RESUMO
Macrodomains are proteins that recognize and hydrolyze ADP ribose (ADPR) modifications of intracellular proteins. Macrodomains are implicated in viral genome replication and interference with host cell immune responses. They are important to the infectious cycle of Coronaviridae and Togaviridae viruses. We describe crystal structures of the conserved macrodomain from the bat coronavirus (CoV) HKU4 in complex with ligands. The structures reveal a binding cavity that accommodates ADPR and analogs via local structural changes within the pocket. Using a radioactive assay, we present evidence of mono-ADPR (MAR) hydrolase activity. In silico analysis presents further evidence on recognition of the ADPR modification for hydrolysis. Mutational analysis of residues within the binding pocket resulted in diminished enzymatic activity and binding affinity. We conclude that the common structural features observed in the macrodomain in a bat CoV contribute to a conserved function that can be extended to other known macrodomains.
Assuntos
Adenosina Difosfato Ribose/química , Coronavirus/enzimologia , Pirofosfatases/química , Proteínas não Estruturais Virais/química , Animais , Sítios de Ligação , Quirópteros , Coronavirus/genética , Cristalografia por Raios X , Hidrólise , Pirofosfatases/genética , Proteínas não Estruturais Virais/genéticaRESUMO
BACKGROUND: We describe the spectrum of ICD-10 classified causes for hospitalisations occurring between 2011 and 2018 in a cohort of people living with HIV (PLHIV). METHODS: This sub-study includes 798 PLHIV participating in the Antiretroviral, Sexual Transmission Risk and Attitudes (ASTRA) questionnaire study who were recruited from a large London centre. A medical record review identified the occurrence and causes of hospitalisation from the date of questionnaire completion (February-December 2011) until 1 June 2018. Up to five causes were classified by an HIV clinician using the ICD-10 system. RESULTS: There were 274 hospitalisations in 153 people (rate = 5.8/100 person-years; 95% CI: 5.1, 6.5). Causes were wide-ranging; the most common were circulatory (16.8%), digestive (13.1%), respiratory (11.7%), infectious diseases (11.0%), injury/poisoning (10.6%), genitourinary diseases (9.9%) and neoplasms (9.1%). A tenth (27/274) of hospitalisations were related to at least one AIDS-defining illness. Median duration of hospitalisation was 5 days (IQR 2-9). At the time of hospitalisation, median CD4 count was high (510 cells/µl; IQR: 315-739), while median CD4 nadir was relatively low (113 cells/µl; IQR: 40-239). At admission, half of individuals (51%) had a previous AIDS-defining illness and 21% had viral load > 50 copies/ml. Individuals admitted for infectious diseases were particularly likely to have unfavourable HIV-related clinical characteristics (low CD4, viral non-suppression, not on antiretroviral therapy (ART), previous AIDS). CONCLUSIONS: In the modern combination antiretroviral therapy era, the spectrum of causes of hospitalisation in PLHIV in the UK is wide-ranging, highlighting the importance of holistic care for PLHIV, including prevention, early detection and treatment of comorbidities.
Assuntos
Infecções por HIV/epidemiologia , Infecções por HIV/etiologia , Hospitalização/estatística & dados numéricos , Adulto , Antirretrovirais/uso terapêutico , Contagem de Linfócito CD4 , Doenças Cardiovasculares/epidemiologia , Estudos de Coortes , Comorbidade , Doenças do Sistema Digestório/epidemiologia , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Infecções/epidemiologia , Londres/epidemiologia , Masculino , Pessoa de Meia-Idade , Neoplasias/epidemiologia , Carga ViralRESUMO
Disclosure of HIV status to family, friends, and a stable partner may be linked to improved health outcomes for people living with HIV. This study assessed whether non-disclosure is associated with psychological symptoms, non-adherence to antiretroviral therapy (ART), and viral load (VL) non-suppression. A total of 3258 HIV-diagnosed individuals in the UK completed the confidential ASTRA study questionnaire (2011-2012). Participants reported whether they told anyone they had HIV; to which confidant(s) (friends, family, work colleagues, stable partner) and to what extent (none, some, most/all). The prevalence and factors associated with non-disclosure were assessed. Associations between non-disclosure and the following factors were established using modified Poisson regression with adjustment for socio-demographic factors (gender, age group, ethnicity), HIV-related factors (time since HIV diagnosis, ART status), and clinic: low social support (score ≤ 12 on modified Duke-UNC FSSQ); depression and anxiety symptoms (≥10 on PHQ-9 and GAD-7 respectively); self-reported ART non-adherence in past 2 weeks/3 months; VL non-suppression (clinic-recorded VL > 50 copies/mL among those who started ART ≥ 6 months ago). Among 3233 participants with disclosure data, the prevalence of non-disclosure to anyone was 16.6 % (n/N = 61/367) among heterosexual men, 15.7 % (98/626) among women, and 5.0 % (113/2240) among MSM. MSM were more likely to disclose to some/all friends compared to family (85.8 vs. 59.9 %) while heterosexuals were less likely to disclose to friends than family (44.1 vs. 61.1 % for men, 57.5 vs. 67.1 % for women). Among 1,631 participants with a stable partner, non-disclosure to a stable partner was 4.9 % for MSM, 10.9 % for heterosexual men, and 13.0 % for women. In adjusted analyses, older age (≥60 years), non-white ethnicity, more recent HIV diagnosis, and not having a stable partner were significantly associated with overall non-disclosure for MSM and heterosexual individuals. The prevalence of low social support was 14.4 %, of depression and anxiety symptoms 27.1 and 22.0 %, respectively, of ART non-adherence 31.8 %, and of viral load non-suppression on ART 9.8 %. There was no evidence that non-disclosure overall (versus disclosure to anyone) was associated with low social support, depression or anxiety symptoms, ART non-adherence or VL non-suppression among MSM or heterosexual individuals. However, compared to MSM who disclosed to 'none' or 'some' friends and family, MSM who disclosed to 'most or all' of their friends and family were more likely to have symptoms of depression (adjusted PR = 1.4, 95 % CI 1.2-1.7), anxiety (1.3, 1.1-1.6), and to report ART non-adherence (1.3, 1.1-1.5). In this large multicentre study of people living with HIV in the UK, non-disclosure was overall low, but higher for heterosexual individuals compared to MSM. Non-disclosure was not associated with higher prevalence of adverse health measures.
Assuntos
Ansiedade/psicologia , Depressão/psicologia , Infecções por HIV/psicologia , Adesão à Medicação , Autorrevelação , Adulto , Antirretrovirais/uso terapêutico , População Negra , Revelação , Etnicidade , Família , Feminino , Amigos , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Heterossexualidade , Humanos , Masculino , Pessoa de Meia-Idade , Parceiros Sexuais , Minorias Sexuais e de Gênero , Apoio Social , Inquéritos e Questionários , Reino Unido , Carga Viral , População BrancaRESUMO
BACKGROUND: The range of combination antiretroviral therapy (cART) regimens available in many middle-income countries differs from those suggested in international HIV treatment guidelines. We compared first-line cART regimens, timing of initiation and treatment outcomes in a middle income setting (HIV Centre, Belgrade, Serbia - HCB) with a high-income country (Royal Free London Hospital, UK - RFH). METHODS: All antiretroviral-naïve HIV-positive individuals from HCB and RFH starting cART between 2003 and 2012 were included. 12-month viral load and CD4 count responses were compared, considering the first available measurement 12-24 months post-cART. The percentage that had made an antiretroviral switch for any reason, or for toxicity and the percentage that had died by 36 months (the latest time at which sufficient numbers remained under follow-up) were investigated using standard survival methods. RESULTS: 361/597 (61 %) of individuals initiating cART at HCB had a prior AIDS diagnosis, compared to 337/1763 (19 %) at RFH. Median pre-ART CD4 counts were 177 and 238 cells/mm(3) respectively (p < 0.0001). The most frequently prescribed antiretrovirals were zidovudine with lamivudine (149; 25 %) and efavirenz [329, 55 %] at HCB and emtricitabine with tenofovir (899; 51 %) and efavirenz [681, 39 %] at RFH. At HCB, a median of 2 CD4 count measurements in the first year of cART were taken, compared to 5 at RFH (p < 0.0001). Median (IQR) CD4 cell increase after 12 months was +211 (+86, +359) and +212 (+105, +318) respectively. 287 (48 %) individuals from HCB and 1452 (82 %) from RFH had an available viral load measurement, of which 271 (94 %) and 1280 (88 %) were <400 copies/mL (p < 0.0001). After 36 months, comparable percentages had made at least one antiretroviral switch (77 % HCB vs. 78 % RFH; p = 0.23). However, switches for toxicity/patient choice were more common at RFH. After 12 and 36 months of cART 3 % and 8 % of individuals died at HCB, versus 2 % and 4 % at RFH (p < 0.0001). CONCLUSION: In middle-income countries, cART is usually started at an advanced stage of HIV disease, resulting in higher mortality rates than in high income countries, supporting improved testing campaigns for early detection of HIV infection and early introduction of newer cART regimens.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Países Desenvolvidos , Países em Desenvolvimento , Fidelidade a Diretrizes/estatística & dados numéricos , Infecções por HIV/tratamento farmacológico , HIV-1 , Padrões de Prática Médica/estatística & dados numéricos , Adulto , Quimioterapia Combinada , Feminino , Seguimentos , Humanos , Londres , Masculino , Pessoa de Meia-Idade , Guias de Prática Clínica como Assunto , Estudos Retrospectivos , Sérvia , Resultado do TratamentoRESUMO
Inteins are nature's escape artists; they facilitate their excision from flanking polypeptides (exteins) concomitant with extein ligation to produce a mature host protein. Splicing requires sequential nucleophilic displacement reactions catalyzed by strategies similar to proteases and asparagine lyases. Inteins require precise reaction coordination rather than rapid turnover or tight substrate binding because they are single turnover enzymes with covalently linked substrates. This has allowed inteins to explore alternative mechanisms with different steps or to use different methods for activation and coordination of the steps. Pressing issues include understanding the underlying details of catalysis and how the splicing steps are controlled.
Assuntos
Inteínas/genética , Modelos Genéticos , Precursores de Proteínas/genética , Processamento de Proteína/genética , Aminoácidos/química , Aminoácidos/genética , Exteínas/genética , Estrutura Molecular , Precursores de Proteínas/química , Proteínas/química , Proteínas/genéticaRESUMO
BACKGROUND: Chronic kidney disease (CKD) is a major health issue for HIV-positive individuals, associated with increased morbidity and mortality. Development and implementation of a risk score model for CKD would allow comparison of the risks and benefits of adding potentially nephrotoxic antiretrovirals to a treatment regimen and would identify those at greatest risk of CKD. The aims of this study were to develop a simple, externally validated, and widely applicable long-term risk score model for CKD in HIV-positive individuals that can guide decision making in clinical practice. METHODS AND FINDINGS: A total of 17,954 HIV-positive individuals from the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study with ≥3 estimated glomerular filtration rate (eGFR) values after 1 January 2004 were included. Baseline was defined as the first eGFR > 60 ml/min/1.73 m2 after 1 January 2004; individuals with exposure to tenofovir, atazanavir, atazanavir/ritonavir, lopinavir/ritonavir, other boosted protease inhibitors before baseline were excluded. CKD was defined as confirmed (>3 mo apart) eGFR ≤ 60 ml/min/1.73 m2. Poisson regression was used to develop a risk score, externally validated on two independent cohorts. In the D:A:D study, 641 individuals developed CKD during 103,185 person-years of follow-up (PYFU; incidence 6.2/1,000 PYFU, 95% CI 5.7-6.7; median follow-up 6.1 y, range 0.3-9.1 y). Older age, intravenous drug use, hepatitis C coinfection, lower baseline eGFR, female gender, lower CD4 count nadir, hypertension, diabetes, and cardiovascular disease (CVD) predicted CKD. The adjusted incidence rate ratios of these nine categorical variables were scaled and summed to create the risk score. The median risk score at baseline was -2 (interquartile range -4 to 2). There was a 1:393 chance of developing CKD in the next 5 y in the low risk group (risk score < 0, 33 events), rising to 1:47 and 1:6 in the medium (risk score 0-4, 103 events) and high risk groups (risk score ≥ 5, 505 events), respectively. Number needed to harm (NNTH) at 5 y when starting unboosted atazanavir or lopinavir/ritonavir among those with a low risk score was 1,702 (95% CI 1,166-3,367); NNTH was 202 (95% CI 159-278) and 21 (95% CI 19-23), respectively, for those with a medium and high risk score. NNTH was 739 (95% CI 506-1462), 88 (95% CI 69-121), and 9 (95% CI 8-10) for those with a low, medium, and high risk score, respectively, starting tenofovir, atazanavir/ritonavir, or another boosted protease inhibitor. The Royal Free Hospital Clinic Cohort included 2,548 individuals, of whom 94 individuals developed CKD (3.7%) during 18,376 PYFU (median follow-up 7.4 y, range 0.3-12.7 y). Of 2,013 individuals included from the SMART/ESPRIT control arms, 32 individuals developed CKD (1.6%) during 8,452 PYFU (median follow-up 4.1 y, range 0.6-8.1 y). External validation showed that the risk score predicted well in these cohorts. Limitations of this study included limited data on race and no information on proteinuria. CONCLUSIONS: Both traditional and HIV-related risk factors were predictive of CKD. These factors were used to develop a risk score for CKD in HIV infection, externally validated, that has direct clinical relevance for patients and clinicians to weigh the benefits of certain antiretrovirals against the risk of CKD and to identify those at greatest risk of CKD.
Assuntos
Fármacos Anti-HIV/efeitos adversos , Infecções por HIV/complicações , Rim/efeitos dos fármacos , Insuficiência Renal Crônica/etiologia , Adulto , Fatores Etários , Fármacos Anti-HIV/uso terapêutico , Contagem de Linfócito CD4 , Tomada de Decisão Clínica , Comorbidade , Feminino , HIV , Infecções por HIV/tratamento farmacológico , Soropositividade para HIV/complicações , Humanos , Incidência , Rim/patologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Insuficiência Renal Crônica/epidemiologia , Risco , Medição de Risco , Fatores SexuaisRESUMO
Bacterial capsular polysaccharides have been used as effective vaccines for adults but infants and seniors respond poorly to these immunogens because pure polysaccharides are unable to activate T-cells resulting in antibodies of low affinity and poor immunological memory. These deficiencies are addressed by conjugate vaccines composed of bacterial polysaccharide covalently attached to protein carriers such as tetanus or diphtheria toxoids. These vaccines activate T-cells and have been hugely effective in reducing the incidence of dangerous infectious diseases such as bacterial meningitis in infants and adults. The methods of conjugation often produce conjugate vaccines that contain polysaccharides with several cross links to one or more protein carrier molecules. Synthesis of large oligosaccharides derivatized to achieve single site attachment to protein has established the minimal size of an oligosaccharide that is required to produce protective antibody. Several examples are described where such oligosaccharides range in size from 8 to 16 monosaccharides. A more limited set of examples show that protective antibodies may be elicited by conjugate vaccines composed of tri and tetrasaccharide epitopes. One example is the ß-mannan present in the phosphomannan glycoprotein of Candida albicans. Reverse engineering a protective antibody that recognizes this oligosaccharide revealed that a disaccharide was the minimal epitope. Further epitope mapping by functional group modification established that the internal mannose residue of a disaccharide was involved in the most important antibody-disaccharide interactions. NMR binding studies in combination with homology modeling of the bound ß-mannan antigen suggested an optimum oligosaccharide for inclusion in a conjugate vaccine. Several conjugate vaccines were developed to test these conclusions. Immunization of mice or rabbits with conjugates containing disaccharide or trisaccharide conjugated to immunogenic proteins followed by live challenge experiments showed that the vaccines reduced fungal burden. The results of these and other studies suggest new approaches to novel synthetic conjugate vaccines.
Assuntos
Antifúngicos/química , Glicoconjugados/química , Vacinas Conjugadas/química , Animais , Anticorpos Monoclonais/imunologia , Antifúngicos/imunologia , Candidíase/prevenção & controle , Desenho de Fármacos , Glicoconjugados/síntese química , Glicoconjugados/uso terapêutico , Glicopeptídeos/química , Glicopeptídeos/uso terapêutico , Humanos , Toxoide Tetânico/química , Vacinação , Vacinas Conjugadas/uso terapêuticoRESUMO
A self-consistent model of ß-mannan oligosaccharides bound to a monoclonal antibody, C3.1, that protects mice against Candida albicans has been developed through chemical mapping, NMR spectroscopic, and computational studies. This antibody optimally binds di- and trisaccharide epitopes, whereas larger oligomers bind with affinities that markedly decrease with increasing chain length. The (1â2)-ß-linked di-, tri-, and tetramannosides bind in helical conformations similar to the solution global minimum. Antibody recognition of the di- and trisaccharide is primarily dependent on the mannose unit at the reducing end, with the hydrophobic face of this sugar being tightly bound. Recognition of a tetrasaccharide involves a frameshift in the ligand interaction, shown by strong binding of the sugar adjacent to the reducing end. We show that frameshifting may also be deliberately induced by chemical modifications. Molecular recognition patterns similar to that of mAb C3.1, determined by saturation transfer difference-NMR, were also observed in polyclonal sera from rabbits immunized with a trisaccharide glycoconjugate. The latter observation points to the importance of internal residues as immunodominant epitopes in (1â2)-ß-mannans and to the viability of a glycoconjugate vaccine composed of a minimal length oligosaccharide hapten.
Assuntos
Anticorpos Monoclonais/imunologia , Candida albicans/metabolismo , Mananas/imunologia , Sequência de Carboidratos , Simulação por Computador , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Mananas/química , Mananas/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
BACKGROUND: The non-nucleoside reverse transcriptase inhibitor (NNRTI), rilpivirine (TMC278; Tibotec Pharmaceuticals, County Cork, Ireland), had equivalent sustained efficacy to efavirenz in a phase 2b trial in treatment-naive patients infected with HIV-1, but fewer adverse events. We aimed to assess non-inferiority of rilpivirine to efavirenz in a phase 3 trial with common background nucleoside or nucleotide reverse transcriptase inhibitors (N[t]RTIs). METHODS: We undertook a 96-week, phase 3, randomised, double-blind, double-dummy, non-inferiority trial in 98 hospitals or medical centres in 21 countries. We enrolled adults (≥18 years) not previously given antiretroviral therapy and with a screening plasma viral load of 5000 copies per mL or more and viral sensitivity to background N(t)RTIs. We randomly allocated patients (1:1) using a computer-generated interactive web-response system to receive oral rilpivirine 25 mg once daily or efavirenz 600 mg once daily; all patients received an investigator-selected regimen of background N(t)RTIs (tenofovir-disoproxil-fumarate plus emtricitabine, zidovudine plus lamivudine, or abacavir plus lamivudine). The primary outcome was non-inferiority (12% margin on logistic regression analysis) at 48 weeks in terms of confirmed response (viral load <50 copies per mL, defined by the intent-to-treat time to loss of virologic response [TLOVR] algorithm) in all patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT00543725. FINDINGS: From May 22, 2008, we screened 947 patients and enrolled 340 to each group. 86% of patients (291 of 340) who received at least one dose of rilpivirine responded, compared with 82% of patients (276 of 338) who received at least one dose of efavirenz (difference 3.5% [95% CI -1.7 to 8.8]; p(non-inferiority)<0.0001). Increases in CD4 cell counts were much the same between groups. 7% of patients (24 of 340) receiving rilpivirine had a virological failure compared with 5% of patients (18 of 338) receiving efavirenz. 4% of patients (15) in the rilpivirine group and 7% (25) in the efavirenz group discontinued treatment due to adverse events. Grade 2-4 treatment-related adverse events were less common with rilpivirine (16% [54 patients]) than they were with efavirenz (31% [104]; p<0.0001), as were rash and dizziness (p<0.0001 for both) and increases in lipid levels were significantly lower with rilpivirine than they were with efavirenz (p<0.0001). INTERPRETATION: Despite a slightly increased incidence of virological failures, a favourable safety profile and non-inferior efficacy compared with efavirenz means that rilpivirine could be a new treatment option for treatment-naive patients infected with HIV-1. FUNDING: Tibotec.
Assuntos
Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Fármacos Anti-HIV/uso terapêutico , Benzoxazinas/uso terapêutico , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Nitrilas/uso terapêutico , Pirimidinas/uso terapêutico , Inibidores da Transcriptase Reversa/uso terapêutico , Síndrome da Imunodeficiência Adquirida/etnologia , Síndrome da Imunodeficiência Adquirida/virologia , Adenina/análogos & derivados , Adenina/uso terapêutico , Adulto , Alcinos , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/efeitos adversos , Benzoxazinas/administração & dosagem , Benzoxazinas/efeitos adversos , Ciclopropanos , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Método Duplo-Cego , Esquema de Medicação , Emtricitabina , Feminino , HIV-1/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Nitrilas/administração & dosagem , Nitrilas/efeitos adversos , Nucleosídeos/uso terapêutico , Nucleotídeos/uso terapêutico , Organofosfonatos/uso terapêutico , Pirimidinas/administração & dosagem , Pirimidinas/efeitos adversos , Rilpivirina , Tenofovir , Resultado do Tratamento , Carga Viral/efeitos dos fármacosRESUMO
The cell wall phosphomannan of Candida species is a complex N-linked glycoprotein with a glycan chain containing predominantly an α-linked mannose backbone with α-mannose branches. A minor ß-mannan component is attached to the branches either via a glycosidic bond (acid stable ß-mannan) or a phosphodiester bond (acid-labile ß-mannan). The α-mannan residues of the cell wall phosphomannan do not afford protective antibody, while the ß-mannan portion is a protective antigen and has become an attractive target as the key epitope of a conjugate vaccine. We report the first synthesis of a tetrasaccharide 1 consisting of a ß1,2-mannopyranosyl trisaccharide linked via a phosphodiester to methyl α-mannopyranoside. This encompasses the attachment site of the acid labile ß-mannan to the α-mannan component of the cell wall phosphomannan. The trisaccharide was formed by an iterative process to first create a ß-glucopyranoside linkage and then epimerize the C-2 center via an oxidation-reduction sequence. The phosphate diester linkage was accessed via an anomeric H-phosphonate. The binding of phosphomannan fragment 1 with the protective antibody C3.1 has been evaluated and compared with a ß-mannotrioside in hapten inhibition experiments. The observed activities are rationalized with a model for docked in the binding site of C3.1.
Assuntos
Candida albicans/química , Mananas/química , Oligonucleotídeos/química , Polissacarídeos/síntese química , Sequência de Carboidratos , Epitopos/química , Dados de Sequência Molecular , Estrutura Molecular , Polissacarídeos/químicaRESUMO
We assessed the efficacy and safety of 10-d monotherapy with the orally administered CCR5 antagonist maraviroc in 63 HIV-1-positive individuals prescreened for the absence of CXCR4-using virus. Maximum reduction in viral load occurred at a median of 10-15 d, with a mean reduction of >or=1.6 log(10) copies/ml at all twice daily doses >or=100 mg. These results provide proof of concept that CCR5 antagonism is a viable antiretroviral therapeutic approach.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Antagonistas dos Receptores CCR5 , Ensaios Clínicos Fase II como Assunto , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Ensaios Clínicos Controlados Aleatórios como Assunto , Fármacos Anti-HIV/efeitos adversos , Fármacos Anti-HIV/uso terapêutico , Área Sob a Curva , Cicloexanos/antagonistas & inibidores , Cicloexanos/uso terapêutico , Relação Dose-Resposta a Droga , Infecções por HIV/sangue , Infecções por HIV/virologia , Humanos , Maraviroc , RNA Viral/sangue , Fatores de Tempo , Resultado do Tratamento , Triazóis/antagonistas & inibidores , Triazóis/uso terapêutico , Carga Viral/estatística & dados numéricosRESUMO
Inteins are single turnover enzymes that splice out of protein precursors during maturation of the host protein (extein). The Cys or Ser at the N terminus of most inteins initiates a four-step protein splicing reaction by forming a (thio)ester bond at the N-terminal splice junction. Several recently identified inteins cannot perform this acyl rearrangement because they do not begin with Cys, Thr, or Ser. This study analyzes one of these, the mycobacteriophage Bethlehem DnaB intein, which we describe here as the prototype for a new class of inteins based on sequence comparisons, reactivity, and mechanism. These Class 3 inteins are characterized by a non-nucleophilic N-terminal residue that co-varies with a non-contiguous Trp, Cys, Thr triplet (WCT) and a Thr or Ser as the first C-extein residue. Several mechanistic differences were observed when compared with standard inteins or previously studied atypical KlbA Ala(1) inteins: (a) cleavage at the N-terminal splice junction in the absence of all standard N- and C-terminal splice junction nucleophiles, (b) activation of the N-terminal splice junction by a variant Block B motif that includes the WCT triplet Trp, (c) decay of the branched intermediate by thiols or Cys despite an ester linkage at the C-extein branch point, and (d) an absolute requirement for the WCT triplet Block F Cys. Based on biochemical data and confirmed by molecular modeling, we propose roles for these newly identified conserved residues, a novel protein splicing mechanism that includes a second branched intermediate, and an intein classification with three mechanistic categories.
Assuntos
DnaB Helicases/classificação , DnaB Helicases/metabolismo , Inteínas/fisiologia , Micobacteriófagos/enzimologia , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína/fisiologia , Sequência de Aminoácidos , Biologia Computacional , Sequência Conservada , DnaB Helicases/genética , Inteínas/genética , Dados de Sequência Molecular , Mutagênese , Micobacteriófagos/genética , Prolina/metabolismo , Processamento de Proteína/efeitos dos fármacos , Compostos de Sulfidrila/farmacologia , TemperaturaRESUMO
BACKGROUND: Nevirapine is metabolized by CYP2B6 and polymorphisms within the CYP2B6 gene partly explain inter-patient variability in pharmacokinetics. The aim of this study was to model the complex relationship between nevirapine exposure, weight and genetics (based on combined analysis of CYP2B6 516Gâ>âT and 983Tâ>âC single nucleotide polymorphisms). METHODS: Non-linear mixed-effects modelling was used to estimate pharmacokinetic parameters from 275 patients. Simulations of the nevirapine concentration profile were performed with dosing regimens of 200 mg twice daily and 400 mg once daily for individuals with body weights of 50, 70 and 90 kg in combination with CYP2B6 genetic variation. RESULTS: A one-compartment model with first-order absorption best described the data. Population clearance was 3.5 L/h with inter-patient variability of 24.6%. 516T homozygosity and 983C heterozygosity were associated with 37% and 40% lower clearance, respectively. Body weight was the only significant demographic factor influencing clearance, which increased by 5% for every 10 kg increase. For individuals with higher body weight, once-daily nevirapine was associated with a greater risk of sub-therapeutic drug exposure than a twice-daily regimen. This risk was offset in individuals who were 516T homozygous or 983C heterozygous in which drug exposure was optimal for â>â 95% of patients with body weight of ≤ 70 kg. CONCLUSIONS: The data suggest that a 400 mg once-daily dose could be implemented in accordance with CYP2B6 polymorphism and body weight. However, the use of nevirapine once daily (immediate release; off-label) in the absence of therapeutic drug monitoring is not recommended due to the risk of inadequate exposure to nevirapine in a high proportion of patients. There are different considerations for the extended-release formulation (nevirapine XR) that demonstrate minimal peak-to-trough fluctuations in plasma nevirapine levels.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/farmacocinética , Hidrocarboneto de Aril Hidroxilases/genética , Infecções por HIV/tratamento farmacológico , Nevirapina/administração & dosagem , Nevirapina/farmacocinética , Oxirredutases N-Desmetilantes/genética , Farmacogenética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Peso Corporal , Citocromo P-450 CYP2B6 , Feminino , Frequência do Gene , Genética Populacional , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Adulto JovemRESUMO
OBJECTIVE: We investigated differences in all-cause hospitalization between key demographic groups among people with HIV in the UK in the current antiretroviral therapy (ART) era. DESIGN/METHODS: We used data from the Royal Free HIV Cohort study between 2007 and 2018. Individuals were classified into five groups: MSM, Black African men who have sex with women (MSW), MSW of other ethnicity, Black African women and women of other ethnicity. We studied hospitalizations during the first year after HIV diagnosis (Analysis-A) separately from those more than one year after diagnosis (Analysis-B). In Analysis-A, time to first hospitalization was assessed using Cox regression adjusted for age and diagnosis date. In Analysis-B, subsequent hospitalization rate was assessed using Poisson regression, accounting for repeated hospitalization within individuals, adjusted for age, calendar year, time since diagnosis. RESULTS: The hospitalization rate was 30.7/100 person-years in the first year after diagnosis and 2.7/100 person-years subsequently; 52% and 13% hospitalizations, respectively, were AIDS-related. Compared with MSM, MSW and women were at much higher risk of hospitalization during the first year [aHR (95% confidence interval, 95% CI): 2.7 (1.7-4.3), 3.0 (2.0-4.4), 2.0 (1.3-2.9), 3.0 (2.0-4.5) for Black African MSW; other ethnicity MSW; Black African women; other ethnicity women respectively, Analysis-A] and remained at increased risk subsequently [corresponding aIRR (95% CI): 1.7 (1.2-2.4), 2.1 (1.5-2.8), 1.5 (1.1-1.9), 1.7 (1.2-2.3), Analysis-B]. CONCLUSION: In this setting with universal healthcare, substantial variation exists in hospitalization risk across demographic groups, both in early and subsequent periods after HIV diagnosis, highlighting the need for targeted interventions.
Assuntos
Infecções por HIV , Minorias Sexuais e de Gênero , Terapia Antirretroviral de Alta Atividade , Estudos de Coortes , Feminino , Infecções por HIV/tratamento farmacológico , Homossexualidade Masculina , Hospitalização , Humanos , MasculinoRESUMO
OBJECTIVE: HIV-remission strategies including kick-and-kill could induce viral transcription and immune-activation in the central nervous system, potentially causing neuronal injury. We investigated the impact of kick-and-kill on plasma neurofilament light (NfL), a marker of neuro-axonal injury, in RIVER trial participants commencing antiretroviral treatment (ART) during primary infection and randomly allocated to ART-alone or kick-and-kill (ART + vaccination + vorinostat (ART + V + V)). DESIGN: Sub-study measuring serial plasma NfL concentrations. METHODS: Plasma NfL (using Simoa digital immunoassay), plasma HIV-1 RNA (using single-copy assay) and total HIV-1 DNA (using quantitative polymerase chain reaction in peripheral CD4+ T-cells) were measured at randomisation (following ≥22 weeks ART), week 12 (on final intervention day in ART + V + V) and week 18 post-randomisation. HIV-specific T-cells were quantified by intracellular cytokine staining at randomisation and week 12. Differences in plasma NfL longitudinally and by study arm were analysed using mixed models and Student's t-test. Associations with plasma NfL were assessed using linear regression and rank statistics. RESULTS: At randomisation, 58 male participants had median age 32 years and CD4+ count 696 cells/µL. No significant difference in plasma NfL was seen longitudinally and by study arm, with median plasma NfL (pg/mL) in ART-only vs ART + V + V: 7.4 vs 6.4, p = 0.16 (randomisation), 8.0 vs 6.9, p = 0.22 (week 12) and 7.1 vs 6.8, p = 0.74 (week 18). Plasma NfL did not significantly correlate with plasma HIV-1 RNA and total HIV-1 DNA concentration in peripheral CD4+ T-cells at any timepoint. While higher HIV-specific T-cell responses were seen at week 12 in ART + V + V, there were no significant correlations with plasma NfL. In multivariate analysis, higher plasma NfL was associated with older age, higher CD8+ count and lower body mass index. CONCLUSIONS: Despite evidence of vaccine-induced HIV-specific T-cell responses, we observed no evidence of increased neuro-axonal injury using plasma NfL as a biomarker up to 18 weeks following kick-and-kill, compared with ART-only.
RESUMO
BACKGROUND: Predictors of hospitalisation in people with HIV (PLHIV) in the contemporary treatment era are not well understood. METHODS: This ASTRA sub-study used clinic data linkage and record review to determine occurrence of hospitalisations among 798 PLHIV from baseline questionnaire (February to December 2011) until 1 June 2018. Associations of baseline social circumstance, socioeconomic, lifestyle, mental health, demographic and clinical factors with repeated all-cause hospitalisation from longitudinal data were investigated using Prentice-Williams-Peterson models. Associations were also assessed in 461 individuals on antiretroviral therapy (ART) with viral load ≤50 copies/ml and CD4 count ≥500 cells/ µl. FINDINGS: Rate of hospitalisation was 5.8/100 person-years (95% CI: 5.1-6.5). Adjusted for age, demographic group and time with diagnosed HIV, the following social circumstance, socioeconomic, lifestyle and mental health factors predicted hospitalisation: no stable partner (adjusted hazard ratio (aHR)=1.59; 95% CI=1.16-2.20 vs living with partner); having children (aHR=1.50; 1.08-2.10); non-employment (aHR=1.56; 1.07-2.27 for unemployment; aHR=2.39; 1.70-3.37 for sick/disabled vs employed); rented housing (aHR=1.72; 1.26-2.37 vs homeowner); not enough money for basic needs (aHR=1.82; 1.19-2.78 vs enough); current smoking (aHR=1.39; 1.02-1.91 vs never); recent injection-drug use (aHR=2.11; 1.30-3.43); anxiety symptoms (aHRs=1.39; 1.01-1.91, 2.06; 1.43-2.95 for mild and moderate vs none/minimal); depressive symptoms (aHRs=1.67; 1.17-2.38, 1.91; 1.30-2.78 for moderate and severe vs none/minimal); treated/untreated depression (aHRs=1.65; 1.03-2.64 for treated depression only, 1.87; 1.39-2.52 for depressive symptoms only; 1.53; 1.05-2.24; for treated depression and depressive symptoms, versus neither). Associations were broadly similar in those with controlled HIV and high CD4. INTERPRETATION: Social circumstance, socioeconomic disadvantage, adverse lifestyle factors and poorer mental health are strong predictors of hospitalisation in PLHIV, highlighting the need for targeted interventions and care. FUNDING: British HIV Association (BHIVA) Research Award (2017); SMR funded by a PhD fellowship from the Royal Free Charity.
RESUMO
The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand beta-sheet holding two alpha-helices of three and four turns that are oriented antiparallel to the beta-strands. Two antiparallel two-strand beta-sheets and two 3(10)-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.
Assuntos
RNA Polimerase Dependente de RNA/química , RNA/metabolismo , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Sítios de Ligação , Espectroscopia de Ressonância Magnética , Conformação Molecular , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The nuclear magnetic resonance (NMR) structure of a central segment of the previously annotated severe acute respiratory syndrome (SARS)-unique domain (SUD-M, for "middle of the SARS-unique domain") in SARS coronavirus (SARS-CoV) nonstructural protein 3 (nsp3) has been determined. SUD-M(513-651) exhibits a macrodomain fold containing the nsp3 residues 528 to 648, and there is a flexibly extended N-terminal tail with the residues 513 to 527 and a C-terminal flexible tail of residues 649 to 651. As a follow-up to this initial result, we also solved the structure of a construct representing only the globular domain of residues 527 to 651 [SUD-M(527-651)]. NMR chemical shift perturbation experiments showed that SUD-M(527-651) binds single-stranded poly(A) and identified the contact area with this RNA on the protein surface, and electrophoretic mobility shift assays then confirmed that SUD-M has higher affinity for purine bases than for pyrimidine bases. In a further search for clues to the function, we found that SUD-M(527-651) has the closest three-dimensional structure homology with another domain of nsp3, the ADP-ribose-1"-phosphatase nsp3b, although the two proteins share only 5% sequence identity in the homologous sequence regions. SUD-M(527-651) also shows three-dimensional structure homology with several helicases and nucleoside triphosphate-binding proteins, but it does not contain the motifs of catalytic residues found in these structural homologues. The combined results from NMR screening of potential substrates and the structure-based homology studies now form a basis for more focused investigations on the role of the SARS-unique domain in viral infection.
Assuntos
Espectroscopia de Ressonância Magnética , RNA Polimerase Dependente de RNA/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Proteínas não Estruturais Virais/química , Ensaio de Desvio de Mobilidade Eletroforética , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , RNA/metabolismo , Proteínas de Ligação a RNA/químicaRESUMO
OBJECTIVES: Guidelines recommend routine testing for latent TB infection (LTBI) in people living with HIV. However there are few cost-effectiveness studies to justify this in contemporary high resource, low TB/HIV incidence settings. We sought to assess the uptake, yield and cost-effectiveness of testing for latent and active TB. METHODS: Adults attending an ambulatory HIV clinic in London, UK were prospectively recruited by stratified selection and tested for TB infection using symptom questionnaires, chest radiograph (CXR), tuberculin skin test (TST), T-Spot.TB and induced sputum. From this, 30 testing strategies were compared in a cost-effectiveness model including probabilistic sensitivity analysis using Monte Carlo simulation. RESULTS: 219 subjects were assessed; 95% were using antiretroviral therapy (ART). Smear negative, culture positive TB was present in 0.9% asymptomatic subjects, LTBI in 9%. Only strategies testing those from subSaharan Africa with a TST or interferon gamma release assay (IGRA) with or without CXR, or testing those from countries with a TB incidence of >40/100,000 with TST alone were cost-effective using a £30,000/QALY threshold. CONCLUSIONS: Cost-effectiveness analysis in an adult HIV cohort with high ART usage suggests there is limited benefit beyond routine testing for latent TB in people from high and possibly medium TB incidence settings.
Assuntos
Infecções por HIV , Tuberculose Latente , Adulto , Análise Custo-Benefício , Infecções por HIV/complicações , Humanos , Testes de Liberação de Interferon-gama , Tuberculose Latente/diagnóstico , Tuberculose Latente/epidemiologia , Londres/epidemiologia , Teste TuberculínicoRESUMO
OBJECTIVE: To investigate the prevalence of widespread pain among people with HIV (PWH) and describe associations with antiretroviral therapy (ART) and markers of HIV disease stage. DESIGN: Cross-sectional analysis of cohort study in the United Kingdom and Ireland. METHODS: Pain information was collected during the baseline visit (conducted from 2013 to 2015) through a self-completed manikin identifying pain at 15 sites from five body regions. Pain was classified as widespread if reported at at least four regions and at least seven sites, or regional otherwise. Chi-squared tests, Kruskal-Wallis tests and ordinal logistic regression were used to consider associations between pain extent and sociodemographic and HIV-related factors. RESULTS: Among the 1207 participants (614 PWHâ≥â50 years, 330 PWHâ<â50 years, 263 HIV-negative controls ≥50 years), pain was most commonly reported at the upper (left: 28.9%, right: 28.0%) and lower (left: 25.7%; right: 24.5%) leg, upper (18.6%) and lower (29.7%) back and shoulders (left: 16.0%; right: 16.8%). Widespread pain was more commonly reported in PWH than in HIV-negative controls (PWHâ≥â50 years: 18.7%; PWHâ<â50 years: 12.7%; HIV-negative ≥50 years: 9.5%) with regional pain reported in 47.6, 44.8 and 49.8%, respectively (global Pâ=â0.001). In multivariable analyses, pain extent was greater in those with lower educational attainment, those exposed to more ART drugs, and those with a higher current CD4 cell count but longer exposure to immunosuppression. CONCLUSION: Widespread pain is commonly reported in PWH and is associated with longer duration of exposure to HIV, immunosuppression and ART. Our findings call for greater awareness, and interventions to support the management, of pain in PWH.