Potent inhibition of microRNA in vivo without degradation

Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2′-fluoro/2′-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies.     

Discovery of amino-1,4-oxazines as potent BACE-1 inhibitors

New amino-1,4-oxazine derived BACE-1 inhibitors were explored and various synthetic routes developed. The binding mode of the inhibitors was elucidated by co-crystallization of 4 with BACE-1 and X-ray analysis. Subsequent optimization led to inhibitors with low double digit nanomolar activity in a biochemical and single digit nanomolar potency in a cellular assays. To assess the inhibitors for their permeation properties and potential to cross the blood-brain-barrier a MDR1-MDCK cell model was successfully applied. Compound 8a confirmed the in vitro results by dose-dependently reducing Aβ levels in mice in an acute treatment regimen.     

Elimination of inhibitory synapses is a major component of adult ocular dominance plasticity

During development, cortical plasticity is associated with the rearrangement of excitatory connections. While these connections become more stable with age, plasticity can still be induced in the adult cortex. Here we provide evidence that structural plasticity of?inhibitory synapses onto pyramidal neurons is?a major component of plasticity in the adult neocortex. In?vivo two-photon imaging was used to monitor the formation and elimination of fluorescently labeled inhibitory structures on pyramidal neurons. We find that ocular dominance plasticity in the adult visual cortex is associated with rapid inhibitory synapse loss, especially of those present on dendritic spines. This occurs not only with monocular deprivation but also with subsequent restoration of binocular vision. We propose that in the adult visual cortex the experience-induced loss of inhibition may effectively strengthen specific visual inputs with limited need for rearranging the excitatory circuitry.     

Antiviral compounds obtained from microalgae commonly used as carotenoid sources

Pressurized liquid extraction (PLE), an environmentally friendly technique, has been used to obtain antiviral compounds from microalgae commonly used as carotenoid sources: Haematococcus pluvialis and Dunaliella salina. The antiviral properties of PLE extracts (hexane, ethanol and water) were evaluated against herpes simplex virus type 1 (HSV-1) at different stages during viral infection. Pretreatment of Vero cells with 75?μg?mL^?1 of H. pluvialis ethanol extract inhibited virus infection by approximately 85%, whereas the same concentration of water and hexane extracts reduced the virus infectivity 75% and 50%, respectively. D. salina extracts were less effective than H. pluvialis extracts and presented a different behaviour since water and ethanol extracts produced a similar virus inhibition (65%). Moreover, H. pluvialis ethanol extract was also the most effective against HSV-1 intracellular replication. The antiviral activity of water PLE extracts was found to correlate with polysaccharides since the polysaccharide-rich fraction isolated from these extracts showed higher antiviral activity than the original water extracts. A gas chromatography-mass spectrometry (GC-MS) characterization of the H. pluvialis ethanol extract showed the antiviral activity of this extract could be partially related with the presence of short-chain fatty acids, although other compounds could be involved in this activity; meanwhile, in the case of D. salina ethanol extract other compounds seemed to be implied, such as: β-ionone, neophytadiene, phytol, palmitic acid and α-linolenic acid. The results demonstrate the use of PLE allows obtaining antiviral compounds from microalgae used as carotenoids sources, which gives the microalgae biomass an added value.     

Prevalence and follow-up of occult HCV infection in an italian population free of clinically detectable infectious liver disease

Background

Occult hepatitis C virus infection (OCI) is a recently described phenomenon characterized by undetectable levels of HCV-RNA in serum/plasma by current laboratory assays, with identifiable levels in peripheral blood mononuclear cells (PBMCs) and/or liver tissue by molecular tests with enhanced sensitivity. Previous results from our group showed an OCI prevalence of 3.3% in a population unselected for hepatic disease. The present study aimed to evaluate OCI prevalence in a larger cohort of infectious liver disease-free (ILDF) subjects. Clinical follow-up of OCI subjects was performed to investigate the natural history of the infection.

Methods and Findings

439 subjects referred to a Turin Blood Bank for phlebotomy therapy were recruited. They included 314 ILDF subjects, 40 HCV-positive subjects and 85 HBV-positive subjects, of whom 7 were active HBV carriers. Six subjects (4/314 ILDF subjects [1.27%] and 2/7 active HBV carriers [28%]) were positive for HCV-RNA in PBMCs, but negative for serological and virological markers of HCV, indicating OCI. HCV genotypes were determined in the PBMCs of 3/6 OCI subjects two had type 1b; the other had type 2a/2c. OCI subjects were followed up for at least 2 years. After 12 months only one OCI persisted, showing a low HCV viral load (3.73×10¹ UI/ml). By the end of follow-up all OCI subjects were negative for HCV. No seroconversion, alteration of liver enzyme levels, or reduction of liver synthesis occurred during follow-up.

Conclusions

This study demonstrated the existence of OCI in ILDF subjects, and suggested a high OCI prevalence among active HBV carriers. Follow-up suggested that OCI could be transient, with a trend toward the decrease of HCV viral load to levels undetectable by conventional methods after 12–18 months. Confirmation studies with a longer follow-up period are needed for identification of the OCI clearance or recurrence rates, and to characterize the viruses involved.     

Mitochondrial aquaporin-8 in renal proximal tubule cells: evidence for a role in the response to metabolic acidosis

Mitochondrial ammonia synthesis in proximal tubules and its urinary excretion are key components of the renal response to maintain acid-base balance during metabolic acidosis. Since aquaporin-8 (AQP8) facilitates transport of ammonia and is localized in inner mitochondrial membrane (IMM) of renal proximal cells, we hypothesized that AQP8-facilitated mitochondrial ammonia transport in these cells plays a role in the response to acidosis. We evaluated whether mitochondrial AQP8 (mtAQP8) knockdown by RNA interference is able to impair ammonia excretion in the human renal proximal tubule cell line, HK-2. By RT-PCR and immunoblotting, we found that AQP8 is expressed in these cells and is localized in IMM. HK-2 cells were transfected with short-interfering RNA targeting human AQP8. After 48 h, the levels of mtAQP8 protein decreased by 53% (P < 0.05). mtAQP8 knockdown decreased the rate of ammonia released into culture medium in cells grown at pH 7.4 (-31%, P < 0.05) as well as in cells exposed to acid (-90%, P < 0.05). We also evaluated mtAQP8 protein expression in HK-2 cells exposed to acidic medium. After 48 h, upregulation of mtAQP8 (+74%, P < 0.05) was observed, together with higher ammonia excretion rate (+73%, P < 0.05). In vivo studies in NH(4)Cl-loaded rats showed that mtAQP8 protein expression was also upregulated after 7 days of acidosis in renal cortex (+51%, P < 0.05). These data suggest that mtAQP8 plays an important role in the adaptive response of proximal tubule to acidosis possibly facilitating mitochondrial ammonia transport.     

Integrating correlation between traits improves spatial predictions of plant functional composition

Functional trait composition is increasingly recognized as key to better understand and predict community responses to environmental gradients. Predictive approaches traditionally model the weighted mean trait values of communities (CWMs) as a function of environmental gradients. However, most approaches treat traits as independent regardless of known tradeoffs between them, which could lead to spurious predictions. To address this issue, we suggest jointly modeling a suit of functional traits along environmental gradients while accounting for relationships between traits. We use generalized additive mixed effect models to predict the functional composition of alpine grasslands in the Guisane Valley (France). We demonstrate that, compared to traditional approaches, joint trait models explain considerable amounts of variation in CWMs, yield less uncertainty in trait CWM predictions and provide more realistic spatial projections when extrapolating to novel environmental conditions. Modeling traits and their co‐variation jointly is an alternative and superior approach to predicting traits independently. Additionally, compared to a ‘predict first, assemble later’ approach that estimates trait CWMs post hoc based on stacked species distribution models, our ‘assemble first, predict later’ approach directly models trait‐responses along environmental gradients, and does not require data and models on species’ distributions, but only mean functional trait values per community plot. This highlights the great potential of joint trait modeling approaches in large‐scale mapping applications, such as spatial projections of the functional composition of vegetation and associated ecosystem services as a response to contemporary global change.