Retinoids control anterior and dorsal properties in the developing forebrain

We have previously shown that retinoic acid (RA) synthesized by the retinaldehyde dehydrogenase 2 (RALDH2) is required in forebrain development. Deficiency in RA due to inactivation of the mouse Raldh2 gene or to complete absence of retinoids in vitamin-A-deficient (VAD) quails, leads to abnormal morphogenesis of various forebrain derivatives. In this study we show that double Raldh2/Raldh3 mouse mutants have a more severe phenotype in the craniofacial region than single null mutants. In particular, the nasal processes are truncated and the eye abnormalities are exacerbated. It has been previously shown that retinoids act mainly on cell proliferation and survival in the ventral forebrain by regulating SHH and FGF8 signaling. Using the VAD quail model, which survives longer than the Raldh-deficient mouse embryos, we found that retinoids act in maintaining the correct position of anterior and dorsal boundaries in the forebrain by modulating FGF8 anteriorly and WNT signaling dorsally. Furthermore, BMP4 and FGF8 signaling are affected in the nasal region and BMP4 is ventrally expanded in the optic vesicle. At the optic cup stage, Pax6, Tbx5 and Bmp4 are ectopically expressed in the presumptive retinal pigmented epithelium (RPE), while Otx2 and Mitf are not induced, leading to a dorsal transdifferentiation of RPE to neural retina. Therefore, besides being required for survival of ventral structures, retinoids are involved in restricting anterior identity in the telencephalon and dorsal identity in the diencephalon and the retina.     

The NLRP3 inflammasome is differentially activated by pneumolysin variants and contributes to host defense in pneumococcal pneumonia

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and sepsis. Pneumococci can be divided into >90 serotypes that show differences in the pathogenicity and invasiveness. We tested the hypotheses that the innate immune inflammasome pathway is involved in fighting pneumococcal pneumonia and that some invasive pneumococcal types are not recognized by this pathway. We show that human and murine mononuclear cells responded to S. pneumoniae expressing hemolytic pneumolysin by producing IL-1β. This IL-1β production depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Some serotype 1, serotype 8, and serotype 7F bacteria, which have previously been associated with increased invasiveness and with production of toxins with reduced hemolytic activity, or bacterial mutants lacking pneumolysin did not stimulate notable IL-1β production. We further found that NLRP3 was beneficial for mice during pneumonia caused by pneumococci expressing hemolytic pneumolysin and was involved in cytokine production and maintenance of the pulmonary microvascular barrier. Overall, the inflammasome pathway is protective in pneumonia caused by pneumococci expressing hemolytic toxin but is not activated by clinically important pneumococcal sequence types causing invasive disease. The study indicates that a virulence factor polymorphism may substantially affect the recognition of bacteria by the innate immune system.     

In Vitro Metabolic and Mitogenic Signaling of Insulin Glargine and Its Metabolites

Background

Insulin glargine (Lantus®) is a long-acting basal insulin analog that demonstrates effective day-long glycemic control and a lower incidence of hypoglycemia than NPH insulin. After subcutaneous injection insulin glargine is partly converted into the two main metabolites M1 ([Gly^A21]insulin) and M2 ([Gly^A21,des-Thr^B30]insulin). The aim of this study was to characterize the glargine metabolites in vitro with regard to their insulin receptor (IR) and IGF-1 receptor (IGF1R) binding and signaling properties as well as their metabolic and mitogenic activities.

Methods

The affinity of human insulin, insulin glargine and its metabolites to the IR isoforms A and B or IGF1R was analyzed in a competitive binding assay using SPA technology. Receptor autophosphorylation activities were studied via In-Cell Western in CHO and MEF cells overexpressing human IR-A and IR-B or IGF1R, respectively. The metabolic response of the insulins was studied as stimulation of lipid synthesis using primary rat adipocytes. Thymidine incorporation in Saos-2 cells was used to characterize the mitogenic activity.

Conclusions

The binding of insulin glargine and its metabolites M1 and M2 to the IR were similar and correlated well with their corresponding autophosphorylation and metabolic activities in vitro. No differences were found towards the two IR isoforms A or B. Insulin glargine showed a higher affinity for IGF1R than insulin, resulting in a lower EC₅₀ value for autophosphorylation of the receptor and a more potent stimulation of thymidine incorporation in Saos-2 cells. In contrast, the metabolites M1 and M2 were significantly less active in binding to and activation of the IGF1R and their mitogenicity in Saos-2 cells was equal to human insulin. These findings strongly support the idea that insulin glargine metabolites contribute with the same potency as insulin glargine to blood glucose control but lead to significantly reduced growth-promoting activity.     

Mechanisms of skeletal muscle aging: insights from Drosophila and mammalian models

A characteristic feature of aged humans and other mammals is the debilitating, progressive loss of skeletal muscle function and mass that is known as sarcopenia. Age-related muscle dysfunction occurs to an even greater extent during the relatively short lifespan of the fruit fly Drosophila melanogaster. Studies in model organisms indicate that sarcopenia is driven by a combination of muscle tissue extrinsic and intrinsic factors, and that it fundamentally differs from the rapid atrophy of muscles observed following disuse and fasting. Extrinsic changes in innervation, stem cell function and endocrine regulation of muscle homeostasis contribute to muscle aging. In addition, organelle dysfunction and compromised protein homeostasis are among the primary intrinsic causes. Some of these age-related changes can in turn contribute to the induction of compensatory stress responses that have a protective role during muscle aging. In this Review, we outline how studies in Drosophila and mammalian model organisms can each provide distinct advantages to facilitate the understanding of this complex multifactorial condition and how they can be used to identify suitable therapies.     

Molecular dynamics simulation approach for the prediction of transmembrane helix–helix heterodimers assembly

Computational methods are useful to identify favorable structures of transmembrane (TM) helix oligomers when experimental data are not available or when they cannot help to interpret helix-helix association. We report here a global search method using molecular dynamics (MD) simulations to predict the structures of transmembrane homo and heterodimers. The present approach is based only on sequence information without any experimental data and is first applied to glycophorin A to validate the protocol and to the HER2-HER3 heterodimer receptor. The method successfully reproduces the experimental structures of the TM domain of glycophorin A (GpA(TM)) with a root mean square deviation of 1.5 A. The search protocol identifies three energetically stable models of the TM domain of HER2-HER3 receptor with favorable helix-helix arrangement, including right-handed and left-handed coiled-coils. The predicted TM structures exhibit the GxxxG-like motif at the dimer interface which is presumed to drive receptor oligomerization. We demonstrate that native structures of TM domain can be predicted without quantitative experimental data. This search protocol could help to predict structures of the TM domain of HER heterodimer family.     

Processing of Drosophila endo-siRNAs depends on a specific Loquacious isoform

Drosophila melanogaster expresses three classes of small RNAs, which are classified according to their mechanisms of biogenesis. MicroRNAs are ∼22–23 nucleotides (nt), ubiquitously expressed small RNAs that are sequentially processed from hairpin-like precursors by Drosha/Pasha and Dcr-1/Loquacious complexes. MicroRNAs usually associate with AGO1 and regulate the expression of protein-coding genes. Piwi-interacting RNAs (piRNAs) of ∼24–28 nt associate with Piwi-family proteins and can arise from single-stranded precursors. piRNAs function in transposon silencing and are mainly restricted to gonadal tissues. Endo-siRNAs are found in both germline and somatic tissues. These ∼21-nt RNAs are produced by a distinct Dicer, Dcr-2, and do not depend on Drosha/Pasha complexes. They predominantly bind to AGO2 and target both mobile elements and protein-coding genes. Surprisingly, a subset of endo-siRNAs strongly depend for their production on the dsRNA-binding protein Loquacious (Loqs), thought generally to be a partner for Dcr-1 and a cofactor for miRNA biogenesis. Endo-siRNA production depends on a specific Loqs isoform, Loqs-PD, which is distinct from the one, Loqs-PB, required for the production of microRNAs. Paralleling their roles in the biogenesis of distinct small RNA classes, Loqs-PD and Loqs-PB bind to different Dicer proteins, with Dcr-1/Loqs-PB complexes and Dcr-2/Loqs-PD complexes driving microRNA and endo-siRNA biogenesis, respectively.     

Copper deficiency induced emphysema is associated with focal adhesion kinase inactivation

Background

Copper is an important regulator of hypoxia inducible factor 1 alpha (HIF-1α) dependent vascular endothelial growth factor (VEGF) expression, and is also required for the activity of lysyl oxidase (LOX) to effect matrix protein cross-linking. Cell detachment from the extracellular matrix can induce apoptosis (anoikis) via inactivation of focal adhesion kinase (FAK).

Methodology

To examine the molecular mechanisms whereby copper depletion causes the destruction of the normal alveolar architecture via anoikis, Male Sprague-Dawley rats were fed a copper deficient diet for 6 weeks while being treated with the copper chelator, tetrathiomolybdate. Other groups of rats were treated with the inhibitor of auto-phosphorylation of FAK, 1,2,4,5-benzenetetraamine tetrahydrochloride (1,2,4,5-BT) or FAK small interfering RNA (siRNA).

Principal Findings

Copper depletion caused emphysematous changes, decreased HIF-1α activity, and downregulated VEGF expression in the rat lungs. Cleaved caspase-3, caspase-8 and Bcl-2 interacting mediator of cell death (Bim) expression was increased, and the phosphorylation of FAK was decreased in copper depleted rat lungs. Administration of 1,2,4,5-BT and FAK siRNA caused emphysematous lung destruction associated with increased expression of cleaved capase-3, caspase-8 and Bim.

Conclusions

These data indicate that copper-dependent mechanisms contribute to the pathogenesis of emphysema, which may be associated with decreased HIF-1α and FAK activity in the lung.     

CAPITULUM PHYLLOTAXIS AND NUMERICAL CANALIZATION IN MICROSERIS PYGMAEA (ASTERACEAE: LACTUCEAE)

The positions at which floret primordia arise in developing capitulum buds of Microseris pygmaea D. Don have been mapped by computer-assisted light microscopy. The primordia can be assigned positions along a basic phyllotactic spiral with a divergence angle of about 137.5°. In addition, there are regular deviations from a spiral arrangement. Typically, the first 26 primordia in phyllotactic sequence are arranged in two concentric circles of 13 primordia with considerable deviations in the divergence angle and in the distances between primordia along a parastichy at positions 13 and 26. This arrangement can be simulated by geometric models that include nearest neighbor packing, together with spiral phyllotaxis. The circular arrangement of peripheral primordia at nearly equal radial distances from the center of the developing capitulum helps to explain the numerical constancy (canalization) of peripheral structures, especially the constant number of 13 inner phyllaries on heads with very different numbers of florets.     

Optimization of the differentiation of human preadipocytes in vitro

This study aimed at developing an optimal protocol for proliferation and differentiation of preadipocytes that is a prerequisite for constructing an ideal biohybrid composed of viable adipose precursor cells in a three-dimensional matrix. Such an implant could represent an adequate solution for correcting soft tissue defects, e.g., extensive deep burns or tumor resections. Preadipocytes were isolated from human subcutaneous adipose tissue samples and cultured in Dulbecco's modified eagle medium (DMEM)/Ham's F12 medium (F12) or OPTIMEM medium with or without the addition of human serum (hS) or fetal calf serum (FCS). The advantages of fibronectin-coated culture dishes for preadipocyte yield after isolation and differentiation were evaluated. After culture expansion, differentiation was induced by insulin, isobutylmethylxanthine, pioglitazone, dexamethasone, and transferrin in the absence of serum. The extent of differentiation was assayed by measuring the activity of glycerophosphate dehydrogenase as well as counting of differentiated versus undifferentiated cells. Our results show that fibronectin coating does not only strongly increase the yield of preadipocytes after isolation from adipose tissue but also significantly enhances differentiation of precursor cells to mature adipocytes. For optimal cell expansion, DMEM/F12 is more promoting than OPTIMEM and culturing with FCS shows a slightly better proliferation compared with hS supplementation. Differentiation, in contrast, is significantly improved when hS is used instead of FCS during proliferation. Our results smooth the way for autologous preadipocyte culturing and show that hS for preadipocyte culturing opens new and promising perspectives for adipose tissue engineering by optimizing in vitro expansion in cell culture and inducing substantial differentiation.     

Steps towards sustainable harvest of Ophiocordyceps sinensis in Bhutan

The insect-pathogenic fungus Ophiocordyceps sinensis (better known as Cordyceps sinensis) is harvested over much of the Himalayan plateau as a highly prized remedy in traditional Oriental medicine. Over the past 10 years its financial value has increased dramatically, with collectors paid as much as US $12,500 kg⁻¹ for top-quality material. This is causing significant distortion to local economies, and there is widespread concern that the current rate of collection is unsustainable. This paper introduces the fungus and its insect hosts, documents some of the biological and social constraints to achieving sustainability, describes the socioeconomic climate within which harvest and sale occurs in Bhutan, and details the measures put in place by the Royal Government of Bhutan to promote wise management of this valuable natural resource.