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Molecular mechanisms controlling functional bacterial chromosome (nucleoid) compaction and organization are surprisingly enigmatic but partly depend on conserved, histone-like proteins HUαα and HUαβ and their interactions that span the nanoscale and mesosc

Extrapulmonary small-cell carcinoma (SCC) is a rare neoplasm that shares certain features with its pulmonary counterpart and occurs predominantly in the gastrointestinal tract (GIT). It is a high-grade and poorly differentiated neuroendocrine tumor, usually diagnosed in advanced stages, with a poor prognosis and few therapeutic options in that setting. This is a case report of a 77-year-old Spanish male patient with localized SCC of the colon, who presented a pathological complete response in the surgical specimen after neoadjuvant chemotherapy with cisplatin and etoposide. To date, 5 years after surgery, the patient remains without evidence of tumor recurrence. As clinical guidelines for the management of this entity are lacking, and therefore its management has not been standardized, an attempt to summarize the current evidence in the literature was made.

Immunotherapy has demonstrated a role in the therapeutic landscape of a small subset of patients with colorectal carcinoma (CRC) that harbor a microsatellite instability (MSI-H) status due to a deficient DNA mismatch repair (dMMR) system. The remarkable responses to immune checkpoint inhibitors (ICIs) are now being tested in the neoadjuvant setting in localized CRC, where the dMMR/MSI-H status can be found in up to 15% of patients, with remarkable results obtained in NICHE2 and 3 trials, among others. This case series aims to report our experience at a tertiary center and provide a comprehensive analysis of the possible questions and challenges to overcome if ICIs were established as standard of care in a neoadjuvant setting, as well as the potential role they may have as conversion therapy not only in locoregional advanced CRC but also in oligometastatic disease.

Introducción: Los resultados clínicos están más condicionados al papel activo del paciente en su autocuidado que por la cualificación profesional, por ello es importante capturar la experiencia del paciente para mejorar la calidad asistencial. Objetivo: Evaluar la experiencia del paciente renal en la atención recibida por los profesionales. Material y Método: Estudio descriptivo transversal en pacientes de Hemodiálisis y consulta de Enfermedad Renal Crónica Avanzada. Se administró el Instrumento de Evaluación de la eXperiencia del PAciente Crónico, 15 ítems que puntuaron de 0 (peor) a 10 (mejor experiencia) y que, midió 3 factores: interacciones productivas, autogestión del paciente y nuevo modelo relacional. Los datos se analizaron con el software R. Resultados: Se incluyeron 76 pacientes. La puntuación de la experiencia del paciente (ítems 1-11) fue de 6,68±1,41 puntos, siendo para cada factor: “interacciones productivas” (9,00±1,37 puntos), “autogestión del paciente” (7,80±1,78 puntos) y “nuevo modelo relacional” (1,86±2,01 puntos). Los ítems 12-15 obtuvieron bajas puntuaciones con valores medios entre 2-4 puntos. Al comparar los pacientes de consulta y hemodiálisis, el grupo hemodiálisis puntuó menos en los factores: “autogestión del paciente” (p=0,01) y “nuevo modelo relacional” (p=0,03); y con respecto a los ítems 12-15, también se obtuvo menor puntuación en el ítem “atención continuada tras un ingreso/urgencia (p=0,04). Conclusiones: La experiencia del paciente renal es positiva en la interacción con los profesionales y en la gestión del autocuidado, pero surgen áreas de mejora como el uso de tecnología digital, compartir experiencias con iguales y, el seguimiento de la salud tras un episodio agudo.

Background: Enteral nutrition interruptions (ENI) are prevalent in the pediatric intensive care unit (PICU), but there is little evidence of their characteristics. Methods: This is a cross-sectional multicenter study including critically ill children on enteral nutrition. ENIs were classified as PICU procedures, procedures performed outside the PICU (PPOP), feeding intolerance and other criteria. The number and features of ENIs were collected. Results: A total of 75 children were enrolled. There were 41 interruptions affecting 37.3% of the patients with a median duration of 5 ± 9.4 h. The most common reason for ENI was PPOP (41.5%), followed by other criteria. Interruptions were considered preventable in 24.4% of the cases, but only eight were compensated. ENIs were more prevalent among children with cardiac disease (p = 0.047), higher PRISM (p = 0.047) and longer PICU stay (p = 0.035). There was association between PRISM and total interruption time (p = 0.02) and lower caloric intake (p = 0.035). Patients with respiratory illness (p = 0.022) and on noninvasive ventilation (p = 0,028) had fewer ENIs. ENI total time was associated with lower caloric (p = 0.001) and protein (p = 0.02) intake. Conclusions: ENIs are prevalent in PICU, especially in children with higher PRISM, longer PICU stays and cardiac disease, and result in lower caloric and protein intake.

Introducción: Las personas con enfermedad renal crónica dializadas demandan mayor cuidado por el manejo complejo de su condición, las múltiples complicaciones asociadas al tratamiento y la necesidad de asumir estilos de vida saludables, para garantizar la efectividad y seguridad de las terapias. Objetivos: Determinar la competencia para el autocuidado de las personas en diálisis, así como su relación con las características sociodemográficas y clínicas. Metodología: Se realizó un estudio observacional descriptivo de corte transversal, en una unidad renal del sur de Colombia. Instrumentos utilizados: Encuesta de caracterización para el cuidado de la persona con enfermedad crónica, Formulario de características clínicas y Cuestionario de Competencia para el cuidado versión-paciente. Resultados: Se incluyeron un total de 200 personas dializadas, con una edad media de 59±13,89 años, el 50,5% mujeres, con pareja estable, apoyo para su cuidado, grado educativo primario, dedicados a labores del hogar, y nivel socioeconómico bajo. El 47,5% evidenció alta competencia para su autocuidado, el 42% una competencia media y el 10,5% baja. Se identificaron asociaciones estadísticamente significativas de la competencia para el autocuidado con las variables asistencia puntual a terapia (p=0,024) y adherencia farmacológica (p=0,001). Conclusiones: La población abordada se caracterizó por vivir en condiciones socioeconómicas que aumentan la vulnerabilidad de la persona dializada, en su mayoría contó con soporte para el cuidado, presentó nivel de competencia para el autocuidado entre alto y medio, relacionado con asistencia puntual a terapia y cumplimiento en adherencia farmacológica.

IMPORTANCE:. A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 is hypothesized to play a role in the pathogenesis of invasive infection, but studies in sepsis are lacking. OBJECTIVES:. To study A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 protein level in pediatric sepsis and to study the association with outcome. DESIGN:. Data from two prospective cohort studies. SETTING AND PARTICIPANTS:. Cohort 1 is from a single-center study involving children admitted to PICU with meningococcal sepsis (samples obtained at three time points). Cohort 2 includes patients from a multicenter study involving children admitted to the hospital with invasive bacterial infections of differing etiologies (samples obtained within 48 hr after hospital admission). MAIN OUTCOMES AND MEASURES:. Primary outcome measure was mortality. Secondary outcome measures were PICU-free days at day 28 and hospital length of stay. RESULTS:. In cohort 1 (n = 59), nonsurvivors more frequently had A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 levels above the detection limit than survivors at admission to PICU (8/11 [73%] and 6/23 [26%], respectively; p = 0.02) and at t = 24 hours (2/3 [67%] and 3/37 [8%], respectively; p = 0.04). In cohort 2 (n = 240), A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 levels in patients within 48 hours after hospital admission were more frequently above the detection limit than in healthy controls (110/240 [46%] and 14/64 [22%], respectively; p = 0.001). Nonsurvivors more often had detectable A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 levels than survivors (16/21 [76%] and 94/219 [43%], respectively; p = 0.003), which was mostly attributable to patients with Neisseria meningitidis. CONCLUSIONS AND RELEVANCE:. In children with bacterial infection, detection of A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 within 48 hours after hospital admission is associated with death, particularly in meningococcal sepsis. Future studies should confirm the prognostic value of A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 and should study pathophysiologic mechanisms.

(1) Background: The COVID-19 pandemic and the implementation of restrictions and nonpharmaceutical interventions (NPIs) changed the trends in respiratory viral circulation and the pattern in pediatric healthcare utilization; (2) Methods: A retrospective, multicenter observational study designed to analyze the impact of the pandemic on pediatric healthcare utilization and the viral circulation pattern in children in a region in Northern Spain was carried out. Viral diagnostics data from all nasal or pharyngeal swabs collected in children in Asturias during the periods of March 2018–September 2019 and March 2020–September 2021 were analyzed, as well as the number of pediatric hospitalizations and emergency visits; (3) Results: A total of 14,640 samples were collected during the pandemic period. Of these, at least one respiratory virus was detected in 2940 (20.1%) while 5568/10,298 samples were positive in the pre-pandemic period (54.1%); p < 0.001. The detection of both enveloped and non-enveloped viruses decreased among periods (p < 0.001). After week 14, 2020, enveloped viruses were no longer detected until one year later, while non-enveloped viruses continued to be detected in children. Overall, a mean of 4946.8 (95% CI 4519.1–5374.4) pediatric emergency visits per month during the period 2018–2019 as compared to 2496.5 (95% CI 2086.4–2906.5) for 2020–2021 occurred (p < 0.001). The mean of pediatric hospitalizations also significantly decreased between periods, as follows: 346.6 (95% CI 313–380.2) in 2018–2019 vs. 161.1 (95% CI 138.4–183.8); p < 0.001; (4) Conclusions: Our study showed a remarkably reduction in pediatric hospitalizations and emergency visits and a change in the pattern of viral circulation during the COVID-19 pandemic in Asturias. The usual seasonal respiratory viruses, namely influenza or RSV were nearly absent in the pediatric population during the pandemic.

Abstract Background Nutritional support is essential in the care of critically ill children since malnutrition in this population is associated with increased morbidity and mortality. Injury in patients admitted to pediatric intensive care units (PICU) results in a catabolic state and augmented protein breakdown, leading to a negative protein balance. Current recommendations about protein prescription in the PICU are fundamentally based on expert opinions, and the minimum threshold is 1.5 g/kg per day of protein, although protein needs could be higher in certain subgroups of patients. The main objectives of the present study are to examine whether the administration of a protein-enriched infant formula increases the serum levels of total proteins, albumin, prealbumin, transferrin, and retinol and improves nitrogen balance and to analyze the effect of the high-protein diet on energy expenditure. A secondary objective is to register possible secondary effects of the protein-enriched diet. Methods A multicenter prospective randomized controlled trial (RCT) will be performed in three hospitals. Patients meeting inclusion criteria will be randomly allocated to one of three enteral feeding formulae with different protein contents. Blood and urine test, nitrogen balance assessment, and energy expenditure testing by indirect calorimetry will be performed at the beginning of the nutrition regimen and at 24 h, 72 h and 5–7 days after initiation. The sample size for this trial is estimated to be 90 participants (about 30 participants in each group). The data analysis will be by intention to treat. Discussion This RCT will provide new data about the amount of protein needed to improve levels of serum protein and nitrogen balance, a surrogate of protein balance, in critically ill infants receiving enteral nutrition. Trial registration ClinicalTrials.gov identifier: NCT03901742. Registered April 1, 2019 – Retrospectively registered.

New and improved energy storage technologies are required to overcome non-dispatchability, which is the main challenge for the successful integration of large shares of renewable energy within energy supply systems. Energy storage is proposed to tackle daily variations on the demand side, i.e., storing low-price energy during off-peak or valley periods for utilization during peak periods. Regarding electrical energy storage, several technologies are available with different potentials for scalability, density, and cost. A recent approach for grid-scale applications is based on transcritical carbon dioxide charge and discharge cycles in combination with thermal energy storage systems. This alternative to pumped-hydro and compressed air energy storage has been discussed in scientific literature, where different configurations have been proposed and their efficiency and costs calculated. The potential of the concept has been demonstrated to be an economical alternative, including hybrid concepts with solar thermal storage. Even at low temperatures, the addition of solar energy has proved to be cost effective. This paper explores the effect of introducing solar-based high temperature heat on the performance of different configurations of “Transcritical carbon dioxide ‒ thermal energy storage system” cycles. A base-cycle with 8-hour discharge time is compared with different layouts. Discussions include details on the models, parametric analyses -including solar technology alternatives-, and simulation results. Round trip efficiency of the base case, without solar support and at pressure ratio of 9.4, is 52%. When solar input is considered, the efficiency is above 60%, increasing the turbine inlet temperature to 950 K. Estimated levelized cost of electricity values are in the range of pumped hydro and compressed air energy storage, 90-140 USD/MWh in agreement with other works on this thermal storage technology. The global analysis shows clear advantages for advancing in the study and definition of this technology for exploitation of synergies at different power ranges, integrated with mid/high temperature solar power plants and with smaller-scale renewable installations.

Objetivo: describir la experiencia de los pacientes que transitan de hemodiálisis a diálisis peritoneal. Método: estudio cualitativo fenomenológico con nueve entrevistas semiestructuradas, entre septiembre de 2017 a mayo de 2018. Criterios de inclusión: autonomía para la diálisis peritoneal, cambio de terapia renal sustitutiva y permanencia mínima de dos meses en la antigua y nueva terapia renal. Criterios de exclusión: hemodiálisis domiciliaria y deterioro psíquico o cognitivo. Un paradigma de codificación deductivo-inductivo reveló cinco categorías principales. Resultados: la transición denotó separarse de una vida dependiente de terceros y experimentar cambios de vida, y ajustes para gestionar la terapia domiciliaria. Conclusión: la experiencia vivida movilizó recursos para adquirir una identidad integradora fluida, renaciendo un rol caracterizado por satisfacción personal y responsabilidad con el autocuidado.

Background: The 5-year survival rate of patients with pancreatic ductal adenocarcinoma (PDAC) is around 5% due to the fact that the majority of patients present with advanced disease that is treatment resistant. Familial pancreatic cancer (FPC) is a rare disorder that is defined as a family with at least two affected first degree relatives, with an estimated incidence of 4%–10%. The genetic basis is unknown in the majority of families although around 10%–13% of families carry germline mutations in known genes associated with hereditary cancer and pancreatitis syndromes. Methods: Panel sequencing was performed of 35 genes associated with hereditary cancer in 43 PDAC cases from families with an apparent hereditary pancreatic cancer syndrome. Findings: Pathogenic variants were identified in 19% (5/26) of PDAC cases from pure FPC families in the genes MLH1, CDKN2A, POLQ and FANCM. Low frequency potentially pathogenic VUS were also identified in 35% (9/26) of PDAC cases from FPC families in the genes FANCC, MLH1, PMS2, CFTR, APC and MUTYH. Furthermore, an important proportion of PDAC cases harboured more than one pathogenic, likely pathogenic or potentially pathogenic VUS, highlighting the multigene phenotype of FPC. Interpretation: The genetic basis of familial or hereditary pancreatic cancer can be explained in 21% of families by previously described hereditary cancer genes. Low frequency variants in other DNA repair genes are also present in 35% of families which may contribute to the risk of pancreatic cancer development. Funding: This study was funded by the Instituto de Salud Carlos III (Plan Estatal de I + D + i 2013–2016): ISCIII (PI09/02221, PI12/01635, PI15/02101 and PI18/1034) and co-financed by the European Development Regional Fund ‘‘A way to achieve Europe’’ (ERDF), the Biomedical Research Network in Cancer: CIBERONC (CB16/12/00446), Red Temática de investigación cooperativa en cáncer: RTICC (RD12/0036/0073) and La Asociación Española contra el Cáncer: AECC (Grupos Coordinados Estables 2016). Keywords: Familial pancreatic cancer, Panel sequencing, DNA repair and hereditary cancer genes, Pathogenic variants

Introducción: El cambio de modalidad de dialítica evidencia un acontecimiento de gran estrés e incertidumbre cuando no es una transferencia deseada, y el cual se describe como un proceso de transición, donde la persona se mueve de un estado de la vida a otro, implicando el dominio de nuevos roles, conocimientos y habilidades para regresar al estado de equilibrio previo al cambio. Objetivos: Describir la experiencia de los pacientes que transitan de diálisis peritoneal a hemodiálisis. Material y Método: Estudio cualitativo, descriptivo, de orientación fenomenológica. Nueve informantes elegidos por muestreo de conveniencia fueron sometidos a entrevista semiestructurada en el domicilio. El desarrollo analítico resultó de un proceso deductivo-inductivo del que surgieron los ejes temáticos en los que se articuló la vivencia de los participantes. Se obtuvo la autorización del Comité Ético y los hallazgos fueron confirmados por los informantes. Resultados: Emergieron 3 temas principales tales como, el significado de la experiencia de transición, las fases del proceso de la transición, y los cambios del ser en transición como fueron las modificaciones físicas, psíquicas, sociales y familiares. Conclusiones: La transición de diálisis peritoneal a hemodiálisis se revela como una vivencia vulnerable que denota cambios en el estado de salud y en las relaciones de roles y habilidades, asimismo, requiere cambio en los patrones de comportamiento de los participantes y, por tanto, resulta en la necesidad de redefinir un sentido de sí mismos.

Introdución: El catéter peritoneal resulta imprescindible para la realización de la diálisis peritoneal. Se han desarrollado distintos modelos y en 1996 apareció el catéter Autoposicionante con la intención de disminuir las disfunciones por desplazamiento gracias al peso de tungsteno de su extremo. Esta complicación condiciona su funcionamiento y la viabilidad de la técnica. CASO CLÍNICO: Presentamos el caso de un varón al que se coloca catéter Swan Neck con funcionamiento inicial bueno, pero que en tres meses necesita dos maniobras alfa por migraciones y termina precisando recambio de catéter, colocándose en esta ocasión un catéter Autoposicionante. Ya durante el periodo de cicatrización presenta nuevo desplazamiento y una tercera maniobra alfa permite reiniciar la técnica. A los tres meses ingresa para estudios por clínica abdominal; en la radiografía de abdomen se observa dilatación de asas, el TAC urgente muestra dilatación de ciego y colon hasta el cruce con el catéter de diálisis peritoneal, llegándose al diagnóstico de obstrucción mecánica de colon por catéter de diálisis. Se realiza laparoscopia y se recoloca la punta en la pelvis. Tras la cirugía el paciente evoluciona favorablemente y a los tres meses recibe trasplante renal de cadáver. A pesar optar por el catéter autoposicionante debido a su menor tasa de desplazamientos, en el caso de nuestro paciente se repite el episodio y además se asocia a una complicación grave.

La gastroparesia es una alteración crónica de la motilidad gástrica en la que se produce un retraso en el vaciamiento en ausencia de una obstrucción mecánica. La desorganización de la peristalsis antral puede ser consecuencia de cualquier desorden que induzca una disfunción neuromuscular del tracto gastrointestinal, ya que el vaciamiento gástrico refleja la coordinación de diferentes regiones del estómago y el duodeno, así como la modulación extrínseca del sistema nervioso central1. Las etiologías más frecuentes son la idiopática, la diabética y la postquirúrgica aunque también se contemplan otras menos habituales entre ellas las que son consecuencia de trastornos de la conducta alimentaria. Los síntomas habituales incluyen plenitud postprandial, saciedad precoz, náuseas, vómitos, malestar abdominal y sensación de distensión. El diagnóstico de certeza tras excluir otras causas como la obstrucción intestinal, se basa en pruebas que demuestren el vaciamiento gástrico enlentecido, siendo el patrón oro el estudio con escintigrafía1,2. Los objetivos del tratamiento son asegurar el adecuado aporte nutricional que se ve amenazado en este contexto, tratar la enfermedad subyacente si es reconocible y aliviar la sintomatología. Como abordaje inicial son básicos los cambios en la dieta y la terapia con fármacos antieméticos y procinéticos; cuando estas medidas fracasan, los vómitos son incoercibles y derivan en alteraciones electrolíticas, es necesario ingreso hospitalario para la realización de maniobras terapéuticas más invasivas. En casos extremos se inicia nutrición enteral o parenteral, para el tratamiento farmacológico se usa la vía intravenosa.

En el curso escolar 2005-2006 iniciaba su andadura la Escuela Superior de Arte Dramático (ESAD) de Galicia, centro público de educación superior dependiente de la Consejería de Cultura, Educación y Ordenación Universitaria, y en el curso escolar 2008-2009 salía la primera promoción de titulados/as superiores en las especialidades de interpretación y dirección escénica, cursando sus estudios al amparo de lo dispuesto en el RD754/1992, que establecía la equivalencia de los estudios superiores de arte dramático a una licenciatura universitaria. Desde entonces, y hasta el curso escolar 2012-2013, terminan sus estudios cinco promociones, lo que implica un total de 110 personas. Actualmente, puesto en marcha el nuevo plan de estudios regulado por el RD 1614/2009, parece oportuno analizar aspectos básicos de la formación y la transición de esas cinco promociones tanto por los datos que puedan aportar en relación a su incorporación a la práctica profesional y a los ámbitos más relevantes, como por la valoración que pueden hacer de su formación y de la utilidad que pueda haber tenido en esa transición a la vida activa. En este contexto nos hemos planteado como objetivos estudiar las características de su proceso de inserción (grado de inserción, condiciones laborales…), y conocer la valoración de la formación recibida en la ESAD así como las necesidades detectadas. Para ello hemos aplicado un cuestionario diseñado ad hoc a los miembros de las promociones de egresados y egresadas, habiendo obtenido un total de 45 respuestas. El perfil de la muestra está definida por ser mayoritariamente mujer (71,4%), con una media de edad de 29,16 (s=6,80) y un rango entre 22 y 59, habiendo realizado las siguientes especialidades: dirección escénica y dramaturgia (21,5%), interpretación (69%), y escenografía (9,5%). De los resultados obtenidos extraemos importantes conclusiones relativas a la formación recibida y al ejercicio profesional. En relación a la formación, se percibe una satisfacción medio-alta, siendo únicamente valorada de forma negativa por un 9,8%; así mismo, sólo un 23,8% considera que la formación recibida en la ESADg atendiendo al desempeño profesional fue insuficiente. No obstante, un 81,1% plantea como mejora en su formación la inclusión de más materias prácticas. En cuanto a la inserción profesional, un 78.57% estaban trabajando, ocupando el 60,6% de las mismas un empleo vinculado con la formación recibida. Las condiciones en las que estaban desarrollando su labor profesional son las siguientes: poseer el Grado en Arte Dramático fue exigido en el 50% de los casos; el 89,5% de los egresados/as encuestados/as afirman que las funciones que desempeñan son acordes a la categoría profesional y la media de ingresos mensuales que declaran es de 1.057,08€; sólo el 10,5% tiene contrato laboral estable y un 37,5% a tiempo completo, y la mayoría tiene un contrato temporal. Los ámbitos profesionales en los que desarrollan su actividad profesional son los de interpretación (54,76%), audiovisual (38,09%), formación (28,57%), o animación (26,19%), mientras que áreas como producción, dirección de escena, gestión, dramaturgia o diseño escénico presentan un porcentaje menor.

Interventional endoscopy is a field that continues to grow rapidly. A novel prototype forward-viewing echoendoscope (FV-EUS) has been recently developed in an attempt to overcome some of the limitations of conventional curved linear-array echoendoscopes (OV-EUS). We present a case of a successful endoscopic ultrasound-guided drainage of a pancreatic pseudocyst using a forward-viewing echoendoscope. Although the use of this newly developed echoendoscope has not yet become widespread, its unique characteristics can help to easily perform routine therapeutic procedures and contribute to the expansion of interventional endoscopic ultrasound.

The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in the APC superfamily is specific for a unique subset of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here, we report two crystal structures of an APC member from Methanococcus maripaludis , the alanine or glycine:cation symporter (AgcS), with l- or d-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. These studies provide initial insights into substrate selectivity in AgcS symporters., Competing Interests: The authors declare no conflict of interest.

The flavin mononucleotide (FMN) riboswitch is an emerging target for the development of novel RNA-targeting antibiotics. We previously discovered an FMN derivative, 5FDQD, that protects mice against diarrhea-causing Clostridium difficile bacteria. Here, we present the structure-based drug design strategy that led to the discovery of this fluoro-phenyl derivative with antibacterial properties. This approach involved the following stages: (1) structural analysis of all available free and bound FMN riboswitch structures; (2) design, synthesis, and purification of derivatives; (3) in vitro testing for productive binding using two chemical probing methods; (4) in vitro transcription termination assays; and (5) resolution of the crystal structures of the FMN riboswitch in complex with the most mature candidates. In the process, we delineated principles for productive binding to this riboswitch, thereby demonstrating the effectiveness of a coordinated structure-guided approach to designing drugs against RNA.

Spider venom toxins, such as Protoxin-II (ProTx-II), have recently received much attention as selective Na v 1.7 channel blockers, with potential to be developed as leads for the treatment of chronic nocioceptive pain. ProTx-II is a 30-amino acid peptide with three disulfide bonds that has been reported to adopt a well-defined inhibitory cystine knot (ICK) scaffold structure. Potential drawbacks with such peptides include poor pharmacodynamics and potential scrambling of the disulfide bonds in vivo. In order to address these issues, in the present study we report the solid-phase synthesis of lanthionine-bridged analogues of ProTx-II, in which one of the three disulfide bridges is replaced with a thioether linkage, and evaluate the biological properties of these analogues. We have also investigated the folding and disulfide bridging patterns arising from different methods of oxidation of the linear peptide precursor. Finally, we report the X-ray crystal structure of ProTx-II to atomic resolution; to our knowledge this is the first crystal structure of an ICK spider venom peptide not bound to a substrate.

Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.

hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method MicroED, we discover that one segment, 19-29 S20G, forms pairs of β-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils. In contrast, a second segment, 15-25 WT, forms non-toxic labile β-sheets. These segments possess different structures and cytotoxic effects, however, both can seed full-length hIAPP, and cause hIAPP to take on the cytotoxic and structural features of that segment. These results suggest that protein segment structures represent polymorphs of their parent protein and that segment 19-29 S20G may serve as a model for the toxic spine of hIAPP., Competing Interests: The authors declare that no competing interests exist.

The electron cryo-microscopy (cryoEM) method MicroED has been rapidly developing. In this review we highlight some of the key steps in MicroED from crystal analysis to structure determination. We compare and contrast MicroED and the latest X-ray based diffraction method the X-ray free-electron laser (XFEL). Strengths and shortcomings of both MicroED and XFEL are discussed. Finally, all current MicroED structures are tabulated with a view to the future., (© 2016 The Protein Society.)

Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined. We show with four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods., Competing Interests: The authors declare no conflict of interest.

Molecular mechanisms controlling functional bacterial chromosome (nucleoid) compaction and organization are surprisingly enigmatic but partly depend on conserved, histone-like proteins HUαα and HUαβ and their interactions that span the nanoscale and mesoscale from protein-DNA complexes to the bacterial chromosome and nucleoid structure. We determined the crystal structures of these chromosome-associated proteins in complex with native duplex DNA. Distinct DNA binding modes of HUαα and HUαβ elucidate fundamental features of bacterial chromosome packing that regulate gene transcription. By combining crystal structures with solution x-ray scattering results, we determined architectures of HU-DNA nucleoproteins in solution under near-physiological conditions. These macromolecular conformations and interactions result in contraction at the cellular level based on in vivo imaging of native unlabeled nucleoid by soft x-ray tomography upon HUβ and ectopic HUα38 expression. Structural characterization of charge-altered HUαα-DNA complexes reveals an HU molecular switch that is suitable for condensing nucleoid and reprogramming noninvasive Escherichia coli into an invasive form. Collective findings suggest that shifts between networking and cooperative and noncooperative DNA-dependent HU multimerization control DNA compaction and supercoiling independently of cellular topoisomerase activity. By integrating x-ray crystal structures, x-ray scattering, mutational tests, and x-ray imaging that span from protein-DNA complexes to the bacterial chromosome and nucleoid structure, we show that defined dynamic HU interaction networks can promote nucleoid reorganization and transcriptional regulation as efficient general microbial mechanisms to help synchronize genetic responses to cell cycle, changing environments, and pathogenesis.

The weak pixel counts surrounding the Bragg spots in a diffraction image are important for establishing a model of the background underneath the peak and estimating the reliability of the integrated intensities. Under certain circumstances, particularly with equipment not optimized for low-intensity measurements, these pixel values may be corrupted by corrections applied to the raw image. This can lead to truncation of low pixel counts, resulting in anomalies in the integrated Bragg intensities, such as systematically higher signal-to-noise ratios. A correction for this effect can be approximated by a three-parameter lognormal distribution fitted to the weakly positive-valued pixels at similar scattering angles. The procedure is validated by the improved refinement of an atomic model against structure factor amplitudes derived from corrected micro-electron diffraction (MicroED) images.

The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.

The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy. As the crystals are thousands of times too small for structure determination by synchrotron X-ray diffraction, we use micro-electron diffraction to determine the structure at atomic resolution. The 1.4 Å resolution structure demonstrates that this method can determine previously unknown protein structures and here yields, to our knowledge, the highest resolution achieved by any cryo-electron microscopy method to date. The structure exhibits protofibrils built of pairs of face-to-face β-sheets. X-ray fibre diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. The NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, presenting opportunities for the design of inhibitors of α-synuclein fibrils.

MicroED, a method at the intersection of X-ray crystallography and electron cryo-microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to determine the atomic structures of several proteins from sub-micron-sized, three-dimensional crystals. A major limiting factor in X-ray crystallography is the requirement for large and well ordered crystals. By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult-to-crystallize samples. This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high-resolution, three-dimensional models by MicroED, and presents some of its unique challenges.

MicroED is a recently developed method that uses electron diffraction for structure determination from very small three-dimensional crystals of biological material. Previously we used a series of still diffraction patterns to determine the structure of lysozyme at 2.9 Å resolution with MicroED (Shi et al., 2013). Here we present the structure of bovine liver catalase determined from a single crystal at 3.2 Å resolution by MicroED. The data were collected by continuous rotation of the sample under constant exposure and were processed and refined using standard programs for X-ray crystallography. The ability of MicroED to determine the structure of bovine liver catalase, a protein that has long resisted atomic analysis by traditional electron crystallography, demonstrates the potential of this method for structure determination.

In bacteria, sulfur metabolism is regulated in part by seven known families of riboswitches that bind S-adenosyl-l-methionine (SAM). Direct binding of SAM to these mRNA regulatory elements governs a downstream secondary structural switch that communicates with the transcriptional and/or translational expression machinery. The most widely distributed SAM-binding riboswitches belong to the SAM clan, comprising three families that share a common SAM-binding core but differ radically in their peripheral architecture. Although the structure of the SAM-I member of this clan has been extensively studied, how the alternative peripheral architecture of the other families supports the common SAM-binding core remains unknown. We have therefore solved the X-ray structure of a member of the SAM-I/IV family containing the alternative "PK-2" subdomain shared with the SAM-IV family. This structure reveals that this subdomain forms extensive interactions with the helix housing the SAM-binding pocket, including a highly unusual mode of helix packing in which two helices pack in a perpendicular fashion. Biochemical and genetic analysis of this RNA reveals that SAM binding induces many of these interactions, including stabilization of a pseudoknot that is part of the regulatory switch. Despite strong structural similarity between the cores of SAM-I and SAM-I/IV members, a phylogenetic analysis of sequences does not indicate that they derive from a common ancestor.

Understanding the biological activities of riboswitches and of RNA in general requires a thorough analysis of both the spatial arrangement of the residues and the dynamics of the structural ensemble. Specifically, evaluating the structural basis for riboswitch function requires analyses of many relevant states that include ligand-bound and -free, high Mg(2+), and quite possibly, the active transcription state, which is challenging to achieve by most methods. Small angle X-ray scattering (SAXS) is an enabling technique for comprehensive analyses of RNA structures in solution. Here, we describe recent SAXS tools and technologies that substantially improve the potential for accurate and comprehensive analyses of flexibility, unstructured elements, conformational selection, and induced fit in RNA function. We note equipment needed plus appropriate annealing and purification procedures. We describe key model-independent parameters (SAXS invariants) which can be used to monitor changes in a particle's thermodynamic state: the Guinier-based Rg, the volume-of-correlation (Vc), the Porod-Debye exponent (P(E)), and the power-law parameter, Q(R), that determines mass directly from the SAXS data. We also consider the value of real-space parameters and of multiphase modeling with MONSA to locate secondary structure elements within SAXS volumetric envelopes. For conformation changes, experiments with nanogold-labeled RNA analyzed using the SAXS structural comparison map and volatility ratio difference metric enable high-throughput evaluation of solution-state conformations. Collectively, the described tools and procedures enable quantitative and comprehensive measures of riboswitch structures with general implications for our views and strategies of RNA structural analysis.

Objective: To establish whether human fallopian tube (FT) epithelium can induce apoptosis in T lymphocytes and endometrial cells., Design: Laboratory-based study., Setting: Hospital., Patient(s): Women undergoing abdominal hysterectomy for FT samples, and women volunteers with and without endometriosis for endometrial biopsies., Intervention(s): FT samples obtained at time of surgery performed in reproductive-aged women with normal menstrual cycles., Main Outcome Measure(s): T lymphocytes or endometrial cells coincubated with FT epithelial cells and assayed for apoptosis by DNA nick-end labeling and caspase-3 activity, with the presence of Fas ligand (FasL) and Fas receptor (FasR) assessed by indirect immunostaining., Result(s): The epithelium of the FT-induced apoptosis in T cells as well as in human endometrial cells. The mechanism probably involves the FasL/FasR system; accordingly, we observed FasL at the apical surface of the epithelium and in the stroma of the FT at all phases of the menstrual cycle except during the early proliferative phase. The endometrial samples from patients with endometriosis did not express FasR and were resistant to apoptosis., Conclusion(s): In both FasR(+) T lymphocytes and endometrial cells, FasL(+) FT cells induce apoptosis. Data suggest that the FT epithelium acts as a barrier to limit the influx of lymphocytes as well as endometrial cells ascending the tube. Failure of these regulatory mechanisms may be related to the development of endometriosis., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Structures of riboswitch receptor domains bound to their effector have shown how messenger RNAs recognize diverse small molecules, but mechanistic details linking the structures to the regulation of gene expression remain elusive. To address this, here we solve crystal structures of two different classes of cobalamin (vitamin B(12))-binding riboswitches that include the structural switch of the downstream regulatory domain. These classes share a common cobalamin-binding core, but use distinct peripheral extensions to recognize different B(12) derivatives. In each case, recognition is accomplished through shape complementarity between the RNA and cobalamin, with relatively few hydrogen bonding interactions that typically govern RNA-small molecule recognition. We show that a composite cobalamin-RNA scaffold stabilizes an unusual long-range intramolecular kissing-loop interaction that controls mRNA expression. This is the first, to our knowledge, riboswitch crystal structure detailing how the receptor and regulatory domains communicate in a ligand-dependent fashion to regulate mRNA expression.

Transport and biosynthesis of folate and its derivatives are frequently controlled by the tetrahydrofolate (THF) riboswitch in Firmicutes. We have solved the crystal structure of the THF riboswitch aptamer in complex with folinic acid, a THF analog. Uniquely, this structure reveals two molecules of folinic acid binding to a single structured domain. These two sites interact with ligand in a similar fashion, primarily through recognition of the reduced pterin moiety. 7-deazaguanine, a soluble analog of guanine, binds the riboswitch with nearly the same affinity as its natural effector. However, 7-deazaguanine effects transcriptional termination to a substantially lesser degree than folinic acid, suggesting that the cellular guanine pool does not act upon the THF riboswitch. Under physiological conditions the ligands display strong cooperative binding, with one of the two sites playing a greater role in eliciting the regulatory response, which suggests that the second site may play another functional role., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The increasing number of RNA crystal structures enables a structure-based approach to the discovery of new RNA-binding ligands. To develop the poorly explored area of RNA-ligand docking, we have conducted a virtual screening exercise for a purine riboswitch to probe the strengths and weaknesses of RNA-ligand docking. Using a standard protein-ligand docking program with only minor modifications, four new ligands with binding affinities in the micromolar range were identified, including two compounds based on molecular scaffolds not resembling known ligands. RNA-ligand docking performed comparably to protein-ligand docking indicating that this approach is a promising option to explore the wealth of RNA structures for structure-based ligand design., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

S-adenosyl-(L)-homocysteine (SAH) riboswitches are regulatory elements found in bacterial mRNAs that up-regulate genes involved in the S-adenosyl-(L)-methionine (SAM) regeneration cycle. To understand the structural basis of SAH-dependent regulation by RNA, we have solved the structure of its metabolite-binding domain in complex with SAH. This structure reveals an unusual pseudoknot topology that creates a shallow groove on the surface of the RNA that binds SAH primarily through interactions with the adenine ring and methionine main chain atoms and discriminates against SAM through a steric mechanism. Chemical probing and calorimetric analysis indicate that the unliganded RNA can access bound-like conformations that are significantly stabilized by SAH to direct folding of the downstream regulatory switch. Strikingly, we find that metabolites bearing an adenine ring, including ATP, bind this aptamer with sufficiently high affinity such that normal intracellular concentrations of these compounds may influence regulation of the riboswitch.

Telomeres must be capped to preserve chromosomal stability. The conserved Stn1 and Ten1 proteins are required for proper capping of the telomere, although the mechanistic details of how they contribute to telomere maintenance are unclear. Here, we report the crystal structures of the C-terminal domain of the Saccharomyces cerevisiae Stn1 and the Schizosaccharomyces pombe Ten1 proteins. These structures reveal striking similarities to corresponding subunits in the replication protein A complex, further supporting an evolutionary link between telomere maintenance proteins and DNA repair complexes. Our structural and in vivo data of Stn1 identify a new domain that has evolved to support a telomere-specific role in chromosome maintenance. These findings endorse a model of an evolutionarily conserved mechanism of DNA maintenance that has developed as a result of increased chromosomal structural complexity.

Purine riboswitches discriminate between guanine and adenine by at least 10,000-fold based on the identity of a single pyrimidine (Y74) that forms a Watson-Crick base pair with the ligand. To understand how this high degree of specificity for closely related compounds is achieved through simple pairing, we investigated their interaction with purine analogs with varying functional groups at the 2- and 6-positions that have the potential to alter interactions with Y74. Using a combination of crystallographic and calorimetric approaches, we find that binding these purines is often facilitated by either small structural changes in the RNA or tautomeric changes in the ligand. This work also reveals that, along with base pairing, conformational restriction of Y74 significantly contributes to nucleobase selectivity. These results reveal that compounds that exploit the inherent local flexibility within riboswitch binding pockets can alter their ligand specificity.

In archaea and eukarya, box C/D ribonucleoprotein (RNP) complexes are responsible for 2'-O-methylation of tRNAs and rRNAs. The archaeal box C/D small RNP complex requires a small RNA component (sRNA) possessing Watson-Crick complementarity to the target RNA along with three proteins: L7Ae, Nop5p, and fibrillarin. Transfer of a methyl group from S-adenosylmethionine to the target RNA is performed by fibrillarin, which by itself has no affinity for the sRNA-target duplex. Instead, it is targeted to the site of methylation through association with Nop5p, which in turn binds to the L7Ae-sRNA complex. To understand how Nop5p serves as a bridge between the targeting and catalytic functions of the box C/D small RNP complex, we have employed alanine scanning to evaluate the interaction between the Pyrococcus horikoshii Nop5p domain and an L7Ae box C/D RNA complex. From these data, we were able to construct an isolated RNA-binding domain (Nop-RBD) that folds correctly as demonstrated by x-ray crystallography and binds to the L7Ae box C/D RNA complex with near wild type affinity. These data demonstrate that the Nop-RBD is an autonomously folding and functional module important for protein assembly in a number of complexes centered on the L7Ae-kinkturn RNP.

A number of RNAs ranging from small helices to large megadalton ribonucleoprotein complexes have been solved to atomic resolution using X-ray crystallography. As with proteins, RNA crystallography involves a number of screening trials in which the concentration of macromolecule, precipitant, salt, and temperature are varied, an approach known as searching "condition space." In contrast to proteins, the nature of base pairing in nucleic acids creates predictable secondary structure that facilitates the rational design of RNA variants, allowing "sequence space" to be screened in parallel. This chapter reviews RNA-specific techniques and considerations for RNA crystallography and presents a complete workflow used by our laboratory for solving RNA structures starting with initial library construction, methods to investigate and improve RNA crystal quality, and finally phase determination and structure solution., (Copyright © 2009 Elsevier Inc. All rights reserved.)