An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA.

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed. To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Quality assessment of orthopedic surgery referral request letters from primary care consultation: Evaluation of a Spanish healthcare area.

BACKGROUND: One of the most requested referrals for specialist consultations from primary care (PC) is orthopedic surgery (OS). The purpose of this work was to analyze the number, characteristics, and quality of PC referral request letters in a Spanish healthcare area to their OS reference department. MATERIALS AND METHODS: The referral rates for 10 years were collected, and a total of 6,225 referral letters issued during the first half of 2019 were analyzed in detail. Gender, age, patient provenance, as well as priority and other process descriptors (type of pathology, suspected diagnosis, exploratory signs, imaging tests) were assessed. A scoring system was developed to evaluate the quality of the referral to an OS consultation: Quality evaluation in OS (QEOS) model. RESULTS: The rate of referral to OS is rising. The mean age of the referred patient was 53 years and 59.3% were women. Degenerative pathologies justified most referral requests (65.7%), most of which related to the spine (24.2%) or the knee (23.2%). In the QEOS analysis of the referral request letters, we noted that only 36.5% described some physical exploration, 32.9% image tool request, 25.8% indicated the pharmacological treatment, and 11.2% subsequent physiotherapy, resulting in a poor average quality of PC to OS patient referral. CONCLUSION: There is a growing demand for patient referral from PC to OS, however, the number, content, and quality of referral request letters varies greatly. The QEOS tool can be the germ of a simple evaluation system that would help in the improvement of the process of continuous care.

Time-Resolved Transcriptional Profiling of Epithelial Cells Infected by Intracellular Acinetobacter baumannii.

The rise in the number of antibiotic-resistant bacteria has become a serious threat to health, making it important to identify, characterize and optimize new molecules to help us to overcome the infections they cause. It is well known that Acinetobacter baumannii has a significant capacity to evade the actions of antibacterial drugs, leading to its emergence as one of the bacteria responsible for hospital and community-acquired infections. Nonetheless, how this pathogen infects and survives inside the host cell is unclear. In this study, we analyze the time-resolved transcriptional profile changes observed in human epithelial HeLa cells after infection by A. baumannii, demonstrating how it survives in host cells and starts to replicate 4 h post infection. These findings were achieved by sequencing RNA to obtain a set of Differentially Expressed Genes (DEGs) to understand how bacteria alter the host cells' environment for their own benefit. We also determine common features observed in this set of genes and identify the protein-protein networks that reveal highly-interacted proteins. The combination of these findings paves the way for the discovery of new antimicrobial candidates for the treatment of multidrug-resistant bacteria.

The impact of antithrombotic therapy on surgical delay and 2-year mortality in older patients with hip fracture: a prospective observational study.

PURPOSE: To analyze the associations of oral anticoagulant and antiplatelet treatments in older patients requiring surgical treatment for hip fracture with mortality, length of stay, waiting time to surgery and postsurgical immediate complications. METHODS: A prospective observational study, including surgically treated hip fracture patients aged 65 years and older. We analyzed admission status variables, and time to surgery, length of stay and immediate surgical and medical complications. We recorded a 2-year survival follow-up. RESULTS: Of the 237 patients studied, 32.5% received antiplatelet treatment and 17.7% received oral anticoagulant treatment. The overall 2-year mortality was 29.1%. The 2-year mortality rate reached the 33.8% for antiplatelet-treated patients (n.s.) and 45.2% for oral anticoagulated patients (p = 0.011). The length of stay increased significantly associated with the oral anticoagulant or high-dose antiplatelet treatment (p < 0.001). The same happened for the time to surgery (p < 0.001), but no delay was observed for patients with low-dose antiplatelet treatment. Oral anticoagulated and antiplatelet-treated patients had a higher Charlson comorbidity index (p = 0.004, p = 0.019) and ASA score (p = 0.006, p = 0.011). Those treatments were also associated with a higher rate of some immediate postsurgical medical complications. We found a relationship between low hemoglobin at admission and high-dose antiplatelet treatment (p = 0.044). CONCLUSIONS: Hip fracture patients following previous oral anticoagulant treatment have a higher 2-year mortality rate. The oral anticoagulant or high-dose antiplatelet treatments are significantly associated with an increase in the length of stay, related to time to surgery, but we could not demonstrate an effect on early mortality.

Centrality in the host-pathogen interactome is associated with pathogen fitness during infection.

To perform their functions proteins must interact with each other, but how these interactions influence bacterial infection remains elusive. Here we demonstrate that connectivity in the host-pathogen interactome is directly related to pathogen fitness during infection. Using Y. pestis as a model organism, we show that the centrality-lethality rule holds for pathogen fitness during infection but only when the host-pathogen interactome is considered. Our results suggest that the importance of pathogen proteins during infection is directly related to their number of interactions with the host. We also show that pathogen proteins causing an extensive rewiring of the host interactome have a higher impact in pathogen fitness during infection. Hence, we conclude that hubs in the host-pathogen interactome should be explored as promising targets for antimicrobial drug design.

A case of necrotizing vasculitis with panniculitis, during sorafenib treatment for hepatocellular carcinoma, appeared in disease progression.

Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and is the third most common cause of death from cancer. Sorafenib is the only drug which improves survival in first line advanced HCC. Sorafenib has been associated with several dermatologic toxicities and toxic effects have been related to a better treatment response. We report the case of a well-circumscribed panniculitis and necrotizing vasculitis due to sorafenib, appeared in disease progression in a man affected by advanced HCC.

New model to explain the EQ-5D VAS in patients who have undergone spinal fusion.

BACKGROUND: In the health sector, the EQ-5D has been widely used to evaluate health-related quality of life, mainly due to its simplicity, robustness and plasticity in calculating years of life adjusted for quality. METHODS: However, its model has remained stable over time and knowledge of the influence of such biodemographical variables as age and gender has not been incorporated. This information, along with the well-known non-linear behavior of the VAS, which measures the self-concept of the 'General State of Health', forms the basis for a new model, which we present here from a sample of patients who were operated on for chronic low back pain. RESULTS: These are obtained using multiple regression analysis are consistent in terms of goodness-of-fit given that this new model is able to explain 91.3% of the self-concept of the 'general state of health'. CONCLUSIONS: Above leads to an improvement of 21.6% compared to the available models more robust and meets most of the restrictions for this type of statistical modeling.

Meningeal carcinomatosis and uterine carcinoma: three different clinical settings and review of the literature.

INTRODUCTION: Leptomeningeal carcinomatosis is a rare metastatic event in gynecological neoplasias, and most cases occur in ovarian cancer. It is extremely infrequent in cervical cancer, and so far, there are not any reports of this complication in association with endometrial cancer. PATIENTS AND METHODS: We report a case of leptomeningeal carcinomatosis secondary to endometrial carcinoma and 2 complex cervix cancer cases. A MEDLINE search was done to review all published cases of this complication in gynecological cancer to identify predictive factors for this diagnosis. RESULTS AND DISCUSSION: Leptomeningeal carcinomatosis is usually diagnosed late in the course of the disease, and most reports concern ovarian cancer patients. The number of cases describing this neurologic complication in cervix cancer is increasing. Gadolinium-enhanced magnetic resonance imaging may be necessary for this diagnosis, because cerebrospinal fluid analysis results may be negative. Most cervix cases had squamous cell (8/14) or neuroendocrine histologic subtype (3/14), and when reported, differentiation was usually poor. The case we report of endometrial carcinoma, unique in the literature, is a serous adenocarcinoma. CONCLUSIONS: A high index of suspicion is necessary, and leptomeningeal carcinomatosis should be considered in patients with unexplained neurologic symptoms whose gynecologic tumors are poorly undifferentiated or have a serous component.