Weekly osimertinib dosing prevents EGFR mutant tumor cells destined to home mouse lungs

The recently conducted ADAURA trial concludes daily dosing of adjuvant osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), improves disease-free survival with stage IB/II/IIIA EGFR -mutated non-small cell lung cancer patients in comparison to placebo. We have developed a preclinical orthotopic mouse model, using luciferase tagged lung adenocarcinoma cells harboring EGFR TKI sensitive exon 19 deletion to model and extend trial implications comparing a weekly vs daily dosing outcome of osimertinib to a first-generation TKI- erlotinib. We find that 100% of mice in both the groups receiving osimertinib daily or weekly before injection of cells show a complete absence of homing of cells in mice''s lungs from day three until day 18 post-injection of cells. On the other hand, 25% and 75% of mice receiving erlotinib daily and weekly before injecting cells show homing of cells to the lungs. The tumors observed in the lungs, when dissected at day 30, confirmed the colonization of the injected cells homing to the organ. Thus, our study establishes the efficacy of pretreatment with osimertinib in reducing tumor cells'' homing to mouse lungs in an in vivo mouse model.     

Diverse viscerotropic isolates of Leishmania all express a highly conserved secretory nuclease during human infections

Previously, we characterized a gene encoding the unique nuclease (LdNuc(s)) from a Sudanese isolate of the human pathogen Leishmania donovani. This parasite secretory enzyme is involved in the salvage of host-derived purines and is constitutively expressed by both developmental forms of the parasite. Currently, we assessed whether an LdNuc(s)-like nuclease was conserved among other geographically disparate isolates of L. donovani and whether this enzyme was produced by intracellular amastigotes during human infections. Using RT-PCR and Southern blotting, we showed that LdNuc(s) gene homologs were present in each of the viscerotropic Leishmania tested (i.e., L. donovani isolates from the Sudan, Ethiopia and India as well as L. infantum). Further results of in situ enzyme activity gel analyses showed that each of these parasite isolates also expressed a released/secreted LdNuc(s)-like nuclease activity. In Western blots, our anti-LdNuc(s) (Sudan) peptide-specific antibody reacted with only a single ~35 kDa protein in each of the viscerotropic Leishmania isolates. Further, the ~35 kDa nuclease secreted by each of these isolates was specifically immunoprecipitated by the anti-LdNuc(s) antibody above. In situ gel analyses showed that each of these immunoprecipitates had LdNuc(s)-like nuclease activity. Moreover, sera from acute visceral leishmaniasis patients from India, Sudan and Brazil all immunoprecipitated an LdNuc(s)-HA expressed nuclease demonstrating, that these patients possessed antibodies against this parasite secretory enzyme. Cumulatively, these results showed that the LdNuc(s) homologs were functionally conserved among geographically disparate visceral Leishmania spp. and that amastigotes of these parasites must produce this nuclease enzyme during the course of human disease.     

Connectivity of Tiger (Panthera tigris) Populations in the Human-Influenced Forest Mosaic of Central India

Today, most wild tigers live in small, isolated Protected Areas within human dominated landscapes in the Indian subcontinent. Future survival of tigers depends on increasing local population size, as well as maintaining connectivity between populations. While significant conservation effort has been invested in increasing tiger population size, few initiatives have focused on landscape-level connectivity and on understanding the effect different landscape elements have on maintaining connectivity. We combined individual-based genetic and landscape ecology approaches to address this issue in six protected areas with varying tiger densities and separation in the Central Indian tiger landscape. We non-invasively sampled 55 tigers from different protected areas within this landscape. Maximum-likelihood and Bayesian genetic assignment tests indicate long-range tiger dispersal (on the order of 650 km) between protected areas. Further geo-spatial analyses revealed that tiger connectivity was affected by landscape elements such as human settlements, road density and host-population tiger density, but not by distance between populations. Our results elucidate the importance of landscape and habitat viability outside and between protected areas and provide a quantitative approach to test functionality of tiger corridors. We suggest future management strategies aim to minimize urban expansion between protected areas to maximize tiger connectivity. Achieving this goal in the context of ongoing urbanization and need to sustain current economic growth exerts enormous pressure on the remaining tiger habitats and emerges as a big challenge to conserve wild tigers in the Indian subcontinent.     

Domain motions of hyaluronan lyase underlying processive hyaluronan translocation

Hyaluronan lyase (Hyal) is a surface enzyme occurring in many bacterial organisms including members of Streptococcus species. Streptococcal Hyal primarily degrades hyaluronan‐substrate (HA) of the extracellular matrix. This degradation appears to facilitate the spread of this bacterium throughout host tissues. Unlike purely endolytic degradation of its other substrates, unsulfated chondroitin or some chondroitin sulfates, the degradation of HA by Hyal proceeds by processive exolytic cleavage of one disaccharide at a time following an initial endolytic cut. Molecular dynamics (MD) studies of Hyal from Streptococcus pneumoniae are presented that address the enzyme's molecular mechanism of action and the role of domain motions for processive functionality. The analysis of extensive sub‐microsecond MD simulations of this enzyme action on HA‐substrates of different lengths and the connection between the domain dynamics of Hyal and the translocation of the HA‐substrate reveals that opening/closing and twisting domain motions of the Hyal are intimately linked to processive HA degradation. Enforced simulations confirmed this finding as the domain motions in SpnHyal were found to be induced by enforced substrate translocation. These results establish the dynamic interplay between Hyal flexibility and substrate translocation and provide insight into the processive mechanism of Hyal. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Cyclosporin A as prophylaxis against graft-versus-host disease in 36 patients.

Oral cyclosporin A was used as prophylaxis against graft-versus-host disease in (a) 31 patients with acute leukaemia or aplastic anaemia given transplants of HLA-matched bone marrow and (b) five patients with inborn errors of metabolism given transplants of haplotype-identical (parental) bone marrow. Twenty-six patients survived longer than two months after the operation. Despite the cyclosporin A, 31 patients (86%) suffered an acute form of graft-versus-host disease and 22 (61%) a chronic form. Nevertheless, the disease was usually treatable with immunosuppressive agents and caused the death of only one patient. Cyclosporin A caused renal toxicity in all cases; occasionally this was associated with a "capillary leak" syndrome, fatal in two patients. In children hypertension, fits, and fluid retention were common side effects. Blood concentrations of cyclosporin A correlated with blood urea values and blood pressure but did not predict the occurrence of graft-versus-host disease. Four different dose schedules were used to find the optimum way to administer this drug. Oral cyclosporin A is extremely effective at reducing the severity of graft-versus-host disease, but prevention of the disease is limited by toxicity of the drug and variable absorption. Better results might be achieved with parenteral administration or by using the drug in combination with other methods.     

Rhythm-Specific Mutations in Drosophila rajasekari Altered Adult Locomotor Activity Rhythm but Not Eclosion Rhythm

The early and late strains for phase angle difference (Φ) of adult locomotor activity in Drosophila rajasekari were developed by artificial selection; these strains differed in Φ, activity pattern, activity level, free-running period (τ) in constant darkness (DD) and light induced phase shifts from those of the wild type (Joshi, 1998). The present studies were designed to determine whether or not the psi-mutations for adult locomotor activity rhythm had also altered the fundamental properties of the eclosion rhythms in these strains. The circadian rhythms of eclosion have been studied in the wild type, the early and late strains. In contrast to the effects on the locomotor activity rhythms in the early and late strains, the psi-mutations have no apparent effect on the eclosion median in light-dark cycles of 12 : 12 h, on τ in DD, light induced phase shifts or subjective light sensitivity in these strains. Thus the psi-mutations for the adult locomotor activity rhythms in D. rajasekari appear to be rhythm-specific mutations altering the locomotor rhythms but not the eclosion rhythms.     

Antifreeze polypeptides from the Newfoundland ocean pout,Macrozoarces americanus: presence of multiple and compositionally diverse components

Summary Eight major antifreeze polypeptides (AFP) were purified from the sera of Newfoundland ocean pout. Except for their approximately identical size (6,000 Dalton), these components were shown to be separate entities by their behaviour on polyacrylamide gel electrophoresis, ion exchange chromatography, gel permeation and reverse phase high performance liquid chromatography. They could also be divided into two cross-reactive, yet distinct, immunological groups. Amino acid analysis demonstrated that ocean pout AFP are different from all of the other antifreezes studied to date. The ocean pout AFP do not contain the abundance of alanine (60 mol%) found in winter flounder and shorthorn sculpin AFP nor the high half-cystine residues (8 mol%) observed in sea raven AFP. It is suggested that ocean pout AFP represent a new type of macromolecular antifreeze.Abbreviations AFGP antifreeze glycoprotein(s) - AFP antifreeze polypeptide(s) - HPLC high performance liquid chromatography - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis     

Tumor suppressor FOXO3 mediates signals from the EGF receptor to regulate proliferation of colonic cells

Epithelial proliferation, critical for homeostasis, healing, and colon cancer progression, is in part controlled by epidermal growth factor receptor (EGFR). Proliferation of colonic epithelia can be induced by Citrobacter rodentium infection, and we have demonstrated that activity of tumor suppressor FOXO3 was attenuated after this infection. Thus the aim of this study was to determine the contribution of FOXO3 in EGFR-dependent proliferation of intestinal epithelia and colon cancer cell lines. In this study we show that, during infection with C. rodentium, EGFR was significantly phosphorylated in colonic mucosa and Foxo3 deficiency in this model lead to an increased number of bromodeoxyuridine-positive cells. In vitro, in human colon cancer cells, increased expression and activation of EGFR was associated with proliferation that leads to FOXO3 phosphorylation (inactivation). Following EGFR activation, FOXO3 was phosphorylated (via phosphatidylinositol 3-kinase/Akt) and translocated to the cytosol where it was degraded. Moreover, inhibition of proliferation by overexpressing FOXO3 was not reversed by the EGFR signaling, implicating FOXO3 as one of the regulators downstream of EGFR. FOXO3 binding to the promoter of the cell cycle inhibitor p27kip1 was decreased by EGFR signaling, suggesting its role in EGFR-dependent proliferation. In conclusion, we show that proliferation in colonic epithelia and colon cancer cells, stimulated by EGFR, is mediated via loss of FOXO3 activity and speculate that FOXO3 may serve as a target in the development of new pharmacological treatments of proliferative diseases.