Identification of NAD interacting residues in proteins

Background: Small molecular cofactors or ligands play a crucial role in the proper functioning of cells. Accurate annotation of their target proteins and binding sites is required for the complete understanding of reaction mechanisms. Nicotinamide adenine dinucleotide (NAD+ or NAD) is one of the most commonly used organic cofactors in living cells, which plays a critical role in cellular metabolism, storage and regulatory processes. In the past, several NAD binding proteins (NADBP) have been reported in the literature, which are responsible for a wide-range of activities in the cell. Attempts have been made to derive a rule for the binding of NAD+ to its target proteins. However, so far an efficient model could not be derived due to the time consuming process of structure determination, and limitations of similarity based approaches. Thus a sequence and non-similarity based method is needed to characterize the NAD binding sites to help in the annotation. In this study attempts have been made to predict NAD binding proteins and their interacting residues (NIRs) from amino acid sequence using bioinformatics tools. Results: We extracted 1556 proteins chains from 555 NAD binding proteins whose structure is available in Protein Data Bank. Then we removed all redundant protein chains and finally obtained 195 non-redundant NAD binding protein chains, where no two chains have more than 40% sequence identity. In this study all models were developed and evaluated using five-fold cross validation technique on the above dataset of 195 NAD binding proteins. While certain type of residues are preferred (e.g. Gly, Tyr, Thr, His) in NAD interaction, residues like Ala, Glu, Leu, Lys are not preferred. A support vector machine (SVM) based method has been developed using various window lengths of amino acid sequence for predicting NAD interacting residues and obtained maximum Matthew's correlation coefficient (MCC) 0.47 with accuracy 74.13% at window length 17. We also developed a SVM based method using evolutionary information in the form of position specific scoring matrix (PSSM) and obtained maximum MCC 0.75 with accuracy 87.25%. Conclusion: For the first time a sequence-based method has been developed for the prediction of NAD binding proteins and their interacting residues, in the absence of any prior structural information. The present model will aid in the understanding of NAD+ dependent mechanisms of action in the cell. To provide service to the scientific community, we have developed a user-friendly web server, which is available from URL http://www.imtech.res.in/raghava/nadbinder/

Cholera- and Anthrax-Like Toxins Are among Several New ADP-Ribosyltransferases

Chelt, a cholera-like toxin from Vibrio cholerae, and Certhrax, an anthrax-like toxin from Bacillus cereus, are among six new bacterial protein toxins we identified and characterized using in silico and cell-based techniques. We also uncovered medically relevant toxins from Mycobacterium avium and Enterococcus faecalis. We found agriculturally relevant toxins in Photorhabdus luminescens and Vibrio splendidus. These toxins belong to the ADP-ribosyltransferase family that has conserved structure despite low sequence identity. Therefore, our search for new toxins combined fold recognition with rules for filtering sequences – including a primary sequence pattern – to reduce reliance on sequence identity and identify toxins using structure. We used computers to build models and analyzed each new toxin to understand features including: structure, secretion, cell entry, activation, NAD+ substrate binding, intracellular target binding and the reaction mechanism. We confirmed activity using a yeast growth test. In this era where an expanding protein structure library complements abundant protein sequence data – and we need high-throughput validation – our approach provides insight into the newest toxin ADP-ribosyltransferases

FTIR Spectroscopy of microalgae as a novel tool for biodiversity studies, species identification, and for the assessment of water quality.

Fourier transform infrared (FTIR) spectrometry was used to study the spectral features of 12 eukaryotic and two prokaryotic species of microalgae. The algae were cultured in liquid media containing either NO3- or NH4+ as the sole N-source; for the NH4+ treatment, the algae were subjected to shortterm (24 h) or long-term (1 month) incubations; for the hypersaline species, cells were also grown in the presence of 2 M NaCl. Over 500 spectra, acquired from at least three distinct cultures for each species, in each growth regime, were subjected to hierarchical cluster analysis (HCA) and were successfully separated according to their taxonomy, showing that the overall spectra were characteristic of each species and that this technique could be fruitfully employed to separate microalgal species living in a similar condition (as would be the case for a natural assemblage). In addition, in most cases, it was possible to differentiate between algae subjected to different growth treatments although belonging to the same species. We also demonstrated that it is possible to accurately identify species and determine the nutritional status of their environment of origin (e.g., N-source), provided that suitable FTIR spectral libraries are available. This study aims to provide the basis for the development of rapid, easy, and inexpensive methods for the evaluation of biodiversity in natural phytoplankton samples and to monitor the water quality of natural environments

Quantitative analysis of Hepatitis B Virus DNA by real-time amplification.

Diagnostic assays allowing the quantification of hepatitis B virus (HBV) DNA over a wide range of concentrations are important for monitoring patients during antiviral therapy. The aim of this study was to develop a new real-time method for HBV DNA quantification. Primers and probe were selected in a highly conserved region of the HBV S gene, and a plasmid containing the pre-S/S region was used as a standard. Linear quantification of the standard was obtained between 10 and 10(9) copies/reaction, with high correlation between ayw and adw genomes (P<0.001). HBV DNA was detected in serial dilutions of a high-titer serum sample with linear results until 2.4 x 10(3) copies/ml. One hundred eight serum samples positive for hepatitis B surface antigen were tested in both the real-time assay and the Digene Hybrid Capture assay (Digene, USA). HBV DNA could be detected by both assays in 70 samples, with significant correlation of results (P<0.001). Results for 38 samples were below the sensitivity limit of the Digene assay, but they could be quantified by the real time polymerase chain reaction assay. These results show that real-time polymerase chain reaction allows sensitive, rapid and linear quantification of HBV DNA in serum

New crystalline form of cabazitaxel, process for the preparation and pharmaceutical compositions thereof

The present invention relates to a new crystalline anhydrous form of Cabazitaxel, to a process for its preparation and to pharmaceutical compositions thereof

Spectroscopic classification of 14 different microalgae species: a first steps towards spectroscopic measurement of phytoplankton diversity

Background: Phytoplankton species composition and abundance may reflect the nutritional status of aquatic environments. Species-specific spectral signatures and spectral features dependent on the nutritional status of cells can be derived by Fourier Transform InfraRed (FTIR) spectroscopy. Aims: This study uses analytical methods to objectively and automatically identify microalga species and their environmen- tal conditions based on FTIR data. Methods: Fourteen species and two growth regimes were used for this study. Each of these species–nutrient combinations constituted a ‘class’. The assignment of a sample to a class was based on the similarity of its FTIR spectrum to known class members, derived from correlation coefficients. To assess to what extent classes could be discriminated and how reliable this discrimination was, each spectrum was correlated with all others. The unknown was assigned to all classes with whose members it had a high similarity. Results: Correct classifications were achieved in >90% cases. Subsequently, an unambiguous classification was performed by assigning an unknown to the class with which it had the highest similarity. Use of derivative spectra for baseline shifts removal increased the success percentage to 94. Conclusions: Chemometric methods applied to FTIR spectra of microalgae allow to discriminate species and nutritional conditions to which the cells had been exposed

Real-time quantitation of hepatitis B virus (HBV) DNA in tumorous and surrounding tissue from patients with hepatocellular carcinoma

Few data are available on the levels of HBV DNA in liver tissue of patients with hepatocellular carcinoma. In this study, HBV DNA was quantitated by a TaqMan real-time PCR method and results were normalised to an endogenous reference gene. The assay could detect reproducibly viral sequences from over 107 to less than 50 copies/mg of liver DNA. The HBV DNA content in liver samples from 11 HBsAg-positive patients (median: 105 copies/mg of DNA) was significantly higher (P<0.001) compared to the viral DNA concentration detected in liver samples from 15 of 25 HBsAg-negative patients (median: 2.6102 copies/mg). A liver DNA amount 1 HBV DNA copy per cell was detected in half of tissue samples from HBsAg-positive patients, and in none from HBsAg-negative ones. Liver tissue HBVDNAcontent was significantly higher in anti- HCV-negative than in anti-HCV-positive cases (P<0.001). These results show that the quantitation of liver HBV DNA by real-time PCR can be useful to understand HBV state in hepatocellular carcinoma and viral interplay in patients with multiple viral infections

Crystalline solvate forms of cabazitaxel

The present invention relates to new crystalline solvate forms of Cabazitaxel and to processes for the preparation thereof

A CRYSTALLINE ANHYDROUS FORM OF CABAZITAXEL, PROCESS FOR THE PREPARATION AND PHARMACEUTICAL COMPOSITIONS THEREOF

The present invention refers to an amorphous form of a thiocolchicine derivative, IDN 5404, to a process for producing it and to pharmaceutical compositions thereof. The amorphous form is characterised by the XRPD pattern, DSC profile and/or TG/DTA profile