Optimizing Optical Flow Cytometry for Cell Volume-Based Sorting and Analysis

Cell size is a defining characteristic central to cell function and ultimately to tissue architecture. The ability to sort cell subpopulations of different sizes would facilitate investigation at genomic and proteomic levels of mechanisms by which cells attain and maintain their size. Currently available cell sorters, however, cannot directly measure cell volume electronically, and it would therefore be desirable to know which of the optical measurements that can be made in such instruments provide the best estimate of volume. We investigated several different light scattering and fluorescence measurements in several different cell lines, sorting cell fractions from the high and low end of distributions, and measuring volume electronically to determine which sorting strategy yielded the best separated volume distributions. Since we found that different optical measurements were optimal for different cell lines, we suggest that following this procedure will enable other investigators to optimize their own cell sorters for volume-based separation of the cell types with which they work

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Geminin deploys multiple mechanisms to regulate Cdt1 before cell division thus ensuring the proper execution of DNA replication

Cdc10-dependent transcript 1 (Cdt1) is an essential DNA replication protein whose accumulation at the end of the cell cycle promotes the formation of pre-replicative complexes and replication in the next cell cycle. Geminin is thought to be involved in licensing replication by promoting the accumulation of Cdt1 in mitosis, because decreasing the Geminin levels prevents Cdt1 accumulation and impairs DNA replication. Geminin is known to inhibit Cdt1 function; its depletion during G2 leads to DNA rereplication and checkpoint activation. Here we show that, despite rapid Cdt1 protein turnover in G2 phase, Geminin promotes Cdt1 accumulation by increasing its RNA and protein levels in the unperturbed cell cycle. Therefore, Geminin is a master regulator of cell-cycle progression that ensures the timely onset of DNA replication and prevents its rereplication

Mice Lacking the p53-Effector Gene Gadd45a Develop a Lupus-Like Syndrome

AbstractThis study addresses the biological function of the p53-effector genes Gadd45a and p21 in the immune system. We find that Gadd45a is a negative regulator of T cell proliferation because, compared to wild-type cells, Gadd45a−/− T cells have a lower threshold of activation and proliferate to a greater extent following primary T cell receptor stimulation. Gadd45a−/− mice develop an autoimmune disease, similar to human systemic lupus erythematosus (SLE), characterized by high titers of anti-dsDNA, anti-ssDNA, and anti-histone autoantibodies, severe hematological disorders, autoimmune glomerulonephritis, and premature death. Here we show that the lack of both Gadd45a and p21 dramatically accelerates the development of autoimmunity observed in each individual single-gene disruption mutant, demonstrating that these genes play nonredundant roles in the immune response

<i>In vivo</i> epigenetic effects induced by engineered nanomaterials: A case study of copper oxide and laser printer-emitted engineered nanoparticles

<p>Evidence continues to grow on potential environmental health hazards associated with engineered nanomaterials (ENMs). While the geno- and cytotoxic effects of ENMs have been investigated, their potential to target the epigenome remains largely unknown. The aim of this study is two-fold: 1) determining whether or not industry relevant ENMs can affect the epigenome <i>in vivo</i> and 2) validating a recently developed <i>in vitro</i> epigenetic screening platform for inhaled ENMs. Laser printer-emitted engineered nanoparticles (PEPs) released from nano-enabled toners during consumer use and copper oxide (CuO) were chosen since these particles induced significant epigenetic changes in a recent <i>in vitro</i> companion study. In this study, the epigenetic alterations in lung tissue, alveolar macrophages and peripheral blood from intratracheally instilled mice were evaluated. The methylation of global DNA and transposable elements (TEs), the expression of the DNA methylation machinery and TEs, in addition to general toxicological effects in the lung were assessed. CuO exhibited higher cell-damaging potential to the lung, while PEPs showed a greater ability to target the epigenome. Alterations in the methylation status of global DNA and TEs, and expression of TEs and DNA machinery in mouse lung were observed after exposure to CuO and PEPs. Additionally, epigenetic changes were detected in the peripheral blood after PEPs exposure. Altogether, CuO and PEPs can induce epigenetic alterations in a mouse experimental model, which in turn confirms that the recently developed <i>in vitro</i> epigenetic platform using macrophage and epithelial cell lines can be successfully utilized in the epigenetic screening of ENMs.</p

Proteomic and Metabolomic Characterization of a Mammalian Cellular Transition from Quiescence to Proliferation

There exist similarities and differences in metabolism and physiology between normal proliferative cells and tumor cells. Once a cell enters the cell cycle, metabolic machinery is engaged to facilitate various processes. The kinetics and regulation of these metabolic changes have not been properly evaluated. To correlate the orchestration of these processes with the cell cycle, we analyzed the transition from quiescence to proliferation of a non-malignant murine pro-B lymphocyte cell line in response to IL-3. Using multiplex mass-spectrometry-based proteomics, we show that the transition to proliferation shares features generally attributed to cancer cells: upregulation of glycolysis, lipid metabolism, amino-acid synthesis, and nucleotide synthesis and downregulation of oxidative phosphorylation and the urea cycle. Furthermore, metabolomic profiling of this transition reveals similarities to cancer-related metabolic pathways. In particular, we find that methionine is consumed at a higher rate than that of other essential amino acids, with a potential link to maintenance of the epigenome

Short-Term Pre-Operative Protein Caloric Restriction in Elective Vascular Surgery Patients: A Randomized Clinical Trial

(1) Background: Vascular surgery operations are hampered by high failure rates and frequent occurrence of peri-operative cardiovascular complications. In pre-clinical studies, pre-operative restriction of proteins and/or calories (PCR) has been shown to limit ischemia-reperfusion damage, slow intimal hyperplasia, and improve metabolic fitness. However, whether these dietary regimens are feasible and safe in the vascular surgery patient population remains unknown. (2) Methods: We performed a randomized controlled trial in patients scheduled for any elective open vascular procedure. Participants were randomized in a 3:2 ratio to either four days of outpatient pre-operative PCR (30% calorie, 70% protein restriction) or their regular ad-libitum diet. Blood was drawn at baseline, pre-operative, and post-operative day 1 timepoints. A leukocyte subset flow cytometry panel was performed at these timepoints. Subcutaneous/perivascular adipose tissue was sampled and analyzed. Follow-up was one year post-op. (3) Results: 19 patients were enrolled, of whom 11 completed the study. No diet-related reasons for non-completion were reported, and there was no intervention group crossover. The PCR diet induced weight loss and BMI decrease without malnutrition. Insulin sensitivity was improved after four days of PCR (p = 0.05). Between diet groups, there were similar rates of re-intervention, wound infection, and cardiovascular complications. Leukocyte populations were maintained after four days of PCR. (4) Conclusions: Pre-operative PCR is safe and feasible in elective vascular surgery patients.ISSN:2072-664