Analysis of purine receptor expression and functionality in alveolar epithelial cells

Despite its fundamental role in providing an extensive surface for gas exchange, the alveolar epithelium (AE) serves as an immunological barrier through, e.g., the release of proinflammatory cytokines and secretion of surfactant to prevent alveolar collapse. Thus, AE is important for sustaining lung homeostasis. Extracellular ATP secreted by alveolar epithelial cells (AECs) is involved in physiological and pathological conditions and acts mainly through the activation of purine receptors (P2Rs). When studying P2R-mediated processes, primary isolated type II AECs (piAECs) still represent the gold standard in in vitro research, although their preparation is time-consuming and requires the sacrifice of many animals. Hence, cultivated immortalized and tumor-derived AEC lines may constitute a valuable alternative. In this work, we examined P2R expression and functionality in piAECs, in immortalized and tumor-derived AEC lines with the purpose of gaining a better understanding of purinergic signaling in different cell systems and assisting researchers in the choice of a suitable cell line with a certain P2R in demand. We combined mRNA and protein analysis to evaluate the expression of P2R. For pharmacological testing, we conducted calcium ([Ca²⁺]) measurements and siRNA receptor knockdown. Interestingly, the mRNA and protein levels of P2Y₂, P2Y_6, and P2X₄ were detected on all cell lines. Concerning functionality, P2XR could be narrowed to L2 and piAECs while P2YR were active in all cell lines.

     

Soil amendment interacts with invasive grass and drought to uniquely influence aboveground versus belowground biomass in aridland restoration

Water‐holding soil amendments such as super‐absorbent polymer (SAP) may improve native species establishment in restoration but may also interact with precipitation or invasive species such as Bromus tectorum L. (cheatgrass or downy brome) to influence revegetation outcomes. We implemented an experiment at two sites in Colorado, U.S.A., in which we investigated the interactions of drought (66% reduction of ambient rainfall), B. tectorum seed addition (BRTE, 465 seeds/m²), and SAP soil amendment (25 g/m²) on initial plant establishment and 3‐year aboveground and belowground biomass and allocation. At one site, SAP resulted in higher native seeded species establishment but only with ambient precipitation. However, by the third year, we detected no SAP effects on native seeded species biomass. Treatments interacted to influence aboveground and belowground biomass and allocation differently. At one site, a SAP × precipitation interaction resulted in lower belowground biomass in plots with SAP and drought (61.7 ± 7.3 g/m²) than plots with drought alone (91.6 ± 18.1 g/m²). At the other site, a SAP × BRTE interaction resulted in higher belowground biomass in plots with SAP and BRTE (56.6 ± 11.2 g/m²) than BRTE alone (35.0 ± 3.7 g/m²). These patterns were not reflected in aboveground biomass. SAP should be used with caution in aridland restoration because initial positive effects may not translate to long‐term benefits, SAP may uniquely influence aboveground versus belowground biomass, and SAP can interact with environmental variables to impact developing plant communities in positive and negative ways.     

Hysteresis in poly‐2′‐deoxycytidine i‐motif folding is impacted by the method of analysis as well as loop and stem lengths

In DNA, i‐motif (iM) folds occur under slightly acidic conditions when sequences rich in 2′‐deoxycytidine (dC) nucleotides adopt consecutive dC self base pairs. The pH stability of an iM is defined by the midpoint in the pH transition (pH_T) between the folded and unfolded states. Two different experiments to determine pH_T values via circular dichroism (CD) spectroscopy were performed on poly‐dC iMs of length 15, 19, or 23 nucleotides. These experiments demonstrate two points: (1) pH_T values were dependent on the titration experiment performed, and (2) pH‐induced denaturing or annealing processes produced isothermal hysteresis in the pH_T values. These results in tandem with model iMs with judicious mutations of dC to thymidine to favor particular folds found the hysteresis was maximal for the shorter poly‐dC iMs and those with an even number of base pairs, while the hysteresis was minimal for longer poly‐dC iMs and those with an odd number of base pairs. Experiments to follow the iM folding via thermal changes identified thermal hysteresis between the denaturing and annealing cycles. Similar trends were found to those observed in the CD experiments. The results demonstrate that the method of iM analysis can impact the pH_T parameter measured, and hysteresis was observed in the pH_T and T_m values.     

ASHG 2020 Curt Stern Award introduction: Fowzan Sami Alkuraya

This article is based on the address given by the author at the 2020 virtual meeting of the American Society of Human Genetics (ASHG) on October 26, 2020. The video of the original address can be found at the ASHG website.     

Correction to: Sex and race differences of cerebrospinal fluid metabolites in healthy individuals

Metabolomics - Metabolomics applications to the aquaculture research are increasing steadily. The use of standardized proton nuclear magnetic resonance (1H NMR) spectroscopy can provide the...     

Pyridostigmine improves cardiac function and rhythmicity through RyR2 stabilization and inhibition of STIM1-mediated calcium entry in heart failure

Heart failure (HF) is characterized by asymmetrical autonomic balance. Treatments to restore parasympathetic activity in human heart failure trials have shown beneficial effects. However, mechanisms of parasympathetic-mediated improvement in cardiac function remain unclear. The present study examined the effects and underpinning mechanisms of chronic treatment with the cholinesterase inhibitor, pyridostigmine (PYR), in pressure overload HF induced by transverse aortic constriction (TAC) in mice. TAC mice exhibited characteristic adverse structural (left ventricular hypertrophy) and functional remodelling (reduced ejection fraction, altered myocyte calcium (Ca) handling, increased arrhythmogenesis) with enhanced predisposition to arrhythmogenic aberrant sarcoplasmic reticulum (SR) Ca release, cardiac ryanodine receptor (RyR2) hyper-phosphorylation and up-regulated store-operated Ca entry (SOCE). PYR treatment resulted in improved cardiac contractile performance and rhythmic activity relative to untreated TAC mice. Chronic PYR treatment inhibited altered intracellular Ca handling by alleviating aberrant Ca release and diminishing pathologically enhanced SOCE in TAC myocytes. At the molecular level, these PYR-induced changes in Ca handling were associated with reductions of pathologically enhanced phosphorylation of RyR2 serine-2814 and STIM1 expression in HF myocytes. These results suggest that chronic cholinergic augmentation alleviates HF via normalization of both canonical RyR2-mediated SR Ca release and non-canonical hypertrophic Ca signaling via STIM1-dependent SOCE.     

Rapid high‐yield N‐acylation of aminothiols: N‐acetylglutathione and N‐acetylhomocysteine and their thiol pKa values

Methodology for the rapid N‐acylation of aminothiols in aqueous solution using procedures commonly employed in biochemical studies is described here. Glutathione disulfide (GSSG) and homocystine were diN‐acetylated in ~100% yield in 0.1 M aqueous NaHCO₃ (pH 8.5) at room temperature by 2.5 equiv of the activated ester, N‐hydroxysulfosuccinimidyl acetate, an efficient water‐soluble acetylating reagent. Following acetone precipitation, diN‐acetylGSSG was further purified and desalted on a strong anion‐exchange (SAX) cartridge. DiN‐acetylhomocystine was simultaneously purified and desalted on a C₁₈ cartridge. The N‐acetylated aminothiols were generated using gel‐immobilized tris(2‐carboxyethyl)phosphine as a reductant, which obviated the need for further purification. Alternatively, disulfide exchange with dissolved dithiothreitol yielded N‐acetylglutathione, which was purified on the SAX cartridge. pH titrations of N‐acetylglutathione (8.99) and N‐acetylhomocysteine (9.66) as well as those of commercially available N‐acetylcysteine (9.53) and N‐acetylpenicillamine (10.21) yielded pK_a(SH) values of importance for biological studies. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

A Metagenomic Framework for the Study of Airborne Microbial Communities

Understanding the microbial content of the air has important scientific, health, and economic implications. While studies have primarily characterized the taxonomic content of air samples by sequencing the 16S or 18S ribosomal RNA gene, direct analysis of the genomic content of airborne microorganisms has not been possible due to the extremely low density of biological material in airborne environments. We developed sampling and amplification methods to enable adequate DNA recovery to allow metagenomic profiling of air samples collected from indoor and outdoor environments. Air samples were collected from a large urban building, a medical center, a house, and a pier. Analyses of metagenomic data generated from these samples reveal airborne communities with a high degree of diversity and different genera abundance profiles. The identities of many of the taxonomic groups and protein families also allows for the identification of the likely sources of the sampled airborne bacteria.     

Microbiota Dynamics in Patients Treated with Fecal Microbiota Transplantation for Recurrent Clostridium difficile Infection

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembraneous colitis and is responsible for a large and increasing fraction of hospital-acquired infections. Fecal microbiota transplantation (FMT) is an alternate treatment option for recurrent C. difficile infection (RCDI) refractory to antibiotic therapy. It has recently been discussed favorably in the clinical and scientific communities and is receiving increasing public attention. However, short- and long-term health consequences of FMT remain a concern, as the effects of the transplanted microbiota on the patient remain unknown. To shed light on microbial events associated with RCDI and treatment by FMT, we performed fecal microbiota analysis by 16S rRNA gene amplicon pyrosequencing of 14 pairs of healthy donors and RCDI patients treated successfully by FMT. Post-FMT patient and healthy donor samples collected up to one year after FMT were studied longitudinally, including one post-FMT patient with antibiotic-associated relapse three months after FMT. This analysis allowed us not only to confirm prior reports that RCDI is associated with reduced diversity and compositional changes in the fecal microbiota, but also to characterize previously undocumented post-FMT microbiota dynamics. Members of the Streptococcaceae, Enterococcaceae, or Enterobacteriaceae were significantly increased and putative butyrate producers, such as Lachnospiraceae and Ruminococcaceae were significantly reduced in samples from RCDI patients before FMT as compared to post-FMT patient and healthy donor samples. RCDI patient samples showed more case-specific variations than post-FMT patient and healthy donor samples. However, none of the bacterial groups were invariably associated with RCDI or successful treatment by FMT. Overall microbiota compositions in post-FMT patients, specifically abundances of the above-mentioned Firmicutes, continued to change for at least 16 weeks after FMT, suggesting that full microbiota recovery from RCDI may take much longer than expected based on the disappearance of diarrheal symptoms immediately after FMT.