Genomic profiling in GIST: Implications in clinical outcome and future challenges.

Gastrointestinal Stromal Tumors (GIST) are the most frequent mesenchymal neoplasia of the digestive tract. Genomic alterations in KIT, PDFGRA, SDH, and BRAF genes are essential in GIST oncogenesis. Therefore, the mutations in these genes have demonstrated clinical implications. Tumors with deletions in KIT-exon 11 or duplications in exon 9 are associated with a worse prognosis. In contrast, KIT-exon 11 substitutions and duplications are associated with a better clinical outcome. Moreover, mutations in Kit exon 9 and 11 are actionable, due to their response to imatinib, while mutations in PDGFRA respond to sunitinib and/or avapritinib. Although, molecular testing on tissue samples is effective; it is invasive, requires adequate amounts of tissue, and a long experimental process is needed for results. In contrast, liquid biopsy has been proposed as a simple and non-invasive method to test biomarkers in cancer. The most common molecule analyzed by liquid biopsy is circulating tumor DNA (ctDNA). GISTs ctDNA testing has been demonstrated to be effective in identifying known and novel KIT mutations that were not detected using traditional tissue DNA testing and have been useful in determining progression risk and response to TKI therapy. This allows the clinician to have an accurate picture of the genetic changes of the tumor over time. In this work, we aimed to discuss the implications of mutational testing in clinical outcomes, the methods to test ctDNA and the future challenges in the establishment of alternatives of personalized medicine.

Penicillin-Binding Proteins and Alternative Dual-Beta-Lactam Combinations for Serious Enterococcus faecalis Infections with Elevated Penicillin MICs.

Ampicillin-ceftriaxone has become a first-line therapy for Enterococcus faecalis endocarditis. We characterized the penicillin-binding protein (PBP) profiles of various E. faecalis strains and tested for synergy to better inform beta-lactam options for the treatment of E. faecalis infections. We assessed the affinity of PBP2B from elevated-MIC strain E. faecalis LS4828 compared to type strain JH2-2 using the fluorescent beta-lactam Bocillin FL. We also characterized pbp4 and pbpA structures and PBP4 and PBP2B expression and used deletion and complementation studies to assess the impact of PBP2B on the levels of resistance. We tested penicillin-susceptible and -resistant E. faecalis isolates against ceftriaxone or ceftaroline combinations with other beta-lactams in 24-h time-kill studies. Two penicillin-susceptible strains (JH2-2 and L2052) had identical pbp sequences and similar PBP expression levels. One reduced-penicillin-susceptibility strain (L2068) had pbp sequences identical to those of the susceptible strains but expressed more PBP4. The second decreased-penicillin-susceptibility strain (LS4828) had amino acid substitutions in both PBP4 and PBP2B and expressed increased quantities of both proteins. PBP2B did not appear to contribute significantly to the elevated beta-lactam MICs. No synergy was demonstrable against the strains with both mutated PBPs and increased expression (L2068 and LS4828). Meropenem plus ceftriaxone or ertapenem plus ceftriaxone demonstrated the most consistent synergistic activity. PBP2B of strain LS4828 does not contribute significantly to reduced penicillin susceptibility. Neither the MIC nor the level of PBP expression correlated directly with the identified synergistic combinations when tested at static subinhibitory concentrations.

Coordinated transient interaction of ZO-1 and afadin is required for pedestal maturation induced by EspF from enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) infection causes a histopathological lesion including recruitment of F-actin beneath the attached bacteria and formation of actin-rich pedestal-like structures. Another important target of EPEC is the tight junction (TJ), and EspF induces displacement of TJ proteins and increased intestinal permeability. Previously, we determined that an EPEC strain lacking EspF did not cause TJ disruption; meanwhile, pedestals were located on the TJ and smaller than those induced by the wild-type strain. Therefore, EspF could be playing an important role in both phenotypes. Here, using different cell models, we found that EspF was essential for pedestal maturation through ZO-1 disassembly from TJ, leading to (a) ZO-1 recruitment to the pedestal structure; no other main TJ proteins were required. Recruited ZO-1 allowed the afadin recruitment. (b) Afadin recruitment caused an afadin-ZO-1 transient interaction, like during TJ formation. (c) Afadin and ZO-1 were segregated to the tip and the stem of pedestal, respectively, causing pedestal maturation. Initiation of these three discrete phases for pedestal maturation functionally and physically required EspF expression. Pedestal maturation process could help coordinate the epithelial actomyosin function by maintaining the actin-rich column composing the pedestal structure and could be important in the dynamics of the pedestal movement on epithelial cells.

Latrophilins: A Neuro-Centric View of an Evolutionary Conserved Adhesion G Protein-Coupled Receptor Subfamily.

The adhesion G protein-coupled receptors latrophilins have been in the limelight for more than 20 years since their discovery as calcium-independent receptors for α-latrotoxin, a spider venom toxin with potent activity directed at neurotransmitter release from a variety of synapse types. Latrophilins are highly expressed in the nervous system. Although a substantial amount of studies has been conducted to describe the role of latrophilins in the toxin-mediated action, the recent identification of endogenous ligands for these receptors helped confirm their function as mediators of adhesion events. Here we hypothesize a role for latrophilins in inter-neuronal contacts and the formation of neuronal networks and we review the most recent information on their role in neurons. We explore molecular, cellular and behavioral aspects related to latrophilin adhesion function in mice, zebrafish, Drosophila melanogaster and Caenorhabditis elegans, in physiological and pathophysiological conditions, including autism spectrum, bipolar, attention deficit and hyperactivity and substance use disorders.

Tight Junction Disruption Induced by Type 3 Secretion System Effectors Injected by Enteropathogenic and Enterohemorrhagic Escherichia coli.

The intestinal epithelium consists of a single cell layer, which is a critical selectively permeable barrier to both absorb nutrients and avoid the entry of potentially harmful entities, including microorganisms. Epithelial cells are held together by the apical junctional complexes, consisting of adherens junctions, and tight junctions (TJs), and by underlying desmosomes. TJs lay in the apical domain of epithelial cells and are mainly composed by transmembrane proteins such as occludin, claudins, JAMs, and tricellulin, that are associated with the cytoplasmic plaque formed by proteins from the MAGUK family, such as ZO-1/2/3, connecting TJ to the actin cytoskeleton, and cingulin and paracingulin connecting TJ to the microtubule network. Extracellular bacteria such as EPEC and EHEC living in the intestinal lumen inject effectors proteins directly from the bacterial cytoplasm to the host cell cytoplasm, where they play a relevant role in the manipulation of the eukaryotic cell functions by modifying or blocking cell signaling pathways. TJ integrity depends on various cell functions such as actin cytoskeleton, microtubule network for vesicular trafficking, membrane integrity, inflammation, and cell survival. EPEC and EHEC effectors target most of these functions. Effectors encoded inside or outside of locus of enterocyte effacement (LEE) disrupt the TJ strands. EPEC and EHEC exploit the TJ dynamics to open this structure, for causing diarrhea. EPEC and EHEC secrete effectors that mimic host proteins to manipulate the signaling pathways, including those related to TJ dynamics. In this review, we focus on the known mechanisms exploited by EPEC and EHEC effectors for causing TJ disruption.

Actin cytoskeleton manipulation by effector proteins secreted by diarrheagenic Escherichia coli pathotypes.

The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology.