Classical methods and perspectives for manipulating the human gut microbial ecosystem.

A healthy Human Gut Microbial Ecosystem (HGME) is a necessary condition for maintaining the orderly function of the whole body. Major alterations in the normal gut microbial composition, activity and functionality (dysbiosis) by an environmental or host-related disruptive event, can compromise metabolic, inflammatory, and neurological processes, causing disorders such as obesity, inflammatory bowel disease, colorectal cancer, and depressive episodes. The restore or the maintaining of the homeostatic balance of Gut Microbiota (GM) populations (eubiosis) is possible through diet, the use of probiotics, prebiotics, antibiotics, and even Fecal Microbiota Transplantation (FMT). Although these "classic methods" represent an effective and accepted way to modulate GM, the complexity of HGME requires new approaches to control it in a more appropriate way. Among the most promising emergent strategies for modulating GM are the use of engineered nanomaterials (metallic nanoparticles (NP), polymeric-NP, quantum dots, micelles, dendrimers, and liposomes); phagotherapy (i.e., phages linked with the CRISPR/Cas9 system), and the use of antimicrobial peptides, non-antibiotic drugs, vaccines, and immunoglobulins. Here we review the current state of development, implications, advantages, disadvantages, and perspectives of the different approaches for manipulating HGME.

Effect of microbiota on the physiology of blood-tissue barriers.

Las uniones estrechas (UE) son estructuras altamente complejas que se localizan en la porción más apical de la membrana basolateral y están compuestas por una serie de proteínas, como claudinas, ocludinas y proteínas de la familia ZO. Las UE restringen el paso de sustancias potencialmente dañinas o microorganismos a lo largo del espacio paracelular, y participan de manera importante en procesos de mecanotransducción y señalización intercelular. Aunque la ultraestructura de las UE les permiten funcionar como una barrera en varios tejidos, como en la barrera hematoencefálica y la barrera hematotesticular, estas son propensas a cambios en su composición, lo cual podría disminuir sus características de permeabilidad. En este sentido, se ha demostrado que ciertos microorganismos enteropatógenos son capaces de desensamblar o modificar las propiedades de permeabilidad de las UE en las barreras hematotisulares. En particular, se ha estudiado cómo la microbiota contribuye a la formación, la función y el mantenimiento de las UE en varios nichos inmunitariamente privilegiados, tales como el tracto gastrointestinal, el sistema nervioso central y los testículos. Por lo tanto, resulta primordial comprender los mecanismos fisiológicos por los cuales la microbiota puede modificar la función de las barreras hematotisulares, con el -objetivo de diseñar nuevas estrategias terapéuticas que mejoren los efectos dañinos de varias enfermedades sobre nichos inmunitariamente privilegiados en el humano.Tight-junctions (TJ) are a highly complex structure located in the most apical portion of the basolateral membrane, composed of series of proteins, such as claudins, occludins and proteins of the ZO family. TJ restrict the passage of potentially harmful substances or microorganisms through paracellular space and participate importantly in the mecanotransduction and intercellular signaling processes. Although the complex structure of TJ, allow them function as barrier in various tissues, such as brain-blood-barrier and testicular-blood-barrier, these barriers are prone to changes decreasing its permeability features. The contribution of microbiota in the formation, function and maintenance of TJ in various immunologically privileged niches, such as gastrointestinal tract, central nervous system and testicles have been recently studied. Nevertheless, it has been demonstrated that certain pathogenic microorganisms are able to disassemble or modify the permeability of TJ in blood-tissue barrier. Thereby, it is central to understand the physiological mechanisms of how microbiota could modify the function of epithelial blood barriers in order to design new therapeutic strategies to ameliorate harmful effects of many human diseases.

Lys48 ubiquitination during the intraerythrocytic cycle of the rodent malaria parasite, Plasmodium chabaudi.

Ubiquitination tags proteins for different functions within the cell. One of the most abundant and studied ubiquitin modification is the Lys48 polyubiquitin chain that modifies proteins for their destruction by proteasome. In Plasmodium is proposed that post-translational regulation is fundamental for parasite development during its complex life-cycle; thus, the objective of this work was to analyze the ubiquitination during Plasmodium chabaudi intraerythrocytic stages. Ubiquitinated proteins were detected during intraerythrocytic stages of Plasmodium chabaudi by immunofluorescent microscopy, bidimensional electrophoresis (2-DE) combined with immunoblotting and mass spectrometry. All the studied stages presented protein ubiquitination and Lys48 polyubiquitination with more abundance during the schizont stage. Three ubiquitinated proteins were identified for rings, five for trophozoites and twenty for schizonts. Only proteins detected with a specific anti- Lys48 polyubiquitin antibody were selected for Mass Spectrometry analysis and two of these identified proteins were selected in order to detect the specific amino acid residues where ubiquitin is placed. Ubiquitinated proteins during the ring and trophozoite stages were related with the invasion process and in schizont proteins were related with nucleic acid metabolism, glycolysis and protein biosynthesis. Most of the ubiquitin detection was during the schizont stage and the Lys48 polyubiquitination during this stage was related to proteins that are expected to be abundant during the trophozoite stage. The evidence that these Lys48 polyubiquitinated proteins are tagged for destruction by the proteasome complex suggests that this type of post-translational modification is important in the regulation of protein abundance during the life-cycle and may also contribute to the parasite cell-cycle progression.

Establishment and characterization of a cell population derived from a dentigerous cyst.

BACKGROUND: Dentigerous cyst (DC) occurs in approximately 20% of jaw cysts, being the second major common odontogenic cyst, after radicular cyst. This oral lesion has the ability to destroy maxillary bones and could be the origin of several odontogenic tumors. However, molecules implicated in its pathogenesis as well as those involved in its neoplastic transformation remain unknown. Here, we established a cell population derived from a DC as an in vitro model for the study of this oral lesion. METHODS: Cell culture was performed from a DC from a 44-year-old male. Cells were cultured at 37°C in DMEM/F12 medium containing 10% fetal bovine serum. Expression of epithelial markers was analyzed by Western blot and immunofluorescence. Ultrastructural characterization was carried out by transmission electron microscopy. Conditioned media were obtained and characterized by zymography and Western blot. RESULTS: Cells showed spindle-shaped morphology, but they express epithelial markers, such as cytokeratins and the odontogenic ameloblast-associated protein. The ultrastructural analysis showed well-formed desmosomes present in adhering contiguous cells, confirming the epithelial lineage of this cell population. Cells also contain several vesicles adjacent to plasma membrane, suggesting an active secretion. Indeed, the analysis of the conditioned medium revealed the presence of several secreted proteins, among them the matrix metalloproteinase-2. CONCLUSIONS: Our work provides a useful model to identify the molecular mechanisms involved in the pathogenesis of DC.

Protein phosphorylation during Plasmodium berghei gametogenesis.

Plasmodium gametogenesis within the mosquito midgut is a complex differentiation process involving signaling mediated by phosphorylation, which modulate metabolic routes and protein synthesis required to complete this development. However, the mechanisms leading to gametogenesis activation are poorly understood. We analyzed protein phosphorylation during Plasmodium berghei gametogenesis in vitro in serum-free medium using bidimensional electrophoresis (2-DE) combined with immunoblotting (IB) and antibodies specific to phosphorylated serine, threonine and tyrosine. Approximately 75 protein exhibited phosphorylation changes, of which 23 were identified by mass spectrometry. These included components of the cytoskeleton, heat shock proteins, and proteins involved in DNA synthesis and signaling pathways among others. Novel phosphorylation events support a role for these proteins during gametogenesis. The phosphorylation sites of six of the identified proteins, HSP70, WD40 repeat protein msi1, enolase, actin-1 and two isoforms of large subunit of ribonucleoside reductase were investigated using TiO2 phosphopeptides enrichment and tandem mass spectrometry. In addition, transient exposure to hydroxyurea, an inhibitor of ribonucleoside reductase, impaired male gametocytes exflagellation in a dose-dependent manner, and provides a resource for functional studies.

Influence of chronic food deprivation on structure-function relationship of juvenile rat fast muscles.

In the present study, we analyze the influence of chronic undernutrition on protein expression, muscle fiber type composition, and fatigue resistance of the fast extensor digitorum longus (EDL) muscle of male juvenile rats (45 ± 3 days of life; n = 25 and 31 rats for control and undernourished groups, respectively). Using 2D gel electrophoresis and mass spectrometry, we identified in undernourished muscles 12 proteins up-regulated (8 proteins of the electron transport chain and the glycolytic pathway, 2 cross-bridge proteins, chaperone and signaling proteins that are related to the stress response). In contrast, one down-regulated protein related to the fast muscle contractile system and two other proteins with no changes in expression were used as charge controls. By means of COX and alkaline ATPase histochemical techniques and low-frequency fatigue protocols we determined that undernourished muscles showed a larger proportion (15% increase) of Type IIa/IId fibers (oxidative-glycolytic) at the expense of Type IIb (glycolytic) fibers (15.5% decrease) and increased fatigue resistance (55.3%). In addition, all fiber types showed a significant reduction in their cross-sectional area (slow: 64.4%; intermediate: 63.9% and fast: 61.2%). These results indicate that undernourished EDL muscles exhibit an increased expression of energy metabolic and myofibrillar proteins which are associated with the predominance of oxidative and Type IIa/IId fibers and to a higher resistance to fatigue. We propose that such alterations may act as protective and/or adaptive mechanisms that counterbalance the effect of chronic undernourishment.