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

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. ...

Morehouse School of Medicine

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.

Une enqu&ecirc;te approfondie d&#39;un probl&egrave;me environnemental au sein de l&#39;h&ocirc;pital g&eacute;n&eacute;ral de Brockville, Ontario, Canada, est indispensable. Ce rapport sp&eacute;cial est par William Roth, Chris Rahm et Bruce G. Fraser--ensemble de Jacques Whitford Environment Limited. William Roth est le premier point de contact pour les clients de l&#39;h&ocirc;pital de Jacques Whitford et est un chef de projet au sein du groupe de g&eacute;nie de l&#39;environnement du Bureau d&#39;Ottawa de JWEL. Chris Rahm est un membre du groupe de mati&egrave;res dangereuses dans le Bureau d&#39;Ottawa de JWEL et &eacute;tait le chef de chantier pour la r&eacute;duction des &eacute;missions de moule au BGH. Bruce Fraser est le directeur technique de qualit&eacute; de l&#39;air pour JWEL et fournit r&eacute;guli&egrave;rement une assistance technique pour le moule des projets men&eacute;s par JWEL.

Sonder et surmonter une menace de moisissure.

Un&#39;indagine approfondita di un problema ambientale all&#39;interno dell&#39;ospedale generale di Brockville, Ontario, Canada, era vitale. Questo rapporto speciale &egrave; da William Roth, Chris Rahm e Bruce G. Fraser - tutti di Jacques Whitford ambiente Limited. William Roth &egrave; il primo punto di contatto per i clienti di ospedale di Jacques Whitford ed &egrave; responsabile del progetto all&#39;interno del gruppo di ingegneria per l&#39;ambiente dell&#39;ufficio di Ottawa di JWEL. Chris Rahm &egrave; un membro del gruppo di materiali pericolosi nell&#39;ufficio di Ottawa di JWEL ed era il supervisore del sito per l&#39;abbattimento di stampo a BGH. Bruce Fraser &egrave; il direttore tecnico azienda della qualit&agrave; dell&#39;aria interna per JWEL e regolarmente fornisce assistenza tecnica per stampo correlati a progetti condotti da JWEL.

Sondaggio e superare una minaccia di stampo.

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.

Nous pr&eacute;sentons deux cas de primaire limit&eacute;e cutan&eacute; p&eacute;rin&eacute;al et g&eacute;nital maladie extra-mammaire de Paget (EMPD) qui ont &eacute;t&eacute; trait&eacute;s avec succ&egrave;s avec l&#39;imiquimod 5 % cr&egrave;me. Gu&eacute;rison clinique et histologique a &eacute;t&eacute; confirm&eacute;e apr&egrave;s 7,5 &agrave; 12 semaines de monoth&eacute;rapie. Traitement associ&eacute; &agrave; la morbidit&eacute; est minime par rapport aux th&eacute;rapies plus envahissantes, et auto-application par le patient am&eacute;liore la commodit&eacute; et l&#39;appel. Imiquimod peut offrir une option th&eacute;rapeutique minimalement invasive et prometteuse pour le traitement de l&#39;EMPD cutan&eacute;e limit&eacute;e. Le diagnostic et la gestion des EMPD et la litt&eacute;rature actuelle concernant la th&eacute;rapie de l&#39;imiquimod sont examin&eacute;es.

Traitement du primaire limit&eacute;e cutan&eacute;e maladie extra-mammaire de Paget avec imiquimod topique en monoth&eacute;rapie : deux rapports de cas.

Vi presentiamo due casi di primario limitato cutaneo perineale e genitale extramammary morbo di Paget (elettrici) che sono stati trattati con successo con imiquimod 5% crema. Cure cliniche e istologiche &egrave; stata confermata dopo 7,5 a 12 settimane di monoterapia. Morbilit&agrave; associato al trattamento &egrave; minima rispetto a terapie pi&ugrave; invasive, e auto-applicazione da parte del paziente migliora comodit&agrave; e fascino. Imiquimod pu&ograve; offrire un minimamente invasiva e promettente opzione terapeutica per il trattamento della limitata elettrici cutanei. La diagnosi e la gestione di elettrici e la letteratura corrente per quanto riguarda la terapia imiquimod sono rivisti.

Trattamento della primaria limitata cutanea extramammary malattia di Paget con imiquimod topico in monoterapia: due case report.

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.

Pour mieux comprendre l&#39;&eacute;mergence de variants du VIH-1 &agrave; la Barbade et la liaison avec les modes de transmission, nous avons analys&eacute; les relations phylog&eacute;n&eacute;tiques et variabilit&eacute; g&eacute;n&eacute;tique parmi les souches de VIH-1 provenant de 36 patients de na&iuml;fs th&eacute;rapie antir&eacute;trovirale en 1996. Seules les variantes de sous-type B &eacute;taient pr&eacute;sents dans cet &eacute;chantillonnage, analyse sur la base de l&#39;enveloppe du VIH-1 (env) C2V3, prot&eacute;ase (PR) et des s&eacute;quences de la transcriptase inverse (RT). La diversit&eacute; g&eacute;n&eacute;tique des s&eacute;quences env &eacute;tait large (13,9 %; intervalle, 5,9-24,9 %), ce qui sugg&egrave;re des introductions multiples de diff&eacute;rentes souches de VIH-1 &agrave; l&#39;&icirc;le. La fr&eacute;quence du sous-type B du VIH-1 des variantes avec des fonctions similaires env V3, y compris les conseils t&eacute;tram&egrave;res, GPGR et GPGK, la suppression de la thr&eacute;onine en position 23, et la substitution de thr&eacute;onine &agrave; l&#39;arginine en position 22, &eacute;tait comparable chez les h&eacute;t&eacute;rosexuels, bisexuels et homosexuels. Analyses des variations PR s&eacute;quences d&#39;acides amin&eacute;s a r&eacute;v&eacute;l&eacute; une absence de mutations conf&eacute;rant la r&eacute;sistance de drogue et une forte pr&eacute;valence (90 %) des mutations secondaires aux positions 36, 63, 71 et 77. Tout en l&#39;occurrence de 361, 63p et 71 t des mutations chez les souches barbadiennes &eacute;tait similaire &agrave; la pr&eacute;valence mondiale pour les variantes de sous-type B, la fr&eacute;quence (64 %) de la mutation de V77I &eacute;tait plus de trois fois celle observ&eacute;e dans le monde entier. Seules deux mutations de la r&eacute;sistance RT antir&eacute;troviral (M41L et T215Y) ont &eacute;t&eacute; observ&eacute;s, tous deux d&#39;un seul patient. Cette analyse g&eacute;n&eacute;tique compl&egrave;te documente une grande diversit&eacute; au sein de VIH-1 de sous-type B &agrave; la Barbade et sugg&egrave;re une absence d&#39;association entre les modes de transmission et de variantes particuli&egrave;res sous-type B.

L&#39;&eacute;pid&eacute;miologie mol&eacute;culaire et la d&eacute;termination de la r&eacute;sistance de drogue du VIH infection type 1 sous-type B &agrave; la Barbade.

Per meglio comprendere la comparsa di varianti di HIV-1 in Barbados e l&#39;associazione con le modalit&agrave; di trasmissione, abbiamo analizzato le relazioni filogenetiche e variabilit&agrave; genetica tra i ceppi di HIV-1 raccolti nel 1996 da 36 pazienti di ingenuo di terapia antiretrovirale. Solo sottotipo B varianti erano presenti in questo campionamento, sulla base di analisi della busta HIV-1 (env) C2V3, proteasi (PR) e sequenze di trascrittasi inversa (RT). La diversit&agrave; genetica delle sequenze env era ampia (13,9%; 5,9-24,9%, gamma), suggerendo pi&ugrave; introduzioni di diversi ceppi di HIV-1 per l&#39;isola. La frequenza del sottotipo B HIV-1 varianti con caratteristiche simili di env V3, comprese le punte tetramerica, GPGR e GPGK, l&#39;eliminazione di treonina in posizione 23, e la sostituzione di treonina a arginina alla posizione 22, era comparabile nei pazienti eterosessuali, bisessuali e omosessuali. Analisi delle variazioni dell&#39;amminoacido in sequenze di PR ha rivelato una mancanza di mutazioni che conferiscono resistenza principale droga e un&#39;elevata prevalenza (90%) di mutazioni secondarie a posizioni 36, 63, 71 e 77. Mentre l&#39;occorrenza di 361, 63P e 71T mutazioni nei ceppi Barbados era simile alla prevalenza globale per varianti di sottotipo B, la frequenza (64%) della mutazione V77I era pi&ugrave; di tre volte che visto in tutto il mondo. Sono state osservate solo due mutazioni della resistenza RT antiretrovirale (M41L e T215Y), sia da un singolo paziente. Questa analisi genetica completa documenti una vasta diversit&agrave; all&#39;interno di HIV-1 sottotipo B in Barbados e suggerisce una mancanza di associazione tra particolare sottotipo B varianti e modalit&agrave; di trasmissione.

L&#39;epidemiologia molecolare e determinazione resistenza droga del HIV tipo 1 infezione sottotipo B in Barbados.

Les tests toxicologiques chez l&#39;animal sont invoqu&eacute; comme un substitut pour les essais cliniques de la plupart des additifs alimentaires. Les deux r&eacute;sultats d&#39;essai clinique humaine et animale sont g&eacute;n&eacute;ralement disponibles pour les additifs directs lorsque les niveaux d&#39;exposition &eacute;lev&eacute;s sont suppos&eacute;s. Limit&eacute; des &eacute;tudes chez l&#39;animal ou r&eacute;sultats d&#39;essais in vitro peuvent &ecirc;tre les seules sources de donn&eacute;es toxicologiques disponibles lorsque les faibles niveaux d&#39;exposition (microg/personne/jour) sont suppos&eacute;s et lorsque aucun effet de l&#39;additif sur la nourriture elle-m&ecirc;me n&#39;est demand&eacute;s. &Eacute;valuation de l&#39;innocuit&eacute; de ces mati&egrave;res pour l&#39;homme n&eacute;cessite math&eacute;matique extrapolation &agrave; partir des effets observ&eacute;s chez les animaux d&#39;essai pour arriver &agrave; des doses journali&egrave;res acceptables (DJA) pour l&#39;homme. Mod&egrave;les d&#39;anatomie peuvent &ecirc;tre utilis&eacute;s pour estimer le poids des tissus et des organes o&ugrave; cette information est manquante et n&eacute;cessaire pour l&#39;&eacute;valuation d&#39;un ensemble de donn&eacute;es. L&#39;effet de la croissance sur l&#39;exposition des tissus cibles pendant les phases critiques du d&eacute;veloppement des organes peut &ecirc;tre &eacute;valu&eacute;e plus pr&eacute;cis&eacute;ment lorsque les mod&egrave;les de croissance et des changements physiologiques connus sont combin&eacute;s avec des r&eacute;sultats pharmacocin&eacute;tiques pour l&#39;esp&egrave;ce. Mod&egrave;les cin&eacute;tiques, lorsqu&#39;il est combin&eacute; avec la propri&eacute;t&eacute; chimique limit&eacute;e, cin&eacute;tique et de donn&eacute;es de distribution, permet souvent de pr&eacute;voir des niveaux plasmatiques et tissulaires d&#39;&eacute;quilibre d&#39;un mat&eacute;riel d&#39;essai dans l&#39;intervalle de doses employ&eacute;es dans les &eacute;tudes de toxicit&eacute; chronique pour aider &agrave; l&#39;interpr&eacute;tation des effets qui sont souvent non lin&eacute;aire par rapport &agrave; la dose administr&eacute;e. Mieux comprendre les raisons de la non lin&eacute;arit&eacute; des effets chez les animaux am&eacute;liore notre confiance dans l&#39;extrapolation &agrave; l&#39;homme.

Utilisation de mod&egrave;les anatomiques et cin&eacute;tiques dans l&#39;&eacute;valuation de l&#39;innocuit&eacute; additive.

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.

Test tossicologici negli animali &egrave; invocata come un surrogato per la sperimentazione clinica della maggior parte degli additivi alimentari. Entrambi risultati di test clinici animale e umano sono generalmente disponibili per additivi diretti quando alti livelli di esposizione sono attesi. Studi limitati su animali o risultati di test in vitro possono essere le uniche fonti di dati tossicologici disponibili sono tenuti bassi livelli di esposizione (microg/persona/giorno) dove e quando sono desiderati effetti dell&#39;additivo sull&#39;alimento stesso. Valutazione della sicurezza di tali materiali per gli esseri umani richiede un&#39;estrapolazione matematica da eventuali effetti osservati negli animali di test per arrivare a dosi giornaliere ammissibili (DGA) per gli esseri umani. Modelli di anatomia possono essere utilizzati per stimare i pesi dei tessuti e organi dove tale informazione &egrave; mancante e necessaria per la valutazione di un set di dati. L&#39;effetto della crescita sull&#39;esposizione del tessuto bersaglio durante fasi critiche dello sviluppo organo pu&ograve; essere valutato con maggiore precisione quando modelli di crescita e cambiamenti fisiologici noti sono combinati con risultati farmacocinetici per test di specie. Modelli di cinetiche, quando combinato con limitate propriet&agrave; chimica, cinetica e dati dei distribuzione, spesso possono essere utilizzati per prevedere livelli di plasma e tessuto steady-state di un materiale di prova nell&#39;intervallo delle dosi impiegate negli studi cronici per aiutare nell&#39;interpretazione degli effetti che sono spesso non lineare rispetto alla dose erogata. Una migliore comprensione delle ragioni per la non linearit&agrave; degli effetti negli animali migliora la nostra fiducia nell&#39;estrapolazione all&#39;uomo.

Utilizzo di modelli anatomici e cinetici nella valutazione della sicurezza di additivo alimentare umana.

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.

Il semble maintenant probable que des oligom&egrave;res solubles du peptide amylo&iuml;de-beta1-42, plut&ocirc;t que les fibrilles insolubles, servent de la neurotoxine primaire dans la maladie d&#39;Alzheimer (ma). Par cons&eacute;quent, compos&eacute;s susceptibles de modifier l&#39;&eacute;tat de l&#39;Assembl&eacute;e de ces oligom&egrave;res (appel&eacute;s ADDLs) peuvent avoir potentiel th&eacute;rapeutique de l&#39;AD. Compos&eacute;s ph&eacute;noliques sont d&#39;un int&eacute;r&ecirc;t particulier pour leur capacit&eacute; &agrave; perturber l&#39;oligom&eacute;risation de b&ecirc;ta-amylo&iuml;de et r&eacute;duire la pathog&eacute;nicit&eacute;. Cette &eacute;tude a port&eacute; sur oleocanthal (OC), un compos&eacute; ph&eacute;nolique naturels trouv&eacute;s dans l&#39;huile d&#39;olive extra vierge. OC a augment&eacute; l&#39;immunor&eacute;activit&eacute; d&iquest;esp&egrave;ces Ab&ecirc;ta soluble, mesur&eacute;e avec les deux s&eacute;quence - et conformation sp&eacute;cifique des anticorps de b&ecirc;ta-amylo&iuml;de, en indiquant les changements dans la structure de l&#39;oligom&egrave;re. Analyse des oligom&egrave;res en pr&eacute;sence de l&#39;OC a montr&eacute; un d&eacute;calage vers le haut MW et une distribution de type &eacute;chelle des sous-esp&egrave;ces de l&#39;ADDL SDS-stable. En comparaison avec le contr&ocirc;le ADDLs, oligom&egrave;res forment en pr&eacute;sence de colocalisation &eacute;quivalente OC (Ab&ecirc;ta-OC) a montr&eacute; aux synapses mais expos&eacute;es plu immunofluorescence par suite de la reconnaissance d&#39;anticorps accrue. Le signal am&eacute;lior&eacute; aux synapses n&#39;&eacute;tait pas d&ucirc; &agrave; une liaison synaptique accrue, comme la d&eacute;tection directe de ADDLs fluorescent marqu&eacute; a montr&eacute; qu&#39;une r&eacute;duction globale de signal plus deux autres en pr&eacute;sence de liaison OC a diminu&eacute; de synapses, accompagnait significativement moins d&eacute;gradation synaptique dos&eacute;e par la perte de drebrin. En outre, le traitement avec OC am&eacute;lior&eacute; clairance d&#39;anticorps de ADDLs. Ces r&eacute;sultats indiquent oleocanthal est capable de modifier l&#39;&eacute;tat d&#39;oligom&eacute;risation des ADDLs tout en prot&eacute;geant les neurones contre les effets de synaptopathological de ADDLs et OC comme un chef de file pour le d&eacute;veloppement en th&eacute;rapeutique de l&#39;AD.

Oligom&egrave;res de b&ecirc;ta-amylo&iuml;de associ&eacute;e &agrave; Alzheimer montrent la structure alt&eacute;r&eacute;e, l&#39;immunor&eacute;activit&eacute; et synaptotoxicity avec de faibles doses d&#39;oleocanthal.

Ora sembra probabile che oligomeri solubili del peptide amiloide-beta1-42, anzich&eacute; fibrille insolubili, agiscono come la neurotossina primario nella malattia di Alzheimer (AD). Di conseguenza, composti in grado di alterare lo stato di assemblaggio di questi oligomeri (denominate ADDLs) possono avere il potenziale per terapie AD. Composti fenolici sono di particolare interesse per la loro capacit&agrave; di interferire con Abeta oligomerizzazione e ridurre la patogenicit&agrave;. Questo studio si &egrave; concentrato su oleocanthal (OC), un composto fenolico che si verificano naturalmente trovato in olio extravergine di oliva. OC aumentata immunoreattivit&agrave; delle specie di Abeta solubile, quando dosati con entrambi sequenza e conformazione-specifici anticorpi Abeta, indicando le modifiche nella struttura oligomero. Analisi di oligomeri in presenza di OC ha mostrato uno spostamento verso l&#39;alto in MW e una scala-come distribuzione delle sottospecie ADDL SDS-stabile. Rispetto al controllo ADDLs, oligomeri formano in presenza di OC (Abeta-OC) ha mostrato presenza equivalente alla sinapsi ma esposto immunofluorescenza maggiore a seguito di riconoscimento di anticorpi maggiore. Il segnale di avanzata a sinapsi non era a causa della maggiore associazione sinaptica, come la rilevazione diretta di fluorescente-etichetta ADDLs ha mostrato che una riduzione complessiva nel segnale ADDL in presenza di associazione OC. diminuito di sinapsi &egrave; stata accompagnata da significativamente meno deterioramento sinaptico dosato dalla perdita di drebrin. Inoltre, il trattamento con OC migliorato liquidazione dell&#39;anticorpo di ADDLs. Questi risultati indicano oleocanthal &egrave; in grado di alterare lo stato di oligomerizzazione di ADDLs, proteggendo i neuroni dagli effetti synaptopathological di ADDLs e suggerire OC come un lead compound per lo sviluppo di terapie AD.

Oligomeri di Abeta associati al morbo di Alzheimer mostrano struttura alterata, immunoreattivit&agrave; e synaptotoxicity con basse dosi di 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.

La prot&eacute;ine Nef VIH-1 est connue pour &ecirc;tre s&eacute;cr&eacute;t&eacute;es, et notre groupe a montr&eacute; que la Nef est s&eacute;cr&eacute;t&eacute;e par les cellules transfect&eacute;es nef et infect&eacute;s par le VIH-1 de l&#39;exosome comme petites v&eacute;sicules (d. 40-100 nm). Le r&ocirc;le de la Nef s&eacute;cr&eacute;t&eacute;e reste &agrave; &ecirc;tre enti&egrave;rement caract&eacute;ris&eacute;e. Ainsi, il est important de caract&eacute;riser la nature des et les m&eacute;canismes r&eacute;gulant la s&eacute;cr&eacute;tion de la Nef. Nous avons &eacute;mis l&#39;hypoth&egrave;se que les domaines structuraux sp&eacute;cifiques sur la prot&eacute;ine Nef interagissent avec les composants de l&#39;endosomale traite des machines, trie Nef vers multiv&eacute;siculaires (MVB) et conditionnement dans les v&eacute;sicules de l&#39;exosome semblable. Pour identifier ces domaines, une s&eacute;rie de mutants s&#39;&eacute;tendant sur la s&eacute;quence enti&egrave;re de nef ont &eacute;t&eacute; faites et clon&eacute;s dans l&#39;expression vector pQB1, qui exprime les mutants comme prot&eacute;ines de fusion de GFP-Nef. Ces structures ont &eacute;t&eacute; utilis&eacute;es dans les essais de transfection transitoire pour identifier les s&eacute;quences n&eacute;cessaires &agrave; la s&eacute;cr&eacute;tion de la prot&eacute;ine de fusion de GFP-Nef. Domaines N-terminaux ont &eacute;t&eacute; identifi&eacute;s comme critiques pour la s&eacute;cr&eacute;tion de la v&eacute;sicule induite par la Nef: (1) un cluster de base de quatre r&eacute;sidus d&#39;arginine (aa 17, 19, 21, 22), (2) le phosphofurin acide cluster s&eacute;quence d&#39;images (PACS ; Glu62-65) et (3) un domaine non d&eacute;finie auparavant s&#39;&eacute;tendant sur les r&eacute;sidus d&#39;acides amin&eacute;s 66-70 (VGFPV), que nous avons nomm&eacute;e la r&eacute;gion de modification de s&eacute;cr&eacute;tion (SMR). Acides amin&eacute;s suppl&eacute;mentaires T44 P25 et 29GVG31 ont &eacute;t&eacute; identifi&eacute;s dans la Nef du VIH-1 comme la r&eacute;gulation de la s&eacute;cr&eacute;tion. Ces r&eacute;sidus n&#39;ont pas &eacute;t&eacute; associ&eacute;s avec d&#39;autres fonctions d&eacute;clar&eacute;es de la Nef. Le domaine de la myristoylation, r&eacute;sidus de lysine ubiquitination et la portion C-terminale de la Nef (aa 71-206) a eu aucun effet sur la s&eacute;cr&eacute;tion. Une s&eacute;quence minimale de Nef VIH-1, comprenant les motifs identifi&eacute;s, suffisait pour la s&eacute;cr&eacute;tion de la v&eacute;sicule induite par la Nef.

Caract&eacute;risation g&eacute;n&eacute;tique de la s&eacute;cr&eacute;tion de v&eacute;sicule induite par la Nef VIH de type 1.

La proteina HIV-1 Nef &egrave; noto per essere secreto, e il nostro gruppo ha dimostrato che il Nef &egrave; secreta dalle cellule transfected nef e HIV-1-infected in piccole vescicole exosome-come (d. 40-100 nm). Il ruolo di Nef secrete rimane completamente essere caratterizzato. Quindi, &egrave; importante caratterizzare la natura di e i meccanismi che regolano la secrezione Nef. Abbiamo ipotizzato che specifici domini strutturali sulla proteina Nef interagiscano con componenti di endosomal traffico di macchine, ordinamento Nef in corpi multivesicular (MVB) e imballaggio in vescicole exosome-like. Per identificare tali domini, una serie di mutanti che attraversa la sequenza intera nef sono stati realizzati e clonato nell&#39;espressione vettoriale pQB1, che esprime i mutanti come proteine di fusione di GFP Nef. Questi costrutti sono stati utilizzati dosaggi di trasfezione transiente per identificare le sequenze necessarie per la secrezione della proteina di fusione GFP-Nef. Domini N-terminale sono stati identificati come critici per la secrezione della vescicola Nef-indotta: (1) un cluster a base di quattro residui di arginina (aa 17, 19, 21, 22), (2) la phosphofurin acida del cluster sequenza (PACS; Glu62-65) e (3) un dominio precedentemente atipiche che abbracciano i residui dell&#39;amminoacido 66-70 (VGFPV), che abbiamo chiamato regione di modificazione della secrezione (SMR). Supplementare di aminoacidi P25, 29GVG31 e T44 sono stati identificati in HIV-1 Nef come che regolano la secrezione. Questi residui non sono stati associati con altre funzioni Nef segnalati. Il dominio myristoylation, residui di lisina ubiquitination e la porzione C-terminale del Nef (aa 71-206) ha avuto alcun effetto sulla secrezione. Una sequenza di HIV-1 Nef minimal, comprendente i motivi identificati, era sufficiente per la secrezione della vescicola Nef-indotta.

Caratterizzazione genetica di secrezione vescicola Nef-indotta di HIV di tipo 1.

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.

La VIH-1 prot&eacute;ine Nef joue un r&ocirc;le actif dans la pathog&eacute;n&egrave;se du sida, par ses nombreuses interactions cellulaires qui facilitent la lib&eacute;ration des particules virales. Cette prot&eacute;ine de 27 kDa est requise pour la maintenance de la r&eacute;plication virale dans le VIH et est &eacute;galement connue pour contribuer &agrave; l&#39;&eacute;vasion immunitaire, blocage de l&#39;apoptose dans les cellules infect&eacute;es par le virus et l&#39;am&eacute;lioration de l&#39;infectiosit&eacute; du virus. Nef s&#39;est av&eacute;r&eacute; &ecirc;tre s&eacute;cr&eacute;t&eacute;e et est pr&eacute;sent sur la surface des cellules infect&eacute;es par le virus. Dans notre laboratoire, des &eacute;tudes r&eacute;centes ont montr&eacute; que la prot&eacute;ine Nef est s&eacute;cr&eacute;t&eacute;e par les cellules transfect&eacute;es nef et infect&eacute;s par le VIH-1 de petites v&eacute;sicules semblables exosome (40-100 nm diam.) qui ne contiennent pas des virions. Nous avons identifi&eacute; trois domaines amino-terminale de la Nef comme n&eacute;cessaire pour la s&eacute;cr&eacute;tion : r&eacute;sidus d&#39;arginine (i) les quatre (17,19,21, 22) comprenant la r&eacute;gion basique ; (ii) la s&eacute;quence de cluster acides phosphofurin (PACS) compos&eacute;e de quatre r&eacute;sidus de l&#39;acide glutamique (61-64) ; (iii) un motif inconnu s&#39;&eacute;tendant sur les r&eacute;sidus d&#39;acides amin&eacute;s 65-69 (VGFPV) que nous avons nomm&eacute;e la r&eacute;gion de modification de s&eacute;cr&eacute;tion (SMR). Dans cette &eacute;tude, nous avons utilis&eacute; l&#39;analyse de donn&eacute;es et s&eacute;quence de phylog&eacute;nie bas&eacute;e sur la population pour caract&eacute;riser la conservation du domaine Nef SMR qui r&eacute;gularise la s&eacute;cr&eacute;tion de la v&eacute;sicule. Nous avons effectu&eacute; dans l&#39;analyse de la chimie computationnelle silico impliquant la mod&eacute;lisation de la structure dynamique mol&eacute;culaire des mutations dans le motif SMR. Analyse de la Nef de patients infect&eacute;s par le VIH-1, y compris les progresseurs lents (SP), longtemps progresseurs de terme (LTP) et long terme non-progresseurs (Progresseurs) a d&eacute;montr&eacute; de conservation de 99 % du motif Nef SMR de s&eacute;quences. Analyse computationnelle, y compris la mod&eacute;lisation de Nef VIH-1 de type sauvage et mutant V66A Nef SMR &agrave; l&#39;aide d&#39;homologie structurale et dynamique mol&eacute;culaire des interactions ligand associ&eacute;e &agrave; des changements structurels importants indiqu&eacute;s chez le mutant de la Nef, favorisant ainsi l&#39;importance du domaine SMR pour la m&eacute;diation de la s&eacute;cr&eacute;tion v&eacute;siculaire Nef.

Validation d&#39;une r&eacute;gion de modification de s&eacute;cr&eacute;tion roman (SMR) de Nef VIH-1 en utilisant l&#39;analyse de s&eacute;quences de cohorte et mod&eacute;lisation mol&eacute;culaire.

La proteina accessoria di HIV-1 Nef svolge un ruolo attivo nella patogenesi dell&#39;AIDS di sue numerose interazioni cellulari che facilitano il rilascio di particelle virali. Questa proteina 27 kDa &egrave; necessaria per la manutenzione della replicazione virale dell&#39;HIV e conosciuta anche a contribuire all&#39;evasione immune, blocco dell&#39;apoptosi nelle cellule infettate da virus e valorizzazione di infettivit&agrave; del virus. NEF ha dimostrato di essere secreta e presente sulla superficie delle cellule infettate da virus. Recenti studi dal nostro laboratorio hanno dimostrato che la proteina Nef &egrave; secreta dalle cellule transfected nef e HIV-1-infected in piccole vescicole exosome-like (diam. di 40-100 nm) che non contengono virioni. Abbiamo identificato tre domini amminico-terminale di Nef come necessarie per la secrezione: residui di arginina (i) i quattro (17,19,21, 22) che compongono la regione di base; (ii) la sequenza di cluster acide phosphofurin (PACS) &egrave; composto da quattro residui di acido glutammico (61-64); (iii) un motivo precedentemente sconosciuto che abbracciano i residui dell&#39;amminoacido 65-69 (VGFPV) che abbiamo chiamato regione di modificazione della secrezione (SMR). In questo studio, abbiamo usato analisi dati e sequenza phylogeny population-based per caratterizzare la conservazione del dominio Nef SMR che regola la secrezione di vescicola. Abbiamo eseguito in silico analisi di chimica computazionale che coinvolgono modellazione struttura dinamica molecolare di mutazioni il motivo SMR. Analisi di Nef da HIV-1-infected patients, lenti progressori (SP), progressori di termine (LTP) lungo e a lungo termine non-progressori (LTNP) ha dimostrati il 99% conservazione del motivo Nef SMR di sequenza. Analisi computazionale compresa la modellazione del selvaggio-tipo HIV-1 Nef e V66A Nef SMR mutante usando omologia strutturale e dinamica molecolare delle interazioni ligando-associati indicati significativi cambiamenti strutturali nel mutante Nef, sostenendo in tal modo l&#39;importanza del dominio SMR per mediare la secrezione della vescicola Nef.

Convalida di una regione di modificazione secrezione romanzo (SMR) di HIV-1 Nef mediante analisi di sequenza di coorte e modellistica molecolare.

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.

William Roth

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Microbiology,Biochemistry,& Immunology,Immunology,Department of Microbiology, Biochemistry, & Immunology,Department of Microbiology, Biochemistry, Immunology

Morehouse School of Medicine

Peptide-based Identification of Functional Motifs and their Binding Partners

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

Department of Microbiology,
Biochemistry,
& Immunology,
Immunology,
Department of Microbiology, Biochemistry, & Immunology,
Department of Microbiology, Biochemistry, Immunology