A novel bicistronic DNA vaccine with enhanced protective immune response against Bacillus anthracis through DNA prime-protein boost vaccination approach.

Anthrax, by Bacillus anthracis, remains a dreadful fatal hazard worldwide. The currently used anthrax vaccines are plagued by numerous issues that limit their widespread use. As an immunization approach targeting both extracellular antigens and toxins of B. anthracis may achieve better sterile immunity, the present investigation designed a bicistronic secretory anti-anthrax DNA vaccine targeting immune response against toxin and cells. The efficacy of the vaccine was compared with monocistronic DNA vaccines and the currently used anthrax vaccine. For this, mice were immunized with the developed vaccine containing pag (encoding protective antigen to block toxin) and eag genes (encoding EA1 to target cells) of B. anthracis through DNA-prime/Protein-boost (D/P) and DNA prime/DNA-boost (D/D) approaches. There was a >2 and > 5 fold increase in specific antibody level by D/D and D/P approaches respectively, on 42nd days post-immunization (dpi). Serum cytokine profiling showed that both Th1 and Th2 immune responses were elicited, with more Th2 responses in D/P strategy. More importantly, challenge with 100 times LD50 of B. anthracis at 42nd dpi exhibited maximum cumulative survival (83.33 %) by bicistronic D/P approach. Remarkably, immunization with EA1 delayed mortality onset in infection. The study forms the first report on complement-dependent bactericidal activity of antiEA1 antibodies. In short, co-immunization of PA and EA1 through the developed bicistronic DNA vaccine would be an effective immunization approach in anthrax vaccination. Further, D/P strategy could enhance vaccine-induced immunity against B. anthracis. Altogether, the study generates certain critical insights having direct applications in next-generation vaccine development against anthrax.

Thickness uniformity measurements and damage threshold tests of large-area GaAs/AlGaAs crystalline coatings for precision interferometry.

Precision interferometry is the leading method for extremely sensitive measurements in gravitational wave astronomy. Thermal noise of dielectric coatings poses a limitation to the sensitivity of these interferometers. To decrease coating thermal noise, new crystalline GaAs/AlGaAs multilayer mirrors have been developed. To date, the surface figure and thickness uniformity of these alternative low-loss coatings has not been investigated. Surface figure errors, for example, cause small angle scattering and thereby limit the sensitivity of an interferometer. Here we measure the surface figure of highly reflective, substrate-transferred, crystalline GaAs/AlGaAs coatings with a custom scanning reflectance system. We exploit the fact that the reflectivity varies with the thickness of the coating. To increase penetration into the coating, we used a 1550 nm laser on a highly reflective coating designed for a center wavelength of 1064 nm. The RMS thickness variation of a two inch optic was measured to be 0.41 ± 0.05 nm. This result is within 10% of the thickness uniformity, of 0.37 nm RMS, achieved with ion-beam sputtered coatings for the aLIGO detector. We additionally measured a lower limit of the laser induced damage threshold of 64 MW/cm 2 for GaAs/AlGaAs coatings at a wavelength of 1064 nm.

Rapid analysis of pharmaceutical drugs using LIBS coupled with multivariate analysis.

Type 2 diabetes drug tablets containing voglibose having dose strengths of 0.2 and 0.3 mg of various brands have been examined, using laser-induced breakdown spectroscopy (LIBS) technique. The statistical methods such as the principal component analysis (PCA) and the partial least square regression analysis (PLSR) have been employed on LIBS spectral data for classifying and developing the calibration models of drug samples. We have developed the ratio-based calibration model applying PLSR in which relative spectral intensity ratios H/C, H/N and O/N are used. Further, the developed model has been employed to predict the relative concentration of element in unknown drug samples. The experiment has been performed in air and argon atmosphere, respectively, and the obtained results have been compared. The present model provides rapid spectroscopic method for drug analysis with high statistical significance for online control and measurement process in a wide variety of pharmaceutical industrial applications.

Exonal switch down-regulates the expression of CD5 on blasts of acute T cell leukaemia.

To date, CD5 expression and its role in acute T cell lymphoblastic leukaemia (T-ALL) have not been studied closely. We observed a significant reduction in surface expression of CD5 (sCD5) on leukaemic T cells compared to autologous non-leukaemic T cells. In this study, we have shown the molecular mechanism regulating the expression and function of CD5 on leukaemic T cells. A total of 250 patients suffering from leukaemia and lymphoma were immunophenotyped. Final diagnosis was based on their clinical presentation, morphological data and flow cytometry-based immunophenotyping. Thirty-nine patients were found to be of ALL-T origin. Amplification of early region of E1A and E1B transcripts of CD5 was correlated with the levels of surface and intracellular expression of CD5 protein. Functional studies were performed to show the effect of CD5 blocking on interleukin IL-2 production and survival of leukaemic and non-leukaemic cells. Lack of expression of sCD5 on T-ALL blasts was correlated closely with predominant transcription of exon E1B and significant loss of exon E1A of the CD5 gene, which is associated with surface expression of CD5 on lymphocytes. High expression of E1B also correlates with increased expression of cytoplasmic CD5 (cCD5) among leukaemic T cells. Interestingly, we observed a significant increase in the production of IL-2 by non-leukaemic T cells upon CD5 blocking, leading possibly to their increased survival at 48 h. Our study provides understanding of the regulation of CD5 expression on leukaemic T cells, and may help in understanding the molecular mechanism of CD5 down-regulation.

Role of folate-homocysteine pathway gene polymorphisms and nutritional cofactors in Down syndrome: A triad study.

STUDY QUESTION: Do gene-gene and gene-environment interactions in folate-homocysteine (Hcy) pathway have a predisposing role for Down syndrome (DS)? SUMMARY ANSWER: The study provides evidence that in addition to advanced age, maternal genotype, micronutrient deficiency and elevated Hcy levels, individually and in combination, are risk factors for Down syndrome. WHAT IS KNOWN ALREADY: Polymorphisms in certain folate-Hcy-pathway genes (especially the T allele of MTHFR C677T), elevated Hcy and poor folate levels in mothers during pregnancy have been shown to be risk factors for Down syndrome in certain Asian populations (including the eastern region of India), while the same SNPs are not a risk factor in European populations. This conflicting situation alludes to differential gene-environment (nutrition) interactions in different populations which needs to be explored. STUDY DESIGN, SIZE, DURATION: Between 2008 and 2012, 151 Down syndrome triads and 200 age-matched controls (Control mothers n = 186) were included in the study. Seven polymorphisms in six genes of folate-Hcy metabolic pathway, along with Hcy, cysteine (Cys), vitamin B12 (vit-B12) and folate levels, were analysed and compared among the case and control groups. PARTICIPANTS/MATERIALS, SETTING, METHODS: Genotyping was performed by the PCR-RFLP technique. Levels of homocysteine and cysteine were measured by HPLC while vitamin B12 and folate were estimated by chemiluminescence. MAIN RESULTS AND THE ROLE OF CHANCE: We demonstrate that polymorphisms in the folate-Hcy pathway genes in mothers collectively constitute a genotypic risk for DS which is effectively modified by interactions among genes and by the environment affecting folate, Hcy and vitamin B12 levels. The study also supports the idea that these maternal risk factors provide an adaptive advantage during pregnancy supporting live birth of the DS child. LIMITATIONS AND REASONS FOR CAUTION: Our inability to obtain genotype and nutritional assessments of unaffected siblings of the DS children was an important limitation of the study. Also, its confinement to a specific geographic region (the eastern part) of India, and relatively small sample size is a limitation. A parallel investigation on another population could add greater authenticity to the data. WIDER IMPLICATIONS OF THE FINDINGS: For mothers genetically susceptible to deliver a DS child (particularly in South Asia), peri-conceptional nutritional supplementation and antenatal care could potentially reduce the risk of a DS child. Additionally, nutritional strategies could possibly be used for better management of the symptoms of DS children. STUDY FUNDING/COMPETING INTERESTS: The work is funded through Programme support for Genetic disorders by Department of Biotechnology, Government of India to R.R. The authors declare no conflict of interest.

Phenotypic expression of blast resistance gene Pi54 is not affected by its chromosomal position.

KEY MESSAGE: This is a novel report in which chromosomal position of the rice blast resistance gene Pi54 was not found to affect significantly the resistance phenotype or morphological traits. Blast disease caused by Magnaporthe oryzae is a serious constraint in rice production at global level. Pi54 gene imparts resistance against M. oryzae. Three different transgenic lines containing Pi54 and its orthologue Pi54rh were shown to be resistant to different races of M. oryzae. To determine the chromosomal location of Pi54 gene in transgenic lines, inverse PCR was performed. Our analysis showed that in two transgenic lines, Pi54 gene was integrated on chromosomes 6 and 10 at 12.94 and 22.30 Mb, respectively. Similarly, Pi54rh allele was integrated on chromosome 1 at 16.25 Mb. The Pi54 gene present on chromosome 6 was located in a non-coding region whereas in the other TP-Pi54 line, the gene was introgressed on chromosome 10 in between the coding region of SAP domain gene. The Pi54rh was also located in the non coding region flanked by the retrotransposon genes. These rice lines were evaluated for eight different traits related to seed and plant morphology and agronomic features for two consecutive years. The transgenic lines containing Pi54 gene have higher tiller number, grain weight, epicotyl length, and yield compared to the non-transgenic control. Multivariate correlation analysis shows that blast resistance was positively correlated with the number of tillers; thousand grain weight and epicotyl length. These results will facilitate precise utilization of Pi54 gene and its orthologue in breeding programs for the development of rice cultivars with broad spectrum and durable resistance to M. oryzae.

Development of a simple method for the rapid identification of organisms causing anthrax by coagglutination test.

A protective antigen (PA) based coagglutination test was optimized in the present study for the specific and sensitive identification of bacteria causing anthrax in a cost effective and less risky manner. The test showed 100% specificity and sensitivity up to 9 × 10(3) formalinized vegetative cells or 11 ng of PA. The optimized test also detected anthrax toxin directly from the serum as well as blood of anthrax infected animals indicating the potential application for direct diagnosis of anthrax under field conditions.

Development of a simple and rapid method for the specific identification of organism causing anthrax by slide latex agglutination.

UNLABELLED: A specific latex agglutination test (LAT) based on anti-PA (protective antigen) antibodies having detection limit of 5 × 10(4) formalin treated Bacillus anthracis cells or 110 ng of PA was optimized in this study. The optimized LAT could detect anthrax toxin in whole blood as well as in serum from the animal models of anthrax infection. The protocol is a simple and promising method for the specific detection of bacteria causing anthrax under routine laboratory, as well as in field, conditions without any special equipments or expertise. SIGNIFICANCE AND IMPACT OF THE STUDY: The article presents the first report of a latex agglutination test for the specific identification of the cultures of bacteria causing anthrax. As the test is targeting one of anthrax toxic protein (PA), this can also be used to determine virulence of suspected organisms. At the same time, the same LAT can be used directly on whole blood or sera samples under field conditions for the specific diagnosis of anthrax.

Functional Outcomes and a Review of Management Options for Revision Shoulder Arthroplasty.

Introduction: Increase in the number of primary shoulder arthroplasty has led to an increase in the number of revisions which presents many complex challenges and often has inferior outcomes. Materials and Methods: Data was collected retrospectively, and patients were classified using Dines classification. Comprehensive case reviews were done to identify preoperative and intra-operative challenges. The primary outcome measure was Oxford shoulder score (OSS). The secondary measures were range of motion (ROM) and patient satisfaction (very satisfied, satisfied, not satisfied or worse). Results: A total of 32 patients were identified with a mean age of 67.64 years and the most common cause of revision was a combination of bone and soft tissue failure (39.3%). All patients (n=8) with hemiarthroplasty had rotator cuff deficiency while patients with resurfacing had both rotator cuff failure and bony erosion. Four patients needed a proximal humeral osteotomy and six patients needed allograft reconstruction of the glenoid for bone loss. Twenty-one shoulders were revised to reverse total shoulder arthroplasty (TSA), 2 to anatomical TSA and 5 were left with cement spacer in situ. Mean duration of follow-up was 41.6 months. Mean OSS at the last follow-up was 26.88 with statistically significant improvement in ROM. There was no statistical difference in clinical outcomes (p>0.05) based on the type of primary prosthesis or cause of revision. A total of 70% patients were pain free. Patients with infection had inferior outcomes with a mean OSS of 17. Conclusion: Management of patients with failed shoulder arthroplasty is often challenging but has good clinical outcome except in infections.

Compositional analysis of copper and iron-based alloys using LIBS coupled with chemometric method.

The present manuscript deals with the utility of the calibration-free LIBS and calibration curve methods for the compositional study of different alloys using laser-induced breakdown spectroscopy (LIBS). In the process of alloying in the smelting industry, metal concentration in different alloys affects the physical and chemical properties of the final products. Therefore, LIBS can be used as an efficient quantitative analysis tool for online monitoring of the quality of the products. This is because LIBS can be performed online, in situ, without any pre-processing, and need no sample preparation for the compositional analysis of any type of materials present in any phase (solid, liquid, gas or even molten alloys in the industries). In the present study, four alloys (three copper and one iron-based alloy) consisting of Cu, Al, Zn, Ni, Fe, Cr and Mn as major and Sn and Si as minor elements were selected for the study using calibration-free laser-induced breakdown spectroscopy (CF-LIBS) and calibration curve method i.e. partial least square regression (PLSR). For the CF-LIBS method, the temporal delay has been optimized in order to satisfy the optically thin and local thermal equilibrium (LTE) condition of the plasma. For the PLSR method, different regions of the strongest emission lines of constituents have been selected for quantitative analysis. The study of time-resolved LIBS spectra and the variation of plasma parameters with respect to the delay time is also discussed. The utility of the combined technique of CF-LIBS with the PLSR method for rapid monitoring and quality assessment of desired material/products without any sample pretreatment, thus reducing the cost of the analysis, is presented in this paper.

Confinement and interaction of single indirect excitons in a voltage-controlled trap formed inside double InGaAs quantum Wells.

Voltage-tunable quantum traps confining individual spatially indirect and long-living excitons are realized by providing a coupled double quantum well with nanoscale gates. This enables us to study the transition from confined multiexcitons down to a single, electrostatically trapped indirect exciton. In the few exciton regime, we observe discrete emission lines identified as resulting from a single dipolar exciton, a biexciton, and a triexciton, respectively. Their energetic splitting is well described by Wigner-like molecular structures reflecting the interplay of dipolar interexcitonic repulsion and spatial quantization.

Current State and Future Perspective of Diabetic Wound Healing Treatment: Present Evidence from Clinical Trials.

Diabetes is a chronic metabolic condition that is becoming more common and is characterised by sustained hyperglycaemia and long-term health effects. Diabetes-related wounds often heal slowly and are more susceptible to infection because of hyperglycaemia in the wound beds. The diabetic lesion becomes harder to heal after planktonic bacterial cells form biofilms. A potential approach is the creation of hydrogels with many functions. High priority is given to a variety of processes, such as antimicrobial, pro-angiogenesis, and general pro-healing. Diabetes problems include diabetic amputations or chronic wounds (DM). Chronic diabetes wounds that do not heal are often caused by low oxygen levels, increased reactive oxygen species, and impaired vascularization. Several types of hydrogels have been developed to get rid of contamination by pathogens; these hydrogels help to clean up the infection, reduce wound inflammation, and avoid necrosis. This review paper will focus on the most recent improvements and breakthroughs in antibacterial hydrogels for treating chronic wounds in people with diabetes. Prominent and significant side effects of diabetes mellitus include foot ulcers. Antioxidants, along with oxidative stress, are essential to promote the healing of diabetic wounds. Some of the problems that can come from a foot ulcer are neuropathic diabetes, ischemia, infection, inadequate glucose control, poor nutrition, also very high morbidity. Given the worrying rise in diabetes and, by extension, diabetic wounds, future treatments must focus on the rapid healing of diabetic wounds.

Proniosomal formulation of curcumin having anti-inflammatory and anti-arthritic activity in different experimental animal models.

Curcumin, the active ingredient of the spice turmeric, has a long history as an herbal remedy for a variety of diseases. Transdermal drug delivery has been recognized as an alternative route to oral delivery. Proniosomes offer a versatile vesicle delivery concept with the potential for drug delivery via the transdermal route. In this study, different proniosomal gel bases were prepared by the ether injection method, using Span 60 and Span 80, Tween 20, cholesterol, and formulation PA2. They were characterized by scanning electron microscopy, revealing vesicular structures, and assessed for stability and effect on in vitro skin permeation using rat skin. Anti-inflammatory and anti-arthritic effects of formulation PA2 and PB1 were compared with a standard market product containing indomethacin. The effect of formulation PA2 and PB1 was evaluated for acute inflammation in carrageenan induced rat paw edema and for chronic inflammation in complete Freud's adjuvant (CFA) induced arthritis in rats. Further histopathological and radiographic evaluation was performed. The investigated curcumin loaded proniosomal formula proved to be non-irritant, non-toxic, but had lower anti-inflammatory and anti-arthritic effects than the marketed indomethacin products.

A Study of Otological Manifestations of Temporal Bone Fractures.

The aim of study was to evaluate various otological manifestations of temporal bone fractures and to suggest their management. This prospective observational study was conducted in a tertiary care hospital of Armed Forces over 2.5 years in 57 cases of temporal bone fracture in patients of age group of 12-59 yrs comprising 49 males and 8 females. Radioimaging was done for diagnosing the fracture, mapping its extent and for clinical correlation. Hearing was assessed by tuning forks, free field hearing and pure tone audiogram as per the fitness of patient. Majority of patients were in the age group 20-40 years who had met with road accidents. Important otological features included Battle sign (24.6%), ear canal laceration (53%), tympanic membrane perforation (7%), haemotymanum (40%), ear discharge (66.7%), hearing loss (63%), vertigo (16%), tinnitus (21%), facial paralysis (68%) and cerebrospinal fluid otorrhea (3.5%). Otic capsule sparing fracture was three times more common than violating ones. Facial palsies were treated with oral steroid with complete recovery in 56.41% cases but three required decompression surgery. 4 patients underwent tympanoplasty. Both the cerebrospinal fluid leaks stopped in 2 weeks time. Facial palsy is as common as hearing loss. TM perforation is as rare as CSF leak. Otic capsule violating fracture line is less common. Hearing loss shows recovery in most of the cases. Delayed onset positional vertigo is more common than acute vertigo. Large numbers of immediate onset complete facial palsy do recover from injury without surgery.

Sudden loss of smell and taste: clinical predictors of coronavirus disease 2019 infection.

OBJECTIVES: This study aimed: to evaluate the association between coronavirus disease 2019 infection and olfactory and taste dysfunction in patients presenting to the out-patient department with influenza-like illness, who underwent reverse transcription polymerase chain reaction testing for coronavirus; and to determine the sensitivity, specificity, and positive and negative predictive values of olfactory and taste dysfunction and other symptoms in these patients. METHODS: Patients presenting with influenza-like illness to the study centre in September 2020 were included in the study. The symptoms of patients who tested positive for coronavirus on reverse transcription polymerase chain reaction testing were compared to those with negative test results. RESULTS: During the study period, 909 patients, aged 12-70 years, presented with influenza-like illness; of these, 316 (34.8 per cent) tested positive for coronavirus. Only the symptoms of olfactory and taste dysfunction were statistically more significant in patients testing positive for coronavirus than those testing negative. CONCLUSION: During the pandemic, patients presenting to the out-patient department with sudden loss of sense of smell or taste may be considered as positive for coronavirus disease 2019, until proven otherwise.

Enrichment of invariant natural killer T cells in the bone marrow of visceral leishmaniasis patients.

Lipid antigens of Leishmania donovani like lipophosphoglycans are shown as a potent ligand for the activation of invariant natural killer T (iNKT) cells. It is reported that activation of iNKT cells augments the disease pathology in experimental visceral leishmaniasis (VL). In this study, we demonstrate the enrichment of iNKT cells in the bone marrow, one of the disease sites among patients with VL.

High concentration of adenosine in human visceral leishmaniasis despite increased ADA and decreased CD73.

Absence of an effective Th-1 response has been demonstrated as a major cause for the disease pathology among patients with visceral leishmaniasis (VL). Defining strategies to prevent the development of Th-2 response and/or initiate/activate effective Th-1 response may be of help to reduce the growing incidence of drug unresponsiveness. Adenosine, which is considered as an endogenous anti-inflammatory agent is generated in injured/inflamed tissues by the enzymatic catabolism of adenosine triphosphate (ATP), and it suppresses inflammatory responses of essentially all immune cells. The extracellular adenosine-producing pathway comprises two major enzymes CD39 (ATP → ADP → AMP) and CD73 (AMP → Adenosine). In contrast, the adenosine-degrading pathway contains only one major enzyme adenosine deaminase (ADA). Our study shows high concentration of adenosine in diseased condition, varying expression of enzyme involved in adenosine-producing (CD73↓) and adenosine-degrading (ADA↑) pathways. These are less studied in infections like VL but are very important in terms of endogenous regulation of immune response among patients.

Anosmia: A Clinical Indicator of COVID-19 Reinfection.

COVID-19, an ongoing world pandemic, is caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). Many organizations have recognized that COVID-19 patients may have sudden loss of smell or taste and have included these symptoms in their diagnostic guidelines. However, the occurrence of anosmia and dysgeusia in COVID-19 reinfection is yet to be ascertained.

Cross-sectional study of kidney stones by laser-induced breakdown spectroscopy.

We performed laser-induced breakdown spectroscopy (LIBS) for the in situ quantitative estimation of elemental constituents distributed in different parts of kidney stones obtained directly from patients by surgery. We did this by focusing the laser light directly on the center, shell, and surface of the stones to find the spatial distribution of the elements inside the stone. The elements detected in the stones were calcium, magnesium, manganese, copper, iron, zinc, strontium, sodium, potassium, carbon, hydrogen, nitrogen, oxygen, phosphorus, sulfur, and chlorine (Cl), etc. We optimized the LIBS signals by varying the laser energy from 10 mJ to 40 mJ to obtain the best signal-to-background and signal-to-noise ratios. We estimated the quantities of different elements in the stones by drawing calibration curves, plotting graphs of the analyte signal versus the absolute concentration of the elements in standard samples. The detection limits of the calibration curves were discussed. The concentrations of the different elements were found to be widely different in different stones found in different age groups of patients. It was observed that stones containing higher amounts of copper also possessed higher amounts of zinc. In general, the concentrations of trace elements present in the kidney stones decreased as we moved from center to shell and surface. Our results also revealed that the concentrations of elements present in the stones increased with the age of the patients. The results obtained from the calibration curves were compared with results from inductively coupled plasma mass spectrometry (ICP-MS). We also used the intensity ratios of different elemental lines to find the spatial distribution of different elements inside the kidney stones.

Investigation of common Indian edible salts suitable for kidney disease by laser induced breakdown spectroscopy.

Salt is an essential and important dietary mineral for maintaining life. Currently, the issue of the potential benefit or damage from salt intake in chronic kidney disease patients is controversial. The attempt of this article is to bring into focus the potential role of elements particularly sodium, Na, and potassium, K, which are the main constituents of dietary salts, in kidney patients by using laser-induced breakdown spectroscopy (LIBS). LIBS spectra of different salt samples have been recorded in the spectral region 200-500 nm with spectral resolution 0.1 nm and in the spectral region 200-900 nm with spectral resolution 0.75 nm. Quantitative elemental study was carried out to determine the constituents of different types of common Indian edible salts by using the calibration-free LIBS method. Our experimental results demonstrate that Saindha salt (commonly known as rock salt) is more beneficial than other edible salts for patients suffering from chronic kidney disease. The results of the quantitative elemental analysis of the salts obtained from LIBS measurements are also compared to atomic absorption spectroscopy (AAS).