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1.
Proc Natl Acad Sci U S A ; 118(38)2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34479991

RESUMO

COVID-19 induces a robust, extended inflammatory "cytokine storm" that contributes to an increased morbidity and mortality, particularly in patients with type 2 diabetes (T2D). Macrophages are a key innate immune cell population responsible for the cytokine storm that has been shown, in T2D, to promote excess inflammation in response to infection. Using peripheral monocytes and sera from human patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and a murine hepatitis coronavirus (MHV-A59) (an established murine model of SARS), we identified that coronavirus induces an increased Mφ-mediated inflammatory response due to a coronavirus-induced decrease in the histone methyltransferase, SETDB2. This decrease in SETDB2 upon coronavirus infection results in a decrease of the repressive trimethylation of histone 3 lysine 9 (H3K9me3) at NFkB binding sites on inflammatory gene promoters, effectively increasing inflammation. Mφs isolated from mice with a myeloid-specific deletion of SETDB2 displayed increased pathologic inflammation following coronavirus infection. Further, IFNß directly regulates SETDB2 in Mφs via JaK1/STAT3 signaling, as blockade of this pathway altered SETDB2 and the inflammatory response to coronavirus infection. Importantly, we also found that loss of SETDB2 mediates an increased inflammatory response in diabetic Mϕs in response to coronavirus infection. Treatment of coronavirus-infected diabetic Mφs with IFNß reversed the inflammatory cytokine production via up-regulation of SETDB2/H3K9me3 on inflammatory gene promoters. Together, these results describe a potential mechanism for the increased Mφ-mediated cytokine storm in patients with T2D in response to COVID-19 and suggest that therapeutic targeting of the IFNß/SETDB2 axis in T2D patients may decrease pathologic inflammation associated with COVID-19.


Assuntos
Coronavirus/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/virologia , Macrófagos/metabolismo , Animais , COVID-19/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Síndrome da Liberação de Citocina , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/genética , Feminino , Histona-Lisina N-Metiltransferase/genética , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , SARS-CoV-2/metabolismo , Transdução de Sinais
2.
Acad Med ; 96(5): 655-660, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33208674

RESUMO

The COVID-19 pandemic has had significant ramifications for provider well-being. During these unprecedented and challenging times, one institution's Department of Surgery put in place several important initiatives for promoting the well-being of trainees as they were redeployed to provide care to COVID-19 patients. In this article, the authors describe these initiatives, which fall into 3 broad categories: redeploying faculty and trainees, ensuring provider safety, and promoting trainee wellness. The redeployment initiatives are the following: reframing the team mindset, creating a culture of grace and forgiveness, establishing a multidisciplinary wellness committee, promoting centralized leadership, providing clear communication, coordinating between departments and programs, implementing phased restructuring of the department's services, establishing scheduling flexibility and redundancy, adhering to training regulations, designating a trainee ombudsperson, assessing physical health risks for high-risk individuals, and planning for structured deimplementation. Initiatives specific to promoting provider safety are appointing a trainee safety advocate, guaranteeing personal protective equipment and relevant information about these materials, providing guidance regarding safe practices at home, and offering alternative housing options when necessary. Finally, the initiatives put in place to directly promote trainee wellness are establishing an environment of psychological safety, providing mental health resources, maintaining the educational missions, solidifying a sense of community by showing appreciation, being attentive to childcare, and using social media to promote community morale. The initiatives to carry out the department's strategy presented in this article, which were well received by both faculty and trainee members of the authors' community, may be employed in other departments and even outside the context of COVID-19. The authors hope that colleagues at other institutions and departments, independent of specialty, will find the initiatives described here helpful during, and perhaps after, the pandemic as they develop their own institution-specific strategies to promote trainee wellness.


Assuntos
COVID-19/epidemiologia , Internato e Residência , Estresse Ocupacional/prevenção & controle , Pandemias , Administração de Recursos Humanos em Hospitais , Centro Cirúrgico Hospitalar/organização & administração , COVID-19/transmissão , Humanos , Transmissão de Doença Infecciosa do Paciente para o Profissional/prevenção & controle , Liderança , Equipamento de Proteção Individual , Admissão e Escalonamento de Pessoal , SARS-CoV-2 , Apoio Social
3.
JCI Insight ; 5(17)2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32879137

RESUMO

Macrophages are a primary immune cell involved in inflammation, and their cell plasticity allows for transition from an inflammatory to a reparative phenotype and is critical for normal tissue repair following injury. Evidence suggests that epigenetic alterations play a critical role in establishing macrophage phenotype and function during normal and pathologic wound repair. Here, we find in human and murine wound macrophages that cyclooxygenase 2/prostaglandin E2 (COX-2/PGE2) is elevated in diabetes and regulates downstream macrophage-mediated inflammation and host defense. Using single-cell RNA sequencing of human wound tissue, we identify increased NF-κB-mediated inflammation in diabetic wounds and show increased COX-2/PGE2 in diabetic macrophages. Further, we identify that COX-2/PGE2 production in wound macrophages requires epigenetic regulation of 2 key enzymes in the cytosolic phospholipase A2/COX-2/PGE2 (cPLA2/COX-2/PGE2) pathway. We demonstrate that TGF-ß-induced miRNA29b increases COX-2/PGE2 production via inhibition of DNA methyltransferase 3b-mediated hypermethylation of the Cox-2 promoter. Further, we find mixed-lineage leukemia 1 (MLL1) upregulates cPLA2 expression and drives COX-2/PGE2. Inhibition of the COX-2/PGE2 pathway genetically (Cox2fl/fl Lyz2Cre+) or with a macrophage-specific nanotherapy targeting COX-2 in tissue macrophages reverses the inflammatory macrophage phenotype and improves diabetic tissue repair. Our results indicate the epigenetically regulated PGE2 pathway controls wound macrophage function, and cell-targeted manipulation of this pathway is feasible to improve diabetic wound repair.


Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus/fisiopatologia , Dinoprostona/farmacologia , Epigênese Genética , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/prevenção & controle , Macrófagos/efeitos dos fármacos , Cicatrização , Idoso , Animais , Ciclo-Oxigenase 2/metabolismo , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Ocitócicos/farmacologia , Fenótipo , Pseudomonas aeruginosa/efeitos dos fármacos , Transdução de Sinais
4.
J Vasc Surg Venous Lymphat Disord ; 8(2): 299-305, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32067731

RESUMO

OBJECTIVE: Venous thromboembolism (VTE) is a common disease with potentially devastating and long-term sequelae, such as pulmonary embolism and post-thrombotic syndrome (PTS). Given the mortality risk, prevalence of VTE, and limited access to diagnostic imaging, clinically relevant biomarkers for diagnosis and prognostication are needed. Therefore, this review aimed to summarize the data on clinically applicable biomarkers that best indicate acute VTE and chronic PTS. METHODS: We reviewed the medical and scientific literature from 2001 to 2019 for VTE biomarkers. Randomized controlled trials, meta-analyses, and review articles were included. Primary basic research papers with no clinical applicability, opinion papers, institutional guidelines, and case reports were excluded. RESULTS: We highlight the diagnostic value of D-dimer alongside other promising biomarkers, including cellular adhesion molecules, P-selectin, cytokines (interleukins 6 and 10), fibrin monomer complexes, and coagulation factors (factor VIII). CONCLUSIONS: High-sensitivity D-dimer remains the most clinically established VTE biomarker. Current research endeavors are under way to identify more precise biomarkers of VTE and PTS.


Assuntos
Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Mediadores da Inflamação/sangue , Síndrome Pós-Trombótica/sangue , Tromboembolia Venosa/sangue , Trombose Venosa/sangue , Animais , Biomarcadores/sangue , Humanos , Síndrome Pós-Trombótica/diagnóstico , Síndrome Pós-Trombótica/epidemiologia , Valor Preditivo dos Testes , Prognóstico , Fatores de Risco , Tromboembolia Venosa/diagnóstico , Tromboembolia Venosa/epidemiologia , Trombose Venosa/diagnóstico , Trombose Venosa/epidemiologia
5.
Semin Vasc Surg ; 32(1-2): 23-26, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31540652

RESUMO

Burnout among vascular surgery trainees is a significant problem and needs to be addressed at the level of the individual, training program duties, and at each institution. The clinical challenges and patient-oriented care required of a vascular surgeon generate a level of stress that requires its recognition and development of coping methods to promote well-being and personal happiness. There are ways to minimize burnout during surgical training, including acknowledgment of its symptoms, mentorship, self-care, and access to resources for stress reduction. Crucial factors in maintaining a positive outlook and a sense of meaningful work are faculty entrustability, receptive leadership, celebrating small victories, and recognition that resiliency is a skill that can be learned. Successful vascular surgeon training is a mission that requires everyone involved to actively promote well-being behavior and a supportive work environment. With appropriate implementation of these practices, our training programs can cultivate surgeons who are competent, compassionate, and committed to advancing vascular care.


Assuntos
Adaptação Psicológica , Esgotamento Profissional/prevenção & controle , Educação de Pós-Graduação em Medicina/métodos , Saúde Mental , Cirurgiões/educação , Cirurgiões/psicologia , Procedimentos Cirúrgicos Vasculares/educação , Esgotamento Profissional/etiologia , Esgotamento Profissional/psicologia , Currículo , Humanos , Relações Interpessoais , Liderança , Mentores , Resiliência Psicológica , Local de Trabalho/psicologia
6.
J Vasc Surg Venous Lymphat Disord ; 7(3): 452-462, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30853559

RESUMO

BACKGROUND: Deep venous thrombosis (DVT) in the pediatric population is rare, occurring in about 10 to 14 out of 10,000 pediatric admissions annually, but with serious consequences such as pulmonary embolism and/or post-thrombotic syndrome. There is a dearth of surgical literature regarding this entity, its pathophysiology, its treatment and its long-term sequelae. METHODS: An extensive search of available surgical and medical literature in Medline, PubMed was obtained by searching terms synonymous with pediatric DVT. Case reports and opinion articles were excluded. Ongoing clinical trials were culled from clinicaltrial.gov by searching for pediatric DVT studies. Institutional guidelines, where available, were included in this summary. RESULTS: We provide a clinically relevant summary with the aims of improving prevention, early identification and treatment of pediatric DVT. CONCLUSIONS: Although rare and frequently with subtle presentations, pediatric DVT can be serious. Early identification and treatment can be instrumental in limiting sequelae and in improving outcomes for these patients.


Assuntos
Trombose Venosa , Adolescente , Idade de Início , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Valor Preditivo dos Testes , Fatores de Risco , Resultado do Tratamento , Trombose Venosa/diagnóstico por imagem , Trombose Venosa/epidemiologia , Trombose Venosa/fisiopatologia , Trombose Venosa/terapia
7.
J Vasc Surg ; 69(2): 532-543, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30683200

RESUMO

BACKGROUND: Groin wound infections represent a substantial source of patients' morbidity and resource utilization. Definitions and reporting times of groin infections are poorly standardized, which limits our understanding of the true scope of the problem and potentially leads to event under-reporting. Our objective was to investigate the timing and variation of groin wound complications after vascular surgery. METHODS: We reviewed all patients who underwent vascular surgery with a groin incision at our institution during 2013 (N = 256; 32% female; mean age, 68.8 years). We analyzed patient- and procedure-level variables. Our primary outcome was any groin complication within 180 days. We classified groin-related events as major (hospital readmission or reoperation for groin wound) or minor (wound opened in clinic, initiation of antibiotics specifically for a groin wound, or new groin hematoma or wound drainage). RESULTS: The Kaplan-Meier estimated rate of groin complications at 180 days was 23% (n = 53/256); 29 (54%) were major and 24 (46%) were minor. The Kaplan-Meier 30-day event rate was 13% for any complication and only 3% for major complications, indicating that most events occurring within the first 30 days did not require readmission or reoperation. By 180 days, the overall complication rate rose to 23% and the major event rate to 14%, indicating that nearly all complications occurring after 30 days required readmission or reoperation. Those with a groin complication more commonly had tissue loss (23% vs 12%; P = .05), underwent infrainguinal bypass (42% vs 22%; P=.004), had a redo incision (32% vs 18%; P = .03), and had a longer operation (77% vs 65% surgery >200 minutes; P = .07). There were no significant differences in patients' comorbidities, skin closure, dressing type, prosthetic implants, hemostatic agents, or discharge status. CONCLUSIONS: Whereas >20% of patients suffered a groin complication, nearly half of these events occurred after 30 days. Standardized reporting measures limited to 30-day events or infection definitions that are limited to the need for antibiotic use may misrepresent the true infection rate and thus highlight the need for uniform reporting standards.


Assuntos
Virilha/irrigação sanguínea , Hematoma/etiologia , Indicadores de Qualidade em Assistência à Saúde/normas , Projetos de Pesquisa/normas , Infecção da Ferida Cirúrgica/etiologia , Procedimentos Cirúrgicos Vasculares/efeitos adversos , Idoso , Idoso de 80 Anos ou mais , Antibacterianos , Confiabilidade dos Dados , Bases de Dados Factuais , Drenagem , Feminino , Hematoma/diagnóstico , Hematoma/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Readmissão do Paciente , Reoperação , Estudos Retrospectivos , Infecção da Ferida Cirúrgica/diagnóstico , Infecção da Ferida Cirúrgica/terapia , Fatores de Tempo , Resultado do Tratamento
8.
J Vasc Surg Venous Lymphat Disord ; 5(2): 257-260, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28214495

RESUMO

Aneurysmal disease of the internal iliac vein is rare, with no standard indication for or accepted modality of treatment. Here we report an instance of unilateral, primary left internal iliac venous aneurysm and associated pelvic venous insufficiency. Following extensive workup for alternative causes, the aneurysm and left gonadal vein were coil embolized with good effect.


Assuntos
Aneurisma Ilíaco/etiologia , Insuficiência Venosa/complicações , Embolização Terapêutica/métodos , Humanos , Aneurisma Ilíaco/diagnóstico por imagem , Aneurisma Ilíaco/terapia , Masculino , Pessoa de Meia-Idade , Pelve/irrigação sanguínea , Flebografia , Insuficiência Venosa/diagnóstico por imagem , Insuficiência Venosa/terapia
9.
Biochemistry ; 53(43): 6776-85, 2014 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-25286246

RESUMO

NEMO is a scaffolding protein that, together with the catalytic subunits IKKα and IKKß, plays an essential role in the formation of the IKK complex and in the activation of the canonical NF-κB pathway. Rational drug design targeting the IKK-binding site on NEMO would benefit from structural insight, but to date, the determination of the structure of unliganded NEMO has been hindered by protein size and conformational heterogeneity. Here we show how the utilization of a homodimeric coiled-coil adaptor sequence stabilizes the minimal IKK-binding domain NEMO(44-111) and furthers our understanding of the structural requirements for IKK binding. The engineered constructs incorporating the coiled coil at the N-terminus, C-terminus, or both ends of NEMO(44-111) present high thermal stability and cooperative melting and, most importantly, restore IKKß binding affinity. We examined the consequences of structural content and stability by circular dichoism and nuclear magnetic resonance (NMR) and measured the binding affinity of each construct for IKKß(701-745) in a fluorescence anisotropy binding assay, allowing us to correlate structural characteristics and stability to binding affinity. Our results provide a method for engineering short stable NEMO constructs to be suitable for structural characterization by NMR or X-ray crystallography. Meanwhile, the rescuing of the binding affinity implies that a preordered IKK-binding region of NEMO is compatible with IKK binding, and the conformational heterogeneity observed in NEMO(44-111) may be an artifact of the truncation.


Assuntos
Quinase I-kappa B/química , Engenharia de Proteínas , Sítios de Ligação , Cristalografia por Raios X , Humanos , Quinase I-kappa B/genética , Espectroscopia de Ressonância Magnética , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína
10.
Proteins ; 82(7): 1370-5, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24375686

RESUMO

We designed and characterized a soluble mimic of the parathyroid hormone (PTH) receptor (PTH1R) that incorporates the N-terminus and third extracellular loop of PTH1R, important for ligand binding. The engineered receptor (PTH1R-NE3) was conceived to enable easy production and the use of standard biochemical and biophysical assays for the screening of competitive antagonists of PTH. We show that PTH1R-NE3 is folded, thermodynamically stable and selectively binds PTH. We also demonstrate the utility of our mimic by identifying a small molecule that competes with PTH in our PTH1R-NE3-based fluorescence polarization assay. Antagonists to PTH1R, a transmembrane protein belonging to the class B G-protein coupled receptor family, may provide new therapeutic options for calcium metabolism diseases like humoral hypercalcemia of malignancy.


Assuntos
Engenharia de Proteínas/métodos , Receptor Tipo 1 de Hormônio Paratireóideo/química , Proteínas Recombinantes de Fusão/química , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade
11.
J Pept Sci ; 19(8): 504-10, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23794508

RESUMO

The parathyroid hormone, PTH, is responsible for calcium and phosphate ion homeostasis in the body. The first 34 amino acids of the peptide maintain the biological activity of the hormone and is currently marketed for calcium imbalance disorders. Although several methods for the production of recombinant PTH(1-34) have been reported, most involve the use of cleavage conditions that result in a modified peptide or unfavorable side products. Herein, we detail the recombinant production of (15) N-enriched human parathyroid hormone, (15) N PTH(1-34), generated via a plasmid vector that gives reasonable yield, low-cost protease cleavage (leaving the native N-terminal serine in its amino form), and purification by affinity and size exclusion chromatography. We characterize the product by multidimensional, heteronuclear NMR, circular dichroism, and LC/MS.


Assuntos
Endopeptidases/química , Hormônio Paratireóideo/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Cromatografia de Afinidade , Escherichia coli , Humanos , Dados de Sequência Molecular , Hormônio Paratireóideo/química , Hormônio Paratireóideo/isolamento & purificação , Estrutura Secundária de Proteína , Proteólise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação
12.
Biochemistry ; 51(41): 8092-9, 2012 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-23002929

RESUMO

The BK polyoma virus is a leading cause of chronic post kidney transplantation rejection. One target for therapeutic intervention is the initial association of the BK virus with the host cell. We hypothesize that the rate of BKV infection can be curbed by competitively preventing viral binding to cells. The X-ray structures of homologous viruses complexed with N-terminal glycoproteins suggest that the BC and HI loops of the viral coat are determinant for binding and thereby infection of the host cell. The large size of the viral coat precludes it from common biophysical and small molecule screening studies. Hence, we sought to develop a smaller protein template incorporating the identified binding loops of the BK viral coat in a manner that adequately mimics the binding characteristics of the BK virus coat protein to cells. Such a mimic may serve as a tool for the identification of inhibitors of BK viral progression. Herein, we report the design and characterization of a reduced-size and soluble template derived from a four-helix protein-TM1526 of Thermatoga maritima archaea bacteria-which maintains the topological display of the BC and HI loops as found in the viral coat protein, VP1, of BKV. We demonstrate that the GT1b and GD1b sialogangliosides, which bind to the VP1 of BKV, also associate with our BKV template. Employing a GFP-tagged template, we show host cell association that is dose dependent and that can be reduced by neuraminidase treatment. These data demonstrate that the BKV template mimics the host cell binding observed for the wild-type virus coat protein VP1.


Assuntos
Vírus BK/patogenicidade , Proteínas do Capsídeo/metabolismo , Nefropatias/virologia , Mimetismo Molecular , Sequência de Aminoácidos , Animais , Vírus BK/metabolismo , Proteínas do Capsídeo/química , Chlorocebus aethiops , Citometria de Fluxo , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Células Vero , Difração de Raios X
13.
J Comb Chem ; 11(1): 34-43, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19105723

RESUMO

For the successful implementation of Distributed Drug Discovery (D(3)) (outlined in the accompanying Perspective), students, in the course of their educational laboratories, must be able to reproducibly make new, high quality, molecules with potential for biological activity. This article reports the successful achievement of this goal. Using previously rehearsed alkylating agents, students in a second semester organic chemistry laboratory performed a solid-phase combinatorial chemistry experiment in which they made 38 new analogs of the most potent member of a class of antimelanoma compounds. All compounds were made in duplicate, purified by silica gel chromatography, and characterized by NMR and LC/MS. As a continuing part of the Distributed Drug Discovery program, a virtual D(3) catalog based on this work was then enumerated and is made freely available to the global scientific community.


Assuntos
Antineoplásicos/síntese química , Pesquisa Biomédica/educação , Descoberta de Drogas/métodos , Melanoma/tratamento farmacológico , Laboratórios , Universidades
14.
J Comb Chem ; 11(1): 14-33, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19105725

RESUMO

Distributed Drug Discovery (D(3)) proposes solving large drug discovery problems by breaking them into smaller units for processing at multiple sites. A key component of the synthetic and computational stages of D(3) is the global rehearsal of prospective reagents and their subsequent use in the creation of virtual catalogs of molecules accessible by simple, inexpensive combinatorial chemistry. The first section of this article documents the feasibility of the synthetic component of Distributed Drug Discovery. Twenty-four alkylating agents were rehearsed in the United States, Poland, Russia, and Spain, for their utility in the synthesis of resin-bound unnatural amino acids 1, key intermediates in many combinatorial chemistry procedures. This global reagent rehearsal, coupled to virtual library generation, increases the likelihood that any member of that virtual library can be made. It facilitates the realistic integration of worldwide virtual D(3) catalog computational analysis with synthesis. The second part of this article describes the creation of the first virtual D(3) catalog. It reports the enumeration of 24,416 acylated unnatural amino acids 5, assembled from lists of either rehearsed or well-precedented alkylating and acylating reagents, and describes how the resulting catalog can be freely accessed, searched, and downloaded by the scientific community.


Assuntos
Aminoácidos/síntese química , Técnicas de Química Combinatória , Descoberta de Drogas/métodos , Alquilantes , Antineoplásicos/síntese química , Descoberta de Drogas/economia , Saúde Global , Disseminação de Informação , Internet
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