells and endothelial cells of the uterus. Using endometrial explant cultures from Day 12 of the estrous cycle, we determined that expression of SLA-DQA and SLA-DQB mRNAs increased in response to interferon-gamma (IFNG), which is produced by pig conceptus trophectoderm between Days 14 and 18 of pregnancy. The abundance of SLA-DQ protein was less in endometria from gilts with conceptuses resulting from somatic cell nuclear transfer compared with endometria from gilts with conceptuses resulting from natural mating. These results support our hypothesis that SLA-DQ is expressed in response to IFNG from the conceptus, and likely regulates immune response at the maternal-fetal interface to support the maintenance of pregnancy in pigs.

Regulation of S100G Expression in the Uterine Endometrium during Early Pregnancy in Pigs.

Calcium ions play an important role in the establishment and maintenance of pregnancy, but molecular and cellular regulatory mechanisms of calcium ion action in the uterine endometrium are not fully understood in pigs. Previously, we have shown that calcium regulatory molecules, transient receptor potential vanilloid type 5 (TRPV6) and calbindin-D9k (S100G), are expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner, and that estrogen of conceptus origin increases endometrial TRPV6 expression. However, regulation of S100G expression in the uterine endometrium and conceptus expression of S100G has been not determined during early pregnancy. Thus, we investigated regulation of S100G expression by estrogen and interleukin-1ß (IL1B) in the uterine endometrium and conceptus expression of S100G during early pregnancy in pigs. We obtained uterine endometrial tissues from day (D) 12 of the estrous cycle and treated with combinations of steroid hormones, estradiol-17ß (E2) and progesterone (P4), and increasing doses of IL1B. Real-time RT-PCR analysis showed that E2 and IL1B increased S100G mRNA levels in the uterine endometrium, and conceptuses expressed S100G mRNA during early pregnancy, as determined by RT-PCR analysis. To determine if endometrial expression of S100G mRNA during the implantation period was affected by the somatic cell nuclear transfer (SCNT) procedure, we compared S100G mRNA levels in the uterine endometrium from gilts with SCNT-derived conceptuses with those from gilts with conceptuses derived from natural mating on D12 of pregnancy. Real-time RT-PCR analysis showed that levels of S100G mRNA in the uterine endometrium from gilts carrying SCNT-derived conceptuses was significantly lower than those from gilts carrying conceptuses derived from natural mating. These results showed that S100G expression in the uterine endometrium was regulated by estrogen and IL1B of conceptus origin, and affected by the SCNT procedure during early pregnancy. These suggest that conceptus signals regulate S100G, an intracellular calcium transport protein, for the establishment of pregnancy in pigs.

Microarray Analysis of Gene Expression in the Uterine Endometrium during the Implantation Period in Pigs.

During embryo implantation in pigs, the uterine endometrium undergoes dramatic morphological and functional changes accompanied with dynamic gene expression. Since the greatest amount of embryonic losses occur during this period, it is essential to understand the expression and function of genes in the uterine endometrium. Although many reports have studied gene expression in the uterine endometrium during the estrous cycle and pregnancy, the pattern of global gene expression in the uterine endometrium in response to the presence of a conceptus (embryo/fetus and associated extraembryonic membranes) has not been completely determined. To better understand the expression of pregnancy-specific genes in the endometrium during the implantation period, we analyzed global gene expression in the endometrium on day (D) 12 and D15 of pregnancy and the estrous cycle using a microarray technique in order to identify differentially expressed endometrial genes between D12 of pregnancy and D12 of the estrous cycle and between D15 of pregnancy and D15 of the estrous cycle. Results showed that the global pattern of gene expression varied with pregnancy status. Among 23,937 genes analyzed, 99 and 213 up-regulated genes and 92 and 231 down-regulated genes were identified as differentially expressed genes (DEGs) in the uterine endometrium on D12 and D15 of pregnancy compared to D12 and D15 of the estrous cycle, respectively. Functional annotation clustering analysis showed that those DEGs included genes involved in immunity, steroidogenesis, cell-to-cell interaction, and tissue remodeling. These findings suggest that the implantation process regulates differential endometrial gene expression to support the establishment of pregnancy in pigs. Further analysis of the genes identified in this study will provide insight into the cellular and molecular bases of the implantation process in pigs.

Salivary lipocalin is uniquely expressed in the uterine endometrial glands at the time of conceptus implantation and induced by interleukin 1beta in pigs.

Uterine secretions are essential for the development of the conceptus during pregnancy. In pigs, various molecules, including transport proteins, growth factors, enzymes, and extracellular matrix proteins, are secreted into the uterine lumen. Our previous work identified salivary lipocalin (SAL1), a steroidal pheromone-binding protein, as present in the porcine uterus. To initiate studies on the role of SAL1 in the porcine uterus, we evaluated 1) the spatial and temporal expression of SAL1 in the uterine endometrium during the estrous cycle and pregnancy, and in the conceptus during early pregnancy; 2) secretion of SAL1 into the uterine lumen on Day (D) 12 of the estrous cycle and pregnancy; and 3) the effects of steroid hormones and cytokines on SAL1 mRNA levels. SAL1 was localized to glandular epithelial cells (GE) in the endometrium during the estrous cycle and pregnancy, with the highest level of SAL1 expression on D12 of pregnancy. In addition, SAL1 protein secretion into the uterine lumen was detected in uterine flushings on D12 of the estrous cycle and pregnancy, with higher levels on D12 of pregnancy. SAL1 protein, but not SAL1 transcript, was also detected in the conceptuses on D12 and D15. In explant culture experiments, SAL1 mRNA levels in the endometrium were increased by interleukin 1beta. The results of a GE- and implantation stage-specific expression and uterine secretion of SAL1 in the porcine uterus suggest that SAL1 present at the maternal-fetal interface may act as a histotroph and play an important role in the establishment of pregnancy.

Extracting regulatory modules from gene expression data by sequential pattern mining.

BACKGROUND: Identifying a regulatory module (RM), a bi-set of co-regulated genes and co-regulating conditions (or samples), has been an important challenge in functional genomics and bioinformatics. Given a microarray gene-expression matrix, biclustering has been the most common method for extracting RMs. Among biclustering methods, order-preserving biclustering by a sequential pattern mining technique has native advantage over the conventional biclustering approaches since it preserves the order of genes (or conditions) according to the magnitude of the expression value. However, previous sequential pattern mining-based biclustering has several weak points in that they can easily be computationally intractable in the real-size of microarray data and sensitive to inherent noise in the expression value. RESULTS: In this paper, we propose a novel sequential pattern mining algorithm that is scalable in the size of microarray data and robust with respect to noise. When applied to the microarray data of yeast, the proposed algorithm successfully found long order-preserving patterns, which are biologically significant but cannot be found in randomly shuffled data. The resulting patterns are well enriched to known annotations and are consistent with known biological knowledge. Furthermore, RMs as well as inter-module relations were inferred from the biologically significant patterns. CONCLUSIONS: Our approach for identifying RMs could be valuable for systematically revealing the mechanism of gene regulation at a genome-wide level.

Mixture-model based estimation of gene expression variance from public database improves identification of differentially expressed genes in small sized microarray data.

MOTIVATION: The small number of samples in many microarray experiments is a challenge for the correct identification of differentially expressed gens (DEGs) by conventional statistical means. Information from public microarray databases can help more efficient identification of DEGs. To model various experimental conditions of a public microarray database, we applied Gaussian mixture model and extracted bi- or tri-modal distributions of gene expression. Prior variance of Baldi's Bayesian framework was estimate for the analysis of the small sample-sized datasets. RESULTS: First, we estimated the prior variance of a gene expression by pooling variances obtained from mixture modeling of large samples in the public microarray database. Then, using the prior variance, we identified DEGs in small sample-sized test datasets using the Baldi's framework. For benchmark study, we generated test datasets having several samples from relatively large datasets. Our proposed method outperformed other benchmark methods in terms of detecting gold-standard DEGs from the test datasets. The results may be a challenging evidence for usage of public microarray databases in microarray data analysis.

Dynamic expression of calcium-regulatory molecules, TRPV6 and S100G, in the uterine endometrium during pregnancy in pigs.

Calcium ions have been implicated in the establishment and maintenance of pregnancy, but the regulatory mechanisms of calcium ions in the uterine endometrium and conceptus are not well understood in pigs. Recently, we showed that TRPV6, a calcium ion channel protein associated with cellular entry of calcium ions, is highly expressed in the uterine endometrium during the implantation period in pigs. In the present study, we investigated spatial and temporal expression and regulation of TRPV6 and S100G, an intracellular calcium-regulatory molecule, in the uterine endometrium during the estrous cycle and pregnancy in pigs. TRPV6 expression was maintained at significantly higher levels in the uterine endometrium during pregnancy compared with levels during the estrous cycle. TRPV6 transcripts and proteins were localized mainly to luminal epithelial cells (LE) and weakly to glandular epithelial cells (GE) and chorionic membrane (CM) during pregnancy. TRPV6 expression was also detected in conceptuses on Day (D) 12 and D15. TRPV6 mRNA levels in the endometrium were increased by estrogen treatment. S100G expression showed a biphasic pattern of increases on D12 of pregnancy and from D60 to term pregnancy, and it localized primarily to LE during early pregnancy and to LE, GE, and CM from D30 to term pregnancy. These results indicate that spatial and temporal expression of TRPV6 and S100G is dynamically regulated in the uterine endometrium during pregnancy and that endometrial regulation of calcium ion concentration by TRPV6 and S100G may be critical for the establishment and maintenance of pregnancy in pigs.

Analysis of hepatic gene expression during fatty liver change due to chronic ethanol administration in mice.

Chronic consumption of ethanol can cause cumulative liver damage that can ultimately lead to cirrhosis. To explore the mechanisms of alcoholic steatosis, we investigated the global intrahepatic gene expression profiles of livers from mice administered alcohol. Ethanol was administered by feeding the standard Lieber-DeCarli diet, of which 36% (high dose) and 3.6% (low dose) of the total calories were supplied from ethanol for 1, 2, or 4 weeks. Histopathological evaluation of the liver samples revealed fatty changes and punctate necrosis in the high-dose group and ballooning degeneration in the low-dose group. In total, 292 genes were identified as ethanol responsive, and several of these differed significantly in expression compared to those of control mice (two-way ANOVA; p<0.05). Specifically, the expression levels of genes involved in hepatic lipid transport and metabolism were examined. An overall net increase in gene expression was observed for genes involved in (i) glucose transport and glycolysis, (ii) fatty acid influx and de novo synthesis, (iii) fatty acid esterification to triglycerides, and (iv) cholesterol transport, de novo cholesterol synthesis, and bile acid synthesis. Collectively, these data provide useful information concerning the global gene expression changes that occur due to alcohol intake and provide important insights into the comprehensive mechanisms of chronic alcoholic steatosis.

Aberrant expression of retinol-binding protein, osteopontin and fibroblast growth factor 7 in the porcine uterine endometrium of pregnant recipients carrying embryos produced by somatic cell nuclear transfer.

The technique of somatic cell nuclear transfer (NT) is a useful tool to produce cloned animals for various purposes, but the efficiency to generate cloned animals using this technique is still very low. To improve the low efficiency in production of cloned pigs it is critical to understand the reprogramming process during development of cloned embryos, but it is also essential to understand the uterine function interacting with the transferred cloned embryos during implantation and placentation. Thus, to understand the uterine responsiveness to NT cloned embryos during pregnancy, we investigated expression of retinol-binding protein (RBP), osteopontin (OPN) and fibroblast growth factor 7 (FGF7), which play important roles in implantation and/or maintenance of pregnancy as a transport protein, an extracellular matrix protein and a growth factor, respectively, in the uterine endometrium in pigs. The uterine tissue samples were obtained by C-section from pigs with NT cloned normal (NT-normal) embryos and NT cloned abnormal (NT-abnormal) embryos and pigs with non-NT (Non-NT) embryos at term. Immunoblot analysis showed that expression of RBP and FGF7 decreased in the uterine endometrium of recipient gilts carrying NT embryos than in the endometrium of gilts carrying Non-NT embryos. Levels of OPN protein of 70 and 45kDa were not different in between the uterine endometrium of gilts carrying Non-NT and NT-normal embryos, but in the uterine endometrium of gilts carrying NT-abnormal embryos 70 and 45kDa OPN proteins increased compared to those in the endometrium of gilts carrying Non-NT embryos. Immunohistochemistry results showed that RBP expression was lower in the endometrial glandular epithelial cells, while OPN expression was higher in the endometrial luminal epithelial cells of the uterus of gilts carrying NT embryos than in the uterus of gilts carrying Non-NT embryos. Results of this study showed that maternal uterine genes were aberrantly expressed in the uterine endometrium of gilts carrying NT cloned embryos in varying degrees depending on the normality of the developing embryos. These results indicate that abnormal maternal-fetal interactions of the uterus carrying the developing NT cloned embryos may cause problems in development of cloned embryos.