Ultrafast interfacial solvation dynamics in specific protein DNA recognition

An overwhelming number of structural and functional studies on specific protein–DNA complexes reveal the existence of water molecules at the interaction interface. What role does the interfacial water molecules play in determining the specificity of association is thus a critical question. Herein, we have explored the dynamical role of minor groove water molecules and DNA side chain flexibility in lambda repressor–operator DNA interaction using well-characterized DNA minor groove binder dye, Hoechst 33258. The most striking finding of our studies reveals that the solvation time scale corresponding to the minor groove water molecules (∼50 ps) and DNA side chain flexibility (∼10 ns) remain unaltered even in protein–DNA complex in comparison to unbound operator DNA. The temperature dependent study further reveals the slower exchange of minor grove water molecules with bulk water in DNA–protein complex in comparison to the unbound DNA. Detailed structural studies including circular dichroism (CD) and Förster resonance energy transfer (FRET) have also been performed to elucidate the interaction between protein and DNA.     

Ursolic acid derivatives from Bangladeshi medicinal plant, Saurauja roxburghii: Isolation and cytotoxic activity against A431 and C6 glioma cell lines

Ursene-type pentacyclic triterpenes, including the ursolic acid, corosolic acid, and a new ursene-type pentacyclic triterpene, 7,24-dihydroxy ursolic acid, were isolated from the methanolic extract of the leaves of the Bangladeshi medicinal plant, Saurauja roxburghii, a higher plant indigenous to South East Asia and some part of North America. They were tested for the cytotoxicity against C6 rat glioma and A431 human epidermoid carcinoma cell lines. Although they have the same ursene-type pentacyclic triterpene core structure, the position and numbers of hydroxyl groups on the terpene structure significantly affected the activity and the selectivity to the cell lines.     

Potency, efficacy and durability of DNA/DNA, DNA/protein and protein/protein based vaccination using gp63 against Leishmania donovani in BALB/c mice

Background

Visceral leishmaniasis (VL) caused by an intracellular protozoan parasite Leishmania, is fatal in the absence of treatment. At present there are no effective vaccines against any form of leishmaniasis. Here, we evaluate the potency, efficacy and durability of DNA/DNA, DNA-prime/Protein-boost, and Protein/Protein based vaccination against VL in a susceptible murine model.

Methods and Findings

To compare the potency, efficacy, and durability of DNA, protein and heterologous prime-boost (HPB) vaccination against Leishmania donovani, major surface glycoprotein gp63 was cloned into mammalian expression vector pcDNA3.1 for DNA based vaccines. We demonstrated that gp63 DNA based vaccination induced immune responses and conferred protection against challenge infection. However, vaccination with HPB approach showed comparatively enhanced cellular and humoral responses than other regimens and elicited early mixed Th1/Th2 responses before infection. Moreover, challenge with parasites induced polarized Th1 responses with enhanced IFN-γ, IL-12, nitric oxide, IgG2a/IgG1 ratio and reduced IL-4 and IL-10 responses compared to other vaccination strategies. Although, vaccination with gp63 DNA either alone or mixed with CpG- ODN or heterologously prime-boosting with CpG- ODN showed comparable levels of protection at short-term protection study, DNA-prime/Protein-boost in presence of CpG significantly reduced hepatic and splenic parasite load by 10⁷ fold and 10¹⁰ fold respectively, in long-term study. The extent of protection, obtained in this study has till now not been achieved in long-term protection through HPB approach in susceptible BALB/c model against VL. Interestingly, the HPB regimen also showed marked reduction in the footpad swelling of BALB/c mice against Leishmania major infection.

Conclusion/Significance

HPB approach based on gp63 in association with CpG, resulted in robust cellular and humoral responses correlating with durable protection against L. donovani challenge till twelve weeks post-vaccination. These results emphasize the potential of DNA-prime/Protein-boost vaccination over DNA/DNA and Protein/Protein based vaccination in maintaining long-term immunity against intracellular pathogen like Leishmania.     

High Capacity and High‐Rate NASICON‐Na3.75V1.25Mn0.75(PO4)3 Cathode for Na‐Ion Batteries via Modulating Electronic and Crystal Structures

Sodium superionic conductor (NASICON) cathodes are attractive for Na‐ion battery applications as they exhibit both high structural stability and high sodium ion mobility. Herein, a comprehensive study is presented on the structural and electrochemical properties of the NASICON‐Na_3+yV_2?yMn_y(PO₄)₃ (0 ≤ y ≤ 1) series. A phase miscibility gap is observed at y = 0.5, defining two solid solution domains with low and high Mn contents. Although, members of each of these domains Na_3.25V_1.75Mn_0.25(PO₄)₃ and Na_3.75V_1.25Mn_0.75(PO₄)₃ reversibly exchange sodium ions with high structural integrity, the activity of the Mn³⁺/Mn²⁺ redox couple is found to be absent and present in the former and latter candidate, respectively. Galvanostatic cycling and rate studies reveal higher capacity and rate capability for the Na_3.75V_1.25Mn_0.75(PO₄)₃ cathode (100 and 89 mA h g^?1 at 1C and 5C rate, respectively) in the Na_3+yV_2?yMn_y(PO₄)₃ series. Such a remarkable performance is attributed to optimum bottleneck size (≈5 Ų) and modulated V‐ and Mn‐redox centers as deduced from Rietveld analysis and DFT calculations, respectively. This study shows how important it is to manipulate electronic and crystal structures to achieve high‐performance NASICON cathodes.     

Refining trophic dynamics through multi‐factor Bayesian mixing models: A case study of subterranean beetles

Food web dynamics are vital in shaping the functional ecology of ecosystems. However, trophic ecology is still in its infancy in groundwater ecosystems due to the cryptic nature of these environments. To unravel trophic interactions between subterranean biota, we applied an interdisciplinary Bayesian mixing model design (multi‐factor BMM) based on the integration of faunal C and N bulk tissue stable isotope data (δ¹³C and δ¹⁵N) with radiocarbon data (Δ¹⁴C), and prior information from metagenomic analyses. We further compared outcomes from multi‐factor BMM with a conventional isotope double proxy mixing model (SIA BMM), triple proxy (δ¹³C, δ¹⁵N, and Δ¹⁴C, multi‐proxy BMM), and double proxy combined with DNA prior information (SIA + DNA BMM) designs. Three species of subterranean beetles (Paroster macrosturtensis, Paroster mesosturtensis, and Paroster microsturtensis) and their main prey items Chiltoniidae amphipods (AM1: Scutachiltonia axfordi and AM2: Yilgarniella sturtensis), cyclopoids and harpacticoids from a calcrete in Western Australia were targeted. Diet estimations from stable isotope only models (SIA BMM) indicated homogeneous patterns with modest preferences for amphipods as prey items. Multi‐proxy BMM suggested increased—and species‐specific—predatory pressures on amphipods coupled with high rates of scavenging/predation on sister species. SIA + DNA BMM showed marked preferences for amphipods AM1 and AM2, and reduced interspecific scavenging/predation on Paroster species. Multi‐factorial BMM revealed the most precise estimations (lower overall SD and very marginal beetles' interspecific interactions), indicating consistent preferences for amphipods AM1 in all the beetles' diets. Incorporation of genetic priors allowed crucial refining of the feeding preferences, while integration of more expensive radiocarbon data as a third proxy (when combined with genetic data) produced more precise outcomes but close dietary reconstruction to that from SIA + DNA BMM. Further multidisciplinary modeling from other groundwater environments will help elucidate the potential behind these designs and bring light to the feeding ecology of one the most vital ecosystems worldwide.     

Structure-based Comparative Analysis and Prediction of N-linked Glycosylation Sites in Evolutionarily Distant Eukaryotes

The asparagine-X-serine/threonine (NXS/T) motif, where X is any amino acid except proline, is the consensus motif for N-linked glycosylation. Significant numbers of high-resolution crystal structures of glycosylated proteins allow us to carry out structural analysis of the N-linked glycosylation sites (NGS). Our analysis shows that there is enough structural information from diverse glycoproteins to allow the development of rules which can be used to predict NGS. A Python-based tool was developed to investigate asparagines implicated in N-glycosylation in five species: Homo sapiens, Mus musculus, Drosophila melanogaster, Arabidopsis thaliana and Saccharomyces cerevisiae. Our analysis shows that 78% of all asparagines of NXS/T motif involved in N-glycosylation are localized in the loop/turn conformation in the human proteome. Similar distribution was revealed for all the other species examined. Comparative analysis of the occurrence of NXS/T motifs not known to be glycosylated and their reverse sequence (S/TXN) shows a similar distribution across the secondary structural elements, indicating that the NXS/T motif in itself is not biologically relevant. Based on our analysis, we have defined rules to determine NGS. Using machine learning methods based on these rules we can predict with 93% accuracy if a particular site will be glycosylated. If structural information is not available the tool uses structural prediction results resulting in 74% accuracy. The tool was used to identify glycosylation sites in 108 human proteins with structures and 2247 proteins without structures that have acquired NXS/T site/s due to non-synonymous variation. The tool, Structure Feature Analysis Tool (SFAT), is freely available to the public at http://hive.biochemistry.gwu.edu/tools/sfat.     

Alterations of <Emphasis Type="Italic">ATM</Emphasis> and <Emphasis Type="Italic">CADM1</Emphasis> in chromosomal 11q22.3–23.2 region are associated with the development of invasive cervical carcinoma

To understand the importance of chr11q22.3–23.2 region in the development of cervical cancer, we have studied the genetic and epigenetic alterations of the candidate genes ATM, PPP2R1B, SDHD and CADM1 in cervical intraepithelial neoplasia (CIN) and cervical carcinoma (CACX) samples. Our study revealed low expression and high alterations (methylation/deletion) (55–59%) of ATM and CADM1 genes along with poor patient outcome. The alterations of ATM and CADM1 are associated with the progression of tumor from CIN to Stage I/II, thus implying their role in early invasiveness. The two genes, PPP2R1B and SDHD, lying in between ATM and CADM1, have low frequency of alterations, and majority of the alterations are in CACX samples, indicating that their alterations might be associated with disease progression. Expressions (mRNA/protein) of the genes showed concordance with their molecular alterations. Significant co-alteration of ATM and CADM1 points to their synergic action for the development of CACX. Mutation is, however, a rare phenomenon for inactivation of ATM. Association between the alteration of ATM and CHEK1 and poor survival of the patients having co-alterations of ATM and CHEK1 points to the DNA damage response pathway disruption in development of CACX. Thus, our data suggest that inactivation of ATM–CHEK1-associated DNA damage response pathway and CADM1-associated signaling network might have an important role in the development of CACX.     

Human urothelial micronucleus assay to assess genotoxic recovery by reduction of arsenic in drinking water: a cohort study in West Bengal, India

Chronic exposure to arsenic through drinking water affects nearly 26 million individuals in West Bengal, India. Cytogenetic biomarkers like urothelial micronucleus (MN) are extensively used to monitor arsenic exposed population. In 2004–2005, 145 arsenic exposed individuals and 60 unexposed controls were surveyed of which 128 exposed individuals and 54 unexposed controls could be followed up in 2010–2011. In 2004–2005, the extent of arsenic content in the drinking water was 348.23 ± 102.67 μg/L, which was significantly lowered to 5.60 ± 10.83 μg/L in 2010–2011. Comparing the data obtained between 2004–2005 and 2010–2011, there was a significant decline in the MN frequency, when assayed in 2010–2011 compared to 2004–2005. Hence, we infer that urothelial MN can be utilized as a good biomarker in detecting remedial effects from toxicity of the low dose of arsenic through drinking water.     

Processing of deoxyuridine mismatches and abasic sites by human immunodeficiency virus type-1 integrase.

We have examined the activities of HIV-1 integrase on substrates containing mismatches, composed of deoxyuridine at different positions in either the processed or nonprocessed strand of viral DNA, within and near the conserved CA dinucleotide of the U5 end of the HIV-1 LTR. Substitution in the processed strand of either the C or A of the CA dinucleotide or of the G 5' to the CA reduced strand transfer six-, three- and seven-fold respectively. 3'-processing was also reduced by substitution at the GC but not at the A. Substitution in the nonprocessed strand of the G nucleotide at the processing site abolished strand transfer while substitution of the T had no effect. DNA binding of HIV-1 integrase was not affected by deoxyuridine substitutions. Deoxyuridine substitution outside the trinucleotide remained compatible with enzyme activity. Enzymatically generated abasic sites were created at each mismatch to determine the effect of a missing base on integrase activity. Consistent with the deoxyuridine mismatch observations, 3'-processing and strand transfer were abolished when the abasic site was substituted for either of the nucleotides of the GCA trinucleotide. Integrase was, however, able to tolerate mismatches within this trinucleotide during the disintegration reaction. Taken together, these results suggest that base-mismatched or base-deleted substrates, which can be created by the proofreading-deficient HIV-1 RT, can be tolerated by HIV-1 integrase when located outside of the GCA trinucleotide at the U5 end of the LTR.