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Percutaneous ablation is recommended in Barcelona Clinic Liver Cancer (BCLC) stage 0/A patients with HCC ≤3 cm as a curative treatment modality alongside surgical resection and liver transplantation. However, trans-arterial chemo-embolisation (TACE) is commonly used in the real-world as an initial treatment in patients with single small HCC in contrast to widely accepted clinical practice guidelines which typically describe TACE as a treatment for intermediate-stage HCC. We performed this real-world propensity-matched multi-centre cohort study in patients with single HCC ≤ 3 cm to assess for differences in survival outcomes between those undergoing initial TACE and those receiving upfront ablation. Patients with a new diagnosis of BCLC 0/A HCC with a single tumour ≤3 cm first diagnosed between 1 January 2016 and 31 December 2020 who received initial TACE or ablation were included in the study. A total of 348 patients were included in the study, with 147 patients receiving initial TACE and 201 patients undergoing upfront ablation. After propensity score matching using key covariates, 230 patients were available for analysis with 115 in each group. There were no significant differences in overall survival (log-rank test p = 0.652) or liver-related survival (log-rank test p = 0.495) over a median follow-up of 43 months. While rates of CR were superior after ablation compared to TACE as a first treatment (74% vs. 56%, p < 0.004), there was no significant difference in CR rates when allowing for further subsequent treatments (86% vs. 80% p = 0.219). In those who achieved CR, recurrence-free survival and local recurrence-free survival were similar (log rank test p = 0.355 and p = 0.390, respectively). Our study provides valuable real-world evidence that TACE when offered with appropriate follow-up treatment is a reasonable initial management strategy in very early/early-stage HCC, with similar survival outcomes as compared to those managed with upfront ablation. Further work is needed to better define the role for TACE in BCLC 0/A HCC.
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Simian immunodeficiency virus (SIV) vaccines based upon 68-1 Rhesus Cytomegalovirus (RhCMV) vectors show remarkable protection against pathogenic SIVmac239 challenge. Across multiple independent rhesus macaque (RM) challenge studies, nearly 60% of vaccinated RM show early, complete arrest of SIVmac239 replication after effective challenge, whereas the remainder show progressive infection similar to controls. Here, we performed viral sequencing to determine whether the failure to control viral replication in non-protected RMs is associated with the acquisition of viral escape mutations. While low level viral mutations accumulated in all animals by 28 days-post-challenge, which is after the establishment of viral control in protected animals, the dominant circulating virus in virtually all unprotected RMs was nearly identical to the challenge stock, and there was no difference in mutation patterns between this cohort and unvaccinated controls. These data definitively demonstrate that viral mutation does not explain lack of viral control in RMs not protected by RhCMV/SIV vaccination. We further demonstrate that during chronic infection RhCMV/SIV vaccinated RMs do not acquire escape mutation in epitopes targeted by RhCMV/SIV, but instead display mutation in canonical MHC-Ia epitopes similar to unvaccinated RMs. This suggests that after the initial failure of viral control, unconventional T cell responses induced by 68-1 RhCMV/SIV vaccination do not exert strong selective pressure on systemically replicating SIV.
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Macaca mulatta , Mutação , Vacinas contra a SAIDS , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinas contra a SAIDS/imunologia , Vacinas contra a SAIDS/genética , Citomegalovirus/imunologia , Citomegalovirus/genética , Replicação Viral/imunologia , Vacinação , Evasão da Resposta Imune/genéticaRESUMO
The management of early-stage hepatocellular carcinoma (HCC) is complex, with multiple treatment strategies available. There is a paucity of literature regarding variations in the patterns of care and outcomes between transplant and non-transplant centres. We conducted this real-world multi-centre cohort study in two liver cancer referral centres with an integrated liver transplant program and an additional eight non-transplant HCC referral centres across Australia to identify variation in patterns of care and key survival outcomes. Patients with stage Barcelona Clinic Liver Cancer (BCLC) 0/A HCC, first diagnosed between 1 January 2016 and 31 December 2020, who were managed at a participating site, were included in the study. Patients were excluded if they had a history of prior HCC or if they received upfront liver transplantation. A total of 887 patients were included in the study, with 433 patients managed at a liver cancer centre with a transplant program (LTC) and 454 patients managed at a non-transplant centre (NTC). Management at an LTC did not significantly predict allocation to resection (adjusted OR 0.75, 95% CI 0.50 to 1.11, p = 0.148). However, in those not receiving resection, LTC and NTC patients were systematically managed differently, with LTC patients five times less likely to receive upfront ablation than NTC patients (adjusted OR 0.19, 95% CI 0.13 to 0.28, p < 0.001), even after adjusting for tumour burden, as well as for age, gender, liver disease aetiology, liver disease severity, and medical comorbidities. LTCs exhibited significantly higher proportions of patients undergoing TACE for every tumour burden category, including those with a single tumour measuring 2 cm or less (p < 0.001). Using multivariable Cox proportional hazards analysis, management at a transplant centre was associated with reduced all-cause mortality (adjusted HR 0.71, 95% CI 0.51 to 0.98, p = 0.036), and competing-risk regression analysis, considering liver transplant as a competing event, demonstrated a similar reduction in risk (adjusted HR 0.70, 95% CI 0.50 to 0.99, p = 0.041), suggesting that the reduced risk of death is not fully explained by higher rates of transplantation. Our study highlights systematic differences in HCC care between large volume liver transplant centres and other sites, which has not previously been well-described. Further work is needed to better define the reasons for differences in treatment allocation and to aim to minimise unwarranted treatment variation to maximise patient outcomes across Australia.
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ECHO (Extensions for Community Healthcare Outcomes) Autism is a telementoring learning model to increase community capacity for autism-related health care. Seventy-seven pediatric providers (mostly primary care, seeing exclusively Medicaid patient populations) enrolled in 1 year of ECHO Autism Washington. Analysis of self-report surveys showed a significant increase in autism diagnoses made by ECHO providers after 1 year, F(1, 65) = 7.52, P = .008. Providers who attended more sessions reported making more diagnoses, F(2, 613.26), P = .045. Of note, autism diagnoses were not externally validated. The total number of reported barriers reduced, F(2, 61) = 13.5), P < .001, and confidence ratings increased F(2, 60) = 24.21, P < .001. The average number of diagnostic referrals from ECHO providers to the state's largest autism specialty clinic significantly reduced, t(43) = 4.23, P < .001, with significantly fewer diagnostic referrals made during and after ECHO training compared with a comparison group of 28 non-ECHO providers, t(58.77) = -3.36, P < .001. Overall, 1 year of ECHO Autism Washington participation led to significant changes in autism diagnostic practices.
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BACKGROUND: The carbon footprint of Canada's health sector is among the worst in the world, responsible for 4·6% of Canada's total greenhouse gas emissions. A quarter of emissions from Canada's health sector are linked to pharmaceuticals, including metered dose inhalers (MDIs). MDIs use propellants, such as hydrofluorocarbons, which act as greenhouse gas emissions and contribute to the health-care sector's overall carbon footprint. The objective of this study was to describe MDI prescribing, dispensing, usage, and waste patterns at The Ottawa Hospital (Ottawa, ON, Canada). Secondary objectives included estimating the monetary and carbon cost of current practice and the potential benefits and costs of switching to the more environmentally friendly dry powder inhalers. METHODS: In this retrospective point-prevalence cohort study, we identified 100 consecutive patients from medical and surgical services at both campuses of The Ottawa Hospital from health records discharged from medical and surgical services and who were prescribed at least one MDI during their admission. Medical records were reviewed and data related to demographics, MDI prescribing, dispensing, usage, and wastage were collected using a pre-piloted electronic case report form. Financial cost was calculated using local costing estimates and carbon cost was calculated using published estimates. FINDINGS: Between Jan 1, 2023, and June 1, 2023, we collected data for 100 eligible patients, of whom 60 (60%) were female and 90 (90%) were admitted to hospital medicine wards (10% from surgical wards). The median length of stay was 7 (range 1-47) days. The most common inpatient diagnoses were respiratory tract infections in 43 (43%) of 100 patients and chronic obstructive pulmonary disease exacerbations in 28 (28%) of 100 patients. The median number of MDIs prescribed during a patients stay was two (range one to 15) and the median number dispensed was one (range one to seven). For formulary options of MDIs, of the 200 (range 30-1400) actuations dispensed per patient, 8% were used, representing 92% wastage. During the audit, 315 MDIs were dispensed in total, of which 97 were not used at all. INTERPRETATION: MDIs are significant contributors to the carbon footprint attributed to pharmaceutical use in hospitals. This study suggests that 90% of MDI doses are wasted, showing that there is substantial room for improvement. FUNDING: None.
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Gases de Efeito Estufa , Humanos , Feminino , Masculino , Estudos de Coortes , Estudos Retrospectivos , Nebulizadores e Vaporizadores , Inaladores Dosimetrados , Hospitais , CarbonoRESUMO
Adults with intellectual and developmental disabilities frequently experience poor life outcomes, with individuals reporting lower levels of social support, relationships, gainful employment, and satisfaction in their quality of life. To ameliorate these outcomes, social skills and social communication interventions aligned with the needs of adults are warranted. This study examined the efficacy of Snack Talk, a supplemental naturalistic visual communication support, with five adults with intellectual and developmental disabilities. Snack Talk was implemented during the midday mealtime, with the goal of increasing conversation engagement. A withdrawal design across participants was used. Results demonstrated increases in conversation engagement and showed meaningful gains for participants in the intervention and postintervention phase compared to baseline.
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The optimal treatment approach in very-early and early-stage hepatocellular carcinoma (HCC) is not precisely defined, and there is ambiguity in the literature around the comparative efficacy of surgical resection versus ablation as curative therapies for limited disease. We performed this real-world propensity-matched, multi-centre cohort study to assess for differences in survival outcomes between those undergoing resection and those receiving ablation. Patients with Barcelona Clinic Liver Cancer (BCLC) 0/A HCC first diagnosed between 1 January 2016 and 31 December 2020 who received ablation or resection as initial treatment were included in the study. A total of 450 patients were included in the study from 10 major liver centres including two transplant centres. Following propensity score matching using key covariates, 156 patients were available for analysis with 78 in each group. Patients who underwent resection had significantly improved overall survival (log-rank test p = 0.023) and local recurrence-free survival (log rank test p = 0.027) compared to those who received ablation. Based on real-world data, our study supports the use of surgical resection in preference to ablation as first-line curative therapy in appropriately selected BCLC 0/A HCC patients.
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Allogeneic hematopoietic stem cell transplantation (alloHSCT) from donors lacking C-C chemokine receptor 5 (CCR5Δ32/Δ32) can cure HIV, yet mechanisms remain speculative. To define how alloHSCT mediates HIV cure, we performed MHC-matched alloHSCT in SIV+, anti-retroviral therapy (ART)-suppressed Mauritian cynomolgus macaques (MCMs) and demonstrated that allogeneic immunity was the major driver of reservoir clearance, occurring first in peripheral blood, then peripheral lymph nodes, and finally in mesenteric lymph nodes draining the gastrointestinal tract. While allogeneic immunity could extirpate the latent viral reservoir and did so in two alloHSCT-recipient MCMs that remained aviremic >2.5 years after stopping ART, in other cases, it was insufficient without protection of engrafting cells afforded by CCR5-deficiency, as CCR5-tropic virus spread to donor CD4+ T cells despite full ART suppression. These data demonstrate the individual contributions of allogeneic immunity and CCR5 deficiency to HIV cure and support defining targets of alloimmunity for curative strategies independent of HSCT.
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Infecções por HIV , Transplante de Células-Tronco Hematopoéticas , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Macaca fascicularis , Carga ViralRESUMO
As the prevalence of autism continues to rise, early childhood programs continue to evolve to meet the needs of children across a spectrum of abilities. To do this, strategies and supports are needed for teachers to engage with children who experience difficulties across developmental domains. Snack Talk, a naturalistic visual communication intervention, focuses on increases in conversation engagement for children with autism and related disabilities during mealtimes. This study examined the effects of the implementation of Snack Talk on increasing teacher engagement in conversation with five preschool children with autism during mealtimes in an EIBI classroom setting. A reversal design was used to analyze the relationship between Snack Talk and teacher conversational engagement with children. Results of this study demonstrated that implementation of Snack Talk increased instances of teacher engagement in conversation with children compared to baseline phases, demonstrating the promise of this intervention supporting students' different levels of support needs in inclusive, blended settings. A functional relationship has been established between baseline and intervention phases and generalization. Limitations and directions for further research are discussed.
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The CCR5-specific antibody Leronlimab is being investigated as a novel immunotherapy that can suppress HIV replication with minimal side effects. Here we studied the virological and immunological consequences of Leronlimab in chronically CCR5-tropic HIV-1 infected humans (n = 5) on suppressive antiretroviral therapy (ART) and in ART-naïve acutely CCR5-tropic SHIV infected rhesus macaques (n = 4). All five human participants transitioned from daily combination ART to self-administered weekly subcutaneous (SC) injections of 350 mg or 700 mg Leronlimab and to date all participants have sustained virologic suppression for over seven years. In all participants, Leronlimab fully occupied CCR5 receptors on peripheral blood CD4+ T cells and monocytes. In ART-naïve rhesus macaques acutely infected with CCR5-tropic SHIV, weekly SC injections of 50 mg/kg Leronlimab fully suppressed plasma viremia in half of the macaques. CCR5 receptor occupancy by Leronlimab occurred concomitant with rebound of CD4+ CCR5+ T-cells in peripheral blood, and full CCR5 receptor occupancy was found in multiple anatomical compartments. Our results demonstrate that weekly, self-administered Leronlimab was safe, well-tolerated, and efficacious for long-term virologic suppression and should be included in the arsenal of safe, easily administered, longer-acting antiretroviral treatments for people living with HIV-1. Trial Registration: ClinicalTrials.gov Identifiers: NCT02175680 and NCT02355184.
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Vírus da Imunodeficiência Símia , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Anti-HIV , Humanos , Macaca mulatta , Receptores CCR5RESUMO
CD8+ T cells are key mediators of antiviral and antitumor immunity. The isolation and study of Ag-specific CD8+ T cells, as well as mapping of their MHC restriction, has practical importance to the study of disease and the development of therapeutics. Unfortunately, most experimental approaches are cumbersome, owing to the highly variable and donor-specific nature of MHC-bound peptide/TCR interactions. Here we present a novel system for rapid identification and characterization of Ag-specific CD8+ T cells, particularly well suited for samples with limited primary cells. Cells are stimulated ex vivo with Ag of interest, followed by live cell sorting based on surface-trapped TNF-α. We take advantage of major advances in single-cell sequencing to generate full-length sequence data from the paired TCR α- and ß-chains from these Ag-specific cells. The paired TCR chains are cloned into retroviral vectors and used to transduce donor CD8+ T cells. These TCR transductants provide a virtually unlimited experimental reagent, which can be used for further characterization, such as minimal epitope mapping or identification of MHC restriction, without depleting primary cells. We validated this system using CMV-specific CD8+ T cells from rhesus macaques, characterizing an immunodominant Mamu-A1*002:01-restricted epitope. We further demonstrated the utility of this system by mapping a novel HLA-A*68:02-restricted HIV Gag epitope from an HIV-infected donor. Collectively, these data validate a new strategy to rapidly identify novel Ags and characterize Ag-specific CD8+ T cells, with applications ranging from the study of infectious disease to immunotherapeutics and precision medicine.
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Linfócitos T CD8-Positivos , Infecções por HIV , Animais , Epitopos , Epitopos de Linfócito T , Macaca mulatta , Receptores de Antígenos de Linfócitos T , Fator de Necrose Tumoral alfaRESUMO
In the absence of a prophylactic vaccine, the use of antiretroviral therapy (ART) as pre-exposure prophylaxis (PrEP) to prevent HIV acquisition by uninfected individuals is a promising approach to slowing the epidemic, but its efficacy is hampered by incomplete patient adherence and ART-resistant variants. Here, we report that competitive inhibition of HIV Env-CCR5 binding via the CCR5-specific antibody Leronlimab protects rhesus macaques against infection following repeated intrarectal challenges of CCR5-tropic SHIVSF162P3. Injection of Leronlimab weekly at 10 mg/kg provides significant but partial protection, while biweekly 50 mg/kg provides complete protection from SHIV acquisition. Tissue biopsies from protected macaques post challenge show complete CCR5 receptor occupancy and an absence of viral nucleic acids. After Leronlimab washout, protected macaques remain aviremic, and adoptive transfer of hematologic cells into naïve macaques does not transmit viral infection. These data identify CCR5 blockade with Leronlimab as a promising approach to HIV prophylaxis and support initiation of clinical trials.
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Receptores CCR5/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/transmissão , Vírus da Imunodeficiência Símia/imunologia , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Feminino , Anticorpos Anti-HIV/farmacologia , Infecções por HIV , Humanos , Macaca mulatta , Masculino , Mucosa , Profilaxia Pré-Exposição , Carga ViralRESUMO
Macaques are physiologically relevant animal models of human immunology and infectious disease that have provided key insights and advanced clinical treatment in transplantation, vaccinology, and HIV/AIDS. However, the small size of macaques is a stumbling block for studies requiring large numbers of cells, such as hematopoietic stem cells (HSCs) for transplantation, antigen-specific lymphocytes for in-depth immunological analysis, and latently-infected CD4+ T-cells for HIV cure studies. Here, we provide a detailed protocol for collection of large numbers of HSCs and T-cells from cynomolgus macaques as small as 3 kg using the Terumo Spectra Optia apheresis system, yielding an average of 5.0 × 109 total nucleated cells from mobilized animals and 1.2 × 109 total nucleated cells from nonmobilized animals per procedure. This report provides sufficient detail to adapt this apheresis technique at other institutions, which will facilitate more efficient and detailed analysis of HSCs and their progeny blood cells.
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Remoção de Componentes Sanguíneos/métodos , Células-Tronco Hematopoéticas/citologia , Linfócitos T/citologia , Animais , Benzilaminas/farmacologia , Creatinina/sangue , Ciclamos/farmacologia , Feminino , Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Macaca fascicularis , MasculinoRESUMO
Despite the success of antiretroviral therapy (ART) to halt viral replication and slow disease progression, this treatment is not curative and there remains an urgent need to develop approaches to clear the latent HIV reservoir. The human IL-15 superagonist N-803 (formerly ALT-803) is a promising anti-cancer biologic with potent immunostimulatory properties that has been extended into the field of HIV as a potential "shock and kill" therapeutic for HIV cure. However, the ability of N-803 to reactivate latent virus and modulate anti-viral immunity in vivo under the cover of ART remains undefined. Here, we show that in ART-suppressed, simian-human immunodeficiency virus (SHIV)SF162P3-infected rhesus macaques, subcutaneous administration of N-803 activates and mobilizes both NK cells and SHIV-specific CD8+ T cells from the peripheral blood to lymph node B cell follicles, a sanctuary site for latent virus that normally excludes such effector cells. We observed minimal activation of memory CD4+ T cells and no increase in viral RNA content in lymph node resident CD4+ T cells post N-803 administration. Accordingly, we found no difference in the number or magnitude of plasma viremia timepoints between treated and untreated animals during the N-803 administration period, and no difference in the size of the viral DNA cell-associated reservoir post N-803 treatment. These results substantiate N-803 as a potent immunotherapeutic candidate capable of activating and directing effector CD8+ T and NK cells to the B cell follicle during full ART suppression, and suggest N-803 must be paired with a bona fide latency reversing agent in vivo to facilitate immune-mediated modulation of the latent viral reservoir.
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Antirretrovirais/administração & dosagem , Linfócitos B/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Interleucina-15/antagonistas & inibidores , Células Matadoras Naturais/efeitos dos fármacos , Proteínas/administração & dosagem , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/fisiopatologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Interleucina-15/genética , Interleucina-15/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Macaca mulatta , Proteínas Recombinantes de Fusão , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/fisiopatologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/fisiologia , Latência Viral/efeitos dos fármacosRESUMO
Allogeneic hematopoietic stem cell transplantation (HSCT) and xenotransplantation are accompanied by viral reactivations and virus-associated complications resulting from immune deficiency. Here, in a Mauritian cynomolgus macaque model of fully MHC-matched allogeneic HSCT, we report reactivations of cynomolgus polyomavirus, lymphocryptovirus, and cytomegalovirus, macaque viruses analogous to HSCT-associated human counterparts BK virus, Epstein-Barr virus, and human cytomegalovirus. Viral replication in recipient macaques resulted in characteristic disease manifestations observed in HSCT patients, such as polyomavirus-associated hemorrhagic cystitis and tubulointerstitial nephritis or lymphocryptovirus-associated post-transplant lymphoproliferative disorder. However, in most cases, the reconstituted immune system, alone or in combination with short-term pharmacological intervention, exerted control over viral replication, suggesting engraftment of functional donor-derived immunity. Indeed, the donor-derived reconstituted immune systems of two long-term engrafted HSCT recipient macaques responded to live attenuated yellow fever 17D vaccine (YFV 17D) indistinguishably from untransplanted controls, mounting 17D-targeted neutralizing antibody responses and clearing YFV 17D within 14 days. Together, these data demonstrate that this macaque model of allogeneic HSCT recapitulates clinical situations of opportunistic viral infections in transplant patients and provides a pre-clinical model to test novel prophylactic and therapeutic modalities.
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Modelos Animais de Doenças , Transplante de Células-Tronco Hematopoéticas , Infecções Oportunistas , Viroses , Aloenxertos , Animais , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Macaca fascicularis , Infecções Oportunistas/virologiaRESUMO
The gene product of the open reading frame Rv3340 from Mycobacterium tuberculosis is annotated as encoding a probable O-acetylhomoserine (OAH) sulfhydrylase (MetC), an enzyme that catalyzes the last step in the biosynthesis of methionine, which is an essential amino acid in bacteria and plants. Following overexpression in Escherichia coli, the M. tuberculosis MetC enzyme was purified and crystallized using the hanging-drop vapor-diffusion method. Native diffraction data were collected from crystals belonging to space group P2(1) and were processed to a resolution of 2.1â Å.
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Carbono-Oxigênio Liases/química , Mycobacterium tuberculosis/enzimologia , Sequência de Aminoácidos , Carbono-Oxigênio Liases/genética , Carbono-Oxigênio Liases/isolamento & purificação , Sequência Conservada , Cristalografia por Raios X , Expressão Gênica , Humanos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
This review outlines known examples of the three-dimensional structures of protein proteinase inhibitors from plants. Three families of enzymes, serine proteinases, carboxypeptidases and cysteine proteinases, are targeted by at least a dozen inhibitor families, with the majority of them adopting the standard mechanism of inhibition towards the serine proteinases. All of the inhibitors discussed maintain compact and stable inhibitory domains that bind to the active site of their target proteinases and prevent access to the substrate molecules. One interesting highlight is the knottin group. Three separate inhibitor families utilize the overall knottin fold in a different way. This fold can accommodate extensive sequence variation and for each of the squash, Mirabilis and Potato carboxypeptidase families, the proteinase-binding residues are found at a different location. Plants have also evolved additional strategies to regulate proteinase activity, such as linking inhibitory domains and targeting multiple enzymes at once. The structural aspects of these strategies are discussed in the review.
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Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Plantas/metabolismoRESUMO
ß-Hexosaminidases (ß-hex) are a group of glycosyl hydrolase isozymes that break down neutral and sialylated glycosphingolipids in the lysosomes, thereby preventing their buildup in neuronal cells. Some mutants of ß-hex have decreased folding stability that results in adult-onset forms of lysosomal storage diseases. However, prevention of the harmful accumulation of glycolipids only requires 10% of wild-type activity. Pyrimethamine (PYR) is a potential pharmacological chaperone that works by stabilizing these mutant enzymes sufficiently to allow more ß-hex to arrive in the lysosome, where it can carry out its function. An X-ray structure of the complex between human ß-hexosaminidase B (HexB) and PYR has been determined to 2.8 Å. PYR binds to the active site of HexB where several favorable van der Waals contacts and hydrogen bonds are introduced. Small adjustments of the enzyme structure are required to accommodate the ligand, and details of the inhibition and stabilization properties of PYR are discussed.
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Modelos Moleculares , Pirimetamina/química , beta-N-Acetil-Hexosaminidases/química , Domínio Catalítico , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Estrutura Molecular , Ligação Proteica , Subunidades Proteicas/química , beta-N-Acetil-Hexosaminidases/antagonistas & inibidoresRESUMO
RATIONALE: Methamphetamine (Meth) is a highly addictive psychostimulant associated with enhanced sexual desire, arousal, and sexual pleasure. Moreover, Meth abuse is frequently linked with the practice of sexual risk behavior and increased prevalence of human immunodeficiency virus. Currently, there is a lack of studies investigating the effects of Meth on maladaptive sexual behavior under controlled experimental settings in animal studies. OBJECTIVE: The overall objective of the current study was to examine the effects of Meth on various aspects of male sexual behavior including maladaptive sex-seeking behavior. METHODS: First, a dose-response curve of the effects of Meth (0, 1, 2, and 4 mg/kg; s.c.) on sexual motivation and performance was conducted in sexually naïve and experienced male rats. Next, the effects of Meth (1 mg/kg; s.c.) on inhibition of maladaptive sexual behavior was tested using a sex aversion conditioning paradigm, in which visceral illness induced by lithium chloride (LiCl) was paired with sexual behavior. RESULTS: Meth administration inhibited sexual performance in a dose-dependent matter as evidenced by the decreased percentages of males that mated and increased latencies to initiate sexual behavior when injected with 2 or 4 mg/kg Meth. Moreover, an acute dose of Meth prior to or following sex aversion conditioning resulted in disrupted conditioned inhibition of sexual behavior. CONCLUSIONS: These data suggest that Meth administration in male rats impairs sexual motivation and performance. In addition, low doses of Meth that do not disrupt sexual function may result in maladaptive seeking of sexual behavior.
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Estimulantes do Sistema Nervoso Central/toxicidade , Metanfetamina/toxicidade , Comportamento Sexual Animal/efeitos dos fármacos , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/administração & dosagem , Relação Dose-Resposta a Droga , Cloreto de Lítio , Masculino , Metanfetamina/administração & dosagem , Ratos , Ratos Sprague-DawleyRESUMO
ATP-citrate lyase (ACLY) catalyzes the conversion of citrate and CoA into acetyl-CoA and oxaloacetate, coupled with the hydrolysis of ATP. In humans, ACLY is the cytoplasmic enzyme linking energy metabolism from carbohydrates to the production of fatty acids. In situ proteolysis of full-length human ACLY gave crystals of a truncated form, revealing the conformations of residues 2-425, 487-750, and 767-820 of the 1101-amino acid protein. Residues 2-425 form three domains homologous to the beta-subunit of succinyl-CoA synthetase (SCS), while residues 487-820 form two domains homologous to the alpha-subunit of SCS. The crystals were grown in the presence of tartrate or the substrate, citrate, and the structure revealed the citrate-binding site. A loop formed by residues 343-348 interacts via specific hydrogen bonds with the hydroxyl and carboxyl groups on the prochiral center of citrate. Arg-379 forms a salt bridge with the pro-R carboxylate of citrate. The pro-S carboxylate is free to react, providing insight into the stereospecificity of ACLY. Because this is the first structure of any member of the acyl-CoA synthetase (NDP-forming) superfamily in complex with its organic acid substrate, locating the citrate-binding site is significant for understanding the catalytic mechanism of each member, including the prototype SCS. Comparison of the CoA-binding site of SCSs with the similar structure in ACLY showed that ACLY possesses a different CoA-binding site. Comparisons of the nucleotide-binding site of SCSs with the similar structure in ACLY indicates that this is the ATP-binding site of ACLY.