Cellular origin and microRNA profiles of circulating extracellular vesicles in different stages of diabetic nephropathy

Background: Diabetic nephropathy (DN) is a major complication of diabetes and the main cause of end-stage renal disease. Extracellular vesicles (EVs) are small cell-derived vesicles that can alter disease progression by microRNA (miRNA) transfer. Methods: In this study, we aimed to characterize the cellular origin and miRNA content of EVs in plasma samples of type 2 diabetes patients at various stages of DN. Type 2 diabetes patients were classified in three groups: normoalbuminuria, microalbuminuria and macroalbuminuria. The concentration and cellular origin of plasma EVs were measured by flow cytometry. A total of 752 EV miRNAs were profiled in 18 subjects and differentially expressed miRNAs were validated. Results: Diabetic patients with microalbuminuria and/or macroalbuminuria showed elevated concentrations of total EVs and EVs from endothelial cells, platelets, leucocytes and erythrocytes compared with diabetic controls. miR-99a-5p was upregulated in macroalbuminuric patients compared with normoalbuminuric and microalbuminuric patients. Transfection of miR-99a-5p in cultured human podocytes downregulated mammalian target of rapamycin (mTOR) protein expression and downregulated the podocyte injury marker vimentin. Conclusions: Type 2 diabetes patients with microalbuminuria and macroalbuminuria display differential EV profiles. miR-99a-5p expression is elevated in EVs from macroalbuminuria and mTOR is its validated mRNA target

Conserved developmental trajectories of the cecal microbiota of broiler chickens in a field study

There is great interest in identifying gut microbiota development patterns and underlying assembly rules that can inform strategies to improve broiler health and performance. Microbiota stratification using community types helps to simplify complex and dynamic ecosystem principles of the intestinal microbiota. This study aimed to identify community types to increase insight in intestinal microbiota variation between broilers and to identify factors that explain this variation. A total of 10 well-performing poultry flocks on four farms were followed. From each flock, the cecal content of nine broilers was collected at 7, 14, and 35 days posthatch. A total of two robust community types were observed using different clustering methods, one of which was dominated by 7-day-old broilers, and one by 35-day-old broilers. Broilers, 14-day-old, were divided across both community types. This is the first study that showed conserved cecal microbiota development trajectories in commercial broiler flocks. In addition to the temporal development with age, the cecal microbiota variation between broilers was explained by the flock, body weight, and the different feed components. Our data support a conserved development of cecal microbiota, despite strong influence of environmental factors. Further investigation of mechanisms underlying microbiota development and function is required to facilitate intestinal health promoting management, diagnostics, and nutritional interventions

Evaluation of a field-deployable Nafion (TM)-based air-drying system for collecting whole air samples and its application to stable isotope measurements of CO2

Atmospheric flask samples are either collected at atmospheric pressure by opening a valve of a pre-evacuated flask or pressurized with the help of a pump to a few bar above ambient pressure. Under humid conditions, there is a risk that water vapor in the sample leads to condensation on the walls of the flask, notably at higher than ambient sampling pressures. Liquid water in sample flasks is known to affect the CO2 mixing ratios and also alters the isotopic composition of oxygen (17O and 18O) in CO2 via isotopic equilibration. Hence, for accurate determination of CO2 mole fractions and its stable isotopic composition, it is vital to dry the air samples to a sufficiently low dew point before they are pressurized in flasks to avoid condensation. Moreover, the drying system itself should not influence the mixing ratio and the isotopic composition of CO2 or that of the other constituents under study. For the Airborne Stable Isotopes of Carbon from the Amazon (ASICA) project focusing on accurate measurements of CO2 and its singly substituted stable isotopologues over the Amazon, an air-drying system capable of removing water vapor from air sampled at a dew point lower than -2 °C, flow rates up to 12 L min-1 and without the need for electrical power was needed. Since to date no commercial air-drying device that meets these requirements has been available, we designed and built our own consumable-free, power-free and portable drying system based on multitube Nafion™ gas sample driers (Perma Pure, Lakewood, USA). The required dry purge air is provided by feeding the exhaust flow of the flask sampling system through a dry molecular sieve (type 3A) cartridge. In this study we describe the systematic evaluation of our Nafion™-based air sample dryer with emphasis on its performance concerning the measurements of atmospheric CO2 mole fractions and the three singly substituted isotopologues of CO2 (16O13C16O, 16O12C17O and 16O12C18O), as well as the trace gas species CH4, CO, N2O and SF6. Experimental results simulating extreme tropical conditions (saturated air at 33 °C) indicated that the response of the air dryer is almost instantaneous and that approximately 85 L of air, containing up to 4 % water vapor, can be processed staying below a -2 °C dew point temperature (at 275 kPa). We estimated that at least eight flasks can be sampled (at an overpressure of 275 kPa) with a water vapor content below -2 °C dew point temperature during a typical flight sampling up to 5 km altitude over the Amazon, whereas the remaining samples would stay well below 5 °C dew point temperature (at 275 kPa). The performance of the air dryer on measurements of CO2, CH4, CO, N2O, and SF6 and the CO2 isotopologues 16O13C16O and 16O12C18O was tested in the laboratory simulating real sampling conditions by compressing humidified air from a calibrated cylinder, after being dried by the air dryer, into sample flasks. We found that the mole fraction and the isotopic composition difference between the different test conditions (including the dryer) and the base condition (dry air, without dryer) remained well within or very close to, in the case of N2O, the World Meteorological Organization recommended compatibility goals for independent measurement programs, proving that the test condition induced no significant bias on the sample measurements

Diurnal variability of atmospheric O-2, CO2, and their exchange ratio above a boreal forest in southern Finland

The exchange ratio (ER) between atmospheric O(2 )and CO2 is a useful tracer for better understanding the carbon budget on global and local scales. The variability of ER (in mol O(2 )per mol CO2) between terrestrial ecosystems is not well known, and there is no consensus on how to derive the ER signal of an ecosystem, as there are different approaches available, either based on concentration (ERatmos) or flux measurements (ERforest). In this study we measured atmospheric O-2 and CO2 concentrations at two heights (23 and 125 m) above the boreal forest in Hyytiala, Finland. Such measurements of O-2 are unique and enable us to potentially identify which forest carbon loss and production mechanisms dominate over various hours of the day. We found that the ERatmos signal at 23 m not only represents the diurnal cycle of the forest exchange but also includes other factors, including entrainment of air masses in the atmospheric boundary layer before midday, with different thermodynamic and atmospheric composition characteristics. To derive ERforest, we infer O(2 )fluxes using multiple theoretical and observation-based micro-meteorological formulations to determine the most suitable approach. Our resulting ERforest shows a distinct difference in behaviour between daytime (0.92 +/- 0.17 mol mol(-1)) and nighttime (1.03 +/- 0.05 mol mol(-1)). These insights demonstrate the diurnal variability of different ER signals above a boreal forest, and we also confirmed that the signals of ERatmos and ERforest cannot be used interchangeably. Therefore, we recommend measurements on multiple vertical levels to derive O-2 and CO2 fluxes for the ERforest signal instead of a single level time series of the concentrations for the ERatmos signal. We show that ERforest can be further split into specific signals for respiration (1.03 +/-; 0.05 mol mol-1) and photosynthesis (0.96 +/- 0.12 molmol(-1)). This estimation allows us to separate the net ecosystem exchange (NEE) into gross primary production (GPP) and total ecosystem respiration (TER), giving comparable results to the more commonly used eddy covariance approach. Our study shows the potential of using atmospheric O-2 as an alternative and complementary method to gain new insights into the different CO2 signals that contribute to the forest carbon budget.Peer reviewe

Segmentation of diagnostic tissue compartments on whole slide images with renal thrombotic microangiopathies (TMAs)

The thrombotic microangiopathies (TMAs) manifest in renal biopsy histology with a broad spectrum of acute and chronic findings. Precise diagnostic criteria for a renal biopsy diagnosis of TMA are missing. As a first step towards a machine learning- and computer vision-based analysis of wholes slide images from renal biopsies, we trained a segmentation model for the decisive diagnostic kidney tissue compartments artery, arteriole, glomerulus on a set of whole slide images from renal biopsies with TMAs and Mimickers (distinct diseases with a similar nephropathological appearance as TMA like severe benign nephrosclerosis, various vasculitides, Bevacizumab-plug glomerulopathy, arteriolar light chain deposition disease). Our segmentation model combines a U-Net-based tissue detection with a Shifted windows-transformer architecture to reach excellent segmentation results for even the most severely altered glomeruli, arterioles and arteries, even on unseen staining domains from a different nephropathology lab. With accurate automatic segmentation of the decisive renal biopsy compartments in human renal vasculopathies, we have laid the foundation for large-scale compartment-specific machine learning and computer vision analysis of renal biopsy repositories with TMAs.Comment: 12 pages, 3 figure

The influence of the accessory genome on bacterial pathogen evolution

Bacterial pathogens exhibit significant variation in their genomic content of virulence factors. This reflects the abundance of strategies pathogens evolved to infect host organisms by suppressing host immunity. Molecular arms-races have been a strong driving force for the evolution of pathogenicity, with pathogens often encoding overlapping or redundant functions, such as type III protein secretion effectors and hosts encoding ever more sophisticated immune systems. The pathogens’ frequent exposure to other microbes, either in their host or in the environment, provides opportunities for the acquisition or interchange of mobile genetic elements. These DNA elements accessorise the core genome and can play major roles in shaping genome structure and altering the complement of virulence factors. Here, we review the different mobile genetic elements focusing on the more recent discoveries and highlighting their role in shaping bacterial pathogen evolution

Effect of TREM-1 blockade and single nucleotide variants in experimental renal injury and kidney transplantation

Renal ischemia reperfusion (IR)-injury induces activation of innate immune response which sustains renal injury and contributes to the development of delayed graft function (DGF). Triggering receptor expressed on myeloid cells-1 (TREM-1) is a pro-inflammatory evolutionary conserved pattern recognition receptor expressed on a variety of innate immune cells. TREM-1 expression increases following acute and chronic renal injury. However, the function of TREM-1 in renal IR is still unclear. Here, we investigated expression and function of TREM-1 in a murine model of renal IR using different TREM-1 inhibitors: LP17, LR12 and TREM-1 fusion protein. In a human study, we analyzed the association of non-synonymous single nucleotide variants in the TREM1 gene in a cohort comprising 1263 matching donors and recipients with post-transplant outcomes, including DGF. Our findings demonstrated that, following murine IR, renal TREM-1 expression increased due to the influx of Trem1 mRNA expressing cells detected by in situ hybridization. However, TREM-1 interventions by means of LP17, LR12 and TREM-1 fusion protein did not ameliorate IR-induced injury. In the human renal transplant cohort, donor and recipient TREM1 gene variant p. Thr25Ser was not associated with DGF, nor with biopsy-proven rejection or death-censored graft failure. We conclude that TREM-1 does not play a major role during experimental renal IR and after kidney transplantation

Biopsy-Controlled Non-Invasive Quantification of Collagen Type VI in Kidney Transplant Recipients:A Post-Hoc Analysis of the MECANO Trial

The PRO-C6 assay, a reflection of collagen type VI synthesis, has been proposed as a non-invasive early biomarker of kidney fibrosis. We aimed to investigate cross-sectional and longitudinal associations between plasma and urine PRO-C6 and proven histological changes after kidney transplantation. The current study is a post-hoc analysis of 94 participants of the MECANO trial, a 24-month prospective, multicenter, open-label, randomized, controlled trial aimed at comparing everolimus-based vs. cyclosporine-based immunosuppression. PRO-C6 was measured in plasma and urine samples collected 6 and 24 months post-transplantation. Fibrosis was evaluated in biopsies collected at the same time points by Banff interstitial fibrosis/tubular atrophy (IF/TA) scoring and collagen staining (Picro Sirius Red; PSR); inflammation was evaluated by the tubulo-interstitial inflammation score (ti-score). Linear regression analyses were performed. Six-month plasma PRO-C6 was cross-sectionally associated with IF/TA score (Std. beta = 0.34), and prospectively with 24-month IF/TA score and ti-score (Std. beta = 0.24 and 0.23, respectively) (p <0.05 for all). No significant associations were found between urine PRO-C6 and any of the biopsy findings. Fibrotic changes and urine PRO-C6 behaved differentially over time according to immunosuppressive therapy. These results are a first step towards non-invasive fibrosis detection after kidney transplantation by means of collagen VI synthesis measurement, and further research is required