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Visualizing scar development using SCAD assay - An <em>Ex-situ</em> skin scarring assay.
No full textThe mammalian global response to sealing deep tissue wounds is through scar formation and tissue contraction, mediated by specialized fascia fibroblasts. Despite the clinical significance of scar formation and impaired wound healing, our understanding of fascia fibroblast dynamics in wound healing is cursory due to the lack of relevant assays that enable direct visualization of fibroblast choreography and dynamics in complex environments such as in skin wounds. This paper presents a protocol to generate ex- situ skin scars using SCAD or "SCar-like tissue in A Dish" that emulate the complex environment of skin wounds. In this assay, 2 mm full-thickness skin is excised and cultured upside down in media for 5 days, during which scars and skin contractures develop uniformly. This methodology, coupled with fibroblast-lineage specific transgenic mouse models, enables visualization of individual fibroblast lineages across the entire wound repair process. Overall, this protocol aids researchers in understanding fundamental processes and mechanisms of wound repair, directly exploring the effects of modulators on wound healing outcomes
Current state-of-the-art of separation methods used in LC-MS based metabolomics and lipidomics.
No full textMetabolomics deals with the large-scale analysis of metabolites, belonging to numerous compound classes and showing an extremely high chemical diversity and complexity. Lipidomics, being a subcategory of metabolomics, analyzes the cellular lipid species. Both require state-of-the-art analytical methods capable of accessing the underlying chemical complexity. One of the major techniques used for the analysis of metabolites and lipids is Liquid Chromatography-Mass Spectrometry (LC-MS), offering both different selectivities in LC separation and high sensitivity in MS detection. Chromatography can be divided into different modes, based on the properties of the employed separation system. The most popular ones are Reversed-Phase (RP) separation for non– to mid-polar molecules and Hydrophilic Interaction Liquid Chromatography (HILIC) for polar molecules. So far, no single analysis method exists that can cover the entire range of metabolites or lipids, due to the huge chemical diversity. Consequently, different separation methods have been used for different applications and research questions. In this review, we explore the current use of LC-MS in metabolomics and lipidomics. As a proxy, we examined the use of chromatographic methods in the public repositories EBI MetaboLights and NIH Metabolomics Workbench. We extracted 1484 method descriptions, collected separation metadata and generated an overview on the current use of columns, eluents, etc. Based on this overview, we reviewed current practices and identified potential future trends as well as required improvements that may allow us to increase metabolite coverage, throughput or both simultaneously
Isolation and direct neuronal reprogramming of mouse astrocytes.
No full textDirect neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties
Streambed microbial communities in the transition zone between groundwater and a first-order stream as impacted by bidirectional water exchange.
No full textThe input of nitrate and other agricultural pollutants in higher-order streams largely derives from first-order streams. The streambed as the transition zone between groundwater and stream water has a decisive impact on the attenuation of such pollutants. This reactivity is not yet well understood for lower-order agricultural streams, which are often anthropogenically altered and lack the streambed complexity allowing for extensive hyporheic exchange. Reactive hot spots in such streambeds have been hypothesized as a function of hydrology, which controls the local gaining (groundwater exfiltration) or losing (infiltration) of stream water. However, streambed microbial communities and activities associated with such reactive zones remain mostly uncharted. In this study, sediments of a first-order agriculturally impacted stream in southern Germany were investigated. Along with a hydraulic dissection of distinct gaining and losing reaches of the stream, community composition and the abundance of bacterial communities in the streambed were investigated using PacBio long-read sequencing of bacterial 16S rRNA gene amplicons, and qPCR of bacterial 16S rRNA and denitrification genes (nirK and nirS). We show that bidirectional water exchange between groundwater and the stream represents an important control for sediment microbiota, especially for nitrate-reducing populations. Typical heterotrophic denitrifiers were most abundant in a midstream net losing section, while up- and downstream net gaining sections were associated with an enrichment of sulfur-oxidizing potential nitrate reducers affiliated with Sulfuricurvum and Thiobacillus spp. Dispersal-based community assembly was found to dominate such spots of groundwater exfiltration. Our results indicate a coupling of N- and S-cycling processes in the streambed of an agricultural first-order stream, and a prominent control of microbiology by hydrology and hydrochemistry in situ. Such detailed local heterogeneities in exchange fluxes and streambed microbiomes have not been reported to date, but seem relevant for understanding the reactivity of lower-order streams
Levels and drivers of urban black carbon and health risk assessment during pre- and COVID19 lockdown in Augsburg, Germany.
No full textThis study aimed to evaluate the levels and phenomenology of equivalent black carbon (eBC) at the city center of Augsburg, Germany (01/2018 to 12/2020). Furthermore, the potential health risk of eBC based on equivalent numbers of passively smoked cigarettes (PSC) was also evaluated, with special emphasis on the impact caused by the COVID19 lockdown restriction measures. As it could be expected, peak concentrations of eBC were commonly recorded in morning (06:00–8:00 LT) and night (19:00–22:00 LT) in all seasons, coinciding with traffic rush hours and atmospheric stagnation. The variability of eBC was highly influenced by diurnal variations in traffic and meteorology (air temperature (T), mixing-layer height (MLH), wind speed (WS)) across days and seasons. Furthermore, a marked “weekend effect” was evidenced, with an average eBC decrease of ∼35% due to lower traffic flow. During the COVID19 lockdown period, an average ∼60% reduction of the traffic flow resulted in ∼30% eBC decrease, as the health risks of eBC exposure was markedly reduced during this period. The implementation of a multilinear regression analysis allowed to explain for 53% of the variability in measured eBC, indicating that the several factors (e.g., traffic and meteorology) may contribute simultaneously to this proportion. Overall, this study will provide valuable input to the policy makers to mitigate eBC pollutant and its adverse effect on environment and human health
The neuropeptide receptor calcitonin receptor-like (CALCRL) is a potential therapeutic target in acute myeloid leukemia.
No full textCalcitonin receptor-like (CALCRL) is a G-protein-coupled neuropeptide receptor involved in the regulation of blood pressure, angiogenesis, cell proliferation, and apoptosis, and is currently emerging as a novel target for the treatment of migraine. This study characterizes the role of CALCRL in acute myeloid leukemia (AML). We analyzed CALCRL expression in collectively more than 1500 well-characterized AML patients from five international cohorts (AMLCG, HOVON, TCGA, Leucegene, and UKM) and evaluated associations with survival. In the AMLCG analytic cohort, increasing transcript levels of CALCRL were associated with decreasing complete remission rates (71.5%, 53.7%, 49.6% for low, intermediate, high CALCRL expression), 5-year overall (43.1%, 26.2%, 7.1%), and event-free survival (29.9%, 15.8%, 4.7%) (all P < 0.001). CALCRL levels remained associated with all endpoints on multivariable regression analyses. The prognostic impact was confirmed in all validation sets. Genes highly expressed in CALCRLhigh AML were significantly enriched in leukemic stem cell signatures and CALCRL levels were positively linked to the engraftment capacity of primary patient samples in immunocompromised mice. CRISPR- Cas9-mediated knockout of CALCRL significantly impaired colony formation in human myeloid leukemia cell lines. Overall, our study demonstrates that CALCRL predicts outcome beyond existing risk factors and is a potential therapeutic target in AML
Differences of nitrogen metabolism in date palm (<em>Phoenix dactylifera</em>) seedlings subjected to water deprivation and salt exposure.
No full textDrought and salt exposure are among the most prevalent and severe abiotic stressors causing serious agricultural yield losses, alone and in combination. Little is known about differences and similarities in the effects of these two stress factors on plant metabolic regulation, particularly on nitrogen metabolism. Here, we studied the effects of water deprivation and salt exposure on water relations and nitrogen metabolites in leaves and roots of date palm seedlings. Both, water deprivation and salt exposure had no significant effects on plant water content or stable carbon (C) and nitrogen (N) isotope signatures. Significant effects of water deprivation on total C and N concentrations were only observed in roots, i.e., decreased total C and increased total N concentrations. Whereas salt exposure initially decreased total C and increased total N concentrations significantly in roots, foliar total C concentration was increased upon prolonged exposure. Initially C/N ratios declined in roots of plants from both treatments and upon prolonged salt exposure also in the leaves. Neither treatment affected soluble protein and structural N concentrations in leaves or roots, but resulted in the accumulation of most amino acids, except for glutamate and tryptophan, which remained stable, and serine, which decreased, in roots. Accumulation of the most abundant amino acids, lysine and proline, was observed in roots under both treatments, but in leaves only upon salt exposure. This finding indicates a similar role of these amino acids as compatible solutes in the roots in response to salt und drought, but not in the leaves. Upon prolonged treatment, amino acid concentrations returned to levels found in unstressed plants in leaves of water deprived, but not salt exposed, plants. The present results show both water deprivation and salt exposure strongly impact N metabolism of date palm seedlings, but in a different manner in leaves and roots
Development and validation of an LC-MS/MS methodology for the quantification of thyroid hormones in dko MCT8/OATP1C1 mouse brain.
No full textThe Allan-Herndon Dudley Syndrome (AHDS) is a rare disease caused by the progressive loss of monocarboxylate transporter 8 (MCT8). In patients with AHDS, the absence of MCT8 impairs transport of thyroid hormones (TH) through the blood brain barrier, leading to a central state of TH deficiency. In mice, the AHDS is mimicked by simultaneous deletion of the TH transporters MCT8 and the solute carrier organic anion transporter family member 1c1 (OATP1C1). To support preclinical mouse studies, an analytical methodology was developed and successfully applied for quantifying selected thyroid hormones in mouse whole brain and in specific regions using liquid chromatography tandem mass-spectrometry (LC-MS/MS). An important requirement for the methodology was its high sensitivity since a very low concentration of THs was expected in MCT8/OATP1C1 double-knockout (dko) mouse brain. Seven THs were targeted: L-thyroxine (T4), 3,3´,5‐triiodo‐L‐thyronine-thyronine (T3), 3,3´,5´‐triiodo‐L‐thyronine-thyronine (rT3), 3,3´‐diiodo‐L‐thyronine (3,3´‐T2, T2), 3,5-diiodo-L-thyronine (rT2, 3,5-T2), 3-iodo-L-thyronine (T1), 3-iodothyronamine (T1AM). Isotope dilution liquid chromatography triple-quadrupole mass spectrometry methodology was applied for detection and quantification. The method was validated in wild-type animals for mouse whole brain and for five different brain regions (hypothalamus, hippocampus, prefrontal cortex, brainstem and cortex). Instrumental calibration curves ranged from 0.35 to 150 pg/µL with good linearity (r2 >0.996). The limit of quantification was from 0.08 to 0.6 pg/mg, with an intra- and inter-day precision of 4.2–14.02% and 0.4–17.9% respectively, and accuracies between 84.9% and 114.8% when the methodology was validated for the whole brain. In smaller, distinct brain regions, intra- and inter-day precision were 0.6–20.7% and 2.5–15.6% respectively, and accuracies were 80.2–128.6%. The new methodology was highly sensitive and allowed for the following quantification in wild-type mice: (i) for the first time, four distinct thyroid hormones (T4, T3, rT3 and 3,3´‐T2) in only approximately 100 mg of mouse brain were detected; (ii) the quantification of T4 and T3 for the first time in distinct mouse brain regions were reported. Further, application of our method to MCT8/OATP1C1 dko mice revealed the expected, relative lack of T3 and T4 uptake into the brain, and confirmed the utility of our analytical method to study TH transport across the blood brain barrier in a preclinical model of central TH deficiency
Optimization of an efficient cell culture hepatitis B infection system for assessment of hepatitis B virus neutralizing monoclonal antibodies.
No full textBackground: Human polyclonal plasma-derived hepatitis B immunoglobulin (HBIG) is currently used for immunoprophylaxis of HBV infection. The development of virus-neutralizing monoclonal antibodies (MAbs) requires the use of optimized cell culture systems supporting HBV infection. Objective: This study aims to optimize the hepatitis B virus infectivity of NTCP-reconstituted HepG2 (HepG2-NTCP) cells to establish an efficient system to evaluate the HBV-neutralizing effect of anti-HBs MAbs. Methods: Serum-derived HBV (sHBV) and cell culture-derived HBV (ccHBV) were simultaneously used for the optimization of HBV infection in HepG2-NTCP cells by applying different modifications. Results: Our results for the first time showed that in addition to human serum, monkey serum could significantly improve ccHBV infection, while fetal and adult bovine serum as well as duck and sheep serum did not have a promotive effect. In addition, sHBV and ccHBV infectivity are largely similar except that adding 5% of PEG, which is commonly used to improve in vitro infection of ccHBV, significantly reduced sHBV infection. We showed that a combination of spinoculation, trypsinization, and also adding human or monkey serum to HBV inoculum could significantly improve the permissivity of HepG2-NTCP cells to HBV infection compared with individual strategies. All anti-HBs MAbs were able to successfully neutralize both ccHBV and sHBV infection in our optimized in vitro system. Conclusion: Our study suggests different strategies for improving ccHBV and sHBV infection in HepG2-NTCP cells. This cell culture-based system allows assessment of HBV neutralizing MAbs and may also prove to be valuable for the analysis of other HBV neutralizing therapeutics
Inflammatory macrophage memory in nonsteroidal anti-inflammatory drug–exacerbated respiratory disease.
No full textBackground: Nonsteroidal anti-inflammatory drug–exacerbated respiratory disease (N-ERD) is a chronic inflammatory condition, which is driven by an aberrant arachidonic acid metabolism. Macrophages are major producers of arachidonic acid metabolites and subject to metabolic reprogramming, but they have been neglected in N-ERD. Objective: This study sought to elucidate a potential metabolic and epigenetic macrophage reprogramming in N-ERD. Methods: Transcriptional, metabolic, and lipid mediator profiles in macrophages from patients with N-ERD and healthy controls were assessed by RNA sequencing, Seahorse assays, and LC-MS/MS. Metabolites in nasal lining fluid, sputum, and plasma from patients with N-ERD (n = 15) and healthy individuals (n = 10) were quantified by targeted metabolomics analyses. Genome-wide methylomics were deployed to define epigenetic mechanisms of macrophage reprogramming in N-ERD. Results: This study shows that N-ERD monocytes/macrophages exhibit an overall reduction in DNA methylation, aberrant metabolic profiles, and an increased expression of chemokines, indicative of a persistent proinflammatory activation. Differentially methylated regions in N-ERD macrophages included genes involved in chemokine signaling and acylcarnitine metabolism. Acylcarnitines were increased in macrophages, sputum, nasal lining fluid, and plasma of patients with N-ERD. On inflammatory challenge, N-ERD macrophages produced increased levels of acylcarnitines, proinflammatory arachidonic acid metabolites, cytokines, and chemokines as compared to healthy macrophages. Conclusions: Together, these findings decipher a proinflammatory metabolic and epigenetic reprogramming of macrophages in N-ERD