Endophytic fungal communities in woody perennials of three tropical forest types of the Western Ghats,southern India

Fungal endophytes of tropical trees are expected to be exceptionally species rich as a consequence of high tree diversity in the tropics and the purported host restriction among the endophytes. Based on this premise, endophytes have been regarded as a focal group for estimating fungal numbers because their possible hyperdiverse nature would reflect significantly global fungal diversity. We present our consolidated ten-year work on 75 dicotyledonous tree hosts belonging to 33 families and growing in three different types of tropical forests of the NBR in the Western Ghats, southern India. We conclude that endophyte diversity in these forests is limited due to loose host affiliations among endophytes. Some endophytes have a wide host range and colonize taxonomically disparate hosts suggesting adaptations in them to counter a variety of defense chemicals in their hosts. Furthermore, such polyphagous endophytes dominate the endophyte assemblages of different tree hosts. Individual leaves may be densely colonized but only by a few endophyte species. It appears that the environment (the type of forest in this case) has a larger role in determining the endophyte assemblage of a plant host than the taxonomy of the host plant. Thus, different tropical plant communities have to be studied for their endophyte diversity to test the generalization that endophytes are hyperdiverse in the tropics, estimate their true species richness, and use them as a predictor group for more accurate assessment of global fungal diversity.     

Magnetic resonance imaging of organic contrast agents in mice: capturing the whole-body redox landscape

Nitroxides are a class of stable free radicals that have several biomedical applications including radioprotection and noninvasive assessment of tissue redox status. For both of these applications, it is necessary to understand the in vivo biodistribution and reduction of nitroxides. In this study, magnetic resonance imaging was used to compare tissue accumulation (concentration) and reduction of two commonly studied nitroxides: the piperidine nitroxide Tempol and the pyrrolidine nitroxide 3-CP. It was found that 3-CP was reduced 3 to 11 times slower (depending on the tissue) than Tempol in vivo and that maximum tissue concentration varies substantially between tissues (0.6-7.2mM). For a given tissue, the maximum concentration usually did not vary between the two nitroxides. Furthermore, using electron paramagnetic resonance spectroscopy, we showed that the nitroxide reduction rate depends only weakly on cellular pO(2) in the oxygen range expected in vivo. These observations, taken with the marked variation in nitroxide reduction rates observed between tissues, suggest that tissue pO(2) is not a major determinant of the nitroxide reduction rate in vivo. For the purpose of redox imaging, 3-CP was shown to be an optimal choice based on the achievable concentrations and bioreduction observed in vivo.     

Development of anti-EGF receptor peptidomimetics (AERP) as tumor imaging agent

EGFR is over-expressed in several solid tumors including breast, prostate, pancreas, and lung cancers and is correlated to the metastasic potential of the tumor. Anti-EGFR receptor-binding peptidomimetics (AERP) were examined to assess the small molecule’s potential use as tumor-specific imaging agents. The aim of this work was to design and characterize the binding specificity of the radiolabeled peptidomimetics to EGFR over-expressing cell lysate and to A431 xenograft tumors. Our newly designed peptidomimetic, AERP, was conjugated to DTPA and labeled with ^99mTc. The in vivo tumor accumulation of [^99mTc] DTPA-AERP-2 was 1.6 ± 0.1 %ID/g and tumor to muscle ratio was 5.5. Our studies suggest that this novel peptidomimetic, AERP-2, warrants further development as an EGFR specific tumor-imaging agent.     

Insights from the analysis of conserved motifs and permitted amino acid exchanges in the human, the fly and the worm GPCR clusters

G-protein coupled receptors (GPCRs) belong to biologically important and functionally diverse and largest super family of membrane proteins. GPCRs retain a characteristic membrane topology of seven alpha helices with three intracellular, three extracellular loops and flanking N' and C' terminal residues. Subtle differences do exist in the helix boundaries (TM-domain), loop lengths, sequence features such as conserved motifs, and substituting amino acid patterns and their physiochemical properties amongst these sequences (clusters) at intra-genomic and inter-genomic level (please re-phrase into 2 statements for clarity). In the current study, we employ prediction of helix boundaries and scores derived from amino acid substitution exchange matrices to identify the conserved amino acid residues (motifs) as consensus in aligned set of homologous GPCR sequences. Co-clustered GPCRs from human and other genomes, organized as 32 clusters, were employed to study the amino acid conservation patterns and species-specific or cluster-specific motifs. Critical analysis on sequence composition and properties provide clues to connect functional relevance within and across genome for vast practical applications such as design of mutations and understanding of disease-causing genetic abnormalities.     

Latency antigen α-crystallin based vaccination imparts a robust protection against TB by modulating the dynamics of pulmonary cytokines

Background

Efficient control of tuberculosis (TB) requires development of strategies that can enhance efficacy of the existing vaccine Mycobacterium bovis Bacille Calmette Guerin (BCG). To date only a few studies have explored the potential of latency-associated antigens to augment the immunogenicity of BCG.

Methods/Principal Findings

We evaluated the protective efficacy of a heterologous prime boost approach based on recombinant BCG and DNA vaccines targeting α-crystallin, a prominent latency antigen. We show that “rBCG prime - DNA boost” strategy (R/D) confers a markedly superior protection along with reduced pathology in comparison to BCG vaccination in guinea pigs (565 fold and 45 fold reduced CFU in lungs and spleen, respectively, in comparison to BCG vaccination). In addition, R/D regimen also confers enhanced protection in mice. Our results in guinea pig model show a distinct association of enhanced protection with an increased level of interleukin (IL)12 and a simultaneous increase in immuno-regulatory cytokines such as transforming growth factor (TGF)β and IL10 in lungs. The T cell effector functions, which could not be measured in guinea pigs due to technical limitations, were characterized in mice by multi-parameter flow cytometry. We show that R/D regimen elicits a heightened multi-functional CD4 Th1 cell response leading to enhanced protection.

Conclusions/Significance

These results clearly indicate the superiority of α-crystallin based R/D regimen over BCG. Our observations from guinea pig studies indicate a crucial role of IL12, IL10 and TGFβ in vaccine-induced protection. Further, characterization of T cell responses in mice demonstrates that protection against TB is predictable by the frequency of CD4 T cells simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and IL2. We anticipate that this study will not only contribute toward the development of a superior alternative to BCG, but will also stimulate designing of TB vaccines based on latency antigens.     

BRAF mutations in cutaneous melanoma are independently associated with age, anatomic site of the primary tumor, and the degree of solar elastosis at the primary tumor site

Oncogenic BRAF mutations are more frequent in cutaneous melanoma occurring at sites with little or moderate sun-induced damage than at sites with severe cumulative solar ultraviolet (UV) damage. We studied cutaneous melanomas from geographic regions with different levels of ambient UV radiation to delineate the relative effects of cumulative UV damage, age, and anatomic site on the frequency of BRAF mutations. We show that BRAF-mutated melanomas occur in a younger age group on skin without marked solar elastosis and less frequently affect the head and neck area, compared to melanomas without BRAF mutations. The findings indicate that BRAF-mutated melanomas arise early in life at low cumulative UV doses, whereas melanomas without BRAF mutations require accumulation of high UV doses over time. The effect of anatomic site on the mutation spectrum further suggests regional differences among cutaneous melanocytes.     

A booster vaccine expressing a latency-associated antigen augments BCG induced immunity and confers enhanced protection against tuberculosis

Background

In spite of a consistent protection against tuberculosis (TB) in children, Mycobacterium bovis Bacille Calmette-Guerin (BCG) fails to provide adequate protection against the disease in adults as well as against reactivation of latent infections or exogenous reinfections. It has been speculated that failure to generate adequate memory T cell response, elicitation of inadequate immune response against latency-associated antigens and inability to impart long-term immunity against M. tuberculosis infections are some of the key factors responsible for the limited efficiency of BCG in controlling TB.

Methods/Principal Findings

In this study, we evaluated the ability of a DNA vaccine expressing α-crystallin- a key latency antigen of M. tuberculosis to boost the BCG induced immunity. ‘BCG prime – DNA boost’ regimen (B/D) confers robust protection in guinea pigs along with a reduced pathology in comparison to BCG vaccination (1.37 log₁₀ and 1.96 log₁₀ fewer bacilli in lungs and spleen, respectively; p<0.01). In addition, B/D regimen also confers enhanced protection in mice. Further, we show that B/D immunization in mice results in a heightened frequency of PPD and antigen specific multi-functional CD4 T cells (3⁺) simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and interleukin (IL)2.

Conclusions/Significance

These results clearly indicate the superiority of α-crystallin based B/D regimen over BCG. Our study, also demonstrates that protection against TB is predictable by an increased frequency of 3⁺ Th1 cells with superior effector functions. We anticipate that this study would significantly contribute towards the development of superior booster vaccines for BCG vaccinated individuals. In addition, this regimen can also be expected to reduce the risk of developing active TB due to reactivation of latent infection.     

Metabolomics in early Alzheimer's disease: identification of altered plasma sphingolipidome using shotgun lipidomics

Background

The development of plasma biomarkers could facilitate early detection, risk assessment and therapeutic monitoring in Alzheimer''s disease (AD). Alterations in ceramides and sphingomyelins have been postulated to play a role in amyloidogensis and inflammatory stress related neuronal apoptosis; however few studies have conducted a comprehensive analysis of the sphingolipidome in AD plasma using analytical platforms with accuracy, sensitivity and reproducibility.

Methods and Findings

We prospectively analyzed plasma from 26 AD patients (mean MMSE 21) and 26 cognitively normal controls in a non-targeted approach using multi-dimensional mass spectrometry-based shotgun lipidomics [1], [2] to determine the levels of over 800 molecular species of lipids. These data were then correlated with diagnosis, apolipoprotein E4 genotype and cognitive performance. Plasma levels of species of sphingolipids were significantly altered in AD. Of the 33 sphingomyelin species tested, 8 molecular species, particularly those containing long aliphatic chains such as 22 and 24 carbon atoms, were significantly lower (p<0.05) in AD compared to controls. Levels of 2 ceramide species (N16:0 and N21:0) were significantly higher in AD (p<0.05) with a similar, but weaker, trend for 5 other species. Ratios of ceramide to sphingomyelin species containing identical fatty acyl chains differed significantly between AD patients and controls. MMSE scores were correlated with altered mass levels of both N20:2 SM and OH-N25:0 ceramides (p<0.004) though lipid abnormalities were observed in mild and moderate AD. Within AD subjects, there were also genotype specific differences.

Conclusions

In this prospective study, we used a sensitive multimodality platform to identify and characterize an essentially uniform but opposite pattern of disruption in sphingomyelin and ceramide mass levels in AD plasma. Given the role of brain sphingolipids in neuronal function, our findings provide new insights into the AD sphingolipidome and the potential use of metabolomic signatures as peripheral biomarkers.