DNA extraction method influences human milk bacterial profiles.

AIMS: To evaluate four DNA extraction methods to elucidate the most effective method for bacterial DNA recovery from human milk (HM). METHODS AND RESULTS: Human milk DNA was extracted using the following methods: (i) Qiagen MagAttract Microbial DNA Isolation Kit (kit QM), (ii) Norgen Milk Bacterial DNA Isolation Kit (kit NM), (iii) Qiagen MagAttract Microbiome DNA/RNA Isolation Kit (kit MM) and (iv) TRIzol LS Reagent (method LS). The full-length 16S rRNA gene was sequenced. Kits MM and method LS were unable to extract detectable levels of DNA in 9/11 samples. Detectable levels of DNA were recovered from all samples using kits NM (mean = 0·68 ng µl-1 ) and QM (mean = 0·55 ng µl-1 ). For kits NM and QM, the greatest number of reads were associated with Staphylococcus epidermidis, Streptococcus vestibularis, Propionibacterium acnes, Veillonella dispar and Rothia mucilaginosa. Contamination profiles varied substantially between kits, with one bacterial species detected in negative extraction controls generated with kit QM and six with kit NM. CONCLUSIONS: Kit QM is the most suitable of the kits tested for the extraction of bacterial DNA from human milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Choice of extraction method impacts the efficiency of bacterial DNA extraction from human milk and the resultant bacterial community profiles generated from these samples.

Impact of expression mode and timing of sample collection, relative to milk ejection, on human milk bacterial DNA profiles.

AIM: To investigate the impact of expression mode: electric breast pump or hand expression, and timing of sample collection: pre- and post-milk ejection on human milk (HM) bacterial DNA profiles. METHODS AND RESULTS: Three HM samples from the same breast were collected from 30 breastfeeding mothers: a pre-milk ejection pump-expressed sample (pre-pump), a post-milk ejection pump-expressed sample (post-pump) and a post-milk ejection hand-expressed sample (post-hand). Full-length 16S rRNA gene sequencing was used to assess milk bacterial DNA profiles. Bacterial profiles did not differ significantly based on mode of expression nor timing of sample collection. No significant differences were detected in the relative abundance of any OTUs based on expression condition (pre-pump/ post-pump and post-pump/post-hand) with univariate linear mixed-effects regression analyses (all P-values > 0·01; α = 0·01). Similarly, no difference in richness was observed between sample types (number of observed OTUs: post-pump/post-hand P = 0·13; pre-pump/post-pump P = 0. 45). CONCLUSION: Bacterial DNA profiles of HM did not differ according to either expression method or timing of sample collection. SIGNIFICANCE AND IMPACT OF THE STUDY: Hand or pump expression can be utilized to collect samples for microbiome studies. This has implications for the design of future HM microbiome studies.

Metabolomics of human intestinal transplant rejection.

Surveillance endoscopy with biopsy is the standard method to monitor intestinal transplant recipients but it is invasive, costly and prone to sampling error. Early noninvasive biomarkers of intestinal rejection are needed. In this pilot study we applied metabolomics to characterize the metabolomic profile of intestinal allograft rejection. Fifty-six samples of ileostomy fluid or stool from 11 rejection and 45 nonrejection episodes were analyzed by ultraperformance liquid chromatography in conjunction with Quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS). The data were acquired in duplicate for each sample in positive ionization mode and preprocessed using XCMS (Scripps) followed by multivariate data analysis. We detected a total of 2541 metabolites in the positive ionization mode (mass 50-850 Daltons). A significant interclass separation was found between rejection and nonrejection. The proinflammatory mediator leukotriene E4 was the metabolite with the highest fold change in the rejection group compared to nonrejection. Water-soluble vitamins B2, B5, B6, and taurocholate were also detected with high fold change in rejection. The metabolomic profile of rejection was more heterogeneous than nonrejection. Although larger studies are needed, metabolomics appears to be a promising tool to characterize the pathophysiologic mechanisms involved in intestinal allograft rejection and potentially to identify noninvasive biomarkers.

Venous thromboembolism prevention through the use of novel Factor Xa inhibitors.

The risk of a venous thromboembolic event is not limited to the pre-hospital, hospital, or immediate post-hospital period. Because of challenges with data collection, the risk of venous thromboembolism (VTE) up to 3 months post-hospitalization for patients with acute and serious medical problems in the setting of chronic disease and/or risk factors for VTE is probably under reported. The growing acceptance and indications of direct oral anticoagulants (DOACs) now includes an indication for VTE prevention for one of the Factor Xa agents. In this paper, the authors explore the issue of VTE in the extended post-hospital period and strategies to provide protection from these morbid and potentially mortal events with oral anticoagulants.

Differences in adipocyte long chain fatty acid uptake in Osborne-Mendel and S5B/Pl rats in response to high-fat diets.

OBJECTIVE: To determine whether strain differences in adipocyte uptake of long chain fatty acids (LCFAs) contribute to differences in weight gain by Osborne-Mendel (OM) and S5B/Pl rats (S) fed a high-fat diet (HFD). SUBJECTS: Ninety-four adult (12-14-week old) male OM and S rats. MEASUREMENTS: Body weight; epididymal fat pad weight; adipocyte size, number, LCFA uptake kinetics; and plasma insulin and leptin during administration of HFD or chow diets (CDs). RESULTS: In both strains, rate of weight gain (RWG) was greater on an HFD than a CD; RWG on an HFD was greater, overall, in OM than S. A significant RWG increase occurred on days 1 and 2 in both strains. It was normalized in S by days 6-9 but persisted at least till day 14 in OM. RWGs were significantly correlated (P<0.001) with the V(max) for saturable adipocyte LCFA uptake (V(max)). In S, an increase in V(max) on day 1 returned to baseline by day 7 and was correlated with both plasma insulin and leptin levels throughout. In OM, a greater increase in V(max) was evident by day 2, and persisted for at least 14 days, during which both insulin and leptin levels remained elevated. Growth in epididymal fat pads on the HFD correlated with body weight, reflecting hypertrophy in OM and both hypertrophy and hyperplasia in S. CONCLUSIONS: (a) Changes in V(max) contribute significantly to changes in RWG on HFDs. (b) There are important strain differences in circulating insulin and leptin responses to an HFD. (c) Both insulin and leptin responses to an HFD are closely correlated with V(max) of adipocyte fatty acid uptake in S animals, but suggest early onset of insulin resistance in OM. Thus, differences in hormonal regulation of adipocyte LCFA uptake may underlie the different responses of OM and S to HFD.

Effects of autonomic stimulation and blockade on signal-averaged P wave duration.

OBJECTIVES: This study sought to evaluate the effects of autonomic stimulation and blockade on the signal-averaged P wave duration. BACKGROUND: Signal-averaged P wave duration has been shown to have prognostic implications for patients prone to develop atrial fibrillation, but autonomic influences on the signal-averaged P wave duration have not been studied. METHODS: In 14 healthy volunteers (8 men, 6 women; mean [ +/- SD] age 28.5 +/- 4.8 years, range 22 to 38), signal-averaged P wave duration was measured on day 1 at baseline, during sympathetic stimulation with infusions of epinephrine (50 ng/kg body weight per min) and isoproterenol (50 ng/kg per min), beta-blockade with propranolol (0.2 mg/kg) and autonomic blockade with propranolol followed by atropine (0.04 mg/kg). On a second day, 10 of the 14 subjects returned for repeat baseline recordings and recordings during parasympathetic blockade with atropine (0.04 mg/kg). Signal averaging was performed using a P wave template. Both unfiltered and filtered (least-squares fit filter with 100-ms window) P wave durations were measured. Day to day and interobserver variability were assessed by calculation of intraclass correlation coefficients. RESULTS: The mean ( +/- SD) baseline filtered P wave duration on day 1 was 141 +/- 10 ms. Isoproterenol infusion significantly shortened the P wave duration to 110 +/- 16 ms (p < 0.001), and epinephrine resulted in significant prolongation to 150 +/- 10 ms (p < 0.05). Beta-adrenergic blockade increased the P wave duration to 153 +/- 10 ms (p < 0.005). Autonomic blockade shortened the P wave duration to 143 +/- 16 ms (p < 0.05 vs. beta-blockade). On the second day, the mean baseline P wave duration was slightly longer (144 +/- 10 ms, p < 0.02). Parasympathetic blockade with atropine resulted in mild shortening of the P wave duration to 136 +/- 15 ms (p < 0.1). Interobserver reproducibility was excellent (intraclass correlation coefficient 0.99). Day to day reproducibility was good (intraclass correlation coefficient 0.56). CONCLUSIONS: The signal-averaged P wave duration is not a fixed variable because it may change significantly under different autonomic conditions. This has important implications for the application of this test to the heterogeneous population susceptible to atrial fibrillation.

Arterial elastase activity after balloon angioplasty and effects of elafin, an elastase inhibitor.

Increased proteolytic activity may be a factor in intimal hyperplasia after balloon angioplasty (BA). The objectives of this study were to assess elastase activity after BA in a rabbit arterial double-injury model and the effects of elastase inhibition. Elastase activity increased immediately after BA, reached an 8-fold peak at 1 week, and declined to baseline levels by 4 weeks. Elastin zymography showed that the elastase activity was associated predominantly with a molecular mass of 25 kDa. Elastase activity was significantly inhibited in vitro by elafin and phenylmethylsulfonyl fluoride, selective inhibitors of serine elastases. A second group of animals was transfected after BA with a plasmid containing the cDNA for either elafin or a control (chloramphenicol acetyltransferase, CAT) construct by using a hemagglutinating virus of Japan-liposome transfection technique. Arterial segments were obtained at 48 hours, 1 week, and 4 weeks to assess transgene expression, arterial wall elastase activity, and intimal cross-sectional area, respectively. Elafin transgene expression was evident at 48 hours and resulted in a significant (80%) inhibition of elastase activity compared with chloramphenicol acetyltransferase-transfected arteries. There was a 43% reduction in intimal cross-sectional area in elafin-transfected arteries (0.28+/-0.22 versus 0.16+/-0.07 mm(2) for CAT-transfected versus elafin-transfected arteries, respectively; P<0.05). These data suggest that an early increase in serine elastase activity after BA contributes to intimal hyperplasia. Serine elastase inhibition may be a potential therapeutic approach to inhibit intimal hyperplasia.

Unmasking of primary hyperaldosteronism by renal transplantation.

BACKGROUND: Primary hyperaldosteronism is an uncommon cause of hypertension in the general population. Given the mechanism of action of aldosterone clinical manifestations may not occur in the setting of end stage renal disease. However, if a successful renal transplant is performed clinical manifestations may occur. METHODS: We present a case of a patient with a preexisting adrenal adenoma who only presented with clinical signs of hyperaldosteronism after renal transplantation. Patients' work-up included plasma aldosterone, plasma renin activity, serum cortisol, and estimation of trans tubular potassium gradient. RESULTS: The patient's serum aldosterone was markedly elevated with a relatively suppressed plasma renin activity. Trans tubular potassium gradient was high in the presence of hypokalemia. CONCLUSION: Previously silent hyperaldosteronism may be unmasked by a successful renal transplant.

A controlled study of longitudinal IQ changes in females and males with fragile X syndrome.

The aim is this study is to compare the longitudinal changes in IQ scores of females and males with fragile X syndrome and controls and to assess the impact on IQ of molecular variations of the FMR-1 gene in males. Medical records from the child development unit at a university-affiliated children's hospital were retrospectively reviewed. Chart review yielded 35 males with fragile X (19 with a fully methylated full mutation, 9 with a mosaic pattern, and 7 with a partially unmethylated full mutation) 16 females with fragile X and a full mutation, 9 female controls, and 9 male controls who had repeated standardized IQ testing separated by 7 months to 13 years. The differences between the first and last IQ scores from the same IQ test were compared by t tests and subsequently by analysis of variance. Overall, a significant IQ decline was seen in 10/35 (28%) of fragile X males, 0/9 (0%) of control males, 6/16 (36%) of fragile X females, and 1/9 (11%) of control females. The initial t tests and analysis of variance showed a significant difference in IQ (p = 0.02) between fragile X males and control males but did not show a significant difference between males and females with fragile X syndrome or between fragile X and control females. When an analysis of covariance was carried out with the initial IQ as a covariable, a significant difference persisted between fragile X and control males, with a greater IQ decline in fragile X males. There were limitations in using the same IQ test. A comparison among the molecular subgroups of males yielded a significant IQ decline in 3/9 (33%) of mosaic males, 6/19 (32%) of fully methylated full mutation males, and 1/7 (14%) of partially methylated full mutation males. An analysis of covariance using the initial IQ and the intertest interval as covariables demonstrated significant differences between the fragile X molecular subgroups and the controls. Our findings show that a substantial percentage of both male and female fragile X patients and female control patients demonstrated significant IQ decline. There was a significant difference in the IQ change between fragile X and control males. There were no significant differences between fragile X and female controls. There were also significant differences in IQ decline among males with different molecular patterns compared with controls. Males with a mosaic pattern versus control males had the most significant decline of the molecular subtypes. Although the numbers were limited, there was no significant IQ decline in males with less than 50% methylation of the full mutation. This suggests that a small amount of FMR-1 protein production, which is often seen in males with less than 50% methylation, protects against significant IQ decline.

Reserpine-induced gastric ulcers: protection by lysosomal stabilization due to zinc.

The effects of graded doses of zinc sulfate pretreatment on reserpine-induced gastric ulceration and on lysosomal fragility both in vivo and in vitro, were studied in rats. Reserpine treatment (5 mg/kg, i.p., 18 h before sacrifice) induced marked gastric glandular ulceration and elicited the release of free beta-glucuronidase from lysosomes in the gastric mucosa. A similar effect on release of this enzyme from isolated rat hepatic lysosomes was observed after in vitro incubation with reserpine. Zinc sulfate (22, 44 or 88 mg/kg, i.p., 30 h before reserpinization, or 10(-3) M in vitro) inhibited the reserpine-induced response, and zinc sulfate alone (10(-11)--10(-3) M) also stabilized lysosomal membrane permeability to beta-glucuronidase. No direct effect of zinc or reserpine on purified beta-glucuronidase activity was observed. In conclusion, it is postulated that the stabilizing effect of zinc on lysosomal membranes, as manifest by reduced release of beta-glucuronidase from isolated lysosomes, is one of the protective mechanisms of zinc against reserpine-induced ulceration.

Leishmania infecting man and wild animals in Saudi Arabia. 4. Canine cutaneous leishmaniasis in the Eastern Province.

Cutaneous leishmaniasis was detected in 4 of 117 feral dogs, none of which presented any evidence of visceral involvement, in the Al-Hassa area of Saudi Arabia where neither Leishmania infantum nor L. tropica are transmitted. The parasites isolated from 3 of the animals were identified by biochemical techniques as L. major, L. arabica and Leishmania zymodeme 62 respectively. Clinically, and in their histology, the lesions closely resembled those caused in man in the same geographical area by L. major. Dogs are probably victims rather than reservoirs of Leishmania.

Autonomous thyroid nodules visualized by positron emission tomography with 18F-fluorodeoxyglucose: a case report and review of the literature.

A 41-year-old white female underwent a positron emission tomography with (18)F-fluorodeoxyglucose (FDG-PET) scan as part of staging follow-up for breast cancer. Focal FDG uptake was noted in the right neck. Further evaluation revealed only thyroid nodules and subclinical hyperthyroidism. A sodium pertechnetate thyroid scan showed two "hot" nodules in the right thyroid lobe corresponding to the focal uptake visualized on FDG-PET scan. Technetium-99m uptake was suppressed in the remaining thyroid gland. The patient underwent a subtotal thyroidectomy. Histopathologic evaluation revealed two hyperplastic nodules in each thyroid lobe. We review the literature with regard to FDG-PET imaging of normal and diseased thyroid tissue.

Studies of zinc and histamine on lysosomal fragility: possible role in stress ulceration.

A combined in vivo and in vitro study was undertaken with rats to test the hypothesis that zinc would protect against cold water immersion--restraint gastric ulcers, and that this phenomenon was mediated in part by stabilization of lysosomal membranes. This postulate was confirmed by observed activity changes in released beta-glucuronidase in mucosal tissue, as well as by dose-response in vitro data on isolated hepatic lysosomes exposed to zinc. Histamine, a known ulcer-enhancing agent, induced the opposite effect and increased the lysosomal release of this marker acid hydrolase.

Influence of diagnostic and therapeutic doses on thyroid remnant ablation rates.

Radioiodine ablation of thyroid tissue after subtotal thyroidectomy has been shown to decrease recurrence in certain subsets of patients with well-differentiated thyroid cancer. In a substantial percentage of cases (20-30%), initial ablation of the thyroid remnant fails, necessitating a second treatment. The factors associated with ablation failure are not fully understood. In particular, it is not certain whether the use of doses higher than 3.70 GBq would result in any additional benefit, or whether there is a 'stunning' effect of the diagnostic dose of 131I on the subsequent ablation rate. A retrospective analysis was performed of all patients (n=389) with well-differentiated thyroid cancer treated at our institution between 1992 and 2001. Remnant ablation success was determined by a whole-body radioiodine scan. The following factors, thought to be associated with thyroid remnant ablation, were studied by logistic regression analysis: age, gender, tumour histology, stage, pre-therapy neck uptake of 131I, diagnostic dose, ablation dose, time between diagnostic and therapeutic dose (T1), time between therapeutic administration and the first follow-up whole-body scan (T2) and the thyroid-stimulating hormone (TSH) level measured at the time of therapy. Follow-up whole-body scans were available in 214 patients. We found no association with age, gender, histology, TSH level, neck uptake, diagnostic dose and successful ablation. The therapeutic dose was the only variable found to be associated with success (odds ratio, 1.96 per 1.85 GBq increment; 95% confidence interval, 1.11-3.46). Our results confirm the presence of a significant percentage of ablation failures (24.4%) despite the use of high ablative doses (3.70-7.40 GBq). Higher therapeutic doses are associated with higher rates of successful ablation, even when administered to patients with more advanced stages. Using our protocol, higher diagnostic doses were not associated with higher rates of ablation failure.

Immunosuppressive potential and pathogenicity of an avian adenovirus isolate involved in hydropericardium syndrome in broilers.

The role of avian adenovirus isolate PARC-1 as an immunosuppressive agent was investigated using a Newcastle disease virus (NDV) vaccine immune response procedure. The immunosuppressive effect on the humoral immune response was investigated up to 21 days after inoculation with adenovirus. Infected chickens showed a serologic response to NDV that was reduced compared with that of the controls. To further investigate the effect of the virus on major lymphoid organs, the pattern of virus dissemination in various organs was studied at various time intervals after inoculation. Spleen, thymus, bursa of Fabricius, and cecal tonsils of broilers were examined using a dot-immunobinding assay. The virus was found to have a predilection for lymphoid organs, and virus from lymphoid organs was capable of producing disease when inoculated into healthy chickens. The relationship of virus predilection to its immunosuppressive effect also was studied.

Phenotypic involvement in females with the FMR1 gene mutation.

Fragile X syndrome is the most common from of inherited mental retardation. Approximately half of females with the full mutation have significant cognitive deficits, whereas females with the premutation do not. Phenotypic effects seen in 281 females (IQs from 64 to 139) were analyzed. Results showed that females with the full mutation differ significantly from controls on selected anthropometric measurements, physical index score, and various behavioral features. Females with the premutation differed significantly from controls in regards to a few anthropometric measurements and the physical index score but not in behavioral features. These results suggest that phenotypic effects of the FMR1 mutation are not only common in females with the full mutation, but in females with the premutation as well.

Patterns of atrial fibrillation at a regional hospital in Saudi Arabia.

The objective of this retrospective study was to report on the clinical presentation, etiology, and laboratory tests of both chronic and acute atrial fibrillation (AF) admitted to the cardiology unit of a teaching hospital in southern Saudi Arabia. We studied 219 records; 132 (60.3%) and 87 (39.7%) had documented chronic AF (group 1), and acute AF (group 2) respectively. The mean age (SD) was significantly higher in group 1 (64.6 [SD 19.4] vs 52.9 [SD 15.6]) (P<0.001). Palpitation, dizziness and syncope were the most frequent symptoms in acute AF, while dyspnea was the most common presentation in the chronic type. On the other hand, heart failure and embolic complications were reported significantly in group 1, but the frequency of acute respiratory problems and acute myocardial infarction was similar in both groups. The most common causes of both types of AF were rheumatic valvular diseases (26%), IHD (24.2%), hypertension (23.7%), and lung diseases (13.2%); however, in 28 patients (12.8%) no cause was detected. The echocardiography findings of chamber dilatation, valve lesions, and depressed left ventricular function were significantly frequent in group 1 (P<0.01). Although rheumatic valvular diseases are still common in Saudi Arabia, ischemic heart disease and hypertension are emerging as important causes of AF in this developing nation, and therefore require prevention and control.

Experimental cholecystitis in goats caused by mature Fasciola gigantica.

Mature Fasciola gigantica obtained from naturally infected cattle were surgically transferred into the gallbladders of six fluke-free goats. Two of the goats died at two and 19 weeks and the remaining four were slaughtered at 12, 48, 48 and 80 weeks after the transfer. Nine of 20 transferred F gigantica were recovered from the gallbladder and common hepatic duct of the experimental goats. The walls of gallbladders and bile ducts were thickened by epithelial proliferation, fibrosis and mononuclear cell infiltration. Large numbers of globule leucocytes were present in the epithelium of gallbladders and common hepatic ducts as well as in minute intrahepatic bile ducts. Very few mast cells were observed in these locations. Globule leucocytes in two goats were positive for argentaffin reaction with Gomori hexamine silver stain. The proliferative cholecystitis in experimental goats was similar to adenomatous cholecystitis in cattle naturally infected with fascioliasis. Globule leucocytes had no obvious deleterious effects on the parasites.

Influence of soybean meal and sorghum grain supplementation on intake, digesta kinetics, ruminal fermentation, site and extent of digestion and microbial protein synthesis in beef steers grazing blue grama rangeland.

Six beef steers (British x Brahman) cannulated at the rumen, duodenum and ileum (avg wt 334 kg) and three mature steers (British x British) cannulated at the esophagus were used in a replicated 3 x 3 latin square design and fed no supplement (C), .5 kg soybean meal (SBM) or .5 kg steam-flaked sorghum grain (SFS).head-1.d-1 (DM basis) while grazing blue grama rangeland. Periods of the latin square included a minimum of 14 d for adaptation and 11 d for esophageal masticate collection and digesta sampling. In September, October and November, respectively, forage collected by esophageally cannulated steers averaged 74.5, 88.8 and 71.0% grasses; 2.06, 1.53 and 1.77% N and 68.3, (P greater than .10) by treatment, but total N intake was greater (P less than .05) for SBM vs C and SFS treatments. No differences (P greater than .10) were detected among treatments in OM, NDF, ADF and N digestibilities in the rumen, small intestine or hindgut, but total tract OM digestibility was greater (P less than .10) for SBM and SFS than for C, and total tract N digestibility was greater (P less than .10) for SBM than for C or SFS. Duodenal ammonia N flow was greater (P less than .05) when SBM was fed that when SFS and C were fed, but microbial N and non-ammonia, non-microbial N flows and microbial efficiency were not altered by treatment. Likewise, ileal N flow was not affected (P greater than .10) by treatment. Particulate passage rate, gastrointestinal mean retention time, forage in vitro OM disappearance and in situ rate of forage NDF digestion also were not affected (P greater than .10) by treatments. Ruminal fluid volume was greater (P less than .05) for SFS vs SBM and C treatments, but no differences were noted in fluid dilution rate. Ruminal fluid ammonia concentration was greater (P less than .05) when SBM was fed than when SFS and C were fed (13.5, 9.9 and 8.7 mg/dl, respectively), whereas pH and total VFA concentrations were not different (P greater than .10). Proportion of acetate in ruminal fluid was less (P less than .10) for SBM and SFS than for C. Small amounts of supplemental SBM and SFS had little effect on forage intake, ruminal fermentation and site of digestion but both increased total tract OM digestion in steers grazing blue grama rangeland.