The endoplasmic reticulum stress sensor IRE1 regulates collagen secretion through the enforcement of the proteostasis factor P4HB/PDIA1 contributing to liver damage and fibrosis.

Collagen is one the most abundant proteins and the main cargo of the secretory pathway, contributing to hepatic fibrosis and cirrhosis due to excessive deposition of extracellular matrix. Here we investigated the possible contribution of the unfolded protein response, the main adaptive pathway that monitors and adjusts the protein production capacity at the endoplasmic reticulum, to collagen biogenesis and liver disease. Genetic ablation of the ER stress sensor IRE1 reduced liver damage and diminished collagen deposition in models of liver fibrosis triggered by carbon tetrachloride (CCl 4 ) administration or by high fat diet. Proteomic and transcriptomic profiling identified the prolyl 4-hydroxylase (P4HB, also known as PDIA1), which is known to be critical for collagen maturation, as a major IRE1-induced gene. Cell culture studies demonstrated that IRE1 deficiency results in collagen retention at the ER and altered secretion, a phenotype rescued by P4HB overexpression. Taken together, our results collectively establish a role of the IRE1/P4HB axis in the regulation of collagen production and its significance in the pathogenesis of various disease states.

Evaluación teórica de la exposición dietaria a la bacteriocina nisina como conservante natural para aderezos de tipo mayonesa vegetal en Chile

Actualmente, existe una necesidad en la industria alimentaria de buscar preservantes de origen natural que tengan una mejor aceptación por el consumidor. Una alternativa interesante que ha emergido en los últimos años es el uso de antimicrobianos naturales, los cuales además de ser efectivos en el control de microorganismos, son inocuos para la salud. La nisina es una bacteriocina producida por bacterias ácido lácticas que se utiliza actualmente como preservante en varios productos alimenticios. En Chile, el uso de la nisina como aditivo está restringido a quesos y cremas naturales o nata cuajada, pero existen otros alimentos que podrían beneficiarse del uso de este preservante, como es el caso de los aderezos tipo mayonesa vegetal. En el presente trabajo, se analiza de manera teórica, como el uso de nisina en estos aderezos impactaría el consumo máximo permitido para este aditivo en la población chilena. Para esto, se analizó cuál es el consumo teórico actual de nisina en Chile, y cómo este se vería afectado si se utilizara nisina como preservante en aderezos tipo mayonesa vegetal. Finalmente, los datos obtenidos se compararon con el valor de ingesta diaria admisible (IDA) para la nisina, evidenciándose que existe una ventana de oportunidad para el uso de nisina en otros productos alimenticios.

Currently, there is a need in the food industry for natural preservatives that have good consumers acceptance. Natural antimicrobials are a novel alternative, effective in controlling microbial growth, and safe to consume. Nisin is a bacteriocin produced by lactic acid bacteria that is currently used as a preservative in several food products. In Chile, nisin is regulated and restricted as an additive to cheeses and natural creams or curds. However, other food products could benefit from using nisin as a preservative, such as plant-based mayonnaise, where the consumer expects to find natural preservatives. In this review, we make a theoretical analysis of how adding nisin to plant-based mayonnaise would impact the daily intake of this additive in the Chilean population. Finally, we compare this data with the acceptable daily intake for nisin to conclude that there is an opportunity for using nisin as a preservative in plant-based mayonnaise without affecting consumers´ health.

Palmitic acid control of ciliogenesis modulates insulin signaling in hypothalamic neurons through an autophagy-dependent mechanism.

Palmitic acid (PA) is significantly increased in the hypothalamus of mice, when fed chronically with a high-fat diet (HFD). PA impairs insulin signaling in hypothalamic neurons, by a mechanism dependent on autophagy, a process of lysosomal-mediated degradation of cytoplasmic material. In addition, previous work shows a crosstalk between autophagy and the primary cilium (hereafter cilium), an antenna-like structure on the cell surface that acts as a signaling platform for the cell. Ciliopathies, human diseases characterized by cilia dysfunction, manifest, type 2 diabetes, among other features, suggesting a role of the cilium in insulin signaling. Cilium depletion in hypothalamic pro-opiomelanocortin (POMC) neurons triggers obesity and insulin resistance in mice, the same phenotype as mice deficient in autophagy in POMC neurons. Here we investigated the effect of chronic consumption of HFD on cilia; and our results indicate that chronic feeding with HFD reduces the percentage of cilia in hypothalamic POMC neurons. This effect may be due to an increased amount of PA, as treatment with this saturated fatty acid in vitro reduces the percentage of ciliated cells and cilia length in hypothalamic neurons. Importantly, the same effect of cilia depletion was obtained following chemical and genetic inhibition of autophagy, indicating autophagy is required for ciliogenesis. We further demonstrate a role for the cilium in insulin sensitivity, as cilium loss in hypothalamic neuronal cells disrupts insulin signaling and insulin-dependent glucose uptake, an effect that correlates with the ciliary localization of the insulin receptor (IR). Consistently, increased percentage of ciliated hypothalamic neuronal cells promotes insulin signaling, even when cells are exposed to PA. Altogether, our results indicate that, in hypothalamic neurons, impairment of autophagy, either by PA exposure, chemical or genetic manipulation, cause cilia loss that impairs insulin sensitivity.

New emerging roles of Polycystin-2 in the regulation of autophagy.

Polycystin-2 (PC2) is a calcium channel that can be found in the endoplasmic reticulum, the plasmatic membrane, and the primary cilium. The structure of PC2 is characterized by a highly ordered C-terminal tail with an EF-motif (calcium-binding domain) and a canonical coiled-coil domain (CCD; interaction domain), and its activity is regulated by interacting partners and post-translational modifications. Calcium mobilization into the cytosol by PC2 has been mainly associated with cell growth and differentiation, and therefore mutations or dysfunction of PC2 lead to renal and cardiac consequences. Interestingly, PC2-related pathologies are usually treated with rapamycin, an autophagy stimulator. Autophagy is an intracellular degradation process where recycling material is sequestered into autophagosomes and then hydrolyzed by fusion with a lysosome. Interestingly, several studies have provided evidence that PC2 may be required for autophagy, suggesting that PC2 maintains a physiologic catabolic state.

New Insights on the Role of Lipid Metabolism in the Metabolic Reprogramming of Macrophages.

Macrophage activation is intimately linked to metabolic reprogramming. Inflammatory (M1) macrophages are able to sustain inflammatory responses and to kill pathogens, mostly by relying on aerobic glycolysis and fatty acid biosynthesis. Glycolysis is a fast way of producing ATP, and fatty acids serve as precursors for the synthesis of inflammatory mediators. On the opposite side, anti-inflammatory (M2) macrophages mediate the resolution of inflammation and tissue repair, switching their metabolism to fatty acid oxidation and oxidative phosphorylation. Over the years, this classical view has been challenged by recent discoveries pointing to a more complex metabolic network during macrophage activation. Lipid metabolism plays a critical role in the activation of both M1 and M2 macrophages. Recent evidence shows that fatty acid oxidation is also essential for inflammasome activation in M1 macrophages, and glycolysis is now known to fuel fatty acid oxidation in M2 macrophages. Ultimately, targeting lipid metabolism in macrophages can improve the outcome of metabolic diseases. Here, we review the main aspects of macrophage immunometabolism from the perspective of the metabolism of lipids. Building a reliable metabolic network during macrophage activation will bring us closer to targeting macrophages for improving human health.

Autophagy Regulates the Liver Clock and Glucose Metabolism by Degrading CRY1.

The circadian clock coordinates behavioral and circadian cues with availability and utilization of nutrients. Proteasomal degradation of clock repressors, such as cryptochrome (CRY)1, maintains periodicity. Whether macroautophagy, a quality control pathway, degrades circadian proteins remains unknown. Here we show that circadian proteins BMAL1, CLOCK, REV-ERBα, and CRY1 are lysosomal targets, and that macroautophagy affects the circadian clock by selectively degrading CRY1. Autophagic degradation of CRY1, an inhibitor of gluconeogenesis, occurs in a diurnal window when rodents rely on gluconeogenesis, suggesting that CRY1 degradation is time-imprinted to maintenance of blood glucose. High-fat feeding accelerates autophagic CRY1 degradation and contributes to obesity-associated hyperglycemia. CRY1 contains several light chain 3 (LC3)-interacting region (LIR) motifs, which facilitate the interaction of cargo proteins with the autophagosome marker LC3. Using mutational analyses, we identified two distinct LIRs on CRY1 that exert circadian glycemic control by regulating CRY1 degradation, revealing LIRs as potential targets for controlling hyperglycemia.

System-wide Benefits of Intermeal Fasting by Autophagy.

Autophagy failure is associated with metabolic insufficiency. Although caloric restriction (CR) extends healthspan, its adherence in humans is poor. We established an isocaloric twice-a-day (ITAD) feeding model wherein ITAD-fed mice consume the same food amount as ad libitum controls but at two short windows early and late in the diurnal cycle. We hypothesized that ITAD feeding will provide two intervals of intermeal fasting per circadian period and induce autophagy. We show that ITAD feeding modifies circadian autophagy and glucose/lipid metabolism that correlate with feeding-driven changes in circulating insulin. ITAD feeding decreases adiposity and, unlike CR, enhances muscle mass. ITAD feeding drives energy expenditure, lowers lipid levels, suppresses gluconeogenesis, and prevents age/obesity-associated metabolic defects. Using liver-, adipose-, myogenic-, and proopiomelanocortin neuron-specific autophagy-null mice, we mapped the contribution of tissue-specific autophagy to system-wide benefits of ITAD feeding. Our studies suggest that consuming two meals a day without CR could prevent the metabolic syndrome.

Enhanced control of Mycobacterium tuberculosis extrapulmonary dissemination in mice by an arabinomannan-protein conjugate vaccine.

Currently there are a dozen or so of new vaccine candidates in clinical trials for prevention of tuberculosis (TB) and each formulation attempts to elicit protection by enhancement of cell-mediated immunity (CMI). In contrast, most approved vaccines against other bacterial pathogens are believed to mediate protection by eliciting antibody responses. However, it has been difficult to apply this formula to TB because of the difficulty in reliably eliciting protective antibodies. Here, we developed capsular polysaccharide conjugates by linking mycobacterial capsular arabinomannan (AM) to either Mtb Ag85b or B. anthracis protective antigen (PA). Further, we studied their immunogenicity by ELISA and AM glycan microarrays and protection efficacy in mice. Immunization with either Abg85b-AM or PA-AM conjugates elicited an AM-specific antibody response in mice. AM binding antibodies stimulated transcriptional changes in Mtb. Sera from AM conjugate immunized mice reacted against a broad spectrum of AM structural variants and specifically recognized arabinan fragments. Conjugate vaccine immunized mice infected with Mtb had lower bacterial numbers in lungs and spleen, and lived longer than control mice. These findings provide additional evidence that humoral immunity can contribute to protection against Mtb.

The Type of Growth Medium Affects the Presence of a Mycobacterial Capsule and Is Associated With Differences in Protective Efficacy of BCG Vaccination Against Mycobacterium tuberculosis.

BACKGROUND: Bacillus Calmette-Guerin (BCG) vaccine is widely used for the prevention of tuberculosis, despite limited efficacy. Most immunological studies of BCG or Mycobacterium tuberculosis strains grow bacteria in the presence of detergent, which also strips the mycobacterial capsule. The impact of the capsule on vaccine efficacy has not been explored. METHODS: We tested the influence of detergent in cultures of BCG and M. tuberculosis strains on the outcome of vaccination experiments on mice and transcriptional responses on M. tuberculosis RESULTS: Vaccination of mice with encapsulated BCG promoted a more potent immune response relative to vaccination with unencapsulated BCG, including higher polysaccharide-specific capsule antibody titers, higher interferon γ and interleukin 17 splenic responses, and more multifunctional CD4(+) T cells. These differences correlated with variability in the bacterial burden in lung and spleen of mice infected with encapsulated or unencapsulated M. tuberculosis The combination of vaccination and challenge with encapsulated strains resulted in the greatest protection efficacy. The transcriptome of encapsulated M. tuberculosis was similar to that of starvation, hypoxia, stationary phase, or nonreplicating persistence. CONCLUSIONS: The presence of detergent in growth media and a capsule on BCG were associated with differences in the outcome of vaccination, implying that these are important variables in immunological studies.

Online Measurement of Real-Time Cytotoxic Responses Induced by Multi-Component Matrices, such as Natural Products, through Electric Cell-Substrate Impedance Sensing (ECIS).

Natural products are complex matrices of compounds that are prone to interfere with the label-dependent methods that are typically used for cytotoxicity screenings. Here, we developed a label-free Electric Cell-substrate Impedance Sensing (ECIS)-based cytotoxicity assay that can be applied in the assessment of the cytotoxicity of natural extracts. The conditions to measure the impedance using ECIS were first optimized in mice immortalized hypothalamic neurons GT1-7 cells. The performance of four natural extracts when tested using three conventional cytotoxicity assays in GT1-7 cells, was studied. Betula pendula (silver birch tree) was found to interfere with all of the cytotoxicity assays in which labels were applied. The silver birch extract was also proven to be cytotoxic and, thus, served as a proof-of-concept for the use of ECIS. The extract was fractionated and the ECIS method permitted the distinction of specific kinetic patterns of cytotoxicity on the fractions as well as the extract's pure constituents. This study offers evidence that ECIS is an excellent tool for real-time monitoring of the cytotoxicity of complex extracts that are difficult to work with using conventional (label-based) assays. Altogether, it offers a very suitable cytotoxicity-screening assay making the work with natural products less challenging within the drug discovery workflow.

Efectividad de la auriculopuntura en el tratamiento de pacientes con hipertensión arterial / Effectiveness of auriculopuncture in the treatment of patients with hypertension

Se realizó un estudio descriptivo y longitudinal de 53 adultos hipertensos pertenecientes al consultorio médico No. 55 del Policlínico Universitario "José Martí Pérez" de Santiago de Cuba, desde febrero hasta septiembre de 2012, con vistas a demostrar la efectividad de la auriculopuntura en el tratamiento de estos pacientes. Se halló predominio de la hipertensión arterial ligera (69,6 %) y del sexo femenino (47,1 %). Luego de aplicada la terapia disminuyeron los síntomas más frecuentes: la cefalea, de 50,9 a 5,6 % y el insomnio, de 20,7 a 1,8 %. Los resultados obtenidos evidenciaron la efectividad de este tratamiento no convencional en 92,4 % de los afectados, a la vez que se logró 96,3 % de satisfacción con el proceder empleado.

A descriptive and longitudinal study of 53 hypertensive adults belonging to the doctor's office No. 55 of "José Martí Pérez" University Polyclinic in Santiago de Cuba was carried out from February to September, 2012, with the objective of demonstrating the effectiveness of the auriculopuncture in the treatment of these patients. A prevalence of the mild hypertension (69.6%), and of the female sex (47.1%) was found. After applying the therapy, the most frequent symptoms decreased: migraine, 50.9 - 5.6% and the insomnia, 20.7 - 1.8%. The obtained results evidenced the effectiveness of this non conventional treatment in 92.4% of the affected patients, at the same time that 96.3% of satisfaction was possible with the used procedure.

Mycobacterial membrane vesicles administered systemically in mice induce a protective immune response to surface compartments of Mycobacterium tuberculosis.

UNLABELLED: Pathogenic and nonpathogenic species of bacteria and fungi release membrane vesicles (MV), containing proteins, polysaccharides, and lipids, into the extracellular milieu. Previously, we demonstrated that several mycobacterial species, including bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis, release MV containing lipids and proteins that subvert host immune response in a Toll-like receptor 2 (TLR2)-dependent manner (R. Prados-Rosales et al., J. Clin. Invest. 121:1471-1483, 2011, doi:10.1172/JCI44261). In this work, we analyzed the vaccine potential of MV in a mouse model and compared the effects of immunization with MV to those of standard BCG vaccination. Immunization with MV from BCG or M. tuberculosis elicited a mixed humoral and cellular response directed to both membrane and cell wall components, such as lipoproteins. However, only vaccination with M. tuberculosis MV was able to protect as well as live BCG immunization. M. tuberculosis MV boosted BCG vaccine efficacy. In summary, MV are highly immunogenic without adjuvants and elicit immune responses comparable to those achieved with BCG in protection against M. tuberculosis. IMPORTANCE: This work offers a new vaccine approach against tuberculosis using mycobacterial MV. Mycobacterium MV are a naturally released product combining immunogenic antigens in the context of a lipid structure. The fact that MV do not need adjuvants and elicit protection comparable to that elicited by the BCG vaccine encourages vaccine approaches that combine protein antigens and lipids. Consequently, mycobacterium MV establish a new type of vaccine formulation.

Exploration of natural compounds as sources of new bifunctional scaffolds targeting cholinesterases and beta amyloid aggregation: the case of chelerythrine.

The presented project started by screening a library consisting of natural and natural based compounds for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity. Active compounds were chemically clustered into groups and further tested on the human cholinesterases isoforms. The aim of the presented study was to identify compounds that could be used as leads to target two key mechanisms associated with the AD's pathogenesis simultaneously: cholinergic depletion and beta amyloid (Aß) aggregation. Berberin, palmatine and chelerythrine, chemically clustered in the so-called isoquinoline group, showed promising cholinesterase inhibitory activity and were therefore further investigated. Moreover, the compounds demonstrated moderate to good inhibition of Aß aggregation as well as the ability to disaggregate already preformed Aß aggregates in an experimental set-up using HFIP as promotor of Aß aggregates. Analysis of the kinetic mechanism of the AChE inhibition revealed chelerythrine as a mixed inhibitor. Using molecular docking studies, it was further proven that chelerythrine binds on both the catalytic site and the peripheral anionic site (PAS) of the AChE. In view of this, we went on to investigate its effect on inhibiting Aß aggregation stimulated by AChE. Chelerythrine showed inhibition of fibril formation in the same range as propidium iodide. This approach enabled for the first time to identify a cholinesterase inhibitor of natural origin-chelerythrine-acting on AChE and BChE with a dual ability to inhibit Aß aggregation as well as to disaggregate preformed Aß aggregates. This compound could be an excellent starting point paving the way to develop more successful anti-AD drugs.

In vivo and in vitro antioxidant activity and hepatoprotective properties of polyphenols from Halimeda opuntia (Linnaeus) Lamouroux.

Antioxidant activity and hepatoprotective properties of the aqueous extract and tetrahydrofuran-extracted phenolic fractions of Halimeda opuntia (Linnaeus) Lamouroux were investigated in rats with chemically induced liver injury. Total polyphenols were determined by using the Folin-Ciocalteau reagent. Liver damage was induced by CCl(4) and assessed by a histological technique. Reverse transcription/polymerase chain reaction (RT/PCR) analysis showed increased superoxide dismutase (SOD) and catalase (CAT) gene expression and activities in the group treated with free phenolic acid (FPA) fractions of H. opuntia, suggesting inducing effects on both enzymes. In addition, rats treated with FPA fractions displayed lower liver thiobarbituric acid reactive substance (TBARS) levels than those observed for rats in the CCl(4)-treated group. These data suggest that the phenolic fractions from H. opuntia may protect the liver against oxidative stress-inducing effects of chemicals by modulating its antioxidant enzymes and oxidative status.

Inhibition of LDL-oxidation and antioxidant properties related to polyphenol content of hydrophilic fractions from seaweed Halimeda Incrassata (Ellis) Lamouroux

LDL oxidation and oxidative stress are closely related to atherosclerosis. Therefore, natural antioxidants have been studied as promising candidates. In the present study, the LDL oxidation inhibition activity of bioactive compounds from Halimeda incrassata seaweed. associated to antioxidant capacity, was evaluated in vitro. Experimental work was conducted with lyophilized aqueous extract and phenolic-rich fractions of the seaweed and their effect on LDL oxidation was evaluated using heparin-precipitated LDL (hep-LDL) with exposure to Cu2+ ions and AAPH as the free radical generator. H. incrassata had a protective effect for hep-LDL in both systems and the presence of phenolic compounds contributed to the activity where phenolic-rich fractions showed significant capacity for inhibition of oxidation mediated by Cu2+ ions. The observed effect could be related to the antioxidant potential of polar fractions evidenced by reducing activity and DPPH• radical scavenging. The results obtained in vitro further support the antioxidant and LDL oxidation inhibition properties of H. incrassata and further knowledge toward future phytotherapeutic application of the seaweed.

A oxidação da LDL e o estresse oxidativo estão intimamente relacionados com a aterosclerose. Por isso, os antioxidantes naturais têm sido estudados como candidatos promissores. No presente trabalho foi avaliada in vitro a capacidade de inibição da oxidação da LDL pelos compostos bioativos da alga Halimeda incrassata em associação à capacidade antioxidante. O trabalho experimental foi conduzido com extratos polares (extrato aquoso liofilizado e frações ricas em fenólicos) e seu efeito na oxidação da LDL foi avaliado usando LDL precipitada com heparina (hep-LDL), oxidada com íons de Cu2+ e AAPH, como geradores de radicais livres. A H. incrassata apresentou efeito protetor para hep-LDL em ambos sistemas e a presença de compostos fenólicos contribuiu para a atividade em que as frações ricas em fenólicos demonstram capacidade significativa em inibir a oxidação mediada pelos íons de Cu2+. O efeito observado deve estar relacionado com o potencial antioxidante das frações polares medido pela atividade redutora e varredura do radical DPPH. Os resultados obtidos demonstram as propriedades antioxidantes e de inibição da oxidação da LDL da H. incrassata e podem contribuir para as evidências de futuras aplicações fitoterapêuticas desta alga.

Las algas marinas como fuentes de fitofármacos antioxidantes: [revisión] / Seaweeds as sources of antioxidant phytomedicines: [review]

Por causa del estrés oxidativo a que se encuentran sometidas constantemente, las algas marinas son organismos que presentan en su composición química cantidades apreciables de compuestos antioxidantes, entre los que se encuentran compuestos lipofílicos como ácidos grasos insaturados, clorofila y carotenos; compuestos hidrofílicos como polifenoles y vitamina C; y polisacáridos...

As seaweeds are constantly exposed to oxidative stress conditions, significant quantities of antioxidant compounds are found in their chemical composition such as lypophilic compounds like unsaturated fatty acids, chlorophyll and carotenoids; hydrophilic compounds like polyphenols and vitamin C, and polysaccharides...