Lactobacillus Plantarum 299v Changes miRNA Expression in the Intestines of Piglets and Leads to Downregulation of LITAF by Regulating ssc-miR-450a

Lactiplantibacillus plantarum subsp. plantarum 299v (L. plantarum 299v) is one of the most important probiotic strains in animal health, but the molecular mechanisms of how it exerts health benefits remain unclear. The purpose of this study was to explore the changes in miRNA expression profiles in the intestinal tissues of piglets by L. plantarum 299v and to explore its possible molecular regulatory mechanism in intestinal function. Neonatal piglets were orally administered L. plantarum 299v daily from 1 to 20 days old, and high-throughput sequencing was conducted to analyse the changes in miRNA expression in the jejunum and ileum. The results showed that 370 known porcine miRNAs were identified from eight libraries. Five miRNAs (ssc-miR-21-5p, -143-3p, -194b-5p, -192, and -126-3p) were highly expressed in the intestinal tissues. There were 15 differentially expressed miRNAs between the control group and the L. plantarum group, and only miR-450a was expressed differentially in both intestinal tissues. KEGG analysis revealed that the target genes of the 15 differentially expressed miRNAs were involved in 37 significantly enriched pathways (P < 0.01). Then, quantitative polymerase chain reaction confirmed that the miRNA expression was corresponded well with those from the sequencing. Luciferase reporter assays verified that lipopolysaccharide-induced TNF-α factor is a target of miR-450a. Our results also showed L. plantarum 299v could influence intestinal function by changing the levels of cytokines via miRNA expression. This is the first study to analyse differential expression miRNA profiles in intestinal tissue after L. plantarum 299v treatment and investigate the molecular regulatory mechanism of functional miRNA.

     

Lactulose and Lactobacillus plantarum,a Potential Complementary Synbiotic To Control Postweaning Colibacillosis in Piglets

The potential of a prebiotic oligosaccharide lactulose, a probiotic strain of Lactobacillus plantarum, or their synbiotic combination to control postweaning colibacillosis in pigs was evaluated using an enterotoxigenic Escherichia coli (ETEC) K88 oral challenge. Seventy-two weanlings were fed four diets: a control diet (CTR), that diet supplemented with L. plantarum (2 × 10¹⁰ CFU · day⁻¹) (LPN), that diet supplemented with 10 g · kg⁻¹ lactulose (LAC), or a combination of the two treatments (SYN). After 7 days, the pigs were orally challenged. Six pigs per treatment were euthanized on days 6 and 10 postchallenge (PC). Inclusion of lactulose improved the average daily gain (ADG) (P < 0.05) and increased lactobacilli (P < 0.05) and the percentage of butyric acid (P < 0.02) in the colon. An increase in the ileum villous height (P < 0.05) and a reduction of the pig major acute-phase protein (Pig-MAP) in serum (P < 0.01) were observed also. The inclusion of the probiotic increased numbers of L. plantarum bacteria in the ileum and colon (P < 0.05) and in the total lactobacilli in the colon and showed a trend to reduce diarrhea (P = 0.09). The concentrations of ammonia in ileal and colonic digesta were decreased (P < 0.05), and the villous height (P < 0.01) and number of ileal goblet cells (P < 0.05) increased, at day 10 PC. A decrease in plasmatic tumor necrosis factor alpha (TNF-α) (P < 0.01) was also seen. The positive effects of the two additives were combined in the SYN treatment, resulting in a complementary synbiotic with potential to be used to control postweaning colibacillosis.     

Immunomodulatory activity of two potential probiotic strains in LPS-stimulated HT-29 cells

The relative expression of mucin, pro- and anti-inflammatory genes besides other signaling molecules in HT-29 cells by two test probiotic strains of Lactobacillus plantarum Lp9 and Lp91 and the reference strain L. plantarum 5276 was evaluated by RT-qPCR using Relative Expression Software Tool qBase-Plus under in vitro simulated gut conditions. Ten house keeping genes were evaluated by using geNorm 3.4 excel based application. The most stable genes were RPL27, ACTB and B2M which were subsequently used for calculating the normalization factor. Under pretreatment conditions (4 h probiotic treatment, followed by lipopolysaccharide challenge for 3 h), all the three strains evoked downregulation of IL-8 expression by ~100 %, while in case of TNF-α, the downregulation of the relative gene expression was at the rate of 98.2, 93.8 and 98.0 % with Lp5276, Lp9 and Lp91, respectively, under the same set of conditions. Lp91 evoked maximum downregulation of IL12p35 and IFN-γ with corresponding fold reduction in relative expression of the two genes by 96.5 and 96.7 % during pre-treatment conditions. However, IL-10 and IFN-α were significantly upregulated to the extent of 8.13 ± 0.36 and 2.62 ± 0.14 fold by Lp91 under the same conditions. Lp9 and Lp91 were also quite effective in inducing the expression of Cox-1 and Cox-2 in HT-29 cells as can be reflected from their ratios, i.e., 5.90 and 6.50 (under pretreatment conditions); 3.79 and 4.36 (under co-culture conditions). Thus, the two putative indigenous L. plantarum strains Lp9 and Lp91 demonstrated immunomodulating functions in HT-29 cells at significant levels under different experimental conditions.

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Biotic elicitor enhanced production of psoralen in suspension cultures of Psoralea corylifolia L.

Cell cultures of Psoralea corylifolia L. were established from the leaf disk derived callus. The effect of different biotic elicitors prepared from the fungal extract (Aspergillus niger and Penicillium notatum), yeast extract and chitosan with different concentrations was studied. The increased synthesis of psoralen in 16-day old cell cultures under 16 h of light and 8 h of dark period was studied. Elicitation of psoralen in A. niger elicitor treated cells was found 9-fold higher over control cells. Treating the cells with P. notatum, yeast extract and chitosan elicitors lead to four to seven-fold higher psoralen accumulation over control cells. The extract of A. niger at 1.0% v/v increased the significant accumulation of psoralen (9850 μg/g DCW) in the cultured cells. Our study clearly shows that all the elicitors had the potential to increase the accumulation of psoralen but the A. niger elicitor at 1.0% v/v induced maximum accumulation.     

Unusual genome complexity in Lactobacillus salivarius JCM1046

Background

Lactobacillus salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable and is often mediated by plasmids or integrative and conjugative elements. L. salivarius strains typically have multireplicon genomes including circular megaplasmids that encode strain-specific traits for intestinal survival and probiotic activity. Linear plasmids are less common in lactobacilli and show a very limited distribution in L. salivarius. Here we present experimental evidence that supports an unusually complex multireplicon genome structure in the porcine isolate L. salivarius JCM1046.

Results

JCM1046 harbours a 1.83 Mb chromosome, and four plasmids which constitute 20% of the genome. In addition to the known 219 kb repA-type megaplasmid pMP1046A, we identified and experimentally validated the topology of three additional replicons, the circular pMP1046B (129 kb), a linear plasmid pLMP1046 (101 kb) and pCTN1046 (33 kb) harbouring a conjugative transposon. pMP1046B harbours both plasmid-associated replication genes and paralogues of chromosomally encoded housekeeping and information-processing related genes, thus qualifying it as a putative chromid. pLMP1046 shares limited sequence homology or gene synteny with other L. salivarius plasmids, and its putative replication-associated protein is homologous to the RepA/E proteins found in the large circular megaplasmids of L. salivarius. Plasmid pCTN1046 harbours a single copy of an integrated conjugative transposon (Tn6224) which appears to be functionally intact and includes the tetracycline resistance gene tetM.

Conclusion

Experimental validation of sequence assemblies and plasmid topology resolved the complex genome architecture of L. salivarius JCM1046. A high-coverage draft genome sequence would not have elucidated the genome complexity in this strain. Given the expanding use of L. salivarius as a probiotic, it is important to determine the genotypic and phenotypic organization of L. salivarius strains. The identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications.

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Lactobacillus plantarum 299v Prevents Caspase-Dependent Apoptosis In Vitro

Selective microbes used as probiotics can enhance epithelial cell protection. We have previously shown that a Lactobacillus plantarum strain 299v (Lp299v) has the ability to induce mucin genes. In the current study, we utilized a cytokine model of inflammation in cell culture to study the modulation of apoptosis by this probiotic. HT-29 cells were pre-incubated with the Lp299v or L. plantarum strain adh- (Lpadh-), a non-adherent derivative of Lp299v. Cells were challenged with a mixture of cytokines (TNF-α, IFN-γ, and IL-1a) to imitate conditions of inflammation. To assess for cell death, we evaluated TUNEL, multi-caspase, and caspase-3 and caspase-7 activity assays. There was a marked decrease in apoptosis as measured by TUNEL⁺ cells in samples pre-treated with Lp299v (18.7 ± 4.1%, p < 0.01) and Lpadh- (16.6 ± 3.2%, p < 0.05) prior to cytokine exposure when compared to cells (43.6 ± 6.2%) exposed to the cytokine mixture. Lp299v pre-incubation with HT-29 cells reduced caspase⁺ cells in the multi-caspase activity assay (3.6 ± 0.6%, p < 0.05) compared to cells exposed to cytokines (68.9 ± 5.1%) whereas Lpadh- did not (46.8 ± 17.5%, p > 0.05). Similarly, caspase-3, caspase-7 activity was also reduced by Lp299v. Selected probiotics may confer an exogenous protective effect at the mucosal–luminal interface for intestinal epithelial cells via alteration of caspase-dependent apoptotic pathways.     

Heat Shock Response in Lactobacillus plantarum

Heat stress resistance and response were studied in strains of Lactobacillus plantarum. Stationary-phase cells of L. plantarum DPC2739 had decimal reduction times (D values) (D value was the time that it took to reduce the number of cells by 1 log cycle) in sterile milk of 32.9, 14.7, and 7.14 s at 60, 72, and 75°C, respectively. When mid-exponential-phase cells were used, the D values decreased. The temperature increases which caused a 10-fold reduction in the D value ranged from 9 to 20°C, depending on the strain. Part of the cell population treated at 72°C for 90 s recovered viability during incubation at 7°C in sterile milk for 20 days. When mid-exponential- or stationary-phase cells of L. plantarum DPC2739 were adapted to 42°C for 1 h, the heat resistance at 72°C for 90 s increased ca. 3 and 2 log cycles, respectively. Heat-adapted cells also showed increased growth at pH 5 and in the presence of 6% NaCl. Two-dimensional gel electrophoresis of proteins expressed by control and heat-adapted cells revealed changes in the levels of expression of 31 and 18 proteins in mid-exponential- and stationary-phase cells, respectively. Twelve proteins were commonly induced. Nine proteins induced in the heat-adapted mid-exponential- and/or stationary-phase cells of L. plantarum DPC2739 were subjected to N-terminal sequencing. These proteins were identified as DnaK, GroEL, trigger factor, ribosomal proteins L1, L11, L31, and S6, DNA-binding protein II HlbA, and CspC. All of these proteins have been found to play a role in the mechanisms of stress adaptation in other bacteria. Antibodies against GroES detected a protein which was induced moderately, while antibodies against DnaJ and GrpE reacted with proteins whose level of expression did not vary after heat adaptation. This study showed that the heat resistance of L. plantarum is a complex process involving proteins with various roles in cell physiology, including chaperone activity, ribosome stability, stringent response mediation, temperature sensing, and control of ribosomal function. The physiological mechanisms of response to pasteurization in L. plantarum are fundamental for survival in cheese during manufacture.     

Dietary polyunsaturated fatty acids and the Pro12Ala polymorphisms of PPARG regulate serum lipids through divergent pathways: a randomized crossover clinical trial

Human and animal studies suggest an interaction between the Pro12Ala polymorphism of PPARG and dietary fat. In this randomized crossover clinical trial, we investigated whether subjects with the Pro12Pro and Ala12Ala genotypes of PPARG respond differently to a diet supplemented with high saturated (SAFA) or polyunsaturated fatty acid (PUFA).We recruited non-diabetic men from a population-based METSIM study (including 10,197 men) to obtain men with the Ala12Ala and the Pro12Pro genotypes matched for age and body mass index. Seventeen men with the Pro12Pro genotype and 14 with the Ala12Ala genotype were randomized to both a PUFA diet and a SAFA diet for 8 weeks in a crossover setting. Serum lipids and adipose tissue mRNA expression were measured during the diet intervention. At baseline, subjects with the Ala12Ala genotype had higher levels of HDL cholesterol and lower levels of LDL cholesterol, total triglycerides, and apolipoprotein B compared to those subjects with the Pro12Pro genotype (P < 0.05, FDR < 0.1). The Ala12Ala genotype also associated with higher mRNA expression of PPARG2, LPIN1, and SREBP-1c compared to participants with the Pro12Pro genotype (FDR < 0.001). On the other hand, PUFA diet resulted in lower levels of fasting glucose, total cholesterol, total triglycerides, and apolipoprotein B (P < 0.05, FDR < 0.1) but did not affect PPARG2 mRNA expression in adipose tissue. We conclude that individuals with the Pro12Pro genotype, with higher triglyceride levels at baseline, are more likely to benefit from the PUFA diet. However, the beneficial effects of dietary PUFA and the Ala12Ala genotype of PPARG on serum lipids are mediated through divergent mechanisms.

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In Vitro Evaluation of the Probiotic Potential of Halotolerant Lactobacilli Isolated from a Ripened Tropical Mexican Cheese

Three halotolerant lactobacilli (Lactobacillus plantarum, L. pentosus, and L. acidipiscis) isolated from a ripened Mexican tropical cheese (double cream Chiapas cheese) were evaluated as potential probiotics and compared with two commercial probiotic strains (L. casei Shirota and L. plantarum 299v) from human origin. All the strains survived the in vitro gastrointestinal simulation from the oral cavity to the ileum. During the stomach simulation, all the strains survived in satiety conditions (60 min, pH 3.0, 3 g/L pepsin, 150 rpm) and only L. pentosus could not survive under fasting conditions (60 min, pH 2.0, 3 g/L pepsin, 150 rpm). All the strains showed a strong hydrophilic character with low n-hexadecane and a variable chloroform affinity. L. plantarum showed a mucin adhesion rate similar to that of L. plantarum 299v and L. casei Shirota, while L. pentosus and L. acidipiscis had a lower mucin adhesion. The isolated halotolerant lactobacilli exhibited similar antimicrobial activity against some gram-positive and gram-negative pathogens in comparison with the two commercial strains. In addition, the proteinaceous character of the antimicrobial agents against the most pathogenic strains was demonstrated. The compounds showed a low molecular weight (less than 10 kDa). Besides, L. plantarum and L. acidipiscis were able to produce the enzyme β-galactosidase. Finally, L. pentosus was able to deconjugate taurocholic, taurodeoxycholic, glycocholic, and glycodeoxycholic acids better than the two commercial strains analyzed. All these results suggest that the halotolerant lactobacilli isolated from this ripened Mexican cheese could be potentially probiotic. This is the first time that halotolerant lactic acid bacteria have been shown to have probiotic properties.     

Protection of human erythrocyte using Crinumasiaticum extract and lycorine from oxidative damage induced by 2-amidinopropane

The intention of this investigation was to evaluate the free radical scavenging activity and erythrocyte protective activity of ethanolic extract of Crinumasiaticum (L) and lycorine. The ethanolic extract of C. asiaticum (L) and lycorine were found to have different levels of antioxidant properties in the test models. Both ethanolic extract of C. asiaticum (L) (0.5–2.5 mg/ml) and lycorine (0.010 mg–0.050 mg/ml) increases the percentage of lipid peroxidation inhibition (26.25 ± 0.23% and 19.25 ± 0.23%) and enhances the free radical scavenging activity (20.92 ± 0.22% and 20.52 ± 0.22%), scavenging of hydrogen peroxide (25.67 ± 0.17% and 23.07 ± 0.3%) superoxide anion scavenging activity (27.69 ± 0.16% and 16.09 ± 0.7%) at concentration of 2.5 and 0.050 mg of C. asiaticum (L) and lycorine, respectively. But compared with tocopherol (P < 0.05) less activity was observed by C. asiaticum (L) and lycorine. The ethanolic extract of C. asiaticum (L) and lycorine were normalized to reduce the level of glutathione and also to sustain the status of protein in erythrocytes during the peroxyl radical [2,2-azobis (2-amidinopropane) dihydrochloride (AAPH)] induced oxidative damage in ex vivo model. The present results of the investigations demonstrated that protective nature of the C. asiaticum (L) and lycorine will be considered as a significant natural antioxidant source.     

A new species of plasmodiidae (Coccidia: Hemosporidia) from the blood of the skink Scincus hemprichii (Scincidae: Reptilia) in Saudi Arabia

Fallisia arabica n. sp. was described from peripheral blood smears of the Skink lizard, Scincus hemprichii from Jazan Province in the southwest of Saudi Arabia. Schizogony and gametogony take place within neutrophils in the peripheral blood of the host. Mature schizont is rosette shaped 17.5 ± 4.1 × 17.0 ± 3.9 μm, with a L/W ratio of 1.03(1.02–1.05) μm and produces 24(18–26) merozoites. Young gametocytes are ellipsoidal, 5.5 ± 0.8 × 3.6 ± 0.5 μm, with a L/W of 1.53(1.44–1.61) μm. Mature macrogametocytes are ellipsoidal, 9.7 ± 1.2 × 7.8 ± 1.0 μm, with a L/W of 1.24(1.21–1.34) μm and microgametocytes are ellipsoidal, 7.0 ± 1.1 × 6.8 ± 0.9 μm. with a L/W of 1.03(1.01–1.10) μm. In comparison to the described Fallisia species, this new taxon has rosette schizonts and is larger than F. dominicensis, in Hispaniola, F. bipocrati, F. poecilopi, in Panama, F. thecadactyli in Venezuela, and F. effusa, F. simplex, F. modesta, in Brazil. F. arabica has fewer merozoites than F. effusa, F. poecilopi, F. thecadactyli and F. siamense in Thailand. This new species has more merozoites than F. dominicensis and F. modesta. All of these species belong to diverse saurian families (Agamidae, Gekkonidae, Polychrotidae, Scincidae and Teiidae) parasitize only thrombocytes or lymphocytes and some species parasitize immature erythroid cells and leucocytes.     

Phylogenetic Study of Methanol Oxidizers from Chilika-Lake Sediments Using Genomic and Metagenomic Approaches

Group-wise diversity of sediment methylotrophs of Chilika lake (Lat. 19°28′–19°54′N; Long. 85°06′–85°35′E) Odisha, India at various identified sites was studied. Both the culturable and unculturable (metagenome) methylotrophs were investigated in the lake sediments employing both mxaF and 16S rRNA genes as markers. ARDRA profiling, 16S rRNA gene sequencing, PAGE profiling of HaeIII, EcoRI restricted mxaF gene and the mxaF gene sequences using culture-dependent approach revealed the relatedness of α-proteobacteria and Methylobacterium, Hyphomicrobium and Ancyclobacter sp. The total viable counts of the culturable aerobic methylotrophs were relatively higher in sediments near the sea mouth (S3; Panaspada), also demonstrated relatively high salinity (0.1 M NaCl) tolerance. Metagenomic DNA from the sediments, amplified using GC clamp mxaF primers and resolved through DGGE, revealed the diversity within the unculturable methylotrophic bacterium Methylobacterium organophilum, Ancyclobacter aquaticus, Burkholderiales and Hyphomicrobium sp. Culture-independent analyses revealed that up to 90 % of the methylotrophs were unculturable. The study enhances the general understandings of the metagenomic methylotrophs from such a special ecological niche.

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Essential Oils,Silver Nanoparticles and Propolis as Alternative Agents Against Fluconazole Resistant Candida albicans,Candida glabrata and Candida krusei Clinical Isolates

Development of effective and safe therapeutic treatment of fungal infections remains one of the major challenge for modern medicine. The aim of presented investigation was to analyze the in vitro antifungal activity of selected essential oils, ethanolic extracts of propolis and silver nanoparticles dropped on TiO₂ against azole-resistant C. albicans (n = 20), C. glabrata (n = 14) and C. krusei (n = 10) clinical isolates. Among tested essential oils, the highest activity has definitely been found in the case of the oil isolated from the bark of Cinnamomum cassia, with MIC and MFC values for all tested strains in the range of 0.0006–0.0097 % (v/v) and 0.0012–0.019 % (v/v), respectively. High activity was also observed for the Lemon, Basil, Thyme, Geranium and Clove (from buds) essential oils. Significant differences in fungicidal activity have been observed in the case of four tested propolis samples. Only one of them revealed high activity, with MFC values in the range from 0.156 to 1.25 % (v/v). Satisfactory fungicidal activity, against C. albicans and C. glabrata isolates, was also observed in the case of silver nanoparticles, however C. krusei isolates were mostly resistant. We also revealed that constituents of most of essential oils and propolis as well as silver nanoparticles are not substrates for drug transporters, which belong to the most important factors affecting resistance of Candida spp. clinical isolates to many of conventional antimycotics. To conclude, the results of our investigation revealed that essential oils, propolis and silver nanoparticles represent high potential for controlling and prevention candidiasis.     

Adenosine 5′-monophosphate ameliorates D-galactosamine/lipopolysaccharide-induced liver injury through an adenosine receptor-independent mechanism in mice

D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced lethality and acute liver failure is dependent on endogenously produced inflammatory cytokines. Adenosine has been proven to be a central role in the regulation of inflammatory response. It is not entirely clear that which adenosine action is actually crucial to limiting inflammatory tissue destruction. Here we showed that GalN/LPS challenge elevated hepatic adenosine and induced lethality in adenosine receptor-deficient mice with equal efficiency as wild-type mice. In GalN/LPS-treated mice, pretreatment with adenosine 5′-monophosphate (5′-AMP) significantly elevated hepatic adenosine level and reduced mortality through decreasing cytokine and chemokine production. In RAW264.7 cells, 5′-AMP treatment inhibited the production of inflammatory cytokines, which is not mediated through adenosine receptors. 5′-AMP failed to attenuate LPS-induced nuclear factor-κB (NF-κB) p65 nuclear translocation, but reduced LPS-induced recruitment of NF-κB p65 to inflammatory gene promoters and decreased LPS-induced enrichment of H3K4 dimethylation at the tumor necrosis factor-α (TNF-α) promoter, which was involved in 5′-AMP-induced elevation of cellular adenosine and a decline of methylation potential. In vitro biochemical analysis revealed that adenosine directly attenuated recruitment of NF-κB to the TNF-α and interleukin-6 promoters. Our findings demonstrate that 5′-AMP-inhibiting inflammatory response is not mediated by adenosine receptors and it may represent a potential protective agent for amelioration of LPS-induced liver injury.     

Mutagenesis of Trichoderma reesei endoglucanase I: impact of expression host on activity and stability at elevated temperatures

Background

Trichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. Lignocellulose hydrolysis at temperatures above the optimum temperature of T. reesei cellulases (~50°C) could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. These potential advantages motivate efforts to engineer T. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60–70°C.

Results

A B-factor guided approach for improving thermostability was used to engineer variants of endoglucanase I (Cel7B) from T. reesei (TrEGI) that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type TrEGI at temperatures ranging from 50–70°C. When expressed in T. reesei, TrEGI variant G230A/D113S/D115T (G230A/D113S/D115T Tr_TrEGI) had a higher apparent melting temperature (3°C increase in T_m) and improved half-life at 60°C (t_1/2 = 161 hr) than the recombinant (T. reesei host) wild-type TrEGI (t_1/2 = 74 hr at 60°C, Tr_TrEGI). Furthermore, G230A/D113S/D115T Tr_TrEGI showed 2-fold improved activity compared to Tr_TrEGI at 65°C on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°C as Tr_TrEGI was at 50°C. The activities and stabilities of the recombinant TrEGI enzymes followed similar trends but differed significantly in magnitude depending on the expression host (Escherichia coli cell-free, Saccharomyces cerevisiae, Neurospora crassa, or T. reesei). Compared to N.crassa-expressed TrEGI, S. cerevisiae-expressed TrEGI showed inferior activity and stability, which was attributed to the lack of cyclization of the N-terminal glutamine in Sc_TrEGI and not to differences in glycosylation. N-terminal pyroglutamate formation in TrEGI expressed in S. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the T. reesei or N. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in GH7 enzymes.

Conclusion

Structure-guided evolution of T. reesei EGI was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. Production of TrEGI enzymes in four hosts highlighted the impact of the expression host and the role of N-terminal pyroglutamate formation on the activity and stability of TrEGI enzymes.

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Effect of early antibiotic intervention on specific bacterial communities and immune parameters in the small intestine of growing pigs fed different protein level diets

Antibiotics are designed to affect gut microbiota and subsequently gut homeostasis. However, limited information exists about short- and long-term effects of early antibiotic intervention (EAI) on gut homeostasis (especially for the small intestine) of pigs following antibiotic withdrawal. We investigated the impact of EAI on specific bacterial communities, microbial metabolites and mucosal immune parameters in the small intestine of later-growth-stage pigs fed with diets differing in CP levels. Eighteen litters of piglets were fed creep feed with or without antibiotics from day 7 to day 42. At day 42, pigs within each group were offered a normal- or low-CP diet. Five pigs per group were slaughtered at days 77 and 120. At day 77, EAI increased Enterobacteriaceae counts in the jejunum and ileum and decreased Bifidobacterium counts in the jejunum and ileum (P < 0.05). Moreover, tryptamine, putrescine, secretory immunoglobulin (Ig) A and IgG concentrations in the ileum and interleukin-10 (IL-10) mRNA and protein levels in the jejunum and ileum were decreased in pigs with EAI (P < 0.05). At day 120, EAI only suppressed Clostridium cluster XIVa counts in the jejunum and ileum (P < 0.05). These results suggest that EAI has a short-term effect on specific bacterial communities, amino acid decarboxylation and mucosal immune parameters in the small intestine (particularly in the ileum). At days 77 and 120, feeding a low-CP diet affected Bifidobacterium, Clostridium cluster IV, Clostridium cluster XIVa and Enterobacteriaceae counts in the jejunum or ileum (P < 0.05). Moreover, feeding a low-CP diet increased the concentrations of Igs in the jejunum and decreased pro-inflammatory cytokines levels in the jejunum and ileum (P < 0.05). At day 120, feeding a low-CP diet increased short-chain fatty acid concentrations, reduced ammonia and spermidine concentrations and up-regulated genes related to barrier function in the jejunum and ileum (P < 0.05). These results suggest that feeding a low-CP diet changes specific bacterial communities and intestinal metabolite concentrations and modifies mucosal immune parameters. These findings contribute to our understanding on the duration of the impact of EAI on gut homeostasis and may provide basis data for nutritional modification in young pigs after antibiotic treatment.     

The optimization of Marasmius androsaceus submerged fermentation conditions in five-liter fermentor

Using desirability function, four indexes including mycelium dry weight, intracellular polysaccharide, adenosine and mannitol yield were uniformed into one expected value (Da) which further served as the assessment criteria. In our present study, Plackett–Burman design was applied to evaluate the effects of eight variables including initial pH, rotating speed, culture temperature, inoculum size, ventilation volume, culture time, inoculum age and loading volume on Da value during Marasmius androsaceus submerged fermentation via a five-liter fermentor. Culture time, initial pH and rotating speed were found to influence Da value significantly and were further optimized by Box–Behnken design. Results obtained from Box–Behnken design were analyzed by both response surface regression (Design-Expert.V8.0.6.1 software) and artificial neural network combining the genetic algorithm method (Matlab2012a software). After comparison, the optimum M. androsaceus submerged fermentation conditions via a five-liter fermentor were obtained as follows: initial pH of 6.14, rotating speed of 289.3 rpm, culture time of 6.285 days, culture temperature of 26 °C, inoculum size of 5%, ventilation volume of 200 L/h, inoculum age of 4 days, and loading volume of 3.5 L/5 L. The predicted Da value of the optimum model was 0.4884 and the average experimental Da value was 0.4760. The model possesses well fitness and predictive ability.     

Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells

Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP₃/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v) FBS. Following stimulation of RTKs (receptor tyrosine kinases), microinjected PI3Kα was recruited to the PM, but oncogenic forms of PI3Kα were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3Kα. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein–protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity.