Characterization and evaluation of lactic acid bacteria candidates for intestinal epithelial permeability and Salmonella Typhimurium colonization in neonatal turkey poults.

The present study evaluated the microbiological properties of three probiotic candidate strains of lactic acid bacteria (LAB) (128; 131; CE11_2), their effect on intestinal epithelial permeability, and their ability to reduce intestinal colonization of Salmonella Typhimurium (ST) individually or as a batch culture in neonatal turkey poults. Isolates were characterized morphologically and identified using 16S rRNA sequence analyses. Each isolate was evaluated for tolerance and resistance to acidic pH, high osmotic NaCl concentrations, and bile salts in broth medium. In vitro assessment of antimicrobial activity against different enteropathogenic bacteria was determined using an overlay technique. In vitro intestinal permeability was evaluated using a stressed Caco-2 cell culture assay treated with/without the probiotic candidates. The in vivo effect of the selected LAB strains on ST cecal colonization was determined in two independent trials with neonatal turkey poults. The results obtained in this study demonstrate the tolerance of LAB candidates to pH 3, a NaCl concentration of 6.5%, and high bile salts (0.6%). All strains evaluated exhibited in vitro antibacterial activity against Salmonella Enteritidis, ST, and Campylobacter jejuni. Candidates 128 and 131 exhibited a coccus morphology and were identified as Enterococcus faecium, and bacterial strain CE11_2 exhibited clusters of cocci-shaped cells and was identified as Pediococcus parvulus. All three candidate probiotics significantly (P < 0.05) increased transepithelial electrical resistance (TEER) in Caco-2 cells following a 3-h incubation period with hydrogen peroxide compared to control and blank groups. The combination of all three candidates as a batch culture exhibited significant efficacy in controlling intestinal colonization of ST in neonatal turkey poults. Evaluation of the combination of these selected LAB strains according to performance and intestinal health parameters of chickens and turkeys are currently in process.

Enhanced expression of proteins involved in energy production and transfer in breast muscle of pedigree male broilers exhibiting high feed efficiency.

In cells with fluctuating energy demand (e.g., skeletal muscle), a transfer system of proteins across the inner and outer mitochondrial membranes links mitochondrial oxidative phosphorylation to cytosolic phosphorylated creatine (PCr) that serves as a phosphate reservoir for rapid repletion of cytosolic adenosine triphosphate (ATP). Crucial proteins of this energy transfer system include several creatine kinase (CK) isoforms found in the cytosol and mitochondria. In a recent proteomic study (Kong et al., 2016), several components of this system were up-regulated in high feed efficiency (FE) compared to low FE breast muscle; notably adenine nucleotide translocase (ANT), voltage dependent activated channel (VDAC), the brain isoform of creatine kinase (CK-B), and several proteins of the electron transport chain. Reexamination of the original proteomic dataset revealed that the expression of two mitochondrial CK isoforms (CKMT1A and CKMT2) had been detected but were not recognized by the bioinformatics program used by Kong et al. (2016a). The CKMT1A isoform was up-regulated (7.8-fold, P = 0.05) in the high FE phenotype but there was no difference in CKMT2 expression (1.1-fold, P = 0.59). From these findings, we hypothesize that enhanced expression of the energy production and transfer system in breast muscle of the high FE pedigree broiler male could be fundamentally important in the phenotypic expression of feed efficiency.

Cell bioenergetics in Leghorn male hepatoma cells and immortalized chicken liver cells in response to 4-hydroxy 2-nonenal-induced oxidative stress.

The major objectives of this study were to compare cell bioenergetics in 2 avian liver cell lines under control conditions and in response to oxidative stress imposed by 4-hydroxy 2-nonenal (4-HNE). Cells in this study were from a chemically immortalized Leghorn male hepatoma (LMH) cell line and a spontaneously immortalized chicken liver (CELi) cell line. Oxygen consumption rate (OCR) was monitored in specialized microtiter plates using an XF24 Flux Analyzer (Seahorse Bioscience, Billerica, MA). Cell bioenergetics was assessed by sequential additions of oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and antimycin-A that enables the determination of a) OCR linked to adenosine triphosphate (ATP) synthase activity, b) mitochondrial oxygen reserve capacity, c) proton leak, and d) nonmitochondrial cytochrome c oxidase activity. Under control (unchallenged) conditions, LMH cells exhibited higher basal OCR and higher OCR attributed to each of the bioenergetic components listed above compared with CELi cells. When expressed as a percentage of maximal OCR (following uncoupling with FCCP), LMH cells exhibited higher OCR due to ATP synthase and proton leak activity, but lower mitochondrial oxygen reserve capacity compared with CELi cells; there were no differences in OCR associated with nonmitochondrial cytochrome c oxidase activity. Whereas the LMH cells exhibited robust ATP synthase activity up to 50 µM 4-HNE, CELi cells exhibited a progressive decline in ATP synthase activity with 10, 20, and 30 µM 4-HNE. The CELi cells exhibited higher mitochondrial oxygen reserve capacity compared with LMH cells with 0 and 20 µM 4-HNE but not with 30 µM 4-HNE. Both cell lines exhibited inducible proton leak in response to increasing levels of 4-HNE that was evident with 30 µM 4-HNE for CELi cells and with 40 and 50 µM 4-HNE in LMH cells. The results of these studies demonstrate fundamental differences in cell bioenergetics in 2 avian liver-derived cell lines under control conditions and in response to oxidative challenge due to 4-HNE.

Cell Biology Symposium: feed efficiency: mitochondrial function to global gene expression.

Understanding the cellular basis of feed efficiency (FE) is instrumental to helping poultry and livestock industries continue to provide high-quality protein for an increasingly crowded world. To understand relationships of FE and gene expression, global RNA transcription was investigated in breast muscle obtained from a male broiler line fed the same diet and individually phenotyped for FE. In these studies, RNA samples obtained from broilers that exhibited either high FE (0.65 ± 0.01) or low FE (0.46 ± 0.01) were analyzed with an Agilent 44K chicken oligoarray. A 1.3-fold cutoff in expression (30% difference between groups) resulted in 782 genes that were differentially expressed (P < 0.05) in muscle between the high- and low-FE phenotypes. Ingenuity Pathway Analysis, an online software program, was used to identify genes, gene networks, and pathways associated with the phenotypic expression of FE. The results indicate that the high-FE phenotype exhibited increased expression of genes associated with 1) signal transduction pathways, 2) anabolic activities, and 3) energy-sensing and energy coordination activities, all of which would likely be favorable to cell growth and development. In contrast, the low-FE broiler phenotype exhibited upregulation of genes 1) associated with actin-myosin filaments, cytoskeletal architecture, and muscle fibers and 2) stress-related or stress-responsive genes. Because the low-FE broiler phenotype exhibits greater oxidative stress, it would appear that the low-FE phenotype is the product of inherent gene expression that is modulated by oxidative stress. The results of these studies begin to provide a comprehensive picture of gene expression in muscle, a major organ of energy demand in an animal, associated with phenotypic expression of FE.

Gene expression in breast muscle associated with feed efficiency in a single male broiler line using a chicken 44K microarray. II. Differentially expressed focus genes.

Global RNA expression in breast muscle obtained from a male broiler line phenotyped for high or low feed efficiency (FE) was investigated using microarray analysis. Microarray procedures and validation were reported previously. By using an overlay function of a software program (Ingenuity Pathway Analysis, IPA) in which canonical pathways are projected onto a set of genes, a subset of 27 differentially expressed focus genes were identified. Focus genes that were upregulated in the high FE phenotype were associated with important signal transduction pathways (Jnk, G-coupled, and retinoic acid) or in sensing cell energy status and stimulating energy production that would likely enhance growth and development of muscle tissue. In contrast, focus genes that were upregulated in the low FE muscle phenotype were associated with cytoskeletal architecture (e.g., actin-myosin filaments), fatty acid oxidation, growth factors, or ones that would likely be induced in response to oxidative stress. The results of this study provide additional information on gene expression and the cellular basis of feed efficiency in broilers.

Gene expression in breast muscle associated with feed efficiency in a single male broiler line using a chicken 44K oligo microarray. I. Top differentially expressed genes.

Global RNA expression in breast muscle obtained from a male broiler line phenotyped for high or low feed efficiency (FE) was investigated. Pooled RNA samples (n = 6/phenotype) labeled with cyanine 3 or cyanine 5 fluorescent dyes to generate cRNA probes were hybridized on a 4 × 44K chicken oligo microarray. Local polynomial regression normalization was applied to background-corrected red and green intensities with a moderated t-statistic. Corresponding P-values were computed and adjusted for multiple testing by false discovery rate to identify differentially expressed genes. Microarray validation was carried out by comparing findings with quantitative reverse-transcription PCR. A 1.3-fold difference in gene expression was set as a cutoff value, which encompassed 20% (782 of 4,011) of the total number of genes that were differentially expressed between FE phenotypes. Using an online software program (Ingenuity Pathway Analysis), the top 10 upregulated genes identified by Ingenuity Pathway Analysis in the high-FE group were generally associated with anabolic processes. In contrast, 7 of the top 10 downregulated genes in the high-FE phenotype (upregulated in the low-FE phenotype) were associated with muscle fiber development, muscle function, and cytoskeletal organization, with the remaining 3 genes associated with self-recognition or stress-responding genes. The results from this study focusing on only the top differentially expressed genes suggest that the high-FE broiler phenotype is derived from the upregulation of genes associated with anabolic processes as well as a downregulation of genes associated with muscle fiber development, muscle function, cytoskeletal organization, and stress response.

Amylolytic hydrolysis of native starch granules affected by granule surface area.

Initial stage of hydrolysis of native starch granules with various amylolytic enzymes, alpha-amylase from Bacillus subtilis, glucoamylase I (GA-I) and II (GA-II) from Aspergillus niger, and beta-amylase from sweet potato showed that the reaction was apparently affected by a specific surface area of the starch granules. The ratios of the reciprocal of initial velocity of each amylolytic hydrolysis for native potato and maize starch to that for rice with the amylolytic enzymes were nearly equivalent to the ratio of surface area per mass of the 2 starch granules to that of rice, that is, 6.94 and 2.25, respectively. Thus, the reciprocal of initial velocity of each enzymatic hydrolysis as expressed in a Lineweaver-Burk plot was a linear function of the reciprocal of surface area for each starch granule. As a result, it is concluded that amylolytic hydrolysis of native starch granules is governed by the specific surface area, not by the mass concentration, of each granule.

Application of the Sleeping Beauty transposon system to avian cells.

We have for the first time assessed the ability of the Sleeping Beauty (SB) transposon system to enhance transgenesis in chicken and turkey cells. The efficiency of transgenesis with a transposon encoding an antibiotic resistance gene was dramatically enhanced 15- to 35-fold when transposase was supplied by co-transfection of immortalized chicken and turkey cells with a construct encoding SB. In contrast, transgenesis of primary chicken embryo fibroblast (CEF) cells was not significantly increased by providing transposase, suggesting that the benefits of transposon-transgenesis in primary avian cells will require the application of more efficient transfection methods, further enhanced SB transposase or an alternative transposon system.

Chicken embryo extract mitigates growth and morphological changes in a spontaneously immortalized chicken embryo fibroblast cell line.

The SC-1 spontaneously immortalized chicken embryo fibroblast (CEF) cell line has been established recently. Although this cell line had been in culture for over 3 yr, its growth rate has remained lower than that of primary CEF cells, and the morphology has not been as uniform as observed in primary cells. In the present study, the SC-1 cell line was treated with chicken embryo extract (CEE) to determine whether growth rates could be increased and cell morphology enhanced. The CEE also was tested on primary CEF cells, another spontaneously immortalized CEF cell line (DF-1), and on 2 other nonvirally and nonchemically immortalized CEF cell lines (BCEFi and HCEFi). Results indicated that concentrations of CEE > or = 100 microg/mL inhibited growth of all cells tested. However, addition of 50 microg of CEE/mL enhanced the growth rate and improved the morphology of the SC-1 cells. Addition of CEE to the other immortal or primary CEF cells did not increase the growth rate or change their morphology. Analysis of mRNA expression revealed that SC-1 cells treated with 50 microg of CEE/mL had lower levels of the p16(INK4a) alternate reading frame sequence (ARF) and E2F-1 than untreated SC-1 cells. The increased growth rate and improved morphology of the SC-1 cells achieved with CEE treatment were retained following removal of CEE, and these improvements should aid in increasing the utility of the SC-1 cell line as a cellular/molecular reagent.

Necrotic cell death by hydrogen peroxide in immortal DF-1 chicken embryo fibroblast cells expressing deregulated MnSOD and catalase.

The reactive oxygen species are known as endogenous toxic oxidant damaging factors in a variety of cell types, and in response, the antioxidant genes have been implicated in cell proliferation, senescence, immortalization, and tumorigenesis. The expression of manganese superoxide dismutase mRNA was shown to increase in most of the immortal chicken embryo fibroblast (CEF) cells tested, while expression of catalase mRNA appeared to be dramatically decreased in all immortal CEF cells compared to their primary counterparts. The expression of copper-zinc superoxide dismutase mRNA was shown to increase slightly in some immortal CEF cells. The glutathione peroxidase expressed relatively similar levels in both primary and immortal CEF cells. As primary and immortal DF-1 CEF cells were treated with 10-100 microM of hydrogen peroxide (concentrations known to be sublethal in human diploid fibroblasts), immortal DF-1 CEF cells were shown to be more sensitive to hydrogen peroxide, and total cell numbers were dramatically reduced when compared with primary cell counterparts. This increased sensitivity to hydrogen peroxide in immortal DF-1 cells occurred without evident changes in either antioxidant gene expression, mitochondrial membrane potential, cell cycle distribution or chromatin condensation. However, the total number of dead cells without chromatin condensation was dramatically elevated in immortal DF-1 CEFs treated with hydrogen peroxide, indicating that the inhibition of immortal DF-1 cell growth by low concentrations of hydrogen peroxide is due to increased necrotic cell death, but not apoptosis. Taken together, our observation suggests that the balanced antioxidant function might be important for cell proliferation in response to toxic oxidative damage by hydrogen peroxide.

Psychometric properties of the VSQLQ in black patients with mild hypertension. Vital Signs Quality of Life Questionnaire.

The Vital Signs Quality of Life Questionnaire (VSQLQ) is a condition- and culture-specific measure designed to assess health-related quality of life (HRQL) in black patients with hypertension. This study examined the instrument's reliability and validity when administered via personal interview to patients with mild systemic hypertension. Data were gathered from 304 black patients during the screening visit of a multicenter trial evaluating the efficacy of candesartan cilexetil (ATACAND). In addition to internal consistency and reproducibility, validity was assessed by correlating the VSQLQ with the Short Form-36 (SF-36). Sensitivity to sociodemographic effects and responsiveness to change was also examined. Cronbach's alpha levels were high (0.90, 0.92, 0.92 for frequency, intensity and combined scores, respectively) and the instrument was stable in patients reporting no health change over 8 weeks (ICC = 0.79, 0.79, and 0.80). Correlations between the VSQLQ and the SF-36 were moderate to high (0.32 to - 0.69) and statistically significant (p < 0.001). VSQLQ scores varied by gender, education, and income (p < 0.05). Patients who reported improvement in their general health status also reported significant improvements on the VSQLQ (n = 90; p < 0.05). Results support the reliability and validity of VSQLQ administered via personal interview to black patients with mild systemic hypertension.

Effects of candesartan cilexetil on health-related quality of life in black patients with systemic hypertension in the ABC Trial.

Tolerability is an important consideration in evaluating a new antihypertensive agent. This can be assessed informally by conventional patient interviews or more formally with the use of validated health-related quality of life (HRQL) measures assessing the patient's perception of the agent's tolerability. HRQL was a secondary end point of a 12-week, multicenter, double-blind, randomized, placebo-controlled study of the efficacy and tolerability of candesartan cilexetil in black patients with systemic hypertension. HRQL was evaluated using the generic Medical Outcomes Study 36-Item Short Form (SF-36) and population- and condition-specific Vital Signs Quality of Life Questionnaire (VSQLQ). Data were gathered via face-to-face interviews at screening, baseline, and weeks 8 and 12. Of the 304 patients randomized, 268 were evaluable for the HRQL analysis. Clinical results, reported in the companion article, found that candesartan cilexetil initiated at 16 mg once daily and titrated to 32 mg once daily as needed, with the subsequent addition of hydrochlorothiazide 12.5 mg as needed, was effective for lowering diastolic and systolic blood pressure and was well tolerated based on office interviews. Analyses of patients' perceptions of tolerability found that HRQL was maintained during the 12-week study period, with no significant differences between treatment and placebo groups at the end of double-blind treatment. These results indicate that the HRQL of black patients with systemic hypertension is maintained during treatment with candesartan cilexetil.

Community-based hypertension control programs that work.

Hypertension is the number one public health problem in the United States, particularly among African Americans. Although the National High Blood Pressure Education Program, started in 1972, led the way to a substantial decrease in morbidity and mortality from this disease, the percentage of African American hypertensives whose conditions are detected, treated, and controlled continues to lag behind that of white hypertensives. Community-based programs at locations where people congregate-for example, churches, barbershops, beauty salons, firehouses, housing projects, and worksites-can play a valuable role in increasing the number of African American hypertensives who receive treatment. Physicians can be a potent force for the development of these programs by acting as consultants to define the scope and function of lay volunteers and by promoting these programs in a variety of other ways.

Assessment of the Vital Signs Quality of Life Questionnaire in three studies on hypertension.

The Vital Signs Quality of Life Questionnaire is a patient survey developed to assess the effects of treatment on 25 variables associated with a sense of well-being and perceptions of general health, mental functioning and social performance. Data collected from this questionnaire in three studies on hypertension were analysed and are presented here. In study 1, low-dose verapamil significantly reduced office and ambulatory diastolic (DBP) and systolic (SBP) blood pressure (P < 0.01) in patients with mild hypertension. In study 2, verapamil and nifedipine significantly and similarly reduced office DBP and SBP in patients with mild to moderate hypertension. In study 3, significantly greater reductions in ambulatory DBP and SBP were observed with verapamil compared with placebo (P < 0.01) in elderly patients with mild to moderate hypertension. Reported adverse experiences were not different from those known to be associated with the study drugs. There was significant improvement in the intensity of quality of life (QOL) variables (P = 0.03) and in the total (sum of frequency and intensity) QOL score (P = 0.02) following treatment in study 1. In study 2, there were no significant changes in QOL scores following treatment with either verapamil or nifedipine. In study 3, QOL scores remained statistically unchanged for both treatment groups; however, there was a trend toward improvement with verapamil and worsening with pacebo. Taken together, the data show no negative effects of verapamil on QOL, even though verapamil demonstrated significant anti-hypertensive effectiveness. The questionnaire displayed a high level reliability, as measured by Cronbach's alpha (0.91 for both frequency and intensity).

A report of 131 cases of endoscopic laser lacrimal surgery.

BACKGROUND: Despite the many advantages of endoscopic laser dacryocystorhinostomy, clinical data have been sparse, and the effectiveness of this procedure has not been fully evaluated. METHODS: Among 127 patients (131 procedures) who underwent endoscopic laser lacrimal surgery, 92 procedures with longer than 3 months of follow-up were evaluated. The authors used endoscopic sinus surgery equipment, radiosurgical instruments, otodrill, microrongeur, holmium:YAG and contact neodymium:YAG lasers. RESULTS: After the primary operation, 71 (77.2%) of 92 cases were symptom-free and had positive results on primary Jones dye test. With simple revision of failed cases, 88 (95.7%) of 92 cases were treated successfully. CONCLUSION: Improvement of surgical technique and use of adequate surgical instruments were essential in increasing the rate of success. Endoscopic laser surgery is useful in treating patients with lacrimal passage obstruction, but a longer period of observation is necessary to confirm its effectiveness.

Comparison of the efficacy and metabolic effects of nicardipine and hydrochlorothiazide in hypertensive black men and women.

A double-blind, randomised, parallel study compared the BP and metabolic responses in black hypertensive patients following monotherapy with nicardipine or hydrochlorothiazide (HCTZ). Following a single-blind placebo wash-out period of 1-4 weeks, the study drug, nicardipine 20-40 mg three times daily or HCTZ 25-100 mg four times daily, was administered in a double-blind fashion for 12 weeks. Doses were titrated to control sitting DBP (< or = 90 mmHg). Both drugs were effective in reducing sitting SBP and DBP as compared with baseline values (nicardipine: 152.5 +/- 1.6/102.0 +/- 0.6, HCTZ: 152.5 +/- 1.5/101.4 +/- 0.5 mmHg). DBP responses (reduction from baseline; nicardipine: -10.9, HCTZ: -12.7 mmHg), and the percentage of patients achieving a response < or = 90 mmHg (nicardipine: 54%, HCTZ: 63%) to the two drugs were similar. Although SBPs at baseline and endpoint (137.3 +/- 1.6 on nicardipine and 132.1 +/- 1.4 mmHg on HCTZ), and the percentage of patients achieving a response < or = 140 mmHg (nicardipine: 70%, HCTZ: 79%), were comparable between the two treatments, the SBP reduction with HCTZ was statistically greater (P = 0.026). A comparison of the metabolic responses in the two treatment groups showed significant differences. Nicardipine caused no significant changes in blood chemistry, whereas HCTZ caused statistically significant decreases (P < 0.001) in sodium and potassium and increases (P < or = 0.01) in glucose, BUN, creatinine, uric acid, cholesterol and LDL compared with baseline. In 12.7% of the patients in the HCTZ group, serum potassium dropped to levels < 3.5 meq/l, which occurred in only 1.4% of the patients who used nicardipine.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the efficacy and tolerability of lisinopril and sustained-release verapamil in black patients with hypertension.

In this clinical study the efficacy and tolerability of lisinopril and sustained-release (SR) verapamil hydrochloride were compared in black patients with mild-to-moderate uncomplicated essential hypertension. The goal of therapy was to achieve a supine diastolic blood pressure (SDBP) of less than 90 mmHg or a greater than or equal to 10-mmHg fall in SDBP. Forty-three patients completed the titration phase, 23 in the lisinopril group and 20 in the verapamil SR group. The mean baseline supine systolic/diastolic blood pressures were 147/98 mmHg for the lisinopril group and 155/100 mmHg for the verapamil SR group. At the end of a two- to eight-week titration period, 87% of the lisinopril-treated patients and 90% of the verapamil SR-treated patients had achieved SDBP control. Six patients were excluded from maintenance therapy: four in the lisinopril group (one because of urticaria and three because of failure to reach goal blood pressure) and two in the verapamil SR group (because of failure to reach goal blood pressure). After eight weeks of maintenance therapy, 68% of the lisinopril-treated patients and 72% of the verapamil SR-treated patients had achieved SDBP control. The mean decreases in SDBP were comparable for both treatment groups. At the end of titration, the mean decreases from baseline were 10.5 mmHg for the lisinopril group and 12.6 mmHg for the verapamil SR group. At the end of maintenance, the mean decreases from baseline were 7.8 mmHg for the lisinopril group and 9.2 mmHg for the verapamil SR group. Adverse experiences were few.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of the efficacy and tolerability of enalapril and sustained-release diltiazem in mild to moderate essential hypertension.

The subjects of this multicenter study were 159 patients aged 21 to 76 years with mild to moderate uncomplicated essential hypertension. The patients were randomly assigned to receive up to 40 mg of enalapril daily or 360 mg of sustained-release diltiazem daily for a titration period of eight weeks and then maintenance doses for four weeks. The treatment goal was a supine diastolic blood pressure of less than 90 mmHg or a fall of at least 10 mmHg from baseline. During titration, 62% of the enalapril-treated patients and 51% of the diltiazem-treated patients reached the treatment goal after two weeks, 82% and 74% after four weeks, 87% and 84% after six weeks, and 92% and 87% after eight weeks. During the maintenance period, 85% of the enalapril-treated and 87% of the diltiazem-treated patients maintained the goal blood pressure. Treatment side effects were reported by 21% of the enalapril-treated patients and 29% of the diltiazem-treated patients; treatment was discontinued in two patients from each group because of side effects. It is concluded that both drugs were generally well tolerated and effective in the treatment of adult mild to moderate essential hypertension.