Identification of the potential regions of Epap-1 that interacts with V3 loop of HIV-1 gp120

Early pregnancy associated protein-1 (Epap-1), a 90 kDa glycoprotein present in first trimester placental tissue, inhibits HIV-1 entry through interaction with HIV-1 gp120 at V3 and C5 regions. In the present study, we have identified the specific 32 mer region of Epap-1 that can interact with V3 loop. This was achieved by docking between Epap-1 molecular model and gp120 and studying the interaction of peptides with gp120 in vitro. Out of four peptides analyzed, two peptides (P-2 and P-3) showed significant interaction with V3 domain (N = 8; N = 7) of gp120. In the studies conducted using soluble gp120 and virus, peptide P-2 has shown conserved interaction at V3 loop regions recognized by 257D and F425 antibodies and higher anti-viral activity. Also, P-2 inhibited cell fusion mediated dye transfer between gp120 expressing HL2/3 and CD4 expressing Sup T1 cells suggesting its inhibition of viral entry, which is further confirmed by its action on HIV infection mediated by Tat activated beta gal expression in TZM-bl cells. Further optimization of P-2 peptide showed that the anti-viral activity and gp120 interaction residues lie in the N-terminal region of the peptide. These results together suggest that P-2 inhibits viral entry through specific interaction at V3 loop region.     

FOXO1 promotes wound healing through the up-regulation of TGF-β1 and prevention of oxidative stress

Keratinocyte mobilization is a critical aspect of wound re-epithelialization, but the mechanisms that control its precise regulation remain poorly understood. We set out to test the hypothesis that forkhead box O1 (FOXO1) has a negative effect on healing because of its capacity to inhibit proliferation and promote apoptosis. Contrary to expectations, FOXO1 is required for keratinocyte transition to a wound-healing phenotype that involves increased migration and up-regulation of transforming growth factor β1 (TGF-β1) and its downstream targets, integrin-α3 and -β6 and MMP-3 and -9. Furthermore, we show that FOXO1 functions in keratinocytes to reduce oxidative stress, which is necessary to maintain cell migration and prevent cell death in a TGF-β1–independent manner. Thus, our studies identify a novel function for FOXO1 in coordinating the response of keratinocytes to wounding through up-regulation of TGF-β1 and other factors needed for keratinocyte migration and protection against oxidative stress, which together promote migration and decrease apoptosis.     

An ecofriendly synthesis and DNA binding interaction study of some pyrazolo [1,5-a]pyrimidines derivatives

The DNA molecule is a target for plethora of anticancer and antiviral drugs that forms covalent and non-covalent adducts with major or minor groove of DNA. In present study we synthesized series of novel Pyrazolo [1,5-a]pyrimidine derivatives. The newly synthesized compounds were characterized by elemental analysis, IR, ¹H NMR, and mass spectral data. The selected compounds were studied for interaction with Calf thymus DNA (CT-DNA) using electronic spectra, viscosity measurement and thermal denaturation studies. Further, molecular interactions were revealed for compound IIIa and IVa by computational methodologies. The preferred mode of ligand binding with double helical DNA as well as preferable DNA groove were explored by molecular docking in different DNA models.     

Modulation of ROS/MAPK signaling pathways by okadaic acid leads to cell death via,mitochondrial mediated caspase-dependent mechanism

Okadaic acid (OA) is a specific and potent protein phosphatase inhibitor and tumor promoter. The present study establishes the role of reactive oxygen species (ROS) and mitogen activated protein kinases in cell death induced by okadaic acid. The study showed that okadaic acid is cytotoxic at 10 nM with an IC50 of 100 nM in U-937 cells. The CVDE assay and mitochondrial dehydrogenase assay showed a time dependent cytotoxicity. The phase contrast visualization of the OA treated cells showed the apoptotic morphology and was confirmed with esterase staining for plasma membrane integrity. OA activated caspases-7, 9 and 3, PARP cleavage and induced nuclear damage in a time and dose dependent manner. Compromised mitochondrial membrane potential, release of cytochrome-c and apoptosis inducing factor confirms the involvement of mitochondria. A time dependent decrease in glutathione levels and a dose dependent increase in ROS with maximum at 30 min were observed. ROS scavenger-N-acetyl cysteine, mitochondrial stabilizer-cyclosporin-A, and broad spectrum caspase inhibitor Z-VAD-FMK inhibited the OA induced caspase-3 activation, DNA damage and cell death but caspase-8 inhibitor had no effect. OA activated p38 MAPK and JNK in a time dependent manner, but not ERK½. MAP kinase inhibitors SB203580, SP600125 and PD98059 confirm the role of p38 MAPK and JNK in OA induced caspase-3 activation and cell death. Over all, our results indicate that OA induces cell death by generation of ROS, and activation of p38 MAPK and JNK, and executed through mitochondrial mediated caspase pathway.     

Influence of recirculation on the performance of anaerobic sequencing batch biofilm reactor (AnSBBR) treating hypersaline composite chemical wastewater

Influence of recirculation on the performance of anaerobic sequencing batch biofilm reactor (AnSBBR) was studied in the process of treating hypersaline (total dissolved inorganic solids (TDIS) approximately 26 g/l) and low biodegradable (BOD/COD approximately 0.3) composite chemical wastewater. Significant enhancement in the substrate removal efficiency and biogas yield was observed after introducing the recirculation to the system. Maximum efficiency (COD removal efficiency - 51%; SDR - 3.14 kg COD/cum-day) was observed at recirculation to feed (R/F) ratio of 2 (OLR - 6.15 kg C OD/cum-day; HLR - 2.30 cum (liquid)/cum day; UFV(A) - 0.023 m/h). Subsequent increase of R/F to 3 (OLR - 6.15 kg COD/cum-day; HLR - 3.07cum (liquid)/cum-day; UFV(A) - 0.035 m/h) resulted in reduction in COD removal efficiency (32%; SDR - 1.97 kg COD/cum-day). The enhanced performance of the system due to the introduction of recirculation was attributed to the improvement in the mass transfer between the substrate present in the bulk liquid and the attached biofilm. The hydrodynamic behavior due to recirculation mode of operation reduced the concentration gradient (substrate inhibition) of substrate and reaction by-products (VFA) resulting in mixed flow conditions.     

Direct shoot bud induction and plant regeneration in <Emphasis Type="Italic">Capsicum frutescens</Emphasis> Mill.: influence of polyamines and polarity

Direct shoot bud induction and plant regeneration was achieved in Capsicum frutescens var. KTOC. Aseptically grown seedling explants devoid of roots, apical meristem and cotyledons were inoculated in an inverted position in medium comprising of Murashige and Skoog (Physiol Plant 15:472–497, 1962) basal medium supplemented with 2-(N-morpholine) ethanesulphonic acid buffer along with 2.28 μM indole-3-acetic acid, 10 μM silver nitrate and either of 13.31–89.77 μM benzyl adenine (BA), 9.29–23.23 μM kinetin, 0.91–9.12 μM zeatin, 2.46–9.84 μM 2-isopentenyl adenine. Profuse shoot bud induction was observed only in explants grown on a media supplemented with BA (26.63 μM) as a cytokinin source and 19.4 ± 4.2 shoot buds per explant was obtained in inverted mode under continuous light. Incorporation of polyamine inhibitors in the culture medium completely inhibited shoothoot bud induction. Incorporation of exogenous polyamines improved the induction of shoot buds under 24 h photoperiod. These buds were elongated in MS medium containing 2.8 μM gibberellic acid. Transfer of these shoots to hormone-free MS medium resulted in rooting and rooted plants were transferred to fields. This protocol can be efficiently used for mass propagation and presumably also for regeneration of genetically transformed C. frutescens.     

Activation of anti-tumor immune response and reduction of regulatory T cells with Mycobacterium indicus pranii (MIP) therapy in tumor bearing mice

Background

Role of immune system in protecting the host from cancer is well established. Growing cancer however subverts immune response towards Th2 type and escape from antitumor mechanism of the host. Activation of both innate and Th1 type response is crucial for host antitumor activity. In our previous study it was found, that Mycobacterium indicus pranii (MIP) also known as M. w induces Th1 type response and activates macrophages in animal model of tuberculosis. Hence, we studied the immunotherapeutic potential of MIP in mouse tumor model and the underlying mechanisms for its antitumor activity.

Methodology and Principal Findings

Tumors were implanted by injecting B16F10 melanoma cells subcutaneously into C57BL/6 mice. Using the optimized dose and treatment regimes, anti-tumor efficacy of heat killed MIP was evaluated. In MIP treated group, tumor appeared in only 50–60% of mice, tumor growth was delayed and tumor volume was less as compared to control. MIP mediated immune activation was analysed in the tumor microenvironment, tumor draining lymph node and spleen. Induction of Th1 response and higher infiltration of immune cells in the tumor microenvironment was observed in MIP treated mice. A large fraction of these immune cells were in activated state as confirmed by phenotypic and functional analysis. Interestingly, percentage of Treg cells in the tumor milieu of treated mice was less. We also evaluated efficacy of MIP along with chemotherapy and found a better response as compared to chemotherapy alone.

Conclusion

MIP therapy is effective in protecting mice from tumor. It activates the immune cells, increases their infiltration in tumor, and abrogates tumor mediated immune suppression.     

Supersensitive detection of T-2 toxin by the in situ synthesized π-conjugated molecularly imprinted nanopatterns. An in situ investigation by surface plasmon resonance combined with electrochemistry

A π-conjugated molecularly imprinted polymer (MIP) with nanopatterns for T-2 toxin (T-2) was prepared on SPR chip by in situ electropolymerization of 3-aminophenylboronicacid (3-APBA) with T-2. The complete removal of T-2 from polymer was confirmed in situ by SPR and EIS and also ex situ by SEM, EDAX, fluorescence microscopy and Raman spectroscopy. SEM image of T-2 MIP exhibited nanopatterns due to imprinting of T-2. The MIP of T-2 showed a linear response for T-2 from 2.1 fM to 33.6 fM with a detection limit of 0.1 fM (0.05 pg/mL). In this study, thermodynamic parameters such as change in Gibb's free energy (ΔG), change in enthalpy (ΔH) and change in entropy (ΔS) were determined and the values revealed that the interaction between T-2 and T-2 MIP as spontaneous, endothermic and entropy driven one. Moreover, interactions of very high concentration of interferents with T-2 MIP showed very less response due to the presence of nanopatterns of T-2 in the T-2 MIP. Equilibrium constant (12.7 fM) obtained in this study indicates the super binding affinity of T-2 with T-2 MIP. Moreover, the present methodology provides an outline to develop field-detection equipment capable of detecting T-2 toxin at or well below the guideline concentrations recommended by American subcommittee on military field drinking water.     

Elotuzumab directly enhances NK cell cytotoxicity against myeloma via CS1 ligation: evidence for augmented NK cell function complementing ADCC

Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1, a cell surface glycoprotein expressed on MM cells. In preclinical models, elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein, we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1–CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary, human MM cells. Taken together, these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone.     

Bioavailability and metabolism of fucoxanthin in rats: structural characterization of metabolites by LC-MS (APCI)

This study reports bioavailability and metabolism of fucoxanthin (FUCO) from brown algae Padina tetrastromatica in rats. Rats were divided into two groups (n = 25/group). Group one was fed basal diet (control) while the group two received retinol deficient diet (RD group) for 8 weeks. After confirmed RD in blood (0.53 μmol/l), rats were further sub-grouped (n = 5/sub group), intubated a dose of FUCO (0.83 μmol) and killed after 0, 2, 4, 6 and 8 h. The plasma levels (area under curve/8 h) of FUCO (fucoxanthinol (FUOH) + amarouciaxanthin (AAx)) was 2.93 (RD group) and 2.74 pmol/dl (control), respectively. No newly formed retinol was detected in RD rats intubated with FUCO. Besides FUOH (m/z 617 (M+H)⁺) and AAx (m/z 617 (M+H^?)⁺), other deacetylated, hydrolyzed and demethylated metabolites of bearing molecular mass at m/z 600.6 (FUOH–H₂O), m/z 597 (AAx–H₂O), m/z 579 (AAx–2H₂O+1), m/z 551 (AAx–2H₂O–2CH₃+2) and m/z 523 (AAx–2H₂O–4CH₃+4) were also detected in plasma and liver by LC-MS (APCI). Although biological functions of FUCO metabolites need thorough investigation, this is the first detailed report on FUCO metabolites in rats.