Isolation and characterization of the first Xylanolytic hyperthermophilic euryarchaeon <i>Thermococcus</i> sp strain 2319x1 and its unusual multidomain glycosidase

Enzymes from (hyper)thermophiles Thermozymes offer a great potential for biotechnological applications. Thermophilic adaptation does not only provide stability towards high temperature but is also often accompanied by a higher resistance to other harsh physicochemical conditions, which are also frequently employed in industrial processes, such as the presence of e.g. denaturing agents as well as low or high pH of the medium. In order to find new thermostable, xylan degrading hydrolases with potential for biotechnological application we used an in situ enrichment strategy incubating Hungate tubes with xylan as the energy substrate in a hot vent located in the tidal zone of Kunashir Island (Kuril archipelago). Using this approach a hyperthermophilic euryarchaeon, designated Thermococcus sp. strain 2319x1, growing on xylan as sole energy and carbon source was isolated. The organism grows optimally at 85°C and pH 7.0 on a variety of natural polysaccharides including xylan, carboxymethyl cellulose (CMC), amorphous cellulose (AMC), xyloglucan, and chitin. The protein fraction extracted from the cells surface with Twin 80 exhibited endoxylanase, endoglucanase and xyloglucanase activities. The genome of Thermococcus sp. strain 2319x1 was sequenced and assembled into one circular chromosome. Within the newly sequenced genome, a gene, encoding a novel type of glycosidase (143 kDa) with a unique five-domain structure, was identified. It consists of three glycoside hydrolase (GH) domains and two carbohydrate-binding modules (CBM) with the domain order GH5-12-12-CBM2-2 (N- to C-terminal direction). The full length protein, as well as truncated versions, were heterologously expressed in Escherichia coli and their activity was analyzed. The full length multidomain glycosidase (MDG) was able to hydrolyze various polysaccharides, with the highest activity for barley β-glucan (β-1,3/1,4-glucoside), followed by that for carboxymethyl cellulose (β-1,4-glucoside), cellooligosaccharides and galactomannan. The results reported here indicate that the modular MDG structure with multiple glycosidase and carbohydrate-binding domains not only extends the substrate spectrum, but also seems to allow the degradation of partially soluble and insoluble polymers in a processive manner. This report highlights the great potential in a multi-pronged approach consisting of a combined in situ enrichment, (comparative) genomics, and biochemistry strategy for the screening for novel enzymes of biotechnological relevance

A mouse model of visual perceptual learning reveals alterations in neuronal coding and dendritic spine density in the visual cortex

Visual perceptual learning (VPL) can improve spatial vision in normally sighted and visually impaired individuals. Although previous studies of humans and large animals have explored the neural basis of VPL, elucidation of the underlying cellular and molecular mechanisms remains a challenge. Owing to the advantages of molecular genetic and optogenetic manipulations, the mouse is a promising model for providing a mechanistic understanding of VPL. Here, we thoroughly evaluated the effects and properties of VPL on spatial vision in C57BL/6J mice using a two-alternative, forced-choice visual water task. Briefly, the mice underwent prolonged training at near the individual threshold of contrast or spatial frequency (SF) for pattern discrimination or visual detection for 35 consecutive days. Following training, the contrast-threshold trained mice showed an 87% improvement in contrast sensitivity (CS) and a 55% gain in visual acuity (VA). Similarly, the SF-threshold trained mice exhibited comparable and long-lasting improvements in VA and significant gains in CS over a wide range of SFs. Furthermore, learning largely transferred across eyes and stimulus orientations. Interestingly, learning could transfer from a pattern discrimination task to a visual detection task, but not vice versa. We validated that this VPL fully restored VA in adult amblyopic mice and old mice. Taken together, these data indicate that mice, as a species, exhibit reliable VPL. Intrinsic signal optical imaging revealed that mice with perceptual training had higher cut-off SFs in primary visual cortex (V1) than those without perceptual training. Moreover, perceptual training induced an increase in the dendritic spine density in layer 2/3 pyramidal neurons of V1. These results indicated functional and structural alterations in V1 during VPL. Overall, our VPL mouse model will provide a platform for investigating the neurobiological basis of VPL

Heroin Inhibits HIV-Restriction miRNAs and Enhances HIV Infection of Macrophages

Although opioids have been extensively studied for their impact on the immune system, limited information is available about the specific actions of opioids on intracellular antiviral innate immunity against HIV infection. Thus, we investigated whether heroin, one of the most abused drugs, inhibits the expression of intracellular HIV restriction microRNA (miRNA) and facilitates HIV replication in macrophages. Heroin treatment of macrophages enhanced HIV replication, which was associated with the downregulation of several HIV restriction miRNAs. These heroin-mediated actions on the miRNAs and HIV could be antagonized by naltrexone, an opioid receptor antagonist. Furthermore, the in vitro negative impact of heroin on HIV-associated miRNAs was confirmed by the in vivo observation that heroin addicts had significantly lower levels of macrophage-derived HIV restriction miRNAs than those in the control subjects. These in vitro and in vivo findings indicate that heroin use compromises intracellular anti-HIV innate immunity, providing a favorable microenvironment for HIV survival in the target cells