Gut microbiome profiles in Thai healthy pregnant women and its association with types of foods.

BACKGROUND: Gut microbiome colonization during early life is significant for immunological and physiological development. Maternal microbiome is associated with proper development of infants. The aim of this study was to determine the gut microbiome profiles among Thai healthy pregnant women and its associated factors. METHODS: A multicenter, open trial prospective study was performed at three hospitals in Northern, Central, and Northeastern regions of Thailand. Thai healthy pregnant women attending antenatal clinics were recruited. Fecal samples of subjects at the third trimester of pregnancy were collected with sterilized techniques. The gut microbiome profiles and bacterial diversity were assessed using 16Ss RNA gene sequencing. Demographic data, dietary intake, and anthropometric data were recorded and analyzed. RESULTS: There were 86 healthy pregnant women. The dominant of gut microbiome profiles were Bacteroidetes and Firmicutes. Pregnant women in the Central region had significantly higher of Ruminococcaceae and Lachnospiraceae than those in other regions (p < 0.001). Pregnant women in the Northern region significantly consumed more glutinous rice than those in other regions (p < 0.001). Glutinous rice intake was positively correlated with Bacteroidetes (rho = 0.405, p = 0.01) and negatively correlated with Firmicutes (rho = - 0.440, p = 0.001). Alpha diversity was not correlated with pre-pregnancy body mass index (BMI) or gestational weight gain. CONCLUSIONS: The gut microbiome profiles mainly found in Thai healthy pregnant women were Bacteroidetes and Firmicutes. The gut microbiome profiles in pregnant women found in this study possibly depended on dietary patterns. Glutinous rice with high amylopectin is probably related to abundance of Bacteroidetes.

Alteration in faecal bile acids, gut microbial composition and diversity after laparoscopic sleeve gastrectomy.

BACKGROUND: Laparoscopic sleeve gastrectomy (LSG) is a well established treatment for severe obesity and type 2 diabetes. Although the gut microbiota is linked to the efficacy of LSG, the underlying mechanisms remain elusive. The effect of LSG for morbid obesity on the gut microbiota and bile acids was assessed here. METHODS: Severely obese subjects who were candidates for LSG were included and followed until 6 months after surgery. The composition and abundance of the microbiota and bile acids in faeces were assessed by 16S ribosomal RNA sequencing, quantitative PCR and liquid chromatography-mass spectrometry. RESULTS: In total, 28 patients with a mean(s.d.) BMI of 44·2(6·6) kg/m2 were enrolled. These patients had achieved excess weight loss of 53·2(19·0) per cent and showed improvement in metabolic diseases by 6 months after LSG, accompanied by an alteration in the faecal microbial community. The increase in α-diversity and abundance of specific taxa, such as Rikenellaceae and Christensenellaceae, was strongly associated with reduced faecal bile acid levels. These changes had a significant positive association with excess weight loss and metabolic alterations. However, the total number of faecal bacteria was lower in patients before (mean(s.d.) 10·26(0·36) log10 cells per g faeces) and after (10·39(0·29) log10 cells per g faeces) operation than in healthy subjects (10·83(0·27) log10 cells per g faeces). CONCLUSION: LSG is associated with a reduction in faecal bile acids and greater abundance of specific bacterial taxa and α-diversity that may contribute to the metabolic changes.

ANTECEDENTES: La gastrectomía vertical laparoscópica (laparoscopic sleeve gastrectomy, LSG) es un tratamiento bien establecido para la obesidad grave y la diabetes tipo 2. Aunque la microbiota intestinal se ha vinculado con la eficacia de LSG, los mecanismos subyacentes siguen siendo poco conocidos. En este estudio se evaluó el efecto de LSG en la obesidad mórbida sobre la microbiota del intestino y de los ácidos biliares (bile acids, BA). MÉTODOS: Tras la aprobación del Comité ético y la obtención del consentimiento informado, los sujetos con obesidad grave que eran candidatos para LSG fueron incluidos en el estudio y seguidos durante 6 meses después de la operación. Se evaluaron la composición y abundancia de la microbiota y BA en las heces mediante secuenciación del gen 16S rRNA, PCR cuantitativa y cromatografía líquida-espectrometría de masas. RESULTADOS: En total, 28 pacientes con una mediana (rango) del IMC de 43,9 kg/m2 (35,0-61,9) fueron reclutados y a los 6 meses tras una LSG, consiguieron una pérdida del exceso de peso de 47,3% (20,7-95,1) y mejoría de las enfermedades metabólicas acompañada de una alteración en la comunidad microbiana fecal. El aumento en la diversidad α y abundancia de especies taxonómicas específicas como Rikenellaceae y Christensenellaceae, se asociaba fuertemente con niveles fecales reducidos de BA. Estos cambios se asociaban de manera positiva y significativa con la pérdida del exceso de peso y las alteraciones metabólicas. Sin embargo, el número total de bacterias fecales en los pacientes fue inferior al de los sujetos sanos (10,84 log10 células/g heces (9,46-11,35)) antes de la operación (10,26 log10 células/g heces (9,44-10,91)) y después de la misma (10,42 log10 células/g heces (9,57-10,96)). CONCLUSIÓN: LSG se asoció con menos BA fecal y mayor abundancia de especies bacterianas específicas y diversidad α lo que puede contribuir a los cambios metabólicos.

The effects of administration of the Lactobacillus gasseri strain CP2305 on quality of life, clinical symptoms and changes in gene expression in patients with irritable bowel syndrome.

AIMS: To clarify the effects of Lactobacillus gasseri CP2305 (CP2305) on quality of life and clinical symptoms and its functional mechanisms in patients with irritable bowel syndrome (IBS). METHODS AND RESULTS: After the patients were administered CP2305 daily for 4 weeks, the IBS-severity index score was significantly improved compared with that of the placebo group, and this improvement was accompanied by a reduction in health-related worry and changes in intestinal microbiota. The gene expression profiling of the peripheral blood leucocytes showed that CP2305 treatment significantly up-regulated genes related to eukaryotic initiation factor 2 (EIF2) signalling. Eighty-two genes were down-regulated in IBS patients compared with healthy controls. The expression of 23 of these genes exhibited a CP2305-dependent increase associated with an improvement in IBS severity. The majority of the restored genes were related to EIF2 signalling. CONCLUSIONS: CP2305 administration is a potential candidate therapeutic option for patients with IBS. SIGNIFICANCE AND IMPACT OF THE STUDY: Although probiotics have been proposed to benefit IBS patients, objective clinical evidence and elucidation of the functional mechanism remain insufficient. Our study demonstrated that CP2305 administration beneficially influences IBS patients in both subjective and objective evaluations, and gene expression profiling provided insights into the functional mechanism.

Axonal TDP-43 aggregates in sporadic amyotrophic lateral sclerosis.

AIMS: Axonal aggregates of phosphorylated (p-) transactive response DNA-binding protein 43 kDa (TDP-43) in sporadic amyotrophic lateral sclerosis (sALS) were examined in relation to propagation of the protein in the nervous system. METHODS: Brains and spinal cords of Japanese patients with sALS and control subjects were examined immunohistochemically using formalin-fixed paraffin-embedded specimens with special reference to the topographical distribution, microscopic features, presynaptic aggregates, and correlation between the aggregates in axons and the clinical course. RESULTS: (i) Aggregates of p-TDP-43 were frequently present in axons of the hypoglossal and facial nerve fibres and the spinal anterior horn cells. (ii) Aggregates of p-TDP-43 in the axons showed two characteristic microscopic features - dash-like granuloreticular aggregates (GRAs) and massive aggregates (MAs). (iii) MAs were surrounded by p-neurofilaments, but p-neurofilament immunnoreactivity decreased at the inside of axons with GRAs. (iv) Patients showing MAs and GRAs had a relatively shorter clinical course than patients without the aggregates. (v) Some neurones in the red nucleus in patients were surrounded by synapses containing p- and p-independent (i)-TDP-43, and almost all neurones had lost their nuclear TDP-43 immunoreactivity; 17% of those neurones in the red nucleus also had TDP-43-immunopositive neuronal cytoplasmic inclusions, but no postsynaptic p-TDP-43 deposition was evident. CONCLUSIONS: There are two types of axonal p-TDP-43 aggregates, MAs and GRAs, located predominantly in the facial and hypoglossal nuclei and anterior horn cells. These aggregates may influence the function of neurones, and presynaptic aggregates of the protein induce loss of p-i-TDP-43 in the nuclei of postsynaptic neurones.

One-year follow-up of zoster-associated pain in 764 immunocompetent patients with acute herpes zoster treated with famciclovir (FAMILIAR study).

BACKGROUND: Herpes zoster (HZ), a reactivation of varicella zoster virus manifested by skin blisters and neuralgia, can lead to postherpetic neuralgia in 10-20% of affected subjects. METHOD: In this study, a cohort of 764 patients with HZ was treated with 1500 mg/day of famciclovir for 7 days, and zoster-associated pain (ZAP) was monitored monthly thereafter for up to 12 months until pain resolution was achieved. Patients were questioned monthly by telephone, and pain was recorded using a numerical rating scale (NRS, 0-10). KEY RESULTS: A total of 751 of 764 (98.3%) patients completed follow-up. The percentage of patients with ZAP was 12.4% at day 90, 7.1% at 6 months and 4.0% at 1 year. After the third month, the NRS were 3 or less in most of the remaining patients with ZAP. Stratified analysis revealed significant persistence of ZAP in patients aged ≥50 years and in those aged ≥65 years, and in patients with either moderate-to-severe skin symptoms or severe pain at the initial consultation.Stratified analyses unexpectedly showed patients who commenced famciclovir at 0-2 days after onset of the eruption had a higher prevalence of ZAP at day 90 than those treated at 3-5 days or ≥6 days after rash onset (P = 0.0164, log-rank test). On further analysis, a higher proportion of patients (45.4%) treated at 0-2 days had moderate to severe symptoms compared with those treated at 3-5 days (40.5%) or ≥6 days (37.0%) (P = 0.0987, Cochran-Armitage test). CONCLUSION & INFERENCE: This study, with an exceptionally high follow-up rate, revealed several new findings, including the influence of disease severity on the delay between the onset of symptoms and seeking medical attention. Six adverse drug reactions were reported in five of 721 patients in the safety analysis, including two severe cases of vomiting and convulsions.

Secukinumab improves hand, foot and nail lesions in moderate-to-severe plaque psoriasis: subanalysis of a randomized, double-blind, placebo-controlled, regimen-finding phase 2 trial.

BACKGROUND: Psoriasis affecting sites such as the hands, feet and nails can be particularly difficult to treat. There are limited data on the efficacy of biological agents to treat these specific localizations. OBJECTIVE: This analysis of a phase 2 regimen-finding study evaluated the efficacy of secukinumab in subjects with moderate-to-severe psoriasis and non-pustular involvement of the hands, feet and/or nails. METHODS: Subjects were randomized (1 : 2 : 2 : 1) to one of three subcutaneous secukinumab 150-mg induction regimens [Single (Week 0), Monthly (Weeks 0, 4, 8), Early (Weeks 0, 1, 2, 4)] or placebo. In the subgroup (n = 131) with hand and/or foot psoriasis [baseline 5-point hand/foot Investigator's Global Assessment (IGA) score ≥2], efficacy was assessed as percentage of subjects achieving an IGA response [a score of 0 (clear) or 1 (minimal) and an improvement of ≥2 points on the 5-point hand/foot scale vs. baseline] at Week 12. In the subgroup (n = 304) with fingernail psoriasis (baseline composite score ≥1), efficacy was assessed as mean percentage change from baseline to Week 12 in a composite score. RESULTS: At Week 12, a markedly higher percentage of subjects with hand and/or foot psoriasis achieved an IGA response with the Early regimen vs. placebo (54.3% vs. 19.2%, P = 0.005). The composite fingernail score improved with the Early and Monthly regimens, but worsened with placebo [percentage mean change from baseline (SE): -19.1% (6.12) and -10.6% (7.06) vs. 14.4% (11.92); P = 0.010 vs. placebo for Early, P = 0.027 for Monthly). Secukinumab was well tolerated. CONCLUSION: Secukinumab demonstrated a beneficial effect on psoriasis of the hands/feet/nails in this short-term assessment.

Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas.

Telomerase is a specialized type of reverse transcriptase which catalyzes the synthesis and extension of telomeric DNA (for review, see ref.1). This enzyme is highly active in most cancer cells, but is inactive in most somatic cells. This striking observation led to the suggestion that telomerase might be important for the continued growth or progression of cancer cells. However, little is known about the molecular mechanism of telomerase activation in cancer cells. Human telomerase reverse transcriptase (hTRT) has recently been identified as a putative human telomerase catalytic subunit. We transfected the gene encoding hTRT into telomerase-negative human normal fibroblast cells and demonstrated that expression of wild-type hTRT induces telomerase activity, whereas hTRT mutants containing mutations in regions conserved among other reverse transcriptases did not. Hepatocellular carcinoma (20 samples) and non-cancerous liver tissues (19 samples) were examined for telomerase activity and expression of hTRT, the human telomerase RNA component (hTR; encoded by TERC) and the human telomerase-associated protein (hTLP1; encoded by TEP1). A significant correlation between hTRT expression and telomerase activity was observed. These results indicate that the hTRT protein is the catalytic subunit of human telomerase, and that it plays a key role in the activation of telomerase in cancer cells.

Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene.

Macular corneal dystrophy (MCD; MIM 217800) is an autosomal recessive hereditary disease in which progressive punctate opacities in the cornea result in bilateral loss of vision, eventually necessitating corneal transplantation. MCD is classified into two subtypes, type I and type II, defined by the respective absence and presence of sulphated keratan sulphate in the patient serum, although both types have clinically indistinguishable phenotypes. The gene responsible for MCD type I has been mapped to chromosome 16q22, and that responsible for MCD type II may involve the same locus. Here we identify a new carbohydrate sulphotransferase gene (CHST6), encoding an enzyme designated corneal N-acetylglucosamine-6-sulphotransferase (C-GlcNAc6ST), within the critical region of MCD type I. In MCD type I, we identified several mutations that may lead to inactivation of C-GlcNAc6ST within the coding region of CHST6. In MCD type II, we found large deletions and/or replacements caused by homologous recombination in the upstream region of CHST6. In situ hybridization analysis did not detect CHST6 transcripts in corneal epithelium in an MCD type II patient, suggesting that the mutations found in type II lead to loss of cornea-specific expression of CHST6.

Topical vitamin D3 analogues induce thymic stromal lymphopoietin and cathelicidin in psoriatic skin lesions.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease of unknown aetiology, and an active form of vitamin D(3) (1α,25-dihydroxyvitamin D(3)) and its analogues (VD3As) are widely used topical reagents for psoriasis treatment. Besides their well-known calcium homeostasis functions, VD3As have been shown to have various immune-modulating effects including the induction of thymic stromal lymphopoietin (TSLP), a master cytokine for inducing Th2 inflammation, in mouse models, but not yet in human psoriasis. VD3As also have been shown to induce cathelicidin, an antimicrobial peptide and strong inducer of innate immunity. Cathelicidin is overexpressed in psoriatic skin lesions; however, its role in this disease seems as yet inconclusive. OBJECTIVES: To clarify whether topical VD3As induce TSLP and cathelicidin, and to examine the modulation of expression patterns of related cytokines in human psoriatic lesions. METHODS: Skin biopsy samples from psoriatic lesions with or without VD3A treatment were subjected to immunohistochemical staining and quantitative reverse transcription-polymerase chain reaction analyses to measure the expression levels of various cytokines. RESULTS: Significantly higher levels of TSLP, thymus and activation-related chemokine and CCR4 expression were observed in VD3A+ skin samples than in VD3A- samples. In contrast, significantly lower levels of interleukin (IL)-12/23 p40, IL-1α, IL-1ß and tumour necrosis factor (TNF)-α expression were observed in the VD3A+ samples than in the VD3A- samples. Expression of cathelicidin was elevated in VD3A+ samples. CONCLUSIONS: Topical VD3As induce TSLP and cathelicidin in psoriatic lesions, resulting in suppression of IL-12/23 p40, IL-1α, IL-1ß and TNF-α, thereby ameliorating psoriatic plaques.

Characterization and identification of weissellicin Y and weissellicin M, novel bacteriocins produced by Weissella hellenica QU 13.

AIMS: To identify and characterize novel bacteriocins from Weissella hellenica QU 13. METHODS AND RESULTS: Weissella hellenica QU 13, isolated from a barrel used to make Japanese pickles, produced two novel bacteriocins termed weissellicin Y and weissellicin M. The primary structures of weissellicins Y and M were determined, and their molecular masses were determined to be 4925·12 and 4968·40 Da, respectively. Analysis of the DNA sequence encoding the bacteriocins revealed that they were synthesized and secreted without N-terminal extensions such as leader sequences or sec signal peptides. Weissellicin M showed significantly high and characteristic homology with enterocins L50A and L50B, produced by Enterococcus faecium L50, while weissellicin Y showed no homology with any other known bacteriocins. Both bacteriocins showed broad antimicrobial spectra, with especially high antimicrobial activity against species, which contaminate pickles, such as Bacillus coagulans, and weissellicin M showed relatively higher activity than weissellicin Y. Furthermore, the stability of weissellicin M against pH and heat was distinctively higher than that of weissellicin Y. CONCLUSIONS: Weissella hellenica QU 13 produced two novel leaderless bacteriocins, weissellicin Y and weissellicin M, and weissellicin M exhibited remarkable potency that could be employed by pickle-producing industry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report, which represents a complete identification and characterization of novel leaderless bacteriocins from Weissella genus.

Expression of laminin 5-γ2 chain in cutaneous squamous cell carcinoma and its role in tumour invasion.

BACKGROUND: Laminin-5 (Ln5), a heterotrimer composed of three chains (α3, ß3, and γ2), is a major component of the basement membrane in most adult tissues. One of the chains, Ln5-γ2, is a marker of invasive tumours because it is frequently expressed as a monomer in malignant tumours. Recent studies from our laboratories detected higher levels of Ln5-γ2 expression in basal cell carcinoma (BCC) than in trichoblastoma. Furthermore, Ln5-γ2 overexpression tended to correlate with aggressiveness in BCC. METHODS: In this study, we compared the expression of Ln5-γ2 in invasive squamous cell carcinoma (SCC, n = 62) of the skin to that in preinvasive Bowen's disease (BD, n = 51), followed by analysis of the role of Ln5-γ2 in cancer invasion in vitro. RESULTS: Immunohistochemically, the proportion of SCC cases (86%) strongly positive for Ln5-γ2 expression was higher than that of BD (16%). Real-time RT-PCR showed Ln5-γ2 overexpression in SCC cell line, A431, compared with normal keratinocyte cell line, HaCaT. Ln5-γ2 monomer and proteolytically cleaved, biologically active fragments of Ln5-γ2 were identified in SCC tumour extracts. In in vitro raft cultures, which simulate in vivo conditions, Ln5-γ2 siRNA significantly suppressed epidermal growth factor (EGF)-stimulated A431 cell invasion. CONCLUSION: Our results indicate that Ln5-γ2 has a role in cutaneous SCC invasion.

The effect of including Lactobacillus reuteri KUB-AC5 during post-hatch feeding on the growth and ileum microbiota of broiler chickens.

The probiotic strain Lactobacillus reuteri KUB-AC5, which was originally isolated from chicken intestine, was fed to newborn broiler chicks for the first week post-hatch. The growth and ileum microbiota of the chickens were carefully monitored for 6 wk. The inclusion of 5 log cfu/g of feed statistically increased the BW gain in the first week compared with that of the control group, but this effect did not continue thereafter. Significant effects on host feed consumption and the feed-to-growth conversion ratio were not detected. The total amount and composition of ileum bacteria were investigated by quantitative PCR and pyrosequencing of the 16S rRNA gene (rDNA), respectively, and were compared between the control and the probiotic-treated groups. The amount of total bacterial 16S rDNA in ileum samples at d 42 was 5 times higher in the probiotic group than in the control, whereas no significant difference was observed at d 21. A composition analysis revealed the establishment of lactobacilli-enriched microbiota in the probiotic-treated chickens at d 42. At this point, the population level and species diversity of lactobacilli were significantly enhanced compared with those of the control group. In addition, Actinobacteria, mainly genera Corynebacterium and Dietzia, were also statistically higher in the probiotic group. However, Proteobacteria, including those of the family Campylobacterales and some other nonbeneficial bacterial groups, were decreased in the probiotic group at the growing stage. Therefore, with probiotic supplementation, it was demonstrated that Lactobacillus reuteri KUB-AC5 in the early post-hatching period had a delayed effect on ileum microbiota, which resulted in the enrichment of potentially beneficial lactobacilli and the suppression of Proteobacteria, including nonbeneficial bacterial groups.

Evaluation of essential and variable residues of nukacin ISK-1 by NNK scanning.

Nukacin ISK-1, a type-A(II) lantibiotic, comprises 27 amino acids with a distinct linear N-terminal and a globular C-terminal region. To identify the positional importance or redundancy of individual residues responsible for nukacin ISK-1 antimicrobial activity, we replaced the native codons of the parent peptide with NNK triplet oligonucleotides in order to generate a bank of nukacin ISK-1 variants. The bioactivity of each peptide variant was evaluated by colony overlay assay, and hence we identified three Lys residues (Lys1, Lys2 and Lys3) that provided electrostatic interactions with the target membrane and were significantly variable. The ring structure of nukacin ISK-1 was found to be crucially important as replacing the ring-forming residues caused a complete loss of bioactivity. In addition to the ring-forming residues, Gly5, His12, Asp13, Met16, Asn17 and Gln20 residues were found to be essential for antimicrobial activity; Val6, Ile7, Val10, Phe19, Phe21, Val22, Phe23 and Thr24 were relatively variable; and Ser4, Pro8, His15 and Ser27 were extensively variable relative to their positions. We obtained two variants, Asp13Glu and Val22Ile, which exhibited a twofold higher specific activity compared with the wild-type and are the first reported type-A(II) lantibiotic mutant peptides with increased potency.

Identification and characterization of novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15.

AIM: To characterize novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15. METHODS AND RESULTS: Leuconostoc pseudomesenteroides QU 15 isolated from Nukadoko (rice bran bed) produced novel bacteriocins. By using three purification steps, four antimicrobial peptides termed leucocin A (ΔC7), leucocin A-QU 15, leucocin Q and leucocin N were purified from the culture supernatant. The amino acid sequences of leucocin A (ΔC7) and leucocin A-QU 15 were identical to that of leucocin A-UAL 187 belonging to class IIa bacteriocins, but leucocin A (ΔC7) was deficient in seven C-terminal residues. Leucocin Q and leucocin N are novel class IId bacteriocins. Moreover, the DNA sequences encoding three bacteriocins, leucocin A-QU 15, leucocin Q and leucocin N were obtained. CONCLUSIONS: These bacteriocins including two novel bacteriocins were identified from Leuc. pseudomesenteroides QU 15. They showed similar antimicrobial spectra, but their intensities differed. The C-terminal region of leucocin A-QU 15 was important for its antimicrobial activity. Leucocins Q and N were encoded by adjacent open reading frames (ORFs) in the same operon, but leucocin A-QU 15 was not. SIGNIFICANCE AND IMPACT OF STUDY: These leucocins were produced concomitantly by the same strain. Although the two novel bacteriocins were encoded by adjacent ORFs, a characteristic of class IIb bacteriocins, they did not show synergistic activity.

Protective effect of Lactobacillus reuteri KUB-AC5 against Salmonella Enteritidis challenge in chickens.

Poultry is an important high-quality food and protein source for humans. However, chicken is considered a primary source of foodborne diseases, especially Salmonella Enteritidis infection. Reducing Salmonella contamination in live poultry will thus lower the risk to consumers. Our previous studies reported that Lactobacillus reuteri KUB-AC5 can produce a substance with antimicrobial activity against pathogenic bacteria, especially Salmonella. In vivo testing revealed that this strain greatly influenced the ileal microbiota by improving chicken gastrointestinal health and inhibiting certain pathogenic bacteria. However, its activity against Salmonella in chicken is unknown. This study investigated the effects of the probiotic L. reuteri KUB-AC5 at various concentrations against Salmonella and the microbiota status in the gastrointestinal tract of broiler chickens. Four treatments groups were used: negative-control group (no Salmonella challenge), positive-control group (Salmonella challenge), and 5 or 7 log cfu probiotic supplementation to Salmonella-challenged chickens. The resultant microbial diversities at the growing and finisher stages were not significantly different among the groups (P>0.05). However, a high dosage of KUB-AC5 maintained similar microbial diversity in Salmonella-challenged chickens as observed in the non-challenged group in the early stage. The exposure Salmonella can affect the microbial diversity that consequently contributes to the disease progression in chicken. Low and high dosages of KUB-AC5 eliminated S. Enteritidis from the ileum and caecum at 14, 21 and 35 days of age. A high-dose of KUB-AC5 also enhanced Lactobacillaceae levels in the growing stage in both the ileum and caecum and suppressed Enterobacteriaceae levels in the finisher stage on day 35, whereas these effects were not observed in the low dose of KUB-AC5 or control groups. These results support the potential value of high-dose L. reuteri KUB-AC5 supplementation for three days after hatching in preventing Salmonella infection in chickens.

Bacteriocin detection by liquid chromatography/mass spectrometry for rapid identification.

AIMS: To establish a new system to detect and identify bacteriocins in the early stage of screening for novel bacteriocins. METHODS AND RESULTS: Liquid chromatography/mass spectrometry (LC/MS) was employed for development of a new system for rapid detection and identification of bacteriocins. The system detected and identified bacteriocins such as nisin and lacticin 481 from 25 microl of culture supernatants of their producing strains by accurate mass determination coupled with simultaneous impurity removal within 40 min. Especially, the system clearly distinguished three nisin variants (A, Z, Q) in culture supernatants of their producing strains, although they have similar structures and molecular masses. Each one-step pretreatment by cell adsorption-desorption or acetone precipitation improved bacteriocin detection dramatically, especially for mundticin KS. This system could be applied for detection and molecular mass determination of novel bacteriocins by extracting bacteriocin-related ions. CONCLUSIONS: The developed system could detect and identify some kinds of bacteriocin from culture supernatants or pretreated samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed system helps us to identify bacteriocins in the early stage of screening without any or with one-step pretreatment. This system is effective on not only detection of known bacteriocins but also identification of novel bacteriocins. Consequently, this system will accelerate discovery of novel bacteriocins.

Description of durancin TW-49M, a novel enterocin B-homologous bacteriocin in carrot-isolated Enterococcus durans QU 49.

AIMS: To characterize the novel bacteriocin produced by Enterococcus durans. METHODS AND RESULTS: Enterococcus durans QU 49 was isolated from carrot and expressed bactericidal activity over 20-43 degrees C. Bacteriocins were purified to homogeneity using the three-step purification method, one of which, termed durancin TW-49M, was an enterocin B-homologous peptide with most identical residues occurring in the N-terminus. Durancin TW-49M was more tolerant in acidic than in alkali. DNA sequencing analysis revealed durancin TW-49M was translated as a prepeptide of the double-glycine type. Durancin TW-49M and enterocin B expressed similar antimicrobial spectra, in which no significant variation due to the diversity in their C-termini was observed. CONCLUSIONS: Durancin TW-49M, a novel nonpediocin-like class II bacteriocin, was characterized to the amino acid and genetic levels. The diverse C-terminal parts of durancin TW-49M and enterocin B were hardly to be suggested as the place determining the target cell specificity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first and comprehensive study of a novel bacteriocin produced by Ent. durans. The high homology at the N-terminal halves between durancin TW-49M and enterocin B makes them suitable to study the structure-function relationship of bacteriocins and their immunity proteins.

Biosynthetic characterization and biochemical features of the third natural nisin variant, nisin Q, produced by Lactococcus lactis 61-14.

AIMS: To characterize the genetic and biochemical features of nisin Q. METHODS AND RESULTS: The nisin Q gene cluster was sequenced, and 11 putative orfs having 82% homology with the nisin A biosynthesis gene cluster were identified. Nisin Q production was confirmed from the nisQ-introduced nisin Z producer. In the reporter assay, nisin Q exhibited an induction level that was threefold lower than that of nisin A. Nisin Q demonstrated an antimicrobial spectrum similar to those of the other nisins. Under oxidizing conditions, nisin Q retained a higher level of activity than nisin A. This higher oxidative tolerance could be attributed to the presence of only one methionine residue in nisin Q, in contrast to other nisins that contain two. CONCLUSIONS: The 11 orfs of the nisin producers were identical with regard to their functions. The antimicrobial spectra of the three natural nisins were similar. Nisin Q demonstrated higher oxidative tolerance than nisin A. SIGNIFICANCE AND IMPACT OF THE STUDY: Genetic and biochemical features of nisin Q are similar to those of other variants. Moreover, owing to its higher oxidative tolerance, nisin Q is a potential alternative for nisin A.

Inhibition of NADPH oxidase 4 activates apoptosis via the AKT/apoptosis signal-regulating kinase 1 pathway in pancreatic cancer PANC-1 cells.

Pancreatic adenocarcinoma is an aggressive human malignancy and is characterized by resistance to apoptosis. Recently, NADPH oxidase (Nox) 4-mediated generation of intracellular reactive oxygen species (ROS) was proposed to confer antiapoptotic activity and thus a growth advantage to pancreatic cancer cells. The signaling mechanism by which Nox4 transmits cell survival signals remains unclear. Here, we show that both a flavoprotein inhibitor, diphenylene iodonium (DPI), and small interfering RNAs designed to target Nox4 mRNA (siNox4RNAs) inhibited superoxide production in PANC-1 pancreatic cancer cells, and depletion of ROS by DPI or siNox4RNAs induced apoptosis. Parallely, DPI treatment and siNox4RNA transfection blocked activation of the cell survival kinase AKT by attenuating phosphorylation of AKT. Furthermore, AKT phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) on Ser-83 was reduced by DPI and siNox4RNAs. When ASK1Ser83Ala (an AKT phosphorylation-defective ASK1 mutant) was introduced into PANC-1 cells, this mutant alone induced apoptosis. But, addition of DPI or co-transfection of siNox4RNA had no additive effect, indicating that the mutant can substitute for these reagents in apoptosis induction. Taken together, these findings suggest that ROS generated by Nox4, at least in part, transmit cell survival signals through the AKT-ASK1 pathway in pancreatic cancer cells and their depletion leads to apoptosis.