The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing

Peer reviewedPublisher PD

The effects of tualang honey on female reproductive organs, tibia bone and hormonal profile in ovariectomised rats - animal model for menopause

<p>Abstract</p> <p>Background</p> <p>Honey is a highly nutritional natural product that has been widely used in folk medicine for a number of therapeutic purposes. We evaluated whether Malaysian Tualang honey (AgroMas, Malaysia) was effective in reducing menopausal syndrome in ovariectomised female rats; an animal model for menopause.</p> <p>Methods</p> <p>The rats were divided into two control groups and three test groups. The control groups were sham-operated (SH) and ovariectomised (OVX) rats. The SH and OVX control rats were fed on 0.5 ml of distill water. The rats in the test groups were fed with low dose 0.2 g/kg (THL), medium dose, 1.0 g/kg (THM) and high dose 2.0 g/kg (THH) of Tualang honey in 0.5 ml of distill water. The administration was given by oral gavage once daily for 2 weeks. The reproductive organs (uterus and vagina), tibia bone and aorta were taken for histopathological examination while serum for hormonal assays.</p> <p>Results</p> <p>Administration of Tualang honey for 2 weeks to ovariectomised rats significantly increased the weight of the uterus and the thickness of vaginal epithelium, restored the morphology of the tibia bones and reduced the body weight compared to rats in the ovariectomised group. The levels of estradiol and progesterone, in honey treated groups were markedly lower than that in the OVX group. At low doses (0.2 g/kg; THL group) of Tualang honey there was an increased in serum free testosterone levels compared to OVX group (P < 0.01). Progesterone concentrations was significantly decreased in the OVX group as compared to SHAM group (P < 0.05).</p> <p>Conclusions</p> <p>Tualang honey was shown to have beneficial effects on menopausal (ovariectomised) rats by preventing uterine atrophy, increased bone density and suppression of increased body weight. Honey could be an alternative to HRT.</p

Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens

<p>Abstract</p> <p>Background</p> <p>The influence of diet on intestinal microflora has been investigated mainly using conventional microbiological approaches. Although these studies have advanced knowledge on human intestinal microflora, it is imperative that new methods are applied to facilitate scientific progress. Culture-independent molecular fingerprinting method of Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) has been used to study microbial communities in a variety of environmental samples. However, these protocols must be optimized prior to their application in order to enhance the quality and accuracy of downstream analyses. In this study, the relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean^®Fecal DNA Isolation Kit, M; QIAamp^®DNA Stool Mini Kit, Q; FastDNA^®SPIN Kit, FSp; FastDNA^®SPIN Kit for Soil, FSo) were evaluated. Further, PCR-DGGE technique was also assessed for its feasibility in detecting differences in human intestinal bacterial fingerprint profiles.</p> <p>Method</p> <p>Total DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons.</p> <p>Results</p> <p>Regardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt) of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences.</p> <p>Conclusion</p> <p>We conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal specimens (10 to 50 mg, wet wt) that can be resolved as bacterial community fingerprints using PCR-DGGE technique. Subsequently, PCR-DGGE technique can be applied for studying variations in human intestinal microbial communities.</p

Evaluating the influence of diet on human intestinal microflora using molecular techniques

Microbial communities in the gut play an important role in human health. Researchers have begun to characterize this complex ecosystem but many fundamental questions including the influence of diet remain unanswered. Previous studies have relied on conventional microbiological methods but these lack sensitivity that is needed to detect changes in such a complex system. The purpose of this study was twofold: to evaluate the feasibility of using a combination of Polymerase Chain Reaction and Denaturing Gel Gradient Electrophoresis (PCR-DGGE) to monitor changes in intestinal microflora and to investigate the influence of diet on microbial communities in the human intestine. Alterations in microbial communities were investigated based on two types of diet modifications: (a) supplementation with a prebiotic dietary component (i.e., soy isolate enriched with high non-digestible oligosaccharides (NDO)) and (b) a controlled diet made up of a set of foods commonly consumed as part of regular diet with no added prebiotics or probiotics. The effects of the controlled (camp) diet were compared with a free-choice diet (non-controlled, home diet) consumed before and between periods of controlled diet. Thus, in the soy NDO study, we attempted to investigate the prebiotic property of the soy isolate, while in the second study, we aimed to evaluate whether variability in the human intestinal microflora profiles can be reduced when consuming a diet made up of the same foods over an extended period of time. Fecal samples were collected from human subjects at regular intervals throughout the study. Total DNA extracted from the fecal samples was used to amplify the bacterial 16S rRNA gene by PCR using domain specific primers. Intestinal microbial communities were profiled using DGGE separation of the PCR amplicons and Bionumerics software was used to compare DGGE bacterial fingerprint profiles. Bands were selected from the DGGE profiles, excised from the respective gels, sequenced for identification and grouped into a phylogenetic tree using neighbor-joining algorithm. In addition, the archaeal community was also studied using similar techniques. Overall, it was found that human intestinal microbial communities were largely made up of profiles composed of 10 to 20 intense bands. There was considerable inter-subject variability in the bacterial community profiles, but a smaller intra-subject variability. In the soy NDO study, the soy isolate was observed to enhance the growth of Bifidobacterium spp. These results provide evidence of the prebiotic effect of the NDO enriched soy isolate. In the study with regular diet, DGGE fingerprint data indicate that bacterial communities can be modified by changing the diet. Specifically, changes in intestinal microbial community profiles were observed before and after participation in a clinical camp. Data indicate that subjects attained a set of DGGE fingerprint profiles as a response to non-controlled diet at home. Within 2 to 4 days of controlled diet at camp, these profiles changed to another set. The camp fingerprint profiles remained as long as their diet remained unchanged. In addition, each individual assumed their own unique fingerprint profiles even though the same types of foods were given to all subjects during camp. In contrast, intestinal archaeal community had a simpler profile composed of 1 to 2 bands. Within an individual, the presence of these bands varied over time regardless of diet. The findings suggest that unlike bacteria that are consistently found in the gut, Archaea appear intermittently. In summary, data from DGGE and sequencing indicate that the composition of the human intestinal microflora can be modified by diet. Sequences obtained from DGGE data showed that special food supplement (soy isolate enriched with NDO) had prebiotic property, whereby it enhanced the growth of Bifidobacterium spp. when administered at a dose of 5 to 15 g/d for 16 days. Further, DGGE fingerprint profiles indicated that the human intestinal microflora can also be modified by regular food items when they are consumed consistently over a period of time. Overall, these data also imply that with optimized conditions and appropriate analytical software, molecular fingerprinting technique such as PCR-DGGE can be applied in clinical studies as a rapid and reliable method to evaluate microbial community changes in the gut in response to dietary treatment

The Utility of Pharmacogenetic-Guided Psychotropic Medication Selection for Pediatric Patients: A Retrospective Study

Background: To describe trends and clinical experiences in applying commercial pharmacogenetic testing among pediatric patients with neuropsychiatric disorders. Methods: Demographic and clinical data of patients receiving GeneSight® testing from January 2015 to November 2016 at an urban pediatric hospital were retrospectively extracted from medical charts. Outcome data included pharmacogenetic test results and medication prescriptions before and after the test. Results: A total of 450 patients (12.1 ± 4.3 years) diagnosed with anxiety disorder, attention deficit hyperactivity disorder, developmental disorders including autism, and/or a mood disorder received testing, and 435 of them were prescribed medications. Comparing data before and after testing, the total number of psychotropic prescriptions were reduced by 27.2% and the number of prescribed medications with severe gene-drug interactions decreased from 165 to 95 (11.4% to 8.9% of total medications prescribed). Approximately 40% of actionable genetic annotation were related to CYP2CD6 and CYP2C19. Patients of Asian descent had significantly higher likelihood than other races of being classified as poor to intermediate metabolizers of antidepressants, mood stabilizers, and antipsychotics (p = 0.008, 0.007, and 0.001, respectively). Diagnoses, including autism spectrum disorder, were not associated with increased risks of severe gene-drug interactions. Conclusions: Pharmacogenetic testing in child and adolescent psychiatry is currently based on few clinically actionable genes validated by CPIC and/or FDA. Although this approach can be moderately utilized to guide psychotropic medication prescribing for pediatric patients with psychiatric disorders, clinicians should cautiously interpret test results while still relying on clinical experience and judgment to direct the final selection of medication