Rescue therapy with upadacitinib in medically refractory pediatric ulcerative colitis.

Approved options for advanced therapy in pediatric inflammatory bowel disease (IBD) are limited. Although Janus kinase (JAK) inhibitors are approved in adult IBD, their benefit in pediatric populations is not yet delineated. We present a 13-year-old female patient with ulcerative colitis (UC) refractory to numerous therapies and courses of prednisone that ultimately responded to a JAK inhibitor. Initial treatment consisted of 5-aminosalicylate and azathioprine. This was changed to adalimumab due to persistent symptoms. Repeat colonoscopy revealed pancolitis, thus she was transitioned to vedolizumab. She was hospitalized twice for uncontrolled symptoms on vedolizumab and subsequent scope showed continued pancolitis. As a result, she transitioned to ustekinumab without symptomatic relief after adjusting to monthly dosing. The family declined colectomy, opting to exhaust all medical therapies. Upadacitinib was started and her symptoms resolved within 1 week, and she remains in steroid-free remission. This case illustrates the possible role of JAK inhibitors in extensively refractory pediatric UC patients before colectomy.

Hemorragia Intracerebral Imprevisible en un Hombre Joven: La Importancia de la Tomografía computarizada craneal post-mortem / Unpredictable Intracerebral Hemorrhage in a Young Male: The Importance of Post-Mortem Cranial Computed Tomography

La muerte súbita es aquella que ocurre dentro de las 24 horas posteriores al inicio de los síntomas y se caracteriza por ser clínicamente inexplicable, inesperada y repentina. Debido a la naturaleza de la muerte súbita, no es posible llegar a un diagnóstico preciso sin una autopsia. En esta comunicación breve, evaluaremos el caso de un empleado de crucero de 33 años, sin historial médico/farmacológico previo, el cual falleció súbitamente mientras reposaba en su camarote. Debido a las sospechas iniciales de una posible muerte causada por una sobredosis de cocaína, se le realizó un panel toxicológico abarcador el cual resultó negativo. Empero, una tomografía computarizada (TC) craneal sin contraste revirtió la hipótesis inicial y la autopsia neuropatológica -sorpresivamente- confirmó que la verdadera causa de muerte fue la ruptura de un aneurisma sacular desconocido en el polígono de Willis.

Sudden death occurs within 24 hours after the onset of symptoms and is characterized by being clinically inexplicable, sudden, and unexpected. Due to the nature of sudden death, it is not possible an accurate diagnosis without performing an autopsy. In this brief communication, we will evaluate the case of a 33-year-old cruise employee, with no prior medical/pharmacological history, who suddenly died while resting in his cabin. Due to initial suspicions of a possible cocaine overdose death, a comprehensive toxicology panel was performed, although yielding a negative result. A cranial computed tomography without contrast reversed the initial hypothesis and the neuropathological autopsy -surprisingly- confirmed that the true cause of death was the rupture of an unknown saccular aneurysm in the Circle of Willis.

Structural basis for cell type specific DNA binding of C/EBPß: The case of cell cycle inhibitor p15INK4b promoter.

C/EBPß is a key regulator of numerous cellular processes, but it can also contribute to tumorigenesis and viral diseases. It binds to specific DNA sequences (C/EBP sites) and interacts with other transcription factors to control expression of multiple eukaryotic genes in a tissue and cell-type dependent manner. A body of evidence has established that cell-type-specific regulatory information is contained in the local DNA sequence of the binding motif. In human epithelial cells, C/EBPß is an essential cofactor for TGFß signaling in the case of Smad2/3/4 and FoxO-dependent induction of the cell cycle inhibitor, p15INK4b. In the TGFß-responsive region 2 of the p15INK4b promoter, the Smad binding site is flanked by a C/EBP site, CTTAA•GAAAG, which differs from the canonical, palindromic ATTGC•GCAAT motif. The X-ray crystal structure of C/EBPß bound to the p15INK4b promoter fragment shows how GCGC-to-AAGA substitution generates changes in the intermolecular interactions in the protein-DNA interface that enhances C/EBPß binding specificity, limits possible epigenetic regulation of the promoter, and generates a DNA element with a unique pattern of methyl groups in the major groove. Significantly, CT/GA dinucleotides located at the 5'ends of the double stranded element maintain local narrowing of the DNA minor groove width that is necessary for DNA recognition. Our results suggest that C/EBPß would accept all forms of modified cytosine in the context of the CpT site. This contrasts with the effect on the consensus motif, where C/EBPß binding is modestly increased by cytosine methylation, but substantially decreased by hydroxymethylation.

DASH diet vs. DASH diet plus physical activity in older patients with type 2 diabetes and high blood pressure: A randomized clinical trial.

BACKGROUND AND AIMS: To evaluate the effect of lifestyle modification by adopting a DASH diet, with and without physical activity guidance, on blood pressure, glycemic control, lipid profile, weight, and body composition in older patients with type 2 diabetes mellitus (T2DM) and hypertension. METHODS AND RESULTS: For this randomized clinical trial, we recruited patients aged 60 years or older with T2DM and uncontrolled hypertension. One group (DASH) received only DASH dietary guidance, while the other group (DASHPED) received dietary guidance and encouragement to walk with a pedometer. Outcomes of interest were (1) blood pressure, (2) physical activity, (3) weight, body mass index (BMI), and body composition, and (4) biochemical variables. Measurements were taken at baseline and 16 weeks after the intervention. We included 35 patients in the analysis. At the end of the study, the DASHPED group had an mean increase in physical activity of 1721 steps/day. Both groups displayed significantly reduced weight, BMI, and waking diastolic pressures on ambulatory blood pressure monitoring after the intervention. A trend of reduced sleeping diastolic pressure was found in the DASHPED group. Changes in weight, BMI, muscle mass, body fat, waist-hip ratio, glycemic control, lipid profile, and insulin sensitivity did not differ between the groups. CONCLUSION: There was no difference in outcomes between the group that only dieted and the group that also performed increased physical activity, despite a significant increase in exercise. This reinforces the importance of dietary changes in immediate blood pressure control.

Automated TTF-1 Immunohistochemistry Assay for the Differentiation of Lung Adenocarcinoma Versus Lung Squamous Cell Carcinoma.

Due to therapeutic advances, the subclassification of non-small cell lung carcinomas (NSCLC) between the adenocarcinomas and squamous cell carcinomas subtypes is essential for the practice of personalized and targeted medicine. The clinical management for these two NSCLC subtypes is different due to their different molecular properties and histological origins. Immunohistochemistry (IHC) markers such is TTF-1 play a key role in the differentiation of lung adenocarcinomas and squamous cell carcinomas. However, immunohistochemistry is a complex process involving many critical steps and the reliability of results depends on the standardization of the assay as well as the appropriate interpretation. Different laboratories use different reagents and different IHC approaches for the detection of TTF-1 in lung cancer tumors. Here we describe an automated IHC protocol used in our laboratory for the detection of TTF-1 in formalin-fixed, paraffin-embedded (FFPE) tissue sections from lung tumors.

Automated Immunohistochemistry Assay for the Detection of Napsin-A in Formalin-Fixed Paraffin-Embedded Tissues from Lung Tumors.

Immunohistochemistry (IHC) enables the selective detection of proteins in cells of formalin-fixed-paraffin-embedded (FFPE) tissue sections. This technique plays a key role in the identification and classification of primary lung cancer tumors through the evaluation of the expression of the aspartic proteinase Napsin-A. However, immunohistochemistry is a complex process involving many critical steps and the lack of standardization as well as inappropriate analytical conditions may contribute to inconsistent results between laboratories. Automated immunohistochemistry addresses this issue by ensuring the quality and the reproducibility of the results among different laboratories. Here we describe an automated IHC protocol used in our laboratory for the detection of Napsin-A in FFPE lung tissue sections.

Intratumoral Injection of Clostridium novyi-NT Spores in Patients with Treatment-refractory Advanced Solid Tumors.

PURPOSE: Intratumorally injected Clostridium novyi-NT (nontoxic; lacking the alpha toxin), an attenuated strain of C. novyi, replicates within hypoxic tumor regions resulting in tumor-confined cell lysis and inflammatory response in animals, which warrants clinical investigation. PATIENTS AND METHODS: This first-in-human study (NCT01924689) enrolled patients with injectable, treatment-refractory solid tumors to receive a single intratumoral injection of C. novyi-NT across 6 dose cohorts (1 × 104 to 3 × 106 spores, 3+3 dose-escalation design) to determine dose-limiting toxicities (DLT), and the maximum tolerated dose. RESULTS: Among 24 patients, a single intratumoral injection of C. novyi-NT led to bacterial spores germination and the resultant lysis of injected tumor masses in 10 patients (42%) across all doses. The cohort 5 dose (1 × 106 spores) was defined as the maximum tolerated dose; DLTs were grade 4 sepsis (n = 2) and grade 4 gas gangrene (n = 1), all occurring in three patients with injected tumors >8 cm. Other treatment-related grade ≥3 toxicities included pathologic fracture (n = 1), limb abscess (n = 1), soft-tissue infection (n = 1), respiratory insufficiency (n = 1), and rash (n = 1), which occurred across four patients. Of 22 evaluable patients, nine (41%) had a decrease in size of the injected tumor and 19 (86%) had stable disease as the best overall response in injected and noninjected lesions combined. C. novyi-NT injection elicited a transient systemic cytokine response and enhanced systemic tumor-specific T-cell responses. CONCLUSIONS: Single intratumoral injection of C. novyi-NT is feasible. Toxicities can be significant but manageable. Signals of antitumor activity and the host immune response support additional studies of C. novyi-NT in humans.

Da relação entre prosódia e compreensão leitora: considerações teóricas, metodológicas e controvérsias / On the relationship between prosody and reading comprehension: theoretical, methodological and controversial considerations / Relación entre prosodia y comprensión lectora: consideraciones teóricas, metodológicas y controvertidas / Sur la relation entre la prosodie et la compréhension en lecture: considérations théoriques, méthodologiques et controversées

Resumo Este artigo, de natureza teórico-metodológica, discute a relação entre prosódia e compreensão leitora a partir de dois enfoques. O primeiro trata dos diferentes recursos metodológicos adotados na investigação dessa complexa relação, e o segundo versa sobre a controvérsia existente quanto à maneira como se configura essa relação. Reflexões como essas permitem aprofundar o conhecimento acerca das características dos diferentes recursos metodológicos adotados na investigação de dado fenômeno, assim como contribuem para esclarecer controvérsias em determinado campo do conhecimento, como é o caso da relação entre prosódia e compreensão leitora, tema ainda aberto a muitas interpretações. As discussões tomam por base pesquisas realizadas com crianças matriculadas no ensino fundamental.

Abstract This theoretical and methodological study discusses the relationship between prosody and reading comprehension from two different approaches: 1) the different methodological resources adopted to investigate this complex relationship; 2) the existing controversy on how this relationship is configured. Reflections such as these allow for an in-depth knowledge on the characteristics of the different methodological resources used to study a given phenomenon and to clarify controversies in certain fields of knowledge, such as the prosody-reading comprehension relationship, a topic still open to different interpretations. The discussions presented here are based on research carried out with elementary school children.

Resumen Este artículo, de carácter teórico-metodológico, analiza la relación entre la prosodia y la comprensión lectora desde dos enfoques. El primer enfoque aborda los diferentes recursos metodológicos adoptados en la investigación de esta compleja relación, mientras que el segundo plantea la controversia existente sobre cómo se configura esta relación. Estas reflexiones nos permiten profundizar nuestro conocimiento acerca de las características de diferentes recursos metodológicos adoptados en la investigación de un fenómeno dado; también contribuye a esclarecer controversias en determinados campos del conocimiento, como la relación entre prosodia y comprensión lectora, tema que aún permanece abierto a diversas interpretaciones. Las discusiones que aquí se presentan están basadas en investigaciones realizadas con niños de escuela primaria.

Résumé Cet étude théorico-méthodologique traite de la relation entre la prosodie et la compréhension en lecture à partir de deux approches différentes: 1) les différentes ressources méthodologiques adoptées pour étudier cette relation complexe; 2) la controverse existante sur la façon dont cette relation est configurée. De telles réflexions permettent d'approfondir la connaissance des caractéristiques des différentes ressources méthodologiques adoptées pour étudier un phénomène donné et de clarifier les controverses dans certains domaines de savoir, comme la relation prosodie-compréhension en lecture, un sujet encore ouvert à différentes interprétations. Les discussions présentées ici sont basées sur des recherches menées auprès d'enfants de l'école primaire.

Association of plasma vitamin D status with lifestyle patterns and ambulatory blood pressure monitoring parameters in patients with type 2 diabetes and hypertension.

AIMS: To evaluate nutritional and metabolic parameters associated with vitamin D status and blood pressure (BP) in type 2 diabetes and hypertensive patients. METHODS: BP evaluated by office and 24-h ambulatory BP monitoring (ABPM). Physical activity was evaluated by daily step count, body composition by DXA, and diet by a food frequency questionnaire. RESULTS: 116 patients were evaluated and median 25-hydroxyvitamin D level was 21 (16-27) ng/ml; 43% deficient (<20 ng/ml). Vitamin D deficiency was associated with higher systolic ABPM (136 ±â€¯10 vs. 130 ±â€¯13 mmHg; P = 0.01) and daytime ABPM (138 ±â€¯11 vs. 133 ±â€¯13 mmHg; P = 0.02), lower step counts (4400 [2700-6600] vs. 6400 [4700-8100] steps/day), lower urinary calcium (47 [32-141] vs. 89 [68-152] mEq), and higher fat mass (31 ±â€¯8 vs. 27 ±â€¯6.5 kg). Milk intake (37 vs. 64%; P = 0.009) and fish (31 vs. 69%; P < 0.001) were lower in deficients. On multivariate analysis, adjusted for fat mass and colder seasons, <5000 steps/day (OR = 3.30; 95%CI 1.34-8.12), no milk/fish intake (OR = 6.56; 95%CI 2.52-17.17), and both (OR = 7.24; 95%CI 2.19-23.90) remained associated with vitamin D deficiency. CONCLUSIONS: Vitamin D deficiency was highly prevalent in patients with hypertension and type 2 diabetes and associated with higher systolic ABPM (daytime and 24-h), less physical activity, and no milk or fish intake.

Protein Adsorption and Layer Formation at the Stainless Steel-Solution Interface Mediates Shear-Induced Particle Formation for an IgG1 Monoclonal Antibody.

Passage of specific protein solutions through certain pumps, tubing, and/or filling nozzles can result in the production of unwanted subvisible protein particles (SVPs). In this work, surface-mediated SVP formation was investigated. Specifically, the effects of different solid interface materials, interfacial shear rates, and protein concentrations on SVP formation were measured for the National Institute of Standards and Technology monoclonal antibody (NISTmAb), a reference IgG1 monoclonal antibody (mAb). A stainless steel rotary piston pump was used to identify formulation and process parameters that affect aggregation, and a flow cell (alumina or stainless steel interface) was used to further investigate the effect of different interface materials and/or interfacial shear rates. SVP particles produced were monitored using flow microscopy or flow cytometry. Neutron reflectometry and a quartz crystal microbalance with dissipation monitoring were used to characterize adsorption and properties of NISTmAb at the stainless steel interface. Pump/shear cell experiments showed that the NISTmAb concentration and interface material had a significant effect on SVP formation, while the effects of interfacial shear rate and passage number were less important. At the higher NISTmAb concentrations, the adsorbed protein became structurally altered at the stainless steel interface. The primary adsorbed layer remained largely undisturbed during flow, suggesting that SVP formation at high NISTmAb concentration was caused by the disruption of patches and/or secondary interactions.

Bracing in Ponseti Clubfoot Treatment: Improving Parental Adherence Through an Innovative Health Education Intervention.

Clubfoot is the most common musculoskeletal birth defect, characterized by abnormal tendon and muscle development, leading to abnormal bone alignment of the feet. The Ponseti method is considered the gold standard in clubfoot treatment, and consists of a series of plaster castings, followed by 4 years of brace use. The most common cause of clubfoot relapse is nonadherence with the bracing protocol by the child's caretakers. The purpose of this study was to design, implement, and evaluate an educational bracing program for parents of children with clubfoot in an effort to improve bracing adherence. The educational bracing program for parents of children with clubfoot was designed with incorporation of findings from previous research, adult teaching methodology, and parental feedback. An educational brochure and a practice doll were created for use in educational sessions with parents during routine treatment visits. Two educational sessions were conducted with a health educator, employing identical questionnaires to assess changes in parental knowledge and skills upon completion of the program. Thirty parents completed the educational bracing program, and the majority reported increased knowledge and self-efficacy regarding the bracing protocol of the Ponseti method. In addition, the health practitioners who conducted the educational sessions witnessed an improved ability of all parents to apply the brace as directed, and to recognize and correct improper fit. Completion of the educational program by the parents resulted in immediate improvements in knowledge and skills related to clubfoot bracing. Given that noncompliance to the bracing protocol is the most common cause of clubfoot relapse, these immediate effects of the educational program are promising not only because they encourage proper brace use, but because these immediate improvements have the potential to reduce future rates of clubfoot relapse.

Interactions of CCAAT/enhancer-binding protein ß with transcriptional coregulators.

CCAAT/enhancer-binding proteins (C/EBPs) are key regulators of numerous cellular processes, including cell proliferation, differentiation, and tumorigenesis. Their biological activities require interactions with several protein partners and the formation of functional multiprotein complexes involved in DNA repair and cell cycle control. Members of the family (C/EBPα, ß, δ, ε, and γ) bind to common DNA sequence motifs as homo- or hetero-dimers and interact with other transcription factors to control transcription of a number of eukaryotic genes. Of particular interest is C/EBPß, which binds to closed chromatin and acts as a pioneering factor for initiating tissue-specific gene expression at several promoters. This review focuses on intermolecular interactions that underlie C/EBPß's ability to regulate chromatin accessibility and directs readers to general reviews describing transcription regulation in eukaryotes.

Elucidation of the structure of retroviral proteases: a reminiscence.

Determinations of only a very few protein structures had consequences comparable to the impact exerted by the structure of the protease encoded by HIV-1, published just over 25 years ago. The structure of this relatively small protein and its cousins from other retroviruses provided a clear target for a spectacularly successful structure-assisted drug design effort that offered new hope for controlling the then-escalating AIDS epidemic. This reminiscence is limited primarily to work conducted at the National Cancer Institute, and is not meant to be a comprehensive history of the field, but is rather an attempt to provide a very personal account of how the structures of this most thoroughly studied crystallographic target were determined.

Use of Milestones and Development of Entrustable Professional Activities in 2 Hematology/Oncology Training Programs.

BACKGROUND: The Next Accreditation System (NAS) increases the focus on educational outcomes and meaningful evaluation of learners. This requires that key clinical faculty develop new assessment formats such as entrustable professional activities (EPAs). OBJECTIVES: To build and develop milestone-based assessment tools supporting 5 EPAs for a hematology/oncology fellow continuity clinic, and to educate key clinical faculty regarding the Clinical Competency Committee (CCC) and the NAS. METHODS: Program directors from 2 hematology/oncology fellowship programs developed 5 EPAs for continuity clinic evaluation supported by milestone-based assessment. The program directors met to create a unified CCC charter. Key clinical faculty helped to develop a milestone-based evaluation of fellow continuity clinic through creation of 5 hematology/oncology-specific EPAs. Formal entrustment regarding EPAs was deliberated by the CCC. RESULTS: A total of 18 fellows were evaluated. Clinical Competency Committee deliberation at each institution took approximately 10 minutes per fellow for discussion and decision regarding entrustment for all 5 EPAs supporting continuity clinic. One-third of postgraduate year (PGY)-4s, 50% of PGY-5s, and 100% of PGY-6s were deemed competent in all 5 EPAs by the CCC. CONCLUSIONS: All hematology/oncology trainees in San Antonio were evaluated using milestone-based assessment for continuity clinic, and entrustment decisions regarding 5 EPAs were made by the CCC. This project may provide other programs with a sound basis for adoption and further development of the next generation of evaluation tools at their institutions. Entrustable professional activities that are rotation specific should be used as a starting point for linking to the competencies, subcompetencies, and the reporting milestones.

Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses.

Species of Clostridium bacteria are notable for their ability to lyse tumor cells growing in hypoxic environments. We show that an attenuated strain of Clostridium novyi (C. novyi-NT) induces a microscopically precise, tumor-localized response in a rat orthotopic brain tumor model after intratumoral injection. It is well known, however, that experimental models often do not reliably predict the responses of human patients to therapeutic agents. We therefore used naturally occurring canine tumors as a translational bridge to human trials. Canine tumors are more like those of humans because they occur in animals with heterogeneous genetic backgrounds, are of host origin, and are due to spontaneous rather than engineered mutations. We found that intratumoral injection of C. novyi-NT spores was well tolerated in companion dogs bearing spontaneous solid tumors, with the most common toxicities being the expected symptoms associated with bacterial infections. Objective responses were observed in 6 of 16 dogs (37.5%), with three complete and three partial responses. On the basis of these encouraging results, we treated a human patient who had an advanced leiomyosarcoma with an intratumoral injection of C. novyi-NT spores. This treatment reduced the tumor within and surrounding the bone. Together, these results show that C. novyi-NT can precisely eradicate neoplastic tissues and suggest that further clinical trials of this agent in selected patients are warranted.

Geographic distribution of risk of death due to homicide in Puerto Rico, 2001–2010 / Distribución geográfica del riesgo de muerte por homicidio en Puerto Rico,2001–2010

Objective. To raise awareness of the impact of homicides in Puerto Rico based on the findings of the spatial and temporal distribution of homicides and the use of firearms, by age and gender, using reports of interpersonal violent deaths from the Institute of Forensic Science (IFS) headquartered in San Juan, Puerto Rico. Methods. This was a descriptive study of all homicide incidents in Puerto Rico reported by the IFS for the period 2001–2010. For each of the 8 542 cases, data analyzed included age, sex, municipality of incident, date of death, and mechanism. Crude sex- and age-specific mortality rates for Puerto Rico and for each municipality per year and for the 10-year period were calculated. Cumulative rate and cumulative risks were estimated and defined as lifetime risk. The relative distribution of cumulative rates for each municipality was categorized into quartiles of highest to lowest risk and displayed as a map. Results. The risk of homicide death among males is 13 times greater than among females. The highest rates were observed among males 20–24 years of age (198.4 homicides per 100 000). In any given year, firearms were used in at least 80% of homicides. The average lifetime risk of homicide death for males is 1 in 34. Conclusions. Young adult males with access to firearms are at greatest risk of homicide in Puerto Rico. Also, highly urbanized municipalities are at highest risk; however, certain nonurban municipalities along the coast also have a very high homicide risk. Top priorities should be applying the WHO “ecological model” for violent injury prevention and establishing a surveillance system that will assist in identifying the role that socioeconomics, illegal firearms trade, and drug trafficking are playing.

Objetivo. Concientizar sobre la repercusión de los homicidios en Puerto Rico con base en los resultados de la distribución espacial y temporal de los homicidios y el uso de las armas de fuego, según la edad y el sexo, a partir de los informes del Instituto de Ciencias Forenses (ICF), con sede en San Juan, Puerto Rico, sobre defunciones por violencia interpersonal. Métodos. Estudio descriptivo de todos los incidentes de homicidio ocurridos en Puerto Rico informados por el ICF durante el período del 2001 al 2010. La edad, el sexo, el municipio del incidente, la fecha de muerte y el mecanismo fueron los datos analizados en cada uno de los 8 542 casos. Se calcularon las tasas brutas de mortalidad específicas de cada sexo y edad en Puerto Rico y en cada municipio, por año y durante el período de 10 años. Se calcularon también las tasas y los riesgos acumulados y se definieron como riesgo durante toda la vida. La distribución relativa de las tasas acumuladas para cada municipio se clasificó en cuartiles, del riesgo más alto al más bajo, y se ilustró en un mapa. Resultados. El riesgo de muerte por homicidio en varones es 13 veces mayor que en mujeres. Las tasas más elevadas se observaron en hombres de 20 a 24 años de edad (198,4 homicidios por 100 000). Cualquiera que fuera el año escogido, en al menos 80% de los homicidios se utilizaron armas de fuego. En varones, el riesgo promedio de morir por homicidio durante toda la vida es de 1/34. Conclusiones. Los jóvenes varones adultos con acceso a las armas de fuego están sometidos a un mayor riesgo de homicidio en Puerto Rico. Además, en los municipios muy urbanizados el riesgo es más alto; sin embargo, en ciertos municipios no urbanos de la costa también hay un riesgo de homicidio muy alto. Las máximas prioridades deben ser aplicar el “modelo ecológico” de la OMS para prevenir las lesiones violentas y establecer un sistema de vigilancia que ayude a determinar la función que desempeñan las condiciones socioeconómicas, el comercio ilegal de armas de fuego y el tráfico de drogas.

Crystal structures of the reverse transcriptase-associated ribonuclease H domain of xenotropic murine leukemia-virus related virus.

The ribonuclease H (RNase H) domain of retroviral reverse transcriptase (RT) plays a critical role in the life cycle by degrading the RNA strands of DNA/RNA hybrids. In addition, RNase H activity is required to precisely remove the RNA primers from nascent (-) and (+) strand DNA. We report here three crystal structures of the RNase H domain of xenotropic murine leukemia virus-related virus (XMRV) RT, namely (i) the previously identified construct from which helix C was deleted, (ii) the intact domain, and (iii) the intact domain complexed with an active site α-hydroxytropolone inhibitor. Enzymatic assays showed that the intact RNase H domain retained catalytic activity, whereas the variant lacking helix C was only marginally active, corroborating the importance of this helix for enzymatic activity. Modeling of the enzyme-substrate complex elucidated the essential role of helix C in binding a DNA/RNA hybrid and its likely mode of recognition. The crystal structure of the RNase H domain complexed with ß-thujaplicinol clearly showed that coordination by two divalent cations mediates recognition of the inhibitor.

Concentrated dispersions of equilibrium protein nanoclusters that reversibly dissociate into active monomers.

Stabilizing proteins at high concentration is of broad interest in drug delivery, for treatment of cancer and many other diseases. Herein, we create highly concentrated antibody dispersions (up to 260 mg/mL) comprising dense equilibrium nanoclusters of protein (monoclonal antibody 1B7, polyclonal sheep immunoglobulin G, and bovine serum albumin) molecules which, upon dilution in vitro or administration in vivo, remain conformationally stable and biologically active. The extremely concentrated environment within the nanoclusters (∼700 mg/mL) provides conformational stability to the protein through a novel self-crowding mechanism, as shown by computer simulation, while the primarily repulsive nanocluster interactions result in colloidally stable, transparent dispersions. The nanoclusters are formed by adding trehalose as a cosolute which strengthens the short-ranged attraction between protein molecules. The protein cluster diameter was reversibly tuned from 50 to 300 nm by balancing short-ranged attraction against long-ranged electrostatic repulsion of weakly charged protein at a pH near the isoelectric point. This behavior is described semiquantitatively with a free energy model which includes the fractal dimension of the clusters. Upon dilution of the dispersion in vitro, the clusters rapidly dissociated into fully active protein monomers as shown with biophysical analysis (SEC, DLS, CD, and SDS-PAGE) and sensitive biological assays. Since the concept of forming nanoclusters by tuning colloid interactions is shown to be general, it is likely applicable to a variety of biological therapeutics, mitigating the need to engineer protein stability through amino acid modification. In vivo subcutaneous injection into mice results in indistinguishable pharmacokinetics versus a standard antibody solution. Stable protein dispersions with low viscosities may potentially enable patient self-administration by subcutaneous injection of antibody therapeutics being discovered and developed.

The early years of retroviral protease crystal structures.

Soon after its discovery, the attempts to develop anti-AIDS therapeutics focused on the retroviral protease (PR)-an enzyme used by lentiviruses to process the precursor polypeptide into mature viral proteins. An urgent need for the three-dimensional structure of PR to guide rational drug design prompted efforts to produce milligram quantities of this enzyme. However, only minute amounts of PR were present in the HIV-1 and HIV-2 viruses, and initial attempts to express this protein in bacteria were not successful. This review describes X-ray crystallographic studies of the retroviral proteases carried out at NCI-Frederick in the late 1980s and early 1990s and puts into perspective the crucial role that the total protein chemical synthesis played in unraveling the structure, mechanism of action, and inhibition of HIV-1 PR. Notably, the first fully correct structure of HIV-1 PR and the first cocrystal structure of its complex with an inhibitor (a substrate-derived, reduced isostere hexapeptide MVT-101) were determined using chemically synthesized protein. Most importantly, these sets of coordinates were made freely available to the research community and were used worldwide to solve X-ray structures of HIV-1 PR complexes with an array of inhibitors and set in motion a variety of theoretical studies. Publication of the structure of chemically synthesized HIV-1 PR complexed with MVT-101 preceded only by six years the approval of the first PR inhibitor as an anti-AIDS drug.

CCAAT/Enhancer-binding protein beta DNA binding is auto-inhibited by multiple elements that also mediate association with p300/CREB-binding protein (CBP).

Signaling through Ras GTPases controls the activity of many transcription factors including CCAAT/enhancer-binding protein (C/EBPbeta), which regulates oncogenic H-Ras(V12)-induced senescence and growth arrest. Here we report that C/EBPbeta (LAP) DNA binding is inhibited by N-terminal sequences and derepressed by oncogenic Ras signaling. Sequence and mutational analyses showed that auto-repression involves two LXXLF (phiXXphiphi)-like motifs (LX1 and LX2) and a third element, auto-inhibitory domain (AID), located within conserved region CR5. LX1 is a critical component of the transactivation domain and has been shown to mediate C/EBPbeta binding to the TAZ2 region of p300/CREB-binding protein coactivators. C/EBPbeta auto-repression also involves a C-terminal regulatory domain (CRD) adjacent to the leucine zipper. CRD contains a third phiXXphiphi motif (LX3) and a short sequence, KQL, which has similarity to a region in the protein-binding site of TAZ2. The C/EBPbeta N- and C-terminal domains physically associate in a manner that requires the basic region and CRD. We propose a model in which the regulatory sequences form a hydrophobic core that reciprocally inhibits DNA binding and transactivation. We also suggest a mechanism for C/EBPbeta derepression involving several recently identified modifications within AID and CRD. Finally, we show that association of activated C/EBPbeta with p300/CREB-binding protein requires the LX2 and AID auto-inhibitory elements. Thus, the N-terminal regulatory elements have dual roles in auto-inhibition and coactivator binding.