Discovery and initial optimization of 5,5′-disubstituted aminohydantoins as potent β-secretase (BACE1) inhibitors

8,8-Diphenyl-2,3,4,8-tetrahydroimidazo[1,5-a]pyrimidin-6-amine (1) was identified through HTS, as a weak (micromolar) inhibitor of BACE1. X-Ray crystallographic studies indicate the 2-aminoimidazole ring forms key H-bonding interactions with Asp32 and Asp228 in the catalytic site of BACE1. Lead optimization using structure-based focused libraries led to the identification of low nanomolar BACE1 inhibitors such as 20b with substituents which extend from the S₁ to the S₃ pocket.     

Bio-ethanol from water hyacinth biomass: An evaluation of enzymatic saccharification strategy

Biomass feedstock having less competition with food crops are desirable for bio-ethanol production and such resources may not be localized geographically. A distributed production strategy is therefore more suitable for feedstock like water hyacinth with a decentralized availability. In this study, we have demonstrated the suitability of this feedstock for production of fermentable sugars using cellulases produced on site. Testing of acid and alkali pretreatment methods indicated that alkali pretreatment was more efficient in making the sample susceptible to enzyme hydrolysis. Cellulase and β-glucosidase loading and the effect of surfactants were studied and optimized to improve saccharification. Redesigning of enzyme blends resulted in an improvement of saccharification from 57% to 71%. A crude trial on fermentation of the enzymatic hydrolysate using the common baker’s yeast Saccharomyces cerevisiae yielded an ethanol concentration of 4.4 g/L.     

Bioactivity-guided isolation of mosquitocidal constituents from the rhizomes of Plumbago capensis Thunb

A bioassay-guided fractionation and chemical examination of chloroform extract of Plumbago capensis roots resulted in isolation and characterization of two new napthaquinone derivatives (4, 8) along with six known compounds (1–3, 5–7). Their structures were determined on the basis of extensive spectroscopic (IR, MS, 1D and 2D NMR) data analysis and by comparison with the literature data. All the compounds were tested for their mosquito larvicidal activity against fourth instar larvae of Aedes aegypti, and compared with that of rotenone. Among the tested compounds, isoshinanolone (3) and plumbagin (1) showed excellent toxicity with LC₅₀ values of 1.26 and 5.43 μg/mL. New compound (8) displayed moderate toxicity against the tested mosquito species.     

Mitochondrial DNA haplogroup ‘R’ is associated with Noonan syndrome of South India

Mutations in PTPN11 gene was responsible for ～50% of the Noonan syndrome (NS), however, we did not find any mutation in PTPN11 in any of seven NS patients analysed. Whereas, the complete mtDNA sequencing revealed 146 mutations, of which five, including one heteroplasmic (A11144R; Thr → Ala) non-synonymous mutation, were novel and exclusively observed in NS patients. Interestingly all the seven probands and their maternal relatives were clustered under a major haplogroup R and its novel sub-haplogroups (R7b1b, R30a1, R30c, T2b7, U9a1) exclusive in NS, therefore we strongly suggest that these haplogroups may influence NS in South Indian populations.     

MVA recombinants expressing the fusion and hemagglutinin genes of PPRV protects goats against virulent challenge

Peste des Petits Ruminants (PPR) is a highly contagious animal disease caused by the Peste des Petits Ruminants virus (PPRV) belonging to the genus morbillivirus and family Paramyxoviridae. The disease results in high morbidity and mortality in goats, sheep and in some small wild ruminants. The presence of large number of small ruminants reared in endemic areas makes PPR a notorious disease threatening the livelihood of poor farmers. Conventional vaccination using a live, attenuated vaccine gives adequate protection but cannot be used in case of eradication of the disease due to difficulty in differentiation of infected animals from the vaccinated ones.     

Kaposi's Sarcoma Associated Herpes Virus (KSHV) Induced COX-2: A Key Factor in Latency,Inflammation, Angiogenesis,Cell Survival and Invasion

Kaposi''s sarcoma (KS), an enigmatic endothelial cell vascular neoplasm, is characterized by the proliferation of spindle shaped endothelial cells, inflammatory cytokines (ICs), growth factors (GFs) and angiogenic factors. KSHV is etiologically linked to KS and expresses its latent genes in KS lesion endothelial cells. Primary infection of human micro vascular endothelial cells (HMVEC-d) results in the establishment of latent infection and reprogramming of host genes, and cyclooxygenase-2 (COX-2) is one of the highly up-regulated genes. Our previous study suggested a role for COX-2 in the establishment and maintenance of KSHV latency. Here, we examined the role of COX-2 in the induction of ICs, GFs, angiogenesis and invasive events occurring during KSHV de novo infection of endothelial cells. A significant amount of COX-2 was detected in KS tissue sections. Telomerase-immortalized human umbilical vein endothelial cells supporting KSHV stable latency (TIVE-LTC) expressed elevated levels of functional COX-2 and microsomal PGE2 synthase (m-PGES), and secreted the predominant eicosanoid inflammatory metabolite PGE2. Infected HMVEC-d and TIVE-LTC cells secreted a variety of ICs, GFs, angiogenic factors and matrix metalloproteinases (MMPs), which were significantly abrogated by COX-2 inhibition either by chemical inhibitors or by siRNA. The ability of these factors to induce tube formation of uninfected endothelial cells was also inhibited. PGE2, secreted early during KSHV infection, profoundly increased the adhesion of uninfected endothelial cells to fibronectin by activating the small G protein Rac1. COX-2 inhibition considerably reduced KSHV latent ORF73 gene expression and survival of TIVE-LTC cells. Collectively, these studies underscore the pivotal role of KSHV induced COX-2/PGE2 in creating KS lesion like microenvironment during de novo infection. Since COX-2 plays multiple roles in KSHV latent gene expression, which themselves are powerful mediators of cytokine induction, anti-apoptosis, cell survival and viral genome maintainence, effective inhibition of COX-2 via well-characterized clinically approved COX-2 inhibitors could potentially be used in treatment to control latent KSHV infection and ameliorate KS.     

Role of activated macrophages in Acanthamoeba keratitis

The purpose of this study was to determine whether activating the conjunctival macrophages would affect the course of Acanthamoeba spp. keratitis in a Chinese hamster model of this disease. Chinese hamster spleen cells were stimulated with concanavalin A (Con A), and interferon gamma (IFN-gamma) -containing supernatants were collected 24 hr later. The IFN-gamma-containing supernatants were loaded into liposomes, which were fed to peritoneal macrophages in vitro. Macrophage activation was assessed by testing for production of nitric oxide (NO) with the use of Griess reagent. Conjunctival macrophages were activated in situ by subconjunctival injection of liposomes containing Con A-activated spleen cell culture supernatants. Control liposomes were loaded with phosphate-buffered saline (PBS). Macrophages exposed to supernatants from Con A-stimulated spleen cells produced 4-fold-higher amounts of NO than unstimulated macrophages. Activation of macrophages via subconjunctival injection of liposomes containing supernatants from Con A-stimulated spleen cell cultures resulted in rapid resolution of the corneal infection. Approximately 80% of animals treated with PBS-containing liposomes demonstrated evidence of corneal disease at day 14 compared to 10% incidence of infection in the Con A-treated group. Moreover, at all time points examined, the clinical appearance of the keratitis in animals treated with liposomes containing Con A supernatant was significantly reduced compared to the group treated with liposomes containing PBS (P < 0.05). Macrophages stimulated with IFN-gamma-containing supernatants killed significant numbers of the trophozoites in vitro (P < 0.05). Killing was inhibited by cytochalasin D, but not by L-N6-1-iminoethyl-L-lysine dihydrochloride (L-NIL), which is a selective inhibitor of inducible NO synthase (INOS).     

Comparative genomic structure of human, dog, and cat MHC: HLA, DLA, and FLA

Comparisons of the genomic structure of 3 mammalian major histocompatibility complexes (MHCs), human HLA, canine DLA, and feline FLA revealed remarkable structural differences between HLA and the other 2 MHCs. The 4.6-Mb HLA sequence was compared with the 3.9-Mb DLA sequence from 2 supercontigs generated by 7x whole-genome shotgun assembly and 3.3-Mb FLA draft sequence. For FLA, we confirm that 1) feline FLA was split into 2 pieces within the TRIM (member of the tripartite motif) gene family found in human HLA, 2) class II, III, and I regions were placed in the pericentromeric region of the long arm of chromosome B2, and 3) the remaining FLA was located in subtelomeric region of the short arm of chromosome B2. The exact same chromosome break was found in canine DLA structure, where class II, III, and I regions were placed in a pericentromeric region of chromosome 12 whereas the remaining region was located in a subtelomeric region of chromosome 35, suggesting that this chromosome break occurred once before the split of felid and canid more than 55 million years ago. However, significant differences were found in the content of genes in both pericentromeric and subtelomeric regions in DLA and FLA, the gene number, and amplicon structure of class I genes plus 2 other class I genes found on 2 additional chromosomes; canine chromosomes 7 and 18 suggest the dynamic nature in the evolution of MHC class I genes.