The neurovascular unit (NVU), composed of endothelial cells, pericytes, juxtaposed astrocytes and microglia together with neurons, is essential for proper central nervous system functioning. The NVU ...critically regulates blood-brain barrier (BBB) function, which is impaired in several neurological diseases and is therefore a key therapeutic target. To understand the extent and cellular source of BBB dysfunction, simultaneous isolation and analysis of NVU cells is needed. Here, we describe a protocol for the EPAM-ia method, which is based on flow cytometry for simultaneous isolation and analysis of endothelial cells, pericytes, astrocytes and microglia. This method is based on differential processing of NVU cell types using enzymes, mechanical homogenization and filtration specific for each cell type followed by combining them for immunostaining and fluorescence-activated cell sorting. The gating strategy encompasses cell-type-specific and exclusion markers for contaminating cells to isolate the major NVU cell types. This protocol takes ~6 h for two sets of one or two animals. The isolation part requires experience in animal handling, fresh tissue processing and immunolabeling for flow cytometry. Sorted NVU cells can be used for downstream applications including transcriptomics, proteomics and cell culture. Multiple cell-type analyses using UpSet can then be applied to obtain robust targets from single or multiple NVU cell types in neurological diseases associated with BBB dysfunction. The EPAM-ia method is also amenable to isolation of several other cell types, including cancer cells and immune cells. This protocol is applicable to healthy and pathological tissue from mouse and human sources and to several cell types compared with similar protocols.
Abstract only Background: Delays in endovascular therapy can lead to worse outcomes in stroke patients. Interfacility delays when transferring patients to thrombectomy-capable hospitals are common. ...We sought to identify causes of such delays and create a standardized transfer process to improve transfer times. Methods: A 15-hospital system formed a multidisciplinary team to assess the current state of DIDO processes from data on previous transfers, evaluate current transfer algorithms, and analyze baseline performance. Individual hospital transfer algorithms were compared. Results: Data was analyzed for 47 patients transferred from 12 facilities to thrombectomy-capable centers from June 2017 to July 2018 who received ischemic stroke treatment. 71% were from a metro area within 50 miles of a thrombectomy-capable hospital and 29% were from outside the metro area, up to 400 miles away. 80% were transported by air by 9 different agencies. DIDO times were: 27% less than 90 min; 27% 90 to 120 min; 44% greater than 120 min. There was minimal opportunity for improvement based on: day of week, NIHSS, and treatment with alteplase. Areas for improvement include: timeliness of door to CT (x-=13 vs 35 min), time to CT read (x-=8 vs 16 min), time to alteplase (x-=48 vs 115 min), and time to transfer (x-=125 vs 148 min), time from arrival to consult with thrombectomy capable centers (x-= 53 vs 86 min), time from consult to dispatch (x-=19 vs 26 min), and time of transfer agency on scene (x-=22 vs 37 min) for metro vs rural hospitals. Conclusions: Identified causes of delays include decision to transfer and initiation of dispatch to coordinate transport. A difference in timeliness initial diagnosis and treatment of patients outside metro areas was identified. Last known well (LKW) to arrival for transferred patients revealed a need to educate sending facilities on the expanded treatment windows- only 3 patients had a LKW to arrival time greater than 4.5 hours.
Podocytes form the kidney filtration barrier and continuously adjust to external stimuli to preserve their integrity even in the presence of inflammation. It was suggested that canonical toll-like ...receptor signaling, mediated by the adaptor protein MYD88, plays a crucial role in initiating inflammatory responses in glomerulonephritis (GN). We explored the influence of podocyte-intrinsic MYD88 by challenging wild-type (WT) and podocyte-specific Myd88 knockout (MyD88
) mice, with a model of experimental GN (nephrotoxic nephritis, NTN). Next-generation sequencing revealed a robust upregulation of inflammatory pathways and changes in cytoskeletal and cell adhesion proteins in sorted podocytes from WT mice during disease. Unchallenged MyD88
mice were healthy and showed no proteinuria, normal kidney function and lacked morphological changes. During NTN, MyD88
exhibited a transient increase in proteinuria in comparison to littermates, while histological damage, podocyte ultrastructure in STED imaging and frequencies of infiltrating immune cells by flow cytometry were unchanged. MYD88-deficiency led to subtle changes in the podocyte transcriptome, without a significant impact on the overall podocyte response to inflammation, presumably through MYD88-independent signaling pathways. In conclusion, our study reveals a comprehensive analysis of podocyte adaptation to an inflammatory environment on the transcriptome level, while MYD88-deficiency had only limited impact on the course of GN suggesting additional signaling through MYD88-independent signaling.
Abstract Background aims The 3-dimensional (3-D) culture of various cell types reflects the in vivo situation more precisely than 2-dimensional (2-D) cell culture techniques. Spheroids as 3-D cell ...constructs have been used in tumor research for a long time. They have also been used to study angiogenic mechanisms, which are essential for the success of many tissue-engineering approaches. Several methods of forming spheroids are known, but there is a lack of systematic studies evaluating the performance of these techniques. Methods We evaluated the performance of the hanging drop technique, carboxymethyl cellulose technique and liquid overlay technique to form both mono- and co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. The performance of the three techniques was evaluated in terms of rate of yield and reproducibility. The size of the generated spheroids was determined systematically. Results The liquid overlay technique was the most suitable for generating spheroids reproducibly. The rate of yield for this technique was between 60% and 100% for monoculture spheroids and 100% for co-culture spheroids. The size of the spheroids could be adjusted easily and precisely by varying the number of seeded cells organized in one spheroid. The formation of co-culture spheroids consisting of three different cell types was possible. Conclusions Our results show that the most suitable technique for forming spheroids can vary from the chosen cell type, especially if primary cells are used. Co-culture spheroids consisting of three different cell types will be used to study angiogenic phenomena in further studies.
The oxidation mechanism of metals depends, among other factors, on the surface integrity. The surface and rim zone properties are often determined by the manufacturing process that was used to ...machine the material. Laser chemical machining (LCM) is a manufacturing process that uses laser radiation as a localized and selective heat source to activate a chemical reaction between an electrolyte and a metallic surface. The objective of this work is first to investigate how different LCM processes affect the rim zone properties of 42CrMo4. For this purpose, the surface chemistry is analyzed by EDS and XPS, phases and residual stresses are determined by XRD, and the morphology is investigated by SEM. Second, the influence of these modified rim zones on the oxidation properties of the steel at 500 °C in air is to be demonstrated in oxidation tests by in situ XRD and subsequent SEM/EDS investigations. A decisive influence of the oxides formed on the surface of 42CrMo4 during LCM in different electrolytes (NaNO3 solution and H3PO4) at two different laser powers on the high-temperature oxidation properties was demonstrated. These oxides were supposed to act as nucleation sites for oxide layer formation at 500 °C and led to an overall increase in oxide layer thickness after high-temperature oxidation compared to non-LCM-processed surfaces.
In this work, we illustrate a method to continuously hyperpolarize a biomolecule, nicotinamide, in water using parahydrogen and signal amplification by reversible exchange (SABRE). Building on the ...preparation procedure described recently by Truong et al. J. Phys. Chem. B, 2014, 118, 13882−13889 , aqueous solutions of nicotinamide and an Ir-IMes catalyst were prepared for low-field NMR and MRI. The 1H-polarization was continuously renewed and monitored by NMR experiments at 5.9 mT for more than 1000 s. The polarization achieved corresponds to that induced by a 46 T magnet (P = 1.6 × 10–4) or an enhancement of 104. The polarization persisted, although reduced, if cell culture medium (DPBS with Ca2+ and Mg2+) or human cells (HL-60) were added, but was no longer observable after the addition of human blood. Using a portable MRI unit, fast 1H-MRI was enabled by cycling the magnetic field between 5 mT and the Earth’s field for hyperpolarization and imaging, respectively. A model describing the underlying spin physics was developed that revealed a polarization pattern depending on both contact time and magnetic field. Furthermore, the model predicts an opposite phase of the dihydrogen and substrate signal after one exchange, which is likely to result in the cancelation of some signal at low field.
Aging causes major alterations of all components of the neurovascular unit and compromises brain blood supply. Here, we tested how aging affects vascular reactivity in basilar arteries from young ...(<10 weeks; y-BA), old (>22 months; o-BA) and old (>22 months) heterozygous MYPT1-T-696A/+ knock-in mice. In isometrically mounted o-BA, media thickness was increased by ∼10% while the passive length tension relations were not altered. Endothelial denudation or pan-NOS inhibition (100 µmol/L L-NAME) increased the basal tone by 11% in y-BA and 23% in o-BA, while inhibition of nNOS (1 µmol/L L-NPA) induced ∼10% increase in both ages. eNOS expression was ∼2-fold higher in o-BA. In o-BA, U46619-induced force was augmented (pEC50 ∼6.9 vs. pEC50 ∼6.5) while responsiveness to DEA-NONOate, electrical field stimulation or nicotine was decreased. Basal phosphorylation of MLC20-S19 and MYPT1-T-853 was higher in o-BA and was reversed by apocynin. Furthermore, permeabilized o-BA showed enhanced Ca2+-sensitivity. Old T-696A/+ BA displayed a reduced phosphorylation of MYPT1-T696 and MLC20, a lower basal tone in response to L-NAME and a reduced eNOS expression. The results indicate that the vascular hypercontractility found in o-BA is mediated by inhibition of MLCP and is partially compensated by an upregulation of endothelial NO release.
Background
Quantitative serological assays detecting response to SARS-CoV-2 are needed to quantify immunity. This study analyzed the performance and correlation of two quantitative anti-S1 assays in ...oligo-/asymptomatic individuals from a population-based cohort.
Methods
In total, 362 plasma samples (108 with reverse transcription-polymerase chain reaction RT-PCR-positive pharyngeal swabs, 111 negative controls, and 143 with positive serology without confirmation by RT-PCR) were tested with quantitative assays (Euroimmun Anti-SARS-CoV-2 QuantiVac enzyme-linked immunosorbent assay EI-S1-IgG-quant) and Roche Elecsys
®
Anti-SARS-CoV-2 S Ro-RBD-Ig-quant), which were compared with each other and confirmatory tests, including wild-type virus micro-neutralization (NT) and GenScript
®
cPass™. Square roots
R
of coefficients of determination were calculated for continuous variables and non-parametric tests were used for paired comparisons.
Results
Quantitative anti-S1 serology correlated well with each other (true positives, 96%; true negatives, 97%). Antibody titers decreased over time (< 30 to > 240 days after initial positive RT-PCR). Agreement with GenScript-cPass was 96%/99% for true positives and true negatives, respectively, for Ro-RBD-Ig-quant and 93%/97% for EI-S1-IgG-quant. Ro-RBD-Ig-quant allowed distinct separation between positives and negatives, and less non-specific reactivity versus EI-S1-IgG-quant. Raw values (95% CI) ≥ 28.7 U/mL (22.6–36.4) for Ro-RBD-Ig-quant and ≥ 49.8 U/mL (43.4–57.1) for EI-S1-IgG-quant predicted NT > 1:5 in 95% of cases.
Conclusions
Our findings suggest both quantitative anti-S1 assays (EI-S1-IgG-quant and Ro-RBD-Ig-quant) may replace direct neutralization assays in quantitative measurement of immune protection against SARS-CoV-2 in certain circumstances. However, although the mean antibody titers for both assays tended to decrease over time, a higher proportion of Ro-RBD-Ig-quant values remained positive after 240 days.
The stress hormone-regulating hypothalamic-pituitary-adrenal (HPA) axis has been implicated in the causality as well as the treatment of depression. To investigate a possible association between ...genes regulating the HPA axis and response to antidepressants and susceptibility for depression, we genotyped single-nucleotide polymorphisms in eight of these genes in depressed individuals and matched controls. We found significant associations of response to antidepressants and the recurrence of depressive episodes with single-nucleotide polymorphisms in FKBP5, a glucocorticoid receptor-regulating cochaperone of hsp-90, in two independent samples. These single-nucleotide polymorphisms were also associated with increased intracellular FKBP5 protein expression, which triggers adaptive changes in glucocorticoid receptor and, thereby, HPA-axis regulation. Individuals carrying the associated genotypes had less HPA-axis hyperactivity during the depressive episode. We propose that the FKBP5 variant-dependent alterations in HPA-axis regulation could be related to the faster response to antidepressant drug treatment and the increased recurrence of depressive episodes observed in this subgroup of depressed individuals. These findings support a central role of genes regulating the HPA axis in the causality of depression and the mechanism of action of antidepressant drugs.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The entry of illegally discharged waste into the sewage system can cause serious damage to sewage pipes and harm the public domain. Besides industrial and communal sources, discharges from ...clandestine laboratories are of special forensic interest. Aim of this study is to investigate the possibility to detect clandestine discharges of possibly hazardous waste directly at the point of discharge. Tests were performed at a facility where real wastewater was pumped in a controlled way through an open sewage pipe above ground. Chloride, ammonia, pH and conductivity electrodes were investigated for their ability to detect discharges of different types of waste. Waste samples were diluted up to 50 times in a static wastewater environment and pH, conductivity and chloride electrodes were able to distinguish all waste dilutions from blank wastewater. These three electrodes were then used for dynamic tests by placing them inside flowing sewage water and discharging different types of liquid waste ten meters upstream of them. Parameters of the waste discharges like volume, time interval and speed of discharge were varied and the responses of the electrodes were collected. The dynamic tests showed that these three electrodes were able to pick up different waste discharges in a real wastewater environment. It was found that a high sampling rate of the sensors would be required to connect a certain discharge to a specific household connection. These findings highlight the possibility to locate illegal discharges, coming from a variety of sources, using the waste discharge itself.
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