Nitric oxide/cGMP signaling inhibits TSH-stimulated iodide uptake and expression of thyroid peroxidase and thyroglobulin mRNA in FRTL-5 thyroid cells.

OBJECTIVE: Nitric oxide (NO) induces morphological and functional alterations in primary cultured thyroid cells. The aim of this paper was to analyze the direct influence of a long-term exposition to NO on parameters of thyroid hormone biosynthesis in FRTL-5 cells. DESIGN: Cells were treated with the NO donor sodium nitroprusside (SNP) for 24-72 h. MAIN OUTCOME: SNP (50-500 micromol/L) reduced iodide uptake in a concentration-dependent manner. The inhibition of iodide uptake increased progressively with time and matched nitrite accumulation. SNP inhibited thyroperoxidase (TPO) and thyroglobulin (TG) mRNA expression in a concentration-dependent manner. SNP enhanced 3',5'-cyclic guanosine monophosphate (cGMP) production. 3',5'-cyclic adenosine phosphate (cAMP) generation was reduced by a high SNP concentration after 48 h. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), a cGMP analog, inhibited iodide uptake as well as TPO and TG mRNA expression. The cGMP-dependent protein kinase (cGK) inhibitor KT-5823 reversed SNP or 8-Br-cGMP-inhibited iodide uptake. Thyroid-stimulating hormone pretreatment for 24-48 h prevented SNP-reduced iodide uptake although nitrite levels remained unaffected. CONCLUSION: These findings favor a long-term inhibitory role of the NO/cGMP pathway on parameters of thyroid hormone biosynthesis. A novel property of NO to inhibit TPO and TG mRNA expression is supported. The NO action on iodide uptake could involve cGK mediation. The long-term inhibition of steps of thyroid hormonogenesis by NO could be of interest in thyroid pathophysiology.     

Adaptive differentiation and rapid evolution of a soil bacterium along a climate gradient

Microbial community responses to environmental change are largely associated with ecological processes; however, the potential for microbes to rapidly evolve and adapt remains relatively unexplored in natural environments. To assess how ecological and evolutionary processes simultaneously alter the genetic diversity of a microbiome, we conducted two concurrent experiments in the leaf litter layer of soil over 18 mo across a climate gradient in Southern California. In the first experiment, we reciprocally transplanted microbial communities from five sites to test whether ecological shifts in ecotypes of the abundant bacterium, Curtobacterium, corresponded to past adaptive differentiation. In the transplanted communities, ecotypes converged toward that of the native communities growing on a common litter substrate. Moreover, these shifts were correlated with community-weighted mean trait values of the Curtobacterium ecotypes, indicating that some of the trait variation among ecotypes could be explained by local adaptation to climate conditions. In the second experiment, we transplanted an isogenic Curtobacterium strain and tracked genomic mutations associated with the sites across the same climate gradient. Using a combination of genomic and metagenomic approaches, we identified a variety of nonrandom, parallel mutations associated with transplantation, including mutations in genes related to nutrient acquisition, stress response, and exopolysaccharide production. Together, the field experiments demonstrate how both demographic shifts of previously adapted ecotypes and contemporary evolution can alter the diversity of a soil microbiome on the same timescale.

Microbial communities respond quickly to environmental change (1, 2). These responses are typically associated with ecological processes; however, the potential for microbes to evolve and adapt to changes in the environment on ecological timescales remains largely unexplored in natural ecosystems. While evolutionary processes are typically considered over longer timescales, the short generation times, large populations, and high mutation rates indicative of microorganisms may allow for rapid adaptation. Laboratory studies have repeatedly demonstrated rapid evolution of bacterial populations (3) with consequences for organismal physiology (4), yet it remains unclear how these in vitro studies extend to in situ communities (5).Both ecological and evolutionary processes likely contribute simultaneously (6, 7) to the response of a microbiome to changing environmental conditions (8). However, separating these processes for bacteria can be difficult as they occur along a continuum of temporal and genetic scales. In terms of ecological processes, microbiome composition can respond demographically, as selective forces promote the growth and survival of differentially adapted taxa within the bacterial community. Certainly, many studies have observed such shifts in taxonomic composition of 16S ribosomal RNA (rRNA)–defined taxa in response to simulated global changes (9), and these responses are considered an ecological process (e.g., species sorting). Few examples, however, link these responses to trait differences among bacterial taxa (10–13), precluding direct insights into whether these ecological shifts are due to adaptive differentiation among taxa as a result of past evolutionary divergence. Concurrently, the same selective forces can also shift the abundance of conspecific strains and alter the allele frequencies of preexisting genetic variation, which at this genetic scale is defined as an evolutionary process (14). Finally, evolution through de novo mutation can provide a new source of genetic variation that may allow for further adaptation to environmental change.In this study, we aimed to capture this continuum of ecological and evolutionary processes that together produce the response of a microbiome’s diversity to environmental change (Fig. 1A). Studying evolution in microbial communities in situ, however, is challenging. For one, variation in highly conserved marker genes used in many microbiome studies (e.g., 16S rRNA) represents distant evolutionary divergences, and thus these regions are too conserved to detect locally adapted lineages (11, 12, 15), let alone recent evolutionary change within communities (16). To overcome this limitation, studies have leveraged shotgun metagenomic data (17, 18) and genome sequences of co-occurring, closely related strains (19, 20) to characterize evolutionary processes (e.g., recombination and gene flow) structuring the genetic diversity of bacterial lineages. However, these studies are also limited by an inherent challenge in microbiome research: delineating population boundaries, the fundamental unit of evolution. While progress has been made in defining microbial species (21–23), the high genetic heterogeneity within diverse microbial communities, such as soils, convolute the boundaries of the fine-scale patterns of genetic diversity within microbial taxonomic units (12). For instance, metagenome-assembled genomes are often composed of a composite of strains forming a large population of mosaic genomes (24) that may not fully capture the diversity of the local population (25). As such, it remains difficult to study evolutionary rates within microbial communities (however, see refs. 26, 27), and the extent and time scale at which evolutionary processes contribute to both standing and new genetic variation relative to ecological processes.Open in a separate windowFig. 1.Microbial community transplant experiment. (A) Changes in microbial community composition can be due to a continuum of ecological and evolutionary processes. For instance, shifts in standing genetic variation can be attributed to both ecological and evolutionary processes depending on the level of biological resolution, while de novo mutations can be a result from evolutionary adaptation. (B) A schematic of the two parallel transplant experiments at the community and strain level. Inoculated litterbags were transplanted to all sites along an elevation gradient that covaried in temperature and precipitation. Site codes: D=Desert; Sc=Scrubland; G=Grassland; P=Pine-Oak; S=Subalpine.Here, we asked the following question: can we characterize the ecological and evolutionary processes that are contributing concurrently to the response of a soil bacterial community to a changing environment? To answer this question, we utilized a field-based experimental approach to quantify the influence of both ecological and evolutionary processes on one focal soil bacterium in its natural environment, the genus Curtobacterium (28). Specifically, we transplanted the bacterium across an elevation gradient on a common resource (leaf litter) substrate (29) to assess its response to new climates in two parallel experiments over the same 18 mo time period (Fig. 1B). In both experiments, we used microbial cages [nylon mesh bags that allow for nutrient transport (30)] to manipulate microbial composition while restricting microbial migration to eliminate the introduction of new alleles and/or variants from dispersal (31). A reciprocal transplant design allowed for direct testing of microbial adaptation to abiotic conditions (i.e., moisture and temperature) in a natural setting.In the first experiment, we conducted a reciprocal transplant of the entire microbial community (32) and tracked the ecological response of Curtobacterium ecotypes (33). A bacterial ecotype is defined as highly clustered genotypic and phenotypic strains occupying the same ecological niche, somewhat equivalent to a eukaryotic species (34). To test the hypothesis that Curtobacterium ecotypes are locally adapted to their climate conditions, we assessed the convergence of ecotype composition in the transplanted communities to that of control communities (those that remained in their native environment; Fig. 1B). We further hypothesized that the demographic shifts were due to differential adaptation to local climates as a result of trait variation among the ecotypes. Thus, we expected that the climate gradient would select for a strong trait–environment relationship (assessed by community-weighted mean (CWM) trait values) as typically observed in plant communities (35, 36).In parallel, we conducted an in situ evolution experiment by transplanting an isogenic Curtobacterium strain across the same gradient to investigate the potential for rapid evolution on the same timescales. We hypothesized that a variety of genomic mutations would be associated with adaptation to local climate conditions. Therefore, we expected fewer genetic changes when the strain was transplanted to its original environment, the midelevation Grassland site, while the extreme sites of the gradient would impose stronger selective pressures resulting in greater genetic changes. We further expected to observe parallel mutations among replicates within a site, which would be indicative of adaptive events (37). Variation in such mutations across sites would suggest selection differences across the climate gradient. Together, the two experiments capture the simultaneous effects of both ecological and evolutionary processes on the response of a soil bacterium to new climates in the field.     

Corticotropin-releasing hormone receptor expression on normal and tumorous human adrenocortical cells

Corticotropin-releasing hormone (CRH) is not only the principal regulator of the central hypothalamic-pituitary-adrenal (HPA) axis but also exerts direct actions on peripheral tissues. We analyzed the expression of CRH receptors in microdissected preparations of normal human adrenal glands and in adrenocortical and adrenomedullary tumors, employing immunohistochemistry, quantitative RT-PCR of microdissected adrenal tissues, and in situ hybridization. The effect of CRH on adrenal steroidogenesis was tested in adrenal cells. Immunoreactive CRH1R was found primarily within the zona reticularis. In addition, we found a higher expression of CRH type-1 and 2 receptors mRNAs in preparations of adrenal cortices as compared to pheochromocytomas, a 6-fold increase in preparations of clinically unapparent adrenocortical adenomas, and a 10- to 60-fold increase in cortisol-producing adrenal adenomas. Stimulation of the adrenal tumor cell line NCI-H295R with CRH elicited a 1.4-fold increase in DHEA secretion. This result could be reproduced in a culture of primary human adrenocortical cells. We conclude that adrenocortical cells exhibit a higher expression of functional CRH receptors than chromaffin cells and that CRH acts on adrenal DHEA production. The data support the assertion of a direct action of CRH on human adrenocortical cells in addition to an intra-adrenal CRH receptor/adrenocorticotropin system. Enhanced CRH1R expression may be involved in adrenocortical tumorigenesis.     

Long‐term effectiveness of recommended boosted protease inhibitor‐based antiretroviral therapy in Europe

Objectives

The aim of the study was to evaluate the long‐term response to antiretroviral treatment (ART) based on atazanavir/ritonavir (ATZ/r)‐, darunavir/ritonavir (DRV/r)‐, and lopinavir/ritonavir (LPV/r)‐containing regimens.

Methods

Data were analysed for 5678 EuroSIDA‐enrolled patients starting a DRV/r‐, ATZ/r‐ or LPV/r‐containing regimen between 1 January 2000 and 30 June 2013. Separate analyses were performed for the following subgroups of patients: (1) ART‐naïve subjects (8%) at ritonavir‐boosted protease inhibitor (PI/r) initiation; (2) ART‐experienced individuals (44%) initiating the new PI/r with a viral load (VL) ≤500 HIV‐1 RNA copies/mL; and (3) ART‐experienced patients (48%) initiating the new PI/r with a VL >500 copies/mL. Virological failure (VF) was defined as two consecutive VL measurements >200 copies/mL ≥24 weeks after PI/r initiation. Kaplan–Meier and multivariable Cox models were used to compare risks of failure by PI/r‐based regimen. The main analysis was performed with intention‐to‐treat (ITT) ignoring treatment switches.

Results

The time to VF favoured DRV/r over ATZ/r, and both were superior to LPV/r (log‐rank test; P < 0.02) in all analyses. Nevertheless, the risk of VF in ART‐naïve patients was similar regardless of the PI/r initiated after controlling for potential confounders. The risk of VF in both treatment‐experienced groups was lower for DRV/r than for ATZ/r, which, in turn, was lower than for LPV/r‐based ART.

Conclusions

Although confounding by indication and calendar year cannot be completely ruled out, in ART‐experienced subjects the long‐term effectiveness of DRV/r‐containing regimens appears to be greater than that of ATZ/r and LPV/r.