MsrA Efflux Pump Inhibitory Activity of Piper cubeba L.f. and its Phytoconstituents against Staphylococcus aureus RN4220

MsrA, an efflux pump belonging to ATP‐binding cassette (ABC) transporter family that conferred resistance to macrolides, was detected in Staphylococcus aureus strains. Herein, we report the isolation of phytoconstituents from Piper cubeba fruit methanol extract and investigated their efflux pump inhibitory potential against S. aureus MsrA pump. Four isolated compounds, viz. pellitorine, sesamin, piperic acid and tetrahydropiperine studied in combination with erythromycin in S. aureus RN4220, exhibited 2–8‐fold reduction in minimum inhibitory concentration (MIC) of erythromycin. Pellitorine and sesamin decreased MIC of erythromycin by 8‐fold. The real‐time fluorometry‐based efflux and accumulation studies of ethidium bromide (EtBr) on S. aureus RN4220 in the presence of these compounds showed reduced efflux and enhanced uptake, thus indicating inhibition of the efflux pump. Pellitorine showed significant post‐antibiotic effect of erythromycin. The results revealed that the primary mechanism of action of these compounds involves steady ATP production impairment.     

Probiotics L. acidophilus and B. clausii Modulate Gut Microbiota in Th1- and Th2-Biased Mice to Ameliorate Salmonella Typhimurium-Induced Diarrhea

Gut microbiota play important role in maintaining health. Probiotics are believed to augment it further. We aimed at comparing effects of probiotics, Lactobacillus acidophilus (LA) and Bacillus clausii (BC) (a) on the gut microbiota abundance and diversity and (b) their contributions to control intestinal dysbiosis and inflammation in Th1- and Th2-biased mice following Salmonella infection. We report how could gut microbiota and the differential immune bias (Th1 or Th2) of the host regulate host responses when challenged with Salmonella typhimurium in the presence and absence of either of the probiotics. LA was found to be effective in ameliorating the microbial dysbiosis and inflammation caused by Salmonella infection, in Th1 (C57BL/6) and Th2 (BALB/c)-biased mouse. BC was able to ameliorate Salmonella-induced dysbiosis and inflammation in Th2 but not in Th1-biased mouse. These results may support probiotics LA as a treatment option in the case of Salmonella infection.

     

Function and regulation of Alx4 in limb development: complex genetic interactions with Gli3 and Shh

The role of the aristaless-related homeobox gene Alx4 in antero-posterior (AP-) patterning of the developing vertebrate limb has remained somewhat elusive. Polydactyly of Alx4 mutant mice is known to be accompanied by ectopic anterior expression of genes like Shh, Fgf4 and 5'Hoxd. We reported previously that polydactyly in Alx4 mutant mice requires SHH signaling, but we now show that in early Alx4-/- limb buds the anterior ectopic expression of Fgf4 and Hoxd13, and therefore disruption of AP-patterning, occurs independently of SHH signaling. To better understand how Alx4 functions in the pathways that regulate AP-patterning, we also studied genomic regulatory sequences that are capable of directing expression of a reporter gene in a pattern corresponding to endogenous Alx4 expression in anterior limb bud mesenchyme. We observed, as expected for authentic Alx4 expression, expansion of reporter construct expression in a Shh-/- background. Total lack of reporter expression in a Gli3-/- background confirms the existence of Gli3-dependent and -independent Alx4 expression in the limb bud. Apparently, these two modules of Alx4 expression are linked to dissimilar functions.     

Design of cyclic and d‐amino acids containing peptidomimetics for inhibition of protein‐protein interactions of HER2‐HER3

HER2 receptors are surface proteins belonging to the epidermal growth factor family of receptors. Their numbers are elevated in breast, lung, and ovarian cancers. HER2‐positive cancers are aggressive, have higher mortality rate, and have a poor prognosis. We have designed peptidomimetics that bind to HER2 and block the HER2‐mediated dimerization of epidermal growth factor family of receptors. Among these, a symmetrical cyclic peptidomimetic (compound 18 ) exhibited antiproliferative activity in HER2‐overexpressing lung cancer cell lines with IC₅₀ values in the nanomolar concentration range. To improve the stability of the peptidomimetic, d ‐amino acids were introduced into the peptidomimetic, and several analogs of compound 18 were designed. Among the analogs of compound 18 , compound 32 , a cyclic, d ‐amino acid‐containing peptidomimetic, was found to have an IC₅₀ value in the nanomolar range in HER2‐overexpressing cancer cell lines. The antiproliferative activity of compound 32 was also measured by using a 3D cell culture model that mimics the in vivo conditions. The binding of compound 32 to the HER2 protein was studied by surface plasmon resonance. In vitro stability studies indicated that compound 32 was stable in serum for 48 hours and intact peptide was detectable in vivo for 12 hours. Results from our studies indicated that 1 of the d ‐amino acid analogs of 18 , compound 32 , binds to the HER2 extracellular domain, inhibiting the phosphorylation of kinase of HER2.     

Use of Fixed Dose Combination (FDC) Drugs in India: Central Regulatory Approval and Sales of FDCs Containing Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), Metformin,or Psychotropic Drugs

Background

In 2012, an Indian parliamentary committee reported that manufacturing licenses for large numbers of fixed dose combination (FDC) drugs had been issued by state authorities without prior approval of the Central Drugs Standard Control Organization (CDSCO) in violation of rules, and considered that some ambiguity until 1 May 2002 about states’ powers might have contributed. To our knowledge, no systematic enquiry has been undertaken to determine if evidence existed to support these findings. We investigated CDSCO approvals for and availability of oral FDC drugs in four therapeutic areas: analgesia (non-steroidal anti-inflammatory drugs [NSAIDs]), diabetes (metformin), depression/anxiety (anti-depressants/benzodiazepines), and psychosis (anti-psychotics).

Methods and Findings

This was an ecologic study with a time-trend analysis of FDC sales volumes (2007–2012) and a cross-sectional examination of 2011–2012 data to establish the numbers of formulations on the market with and without a record of CDSCO approval (“approved” and “unapproved”), their branded products, and sales volumes. Data from the CDSCO on approved FDC formulations were compared with sales data from PharmaTrac, a database of national drug sales. We determined the proportions of FDC sales volumes (2011–2012) arising from centrally approved and unapproved formulations and from formulations including drugs banned/restricted internationally. We also determined the proportions of centrally approved and unapproved formulations marketed before and after 1 May 2002, when amendments were made to the drug rules. FDC approvals in India, the United Kingdom (UK), and United States of America (US) were compared.For NSAID FDCs, 124 formulations were marketed, of which 34 (27%) were centrally approved and 90 (73%) were unapproved; metformin: 25 formulations, 20 (80%) approved, five (20%) unapproved; anti-depressants/benzodiazepines: 16 formulations, three (19%) approved, 13 (81%) unapproved; anti-psychotics: ten formulations, three (30%) approved, seven (70%) unapproved. After 1 May 2002, the proportions of approved FDC formulations increased for NSAIDs (26%/28%) and anti-psychotics (0%/38%) and decreased for metformin (100%/75%) and anti-depressants/benzodiazepines (20%/18%), and the overall proportion approved remained similar before and after that date.FDC formulations gave rise to multiple branded products, ranging from 211 anti-psychotic FDC products from ten formulations to 2,739 NSAID FDC products from 124 formulations. The proportions of FDC sales volumes arising from unapproved formulations were as follows: anti-depressants/benzodiazepines, 69%; anti-psychotics, 43%; NSAIDs, 28%; and metformin, 0.4%. Formulations including drugs banned/restricted internationally comprised over 12% of NSAID FDC sales and 53% of anti-psychotic FDC sales. Across the four therapeutic areas, 14 FDC formulations were approved in the UK and 22 in the US.

Conclusions

There was evidence supporting concerns about FDCs. Metformin excepted, substantial numbers of centrally unapproved formulations for NSAID, anti-depressant/benzodiazepine, and anti-psychotic FDCs were marketed; sales volumes were high. The legal need for central approval of new drugs before manufacture has been in place continuously since 1961, including for FDCs meeting the applicable legal test. Proportions of centrally unapproved formulations after 1 May 2002 did not decrease overall, and no ambiguity was found about states’ licensing powers. Unapproved formulations should be banned immediately, prioritising those withdrawn/banned internationally and undertaking a review of benefits and risks for patients in ceasing or switching to other medicines. Drug laws need to be amended to ensure the safety and effectiveness of medicines marketed in India.     

Targeting Colon Cancer Cells with Pyrazino-Imidazolinone Derivatives: Synthesis,Molecular Docking,and in Vitro Evaluation of Anti-Proliferative and Pro-Apoptotic Activities

We report the synthesis, spectroscopic characterization, molecular docking and biological evaluation of nine pyrazino-imidazolinone derivatives. These derivatives were evaluated for their anticancer activity against three cancer cell lines: 518A2 melanoma, HCT-116, and HCT-116 p53 knockout mutant colon carcinoma. The MTT assay was employed to assess their effectiveness. Among the nine compounds tested, four compounds (5 a, 5 d, 5 g, and 5 h) exhibited promising antiproliferative activity specifically against HCT-116 p53-negative cells (IC₅₀ 0.23, 0.20, 2.07 and 58.75 μM, respectively). Interestingly, treatment with the 3,4-dimethoxyphenyl derivative 5a resulted in a significant increase (199 %) in caspase activity in HCT-116 p53-negative cells compared to untreated cells while the bromo-pyrazine derivative 5d demonstrated (190 %) increase. These findings suggest that compounds 5a and 5 d induce p53-independent apoptotic cell death. Additionally, in silico molecular docking studies with EGFR and tyrosinase proteins indicated that compounds 5 d and 5 e have the potential to bind to important anticancer drug targets.     

Degrons of yeast and mammalian ornithine decarboxylase enzymes make potent combination for regulated targeted protein degradation

Elevation of polyamine levels in eukaryotes leads to rapid degradation of ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis pathway. ODC in yeast (yODC) has two domains, the Nα/β domain consisting of α/β barrel domain (α/β) preceded by an overhang of 50 residues at its N-terminus (N50) and β sheet domain at its C-terminus. Two degradation determinant signals or degrons in yODC sequence, namely the N50 and the antizyme-binding element (AzBE) housed in the α/β domain, are responsible for its degradation by proteasomes. Antizyme (Az) induced under polyamine excess binds to AzBE and delivers ODC to proteasome, while the N50 threads the protein into proteasome. It was previously reported by us that the peptide Nα/β of yODC acts as an independent transplantable degron, whose action can be modulated with the help of antizyme by varying polyamine levels. Mammalian ODC (mODC), in spite of its 40% sequence homology with yODC, is devoid of N50 of yODC and instead sports a C-terminal tail of 37 residues (CmODC). CmODC acts as an independent transplantable degron with no equivalent in yODC. The present study investigates the merits of employing the two degrons Nα/β and CmODC together for targeted protein degradation by expressing them in a chimeric fusion with green fluorescent protein (GFP). Our results establish that under the regulation of antizyme, the signals Nα/β and CmODC acting together enhance degradation better than either degron in isolation. The combination of Nα/β and CmODC can be employed to study the function of novel proteins through their rapid removal.

     

Patterns of species discovery in the Western Ghats,a megadiversity hot spot in India

Even since Linnaeus, naturalists and taxonomists have been systematically describing species new to science. Besides indicating gaps in taxonomic effort, understanding the temporal patterns of species discovery could help in identifying drivers that determine discovery. In this study we report the patterns of discovery of eight taxa — birds, butterflies, frogs, tiger beetles, grasses, asters, ferns and orchids — in the Western Ghats, a megadiversity centre in India. Our results indicate that the discovery curves for birds and butterflies have been saturated while those for frogs and grasses continue to increase. Within each taxon, the major drivers of discovery were commonness of the species and their size. The average years taken for discovery across taxa were directly related to the per cent endemicity and species richness of the taxa. We discuss the trajectories of discovery with respect to rarity or endemicity of the species and life history features, and the implications these might have for strategizing the discovery process in India.