peptide-based-identification-functional-motifs-their-binding

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

isolation-of-exosomes-from-the-plasma-of-hiv-1-positive-individuals

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Thorough investigation of an environmental problem within Brockville General Hospital, Ontario, Canada, was vital. This special report is by William Roth, Chris Rahm and Bruce G. Fraser--all of Jacques Whitford Environment Limited. William Roth is the first point of contact for Jacques Whitford's hospital clients and is a project manager within the Environmental Engineering group of JWEL's Ottawa office. Chris Rahm is a member of the Hazardous Materials group in JWEL's Ottawa office and was the site supervisor for the mould abatement at BGH. Bruce Fraser is the corporate technical director of indoor air quality for JWEL and regularly provides technical assistance for mould related projects carried out by JWEL.

Probing and overcoming a mould menace.

We present two cases of primary limited cutaneous perineal and genital extramammary Paget's disease (EMPD) that were successfully treated with imiquimod 5% cream. Clinical and histologic cure was confirmed after 7.5 to 12 weeks of monotherapy. Treatment-associated morbidity is minimal compared with more invasive therapies, and self-application by the patient improves convenience and appeal. Imiquimod may offer a minimally invasive and promising therapeutic option for the treatment of limited cutaneous EMPD. The diagnosis and management of EMPD and the current literature regarding imiquimod therapy are reviewed.

Treatment of primary limited cutaneous extramammary Paget's disease with topical imiquimod monotherapy: two case reports.

To better understand the emergence of HIV-1 variants in Barbados and the association with transmission modes, we analyzed phylogenetic relationships and genetic variability among HIV-1 strains collected in 1996 from 36 antiretroviral therapy-naive patients. Only subtype B variants were present in this sampling, based on analysis of HIV-1 envelope (env) C2V3, protease (PR), and reverse transcriptase (RT) sequences. The genetic diversity of env sequences was broad (13.9%; range, 5.9-24.9%), suggesting multiple introductions of distinct HIV-1 strains to the island. The frequency of subtype B HIV-1 variants with similar env V3 features, including the tetrameric tips, GPGR and GPGK, the threonine deletion at position 23, and the substitution of threonine to arginine at position 22, was comparable in heterosexual, bisexual, and homosexual patients. Analyses of amino acid variations in PR sequences revealed a lack of major drug resistance-conferring mutations and a high (90%) prevalence of secondary mutations at positions 36, 63, 71, and 77. While the occurrence of 361, 63P, and 71T mutations in Barbadian strains was similar to the global prevalence for subtype B variants, the frequency (64%) of the V77I mutation was more than three times that seen worldwide. Only two RT antiretroviral resistance mutations (M41L and T215Y) were observed, both from a single patient. This comprehensive genetic analysis documents a broad diversity within HIV-1 subtype B in Barbados and suggests a lack of association between particular subtype B variants and transmission modes.

The molecular epidemiology and drug resistance determination of HIV type 1 subtype B infection in Barbados.

Toxicological testing in animals is relied upon as a surrogate for clinical testing of most food additives. Both animal and human clinical test results are generally available for direct additives when high levels of exposure are expected. Limited animal studies or in vitro test results may be the only sources of toxicological data available when low levels of exposure (microg/person/day) are expected and where no effects of the additive on the food itself are desired. Safety assessment of such materials for humans requires mathematical extrapolation from any effects observed in test animals to arrive at acceptable daily intakes (ADIs) for humans. Models of anatomy may be used to estimate tissue and organ weights where that information is missing and necessary for evaluation of a data set. The effect of growth on target tissue exposure during critical phases of organ development can be more accurately assessed when models of growth and known physiological changes are combined with pharmacokinetic results for test species. Kinetic models, when combined with limited chemical property, kinetic, and distribution data, can often be used to predict steady-state plasma and tissue levels of a test material over the range of doses employed in chronic studies to aid in interpretation of effects that are often nonlinear with respect to delivered dose. A better understanding of the reasons for nonlinearity of effects in animals improves our confidence in extrapolation to humans.

Use of anatomical and kinetic models in the evaluation of human food additive safety.

It now appears likely that soluble oligomers of amyloid-beta1-42 peptide, rather than insoluble fibrils, act as the primary neurotoxin in Alzheimer's disease (AD). Consequently, compounds capable of altering the assembly state of these oligomers (referred to as ADDLs) may have potential for AD therapeutics. Phenolic compounds are of particular interest for their ability to disrupt Abeta oligomerization and reduce pathogenicity. This study has focused on oleocanthal (OC), a naturally-occurring phenolic compound found in extra-virgin olive oil. OC increased the immunoreactivity of soluble Abeta species, when assayed with both sequence- and conformation-specific Abeta antibodies, indicating changes in oligomer structure. Analysis of oligomers in the presence of OC showed an upward shift in MW and a ladder-like distribution of SDS-stable ADDL subspecies. In comparison with control ADDLs, oligomers formed in the presence of OC (Abeta-OC) showed equivalent colocalization at synapses but exhibited greater immunofluorescence as a result of increased antibody recognition. The enhanced signal at synapses was not due to increased synaptic binding, as direct detection of fluorescently-labeled ADDLs showed an overall reduction in ADDL signal in the presence of OC. Decreased binding to synapses was accompanied by significantly less synaptic deterioration assayed by drebrin loss. Additionally, treatment with OC improved antibody clearance of ADDLs. These results indicate oleocanthal is capable of altering the oligomerization state of ADDLs while protecting neurons from the synaptopathological effects of ADDLs and suggest OC as a lead compound for development in AD therapeutics.

Alzheimer's-associated Abeta oligomers show altered structure, immunoreactivity and synaptotoxicity with low doses of oleocanthal.

The HIV-1 Nef protein is known to be secreted, and our group has shown that Nef is secreted from nef-transfected and HIV-1-infected cells in small exosome-like vesicles (d. 40-100 nm). The role of secreted Nef remains to be fully characterized. Thus, it is important to characterize the nature of and the mechanisms regulating Nef secretion. We hypothesized that specific structural domains on the Nef protein interact with components of the endosomal trafficking machinery, sorting Nef into multivesicular bodies (MVB) and packaging it in exosome-like vesicles. To identify those domains, a series of mutants spanning the entire nef sequence were made and cloned into the expression vector pQB1, which expresses the mutants as Nef-GFP fusion proteins. These constructs were used in transient transfection assays to identify sequences necessary for secretion of the Nef-GFP fusion protein. N-terminal domains were identified as critical for Nef-induced vesicle secretion: (1) a basic cluster of four arginine residues (aa 17, 19, 21, 22), (2) the phosphofurin acidic cluster sequence (PACS; Glu62-65), and (3) a previously uncharacterized domain spanning amino acid residues 66-70 (VGFPV), which we named the secretion modification region (SMR). Additional amino acids P25, 29GVG31, and T44 were identified in HIV-1 Nef as regulating its secretion. These residues have not been associated with other reported Nef functions. The myristoylation domain, ubiquitination lysine residues, and the C-terminal portion of Nef (aa 71-206) had no effect on secretion. A minimal HIV-1 Nef sequence, comprising the identified motifs, was sufficient for Nef-induced vesicle secretion.

Genetic characterization of HIV type 1 Nef-induced vesicle secretion.

The HIV-1 accessory protein Nef plays an active role in the pathogenesis of AIDS by its numerous cellular interactions that facilitate the release of virus particles. This 27Â kDa protein is required for maintenance of the viral replication in HIV, and is also known to contribute to immune evasion, blocking of apoptosis in virus-infected cells and enhancement of virus infectivity. Nef has been shown to be secreted and is present on the surface of virus-infected cells. Recent studies from our laboratory have shown that the Nef protein is secreted from nef-transfected and HIV-1-infected cells in small exosome-like vesicles (40-100Â nm diam.) that do not contain virions. We have identified three amino-terminal domains of Nef as necessary for secretion: (i) the four arginine residues (17,19,21, 22) comprising the basic region; (ii) the phosphofurin acidic cluster sequence (PACS) composed of four glutamic acid residues (61-64); (iii) a previously unknown motif spanning amino acid residues 65-69 (VGFPV) which we named the secretion modification region (SMR). In this study, we have used population-based phylogeny data and sequence analysis to characterize the conservation of the Nef SMR domain that regulates vesicle secretion. We have performed in silico computational chemistry analysis involving molecular dynamic structure modeling of mutations in the SMR motif. Sequence analysis of Nef from HIV-1-infected patients, including slow progressors (SP), long term progressors (LTP) and long term non-progressors (LTNP) demonstrated 99Â % conservation of the Nef SMR motif. Computational analysis including modeling of wild-type HIV-1 Nef and V66A Nef SMR mutant using structural homology and molecular dynamics of ligand-associated interactions indicated significant structural changes in the Nef mutant, thus supporting the importance of the SMR domain for mediating Nef vesicle secretion.

Validation of a novel secretion modification region (SMR) of HIV-1 Nef using cohort sequence analysis and molecular modeling.

The HIV/AIDS data from the national surveillance systems of China and the United States from 1985 to 2014 were compared to characterize the HIV/AIDS epidemic in both countries. The current estimated national HIV prevalence rate in China and the United States are 0.0598% and 0.348%, respectively. In the United States, the annual number of new HIV infections has remained relatively stable (~50,000 each year) and has shown a downward trend in recent years. The Chinese national HIV prevalence is still low, and new HIV infections have been contained at a low level (50,000-100,000 each year). However, the epidemic has showed an increasing trend since 2012. By risk group, in both countries, men who have sex with men (MSM), heterosexual sex, and injection drug use (IDU) are the most common modes of transmission of new HIV infections. However, in the United States, MSM is the dominant transmission route, accounting for >60% of new infections; whereas in China, heterosexual sex has now become the dominant route, also accounting for >60% of new infections. A rapid increase in the proportion of HIV cases that were attributed to MSM and an obvious decrease in the proportion of HIV cases attributed to IDU in China in recent years imply that the China's epidemic is still evolving, to some extent, copying what was experienced in the United States. By age group, the proportions of HIV cases that were attributed to the age group 25-59 were comparable between the two countries. However, the United States had a higher proportion of cases that were attributed to age groups 15-19 and 20-24 than China, indicating that youth account for more infections in the United States. One other fact worth noting: in China there is a significant increase in the number of HIV new infections in individuals over 50 years of age, which results in much higher proportion of cases that were attributed to age groups 60-64 and over 65 in China than those in the United States. By race/ethnicity, in the United States, Blacks/African Americans continue to experience the most severe HIV burden, followed by Hispanics/Latinos. In China, no official data on race/ethnicity disparities are currently available. Thus, region, risk group, age are important factors in the HIV epidemics in both countries.

Characterizing the HIV/AIDS Epidemic in the United States and China.

Exosomes are small membrane-bound vesicles secreted by cells that function to shuttle RNA and proteins between cells. To examine the role of exosomal micro RNA (miRNA) during the early stage of HIV-1 infection we characterized miRNA in exosomes from HIV-infected macrophages, compared with exosomes from non-infected macrophages. Primary human monocytes from uninfected donors were differentiated to macrophages (MDM) which were either mock-infected or infected with the macrophage-tropic HIV-1 BaL strain. Exosomes were recovered from culture media and separated from virus particles by centrifugation on iodixanol density gradients. The low molecular weight RNA fraction was prepared from purified exosomes. After pre-amplification, RNA was hybridized to microarrays containing probes for 1200 miRNA species of known and unknown function. We observed 48 miRNA species in both infected and uninfected MDM exosomes. Additionally, 38 miRNAs were present in infected-cell exosomes but not uninfected-cell exosomes. Of these, 13 miRNAs were upregulated in exosomes from HIV-infected cells, including 4 miRNA species that were increased by more than 10-fold. Though numerous miRNA species have been identified in HIV-infected cells, relatively little is known about miRNA content in exosomes from these cells. In the future, we plan to investigate whether the upregulated miRNA species we identified are increased in exosomes from HIV-1-positive patients.

ArticleTotal : 2	Year
Publication title Cited by 2	2013
Publication title Cited by 51	2016

Article	Year
Probing and overcoming a mould menace. Health estate\| PubMed ID: 12094779	2002
Treatment of primary limited cutaneous extramammary Paget's disease with topical imiquimod monotherapy: two case reports. Journal of the American Academy of Dermatology\| PubMed ID: 12271284	2002
The molecular epidemiology and drug resistance determination of HIV type 1 subtype B infection in Barbados. AIDS research and human retroviruses\| PubMed ID: 12816080	2003
Use of anatomical and kinetic models in the evaluation of human food additive safety. The AAPS journal\| PubMed ID: 16353912	2005
Alzheimer's-associated Abeta oligomers show altered structure, immunoreactivity and synaptotoxicity with low doses of oleocanthal. Toxicology and applied pharmacology\| PubMed ID: 19631677	2009
Genetic characterization of HIV type 1 Nef-induced vesicle secretion. AIDS research and human retroviruses\| PubMed ID: 20156100	2010
Validation of a novel secretion modification region (SMR) of HIV-1 Nef using cohort sequence analysis and molecular modeling. Journal of molecular modeling\| PubMed ID: 22643973	2012
Characterizing the HIV/AIDS Epidemic in the United States and China. International journal of environmental research and public health\| PubMed ID: 26703667	2015
Micro RNA in Exosomes from HIV-Infected Macrophages. International journal of environmental research and public health\| PubMed ID: 26703692	2015

William Roth

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