Objective Participation of the Fe‐S‐IBG subsystem in the assembly of the [2Fe‐2S] cluster into the bc1 complex. Methods Mutants of S. cerevisiae: Δiba57, Δisa1, Δgrx5, and Δrip1 were grown in YPD medium at 30°C. Mitochondria were isolated by differential centrifugation according to Auchére et al. (2008). Respiratory complex and aconitase activities were evaluated according to Gomez et al. (2014). Mitochondria were treated with digitonin to evaluate the formation of respiratory supercomplexes by electrophoresis in Polyacrylamide Blue Native Gels (BN‐PAGE), and after 2‐dimensional SDS‐PAGE analysis according to Schagger et al. (2000). Detection of the Rip1p subunit was performed by Western blot using the anti‐Rip1p monoclonal antibody at 1:20,000 dilution and developed with anti‐mouse IgG HRP‐conjugate. Abstract The iron‐sulfur clusters [Fe‐S] are inorganic cofactors contained in enzymes involved in multiple cellular processes. In eukaryotes, [Fe‐S] synthesis mainly occurs in the mitochondrial matrix by the biogenesis of Iron Sulfur Cluster system (ISC). This system involves two steps: the novo assembly of the [Fe–S] clusters on scaffold proteins, and the transference of the [Fe–S] cluster to target apo‐proteins. These steps involve the participation of several proteins, which perform specific reactions. The ISC subsystem Fe‐S‐IBG conformed by the Grx5p, Isa1p, Isa2p, and Iba57p proteins is essential for the synthesis of the [4Fe‐4S] clusters. Yeast mutants in the Fe‐S‐IBG subsystem showed deficiency in respiration and excessive ROS generation was found, similarly to the mutant Δrip1, which gene encodes for the Rieske subunit of cytochrome bc1. In this work, the activity of the cytochrome bc1 in mitochondria from the Δiba57,Δisa1, Δgrx5, and Δrip1 mutants strains were determined. Results showed that the cytochrome bc1 complex in mitochondria from the Δiba57,Δisa1, and Δrip1 mutants was dysfunctional; whereas in the Δgrx5, the cytochrome bc1 activity was partially decreased in comparison with the WT strain. BN‐PAGE analysis shows that the respiratory complex lost the capacity to form supercomplexes in mitochondria from the Δiba57 and Δisa1 mutants, Immunodetection assays showed that Rip1p was absent in the iba57Δ and Δisa1 mutants; whereas the Δgrx5 mutant shows an unaltered pattern profile of the supercomplexes bands in BN‐PAGE, without the loss of Rieske protein. In conclusion, results indicate that the Isa1p and Iba57p proteins are involved in the insertion of the [2Fe‐2S] cluster on the Rip1p apoprotein, suggesting that the Fe‐S‐IBG subsystem is not exclusive for the maturation/assembly of proteins with [4Fe‐4S] clusters, but also is involved in [2Fe‐2S] clusters assembly such as occurred in the Rieske subunit.

Objective Evaluation of the anti‐proliferative mechanisms in HeLa cells cultures by cyclodipeptides from Pseudomonas aeruginosa PAO1. Methods Cyclodipeptides from P. aeruginosa PAO1 (PAO1‐CDPs) cultures were obtained by solvent extraction according to Ortiz‐Castro et al. (2011). Cell viability of HeLa cells was determined by the MTT method and apoptosis using Annexin‐V according to Vázquez‐Rivera et al. (2014). Mitochondrial membrane potential determination and real‐time quantification of superoxide was carried out by FACS according to Gómez et al. 2014. Immunodetection was conduced using human antibodies: anti‐Akt, anti‐Akt‐phosphorylated (Ser473), anti‐p70S6 kinase, anti‐phosphorylated‐p70S6 kinase (Thr 389); developed using Goat anti‐Rabbit IgG HRP‐conjugate. Abstract Pseudomonas aeruginosa PAO1 produces the cyclodipeptides cyclo(L‐Pro‐L‐Tyr), cyclo(L‐Pro‐L‐Phe), and cyclo(L‐Pro‐L‐Val), whose effects have been implicated in inhibition of proliferation in human tumor cell lines. Our purpose was to elucidate the anti‐proliferative mechanisms caused by the PAO1‐CDPs in HeLa cells cultures. The results indicate that the PAO1‐CDPs both individually as in mixture, inhibited HeLa cell proliferation by arresting the cell cycle at the G0–G1 transition. The PAO1‐CDPs mixture promoted cell death in HeLa line by a dose‐dependent manner, showing half‐lethal dose (LD50) of 60 μM and inducing apoptosis at 0.6 μM. Moreover, PAO1‐CDPs showed a higher pro‐apoptotic activity (~10 fold) than their synthetic analogs. Subsequently, the mitochondrial membrane potential was arrested by the PAO1‐CDPs and induced apoptosis by caspase‐9‐dependent pathway. The anti‐proliferative mechanism in HeLa cells involves inhibition of phosphorylation of both the Akt‐S473 and S6k‐T389 protein kinases, showing a cyclic behavior of their phosphorylation in a time and concentration‐dependent fashion. Conclusions Our findings indicate that the PI3K/Akt/mTOR/S6k signaling pathway blocking is involved in the anti‐proliferative effect of the PAO1‐CDPs.

El ojo es capaz de regular la respuesta inflamatoria activando una forma de tolerancia específica y sistémica para antígenos que son inoculados en su interior. Esta capacidad puede ser aprovechada para modular la respuesta inflamatoria en enfermedades degenerativas. Sin embargo, se desconoce el tiempo de permanencia de la inmunotolerancia y si ésta depende de la edad de inducción. El objetivo es evaluar la permanencia de inmuno-tolerancia a albúmina de suero bovino (BSA, por sus siglas en inglés). Inoculamos BSA en la cámara anterior del ojo de ratas juveniles y adultas. Estos animales fueron posteriormente inmunizados con BSA para evaluar la inmunotolerancia sistémica y específica al antígeno a lo largo del tiempo. El resultado fue que tanto ratas juveniles como adultas fueron tolerizadas a BSA (t(5):3.102, p < 0.05; t(8):3.278, p < 0.001) y este estado se mantuvo al menos hasta los cinco meses posinducción en ratas juveniles (t(7):11.704, p < 0.001) y hasta los 4 meses en ratas adultas (t(5):3.278, p < 0.05).

Objective Determination of the molecular mechanism involved in the virulence modulation of Pseudomonas aeruginosa PAO1 involving the pvdI and pvdL genes. Methods Supernatants of the P. aeruginosa PAO1 strain and mutants pvdI, pvdJ, and pvdL were obtained from LB liquid medium after 24 h of growth. Evaluation of virulence factors: biofilm was determined by growth of P. aeruginosa strains in 96‐well plates using violet crystal staining. Rhamnolipids production was determined by quantification of rhamnose by the orcinol method. Survival assays were carried out in Caenorhabditis elegans using supernatants of PAO1 strains. Abstract Pseudomonas aeruginosa is an opportunistic pathogenic bacterium for humans, animals and plants. This bacterium possesses a system of perception of bacterial quorum (quorum sensing, QS), which modulates several virulence factors. The LasI/LasR QS system modifies the production of the cyclodipeptides (CDPs) cyclo(L‐Pro‐L‐Val), cyclo(L‐Pro‐L‐Phe) and cyclo(L‐Pro‐L‐Tyr). Although CDPs are molecules with bioactive properties, low elements concern in the mechanism of biosynthesis in bacteria and their role in communication with host cells. In addition, it has been described that the CDPs production in P. aeruginosa mainly depends of the multi‐modular non‐ribosomal peptide synthetases (MM‐NRPS), whose products such as the aeruginaldehyde has been involved in the bacterial QS mechanisms. In this study, was found that levels of pyocyanin (a virulence factor in P. aeruginosa), it was increased in mutant strains in the pvdJ gene, which encodes for a NPRS. The pvd gene cluster is directly related to the pioverdin synthesis, which is a fluorescent peptide‐siderophore involved in the iron acquisition, being essential in the bacterial pathogenicity on the host. Therefore, is important to elucidate the mechanism by which the CDPs of P. aeruginosa PAO1 contribute to its pathogenesis and/or virulence. Production of several virulence factors in P. aeruginosa PAO1 with different single and double mutants in pvdI, pvdJ and pvdL genes were compared. Mutants in the pvdI, pvdL and pvdL/pvdI genes decrease 50% the production of biofilm respect to the WT strain. Likewise, when evaluating rhamnolipids amount, the production of rhamnolipids was significantly increased in the single mutants pvdJ and pvdL compared with the WT strain, but not modified in the double mutant pvdJ/pvdL. Survival in the C. elegans worms indicated that the pvdJ mutant increase the death of the nematode 40% than the WT. The results suggest that the pvd gene cluster was involved in the production of CDPs in P. aeruginosa PAO1 and may to regulate the production of virulence factors (biofilm and rhamnolipids), impacting its pathogenicity on the nematode in vivo model.