Enzymatic Formulation Capable of Degrading Scrapie Prion under Mild Digestion Conditions

The prion agent is notoriously resistant to common proteases and conventional sterilisation procedures. The current methods known to destroy prion infectivity such as incineration, alkaline and thermal hydrolysis are harsh, destructive, environmentally polluting and potentially hazardous, thus limit their applications for decontamination of delicate medical and laboratory devices, remediation of prion contaminated environment and for processing animal by-products including specified risk materials and carcases. Therefore, an environmentally friendly, non-destructive enzymatic degradation approach is highly desirable. A feather-degrading Bacillus licheniformis N22 keratinase has been isolated which degraded scrapie prion to undetectable level of PrP^Sc signals as determined by Western Blot analysis. Prion infectivity was verified by ex vivo cell-based assay. An enzymatic formulation combining N22 keratinase and biosurfactant derived from Pseudomonas aeruginosa degraded PrP^Sc at 65°C in 10 min to undetectable level -. A time-course degradation analysis carried out at 50°C over 2 h revealed the progressive attenuation of PrP^Sc intensity. Test of residual infectivity by standard cell culture assay confirmed that the enzymatic formulation reduced PrP^Sc infectivity to undetectable levels as compared to cells challenged with untreated standard scrapie sheep prion (SSBP/1) (p-value = 0.008 at 95% confidence interval). This novel enzymatic formulation has significant potential application for prion decontamination in various environmentally friendly systems under mild treatment conditions.     

Characterization of macroautophagic flux in vivo using a leupeptin-based assay

Macroautophagy is a highly conserved catabolic process that is crucial for organ homeostasis in mammals. However, methods to directly measure macroautophagic activity (or flux) in vivo are limited. In this study we developed a quantitative macroautophagic flux assay based on measuring LC3b protein turnover in vivo after administering the protease inhibitor leupeptin. Using this assay we then characterized basal macroautophagic flux in different mouse organs. We found that the rate of LC3b accumulation after leupeptin treatment was greatest in the liver and lowest in spleen. Interestingly we found that LC3a, an ATG8/LC3b homologue and the LC3b-interacting protein p62 were degraded with similar kinetics to LC3b. However, the LC3b-related proteins GABARAP and GATE-16 were not rapidly turned over in mouse liver, implying that different LC3b homologues may contribute to macroautophagy via distinct mechanisms. Nutrient starvation augmented macroautophagic flux as measured by our assay, while refeeding the animals after a period of starvation significantly suppressed flux. We also confirmed that beclin 1 heterozygous mice had reduced basal macroautophagic flux compared to wild-type littermates. These results illustrate the usefulness of our leupeptin-based assay for studying the dynamics of macroautophagy in mice.     

Bioluminescent mammalian cells grown in sponge matrices to monitor immune rejection

The growth and bioluminescence of cells seeded in collagen and gelatin sponge matrices were compared in vitro under different conditions, and immune rejection was quantified and visualized directly in situ based on loss of bioluminescence activity. Mammalian cells expressing a Renilla luciferase complementary deoxyribonucleic acid (cDNA) were used to seed collagen and gelatin sponge matrices soaked in either polylysine or gelatin to determine optimal growth conditions in vitro. The sponges were incubated in tissue culture plates for 3 weeks and received 2, 9, or 15 injections of coelenterazine. Measurements of bioluminescence activity indicated that gelatin sponges soaked in gelatin emitted the highest levels of light emission, multiple injections of coelenterazine did not affect light emission significantly, and light emission from live cells grown in sponges could be measured qualitatively but not quantitatively. Histologic analysis of sponge matrices cultured in vitro showed that cells grew best in gelatin matrices. Visualization of subcutaneously implanted sponges in mice showed accelerated loss of light emission in immunocompetent BALB/c mice compared with immunodeficient BALB/c-scid mice, which was associated with increased cell infiltration. Our results indicate that sponge matrices carrying bioluminescent mammalian cells are a valid model system to study immune rejection in situ.     

The Impact of Stressful Life Events on Excessive Alcohol Consumption in the French Population: Findings from the GAZEL Cohort Study

Background

Major life changes may play a causative role in health through lifestyle factors, such as alcohol. The objective was to examine the impact of stressful life events on heavy alcohol consumption among French adults.

Methods

Trajectories of excessive alcohol consumption in 20,625 employees of the French national gas and electricity company for up to 5 years before and 5 years after an event, with annual measurements from 1992. We used repeated measures analysis of time series data indexed to events, employing generalized estimating equations.

Results

For women, excessive alcohol use increased before important purchase (p = 0.021), children leaving home (p<0.001), and death of loved ones (p = 0.03), and decreased before widowhood (p = 0.015); in the year straddling the event, increased consumption was observed for important purchase (p = 0.018) and retirement (p = 0.002); at the time of the event, consumption decreased for marriage (p = 0.002), divorce, widowhood, and death of loved one (all p<0.001), and increased for retirement (p = 0.035). For men, heavy alcohol consumption increased in the years up to and surrounding the death of loved ones, retirement, and important purchase (all p<0.001), and decreased after (all p<0.001, except death of loved one: p = 0.006); at the time of the event, consumption decreased for all events except for children leaving home and retirement, where we observed an increase (all p<0.001). For women and men, heavy alcohol consumption decreased prior to marriage and divorce and increased after (all p<0.001, except for women and marriage: p = 0.01).

Conclusion

Stressful life events promote healthy and unhealthy alcohol consumption. Certain events impact alcohol intake temporarily while others have longer-term implications. Research should disentangle women''s and men''s distinct perceptions of events over time.     

Left ventricular geometric patterns in newly presenting nigerian hypertensives: An echocardiographic study

Background

In recent years a number of algorithms for cardiovascular risk assessment has been proposed to the medical community. These algorithms consider a number of variables and express their results as the percentage risk of developing a major fatal or non-fatal cardiovascular event in the following 10 to 20 years

Discussion

The author has identified three major pitfalls of these algorithms, linked to the limitation of the classical statistical approach in dealing with this kind of non linear and complex information. The pitfalls are the inability to capture the disease complexity, the inability to capture process dynamics, and the wide confidence interval of individual risk assessment. Artificial Intelligence tools can provide potential advantage in trying to overcome these limitations. The theoretical background and some application examples related to artificial neural networks and fuzzy logic have been reviewed and discussed.

Summary

The use of predictive algorithms to assess individual absolute risk of cardiovascular future events is currently hampered by methodological and mathematical flaws. The use of newer approaches, such as fuzzy logic and artificial neural networks, linked to artificial intelligence, seems to better address both the challenge of increasing complexity resulting from a correlation between predisposing factors, data on the occurrence of cardiovascular events, and the prediction of future events on an individual level.     

The influence of sawmill wood wastes on the distribution and population of macroinvertebrates at Benin River, Niger Delta area, Nigeria

The impact of sawmill wood wastes on the distribution of benthic macroinvertebrates at the Sapele section of Benin River, Niger Delta, Nigeria, was investigated from March 2005 to August 2005. A total of 434 individuals were collected by kick-sampling method, representing 21 taxa of benthic macroinvertebrates. Three stations, 1, 2, and 3, were selected from upstream of the site, receiving wood wastes discharge, the impacted site and its down stream, respectively. Among the water quality variables, conductivity, dissolved oxygen, biochemical oxigen demand (BOD(5)), nitrate-nitrogen, phosphate-phosphorus, transparency, and alkalinity were significantly different (P<0.05) among the stations. Orthogonal comparison by Duncan's multiple range test showed that station 2 (the impacted site) was the cause of the difference. More sensitive species such as Ephemeroptera or Plecoptera were completely absent from station 2, the impacted site. Species abundance was similar in station 1 and 3, indicating that the wood wastes must have adversely affected the distribution of these macroinvertebrates, especially the intolerant species. The wood waste discharge not only altered the water chemistry, but also stimulated the abundance of less-sensitive macroinvertebrate species.     

Genome-wide association study for agronomic and physiological traits in spring wheat evaluated in a range of heat prone environments

Key message

We identified 27 stable loci associated with agronomic traits in spring wheat using genome-wide association analysis, some of which confirmed previously reported studies. GWAS peaks identified in regions where no QTL for grain yield per se has been mapped to date, provide new opportunities for gene discovery and creation of new cultivars with desirable alleles for improving yield and yield stability in wheat.

Abstract

We undertook large-scale genetic analysis to determine marker-trait associations (MTAs) underlying agronomic and physiological performance in spring wheat using genome-wide association studies (GWAS). Field trials were conducted at seven sites in three countries (Sudan, Egypt, and Syria) over 2–3 years in each country. Twenty-five agronomic and physiological traits were measured on 188 wheat genotypes. After correcting for population structure and relatedness, a total of 245 MTAs distributed over 66 loci were associated with agronomic traits in individual and mean performance across environments respectively; some of which confirmed previously reported loci. Of these, 27 loci were significantly associated with days to heading, thousand kernel weight, grain yield, spike length, and leaf rolling for mean performance across environments. Despite strong QTL by environment interactions, eight of the loci on chromosomes 1A, 1D, 5A, 5D, 6B, 7A, and 7B had pleiotropic effects on days to heading and yield components (TKW, SM^?2, and SNS). The winter-type alleles at the homoeologous VRN1 loci significantly increased days to heading and grain yield in optimal environments, but decreased grain yield in heat prone environments. Top 20 high-yielding genotypes, ranked by additive main effects and multiplicative interaction (AMMI), had low kinship relationship and possessed 4–5 favorable alleles for GY MTAs except two genotypes, Shadi-4 and Qafzah-11/Bashiq-1–2. This indicated different yield stability mechanisms due to potentially favorable rare alleles that are uncharacterized. Our results will enable wheat breeders to effectively introgress several desirable alleles into locally adapted germplasm in developing wheat varieties with high yield stability and enhanced heat tolerance.

     

Long‐Term Stability of Primary Rat Hepatocytes in Micropatterned Cocultures

Primary hepatocytes display functional and structural instability in standard monoculture systems. We have previously developed a model in which primary hepatocytes are organized in domains of empirically optimized dimensions and surrounded by murine embryonic fibroblasts (HepatoPac?). Here, we assess the long‐term phenotype of freshly isolated and cryopreserved rat hepatocytes in a 96‐well HepatoPac format. The viability, cell polarity (actin microfilaments, bile canaliculi), and functions (albumin, urea, Phase I/II enzymes, transporters) of fresh and cryopreserved rat hepatocytes were retained in HepatoPac at similar levels for at least 4 weeks as opposed to rapidly declining over 5 days in collagen/Matrigel? sandwich cultures. Pulse or continuous exposure of rat HepatoPac to GW‐7647, a selective agonist of PPARα, caused reproducible induction of CYP4A1 and 3‐hydroxy‐3‐methylglutaryl‐CoA synthase over 4 weeks. In conclusion, rat HepatoPac in a 96‐well format can be used for chronic dosing of highly functional hepatocytes and assessment of perturbed hepatocellular pathways. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:204‐212, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21469     

Dermatophytosis among children of Fulani/Hausa herdsmen living in southeastern Nigeria

BackgroundDermatophytic infections are a worldwide public health problem. In Nigeria, children of nomadic herdsmen are thought to be more at risk because of their early and continuous exposure to different kinds of animals. However, little is known about the level of infection in these children in southeastern Nigeria or elsewhere within the West African sub-region.ObjectiveThis study investigated dermatophytic infections among children of nomadic herdsmen living in isolated camps in southeastern Nigeria from October 2008 to May 2009.Methods390 children aged between 4 and 17 years with clinically suggestive lesions were sampled. Isolates were identified by microscopy, culture or both. Biochemical tests and sequencing of the ITS2 region of the ribosomal DNA were used to identify isolates with atypical morphology.Results162 children were confirmed to be infected. Ten different species of dermatophytes were recovered with Trichophyton mentagrophytes showing the highest incidence. There was a significant difference (P < 0.05) in the frequency of isolates recovered among the different age groups screened. No significant (P > 0.05) observations were found according to gender or location of the two camps investigated.ConclusionThis study suggests that tinea capitis is the predominant clinical type of dermatophytosis among children exposed to different kinds of animals based on parental nomadic lifestyle. It also dominated in children with mixed infections in different anatomical sites. The high prevalence of 41.25% suggests that animal to child transmission may be very common.     

Early onset of neurological features differentiates two outbreaks of Lassa fever in Ebonyi state,Nigeria during 2017–2018

Lassa fever (LF) is an acute viral haemorrhagic illness with various non-specific clinical manifestations. Neurological symptoms are rare at the early stage of the disease, but may be seen in late stages, in severely ill patients.The aim of this study was to describe the epidemiological evolution, socio-demographic profiles, clinical characteristics, and outcomes of patients seen during two Lassa fever outbreaks in Ebonyi State, between December 2017 and December 2018.Routinely collected clinical data from all patients admitted to the Virology Centre of the hospital during the period were analysed retrospectively. Out of a total of 83 cases, 70(84.3%) were RT-PCR confirmed while 13 (15.7%) were probable cases. Sixty-nine (83.1%) patients were seen in outbreak 1 of whom 53.6% were urban residents, while 19%, 15%, and 10% were farmers, students and health workers respectively. There were 14 (16.8%) patients, seen in second outbreak with 92.9% rural residents. There were differences in clinical symptoms, signs and laboratory findings between the two outbreaks. The case fatality rates were 29.9% in outbreak 1 and 85.7% for outbreak 2. Neurological features and abnormal laboratory test results were associated with higher mortality rate, seen in outbreak 2. This study revealed significant differences between the two outbreaks. Of particular concern was the higher case fatality during the outbreak 2 which may be from a more virulent strain of the Lassa virus. This has important public health implications and further molecular studies are needed to better define its characteristics.