Fused tricyclic indoles as S1P₁ agonists with robust efficacy in animal models of autoimmune disease

Two series of fused tricyclic indoles were identified as potent and selective S1P(1) agonists. In vivo these agonists produced a significant reduction in circulating lymphocytes which translated into robust efficacy in several rodent models of autoimmune disease. Importantly, these agonists were devoid of any activity at the S1P(3) receptor in vitro, and correspondingly did not produce S1P(3) mediated bradycardia in telemeterized rat.     

Evolution of the Alx homeobox gene family: parallel retention and independent loss of the vertebrate Alx3 gene

The Alx gene family is implicated in craniofacial development and comprises two to four homeobox genes in each vertebrate genome analyzed. Using phylogenetics and comparative genomics, we show that the common ancestor of jawed vertebrates had three Alx genes descendent from the two-round genome duplications (Alx1, Alx3, Alx4), compared with a single amphioxus gene. Later in evolution one of the paralogues, Alx3, was lost independently from at least three different vertebrate lineages, whereas Alx1 and Alx4 were consistently retained. Comparison of spatial gene expression patterns reveals that the three mouse genes have equivalent craniofacial expression to the two chick and frog genes, suggesting that redundancy compensated for gene loss. We suggest that multiple independent loss of one Alx gene was predisposed by extensive and persistent overlap in gene expression between Alx paralogues. Even so, it is unclear whether it was coincidence or evolutionary bias that resulted in the same Alx gene being lost on each occasion, rather than different members of the gene family.     

Foraging Behaviour and Landscape Utilisation by the Endangered Golden-Crowned Flying Fox (Acerodon jubatus), The Philippines

Species of Old World fruit-bats (family Pteropodidae) have been identified as the natural hosts of a number of novel and highly pathogenic viruses threatening livestock and human health. We used GPS data loggers to record the nocturnal foraging movements of Acerodon jubatus, the Golden-crowned flying fox in the Philippines to better understand the landscape utilisation of this iconic species, with the dual objectives of pre-empting disease emergence and supporting conservation management. Data loggers were deployed on eight of 54 A. jubatus (two males and six females) captured near Subic Bay on the Philippine island of Luzon between 22 November and 2 December 2010. Bodyweight ranged from 730 g to 1002 g, translating to a weight burden of 3–4% of bodyweight. Six of the eight loggers yielded useful data over 2–10 days, showing variability in the nature and range of individual bat movements. The majority of foraging locations were in closed forest and most were remote from evident human activity. Forty-six discrete foraging locations and five previously unrecorded roost locations were identified. Our findings indicate that foraging is not a random event, with the majority of bats exhibiting repetitious foraging movements night-to-night, that apparently intact forest provides the primary foraging resource, and that known roost locations substantially underestimate the true number (and location) of roosts. Our initial findings support policy and decision-making across perspectives including landscape management, species conservation, and potentially disease emergence.     

Delineation of lipopolysaccharide (LPS)-binding sites on hemoglobin: from in silico predictions to biophysical characterization

Hemoglobin (Hb) functions as a frontline defense molecule during infection by hemolytic microbes. Binding to LPS induces structural changes in cell-free Hb, which activates the redox activity of the protein for the generation of microbicidal free radicals. Although the interaction between Hb and LPS has implications for innate immune defense, the precise LPS-interaction sites on Hb remain unknown. Using surface plasmon resonance, we found that both the Hb α and β subunits possess high affinity LPS-binding sites, with K(D) in the nanomolar range. In silico analysis of Hb including phospho-group binding site prediction, structure-based sequence comparison, and docking to model the protein-ligand interactions showed that Hb possesses evolutionarily conserved surface cationic patches that could function as potential LPS-binding sites. Synthetic Hb peptides harboring predicted LPS-binding sites served to validate the computational predictions. Surface plasmon resonance analysis differentiated LPS-binding peptides from non-binders. Binding of the peptides to lipid A was further substantiated by a fluorescent probe displacement assay. The LPS-binding peptides effectively neutralized the endotoxicity of LPS in vitro. Additionally, peptide B59 spanning residues 59-95 of Hbβ attached to the surface of Gram-negative bacteria as shown by flow cytometry and visualized by immunogold-labeled scanning electron microscopy. Site-directed mutagenesis of the Hb subunits further confirmed the function of the predicted residues in binding to LPS. In summary, the integration of computational predictions and biophysical characterization has enabled delineation of multiple LPS-binding hot spots on the Hb molecule.     

Homologous and heterologous desensitization of guanylyl cyclase-B signaling in GH3 somatolactotropes

The guanylyl cyclases, GC-A and GC-B, are selective receptors for atrial and C-type natriuretic peptides (ANP and CNP, respectively). In the anterior pituitary, CNP and GC-B are major regulators of cGMP production in gonadotropes and yet mouse models of disrupted CNP and GC-B indicate a potential role in growth hormone secretion. In the current study, we investigate the molecular and pharmacological properties of the CNP/GC-B system in somatotrope lineage cells. Primary rat pituitary and GH3 somatolactotropes expressed functional GC-A and GC-B receptors that had similar EC₅₀ properties in terms of cGMP production. Interestingly, GC-B signaling underwent rapid homologous desensitization in a protein phosphatase 2A (PP2A)-dependent manner. Chronic exposure to either CNP or ANP caused a significant down-regulation of both GC-A- and GC-B-dependent cGMP accumulation in a ligand-specific manner. However, this down-regulation was not accompanied by alterations in the sub-cellular localization of these receptors. Heterologous desensitization of GC-B signaling occurred in GH3 cells following exposure to either sphingosine-1-phosphate or thyrotrophin-releasing hormone (TRH). This heterologous desensitization was protein kinase C (PKC)-dependent, as pre-treatment with GF109203X prevented the effect of TRH on CNP/GC-B signaling. Collectively, these data indicate common and distinct properties of particulate guanylyl cyclase receptors in somatotropes and reveal that independent mechanisms of homologous and heterologous desensitization occur involving either PP2A or PKC. Guanylyl cyclase receptors thus represent potential novel therapeutic targets for treating growth-hormone-associated disorders.     

Metabolic shifts toward glutamine regulate tumor growth,invasion and bioenergetics in ovarian cancer

Glutamine can play a critical role in cellular growth in multiple cancers. Glutamine‐addicted cancer cells are dependent on glutamine for viability, and their metabolism is reprogrammed for glutamine utilization through the tricarboxylic acid (TCA) cycle. Here, we have uncovered a missing link between cancer invasiveness and glutamine dependence. Using isotope tracer and bioenergetic analysis, we found that low‐invasive ovarian cancer (OVCA) cells are glutamine independent, whereas high‐invasive OVCA cells are markedly glutamine dependent. Consistent with our findings, OVCA patients’ microarray data suggest that glutaminolysis correlates with poor survival. Notably, the ratio of gene expression associated with glutamine anabolism versus catabolism has emerged as a novel biomarker for patient prognosis. Significantly, we found that glutamine regulates the activation of STAT3, a mediator of signaling pathways which regulates cancer hallmarks in invasive OVCA cells. Our findings suggest that a combined approach of targeting high‐invasive OVCA cells by blocking glutamine's entry into the TCA cycle, along with targeting low‐invasive OVCA cells by inhibiting glutamine synthesis and STAT3 may lead to potential therapeutic approaches for treating OVCAs.     

A Systematic Approach to Capacity Strengthening of Laboratory Systems for Control of Neglected Tropical Diseases in Ghana,Kenya, Malawi and Sri Lanka

Background

The lack of capacity in laboratory systems is a major barrier to achieving the aims of the London Declaration (2012) on neglected tropical diseases (NTDs). To counter this, capacity strengthening initiatives have been carried out in NTD laboratories worldwide. Many of these initiatives focus on individuals'' skills or institutional processes and structures ignoring the crucial interactions between the laboratory and the wider national and international context. Furthermore, rigorous methods to assess these initiatives once they have been implemented are scarce. To address these gaps we developed a set of assessment and monitoring tools that can be used to determine the capacities required and achieved by laboratory systems at the individual, organizational, and national/international levels to support the control of NTDs.

Methodology and principal findings

We developed a set of qualitative and quantitative assessment and monitoring tools based on published evidence on optimal laboratory capacity. We implemented the tools with laboratory managers in Ghana, Malawi, Kenya, and Sri Lanka. Using the tools enabled us to identify strengths and gaps in the laboratory systems from the following perspectives: laboratory quality benchmarked against ISO 15189 standards, the potential for the laboratories to provide support to national and regional NTD control programmes, and the laboratory''s position within relevant national and international networks and collaborations.

Conclusion

We have developed a set of mixed methods assessment and monitoring tools based on evidence derived from the components needed to strengthen the capacity of laboratory systems to control NTDs. Our tools help to systematically assess and monitor individual, organizational, and wider system level capacity of laboratory systems for NTD control and can be applied in different country contexts.