Syntaxin 18 regulates the DNA damage response and epithelial-to-mesenchymal transition to promote radiation resistance of lung cancer

Radiotherapy is an important modality in lung cancer treatment. Despite advances in treatment planning and dose delivery, patient benefit is still limited by in-field relapse and metastatic recurrence. Simultaneous application of cisplatinum-based chemotherapy leads to moderately improved outcomes, thus providing proof-of-concept for radiosensitization strategies in lung cancer. In an unbiased functional genetic screen for radiosensitization targets in lung cancer, we identified syntaxin 18, a protein involved in retrograde vesicular transport between the Golgi apparatus and endoplasmic reticulum, as mediator of radioresistance. Downregulation of endogenous syntaxin 18 specifically reduced clonogenic survival of radioresistant and radiosensitive lung cancer cells following X-radiation. Gene expression programs regulating DNA repair, mitotic checkpoints and mitosis were altered in isogenic cells with reduced syntaxin 18 expression. Functionally, this translated into impaired DNA damage-induced cell cycle checkpoints leading to cell death by mitotic catastrophe. Interestingly, downregulation of syntaxin 18 in lung cancer cells also impaired expression of markers of epithelial-mesenchymal-transition, and reduced migration and invasion capacity. These findings suggest that syntaxin 18 is a key player regulating genes responsible for controlling the growth of the primary tumor as well as metastases upon radiotherapy of lung cancer. They provide a promising lead for biologically rational radiosensitization strategies impacting on radiation-induced cell death as well as metastasis.Subject terms: Radiotherapy, Non-small-cell lung cancer     

Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification

Loa loa infections have emerged as a serious public health problem in patients co-infected with Onchocerca volvulus or Wuchereria bancrofti because of severe adverse neurological reactions after treatment with ivermectin. Accurate diagnostic tests are needed for careful mapping in regions where mass drug administration is underway. Loop-mediated isothermal amplification (LAMP) has become a widely adopted screening method because of its operational simplicity, rapidity and versatility of visual detection readout options. Here, we present a multi-step bioinformatic pipeline to generate diagnostic candidates suitable for LAMP and experimentally validate this approach using one of the identified candidates to develop a species-specific LAMP assay for L. loa. The pipeline identified ~140 new L. loa specific DNA repeat families as putative biomarkers of infection. The consensus sequence of one family, repeat family 4 (RF4), was compiled from ~ 350 sequences dispersed throughout the L. loa genome and maps to a L. loa-specific region of the long terminal repeats found at the boundaries of Bel/Pao retrotransposons. PCR and LAMP primer sets targeting RF4 specifically amplified L. loa but not W. bancrofti, O. volvulus, Brugia malayi, human or mosquito DNA. RF4 LAMP detects the DNA equivalent of one microfilaria (100 pg) in 25–30 minutes and as little as 0.060 pg of L. loa DNA (~1/1600^th of a microfilaria) purified from spiked blood samples in approximately 50 minutes. In summary, we have successfully employed a bioinformatic approach to mine the L. loa genome for species-specific repeat families that can serve as new DNA biomarkers for LAMP. The RF4 LAMP assay shows promise as a field tool for the implementation and management of mass drug administration programs and warrants further testing on clinical samples as the next stage in development towards this goal.     

Tyrosine Kinase Inhibitors Induce Down-Regulation of c-Kit by Targeting the ATP Pocket

The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-β-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket.     

The model squid–vibrio symbiosis provides a window into the impact of strain- and species-level differences during the initial stages of symbiont engagement

Among horizontally acquired symbioses, the mechanisms underlying microbial strain- and species-level specificity remain poorly understood. Here, confocal-microscopy analyses and genetic manipulation of the squid–vibrio association revealed quantitative differences in a symbiont's capacity to interact with the host during initial engagement. Specifically, dominant strains of Vibrio fischeri, ‘D-type’, previously named for their dominant, single-strain colonization of the squid's bioluminescent organ, were compared with ‘S-type’, or ‘sharing’, strains, which can co-colonize the organ. These D-type strains typically: (i) formed aggregations of 100s–1000s of cells on the light-organ surface, up to 3 orders of magnitude larger than those of S-type strains; (ii) showed dominance in co-aggregation experiments, independent of inoculum size or strain proportion; (iii) perturbed larger areas of the organ's ciliated surface; and, (iv) appeared at the pore of the organ approximately 4×s more quickly than S-type strains. At least in part, genes responsible for biofilm synthesis control the hyperaggregation phenotype of a D-type strain. Other marine vibrios produced relatively small aggregations, while an array of marine Gram-positive and -negative species outside of the Vibrionaceae did not attach to the organ's surface. These studies provide insight into the impact of strain variation on early events leading to establishment of an environmentally acquired symbiosis.     

Hepatitis C Virus Increases Occludin Expression via the Upregulation of Adipose Differentiation-Related Protein

The hepatitis C virus (HCV) life cycle is closely associated with lipid metabolism. In particular, HCV assembly initiates at the surface of lipid droplets. To further understand the role of lipid droplets in HCV life cycle, we assessed the relationship between HCV and the adipose differentiation-related protein (ADRP), a lipid droplet-associated protein. Different steps of HCV life cycle were assessed in HCV-infected human Huh-7 hepatoma cells overexpressing ADRP upon transduction with a lentiviral vector. HCV infection increased ADRP mRNA and protein expression levels by 2- and 1.5-fold, respectively. The overexpression of ADRP led to an increase of (i) the surface of lipid droplets, (ii) the total cellular neutral lipid content (2.5- and 5-fold increase of triglycerides and cholesterol esters, respectively), (iii) the cellular free cholesterol level (5-fold) and (iv) the HCV particle production and infectivity (by 2- and 3.5-fold, respectively). The investigation of different steps of the HCV life cycle indicated that the ADRP overexpression, while not affecting the viral replication, promoted both virion egress and entry (~12-fold), the latter possibly via an increase of its receptor occludin. Moreover, HCV infection induces an increase of both ADRP and occludin expression. In HCV infected cells, the occludin upregulation was fully prevented by the ADRP silencing, suggesting a specific, ADRP-dependent mechanism. Finally, in HCV-infected human livers, occludin and ADRP mRNA expression levels correlated with each other. Alltogether, these findings show that HCV induces ADRP, which in turns appears to confer a favorable environment to viral spread.     

In vitro evaluation of new terpenoid derivatives against Leishmania infantum and Leishmania braziliensis

The activity of five (1-5) abietane phenol derivatives against Leishmania infantum and Leishmania braziliensis was studied using promastigotes and axenic and intracellular amastigotes. Infectivity and cytotoxicity tests were performed with J774.2 macrophage cells using Glucantime as a reference drug. The mechanisms of action were analysed by performing metabolite excretion and transmission electron microscopy ultrastructural studies. Compounds 1-5 were more active and less toxic than Glucantime. The infection rates and mean number of parasites per cell observed in amastigote experiments showed that derivatives 2, 4 and 5 were the most effective against both L. infantum and L. braziliensis. The ultrastructural changes observed in the treated promastigote forms confirmed that the greatest cell damage was caused by the most active compound (4). Only compound 5 caused changes in the nature and amounts of catabolites excreted by the parasites, as measured by 1H nuclear magnetic resonance spectroscopy. All of the assayed compounds were active against the two Leishmania species in vitro and were less toxic in mammalian cells than the reference drug.     

Physiological type I collagen organization induces the formation of a novel class of linear invadosomes

Invadosomes are F-actin structures capable of degrading the matrix through the activation of matrix metalloproteases. As fibrillar type I collagen promotes pro-matrix metalloproteinase 2 activation by membrane type 1 matrix metalloproteinase, we aimed at investigating the functional relationships between collagen I organization and invadosome induction. We found that fibrillar collagen I induced linear F-actin structures, distributed along the fibrils, on endothelial cells, macrophages, fibroblasts, and tumor cells. These structures share features with conventional invadosomes, as they express cortactin and N-WASP and accumulate the scaffold protein Tks5, which proved essential for their formation. On the basis of their ability to degrade extracellular matrix elements and their original architecture, we named these structures "linear invadosomes." Interestingly, podosomes or invadopodia were replaced by linear invadosomes upon contact of the cells with fibrillar collagen I. However, linear invadosomes clearly differ from classical invadosomes, as they do not contain paxillin, vinculin, and β1/β3 integrins. Using knockout mouse embryonic fibroblasts and RGD peptide, we demonstrate that linear invadosome formation and activity are independent of β1 and β3 integrins. Finally, linear invadosomes also formed in a three-dimensional collagen matrix. This study demonstrates that fibrillar collagen I is the physiological inducer of a novel class of invadosomes.     

In vitro and in vivo antiparasital activity against Trypanosoma cruzi of three novel 5-methyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one-based complexes

Conventional reactions of the versatile multidentate ligand 5-methyl-1,2,4-triazolo[1,5-a] pyrimidin-7(4H)-one (HmtpO) with metallic(II) perchlorate salts lead to three novel multidimensional complexes [Cu(HmtpO)₂(H₂O)₃](ClO₄)₂·H₂O (1), {[Cu(HmtpO)₂(H₂O)₂](ClO₄)₂ ·2HmtpO}_n (2) and {[Co(HmtpO)(H₂O)₃](ClO₄)₂·2H₂O}_n (3). We have tested the antiparasital activity in vitro and in vivo of the three new complexes against Trypanosoma cruzi showing very promising results and overcoming clearly the reference drug commonly used for the Chagas disease treatment, benznidazole.     

Comparative anatomical analyses of the forearm muscles of Cebus libidinosus (Rylands et al. 2000): manipulatory behavior and tool use

The present study describes the flexor and extensor muscles in Cebus libidinosus' forearm and compares them with those from humans, chimpanzees and baboons. The data is presented in quantitative anatomical indices for similarity. The capuchin forearm muscles showed important similarities with chimpanzees and humans, particularly those that act on thumb motion and allow certain degree of independence from other hand structures, even though their configuration does not enable a true opposable thumb. The characteristics of Cebus' forearm muscles corroborate the evolutionary convergence towards an adaptive behavior (tool use) between Cebus genus and apes.