Functional analysis of RPS27 mutations and expression in melanoma.

Next-generation sequencing has enabled genetic and genomic characterization of melanoma to an unprecedent depth. However, the high mutational background plus the limited depth of coverage of whole-genome sequencing performed on cutaneous melanoma samples make the identification of novel driver mutations difficult. We sought to explore the somatic mutation portfolio in exonic and gene regulatory regions in human melanoma samples, for which we performed targeted sequencing of tumors and matched germline DNA samples from 89 melanoma patients, identifying known and novel recurrent mutations. Two recurrent mutations found in the RPS27 promoter associated with decreased RPS27 mRNA levels in vitro. Data mining and IHC analyses revealed a bimodal pattern of RPS27 expression in melanoma, with RPS27-low patients displaying worse prognosis. In vitro characterization of RPS27-high and RPS27-low melanoma cell lines, as well as loss-of-function experiments, demonstrated that high RPS27 status provides increased proliferative and invasive capacities, while low RPS27 confers survival advantage in low attachment and resistance to therapy. Additionally, we demonstrate that 10 other cancer types harbor bimodal RPS27 expression, and in those, similarly to melanoma, RPS27-low expression associates with worse clinical outcomes. RPS27 promoter mutation could thus represent a mechanism of gene expression modulation in melanoma patients, which may have prognostic and predictive implications.

Transcriptomic determinants of the response of ST-111 Pseudomonas aeruginosa AG1 to ciprofloxacin identified by a top-down systems biology approach.

Pseudomonas aeruginosa is an opportunistic pathogen that thrives in diverse environments and causes a variety of human infections. Pseudomonas aeruginosa AG1 (PaeAG1) is a high-risk sequence type 111 (ST-111) strain isolated from a Costa Rican hospital in 2010. PaeAG1 has both blaVIM-2 and blaIMP-18 genes encoding for metallo-ß-lactamases, and it is resistant to ß-lactams (including carbapenems), aminoglycosides, and fluoroquinolones. Ciprofloxacin (CIP) is an antibiotic commonly used to treat P. aeruginosa infections, and it is known to produce DNA damage, triggering a complex molecular response. In order to evaluate the effects of a sub-inhibitory CIP concentration on PaeAG1, growth curves using increasing CIP concentrations were compared. We then measured gene expression using RNA-Seq at three time points (0, 2.5 and 5 h) after CIP exposure to identify the transcriptomic determinants of the response (i.e. hub genes, gene clusters and enriched pathways). Changes in expression were determined using differential expression analysis and network analysis using a top-down systems biology approach. A hybrid model using database-based and co-expression analysis approaches was implemented to predict gene-gene interactions. We observed a reduction of the growth curve rate as the sub-inhibitory CIP concentrations were increased. In the transcriptomic analysis, we detected that over time CIP treatment resulted in the differential expression of 518 genes, showing a complex impact at the molecular level. The transcriptomic determinants were 14 hub genes, multiple gene clusters at different levels (associated to hub genes or as co-expression modules) and 15 enriched pathways. Down-regulation of genes implicated in several metabolism pathways, virulence elements and ribosomal activity was observed. In contrast, amino acid catabolism, RpoS factor, proteases, and phenazines genes were up-regulated. Remarkably, > 80 resident-phage genes were up-regulated after CIP treatment, which was validated at phenomic level using a phage plaque assay. Thus, reduction of the growth curve rate and increasing phage induction was evidenced as the CIP concentrations were increased. In summary, transcriptomic and network analyses, as well as the growth curves and phage plaque assays provide evidence that PaeAG1 presents a complex, concentration-dependent response to sub-inhibitory CIP exposure, showing pleiotropic effects at the systems level. Manipulation of these determinants, such as phage genes, could be used to gain more insights about the regulation of responses in PaeAG1 as well as the identification of possible therapeutic targets. To our knowledge, this is the first report of the transcriptomic analysis of CIP response in a ST-111 high-risk P. aeruginosa strain, in particular using a top-down systems biology approach.

Epigenetic Silencing of CDR1as Drives IGF2BP3-Mediated Melanoma Invasion and Metastasis.

Metastasis is the primary cause of death of cancer patients. Dissecting mechanisms governing metastatic spread may uncover important tumor biology and/or yield promising therapeutic insights. Here, we investigated the role of circular RNAs (circRNA) in metastasis, using melanoma as a model aggressive tumor. We identified silencing of cerebellar degeneration-related 1 antisense (CDR1as), a regulator of miR-7, as a hallmark of melanoma progression. CDR1as depletion results from epigenetic silencing of LINC00632, its originating long non-coding RNA (lncRNA) and promotes invasion in vitro and metastasis in vivo through a miR-7-independent, IGF2BP3-mediated mechanism. Moreover, CDR1as levels reflect cellular states associated with distinct therapeutic responses. Our study reveals functional, prognostic, and predictive roles for CDR1as and expose circRNAs as key players in metastasis.

Essential Domain-Dependent Roles Within Soluble IgG for in vivo Superantigen Properties of Staphylococcal Protein A: Resolving the B-Cell Superantigen Paradox.

Staphylococcus aureus is a common commensal and frequent opportunistic pathogen that causes invasive infections that often recur. Co-evolution with the host has led to the development of toxins that affect diverse immune cell types. Recent reports have highlighted the contributions of staphylococcal protein A (SpA). This small oligomeric secreted protein contains 4-5 homologous domains with two distinct immunoglobulin-binding sites; one for IgG Fc domains, while a separate site binds an evolutionarily conserved surface on Fab encoded by VHIII clan related genes. The Fab-binding site has been implicated in in vivo supraclonal VHIII-BCR targeted B-cell depletion by an activation induced death pathway. Yet the concept of a superantigen for B lymphocytes poses a seeming paradox. Unlike TCR that are expressed only in a membrane-associated form, BCR are expressed in both a membrane BCR form and in secreted Ig forms, which permeate virtually every part of the body at high levels. We therefore asked, why circulating immunoglobulin do not block the superantigen properties of SpA? Herein, we show that soluble IgG molecules are not in vivo inhibitors of these B-cell superantigen effects but are instead essential for potentiating these properties. We also show that the Fc subclass of circulating IgG is an indirect critical determinant of the B-cell superantigen effect. In contrast, host FcγR and complement are not required for SpA mediated in vivo B-cell depletion. Unexpectedly, after VHIII-IgG2a pretreatment SpA challenge resulted in fatal anaphylactic reactions, which we speculate may have involved FcγR interactions with mast cells and basophils. Cumulatively, our findings illuminate a cunning and potent molecular strategy by which a bacterial toxin effectively confounds the contributions of host B-lymphocytes to immune defenses.

Activación inmune por productos finales de glicación avanzada

Con el objetivo de revisar los avances recientes en la biología de los productos de glica- ción avanzada y su papel en diversas patologías de alta relevancia en salud pública, se realizó una búsqueda dirigida de la bibliografía entre los años 2000 al 2021 en la base de datos PubMed (NCBI) y se usaron como palabras clave "advanced glycation end pro- ducts". Se ha demostrado que el receptor de productos de glicación avanzada induce una activación sostenida del factor de transcripción proinflamatorio NF-kB y suprime una serie de funciones autorreguladoras endógenas. Este efecto influye negativamente en una gran variedad de patologías como diabetes, autoinmunidad, neurodegeneración y enfermedades infecciosas. La acumulación tisular de productos de glicación avanzada está relacionada con procesos de inflamación crónica y disfunción celular, y constituye un blanco prometedor para el diseño de tratamientos enfocados en esta vía de señali- zación. Actualmente se realizan múltiples ensayos clínicos para determinar su utilidad como marcador de lesiones pulmonares en COVID-19.

To review recent advances in the biology of advanced glycation endproducts and its role in diverse pathologies of high relevance for public health, a survey of the literature from the years 2000 to 2021 was performed in PubMed (NCBI) using the key words "advanced glycation endproducts". It has been demonstrated that the receptor for advanced glyca- tion endproducts (RAGE) induces a sustained activation of the pro-inflammatory tran- scription factor NF-kB and suppresses a series of endogenous self-regulating functions. This affects negatively on a wide variety of pathological conditions such as diabetes, au- toimmunity, neurodegeneration and infectious disease. Tissular accumulation of AGEs is linked to chronic inflammation and cellular dysfunction and constitutes a promising target for the design of treatments focused on this signaling pathway. Multiple clinical trials are currently underway to define its usefulness as a marker of pulmonary lesions in COVID-19.