Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2

IntroductionReliable and accurate methods for probiotic identification and enumeration, at the strain level plays a major role in confirming product efficacy since probiotic health benefits are strain-specific and dose-dependent. In this study, real-time PCR methods were developed for strain specific identification and enumeration of L. paracasei 8700:2, a probiotic strain that plays a role in fighting the common cold.MethodsThe assay was designed to target a unique region in L. paracasei 8700:2 genome sequence to achieve strain level specificity. The identification assay was evaluated for specificity and sensitivity. The enumeration viability real-time PCR (v-qPCR) method was first optimized for the viability treatment, then the method was evaluated for efficiency, limit of quantification, precision, and its performance was compared to plate count (PC) and viability droplet digital PCR (v-ddPCR) methods.ResultsThe identification method proved to be strain specific and highly sensitive with a limit of detection of 0.5 pg of DNA. The optimal viability dye (PMAxx) concentration was 50 μM. The method was efficient (> 90% with R2 values > 0.99), with a linear dynamic range between 6*102 and 6*105 copies. The method was highly precise with a relative standard deviation below 5%. The Pearson correlation coefficient (r) was 0.707 for PC and v-qPCR methods, and 0.922 for v-qPCR and v-ddPCR. Bland-Altman method comparison showed that v-qPCR always gave higher values compared to PC method (relative difference ranging from 119% to 184%) and showed no consistent trend (relative difference ranging from −20% to 22%) when comparing v-qPCR and v-ddPCR methods.DiscussionThe difference between PC and v-PCR methods can potentially be attributed to the proportion of cells that exist in a viable but non culturable (VBNC) state, which can be count by v-PCR but not with PC. The developed v-qPCR method was confirmed to be strain specific, sensitive, efficient, with low variance, able to count VBNC cells, and has shorter time to results compared to plate count methods. Thus, the identification and enumeration methods developed for L. paracasei 8700:2 will be of great importance to achieve high quality and efficacious probiotic products

What proportion of adult allergy referrals to secondary care could be dealt with in primary care by a GP with special interest?

Background: The concept of a General Practitioner with Special Interest (GPwSI) was first proposed in the 2000 National Health Service Plan, as a way of providing specialised treatment closer to the patient’s home and reducing hospital waiting times. Given the patchy and inadequate provision of allergy services in the UK the introduction of GPwSIs might reduce the pressure on existing specialist services. Objectives: This study assessed what proportion of referrals to a specialist allergy clinic could be managed in a GPwSI allergy service with a predefined range of facilities and expertise (accurate diagnosis and management of allergy; skin prick testing; provision of advice on allergen avoidance; ability to assess suitability for desensitisation). Methods: 100 consecutive GP referrals to a hospital allergy clinic were reviewed to determine whether patients could be seen in a community-based clinic led by a general practitioner with special interest (GPwSI) allergy. The documentation relating to each referral was independently assessed by three allergy specialists. The referrals were judged initially on the referral letter alone and then re-assessed with the benefit of information summarised in the clinic letter, to determine whether appropriate triage decisions could be made prospectively. The proportion of referrals suitable for a GPwSI was calculated and their referral characteristics identified. Results: 29 % referrals were judged unanimously appropriate for management by a GPwSI and an additional 30 % by 2 of the 3 reviewers. 18 % referrals were unsuitable for a GPwSI service because of the complexity of the presenting problem, patient co-morbidity or the need for specialist knowledge or facilities. Conclusions and clinical relevance: At least a quarter, and possibly half, of allergy referrals to our hospital-based service could be dealt with in a GPwSI clinic, thereby diversifying the patient pathway, allowing specialist services to focus on more complex cases and reducing the waiting time for first appointments

The power of DNA based methods in probiotic authentication

IntroductionThe global probiotic market is growing rapidly, and strict quality control measures are required to ensure probiotic product efficacy and safety. Quality assurance of probiotic products involve confirming the presence of specific probiotic strains, determining the viable cell counts, and confirming the absence of contaminant strains. Third-party evaluation of probiotic quality and label accuracy is recommended for probiotic manufacturers. Following this recommendation, multiple batches of a top selling multi-strain probiotic product were evaluated for label accuracy.MethodsA total of 55 samples (five multi-strain finished products and 50 single-strain raw ingredients) containing a total of 100 probiotic strains were evaluated using a combination of molecular methods including targeted PCR, non-targeted amplicon-based High Throughput Sequencing (HTS), and non-targeted Shotgun Metagenomic Sequencing (SMS).ResultsTargeted testing using species-specific or strain-specific PCR methods confirmed the identity of all strains/species. While 40 strains were identified to strain level, 60 strains were identified to species level only due to lack of strain-specific identification methods. In amplicon based HTS, two variable regions of 16S rRNA gene were targeted. Based on V5–V8 region data, ~99% of total reads per sample corresponded to target species, and no undeclared species were detected. Based on V3–V4 region data, ~95%–97% of total reads per sample corresponded to target species, while ~2%–3% of reads matched undeclared species (Proteus species), however, attempts to culture Proteus confirmed that all batches were free from viable Proteus species. Reads from SMS assembled to the genomes of all 10 target strains in all five batches of the finished product.DiscussionWhile targeted methods enable quick and accurate identification of target taxa in probiotic products, non-targeted methods enable the identification of all species in a product including undeclared species, with the caveats of complexity, high cost, and long time to result

Visceral fat mass as a novel risk factor for predicting gestational diabetes in obese pregnant women

Objective To develop a model to predict gestational diabetes mellitus incorporating classical and a novel risk factor, visceral fat mass. Methods Three hundred two obese non-diabetic pregnant women underwent body composition analysis at booking by bioimpedance analysis. Of this cohort, 72 (24%) developed gestational diabetes mellitus. Principal component analysis was initially performed to identify possible clustering of the gestational diabetes mellitus and non-GDM groups. A machine learning algorithm was then applied to develop a GDM predictive model utilising random forest and decision tree modelling. Results The predictive model was trained on 227 samples and validated using an independent testing subset of 75 samples where the model achieved a validation prediction accuracy of 77.53%. According to the decision tree developed, visceral fat mass emerged as the most important variable in determining the risk of gestational diabetes mellitus. Conclusions We present a model incorporating visceral fat mass, which is a novel risk factor in predicting gestational diabetes mellitus in obese pregnant wome

Metformin versus placebo in obese pregnant women without diabetes mellitus

Background: Obesity is associated with increased risk of adverse pregnancy outcomes. Lifestyle intervention studies have not improved outcome. Metformin improves insulin sensitivity and leads to less weight gain. Methods: Our double-blind placebo-controlled trial randomized non-diabetic pregnant women with a body mass index >35 kg/m2 to metformin or placebo from 12-18 weeks’ gestation until delivery. Primary outcome was median neonatal birth weight z-score reduction by 0.3 standard deviations (equivalent to a 50% reduction in incidence of large-for-gestational-age neonates from 20% to 10%). Secondary outcomes included maternal gestational weight gain and incidence of gestational diabetes, and preeclampsia, as well as adverse neonatal outcomes. Women were randomized, by computer generated random numbers, to either daily metformin 3.0 grams (n=225) or to placebo (n=225). Analysis was by intention to treat. Results: Fifty women withdrew consent, leaving 202 in the metformin group and 198 in the placebo group. There was no significant difference in median neonatal birth weight z-score (metformin: 0.05, IQR -0.71 to 0.92; placebo: 0.17, IQR -0.62 to 0.89; p=0.655). In the metformin group, compared to placebo, median maternal gestational weight gain was lower (4.6 kg, IQR 1.3-7.2 vs. 6.3 kg, IQR 2.9-9.2, p<0.0001) and incidence of preeclampsia was lower (3.0% vs 11.3%; odds ratio 0.24, 95% CI 0.10-0.61; p=0.001), incidence of side effects was higher; there were no significant differences in gestational diabetes, large for gestational age neonates and adverse neonatal outcomes. Conclusions: In non-diabetic women with BMI >35 kg/m2, antenatal administration of metformin reduces maternal weight gain but not neonatal birth weight. (ClinicalTrials.gov number, NCT01273584

Enhancing radial distribution system performance by optimal placement of DSTATCOM

In this paper, A novel modified optimization method was used to find the optimal location and size for placing distribution Static Compensator in the radial distribution test feeder in order to improve its performance by minimizing the total power losses of the test feeder, enhancing the voltage profile and reducing the costs. The modified grey wolf optimization algorithm is used for the first time to solve this kind of optimization problem. An objective function was developed to study the radial distribution system included total power loss of the system and costs due to power loss in system. The proposed method is applied to two different test distribution feeders (33 bus and 69 bus test systems) using different Dstatcom sizes and the acquired results were analyzed and compared to other recent optimization methods applied to the same test feeders to ensure the effectiveness of the used method and its superiority over other recent optimization mehods. The major findings from obtained results that the applied technique found the most minimized total power loss in system ,the best improved voltage profile and most reduction in costs due power loss compared to other methods

Piezoelastic PVDF/TPU nanofibrous composite membrane: Fabrication and characterization

Poly (vinylidene fluoride) nanofibers (PVDF NFs) have been extensively used in energy harvesting applications due to their promising piezoresponse characteristics. However, the mechanical properties of the generated fibers are still lacking. Therefore, we are presenting in this work a promising improvement in the elasticity properties of PVDF nanofibrous membrane through thermoplastic polyurethane (TPU) additives. Morphological, physical, and mechanical analyses were performed for membranes developed from different blend ratios. Then, the impact of added weight ratio of TPU on the piezoelectric response of the formed nanofibrous composite membranes was studied. The piezoelectric characteristics were studied through impulse loading testing where the electric voltage had been detected under applied mass weights. Piezoelectric characteristics were investigated further through a pressure mode test the developed nanofibrous composite membranes were found to be mechanically deformed under applied electric potential. This work introduces promising high elastic piezoelectric materials that can be used in a wide variety of applications including energy harvesting, wearable electronics, self-cleaning filters, and motion/vibration sensors. - 2019 by the authors.The project was funded “partially” by Kuwait Foundation for the Advancement of Sciences under project code: PN17-35EE-02

Metformin use in obese mothers is associated with improved cardiovascular profile in the offspring

BACKGROUND: Maternal obesity increases the risk for pregnancy complications and adverse neonatal outcome and it has also been associated with long lasting adverse effects in the offspring, including increased body fat mass, insulin resistance and increased risk for premature cardiovascular disease. Lifestyle interventions in pregnancy have produced none or modest effects in reducing adverse pregnancy outcomes in obese mothers. Metformin use in Obese Pregnant women trial was associated with reduced adverse pregnancy outcomes and had no effect on birthweight. However, the long-term implications of metformin on the health of offspring remain unknown. OBJECTIVE: The purpose of this study was to assess whether prenatal exposure to metformin can improve the cardiovascular profile and body composition in the offspring of obese mothers. STUDY DESIGN: In 151 children from the Metformin use in Obese-Pregnant women trial we measured body composition, peripheral blood pressure and arterial pulse wave velocity. Central hemodynamics (central blood pressure and augmentation index) were estimated using the Vicorder device. Left ventricular cardiac function and structure were assessed by echocardiography. RESULTS: Children were 3.9±1.0 years of age and 77 were exposed to metformin prenatally. There was no significant difference in peripheral blood pressure, arterial stiffness and body composition apart from gluteal and tricep circumferences which were lower in the metformin group (p<0.05). The metformin, compared to the placebo group, had lower central hemodynamics (mean adjusted decrease - 0.707mmHg for aortic systolic blood pressure, -1.65mmHg for aortic pulse pressure and -2.68% for augmentation index, p<0.05 for all) and lower left ventricular diastolic function (adjusted difference in left atrial area -0.525cm2, in isovolumic relaxation time -0.324msec and in pulmonary venous systolic wave 2.97cm/s, p<0.05 for all). There were no significant differences in metabolic profile between the groups. CONCLUSION: Children of obese mothers who were prenatally exposed to metformin, compared to those exposed to placebo, have lower central hemodynamic and cardiac diastolic indices. These results suggest that administration of metformin in obese pregnant women may potentially have a beneficial cardiovascular effect for their offspring

CHANGES IN SOME BIOPHYSICAL AND BIOCHEMICAL PARAMETERS IN BLOOD AND URINE OF WORKERS CHRONICALLY EXPOSED TO BENZENE

Objective: Benzene may occur naturally as a component of petroleum, or may be manufactured synthetically. It is found in the environment as a contaminant from both human activities and natural processes, posing serious bio-hazards from chronic exposure.Methods: A total of 330 individual were enrolled to study possible health hazards of benzene contamination; 265 males occupationally chronically exposed to low levels of benzene in their daily activity were compared to 65 healthy individuals of the same socio-economic standard. Benzene workers were divided between 45 workers in printing shops, 70 subjects dealing with benzene containing paints (painters), 75 subjects working in professions related to automotive work (autoworkers) and 75 car drivers.Results: benzene itself was not detected in blood or urine of all participants, but the levels of its metabolites; phenol and t,t-muconic acid, were higher in the blood and urine samples in the group of benzene-exposed workers. The results also indicate that individuals in this group are under oxidative stress. However, neither the determined liver function nor the kidney function tests showed significant deviation from controls. However, the results of the biophysical hematological parameters, including the degree of hemolysis, blood viscosity, RBCs aggregation and form factor were significantly deviated from normal.Conclusion: The deviation of the determined biochemical and biophysical parameters from normal may predispose such workers to a variety of health problems. Early correction of the oxidative stress and the hematological parameters and improvement of working conditions are necessary to prevent their progress to more serious health conditions, especially in children and young adolescents working under similar conditions.Running Title : Chronic exposure to benzene in work plac