lamB Gene Research Articles

The impact of exogenous vasoactive intestinal polypeptide on inflammatory responses and mRNA expression of tight junction genes in lambs fed a high-grain diet.

This study assessed the impact of administering vasoactive intestinal polypeptide (VIP) on inflammation and intestinal VIP and tight junction mRNA expression in lambs fed grain-based finishing diets. Sixteen wether lambs (69.6 ± 1.9kg) were individually housed, adapted to a corn-based diet containing no forage, and randomly assigned to 2 treatment groups. Lambs were intraperitoneally injected every other day for 28 d with either saline (0.9% NaCl) with no VIP (n = 8; control) or saline with VIP (n = 8; 1.3 nmol/kg BW). Blood samples were collected weekly for analysis of cytokine concentrations, and on days 0 and 28 for lipopolysaccharide (LPS), and LPS-binding protein (LBP) concentrations. Upon completion of the treatment period, lambs were euthanized and gastrointestinal tissues, including rumen, jejunum, cecum, and colon samples, were collected for analysis of the expression of tight junction mRNA (claudin-1, claudin-4, occludin, and ZO-1), endogenous VIP, and VIP receptor (VPAC-1). No treatment effects (P ≥ 0.38) were observed for VIP and VPAC-1 mRNA expression in the colon. Supplementation with VIP did not influence (P ≥ 0.28) the expression of claudin-1, claudin-4, occludin, and ZO-1 tight junction mRNA in the rumen, jejunum, cecum, and colon. Lambs treated with VIP had greater (P ≤ 0.01) plasma concentrations of the anti-inflammatory cytokines, IL-10 and IL-36RA. There were treatment-by-day interactions observed (P ≤ 0.02) for concentrations of the pro-inflammatory cytokines, MIP-1α and MIP-1β. Lambs that did not receive VIP had greater serum concentrations of LPS (P = 0.05) than the lambs receiving VIP. These data suggest that VIP administration may not influence tight junction mRNA expression but may decrease LPS concentrations and thus inflammation in lambs fed a grain-based diet.

Immunoprophylactic potential of recombinant outer membrane protein of Salmonella enterica serovar Typhi against enteric fever in BALB/c mice

ObjectivesThis study was design to explore the immunogenic behavior of LamB and SpaO outer membrane protein of Salmonella serovar Typhi against enteric fever in BALB/c mice. MethodsSpaO or LamB genes of locally isolated strain of S.Typhi was amplified, cloned and expressed. Recombinant protein SpaO and LamB were purified and lyophilized to prepare aluminum hydroxide Gel precipitated vaccine. Clinical trials were performed in BALB/c mice. The Immunoprophylactic potential of recombinant SpaO or LamB alone and in combination at different concentrations were determined using Complement fixation test along with whole culture attenuated and commercial vaccine. ResultsThe immune response of rSpaO and rLamB proteins in combination at 500 µg/ml was significantly high at 48th day and maintained until 60thday. Protective immune potential of recombinant monovalent and multivalent vaccine assessed upon challenge in mice with 109 cells of S.Typhi virulent culture. Out of five, no mice could survive in control group although rSpaO and rLamB in combination at 500 µg/ml conferred 80 % protection. Whereas the monovalent rSpaO and rLamB vaccine elucidate protection 60 % or 40 % respectively. ConclusionFrom this study, it is conclude that combine vaccine rSpaO and rLamB effectively enhanced the immunological response and withstand the challenge of the causative agent more effectively as compared to the monovalent vaccines.

RETRACTED: Dietary cysteamine addition remarkably improved the growth performance and increased carcass protein deposition via inhibiting proteolysis-related genes in feedlot lambs

Cysteamine (CS), a metabolic product of coenzyme A in mammals, has been increasingly employed in China as a potential growth promoter in cattle and lamb feedlotting. This study was conducted to investigate the potential effects of CS on apparent nutrient digestibility, rumen methane fermentation, N metabolism, and slaughter performance. The experiment involved 48 male Dorper-Hu hybrid lambs with an initial live body weight of 33.32 ± 0.94 kg. These lambs were assigned to four dietary CS addition treatment groups with diets consisting of foxtail millet silage, corn straw, corn grain, soybean meal rapeseed meal, and different concentrations of CS (0, 20, 40, and 60 mg/kg body weight). The results showed that increasing dietary CS concentrations led to a linear increase in dry matter intake and average daily weight gain, as well as a quadratic increase in the apparent digestibility of dry matter, organic matter and crude protein (P < 0.05). Furthermore, increasing dietary CS concentrations linearly increased free fatty acids, growth hormone and insulin-like growth factor-I, while linearly decreased somatostatin levels (P < 0.01). In addition, increasing dietary CS concentrations linearly increased propionate but decreased acetate and methane prediction by 7.9–15.2% (P < 0.05). Based on the N balance measurement, increasing dietary CS concentrations quadratically increased daily N intake by 12.3% and daily N retention by 8.1–34.8% via reducing N loss efficiency in urine by 13.5% and in feces by 14.8% (P < 0.01). Dietary CS addition linearly increased the meat-to-bone ratio by 8.0–23.1% and lean yield by 6.4–22.2%; however, it decreased back tissue thickness (GR) by 8.7–11.3% and the fat-to-net meat yield ratio by 2.2–13.0% (P < 0.05). Furthermore, increasing dietary CS concentrations linearly increased the mRNA relative expression of insulin-like growth factors - I and protein kinase B (P < 0.05), while linearly decreased the relative mRNA expression of forkhead box protein o4, muscle ring finger 1, and muscle atrophy F-Box (P < 0.05). Cysteamine increased weight gain (+22.1%) via increasing feed intake and inhibiting somatostatin levels, and 40 mg/kg BW was determined to be the optimal dose for feeding. Furthermore, CS increased carcass protein deposition and decreased GR by inhibiting proteolysis, which improved carcass performance.

Carcase traits, meat quality, and lipogenic gene expression in muscle of lambs fed wheat bran feruloyl oligosaccharides

This study investigated the effects of dietary feruloyl oligosaccharides derived from fermented wheat bran (FOs-FWB) supplementation on carcase traits, meat quality, muscle fatty acid composition, and the expression of lipogenic genes in lambs. In a completely randomised design, 50 Dorper × thin-tailed Han lambs (20.21 ± 3.36 kg) were randomly divided into five treatments with ten replications. The lambs in the control group were fed a basal diet, while those in the experimental groups were fed the basal diet supplemented with 50, 100, 200, and 400 mg/kg FOs-FWB. The shear forces of longissimus dorsi (LD) in lambs fed 50 and 100 mg/kg FOs-FWB were significantly lower than that of the control group (p < .05). Lambs fed with 200 mg/kg FOs-FWB had higher crude protein and lower fat contents in LD muscle (p < .05), the ash content in LD muscle was increased by FOs-FWB supplementation (p < .05). Lambs fed 50 mg/kg FOs-FWB had significantly higher proportions of C18:1n − 9, C20:1, C18:3n − 3, C18:3n − 6 and total n − 3 (p < .05). The proportion of C18:3n − 6 was significantly increased by 100 mg/kg FOs-FWB supplementation (p < .05). Feeding 200 mg/kg FOs-FWB significantly decreased proportions of C14:0 and total saturated fatty acid (SFA), but increased proportions of C18:1n − 9, C18:2n − 6, total monounsaturated fatty acid (MUFA) and unsaturated fatty acid (UFA) to SFA ratio (p < .05). Moreover, increases in the proportion of C22:6n − 3 and total polyunsaturated fatty acid (PUFA) were observed in lambs fed FOs-FWB (p < .05). The relative expression levels of ACC, FAS, and SCD genes in the LD muscle of lambs fed 200 and 400 mg/kg FOs-FWB were higher than the control group (p < .05). Dietary supplementation with 100, 200, 400 mg/kg FOs-FWB significantly increased the relative mRNA expression level of LPL (p < .05). Furthermore, a decrease in the relative mRNA expression level of PPARγ was observed in lambs fed 50 mg/kg FOs-FWB (p < .05). In conclusion, dietary supplementation with FOs-FWB reduced shear force, enhanced intramuscular fat content and PUFAs proportion and stimulated lipogenic gene expression in LD muscle of lambs, especially at a level of 200 mg/kg addition. Highlights Dietary FOs-FWB improved the tenderness of the meat. Dietary supplementation with 200 mg/kg FOs-FWB increased the content of intramuscular fat and PUFAs, as well as the ratio of UFA to SFA. Dietary FOs-FWB affected the expression of genes related to lipid metabolism.

Differential expression of outer membrane proteins and quinolone resistance determining region mutations can lead to ciprofloxacin resistance in Salmonella Typhi

Multi-drug resistance in Salmonella Typhi remains a public health concern globally. This study aimed to investigate the function of quinolone resistance determining region (QRDR) of gyrA and parC in ciprofloxacin (CIP) resistant isolates and examine the differential expression of outer membrane proteins (OMPs) on exposure to sub-lethal concentrations of CIP in S. Typhi. The CIP-resistant isolates were screened for mutations in the QRDR and analyzed for bacterial growth. Furthermore, major OMPs encoding genes such as ompF, lamB, yaeT, tolC, ompS1, and phoE were examined for differential expression under the sub-lethal concentrations of CIP by real-time PCR and SDS-PAGE. Notably, our study has shown a single-point mutation in gyrA at codon 83 (Ser83-tyrosine and Ser83-phenylalanine), also the rare amino acid substitution in parC gene at codon 80 (Glu80-glycine) in CIP-resistant isolates. Additionally, CIP-resistant isolates showed moderate growth compared to susceptible isolates. Although most of the OMP-encoding genes (tolC, ompS1, and phoE) showed some degree of upregulation, a significant level of upregulation (p < 0.05) was observed only for yaeT. However, ompF and lamB genes were down-regulated compared to CIP-susceptible isolates. Whereas OMPs profiling using SDS-PAGE did not show any changes in the banding pattern. These results provide valuable information on the QRDR mutation, and the difference in the growth, and expression of OMP-encoding genes in resistant and susceptible isolates of S. Typhi. This further provides insight into the involvement of QRDR mutation and OMPs associated with CIP resistance in S. Typhi.

Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi

We optimized the expression and purification of outer membrane proteins SpaO and LamB from Salmonella typhi. We investigated various factors in the expression and purification processes, including the use of isopropyl β-d-1 thiogalactopyranoside (IPTG), imidazole, and urea. First, PCR amplification was carried out on SpaO and LamB genes. The genes were then cloned in pTZ57R/T, and then expressed in pET28a vector and transformed into Escherichia coli BL21 (DE3). Gene insertion was confirmed by enzymatic digestion with NdeI and XhoI. Inclusion bodies expressing recombinant SpaO and LamB were induced with 200 and 400 µL 0.5 mM IPTG, respectively. The formed protein inclusion bodies were then isolated from the pellet and solubilized in IB buffer containing 8 M urea for SpaO and 6 M urea for LamB. Proteins were refolded by dialysis in 3M urea. Purified proteins with nickel-nitrilotriacetic acid affinity chromatography and eluted with buffer containing 250 mM imidazole for SpaO and 150 mM imidazole for LamB. The protein expression profiles were analyzed by SDS-PAGE, which identified the 33 and 49 kDa bands corresponding to rSpaO and rLamB. Western blotting Purification was carried out by nickel affinity resin with 250 mM and 150 mM imidazole for rSpaO and rLamB and refolded through stepwise dialysis with anti-His tag antibodies confirmed their expression. These optimized methods can be used to generate recombinant proteins for the development of future vaccines.

Influence of Cold Environments on Growth, Antioxidant Status, Immunity and Expression of Related Genes in Lambs.

Simple SummaryIn the scenario of global climatic change, extreme weather is a severe threat to organisms worldwide. For the purposes of dissecting the potential basis of cold environment stress responses and adaptation in lambs in cold climates, we had used cold environments with different temperature gradients to assess the effect of cold environments on growth, antioxidant status and immune function in lambs. The experimental results showed that the growth, antioxidant status and immune capacity of lambs were remarkably inhibited cold environments.Cold climates may be a risk to the health and welfare of lambs during winter because cold environments alter the physiological processes of lambs, and we used cold environments with three different temperature gradients—an indoor heating control group (IHC) using electric heating; an indoor temperature group (IT) with intermittent and slight degrees of stimulation of coldness; an outdoor temperature group (OT) exposed to cold environments in an external natural environment. The results showed that the lambs in the OT group had a greater decrease in the average daily gain (ADG) and increase in the average daily feed intake (ADFI) and the feed-to-gain ratio (F:G) compared to the other two groups. The decrease in immunoglobulin A (IgA) and interleukin 2 (IL-2) contents and IL-2 gene expression, and the increase in tumor necrosis factor α (TNF-α) content and TNF-α and nuclear factor kappa-B p65 (NF-κB p65) gene expressions in the OT group suggested that the lambs had a compromised immune status in cold environments. Moreover, the decrease in catalase (CAT), glutathione peroxidase (GPx), total superoxide dismutase (T-SOD), and total antioxidant capacity (T-AOC) levels, and CAT, GPx, SOD1, SOD2, and nuclear factor-erythroid 2-related factor 2 (Nrf2) gene expressions, and the increase in malondialdehyde (MDA) in the OT group suggested that the lambs had a lower antioxidant defense capacity in cold environments. Thus, in extreme cold, lambs kept outdoors could reduce growth, immune function and antioxidant status. However, shelter feeding in winter could relieve the stress of cold environments on lambs, and housing with heating equipment was more conducive to the improve growth, immune, and antioxidant function of the lambs.

Haemonchus contortus infection induces a variable immune response in resistant and susceptible Pelibuey sheep

The immune response and phenotypic characteristics of Pelibuey lambs were analysed after the induction of a Haemonchus contortus trickle infection. Male lambs (n = 29; 20 kg live weight) were infected with 100 H. contortus infective larvae per kg of live weight on day 3, 5 and 7 of the experiment. The number of eggs per gram (epg), seven haematological parameters and the immunoglobulin A (IgA) level were analysed for 56 experimental days. In addition, histopathological samples from the fundic abomasal region and the relative expression of 10 immune-related genes from 15 infected and three non-infected lambs were analysed at day 0 and 49 of the experiment. The epg count and some haematological parameters (leucocytes, red blood cells, haemoglobin and total protein) with statistically significant differences (P < 0.01) were used to identify nine resistant and 20 susceptible lambs (1166 ± 1071 and 3171 ± 1463 epg, respectively). Moreover, acute infiltration of immune cells and parasitic granuloma formation were observed in susceptible lambs; the resistant group had moderate inflammatory cell infiltration. With respect to relative gene expression, resistant lambs showed upregulation (P < 0.001) of 10 genes, from 2.2 to 15.99 fold. Moreover, there was a strong indirect correlation (P < 0.05) between the epg count and interleukin 5 (IL5) gene expression. By contrast, there was an average 0.34 fold downregulation in nine of the immune-related genes (P ≤ 0.05) in susceptible lambs (the only exception was Fc fragment of IgE receptor Ia [FCER1A] upregulation). In addition, there was a direct correlation (P ≤ 0.05) between the epg count and the expression of IL8, which encodes an inflammatory chemokine. In conclusion, this study showed differential IL5 and IL8 gene expression during haemonchosis in resistant and susceptible Pelibuey lambs, respectively, together with a variable immune response based on histopathological and haematological parameters.

Mutations in porin LamB contribute to ceftazidime-avibactam resistance in KPC-producing Klebsiella pneumoniae

Ceftazidime-avibactam (CAZ-AVI) shows promising activity against carbapenem-resistant Klebsiella pneumoniae (CRKP), however, CAZ-AVI resistance have emerged recently. Mutations in KPCs, porins OmpK35 and/or OmpK36, and PBPs are known to contribute to the resistance to CAZ-AVI in CRKP. To identify novel CAZ-AVI resistance mechanism, we generated 10 CAZ-AVI-resistant strains from 14 CAZ-AVI susceptible KPC-producing K. pneumoniae (KPC-Kp) strains through in vitro multipassage resistance selection using low concentrations of CAZ-AVI. Comparative genomic analysis for the original and derived mutants identified CAZ-AVI resistance-associated mutations in KPCs, PBP3 (encoded by ftsI), and LamB, an outer membrane maltoporin. CAZ-AVI susceptible KPC-Kp strains became resistant when complemented with mutated bla KPC genes. Complementation experiments also showed that a plasmid borne copy of wild-type lamB or ftsI gene reduced the MIC value of CAZ-AVI in the induced resistant strains. In addition, bla KPC expression level increased in four of the six CAZ-AVI-resistant strains without KPC mutations, indicating a probable association between increased bla KPC expression and increased resistance in these strains. In conclusion, we here identified a novel mechanism of CAZ-AVI resistance associated with mutations in porin LamB in KPC-Kp.

Effects of heat stress on antioxidant status and immune function and expression of related genes in lambs.

The study was conducted to evaluate the effects of heat stress on antioxidant status, immune response, and related gene expression of lambs. Eighteen male lambs were randomly allocated into three treatment groups that were as follows: indoor temperature control group furnished with an air-conditioner (ITC), indoor temperature non-control group suffered intermittent and varying degrees of heat stress (ITNC), outdoor temperature non-control group in the external natural environment (OTNC). ITNC group presented a more severe and prolonged exposure to thermal stress than the other two groups. The trial lasted 28 days. Blood samples were collected on days 14 and 28 to analyze total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GPx) activities, malondialdehyde (MDA) concentrations, total antioxidant capacity (T-AOC), interleukin-1 (IL-1), interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) concentrations and gene expressions of SOD1, SOD2, GPx, CAT, nuclear factor erythroid 2-related factor 2 (Nrf2), IL-1β, IL-2, and TNF-α. Results showed that on day 14 an elevated temperature reduced (p < 0.05) the level of GPx, T-SOD, T-AOC, and IgG, whereas significantly increased (p < 0.05) CAT, MDA, IgA, and TNF-α levels. Gene expressions of SOD1 and GPx were down-regulated (p < 0.05). On day 28, ITNC group significantly decreased (p < 0.05) CAT, GPx, T-SOD, T-AOC, IgG, and IL-2 levels and increased (p < 0.05) MDA, IL-1, and TNF-α levels, accompanying by the reducing or increasing (p < 0.05) of their relative gene expression apart from CAT and IL-1β gene (p > 0.10). In addition, in ITNC and OTNC groups, the level of CAT, T-SOD, T-AOC, IgG, IgM, and IL-2 and the expression of CAT, SOD2, and IL-2 reduced (p < 0.05), whereas IL-1 and TNF-α levels and IL-1β expression increased (p < 0.05) on day 28 compared with day 14. In ITC group, the level of IgA, IL-1, and TNF-α and the expression of IL-1β and TNF-α increased (p < 0.05), while the content of IgG and IgM reduced (p < 0.05) on day 28 compared with day 14. These results indicated that heat stress negatively affected the antioxidant status and immune response of lambs, and the negative effects of heat stress are not only related to the stress duration but also associated with the stress severity.

Expression of LamB Vaccine Antigen in Wolffia globosa (Duck Weed) Against Fish Vibriosis.

Vibriosis is a commonly found bacterial disease identified among fish and shellfish cultured in saline waters. A multitude of Vibrio species have been identified as the causative agents. LamB, a member of outer membrane protein (OMPs) family of these bacteria is conserved among all Vibrio species and has been identified as an efficient vaccine candidate against vibriosis. Rootless duckweed (Wolffia) is a tiny, edible aquatic plant possessing characteristics suitable for the utilization as a bioreactor. Thus, we attempted to express a protective edible vaccine antigen against fish vibriosis in nuclear-transformed Wolffia. We amplified LamB gene from virulent Vibrio alginolyticus and it was modified to maximize the protein expression level and translocate the protein to the endoplasmic reticulum (ER) in plants. It was cloned into binary vector pMYC under the control of CaMV 35S promoter and introduced into Wolffia globosa by Agrobacterium-mediated transformation. Integration and expression of the LamB gene was confirmed by genomic PCR and RT-PCR. Western blot analysis revealed accumulation of the LamB protein in 8 transgenic lines. The cross-protective property of transgenic Wolffia was evaluated by orally vaccinating zebrafish through feeding fresh transgenic Wolffia and subsequently challenging with virulent V. alginolyticus. High relative percent survival (RPS) of the vaccinated fish (63.3%) confirmed that fish immunized with transgenic Wolffia were well-protected from Vibrio infection. These findings suggest that Wolffia expressed LamB could serve as an edible plant-based candidate vaccine model for fish vibriosis and feasibility of utilizing Wolffia as bioreactor to produce edible vaccines.

Maternal supplementation with fish oil modulates inflammation-related MicroRNAs and genes in suckling lambs

Dietary n-3 long-chain fatty acids (n-3 LCFA) have been shown to modify lipid metabolism and immune function. The objective of this study was to evaluate the effect of periparturient fish oil (FO) supplementation on the inflammation and metabolic health of ewes and their lambs at a molecular level. Prepartum ewes were fed control diet (CON, n = 12) or CON supplemented with 2% DM of calcium soap of FO (n = 12) from 28 days before until 21 days after parturition. The ewes were evaluated for plasma metabolites and milk composition. The experiment was followed by analyzing the relative transcript abundance of circulating microRNAs (miRNAs) in plasma and targeted miRNA/mRNA expression in peripheral blood mononuclear cells (PBMCs) in both ewes and lambs. FO treatment decreased prepartum feed intake (1812 ± 35 vs 1674 ± 33 g/day, P < 0.01), whereas the influence on plasma metabolites was negligible. Dietary FO supplementation decreased milk fat percentage (8.82 ± 0.49 vs 7.03 ± 0.45, P = 0.02) and reduced milk n-6/n-3 (P < 0.05). Also, it altered the expression of plasma-circulating miRNAs in both ewe and lamb (P < 0.05). Furthermore, maternal nutrition of FO downregulated the relative expression of miR-33a and miR-146b and transcript abundance of genes IL-1β (0.41-fold) and NF-κB (0.25-fold) in lambs' PBMC. In conclusion, results showed that FO supplementation starting antepartum affects milk composition and circulating miRNA in dams and the inflammatory markers in lambs delivered by the supplemented ewes. These may provide a strategy to maintain immune balance during gestation and develop the immune system in lambs.

Effects of postbiotic supplementation on growth performance, ruminal fermentation and microbial profile, blood metabolite and GHR, IGF-1 and MCT-1 gene expression in post-weaning lambs

BackgroundPostbiotics have been established as potential feed additive to be used in monogastric such as poultry and swine to enhance health and growth performance. However, information on the postbiotics as feed additive in ruminants is very limited. The aim of this study was to elucidate the effects of supplementation of postbiotics in newly-weaned lambs on growth performance, digestibility, rumen fermentation characteristics and microbial population, blood metabolite and expression of genes related to growth and volatile fatty acid transport across the rumen epithelium.ResultsPostbiotic supplementation increased weight gain, feed intake, nutrient intake and nutrient digestibility of the lambs. No effect on ruminal pH and total VFA, whereas butyrate and ruminal ammonia-N concentration were improved. The lambs fed with postbiotics had higher blood total protein, urea nitrogen and glucose. However, no difference was observed in blood triglycerides and cholesterol levels. Postbiotics increased the population of fibre degrading bacteria but decreased total protozoa and methanogens in rumen. Postbiotics increased the mRNA expression of hepatic IGF-1 and ruminal MCT-1.ConclusionsThe inclusion of postbiotics from L. plantarum RG14 in newly-weaned lambs improved growth performance, nutrient intake and nutrient digestibility reflected from better rumen fermentation and microbial parameters, blood metabolites and upregulation of growth and nutrient intake genes in the post-weaning lambs.

Maternal folic acid supplementation modulates the growth performance, muscle development and immunity of Hu sheep offspring of different litter size

It is generally accepted that the phenotype and gene expression pattern of the offspring can be altered by maternal folic acid (FA) supplementation during the gestation period. The aims of this study were to investigate the effects of maternal FA supplementation on the growth performance, muscle development and immunity of newborn lambs of different litter size. According to litter size (twins, TW; triplets, TR) and maternal dietary FA supplementation levels (control, C; 16 or 32 mg·kg−1 FA supplementation, F16 and F32), neonatal lambs were randomly divided into six groups (TW-C, TW-F16, TW-F32, TR-C, TR-F16 and TR-F32). After farrowing, the birth weight in TW was higher than that in the TR group, and increased with FA supplementation of their mothers (P<.05). Folate, IGF-I, IgM and IgA concentrations of newborn lambs showed a litter size and FA supplementation interaction (P<.05). FA supplementation also increased diameter, area, perimeter and DNA content of the longissimus dorsi muscle of the lambs (P<.05) regardless of the litter size. Transcriptome analysis of the longissimus dorsi muscle revealed differentially expressed genes with dietary FA supplementation enriched in immunity- and cell development-related genes. Furthermore, FA supplementation upregulated the expression of myogenesis-related genes, while downregulated those involved in the inhibition of muscle development. In addition, immunity-related genes in the neonatal lambs showed lower expression levels in response to maternal dietary FA supplementation. Overall, maternal FA supplementation during gestation could increase the offspring's birth weight and modulate its muscle development and immunity.

Maltoporin (LamB protein) contributes to the virulence and adhesion of Aeromonas veronii TH0426.

Aeromonas veronii is one of the main pathogens causing freshwater fish sepsis and ulcer syndrome. More and more cases have shown that it has become an important zoonotic and aquatic agent. In this study, a A.veronii TH0426 mutant strain (ΔlamB) with an in-frame deletion removed nucleotides 10-1,296 of the lamB gene was firstly constructed to investigate its functions. The results showed that the LD50 value of the mutant ΔlamB to zebrafish and mice was 13.7-fold and 5.6-fold higher than those of the wild-type strain, respectively. The toxicity of wild-type strain to EPC cells was 2.1-fold and threefold higher than those of ∆lamB when infected for 1 and 2hr. Furthermore, the ability of biofilm formation and the adhesion and invasion to EPC cells of ∆lamB significantly decreased for 5.6-fold and 1.8-fold separately. In addition, motility detection result indicated that ∆lamB lost the swimming ability. The results of flagellar staining and TEM demonstrated that the flagella of ∆lamB were shed. In general, the deletion of lamB gene caused a significant decrease in the virulence and adhesion of A.veronii TH0426, and it can be known that the lamB gene of A.veronii plays a crucial role in the pathogenesis.

MP08-20 RAPID PHENOTYPING OF GENITOURINARY ANOMALIES IN MICE USING MICRO-CT

You have accessJournal of UrologyImaging/Radiology: Uroradiology I1 Apr 2017MP08-20 RAPID PHENOTYPING OF GENITOURINARY ANOMALIES IN MICE USING MICRO-CT Gene Huang, Marisol O'Neill, Meade Haller, Carolina Jorgez, and D.J. Lamb Gene HuangGene Huang More articles by this author , Marisol O'NeillMarisol O'Neill More articles by this author , Meade HallerMeade Haller More articles by this author , Carolina JorgezCarolina Jorgez More articles by this author , and D.J. LambD.J. Lamb More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.307AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Mouse models serve as an excellent tool for studying human disease. Yet, the small size of the mouse presents technical challenges in characterizing organ defects. The use of micro-computed tomography (micro-CT) in biomedical research has historically been limited to studying osseous structures. However, development of iodine staining techniques has allowed for improved ex vivo study of soft tissue structures. Images acquired through micro-CT allow for limitless virtual sectioning (as thin as 2 µm), a distinct advantage over traditional tissue sectioning. In this study, we demonstrate the successful use of iodine staining and micro-CT to rapidly phenotype genitourinary (GU) anomalies in mice. METHODS Mice GU specimens were excised, fixed in formalin, and dehydrated in 70% ethanol. Iodine staining was performed by soaking the tissue in 0.1 N iodine (Fluka). The specimen was suspended in agar in preparation for imaging. Images were acquired using the SkyScan 1272 High-Resolution X-Ray Microtomograph (Bruker microCT, Kontich, Belgium). A 0.5 µm aluminum filter was utilized. Imaging parameters were 5 µm and 11 µm pixel size for penis and kidney specimens, respectively, and 2016 x 1344 resolution. Images were reconstructed using NRecon (Bruker microCT) and visualized using 3D Slicer v4 (slicer.org). RESULTS Both embryonic and adult mice GU specimens were imaged. Micro-CT scan time varied with specimen size and desired resolution, but was at longest 120 minutes. Normal and pathologic GU phenotypes were characterized on three-dimensional, reconstructed images. Embryonic GU systems were examined, including a number of hydronephrotic kidneys (top left). Normal, hydronephrotic, duplex (top right), and polycystic kidneys (bottom left) were identified in adult specimens. Adult penile specimens (bottom right) were reconstructed to allow for morphometric measurements. CONCLUSIONS We demonstrate successful generation of three-dimensional, high-resolution, contrast-enhanced images of GU organs in the murine model using micro-CT. The use of micro-CT possesses a vast potential in rapid phenotyping and study of GU anomalies in the murine model. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e99 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Gene Huang More articles by this author Marisol O'Neill More articles by this author Meade Haller More articles by this author Carolina Jorgez More articles by this author D.J. Lamb More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Evaluation of Polymerase Chain Reaction for Detecting Coliform Bacteria in Drinking Water Sources.

Background:Coliform bacteria are used as indicator organisms for detecting fecal pollution in water. Traditional methods including microbial culture tests in lactose-containing media and enzyme-based tests for the detection of β-galactosidase; however, these methods are time-consuming and less specific. The aim of this study was to evaluate polymerase chain reaction (PCR) for detecting coliform.Materials and Methods:Totally, 100 of water samples from Isfahan drinking water source were collected. Coliform bacteria and Escherichia coli were detected in drinking water using LacZ and LamB genes in PCR method performed in comparison with biochemical tests for all samples.Results:Using phenotyping, 80 coliform isolates were found. The results of the biochemical tests illustrated 78.7% coliform bacteria and 21.2% E. coli. PCR results for LacZ and LamB genes were 67.5% and 17.5%, respectively.Conclusion:The PCR method was shown to be an effective, sensitive, and rapid method for detecting coliform and E. coli in drinking water from the Isfahan drinking water sources.

Brief neonatal nutritional supplementation has sex-specific effects on glucose tolerance and insulin regulating genes in juvenile lambs.

The nutritional plane and composition during fetal life can impact upon growth and epigenetic regulation of genes affecting pancreatic β-cell development and function. However, it is not clear whether β-cell development can be altered by nutritional factors or growth rate after birth. We therefore investigated the effect of neonatal nutritional supplements on growth, glucose tolerance, and pancreatic development in lambs. Newborn lambs were randomized to daily nutritional supplements, calculated to increase macronutrient intake to a similar degree as human breast milk fortifier, or an equivalent volume of water, for 2 wk while continuing to suckle ewe milk. Intravenous glucose tolerance test (IVGTT) was performed at 4 mo of age, and pancreata collected for molecular analysis. Supplemented lambs had slower weight gain than controls. In supplemented lambs, insulin response to IVGTT was increased in males but decreased in females, compared to same sex controls, and was unrelated to growth rate. mRNA expression of key genes in β-cell development showed sexually dimorphic effects. Epigenetic change occurred in the promotor region of PDX1 gene with decreased suppression and increased activation marks in supplemented lambs of both sexes. Nutritional interventions in early life have long-term, sex-specific effects on pancreatic function.

Effect of dietary energy and protein restriction followed by realimentation on pituitary mRNA expression of growth hormone and related genes in lambs

The objective of the current study was to evaluate the influence of dietary energy and protein restriction followed by realimentation on pituitary growth hormone (GH), growth hormone-releasing hormone receptor (GHRHR), GH secretagogue receptor (GHSR), somatostatin receptor 1 (SSTR1), somatostatin receptor 2 (SSTR2), insulin-like growth factor 1 receptor (IGF-1R), and IGF-2 mRNA expression in lambs. Forty 3-month-old native Mongolian wether lambs were selected, and randomly assigned to control group (CON), moderately energy-restricted group (MR1), moderately protein-restricted group (MR2), or severely energy- and protein-restricted group (SR). The feed restriction stage lasted for 60 days and then all groups were fed the same diet for 90 days (realimentation stage). Pituitaries were collected from four lambs in each group at the end of the two stages. Pituitary gene expression was determined by quantitative real-time reverse transcription PCR. At the end of the restriction stage, the body weight of lambs in all restricted groups was significantly lower than that in the CON group. Pituitary GH, GHRHR, GHSR and IGF-2 expression in lambs in the SR group was greater than that in the CON group (P<0.05), whereas the mRNA expression of SSTR1, SSTR2 and IGF-1R did not change (P≥0.05). At the end of the realimentation stage, the body weight of lambs in the MR1 and MR2 groups was not different from that of lambs in the CON group (P>0.05). However, the body weight of lambs in the SR group was still lower than that in the CON group (P<0.05). The pituitary GH expression of lambs in the SR group was still greater (P<0.05) than that of the CON group, whereas expression of the other genes had returned to control levels. No differences were found in pituitary GH and related gene expression in lambs in the MR1 and MR2 groups compared with those in the CON group throughout the entire experiment (P>0.05). Our results indicated that moderate dietary energy and protein restriction did not influence pituitary GH and related gene expression. Only severe energy and protein restriction caused a significant increase in pituitary GH transcription, which lasted throughout the realimentation stage.

The outer membrane protein, LamB (maltoporin), is a versatile vaccine candidate among the Vibrio species

Maltoporin (LamB) is a family of outer membrane proteins. There has been no report of immunological characteristics of LamB in the Vibrio species so far. In this study, lamB genes from eight Vibrio strains were cloned and sequenced. The bioinformatics analysis indicated that sequence similarities of LamB proteins were ranged from 46.7% to 81.1%. Further, the result showed that their antigenic epitopes were highly conserved implying that LamB might be a shared antigen among Vibrios. The Western blot of rabbit sera against recombinant LamB from V. alginolyticus ATCC 33787 with cell lysate of 18 Vibrio strains showed cross-recognition. Bands observed on cell lysate of Vibrio strains immunoblotted with the anti-LamB sera ranged between 40 and 49kDa. The Whole-cell ELISA assay further confirmed that the antisera of recombinant LamB recognized the tested Vibrio strains indicating the surface-exposed of LamB. Finally, the cross-protective property of recombinant LamB was evaluated through vaccination and subsequent challenge with heterogeneous virulent Vibrio strains in zebrafish. Recorded relative percent survival (RPS) of the vaccinated group varied from 54.1% to 77.8%, showing that zebrafish were protected from Vibrio infection after immunization with LamB protein. The cumulative evidences in this study suggested that LamB was a conserved antigen among tested Vibrio species and might be a potentially versatile vaccine candidate for the prevention of Vibriosis.