Nanoparticle delivery of VEGF and SDF-1α as an approach for treatment of pulmonary arterial hypertension.

Endothelial dysfunction is an underlying mechanism for the development of pulmonary arterial hypertension (PAH). Vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF) may help repair the dysfunctional endothelium and provide treatment for PAH. To examine this possibility, nanoparticles carrying human recombinant VEGF and SDF (VEGFNP and SDFNP) were aerosolized into the lungs of nude rats at Day 14 after monocrotaline (MCT) injection and analyses were performed at Day 28. The data show that the VEGFNP/SDFNP delivery led to a lower pulmonary arterial pressure and prevented right ventricular hypertrophy in the MCT rats: the right ventricular systolic pressure of the control, MCT, and MCT + VEGFNP/SDFNP treatment groups were 29±2, 70±9, and 44±5 (mean±SD) mmHg, respectively; the pulmonary vascular resistance indices of the groups were 0.6±0.3, 3.2±0.7, and 1.7±0.5, respectively; and the Fulton indices [-RV/(LV + Septum)] were 0.22±0.01, 0.44±0.07, and 0.23±0.02, respectively. The VEGFNP/SDFNP delivery delayed the thickening of distal pulmonary vessels: the number of nearly occluded vessels in a whole lung section from the MCT and MCT + VEGFNP/SDFNP groups were 46±12 and 2±3, respectively. Gene expression analysis of the endothelial cell markers, VE-cadherin, KDR, BMPR2, and eNOS, and smooth cell markers, SM-MHC and α-SMA, indicated significant loss of distal pulmonary vessels in the MCT- treated rats. VEGFNP/SDFNP delivery did not recover the loss, but significantly increased eNOS and decreased α-SMA expression in the MCT-treated lungs. Thus, the therapeutic effect of VEGFNP/SDFNP may be mediated by improving/repairing endothelial function in the PAH lungs.

Regulation of cancer cell ferroptosis by PTRF/Cavin-1.

Ovarian cancer, marked by high rate of recurrence, novel therapeutic strategies are needed to improve patient outcome. One of the potential strategies is inducing ferroptosis in ovarian cancer cells. Ferroptosis is an iron-dependent, lipid peroxidation-driven mode of cell death primarily occurring on the cell membrane. PTRF, an integral component of the caveolae structures located on the cell membrane, is involved in a multitude of physiological processes, including but not limited to, endocytosis, signal transduction, and lipid metabolism. This study elucidates the relationship between PTRF and ferroptosis in ovarian cancer, offering a fresh perspective for the development of new therapeutic strategies. We knocked down PTRF employing siRNA in the ovarian cancer cell lines HEY and SKOV3, following which we stimulated ferroptosis with Erastin (Era). Our research indicates that the lack of PTRF sensitizes cancer cells to ferroptosis, likely by altering membrane stability and tension, thereby affecting signal pathways related to ferroptosis, such as lipid and atherosclerosis, fluid shear stress, and atherosclerosis. Our findings provide new insights for developing new treatments for ovarian cancer.

Identification of IRAK1BP1 as a candidate prognostic factor in lung adenocarcinoma.

Introduction: Lung cancer is one of the major causes of cancer-related mortality worldwide. High-throughput RNA sequencing (RNA-seq) of surgically removed tumors has been used to identify new biomarkers of lung cancer; however, contamination by non-tumor cells in the tumor microenvironment significantly interferes with the search for novel biomarkers. Tumor organoids, as a pre-clinical cancer model, exhibit similar molecular characteristics with tumor samples while minimizing the interference from other cells. Methods and Results: Here we analyzed six RNA-seq datasets collected from different organoid models, in which cells with oncogenic mutations were reprogrammed to mimic lung adenocarcinoma (LUAD) tumorigenesis. We uncovered 9 LUAD-specific biomarker genes by integrating transcriptomic data from multiple sources, and identified IRAK1BP1 as a novel predictor of LUAD disease outcome. Validation with RNA-seq and microarray data collected from multiple patient cohorts, as well as patient-derived xenograft (PDX) and lung cancer cell line models confirmed that IRAK1BP1 expression was significantly lower in tumor cells, and had no correlation with known markers oflung cancer prognosis. In addition, loss of IRAK1BP1 correlated with the group of LUAD patients with worse survival; and gene-set enrichment analysis using tumor and cell line data implicated that high IRAK1BP1 expression was associated with suppression of oncogenic pathways. Discussion: In conclusion, we demonstrate that IRAK1BP1 is a promising biomarker of LUAD prognosis.

HucMSC-EVs Facilitate In Vitro Development of Maternally Aged Preantral Follicles and Oocytes.

Follicle developmental capacity and oocyte quality decline with advanced maternal age. Extracellular vesicles from human umbilical cord mesenchymal stem cells (HucMSC-EVs) act as a potential therapeutic product in the treatment of age-related ovarian dysfunction. In vitro culture (IVC) of preantral follicles is a useful method for understanding the mechanism of follicle development and is a promising means for improving female fertility. However, whether HucMSC-EVs have beneficial effects on aged follicle development during IVC has not yet been reported. Our research demonstrated that follicular development with single-addition withdrawal of HucMSC-EVs was better than that with continuous treatment with HucMSC-EVs. HucMSC-EVs facilitated the survival and growth of follicles, promoted the proliferation of granulosa cells (GCs), and improved the steroid hormone secretion of GCs during IVC of aged follicles. Both GCs and oocytes could uptake HucMSC-EVs. Moreover, we observed elevated cellular transcription in GCs and oocytes after treatment with HucMSC-EVs. The RNA sequencing (RNA-seq) results further validated that the differentially expressed genes are related to the promotion of GC proliferation, cell communication, and oocyte spindle organization. Additionally, the aged oocytes displayed a higher maturation rate, presented less aberrant spindle morphology, and expressed a higher level of the antioxidant protein Sirtuin 1 (SIRT1) after treatment with HucMSC-EVs. Our findings suggested that HucMSC-EVs can improve the growth and quality of aged follicles and oocytes in vitro through the regulation of gene transcription, which provides evidence for HucMSC-EVs as potential therapeutic reagents to restore female fertility with advanced age.

Asymmetric Cell Division and Tumor Heterogeneity.

Asymmetric cell division (ACD) gives rise to two daughter cells with different fates after mitosis and is a fundamental process for generating cell diversity and for the maintenance of the stem cell population. The cancer stem cell (CSC) theory suggests that CSCs with dysregulated self-renewal and asymmetric cell division serve as a source of intra-tumoral heterogeneity. This heterogeneity complicates the diagnosis and treatment of cancer patients, because CSCs can give rise to aggressive clones that are metastatic and insensitive to multiple drugs, or to dormant tumor cells that are difficult to detect. Here, we review the regulatory mechanisms and biological significance of asymmetric division in tumor cells, with a focus on ACD-induced tumor heterogeneity in early tumorigenesis and cancer progression. We will also discuss how dissecting the relationship between ACD and cancer may help us find new approaches for combatting this heterogeneity.

The de Winter electrocardiographic pattern evolves to ST elevation in acute total left main occlusion: A case series.

The de Winter electrocardiographic (ECG) pattern was characterized by upsloping ST-segment depressions, tall and positive symmetrical T waves in precordial leads. This rare ECG pattern was recognized as an indication of proximal left anterior descending artery occlusion. Less commonly, this ECG pattern was reported in association with occlusion of other coronary artery segments. We present three cases of the de Winter pattern associated with acute total left main occlusion. This pattern may evolve to ST elevation within hours of presentation. Widespread upsloping ST-segment depressions from V2 -V6 , centered on V5 were observed in these patients.

TGF-ß Signaling and Resistance to Cancer Therapy.

The transforming growth factor ß (TGF-ß) pathway, which is well studied for its ability to inhibit cell proliferation in early stages of tumorigenesis while promoting epithelial-mesenchymal transition and invasion in advanced cancer, is considered to act as a double-edged sword in cancer. Multiple inhibitors have been developed to target TGF-ß signaling, but results from clinical trials were inconsistent, suggesting that the functions of TGF-ß in human cancers are not yet fully explored. Multiple drug resistance is a major challenge in cancer therapy; emerging evidence indicates that TGF-ß signaling may be a key factor in cancer resistance to chemotherapy, targeted therapy and immunotherapy. Finally, combining anti-TGF-ß therapy with other cancer therapy is an attractive venue to be explored for the treatment of therapy-resistant cancer.

A crosslinked dextran sulfate-chitosan nanoparticle for delivery of therapeutic heparin-binding proteins.

Negatively charged dextran sulfate (DS)-chitosan nanoparticles (DSCS NPs) contain a DS outer shell with binding properties similar to those of heparin and are useful for the incorporation and delivery of therapeutic heparin-binding proteins. These particles, however, are unstable in physiological salt solutions due to their formation through electrostatic interactions. In the present study, a method was developed to covalently crosslink chitosan in the core of the DSCS NP with a short chain dicarboxylic acid (succinate), while leaving the outer shell of the particle untouched. The crosslinked particles, XDSCS NPs, are stable in NaCl solutions up to 3 M. XDSCS NPs were able to incorporate heparin-binding proteins (VEGF and SDF-1α) rapidly and efficiently, and maintain the full biological activity of the proteins. The incorporated proteins were not released from the particles after a 14-day incubation period at 37 °C in PBS, but retained the same activity as those stored at 4 °C. When aerosolized for delivery to the lungs of rats, XDSCS NP-incorporated SDF-1α showed a ∼17-fold greater retention time compared to that of free protein. These properties suggest that XDSCS NPs could be beneficial for the delivery of therapeutic heparin-binding proteins to achieve sustained in vivo effects.

Prognostic Significance of Preprocedural N-Terminal Pro-B-Type Natriuretic Peptide Assessment in Diabetic Patients With Multivessel Coronary Disease Undergoing Revascularization.

Background and Aims: The N-terminal pro-B-type natriuretic peptide (NT-proBNP) may predict adverse cardiovascular outcomes in patients with diabetes. However, its prognostic value in patients with multivessel disease (MVD) undergoing coronary revascularization remains unclear. This study aimed to evaluate the prognostic significance of preprocedural NT-proBNP levels in diabetic patients with MVD undergoing coronary revascularization. Methods: A total of 886 consecutive diabetic patients with MVD who underwent coronary revascularization were enrolled in this study. Patients were divided into quartiles according to their pre-procedural NT-proBNP levels. Kaplan-Meier curves and Cox regression analyses were performed to evaluate the risk of cardiovascular events, including all-cause death, cardiovascular death, myocardial infarction (MI), stroke, and major adverse cardiovascular events (MACE), according to the NT-proBNP quartiles. Results: During a median follow-up period of 4.2 years, 111 patients died (with 82 being caused by cardiovascular disease), 133 had MI, 55 suffered from stroke, and 250 experienced MACE. Kaplan-Meier curves demonstrated that NT-proBNP levels were significantly associated with higher incidences of all-cause death, cardiovascular death, MI, and MACE (log-rank test, P < 0.001, respectively). Multivariate Cox regression analysis revealed that NT-proBNP level was an independent predictor of adverse outcomes, including all-cause death (HR, 1.968; 95% CI, 1.377-2.812; P < 0.001), cardiovascular death (HR, 1.940; 95% CI, 1.278-2.945; P = 0.002), MI (HR, 1.722; 95% CI, 1.247-2.380; P = 0.001), and MACE (HR, 1.356; 95% CI, 1.066-1.725; P = 0.013). The role of NT-proBNP in predicting adverse outcomes was similar in patients with stable angina pectoris and acute coronary syndrome. Moreover, preprocedural NT-proBNP alone discriminated against the SYNTAX II score for predicting all-cause death [area under the curve (AUC), 0.662 vs. 0.626, P = 0.269], cardiovascular death (AUC, 0.680 vs. 0.622, P = 0.130), MI (AUC, 0.641 vs. 0.579, P = 0.050), and MACE (AUC, 0.593 vs. 0.559, P = 0.171). The addition of NT-proBNP to the SYNTAX II score showed a significant net reclassification improvement, integrated discrimination improvement, and improved C-statistic (all P < 0.05). Conclusion: NT-proBNP levels were an independent prognostic marker for adverse outcomes in diabetic patients with MVD undergoing coronary revascularization, suggesting that preprocedural NT-proBNP measurement might help in the risk stratification of high-risk patients.

Retinal Protection by Sustained Nanoparticle Delivery of Oncostatin M and Ciliary Neurotrophic Factor Into Rodent Models of Retinal Degeneration.

Purpose: Retinitis pigmentosa (RP) is caused by mutations in more than 60 genes. Mutation-independent approaches to its treatment by exogeneous administration of neurotrophic factors that will preserve existing retinal anatomy and visual function are a rational strategy. Ciliary neurotrophic factor (CNTF) and oncostatin M (OSM) are two potent survival factors for neurons. However, growth factors degrade rapidly if administered directly. A sustained delivery of growth factors is required for translating their potential therapeutic benefit into patients. Methods: Stable and biocompatible nanoparticles (NP) that incorporated with CNTF and OSM (CNTF- and OSM-NP) were formulated. Both NP-trophic factors were tested in vitro using photoreceptor progenitor cells (PPC) and retinal ganglion progenitor cells (RGPC) derived from induced pluripotent stem cells and in vivo using an optic nerve crush model for glaucoma and the Royal College of Surgeons rat, model of RP (n = 8/treatment) by intravitreal delivery. Efficacy was evaluated by electroretinography and optokinetic response. Retinal histology and a whole mount analysis were performed at the end of experiments. Results: Significant prosurvival and pro-proliferation effects of both complexes were observed in both photoreceptor progenitor cells and RGPC in vitro. Importantly, significant RGC survival and preservation of vision and photoreceptors in both complex-treated animals were observed compared with control groups. Conclusions: These results demonstrate that NP-trophic factors are neuroprotective both in vitro and in vivo. A single intravitreal delivery of both NP-trophic factors offered neuroprotection in animal models of retinal degeneration. Translational Relevance: Sustained nanoparticle delivery of neurotrophic factors may offer beneficial effects in slowing down progressive retinal degenerative conditions, including retinitis pigmentosa, age-related macular degeneration, and glaucoma.

Therapeutic effects of the TST36 stapler on rectocele combined with internal rectal prolapse.

BACKGROUND: The most common causes of outlet obstructive constipation (OOC) are rectocele and internal rectal prolapse. The surgical methods for OOC are diverse and difficult, and the postoperative complications and recurrence rate are high, which results in both physical and mental pain in patients. With the continuous deepening of the surgeon's concept of minimally invasive surgery and continuous in-depth research on the mechanism of OOC, the treatment concepts and surgical methods are continuously improved. AIM: To determine the efficacy of the TST36 stapler in the treatment of rectocele combined with internal rectal prolapse. METHODS: From January 2017 to July 2019, 49 female patients with rectocele and internal rectal prolapse who met the inclusion criteria were selected for treatment using the TST36 stapler. RESULTS: Forty-five patients were cured, 4 patients improved, and the cure rate was 92%. The postoperative obstructed defecation syndrome score, the defecation frequency score, time/straining intensity, and sensation of incomplete evacuation were significantly decreased compared with these parameters before treatment, and the differences were statistically significant (P < 0.05). The postoperative anal canal resting pressure and maximum squeeze pressure in patients decreased compared with before treatment, and the differences were statistically significant (P < 0.05). The initial and maximum defecation thresholds after surgery were significantly lower than those before treatment, and the differences were statistically significant (P < 0.05). The postoperative ratings of rectocele, resting phase, and defecation phase in these patients were significantly decreased compared with those before treatment, and the differences were statistically significant (P < 0.05). CONCLUSION: The TST36 stapler is safe and effective in treating rectocele combined with internal rectal prolapse and is worth promoting in clinical work.

Oligomerization of Lrrk controls actin severing and α-synuclein neurotoxicity in vivo.

BACKGROUND: Mutations in LRRK2 are the most common cause of familial Parkinson's disease and typically cause disease in the context of abnormal aggregation and deposition of α-synuclein within affected brain tissue. METHODS: We combine genetic analysis of Lrrk-associated toxicity in a penetrant Drosophila model of wild type human α-synuclein neurotoxicity with biochemical analyses and modeling of LRRK2 toxicity in human neurons and transgenic mouse models. RESULTS: We demonstrate that Lrrk and α-synuclein interact to promote neuronal degeneration through convergent effects on the actin cytoskeleton and downstream dysregulation of mitochondrial dynamics and function. We find specifically that monomers and dimers of Lrrk efficiently sever actin and promote normal actin dynamics in vivo. Oligomerization of Lrrk, which is promoted by dominant Parkinson's disease-causing mutations, reduces actin severing activity in vitro and promotes excess stabilization of F-actin in vivo. Importantly, a clinically protective Lrrk mutant reduces oligomerization and α-synuclein neurotoxicity. CONCLUSIONS: Our findings provide a specific mechanistic link between two key molecules in the pathogenesis of Parkinson's disease, α-synuclein and LRRK2, and suggest potential new approaches for therapy development.

NEDD9 Is a Novel and Modifiable Mediator of Platelet-Endothelial Adhesion in the Pulmonary Circulation.

Rationale: Data on the molecular mechanisms that regulate platelet-pulmonary endothelial adhesion under conditions of hypoxia are lacking, but may have important therapeutic implications. Objectives: To identify a hypoxia-sensitive, modifiable mediator of platelet-pulmonary artery endothelial cell adhesion and thrombotic remodeling. Methods: Network medicine was used to profile protein-protein interactions in hypoxia-treated human pulmonary artery endothelial cells. Data from liquid chromatography-mass spectrometry and microscale thermophoresis informed the development of a novel antibody (Ab) to inhibit platelet-endothelial adhesion, which was tested in cells from patients with chronic thromboembolic pulmonary hypertension (CTEPH) and three animal models in vivo. Measurements and Main Results: The protein NEDD9 was identified in the hypoxia thrombosome network in silico. Compared with normoxia, hypoxia (0.2% O2) for 24 hours increased HIF-1α (hypoxia-inducible factor-1α)-dependent NEDD9 upregulation in vitro. Increased NEDD9 was localized to the plasma-membrane surface of cells from control donors and patients with CTEPH. In endarterectomy specimens, NEDD9 colocalized with the platelet surface adhesion molecule P-selectin. Our custom-made anti-NEDD9 Ab targeted the NEDD9-P-selectin interaction and inhibited the adhesion of activated platelets to pulmonary artery endothelial cells from control donors in vitro and from patients with CTEPH ex vivo. Compared with control mice, platelet-pulmonary endothelial aggregates and pulmonary hypertension induced by ADP were decreased in NEDD9-/- mice or wild-type mice treated with the anti-NEDD9 Ab, which also decreased chronic pulmonary thromboembolic remodeling in vivo. Conclusions: The NEDD9-P-selectin protein-protein interaction is a modifiable target with which to inhibit platelet-pulmonary endothelial adhesion and thromboembolic vascular remodeling, with potential therapeutic implications for patients with disorders of increased hypoxia signaling pathways, including CTEPH.

Comparing Clinicopathologic Features and Surgical Treatment of Premenopausal Breast Cancer across Italy and China: Report from a Medical Exchange Program.

BACKGROUND: This study investigated the differences in clinicopathologic features and surgical treatment between an Italian and a Chinese cohort of premenopausal women with breast cancer, and highlighted the potential advantages of international medical exchange projects. METHODS: Premenopausal women who underwent surgical treatment between 2012 and 2016 at one Italian and one Chinese institution participating in a medical exchange program were compared. Factors associated with the probability to receive mastectomy were determined via logistic analysis. Changes in surgical management at the Chinese institution in the period 2018-2019, after the exchange program, were also evaluated. RESULTS: A total of 505 patients, 318 from Italy and 187 from China, were evaluated. The Chinese patients had more frequently advanced-stage tumours, large tumour size (30.9 vs. 18.1 mm, p < 0.01), invasive carcinoma (92.5 vs. 83.3%, p < 0.01), positive axillary lymph nodes (54.5 vs. 27.4%, p < 0.01), Her-2 positivity (36.4 vs. 22.0%, p < 0.01), and high proliferative index (55.1 vs. 30.2%, p < 0.01). Positive oestrogen receptor status and rates of triple-negative breast cancer did not differ (77.0 vs. 69.5%, p = 0.09 and 14.2 vs. 16%, p = 0.56, respectively). Mastectomy rates were higher among Chinese women (85 vs. 41%, p < 0.001), whereas use of sentinel node biopsy was more frequent among Italian women (77 vs. 33%, p < 0.001). Chinese women had more than 4-fold higher risk of receiving mastectomy. In the last 2 years, the rates of breast-conserving surgery and sentinel node biopsy at the Chinese institution increased from 15 to 23%, and from 33 to 42%, respectively. CONCLUSIONS: Tumour features and surgical strategies for premenopausal breast cancer may differ significantly between Italy and China. Since the international exchange program, patients from the Chinese institution have been offered more frequently less invasive surgery. International exchange programs can help in designing epidemiological studies which may be useful for strategies to improve breast cancer management and control.

Triglyceride-glucose index predicts adverse cardiovascular events in patients with diabetes and acute coronary syndrome.

BACKGROUND: The triglyceride-glucose index (TyG index) has been regarded as a reliable alternative marker of insulin resistance and an independent predictor of cardiovascular outcomes. Whether the TyG index predicts adverse cardiovascular events in patients with diabetes and acute coronary syndrome (ACS) remains uncertain. The aim of this study was to investigate the prognostic value of the TyG index in patients with diabetes and ACS. METHODS: A total of 2531 consecutive patients with diabetes who underwent coronary angiography for ACS were enrolled in this study. Patients were divided into tertiles according to their TyG index. The primary outcomes included the occurrence of major adverse cardiovascular events (MACEs), defined as all-cause death, non-fatal myocardial infarction and non-fatal stroke. The TyG index was calculated as the ln (fasting triglyceride level [mg/dL] × fasting glucose level [mg/dL]/2). RESULTS: The incidence of MACE increased with TyG index tertiles at a 3-year follow-up. The Kaplan-Meier curves showed significant differences in event-free survival rates among TyG index tertiles (P = 0.005). Multivariate Cox hazards regression analysis revealed that the TyG index was an independent predictor of MACE (95% CI 1.201-1.746; P < 0.001). The optimal TyG index cut-off for predicting MACE was 9.323 (sensitivity 46.0%; specificity 63.6%; area under the curve 0.560; P = 0.001). Furthermore, adding the TyG index to the prognostic model for MACE improved the C-statistic value (P = 0.010), the integrated discrimination improvement value (P = 0.001) and the net reclassification improvement value (P = 0.019). CONCLUSIONS: The TyG index predicts future MACE in patients with diabetes and ACS independently of known cardiovascular risk factors, suggesting that the TyG index may be a useful marker for risk stratification and prognosis in patients with diabetes and ACS.

Type 2 diabetes impacts colorectal adenoma detection in screening colonoscopy.

BACKGROUND: Diabetes is associated with an increased risk of colorectal cancer (CRC). We conducted a retrospective analysis of adenoma detection rates (ADR) in initial screening colonoscopies to further investigate the role of diabetes in adenoma detection. METHODS: A chart review was performed on initial average risk screening colonoscopies (ages 45-75) during 2012-2015. Data collected included basic demographics, insurance, BMI, family history of CRC, smoking, diabetes, and aspirin use. Multivariable generalized linear mixed models for binary outcomes were used to examine the relationship between diabetes and variables associated with CRC risk and ADR. RESULTS: Of 2865 screening colonoscopies, 282 were performed on patients with type 2 diabetes (T2DM). Multivariable analysis suggested that T2DM (OR = 1.49, 95% CI:1.13-1.97, p = 0.0047) was associated with an increased ADR, as well as smoking, older age, higher BMI and male sex (all p < 0.05). For patients with T2DM, those not taking diabetes medications were more likely to have an adenoma than those taking medication (OR = 2.38, 95% CI:1.09-5.2, p = 0.03). CONCLUSION: T2DM has an effect on ADR after controlling for multiple confounding variables. Early interventions for prevention of T2DM and prescribing anti-diabetes medications may reduce development of colonic adenomas and may contribute to CRC prevention.

Reaction rate of pyruvate and hydrogen peroxide: assessing antioxidant capacity of pyruvate under biological conditions.

Pyruvate, a pivotal glucose metabolite, is an α-ketoacid that reacts with hydrogen peroxide (H2O2). Its pharmacological precursor, ethyl pyruvate, has shown anti-inflammatory/anti-tissue injury effects in various animal models of disease, but failed in a multicenter clinical trial. Since rodents, but not humans, can convert ethyl pyruvate to pyruvate in blood plasma, this additional source of extracellular pyruvate may have contributed to the discrepancy between the species. To examine this possibility, we investigated the kinetics of the reaction under biological conditions and determined the second order rate constant k as 2.360 ± 0.198 M-1 s-1. We then calculated the time required for H2O2 elimination by pyruvate. The results show that, with an average intracellular concentration of pyruvate (150 µM), elimination of 95% H2O2 at normal to pathological concentrations (0.01-50 µM) requires 141-185 min (2.4-3 hour). With 1,000 µM pyruvate, a concentration that can only exist extracellularly or in cell culture media, 95% elimination of H2O2 at 5-200 µM requires 21-25 min. We conclude that intracellular pyruvate, or other α-ketoacids, whose endogenous concentration is controlled by metabolism, have little role in H2O2 clearance. An increased extracellular concentration of pyruvate, however, does have remarkable peroxide scavenging effects, considering minimal peroxidase activity in this space.

Development and validation of the PET-CT score for diagnosis of malignant pleural effusion.

PURPOSE: Although some parameters of positron emission tomography with 18F-fluorodeoxyglucose (18F-FDG) and computed tomography (PET-CT) are somehow helpful in differentiating malignant pleural effusion (MPE) from benign effusions, no individual parameter offers sufficient evidence for its implementation in the clinical practice. The aim of this study was to establish the diagnostic accuracy of a scoring system based on PET-CT (the PET-CT score) in diagnosing MPE. METHODS: One prospective derivation cohort of patients with pleural effusions (84 malignant and 115 benign) was used to develop the PET-CT score for the differential diagnosis of malignant pleural effusion. The PET-CT score was then validated in another independent prospective cohort (n = 74). RESULTS: The PET-CT parameters developed for discriminating MPE included unilateral lung nodules and/or masses with increased 18F-FDG uptake (3 points); extrapulmonary malignancies (3 points); pleural thickening with increased 18F-FDG uptake (2 points); multiple nodules or masses (uni- or bilateral lungs) with increased 18F-FDG uptake (1 point); and increased pleural effusion 18F-FDG uptake (1 point). With a cut-off value of 4 points in the derivation cohort, the area under the curve, sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio of the PET-CT score to diagnose MPE were 0.949 (95% CI: 0.908-0.975), 83.3% (73.6%-90.6%), 92.2% (85.7%-96.4%), 10.7 (5.6-20.1), and 0.2 (0.1-0.3), respectively. CONCLUSIONS: A simple-to-use PET-CT score that uses PET-CT parameters was developed and validated. The PET-CT score can help physicians to differentiate MPE from benign pleural effusions.

Isolating pulmonary microvascular endothelial cells ex vivo: Implications for pulmonary arterial hypertension, and a caution on the use of commercial biomaterials.

Transcriptomic analysis of pulmonary microvascular endothelial cells from experimental models offers insight into pulmonary arterial hypertension (PAH) pathobiology. However, culturing may alter the molecular profile of endothelial cells prior to analysis, limiting the translational relevance of results. Here we present a novel and validated method for isolating RNA from pulmonary microvascular endothelial cells (PMVECs) ex vivo that does not require cell culturing. Initially, presumed rat PMVECs were isolated from rat peripheral lung tissue using tissue dissociation and enzymatic digestion, and cells were cultured until confluence to assess endothelial marker expression. Anti-CD31, anti-von Willebrand Factor, and anti-α-smooth muscle actin immunocytochemistry/immunofluorescence signal was detected in presumed rat PMVECs, but also in non-endothelial cell type controls. By contrast, flow cytometry using an anti-CD31 antibody and isolectin 1-B4 (from Griffonia simplicifolia) was highly specific for rat PMVECs. We next developed a strategy in which the addition of an immunomagnetic selection step for CD31+ cells permitted culture-free isolation of rat PMVECs ex vivo for RNA isolation and transcriptomic analysis using fluorescence-activated cell sorting. Heterogeneity in the validity and reproducibility of results using commercial antibodies against endothelial surface markers corresponded to a substantial burden on laboratory time, labor, and scientific budget. We demonstrate a novel protocol for the culture-free isolation and transcriptomic analysis of rat PMVECs with translational relevance to PAH. In doing so, we highlight wide variability in the quality of commonly used biological reagents, which emphasizes the importance of investigator-initiated validation of commercial biomaterials.