DNA clamp function of the monoubiquitinated Fanconi anaemia ID complex.

The ID complex, involving the proteins FANCI and FANCD2, is required for the repair of DNA interstrand crosslinks (ICL) and related lesions1. These proteins are mutated in Fanconi anaemia, a disease in which patients are predisposed to cancer. The Fanconi anaemia pathway of ICL repair is activated when a replication fork stalls at an ICL2; this triggers monoubiquitination of the ID complex, in which one ubiquitin molecule is conjugated to each of FANCI and FANCD2. Monoubiquitination of ID is essential for ICL repair by excision, translesion synthesis and homologous recombination; however, its function remains unknown1,3. Here we report a cryo-electron microscopy structure of the monoubiquitinated human ID complex bound to DNA, and reveal that it forms a closed ring that encircles the DNA. By comparison with the structure of the non-ubiquitinated ID complex bound to ICL DNA-which we also report here-we show that monoubiquitination triggers a complete rearrangement of the open, trough-like ID structure through the ubiquitin of one protomer binding to the other protomer in a reciprocal fashion. These structures-together with biochemical data-indicate that the monoubiquitinated ID complex loses its preference for ICL and related branched DNA structures, and becomes a sliding DNA clamp that can coordinate the subsequent repair reactions. Our findings also reveal how monoubiquitination in general can induce an alternative protein structure with a new function.

Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a di-heptamer macromolecular assembly.

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.

Proteomic Analysis of Arachis hypogaea Seeds from Different Maturity Classes.

Physiological maturity impacts seed quality through various mechanisms including vigor, desiccation tolerance, dormancy induction, synthesis of raw materials (including seed storage proteins), and the reorganization of metabolisms. Peanut seed development can be classified into seven classes with four incremental stages per class. Based on the mesocarp color, the final three stages are commonly referred to as "orange", "brown", and "black". In 2017, freshly harvested pods from one genotype of runner market-type peanuts grown under conventional practices were obtained from the University of Georgia research facility. The pods were removed from the plant material and 'pod blasted' to reveal the mesocarp. After separation, the remainder of the pod outer layer was removed, and the seeds were segregated for proteomic analysis. The raw peanuts were analyzed by bottom-up LC-MS/MS proteomics, which was conducted by the Proteomics Resource Center at the Rockefeller University, to identify the significant protein composition differences in each maturity class. The proteomic data revealed differentially expressed proteins as a function of maturity class with multiple functions including plant defense, metabolism, cell signaling, nutrient accumulation, and packaging. Understanding the processes needed for seed maturation will enable peanut scientists to evaluate the traits needed for robust germination, hardiness of the seed in response to disease, and nutrient quality.

The lipid droplet protein DHRS3 is a regulator of melanoma cell state.

Lipid droplets are fat storage organelles composed of a protein envelope and lipid rich core. Regulation of this protein envelope underlies differential lipid droplet formation and function. In melanoma, lipid droplet formation has been linked to tumor progression and metastasis, but it is unknown whether lipid droplet proteins play a role. To address this, we performed proteomic analysis of the lipid droplet envelope in melanoma. We found that lipid droplet proteins were differentially enriched in distinct melanoma states; from melanocytic to undifferentiated. DHRS3, which converts all-trans-retinal to all-trans-retinol, is upregulated in the MITFLO/undifferentiated/neural crest-like melanoma cell state and reduced in the MITFHI/melanocytic state. Increased DHRS3 expression is sufficient to drive MITFHI/melanocytic cells to a more undifferentiated/invasive state. These changes are due to retinoic acid mediated regulation of melanocytic genes. Our data demonstrate that melanoma cell state can be regulated by expression of lipid droplet proteins which affect downstream retinoid signaling.

The Lipid Droplet Protein DHRS3 Is a Regulator of Melanoma Cell State.

Lipid droplets are fat storage organelles composed of a protein envelope and lipid-rich core. Regulation of this protein envelope underlies differential lipid droplet formation and function. In melanoma, lipid droplet formation has been linked to tumor progression and metastasis, but it is unknown whether lipid droplet proteins play a role. To address this, we performed proteomic analysis of the lipid droplet envelope in melanoma. We found that lipid droplet proteins were differentially enriched in distinct melanoma states; from melanocytic to undifferentiated. DHRS3, which converts all-trans-retinal to all-trans-retinol, is upregulated in the MITFLO/undifferentiated/neural crest-like melanoma cell state and reduced in the MITFHI/melanocytic state. Increased DHRS3 expression is sufficient to drive MITFHI/melanocytic cells to a more undifferentiated/invasive state. These changes are due to retinoic acid-mediated regulation of melanocytic genes. Our data demonstrate that melanoma cell state can be regulated by expression of lipid droplet proteins which affect downstream retinoid signaling.

Optimization of ultracentrifugation-based method to enhance the purity and proteomic profiling depth of plasma-derived extracellular vesicles and particles.

Circulating extracellular vesicles and particles (EVPs) are being investigated as potential biomarkers for early cancer detection, prognosis, and disease monitoring. However, the suboptimal purity of EVPs isolated from peripheral blood plasma has posed a challenge of in-depth analysis of the EVP proteome. Here, we compared the effectiveness of different methods for isolating EVPs from healthy donor plasma, including ultracentrifugation (UC)-based protocols, phosphatidylserine-Tim4 interaction-based affinity capture (referred to as "PS"), and several commercial kits. Modified UC methods with an additional UC washing or size exclusion chromatography step substantially improved EVP purity and enabled the detection of additional proteins via proteomic mass spectrometry, including many plasma membrane and cytoplasmic proteins involved in vesicular regulation pathways. This improved performance was reproduced in cancer patient plasma specimens, resulting in the identification of a greater number of differentially expressed EVP proteins, thus expanding the range of potential biomarker candidates. However, PS and other commercial kits did not outperform UC-based methods in improving plasma EVP purity. PS yielded abundant contaminating proteins and a biased enrichment for specific EVP subsets, thus unsuitable for proteomic profiling of plasma EVPs. Therefore, we have optimized UC-based protocols for circulating EVP isolation, which enable further in-depth proteomic analysis for biomarker discovery.

Hippocampal γCaMKII dopaminylation promotes synaptic-to-nuclear signaling and memory formation.

Protein monoaminylation is a class of posttranslational modification (PTM) that contributes to transcription, physiology and behavior. While recent analyses have focused on histones as critical substrates of monoaminylation, the broader repertoire of monoaminylated proteins in brain remains unclear. Here, we report the development/implementation of a chemical probe for the bioorthogonal labeling, enrichment and proteomics-based detection of dopaminylated proteins in brain. We identified 1,557 dopaminylated proteins - many synaptic - including γCaMKII, which mediates Ca2+-dependent cellular signaling and hippocampal-dependent memory. We found that γCaMKII dopaminylation is largely synaptic and mediates synaptic-to-nuclear signaling, neuronal gene expression and intrinsic excitability, and contextual memory. These results indicate a critical role for synaptic dopaminylation in adaptive brain plasticity, and may suggest roles for these phenomena in pathologies associated with altered monoaminergic signaling.

Structure of the FA core ubiquitin ligase closing the ID clamp on DNA.

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand crosslinks. Central to the pathway is the FA core complex, a ubiquitin ligase of nine subunits that monoubiquitinates the FANCI-FANCD2 (ID) DNA clamp. The 3.1 Å structure of the 1.1-MDa human FA core complex, described here, reveals an asymmetric assembly with two copies of all but the FANCC, FANCE and FANCF subunits. The asymmetry is crucial, as it prevents the binding of a second FANCC-FANCE-FANCF subcomplex that inhibits the recruitment of the UBE2T ubiquitin conjugating enzyme, and instead creates an ID binding site. A single active site then ubiquitinates FANCD2 and FANCI sequentially. We also present the 4.2-Å structures of the human core-UBE2T-ID-DNA complex in three conformations captured during monoubiquitination. They reveal the core-UBE2T complex remodeling the ID-DNA complex, closing the clamp on the DNA before ubiquitination. Monoubiquitination then prevents clamp opening after release from the core.

Factors associated with high-grade Erectile Dysfunction (ED) in tuberculosis patients from a hospital in Lima, Peru. / Factores asociados a Disfunción Eréctil (DE) alto grado en pacientes con tuberculosis de un Hospital de Lima, Perú.

OBJECTIVE: To determine the prevalence and factors associated with high-grade ED in Lima, Peru. MATERIAL AND METHODS: Analytical cross-sectional study in tuberculosis patients treated in an out patient clinic of a public hospital in Lima, Peru in 2018. High grade SD was the dependent variable, using the IIEF-5 questionnaire and the independent variables were sexual orientation, history of previous pathology, location of tuberculosis, type of treatment scheme and presence of hemoptysis. Prevalence ratios (PR) were estimated using simple and multiple regression models. RESULTS: Of 189 patients, the majority presented pulmonary tuberculosis (98.9%), overweight (25.9%) and just over half had high-grade ED (52.9%). In the simple regression it was found that the factors associated with presenting high-grade SD were age in years (RP=1.11), reporting diabetes (RP=2.09), having multi-drug resistant TB (RP=1.51) and presenting a treatment time from 1 year to more (RP=1.87). In the multiple regression, the variables that were associated with a higher frequency of high-grade TB were age in years (RP=1.11), a history of diabetes (RP=1.66) and having MDR TB (RP=2.04). CONCLUSIONS: This study suggests a high prevalence of ED in patients with TB, where four out of ten presented high-grade ED. Being older, having a history of diabetes and using an MDR treatment were positively associated with the development of high-grade ED. Studies with designs close to causality are required to know the real magnitude of the influence of clinical and therapeutic characteristics on the development of high-grade ED.

OBJETIVO: Determinar la prevalencia y factores asociados a DE (disfunción eréctil) alto grado en Lima, Perú.MATERIAL Y MÉTODOS: Estudio transversal analítico en pacientes tuberculosos atendidos en consultorio externo de un hospital público de Lima, Perú en el 2018. La DE alto grado fue la variable dependiente, utilizando el cuestionario IIEF-5 y las variables independientes fueron la orientación sexual, historia de patología previa, localización de tuberculosis, tipo de esquema de tratamiento y presencia de hemoptisis. Se estimaron razones de prevalencia (RP) utilizando modelos de regresión simple y múltiple. RESULTADOS: De 189 pacientes, la mayoría presentaba tuberculosis pulmonar (98,9%), sobrepeso (25,9%) y poco más de la mitad tuvo DE alto grado (52,9%). En la regresión simple se encontró que los factores asociados a presentar DE alto grado fueron la edad en años (RP=1,11), reportar diabetes (RP=2,09), tener TB multidrogo-resistente (RP=1,51) y presentar un tiempo de tratamiento de 1 año a más (RP=1,87). En la regresión múltiple, las variables que se asociaron a una mayor frecuencia de TB alto grado fueron la edad en años (RP=1,11), el antecedente de diabetes (RP=1,66) y tenerTB MDR (tuberculosis multirresistente) (2,04). CONCLUSIONES: Este estudio sugiere una alta prevalencia de DE en pacientes con TB, donde cuatro de cada diez presentaron DE alto grado. Tener mayor edad, presentar antecedente de diabetes y utilizar untratamiento MDR se asociaron positivamente al desarrollo de DE alto grado. Se requieren estudios con diseños cercanos hacia la causalidad para conocer la magnitud real de la influencia de características clínicas y terapéuticas en el desarrollo de DE alto grado.

Violencia durante el enamoramiento en estudiantes universitarios

RESUMEN Introducción: Siete de cada diez mujeres peruanas entre 15 a 49 años reportan que fueron víctimas de violencia por su pareja alguna vez. Los estudios en Perú acerca de la violencia en el enamoramiento, son escasos; muchos no incluyen ambos sexos o a la población universitaria. Objetivo: Determinar la frecuencia de los tipos de violencia, así como las conductas reportadas durante el enamoramiento en estudiantes universitarios. Métodos: Se realizó un estudio transversal descriptivo en 71 estudiantes de una universidad en Perú; se usó un instrumento elaborado por el Ministerio de la Mujer y Poblaciones Vulnerables del país, para medir los tipos de violencia (física, psicológica y sexual) y las conductas asociadas y ejercidas por la pareja. Resultados: Del total, el 85,5 % reportó padecer de algún tipo de violencia. El 57,7 % de la muestra reportó violencia psicológica, mientras que el 7 % mencionó ser víctima de violencia física, psicológica y sexual. El 11,3 % reportó recibir palabras insinuantes al sexo por parte de su pareja. Entre quienes recibieron simultáneamente violencia física, psicológica y sexual, el 80 % fueron mujeres. Conclusiones: Los estudiantes sufren algún tipo de violencia y la mitad es violentada psicológicamente. Una mayor frecuencia de violencia se reporta en estudiantes mujeres.

ABSTRACT Introduction: Seven out of ten Peruvian women between the ages of 15 and 49 reported that they were victims of intimate partner violence at some time. Studies on dating violence in Peru are scarce, and many do not include both sexes and the university population. Objective: To identify the frequency of the types of violence, as well as the behaviors reported during dating in university students. Methods: A descriptive cross-sectional study was conducted in 71 students from a university in Peru, using an instrument created by the Peruvian Ministry of Women and Vulnerable Populations to measure the types of violence (physical, psychological, and sexual) and associated behaviors by partners. Results: Of all participants, 85,5 % reported suffering from some type of violence. Also, 57,7 % reported psychological violence, while 7 % indicated to be a victim of physical, psychological, and sexual violence. Receiving words suggestive of sex from their partner was reported in 11,3 %. Among those who received simultaneous physical, psychological, and sexual violence, 80 % were women. Conclusions: Students suffer some type of violence and half of them are psychologically abused. A higher frequency of violence is reported in female students. Effective mental health counseling is recommended to help relevant institutions to better address dating violence.

Problemas de visión en personas mayores de 50 años en el Perú: Estudio poblacional / Vision problems in people over 50 years old in Peru: A population-based study

RESUMEN Objetivo: Evaluar el antecedente de catarata y problemas de visión en personas mayores de 50 años en el Perú. Métodos: Análisis de base de datos secundaria de la Encuesta Demográfica y de Salud Familiar (ENDES) 2016. Incluimos 7970 adultos mayores de 50 años. Evaluamos los indicadores de interés a nivel nacional y regional. Asimismo, analizamos factores relacionados a estos eventos mediante un modelo multivariado de Poisson. Resultados: Se evidenció un 13,6 % (IC95 %: 12,8-14,3 %) de antecedente de catarata, 20,4 % (IC95 %: 19,5-21,3 %) de problemas de visión de larga distancia (PVLD) y 29,4 % (IC95 %: 28,4-30,4 %) de problemas de visión de corta distancia (PVCD). En el modelo multivariado observamos que las personas con antecedente de hipertensión arterial, antecedente de diabetes y los grupos de edad mayores tenían una probabilidad significativamente mayor de reportar antecedentes de cataratas y problemas de visión. Observamos también que las personas con mejor estado económico presentaron menor frecuencia de PVLD y PVCD. Conclusión: El antecedente de catarata y los problemas de visión en la población estudiada varían de acuerdo a características sociodemográficas y geográficas. La identificación de factores relacionados a estos eventos de interés permite proponer mejores intervenciones para el control de estos problemas.

ABSTRACT Objective: To evaluate the history of cataract and vision problems in people over 50 years of age in Peru. Methods: Secondary database analysis of the Demographic and Family Health Survey (ENDES) 2016. We included 7970 adults over 50 years of age. We evaluated the indicators of interest at the national and regional level. Likewise, we analyzed factors related to these events using a multivariate Poisson model. Results: 13.6% (95% CI: 12.8-14.3%) had a history of cataract, 20.4% (95%CI: 19.5-21.3%) had long-distance vision problems. (LDVP) and 29.4% (95%CI: 28.4-30.4%) of short-distance vision problems (SDVP). In the multivariate model, we observed that people with a history of high blood pressure, a history of diabetes, and older age groups were significantly more likely to report a history of cataracts and vision problems. We also observed that people with a better economic status had a lower frequency of LDVP and SDVP. Conclusion: The history of cataract and vision problems in the studied population vary according to sociodemographic and geographic characteristics. The identification of factors related to these events of interest allows us to propose better interventions to control these problems.

Aspectos básicos sobre la lectura de revisiones sistemáticas y la interpretación de meta-análisis / Some basic concepts about reading systematic reviews and interpreting meta-analysis

Las revisiones sistemáticas (RS) son estudios que buscan resumir la evidencia disponible sobre una pregunta de investigación, para lo cual pueden usar estrategias estadísticas conocidas como meta-análisis (MA). En la actualidad, las RS son fundamentales para tomar decisiones basadas en evidencias, por lo cual resulta de suma importancia que los profesionales de la salud sepan enfrentarse a este tipo de estudios. Por ello, el presente artículo tiene por objetivo familiarizar al lector con los aspectos básicos para realizar una correcta lectura de RS e interpretación de MA, para lo cual se utiliza un ejemplo hipotético de una condición ficticia llamada "Síndrome del glotón". Asimismo, se aborda la interpretación de la certeza de la evidencia según la metodología Grading of Recommendations Assessment, Development, and Evaluation (GRADE).

Systematic reviews (SR) are studies that seek to summarize the available evidence regarding a research question, for which they can use statistical strategies known as meta-analysis (MA). Currently, SRs are fundamental to making evidence-based decisions, which is why it is very important for health professionals to know how to face this type of studies. Therefore, this article aims to familiarize the reader with the basic concepts to make a correct appraisal of SRs and interpretation of MAs, for which a hypothetical example of a fictitious condition called "Glutton Syndrome" is used. In addition, the interpretation of the certainty of the evidence according to the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology is addressed.