RP-HPLC separation of interconvertible rotamers of a 5-tetrahydroisoquinolin-6-yl-pyridin-3-yl acetic acid derivative and confirmation by VT NMR and DFT study.

Due to emergence of drug resistance and drug tolerability, there is urgent need for discovery of new chemical entity for the treatment of HIV infection. As a part of in-house small molecule drug discovery program for HIV infection, sodium-2-(tert-butoxy)- 2-(5-(2-(2-chloro-6-methylbenzyl)- 1,2,3,4-tetrahydroisoquinolin-6-yl)- 4-(4,4-dimethylpiperidin-1-yl)- 2,6-dimethylpyridin-3-yl) acetate (SCMTDDA) was prepared as an intermediate for the synthesis of an API, designed as a HIV-1 integrase inhibitor. Initially, the final compound was isolated as a mixture of rotamers. In the current study, we have developed a simple and efficient achiral, reversed phase (RP) HPLC method to separate the interconvertible rotamers of SCMTDDA. The effect of several parameters, including stationary phase, buffer, modifiers and column temperature, were optimized for the chromatographic separation and it was observed that best separation was achieved on a SunFire C18 column using TFA/acetonitrile (ACN) - methanol (MeOH) (1:1 v/v) as the mobile phase at 10 0C. The chromatographic observations were complemented with variable-temperature NMR and energy barrier calculations using density functional theory (DFT).

Complete anatomic reduction and monosegmental fusion for lumbar spondylolisthesis of Grade II and higher: use of the minimally invasive "rocking" technique.

OBJECTIVE Different surgical approaches have been described for treatment of spondylolisthesis, including in situ fusions, reductions of various degrees, and inclusion of healthy adjacent segments into the fusion construct. To the authors' knowledge, there are only sparse reports describing consistent complete reduction and monosegmental transforaminal lumbar interbody fusion for spondylolisthesis using a minimally invasive technique. The authors assess the efficacy of this technique in the reduction of local deformity and correction of overall sagittal profile in single-level spondylolisthesis. METHODS This cohort study consists of a total of 36 consecutive patients treated over a period of 6 years. Patients with varying grades of lumbar spondylolisthesis (29 Meyerding Grade II and 7 Meyerding Grade III) were treated with operative reduction via minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) in which the "rocking" technique was used. The clinical outcomes were measured using the visual analog scale (VAS) for pain and the Revised Oswestry Disability Index (ODI) for low-back pain/dysfunction. Meyerding grade, pelvic incidence (PI), lumbar lordosis (LL), disc space angle (DSA), pelvic tilt (PT), and sacral slope (SS) were assessed to measure the radiological outcomes. These were reviewed for each patient for a minimum of 2 years. RESULTS At most recent follow-up, 94% of patients were pain free. There were 2 patients (6%) who had moderate pain (which corresponded to higher-grade of listhesis), but all showed an improvement in pain scores (p < 0.05). The mean VAS score improved from 6.5 (SD 1.5) preoperatively to 1.6 (SD 1.3) and the mean ODI score improved from 53.7 (SD 13.1) preoperatively to 22.5 (SD 15.5) at 2-year follow-up. All radiological parameters improved following surgery. Most significant improvement was noted for LL, DSA, and SS. Both LL and SS were found to decrease, while DSA increased postoperatively. PI remained relatively unchanged, while PT showed a mild increase, which was not significant. Good fusion was achieved with implants in situ at 2-year follow-up. A 100% complete reduction of all grades of spondylolisthesis was achieved. The overall sagittal profile improved dramatically. No major perioperative complications were encountered. CONCLUSIONS Minimally invasive monosegmental TLIF for spondylolisthesis reduction using this rocking technique is effective in the treatment of various grades of spondylolisthesis. Consistent complete reduction of the slippage as well as excellent correction of overall sagittal profile can be achieved, and the need for including healthy adjacent segments in the fusion construct can be avoided.

Molecular regulation of santalol biosynthesis in Santalum album L.

Santalum album L. commonly known as East-Indian sandal or chandan is a hemiparasitic tree of family santalaceae. Santalol is a bioprospecting molecule present in sandalwood and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. Santalol is a sesquiterpene synthesized through mevalonate or non-mevalonate pathways. First step of santalol biosynthesis involves head to tail condensation of isopentenyl pyrophosphate (IPP) with its allylic co-substrate dimethyl allyl pyrophosphate (DMAPP) to produce geranyl pyrophosphate (GPP; C10 - a monoterpene). GPP upon one additional condensation with IPP produces farnesyl pyrophosphate (FPP; C15 - an open chain sesquiterpene). Both the reactions are catalyzed by farnesyl diphosphate synthase (FDS). Santalene synthase (SS), a terpene cyclase catalyzes cyclization of open ring FPP into a mixture of cyclic sesquiterpenes such as α-santalene, epi-ß-santalene, ß-santalene and exo bergamotene, the main constituents of sandal oil. The objective of the present work was to generate a comprehensive knowledge on the genes involved in santalol production and study their molecular regulation. To achieve this, sequences encoding farnesyl diphosphate synthase and santalene synthase were isolated from sandalwood using suppression subtraction hybridization and 2D gel electrophoresis technology. Functional characterization of both the genes was done through enzyme assays and tissue-specific expression of both the genes was studied. To our knowledge, this is the first report on studies on molecular regulation, and tissue-specific expression of the genes involved in santalol biosynthesis.

Expression and lytic efficacy assessment of the Staphylococcus aureus phage SA4 lysin gene.

Treatment of bovine mastitis caused by Staphylococcus (S.) aureus is becoming very difficult due to the emergence of multidrug-resistant strains. Hence, the search for novel therapeutic alternatives has become of great importance. Consequently, bacteriophages and their endolysins have been identified as potential therapeutic alternatives to antibiotic therapy against S. aureus. In the present study, the gene encoding lysin (LysSA4) in S. aureus phage SA4 was cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clone revealed a single 802-bp open reading frame encoding a artial protein with a calculated mass of 30 kDa. Results of this analysis also indicated that the LysSA4 sequence shared a high homology with endolysin of the GH15 phage and other reported phages. The LysSA4 gene of the SA4 phage was subsequently expressed in Escherichia coli. Recombinant LysSA4 induced the lysis of host bacteria in a spot inoculation test, indicating that the protein was expressed and functionally active. Furthermore, recombinant lysin was found to have lytic activity, albeit a low level, against mastitogenic Staphylococcus isolates of bovine origin. Data from the current study can be used to develop therapeutic tools for treating diseases caused by drug-resistant S. aureus strains.