Structural and biophysical analysis of cytochrome P450 2C9*14 and *27 variants in complex with losartan.

The human cytochrome P450 (CYP) 1, 2 and 3 families of enzymes are responsible for the biotransformation of a majority of the currently available pharmaceutical drugs. The highly polymorphic CYP2C9 predominantly metabolizes many drugs including anticoagulant S-warfarin, anti-hypertensive losartan, anti-diabetic tolbutamide, analgesic ibuprofen, etc. There are >80 single nucleotide changes identified in CYP2C9, many of which significantly alter the clearance of important drugs. Here we report the structural and biophysical analysis of two polymorphic variants, CYP2C9*14 (Arg125His) and CYP2C9*27 (Arg150Leu) complexed with losartan. The X-ray crystal structures of the CYP2C9*14 and *27 illustrate the binding of two losartan molecules, one in the active site near heme and another on the periphery. Both losartan molecules are bound in an identical conformation to that observed in the previously solved CYP2C9 wild-type complex, however, the number of losartan differs from the wild-type structure, which showed binding of three molecules. Additionally, isothermal titration calorimetry experiments reveal a lower binding affinity of losartan with *14 and *27 variants when compared to the wild-type. Overall, the results provide new insights into the effects of these genetic polymorphisms and suggests a possible mechanism contributing to reduced metabolic activity in patients carrying these alleles.

Biophysical interaction between lanthanum chloride and (CG)n or (GC)n repeats: A reversible B-to-Z DNA transition.

The transition from right-handed to left-handed DNA is not only acts as the controlling factor for switching gene expression but also has equal importance in designing nanomechanical devices. The (CG)n and (GC)n repeat sequences are well known model molecules to study B-Z transition in the presence of higher concentration of monovalent cations. In this communication, we report a cyclic transition in (CG)6 DNA using millimolar concentration of trivalent lanthanide salt LaCl3. The controlled and reversible transition was seen in (CG)12, and (GC)12 DNA employing CD spectroscopy. While LaCl3 failed to induce B-Z transition in shorter oligonucleotides such as (CG)3 and (GC)3, a smooth B-Z transition was recorded for (CG)6, (CG)12 and (GC)12 sequences. Interestingly, the phenomenon was reversible (Z-B transition) with addition of EDTA. Particularly, two rounds of cyclic transition (B-Z-B-Z-B) have been noticed in (CG)6 DNA in presence of LaCl3 and EDTA which strongly suggest that B-Z transition is reversible in short repeat sequences. Thermal melting and annealing behaviour of B-DNA are reversible while the thermal melting of LaCl3-induced Z-DNA is irreversible which suggest a stronger binding of LaCl3 to the phosphate backbone of Z-DNA. This was further supported by isothermal titration calorimetric study. Molecular dynamics (MD) simulation indicates that the mode of binding of La3+ (of LaCl3) with d(CG)8.d(CG)8 is through the minor groove, wherein, 3 out of 11 La3+ bridge the anionic oxygens of the complementary strands. Such a tight coordination of La3+ with the anionic oxygens at the minor groove surface may be the reason for the experimentally observed irreversibility of LaCl3-induced Z-DNA seen in longer DNA fragments. Thus, these results indicate LaCl3 can easily be adopted as an inducer of left-handed DNA in other short oligonucleotides sequences to facilitate the understanding of the molecular mechanism of B-Z transition.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays.

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

Safety and efficacy of total intraluminal topical anaesthesia for ureteroscopy. Is success worth the pain?

BACKGROUND: Ureteroscopic management of ureteral calculi using topical anaesthesia has been described. Most studies topically anaesthetized the urethra or urinary bladder by instilling 2% of plain lignocaine. In addition to the success rate, the pain perception in these studies was reported subjectively using non-standard criteria. Topical anaesthesia of the ipsilateral ureter and the pelvicalyceal system (PCS), in addition to urethra and urinary bladder, and use of alkalinised lignocaine, for enhanced effect, has not been reported for ureteroscopy. Using these conceptual alterations, we tested the safety and efficacy of performing ureteroscopy and laser lithotripsy under our technique of total intraluminal topical anaesthesia (TILTA). METHODS: One hundred sixty-eight patients underwent ureteroscopic laser lithotripsy by topical instillation of alkalinised lignocaine into the urinary bladder and ipsilateral ureter and PCS. Self-assessed pain perception and changes in vital parameters were objectively scored at various times. The success rate, reasons for failure, maximum pain scores, complications, and willingness to undergo repeat procedure were recorded. RESULTS: The success rate was 91.3% with a mean duration of 14.7 minutes Double J Stent (DJS) was placed in 38.1%. 61.3% patients did not need any postoperative injectable analgesic, with 11.3% requiring more than a single dose. The intraoperative changes in vitals initially and at the height of pain were not statistically significant (p>0.05). Self-assessed median pain scores between intravenous access placement and ureteroscopy were significantly different (p<0.05). CONCLUSIONS: Ureteroscopy, effectively performed under TILTA, is safe with a high success rate. The acceptable pain tolerance and the shortened convalescence in addition makes the procedure's success worth the pain.

Immunocytochemistry using Liquid-based Cytology: A Tool in Hormone Receptor Analysis of Breast Cancer.

CONTEXT: Efficacy of immunocytochemistry (ICC) in determining molecular biomarkers like estrogen receptor (ER), progesterone receptors (PRs), and human epidermal growth factor receptor-2 (HER2). AIMS: To evaluate biomarkers using ICC in breast cancer as per American Society of Clinical Oncology/College of American Pathology (ASCO/CAP) guidelines. SETTINGS AND DESIGN: The study was conducted over a period of 2 years from September 2012 to August 2014 and is the first such study in eastern India. MATERIALS AND METHODS: Fine needle aspiration cytology (FNAC) was done for suspected cases of breast cancers and slides were prepared using ThinPrep (TP) technology of liquid-based cytology (LBC) for ICC and corresponding biopsy specimens were processed as formalin fixed paraffin embedded (FFPE) sections for comparison. Both the LBC slides and tissue sections were subjected to immunostaining for ER, PR, and HER2. ICC was evaluated by Allred Scoring and IHC by Quick Scoring. STATISTICAL ANALYSIS USED: Statistical analysis done by Wilconxon Signed rank test on the SPSS program, Chicago, Illinois, USA. The results of ICC and IHC were compared by evaluation of sensitivity, specificity, kappa-value (k-value), positive predictive value (PPV), and negative predictive value (NPV). RESULTS: The comparison of ICC with immunohistochemistry (IHC), ER, and PR showed very good correspondence rate, sensitivity, specificity, NPV, PPV, and agreement with k-value; whereas for HER2 the results were only good. CONCLUSION: ICC using LBC can be a useful tool in assessing biomarkers in advanced cases of breast cancer where surgery is not possible or cases where ASCO/CAP guidelines for management are not followed.