Chronic rapamycin treatment or lack of S6K1 does not reduce ribosome activity in vivo

Reducing activity of the mTORC1/S6K1 pathway has been shown to extend lifespan in both vertebrate and invertebrate models. For instance, both pharmacological inhibition of mTORC1 with the drug rapamycin or S6K1 knockout extends lifespan in mice. Since studies with invertebrate models suggest that reducing translational activity can increase lifespan, we reasoned that the benefits of decreased mTORC1 or S6K1 activity might be due, at least in part, to a reduction of general translational activity. Here, we report that mice given a single dose of rapamycin have reduced translational activity, while mice receiving multiple injections of rapamycin over 4 weeks show no difference in translational activity compared with vehicle-injected controls. Furthermore, mice lacking S6K1 have no difference in global translational activity compared with wild-type littermates as measured by the percentage of ribosomes that are active in multiple tissues. Translational activity is reduced in S6K1-knockout mice following single injection of rapamycin, demonstrating that rapamycin’s effects on translation can occur independently of S6K1. Taken together, these data suggest that benefits of chronic rapamycin treatment or lack of S6K1 are dissociable from potential benefits of reduced translational activity, instead pointing to a model whereby changes in translation of specific subsets of mRNAs and/or translation-independent effects of reduced mTOR signaling underlie the longevity benefits.     

Plasma High Sensitivity Troponin T Levels in Adult Survivors of Childhood Leukaemias: Determinants and Associations with Cardiac Function

Background

We sought to quantify plasma high sensitivity cardiac troponin (hs-cTnT) levels, their determinants, and their associations with left ventricular (LV) myocardial deformation in adult survivors of childhood acute leukaemias.

Methods and Results

One hundred adult survivors (57 males) of childhood acute leukaemias, aged 24.1±4.2 years, and 42 age-matched controls (26 males) were studied. Plasma cTnT was determined using a highly sensitive assay. Genotyping of NAD(P)H oxidase and multidrug resistance protein polymorphisms was performed. Left ventricular function was assessed by conventional, three-dimensional, and speckle tracking echocardiography. The medians (interquartile range) of hs-cTnT in male and female survivors were 4.9 (4.2 to 7.2) ng/L and 1.0 (1.0 to 3.5) ng/L, respectively. Nineteen survivors (13 males, 6 females) (19%) had elevated hs-cTnT (>95^th centile of controls). Compared to those without elevated hs-TnT levels, these subjects had received larger cumulative anthracycline dose and were more likely to have leukaemic relapse, stem cell transplant, and cardiac irradiation. Their LV systolic and early diastolic myocardial velocities, isovolumic acceleration, and systolic longitudinal strain rate were significantly lower. Survivors having CT/TT at CYBA rs4673 had higher hs-cTnT levels than those with CC genotype. Functionally, increased hs-cTnT levels were associated with worse LV longitudinal systolic strain and systolic and diastolic strain rates.

Conclusions

Increased hs-cTnT levels occur in a significant proportion of adult survivors of childhood acute leukaemias and are associated with larger cumulative anthracycline dose received, history of leukaemic relapse, stem cell transplant, and cardiac irradiation, genetic variants in free radical metabolism, and worse LV myocardial deformation.     

Changes in the Secretory Profile of NSCLC-Associated Fibroblasts after Ablative Radiotherapy: Potential Impact on Angiogenesis and Tumor Growth

In the context of radiotherapy, collateral effects of ablative doses of ionizing radiation (AIR) on stromal components of tumors remains understudied. In this work, cancer-associated fibroblasts (CAFs) isolated from freshly resected human lung tumors were exposed to AIR (1x 18 Gy) and analyzed for their release of paracrine factors. Inflammatory mediators and regulators of angiogenesis and tumor growth were analyzed by multiplex protein assays in conditioned medium (CM) from irradiated and non-irradiated CAFs. Additionally, the profile of secreted proteins was examined by proteomics. In functional assays, effects of CAF-CM on proliferative and migratory capacity of lung tumor cells (H-520/H-522) and human umbilical vein endothelial cells (HUVECs) and their tube-forming capacity were assessed. Our data show that exposure of CAFs to AIR results in 1) downregulated release of angiogenic molecules such as stromal cell-derived factor-1, angiopoietin, and thrombospondin-2 (TSP-2); 2) upregulated release of basic fibroblast growth factor from most donors; and 3) unaffected expression levels of hepatocyte growth factor, interleukin-6 (IL-6), IL-8, IL-1β, and tumor necrosis factor-α. CM from irradiated and control CAFs did not affect differently the proliferative or migratory capacity of tumor cells (H-520/H-522), whereas migratory capacity of HUVECs was partially reduced in the presence of irradiated CAF-CM. Overall, we conclude that AIR mediates a transformation on the secretory profile of CAFs that could influence the behavior of other cells in the tumor tissue and hence guide therapeutic outcomes. Downstream consequences of the changes observed in this study merits further investigations.     

Role of Baseline Antral Follicle Count and Anti-Mullerian Hormone in Prediction of Cumulative Live Birth in the First In Vitro Fertilisation Cycle: A Retrospective Cohort Analysis

Objective

This retrospective study determined for the first time the role of baseline antral follicle count (AFC) and serum anti-Mullerian hormone (AMH) level in the first in-vitro fertilisation (IVF) cycle in predicting cumulative live birth from one stimulation cycle.

Methods

We studied 1,156 women (median age 35 years) undergoing the first IVF cycle. Baseline AFC and AMH level on the day before ovarian stimulation were analysed. The main outcome measure was cumulative live birth in the fresh plus all the frozen embryo transfers after the same stimulation cycle.

Results

Serum AMH was significantly correlated with AFC. Both AMH and AFC showed significant correlation with age and ovarian response in the stimulated cycle and total number of transferrable embryos. Baseline AFC and serum AMH were significantly higher in subjects attaining a live birth than those who did not in the fresh stimulated cycle, as well as those attaining cumulative live birth. There was a significant trend of higher cumulative live birth rate in women with higher AMH or AFC. However, logistic regression revealed that both AMH and AFC were not significant predictors of cumulative live birth after adjusting for age and number of embryos available for transfer. Considering only one single predictor, the areas under the ROC curves for AMH (0.646, 95% CI 0.616–0.675) and age (0.648, 95% CI 0.618–0.677) were slightly higher than that for AFC (0.617, 95% CI 0.587–0.647) in predicting cumulative live birth. However, a model combining AMH (with or without AFC) and age of the women only classified an addition of less than 2% of subjects correctly compared to the model with age alone.

Conclusion

Baseline AFC and serum AMH have only modest predictive performance on the occurrence of cumulative live birth, and may not give additional value on top of the women''s age.     

Thrombospondin-5 and fluvastatin promote angiogenesis and are protective against endothelial cell apoptosis

The thrombospondins (TSPs), multifunctional matricellular proteins, are known mediators of endothelial cell (EC) angiogenesis and apoptosis. TSP-1, an antiangiogenic molecule, is important in the progression of vascular disease, in part by inducing EC apoptosis. TSP-2, although less studied, also induces EC apoptosis and inhibits angiogenesis. The effects of TSP-5 are largely unexplored in ECs, but TSP-5 is believed to be protective against arterial disease. Statin drugs have been shown to have beneficial pleiotropic effects, including decreasing EC apoptosis, increasing angiogenesis, and blocking TSP signaling. We hypothesized TSP-5 will be proangiogenic and antiapoptotic, and statin pretreatment would reverse the proapoptotic and antiangiogenic phenotype of TSP-1 and TSP-2. ECs were exposed to serum-free medium, TSP-1, TSP-2, or TSP-5 with or without fluvastatin pretreatment. Quantitative real-time polymerase chain reaction was performed on 96 apoptosis and 96 angiogenesis-related genes using microfluidic card assays. Angiogenesis was measured using Matrigel assays, while apoptosis was measured by fluorescent caspase assay. TSP-5 suppressed apoptotic genes and had a mixed effect on the angiogenic genes; however, TSP-5 did not alter apoptois but was proangiogenic. Pretreatment with fluvastatin downregulated proapoptotic genes and apoptosis and upregulated proangiogenic genes and angiogenesis. Findings indicate TSP-5 and fluvastatin have a protective effect on ECs, being proangiogenic and reversing the antiangiogenic effects of TSP-1 and TSP-2. In conclusion, TSP-5 and fluvastatin may be beneficial for inducing angiogenesis in the setting of ischemia.     

Thermal environment shapes cuticle melanism and melanin-based immunity in the ground cricket Allonemobius socius

The thermal melanin hypothesis posits that ectothermic individuals of larger size or from colder environments exhibit darker cuticles due to melanin’s efficacy in absorbing solar radiation. However, melanin is also a crucial component of arthropod immunity. Thus, thermal selection for increased cuticle darkness may profoundly influence melanin-based immune function. In this study, we address the relationships between the thermal environment (season length), cuticular melanism and two aspects of melanin-based immunity across nine thermally distinct populations of the cricket Allonemobius socius. We found that season length (i.e. degree days) and body size had a positive association with cuticle melanism in both sexes across populations, supporting the thermal melanism hypothesis. Despite their smaller size, males were found to have darker cuticles and superior melanin-based immunity. This pattern may be the result of additional selection on males due to sex-specific temperature-dependent activities, such as male calling song. Perhaps most interestingly, we found that short season length populations (i.e. colder) exhibited a greater phenoloxidase activity (aspect of the melanin-based immune system) in addition to darker cuticles in both sexes. This pattern is consistent with direct thermal selection on cuticular color, coupled with indirect selection on melanin-based immunity due to pleiotropy. Thus, thermal selection on cuticle darkness appears to indirectly shape the evolution of pathogen resistance in this system, and potentially for other terrestrial arthropod systems whose ranges encompass a significant thermal gradient.     

Development of secondary sexual characters in the seabob shrimp Xiphopenaeus kroyeri (Heller 1862) (Crustacea,Decapoda, Penaeidae): a scanning electron microscope study

The development of secondary sexual characters, the petasma, and thelycum growth were studied in Xiphopenaeus kroyeri. In adult females, the thelycum is a single plate and its anterolateral portion is characterized by a reduced hood. The aperture resembles a transverse ridge. In immature stages, the ridge has a space between the plates, which becomes narrower as it reaches the end of development. The female gonopore is ‘comma’ shaped. In adult males, the endopods of the petasma are linked at the dorsomedial margin by a large quantity of cincinnuli. In juveniles, cincinnuli gradually increase in number until they join both endopods. At the end of development the petasma is T-shaped. The male gonopore is C-shaped. The relative growth of the petasma total length versus juvenile body length showed a highly positive allometry, whereas in adults the growth was isometric. For the relationship carapace length versus thelycum width, the juvenile phase of females is characterized by an isometry and the adult phase by a negative allometry.     

The Trypsin Inhibitor Panulirin Regulates the Prophenoloxidase-activating System in the Spiny Lobster Panulirus argus

The melanization reaction promoted by the prophenoloxidase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms that are still not fully understood. We report here the finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined by combining Edman degradation/N-terminal sequencing and electrospray ionization-MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin (K_i = 8.6 nm) with a notable specificity because it does not inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin, and plasmin. The removal of panulirin from the lobster hemocyte lysate leads to an increase in phenoloxidase response to LPS. Likewise, the addition of increasing concentrations of panulirin to a lobster hemocyte lysate, previously depleted of trypsin-inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme.     

Ubiquitin-specific Peptidase 8 (USP8) Regulates Endosomal Trafficking of the Epithelial Na+ Channel

Ubiquitination plays a key role in trafficking of the epithelial Na⁺ channel (ENaC). Previous work indicated that ubiquitination enhances ENaC endocytosis and sorting to lysosomes for degradation. Moreover, a defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. In this work, we identified a role for USP8 in the control of ENaC ubiquitination and trafficking. USP8 increased ENaC current in Xenopus oocytes and collecting duct epithelia and enhanced ENaC abundance at the cell surface in HEK 293 cells. This resulted from altered endocytic sorting; USP8 abolished ENaC degradation in the endocytic pathway, but it had no effect on ENaC endocytosis. USP8 interacted with ENaC, as detected by co-immunoprecipitation, and it deubiquitinated ENaC. Consistent with a functional role for deubiquitination, mutation of the cytoplasmic lysines of ENaC reduced the effect of USP8 on ENaC cell surface abundance. In contrast to USP8, USP2-45 increased ENaC surface abundance by reducing endocytosis but not degradation. Thus, USP8 and USP2-45 selectively modulate ENaC trafficking at different steps in the endocytic pathway. Together with previous work, the data indicate that the ubiquitination state of ENaC is critical for the regulation of epithelial Na⁺ absorption.