Dynamic regulation of CTCF stability and sub-nuclear localization in response to stress.

The nuclear protein CCCTC-binding factor (CTCF) has diverse roles in chromatin architecture and gene regulation. Functionally, CTCF associates with thousands of genomic sites and interacts with proteins, such as cohesin, or non-coding RNAs to facilitate specific transcriptional programming. In this study, we examined CTCF during the cellular stress response in human primary cells using immune-blotting, quantitative real time-PCR, chromatin immunoprecipitation-sequence (ChIP-seq) analysis, mass spectrometry, RNA immunoprecipitation-sequence analysis (RIP-seq), and Airyscan confocal microscopy. Unexpectedly, we found that CTCF is exquisitely sensitive to diverse forms of stress in normal patient-derived human mammary epithelial cells (HMECs). In HMECs, a subset of CTCF protein forms complexes that localize to Serine/arginine-rich splicing factor (SC-35)-containing nuclear speckles. Upon stress, this species of CTCF protein is rapidly downregulated by changes in protein stability, resulting in loss of CTCF from SC-35 nuclear speckles and changes in CTCF-RNA interactions. Our ChIP-seq analysis indicated that CTCF binding to genomic DNA is largely unchanged. Restoration of the stress-sensitive pool of CTCF protein abundance and re-localization to nuclear speckles can be achieved by inhibition of proteasome-mediated degradation. Surprisingly, we observed the same characteristics of the stress response during neuronal differentiation of human pluripotent stem cells (hPSCs). CTCF forms stress-sensitive complexes that localize to SC-35 nuclear speckles during a specific stage of neuronal commitment/development but not in differentiated neurons. We speculate that these particular CTCF complexes serve a role in RNA processing that may be intimately linked with specific genes in the vicinity of nuclear speckles, potentially to maintain cells in a certain differentiation state, that is dynamically regulated by environmental signals. The stress-regulated activity of CTCF is uncoupled in persistently stressed, epigenetically re-programmed "variant" HMECs and certain cancer cell lines. These results reveal new insights into CTCF function in cell differentiation and the stress-response with implications for oxidative damage-induced cancer initiation and neuro-degenerative diseases.

Modular nonlinear hybrid plasmonic circuit.

Photonic integrated circuits (PICs) are revolutionizing nanotechnology, with far-reaching applications in telecommunications, molecular sensing, and quantum information. PIC designs rely on mature nanofabrication processes and readily available and optimised photonic components (gratings, splitters, couplers). Hybrid plasmonic elements can enhance PIC functionality (e.g., wavelength-scale polarization rotation, nanoscale optical volumes, and enhanced nonlinearities), but most PIC-compatible designs use single plasmonic elements, with more complex circuits typically requiring ab initio designs. Here we demonstrate a modular approach to post-processes off-the-shelf silicon-on-insulator (SOI) waveguides into hybrid plasmonic integrated circuits. These consist of a plasmonic rotator and a nanofocusser, which generate the second harmonic frequency of the incoming light. We characterize each component's performance on the SOI waveguide, experimentally demonstrating intensity enhancements of more than 200 in an inferred mode area of 100 nm2, at a pump wavelength of 1320 nm. This modular approach to plasmonic circuitry makes the applications of this technology more practical.

Novel Third-Generation EGFR Tyrosine Kinase Inhibitors and Strategies to Overcome Therapeutic Resistance in Lung Cancer.

EGFR-activating mutations are observed in approximately 15% to 20% of patients with non-small cell lung cancer. Tyrosine kinase inhibitors have provided an illustrative example of the successes in targeting oncogene addiction in cancer and the role of tumor-specific adaptations conferring therapeutic resistance. The compound osimertinib is a third-generation tyrosine kinase inhibitor, which was granted full FDA approval in March 2017 based on targeting EGFR T790M resistance. The compound has received additional FDA approval as first-line therapy with improvement in progression-free survival by suppressing the activating mutation and preventing the rise of the dominant resistance clone. Drug development has been breathtaking in this space with other third-generation compounds at various stages of development: rociletinib (CO-1686), olmutinib (HM61713), nazartinib (EGF816), naquotinib (ASP8273), mavelertinib (PF-0647775), and AC0010. However, therapeutic resistance after the administration of third-generation inhibitors is complex and not fully understood, with significant intertumoral and intratumoral heterogeneity. Repeat tissue and plasma analyses on therapy have revealed insights into multiple mechanisms of resistance, including novel second site EGFR mutations, activated bypass pathways such as MET amplification, HER2 amplification, RAS mutations, BRAF mutations, PIK3CA mutations, and novel fusion events. Strategies to understand and predict patterns of mutagenesis are still in their infancy; however, technologies to understand synthetically lethal dependencies and track cancer evolution through therapy are being explored. The expansion of combinatorial therapies is a direction forward targeting minimal residual disease and bypass pathways early based on projected resistance.

Characterization of cellular states of CHO-K1 suspension cell culture through cell cycle and RNA-sequencing profiling.

Chinese hamster ovary (CHO) derived cell lines are the preferred host system for the production of therapeutic proteins. The aim of this work was to explore the regulation of suspension-adapted CHO-K1 host cell line bioprocesses, especially under a temperature gradient from 37 °C to 31 °C. We analyzed cell cycle behavior through flow cytometry of propidium iodide stained cells and high throughput transcriptome dynamics by RNA sequencing. We found a cell culture state characterized by G0/G1 synchronization, mainly during the late exponential growth phase and towards the last days of the stationary phase. We successfully identified key genes and pathways connected with the particular culture states, such as response to low temperature, modulation of the cell cycle, regulation of DNA replication and repair, apoptosis, among others. The most important gene expression changes occurred throughout the stationary phase when gene up-regulation markedly prevailed. Our RNA-seq data analysis enabled the identification of target genes for mechanism-based cell line engineering and bioprocess modification, an essential step to translate gene expression data from CHO-K1 host cells into bioprocess-related knowledge. Further efforts aim at increasing desirable phenotypes of CHO cells, and promoting efficient production of high quality therapeutic proteins can highly benefit from this type of studies.

Hematoma retroperitoeneal posterior a histerectomía vaginal con colporrafia anterior y posterior en Bogotá (Colombia): reporte de un caso y revisión de la literatura / Retroperitoneal haematoma following vaginal hysterectomy with anterior and posterior vaginal wall repair in Bogotá, Colombia: Case report and review of the literature

Objetivo: reportar el caso de una paciente con hematoma retroperitoneal posterior a cirugía de corrección de prolapso por vía vaginal, y realizar revisión de la literatura médica sobre la anatomía del espacio retroperitoneal, la etiología, el diagnóstico, manejo y pronóstico del hematoma retroperitoneal posoperatorio en ginecología.Materiales y métodos: se presenta el caso de una paciente con prolapso vaginal estadio II, a quien se le practicó histerectomía vaginal con colporrafia anterior y posterior, quien presentó como complicación un hematoma retroperitoneal. La paciente fue intervenida en el Hospital Universitario de La Samaritana, hospital público de tercer nivel de complejidad, centro de referencia de Cundinamarca, ubicado en Bogotá (Colombia). Se realizó una búsqueda de literatura médica en las bases de dados Medline vía PubMed, Jstor y Lilacs, con terminología MeSH "hysterectomy vaginal" y "retroperitoneal hematoma". La búsqueda se limitó a los idiomas inglés y español entre los años 1980 a 2015.Resultados: se encontraron 15 artículos de los cuales 3 describen casos secundarios a procedimientos ginecológicos. La revisión final se conforma de: 4 revisiones de tema, 8 reportes de caso, 2 series de casos, 1 estudio observacional descriptivo.Conclusión: el hematoma retroperitoneal es una rara entidad que requiere de un alto índice de sospecha clínica para su diagnóstico. Existen varias herramientas diagnósticas, siendo la tomografía computarizada la de mayor utilidad. El manejo debe individualizarse a cada caso. En paciente estable se puede realizar manejo conservador con éxito o intervenciones endovasculares. Sin embargo, en pacientes inestables hemodinámicamente, la laparotomía es la conducta más recomendada.

Objective: To report the case of a patient who developed a retroperitoneal haematoma following prolapse correction surgery through the vaginal approach; and to review the medical literature relating to the anatomy of the retroperitoneal space and the aetiology, diagnosis, management and prognosis of postoperative retroperitoneal haematoma in gynaecology.Materials and methods: Case presentation of a patient with Stage II vaginal prolapse, who was undergone for vaginal hysterectomy with anterior and posterior vaginal wall repair. She had a retroperitoneal haematoma complication. The patien underwent surgery at Samaritana University Hospital, a level III public hospital and referral centre for the Cundinamarca region, located in Bogotá (Colombia). A search in the literature was conducted in the Medline database through PubMed, Jstor and Lilacs using the MeSH terms "vaginal hysterectomy" and "retroperitoneal haematoma". The search was limited to publications in English and Spanish between 1980 and 2015.Results: Overall, 15 articles were found, 3 of which describe cases secondary to gynaecological procedures. The final review consisted of 4 topic reviews, 8 case reports 2 case series and 1 observational descriptive study.Conclusion: Retroperitoneal haematoma is a rare clinical finding and diagnosis requires a high level of clinical suspicion. Several diagnostic tools are available, computed tomography being the most useful. Management has to be individualized in each case. If the patient is stable, the treatments of choice include conservative management, which can be successful, or endovascular interventions. However, in haemodynamically unstable patients, laparotomy is the most recommended treatment approach.

Single-cell analyses of transcriptional heterogeneity during drug tolerance transition in cancer cells by RNA sequencing.

The acute cellular response to stress generates a subpopulation of reversibly stress-tolerant cells under conditions that are lethal to the majority of the population. Stress tolerance is attributed to heterogeneity of gene expression within the population to ensure survival of a minority. We performed whole transcriptome sequencing analyses of metastatic human breast cancer cells subjected to the chemotherapeutic agent paclitaxel at the single-cell and population levels. Here we show that specific transcriptional programs are enacted within untreated, stressed, and drug-tolerant cell groups while generating high heterogeneity between single cells within and between groups. We further demonstrate that drug-tolerant cells contain specific RNA variants residing in genes involved in microtubule organization and stabilization, as well as cell adhesion and cell surface signaling. In addition, the gene expression profile of drug-tolerant cells is similar to that of untreated cells within a few doublings. Thus, single-cell analyses reveal the dynamics of the stress response in terms of cell-specific RNA variants driving heterogeneity, the survival of a minority population through generation of specific RNA variants, and the efficient reconversion of stress-tolerant cells back to normalcy.

Coordinate transcriptional and translational repression of p53 by TGF-ß1 impairs the stress response.

Cellular stress results in profound changes in RNA and protein synthesis. How cells integrate this intrinsic, p53-centered program with extracellular signals is largely unknown. We demonstrate that TGF-ß1 signaling interferes with the stress response through coordinate transcriptional and translational repression of p53 levels, which reduces p53-activated transcription, and apoptosis in precancerous cells. Mechanistically, E2F-4 binds constitutively to the TP53 gene and induces transcription. TGF-ß1-activated Smads are recruited to a composite Smad/E2F-4 element by an E2F-4/p107 complex that switches to a Smad corepressor, which represses TP53 transcription. TGF-ß1 also causes dissociation of ribosomal protein RPL26 and elongation factor eEF1A from p53 mRNA, thereby reducing p53 mRNA association with polyribosomes and p53 translation. TGF-ß1 signaling is dominant over stress-induced transcription and translation of p53 and prevents stress-imposed downregulation of Smad proteins. Thus, crosstalk between the TGF-ß and p53 pathways defines a major node of regulation in the cellular stress response, enhancing drug resistance.

Tubulin binding, protein-bound conformation in solution, and antimitotic cellular profiling of noscapine and its derivatives.

Noscapine and its 7-hydroxy and 7-amino derivatives were characterized for their binding to tubulin. A solution NMR structure of these compounds bound to tubulin shows that noscapine and its 7-aniline derivative do not compete for the same binding site nor does its small molecule crystal structure match its tubulin-bound conformation. These compounds were also tested for their antiproliferative effects on a panel hepatocellular carcinoma cell lines.

Physical activity and transcutaneous oxygen pressure in men with spinal cord injury.

This pilot study proposed a method for assessing the status of vascular flow measured by transcutaneous oxygen pressure (TcPO2) in the area of the ischium in people with spinal cord injury (SCI). In a sample of 38 men (two groups: 12 physically active and 26 sedentary) with thoracic SCI, the distribution of the physiological response of the tissues under load during sitting was assessed through analysis of ischium TcPO2 values obtained by an oximeter. TcPO2 baseline, recovery time of TcPO2 after sitting (Trec), the percentage of TcPO2 (%TcPO2) of maximum pressure TcPO2, and mechanic maximal pressure (Pmax) were evaluated. Trec in the physically active group was significantly lower (p < 0.05) than in the sedentary group. Likewise, significant differences in %TcPO2 between groups (p < 0.05) were also found. We concluded that the physiological response of the tissues under an individual with SCI's own weight resulting from prolonged sitting is better in those who are physically active.

A new role for the Kruppel-like transcription factor KLF6 as an inhibitor of c-Jun proto-oncoprotein function.

Kruppel-like transcription factors (KLFs) represent one of the most diverse set of regulators in vertebrate organisms. KLF family members are involved in cell proliferation and differentiation control in normal as well as in pathological situations. Here, we demonstrate that KLF6 behaves as a functional antagonist of the c-Jun proto-oncoprotein. Thus, KLF6 overexpression downregulated c-Jun-dependent transcription and a physical interaction between c-Jun and KLF6 was detected. Moreover, cell proliferation induced by c-Jun was significantly decreased by KLF6. The inhibition of c-Jun functions correlates directly with c-Jun protein degradation induced by KLF6. We also show that all KLF6 effects on c-Jun were largely dependent on phorbol ester (TPA/ionomycin) extracellular stimulation, which enhanced KLF6 nuclear translocation and transcriptional activity and modified its phosphorylation status. Our data are consistent with a novel mechanism of KLF6's role as an inhibitor of cell proliferation by counteracting the function of the c-Jun proto-oncoprotein involving enhanced c-Jun degradation by the proteasome-dependent pathway, and further reinforces KLF6 as a potential tumor suppressor gene product.