Characterization of the cell of origin for small cell lung cancer

Small cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer that affects more than 200,000 people worldwide every year with a very high mortality rate. Here, we used a mouse genetics approach to characterize the cell of origin for SCLC; in this mouse model, tumors are initiated by the deletion of the Rb and p53 tumor suppressor genes in the lung epithelium of adult mice. We found that mouse SCLCs often arise in the lung epithelium, where neuroendocrine cells are located, and that the majority of early lesions were composed of proliferating neuroendocrine cells. In addition, mice in which Rb and p53 are deleted in a variety of non-neuroendocrine lung epithelial cells did not develop SCLC. These data indicate that SCLC likely arises from neuroendocrine cells in the lung.Key words: Rb, p53, SCLC, cell of origin, cancer, lung, neuroendocrine     

Human apolipoprotein A-I-derived amyloid: its association with atherosclerosis

Amyloidoses constitute a group of diseases in which soluble proteins aggregate and deposit extracellularly in tissues. Nonhereditary apolipoprotein A-I (apoA-I) amyloid is characterized by deposits of nonvariant protein in atherosclerotic arteries. Despite being common, little is known about the pathogenesis and significance of apoA-I deposition. In this work we investigated by fluorescence and biochemical approaches the impact of a cellular microenvironment associated with chronic inflammation on the folding and pro-amyloidogenic processing of apoA-I. Results showed that mildly acidic pH promotes misfolding, aggregation, and increased binding of apoA-I to extracellular matrix elements, thus favoring protein deposition as amyloid like-complexes. In addition, activated neutrophils and oxidative/proteolytic cleavage of the protein give rise to pro amyloidogenic products. We conclude that, even though apoA-I is not inherently amyloidogenic, it may produce non hereditary amyloidosis as a consequence of the pro-inflammatory microenvironment associated to atherogenesis.     

The Role of Ph Fronts in Tissue Electroporation Based Treatments

Treatments based on electroporation (EP) induce the formation of pores in cell membranes due to the application of pulsed electric fields. We present experimental evidence of the existence of pH fronts emerging from both electrodes during treatments based on tissue EP, for conditions found in many studies, and that these fronts are immediate and substantial. pH fronts are indirectly measured through the evanescence time (ET), defined as the time required for the tissue buffer to neutralize them. The ET was measured through a pH indicator imaged at a series of time intervals using a four-cluster hard fuzzy-c-means algorithm to segment pixels corresponding to the pH indicator at every frame. The ET was calculated as the time during which the number of pixels was 10% of those in the initial frame. While in EP-based treatments such as reversible (ECT) and irreversible electroporation (IRE) the ET is very short (though enough to cause minor injuries) due to electric pulse characteristics and biological buffers present in the tissue, in gene electrotransfer (GET), ET is much longer, enough to denaturate plasmids and produce cell damage. When any of the electric pulse parameters is doubled or tripled the ET grows and, remarkably, when any of the pulse parameters in GET is halved, the ET drops significantly. Reducing pH fronts has relevant implications for GET treatment efficiency, due to a substantial reduction of plasmid damage and cell loss.     

Unravelling the distinctive craniomandibular morphology of the Plio-Pleistocene Eumysops in the evolutionary setting of South American octodontoid rodents (Hystricomorpha)

Echimyidae is a species-rich clade of Neotropical rodents, which diversified in association with forested biomes. Since the late Miocene, a few lineages from southern South America have been adapted to open environments. Eumysops is one of these southern echimyids, and its peculiar craniomandibular morphology has been assumed to be a result of adaptation to open environments. We performed a geometric morphometric analysis of craniomandibular shape variation to explore whether, as suspected, Eumysops is divergent from other echimyids and octodontoids. In addition, we explored whether deterministic factors driven by different ecological dimensions can explain the diversification of shape among octodontoids. We found that craniomandibular shape variation in octodontoids was related to ecological variables. Comparing competing evolutionary models suggested that the input of selective factors play a key role in octodontoid craniomandibular shape diversification; habitat and habits were found to be the most influential factors. In the analysed morphospaces, Eumysops was located distant from other echimyids due to its distinctive traits, especially wide and posteriorly displaced orbits, and related low craniomandibular joint. Divergent orbits and resulting wider panoramic vision support the interpretation of Eumysops as an open-habitat specialist echimyid. But what is more relevant, is that Eumysops occupied a sector of the octodontoid cranial morphospace not filled by living representatives; this highlights the contribution of fossils in providing key information on the specialization boundaries explored by a clade throughout its history.     

Role of Rab1b in COPII dynamics and function

In eukaryotic cells, proteins destined for secretion are translocated into the endoplasmic reticulum (ER) and packaged into so-called COPII-coated vesicles. In the ER exit sites (ERES), COPII has the capacity of deforming the lipid bilayer, where it modulates the selective sorting and concentration of cargo proteins. In this study, we analyze the involvement of Rab1b in COPII dynamics and function by expressing either the Rab1b negative-mutant (Rab1N121I) or the Rab1b GTP restricted mutant (Rab1Q67L), or performing short interference RNA-based knockdown. We show that Rab1b interacts with the COPII components Sec23, Sec24 and Sec31 and that Rab1b inhibition changes the COPII phenotype. FRAP assays reveal that Rab1b modulates COPII association/dissociation kinetics at the ERES interface. Furthermore, Rab1b inhibition delays cargo sorting at the ER exit sites. We postulate that Rab1b is a key regulatory component of COPII dynamics and function.     

Use of selective capture of transcribed sequences to identify genes preferentially expressed by Streptococcus suis upon interaction with porcine brain microvascular endothelial cells

By using the selective capture of transcribed sequences (SCOTS) approach, we identified 28 genes preferentially expressed by the major swine pathogen and zoonotic agent Streptococcus suis upon interaction with porcine brain microvascular endothelial cells. Several of these genes may be considered new S. suis candidate virulence factors. Results from this study demonstrate the suitability of SCOTS for the elucidation of gene expression in streptococcal species and may contribute to a better understanding of the pathogenesis of S. suis infections.     

Use of Selective Capture of Transcribed Sequences To Identify Genes Preferentially Expressed by Streptococcus suis upon Interaction with Porcine Brain Microvascular Endothelial Cells

By using the selective capture of transcribed sequences (SCOTS) approach, we identified 28 genes preferentially expressed by the major swine pathogen and zoonotic agent Streptococcus suis upon interaction with porcine brain microvascular endothelial cells. Several of these genes may be considered new S. suis candidate virulence factors. Results from this study demonstrate the suitability of SCOTS for the elucidation of gene expression in streptococcal species and may contribute to a better understanding of the pathogenesis of S. suis infections.     

SUMOylation and deimination of proteins: Two epigenetic modifications involved in Giardia encystation

SUMOylation, a posttranslational modification of proteins, has been recently described as vital in eukaryotic cells. In a previous work, we analyzed the role of SUMO protein and the genes encoding the putative enzymes of the SUMOylation pathway in the parasite Giardia lamblia. Although we observed several SUMOylated proteins, only the enzyme Arginine Deiminase (ADI) was confirmed as a SUMOylated substrate. ADI is involved in the survival of the parasite and, besides its role in ATP production, it also catalyzes the modification of arginine residues to citrulline in the cytoplasmic tail of surface proteins. During encystation, however, ADI translocates to the nuclei and downregulates the expression of the Cyst Wall Protein 2 (CWP2). In this work, we made site-specific mutation of the ADI SUMOylation site (Lys101) and observed that transgenic trophozoites did not translocate to the nuclei at the first steps of encystation but shuttled in the nuclei late during this process through classic nuclear localization signals. Inside the nuclei, ADI acts as a peptidyl arginine deiminase, being probably involved in the downregulation of CWPs expression and cyst wall formation. Our results strongly indicate that ADI plays a regulatory role during encystation in which posttranslational modifications of proteins are key players.     

Functional and structural characterization of the catalytic domain of the starch synthase III from Arabidopsis thaliana

Glycogen and starch are the major energy storage compounds in most living organisms. The metabolic pathways leading to their synthesis involve the action of several enzymes, among which glycogen synthase (GS) or starch synthase (SS) catalyze the elongation of the alpha-1,4-glucan backbone. At least five SS isoforms were described in Arabidopsis thaliana; it has been reported that the isoform III (SSIII) has a regulatory function on the synthesis of transient plant starch. The catalytic C-terminal domain of A. thaliana SSIII (SSIII-CD) was cloned and expressed. SSIII-CD fully complements the production of glycogen by an Agrobacterium tumefaciens glycogen synthase null mutant, suggesting that this truncated isoform restores in vivo the novo synthesis of bacterial glycogen. In vitro studies revealed that recombinant SSIII-CD uses with more efficiency rabbit muscle glycogen than amylopectin as primer and display a high apparent affinity for ADP-Glc. Fold class assignment methods followed by homology modeling predict a high global similarity to A. tumefaciens GS showing a fully conservation of the ADP-binding residues. On the other hand, this comparison revealed important divergences of the polysaccharide binding domain between AtGS and SSIII-CD.     

Signaling complexes of voltage-gated calcium channels and G protein-coupled receptors

Activation of opioid or opioid-receptor-like (ORL1 a.k.a. NOP or orphanin FQ) receptors mediates analgesia through inhibition of N-type calcium channels in dorsal root ganglion (DRG) neurons (1, 2). Unlike the three types of classical mu, delta, and kappa opioid receptors, ORL1 mediates an agonist-independent inhibition of N-type calcium channels. This is mediated via the formation of a physical protein complex between the receptor and the channel, which in turn allows the channel to effectively sense a low level of constitutive receptor activity (3). Further inhibition of N-type channel activity by activation of other G protein-coupled receptors is thus precluded. ORL1 receptors, however, also undergo agonist-induced internalization into lysosomes, and channels thereby become cointernalized in a complex with ORL1. This then results in removal of N-type channels from the plasma membrane and reduced calcium entry (4). Similar signaling complexes between N-type channels and GABA(B) receptors have been reported (5). Moreover, both L-type and P/Q-type channels appear to be able to associate with certain types of G protein-coupled receptors (6, 7). Hence, interactions between receptors and voltage-gated calcium channels may be a widely applicable means to optimize receptor channel coupling.