Grain size stabilization of Fe and Fe-Ni nanostructures developed by mechanical alloying

The mechanical alloying (MA) technique has been used to prepare nanocrystalline Fe, Fe-5wt. %Ni, 7 wt. % Ni and 10 wt. % Ni alloys from elemental powder mixtures. Fe-5wt. %Ni and Fe-7 wt. % Ni were detected to form complete solid solution after MA for 1500 min. as per the X-ray diffraction (XRD) analysis. Out these two solid solutions, Fe-7 wt. % Ni alloy was chosen to investigate its grain size stabilization by addition of minute amount of yttrium. Fe is also stabilized by yttrium. All the alloys were annealed at different temperatures up to 1200ºC under high purity argon atmosphere. XRD analysis and microhardness measurement were carried out for all the specimens to analyze their stability after annealing. XRD analysis was carried out to measure the lattice parameter variation due to alloying, which is a vital data to detect whether the solid solution has been formed. Crystallite size was calculated by using Scherer formula and the value of the as-milled sample was below 15 nm. Atomic force microscopy would also be carried for selected specimens to investigate the microstructural features. The experiment showed yttrium stabilizes Fe and Fe-Ni grain size at higher temperature in nano range. Annealing decreases lattice strain for annealing temperature below 900°C but it suddenly increases while annealing at higher temperature for ball milled Fe. With the increase in Ni content upto 5 wt.% lattice strain increases for all annealing temperature except at 700°C and 1200°C. This in term increases its hardness and hence other mechanical properties. Stable crystallite size (less than 100 nm) and constant microhardness of the annealed samples can dictate its usefulness for high strength and other functional applications. Hardness of as milled sample of stabilized Fe obtained to be 9.933 GPa and of Fe‐7Ni to be 5.99 GPa. Crystallite size increases with increase in temperature. It becomes stable at higher temperature for Fe-Ni

Antibacterial, antioxidant and phytochemical screening of Hibiscus rosa sinensis, Acorus calamus, Cucurbita maxima and Moringa oliefera

A large number of the plants are claimed to possess the antibiotic properties in the traditional system and are also used extensively by the tribal people throughout the world. It is now mostly thought that nature has given the cure of every disease in one form or another. Plants have been known to cure people from various diseases in Ayurveda. Effects of crude extracts of the petals of Hibiscus rosa sinensis, cucurbita maxima, leaves of Moringa oliefera and rhizome of Acorus calamus were studied for the antibacterial and antioxidant activity. The research showed that the methanolic extract were more effective than the aqueous extract. The antioxidant activity of the four samples were carried out using DPPH which showed that out of the four plants Moringa oliefera and Acorus Calamus has better antioxidant properties which could be very useful against reactive oxygen species that are formed during oxidative stress

Visualization of Intracellular Transport of Vesicular Stomatitis Virus Nucleocapsids in Living Cells

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the viral RNA polymerase. In previous studies, we demonstrated that insertion of 19 amino acids in the hinge region of the protein had no significant effect on P protein function. In the present study, we inserted full-length enhanced green fluorescent protein (eGFP) in frame into the hinge region of P and show that the fusion protein (PeGFP) is functional in viral genome transcription and replication, albeit with reduced activity. A recombinant vesicular stomatitis virus encoding PeGFP in place of the P protein (VSV-PeGFP), which possessed reduced growth kinetics compared to the wild-type VSV, was recovered. Using the recombinant VSV-PeGFP, we show that the viral replication proteins and the de novo-synthesized RNA colocalize to sites throughout the cytoplasm, indicating that replication and transcription are not confined to any particular region of the cytoplasm. Real-time imaging of the cells infected with the eGFP-tagged virus revealed that, following synthesis, the nucleocapsids are transported toward the cell periphery via a microtubule (MT)-mediated process, and the nucleocapsids were seen to be closely associated with mitochondria. Treatment of cells with nocodazole or Colcemid, drugs known to inhibit MT polymerization, resulted in accumulation of the nucleocapsids around the nucleus and also led to inhibition of infectious-virus production. These findings are compatible with a model in which the progeny viral nucleocapsids are transported toward the cell periphery by MT and the transport may be facilitated by mitochondria

Migration of cytotoxic lymphocytes in cell cycle permits local MHC I–dependent control of division at sites of viral infection

Virus-specific cytotoxic CD8+ T cells are in cell cycle as they transit from lymphoid tissues to sites of infection

Role of vesicular stomatitis virus nucleocapsid (N) protein in regulating N-RNA template functions

The nucleocapsid (N) protein and the phosphoprotein (P) of VSV play critical roles in viral gene expression. The N protein encapsidates the viral RNA to form N-RNA or nucleocapsid (NC) complex that serves as the template for genome transcription and replication by the viral RNA-dependent RNA polymerase (RdRp), a complex of the viral P and L proteins. To examine the NCs functions in infected cells, the NCs were fluorescently tagged. This was attempted by replacing the coding regions of VSV N and P proteins with those for enhanced green fluorescence protein-fused N (NeGFP) or eGFP-fused P (PeGFP) proteins. Although multiple attempts to recover recombinant VSV encoding NeGFP were unsuccessful, recombinant VSV encoding PeGFP (VSV-PeGFP) was readily recovered. The NCs of VSV-PeGFP could be visualized as green in infected cell cytoplasm since PeGFP was associated with the NCs. Using VSV-PeGFP, we demonstrated that the cellular microtubules are involved in intracellular trafficking of newly synthesized viral NCs. To examine the role of the N protein in template functions of N-RNA complex, we identified a highly conserved region spanning residues (282)GLSSKSPYSS(291) in the N protein of Rhabdoviruses and subjected it to alanine-scanning mutagenesis. Analysis of the mutants demonstrated that the tyrosine residue at position 289 (Y289) is critical for replication functions of the protein. To determine the molecular basis of the defect in Y289A mutant in replication, we examined the interaction of the N protein with itself, with the P protein, and its ability to encapsidate the viral RNA. The mutant Y289A was found to interact with the N protein efficiently but its interaction with the P protein or with the viral RNA was defective. We have also observed that several single amino acid substitutions in this highly conserved region of N, rendered the resulting nucleocapsid template nonfunctional in transcription without adversely affecting replication functions. These results suggest that the structure of the N protein and the resulting N-RNA complex regulate the viral template functions in transcription and replication

Ethosomal Nano-carriers Encapsulating Novel Green Synthesized AgNPs and Sericin Protein as Potential Therapeutics against Skin Carcinoma

UV radiations are the main etiological factor that contributes to the induction of skin carcinoma. The incidence of skin carcinoma is increasing at an alarming rate both in the Indian and worldwide scenario. Due to the ignorance and socioeconomic conditions, most of the non-melanoma skin cancer (NMSC) cases are not registered in the national and state cancer registry. Therefore, development of new therapeutics having higher efficacy along with skin regenerative properties is one of the important issues in today’s rapid and progressive world. In this study, novel and economic resources were selected for developing patient-friendly non-toxic therapeutics against skin carcinoma. Four chitosan formulations were prepared using silver nanoparticles (AgNPs) synthesized through biological routes and silk sericin protein. Similarly, four ethosomal formulations were prepared by encapsulating the previously prepared chitosan formulations to overcome the barrier function provided by the stratum corneum. The prepared formulations were characterized through high throughput techniques such as DLS, FE-SEM, TEM, ATR-FT-IR, and XRD. The hemolysis and hemagglutination assays proved the hemocompatibility of the prepared formulations upon incubation with RBCs. The MTT assay on A431 skin carcinoma and normal HaCaT cells showed their dose-dependent cytotoxicity. DCFH-DA, Rhodamine 123 staining provided an insight into the in vitro ROS production and mitochondrial membrane depolarizing capabilities of the prepared formulations. The morphological changes in the cell membrane, depolarization of mitochondrial membrane potential, DNA fragmentation, and annexin V-FITC staining confirms apoptosis being the primary cause of death in the chitosan and ethosomes treated samples. The time-dependent increase in intracellular production of superoxide and nitric oxide radicals confirmed that ROS are the primary source for triggering the cascade for the destruction of the cancer cells. The in vivo immune response studies in mice model showed the triggering of T cell markers in response to the exposure of prepared chitosan and ethosomal formulation

Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells▿

A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-ΔM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-ΔM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min

Single-Amino-Acid Alterations in a Highly Conserved Central Region of Vesicular Stomatitis Virus N Protein Differentially Affect the Viral Nucleocapsid Template Functions▿

The nucleocapsid protein (N) of vesicular stomatitis virus and other rhabdoviruses plays a central role in the assembly and template functions of the viral N-RNA complex. The crystal structure of the viral N-RNA complex suggests that the central region of the N protein interacts with the viral RNA. Sequence alignment of rhabdovirus N proteins revealed several highly conserved regions, one of which spanned residues 282 to 291 (GLSSKSPYSS) in the central region of the molecule. Alanine-scanning mutagenesis of this region suggested that replacement of the tyrosine residue at position 289 (Y289) with alanine resulted in an N-RNA template that is nonfunctional in viral genome replication and transcription. To establish the molecular basis of this defect, our further studies revealed that the Y289A mutant maintained its interaction with other N protein molecules but that its interactions with the P protein or with the viral RNA were defective. Replacement of Y289 with other aromatic, polar, or large amino acids indicated that the hydrophobic and aromatic nature of this position in the N protein is functionally important and that a larger aromatic residue is less favorable. Interestingly, we have observed that several single-amino-acid substitutions in this highly conserved region of the molecule rendered the nucleocapsid template nonfunctional in transcription without adversely affecting the replication functions. These results suggest that the structure of the N protein and the resulting N-RNA complex, in part, regulate the viral template functions in transcription and replication

IFN-I Independent Antiviral Immune Response to Vesicular Stomatitis Virus Challenge in Mouse Brain

Type I interferon (IFN-I) plays a pivotal role during viral infection response in the central nervous system (CNS). The IFN-I can orchestrate and regulate most of the innate immune gene expression and myeloid cell dynamics following a noncytopathic virus infection. However, the role of IFN-I in the CNS against viral encephalitis is not entirely clear. Here we have implemented the combination of global differential gene expression profiling followed by bioinformatics analysis to decipher the CNS immune response in the presence and absence of the IFN-I signaling. We observed that vesicular stomatitis virus (VSV) infection induced 281 gene changes in wild-type (WT) mice primarily associated with IFN-I signaling. This was accompanied by an increase in antiviral response through leukocyte vascular patrolling and leukocyte influx along with the expression of potent antiviral factors. Surprisingly, in the absence of the IFN-I signaling (IFNAR−/− mice), a significantly higher (1357) number of genes showed differential expression compared to the WT mice. Critical candidates such as IFN-γ, CCL5, CXCL10, and IRF1, which are responsible for the recruitment of the patrolling leukocytes, are also upregulated in the absence of IFN-I signaling. The computational network analysis suggests the presence of the IFN-I independent pathway that compensates for the lack of IFN-I signaling in the brain. The analysis shows that TNF-α is connected maximally to the networked candidates, thus emerging as a key regulator of gene expression and recruitment of myeloid cells to mount antiviral action. This pathway could potentiate IFN-γ release; thereby, synergistically activating IRF1-dependent ISG expression and antiviral response