摘要： Background and Aims The mechanism underlying HCC metastasis remains unclear, many oncogenes are known to regulate this process. However, the role of alternative splicing (AS) in pro-metastatic HCC is poorly understood. Approach and Results By performing RNA sequencing on nine pairs of primary HCC tissues with extrahepatic metastasis (EHMH) and nine pairs of metastasis-free HCC (MFH) tissues, we depicted the AS landscape in HCC and found a higher frequency of AS events in EHMH compared with MFH. Moreover, 28 differentially expressed splicing regulators were identified in EHMH compared with MFH. Among these, DEAD-box RNA helicase 17 (DDX17) was significantly up-regulated in EHMH and was strongly associated with patient outcome. Functional studies indicated that DDX17 knockout inhibited the degradation of the extracellular matrix, and diminished the invasive ability of HCC cells. A significant reduction in lung metastasis induced by DDX17 deficiency was also demonstrated in a diethylnitrosamine-induced DDX17(HKO) mouse model. Mechanistically, high DDX17 induced intron 3 retention of PXN-AS1 and produced a transcript (termed PXN-AS1-IR3). The transcript PXN-AS1-IR3 acted as an important promoter of HCC metastasis by inducing MYC transcription activation via recruiting the complex of testis expressed 10 and p300 to the MYC enhancer region, which led to transcriptional activation of several metastasis-associated downstream genes. Finally, the PXN-AS1-IR3 level was significantly higher in serum and HCC tissues with extrahepatic metastasis. Conclusions DDX17 and PXN-AS1-IR3 act as important metastatic promoters by modulating MYC signaling, suggesting that DDX17 and PXN-AS1-IR3 may be potential prognostic markers for metastatic HCC.

关键词： COVID-19   intensive care   nurses   nursing workforce   qualitative  

摘要： Aims To understand how COVID-19 affected nurse staffing in intensive care units (ICUs) in England, and to identify factors that influenced, and were influenced by, pandemic staffing models. Design Exploratory qualitative study. Methods Semi-structured, online interviews conducted July-September 2020 with regional critical care leaders including policy leads (n = 4) and directors/lead nurses (n = 10) across critical care networks in England. Findings The six themes emerging from the framework analysis illustrate how the pre-pandemic ICU culture influenced ICU staffing models during the pandemic. Changes in staffing impacted on the workforce and the care delivered, whilst it was necessary to learn from, and adjust to, a rapidly changing situation. Variation across and between networks necessitated variation in responses. The overwhelming outcome was that the pandemic has challenged the central tenets of ICU nurse staffing. Conclusions Pandemic nurse staffing models resulted in changes to ICU skill-mix and staffing numbers. Factors such as the impact of nurse staffing on care practices and on the workforce need to be taken into account when developing and testing future nurse staffing models for ICU. The extent to which ICUs will return to former staffing models is not yet known but there seems to be an appetite for change. Impact In common with many countries, nurse staffing in English ICUs was adapted to address surge requirements during the COVID-19 pandemic. Findings highlight the challenge COVID-19 presented to pre-pandemic ICU nurse staffing guidelines, the impact on patient and staff well-being and the potential legacy for future staffing models. Study findings have implications for ICU nurse managers, researchers and policy makers: nurse staffing models need to be adaptable to the local context of care and future research should investigate the impact of different models on patients, staff and health service outcomes.

关键词： Loss network   dynamic alternative routing   metastability   mixing times  

摘要： Consider N stations interconnected with links, each of capacity K, forming a complete graph. Calls arrive to each link at rate lambda and depart at rate 1. If a call arrives to a link xy, connecting stations x and y, which is at capacity, then a third station z is chosen uniformly at random and the call is attempted to be routed via z: if both links xz and zy have spare capacity, then the call is held simultaneously on these two;otherwise the call is lost. We analyse an approximation of this model. We show rigorously that there are three phases according to the traffic intensity alpha := lambda/K: for alpha is an element of (0, alpha(c)) boolean OR (1, infinity), the system has mixing time logarithmic in the number of links n := ((N)(2));for alpha is an element of (alpha(c), 1) the system has mixing time exponential in n, the number of links. Here alpha(c) := 1/3(5 root 10 - 13) approximate to 0.937 is an explicit critical threshold with a simple interpretation. We also consider allowing multiple rerouting attempts. This has little effect on the overall behaviour;it does not remove the metastability phase. Finally, we add trunk reservation: in this, some number sigma of circuits are reserved;a rerouting attempt is only accepted if at least sigma + 1 circuits are available. We show that if sigma is chosen sufficiently large, depending only on alpha, not K or n, then the metastability phase is removed.

关键词： Stem cell homing   Engraftment   The alternative pathway of the complement cascade   Factor B   Nlrp3 inflammasome   Proteomics  

摘要： We reported in the past that activation of the third (C3) and fifth element (C5) of complement cascade (ComC) is required for a proper homing and engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs). Since myeloablative conditioning for transplantation triggers in recipient bone marrow (BM) state of sterile inflammation, we have become interested in the role of complement in this process and the potential involvement of alternative pathway of ComC activation. We noticed that factor B deficient mice (FB-KO) that do not activate properly alternative pathway, engraft poorly with BM cells from normal wild type (WT) mice. We observed defects both in homing and engraftment of transplanted HSPCs. To shed more light on these phenomena, we found that myeloablative lethal irradiation conditioning for transplantation activates purinergic signaling, ComC, and Nlrp3 inflammasome in WT mice, which is significantly impaired in FB-KO animals. Our proteomics analysis revealed that conditioned for transplantation lethally irradiated FB-KO compared to normal control animals have lower expression of several proteins involved in positive regulation of cell migration, transendothelial migration, immune system, cellular signaling protein, and metabolic pathways. Overall, our recent study further supports the role of innate immunity in homing and engraftment of HSPCs.

关键词： Surgical Oncology   Oncology   Surgery  

摘要： Background Lymph node ratio (LNR) and the log odds of positive lymph nodes (LODDS) have been proposed as alternative lymph node (LN) classification schemes. Various cut-off values have been defined for each system, with the question of the most appropriate for patients with medullary thyroid cancer (MTC) still remaining open. We aimed to retrospectively compare the predictive impact of different LN classification systems and to define the most appropriate set of cut-off values regarding accurate evaluation of overall survival (OS) in patients with MTC. Methods 182 patients with MTC who were operated on between 1985 and 2018 were extracted from our medical database. Cox proportional hazards regression models and C-statistics were performed to assess the discriminative power of 28 LNR and 28 LODDS classifications and compare them with the N category according to the 8th edition of the AJCC/UICC TNM classification in terms of discriminative power. Regression models were adjusted for age, sex, T category, focality, and genetic predisposition. Results High LNR and LODDS are associated with advanced T categories, distant metastasis, sporadic disease, and male gender. In addition, among 56 alternative LN classifications, only one LNR and one LODDS classification were independently associated with OS, regardless of the presence of metastatic disease. The C-statistic demonstrated comparable results for all classification systems showing no clear superiority over the N category. Conclusion Two distinct alternative LN classification systems demonstrated a better prognostic performance in MTC patients than the N category. However, larger scale studies are needed to further verify our findings.

关键词： COVID-19   Medical education   e-learning   Learning behavior   Lectures   Remote learning  

摘要： Purpose The onset of the COVID-19 pandemic posed an eminent challenge for medical teachers worldwide. Face-to-face lectures and seminars were no longer possible, and alternatives had to be found. E-learning concepts quickly emerged as the only practicable solutions and also offered the opportunity to evaluate whether traditional face-to-face lectures could be translated into an online format, independent of the COVID-19 pandemic. Methods We offered an e-learning program consisting of lecture notes, screencasts with audio narration, and online webinars that covered topics normally taught in traditional lectures and seminars. To evaluate the learning behavior and quality of our e-learning program, we drafted a questionnaire that students completed at the end of the 2020 summer semester that had been designed to enable a comparative analysis of the different e-learning modules. Results Voluntary participation in the online courses was high. Survey analysis revealed high satisfaction with and a distinctive preference for the format, even under regular, COVID-19-independent conditions. In general, a positive appraisal of e-learning-especially as a substitute for regular lectures-was found. Students also reported higher studying efficiency. Exam results were equal to those of previous semesters. Conclusion Both acceptance of and satisfaction with our e-learning modules were high, and students displayed increased demand for this kind of e-learning format. We, therefore, conclude that e-learning offerings could serve as reasonable, efficient, student-orientated substitutes for certain medical courses, especially lectures. These curricular adaptations would correlate with the high digitalization seen in students' everyday lives. This correlation may also hold true independent of the ongoing COVID-19 pandemic.

关键词： Cottonseed protein concentrate   Largemouth bass   Paraprobiotic   Gut health   Gut microbiome  

摘要： Cottonseed protein concentrate (CPC) is a potential fishmeal alternative but may negatively influence the gut health of fish. A specific paraprobiotic consisting of multi-strain yeast fractions (MsYF) was recently shown to improve gut health via immune modulation. This study aimed to evaluate the effects of MsYF on the growth performance, gut health and microbiome of largemouth bass fed a diet using CPC as 40% fishmeal alternative. Juvenile largemouth bass (body-weight = 31 g) were fed for 65 days with three iso-nitrogen and isoenergetic experimental diets: 1/ one Control diet (50% fishmeal), 2/ one low fishmeal diet (LFM;30% fishmeal, 23.5% CPC) and 3/ one LFM diet + MsYF at 800 mg/kg (LFMY). The highest growth performance, estimated as specific growth rate and protein efficiency ratio, was observed in the LFMY followed by the LFM. Histopathology identified some specimens with distal intestinal lesions in the Control and LFM groups but not of the LFMY group. The gene expression level of il-10 (anti-inflammatory) was up-regulated in the LFM and LFMY groups but only LFMY down-regulated il-1 beta (pro-inflammatory) and hypoxia-inducible factor 1-alpha (hif1 alpha) protein expression level compared to the Control group. Further, LFM and LFMY up-regulated occ-1b (tight-junction) and slc15a2 (peptide transporter) suggesting enhanced intestinal permeability and peptide transport compared to the Control group. Moreover, dietary CPC increased the abundance of genus Clostridium_sensu_stricto_12 and decreased the abundance of genus Escherichia-Shigella compared to the Control group, while the inclusion of MsYF increased the abundance of the potentially beneficial genus Cetobacterium compared to the LFM group. Our results showed 40% substitution of fishmeal protein by CPC could enhance growth performance and regulate the intestinal barrier and peptide transport relevant functions in largemouth bass. Moreover, in a low fishmeal basal diet, MsYF supplementation could increase gr

关键词： calcium   alternative splicing   embryo   protein isoforms   amino acids   exons   skeletal muscles   cell lines   tissue membrane   calcium channels   alanine   sensor   voltage gated calcium channel  

摘要： The skeletal muscle voltage-gated calcium channel (Ca(v)1.1) primarily functions as a voltage sensor for excitation-contraction coupling. Conversely, its ion-conducting function is modulated by multiple mechanisms within the pore-forming a is subunit and the auxiliary alpha(2)delta-1 and gamma(1) subunits. In particular, developmentally regulated alternative splicing of exon 29, which inserts 19 amino acids in the extracellular IVS3-S4 loop of Ca(v)1.1a, greatly reduces the current density and shifts the voltage dependence of activation to positive potentials outside the physiological range. We generated new HEK293 cell lines stably expressing alpha(2)delta-1, beta(3), and STAC3. When the adult (Ca(v)1.1a) and embryonic (Ca(v)1.1e) splice variants were expressed in these cells, the difference in the voltage dependence of activation observed in muscle cells was reproduced, but not the reduced current density of Ca(v)1.1a. Only when we further coexpressed the gamma(1) subunit was the current density of Ca(v)1.1a, but not that of Ca(v)1.1e, reduced by >50%. In addition, gamma(1) caused a shift of the voltage dependence of inactivation to negative voltages in both variants. Thus, the current-reducing effect of gamma(1), unlike its effect on inactivation, is specifically dependent on the inclusion of exon 29 in Ca(v)1.1a. Molecular structure modeling revealed several direct ionic interactions between residues in the IVS3-S4 loop and the y i subunit. However, substitution of these residues by alanine, individually or in combination, did not abolish the gamma(1)-dependent reduction of current density, suggesting that structural rearrangements in Ca(v)1.1a induced by inclusion of exon 29 may allosterically empower the gamma(1) subunit to exert its inhibitory action on Ca-v1.1 calcium currents.

摘要： Alternative polyadenylation (APA) plays an important role in gene regulation. With the recent application of novel sequencing technology in APA profiling, an ever-increasing number of APA genes/sites have been identified. However, the phenotypic relevance of most of these APA isoforms remains elusive, which is largely due to the lack of a convenient genetics tool for APA interference. To address this issue, herein, an efficient method is developed based on the CRISPR-dCas13 system, termed as CRISPR-iPAS. Out of eight different dCas13 proteins, Porphyromonas gulae (Pgu) dCas13b, is identified as the most effective one in blocking the usage of the polyadenylation site (PAS). With guide RNAs targeting at core regulatory elements, dPguCas13b enabled APA regulation of endogenous genes with different APA types, including tandem 3 ' UTR, alternative terminal exon, as well as intronic PAS. Finally, we demonstrated that the proposed APA perturbation tool could be used to investigate the functional relevance of APA isoforms.

摘要： Most human protein-coding genes produce alternative polyadenylation (APA) isoforms that differ in 3 ' UTR size or, when coupled with splicing, have variable coding sequences. APA is an important layer of gene expression program critical for defining cell identity. Here, by using a catalytically dead Cas9 and coupling its target site with polyadenylation site (PAS), we develop a method, named CRISPRpas, to alter APA isoform abundance. CRISPRpas functions by enhancing proximal PAS usage, whose efficiency is influenced by several factors, including targeting strand of DNA, distance between PAS and target sequence and strength of the PAS. For intronic polyadenylation (IPA), splicing features, such as strengths of 5 ' splice site and 3 ' splice site, also affect CRISPRpas efficiency. We show modulation of APA of multiple endogenous genes, including IPA of PCF11, a master regulator of APA and gene expression. In sum, CRISPRpas offers a programmable tool for APA regulation that impacts gene expression.