Genomic changes associated with antigenic variation of visna virus durig persistent infection.

Visna virus undergoes antigenic change during persistent infection of sheep. Antigenic variants of visna virus were compared by using the genomic RNA and analyzing the large RNase T1-resistant oligonucleotides. Mutants isolated from a persistently infected sheep contained a small number of changes in their oligonucleotide patterns when compared with parental virus. To determine whether the changes in the nucleotide structure were clustered in one region of the genome, we determined the order of the oligonucleotides of the parental and mutant RNAs along the genome with respect to the 3' polyadenylylated end. All but one difference between the parental strain and the antigenic mutant used for mapping were located within 2 kilobases from the 3' terminus. Nucleotide sequence analyses showed that several of the oligonucleotides that differed in the parental and mutant RNAs could be accounted for by single base changes.     

Cross-Protection of Influenza A Virus Infection by a DNA Aptamer Targeting the PA Endonuclease Domain

Amino acid residues in the N-terminal of the PA subunit (PA_N) of the influenza A virus polymerase play critical roles in endonuclease activity, protein stability, and viral RNA (vRNA) promoter binding. In addition, PA_N is highly conserved among different subtypes of influenza virus, which suggests PA_N to be a desired target in the development of anti-influenza agents. We selected DNA aptamers targeting the intact PA protein or the PA_N domain of an H5N1 virus strain using systematic evolution of ligands by exponential enrichment (SELEX). The binding affinities of selected aptamers were measured, followed by an evaluation of in vitro endonuclease inhibitory activity. Next, the antiviral effects of enriched aptamers against influenza A virus infections were examined. A total of three aptamers targeting PA and six aptamers targeting PA_N were selected. Our data demonstrated that all three PA-selected aptamers neither inhibited endonuclease activity nor exhibited antiviral efficacy, whereas four of the six PA_N-selected aptamers inhibited both endonuclease activity and H5N1 virus infection. Among the four effective aptamers, one exhibited cross-protection against infections of H1N1, H5N1, H7N7, and H7N9 influenza viruses, with a 50% inhibitory concentration (IC₅₀) of around 10 nM. Notably, this aptamer was identified at the 5th round but disappeared after the 10th round of selection, suggesting that the identification and evaluation of aptamers at early rounds of selection may be highly helpful for screening effective aptamers. Overall, our study provides novel insights for screening and developing effective aptamers for use as anti-influenza drugs.     

Mupirocin resistance in nasal carriage of Staphylococcus aureus among healthcare workers of a tertiary care rural hospital

Introduction:

Mupirocin (pseudomonic acid A) is a topical antimicrobial agent with excellent antistaphylococcal and antistreptococcal activity. A nasal formulation is approved by the United States Food and Drug Administration for eradicating nasal carriage in adult patients as well as in health care personnel. Resistance to mupirocin has already been reported worldwide. The increasing prevalence of mupirocin resistance among Staphylococcus aureus and coagulase-negative Staphylococcus (CoNS) species could be an important threat to the future use of mupirocin against methicillin-resistant S. aureus (MRSA). Thus, this study was carried out to find the prevalence of mupirocin resistance in S. aureus and CoNS by disc diffusion and to determine the rates of high-level and low-level mupirocin resistance in S. aureus and CoNS by disc diffusion.

Materials and Methods:

A total of 140 healthcare workers (HCWs) (doctor, nursing staff, housekeeping staff) were randomly selected. S. aureus and CoNS isolates were tested for mupirocin resistance by the disk diffusion method using 5 μg and 200 μg mupirocin discs. MRSA isolates were tested for antibiotics by Kirby-Bauer disc-diffusion method as per Clinical and Laboratory Standards Institute guidelines.

Results:

Out of 140 nasal swabs collected from HCWs, S. aureus was isolated in 38 (27.14%), and CoNS was isolated in 73 (52.14%). MRSA was isolated in 20 (14.28%) and methicillin-resistant coagulase-negative Staphylococci (MRCoNS) in 34 (24.29%. Methicillin-sensitive S. aureus (MSSA) and MSCoNS isolates were 100% sensitive to mupirocin, but two isolates from MRSA (1.43%) and five from MRCoNS (3.57%) were mupirocin resistant.

Conclusion:

The presence of mupirocin resistance in MRSA and MRCoNS is a cause for concern. It could be limited by regular surveillance and effective infection control initiatives so to inform health care facilities to guide therapeutic and prophylactic use of mupirocin.     

Screw-worm myiasis of prolapsed rectum

Background

Wound myiasis in the Indian subcontinent is most commonly caused by old world screw-worm (Chrysomya bezziana).

Case Report and management

A 4-yearold malnourished girl presented with full thickness rectal prolapse following acute diarrhea with a large wound and screwworm myiasis of the rectum. Turpentine oil was applied to immobilize the maggots followed by manual extraction. Prolapse was successfully treated by manual reduction followed by strapping of the buttocks.

Outcome

Child was thriving well and gained 2 kg weight in follow up after two weeks.

Message

Parents should be educated about taking care of prolapsed rectum.     

Clioquinol promotes the degradation of metal-dependent amyloid-β (Aβ) oligomers to restore endocytosis and ameliorate Aβ toxicity

Alzheimer’s disease (AD) is a common, progressive neurodegenerative disorder without effective disease-modifying therapies. The accumulation of amyloid-β peptide (Aβ) is associated with AD. However, identifying new compounds that antagonize the underlying cellular pathologies caused by Aβ has been hindered by a lack of cellular models amenable to high-throughput chemical screening. To address this gap, we use a robust and scalable yeast model of Aβ toxicity where the Aβ peptide transits through the secretory and endocytic compartments as it does in neurons. The pathogenic Aβ 1–42 peptide forms more oligomers and is more toxic than Aβ 1–40 and genome-wide genetic screens identified genes that are known risk factors for AD. Here, we report an unbiased screen of ∼140,000 compounds for rescue of Aβ toxicity. Of ∼30 hits, several were 8-hydroxyquinolines (8-OHQs). Clioquinol (CQ), an 8-OHQ previously reported to reduce Aβ burden, restore metal homeostasis, and improve cognition in mouse AD models, was also effective and rescued the toxicity of Aβ secreted from glutamatergic neurons in Caenorhabditis elegans. In yeast, CQ dramatically reduced Aβ peptide levels in a copper-dependent manner by increasing degradation, ultimately restoring endocytic function. This mirrored its effects on copper-dependent oligomer formation in vitro, which was also reversed by CQ. This unbiased screen indicates that copper-dependent Aβ oligomer formation contributes to Aβ toxicity within the secretory/endosomal pathways where it can be targeted with selective metal binding compounds. Establishing the ability of the Aβ yeast model to identify disease-relevant compounds supports its further exploitation as a validated early discovery platform.Alzheimer’s disease (AD) is a common and devastating neurodegenerative disorder that is projected to increase in frequency as our population ages. The lack of disease-modifying therapies requires new approaches to address the underlying mechanisms of cellular dysfunction and identify potential therapeutics.The amyloid-β peptide (Aβ) plays a major role in AD and ultimately leads to neuronal death and cognitive impairment (1). Aβ peptides of ∼40 aa are generated by the successive cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. Mutations in APP or γ-secretase cause familial AD and bias APP cleavage toward a 42-aa Aβ peptide that predominates in Aβ plaques, is more aggregation prone, and is toxic to neurons (2, 3). Although Aβ plaques are a common, conspicuous feature of pathology in diseased brains, increasing evidence suggests that small oligomers of Aβ are the most toxic species (4, 5).The conservation of protein homeostasis mechanisms—such as protein trafficking and chaperone networks—among all eukaryotes makes yeast a powerful discovery platform for modeling the cellular toxicities caused by neurodegenerative disease proteins (6). In neurons, the cleavage of APP to generate the Aβ peptide occurs within the secretory and endosomal pathways (7). APP is trafficked through the secretory pathway to the plasma membrane and subsequently internalized and recycled through endosomal vesicles and the trans-Golgi network back to the plasma membrane (7). During this recycling, the Aβ peptide is liberated from APP by β/γ-secretases, thus enabling the peptide to interact with multiple membranous compartments within the cell.We have taken advantage of the great conservation of the secretory and endocytic pathways between yeast and neurons to study Aβ in a simple, highly tractable model organism—budding yeast. By expressing Aβ as a fusion to an endoplasmic reticulum (ER) targeting signal, we have mimicked in yeast the multicompartmental distribution of Aβ (8). This approach bypasses the need to recapitulate the entire APP processing pathway and generates an Aβ peptide with exactly the same sequence as is found in the human brain. The ER targeting signal directs cotranslational transport of the primary translation product into the ER, where the signal sequence is removed by signal peptidase. The peptide then transits through the secretory pathway and is secreted from the cell. In yeast, the cell wall prevents secreted Αβ from diffusing away, allowing it to interact with the plasma membrane and undergo endocytosis. As in the human nervous system (2), the aggregation-prone 42-aa peptide is more prone to forming oligomeric species than the 40-aa peptide (9) and is more toxic (8).This model allowed us to take advantage of yeast genetics to perform a completely unbiased screen of the yeast genome for suppressers or enhancers of Aβ toxicity. Of the ∼6,000 genes we tested, we recovered only a handful of modifiers. There are ∼25,000 genes in the human genome and less than 20 (10) have been shown to confer risk for AD. However, several of the yeast genes that alter Aβ toxicity are either direct homologs or interacting partners of human risk factors (8, 10). For example, YAP1802, is the yeast homolog of PICALM, one the most highly validated risk factors for AD (10, 11); INP52, is homologous to SYNJ1, which interacts with the risk factor BIN1 (12, 13); and SLA1 is homologous to SH3KBP1, which interacts with the risk factor CD2AP (14–16). All of these proteins are involved in clathrin-mediated endocytosis in yeast and humans. In addition to ameliorating the toxicity of Aβ in yeast, these proteins reduced Aβ toxicity in both Caenorhabditis elegans and rat cortical neuronal models (8). The recovery of genes that promote clathrin-mediated endocytosis in unbiased genome-wide screens suggested that Aβ poisons this process (8). Indeed, the peptide compromised endocytosis of a transmembrane receptor (Ste3), an activity crucial for neuronal function. Importantly, the mechanism of action of these risk factors had not previously been linked to Aβ. Thus, the yeast model has already provided key insights on the nature of Aβ’s cellular toxicity in the human brain.In this study, we used our yeast Aβ model to identify small molecules that ameliorate toxicity. In an extremely stringent and unbiased screen of 140,000 compounds, we identified a small number of cytoprotective compounds, including 8-hydroxyquinolines (8-OHQs). Members of this family bind metals and are among the few compounds that have been shown to alleviate Aβ toxicity in mouse models of AD (17, 18), and to show early potential as an AD therapeutic (19). Here, we investigate the mechanism of action for the most efficacious member of this family, clioquinol (CQ).     

Effects of temperature on urinary corticosterone metabolite responses to short-term capture and handling stress in the cane toad (Rhinella marina)

Extreme temperature can cause metabolic, immune and behavioural changes in amphibians. Short-term stress hormonal response via increased secretion of corticosterone enables amphibians to make necessary physiological and behavioural adjustments for coping with stressors. The effect of temperature on short-term corticosterone responses has not been studied in amphibians. In this study, this relationship was evaluated in adult male cane toads (Rhinella marina). We acclimated male toads (n=24 toads per group) at low, medium and high temperature (15, 25 or 35°C) under controlled laboratory conditions for a 14day period. After thermal acclimation, short-term corticosterone responses were evaluated in the toads subjected to a standard capture and handling stress protocol over a 24h period. Corticosterone metabolites in toad urine were measured via enzyme-immunoassay. During acclimation, mean baseline urinary corticosterone level increased after transfer of the toads from wild into captivity and returned to baseline on day 14 of acclimation for each of the three temperatures. At the end of the 14days of thermal acclimation period, baseline corticosterone level were highest for toad group at 35°C and lowest at 15°C. All toads generated urinary corticosterone responses to the standard capture and handling stressor for each temperature. Both individual and mean short-term corticosterone responses of the toads were highest at 35°C and lowest at 15°C. Furthermore, Q(10) values (the factor by which the reaction rate increases when the temperature is raised by 10°) were calculated for mean corrected integrated corticosterone responses as follows; (15-35°C) Q(10)=1.51, (15-25°C) Q(10)=1.60; (25-35°C) Q(10)=1.43. Both total and corrected integrated corticosterone responses were highest for toads at 35°C followed by 25°C and lowest for the 15°C toad group. Overall, the results have demonstrated the thermodynamic response of corticosterone secretion to short-term capture and handling stress in an amphibian species.