Ras hyperactivation versus overexpression : Lessons from Ras dynamics in Candida albicans

We thank Prof. Neta Dean for the CIp10ADH1-Cherry plasmid and Prof. Aaron Mitchell for the BWP17 strain. We gratefully acknowledge Prof. Sudipta Maiti, TIFR, Mumbai, India for providing the data acquisition software. We also appreciate the feedback and discussions with Dr. Rohini Muthuswami, SLS, JNU as well as from the Protein Society group, New Delhi while this study was taking shape. We thank Prof. Alok Bhattacharya for Cytochalasin D. The GC-MS and fluorescence lifetime measurements were carried out at the Advanced Instrumentation Research Facility (AIRF), JNU. Confocal images were recorded either at the central instrumentation facility (CIF), SLS, JNU or at AIRF, JNU. This work was supported by project grants from Department of Biotechnology (DBT, Project grant no. BT/PR20410/BRB/10/1542/2016) and Department of Science and Technology (DST, Project grant no. SB/SO/BB-011/2014), India to S.S.K; and project grants from Department of Information Technology, (DIT, Project grant no. 12(4)/2007-PDD), India to S.S. for FCS setup. In addition, both S.S. and S.S.K. thank DBT-BUILDER for funding support (Project grant no. BT/PR5006/INF/153/2012). S.S.K. also acknowledges funding support from UGC Resource Networking grant to the School of Life Sciences. We thank DST-PURSE and JNU for assistance with funding for publication. G.S.V. and S.C.S. received a fellowship from UGC; V.A.P., B.Y., P.J., N.P., M.F.K. acknowledge CSIR for fellowships. S.L.S. received a fellowship from ICMR. D.T.H. and M.F.K. thank DBT-BUILDER for funding.Peer reviewedPublisher PD

MKKK20 works as an upstream triple-kinase of MKK3-MPK6-MYC2 module in Arabidopsis seedling development

The mitogen-activated protein kinase (MAPK) cascade is involved in several signal transduction processes in eukaryotes. Here, we report a mechanistic function of MAP kinase kinase kinase 20 (MKKK20) in light signal transduction pathways. We show that MKKK20 acts as a negative regulator of photomorphogenic growth at various wavelengths of light. MKKK20 not only regulates the expression of light signaling pathway regulatory genes but also gets regulated by the same pathway genes. The atmyc2 mkkk20 double mutant analysis shows that MYC2 works downstream to MKKK20 in the regulation of photomorphogenic growth. MYC2 directly binds to the promoter of MKKK20 to modulate its expression. The protein-protein interaction study indicates that MKKK20 physically interacts with MYC2, and this interaction likely suppresses the MYC2-mediated promotion of MKKK20 expression. Further, the protein phosphorylation studies demonstrate that MKKK20 works as the upstream kinase of MKK3-MPK6-MYC2 module in photomorphogenesis

Mul-IBS: A Multivariate Identity-Based Signature Scheme Compatible with IoT-based NDN Architecture

It has been forty years since the TCP/IP protocol blueprint, which is the core of modern worldwide Internet, was published. Over this long period, technology has made rapid progress. These advancements are slowly putting pressure and placing new demands on the underlying network architecture design. Therefore, there was a need for innovations that can handle the increasing demands of new technologies like IoT while ensuring secrecy and privacy. It is how Named Data Networking (NDN) came into the picture. NDN enables robust data distribution with interest-based content retrieval and leave-copy-everywhere caching policy. Even though NDN has surfaced as a future envisioned and decisive machinery for data distribution in IoT, it suffers from new data security challenges like content poisoning attacks. In this attack, an attacker attempts to introduce poisoned content with an invalid signature into the network. Given the circumstances, there is a need for a cost-effective signature scheme, requiring inexpensive computing resources and fast when implemented. An identity-based signature scheme (IBS) seems to be the natural choice to address this problem. Herein, we present an IBS, namely Mul-IBS relying on multivariate public key cryptography (MPKC), which leads the race among the post-quantum cryptography contenders. A 5-pass identification scheme accompanying a safe and secure signature scheme based on MPKC works as key ingredients of our design. Our Mul-IBS attains optimal master public key size, master secret key size, and user’s secret key size in the context of multivariate identity-based signatures. The proposed scheme Mul-IBS is proven to be secure in the model “existential unforgeability under chosen-message and chosen identity attack (uf-cma)” contingent upon the fact that Multivariate Quadratic (MQ) problem is NP-hard. The proposed design Mul-IBS can be utilized as a crucial cryptographic building block to build a robust and resilient IoT-based NDN architecture

Thermostable Direct Hemolysin Downregulates Human Colon Carcinoma Cell Proliferation with the Involvement of E-Cadherin, and β-Catenin/Tcf-4 Signaling

BACKGROUND: Colon cancers are the frequent causes of cancer mortality worldwide. Recently bacterial toxins have received marked attention as promising approaches in the treatment of colon cancer. Thermostable direct hemolysin (TDH) secreted by Vibrio parahaemolyticus causes influx of extracellular calcium with the subsequent rise in intracellular calcium level in intestinal epithelial cells and it is known that calcium has antiproliferative activity against colon cancer. KEY RESULTS: In the present study it has been shown that TDH, a well-known traditional virulent factor inhibits proliferation of human colon carcinoma cells through the involvement of CaSR in its mechanism. TDH treatment does not induce DNA fragmentation, nor causes the release of lactate dehydrogenase. Therefore, apoptosis and cytotoxicity are not contributing to the TDH-mediated reduction of proliferation rate, and hence the reduction appears to be caused by decrease in cell proliferation. The elevation of E-cadherin, a cell adhesion molecule and suppression of β-catenin, a proto-oncogene have been observed in presence of CaSR agonists whereas reverse effect has been seen in presence of CaSR antagonist as well as si-RNA in TDH treated cells. TDH also triggers a significant reduction of Cyclin-D and cdk2, two important cell cycle regulatory proteins along with an up regulation of cell cycle inhibitory protein p27(Kip1) in presence of CaSR agonists. CONCLUSION: Therefore TDH can downregulate colonic carcinoma cell proliferation and involves CaSR in its mechanism of action. The downregulation occurs mainly through the involvement of E-cadherin-β-catenin mediated pathway and the inhibition of cell cycle regulators as well as upregulation of cell cycle inhibitors

Confined environment facilitates stacked conformations in Holliday Junction

Holliday Junction (HJ) is an important intermediate in homologous recombination and is involved in DNA break repair. It is a highly dynamic structure and transitions between two stacked isomers through an intermediate open conformer. However, the insight into the effect of environmental confinement on HJ conformation population is incomplete. The understanding would be an important bridge between studies generally carried out in aqueous buffers and confined environments, as found in vivo. By employing fluorescence-based techniques we investigated the effect of confinement on HJ conformation using reverse micelle encapsulation as a model for confined space. We observed that inside the confinement of reverse micelle, HJ prefers to adopt stacked conformation. Most strikingly, even at lower divalent cation concentrations, the preference for stacked conformation is prevalent over an open conformation. The conformational heterogeneity also reduces with increasing degree of confinement. Our finding suggests that such confinement-induced changes in the conformer population might influence the interaction and activity of the HJ-recognizing proteins in the cellular environment

Outer Membrane Protein A (OmpA) of Shigella flexneri 2a, Induces Protective Immune Response in a Mouse Model

Background: In our earlier studies 34 kDa outer membrane protein (OMP) of Shigella flexneri 2a has been identified as an efficient immunostimulant. Key Results: In the present study MALDI-TOF MS analysis of the purified 34 kDa OMP of Shigella flexneri 2a shows considerable sequence homology (Identity 65%) with the OmpA of S. flexneri 2a. By using the specific primers, the gene of interest has been amplified from S. flexneri 2a (N.Y-962/92) genomic DNA, cloned in pET100/D-TOPOH vector and expressed using induction with isopropyl thiogalactoside (IPTG) for the first time. Immunogenicity and protective efficacy of the recombinant OmpA has been evaluated in an intranasally immunized murine pulmonary model. The recombinant protein induces significantly enhanced protein specific IgG and IgA Abs in both mucosal and systemic compartments and IgA secreting cells in the systemic compartment (spleen). The mice immunized with OmpA have been protected completely from systemic challenge with a lethal dose of virulent S. flexneri 2a. Immunization with the protein causes mild polymorphonuclear neutrophil infiltration in the lung, without inducing the release of large amounts of proinflammatory cytokines. Conclusion: These results suggest that the OmpA of S. flexneri 2a can be an efficacious mucosal immunogen inducing protective immune responses. Our findings also demonstrate that antibodies and Th1 immune response may be associate

Understanding growth kinetics of nanorods in microemulsion: a combined fluorescence correlation spectroscopy, dynamic light scattering, and electron microscopy study

Even though nanostructures of various shapes and sizes can be controlled by microemulsions, there is substantial difficulty in understanding their growth mechanism. The evolution of nanostructures from the time of mixing of reactants to their final stage is a heterogeneous process involving a variety of intermediates. To obtain a deeper insight into these kinetic steps, we studied the slow growth kinetics (extending over eight days) of iron oxalate nanorods inside the polar core of water-in-oil microemulsion droplets made of cetyltrimethylammonium bromide/1-butanol/isooctane. Fluorescence correlation spectroscopy (FCS), dynamic light scattering (DLS), and transmission electron microscopy (TEM) have been employed to monitor the nanostructure growth at (near) the single-droplet level and in an ensemble. Analyzing FCS data with suitable kinetic model we obtain transient dimer lifetime (28 μs) and the droplet fusion rates (and fusion tendency) on each day as the reaction proceeds. The droplet fusion rate is found to directly control the nanorod growth in microemulsion solution and attains its maximum value (3.55 × 10⁴ s^–1) on day 6, when long nanorods are found in TEM data, implying that more and more reactants are fed into the growing system at this stage. Combining FCS, DLS, and TEM results, we find three distinct periods in the entire growth process: a long nucleation-dominant nanoparticle growth period which forms nanoparticles of critical (average) size of ∼53 nm, followed by a short period where isotropic nanoparticles switch to anisotropic growth to form nanorods, and finally elongation of nanorods and growth (and shrinking) of nanoparticles

Hydrothermal synthesis of defect-induced pristine alpha-NaCe(WO4)(2): a novel material for solid state lighting and gas sensing

Triclinic NaCe(WO4)(2) with oxygen monovacancies and divacancies has been successfully prepared via a facile cetyltrimethyl ammonium bromide (CTAB)-assisted hydrothermal technique. X-ray diffraction, scanning electron microscopy, and transmission electron microscopy have been employed to determine the unit cell and microstructure of the NaCe(WO4)(2). The oxygen vacancies, structural distortion etc. have been investigated using Fourier-transform infrared, Raman and X-ray photoelectron spectroscopies. The synthesized samples exhibit an intense blue emission at 434 nm due to the 5d-4f transition of Ce3+ within the CeO8 dodecahedra, while the emission at 485 nm is ascribed to the 5d-4f transition within CeO7. It has also been identified that two emissions at 451 and 520 nm come from CeO6. Additionally, we find that the temperature of the hydrothermal reaction guides the formation of CeO7 and CeO6. In contrast to a previous ethylenediamine tetraacetic acid (EDTA)-assisted synthesis of NaCe(WO4)(2) that results in a predominant green emission, our samples exhibit strong violet emissions indicating that less CeO7 and CeO6 is formed when using CTAB. We have also conducted ab initio calculations using density-functional theory, which reveals that the valence and conduction bands comprise of the O(2)p orbitals and a O(2)p-Ce 5d hybridization, respectively. The Ce(5)dz(2), 5dyz and 5dxz orbitals mostly facilitate the 5d-4f transition within the CeO7 and CeO6 polyhedra. Commission Internationale de I'Eclairage coordinates are found in the blue region with a correlated color temperature (CCT) of similar to 7715 K indicating the potential for a-NaCe(WO4)(2) to be used in cold solid state lighting applications. Finally, we also observe that the oxygen vacancies can act as active centers for the adsorption of molecular oxygen, which in consequence leads NaCe(WO4) 2 to have gas sensing properties

Thermostable Direct Hemolysin Downregulates Human Colon Carcinoma Cell Proliferation With the Involvement of E-Cadherin, and β-Catenin/Tcf-4 Signaling

Background: Colon cancers are the frequent causes of cancer mortality worldwide. Recently bacterial toxins have received marked attention as promising approaches in the treatment of colon cancer. Thermostable direct hemolysin (TDH) secreted by Vibrio parahaemolyticus causes influx of extracellular calcium with the subsequent rise in intracellular calcium level in intestinal epithelial cells and it is known that calcium has antiproliferative activity against colon cancer. Key Results: In the present study it has been shown that TDH, a well-known traditional virulent factor inhibits proliferation of human colon carcinoma cells through the involvement of CaSR in its mechanism. TDH treatment does not induce DNA fragmentation, nor causes the release of lactate dehydrogenase. Therefore, apoptosis and cytotoxicity are not contributing to the TDH-mediated reduction of proliferation rate, and hence the reduction appears to be caused by decrease in cell proliferation. The elevation of E-cadherin, a cell adhesion molecule and suppression of β-catenin, a proto-oncogene have been observed in presence of CaSR agonists whereas reverse effect has been seen in presence of CaSR antagonist as well as si-RNA in TDH treated cells. TDH also triggers a significant reduction of Cyclin-D and cdk2, two important cell cycle regulatory proteins along with an up regulation of cell cycle inhibitory protein p27Kip1 in presence of CaSR agonists. Conclusion: Therefore TDH can downregulate colonic carcinoma cell proliferation and involves CaSR in its mechanism of action. The downregulation occurs mainly through the involvement of E-cadherin-β-catenin mediated pathway and the inhibition of cell cycle regulators as well as upregulation of cell cycle inhibitors. © 2011 Chowdhury et al

Characterization of the recombinant his-tag OmpA.

<p><b>A:</b> Immunoblotting analysis of the OmpA using mouse polyclonal antibody raised against the recombinant OmpA. Lane 1, Lonza ProSieve® Color Protein molecular weight marker; lane 2, recombinant BL21 Star™(DE3) <i>E. Coli</i> with 0.25 mM IPTG; lane 3, <i>S. flexneri 2a</i> whole-cell lysate. <b>B:</b> Immunoblot analysis of whole-cell preparations of <i>Shigella</i> spp., with <i>S. flexneri</i> 2a OmpA murine antiserum. <i>S. flexneri</i> 2a OmpA antiserum was used to probe the whole-cell antigen of <i>S. flexneri</i> 2a (lane 1), <i>S. boydii</i> (lane 2), <i>S. sonnei</i> (lane 3), <i>S. dysenteriae</i> type 1 (lane 4). Molecular mass standards are noted on the left. C: Western blot analysis of recombinant his-tag OmpA of <i>S. flexneri</i> 2a with convalescent-phase serum of shigellosis patients. OmpA of <i>S. flexneri</i> 2a was electrophoresed and transferred to nitrocellulose membrane and finally probed with 21-day-old convalescent sera obtained from two patients (lane 1 to 3). Lane 1. Normal serum, Lane 2. Patient-1 serum, Lane 3. Patient-2 serum. Western blot analysis of recombinant OmpA of <i>S. flexneri</i> 2a with antisera from virulent <i>Shigella flexneri</i> 2a challenged mice (lane 4) and immunoblot analysis of recombinant his-tag OmpA antiserum with the whole-cell lysate of enteropathogenic <i>E. coli</i> O115 (lane 5). Molecular mass standards are noted on the left (Lonza ProSieve® Color Protein molecular weight marker).</p