Molecular analysis of the bacterial microbiome in the forestomach fluid from the dromedary camel (Camelus dromedarius)

Rumen microorganisms play an important role in ruminant digestion and absorption of nutrients and have great potential applications in the field of rumen adjusting, food fermentation and biomass utilization etc. In order to investigate the composition of microorganisms in the rumen of camel (Camelus dromedarius), this study delves in the microbial diversity by culture-independent approach. It includes comparison of rumen samples investigated in the present study to other currently available metagenomes to reveal potential differences in rumen microbial systems. Pyrosequencing based metagenomics was applied to analyze phylogenetic and metabolic profiles by MG-RAST, a web based tool. Pyrosequencing of camel rumen sample yielded 8,979,755 nucleotides assembled to 41,905 sequence reads with an average read length of 214 nucleotides. Taxonomic analysis of metagenomic reads indicated Bacteroidetes (55.5 %), Firmicutes (22.7 %) and Proteobacteria (9.2 %) phyla as predominant camel rumen taxa. At a finer phylogenetic resolution, Bacteroides species dominated the camel rumen metagenome. Functional analysis revealed that clustering-based subsystem and carbohydrate metabolism were the most abundant SEED subsystem representing 17 and 13 % of camel metagenome, respectively. A high taxonomic and functional similarity of camel rumen was found with the cow metagenome which is not surprising given the fact that both are mammalian herbivores with similar digestive tract structures and functions. Combined pyrosequencing approach and subsystems-based annotations available in the SEED database allowed us access to understand the metabolic potential of these microbiomes. Altogether, these data suggest that agricultural and animal husbandry practices can impose significant selective pressures on the rumen microbiota regardless of rumen type. The present study provides a baseline for understanding the complexity of camel rumen microbial ecology while also highlighting striking similarities and differences when compared to other animal gastrointestinal environments.     

Ab initio structure of human seminal plasma prostatic inhibin gives significant insight into its biological functions

Human seminal plasma prostatic inhibin (HSPI) is a protein isolated from the human prostate gland. Despite its profound biomedical and biotechnological importance, the 3D structure of this protein of 94 amino acids remains undeciphered. The difficulties in extracting it in pure form and crystallizing it have restrained the determination of its structure experimentally. The homology-based computational methods are also not applicable, as HSPI lacks sufficient sequence homology with known structures in the protein data banks. We have predicted the structure of HSPI by a knowledge-based method using nonparametric multivariate statistical techniques. Stereochemical and other standard validation tests confirm this to be a well-refined structure. The biophysical properties exhibited by this structure are in good agreement with the NMR experimental observations. Docking and other computational studies on this structure provide significant explanation and insight into its binding activities and related biological and immunogenic functions and offer new directions for its potential applications.     

In vivo administration of D609 leads to protection of subsequently isolated gerbil brain mitochondria subjected to in vitro oxidative stress induced by amyloid beta-peptide and other oxidative stressors: relevance to Alzheimer's disease and other oxidative stress-related neurodegenerative disorders

Tricyclodecan-9-yl-xanthogenate (D609) has in vivo and in vitro antioxidant properties. D609 mimics glutathione (GSH) and has a free thiol group, which upon oxidation forms a disulfide. The resulting dixanthate is a substrate for glutathione reductase, regenerating D609. Recent studies have also shown that D609 protects brain in vivo and neuronal cultures in vitro against the potential Alzheimer's disease (AD) causative factor, Abeta(1-42)-induced oxidative stress and cytotoxicity. Mitochondria are important organelles with both pro- and antiapoptotic factor proteins. The present study was undertaken to test the hypothesis that intraperitoneal injection of D609 would provide neuroprotection against free radical-induced, mitochondria-mediated apoptosis in vitro. Brain mitochondria were isolated from gerbils 1 h post injection intraperitoneally (ip) with D609 and subsequently treated in vitro with the oxidants Fe(2+)/H(2)O(2) (hydroxyl free radicals), 2,2-azobis-(2-amidinopropane) dihydrochloride (AAPH, alkoxyl and peroxyl free radicals), and AD-relevant amyloid beta-peptide 1-42 [Abeta(1-42)]. Brain mitochondria isolated from the gerbils previously injected ip with D609 and subjected to these oxidative stress inducers, in vitro, showed significant reduction in levels of protein carbonyls, protein-bound hydroxynonenal [a lipid peroxidation product], 3-nitrotyrosine, and cytochrome c release compared to oxidant-treated brain mitochondria isolated from saline-injected gerbils. D609 treatment significantly maintains the GSH/GSSG ratio in oxidant-treated mitochondria. Increased activity of glutathione S-transferase, glutathione peroxidase, and glutathione reductase in brain isolated from D609-injected gerbils is consistent with the notion that D609 acts like GSH. These antiapoptotic findings are discussed with reference to the potential use of this brain-accessible glutathione mimetic in the treatment of oxidative stress-related neurodegenerative disorders, including AD.     

CDK5 interacts with Slo and affects its surface expression and kinetics through direct phosphorylation

Large-conductance calcium-activated potassium (BK) channels are ubiquitous and play an important role in a number of diseases. In hair cells of the ear, they play a critical role in electrical tuning, a mechanism of frequency discrimination. These channels show variable kinetics and expression along the tonotopic axis. Although the molecular underpinnings to its function in hair cells are poorly understood, it is established that BK channels consist of a pore-forming α-subunit (Slo) and a number of accessory subunits. Here we identify CDK5, a member of the cyclin-dependent kinase family, as an interacting partner of Slo. We show CDK5 to be present in hair cells and expressed in high concentrations in the cuticular plate and in the circumferential zone. In human embryonic kidney cells, we show that CDK5 inhibits surface expression of Slo by direct phosphorylation of Slo. Similarly, we note that CDK5 affects Slo voltage activation and deactivation kinetics, by a direct phosphorylation of T847. Taken together with its increasing expression along the tonotopic axis, these data suggest that CDK5 likely plays a critical role in electrical tuning and surface expression of Slo in hair cells.     

Antitumor activity of Noscapine in combination with Doxorubicin in triple negative breast cancer

Background

The aim of this study was to investigate the anticancer activity and mechanism of action of Noscapine alone and in combination with Doxorubicin against triple negative breast cancer (TNBC).

Methods

TNBC cells were pretreated with Noscapine or Doxorubicin or combination and combination index values were calculated using isobolographic method. Apoptosis was assessed by TUNEL staining. Female athymic Nu/nu mice were xenografted with MDA-MB-231 cells and the efficacy of Noscapine, Doxorubicin and combination was determined. Protein expression, immunohistochemical staining were evaluated in harvested tumor tissues.

Results

Noscapine inhibited growth of MDA-MB-231 and MDA-MB-468 cells with the IC₅₀ values of 36.16±3.76 and 42.7±4.3 µM respectively. The CI values (<0.59) were suggestive of strong synergistic interaction between Noscapine and Doxorubicin and combination treatment showed significant increase in apoptotic cells. Noscapine showed dose dependent reduction in the tumor volumes at a dose of 150–550 mg/kg/day compared to controls. Noscapine (300 mg/kg), Doxorubicin (1.5 mg/kg) and combination treatment reduced tumor volume by 39.4±5.8, 34.2±5.7 and 82.9±4.5 percent respectively and showed decreased expression of NF-KB pathway proteins, VEGF, cell survival, and increased expression of apoptotic and growth inhibitory proteins compared to single-agent treatment and control groups.

Conclusions

Noscapine potentiated the anticancer activity of Doxorubicin in a synergistic manner against TNBC tumors via inactivation of NF-KB and anti-angiogenic pathways while stimulating apoptosis. These findings suggest potential benefit for use of oral Noscapine and Doxorubicin combination therapy for treatment of more aggressive TNBC.     

Influence of medium composition on callus induction and camptothecin(s) accumulation in Nothapodytes foetida

Camptothecin(s) production was examined in callus cultures derived from cotyledons of Nothapodytes foetida (Weigh) Sleumer. The calluses were grown on various combinations of Murashige and Skoog's basal media supplemented with auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a-napthalene acetic acid (NAA) and indole-3-acetic acid (IAA) with 6-benzyl aminopurine (BA)/kinetin in different concentrations. The presence of camptothecin (CPT) and 9-methoxycamptothecin (9-OMeCPT) were analyzed by HPLC in relation to the media composition. Hyper production of CPT(1.306% on dry wt. basis) was observed with a combination of 2,4-D with BA and 2,4,5-T and NAA in 1-month-old callus.     

Coalescent Method in Conjunction with Niche Modeling Reveals Cryptic Diversity among Centipedes in the Western Ghats of South India

Background

There has been growing interest in integrative taxonomy that uses data from multiple disciplines for species delimitation. Typically, in such studies, monophyly is taken as a proxy for taxonomic distinctiveness and these units are treated as potential species. However, monophyly could arise due to stochastic processes. Thus here, we have employed a recently developed tool based on coalescent approach to ascertain the taxonomic distinctiveness of various monophyletic units. Subsequently, the species status of these taxonomic units was further tested using corroborative evidence from morphology and ecology. This inter-disciplinary approach was implemented on endemic centipedes of the genus Digitipes (Attems 1930) from the Western Ghats (WG) biodiversity hotspot of India. The species of the genus Digitipes are morphologically conserved, despite their ancient late Cretaceous origin.

Principal Findings

Our coalescent analysis based on mitochondrial dataset indicated the presence of nine putative species. The integrative approach, which includes nuclear, morphology, and climate datasets supported distinctiveness of eight putative species, of which three represent described species and five were new species. Among the five new species, three were morphologically cryptic species, emphasizing the effectiveness of this approach in discovering cryptic diversity in less explored areas of the tropics like the WG. In addition, species pairs showed variable divergence along the molecular, morphological and climate axes.

Conclusions

A multidisciplinary approach illustrated here is successful in discovering cryptic diversity with an indication that the current estimates of invertebrate species richness for the WG might have been underestimated. Additionally, the importance of measuring multiple secondary properties of species while defining species boundaries was highlighted given variable divergence of each species pair across the disciplines.