Design and Optimization of a Mycoplasma Detection Assay

Mycoplasma are among the smallest free living microorganisms. These bacteria grow slowly, lack a rigid cell wall and are not eliminated by filter sterilization methods used in tissue culture. Mycoplasma infection affects biochemical and genetic aspects of cultured cells, resulting in experimental inconsistency. Therefore, it is necessary to establish routine testing for mycoplasma contamination in tissue culture laboratories. Our goal is to develop a reliable and cost-effective test for mycoplasma in cell culture based on established methods found in literature. We first cloned and sequenced a PCR product from a commercial mycoplasma detection kit. Sequencing revealed the 16s rRNA as the target for mycoplasma detection; we confirmed this target by conducting a literature search. PCR primers were designed using 16s rRNA gene as a target. We set-up reactions and optimized conditions for the real-time PCR assay to detect the target and confirmed amplicon size with agarose gel electrophoresis. We identified that 56oC was the best temperature for the PCR and found that agarose gel electrophoresis was a better detection method because it identified the size to confirm the proper product. The primers we ordered to develop this assay produce the proper band; however, results of several assays have been inconsistent as sometimes a known positive sample fails to amplify. As well, in several PCR reactions the negative showed a signal. The overall reaction needs improvements to have greater reliability and to eliminate all sources of contamination. Research is continuing results are not final

Preliminary soil and groundwater assessment of the Mantinea Development area, East Kimberley, Western Australia

In 2008, the Ord Stage 2 or ‘Ord East Kimberley Expansion Project’ was initiated by the Western Australian Government. The goal of the project was to advance development in the East Kimberley and to bring to market the Weaber Plain (Goomig Farmlands) and Knox Plain. In addition to the existing Ord River Irrigation Area (ORIA) Stage 1 (14 000ha), current Goomig and proposed Knox Plain areas (14 300ha), an estimated 50 000ha of land has potential for irrigation in the region. One of the areas proposed for future development is the Mantinea Development area. It is located on the south bank of the Ord River, 30km from Kununurra. It is north-west of the existing ORIA, which is located on the Ivanhoe and Packsaddle plains, and south-west of the most recent Stage 2 Goomig Farmlands, released in 2012. For the Mantinea Development, the state government is seeking a proponent from the private sector to manage the development process and operate the new farmlands created. The potential development area covers about 9500ha, 430ha of which has been set aside in the Mantinea conservation excision. To support the development process, DAFWA has reviewed relevant soil and water data and undertaken a preliminary assessment of that information. The Mantinea Flat – Carlton Plain area was identified as potentially suitable for irrigated agriculture by the Department of Agriculture and CSIRO as early as 1944, following soil surveys of about 12 000ha in the area. Preliminary soil survey results show that the soils of the proposed development area comprise a mixture of modern alluvial sediments, from fine cracking clays (1500ha, 17%) to sands and loams (6600ha, 73%), and stony soils (800ha, 10%). From this limited data, 4796ha (53%) of the proposed development area has a potentially high capability for irrigated agriculture, 2876ha (32%) is potentially capable but requires further investigation, and 1395ha (15%) of the area has a low capability. Areas assessed as having low capability for irrigated agriculture predominantly have salinity risk, poor drainage, shallow basement or unsuitable soil types

Recruitment Patterns of Juvenile Fish at an Artificial Reef Area in the Gulf of Mexico

In 2011 the Texas Parks and Wildlife Department and the Coastal Conservation Association of Texas deployed over 4,000 concrete culverts in a designated artificial reef area off Port Mansfield, Texas, to enhance habitat for sport fish species in the Gulf of Mexico. A study was conducted to assess juvenile fish recruitment at varying culvert densities within the artificial reef area. Standard monitoring units for the recruitment of reef fish (SMURFs) were used to sample juvenile fish, and these collections were compared with visual scuba surveys. The 0.027-m3 SMURFs were placed at four different culvert densities (0, 1–50, 51–100, and 101+ culverts in a 30-m radius) and sampled during 2013 to 2014. Measurements of rugosity, vertical relief, and percent cover were collected to elucidate factors that drive juvenile recruitment. Average species richness was highest at sites with no culverts and lowest at dense culvert sites. Species compositions were significantly different between sampling locations with no culverts and all locations with culverts; average similarity was 33.8%. Belted Sandfish Serranus subligarius was the most ubiquitous species among all sampling locations. Visual scuba surveys at the same locations showed lower diversity indices of the juvenile fish community than observed by SMURFs and were only 14% similar to the community sampled by the SMURFs. These findings suggest SMURFs are a more effective tool for examining juvenile fish at an artificial reef due to the cryptic nature of juveniles and the low visibility around shallow reefs. Additionally, commercially important Yellowedge Grouper Hyporthodus flavolimbatus, Warsaw Grouper H. nigritus, and Red Snapper Lutjanus campechanus juveniles were only found at SMURFs at sampling locations with no culverts. The presence of juveniles of these key species suggest that fisheries management may benefit from creating low-relief reef patches away from the main reef where juvenile fish can recruit and grow

Combined Effects of Legumes with Phosphorus Fertilizer on Nutrient Balances and Gross Margins in Maize (Zea mays L.) systems of Kabete sub-County, Kenya

Calculation of soil nutrient balances and gross margins (GM) is imperative in ascertaining effect of innovative technologies on soil fertility and farm profitability. A field experiment to evaluate effect of combined legumes and phosphorus fertilizer on soil N, P and K balances and crop GM in maize (Zea mays L.) systems was set up in Kabete Division, Kenya, in the long and short rainy seasons of 2012. The experimental set up was a randomized complete block design (RCBD) with a split plot arrangement. The main plots comprised cropping systems; (i) monocropping (sole maize), (ii) intercropping [white lupin (Lupinus albus L.)/maize (L/M) and chickpea (Cicer arietinum L.)/maize (CP/M)], and (iii) rotation [white lupin-maize (L-M) and chickpea-maize (CP-M)]. The split plots were phosphorus (P) fertilizers; Minjingu phosphate rock (MPR) and triple superphosphate (TSP), and (iii) no P fertilizer applied (CTRL). Soil N, P and K balances and gross margins were analyzed at plot level using NUTrient MONitoring (NUTMON - now known as MonQi) Tool box. Nutrient balances were negative across cropping systems and P sources except for K in M/CP (CTRL and TSP) intercrop. Significantly less negative N balances were obtained in maize monocrop (MPR), CP/M (CTRL) intercrop, CP-M (TSP) rotation, and L/M (MPR) intercrop. L/M (CTRL and TSP) intercrop and L-M (CTRL and TSP) rotation recorded more negative (highest losses) N balances. Across P sources, the maize monocrop, M/L intercrop and L-M rotation had significantly more negative P balances, than CP-M rotation and M/CP intercrop. P balances, across P fertilizers, were significantly less negative in M/CP compared to M/L intercrop. Less negative P balances were recorded in CTRL treatment compared to TSP and MPR across cropping systems. M/L (CTRL and TSP) intercrop system had pronounced negative K balances. In the rotation systems, significantly less negative balances were observed when maize was rotated with chickpea compared to lupin across all P sources. Pronounced GMs were realized in M/L intercrop (TSP) followed by L-M (TSP) and lowest in M/L (TSP and CTRL). The N, P and K nutrient balances in response to P sources and cropping systems exhibited a negative relationship with crop GM. The positive GMs obtained were thus at the expense of soil nutrient mining as treatments with high nutrient losses, case for N and P, had the highest GMs. Considering nutrient balance studies alongside economic analysis has thus demonstrated the hidden environmental costs in the positive crop GMs and by extension the efficiency of such production systems. As a result, increased GMs under introduced technologies are not sustainable unless the same is matched with adequate nutrient replenishments to balance those lost through harvested products and other nutrient loss pathways. Farmers would, actually, go for those technologies that not only maximize yields but also accrue high profits. In the context of this study, and in order of GM (from highest) analysis, M/L intercrop, maize monocrop and L-M rotation with application of TSP are such technologies. In the long-run however these technologies will prove untenable due to nutrient mining. Nonetheless to guarantee efficient production and sustainable maize systems, following application of P fertilizer and legume integration, it is important that profits accrued from farm sales be used to purchase fertilizers and/or support practices geared towards replenishing mined soil nutrients. This way farm profits realized will not be at the expense of nutrient mining. Keywords: Cropping systems; gross margins; Kabete sub-County; MonQi; Nutrient Balances; Rock phosphates

Correlation between CD4 T cell counts and virus compartmentalization in genital and systemic compartments of HIV-infected females

AbstractThe majority of infection by the human immunodeficiency virus (HIV-1) across the world occurs by heterosexual transmission and is likely mediated by virus present in genital secretions. In spite of this, infection is followed by clinical markers of the virus present in blood, which may not be representative of the virus involved in transmission. In fact, several studies have demonstrated that the genital tract represents a unique compartment for the virus. We assessed the relationship between immune system integrity, represented by CD4+ T cell counts, and the maintenance of viral compartmentalization between plasma and vaginal fluid virus in treatment naïve women from the Dominican Republic infected by the heterosexual transmission route. We cloned and sequenced cell free virus from plasma and genital fluid samples from six women to assess viral evolution, phylogenetic relatedness, and calculated co-receptor use for the C2V3 region of the envelope. Our analyses demonstrated plasma and vaginal fluid virus compartments remained intact only in samples from women with CD4+ T cell counts over 350cells/μl. The majority of viral forms were predicted to use the CCR5 co-receptor, although several dual tropic forms were also identified. None of the clones were found to use the CXCR4 co-receptor even though many of the patients showed severe disease. Our findings lend further support to the role of an intact immune system in maintaining compartmentalization across blood and genital quasispecies and provide a compelling rationale to specifically consider genital tract viral forms in therapeutic and vaccine research

The Effect of Endogenous Expression of HIV-1 gp120 on Glutamate Metabolism in Human Astrocytes

Human immunodeficiency virus (HIV) infection is a global epidemic that targets the immune system. HIV infects white blood cells and spreads throughout the entire body via blood stream and makes its way to the brain. HIV infection in the brain may lead to HIV associated neurocognitive disorders (HAND). To be able to address this problem, we have to better understand how HIV infection damages neurons. We hypothesize that gp120 causes neurotoxicity in the cells by inhibiting the conversion of glutamate to glutamine by glutaminase. As a result, glutamate concentrations will build up both inside and outside the cell causing excitatory neurotoxicity. To better understand this process, we transfected human astrocytes (U87MG cells) with mock (control), an empty vector (control), and with gp120 plasmid. Seventy-two hours post transfection, the cells were collected and run through a series of tests including SDS-PAGE/Western Blot and qRT-PCR to assess protein and mRNA levels of glutaminase and gp120. We expect production of gp120 by astrocytes to lead to a decrease in expression of glutaminase. This would inhibit the process of converting glutamate to glutamine and explain how excess of glutamate accumulates inside and outside of the cell causing neurotoxicity and cell death. In conclusion, we expect to find a direct relationship between gp120 and the glutamate metabolism in human astrocytes. Understanding the effect gp120 has on neurons will help develop more effective treatments to better fight the virus

Pediatric Feeding Disorder: Consensus Definition and Conceptual Framework

Pediatric feeding disorders (PFDs) lack a universally accepted definition. Feeding disorders require comprehensive assessment and treatment of 4 closely related, complementary domains (medical, psychosocial, and feeding skill-based systems and associated nutritional complications). Previous diagnostic paradigms have, however, typically defined feeding disorders using the lens of a single professional discipline and fail to characterize associated functional limitations that are critical to plan appropriate interventions and improve quality of life. Using the framework of the World Health Organization International Classification of Functioning, Disability, and Health, a unifying diagnostic term is proposed: “Pediatric Feeding Disorder” (PFD), defined as impaired oral intake that is not age-appropriate, and is associated with medical, nutritional, feeding skill, and/or psychosocial dysfunction. By incorporating associated functional limitations, the proposed diagnostic criteria for PFD should enable practitioners and researchers to better characterize the needs of heterogeneous patient populations, facilitate inclusion of all relevant disciplines in treatment planning, and promote the use of common, precise, terminology necessary to advance clinical practice, research, and health-care policy

Immunogenicity of a low-dose diphtheria, tetanus and acellular pertussis combination vaccine with either inactivated or oral polio vaccine compared to standard-dose diphtheria, tetanus, acellular pertussis when used as a pre-school booster in UK children : a 5-year follow-up of a randomised controlled study

This serological follow up study assessed the kinetics of antibody response in children who previously participated in a single centre, open-label, randomised controlled trial of low-dose compared to standard-dose diphtheria booster preschool vaccinations in the United Kingdom (UK). Children had previously been randomised to receive one of three combination vaccines: either a combined adsorbed tetanus, low-dose diphtheria, 5-component acellular pertussis and inactivated polio vaccine (IPV) (Tdap-IPV, Repevax(®); Sanofi Pasteur MSD); a combined adsorbed tetanus, low-dose diphtheria and 5-component acellular pertussis vaccine (Tdap, Covaxis(®); Sanofi Pasteur MSD) given concomitantly with oral polio vaccine (OPV); or a combined adsorbed standard-dose diphtheria, tetanus, 2-component acellular pertussis and IPV (DTap-IPV, Tetravac(®); Sanofi Pasteur MSD). Blood samples for the follow-up study were taken at 1, 3 and 5 years after participation in the original trial (median, 5.07 years of age at year 1), and antibody persistence to each vaccine antigen measured against defined serological thresholds of protection. All participants had evidence of immunity to diphtheria with antitoxin concentrations greater than 0.01IU/mL five years after booster vaccination and 75%, 67% and 79% of children who received Tdap-IPV, Tdap+OPV and DTap-IPV, respectively, had protective antitoxin levels greater than 0.1IU/mL. Long lasting protective immune responses to tetanus and polio antigens were also observed in all groups, though polio responses were lower in the sera of those who received OPV. Low-dose diphtheria vaccines provided comparable protection to the standard-dose vaccine and are suitable for use for pre-school booster vaccination