G-protein coupled receptor kinase 5 mediates lipopolysaccharide-induced NFκB activation in primary macrophages and modulates inflammation in vivo in mice

G-protein coupled receptor kinase-5 (GRK5) is a serine/threonine kinase discovered for its role in the regulation of G-protein coupled receptor signaling. Recent studies have shown that GRK5 is also an important regulator of signaling pathways stimulated by non-GPCRs. This study was undertaken to determine the physiological role of GRK5 in Toll-like receptor-4-induced inflammatory signaling pathways in vivo and in vitro. Using mice genetically deficient in GRK5 (GRK5(-/-) ) we demonstrate here that GRK5 is an important positive regulator of lipopolysaccharide (LPS, a TLR4 agonist)-induced inflammatory cytokine and chemokine production in vivo. Consistent with this role, LPS-induced neutrophil infiltration in the lungs (assessed by myeloperoxidase activity) was markedly attenuated in the GRK5(-/-) mice compared to the GRK5(+/+) mice. Similar to the in vivo studies, primary macrophages from GRK5(-/-) mice showed attenuated cytokine production in response to LPS. Our results also identify TLR4-induced NFκB pathway in macrophages to be selectively regulated by GRK5. LPS-induced IκBα phosphorylation, NFκB p65 nuclear translocation, and NFκB binding were markedly attenuated in GRK5(-/-) macrophages. Together, our findings demonstrate that GRK5 is a positive regulator of TLR4-induced IκBα-NFκB pathway as well as a key modulator of LPS-induced inflammatory response.     

Niclosamide-loaded nanoparticles disrupt Candida biofilms and protect mice from mucosal candidiasis

Candida albicans biofilms are a complex multilayer community of cells that are resistant to almost all classes of antifungal drugs. The bottommost layers of biofilms experience nutrient limitation where C. albicans cells are required to respire. We previously reported that a protein Ndu1 is essential for Candida mitochondrial respiration; loss of NDU1 causes inability of C. albicans to grow on alternative carbon sources and triggers early biofilm detachment. Here, we screened a repurposed library of FDA-approved small molecule inhibitors to identify those that prevent NDU1-associated functions. We identified an antihelminthic drug, Niclosamide (NCL), which not only prevented growth on acetate, C. albicans hyphenation and early biofilm growth, but also completely disengaged fully grown biofilms of drug-resistant C. albicans and Candida auris from their growth surface. To overcome the suboptimal solubility and permeability of NCL that is well known to affect its in vivo efficacy, we developed NCL-encapsulated Eudragit EPO (an FDA-approved polymer) nanoparticles (NCL-EPO-NPs) with high niclosamide loading, which also provided long-term stability. The developed NCL-EPO-NPs completely penetrated mature biofilms and attained anti-biofilm activity at low microgram concentrations. NCL-EPO-NPs induced ROS activity in C. albicans and drastically reduced oxygen consumption rate in the fungus, similar to that seen in an NDU1 mutant. NCL-EPO-NPs also significantly abrogated mucocutaneous candidiasis by fluconazole-resistant strains of C. albicans, in mice models of oropharyngeal and vulvovaginal candidiasis. To our knowledge, this is the first study that targets biofilm detachment as a target to get rid of drug-resistant Candida biofilms and uses NPs of an FDA-approved nontoxic drug to improve biofilm penetrability and microbial killing.

This study shows that encapsulation of the antiparasitic drug Niclosamide in nanoparticles can enhance its pharmaco-availability, prevent the growth and filamentation of Candida, and enhance biofilm penetrability and detachment, both in vitro and in two mouse models of mucosal candidiasis.     

Atypical ubiquitylation - the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages

Ubiquitylation is one of the most abundant and versatile post-translational modifications (PTMs) in cells. Its versatility arises from the ability of ubiquitin to form eight structurally and functionally distinct polymers, in which ubiquitin moieties are linked via one of seven Lys residues or the amino terminus. Whereas the roles of Lys48- and Lys63-linked polyubiquitin in protein degradation and cellular signalling are well characterized, the functions of the remaining six 'atypical' ubiquitin chain types (linked via Lys6, Lys11, Lys27, Lys29, Lys33 and Met1) are less well defined. Recent developments provide insights into the mechanisms of ubiquitin chain assembly, recognition and hydrolysis and allow detailed analysis of the functions of atypical ubiquitin chains. The importance of Lys11 linkages and Met1 linkages in cell cycle regulation and nuclear factor-κB activation, respectively, highlight that the different ubiquitin chain types should be considered as functionally independent PTMs.     

Microbiology of Lonar Lake and other soda lakes

Soda lakes are saline and alkaline ecosystems that are believed to have existed throughout the geological record of Earth. They are widely distributed across the globe, but are highly abundant in terrestrial biomes such as deserts and steppes and in geologically interesting regions such as the East African Rift valley. The unusual geochemistry of these lakes supports the growth of an impressive array of microorganisms that are of ecological and economic importance. Haloalkaliphilic Bacteria and Archaea belonging to all major trophic groups have been described from many soda lakes, including lakes with exceptionally high levels of heavy metals. Lonar Lake is a soda lake that is centered at an unusual meteorite impact structure in the Deccan basalts in India and its key physicochemical and microbiological characteristics are highlighted in this article. The occurrence of diverse functional groups of microbes, such as methanogens, methanotrophs, phototrophs, denitrifiers, sulfur oxidizers, sulfate reducers and syntrophs in soda lakes, suggests that these habitats harbor complex microbial food webs that (a) interconnect various biological cycles via redox coupling and (b) impact on the production and consumption of greenhouse gases. Soda lake microorganisms harbor several biotechnologically relevant enzymes and biomolecules (for example, cellulases, amylases, ectoine) and there is the need to augment bioprospecting efforts in soda lake environments with new integrated approaches. Importantly, some saline and alkaline lake ecosystems around the world need to be protected from anthropogenic pressures that threaten their long-term existence.     

Multiplication of Eucalyptus citriodora (L) Hook Through Shoot Bud Induction on the Internodal Portions of Mature Tree Explants

Nodal explants of Eucalyptus citriodora responded better for high frequency bud break and fast growth in liquid agitated cultures with 4.4 μM benzyladenine (BA) and 5.37 μM α-naphthalene acetic acid (NAA) rather than on semisolid medium. On transfer to MS medium with 1.11 μM BA and 5.37 μM NAA the sprouted axillary buds showed further elongation growth alongwith a slight callus and formation of globular structures on the internodal regions. The anatomy of these globular structures revealed that they are shoot buds. These buds elongated into shoots on MS medium with low levels of BA.     

Comparison of 16S rRNA gene sequences of genus <Emphasis Type="Italic">Methanobrevibacter</Emphasis>

Background  

The phylogeny of the genus Methanobrevibacter was established almost 25 years ago on the basis of the similarities of the 16S rRNA oligonucleotide catalogs. Since then, many 16S rRNA gene sequences of newly isolated strains or clones representing the genus Methanobrevibacter have been deposited. We tried to reorganize the 16S rRNA gene sequences of this genus and revise the taxonomic affiliation of the isolates and clones representing the genus Methanobrevibacter.     

Further Optimization of Detritylation in Solid-Phase Oligodeoxyribonucleotide Synthesis

Various conditions for optimum detritylation (i.e., the removal of 5′-O-trityl protecting groups) during solid-phase synthesis of oligodeoxyribonucleotides were investigated. Di- and tri-chloroacetic acids of variable concentrations were used to study the removal of the 4,4′-dimethoxytrityl (DMTr) group. It was found that the DMTr group could be completely removed under much milder acidic conditions than what are currently used for automated solid-phase synthesis. The 2,7-dimethylpixyl (DMPx) is proposed as an alternative and more readily removable group for the protection of the 5′-OH functions both in solid- and solution-phase synthesis. The improved detritylation conditions are expected to minimize the waste and offer a protocol for incorporation of acid sensitive building-blocks in oligonucleotides.     

Frankixalus,a New Rhacophorid Genus of Tree Hole Breeding Frogs with Oophagous Tadpoles

Despite renewed interest in the biogeography and evolutionary history of Old World tree frogs (Rhacophoridae), this family still includes enigmatic frogs with ambiguous phylogenetic placement. During fieldwork in four northeastern states of India, we discovered several populations of tree hole breeding frogs with oophagous tadpoles. We used molecular data, consisting of two nuclear and three mitochondrial gene fragments for all known rhacophorid genera, to investigate the phylogenetic position of these new frogs. Our analyses identify a previously overlooked, yet distinct evolutionary lineage of frogs that warrants recognition as a new genus and is here described as Frankixalus gen. nov. This genus, which contains the enigmatic ‘Polypedates’ jerdonii described by Günther in 1876, forms the sister group of a clade containing Kurixalus, Pseudophilautus, Raorchestes, Mercurana and Beddomixalus. The distinctiveness of this evolutionary lineage is also corroborated by the external morphology of adults and tadpoles, adult osteology, breeding ecology, and life history features.