Application of an electro elution system for direct purification of linear covalently closed DNA fragments.

Gene therapy is a powerful treatment modality. Non-viral gene therapy vectors power one arm of this important approach, due to their enhanced safety profile compared to their viral counterparts. New non-viral approaches continue to be developed, but purification can bottleneck the scaleup and cost-effectiveness and quality of some of these advanced vectors. We require more advanced purification and separation techniques compared to conventional methods to maximize resolution in a scalable manner. The Prep Cell system is a continuous electro elution system that contains a circular gel casting tube where DNA mixtures can be run through and subsequently migrate into an elution chamber, to be eluted by a peristaltic pump. This DNA separation and purification process confers advantages over other conventional methods, including i) the elimination of multiple downstream purification process requirements; ii) its ability to be applied in mid-scale settings, and iii), its high-resolution power. In this study, we assessed the ability of this Prep Cell Model 491 system to purify a novel type of non-viral linear covalently closed (LCC) DNA minivector (ministring DNA) from its precursor parent plasmid DNA and process by-product DNA species by analyzing for effective separation via agarose gel electrophoresis, recovery yield, single enzyme digestion, and quality control assessments. Overall, effective separation and resolution of mini-DNA vectors was obtained using the Prep Cell system, conferring its potential to be applied towards mid-scale purification of DNA vectors for a variety of research, and eventually, clinical applications.

Development of a numerical model for sector-average plume gamma dose and its validation with dose rate measurements at Kalpakkam NPP site, India.

A Gaussian Plume based simple numerical model, named DIFFUSE is developed to simulate the long-term sector-average plume gamma dose due to radioactive plume released during normal operation of nuclear facilities. DIFFUSE calculates site specific joint frequency distributions of wind speed, wind direction and atmospheric stability using micrometeorological observations. It performs the finite sector-average dose integration for any stack height and gamma energy using Simpson's 1/3rd method with sufficient computational efficiency within the site boundary up to 2 km. Plume dose contribution to the main plume sector from nearest and next nearest side plume sectors is also calculated. DIFFUSE is validated with a 3-month long, starting from February 2021 to April 2021, dose rate observation data during operational releases from 100 m stack of Madras Atomic Power Station, Kalpakkam, India. Meteorological data from onsite 50 m tower and continuous dose rate observation from two sets of Autonomous Gamma Dose Logger (AGDL) detectors, namely n-AGDLs and r-AGDLs, placed in two different configurations along the geometric arcs of wind sectors around the stack are used. Simulated doses are compared with look-up table based dose estimates by Hukkoo et al. (1988). Linear spatial averaging of cumulative AGDL doses on a sector arc is used as measured sector-average dose for model validation. Simulations performed for both n-AGDL and r-AGDL configurations show DIFFUSE estimated doses are ∼37% lower and Hukkoo estimated doses are at least ∼50% lower than the measured doses. Statistical analysis of DIFFUSE simulated doses shows a statistical correlation of R2∼98.3%, slope of the fit ∼1.36 for n-AGDL setup and R2∼75.3%, slope of the fit ∼1.57 for r-AGDL setup. Overall, DIFFUSE produces conservative doses compared to look-up table based doses as required by regulatory bodies.

Chromosome length ratio as a biomarker of DNA damage in cells exposed to high dose ionizing radiation.

The premature chromosome condensation (PCC) assay is considered as complementary bio-dosimetry tool for chromosome aberration assay and the PCC assay can be used to estimate high dose exposure. Though the PCC ring is considered as prospective biomarker, chromosome length ratio (ratio of longest and shortest chromosome length in PCC spreads) of chemically induced PCC is shown to be very good indicator of ionizing radiation. In view of this, an in-vitro study has been performed using PCC assay to suggest chromosome length ratio (LR) as potential bio-dosimeter induced by high dose ionizing radiation. Blood samples were collected from healthy subjects (n = 3) after prior consent and irradiated to ten different doses ranging between 0 and 20 Gy using 6 MV LINAC X-rays with dose rate of 5.6 Gy/min. Irradiated lymphocytes were cultured and calyculin induced PCC spreads were prepared. PCC spreads were captured using image analysis system and chromosome lengths were measured using open-source ImageJ software. For each dose, LR for 50 chromosome spreads were computed and mean LR value was calculated. LR varies between 6.0 ± 0.08 and 23.6 ± 0.55 for the dose range between 2 and 20 Gy. The dose response curve for LR was observed to be linear with y = 1.02x + 3.36, R2 = 0.97. Linear dose response relationship obtained in the present study confirms the prospective use of LR measurement. This study is first of its kind to examine chromosome length ratio as a biomarker of DNA damage in cells exposed to high dose X-ray exposure.

Multicenter Evaluation of the Simplexa VZV Direct Assay for Detection of Varicella-Zoster Virus in Cerebrospinal Fluid and Lesion-Swab Specimens.

Varicella-zoster virus (VZV) is the etiologic agent of varicella (chickenpox) and herpes zoster (shingles) infections commonly involving skin, mucous membranes, and less frequently the central nervous system. Traditional methods for the laboratory diagnosis of these infections are time-consuming, labor-intensive, and often insensitive. As such, these tests are being replaced by more sensitive and rapid molecular methods. This study evaluated the performance of two different molecular assays, the Simplexa VZV Direct and Simplexa VZV Swab Direct, to detect VZV DNA in cerebrospinal fluid (CSF) and lesion-swab specimens, respectively. The Simplexa VZV Direct and Simplexa VZV Swab Direct assays were compared against individual composite reference methods that varied depending on the sample cohort examined. A total of 883 CSF and 452 cutaneous and mucocutaneous prospective, retrospective, and contrived specimens were evaluated in this multicenter study. The results of this study showed that the Simplexa assays demonstrated near perfect agreement (k = 0.98) compared to the composite reference methods for the detection of VZV in CSF and lesion swab specimens. A further comparison between the standard of care molecular assays employed at the site of specimen collection and the Simplexa assays demonstrated excellent agreement (k = 1.0). The Simplexa assays offer rapid and reliable alternatives for the detection of VZV in certain clinical specimens without the need for nucleic acid extraction.

Distribution, annual committed effective dose, and health safety assessment of 210Po in marine biota from Kalpakkam coast, Bay of Bengal.

Seafood, intertidal biota, beach sediment, and seawater from Kalpakkam coast, Bay of Bengal were analyzed for 210Po to evaluate the internal exposure and other radiological safety aspects. Kalpakkam houses various nuclear power generation facilities on its coast. The activity concentration of 210Po was more pronounced in the intertidal organisms. Pelagic planktivorous fishes have the highest activity of the non-technogenic radionuclide, followed by the detrital feeders, benthic planktivores, benthic carnivores, and pelagic carnivore fishes. The affinity of 210Po to organic detrital matter and planktonic organisms has led to a higher accumulation of radionuclide in planktivorous fishes. Activity concentration of 210Po in seafood ranged between 1.13 ± 0.3 and 96.71 ± 1.6 Bq kg-1 (Becquerel/kilogram). In seaweeds and gastropods, it ranged from 2.09 ± 0.2 to 8.21 ± 0.6 and from 9.31 ± 0.7 to 21.58 ± 1.2 Bq kg-1, respectively. The committed effective dose (CED) of 210Po in seafood varied from 31.18 to 456.68 µSv yr-1 (microSievert/year). Radiological hazard parameters, such as activity intake, CED in consumption, of the seafood from this coast are within the acceptable levels prescribed by the International Commission on Radiological Protection and US Environmental Protection Agency.

Construction of dose response curve for 6 MV LINAC X-rays using Premature Chromosome Condensation assay for radiation dosimetry.

Quantification of chromosomal aberrations in the exposed personnel blood samples is considered as a 'gold standard' and sensitive biomarker in biological dosimetry. Despite technological developments, culture of cells for 48-52 h remains an unmet need in case of triage biodosimetry. Moreover, it is difficult to get sufficient number of metaphase spreads for scoring after high doses of exposures. The technique which causes condensation of chromatin before mitosis using biological or chemical agent is named as Premature Chromosome Condensation (PCC) assay. This assay is considered as an alternative to chromosome aberration assay, particularly at high acute doses of low and high LET radiation. To establish the PCC assay, blood samples were collected from healthy non-smoking individuals (n = 3) and exposed to various doses (0-20 Gy) of 6 MV X-rays at a dose rate of 5.6 Gy/min, using a high energy Linear accelerator (LINAC). Irradiated blood samples were subjected to Calyculin-A induced PCC. About 500 cells or more than 100 Ring Chromosomes (RC) were scored at each dose. Dicentric chromosomes (DC) and acentric fragments were also scored at each dose; the number of chromosomal aberrations in G1, M, G2/M and M/A phase of cell cycle were recorded and the frequency was used to construct the dose response curve. A dose dependent increase in RC and DC frequency were observed with a slope of 0.049 ± 0.002 and 0.30 ± 0.02 respectively. This study is first of its kind to construct a dose response curve for LINAC X-rays using a PCC assay.

Shape-tuned, surface-active and support-free silver oxygen reduction electrocatalyst enabled high performance fully non-PGM alkaline fuel cell.

Exploring non-platinum group metal (n-PGM) based efficient oxygen reduction reaction (ORR) electro-catalysts is highly important for realizing advancement in sustainable next generation-alkaline anion exchange membrane fuel cells (AAEMFCs). Herein, we demonstrate a new "hierarchical shape tuning approach" for the synthesis of controlled sized and shaped non-PGM based Ag ORR electro-catalysts with surface active nano-islands. Hierarchical shapes ranging from spherical (S-AgNs), worm-in-sphere, sphere-in-worm and vermiform (worm-like) Ag nanostructures (V-AgNs) were obtained by precisely varying the ratios of capping agent to dual reducing agents in water at ambient conditions. Compared to S-AgNs, V-AgNs revealed a higher mass normalized ORR Tafel activity (0.303 A mgAg -1 at 0.9 V), onset (1.06 V) and half wave (0.78 V) potentials and higher retention of limiting current density (>88%) after 5000 cycles in 0.5 M potassium hydroxide (KOH) solution attributable to their unique worm like morphology with surface active nano-islands and support free-nature enabled better catalyst utilization. In a fully "non-PGM AAEMFC" (n-PAAEMFC), V-AgNs exhibited the highest fuel cell activity of 115.6 mW cm-2 and stable short-term durability (∼240 h) compared to S-AgNs (41.3 mW cm-2) and previously reported fully n-PAAEMFCs indicating their potential use in next-generation alkaline fuel cells.

Unravelling the utility of modern sulfonylureas from cardiovascular outcome trials and landmark trials: expert opinion from an international panel.

AIM: The primary objective of this review is to develop practice-based expert group opinions on the cardiovascular (CV) safety and utility of modern sulfonylureas (SUs) in cardiovascular outcome trials (CVOTs). BACKGROUND: The United States Food and Drug Administration issued new guidance to the pharmaceutical industry in 2008 regarding the development of new antihyperglycemic drugs. The guidance expanded the scope for the approval of novel antihyperglycemic drugs by mandating CVOTs for safety. A few long-term CVOTs on dipeptidyl peptidase 4 inhibitors, glucagon-like peptide 1 receptor agonists, and sodium-glucose cotransporter 2 inhibitors have been completed, while others are ongoing. SUs, which constitute one of the key antihyperglycemic agents used for the management of type 2 diabetes mellitus (T2DM), have been used as comparator agents in several CVOTs. However, the need for CVOTs on modern SUs remains debatable. In this context, a multinational group of endocrinologists convened for a meeting and discussed the need for CVOTs of modern SUs to evaluate their utility in the management of patients with T2DM. At the meeting, CVOTs of modern SUs conducted to date and the hypotheses derived from the results of these trials were discussed. REVIEW RESULTS: The expert group analyzed the key trials emphasizing the CV safety of modern SUs and also reviewed the results of various CVOTs in which modern SUs were used as comparators. Based on literature evidence and individual clinical insights, the expert group opined that modern SUs are cardiosafe and that since they have been used as comparators in other CVOTs, CVOTs of SUs are not required. CONCLUSION: Modern SUs can be considered a cardiosafe option for the management of patients with diabetes mellitus and CV disease; thus CVOTs among individuals with T2DM are not required.

Fixed-dose combination in management of type 2 diabetes mellitus: Expert opinion from an international panel.

Type 2 diabetes mellitus (T2DM) is a progressive disease with multifactorial etiology. The first-line therapy includes monotherapy (with metformin), which often fails to provide effective glycemic control, necessitating the addition of add-on therapy. In this regard, multiple single-dose agents formulated as a single-dose form called fixed-dose combinations (FDCs) have been evaluated for their safety, efficacy, and tolerability. The primary objective of this review is to develop practice-based expert group opinion on the current status and the causes of concern regarding the irrational use of FDCs, in Indian settings. After due discussions, the expert group analyzed the results from several clinical evidence in which various fixed combinations were used in T2DM management. The panel opined that FDCs (double or triple) improve patient adherence, reduce cost, and provide effective glycemic control and, thereby, play an important role in the management of T2DM. The expert group strongly recommended that the irrational metformin FDC's, banned by Indian government, should be stopped and could be achieved through active participation from the government, regulatory bodies, and health ministry, and through continuous education of primary care physicians and pharmacists. In T2DM management, FDCs play a crucial role in achieving glycemic targets effectively. However, understanding the difference between rational and irrational FDC combinations is necessary from the safety, efficacy, and tolerability perspective. In this regard, primary care physicians will have to use a multistep approach so that they can take informed decisions.

Rhodium(III) complexes derived from complexation of metal with azomethine linkage of chitosan biopolymer Schiff base ligand: Spectral, thermal, morphological and electrochemical studies.

Rhodium(III) complexes such as [Rh(Chi4Hy3mb)(H2O)2]Cl2, [Rh(Chi2Hymb)(H2O)2]Cl2, and [Rh(Chi2Hy3mb)(H2O)2]Cl2 were synthesized by metal chelation/complexation with chitosan Schiff base ligands. Stable Schiff base ligands were prepared by chemical modification of chitosan with aromatic aldehydes such as vanillin, salicylaldehyde and orthovanillin. These chitosan based Schiff base ligands were performed as bidentate ligands through azomethine nitrogen atom and methoxy/hydroxy oxygen atom. These bidentate ligands were favoured to the formation of stable coordination complex with metal ions. The series of Rhodium(III) complexes were characterized by Elemental analysis, FT-IR, UV-Vis spectroscopy, P-XRD and Thermo-gravimetric analysis (TGA). The electrochemical property of Rhodium(III) complexes were analyzed by cyclic voltametry.

UNCERTAINTY EVALUATION IN DOSE ASSESSMENT DUE TO SPATIAL DISTRIBUTION OF NORM IN BEACH SANDS OF SOUTHEAST COAST OF INDIA: A PROBABILISTIC APPROACH.

A study on radionuclide distribution along coastal stretch in high background areas is constantly attracted to many researchers around the world. In the present study, the concentration of radionuclides in beach sands along the southeast coastal region of India has been studied in detail. Concentration of radionuclides is measured using gamma spectrometry at 148 pre-determined locations. It is found that 238U and 232Th concentrations are varying in the range from 3 (BDL) to 298.9 and 1150.2 Bq kg-1, respectively. Concentration of 40K in beach sand varied from 11 to 1743.4 Bq kg-1. The measured activity levels of radionuclides were compared with earlier studies carried out by various researchers in the same region under study. The related radiological parameters of interest were also determined and compared with the internationally recommended values. With an increase in emphasis on quality in dose assessment methods, dose to human population living in the vicinity requires uncertainty evaluation, which is carried out by taking into account variation in the activity values. This study would serve as a baseline radiological mapping for the southeast coast region of India for environmental impact assessment prior to plan of nuclear facility in the future.

A REVIEW ON 210Po AND 210Pb IN INDIAN SEAFOOD AND DOSE TO CONSUMERS.

India is the second largest seafood producer in the world marketing more than 7000 edible species. In this regard, commendable studies have been conducted since the 1970s by different research groups and more data are reported. In this work, all the studies on 210Po and 210Pb measurements in abiotic and biotic components of India have been combined and reviewed. The concentrations of 210Po and 210Pb are estimated by radiochemical separation followed by alpha counting. Grain size, season, Tsunami waves and place of sampling have a decisive bearing on 210Po and 210Pb concentration in abiotic components. Plankton shows a higher Kd factor (104) compared to seaweeds and sea grass (103). Pond ecosystems recorded the highest Kd factor (4·3 × 104) compared to other freshwater and marine ecosystems. Bioaccumulation of 210Po and 210Pb varies with respect to variety of seafood. The committed effective dose (CED) calculated for shellfish species maintained a higher range of 2.5 × 10-2 mSv/y to 9.8 × 10-1 mSv/y and for fish species fluctuated from 3.8 × 10-4 mSv/y to 2.0 × 10-1 mSv/y. The studies conducted so far are scattered, and need to be gathered for future reference and planning (i.e. There is not much information available for Kerala, Karnataka, Maharashtra, Gujarat, Andhra, West Bengal and Odessa coast). Therefore, it is strongly recommended that further and more complete research is undertaken to study the bioaccumulation of 210Po and 210Pb from seafood. Overall, the present review concludes that Indian seafood is radiologically safe.

Utilization of induction furnace steel slag in concrete as coarse aggregate for gamma radiation shielding.

The present investigation highlights the utilization of Induction Furnace (IF) steel slag as a partial replacement for coarse aggregate in concrete. The concrete blocks of size 150 × 150 × 50 mm were casted and the effect of density, compressive strength, linear attenuation coefficient, Gamma Attenuation Factor (GAF) and Half Value Layer (HVL) has been explored. Gamma attenuation measurements were carried out using NaI (Tl) based gamma detector. Gamma ray source 60Co emitting two gamma energies 1.17 and 1.33 MeV was used for radiation measurements. The results of this present study shows that 50% of IF steel slag replacement increases the density (2.81 g/cm3) and compressive strength (29.11 N/mm2). The Linear attenuation coefficient (0.1953 cm-1-0.2236 cm-1) and GAF (0.6343-0.6710) is higher at 50% replacement of IF steel slag than conventional concrete. HVL values were also having positive impact in 50% replacement of IF steel slag (3.10 cm) in concrete than conventional concrete (3.55 cm). The partial replacement of IF steel slag as coarse aggregate in concrete is effective in gamma shielding.

A multicentre study evaluating the risk and prevalence of diabetic retinopathy in children and young people with type 1 diabetes mellitus.

INTRODUCTION: There is currently limited data published on the prognostic factors and prevalence of diabetic eye disease in children and young people (CYP) with Type 1 diabetes mellitus (T1DM), yet diabetic retinopathy remains one of the commonest causes of blindness in young adults. The aim of this study was to determine the risk and prevalence of diabetic retinopathy (DR) and to evaluate the risk factors associated with diabetic retinopathy in CYP with T1DM. METHODS: All CYP with Type 1 diabetes between 12 and 18 years of age, registered with the regional diabetic retinopathy screening programme were evaluated from 2012 to 2013 in four diabetes units within the north west region of the United Kingdom. Patients who had evidence of diabetic retinopathy were reviewed to identify risk factors for presence or absence of diabetic retinopathy. RESULTS: 237 patients between the ages 12-18 years were included in the. The prevalence of diabetic retinopathy was 11%. Out of 27 patients with evidence of diabetic retinopathy, 44% were reported as background changes and 56% had pre-proliferative DR. Significant risk factors for diabetic retinopathy disease in the population using univariate analyses were duration of diabetes, puberty, age at diagnosis and mean HbA1c in the preceding 12 months. Multivariate logistic regression analysis found age of diagnosis (p = 0.04) and mean HbA1c as significant independent risk factors for presence of diabetic retinopathy (p = 0.02). CONCLUSIONS: The prevalence of diabetic retinopathy in this paediatric population was 11%. Early age at diagnosis and poor metabolic control are independent risk factors for diabetic retinopathy.

Dysregulation of miR-146a by periodontal pathogens: A risk for acute coronary syndrome.

BACKGROUND: Periodontitis is a polymicrobial, chronic inflammatory disease leading to loss of tooth-supporting structures. The bacteremia, endotoxemia, and systemic low-grade inflammation associate periodontitis with systemic illnesses such as diabetes mellitus and coronary artery disease. Periodontal pathogens have been detected from atheromatous plaque by amplification of the genetic material by using specific oligonucleotide primers in polymerase chain reaction. Though the association between periodontitis and cardiovascular diseases has been ascertained by systematic reviews and meta-analyses, its pathophysiology is not lucid. MicroRNAs are currently implicated in the regulation of many cellular processes including inflammation and may play a vital role in our understanding of this disease association. In this case-control study, we explored the role of the inflammatory microRNA, miR-146a, in acute coronary syndrome (ACS) subjects with and without chronic periodontitis (CP) and its regulation of the innate immune host response to periodontal pathogens. METHODS: Three groups each comprising 66 patients each, namely group 1 (ACS patients without CP), group 2 (ACS patients with CP) and group 3 (CP only) formed the study population. Subgingival plaque samples and serum samples were subjected to quantitative Polymerase Chain Reaction (qPCR) for detection of Porphyromonas gingivalis, a keystone pathogen and to assess the levels of circulating miR-146a and associated proinflammatory cytokines. RESULTS: miR-146a associated significantly in group 2 subjects with an odds ratio 1.434, 95% confidence interval 1.013-2.030, P < 0.042, and a predictive percentage of 83.3% and group 1 with a predictive percentage of 76.0.% The associated cytokines interleukin-6 (IL-6), tumor necrosis factor-α, and IL-1ß also showed an upregulation with statistical significance (P < 0.05). CONCLUSION: microRNA-146a is a key molecule associating periodontitis with acute coronary syndrome.

Benefits of a Physician-Scientist-Tailored Curriculum at a Medical Student-Run Free Clinic.

We designed a curriculum to help sustain pre-clinical knowledge for MD/PhD trainees in their graduate training phase while securing regular providers for our student-run free clinic. MD/PhD involvement increased from 1.91 to 2.45 students-per-clinic-day following implementation, and participants affirmed increased preparedness and comfort entering medical school clinical clerkships.

Understanding the enhanced electrical properties of free-standing graphene paper: the synergistic effect of iodide adsorption into graphene.

Free-standing graphene (FSG) paper plays a vital role in a wide variety of applications as an electrode material. Specifically, the electrical properties of FSG are the most important factor affecting its use as an electrode material. Herein, the vacuum filtration technique is utilized to fabricate GO paper, which is then reductively treated with HI. Initially, the electrical conductivity is measured for GO papers with different thicknesses by varying the concentration of GO precursor as well as the reduction time. The FSG paper with a thickness of 3 microns exhibits the lowest sheet resistance and further characterization is carried out to reveal the origin of this enhancement of electrical properties. The low resistance is attributed to its crystalline nature, stacking height (L c), in-plane crystallite size (L a) and defect density (n D). Meanwhile, iodide ions intercalated into the graphene layers act as hole-carriers, and their intercalation is favoured over adsorption at the surface.

Evaluation of periodontal status and detection of Dialister pneumosintes in cerebral palsy individuals: A Case-Control study.

BACKGROUND: The worldwide prevalence of cerebral palsy among live births is estimated to be between 1.9 and 3.6/1000. The presence of periodontal disease in cerebral palsy children typically is due to bacterial plaque accumulation caused by their inability to correctly clean their own teeth, difficulties in chewing and swallowing food, and improper movements of masticatory muscles and tongue muscles. OBJECTIVES: The objective of this study is to estimate the periodontal status in cerebral palsy individuals and evaluate the presence of Dialister pneumosintes. MATERIALS AND METHODS: Thirty cerebral palsy children from the Spastics Society of Tamilnadu with signs of periodontitis were compared with the same number of age- and gender-matched controls for oral hygiene and periodontal parameters. Subgingival plaque samples were screened for the presence of respiratory pathogen D. pneumosintes by polymerase chain reaction (PCR). RESULTS: A variation was noted between types of cerebral palsy individuals with a mean probing pocket depth value of 6 in spastic type, 4.86 in the ataxic, and 4.3 in the dyskinetic. Clinical attachment level varied from 6.71 in spastic to 5.43 in ataxic and 3.50 in dyskinetic. Oral hygiene index-simplified ranged from 2.764 in spastic to 2.25 in ataxic and 1.41 in dyskinetic. PCR results indicated 25% and 21.7% positivity for D. pneumosintes among cerebral palsy and control group, respectively. The odds ratio calculated to estimate the risk of periodontitis due to D. pneumosintes was 0.765. CONCLUSION: It was concluded that oral hygiene status and severity of periodontitis worsens as the rigidity and muscle tone limiting limb movement increases in cerebral palsy individuals.