Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies.

Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.

The human body contains around 20,000 different proteins that perform a myriad of essential roles. To understand how these proteins work in healthy individuals and during disease, we need to know their precise locations inside cells and how these locations may change in different situations. Genetic tools known as fluorescent proteins are often used as tags to study the location of specific proteins of interest within cells. When exposed to light, the fluorescent proteins emit specific colours of light that can be observed using microscopes. In a fluorescent protein system known as split mNeonGreen, researchers insert the DNA encoding two fragments of a fluorescent protein (one large, one small) separately into cells. The large fragment can be found throughout the cell, while the small fragment is attached to specific host proteins. When the two fragments meet, they assemble into the full mNeonGreen protein and can fluoresce. Researchers can use split mNeonGreen and other similar systems to generate large libraries of cells where the small fragment of a fluorescent protein is attached to thousands of different host proteins. However, so far these libraries are restricted to a handful of different types of cells. To address this challenge, Husser et al. inserted the DNA encoding the large fragment of mNeonGreen into human cells known as induced pluripotent stem cells, which are able to give rise to any other type of human cell. This then enabled the team to quickly and efficiently generate a library of stem cells that express the small fragment of mNeonGreen attached to different host proteins. Further experiments studied the locations of host proteins in the stem cells just before they divided into two cells. This suggested that there are differences between how induced pluripotent stem cells and other types of cells divide. In the future, the cells and the method developed by Husser et al. may be used by other researchers to create atlases showing where human proteins are located in many other types of cells.

Cost of care pathways before and after appropriate and inappropriate transfers to the emergency department among nursing home residents: results from the FINE study.

BACKGROUND: Transfers of nursing home (NH) residents to the emergency department (ED) is frequent. Our main objective was to assess the cost of care pathways 6 months before and after the transfer to the emergency department among NH residents, according to the type of transfer (i.e. appropriate or inappropriate). METHODS: This was a part of an observational, multicenter, case-control study: the Factors associated with INappropriate transfer to the Emergency department among nursing home residents (FINE) study. Sixteen public hospitals of the former Midi-Pyrénées region participated in recruitment, in 2016. During the inclusion period, all NH residents arriving at the ED were included. A pluri-disciplinary team categorized each transfer to the ED into 2 groups: appropriate or inappropriate. Direct medical and nonmedical costs were assessed from the French Health Insurance (FHI) perspective. Healthcare resources were retrospectively gathered from the FHI database and valued using the tariffs reimbursed by the FHI. Costs were recorded over a 6-month period before and after transfer to the ED. Other variables were used for analysis: sex, age, Charlson score, season, death and presence inside the NH of a coordinating physician or a geriatric nursing assistant. RESULTS: Among the 1037 patients initially included in the FINE study, 616 who were listed in the FHI database were included in this economic study. Among them, 132 (21.4%) had an inappropriate transfer to the ED. In the 6 months before ED transfer, total direct costs on average amounted to 8,145€ vs. 6,493€ in the inappropriate and appropriate transfer groups, respectively. In the 6 months after ED transfer, they amounted on average to 9,050€ vs. 12,094€. CONCLUSIONS: Total costs on average are higher after transfer to the ED, but there is no significant increase in healthcare expenditure with inappropriate ED transfer. Support for NH staff and better pathways of care could be necessary to reduce healthcare expenditures in NH residents. TRIAL REGISTRATION: clinicaltrials.gov, NCT02677272.

Technical and analytical approach to biventricular pressure-volume loops in swine including a completely endovascular, percutaneous closed-chest large animal model.

Pressure-volume (PV) loop analysis is a sophisticated invasive approach to quantifying load-dependent and independent measures of cardiac function. Biventricular (BV) PV loops allow left and right ventricular function to be quantified simultaneously and independently, which is important for conditions and certain physiologic states, such as ventricular decoupling or acute physiologic changes. BV PV loops can be performed in an entirely endovascular, percutaneous, and closed-chest setting. This technique is helpful in a survival animal model, as a percutaneous monitoring system during endovascular device experiments, or in cases where chest wall compliance is being tested or may be a confounder. In this article, we describe the end-to-end implementation of a completely endovascular, totally percutaneous, and closed-chest large animal model to obtain contemporaneous BV PV loops in 40 to 70 kg swine. We describe the associated surgical and technical challenges and our solutions to obtaining endovascular BV PV loops, closed-chest cardiac output, and stroke volume (including validation of the correction factor necessary for thermodilution), as well as how to perform endovascular inferior vena cava occlusion in this swine model. We also include techniques for data acquisition and analysis that are required for this method.

The combination of cigarette smoke and solar rays causes effects similar to skin aging in a bilayer skin model.

Skin aging is a multifactorial process influenced by internal and external factors. The contribution of different environmental factors has been well established individually in the last few years. On the one hand, man is rarely exposed to a single factor, and on the other hand, there is very little knowledge about how these extrinsic factors may interact with each other or even how the skin may react to chronic exposure. This study aimed to evaluate the effect on skin aging of a chronic co-exposure of tissue-engineered skin substitutes to cigarette smoke extract (CSE) and solar simulator light (SSL). Skin substitutes were reconstructed according to the self-assembly method and then exposed to CSE followed by irradiation with SSL simultaneously transmitting UVA1, visible light and infrared. When skin substitutes were chronically exposed to CSE and SSL, a significant decrease in procollagen I synthesis and the inhibition of Smad2 phosphorylation of the TGF-ß signaling pathway were observed. A 6.7-fold increase in MMP-1 activity was also observed when CSE was combined with SSL, resulting in a decrease in collagen III and collagen IV protein expression. The secretory profile resulting from the toxic synergy was investigated and several alterations were observed, notably an increase in the quantities of pro-inflammatory cytokines. The results also revealed the activation of the ERK1/2 (3.4-fold) and JNK (3.3-fold) pathways. Taken together, the results showed that a synergy between the two environmental factors could provoke premature skin aging.

Towards the Identification of Biomarkers for Muscle Function Improvement in Myotonic Dystrophy Type 1.

BACKGROUND: Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults. In DM1 patients, skeletal muscle is severely impaired, even atrophied and patients experience a progressive decrease in maximum strength. Strength training for these individuals can improve their muscle function and mass, however, the biological processes involved in these improvements remain unknown. OBJECTIVE: This exploratory study aims at identifying the proteomic biomarkers and variables associated with the muscle proteome changes induced by training in DM1 individuals. METHODS: An ion library was developed from liquid chromatography-tandem mass spectrometry proteomic analyses of Vastus Lateralis muscle biopsies collected in 11 individuals with DM1 pre-and post-training. RESULTS: The proteomic analysis showed that the levels of 44 proteins were significantly modulated. A literature review (PubMed, UniProt, PANTHER, REACTOME) classified these proteins into biological sub-classes linked to training-induced response, including immunity, energy metabolism, apoptosis, insulin signaling, myogenesis and muscle contraction. Linear models identified key variables explaining the proteome modulation, including atrophy and hypertrophy factors. Finally, six proteins of interest involved in myogenesis, muscle contraction and insulin signaling were identified: calpain-3 (CAN3; Muscle development, positive regulation of satellite cell activation), 14-3-3 protein epsilon (1433E; Insulin/Insulin-like growth factor, PI3K/Akt signaling), myosin-binding protein H (MYBPH; Regulation of striated muscle contraction), four and a half LIM domains protein 3 (FHL3; Muscle organ development), filamin-C (FLNC; Muscle fiber development) and Cysteine and glycine-rich protein 3 (CSRP3). CONCLUSION: These findings may lead to the identification for DM1 individuals of novel muscle biomarkers for clinical improvement induced by rehabilitation, which could eventually be used in combination with a targeted pharmaceutical approach to improving muscle function, but further studies are needed to confirm those results.

Toxic Interaction Between Solar Radiation and Cigarette Smoke on Primary Human Keratinocytes.

Solar radiation and cigarette smoke are two environmental risk factors known to affect skin integrity. Although the toxic effects of these factors on skin have been widely studied separately, few studies have focused on their interaction. The objective of this study was to evaluate and understand the synergistic harmful effects of cigarette smoke and solar rays on human primary keratinocytes. The keratinocytes were exposed to cigarette smoke extract (CSE) and then irradiated with a solar simulator light (SSL). The viability, as determined by measuring metabolic activity of skin cells, and the levels of global reactive oxygen species (ROS) were evaluated after exposure to CSE and SSL. The combination of 3% CSE with 29 kJ m-2 UVA caused a decrease of 81% in cell viability, while with 10% to 20% CSE, the cell viability was null. This phototoxicity was accompanied by an increase in singlet oxygen but a decrease in type I ROS when CSE and SSL were combined in vitro. Surprisingly, an increase in the CSE's total antioxidant capacity was also observed. These results suggest a synergy between the two environmental factors in their effect on skin cells, and more precisely a phototoxicity causing a drastic decrease in cell viability.

Cytokinetic diversity in mammalian cells is revealed by the characterization of endogenous anillin, Ect2 and RhoA.

Cytokinesis is required to physically separate the daughter cells at the end of mitosis. This crucial process requires the assembly and ingression of an actomyosin ring, which must occur with high fidelity to avoid aneuploidy and cell fate changes. Most of our knowledge of mammalian cytokinesis was generated using over-expressed transgenes in HeLa cells. Over-expression can introduce artefacts, while HeLa are cancerous human cells that have lost their epithelial identity, and the mechanisms controlling cytokinesis in these cells could be vastly different from other cell types. Here, we tagged endogenous anillin, Ect2 and RhoA with mNeonGreen and characterized their localization during cytokinesis for the first time in live human cells. Comparing anillin localization in multiple cell types revealed cytokinetic diversity with differences in the duration and symmetry of ring closure, and the timing of cortical recruitment. Our findings show that the breadth of anillin correlates with the rate of ring closure, and support models where cell size or ploidy affects the cortical organization, and intrinsic mechanisms control the symmetry of ring closure. This work highlights the need to study cytokinesis in more diverse cell types, which will be facilitated by the reagents generated for this study.

Co-modulation of T cells and B cells enhances the inhibition of inflammation in experimental hypersensitivity pneumonitis.

BACKGROUND: Hypersensitivity pneumonitis (HP) is an interstitial lung disease characterized by antigen-triggered neutrophilic exacerbations. Although CD4+ T cells are sufficient for HP pathogenesis, this never translated into efficient T cell-specific therapies. Increasing evidence shows that B cells also play decisive roles in HP. Here, we aimed to further define the respective contributions of B and T cells in subacute experimental HP. METHODS: Mice were subjected to a protocol of subacute exposure to the archaeon Methanosphaera stadmanae to induce experimental HP. Using models of adoptive transfers of B cells and T cells in Rag1-deficient mice and of B cell-specific S1P1 deletion, we assessed the importance of B cells in the development of HP by evaluating inflammation in bronchoalveolar lavage fluid. We also aimed to determine if injected antibodies targeting B and/or T cells could alleviate HP exacerbations using a therapeutic course of intervention. RESULTS: Even though B cells are not sufficient to induce HP, they strongly potentiate CD4+ T cell-induced HP­associated neutrophilic inflammation in the airways. However, the reduction of 85% of lung B cells in mice with a CD19-driven S1P1 deletion does not dampen HP inflammation, suggesting that lung B cells are not necessary in large numbers to sustain local inflammation. Finally, we found that injecting antibodies targeting B cells after experimental HP was induced does not dampen neutrophilic exacerbation. Yet, injection of antibodies directed against B cells and T cells yielded a potent 76% inhibition of neutrophilic accumulation in the lungs. This inhibition occurred despite partial, sometimes mild, depletion of B cells and T cells subsets. CONCLUSIONS: Although B cells are required for maximal inflammation in subacute experimental HP, partial reduction of B cells fails to reduce HP-associated inflammation by itself. However, co-modulation of T cells and B cells yields enhanced inhibition of HP exacerbation caused by an antigenic rechallenge.

Role of progression of training volume on intramuscular adaptations in patients with chronic obstructive pulmonary disease.

Introduction: Quadriceps dysfunction is a common systemic manifestation of chronic obstructive pulmonary disease (COPD), for which treatment using resistance training is highly recommended. Even though training volume is suggested to be a key explanatory factor for intramuscular adaptation to resistance training in healthy older adults, knowledge is scarce on the role of progression of training volume for intramuscular adaptations in COPD. Methods: This study was a sub-analysis of a parallel-group randomized controlled trial. Thirteen patients with severe to very severe COPD (median 66 yrs, forced expiratory volume in 1 s 44% predicted) performed 8 weeks of low-load resistance training. In a post hoc analysis, they were divided into two groups according to their training volume progression. Those in whom training volume continued to increase after the first 4 weeks of training outlined the continued progression group (n = 9), while those with limited increase (<5%) or even reduction in training volume after the initial 4 weeks composed the discontinued progression group (n = 4). Fiber-type distribution and oxidative muscle protein levels, i.e., citrate synthase (CS), hydroxyacyl-coenzyme A dehydrogenase (HADH), mitochondrial transcription factor A (TfAM) as well as quadriceps endurance measures (total work from elastic band and isokinetic knee extension tests), were assessed before and after the intervention period. Results: The continued progression group sustained their training volume progression during weeks 5-8 compared to weeks 1-4 (median +25%), while the discontinued progression group did not (median -2%) (p = 0.007 between groups). Compared with baseline values, significant between-group differences in fiber type distribution and TfAM muscle protein levels (range ± 17-62%, p < 0.05) and in individual responses to change in Type I and Type IIa fiber type proportion, CS, HADH, and TfAM muscle protein levels outcomes (median 89 vs. 50%, p = 0.001) were seen in favor of the continued progression group. Moreover, only the continued progression group had a significant increase in HADH muscle protein levels (+24%, p = 0.004), elastic band (+56%, p = 0.004) and isokinetic (+7%, p = 0.004) quadriceps endurance, but the between-group differences did not reach statistical significance (range 14-29%, p = 0.330-1.000). Discussion: The novel findings of the current study were that patients with COPD who had a continued progression of training volume across the 8-weeks intervention had an increased proportion of Type I fibers, and TfAM muscle protein levels and decreased proportion of Type II fibers compared to those that did not continue to progress their training volume after the initial weeks. Additionally, HADH muscle protein levels and quadriceps endurance measurements only improved in the continued progression group, although no significant between-group differences were seen. These findings highlight the importance of continued progression of training volume during resistive training to counteract quadriceps dysfunction within the COPD population. Still, considering the small sample size and the post hoc nature of our analyses, these results should be interpreted cautiously, and further research is necessary.

Malat1 deficiency prevents hypoxia-induced lung dysfunction by protecting the access to alveoli.

Hypoxia is common in lung diseases and a potent stimulator of the long non-coding RNA Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1). Herein, we investigated the impact of Malat1 on hypoxia-induced lung dysfunction in mice. Malat1-deficient mice and their wild-type littermates were tested after 8 days of normoxia or hypoxia (10% oxygen). Hypoxia decreased elastance of the lung by increasing lung volume and caused in vivo hyperresponsiveness to methacholine without altering the contraction of airway smooth muscle. Malat1 deficiency also modestly decreased lung elastance but only when tested at low lung volumes and without altering lung volume and airway smooth muscle contraction. The in vivo responsiveness to methacholine was also attenuated by Malat1 deficiency, at least when elastance, a readout sensitive to small airway closure, was used to assess the response. More impressively, in vivo hyperresponsiveness to methacholine caused by hypoxia was virtually absent in Malat1-deficient mice, especially when hysteresivity, a readout sensitive to small airway narrowing heterogeneity, was used to assess the response. Malat1 deficiency also increased the coefficient of oxygen extraction and decreased ventilation in conscious mice, suggesting improvements in gas exchange and in clinical signs of respiratory distress during natural breathing. Combined with a lower elastance at low lung volumes at baseline, as well as a decreased propensity for small airway closure and narrowing heterogeneity during a methacholine challenge, these findings represent compelling evidence suggesting that the lack of Malat1 protects the access to alveoli for air entering the lung.

Smoking status impacts treatment efficacy in smoke-induced lung inflammation: A pre-clinical study.

Rationale: Smoking status and smoking history remain poorly accounted for as variables that could affect the efficacy of new drugs being tested in chronic obstructive pulmonary disease (COPD) patients. As a proof of concept, we used a pre-clinical model of cigarette smoke (CS) exposure to compare the impact of treatment during active CS exposure or during the cessation period on the anti-inflammatory effects IL-1α signaling blockade. Methods: Mice were exposed to CS for 2 weeks, followed by a 1-week cessation, then acutely re-exposed for 2 days. Mice were treated with an anti-IL-1α antibody either during CS exposure or during cessation and inflammatory outcomes were assessed. Results: We found that mice re-exposed to CS displayed reduced neutrophil counts and cytokine levels in the bronchoalveolar lavage (BAL) compared to mice exposed only acutely. Moreover, we found that treatment with an anti-IL-1α antibody during the initial CS exposure delayed inflammatory processes and interfered with pulmonary adaptation, leading to rebound pulmonary neutrophilia, increased BAL cytokine secretion (CCL2) and upregulated Mmp12 expression. Conversely, administration of anti-IL-1α during cessation had the opposite effect, improving BAL neutrophilia, decreasing CCL2 levels and reducing Mmp12 expression. Discussion: These results suggest that pulmonary adaptation to CS exposure dampens inflammation and blocking IL-1α signaling during CS exposure delays the inflammatory response. More importantly, the same treatment administered during cessation hastens the return to pulmonary inflammatory homeostasis, strongly suggesting that smoking status and treatment timing should be considered when testing new biologics in COPD.

Increase in FeNO Levels Following IL5/IL5R-Targeting Therapies in Severe Asthma: A Case Series.

Purpose: Monoclonal antibodies targeting interleukin-5 (IL5) and its receptor (IL5R), used for severe asthma treatment, reduce eosinophils to almost complete depletion. Fractional exhaled nitric oxide (FeNO), a surrogate marker of eosinophilic airway inflammation, is expected to decrease after their initiation. Our center noticed increased FeNO levels in a few patients in whom anti-IL5/IL5R therapy was initiated. Limited data are available on the kinetics of T2 inflammation biomarkers after initiation of a biologic in that population. This study aims to identify if a subgroup of severe asthma patients experiences increased FeNO levels after initiation of anti-IL5/IL5R therapy and to describe their clinical characteristics. Patients and Methods: This is a retrospective case series of 5 patients on Benralizumab (4M:1F) and 8 on Mepolizumab (5M:3F) who showed a significant increase in FeNO (>20% AND >25 ppb) following initiation of an anti-IL5/IL5R treatment. Clinical data, expiratory flows, and inflammation were extracted from the patients' chart at initiation of treatment (T0), 3 months (T1) and 12 months (T2) post-treatment. Descriptive statistics were used. Results: In patients treated with Benralizumab, the increase in FeNO was observed between T0 and T1 (mean delta = 82 ± 72 ppb) with a subsequent decrease (N = 3). In most patients taking Mepolizumab (N = 6), the FeNO increase was observed between T1 and T2 (mean delta = 57 ± 35 ppb). Under treatment, no Benralizumab patient experienced asthma exacerbation while two on Mepolizumab did. All patients had a significant decrease in blood eosinophils. Conclusion: Although initiation of anti-IL5/IL5R may cause a transient rise in FeNO levels in a subgroup of patients, it does not appear to affect clinical outcomes. A compensatory mechanism involving other inflammatory pathways such as IL13 or IL4, both involved in FeNO production, could theoretically explain these findings. Further investigation is needed to elucidate the actual underlying mechanisms.

Recombinant human ß-defensin 2 delivery improves smoking-induced lung neutrophilia and bacterial exacerbation.

Treatment of the cigarette smoke-associated lung diseases, such as chronic obstructive pulmonary disease (COPD), has largely focused on broad-spectrum anti-inflammatory therapies. However, these therapies, such as high-dose inhaled corticosteroids, enhance patient susceptibility to lung infection and exacerbation. Our objective was to assess whether the cationic host defense peptide, human ß-defensin 2 (hBD-2), can simultaneously reduce pulmonary inflammation in cigarette smoke-exposed mice while maintaining immune competence during bacterial exacerbation. Mice were exposed to cigarette smoke acutely (4 days) or chronically (5 days/wk for 7 wk) and administered hBD-2 intranasally or by gavage. In a separate model of acute exacerbation, chronically exposed mice treated with hBD-2 were infected with nontypeable Haemophilus influenzae before euthanasia. In the acute exposure model, cigarette smoke-associated pulmonary neutrophilia was significantly blunted by both local and systemic hBD-2 administration. Similarly, chronically exposed mice administered hBD-2 therapeutically exhibited reduced pulmonary neutrophil infiltration and downregulated proinflammatory signaling in the lungs compared with vehicle-treated mice. Finally, in a model of acute bacterial exacerbation, hBD-2 administration effectively limited neutrophil infiltration in the lungs while markedly reducing pulmonary bacterial load. This study shows that hBD-2 treatment can significantly attenuate lung neutrophilia induced by cigarette smoke exposure while preserving immune competence and promoting an appropriate host-defense response to bacterial stimuli.

HER2 testing in metastatic breast cancer - Is reflex ISH testing necessary on HER2 IHC-equivocal (2+) cases?

Current guidelines recommend HER2 testing on all primary invasive breast cancers and re-biopsy at disease relapse. The discordance rate between HER2-negative primaries and HER2 IHC2+ metastases that are ISH-amplified is unknown. We hypothesize that the majority of such cases are non-amplified. ISH testing is time-consuming and resource-intensive, and there may be situations where it is unnecessary. A retrospective review of IHC2+ metastatic lesions assessed with ISH at our center from 2013 to 2021 was undertaken. 105 cases were identified after exclusion of cases missing HER2 results, with primaries of unconfirmed origin, and cases of synchronous primary and metastatic disease. IHC and ISH results were recorded with detailed slide review of discordant cases. 91/105 metastases had HER2-negative primaries (87%). A metastasis was significantly more likely to be HER2-negative when the primary was HER2-negative (93%) versus positive (43%) (p < 0.0001). 54/91 primaries were IHC2+/ISH-non-amplified, and 50/54 (93%) corresponding metastases had identical results. Of the 37 HER2-negative primaries that were IHC0/1+, 35 (95%) corresponding metastases were ISH-non-amplified. Six metastases in cases with HER2-negative primaries were discordant. Characteristics of metastases suggesting ISH testing was warranted to assess for discordance included IHC heterogeneity, morphological discordance, increased staining of moderate intensity, and ER/PR discordance. One or more of these factors were present in all discordant metastases. Our results suggest selective ISH testing on HER2 IHC2+ breast cancer metastases in the context of HER2-negative primary disease may be appropriate when there is careful review of the IHC. Validation of our findings awaits further studies with larger sample sizes.

Glycerol contained in vaping liquids affects the liver and aspects of energy homeostasis in a sex-dependent manner.

Vaping is increasingly popular among the young and adult population. Vaping liquids contained in electronic cigarettes (e-cigarettes) are mainly composed of propylene glycol and glycerol, to which nicotine and flavors are added. Among several biological processes, glycerol is a metabolic substrate used for lipid synthesis in fed state as well as glucose synthesis in fasting state. We aimed to investigate the effects of glycerol e-cigarette aerosol exposure on the aspects of glycerol and glucose homeostasis. Adult and young male and female mice were exposed to e-cigarette aerosols with glycerol as vaping liquid using an established whole-body exposure system. Mice were exposed acutely (single 2-h exposure) or chronically (2 h/day, 5 days/week for 9 weeks). Circulating glycerol and glucose levels were assessed and glycerol as well as glucose tolerance tests were performed. The liver was also investigated to assess changes in the histology, lipid content, inflammation, and stress markers. Lung functions were also assessed as well as hepatic mRNA expression of genes controlling the circadian rhythm. Acute exposure to glycerol aerosols generated by an e-cigarette increased circulating glycerol levels in female mice. Increased hepatic triglyceride and phosphatidylcholine concentrations were observed in female mice with no increase in circulating alanine aminotransferase or evidence of inflammation, fibrosis, or endoplasmic reticulum stress. Chronic exposure to glycerol e-cigarette aerosols mildly impacted glucose tolerance test in young female and male mice. Fasting glycerol, glucose, and insulin remained unchanged. Increased pulmonary resistance was observed in young male mice. Taken together, this study shows that the glycerol contained in vaping liquids can affect the liver as well as the aspects of glucose and glycerol homeostasis. Additional work is required to translate these observations to humans and determine the biological and potential pathological impacts of these findings.

Revisiting the role of pulmonary surfactant in chronic inflammatory lung diseases and environmental exposure.

Pulmonary surfactant is a crucial and dynamic lung structure whose primary functions are to reduce alveolar surface tension and facilitate breathing. Though disruptions in surfactant homeostasis are typically thought of in the context of respiratory distress and premature infants, many lung diseases have been noted to have significant surfactant abnormalities. Nevertheless, preclinical and clinical studies of pulmonary disease too often overlook the potential contribution of surfactant alterations - whether in quantity, quality or composition - to disease pathogenesis and symptoms. In inflammatory lung diseases, whether these changes are cause or consequence remains a subject of debate. This review will outline 1) the importance of pulmonary surfactant in the maintenance of respiratory health, 2) the diseases associated with primary surfactant dysregulation, 3) the surfactant abnormalities observed in inflammatory pulmonary diseases and, finally, 4) the available research on the interplay between surfactant homeostasis and smoking-associated lung disease. From these published studies, we posit that changes in surfactant integrity and composition contribute more considerably to chronic inflammatory pulmonary diseases and that more work is required to determine the mechanisms underlying these alterations and their potential treatability.

Characterization of a recently synthesized microtubule-targeting compound that disrupts mitotic spindle poles in human cells.

We reveal the effects of a new microtubule-destabilizing compound in human cells. C75 has a core thienoisoquinoline scaffold with several functional groups amenable to modification. Previously we found that sub micromolar concentrations of C75 caused cytotoxicity. We also found that C75 inhibited microtubule polymerization and competed with colchicine for tubulin-binding in vitro. However, here we found that the two compounds synergized suggesting differences in their mechanism of action. Indeed, live imaging revealed that C75 causes different spindle phenotypes compared to colchicine. Spindles remained bipolar and collapsed after colchicine treatment, while C75 caused bipolar spindles to become multipolar. Importantly, microtubules rapidly disappeared after C75-treatment, but then grew back unevenly and from multiple poles. The C75 spindle phenotype is reminiscent of phenotypes caused by depletion of ch-TOG, a microtubule polymerase, suggesting that C75 blocks microtubule polymerization in metaphase cells. C75 also caused an increase in the number of spindle poles in paclitaxel-treated cells, and combining low amounts of C75 and paclitaxel caused greater regression of multicellular tumour spheroids compared to each compound on their own. These findings warrant further exploration of C75's anti-cancer potential.

Adnexectomy by vaginal Natural Orifice Transluminal Endoscopic Surgery versus laparoscopy: results of a first randomised controlled trial (NOTABLE trial).

OBJECTIVE: To compare adnexectomy by vaginal Natural Orifice Transluminal Endoscopic Surgery (vNOTES) versus laparoscopy. DESIGN: Parallel group, 1:1 single-centre single-blinded randomised trial, designed as non-inferiority study with a margin of 15%. SETTING: Belgian teaching hospital. POPULATION: Non-pregnant non-virgin women with an intact uterus and without obliteration of the pouch of Douglas scheduled to undergo removal of an adnexal mass assessed to be benign on ultrasound by IOTA criteria. METHODS: Randomisation to laparoscopy (control group) or vNOTES (experimental group). Stratification according to adnexal size. Blinding of participants and outcome assessors by sham incisions. MAIN OUTCOME MEASURES: The primary outcome measure was adnexectomy by the allocated technique. Secondary outcomes included duration of surgery, pain scores and analgesics used, quality of life and adverse events. RESULTS: We randomly assigned 67 participants (34 to the vNOTES group and 33 to the laparoscopy group). The primary end point was always reached in both groups: there were no conversions. We performed a sensitivity analysis for the primary outcome, assuming one conversion in the vNOTES group and no conversions in the laparoscopy group: the one-sided 95% upper limit for the differences in proportions of conversion was estimated as 13%, which is below the predefined non-inferiority margin of 15%. The secondary outcomes demonstrated a shorter duration of surgery, lower pain scores, lower total dose of analgesics and a trend for more adverse events in the vNOTES group. CONCLUSIONS: vNOTES is non-inferior to laparoscopy for a successful adnexectomy without conversion. vNOTES allowed shorter operating times and less postoperative pain but there was a trend for more adverse events.

CRISPR-Cas tools to study gene function in cytokinesis.

Cytokinesis is the process that separates a cell into two daughter cells at the end of mitosis. Most of our knowledge of cytokinesis comes from overexpression studies, which affects our interpretation of protein function. Gene editing can circumvent this issue by introducing functional mutations or fluorescent probes directly into a gene locus. However, despite its potential, gene editing is just starting to be used in the field of cytokinesis. Here, we discuss the benefits of using gene editing tools for the study of cytokinesis and highlight recent studies that successfully used CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) technology to answer critical questions regarding the function of cytokinesis proteins. We also present methodologies for editing essential genes and discuss how CRISPR interference (CRISPRi) and activation (CRISPRa) can enable precise control of gene expression to answer important questions in the field. Finally, we address the need for gene editing to study cytokinesis in more physiologically relevant contexts. Therefore, this Review provides a roadmap for gene editing to be used in the study of cytokinesis and other cellular processes.