Genome organization around nuclear speckles drives mRNA splicing efficiency.

The nucleus is highly organized, such that factors involved in the transcription and processing of distinct classes of RNA are confined within specific nuclear bodies1,2. One example is the nuclear speckle, which is defined by high concentrations of protein and noncoding RNA regulators of pre-mRNA splicing3. What functional role, if any, speckles might play in the process of mRNA splicing is unclear4,5. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs and higher co-transcriptional splicing levels than genes that are located farther from nuclear speckles. Gene organization around nuclear speckles is dynamic between cell types, and changes in speckle proximity lead to differences in splicing efficiency. Finally, directed recruitment of a pre-mRNA to nuclear speckles is sufficient to increase mRNA splicing levels. Together, our results integrate the long-standing observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a crucial role for dynamic three-dimensional spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.

ΔNp63 regulates MDSC survival and metabolism in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) contributes greatly to mortality of breast cancer, demanding new targetable options. We have shown that TNBC patients have high ΔNp63 expression in tumors. However, the function of ΔNp63 in established TNBC is yet to be explored. In current studies, targeting ΔNp63 with inducible CRISPR knockout and Histone deacetylase inhibitor Quisinostat showed that ΔNp63 is important for tumor progression and metastasis in established tumors by promoting myeloid-derived suppressor cell (MDSC) survival through tumor necrosis factor alpha. Decreasing ΔNp63 levels are associated with decreased CD4+ and FOXP3+ T-cells but increased CD8+ T-cells. RNA sequencing analysis indicates that loss of ΔNp63 alters multiple MDSC properties such as lipid metabolism, chemotaxis, migration, and neutrophil degranulation besides survival. We further demonstrated that targeting ΔNp63 sensitizes chemotherapy. Overall, we showed that ΔNp63 reprograms the MDSC-mediated immunosuppressive functions in TNBC, highlighting the benefit of targeting ΔNp63 in chemotherapy-resistant TNBC.

Denaturing purifications demonstrate that PRC2 and other widely reported chromatin proteins do not appear to bind directly to RNA in vivo.

Polycomb repressive complex 2 (PRC2) is reported to bind to many RNAs and has become a central player in reports of how long non-coding RNAs (lncRNAs) regulate gene expression. Yet, there is a growing discrepancy between the biochemical evidence supporting specific lncRNA-PRC2 interactions and functional evidence demonstrating that PRC2 is often dispensable for lncRNA function. Here, we revisit the evidence supporting RNA binding by PRC2 and show that many reported interactions may not occur in vivo. Using denaturing purification of in vivo crosslinked RNA-protein complexes in human and mouse cell lines, we observe a loss of detectable RNA binding to PRC2 and chromatin-associated proteins previously reported to bind RNA (CTCF, YY1, and others), despite accurately mapping bona fide RNA-binding sites across others (SPEN, TET2, and others). Taken together, these results argue for a critical re-evaluation of the broad role of RNA binding to orchestrate various chromatin regulatory mechanisms.

Venopulmonary Artery Extracorporeal Life Support (VPa ECMO): A Novel Strategy for Refractory Hypoxemia Complicating VV ECMO.

Refractory hypoxemia (RH) during venovenous extracorporeal membrane oxygenation (VV ECMO) support is a complex problem that limits the benefit of this therapy. The need for sustained deep sedation and delays in active rehabilitation are considered as a direct consequence of RH. Changing from VV ECMO to a configuration that returns the flow to pulmonary artery, such as venopulmonary extracorporeal membrane oxygenation (VPa ECMO) may decrease recirculation and improve systemic oxygen delivery. We present a retrospective report that describes the impact of VPa ECMO on oxygenation during sedation withdrawal in 41 patients who received VV ECMO for coronavirus disease 2019 (COVID-19). We evidenced that arterial oxygen pressure (PaO 2 ) increased from 68 to 112.3 mm Hg ( p = 0.001) with a reduction of ECMO flow (5.7-4.8 L/m; p = 0.001). Other findings included lower rates of depth sedation (Richmond Agitation Sedation Scale [RASS] ≤3, 37-63%; p = 0.007) and lower requirement inotropic support assessed by LVIS score (4.7-1.1; p = 0.005). Discharge survival was 54% with a sustained benefit until day 79. This cannulation strategy improved effectively PaO 2 in this cohort, it may be an alternative in patients with RH in VV ECMO.

INVITED REVIEW: Ketone biology: the shifting paradigm of ketones and ketosis in the dairy cow.

Ketosis is currently regarded as a major metabolic disorder of dairy cows, reflective of the animal's efforts to adapt to energy deficit while transitioning into lactation. Currently viewed as a pathology by some, ketosis is associatively implicated in milk production losses and peripartal health complications that increase the risk of early removal of cows from the herd, thus carrying economic losses for dairy farmers, and jeopardizing the sustainability of the dairy industry. Despite decades of intense research in the mitigation of ketosis and its sequelae, our ability to lessen its purported impacts remains limited. Moreover, the association of ketosis to reduced milk production and peripartal disease is often erratic and likely mired by concurrent potential confounders. In this review we discuss the potential reasons for these apparent paradoxes in the light of currently available evidence, with a focus on the limitations of observational research, and the necessary steps to unambiguously identify the impacts of ketosis on cow health and performance via controlled randomized experimentation. A nuanced perspective is proposed that considers the dissociation of ketosis -as a disease- from healthy hyperketonemia. Furthermore, in consideration of a growing body of evidence that highlights positives roles of ketones in the mitigation of metabolic dysfunction and chronic diseases, we consider the hypothetical functions of ketones as health-promoting metabolites and ponder on their potential usefulness to enhance dairy cow health and productivity.

SPIDR: a highly multiplexed method for mapping RNA-protein interactions uncovers a potential mechanism for selective translational suppression upon cellular stress.

RNA binding proteins (RBPs) play crucial roles in regulating every stage of the mRNA life cycle and mediating non-coding RNA functions. Despite their importance, the specific roles of most RBPs remain unexplored because we do not know what specific RNAs most RBPs bind. Current methods, such as crosslinking and immunoprecipitation followed by sequencing (CLIP-seq), have expanded our knowledge of RBP-RNA interactions but are generally limited by their ability to map only one RBP at a time. To address this limitation, we developed SPIDR (Split and Pool Identification of RBP targets), a massively multiplexed method to simultaneously profile global RNA binding sites of dozens to hundreds of RBPs in a single experiment. SPIDR employs split-pool barcoding coupled with antibody-bead barcoding to increase the throughput of current CLIP methods by two orders of magnitude. SPIDR reliably identifies precise, single-nucleotide RNA binding sites for diverse classes of RBPs simultaneously. Using SPIDR, we explored changes in RBP binding upon mTOR inhibition and identified that 4EBP1 acts as a dynamic RBP that selectively binds to 5'-untranslated regions of specific translationally repressed mRNAs only upon mTOR inhibition. This observation provides a potential mechanism to explain the specificity of translational regulation controlled by mTOR signaling. SPIDR has the potential to revolutionize our understanding of RNA biology and both transcriptional and post-transcriptional gene regulation by enabling rapid, de novo discovery of RNA-protein interactions at an unprecedented scale.

Treatment of neovascular age-related macular degeneration within 48 h from diagnosis improves long-term functional outcome.

PURPOSE: To evaluate long-term visual and anatomical outcomes in neovascular age-related macular degeneration (nAMD) patients treated with anti-vascular endothelial growth factor (VEGF) agents depending on the time delay from confirmed diagnosis to treatment initiation. MATERIALS AND METHODS: Seventy-three nAMD patients (73 eyes) treated with anti-VEGF agents for 12 months using the pro re nata regimen were included in this retrospective longitudinal study. Patients were split into 3 groups according to the time from diagnosis to first anti-VEGF injection: < 48 h (group 1); 48 h-7 days (group 2); > 7 days (group 3). Decimal best-corrected visual acuity (VA) and macular thickness (MT) were recorded at baseline and 1-2-, 3-4-, 6- and 12-month later. Furthermore, age, gender as well as the applied treatment and number of injections after 12 months of treatment were also registered and compared. RESULTS: Long-term effect of the treatment demonstrated enhanced VA in group 1 patients compared with the rest of groups after 1-2-, 6-, and 12-month follow-up (P < 0.05). Positive effects of early treatment were additionally corroborated by the augmented percentage of patients with normal VA in the group 1 respect to the rest of groups over studied time points (P < 0.05). Moreover, the VA gain in nAMD at group 1 was obtained with a mean of 3.7 intravitreal injections over 1-year follow-up period. Regarding MT, non-significant difference was observed among groups. CONCLUSIONS: An early initial treatment with VEGF inhibitors is critical to achieve the best functional benefits of this therapy in new-onset nAMD patients.

3D genome organization around nuclear speckles drives mRNA splicing efficiency.

The nucleus is highly organized such that factors involved in transcription and processing of distinct classes of RNA are organized within specific nuclear bodies. One such nuclear body is the nuclear speckle, which is defined by high concentrations of protein and non-coding RNA regulators of pre-mRNA splicing. What functional role, if any, speckles might play in the process of mRNA splicing remains unknown. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs, and higher co-transcriptional splicing levels relative to genes that are located farther from nuclear speckles. We show that directed recruitment of a pre-mRNA to nuclear speckles is sufficient to drive increased mRNA splicing levels. Finally, we show that gene organization around nuclear speckles is highly dynamic with differential localization between cell types corresponding to differences in Pol II occupancy. Together, our results integrate the longstanding observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a critical role for dynamic 3D spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.

ChIP-DIP: A multiplexed method for mapping hundreds of proteins to DNA uncovers diverse regulatory elements controlling gene expression.

Gene expression is controlled by the dynamic localization of thousands of distinct regulatory proteins to precise regions of DNA. Understanding this cell-type specific process has been a goal of molecular biology for decades yet remains challenging because most current DNA-protein mapping methods study one protein at a time. To overcome this, we developed ChIP-DIP (ChIP Done In Parallel), a split-pool based method that enables simultaneous, genome-wide mapping of hundreds of diverse regulatory proteins in a single experiment. We demonstrate that ChIP-DIP generates highly accurate maps for all classes of DNA-associated proteins, including histone modifications, chromatin regulators, transcription factors, and RNA Polymerases. Using these data, we explore quantitative combinations of protein localization on genomic DNA to define distinct classes of regulatory elements and their functional activity. Our data demonstrate that ChIP-DIP enables the generation of 'consortium level', context-specific protein localization maps within any molecular biology lab.

Novel Treatments for Age-Related Macular Degeneration: A Review of Clinical Advances in Sustained Drug Delivery Systems.

In recent years, the number of patients with ocular diseases is increasing as a consequence of population aging. Among them, one of the most common is the age-related macular degeneration (AMD), a condition that leads to vision loss if it is not treated. AMD is a multifactorial disorder with two advanced forms, dry and neovascular AMD. Currently, although there is no approved therapy that significantly impacts dry AMD progression, several pharmacologic therapies exist for neovascular AMD. Notwithstanding, evidence suggests a suboptimal result in a high number of patients receiving these therapeutic options. Consequently, finding effective strategies is not only a still unmet medical need in dry AMD but also in neovascular AMD. This underlines the need for new drug delivery technologies that can improve the pharmacological action and drug concentration at the target sites. In this regard, sustained drug delivery systems are presented as the most promising therapeutic options in AMD patients. This review summarized the pathogenesis and the current treatment options for AMD, focusing on the emerging ocular sustained drug delivery approaches undergoing clinical trials.

Xist spatially amplifies SHARP/SPEN recruitment to balance chromosome-wide silencing and specificity to the X chromosome.

Although thousands of long non-coding RNAs (lncRNAs) are encoded in mammalian genomes, their mechanisms of action are poorly understood, in part because they are often expressed at lower levels than their proposed targets. One such lncRNA is Xist, which mediates chromosome-wide gene silencing on one of the two X chromosomes (X) to achieve gene expression balance between males and females. How a limited number of Xist molecules can mediate robust silencing of a much larger number of target genes while maintaining specificity exclusively to genes on the X within each cell is not well understood. Here, we show that Xist drives non-stoichiometric recruitment of the essential silencing protein SHARP (also known as SPEN) to amplify its abundance across the inactive X, including at regions not directly occupied by Xist. This amplification is achieved through concentration-dependent homotypic assemblies of SHARP on the X and is required for chromosome-wide silencing. Expression of Xist at higher levels leads to increased localization at autosomal regions, demonstrating that low levels of Xist are critical for ensuring its specificity to the X. We show that Xist (through SHARP) acts to suppress production of its own RNA which may act to constrain overall RNA levels and restrict its ability to spread beyond the X. Together, our results demonstrate a spatial amplification mechanism that allows Xist to achieve two essential but countervailing regulatory objectives: chromosome-wide gene silencing and specificity to the X. This suggests a more general mechanism by which other low-abundance lncRNAs could balance specificity to, and robust control of, their regulatory targets.

Manejo de fracturas abiertas de tibia en Cuba / Management of Open Tibial Fractures in Cuba

Introducción: Las fracturas abiertas de tibia son un subconjunto de la carga de traumatismos en América Latina. Se examinaron cuestiones relacionadas con el tratamiento potencialmente críticas en Cuba, país con recursos limitados, pero con un programa nacional de salud estandarizado, coherencia en educación y similitudes de programas de posgrado. Objetivos: Describir los patrones de tratamiento de la fractura abierta de tibia en Cuba, y comparar las características del manejo agudo y tardío en siete provincias del país. Métodos: Se encuestaron 67 cirujanos ortopédicos para evaluar cuatro aspectos en el tratamiento de la fractura abierta: profilaxis antibiótica, irrigación y desbridamiento, estabilización y tratamiento de heridas. Se utilizó el método de muestreo por conveniencia para identificar a los cirujanos y el análisis se realizó mediante la prueba exacta de Fisher (p < 0,05). Resultados: Se administraron antibióticos posoperatorios durante más de 72 horas para las fracturas GA-I/II (49 por ciento) y las fracturas GA-III (70 por ciento). Los cirujanos de La Habana (n= 32) utilizaron con más frecuencia la fijación interna primaria para las fracturas GA-I/II, que los cirujanos en las restantes provincias (n= 35) (64,3 porciento vs. 30,3 por ciento, p= 0,008). Los cirujanos de otras provincias realizaron cierre primario en el momento de la fijación definitiva de fracturas GA-I /II con más frecuencia que los de La Habana (62,9 por ciento vs. 32,3 por ciento, p= 0,013). Para fracturas GA-III, la mayoría de los cirujanos habaneros (88,6 %), al igual que los de las restantes provincias (96,8 por ciento) prefirieron realizar cierre diferido.Conclusiones: El tratamiento de fracturas abiertas de tibia en Cuba es generalmente consistente con otros países de América Latina. Se describen las características del manejo de fracturas abiertas de tibia en Cuba y se comparan las diferencias en los métodos de estabilización y tratamiento de heridas entre provincias, lo cual resulta útil para evaluar si son resultado de diferencias en la práctica quirúrgica, o en la disponibilidad de recursos. Esto representa una ayuda al abordar las formas de optimizar la atención al paciente, a través de la capacitación especializada y la asignación de los recursos(AU)

Introduction: Open tibia fractures are a significant subset of the overall trauma burden in Latin America. Latin American countries vary in their access to orthopaedic care resources, and country-specific orthopaedic recommendations are necessary. Cuba, a country with limited resources, has a standardized national health program, consistencies in education, and similarities across post-graduate training programs. This study aimed to identify management preferences for open tibia factures in Cuba. Objectives: To describe the treatment of open tibial fractures in Cuba, and to compare the characteristics of acute and delayed management across seven Cuban provinces. Methods: Sixty-seven orthopaedic surgeons were surveyed to evaluate four aspects of open fracture management, regarding antibiotic prophylaxis, irrigation and debridement, stabilization, and wound management. The convenience sampling method was used to identify surgeons and the analysis was performed using Fisher's exact test (p <0.05). Results: Postoperative antibiotics were administered for more than 72 hours for GA-I / II fractures (49 pecent) and GA-III fractures (70 percent). Surgeons in Havana (n = 32) used primary internal fixation for GA-I / II fractures more frequently than surgeons in the remaining provinces (n = 35) (64.3 pecent vs. 30.3 percent p = 0.008). Surgeons from other provinces performed primary closure at the time of definitive fixation of GA-I / II fractures more frequently than those from Havana (62.9 percent vs. 32.3 percent, p = 0.013). For GA-III fractures, the majority of Havana surgeons (88.6 percent), as well as those of the remaining provinces (96.8 percent) preferred to perform deferred closure. Conclusions: The treatment of open tibial fractures in Cuba is generally consistent with other Latin American countries. The characteristics of the management of open tibial fractures in Cuba are described and differences in wound stabilization and treatment methods between provinces are compared, which is useful to assess whether they are the result of differences in surgical practice, or in availability of resources. This is helpful in addressing ways to optimize patient care through specialized training and resource allocation(AU)

RNA promotes the formation of spatial compartments in the nucleus.

RNA, DNA, and protein molecules are highly organized within three-dimensional (3D) structures in the nucleus. Although RNA has been proposed to play a role in nuclear organization, exploring this has been challenging because existing methods cannot measure higher-order RNA and DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the spatial organization of RNA and DNA. These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions.

Fear is the Path to the Dark Side: Unsafe Delivery, One of the Consequences of Fear of the SARS-CoV-2 Pandemic, A Case Report.

Indirect effects of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic are difficult to calculate. Fear of intrahospital infection has led to a decrease in the use of emergency services and the performance of elective procedures. Several low- and middle-income countries have seen the number of institutional deliveries reduced, even in the absence of a follow-up program for home births. We present the case of a patient with adequate prenatal care and an institutional delivery plan who, due to the SARS-CoV-2 pandemic, chose to have a home delivery with unsafe conditions. The lack of supervision by health personnel and the absence of an immediate consultation plan facilitated the presentation of postpartum hemorrhage and poor neonatal results. Little attention has been paid during the pandemic to pregnant women who decide to have their birth at home. A broad discussion is necessary in this regard, to regain the confidence of the population and strengthen institutional births, or to strengthen midwife-assisted home births programs. Patients' fear to acquiring SARS-CoV-2 infection inside hospitals is a factor that must be taken into account in prenatal care programs.

Dll1+ quiescent tumor stem cells drive chemoresistance in breast cancer through NF-κB survival pathway.

Development of chemoresistance in breast cancer patients greatly increases mortality. Thus, understanding mechanisms underlying breast cancer resistance to chemotherapy is of paramount importance to overcome this clinical challenge. Although activated Notch receptors have been associated with chemoresistance in cancer, the specific Notch ligands and their molecular mechanisms leading to chemoresistance in breast cancer remain elusive. Using conditional knockout and reporter mouse models, we demonstrate that tumor cells expressing the Notch ligand Dll1 is important for tumor growth and metastasis and bear similarities to tumor-initiating cancer cells (TICs) in breast cancer. RNA-seq and ATAC-seq using reporter models and patient data demonstrated that NF-κB activation is downstream of Dll1 and is associated with a chemoresistant phenotype. Finally, pharmacological blocking of Dll1 or NF-κB pathway completely sensitizes Dll1+ tumors to chemotherapy, highlighting therapeutic avenues for chemotherapy resistant breast cancer patients in the near future.

Artrodesis transcalcánea con injerto libre del peroné por seudoartrosis de tibia distal / Transcalcaneal Arthrodesis with Free Fibula Graft for Distal Tibial Nonunion

RESUMEN Introducción: Las fracturas abiertas del tercio distal de tibia o pilón son poco frecuentes, en nuestro medio se producen por traumas de alta energía como los accidentes de tránsito, y pueden ser de distintos grados según su envergadura. Entre las complicaciones frecuentes están la seudoartrosis, deformidades y artritis postraumática. Cuando el dolor es refractario a los analgésicos están indicadas las artrodesis. Objetivo: Presentar los resultados del tratamiento realizado en un paciente con seudoartrosis distal de tibia y artritis postraumática del tobillo, dolorosa, con gran lesión de partes blandas, por lo que fue imposible realizar los procedimientos quirúrgicos habituales. Presentación del caso: Se realizó artrodesis de las articulaciones tibio-peronea-astragalina-calcánea, mediante un injerto libre del peroné autólogo, compresión, y estabilización con un fijador externo RALCA®; se asoció un campo electromagnético pulsátil para acelerar la formación del callo óseo y disminuir el dolor posquirúrgico. Durante dos años se le hizo seguimiento. Conclusiones: Se logró el objetivo del tratamiento al fusionar la articulación tibiotarsiana, comenzar el apoyo precoz y su capacidad funcional. Los resultados demuestran además los beneficios de la compresión realizada con los fijadores externos en las artrodesis; el uso del campo electromagnético asociado aceleró la osteogénesis, se consiguió la consolidación ósea, la estabilización, disminuyó el edema y el dolor, además la reincorporación del paciente a la sociedad. No se encontró en la bibliografía revisada otra técnica quirúrgica similar(AU)

ABSTRACT Introduction: Open fractures of the distal third of the tibia or pilon are rare, in our environment they are caused by high-energy traumas such as traffic accidents, and can be of different degrees depending on their size. Common complications include nonunion, deformities, and post-traumatic arthritis. When pain is refractory to analgesics, arthrodesis is indicated. Objective: To report the results of the treatment carried out in a patient with distal tibial pseudoarthrosis and post-traumatic arthritis of the ankle, painful, with a large soft tissue injury, which made it impossible to perform the usual surgical procedures. Case report: Arthrodesis of the tibiofibular-talar-calcaneal joints was performed, using a free graft of the autologous fibula, compression, and stabilization with a RALCA® external fixator. A pulsatile electromagnetic field was associated to accelerate bone callus formation and reduce postoperative pain. This patient was followed up for two years. Conclusions: The treatment objective was achieved by fusing the tibiotarsal joint, by starting early support and functional capacity. The results also prove the benefits of compression performed with external fixators in arthrodesis. The use of the associated electromagnetic field accelerated osteogenesis, bone consolidation and stabilization were achieved, edema and pain decreased, as well as the patient's reincorporation into society. No other similar surgical technique was found in the reviewed literature(AU)

Guía práctica para la confección del expediente de Especialista de Segundo grado / Practical Guide for Preparing the File for Second Degree Specialist

El especialista de Segundo grado es un profesional de las ciencias de la salud que demuestra un dominio de excelencia en su especialidad con alto nivel de conocimientos y competencia profesional lo que debe quedar demostrado ante un tribunal de especialización de segundo grado. En este sentido, por Acuerdo del Consejo de Estado de fecha 29 de junio de 2009 se emite la resolución 132, que aprobó el Reglamento para la obtención del Segundo grado en las ciencias de la salud, y que dispone en cuatro capítulos los siguientes aspectos: los requisitos y formalidades para la obtención del título, la tramitación de las solicitudes, la constitución y funcionamiento de los tribunales y las reclamaciones ante el viceministro para la Docencia y la Investigación en el caso de desacuerdo del aspirante con el dictamen del tribunal evaluado....