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BACKGROUND: In response to critical gaps in medical oxygen access, 2 pressure swing adsorption (PSA) oxygen production centers were established using an ecosystem-strengthening strategy in Amhara, Ethiopia, in 2019. A qualitative study was conducted to assess enablers and bottlenecks to oxygen access at the hospital level after installation. METHODS: A variety of hospital staff (clinicians, biomedical professionals, hospital administrators, and procurement teams) across 13 hospitals procuring oxygen from the plants participated in comprehensive, semistructured focus group discussions. A thematic framework analysis approach was used to identify key themes. FINDINGS: A total of 101 individuals participated in 26 focus groups in 2021, 2 years after plants were installed. Primary themes were accessibility of supply, affordability, and hospital readiness. Respondents indicated a substantial increase in their hospital's ability to access lower-cost oxygen, with many attributing this to the locality of plants and reduced transportation barriers. However, other challenges persisted, and the emergence of COVID-19 1 year after plant installation and a civil conflict exacerbated supply shortages. Investments in equipment, supplies, and training optimized clinical utilization of oxygen and were highlighted as a need for ongoing investment. CONCLUSION: To achieve maximum impact, investments in large-scale oxygen systems must be accompanied by strategic plans to transport oxygen, reduce costs to hospitals, and provide support to clinical teams through equipment, supply procurement, and clinical training. These findings support comprehensive ecosystem approaches to strengthening oxygen access for sustainable impact.
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OBJECTIVE: To examine reported cases of abuse in long-term care (LTC) homes in the province of Ontario, Canada, to determine the extent and nature of abuse experienced by residents between 2019 and 2022. DESIGN: A qualitative mixed methods study was conducted using document analysis and descriptive statistics. Three data sources were analysed: LTC legislation, inspection reports from a publicly available provincial government administrative database and articles published by major Canadian newspapers. A data extraction tool was developed that included variables such as the date of inspection, the type of inspection, findings and the section of legislation cited. Descriptive analyses, including counts and percentages, were calculated to identify the number of incidents and the type of abuse reported. RESULTS: According to legislation, LTC homes are required to protect residents from physical, sexual, emotional, verbal or financial abuse. The review of legislation revealed that inspectors are responsible for ensuring homes comply with this requirement. An analysis of their reports identified that 9% (781) of overall inspections included findings of abuse. Physical abuse was the most common type (37%). Differences between the frequency of abuse across type of ownership, location and size of the home were found. There were 385 LTC homes with at least one reported case of abuse, and 55% of these homes had repeated incidents. The analysis of newspaper articles corroborated the findings of abuse in the inspection reports and provided resident and family perspectives. CONCLUSIONS: There are substantial differences between legislation intended to protect LTC residents from abuse and the abuse occurring in LTC homes. Strategies such as establishing a climate of trust, investing in staff and leadership, providing standardised education and training and implementing a quality and safety framework could improve the care and well-being of LTC residents.
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Abuso de Ancianos , Cuidados a Largo Plazo , Casas de Salud , Investigación Cualitativa , Humanos , Cuidados a Largo Plazo/estadística & datos numéricos , Cuidados a Largo Plazo/normas , Cuidados a Largo Plazo/métodos , Casas de Salud/estadística & datos numéricos , Casas de Salud/normas , Casas de Salud/organización & administración , Ontario , Abuso de Ancianos/estadística & datos numéricos , Abuso de Ancianos/legislación & jurisprudencia , Abuso de Ancianos/prevención & control , Anciano , Femenino , MasculinoRESUMEN
Off-the-shelf immunotherapeutics that suppress tumor growth and provide durable protection against relapse could enhance cancer treatment. We report preclinical studies on a CD33 x CD3 bivalent bispecific diabody, AMV564, that not only suppresses tumor growth, but also facilitates memory responses in a mouse model of acute myelogenous leukemia (AML). Mechanistically, a single 5-day treatment with AMV564 seems to reduce tumor burden by redirection of T cells, providing a time window for allogeneic or other T cells that innately recognize tumor antigens to become activated and proliferate. When the concentration of bispecific becomes negligible, the effector: target ratio has also shifted, and these activated T cells mediate long-term tumor control. To test the efficacy of AMV564 in vivo, we generated a CD33+ MOLM13CG bioluminescent human cell line and optimized conditions needed to control these cells for 62 days in vivo in NSG mice. Of note, not only did MOLM13CG become undetectable by bioluminescence imaging in response to infusion of human T cells plus AMV564, but also NSG mice that had cleared the tumor also resisted rechallenge with MOLM13CG in spite of no additional AMV564 treatment. In these mice, we identified effector and effector memory human CD4+ and CD8+ T cells in the peripheral blood immediately prior to rechallenge that expanded significantly during the subsequent 18 days. In addition to the anti-tumor effects of AMV564 on the clearance of MOLM13CG cells in vivo, similar effects were seen when primary CD33+ human AML cells were engrafted in NSG mice even when the human T cells made up only 2% of the peripheral blood cells and AML cells made up 98%. These studies suggest that AMV564 is a novel and effective bispecific diabody for the targeting of CD33+ AML that may provide long-term survival advantages in the clinic.
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Anticuerpos Biespecíficos , Complejo CD3 , Memoria Inmunológica , Leucemia Mieloide Aguda , Lectina 3 Similar a Ig de Unión al Ácido Siálico , Animales , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/tratamiento farmacológico , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/inmunología , Ratones , Complejo CD3/inmunología , Memoria Inmunológica/efectos de los fármacos , Línea Celular Tumoral , Linfocitos T/inmunología , Linfocitos T/efectos de los fármacosRESUMEN
BH3-mimetics represent promising anti-cancer agents in tumors that rely on the anti-apoptotic function of B-Cell Lymphoma 2 (BCL2) proteins, particularly in leukemia and lymphoma cells primed for apoptosis. Mechanistically, BH3-mimetics may displace pro-apoptotic binding partners thus inducing BAX/BAK-mediated mitochondrial permeabilization followed by cytochrome c release, activation of the caspase cascade and apoptosis. Here, we describe a novel mode of caspase-independent cell death (CICD) induced by BH3-mimetics in a subset of diffuse large B-cell lymphoma (DLBCL) cells. Of note, rather than occurring via necroptosis, CICD induced immediately after mitochondrial permeabilization was associated with transcriptional reprogramming mediated by activation of c-Jun N-terminal Kinase (JNK) signaling and Activator Protein 1 (AP1). Thereby, CICD resulted in the JNK/AP1-mediated upregulation of inflammatory chemokines and increased migration of cytotoxic Natural Killer (NK) cells. Taken together, our study describes a novel mode of CICD triggered by BH3-mimetics that may alter the immune response towards dying cells.
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Antineoplásicos , Linfoma de Células B Grandes Difuso , Humanos , Proteína X Asociada a bcl-2/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Apoptosis , Antineoplásicos/farmacología , Caspasas , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Línea Celular , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Single case studies of extraordinary disease resilience may provide therapeutic insight into conditions for which no definitive treatments exist. An otherwise healthy 35-year-old man (patient-R) with the canonical pathogenic ACVR1R206H variant and the classic congenital great toe malformation of fibrodysplasia ossificans progressiva (FOP) had extreme paucity of post-natal heterotopic ossification (HO) and nearly normal mobility. We hypothesized that patient-R lacked a sufficient post-natal inflammatory trigger for HO. A plasma biomarker survey revealed a reduction in total matrix metalloproteinase-9 (MMP-9) compared to healthy controls and individuals with quiescent FOP. Whole exome sequencing identified compound heterozygous variants in MMP-9 (c.59C > T, p.A20V and c.493G > A, p.D165N). Structural analysis of the D165N variant predicted both decreased MMP-9 secretion and activity that were confirmed by enzyme-linked immunosorbent assay and gelatin zymography. Further, human proinflammatory M1-like macrophages expressing either MMP-9 variant produced significantly less Activin A, an obligate ligand for HO in FOP, compared to wildtype controls. Importantly, MMP-9 inhibition by genetic, biologic, or pharmacologic means in multiple FOP mouse models abrogated trauma-induced HO, sequestered Activin A in the extracellular matrix (ECM), and induced regeneration of injured skeletal muscle. Our data suggest that MMP-9 is a druggable node linking inflammation to HO, orchestrates an existential role in the pathogenesis of FOP, and illustrates that a single patient's clinical phenotype can reveal critical molecular mechanisms of disease that unveil novel treatment strategies.
A healthy 35-year-old man (patient-R) with the classic fibrodysplasia ossificans progressiva (FOP) mutation and the congenital great toe malformation of FOP had extreme lack of heterotopic ossification (HO) and nearly normal mobility. We hypothesized that patient-R lacked a sufficient inflammatory trigger for HO. Blood tests revealed a reduction in the level of an inflammatory protein called matrix metalloproteinase-9 (MMP-9) compared to other individuals with FOP as well as healthy controls. DNA analysis in patient-R identified mutations in MMP-9, one of which predicted decreased activity of MMP-9 which was confirmed by further testing. Inflammatory cells (macrophages) expressing the MMP-9 mutations identified in patient-R produced significantly less Activin A, an obligate stimulus for HO in FOP. In order to determine if MMP-9 deficiency was a cause of HO prevention in FOP, we inhibited MMP-9 activity by genetic, biologic, or pharmacologic means in FOP mouse models and showed that MMP-9 inhibition prevented or dramatically decreased trauma-induced HO in FOP, locked-up Activin A in the extracellular matrix, and induced regeneration of injured skeletal muscle. Our data show that MMP-9 links inflammation to HO and illustrate that one patient's clinical picture can reveal critical molecular mechanisms of disease that unveil new treatment strategies.
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Receptores de Activinas Tipo I , Metaloproteinasa 9 de la Matriz , Miositis Osificante , Adulto , Animales , Humanos , Masculino , Ratones , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/metabolismo , Receptores de Activinas Tipo I/deficiencia , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Miositis Osificante/genética , Miositis Osificante/patología , Miositis Osificante/metabolismo , Osificación Heterotópica/patología , Osificación Heterotópica/genética , Osificación Heterotópica/metabolismoRESUMEN
Health systems worldwide are at a critical juncture due to an increasing demand for health services and a diminishing pool of health human resources. While COVID-19 exacerbated nursing deficits, the need to strengthen and sustain the health workforce in Canada was evident decades prior and supported by numerous studies that warned of significant shortages. Post pandemic, building health system capacity has become paramount. This article examines innovative nursing employment initiatives in Canada. It provides a snapshot of federal, provincial and territorial approaches, with a particular focus on Internationally Educated Nurses (IENs) due to burgeoning interest in and competition for their skills and services. However, recognizing that health human resource planning is a persistent challenge, further initiatives are suggested. These include complementary policy development to improve retention and policy frameworks that support proactive long-term strategies to address the cyclical shortage of nurses.
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COVID-19 , Empleo , Canadá , Humanos , Fuerza Laboral en Salud , SARS-CoV-2 , Pandemias , Enfermeras InternacionalesRESUMEN
Regulation of myeloid cell activity is critical for successful myelin regeneration (remyelination) in demyelinating diseases, such as multiple sclerosis (MS). Here, we show aromatic alpha-keto acids (AKAs) generated from the amino acid oxidase, interleukin-4 induced 1 (IL4I1), promote efficient remyelination in mouse models of MS. During remyelination, myeloid cells upregulated the expression of IL4I1. Conditionally knocking out IL4I1 in myeloid cells impaired remyelination efficiency. Mice lacking IL4I1 expression exhibited a reduction in the AKAs, phenylpyruvate, indole-3-pyruvate, and 4-hydroxyphenylpyruvate, in remyelinating lesions. Decreased AKA levels were also observed in people with MS, particularly in the progressive phase when remyelination is impaired. Oral administration of AKAs modulated myeloid cell-associated inflammation, promoted oligodendrocyte maturation, and enhanced remyelination in mice with focal demyelinated lesions. Transcriptomic analysis revealed AKA treatment induced a shift in metabolic pathways in myeloid cells and upregulated aryl hydrocarbon receptor activity in lesions. Our results suggest myeloid cell-associated aromatic amino acid metabolism via IL4I1 produces AKAs in demyelinated lesions to enable efficient remyelination. Increasing AKA levels or targeting related pathways may serve as a strategy to facilitate the regeneration of myelin in inflammatory demyelinating conditions.
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BACKGROUND: Recent studies indicate that melphalan percutaneous hepatic perfusion (M-PHP) for liver metastases from ocular melanoma (mUM) improves survival. Importantly, this benefit must be carefully balanced with changes in a patient's quality of life (QoL). This study examines the QoL changes post-M-PHP. METHODS: Retrospective analysis of the change in QoL using the Functional Assessment of Cancer Therapy-General (FACT-G) with mUM patients receiving M-PHP ( n â =â 20). The FACT-G scores, which comprise physical (PWB), social (SWB), emotional (EWB) and functional (FWB) wellbeing were measured pre-procedure and at day 1, day of discharge (mean = 2.4 days), 7, 14 and 28 days after M-PHP therapy. Wilcoxon signed-rank test gauged QoL domain changes. RESULTS: Baseline FACT-G median (IQR) scores were 101.8 (21.8). QoL scoring significantly decreased immediately after the procedure [day 1; 85 (27.5); P â =â 0.002] and gradually improved over time. By day 28, QoL almost returned to pre-procedure levels [100.3 (13.8); P â =â 0.31]. Subscore analysis revealed that the initial drop in QoL at day 1 post-procedure was attributable to the PWB (28 vs. 24; P â =â 0.001) and FWB domains (26 vs. 18.5; P â <â 0.001). By day 28 there was a statistically significant improvement in EWB ( P â =â 0.01). CONCLUSION: QoL following M-PHP decreases immediately after therapy and is not significantly different from baseline by the day of discharge. By day 28 there is improved emotional well-being. This study could help to optimize the time between treatment cycles when combined with toxicity data and blood count recovery.
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Melanoma , Neoplasias Primarias Secundarias , Neoplasias Cutáneas , Neoplasias de la Úvea , Humanos , Melanoma/tratamiento farmacológico , Calidad de Vida , Melfalán/uso terapéutico , Estudios Retrospectivos , PerfusiónRESUMEN
BACKGROUND: Strengthening health systems through planned safety and quality improvement initiatives is an imperative to achieve more equitable, resilient, and effective care. And yet, years of organizational behavior research demonstrate that change initiatives often fall short because managers fail to account for organizational readiness for change. This finding remains true especially among surgical safety and quality improvement initiatives in low-income countries and middle-income countries. In this study, our aim was to psychometrically assess the construct validity and internal consistency of the Safe Surgery Organizational Readiness Tool (SSORT), a short survey tool designed to provide change leaders with insight into facility infrastructure that supports learning and readiness to undertake change. MATERIALS AND METHODS: To demonstrate generalizability and achieve a large sample size ( n =1706) to conduct exploratory factor analysis (EFA) and confirmatory factor analysis (CFA), a collaboration between seven surgical and anesthesia safety and quality improvement initiatives was formed. Collected survey data from health care workers were divided into pilot, exploration, and confirmation samples. The pilot sample was used to assess feasibility. The exploration sample was used to conduct EFA, while the confirmation sample was used to conduct CFA. Factor internal consistency was assessed using Cronbach's alpha coefficient. RESULTS: Results of the EFA retained 9 of the 16 proposed factors associated with readiness to change. CFA results of the identified 9 factor model, measured by 28 survey items, demonstrated excellent fit to data. These factors (appropriateness, resistance to change, team efficacy, team learning orientation, team valence, communication about change, learning environment, vision for sustainability, and facility capacity) were also found to be internally consistent. CONCLUSION: Our findings suggest that communication, team learning, and supportive environment are components of change readiness that can be reliably measured prior to implementation of projects that promote surgical safety and quality improvement in low-income countries and middle-income countries. Future research can link performance on identified factors to outcomes that matter most to patients.
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Gestión del Cambio , Personal de Salud , Humanos , Psicometría , Estudios Transversales , Encuestas y Cuestionarios , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Failure rates on medical specialist final summative examinations in Australia are high, regardless of speciality. Examination failure can have detrimental psycho-social, financial and job security effects on the trainee, while delays in completion of training adversely impacts workforce growth and health outcomes for the community. The study aimed to explore the preparation factors that contribute to ophthalmology trainee success in their final summative examination. METHODS: Semi-structured in-depth interviews were conducted with 29 participants via telephone or Zoom with ophthalmology trainees and Fellows. To be eligible, interviewees had to have sat the Royal Australian and New Zealand College of Ophthalmologists Advanced Clinical Examination (RACE) within the past five years or were providing supervision to trainees preparing for RACE. Interviews were audio-recorded, transcribed and thematically analysed. RESULTS: Examination success was underpinned by six themes relating to preparation: (i) 'Those who fail to plan, plan to fail', which related to development and adherence to a study plan; (ii) 'It takes a village' encompassed trainees establishing and activating personal and professional supports; (iii) 'Get to know your opponent', which encompassed developing an understanding of the examination construct, format and requirements; (iv) 'There is no substitute for hard work', which related to intensive study over a period of 12-18 months; (v) 'Keep pace with the herd', which referred to benchmarking preparation efforts and progress against peers; and (vi) 'Don't jump the gun', which related to ensuring readiness to sit. CONCLUSIONS: Maximising medical specialist examination pass rates is in the best interest of trainees, training Colleges, health care systems and communities. Recognising and facilitating preparation approaches that foster success in final summative examinations are the collective responsibility of trainees, specialist training Colleges, training networks and health systems. Trainees need to plan for examination success, be self-determined to commit to intensive study over an extended time period and be realistic about their readiness to sit.
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Curriculum , Educación de Postgrado en Medicina , Humanos , Australia , Evaluación Educacional , Nueva ZelandaRESUMEN
[This corrects the article DOI: 10.3389/fpubh.2023.1195751.].
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PURPOSE: To describe parental experiences in the neonatal intensive care unit (NICU) during the COVID-19 restrictions. We explore what parents found most challenging, the impact these restrictions had on them and their infant, and how they coped. METHODS: A cross-sectional online survey was completed by parents of infants who required care in a Canadian NICU during the early period of the COVID-19 pandemic. Data from 3 questions were coded using thematic analysis. RESULTS: Participants ( n = 161) were primarily mothers (93%), with an average length of stay of 32.1 days. Three themes were identified from responses: (1) emotional and physical closeness of the parents to their infant; (2) physical and psychosocial well-being of the infant and parent; and (3) how parents coped, and strategies for moving forward. Parents reported that parental restriction policies adversely impacted their perceived physical and emotional closeness with their infant and their infant's physical and psychosocial well-being. Parents reported that being able to be present with their infant, having their partner able to be present with them, and effective communication helped them cope. CONCLUSION: Despite the need for some restrictive policies to control the spread of the virus, the benefits and risks to the overall well-being of the parents and infants must be weighed.
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COVID-19 , Unidades de Cuidado Intensivo Neonatal , Recién Nacido , Lactante , Femenino , Humanos , Estudios Transversales , Pandemias , COVID-19/epidemiología , Canadá , Padres/psicologíaRESUMEN
Background: COVID-19 has dramatically affected the delivery of health care and technical assistance. This is true in Tanzania, where maternal mortality and surgical infection rates are significantly higher than in high-income countries. This paper describes lessons learned about the optimal application of in-person and virtual mentorship in the Safe Surgery 2020 program to improve the quality of surgical services in Tanzania before and after the COVID-19 pandemic. Methods: From January 2018 through December 2020, Safe Surgery 2020 supported 40 health facilities in Tanzania's Lake Zone to improve the quality of surgical care. A blended surgical mentorship model, employing both onsite and virtual mentorship, was central to the program's capacity development approach. With COVID-19, the program pivoted to full virtual mentorship. Through continuous learning and adaptation processes, including a human-centered design workshop, surveys assessing mentors' confidence with different competencies, and focus group discussions with mentors, mentees and safe surgery program staff, the program distilled the optimal use of mentorship models. Results: Developing complex surgical skills, addressing contextual considerations, problem-solving, and building trusting relationships were best suited to in-person mentorship, whereas virtual mentorship was most effective in supporting mentees' quality improvement projects, data use, case discussions, and reinforcing clinical practices. Leading successful virtual learning required enhanced facilitation skills and active engagement of health facility leadership. Conclusions: In-person and virtual mentorship offer distinct benefits and complement each other when combined. Investing more in-person mentorship at the beginning of programs allows for the establishment of trust that is foundational to effective mentorship.
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Introduction: Vaccine hesitancy is a global health threat undermining control of many vaccine-preventable diseases. Patient-level education has largely been ineffective in reducing vaccine concerns and increasing vaccine uptake. We built and evaluated a personalized vaccine risk communication website called LetsTalkShots in English, Spanish and French (Canadian) for vaccines across the lifespan. LetsTalkShots tailors lived experiences, credible sources and informational animations to disseminate the right message from the right messenger to the right person, applying a broad range of behavioral theories. Methods: We used mixed-methods research to test our animation and some aspects of credible sources and personal narratives. We conducted 67 discussion groups (n = 325 persons), stratified by race/ethnicity (African American, Hispanic, and White people) and population (e.g., parents, pregnant women, adolescents, younger adults, and older adults). Using a large Ipsos survey among English-speaking respondents (n = 2,272), we tested animations aligned with vaccine concerns and specific to population (e.g., parents of children, parents of adolescents, younger adults, older adults). Results: Discussion groups provided robust feedback specific to each animation as well as areas for improvements across animations. Most respondents indicated that the information presented was interesting (85.5%), clear (96.0%), helpful (87.0%), and trustworthy (82.2%). Discussion: Tailored vaccine risk communication can assist decision makers as they consider vaccination for themselves, their families, and their communities. LetsTalkShots presents a model for personalized communication in other areas of medicine and public health.
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Comunicación , Vacunación , Vacunas , Adolescente , Anciano , Niño , Femenino , Humanos , Embarazo , Negro o Afroamericano , Canadá , Medicina de Precisión , Vacilación a la Vacunación , Riesgo , Salud Pública , Promoción de la Salud , Educación en Salud/métodos , Hispánicos o Latinos , Blanco , Adulto Joven , PadresRESUMEN
Nucleases are strictly regulated and often localized in the cell to avoid the uncontrolled degradation of DNA and RNA. Here, a new type of nuclease complex, composed of RecJ3, RecJ4, and aRNase J, was identified through its ATP-dependent association with the ubiquitin-like SAMP1 and AAA-ATPase Cdc48a. The complex was discovered in Haloferax volcanii, an archaeon lacking an RNA exosome. Genetic analysis revealed aRNase J to be essential and RecJ3, RecJ4, and Cdc48a to function in the recovery from DNA damage including genotoxic agents that generate double-strand breaks. The RecJ3:RecJ4:aRNase J complex (isolated in 2:2:1 stoichiometry) functioned primarily as a 3'-5' exonuclease in hydrolyzing RNA and ssDNA, with the mechanism non-processive for ssDNA. aRNase J could also be purified as a homodimer that catalyzed endoribonuclease activity and, thus, was not restricted to the 5'-3' exonuclease activity typical of aRNase J homologs. Moreover, RecJ3 and RecJ4 could be purified as a 560-kDa subcomplex in equimolar subunit ratio with nuclease activities mirroring the full RecJ3/4-aRNase J complex. These findings prompted reconstitution assays that suggested RecJ3/4 could suppress, alter, and/or outcompete the nuclease activities of aRNase J. Based on the phenotypic results, this control mechanism of aRNase J by RecJ3/4 is not necessary for cell growth but instead appears important for DNA repair. IMPORTANCE Nucleases are critical for various cellular processes including DNA replication and repair. Here, a dynamic type of nuclease complex is newly identified in the archaeon Haloferax volcanii, which is missing the canonical RNA exosome. The complex, composed of RecJ3, RecJ4, and aRNase J, functions primarily as a 3'-5' exonuclease and was discovered through its ATP-dependent association with the ubiquitin-like SAMP1 and Cdc48a. aRNase J alone forms a homodimer that has endonuclease function and, thus, is not restricted to 5'-3' exonuclease activity typical of other aRNase J enzymes. RecJ3/4 appears to suppress, alter, and/or outcompete the nuclease activities of aRNase J. While aRNase J is essential for growth, RecJ3/4, Cdc48a, and SAMPs are important for recovery against DNA damage. These biological distinctions may correlate with the regulated nuclease activity of aRNase J in the RecJ3/4-aRNaseJ complex.
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Haloferax volcanii , Haloferax volcanii/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Fosfodiesterasa I/genética , Fosfodiesterasa I/metabolismo , Ubiquitina/metabolismo , Daño del ADN , Exonucleasas/genética , Exonucleasas/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , ARN/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
In healthy cells, pro- and anti-apoptotic BCL2 family and BH3-only proteins are expressed in a delicate equilibrium. In contrast, this homeostasis is frequently perturbed in cancer cells due to the overexpression of anti-apoptotic BCL2 family proteins. Variability in the expression and sequestration of these proteins in Diffuse Large B cell Lymphoma (DLBCL) likely contributes to variability in response to BH3-mimetics. Successful deployment of BH3-mimetics in DLBCL requires reliable predictions of which lymphoma cells will respond. Here we show that a computational systems biology approach enables accurate prediction of the sensitivity of DLBCL cells to BH3-mimetics. We found that fractional killing of DLBCL, can be explained by cell-to-cell variability in the molecular abundances of signaling proteins. Importantly, by combining protein interaction data with a knowledge of genetic lesions in DLBCL cells, our in silico models accurately predict in vitro response to BH3-mimetics. Furthermore, through virtual DLBCL cells we predict synergistic combinations of BH3-mimetics, which we then experimentally validated. These results show that computational systems biology models of apoptotic signaling, when constrained by experimental data, can facilitate the rational assignment of efficacious targeted inhibitors in B cell malignancies, paving the way for development of more personalized approaches to treatment.
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Linfoma de Células B Grandes Difuso , Proteínas Proto-Oncogénicas c-bcl-2 , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Simulación por Computador , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/patologíaRESUMEN
BACKGROUND: Magnetic resonance imaging (MRI) tagging techniques have been applied to the GI tract to assess bowel contractions and content mixing. We aimed to evaluate the dependence of a tagging measurement (for assessing chyme mixing) on inter-observer variability in both the ascending colon (AC) and descending colon (DC) and to investigate the temporal variation and hence reliability of the colonic tagging technique by acquiring multiple measurements over time on healthy participants. METHODS: Two independent datasets of healthy adults were used for the retrospective inter-observer variability (Study 1: 13 datasets and Study 2: 31 datasets), and ten participants were scanned for the prospective temporal variation study following a 1 L mannitol oral preparation. All colonic tagging data were acquired on 3 T MRI scanners. The mean and the standard deviation (SD) maps were generated pixel-by-pixel using custom-written software in MATLAB. The colonic regions of interest were defined using MIPAV software. Bland-Altman plots and scatter plots were used for the inter-observer variability. The mean and SD of all repeated measures for each subject were calculated along with a one-way ANOVA to test for variations with time. RESULTS: Scatter plots and Bland-Altman plots showed a large range of data with low variation and small limits of agreements (<5% CoV). The intraclass correlation coefficient of inter-rater reliability was excellent and 0.97 or above for the AC and DC measurements for both datasets. The temporal variation study shows that there was no significant difference found between the multiple measures with time (p = 0.53, one-way repeated measures ANOVA test). CONCLUSIONS: MRI tagging technique can provide an assessment of colonic chyme mixing. The inter-observer study data showed high inter-rater agreement. The temporal variation study showed some individual variations with time suggesting multiple measurements may be needed to increase accuracy.
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Imagen por Resonancia Magnética , Adulto , Humanos , Voluntarios Sanos , Estudios Prospectivos , Estudios Retrospectivos , Variaciones Dependientes del Observador , Reproducibilidad de los Resultados , Imagen por Resonancia Magnética/métodosRESUMEN
Amide hydrogen/deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for analyzing the conformational dynamics of proteins in a solution. Current conventional methods have a measurement limit starting from several seconds and are solely reliant on the speed of manual pipetting or a liquid handling robot. Weakly protected regions of polypeptides, such as in short peptides, exposed loops and intrinsically disordered the protein exchange on the millisecond timescale. Typical HDX methods often cannot resolve the structural dynamics and stability in these cases. Numerous academic laboratories have demonstrated the considerable utility of acquiring HDX-MS data in the sub-second regimes. Here, we describe the development of a fully automated HDX-MS apparatus to resolve amide exchange on the millisecond timescale. Like conventional systems, this instrument boasts automated sample injection with software selection of labeling times, online flow mixing and quenching, while being fully integrated with a liquid chromatography-MS system for existing standard "bottom-up" workflows. HDX-MS's rapid exchange kinetics of several peptides demonstrate the repeatability, reproducibility, back-exchange, and mixing kinetics achieved with the system. Comparably, peptide coverage of 96.4% with 273 peptides was achieved, supporting the equivalence of the system to standard robotics. Additionally, time windows of 50 ms-300 s allowed full kinetic transitions to be observed for many amide groups; especially important are short time points (50-150 ms) for regions that are likely highly dynamic and solvent- exposed. We demonstrate that information on structural dynamics and stability can be measured for stretches of weakly stable polypeptides in small peptides and in local regions of a large enzyme, glycogen phosphorylase.