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.
•Mutation of SUMO site lysine 101 alters the subcellular localization of ADI.•Nuclear localization signals are involved in the translocation of ADI to the nuclei.•ADI acts as a peptidyl arginine deiminase.•ADI is involved in the down-regulation of Giardia encystation.
Vesicles formed by phospholipids are promising candidates for drug delivery. It is known that the lipid composition affects properties such as the rigidity–fluidity of the membrane and that it ...influences the bilayer permeability, but sometimes sophisticated techniques are selected to monitor them. In this work, we study the bilayer of different unilamellar vesicles composed of different lipids (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC, and lecithin) and diverse techniques such as extruder and electrospun templates and using 6-propionyl-2-(N,N-dimethyl) aminonaphthalene (PRODAN) and its photophysics. Moreover, we were able to monitor the influence of cholesterol on the bilayers. We demonstrate that the bilayer properties can be evaluated using the emission feature of the molecular probe PRODAN. This fluorescent probe gives relevant information on the polarity and fluidity of the microenvironment for unilamellar vesicles formed by two different methods. The PRODAN emission at 434 nm suggests that the bilayer properties significantly change if DOPC or lecithin is used in the vesicle preparation especially in their fluidity. Moreover, cholesterol induces alterations in the bilayer’s structural and microenvironmental properties to a greater or lesser degree in both vesicles. Thus, we propose an easy and elegant way to evaluate physicochemical properties, which is fundamental for manufacturing vesicles as a drug delivery system, simply by monitoring the molecular probe emission band centered at 434 nm, which corresponds to the PRODAN species deep inside the bilayer.
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•Blocking endocytosis triggers cell differentiation in Giardia trophozoites.•Inhibition of cholesterol entry is not sufficient to complete encystation.•Decreasing expression of the ...medium subunit Glµ2 produced immature cysts when induced to encyst.•GlAP2 is necessary to complete the final steps of encystation.
The protozoan parasite Giardia lamblia acquires cholesterol from the environment since it is unable to synthesise cholesterol de novo and this is vital for trophozoite growth. Conversely, the lack of cholesterol was described as an essential event to trigger encystation, the differentiation of trophozoites to mature cysts. During the G. lamblia cell cycle, cholesterol is acquired as a free molecule as well as through receptor-mediated endocytosis (RME) of lipoproteins. In this work, we describe the involvement of RME in the cell differentiation process of G. lamblia. We found that a reduction in the expression of the medium subunit (Glµ2) of the giardial adaptin protein GlAP2 impaired RME, triggering the process of encystation in growing cells. Contrary to expectations, decreasing Glµ2 expression produced a cohort of trophozoites that yielded significantly less mature cysts when cells were induced to encyst. Analysis of the subcellular localization of Glµ2 and the cyst wall protein 1 (CWP1) during encystation was later performed, to dissect the process. Our results showed, on one hand, that blocking RME by inhibiting Glµ2 expression, and probably cholesterol entry, is sufficient to induce cell differentiation but not to complete the process of encystation. On the other hand, we observed that GlAP2 is necessary to accomplish the final steps of encystation by sorting CWP1 to the plasma membrane for cyst wall formation. The understanding of the mechanisms involved in cyst formation should provide novel insights into the control of giardiasis, an endemic worldwide neglected disease.
ANRIL (Antisense Noncoding RNA in the INK4 Locus), also named CDKN2B-AS1, is a long non-coding RNA with outstanding functions that regulates genes involved in atherosclerosis development. ANRIL ...genotypes and the expression of linear and circular isoforms have been associated with coronary artery disease (CAD). The CDKN2A and the CDKN2B genes at the CDKN2A/B locus encode the Cyclin-Dependent Kinase inhibitor protein (CDKI) p16INK4a and the p53 regulatory protein p14ARF, which are involved in cell cycle regulation, aging, senescence, and apoptosis. Abnormal ANRIL expression regulates vascular endothelial growth factor (VEGF) gene expression, and upregulated Vascular Endothelial Growth Factor (VEGF) promotes angiogenesis by activating the NF-κB signaling pathway. Here, we explored associations between determinations of the linear, circular, and linear-to-circular ANRIL gene expression ratio, CDKN2A, VEGF and its receptor kinase insert domain-containing receptor (KDR) and cardiovascular risk factors and all-cause mortality in high-risk coronary patients before they undergo coronary artery bypass grafting surgery (CABG). We found that the expression of ANRIL isoforms may help in the prediction of CAD outcomes. Linear isoforms were correlated with a worse cardiovascular risk profile while the expression of circular isoforms of ANRIL correlated with a decrease in oxidative stress. However, the determination of the linear versus circular ratio of ANRIL did not report additional information to that determined by the evaluation of individual isoforms. Although the expressions of the VEFG and KDR genes correlated with a decrease in oxidative stress, in binary logistic regression analysis it was observed that only the expression of linear isoforms of ANRIL and VEGF significantly contributed to the prediction of the number of surgical revascularizations.
To date, eight assemblages of Giardia lamblia have been described, but only assemblages A and B are known to infect humans. Despite the fact that the genomic, biological, and clinical differences ...found between these two assemblages has raised the possibility that they may be considered different species, there is relatively limited information on their phenotypic differences. In the present study, we developed monoclonal antibodies against alpha-1 and beta giardin, two immunodominant proteins produced during G. lamblia infection, and studied their expression and localization in WB (assemblage A) and GS trophozoites (assemblage B).
The polyclonal antibodies generated against WB trophozoites, particularly those recognizing intracellular proteins as well as the proteins present at the plasma membrane (variable-specific surface proteins), showed cross-reactivity with intracellular proteins in GS trophozoites. The use of monoclonal antibodies against beta giardin indicated ventral disc localization, particularly at the periphery in WB trophozoites. Interestingly, although beta giardin was also restricted to the ventral disc in GS trophozoites, the pattern of localization clearly differed in this assemblage. On the other hand, monoclonal antibodies against alpha-1 giardin showed plasma membrane localization in both assemblages with the bare area of GS trophozoites also being distinguished. Moreover, the same localization at the plasma membrane was observed in Portland-1 (Assemblage A) and in P15 (Assemblage E) trophozoites.
We found differences in localization of the beta giardin protein between assemblages A and B, but the same pattern of localization of alpha-1 giardin in strains from Assemblages A, B and E. These findings reinforce the need for more studies based on phenotypic characteristics in order to disclose how far one assemblage is from the other.
The functional renal epithelium is composed of differentiated and polarized tubular cells with a strong actin cortex and specialized cell-cell junctions. If, under pathological conditions, these ...cells have to resist higher kidney osmolarity, they need to activate diverse mechanisms to survive external nephrotoxic agents such as inflammation and oxidative stress. Wine pomace polyphenols exert protective effects on renal cells. In this study, two wine-pomace products and their protective effects upon promotion and preservation of normal cell differentiation and attenuation of oxalate-induced type II epithelial mesenchymal transition (EMT) are evaluated. Treatment with gastrointestinal and colonic bioavailable fractions from red (rWPP) and white (wWPP) wine pomaces, both in the presence and the absence of oxalate, showed similar cell numbers and nuclear size than the non-treated differentiated MDCK cells. Immunofluorescence analysis showed the reduction of morphological changes and the preservation of cellular junctions for the rWPP and wWPP pre-treatment of cells exposed to oxalate injury. Hence, both rWPP and wWPP attenuated oxalate type II EMT in MDCK cells that conserved their epithelial morphology and cellular junctions through the antioxidant activities of grape pomace polyphenols.
Food Science; Cell Biology; Plant Products; Cell Differentiation; Cytoskeleton; Natural product; Polyphenol; Antioxidant; Cancer Research; Renal System; wine pomace; polyphenols; EMT; oxalate; E-cadherin
Hypothyroxinemia (Hpx) is a thyroid hormone deficiency (THD) condition highly frequent during pregnancy, which although asymptomatic for the mother, it can impair the cognitive function of the ...offspring. Previous studies have shown that maternal hypothyroidism increases the severity of experimental autoimmune encephalomyelitis (EAE), an autoimmune disease model for multiple sclerosis (MS). Here, we analyzed the immune response after EAE induction in the adult offspring gestated in Hpx. Mice gestated in Hpx showed an early appearance of EAE symptoms and the increase of all parameters of the disease such as: the pathological score, spinal cord demyelination, and immune cell infiltration in comparison to the adult offspring gestated in euthyroidism. Isolated CD4
CD25
T cells from spleen of the offspring gestated in Hpx that suffer EAE showed reduced capacity to suppress proliferation of effector T cells (T
) after being stimulated with anti-CD3 and anti-CD28 antibodies. Moreover, adoptive transfer experiments of CD4
CD25
T cells from the offspring gestated in Hpx suffering EAE to mice that were induced with EAE showed that the receptor mice suffer more intense EAE pathological score. Even though, no significant differences were detected in the frequency of T
cells and IL-10 content in the blood, spleen, and brain between mice gestated in Hpx or euthyroidism, T cells CD4
CD25
from spleen have reduced capacity to differentiate
to T
and to produce IL-10. Thus, our data support the notion that maternal Hpx can imprint the immune response of the offspring suffering EAE probably due to a reduced capacity to trigger suppression. Such "imprints" on the immune system could contribute to explaining as to why adult offspring gestated in Hpx suffer earlier and more intense EAE.
Intensive Care to facilitate Organ Donation (ICOD) consists of the initiation or continuation of intensive care measures in patients with a devastating brain injury (DBI) in whom curative treatment ...is deemed futile and death by neurological criteria (DNC) is foreseen, to incorporate organ donation into their end-of-life plans. In this study we evaluate the outcomes of patients subject to ICOD and identify radiological and clinical factors associated with progression to DNC. In this first prospective multicenter study we tested by multivariate regression the association of clinical and radiological severity features with progression to DNC. Of the 194 patients, 144 (74.2%) patients fulfilled DNC after a median of 25 h (95% IQR: 17-44) from ICOD onset. Two patients (1%) shifted from ICOD to curative treatment, both were alive at discharge. Factors associated with progression to DNC included: age below 70 years, clinical score consistent with severe brain injury, instability, intracranial hemorrhage, midline shift ≥5 mm and certain types of brain herniation. Overall 151 (77.8%) patients progressed to organ donation. Based on these results, we conclude that ICOD is a beneficial and efficient practice that can contribute to the pool of deceased donors.
Neural stem cells continuously generate newborn neurons that integrate into and modify neural circuitry in the adult hippocampus. The molecular mechanisms that regulate or perturb neural stem cell ...proliferation and differentiation, however, remain poorly understood. Here, we have found that mouse hippocampal radial glia-like (RGL) neural stem cells express the synaptic cochaperone cysteine string protein-α (CSP-α). Remarkably, in CSP-α knockout mice, RGL stem cells lose quiescence postnatally and enter into a high-proliferation regime that increases the production of neural intermediate progenitor cells, thereby exhausting the hippocampal neural stem cell pool. In cell culture, stem cells in hippocampal neurospheres display alterations in proliferation for which hyper-activation of the mechanistic target of rapamycin (mTOR) signaling pathway is the primary cause of neurogenesis deregulation in the absence of CSP-α. In addition, RGL cells lose quiescence upon specific conditional targeting of CSP-α in adult neural stem cells. Our findings demonstrate an unanticipated cell-autonomic and circuit-independent disruption of postnatal neurogenesis in the absence of CSP-α and highlight a direct or indirect CSP-α/mTOR signaling interaction that may underlie molecular mechanisms of brain dys-function and neurodegeneration.
The present study evaluates the quality of life of 1,139 surviving victims of the Colombian armed conflict benefited from some actions of reparation, through a brief sociodemographic and ...victimological survey and the WHO quality of life (WHOQOL-BREF) questionnaire, which takes into account perceived physical and mental health, social relations, and environmental factors. The results show that the quality of life of the victims of the armed conflict, once peace agreements had been reached, remained low or medium-low. The number of victimizing events suffered and the time elapsed have little effect on the quality of life, with type of trauma being more relevant. Land-related experiences, such as expropriation, had fundamentally an influence on environmental factors. Kidnappings, sexual assaults, and events where minors were involved affected mental health. Victims of sexual assault were those whose quality of life was most affected and torture victims were those with a lower index of global quality of life. Simple passing of time did not mean quality of life increase with specific interventions being needed to repair damage. To repair the damage caused, special attention should be paid to the sociodemographic characteristics of each victim, the type of aggression suffered, and their needs in the areas of physical and mental health, and economic resources.
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