RESUMO
Objective: To document Grenadian women's knowledge about cervical cancer and human papillomavirus (HPV) infection, as well as their attitudes towards primary cervical cancer screening methods. Methods: In this qualitative study, we used focus groups in Grenada to gather information concerning women's knowledge about, attitudes towards and perceptions of screening for cervical cancer and general knowledge about HPV. Ten focus groups comprising 73 participants representing 5 of the 6 parishes in Grenada were conducted with women aged 19-59. Participants were asked about pelvic exams, Pap smears, HPV, reasons for seeking or avoiding cervical cancer screening and how different modalities of testing might affect their decision-making. Responses were then coded and organized into common themes. Results: While many respondents had heard of HPV, far fewer knew about its causative role in cervical cancer, how to prevent HPV infection or testing for the high-risk HPV types that cause almost all cases of cervical cancer. Many participants were aware that cervical cancer screening was beneficial, but numerous barriers to obtaining that screening were noted, including concerns about privacy and stigma, potential discomfort, and the cost and inconvenience involved. Conclusions: Our findings have implications for future cervical cancer screening efforts in Grenada. Central to these efforts should be a focus on educating Grenadians about the role of HPV infection in cervical cancer and the importance of early detection through screening. In addition, addressing issues of stigma and privacy are key to eliminating cervical cancer in Grenada.
RESUMO
BACKGROUND: Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoeba Dictyostelium discoideum, a eukaryotic microbe widely used to study diverse questions in the cell, developmental and evolutionary biology. RESULTS: We describe REMI-seq, an adaptation of Tn-seq, which allows high throughput, en masse, and quantitative identification of the genomic site of insertion of a drug resistance marker after restriction enzyme-mediated integration. We use REMI-seq to develop tools which greatly enhance the efficiency with which the sequence, transcriptome or proteome variation can be linked to phenotype in D. discoideum. These comprise (1) a near genome-wide resource of individual mutants and (2) a defined pool of 'barcoded' mutants to allow large-scale parallel phenotypic analyses. These resources are freely available and easily accessible through the REMI-seq website that also provides comprehensive guidance and pipelines for data analysis. We demonstrate that integrating these resources allows novel regulators of cell migration, phagocytosis and macropinocytosis to be rapidly identified. CONCLUSIONS: We present methods and resources, generated using REMI-seq, for high throughput gene function analysis in a key model system.
Assuntos
Dictyostelium , Dictyostelium/genética , Genoma , Genômica , TecnologiaRESUMO
Induction of pyrimidine dimers in DNA by solar UV radiation has drastic effects on microorganisms. To better define the nature of these DNA photoproducts in marine bacterioplankton and eukaryotes, a study was performed during a cruise along a latitudinal transect in the Pacific Ocean. The frequency of all possible cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts (64PPs) and their related Dewar valence isomers (DEWs) was determined by high-performance liquid chromatography-mass spectrometry. Studied samples were bacterioplankton and eukaryotic fractions isolated from sea water either collected before sunrise or exposed to ambient sunlight from sunrise to sunset. Isolated DNA dosimeters were also exposed to daily sunlight for comparison purposes. A first major result was the observation in all samples of large amounts of DEWs, a class of photoproducts rarely considered outside photochemical studies. Evidence was obtained for a major role of UVA in the formation of these photoisomerization products of 64PPs. Considerations on the ratio between the different classes of photoproducts in basal and induced DNA damage suggests that photoenzymatic repair (PER) is an important DNA repair mechanism used by marine microorganisms occupying surface seawater in the open ocean. This result emphasizes the biological role of DEWs which are very poor substrate for PER.
Assuntos
Adutos de DNA/genética , Dímeros de Pirimidina/genética , Microbiologia da Água , Cromatografia Líquida de Alta Pressão , Cianobactérias/genética , Cianobactérias/efeitos da radiação , Adutos de DNA/isolamento & purificação , Dano ao DNA , Reparo do DNA , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Isomerismo , Oceano Pacífico , Fitoplâncton/genética , Fitoplâncton/efeitos da radiação , Água do Mar/microbiologia , Luz Solar , Espectrometria de Massas em Tandem , Raios UltravioletaRESUMO
Wastewater serves as an important reservoir of antimicrobial resistance (AMR), and its surveillance can provide insights into population-level trends in AMR to inform public health policy. This study compared two common high-throughput screening approaches, namely (i) high-throughput quantitative PCR (HT qPCR), targeting 73 antimicrobial resistance genes, and (ii) metagenomic sequencing. Weekly composite samples of wastewater influent were taken from 47 wastewater treatment plants (WWTPs) across Wales, as part of a national AMR surveillance programme, alongside 4 weeks of daily wastewater effluent samples from a large municipal hospital. Metagenomic analysis provided more comprehensive resistome coverage, detecting 545 genes compared to the targeted 73 genes by HT qPCR. It further provided contextual information critical to risk assessment (i.e. potential bacterial hosts). In contrast, HT qPCR exhibited higher sensitivity, quantifying all targeted genes including those of clinical relevance present at low abundance. When limited to the HT qPCR target genes, both methods were able to reflect the spatiotemporal dynamics of the complete metagenomic resistome, distinguishing that of the hospital and the WWTPs. Both approaches revealed correlations between resistome compositional shifts and environmental variables like ammonium wastewater concentration, though differed in their interpretation of some potential influencing factors. Overall, metagenomics provides more comprehensive resistome profiling, while qPCR permits sensitive quantification of genes significant to clinical resistance. We highlight the importance of selecting appropriate methodologies aligned to surveillance aims to guide the development of effective wastewater-based AMR monitoring programmes.
Assuntos
Metagenômica , Águas Residuárias , Águas Residuárias/microbiologia , Metagenômica/métodos , Farmacorresistência Bacteriana/genética , Reação em Cadeia da Polimerase em Tempo Real , Monitoramento Ambiental/métodos , Bactérias/genética , Bactérias/efeitos dos fármacosRESUMO
Many human diseases, arising from mutations of disease susceptibility genes (genetic diseases), are also associated with viral infections (virally implicated diseases), either in a directly causal manner or by indirect associations. Here we examine whether viral perturbations of host interactome may underlie such virally implicated disease relationships. Using as models two different human viruses, Epstein-Barr virus (EBV) and human papillomavirus (HPV), we find that host targets of viral proteins reside in network proximity to products of disease susceptibility genes. Expression changes in virally implicated disease tissues and comorbidity patterns cluster significantly in the network vicinity of viral targets. The topological proximity found between cellular targets of viral proteins and disease genes was exploited to uncover a novel pathway linking HPV to Fanconi anemia.
Assuntos
Doença/etiologia , Modelos Biológicos , Viroses/complicações , Biologia Computacional , Doença/genética , Anemia de Fanconi/etiologia , Anemia de Fanconi/genética , Anemia de Fanconi/virologia , Predisposição Genética para Doença , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/patogenicidade , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/patogenicidade , Humanos , Mapas de Interação de Proteínas , Proteínas Virais/metabolismoRESUMO
Cervical carcinomas are initiated through a series of well-defined stages that rely on the expression of human papillomavirus (HPV) oncogenes. A panel of 100 small hairpin RNAs that target essential kinases in many tumor types was used to study the stepwise appearance of kinase requirements during cervical tumor development. Twenty-six kinases were commonly required in three cell lines derived from frank carcinomas, and each kinase requirement was traced to the specific stage in which the requirement emerged. Six kinases became required following HPV-induced immortalization, and the requirement for two kinases, SGK2 and PAK3, was mapped to the inactivation of p53 in primary human epithelial cells. Loss of the p53 tumor suppressor in other primary epithelial cells also induced dependence on SGK2 and PAK3. Hence, SGK2 and PAK3 provide important cellular functions following p53 inactivation, fulfilling the classical definition of synthetic lethality; loss of p53, SGK2, or PAK3 alone has little effect on cell viability, whereas loss of p53 together with either SGK2 or PAK3 loss leads to cell death. Whereas tumor suppressor gene mutations are not directly druggable, other proteins or pathways that become obligatory to cell viability following tumor suppressor loss provide theoretical targets for tumor suppressor-specific drug discovery efforts. The kinases SGK2 and PAK3 may thus represent such targets for p53-specific drug development.
Assuntos
Genes Supressores de Tumor , Genes p53 , Proteínas/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Carcinoma/genética , Carcinoma/virologia , Feminino , Humanos , Masculino , Papillomaviridae/genética , Papillomaviridae/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas/genética , Proteína Supressora de Tumor p53/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologia , Quinases Ativadas por p21RESUMO
Peers are a powerful resource in the learning process. Peer-assisted learning (PAL) is a high value activity for the peer and the coach. We report here a PAL activity focused on resilience and well-being for first-year undergraduate medical students. The model and lessons learned are described.
RESUMO
Small group, case-based learning (CBL) is an integral component of many pre-clerkship undergraduate medical education (UME) curricula. We report here an institutional process for curating a catalog of CBL cases utilized in a pre-clerkship curriculum, providing a practical guide for faculty. We describe the structured revision process conducted by a team of foundational and clinical science faculty, which incorporates student and faculty feedback. Revisions take into account core attributes of a case catalog, producing a collection of cases that are more relevant and instructional, realistic, challenging, consistent, current, diverse and inclusive, patient-centered, and mission-centered. Measurable outcomes after implementation of this process include increased focus on primary care as well as humanization and diversification of the case patients.
RESUMO
Gender gaps in academic performance have been reported at a variety of educational levels including several national standardized exams for medical education, with men scoring higher than women. These gaps potentially impact medical school acceptance and residency matching and may be influenced by curricular design. Performance data for our 4-year integrated hybrid curriculum, which features a large proportion of active learning, revealed a gender gap with men performing better early in the curriculum and on the first national standardized exam. This gap in performance almost entirely disappeared for years 2-4 of the curriculum and the second national standardized exam.
RESUMO
New Jersey was among the first states impacted by the COVID-19 pandemic, with one of the highest overall death rates in the nation. Nevertheless, relatively few reports have been published focusing specifically on New Jersey. Here we report on molecular, clinical, and epidemiologic observations, from the largest healthcare network in the state, in a cohort of vaccinated and unvaccinated individuals with laboratory-confirmed SARS-CoV-2 infection. We conducted molecular surveillance of SARS-CoV-2-positive nasopharyngeal swabs collected in nine hospitals from December 2020 through June 2022, using both whole genome sequencing (WGS) and a real-time RT-PCR screening assay targeting spike protein mutations found in variants of concern (VOCs) within our region. De-identified clinical data were obtained retrospectively, including demographics, COVID-19 vaccination status, ICU admission, ventilator support, mortality, and medical history. Statistical analyses were performed to identify associations between SARS-CoV-2 variants, vaccination status, clinical outcomes, and medical risk factors. A total of 5007 SARS-CoV-2-positive nasopharyngeal swabs were successfully screened and/or sequenced. Variant screening identified three predominant VOCs, including Alpha (n = 714), Delta (n = 1877), and Omicron (n = 1802). Omicron isolates were further sub-typed as BA.1 (n = 899), BA.2 (n = 853), or BA.4/BA.5 (n = 50); the remaining 614 isolates were classified as "Other". Approximately 31.5% (1577/5007) of the samples were associated with vaccine breakthrough infections, which increased in frequency following the emergence of Delta and Omicron. Severe clinical outcomes included ICU admission (336/5007 = 6.7%), ventilator support (236/5007 = 4.7%), and mortality (430/5007 = 8.6%), with increasing age being the most significant contributor to each (p < 0.001). Unvaccinated individuals accounted for 79.7% (268/336) of ICU admissions, 78.3% (185/236) of ventilator cases, and 74.4% (320/430) of deaths. Highly significant (p < 0.001) increases in mortality were observed in individuals with cardiovascular disease, hypertension, cancer, diabetes, and hyperlipidemia, but not with obesity, thyroid disease, or respiratory disease. Significant differences (p < 0.001) in clinical outcomes were also noted between SARS-CoV-2 variants, including Delta, Omicron BA.1, and Omicron BA.2. Vaccination was associated with significantly improved clinical outcomes in our study, despite an increase in breakthrough infections associated with waning immunity, greater antigenic variability, or both. Underlying comorbidities contributed significantly to mortality in both vaccinated and unvaccinated individuals, with increasing risk based on the total number of comorbidities. Real-time RT-PCR-based screening facilitated timely identification of predominant variants using a minimal number of spike protein mutations, with faster turnaround time and reduced cost compared to WGS. Continued evolution of SARS-CoV-2 variants will likely require ongoing surveillance for new VOCs, with real-time assessment of clinical impact.
Assuntos
COVID-19 , Humanos , COVID-19/epidemiologia , COVID-19/prevenção & controle , SARS-CoV-2/genética , New Jersey/epidemiologia , Vacinas contra COVID-19 , Pandemias , Estudos Retrospectivos , Glicoproteína da Espícula de Coronavírus , Infecções IrruptivasRESUMO
Natural selection should favour generalist predators that outperform specialists across all prey types. Two genetic solutions could explain why intraspecific variation in predatory performance is, nonetheless, widespread: mutations beneficial on one prey type are costly on another (antagonistic pleiotropy), or mutational effects are prey-specific, which weakens selection, allowing variation to persist (relaxed selection). To understand the relative importance of these alternatives, we characterised natural variation in predatory performance in the microbial predator Dictyostelium discoideum. We found widespread nontransitive differences among strains in predatory success across different bacterial prey, which can facilitate stain coexistence in multi-prey environments. To understand the genetic basis, we developed methods for high throughput experimental evolution on different prey (REMI-seq). Most mutations (~77%) had prey-specific effects, with very few (~4%) showing antagonistic pleiotropy. This highlights the potential for prey-specific effects to dilute selection, which would inhibit the purging of variation and prevent the emergence of an optimal generalist predator.
Assuntos
Dictyostelium/genética , Comportamento Alimentar , Bactérias/metabolismo , Evolução Biológica , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/fisiologia , Cadeia Alimentar , MutaçãoRESUMO
The equilibrium partitioning sediment benchmarks (ESBs) derived by the US Environmental Protection Agency (USEPA) in 2005 provide a mechanistic framework for understanding metal bioavailability in sediments by considering equilibrium partitioning (EqP) theory, which predicts that metal bioavailability in sediments is determined largely by partitioning to sediment particles. Factors that favor the partitioning of metals to sediment particles, such as the presence of acid volatile sulfide (AVS) and sediment organic matter, reduce metal bioavailability to benthic organisms. Because ESBs link metal bioavailability to partitioning to particles, they also predict that measuring metals in porewater can lead to a more accurate assessment of bioavailability and toxicity to benthic organisms. At the time of their development, sediment ESBs based on the analysis of porewater metal concentrations were limited to comparison with hardness-dependent metals criteria for the calculation of interstitial water benchmark units (IWBUs). However, the multimetal biotic ligand model (mBLM) provides a more comprehensive assessment of porewater metal concentrations, because it considers factors in addition to hardness, such as pH and dissolved organic carbon, and allows for interactions between metals. To evaluate the utility of the various sediment and porewater ESBs, four Hyalella azteca bioassay studies were identified that included sediment and porewater measurements of metals and porewater bioavailability parameters. Evaluations of excess simultaneously extracted metals, IWBUs, and mBLM toxic units (TUs) were compared among the bioassay studies. For porewater, IWBUs and mBLM TUs were calculated using porewater metal concentrations from samples collected using centrifugation and peepers. The percentage of correct predictions of toxicity was calculated for each benchmark comparison. The mBLM-based assessment using peeper data provided the most accurate predictions for the greatest number of samples among the evaluation methods considered. This evaluation demonstrates the value of porewater-based evaluations in conjunction with sediment chemistry in understanding toxicity observed in bioassay studies. Integr Environ Assess Manag 2022;18:1335-1347. © 2021 SETAC.
Assuntos
Sedimentos Geológicos , Poluentes Químicos da Água , Benchmarking , Disponibilidade Biológica , Sedimentos Geológicos/química , Ligantes , Metais/análise , Metais/toxicidade , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidadeRESUMO
The US Environmental Protection Agency Procedures for the Derivation of Equilibrium Partitioning Sediment Benchmarks (ESBs) for the Protection of Benthic Organisms: Metal Mixtures (Cadmium, Copper, Lead, Nickel, Silver and Zinc) equilibrium partitioning approach causally link metal concentrations and toxicological effects; they apply to sediment and porewater (i.e., interstitial water). The evaluation of bioavailable metal concentrations in porewater, using tools such as the biotic ligand model, provides an advancement that complements sediment-based evaluations. However, porewater characterization is less commonly performed in sediment bioassays than sediment chemistry characterization due to the difficulty and expense of porewater collection as well as concerns about interpretation of porewater data. This study discusses the advantages and disadvantages of different porewater extraction methods for analysis of metals and bioavailability parameters during laboratory sediment bioassays, with a focus on peepers and centrifugation. The purpose is to provide recommendations to generate bioassay porewater data of sufficient quality for use in risk-based decision-making, such as for regulated cleanup actions. Comparisons of paired data from previous bioassay studies indicate that metal porewater concentrations collected via centrifugation tend to be higher than those collected via peepers. However, centrifugation disrupts the redox status of the sediment; also, metal concentrations can vary markedly based on centrifugation conditions. Data to compare the concentrations of peeper- and centrifugation-collected bioavailability parameters (e.g., major ions, pH) are much more limited, but indicate smaller differences than those observed for metal concentrations. While peepers can be sampled without altering the redox status of the porewater, the small volume of porewater peepers collected is enough for metal concentration analysis, but insufficient for analysis of all metal bioavailability parameters. Given the benefits of metal collection via peepers, it is optimal to use centrifugation and peepers in tandem for bioassay porewater collection to improve bioavailability predictions. Environ Assess Manag 2022;18:1321-1334. © 2021 SETAC.
Assuntos
Sedimentos Geológicos , Poluentes Químicos da Água , Bioensaio , Cobre/análise , Monitoramento Ambiental/métodos , Sedimentos Geológicos/química , Metais/análise , Metais/toxicidade , Poluentes Químicos da Água/análiseRESUMO
Human papillomavirus (HPV) oncoproteins subvert cellular signaling pathways, including kinase pathways, during the carcinogenic process. To identify kinases targeted by the HPV16 E7 oncoprotein, shRNA kinase screens were performed in RKO colorectal carcinoma cell lines that differ only in their expression of HPV16 E7. Our screens identified kinases that were essential for the survival of RKO cells, but not essential for RKO cells expressing HPV16 E7. These kinases include CDK6, ERBB3, FYN, AAK1, and TSSK2. We show that, as predicted, CDK6 knockdown inhibits pRb phosphorylation and induces S-phase depletion, thereby inhibiting cell viability. Knockdown of ERBB3, FYN, AAK1, and TSSK2 induces a similar loss of cell viability through an unknown mechanism. Expression of the HPV16 E7 oncoprotein, known to bind and degrade pRb, relieves the requirement of these kinases. These studies demonstate that expression of a single oncoprotein can dramatically alter kinase sensitivity in human cells. The shRNA screens used here perform analogously to genetic interaction screens commonly used in genetically tractable organisms such as yeast, and thus represent an exciting method for unbiased identification of cellular signaling pathways targeted by cancer mutations.
Assuntos
Proteínas Oncogênicas Virais/farmacologia , Fosfotransferases/fisiologia , Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Neoplasias/patologia , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus , Fosfotransferases/análise , Fosfotransferases/genética , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Transdução de SinaisRESUMO
shRNA loss-of-function screens were used to identify kinases that were rate-limiting for promoting cell proliferation and survival. Here, we study the differences in kinase requirements among various human cells, including freshly prepared primary cells, isogenic cells, immortalized cells, and cancer cell lines. Closely related patterns of kinase requirements among the various cell types were observed in three cases: (i) in repeat experiments using the same cells, (ii) with multiple populations of freshly prepared primary epithelial cells isolated from the same tissue source, and (iii) between nearly isogenic cells that differ from each other by the expression of a single gene. Other commonly used cancer cell lines were distinct from one another, even when they were isolated from similar tumor types. Even primary cells of different lineages isolated from the same tissue source showed many differences. The differences in kinase requirements among cell lines observed in this study suggest that the control of proliferation and survival may be significantly different between cell lines and that simple comparisons from any one cell to another may be misleading. Although the regulation of cell proliferation and survival are heavily studied areas, we did not see a bias in these screens toward the identification of previously known and well studied kinases, suggesting that our knowledge of molecular events in these areas is still meager.
Assuntos
Células/enzimologia , Fosfotransferases/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Células/citologia , Células Cultivadas , Inativação Gênica , Humanos , Fosfotransferases/análise , Fosfotransferases/genética , RNA Interferente PequenoRESUMO
INTRODUCTION: In recent years, there has been a growing desire to address issues related to menstruation, particularly for adolescent girls. In low-income and middle-income countries, prior literature review of the adolescent menstrual experience suggests the need for further research into the impact and efficacy of interventions with this population. There is evidence to suggest the need for initiatives and research in higher-income countries like the USA. To date, the body of research on adolescent menstrual experience in the USA remains uncharacterised. Therefore, we propose a scoping review of the literature on this subject to better inform on areas for future primary study. METHODS AND ANALYSES: Using the framework proposed by Arksey and O'Malley and expounded on by Levac et al and the Joanna Briggs Institute, we will search electronic databases (MEDLINE, CINAHL, PsycINFO, Web of Science, ProQuest Public Health Database, Social Science Citation Index, Social Services Abstracts and SocINDEX) and grey literature for relevant studies in consultation with experienced librarians. The abstracts and full-text from each reference will be screened by two independent reviewers for inclusion. Bibliographic data, study characteristics and themes will be extracted from studies selected for inclusion using a rubric created by the research team. Findings will be summarised and a list of subject areas for future primary research will be generated in consultation with stakeholders. The review will be conducted using the Preferred Reporting Items from Systematic Reviews and Meta-Analyses extension for Scoping Reviews guidelines. ETHICS AND DISSEMINATION: Formal ethics training for this study is not required, as the research team will review publicly available studies. Stakeholders working in adolescent and menstrual health were consulted in designing this review. We will share key findings with stakeholders and in scholarly journals at the conclusion of the review.
Assuntos
Atenção à Saúde , Menstruação , Adolescente , Feminino , Humanos , Grupos Populacionais , Pobreza , Projetos de Pesquisa , Literatura de Revisão como Assunto , Estados UnidosRESUMO
Coupling the activities of normally disparate proteins into one functional unit has significant potential in terms of constructing novel switching components for synthetic biology or as biosensors. It also provides a means of investigating the basis behind transmission of conformation events between remote sites that is integral to many biological processes, including allostery. Here we describe how the structures and functions of two normally unlinked proteins, namely, the heme binding capability of cytochrome b(562) and the antibiotic degrading beta-lactamase activity of TEM, have been coupled using a directed evolution domain insertion approach. The important small biomolecule heme directly modulates in vivo and in vitro the beta-lactamase activity of selected integral fusion proteins. The presence of heme decreased the concentration of ampicillin tolerated by Escherichia coli and the level of in vitro hydrolysis of nitrocefin by up to 2 orders of magnitude. Variants with the largest switching magnitudes contained insertions at second-shell sites that abut key catalytic residues. Spectrophotometry confirmed that heme bound to the integral fusion proteins in a manner similar to that of cytochrome b(562). Circular dichroism suggested that only subtle structural changes rather than gross folding-unfolding events were responsible for modulating beta-lactamase activity, and size exclusion chromatography confirmed that the integral fusion proteins remained monomeric in both the apo and holo forms. Thus, by sampling a variety of insertion positions and linker sequences, we are able to couple the functions of two unrelated proteins by domain insertion.
Assuntos
Grupo dos Citocromos b/metabolismo , Proteínas de Escherichia coli/metabolismo , Heme/metabolismo , Ampicilina , Cefalosporinas/metabolismo , Evolução Molecular Direcionada , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/genética , Heme/farmacologia , Ligação Proteica , Conformação Proteica , Análise Espectral , beta-Lactamases/metabolismoRESUMO
Pokeweed antiviral protein (PAP) is a type I ribosomal inactivating protein (RIP). PAP binds to and depurinates the sarcin/ricin loop (SRL) of ribosomal RNA resulting in the cessation of protein synthesis. PAP has also been shown to bind to mRNA cap analogs and depurinate mRNA downstream of the cap structure. The biological role of cap binding and its possible role in PAP activity are not known. Here we show the first direct quantitative evidence for PAP binding to the cap analog m(7)GTP. We report a binding affinity of 43.3+/-0.1 nM at 25 degrees C as determined by fluorescence quenching experiments. This is similar to the values reported for wheat cap-binding proteins eIFiso4E and eIFiso4F. van't Hoff analysis of m(7)GTP-PAP equilibrium reveals a binding reaction that is enthalpy driven and entropy favored with TDeltaS degrees contributing 15% to the overall value of DeltaG degrees . This is in contrast to the wheat cap-binding proteins which are enthalpically driven in the DeltaG degrees for binding. Competition experiments indicate that ATP and GTP compete for the cap-binding site on PAP with slightly different affinities. Fluorescence studies of PAP-eIFiso4G binding reveal a protein-protein interaction with a K(d) of 108.4+/-0.3 nM. eIFiso4G was shown to enhance the interaction of PAP with m(7)GTP cap analog by 2.4-fold. These results suggest the involvement of PAP-translation initiation factor complexes in RNA selection and depurination.
Assuntos
Nucleotídeos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Phytolacca americana/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Sítios de Ligação , Cinética , Modelos Biológicos , Nucleotídeos/química , Ligação Proteica , Espectrometria de Fluorescência , TemperaturaRESUMO
The protein translocation channel at the plastid outer envelope membrane, Toc75, is essential for the viability of plants from the embryonic stage. It is encoded in the nucleus and is synthesized with a bipartite transit peptide that is cleaved during maturation. Despite its important function, the molecular mechanism and the biological significance of the full maturation of Toc75 remain unclear. In this study, we show that a type I signal peptidase (SPase I) is responsible for this process. First, we demonstrate that a bacterial SPase I converted Toc75 precursor to its mature form in vitro. Next, we show that disruption of a gene encoding plastidic SPase I (Plsp1) resulted in the accumulation of immature forms of Toc75, severe reduction of plastid internal membrane development, and a seedling lethal phenotype. These phenotypes were rescued by the overexpression of Plsp1 complementary DNA. Plsp1 appeared to be targeted both to the envelope and to the thylakoidal membranes; thus, it may have multiple functions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Membrana/metabolismo , Plastídeos/enzimologia , Precursores de Proteínas/metabolismo , Serina Endopeptidases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Membrana Nuclear/enzimologia , Plantas Geneticamente Modificadas , Precursores de Proteínas/genética , Processamento de Proteína Pós-Traducional , Plântula/enzimologia , Plântula/genética , Serina Endopeptidases/genética , Tilacoides/enzimologia , Tilacoides/ultraestruturaRESUMO
Trinucleotide exchange (TriNEx) is a method for generating novel molecular diversity during directed evolution by random substitution of one contiguous trinucleotide sequence for another. Single trinucleotide sequences were deleted at random positions in a target gene using the engineered transposon MuDel that were subsequently replaced with a randomized trinucleotide sequence donated by the DNA cassette termed SubSeq(NNN). The bla gene encoding TEM-1 beta-lactamase was used as a model to demonstrate the effectiveness of TriNEx. Sequence analysis revealed that the mutations were distributed throughout bla, with variants containing single, double and triple nucleotide changes. Many of the resulting amino acid substitutions had significant effects on the in vivo activity of TEM-1, including up to a 64-fold increased activity toward ceftazidime and up to an 8-fold increased resistance to the inhibitor clavulanate. Many of the observed amino acid substitutions were only accessible by exchanging at least two nucleotides per codon, including charge-switch (R164D) and aromatic substitution (W165Y) mutations. TriNEx can therefore generate a diverse range of protein variants with altered properties by combining the power of site-directed saturation mutagenesis with the capacity of whole-gene mutagenesis to randomly introduce mutations throughout a gene.