Organocatalytic (3+3)-cycloaddition of ortho-substituted phenyl nitrones with aryl cyclopropane carbaldehydes: a facile access to enantioenriched 1,2-oxazinanes.

The first asymmetric (3+3)-cycloaddition of ortho-substituted phenyl nitrones with aryl cyclopropane carbaldehydes has been demonstrated by secondary amine catalysts. While the other ortho-substituents gave 1,2-oxazinanes, ortho-hydroxy ones provided a novel class of tetrahydrochromeno-1,2-oxazine cores via rare 1,3-aryl migration, followed by cyclization. An unusual type of asymmetric approach was also recognized.

Catalytic Atroposelective Aerobic Oxidation Approaches to Axially Chiral Molecules.

A copper and chiral nitroxide co-catalyzed aerobic enantioselective oxidation process has been developed that allows access to axially chiral molecules. Two complementary atroposelective approaches, oxidative kinetic resolution (OKR) and desymmetrization, were studied using ambient air as the stoichiometric terminal oxidant. OKR of rac-N-arylpyrrole alcohols and rac-biaryl alcohols affords the optically pure compounds with er up to 3.5:96.5 and 5.5:94.5, respectively. Desymmetrization of prochiral diols provides axially chiral biaryl compounds with er up to 99:1.

Appraisal of the Possible Role of PPARγ Upregulation by CLA of Probiotic Pediococcus pentosaceus GS4 in Colon Cancer Mitigation.

The prevalence of colon cancer (CC) is increasing at the endemic scale, which is accompanied by subsequent morbidity and mortality. Although there have been noteworthy achievements in the therapeutic strategies in recent years, the treatment of patients with CC remains a formidable task. The current study focused on to study role of biohydrogenation-derived conjugated linoleic acid (CLA) of probiotic Pediococcus pentosaceus GS4 (CLAGS4) against CC, which induced peroxisome proliferator-activated receptor gamma (PPARγ) expression in human CC HCT-116 cells. Pre-treatment with PPARγ antagonist bisphenol A diglycidyl ether has significantly reduced the inhibitory efficacy of enhanced cell viability of HCT-116 cells, suggesting the PPARγ-dependent cell death. The cancer cells treated with CLA/CLAGS4 demonstrated the reduced level of Prostaglandin E2 PGE2 in association with reduced COX-2 and 5-LOX expressions. Moreover, these consequences were found to be associated with PPARγ-dependent. Furthermore, delineation of mitochondrial dependent apoptosis with the help of molecular docking LigPlot analysis showed that CLA can bind with hexokinase-II (hHK-II) (highly expressed in cancer cells) and that this association underlies voltage dependent anionic channel to open, thereby causing mitochondrial membrane depolarization, a condition that initiates intrinsic apoptotic events. Apoptosis was further confirmed by annexin V staining and elevation of caspase 1p10 expression. Taken all together, it is deduced that, mechanistically, the upregulation of PPARγ by CLAGS4 of P. pentosaceus GS4 can alter cancer cell metabolism in association with triggering apoptosis in CC.

2-Nitro-cyclopropyl-1-carbonyl Compounds from Unsaturated Carbonyl Compounds and Nitromethane via Enolonium Species.

2-Nitrocyclopropanes bearing ketones, amides, esters, and carboxylic acids in the 1 position may be accessed as single diastereoisomers in one operation from the corresponding unsaturated carbonyl compounds. The source of the nitro-methylene component is nitromethane. The reaction proceeds at room temperature under mild conditions. The products may be converted into, e.g., cyclopropyl-amino acids in a single step. Both nitrocyclopropanes and amino-cyclopropanes are unique moieties found in biologically active compounds and natural products.

Palladium-catalyzed selective C-C bond cleavage and stereoselective alkenylation between cyclopropanol and 1,3-diyne: one-step synthesis of diverse conjugated enynes.

The stereoselective synthesis of 1,3-enynes from 1,3-diynes is demonstrated by palladium-catalyzed selective C-C bond cleavage of cyclopropanol. Exclusive formation of mono-alkenylated adducts was achieved by eliminating the possibility of di-functionalization with high stereoselectivity. Indeed, this protocol worked very well with electronically and sterically diverse substrates. Several studies, including deuterium labeling experiments and intermolecular competitive experiments, were carried out to understand the mechanistic details. The atomic-level mechanism followed in the catalytic process was also validated using DFT calculations, and the rate-controlling states in the catalytic cycle were identified. Furthermore, preliminary mechanistic investigations with radical scavengers revealed the non-involvement of the radical pathway in this transformation.

Ruthenium-Catalyzed Regioselective C(sp2)-H Activation/Annulation of N-(7-Azaindole)amides with 1,3-Diynes Using N-Amino-7-azaindole as the N,N-Bidentate Directing Group.

The ruthenium(II)-catalyzed regioselective annulation of N-(7-azaindole)amides with 1,3-diynes has been demonstrated. Bioactive N-amino-7-azaindole has been used as a new bidentate directing group to furnish an array of 3-alkynylated isoquinolones. Furthermore, the developed protocol works efficiently for both aryl- and heteroaryl-substituted amides producing a range of pharmacologically useful 7-azaindole-based isoquinolones with a wide range of functionality.

Relieving the stress together: annulation of two different strained rings towards the formation of biologically significant heterocyclic scaffolds.

Small carbo- and heterocycles have become versatile building blocks owing to their intrinsic ring strain and ease of synthesis. However, the traditional approaches of heterocycle synthesis involved the combination of one strained-carbocycle or heterocycle with one unsaturated molecule. On the contrary, there is an exciting possibility of combining two different strained rings to furnish varieties of heterocycles, where one of the strained rings can act as a valuable alternative to the unsaturated molecule. These strategies are also useful to access multi-functionalized rings. Despite these distinctive synthetic benefits, this chemistry has not drawn considerable attention of the community. In this minireview, we explicitly choose this topic to reveal the unexplored possibilities with these different strained rings. This minireview provides comprehensive details with the mechanistic rationale about the reactivity of these pairs of small rings when they are allowed to react together in the presence of different Lewis acids. Subsequently, it will also open a new avenue for heterocycle synthesis.

Ruthenium-Catalyzed Cross Dehydrogenative Annulation of N-(7-Azaindole)benzamides with Maleimides: One-Step Access to Highly Functionalized Pyrroloisoquinoline.

A ruthenium(II)-catalyzed cross dehydrogenative annulation of N-(7-azaindole)benzamides with maleimides has been demonstrated. Herein, we have explored the use of N-amino-7-azaindole as a new bidentate directing group for dehydrogenative [4 + 2] annulation. This method works efficiently, affording a variety of pharmacologically useful 7-azaindole-based isoquinolones and showing a wide functional group tolerance.

Probiotic Enterococcus faecalis AG5 mitigated high fat diet induced obesity and produced propionic acid stimulated apoptosis in 3T3-L1 pre-adipocyte.

AIMS: Probiotics and their metabolites (SCFA) can regulate energy homeostasis. The present study thus evaluates the synergistic effect of probiotic Enterococcus faecalis AG5 on HFD induced obesity and the role of propionic acid (PA) in apoptosis induction of 3T3-L1 pre-adipocyte. METHODS: Male Wistar rats (n = 24) were used to develop an HFD induced obesity model for 24 weeks. The effect of the orally administered probiotic AG5 (18th-24th weeks, 1 × 109 CFU/ml) was evaluated using physiological, biochemical, anthropometry, histopathological and serological analyses. Apoptosis in 3T3-L1 pre-adipocyte was assessed using flow cytometry, protein expression of PPARγ, 5-LOX, NF-κB, p-AKT, caspase 10 and detection of caspase 3/7 by Immunofluorescence confirmed the apoptosis induced by PA. KEY FINDINGS: Probiotic AG5 significantly reduced body weight, BMI, serum cholesterol, triglycerides (p < 0.05) and improved HDL, insulin and leptin but lowered LDL and VLDL (p > 0.05). An inflammatory response was reduced as evident by TNF-α IHC. AG5 reduced adipocyte hypertrophy and fatty acid accumulation. Flow cytometry confirmed late apoptosis in PA-AG5 and standard PA treated 3T3-L1 cells. 5-LOX inhibition is associated with apoptosis induction, and increased caspase 1p 10 is related to cell death initiation. The study initially showed a low PPARγ activity inhibiting 5-LOX which may relate to adipose apoptosis. Finally caspase 3/7 detection using immunofluorescence proved the role of PA in adipocyte apoptosis. SIGNIFICANCE: The present study is a novel approach towards obesity mitigation involving adipocyte apoptosis. The role of SCFA in adipocyte apoptosis is very limited which can prove to be novel therapeutic approach in the future.

Cell adherence efficacy of probiotic Pediococcus pentosaceus GS4 (MTCC 12683) and demonstrable role of its surface layer protein (Slp).

The current study examined the cell adherence property of probiotic Pediococcus pentosaceus GS4 (MTCC12683) with the characterization and functionality in adherence of its surface layer protein (GS4-Slp). The Slp of P. pentosaceus GS4 was extracted purified and detected using SDS-PAGE (98 kDa) and size exclusion chromatography. The cell adherence property of probiotic GS4 (Slp+/Slp-) was evaluated on buccal cells and HCT-116. Purified Slp was found neutralized with raised anti-Slp showing reduced adherence to HCT-116 as evident from SEM analysis. The structure of GS4-Slp was determined by MALDI-TOF analysis, CD analysis, atomic force microscopy (AFM), and FT-IR spectrometry. In Silico approach revealed its indirect similarity with cell membrane protein of Helicobacter pylori. Results thus reveal that GS4 has the potential of the production of 98 kDa Slp which facilitates the cell adherence property. This added probiotic attribute will enhance the probiotic potentials of P. pentosaceus GS4 to use it biotechnologically. SIGNIFICANCE: Probiotic Pediococcus pentosaceus GS4 facilitates demonstrable colonization by the elaboration of Slp. This property imparts a value to the strain and claims to be more useful biotechnologically.

Rhodium-Catalyzed Room Temperature C-C Activation of Cyclopropanol for One-Step Access to Diverse 1,6-Diketones.

A rhodium-catalyzed room temperature C-C activation of cyclopropanol has been demonstrated for the single-step synthesis of a range of electronically and sterically distinct 1,6-diketones. This reaction proceeds efficiently in shorter reaction time following a highly atom-economical pathway. To illustrate the synthetic potential of 1,6-diketones, aldol and macrocyclization reactions have been successfully demonstrated. Preliminary mechanistic studies revealed the involvement of nonradical pathways.

Assessment of bacterial viability: a comprehensive review on recent advances and challenges.

Assessing bacterial contamination in environmental samples is critical in determining threats to public health. The classical methods are time-consuming and only recognize species that grow easily on culture media. Viable but non-culturable (VBNC) bacteria are a possible threat that may resuscitate and cause infections. Recent dye-based screening techniques employ nucleic acid dyes such as ethidium monoazide (EMA) and propidium monoazide (PMA), along with many fluorescent dyes, which are an effective alternative for viability assessment. The measurement of cellular metabolism, heat flow and ATP production has also been widely applied in detection approaches. In addition, RNA-based detection methods, including nucleic acid sequence-based amplification (NASBA), have been applied for bacterial pathogen determination. Stable isotope probing using 13C, 15 N and 18O, which are mobilized by microbes, can also be used for effective viability assessment. Future detection tools, such as microarrays, BioNEMS and BioMEMS, which are currently being validated, might offer better microbial viability detection.

Probiotic Enterococcus faecalis AG5 effectively assimilates cholesterol and produces fatty acids including propionate.

Probiotics are known for their health-promoting abilities through the production of various metabolites, and also benefits are membrane associated. Probiotic Enterococcus faecalis AG5 has been previously assessed for its probiotic properties and this study further strengthens that claim. Strain AG5 was inoculated with 0.3% sodium glychocholate (glycine conjugated) and bile salt mixture for different time periods. The percentage assimilation of cholesterol ranged from 14% to 33.1% in the bile salt mixture, but was slightly higher, ranging from 16.55% to 53.1%, in sodium thioglycocholate alone. Similarly, the bile salt hydrolysis activity was measured in terms of cholic acid release at different intervals. The overall range of cholic acid released ranged from 70.4 to 479 µg/mL. Fatty acid production is also an important criterion for a probiotic selection and it was found that the strain produced several long-chain fatty acids (LCFA) such as tetradecanoic acid, hexadecanoic acid, octadecanoic acid and bis(6-methylheptyl) phthalate. Besides LCFA, AG5 produced short-chain fatty acids (SCFA) such as propionic acid, which was estimated comparing liquid chromatography and mass spectrometry with standard SCFA. Thus the ability of deconjugation of bile salt and SCFA production is a value-added property of probiotic AG5, with the promise of being beneficial for human health.

Purification and characterization of pediocin from probiotic Pediococcus pentosaceus GS4, MTCC 12683.

Pediococcus pentosaceus GS4 (MTCC 12683), a probiotic lactic acid bacterium (LAB), was found to produce bacteriocin in spent culture. Antibacterial and antagonistic potential of this bacteriocin against reference strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 25619), and Listeria monocytogenes (ATCC 15313) was proven by double-layer and well diffusion methods wherein nisin and ampicillin were used as positive controls. Bacteriocin in supernatant was purified and analyzed by SDS-PAGE, RP-HPLC, and circular dichroism (CD). The physico-chemical properties of purified bacteriocin were characterized being treated at different temperatures (30 to 110 °C), pH (3.0 to 12.0), with different enzymes (α-amylase, pepsin, and lysozyme), and organic solvents (hexane, ethanol, methanol, and acetone) respectively. The molar mass of bacteriocin (named pediocin GS4) was determined as 9.57 kDa. The single peak appears at the retention time of 2.403 with area amounting to 25.02% with nisin as positive control in RP-HPLC. CD analysis reveals that the compound appears to have the helix ratio of 40.2% with no beta sheet. The antibacterial activity of pediocin GS4 was optimum at 50 °C and at pH 5.0 and 7.0. The pediocin GS4 was not denatured by the treatment of amylase and lysozyme but was not active in the presence of organic solvents. This novel bacteriocin thus m ay be useful in food and health care industry.

Antimycobacterial potentials of quercetin and rutin against Mycobacterium tuberculosis H37Rv.

Quercetin and rutin, two flavonoids were examined for antimycobacterial activities against M. tuberculosis H37Rv (ATCC 27294). The quercetin exhibited (99.30 ± 0.268%) in (LRP) assay at 200 µg/ml and 56.21 ± 0.97% inhibition in (BMD) at 50 µg/ml, whereas rutin exhibited (90.40 ± 0.68%) in LRP assay at 200 µg/ml and 56.10 ± 0.67% inhibition in BMD at 50 µg/ml. The minimum inhibitory concentration (MIC) was found to be 6.25 µg ml-1 and 25 µg ml-1 respectively. The current investigation suggests that quercetin has better inhibitory activity than rutin.

Detection of parvovirus B19 in selected high-risk patient groups & their phylogenetic & selection analysis.

Background & objectives: Human parvovirus B19V (B19V) is known to be associated with erythema infectiosum commonly in children, aplastic crisis, especially in persons with underlying haemolytic disorders, hydrops fetalis in pregnancies and arthritis. This cross-sectional study was aimed to determine the presence of B19V infection in childhood febrile illnesses, association of B19V with arthropathies and in adult patients with end-stage renal disease (ESRD) on dialysis. The genetic diversity among the sequences was also analysed. Methods: A nested polymerase chain reaction (nPCR) assay was used for B19V DNA targeting VP1/VP2 region and used for testing 618 patients and 100 healthy controls. Phylogenetic analysis on nucleotide and amino acid sequences was carried out to compare our sequences with other Indian strains and global strains. Results: Among 618 samples tested, seven (1.13%) were found positive. The phylogenetic analysis revealed that all the seven sequences belonged to genotype 1 and showed low genetic diversity. The clustering pattern of seven sequences was similar both by nucleotide and by predicted amino acid sequences. The fixed effects likelihood analysis showed no positive or negatively selected sites. Interpretation & conclusions: Seven samples (4 from non-traumatic arthropathies, 2 from patients with ESRD and 1 from febrile illness patient) were found positive by nPCR. When our seven sequences were compared with global strains, the closest neighbour was other Indian strains followed by the Tunisian strains.

Characterization of Functional, Safety, and Probiotic Properties of Enterococcus faecalis AG5 Isolated From Wistar Rat, Demonstrating Adherence to HCT 116 Cells and Gastrointestinal Survivability.

Gut microbiota remains a prominent source for a diverse range of potential probiotics. In this context, the current study explored the rectal region of experimental Wistar rat for the isolation of potent probiotic. Sixteen lactic acid bacteria (LAB), from rectal swab of Wistar rats, were subjected to evaluation of probiotic properties. Among all, AG5 was found unique with consistent probiotic properties and was further identified as Enterococcus faecalis AG5 (NCBI accession number KT248537) using 16S rDNA sequencing, followed by BLAST analysis. Since the Enterococci strains inhibit various food-borne pathogens efficiently while proving itself as a safe probiotic candidate, the study further evaluated the safety of the strain AG5 using primer specific PCR amplification which revealed the existence of gene encoding gelE, asa1, efaA, ace, vanA, and vanB and negative for cylA, hyl, and esp respectively. SEM analysis confirmed the adherence ability of AG5 to HCT 116 cells. Adherence was found to be non-colonial and scattered manner. Furthermore, the strain demonstrated a significant survivability during simulated gastrointestinal transit. Taken together, the E. faecalis AG5 was found potential probiotic candidate with future implication in both food and health industry.

Prediction of Pan-Specific B-Cell Epitopes From Nucleocapsid Protein of Hantaviruses Causing Hantavirus Cardiopulmonary Syndrome.

Hantaviruses are emerging viral pathogens that causes hantavirus cardiopulmonary syndrome (HCPS) in the Americas, a severe, sometimes fatal, respiratory disease in humans with a case fatality rate of ≥50%. IgM and IgG-based serological detection methods are the most common approaches used for laboratory diagnosis of hantaviruses. Such emerging viral pathogens emphasizes the need for improved rapid diagnostic devices and vaccines incorporating pan-specific epitopes of genotypes. We predicted linear B-cell epitopes for hantaviruses that are specific to genotypes causing HCPS in humans using in silico prediction servers. We modeled the Andes and Sin Nombre hantavirus nucleocapsid protein to locate the identified epitopes. Based on the mean percent prediction probability score, epitope IMASKSVGS/TAEEKLKKKSAF was identified as the best candidate B-cell epitope specific for hantaviruses causing HCPS. Promiscuous epitopes were identified in the C-terminal of the protein. Our study for the first time has reported pan-specific B-cell epitopes for developing immunoassays in the detection of antibodies to hantaviruses causing HCPS. Identification of epitopes with pan-specific recognition of all genotypes causing HCPS could be valuable for the development of immunodiagnositic tools toward pan-detection of hantavirus antibodies in ELISA. J. Cell. Biochem. 118: 2320-2324, 2017. © 2017 Wiley Periodicals, Inc.

Development & standardization of an in-house IgM indirect ELISA for the detection of parvovirus B19 infections.

BACKGROUND & OBJECTIVES: Parvovirus B19 infections occur worldwide; the infection is acquired early in childhood but could occur later. B19 is reported to cause infection in childhood febrile illnesses, and arthropathies in adults and children and in end-stage renal disease (ESRD) seen in adults. This study was designed to develop an in-house IgM indirect ELISA for serological screening among patients and controls, and to compare ELISA results with those of nested polymerase chain reaction (nPCR) assay. METHODS: An in-house IgM indirect ELISA was standardized using peptide sequence of VP1/VP2 region of parvovirus B19. A total of 201 children and adult with febrile illnesses, 216 individuals with non-traumatic arthropathies, 201 cases of chronic anaemia associated with ESRD and 100 healthy controls were tested. Serum was separated from the blood and subsequently used for DNA extraction. The nested polymerase chain reaction (nPCR) for the detection of B19V DNA was performed using primers targeting the overlapping region of VP1/VP2 capsid protein genes. RESULTS: A total of 618 samples were tested for parvovirus B19 by an in-house IgM indirect ELISA. Among these samples, six were positive by in-house ELISA. The inter-rater agreement between ELISA and PCR assays was calculated using kappa coefficient analysis. The value of κ was 0.77 and the strength of agreement was 'good' (P<0.001). INTERPRETATION & CONCLUSIONS: The in-house IgM indirect ELISA was found to be simple with high sensitivity and specificity when compared with nPCR and could be used as an alternative to expensive commercial kits in resource-poor settings.

Prediction of B Cell Epitopes Among Hantavirus Strains Causing Hemorragic Fever With Renal Syndrome.

Hantavirus infections are now recognized to be a global problem. The hantaviruses include several genotypic variants of the virus with different distributions in varying geographical regions. The virus genotypes seem to segregate in association with certain manifestations specific for each syndrome. They primarily include HFRS, HCPS, febrile illness with or without mild involvement of renal diseases. In the course of our study on hantavirus etiology of febrile illnesses, we recovered a hantavirus strain identified by nPCR. This has been sequenced to be Hantaan-like virus (partial S segment). The current manuscript is focused on understanding the N protein coded by S segment in terms of variation of amino acid sequences of the virus genotypes associated with HFRS. The diagnosis of this infection is achieved by PCR testing of serum/plasma or demonstration of IgM/IgG in serum. The limitations of PCR are temporal often not positive after 7 days of onset of infection. IgM detection is possible around this period and up to 21 days. IgG detection is less definitive in acute infections. Here, we report characterization of the sequence diversity of HFRS strains, 3D structure of Hantaan N protein, and B-cell epitopes on this molecule. We predicted a 20 amino acid sequence length peptide by using BepiPred online server in IEDB analysis resource program. We suggest this peptide may be used for development of geographic region-specific immunoassays like EIAs for antibody detection, monoclonal antibody development, and immunoblots (line immunoassay). J. Cell. Biochem. 118: 1182-1188, 2017. © 2016 Wiley Periodicals, Inc.