An evaluation of sorter induced cell stress (SICS) on peripheral blood mononuclear cells (PBMCs) after different sort conditions - are your sorted cells getting SICS?

Flow cytometry and fluorescence-activated cell sorting have become invaluable tools to analyze and isolate specific cell populations in a wide range of biomedical research and clinical applications. In countless approaches worldwide, scientists are using single cell analyses to better understand the significance and variation within different cellular populations, and fluorescence-activated cell sorting has become a major technique for cell isolation in both basic and clinical research. However, majority of available cell sorters are pressurized, droplet-based systems, which apply significant environmental pressure and shear stress to cells during sorting. Recently, the flow cytometry community has become increasingly aware about the potential negative effects this could have on sorted cells and the term "sorter induced cell stress" (SICS) has been proposed. However, up to date only a limited number of studies have investigated the effects of cell sorting on cell viability and function. Therefore, solid data on the effects of sheath pressure and nozzle size on survival and function of sorted cells are surprisingly rare. With this in mind, we sorted "CD4 " T-cells and "live" cells from human peripheral blood mononuclear cells (PBMCs) at different sort conditions and analyzed their quality before and after sorting in a series of assays. Here we present our findings in reference to cell viability and cell proliferation following sorting on different instruments (BD FACSAria III SORP and BD FACSJazz), utilizing different nozzle sizes (70 to 100 μm) and sheath pressure settings (20 to 70 psi). The results show no significant differences in cell viability and proliferation after the different tested sort conditions, but rather differences between individual experiments. These findings are evaluated and their potential significance in cell sorting experiments is discussed. [Abstract copyright: Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.

Chocolate consumption and incident heart failure

BACKGROUND: We aimed to examine the association between chocolate intake and the risk of incident heart failure in a UK general population. We conducted a systematic review and meta-analysis to quantify this association. METHODS AND RESULTS: We used data from a prospective population-based study, the European Prospective Investigation into Cancer (EPIC)-Norfolk cohort. Chocolate intake was quantified based on a food frequency questionnaire obtained at baseline (1993-1997) and incident heart failure was ascertained up to March 2009. We supplemented the primary data with a systematic review and meta-analysis of studies which evaluated risk of incident heart failure with chocolate consumption. A total of 20,922 participants (53% women; mean age 58 ± 9 years) were included of whom 1101 developed heart failure during the follow up (mean 12.5 ± 2.7 years, total person years 262,291 years). After adjusting for lifestyle and dietary factors, we found 19% relative reduction in heart failure incidence in the top (up to 100 g/d) compared to the bottom quintile of chocolate consumption (HR 0.81 95%CI 0.66-0.98) but the results were no longer significant after controlling for comorbidities (HR 0.87 95%CI 0.71-1.06). Additional adjustment for potential mediators did not attenuate the results further. We identified five relevant studies including the current study (N = 75,408). The pooled results showed non-significant 19% relative risk reduction of heart failure incidence with higher chocolate consumption (HR 0.81 95%CI 0.66-1.01). CONCLUSIONS: Our results suggest that higher chocolate intake is not associated with subsequent incident heart failure.The EPIC-Norfolk study was supported by grants from the Medical Research Council and Cancer Research UK.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.numecd.2016.01.00

A Summary of the Inaugural WHO Classification of Pediatric Tumors: Transitioning from the Optical into the Molecular Era

Pediatric tumors are uncommon, yet are the leading cause of cancer-related death in childhood. Tumor types, molecular characteristics, and pathogenesis are unique, often originating from a single genetic driver event. The specific diagnostic challenges of childhood tumors led to the development of the first World Health Organization (WHO) Classification of Pediatric Tumors. The classification is rooted in a multilayered approach, incorporating morphology, IHC, and molecular characteristics. The volume is organized according to organ sites and provides a single, state-of-the-art compendium of pediatric tumor types. A special emphasis was placed on “blastomas,” which variably recapitulate the morphologic maturation of organs from which they originate

Birthweight and risk markers for type 2 diabetes and cardiovascular disease in childhood: the Child Heart and Health Study in England (CHASE).

AIMS/HYPOTHESIS: Lower birthweight (a marker of fetal undernutrition) is associated with higher risks of type 2 diabetes and cardiovascular disease (CVD) and could explain ethnic differences in these diseases. We examined associations between birthweight and risk markers for diabetes and CVD in UK-resident white European, South Asian and black African-Caribbean children. METHODS: In a cross-sectional study of risk markers for diabetes and CVD in 9- to 10-year-old children of different ethnic origins, birthweight was obtained from health records and/or parental recall. Associations between birthweight and risk markers were estimated using multilevel linear regression to account for clustering in children from the same school. RESULTS: Key data were available for 3,744 (66%) singleton study participants. In analyses adjusted for age, sex and ethnicity, birthweight was inversely associated with serum urate and positively associated with systolic BP. After additional height adjustment, lower birthweight (per 100 g) was associated with higher serum urate (0.52%; 95% CI 0.38, 0.66), fasting serum insulin (0.41%; 95% CI 0.08, 0.74), HbA1c (0.04%; 95% CI 0.00, 0.08), plasma glucose (0.06%; 95% CI 0.02, 0.10) and serum triacylglycerol (0.30%; 95% CI 0.09, 0.51) but not with BP or blood cholesterol. Birthweight was lower among children of South Asian (231 g lower; 95% CI 183, 280) and black African-Caribbean origin (81 g lower; 95% CI 30, 132). However, adjustment for birthweight had no effect on ethnic differences in risk markers. CONCLUSIONS/INTERPRETATION: Birthweight was inversely associated with urate and with insulin and glycaemia after adjustment for current height. Lower birthweight does not appear to explain emerging ethnic difference in risk markers for diabetes

Methylation array profiling of adult brain tumours: diagnostic outcomes in a large, single centre

The introduction of the classification of brain tumours based on their DNA methylation profile has significantly changed the diagnostic approach for cases with ambiguous histology, non-informative or contradictory molecular profiles or for entities where methylation profiling provides useful information for patient risk stratification, for example in medulloblastoma and ependymoma. We present our experience that combines a conventional molecular diagnostic approach with the complementary use of a DNA methylation-based classification tool, for adult brain tumours originating from local as well as national referrals. We report the frequency of IDH mutations in a large cohort of nearly 1550 patients, EGFR amplifications in almost 1900 IDH-wildtype glioblastomas, and histone mutations in 70 adult gliomas. We demonstrate how additional methylation-based classification has changed and improved our diagnostic approach. Of the 325 cases referred for methylome testing, 179 (56%) had a calibrated score of 0.84 and higher and were included in the evaluation. In these 179 samples, the diagnosis was changed in 45 (25%), refined in 86 (48%) and confirmed in 44 cases (25%). In addition, the methylation arrays contain copy number information that usefully complements the methylation profile. For example, EGFR amplification which is 95% concordant with our Real-Time PCR-based copy number assays. We propose here a diagnostic algorithm that integrates histology, conventional molecular tests and methylation arrays

Cellular development and evolution of the mammalian cerebellum

\ua9 2023, The Author(s).The expansion of the neocortex, a hallmark of mammalian evolution 1,2, was accompanied by an increase in cerebellar neuron numbers 3. However, little is known about the evolution of the cellular programmes underlying the development of the cerebellum in mammals. In this study we generated single-nucleus RNA-sequencing data for around 400,000 cells to trace the development of the cerebellum from early neurogenesis to adulthood in human, mouse and the marsupial opossum. We established a consensus classification of the cellular diversity in the developing mammalian cerebellum and validated it by spatial mapping in the fetal human cerebellum. Our cross-species analyses revealed largely conserved developmental dynamics of cell-type generation, except for Purkinje cells, for which we observed an expansion of early-born subtypes in the human lineage. Global transcriptome profiles, conserved cell-state markers and gene-expression trajectories across neuronal differentiation show that cerebellar cell-type-defining programmes have been overall preserved for at least 160 million years. However, we also identified many orthologous genes that gained or lost expression in cerebellar neural cell types in one of the species or evolved new expression trajectories during neuronal differentiation, indicating widespread gene repurposing at the cell-type level. In sum, our study unveils shared and lineage-specific gene-expression programmes governing the development of cerebellar cells and expands our understanding of mammalian brain evolution

Anti-inflammatory and antioxidant effects of Tualang honey in alkali injury on the eyes of rabbits: Experimental animal study

<p>Abstract</p> <p>Background</p> <p>Alkali injury is one of the most devastating injuries to the eye. It results in permanent unilateral or bilateral visual impairment. Chemical eye injury is accompanied by an increase in the oxidative stress. Anti-inflammatory and antioxidant agents play a major role in the treatment of chemical eye injuries. The purpose of this study is to evaluate the anti-inflammatory (clinical and histopathological) and antioxidant effects of Tualang honey versus conventional treatment in alkali injury on the eyes of rabbits.</p> <p>Methods</p> <p>A preliminary study was carried out prior to the actual study to establish the alkali chemical injury on rabbit's cornea and we found that alkali chemical injury with 2 N NaOH showed severe clinical inflammatory features. In actual study, alkali injury with 2 N NaOH was induced in the right eye of 10 New Zealand White rabbits' cornea. The rabbits were divided into two groups, Group A was given conventional treatment and Group B was treated with both topical and oral Tualang honey. Clinical inflammatory features of the right eye were recorded at 12 hours, 24 hours, 72 hours, 5^thday and 7^thday post induction of alkali burn on the cornea. The histopathological inflammatory features of the right corneas of all rabbits were also evaluated on day-7. The level of total antioxidant status and lipid peroxidation products in the aqueous humour, vitreous humour and serum at day-7 were estimated biochemically. Fisher's Exact, Chi-Square and Mann-Whitney test were used to analyse the data.</p> <p>Results</p> <p>There was no statistically significant difference in clinical inflammatory features (p > 0.05) between honey treated and the conventional treated group at different times of examination. Histopathological examination of the cornea showed the number of polymorphonuclear leucocytes was below 50 for both groups (mild grade). There was also no significant difference in the level of total antioxidant status as well as lipid peroxidation products in aqueous humour (p = 0.117, p = 0.382 respectively), vitreous humour (p = 0.917, p = 0.248 respectively) and serum (p = 0.917, p = 0.332 respectively) between honey treated and the conventional treated group.</p> <p>Conclusion</p> <p>Tualang honey has almost the equal effects when compared with the conventional treatment in treating alkali injury on rabbit's eye. Future research with more number of rabbits and control group is warranted to explore the anti-inflammatory and antioxidant effects of Tualang honey.</p

Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility.

BACKGROUND: Gametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown. METHODS: Plasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163. RESULTS: 615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway. CONCLUSIONS: This study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization

N-α-PGP and PGP, potential biomarkers and therapeutic targets for COPD

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