Loss of glucocorticoid rhythm induces an osteoporotic phenotype in female mice

Glucocorticoid (GC)‐induced osteoporosis is a widespread health problem that is accompanied with increased fracture risk. Detrimental effects of anti‐inflammatory GC therapy on bone have been ascribed to the excess in GC exposure, but it is unknown whether there is also a role for disruption of the endogenous GC rhythm that is inherent to GC therapy. To investigate this, we implanted female C57Bl/6J mice with slow‐release corticosterone (CORT) pellets to blunt the rhythm in CORT levels without inducing hypercortisolism. Flattening of CORT rhythm reduced cortical and trabecular bone volume and thickness, whilst bone structure was maintained in mice injected with supraphysiologic CORT at the time of their endogenous GC peak. Mechanistically, mice with a flattened CORT rhythm showed disrupted circadian gene expression patterns in bone, along with changes in circulating bone turnover markers indicative of a negative balance in bone remodelling. Indeed, double calcein labelling of bone in vivo revealed a reduced bone formation in mice with a flattened CORT rhythm. Collectively, these perturbations in bone turnover and structure decreased bone strength and stiffness, as determined by mechanical testing. In conclusion, we demonstrate for the first time that flattening of the GC rhythm disrupts the circadian clock in bone and results in an osteoporotic phenotype in mice. Our findings indicate that at least part of the fracture risk associated with GC therapy may be the consequence of a disturbed GC rhythm, rather than excess GC exposure alone, and that a dampened GC rhythm may contribute to the age‐related risk of osteoporosis.     

Oxygen concentration drives local adaptation in the greenlip abalone (Haliotis laevigata)

Understanding adaptation has become one of the major biological questions especially in the light of rapid environmental changes induced by climate change. Ocean temperatures are rising which triggers massive changes in water chemistry and thereby alters the living environment of all marine organisms. Studying adaptation, however, can be tricky because spatial genetic patterns might also occur due to random effects, for example, genetic drift. Genetic drift is reduced in very large and well‐connected populations, such as in broadcast marine spawning organisms. Here, spatial genetic divergence is likely to be produced by selection. In this issue of Molecular Ecology, Sandoval‐Castillo et al. (2018) investigated patterns of spatial genetic divergence and their association with environmental factors in the greenlip abalone (Haliotis laevigata). This commercially important species of mollusc is a broadcast spawner with large population sizes, rendering genetic drift an unlikely factor in the genetic divergence of wild populations. Sandoval‐Castillo et al. (2018) used a ddRAD genomic approach to test for genetic divergence between sampled populations while also measuring different environmental factors, for example, water temperature and oxygen content. The majority of identified SNPs was putatively neutral and showed only low levels of genetic divergence between field sites. However, 323 candidate adaptive markers were identified that clearly separated the individuals into five different clusters. These genetic clusters correlated with environmental clusters mainly determined by water temperature and (correlated) oxygen concentration. Gene annotation of the candidate SNPs revealed a large proportion of loci being involved in biological processes influenced by oxygen availability. The study by Sandoval‐Castillo et al. (2018) in this issue of Molecular Ecology exemplifies the benefits of combining genomic studies with ecological data. It is a great starting point for more detailed (gene function, physiology) as well as broader (biodiversity) investigations that might help us to better understand adaptation and predict ecosystems' resilience and resistance to environmental disturbances. In addition, this information can be applied to implement optimal conservation regime policies and sustainable harvesting strategies, hopefully protecting biodiversity as well as commercial interests in marine life.     

Body temperature variation controls pre-mRNA processing and transcription of antiviral genes and SARS-CoV-2 replication

Antiviral innate immunity represents the first defense against invading viruses and is key to control viral infections, including SARS-CoV-2. Body temperature is an omnipresent variable but was neglected when addressing host defense mechanisms and susceptibility to SARS-CoV-2 infection. Here, we show that increasing temperature in a 1.5°C window, between 36.5 and 38°C, strongly increases the expression of genes in two branches of antiviral immunity, nitric oxide production and type I interferon response. We show that alternative splicing coupled to nonsense-mediated decay decreases STAT2 expression in colder conditions and suggest that increased STAT2 expression at elevated temperature induces the expression of diverse antiviral genes and SARS-CoV-2 restriction factors. This cascade is activated in a remarkably narrow temperature range below febrile temperature, which reflects individual, circadian and age-dependent variation. We suggest that decreased body temperature with aging contributes to reduced expression of antiviral genes in older individuals. Using cell culture and in vivo models, we show that higher body temperature correlates with reduced SARS-CoV-2 replication, which may affect the different vulnerability of children versus seniors toward severe SARS-CoV-2 infection. Altogether, our data connect body temperature and pre-mRNA processing to provide new mechanistic insight into the regulation of antiviral innate immunity.     

Selenate reduction rates and kinetics across depth in littoral sediment of the Salton Sea,California

To predict selenium cycling in sediments, it is crucial to identify and quantify the processes leading to selenium sequestration in sediments. More specifically, it is essential to obtain environmentally-relevant kinetic parameters for selenium reduction and information on how they spatially vary in sediments. The Salton Sea (California, USA) is an ideal model system to examine selenium processes in sediments due to its semi-enclosed conditions and increasing selenium concentration over the last century. Selenium enters the Salton Sea mainly as selenate and might be sequestered in the sediment through microbial reduction. To determine the potential selenium sequestration of Salton Sea littoral sediments and which sediment properties are controlling selenate reduction kinetics, we determined the centimeter-scale vertical distribution of potential selenate reduction rates and apparent kinetic parameters (maximum selenate reduction rates, V_max, and selenate half-saturation concentration, K_m) using flow-through reactor (FTR) experiments. We compared sediments from two littoral sites (South and North) and four depth intervals (0–2, 2–4, 4–6 and 6–8 cm). Furthermore, we characterized the selenium fractions in the sediment recovered from the FTR experiments to identify the processes leading to the sequestration of selenium. Our results reveal higher potential for selenium reduction and sequestration in the topmost sediment (0–2 cm) suggesting that microorganisms inhabiting surface sediment are well adapted to reduce selenate entering the Salton Sea. As apparent K_m values (103–2144 µM) exceed the average selenium concentration in the overlying water (6–25 nM), in situ selenate reduction is limited by the low availability of selenate and the resident selenate-reducing microorganisms operate well below their V_max (11 and 43 nmol cm^?3 h^?1). Selenium speciation after FTR experiments confirms the primary sequestration of reduced biomass-associated and elemental selenium (68–99% of total selenium) in the sediment. Further, the absence of correlation between the tested sediment physical (porosity, bulk density, clay content), chemical (C_org, N_tot, total selenium content) and biological characteristics (abundance of culturable selenate-reducers) with the kinetic parameters of selenate reduction indicates that these sediment characteristics cannot be used as predictors of apparent V_max or K_m. Conclusively, microbial selenate reduction is an important, if not the primary process, leading to the sequestration of reduced selenium in the Salton Sea sediments and making the surficial Salton Sea sediments an important selenium sink.     

Repeated H2O2 exposure drives cell cycle progression in an in vitro model of ulcerative colitis

The production of hydrogen peroxide (H₂O₂) drives tumourigenesis in ulcerative colitis (UC). Recently, we showed that H₂O₂ activates DNA damage checkpoints in human colonic epithelial cells (HCEC) through c‐Jun N‐terminal Kinases (JNK) that induces p21^WAF1. Moreover, caspases circumvented the G1/S and intra‐S checkpoints, and cells accumulated in G2/M. The latter observation raised the question of whether repeated H₂O₂ exposures alter JNK activation, thereby promoting a direct passage of cells from G2/M arrest to driven cell cycle progression. Here, we report that increased proliferation of repeatedly H₂O₂‐exposed HCEC cells (C‐cell cultures) was associated with (i) increased phospho‐p46 JNK, (ii) decreased total JNK and phospho‐p54 JNK and (iii) p21^WAF1 down‐regulation. Altered JNK activation and p21^WAF1 down‐regulation were accompanied by defects in maintaining G2/M and mitotic spindle checkpoints through adaptation, as well as by apoptosis resistance following H₂O₂ exposure. This may cause increased proliferation of C‐cell cultures, a defining initiating feature in the inflammation‐carcinoma pathway in UC. We further suggest that dysregulated JNK activation is attributed to a non‐apoptotic function of caspases, causing checkpoint adaptation in C‐cell cultures. Additionally, loss of cell‐contact inhibition and the overcoming of senescence, hallmarks of cancer, contributed to increased proliferation. Furthermore, there was evidence that p54 JNK inactivation is responsible for loss of cell‐contact inhibition. We present a cellular model of UC and suggest a sinusoidal pattern of proliferation, which is triggered by H₂O₂‐induced reactive oxygen species generation, involving an interplay between JNK activation/inactivation, p21^WAF1, c‐Fos, c‐Jun/phospho‐c‐Jun, ATF2/phospho‐ATF2, β‐catenin/TCF4‐signalling, c‐Myc, CDK6 and Cyclin D2, leading to driven cell cycle progression.     

Phospho-NHE3 forms membrane patches and interacts with beta-actin to sense and maintain constant direction during cell migration

The Na(+)/H(+) exchanger NHE3 colocalizes with beta-actin at the leading edge of directionally migrating cells. Using human osteosarcoma cells (SaOS-2), rat osteoblasts (calvaria), and human embryonic kidney (HEK) cells, we identified a novel role for NHE3 via beta-actin in anode and cathode directed motility, during electrotaxis. NHE3 knockdown by RNAi revealed that NHE3 expression is required to achieve constant directionality and polarity in migrating cells. Phosphorylated NHE3 (pNHE3) and beta-actin complex formation was impaired by the NHE3 inhibitor S3226 (IC50 0.02 µM). Fluorescence cross-correlation spectroscopy (FCCS) revealed that the molecular interactions between NHE3 and beta-actin in membrane protrusions increased 1.7-fold in the presence of a directional cue and decreased 3.3-fold in the presence of cytochalasin D. Data from flow cytometric analysis showed that membrane potential of cells (V_mem) decreases in directionally migrating, NHE3-deficient osteoblasts and osteosarcoma cells whereas only V_mem of wild type osteoblasts is affected during directional migration. These findings suggest that pNHE3 has a mechanical function via beta-actin that is dependent on its physiological activity and V_mem. Furthermore, phosphatidylinositol 3,4,5-trisphosphate (PIP3) levels increase while PIP2 remains stable when cells have persistent directionality. Both PI3 kinase (PI3K) and Akt expression levels change proportionally to NHE3 levels. Interestingly, however, the content of pNHE3 level does not change when PI3K/Akt is inhibited. Therefore, we conclude that NHE3 can act as a direction sensor for cells and that NHE3 phosphorylation in persistent directional cell migration does not involve PI3K/Akt during electrotaxis.     

The anti-angiogenic factor PEDF is present in the human heart and is regulated by anoxia in cardiac myocytes and fibroblasts

Cardiac diseases such as myocardial infarction and heart failure are among the leading causes of death in western societies. Therapeutic angiogenesis has been suggested as a concept to combat these diseases. The biology of angiogenic factors expressed in the heart such as vascular endothelial growth factor (VEGF) is well studied, whereas data on anti-angiogenic mediators in the heart are scarce. Here we study the expression of the anti-angiogenic factor pigment epithelium-derived factor (PEDF) in the human heart and in human cardiac cells. PEDF expression could be detected in human cardiac tissue on the protein and mRNA levels. PEDF mRNA levels were significantly lower in explanted human ischemic hearts as compared to healthy hearts. Our in vitro experiments showed that human adult cardiac myocytes and fibroblasts constitutively secrete PEDF. In addition to anoxic conditions, cobalt chloride, 2,2'dipyridyl and dimethoxally glycine, which stabilize hypoxia inducible factor-α decreased PEDF expression. Furthermore we show that PEDF inhibits VEGF-induced sprouting. We have identified PEDF in healthy and ischemic human hearts and we show that PEDF expression is down-regulated by low oxygen levels. Therefore, we suggest a role for PEDF in the regulation of angiogenesis in the heart and propose PEDF as a possible therapeutic target in heart disease.     

A marine Mesorhizobium sp. produces structurally novel long-chain N-acyl-L-homoserine lactones

Our study focused on a Mesorhizobium sp. that is phylogenetically affiliated by 16S rRNA gene sequence to other marine and saline bacteria of this genus. Liquid chromatography-mass spectrometry investigations of the extract obtained from solid-phase extraction of cultures of this bacterium indicated the presence of several N-acyl homoserine lactones (AHLs), with chain lengths of C(10) to C(16). Chromatographic separation of the active bacterial extract yielded extraordinarily large amounts of two unprecedented acylated homoserine lactones, 5-cis-3-oxo-C(12)-homoserine lactone (5-cis-3-oxo-C(12)-HSL) (compound 1) and 5-cis-C(12)-HSL (compound 2). Quorum-sensing activity of compounds 1 and 2 was shown in two different biosensor systems [Escherichia coli MT102(pSB403) and Pseudomonas putida F117(pKR-C12)]. Furthermore, it was shown that both compounds can restore protease and pyoverdin production of an AHL-deficient Pseudomonas aeruginosa PAO1 lasI rhlI double mutant, suggesting that these signal molecules maybe used for intergenus signaling. In conclusion, these data indicate that the quorum-sensing activity of compounds 1 and 2 is modulated by the chain length and functional groups of the acyl moiety. Additionally, compound 1 showed antibacterial and cytotoxic activities.