RESUMO
Pyrococcus furiosus is a hyperthermophilic archaeon with three effector CRISPR complexes (types I-A, I-B, and III-B) that each employ crRNAs derived from seven CRISPR arrays. Here, we investigate the CRISPR adaptation response to a newly discovered and self-transmissible plasmid, pT33.3. Transconjugant strains of Pyrococcus furiosus exhibited dramatically elevated levels of new spacer integration at CRISPR loci relative to the strain harboring a commonly employed, laboratory-constructed plasmid. High-throughput sequence analysis demonstrated that the vast majority of the newly acquired spacers were preferentially selected from DNA surrounding a particular region of the pT33.3 plasmid and exhibited a bi-directional pattern of strand bias that is a hallmark of primed adaptation by type I systems. We observed that one of the CRISPR arrays of our Pyrococcus furiosus laboratory strain encodes a spacer that closely matches the region of the conjugative plasmid that is targeted for adaptation. The hyper-adaptation phenotype was found to strictly depend both on the presence of this single matching spacer as well as the I-B effector complex, known to mediate primed adaptation. Our results indicate that Pyrococcus furiosus naturally encountered this conjugative plasmid or a related mobile genetic element in the past and responds to reinfection with robust primed adaptation.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Pyrococcus furiosus , Pyrococcus furiosus/genética , Sistemas CRISPR-Cas , Plasmídeos/genética , DNA/genéticaRESUMO
CRISPR-Cas adaptive immune systems are used by prokaryotes to defend against invaders like viruses and other mobile genetic elements. Immune memories are stored in the form of 'spacers' which are short DNA sequences that are captured from invaders and added to the CRISPR array during a process called 'adaptation'. Spacers are transcribed and the resulting CRISPR (cr)RNAs assemble with different Cas proteins to form effector complexes that recognize matching nucleic acid and destroy it ('interference'). Adaptation can be 'naïve', i.e. independent of any existing spacer matches, or it can be 'primed', i.e. spurred by the crRNA-mediated detection of a complete or partial match to an invader sequence. Here we show that primed adaptation occurs in Pyrococcus furiosus. Although P. furiosus has three distinct CRISPR-Cas interference systems (I-B, I-A and III-B), only the I-B system and Cas3 were necessary for priming. Cas4, which is important for selection and processing of new spacers in naïve adaptation, was also essential for priming. Loss of either the I-B effector proteins or Cas3 reduced naïve adaptation. However, when Cas3 and all crRNP genes were deleted, uptake of correctly processed spacers was observed, indicating that none of these interference proteins are necessary for naïve adaptation.
Assuntos
Adaptação Fisiológica/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/genética , DNA/metabolismo , Pyrococcus furiosus/genética , Pyrococcus furiosus/imunologia , Pareamento de Bases , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/imunologia , DNA Helicases/metabolismo , Mutação , Hibridização de Ácido Nucleico , Plasmídeos/genética , Plasmídeos/metabolismo , Pyrococcus furiosus/metabolismo , RNA/genética , RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/imunologia , Ribonucleoproteínas/metabolismoRESUMO
The purpose of this study was to first characterize the prevalence of recall, recognition, and knowledge of colon cancer screening tests and guidelines (collectively, "awareness") among non-Hispanic black (NHB) and NH white (NHW) urban colon cancer patients. Second, we sought to examine whether awareness was associated with mode of cancer detection. Low awareness regarding colon cancer screening tests and guidelines may explain low screening rates and high prevalence of symptomatic detection. We examined recall, recognition, and knowledge of colorectal cancer (CRC) screening tests and guidelines and their associations with mode of cancer detection (symptomatic versus screen-detected) in 374 newly diagnosed NHB and NHW patients aged 45-79. Patients were asked to name or describe any test to screen for colon cancer (recall); next, they were given descriptions of stool testing and colonoscopy and asked if they recognized each test (recognition). Lastly, patients were asked if they knew the screening guidelines (knowledge). Overall, awareness of CRC screening guidelines was low; just 20% and 13% of patients knew colonoscopy and fecal test guidelines, respectively. Awareness of CRC screening tests and guidelines was especially low among NHB males, socioeconomically disadvantaged individuals, and those diagnosed at public healthcare facilities. Inability to name or recall a single test was associated with reduced screen-detected cancer compared with recall of at least one test (36% vs. 22%, p = 0.01). Low awareness of CRC screening tests is a risk factor for symptomatic detection of colon cancer.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Neoplasias do Colo/diagnóstico , Colonoscopia , Neoplasias Colorretais/diagnóstico , Detecção Precoce de Câncer , Humanos , Masculino , Programas de Rastreamento , Sangue Oculto , Fatores SexuaisRESUMO
The specific labelling of proteins in recent years has made use of self-labelling proteins, such as the SNAP-tag® and the Halotag®. These enzymes, by their nature or suitably engineered, have the ability to specifically react with their respective substrates, but covalently retaining a part of them in the catalytic site upon reaction. This led to the synthesis of substrates conjugated with, e.g., fluorophores (proposing them as alternatives to fluorescent proteins), but also with others chemical groups, for numerous biotechnological applications. Recently, a mutant of the OGT from Saccharolobus solfataricus (H5) very stable to high temperatures and in the presence of physical and chemical denaturing agents has been proposed as a thermostable SNAP-tag® for in vivo and in vitro harsh reaction conditions. Here, we show two new thermostable OGTs from Thermotoga neapolitana and Pyrococcus furiosus, which, respectively, display a higher catalytic activity and thermostability respect to H5, proposing them as alternatives for in vivo studies in these extreme model organisms.
Assuntos
Biotecnologia , Estabilidade Enzimática , Temperatura Alta , Pyrococcus furiosusRESUMO
Background: Wastewater-based surveillance is an important tool for monitoring the COVID-19 pandemic. However, it remains challenging to translate wastewater SARS-CoV-2 viral load to infection number, due to unclear shedding patterns in wastewater and potential differences between variants. Objectives: We utilized comprehensive wastewater surveillance data and estimates of infection prevalence (i.e., the source of the viral shedding) available for New York City (NYC) to characterize SARS-CoV-2 fecal shedding pattern over multiple COVID-19 waves. Methods: We collected SARS-CoV-2 viral wastewater measurements in NYC during August 31, 2020 - August 29, 2023 ( N = 3794 samples). Combining with estimates of infection prevalence (number of infectious individuals including those not detected as cases), we estimated the time-lag, duration, and per-infection fecal shedding rate for the ancestral/Iota, Delta, and Omicron variants, separately. We also developed a procedure to identify occasions with intensified transmission. Results: Models suggested fecal viral shedding likely starts around the same time as and lasts slightly longer than respiratory tract shedding. Estimated fecal viral shedding rate was highest during the ancestral/Iota variant wave, at 1.44 (95% CI: 1.35 - 1.53) billion RNA copies in wastewater per day per infection (measured by RT-qPCR), and decreased by â¼20% and 50-60% during the Delta wave and Omicron period, respectively. We identified around 200 occasions during which the wastewater SARS-CoV-2 viral load exceeded the expected level in any of 14 sewersheds. These anomalies disproportionally occurred during late January, late April - early May, early August, and from late-November to late-December, with frequencies exceeding the expectation assuming random occurrence ( P < 0.05; bootstrapping test). Discussion: These estimates may be useful in understanding changes in underlying infection rate and help quantify changes in COVID-19 transmission and severity over time. We have also demonstrated that wastewater surveillance data can support the identification of time periods with potentially intensified transmission.
RESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been widespread since 2020 and will likely continue to cause substantial recurring epidemics. However, understanding the underlying infection burden (i.e., including undetected asymptomatic/mild infections) and dynamics, particularly since late 2021 when the Omicron variant emerged, is challenging due to the potential for asymptomatic and repeat SARS-CoV-2 infection, changes in testing practices, and changes in disease reporting. Here, we leverage extensive surveillance data available in New York City (NYC) and a comprehensive model-inference system to reconstruct SARS-CoV-2 dynamics therein from the pandemic onset in March 2020 to August 2023, and further validate the estimates using independent wastewater surveillance data. The validated model-inference estimates indicate a very high infection burden totaling twice the population size (>5 times documented case count) but decreasing infection-fatality risk (a >10-fold reduction) during the first 3.5 years. The detailed estimates also reveal highly complex variant dynamics and immune landscape, changing virus transmissibility, and higher infection risk during winter in NYC over this time period. These transmission dynamics and drivers, albeit based on data in NYC, may be relevant to other populations and inform future planning to help mitigate the public health burden of SARS-CoV-2.
RESUMO
CRISPR-Cas systems provide heritable immunity against viruses and other mobile genetic elements by incorporating fragments of invader DNA into the host CRISPR array as spacers. Integration of new spacers is localized to the 5' end of the array, and in certain Gram-negative Bacteria this polarized localization is accomplished by the integration host factor. For most other Bacteria and Archaea, the mechanism for 5' end localization is unknown. Here we show that archaeal histones play a key role in directing integration of CRISPR spacers. In Pyrococcus furiosus, deletion of either histone A or B impairs integration. In vitro, purified histones are sufficient to direct integration to the 5' end of the CRISPR array. Archaeal histone tetramers and bacterial integration host factor induce similar U-turn bends in bound DNA. These findings indicate a co-evolution of CRISPR arrays with chromosomal DNA binding proteins and a widespread role for binding and bending of DNA to facilitate accurate spacer integration.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Histonas , Histonas/genética , Archaea/genética , Fatores Hospedeiros de Integração , DNA , BactériasRESUMO
Colorectal cancer (CRC) remains the third most commonly diagnosed cancer and the third leading cause of cancer-related death in the USA. CRC can be prevented through regular screening and removal of precancerous polyps. However, roughly one third of eligible adults in the USA are not up to date with recommended CRC screening. To increase timely CRC screening uptake in the USA, in 2014, the National Colorectal Cancer Roundtable (NCCRT) launched 80% by 2018. This multilevel effort involved more than 1,500 pledged organizations targeting patients, providers, health care systems, and policymakers to increase U.S. CRC screening rates to 80% by 2018. Concurrent with this campaign, between 2012 and 2018, CRC screening rates increased nationwide by 3.6% from 65.2% to 68.8%, meaning that about 9.3 million more U.S. adults are being screened. NCCRT attributes these successes to widespread implementation of center- and system-wide evidence-based interventions to increase screening uptake, including direct patient communication, provider reminders via electronic health records, and patient navigation, among others. Moving beyond 2018, NCCRT has rebranded the initiative as the 80% Pledge and has since identified several targeted campaigns, including increased outreach to Hispanics, Latinos, and Asians, whose CRC screening uptake remains less than 50%; encouragement of Medicaid outreach activities around CRC screening in all 50 states; and advocacy for screening right at 50 years of age. Society of Behavioral Medicine continues to support NCCRT and encourages policymakers to do the same by taking legislative action to assure funding for Medicaid outreach, research innovations, and clinical quality improvement that supports the 80% Pledge.
Assuntos
Medicina do Comportamento , Neoplasias Colorretais , Navegação de Pacientes , Adulto , Neoplasias Colorretais/diagnóstico , Detecção Precoce de Câncer , Humanos , Programas de Rastreamento , Estados UnidosRESUMO
The protozoan parasite Toxoplasma gondii is capable of infecting all warm-blooded animals and humans. Infectious, transmissible forms of the parasite include oocysts produced by the sexual cycle within the definitive feline host and tissue cysts that form Toxoplasma in the central nervous system and muscle during the asexual cycle within all chronically infected warm-blooded hosts. These tissue cysts are populated with slow-growing bradyzoites, which until recently have been thought to be dormant entities in the context of immune sufficiency. Reactivation to active growth during immune suppression is of critical clinical importance. However, little is known about tissue cysts or the bradyzoites they house, as the diversity of tissue cysts cannot be replicated in cell culture systems. This protocol for optimization of tissue cyst purification from the brains of infected mice using Percoll gradients provides an efficient means to recover in vivo-derived tissue cysts that can be applied to imaging, cell biological, biochemical, transcriptomic, and proteomic analyses. © 2017 by John Wiley & Sons, Inc.
Assuntos
Encéfalo/parasitologia , Centrifugação com Gradiente de Concentração/métodos , Oocistos/isolamento & purificação , Parasitologia/métodos , Toxoplasma/isolamento & purificação , Animais , Camundongos , Povidona , Dióxido de Silício , Toxoplasmose Animal/parasitologiaRESUMO
Although colorectal cancer (CRC) is largely preventable through identification of pre-cancerous polyps through various screening modalities, morbidity and mortality rates remain a challenge, especially in African-American, Latino, low-income and uninsured/underinsured patients. Barriers to screening include cost, access to health care facilities, lack of recommendation to screen, and psychosocial factors such as embarrassment, fear of the test, anxiety about testing preparation and fear of a cancer diagnosis. Various intervention approaches to improve CRC screening rates have been developed. However, comparative effectiveness research (CER) to investigate the relative performance of different approaches has been understudied, especially across different real-life practice settings. Assessment of differential efficacy across diverse vulnerable populations is also lacking. The current paper describes the rationale and design for the Mile Square Colorectal Cancer Screening, Awareness and Referral and Education Project (Mi-CARE), which aims to increase CRC screening rates in 3 clinics of a large Federally Qualified Health Center (FQHC) by reducing prominent barriers to screening for low-income, minority and underserved patients. Patients attending these clinics will receive one of three interventions to increase screening uptake: lay patient navigator (LPN)-based navigation, provider level navigation, or mailed birthday CRC screening reminders. The design of our program allows for comparison of the effectiveness of the tailored interventions across sites and patient populations. Data from Mi-CARE may help to inform the dissemination of tailored interventions across FQHCs to reduce health disparities in CRC.
Assuntos
Neoplasias Colorretais/diagnóstico , Etnicidade , Conhecimentos, Atitudes e Prática em Saúde , Disparidades em Assistência à Saúde/etnologia , Educação de Pacientes como Assunto/métodos , Navegação de Pacientes/métodos , Encaminhamento e Consulta , Negro ou Afro-Americano , Idoso , Colonoscopia , Pesquisa Comparativa da Efetividade , Detecção Precoce de Câncer , Fezes/química , Feminino , Hemoglobinas/análise , Hispânico ou Latino , Humanos , Imunoquímica , Masculino , Pessoa de Meia-Idade , Grupos Minoritários , Sangue Oculto , Pobreza , Populações VulneráveisRESUMO
PURPOSE OF REVIEW: Despite over a third of the world's population being chronically infected with Toxoplasma gondii, little is known about this largely asymptomatic phase of infection. This stage is mediated in vivo by bradyzoites within tissue cysts. The absence of overt symptoms has been attributed to the dormancy of bradyzoites. In this review, we reexamine the conventional view of chronic toxoplasmosis in light of emerging evidence challenging both the nature of dormancy and the consequences of infection in the CNS. RECENT FINDINGS: New and emerging data reveal a previously unrecognized level of physiological and replicative capacity of bradyzoites within tissue cysts. These findings have emerged in the context of a reexamination of the chronic infection in the brain that correlates with changes in neuronal architecture, neurochemistry, and behavior that suggest that the chronic infection is not without consequence. SUMMARY: The emerging data driven by the development of new approaches to study the progression of chronic toxoplasma infection reveals significant physiological and replicative capacity for what has been viewed as a dormant state. The emergence of bradyzoite and tissue cyst biology from what was viewed as a physiological "black box" offers exciting new areas for investigation with direct implications on the approaches to drug development targeting this drug-refractory state. In addition, new insights from studies on the neurobiology on chronic infection reveal a complex and dynamic interplay between the parasite, brain microenvironment, and the immune response that results in the detente that promotes the life-long persistence of the parasite in the host.