A functional genomic approach to identify reference genes for human pancreatic beta cell real-time quantitative RT-PCR analysis

Exposure of human pancreatic beta cells to pro-inflammatory cytokines or metabolic stressors is used to model events related to type 1 and type 2 diabetes, respectively. Quantitative real-time PCR is commonly used to quantify changes in gene expression. The selection of the most adequate reference gene(s) for gene expression normalization is an important pre-requisite to obtain accurate and reliable results. There are no universally applicable reference genes, and the human beta cell expression of commonly used reference genes can be altered by different stressors. Here we aimed to identify the most stably expressed genes in human beta cells to normalize quantitative real-time PCR gene expression. We used comprehensive RNA-sequencing data from the human pancreatic beta cell line EndoC-βH1, human islets exposed to cytokines or the free fatty acid palmitate in order to identify the most stably expressed genes. Genes were filtered based on their level of significance (adjusted P-value >0.05), fold-change (|fold-change| <1.5) and a coefficient of variation <10%. Candidate reference genes were validated by quantitative real-time PCR in independent samples. We identified a total of 264 genes stably expressed in EndoC-βH1 cells and human islets following cytokines–or palmitate-induced stress, displaying a low coefficient of variation. Validation by quantitative real-time PCR of the top five genes ARF1, CWC15, RAB7A, SIAH1 and VAPA corroborated their expression stability under most of the tested conditions. Further validation in independent samples indicated that the geometric mean of ACTB and VAPA expression can be used as a reliable normalizing factor in human beta cells

The Fourteenth Data Release of the Sloan Digital Sky Survey: First Spectroscopic Data from the extended Baryon Oscillation Spectroscopic Survey and from the second phase of the Apache Point Observatory Galactic Evolution Experiment

The fourth generation of the Sloan Digital Sky Survey (SDSS-IV) has been in operation since July 2014. This paper describes the second data release from this phase, and the fourteenth from SDSS overall (making this, Data Release Fourteen or DR14). This release makes public data taken by SDSS-IV in its first two years of operation (July 2014-2016). Like all previous SDSS releases, DR14 is cumulative, including the most recent reductions and calibrations of all data taken by SDSS since the first phase began operations in 2000. New in DR14 is the first public release of data from the extended Baryon Oscillation Spectroscopic Survey (eBOSS); the first data from the second phase of the Apache Point Observatory (APO) Galactic Evolution Experiment (APOGEE-2), including stellar parameter estimates from an innovative data driven machine learning algorithm known as "The Cannon"; and almost twice as many data cubes from the Mapping Nearby Galaxies at APO (MaNGA) survey as were in the previous release (N = 2812 in total). This paper describes the location and format of the publicly available data from SDSS-IV surveys. We provide references to the important technical papers describing how these data have been taken (both targeting and observation details) and processed for scientific use. The SDSS website (www.sdss.org) has been updated for this release, and provides links to data downloads, as well as tutorials and examples of data use. SDSS-IV is planning to continue to collect astronomical data until 2020, and will be followed by SDSS-V.Comment: SDSS-IV collaboration alphabetical author data release paper. DR14 happened on 31st July 2017. 19 pages, 5 figures. Accepted by ApJS on 28th Nov 2017 (this is the "post-print" and "post-proofs" version; minor corrections only from v1, and most of errors found in proofs corrected

Increased 30-Day Mortality in Very Old ICU Patients with COVID-19 Compared to Patients with Respiratory Failure without COVID-19

Purpose: The number of patients ≥ 80 years admitted into critical care is increasing. Coronavirus disease 2019 (COVID-19) added another challenge for clinical decisions for both admission and limitation of life-sustaining treatments (LLST). We aimed to compare the characteristics and mortality of very old critically ill patients with or without COVID-19 with a focus on LLST. Methods: Patients 80 years or older with acute respiratory failure were recruited from the VIP2 and COVIP studies. Baseline patient characteristics, interventions in intensive care unit (ICU) and outcomes (30-day survival) were recorded. COVID patients were matched to non-COVID patients based on the following factors: age (± 2 years), Sequential Organ Failure Assessment (SOFA) score (± 2 points), clinical frailty scale (± 1 point), gender and region on a 1:2 ratio. Specific ICU procedures and LLST were compared between the cohorts by means of cumulative incidence curves taking into account the competing risk of discharge and death. Results: 693 COVID patients were compared to 1393 non-COVID patients. COVID patients were younger, less frail, less severely ill with lower SOFA score, but were treated more often with invasive mechanical ventilation (MV) and had a lower 30-day survival. 404 COVID patients could be matched to 666 non-COVID patients. For COVID patients, withholding and withdrawing of LST were more frequent than for non-COVID and the 30-day survival was almost half compared to non-COVID patients. Conclusion: Very old COVID patients have a different trajectory than non-COVID patients. Whether this finding is due to a decision policy with more active treatment limitation or to an inherent higher risk of death due to COVID-19 is unclear.info:eu-repo/semantics/publishedVersio

Elective cancer surgery in COVID-19-free surgical pathways during the SARS-CoV-2 pandemic: An international, multicenter, comparative cohort study

PURPOSE As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19–free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19–free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19–free surgical pathways. Patients who underwent surgery within COVID-19–free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19–free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score–matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19–free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION Within available resources, dedicated COVID-19–free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.

PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

The Fourteenth Data Release of the Sloan Digital Sky Survey: First Spectroscopic Data from the Extended Baryon Oscillation Spectroscopic Survey and from the Second Phase of the Apache Point Observatory Galactic Evolution Experiment

The fourth generation of the Sloan Digital Sky Survey (SDSS-IV) has been in operation since 2014 July. This paper describes the second data release from this phase, and the 14th from SDSS overall (making this Data Release Fourteen or DR14). This release makes the data taken by SDSS-IV in its first two years of operation (2014–2016 July) public. Like all previous SDSS releases, DR14 is cumulative, including the most recent reductions and calibrations of all data taken by SDSS since the first phase began operations in 2000. New in DR14 is the first public release of data from the extended Baryon Oscillation Spectroscopic Survey; the first data from the second phase of the Apache Point Observatory (APO) Galactic Evolution Experiment (APOGEE-2), including stellar parameter estimates from an innovative data-driven machine-learning algorithm known as "The Cannon"; and almost twice as many data cubes from the Mapping Nearby Galaxies at APO (MaNGA) survey as were in the previous release (N = 2812 in total). This paper describes the location and format of the publicly available data from the SDSS-IV surveys. We provide references to the important technical papers describing how these data have been taken (both targeting and observation details) and processed for scientific use. The SDSS web site (www.sdss.org) has been updated for this release and provides links to data downloads, as well as tutorials and examples of data use. SDSS-IV is planning to continue to collect astronomical data until 2020 and will be followed by SDSS-V

Pro-inflammatory cytokines induce cell death, inflammatory responses, and endoplasmic reticulum stress in human iPSC-derived beta cells

BACKGROUND: Adult human pancreatic beta cells are the "gold standard" for studies on diabetes pathogenesis, but their use is limited by insufficient availability and variable quality. An important effort has recently taken place to differentiate beta cells from human induced pluripotent stem cells (iPSCs) and validate their use for diabetes research. We presently used a 7-stage protocol to generate beta cells from human iPSC and evaluated whether these cells are responsive to the pro-inflammatory cytokines (IFNγ, IL-1β, or IFNα) that play a role in type 1 diabetes. METHODS: The iPSC-derived islet-like cell clusters contained 40-50% beta and 10-15% alpha cells and expressed the receptors for IFNγ, IL-1β, or IFNα. Cells were exposed to either IFNγ (1000 U/mL) + IL-1β (50 U/mL) or IFNα alone (2000 U/mL) for 24/48 h. Apoptosis was quantified using Hoechst/propidium iodide staining or the RealTime Glo Apoptosis Kit (Promega). After treatment, CXCL10 secretion was quantified by ELISA. The expression of multiples genes (Ins, Gcg, Nkx2.2, Nkx6.1, Pdx1, Mafa, BiP, Chop, Atf3, CXCL10, CXCL9, CCL5, and HLA-ABC) was quantified by RT-qPCR. Phosphorylation state and total expression of STAT1/STAT2, as well as expression of PDL1 and of the ER chaperone BiP, were quantified by Western blotting. The co-localization of HLA-ABC or cleaved caspase-3 and Ins/Gcg expression was assessed by immunohistochemistry. The presence of HLA-ABC at the plasma membrane was measured by flow cytometry. RESULTS: IFNγ + IL-1β and IFNα induced apoptosis of the cells after 48 h of exposure. Cleaved caspase-3 co-localized mostly but not exclusively with Ins+ cells. Exposure to IFNγ + IL-1β induced a pro-inflammatory phenotype, including increased CXCL10, CXCL9, and CCL5 expression; CXCL10 secretion; and HLA-ABC expression. HLA overexpression was confirmed at the protein level by Western blotting and flow cytometry. Exposure to IFNγ + IL-1β (but not IFNα) also induced beta cell dedifferentiation and endoplasmic reticulum stress (increase in BiP, Chop, and Atf3 mRNA expression). Phosphorylation of STAT1 was stimulated already after 1 h by IFNγ + IL-1β and IFNα, while phosphorylation of STAT2 was only activated by IFNα at 1-4 h. PDL1 expression was increased by both IFNγ + IL-1β and IFNα. CONCLUSIONS: Our data show that human iPSC-derived beta cells respond to pro-inflammatory cytokines IL-1β + IFNγ and IFNα, by activating the same pathogenic processes as adult human primary beta cells. These cells thus represent a valuable tool for future research on the pathogenesis of type 1 diabetes

Transcription and splicing regulation by NLRC5 shape the interferon response in human pancreatic 0 cells

IFNa is a key regulator of the dialogue between pancreatic 0 cells and the immune system in early type 1 diabetes (T1D). IFNa up-regulates HLA class I expression in human 0 cells, fostering autoantigen presentation to the immune system. We observed by bulk and single-cell RNA sequencing that exposure of human induced pluripotentderived islet-like cells to IFNa induces expression of HLA class I and of other genes involved in antigen presentation, including the transcriptional activator NLRC5. We next evaluated the global role of NLRC5 in human insulinproducing EndoC-0H1 and human islet cells by RNA sequencing and targeted gene/protein determination. NLRC5 regulates expression of HLA class I, antigen presentation-related genes, and chemokines. NLRC5 also mediates the effects of IFNa on alternative splicing, a generator of 0 cell neoantigens, suggesting that it is a central player of the effects of IFNa on 0 cells that contribute to trigger and amplify autoimmunity in T1D

Transcription and splicing regulation by NLRC5 shape the interferon response in human pancreatic 0 cells

IFNa is a key regulator of the dialogue between pancreatic 0 cells and the immune system in early type 1 diabetes (T1D). IFNa up-regulates HLA class I expression in human 0 cells, fostering autoantigen presentation to the immune system. We observed by bulk and single-cell RNA sequencing that exposure of human induced pluripotentderived islet-like cells to IFNa induces expression of HLA class I and of other genes involved in antigen presentation, including the transcriptional activator NLRC5. We next evaluated the global role of NLRC5 in human insulinproducing EndoC-0H1 and human islet cells by RNA sequencing and targeted gene/protein determination. NLRC5 regulates expression of HLA class I, antigen presentation-related genes, and chemokines. NLRC5 also mediates the effects of IFNa on alternative splicing, a generator of 0 cell neoantigens, suggesting that it is a central player of the effects of IFNa on 0 cells that contribute to trigger and amplify autoimmunity in T1D.Therapeutic cell differentiatio