A novel prokaryotic vector for identification and selection of recombinants: Direct use of the vector for expression studies in E. coli

<p>Abstract</p> <p>Background</p> <p>The selection of bacterial recombinants that harbour a desired insert, has been a key factor in molecular cloning and a series of screening procedures need to be performed for selection of clones carrying the genes of interest. The conventional cloning techniques are reported to have problems such as screening high number of colonies, generation of false positives, setting up of control ligation mix with vector alone etc.</p> <p>Results</p> <p>We describe the development of a novel dual cloning/expression vector, which enables to screen the recombinants directly and expression of the gene of interest. The vector contains Green fluorescence protein (GFP) as the reporter gene and is constructed in such a way that the <it>E. coli </it>cells upon transformation with this vector does not show any fluorescence, but readily fluoresce upon insertion of a foreign gene of interest. The same construct could be easily used for screening of the clones and expression studies by mere switching to specific hosts.</p> <p>Conclusions</p> <p>This is the first vector reported that takes the property of colour or fluorescence to be achieved only upon cloning while all the other vectors available commercially show loss of colour or loss of fluorescence upon cloning. As the fluorescence of GFP depends on the solubility of the protein, the intensity of the fluorescence would also indicate the extent of solubility of the expressed target protein.</p

Molecular characterization of typing and subtyping of Staphylococcal cassette chromosome SCCmec types I to V in methicillin-resistant Staphylococcus aureus from clinical isolates from COVID-19 patients

Background and Objectives: Methicillin resistance is acquired by the bacterium due to mecA gene which codes for penicillin-binding protein (PBP2a) having low affinity for β-lactam antibiotics. mecA gene is located on a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec). SCCmec genomic island comprises two site-specific recombinase genes namely ccrA and ccrB [cassette chromosome recombinase] accountable for mobility. Currently, SCCmec elements are classified into types I, II, III, IV and V based on the nature of the mec and ccr gene complexes and are further classified into subtypes according to variances in their J region DNA. SSCmec type IV has been found in community-acquired isolates with various genetic backgrounds. The present study was undertaken to categorize the types of SCCmec types and subtypes I, II, III, IVa, b, c, d, and V and PVL genes among clinical MRSA isolates from COVID-19 confirmed cases. Materials and Methods: Based on the Microbiological and Molecular (mecA gene PCR amplification) confirmation of MRSA isolated from 500 MRSA SCCmec clinical samples, 144 cultures were selected for multiplex analysis. The multiplex PCR method developed by Zhang et al. was adapted with some experimental alterations to determine the specific type of these isolates. Results: Of the total 500 MRSA, 144 MRSA (60 were CA-MRSA and 84 were HA-MRSA) were selected for characterization of novel multiplex PCR assay for SSCmec Types I to V in MRSA. Molecular characterization of multiplex PCR analysis revealed results compare to the phenotypic results. Of the 60 CA-MRSA; in 56 MRSA strains type IVa was found and significantly defined as CA-MRSA while 4 strains showed mixed gens subtypes. Type II, III, IA, and V were present in overall 84 HA-MRSA. Molecular subtyping was significantly correlated to define molecularly as CA-MRSA and HA-MRSA however 15 (10%) strains showed mixed genes which indicates the alarming finding of changing epidemiology of CA-MRSA and HA-MRSA as well. Conclusion: We have all witnessed of COVID-19 pandemic, and its mortality was mostly associated with co-morbid conditions and secondary infections of MDR pathogens. Rapid detections of causative agents of these superbugs with their changing epidemiology by investing in typing and subtyping clones are obligatory. We have described an assay designed for targeting SSCmec types and subtypes I, II, III, IVa,V according to the current updated SCCmec typing system. Changing patterns of molecular epidemiology has been observed by this newly described assay

Long-term evaluation of a course on evidence-based public health in the U.S. and Europe

The evidence-based public health course equips public health professionals with skills and tools for applying evidence-based frameworks and processes in public health practice. To date, training has included participants from all the 50 U.S. states, 2 U.S. territories, and multiple other countries besides the U.S. This study pooled follow-up efforts (5 surveys, with 723 course participants, 2005-2019) to explore the benefits, application, and barriers to applying the evidence-based public health course content. All analyses were completed in 2020. The most common benefits (reported by \u3e80% of all participants) were identifying ways to apply knowledge in their work, acquiring new knowledge, and becoming a better leader who promotes evidence-based approaches. Participants most frequently applied course content to searching the scientific literature (72.9%) and least frequently to writing grants (42.7%). Lack of funds for continued training (35.3%), not having enough time to implement evidence-based public health approaches (33.8%), and not having coworkers trained in evidence-based public health (33.1%) were common barriers to applying the content from the course. Mean scores were calculated for benefits, application, and barriers to explore subgroup differences. European participants generally reported higher benefits from the course (mean difference=0.12, 95% CI=0.00, 0.23) and higher frequency of application of the course content to their job (mean difference=0.17, 95% CI=0.06, 0.28) than U.S. participants. Participants from later cohorts (2012-2019) reported more overall barriers to applying course content in their work (mean difference=0.15, 95% CI=0.05, 0.24). The evidence-based public health course represents an important strategy for increasing the capacity (individual skills) for evidence-based processes within public health practice. Organization-level methods are also needed to scale up and sustain capacity-building efforts

Evaluating a train-the-trainer approach for improving capacity for evidence-based decision making in public health

BACKGROUND: Evidence-based public health gives public health practitioners the tools they need to make choices based on the best and most current evidence. An evidence-based public health training course developed in 1997 by the Prevention Research Center in St. Louis has been taught by a transdisciplinary team multiple times with positive results. In order to scale up evidence-based practices, a train-the-trainer initiative was launched in 2010. METHODS: This study examines the outcomes achieved among participants of courses led by trained state-level faculty. Participants from trainee-led courses in four states (Indiana, Colorado, Nebraska, and Kansas) over three years were asked to complete an online survey. Attempts were made to contact 317 past participants. One-hundred forty-four (50.9 %) reachable participants were included in analysis. Outcomes measured include frequency of use of materials, resources, and other skills or tools from the course; reasons for not using the materials and resources; and benefits from attending the course. Survey responses were tabulated and compared using Chi-square tests. RESULTS: Among the most commonly reported benefits, 88 % of respondents agreed that they acquired knowledge about a new subject, 85 % saw applications for the knowledge to their work, and 78 % agreed the course also improved abilities to make scientifically informed decisions at work. The most commonly reported reasons for not using course content as much as intended included not having enough time to implement evidence-based approaches (42 %); other staff/peers lack training (34 %); and not enough funding for continued training (34 %). The study findings suggest that utilization of course materials and teachings remains relatively high across practitioner groups, whether they were taught by the original trainers or by state-based trainers. CONCLUSIONS: The findings of this study suggest that train-the-trainer is an effective method for broadly disseminating evidence-based public health principles. Train-the-trainer is less costly than the traditional method and allows for courses to be tailored to local issues, thus making it a viable approach to dissemination and scale up of new public health practices. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12913-015-1224-2) contains supplementary material, which is available to authorized users

A survey tool for measuring evidence-based decision making capacity in public health agencies

BACKGROUND: While increasing attention is placed on using evidence-based decision making (EBDM) to improve public health, there is little research assessing the current EBDM capacity of the public health workforce. Public health agencies serve a wide range of populations with varying levels of resources. Our survey tool allows an individual agency to collect data that reflects its unique workforce. METHODS: Health department leaders and academic researchers collaboratively developed and conducted cross-sectional surveys in Kansas and Mississippi (USA) to assess EBDM capacity. Surveys were delivered to state- and local-level practitioners and community partners working in chronic disease control and prevention. The core component of the surveys was adopted from a previously tested instrument and measured gaps (importance versus availability) in competencies for EBDM in chronic disease. Other survey questions addressed expectations and incentives for using EBDM, self-efficacy in three EBDM skills, and estimates of EBDM within the agency. RESULTS: In both states, participants identified communication with policymakers, use of economic evaluation, and translation of research to practice as top competency gaps. Self-efficacy in developing evidence-based chronic disease control programs was lower than in finding or using data. Public health practitioners estimated that approximately two-thirds of programs in their agency were evidence-based. Mississippi participants indicated that health department leaders' expectations for the use of EBDM was approximately twice that of co-workers' expectations and that the use of EBDM could be increased with training and leadership prioritization. CONCLUSIONS: The assessment of EBDM capacity in Kansas and Mississippi built upon previous nationwide findings to identify top gaps in core competencies for EBDM in chronic disease and to estimate a percentage of programs in U.S. health departments that are evidence-based. The survey can serve as a valuable tool for other health departments and non-governmental organizations to assess EBDM capacity within their own workforce and to assist in the identification of approaches that will enhance the uptake of EBDM processes in public health programming and policymaking. Localized survey findings can provide direction for focusing workforce training programs and can indicate the types of incentives and policies that could affect the culture of EBDM in the workplace

Frequency of Arrhythmias and Postural Orthostatic Tachycardia Syndrome in Patients With Marfan Syndrome: A Nationwide Inpatient Study.

Background Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder affecting multiple systems, particularly the cardiovascular system. The leading causes of death in MFS are aortopathies and valvular disease. We wanted to identify the frequency of arrhythmia and postural orthostatic tachycardia syndrome, length of hospital stay, health care-associated costs (HAC), and in-hospital mortality in patients with MFS. Methods and Results The National Inpatient Sample database from 2005 to 2014 was queried using International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes for MFS and arrhythmias. Patients were classified into subgroups: supraventricular tachycardia, ventricular tachycardia (VT), atrial fibrillation, atrial flutter, and without any type of arrhythmia. Data about length of stay, HAC, and in-hospital mortality were also abstracted from National Inpatient Sample database. Adjusted HAC was calculated as multiplying HAC and cost-to-charge ratio; 12 079 MFS hospitalizations were identified; 1893 patients (15.7%) had an arrhythmia; and 4.9% of the patients had postural orthostatic tachycardia syndrome. Median values of length of stay and adjusted HAC in VT group were the highest among the groups (VT: 6 days, $18 975.8; supraventricular tachycardia: 4 days,$11 906.6; atrial flutter: 4 days, $11 274.5; atrial fibrillation: 5 days,$10431.4; without any type of arrhythmia: 4 days, $8336.6; both P=0.0001). VT group had highest in-patient mortality (VT: 5.3%, atrial fibrillation: 4.1%, without any type of arrhythmia: 2.1%, atrial flutter: 1.7%, supraventricular tachycardia: 0%; P<0.0001) even after adjustment for potential confounders (without any type of arrhythmia versus VT; odds ratio [95% CI]: 3.18 [1.62-6.24], P=0.001). Conclusions Arrhythmias and postural orthostatic tachycardia syndrome in MFS were high and associated with increased length of stay, HAC, and in-hospital mortality especially in patients with VT

Influence of Primary Care Physician Availability and Socioeconomic Deprivation on Breast Cancer from 1988 to 2008: A Spatio-Temporal Analysis

Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer death among women in the United States. It is unclear how county-level primary care physician (PCP) availability and socioeconomic deprivation affect the spatial and temporal variation of breast cancer incidence and mortality.We used the 1988-2008 public-use county-based data from nine Surveillance, Epidemiology, and End Results (SEER) programs to analyze the temporal and spatial disparity of PCP availability and socioeconomic deprivation on early-stage incidence, advanced-stage incidence and breast cancer mortality. The spatio-temporal analysis was implemented by a novel structural additive modeling approach.Greater PCP availability was significantly associated with higher early-stage incidence, advanced-stage incidence and mortality during the entire study period while socioeconomic deprivation was significantly negatively associated with early-stage incidence, advanced-stage incidence, and mortality up to 1992. However, the observed influence of PCP availability and socioeconomic deprivation varied by county.We showed important associations of PCP availability and socioeconomic deprivation with the three breast cancer indicators. However, the effect of these associations varied over time and across counties. The association of PCP availability and socioeconomic deprivation was stronger in selected counties

Individual and setting level predictors of the implementation of a skin cancer prevention program: a multilevel analysis

<p>Abstract</p> <p>Background</p> <p>To achieve widespread cancer control, a better understanding is needed of the factors that contribute to successful implementation of effective skin cancer prevention interventions. This study assessed the relative contributions of individual- and setting-level characteristics to implementation of a widely disseminated skin cancer prevention program.</p> <p>Methods</p> <p>A multilevel analysis was conducted using data from the Pool Cool Diffusion Trial from 2004 and replicated with data from 2005. Implementation of Pool Cool by lifeguards was measured using a composite score (implementation variable, range 0 to 10) that assessed whether the lifeguard performed different components of the intervention. Predictors included lifeguard background characteristics, lifeguard sun protection-related attitudes and behaviors, pool characteristics, and enhanced (<it>i.e</it>., more technical assistance, tailored materials, and incentives are provided) versus basic treatment group.</p> <p>Results</p> <p>The mean value of the implementation variable was 4 in both years (2004 and 2005; SD = 2 in 2004 and SD = 3 in 2005) indicating a moderate implementation for most lifeguards. Several individual-level (lifeguard characteristics) and setting-level (pool characteristics and treatment group) factors were found to be significantly associated with implementation of Pool Cool by lifeguards. All three lifeguard-level domains (lifeguard background characteristics, lifeguard sun protection-related attitudes and behaviors) and six pool-level predictors (number of weekly pool visitors, intervention intensity, geographic latitude, pool location, sun safety and/or skin cancer prevention programs, and sun safety programs and policies) were included in the final model. The most important predictors of implementation were the number of weekly pool visitors (inverse association) and enhanced treatment group (positive association). That is, pools with fewer weekly visitors and pools in the enhanced treatment group had significantly higher program implementation in both 2004 and 2005.</p> <p>Conclusions</p> <p>More intense, theory-driven dissemination strategies led to higher levels of implementation of this effective skin cancer prevention program. Issues to be considered by practitioners seeking to implement evidence-based programs in community settings, include taking into account both individual-level and setting-level factors, using active implementation approaches, and assessing local needs to adapt intervention materials.</p

Study and characetrization of plastic encapsulated packages for MEMS

Technological advancement has thrust MEMS design and fabrication into the forefront of modern technologies. It has become sufficiently self-sustained to allow mass production. The limiting factor which is stalling commercialization of MEMS is the packaging and device reliability. The challenging issues with MEMS packaging are application specific. The function of the package is to give the MEMS device mechanical support, protection from the environment, and electrical connection to other devices in the system. The current state of the art in MEMS packaging transcends the various packaging techniques available in the integrated circuit (IC) industry. At present the packaging of MEMS includes hermetic ceramic packaging and metal packaging with hermetic seals. For example the ADXL202 accelerometer from the Analog Devices. Study of the packaging methods and costs show that both of these methods of packaging are expensive and not needed for majority of MEMS applications. Due to this the cost of current MEMS packaging is relatively high, as much as 90% of the finished product. Reducing the cost is therefore of the prime concern. This Thesis explores the possibility of an inexpensive plastic package for MEMS sensors like accelerometers, optical MEMS, blood pressure sensors etc. Due to their cost effective techniques, plastic packaging already dominates the IC industry. They cost less, weigh less, and their size is small. However, porous nature of molding materials allows penetration of moisture into the package. The Thesis includes an extensive study of the plastic packaging and characterization of three different plastic package samples. Polymeric materials warp upon absorbing moisture, generating hygroscopic stresses. Hygroscopic stresses in the package add to the thermal stress due to high reflow temperature. Despite this, hygroscopic characteristics of the plastic package have been largely ignored. To facilitate understanding of the moisture absorption, an analytical model is presented in this Thesis. Also, an empirical model presents, in this Thesis, the parameters affecting moisture ingress. This information is important to determine the moisture content at a specific time, which would help in assessing reliability of the package. Moisture absorption is modeled using the single phase absorption theory, which assumes that moisture diffusion occurs freely without any bonding with the resin. This theory is based on the Fick’s Law of diffusion, which considers that the driving force of diffusion is the water concentration gradient. A finite difference simulation of one-dimensional moisture diffusion using the Crank-Nicolson implicit formula is presented. Moisture retention causes swelling of compounds which, in turn, leads to warpage. The warpage induces hygroscopic stresses. These stresses can further limit the performance of the MEMS sensors. This Thesis also presents a non invasive methodology to characterize a plastic package. The warpage deformations of the package are measured using Optoelectronic holography (OEH) methodology. The OEH methodology is noninvasive, remote, and provides results in full-field-of-view. Using the quantitative results of OEH measurements of deformations of a plastic package, pressure build up can be calculated and employed to assess the reliability of the package

Lipoma of small intestine

Lipomas of the gastrointestinal tract (GIT) are rare. They are usually submucosal, but rarely involve the muscle layer or serosa. Most ot submucosal lipomas of GIT are present in the colon (65-75%), small intestine (25%) and rarely in stomach or jejunum. These lesions are usually asymptomatic and rarely are incidentally found in autopsies. During lifetime, they are detected when they cause intussusceptions and obstructive symptoms. These lesions can also rarely become ulcerated causing symptoms that come to notice. The present case report is of submucous lipoma in the terminal ileum. A 45-year-old female came complaining of pain in the abdomen, gradually increasing over a period of 4 days. Computerized tomography scan revealed a mass in the submucosa. She was operated, and histopathology examination confirmed the mass as lipoma in the submucosa as well as within the muscle layer of the small intestine