Fumaric acid incorporated Ag/agar-agar hybrid hydrogel: A multifunctional avenue to tackle wound healing.

Wound and its treatment is one of the major health concerns throughout the globe. Various extrinsic and intrinsic factors can influence the dynamics of healing mechanism. One such extrinsic factor is moist environment in wound healing. The advantages of optimum hydration in wound healing are enhanced autolytic debridement, angiogenesis and accelerated cell proliferation and collagen formation. But hydrated wounds often end up with patient's uncomfortability, associated infection, and tissue lipid peroxidation. Healing process prefers antimicrobial, anti-inflammatory and optimum moist microenvironment. Here, we have synthesized fumaric acid incorporated agar-silver hydrogel (AA-Ag-FA); characterized by UV-Visible spectroscopy, FTIR spectroscopy and TEM. The surface morphology is evaluated through SEM. The size of the silver nanoparticles (Ag NPs) was found to be 10-15 nm. The hydrogel shows potential antibacterial effect against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa which are predominantly responsible for wound infection. The gel shows reasonable antioxidant property evaluated through 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Topical application of the gel on the wound site heals the wound at much faster rate even compared to standard (Mega heal, Composition: Colloidal silver 32 ppm hydrogel) gel. Histological analysis reveals better tissue proliferation (i.e. epithelialization), more granulation tissue formation, neovascularisation, fibroblast and mature collagen bundles. The lipid peroxidation of wound tissue estimated through malondialdehyde (MDA) assay was found to be reasonably less when treated with AA-Ag-FA hydrogel compared to standard (Mega heal). Cytotoxicity of the samples tested through MTT assay and live-dead cell staining shows its nontoxic biocompatibility nature. In our hydrogel scaffold, the bio-degradable agar-agar provides the moist environment; the Ag NPs inside the gel acts as bactericidal agent and fumaric acid facilities the antioxidant and angiogenesis path implicitly.

Enhanced antimicrobial activity of peptide-cocktails against common bacterial contaminants of ex vivo stored platelets.

Bacterial contamination of blood components such as ex vivo-stored platelets is a major safety risk in transfusion medicine. We have recently shown that synthetic antimicrobial peptides named PD1-PD4 derived from the thrombin-induced human platelet-derived antimicrobial proteins, and repeats of Arg-Trp (RW1-RW5) demonstrate microbicidal activity against selected bacteria and viruses. In the present study, we selected PD3, PD4, RW2, RW3 and RW4 and evaluated each individual peptide and their various combinations to see whether the cocktail regimen enhances the antimicrobial activity above and over the individual peptides. Stored platelet or plasma samples spiked with known titres of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Bacillus cereus were treated with either individual peptides or with peptides in various combinations. Analyses revealed that individual peptides show moderate microbicidal activity (10- to 100-fold reduction) against the tested bacteria relative to their combined regimen. The peptide combinations (RW2 + RW4, RW2 + RW3 + RW4 and PD4 + RW3 + RW4) on the other hand enhanced the microbicidal activity (c.10 000-fold reduction) and revealed a minimal inhibitory concentration of 5 µM. Time-kill kinetics indicated that these three peptide combinations exhibited enhanced antimicrobial activity bringing about a 100-fold reduction of bacterial titres within 20 min of incubation. The present study therefore demonstrates the synergistic effect of antimicrobial peptides when used in combinations and provides a proof-of-concept of its potential application as a molecular tool towards pathogen reduction and further extends the possibility of using peptide combinatorial therapeutics as broad-spectrum antibiotics or as alternatives to combat drug-resistant bacteria.

Pneumonitis following diesel fuel siphonage.

Petroleum diesel is a complex mixture of liquid hydrocarbons and mainly used as fuel in transport vehicles. The practice of manual siphoning of diesel from fuel tanks is common in developing countries but hydrocarbon pneumonitis due to diesel siphonage is rarely reported. We report pneumonitis following diesel fuel siphonage in a 30-year-old driver. Initially patient had severe nausea and vomiting followed by chest pain and breathlessness after three days. In our case, induced sputum was diagnostic as against majority of cases where bronchoscopy was used as diagnostic tool. Recovery was complete with medical treatment.

Nimbolide a limonoid from Azadirachta indica inhibits proliferation and induces apoptosis of human choriocarcinoma (BeWo) cells.

We investigated the cytotoxic effects of nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica) on human choriocarcinoma (BeWo) cells. Treatment with nimbolide resulted in dose- and time-dependent inhibition of growth of BeWo cells with IC(50) values of 2.01 and 1.19 microM for 7 and 24 h respectively, accompanied by downregulation of proliferating cell nuclear antigen. Examination of nuclear morphology revealed fragmentation and condensation indicating apoptosis. Increase in the generation of reactive oxygen species (ROS) that was reversed by addition of reduced glutathione suggested ROS involvement in the cytotoxicity of nimbolide. A decrease in Bcl-2/Bax ratio with increased expression of Apaf-1 and caspase-3, and cleavage of poly(ADP-ribose) polymerase provide compelling evidence that nimbolide-induced apoptosis is mediated by the mitochondrial pathway. The results of the present study suggest that nimbolide has immense potential in cancer prevention and therapy based on its antiproliferative and apoptosis inducing effects.

In vitro antioxidative potential of lactoferrin and black tea polyphenols and protective effects in vivo on carcinogen activation, DNA damage, proliferation, invasion, and angiogenesis during experimental oral carcinogenesis.

The present study was designed to evaluate the in vitro antioxidant potential of bovine lactoferrin (bLF) and black tea polyphenols [Polyphenon-B (P-B)] as well as in vivo inhibitory effects on the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinomas. Antioxidant activity was screened using a panel of assays including 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), hydroxyl radical anion (OH*), superoxide anion (O2*-), and nitric oxide (NO) radical scavenging assays as well as assay for reducing power. The chemopreventive potential of bLF and P-B was assessed in the HBP model based on the modulatory effects on DMBA-induced oxidative DNA damage as well as the expression of proteins associated with carcinogen activation (CYP1A1, CYP1B1), cell proliferation [cyclin D1, proliferating cell nuclear antigen (PCNA), glutathione S-transferase pi (GST-P)], angiogenesis [vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR1)], and invasion and metastasis [matrix metalloproteinase-9 (MMP-9) and tissue inhibitors of MMP-2 (TIMP-2)]. Both bLF and P-B showed high radical scavenging activity and reductive potential. Although administration of bLF and P-B alone suppressed DMBA-induced HBP tumors, combined administration of bLF and P-B was more effective in inhibiting HBP carcinogenesis by inhibiting oxidative DNA damage, carcinogen activation, cell proliferation, invasion, and angiogenesis. Our study suggests that the antioxidative property of bLF and P-B may be responsible for chemoprevention of HBP carcinogenesis by modulating multiple molecular targets.

Pretreatment with black tea polyphenols modulates xenobiotic-metabolizing enzymes in an experimental oral carcinogenesis model.

The objective of this study was to evaluate the chemopreventive potential of the black tea polyphenols Polyphenon-B and BTF-35 during the preinitiation phase of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Hamsters were divided into six groups. Animals in groups 2 and 3 received diet containing Polyphenon-B and BTF-35, respectively, 4 weeks before carcinogen administration when they were 6 weeks of age and continued until the final exposure to carcinogen. At 10 weeks of age, animals in groups 1, 2, and 3 were painted with 0.5% DMBA three times a week for 14 weeks. Animals in groups 4 and 5 were given Polyphenon-B and BTF-35 alone, respectively, as in groups 2 and 3. Animals in group 6 served as control. All the animals were sacrificed after an experimental period of 18 weeks. Phase I and phase II xenobiotic-metabolizing enzymes and 8-hydroxy-deoxyguanosine (8-OH-dG) in the buccal pouch and liver were used as biomarkers of chemoprevention. Hamsters painted with DMBA showed increased expression of 8-OH-dG and enhanced activities of phase I (CYP450; total as well as CYP1A1, 1A2, and 2B isoforms and cytochrome b5) and phase II (GST and quinone reductase) xenobiotic-metabolizing enzymes with increased immunohistochemical expression of CYP1A1, and CYP1B1 isoforms in the buccal pouch. This was accompanied by increased phase I and decreased phase II enzyme activities in the liver. Administration of Polyphenon-B and BTF-35 significantly decreased tumor incidence, oxidative DNA damage, phase I enzyme activities as well as expression of CYP1A1 and CYP1B1 isoforms, while enhancing phase II enzyme activities in the buccal pouch and liver. Our results provide a mechanistic basis for the chemopreventive potential of black tea polyphenols. Furthermore, the greater efficacy of BTF-35 in chemoprevention of HBP carcinomas via inhibition of oxidative DNA damage and modulation of xenobiotic-metabolizing enzymes may have a major impact in human oral cancer prevention.

Combination chemoprevention of hamster buccal pouch carcinogenesis by bovine milk lactoferrin and black tea polyphenols.

Combination chemoprevention is a promising approach for oral cancer prevention. The authors evaluated the combined chemopreventive effects of bovine milk lactoferrin (bLF) and black tea polyphenols (Polyphenon-B) in a clinically relevant in vivo model of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Although dietary administration of bLF and Polyphenon-B alone significantly reduced the tumor incidence, combined administration of bLF and polyphenon-B was more effective in inhibiting DMBA-induced genotoxicity and development of HBP carcinomas by modulation of carcinogen-metabolizing enzymes and cellular redox status. These results suggest that a "designer item" approach will be useful for human oral cancer prevention strategies.

In vitro evaluation of the anticancer effect of lactoferrin and tea polyphenol combination on oral carcinoma cells.

We investigated the anticancer effects of green and black tea polyphenols alone and in combination with bovine milk lactoferrin (bLF) on human tongue squamous carcinoma (CAL-27) and normal human gingival fibroblast (HGF) cells. Both green (Polyphenon-E;P-E) and black tea polyphenols (Polyphenon-B;P-B) preferentially inhibit the growth of CAL-27 cells in a dose-dependent manner. Based on the IC(50) values, P-E was found to be more effective than P-B and the combination of P-E and bLF (1:2 ratio) exhibited synergistic inhibition of CAL-27 cells. Analysis of the mechanism revealed nuclear fragmentation and condensation with appearance of the A(o) peak indicative of apoptosis. Furthermore, tea polyphenols transduced the apoptosis signal via generation of reactive oxygen species and decrease in the Bcl-2/Bax ratio thereby inducing mitochondrial permeability transition with consequent activation of caspase-3. Overall, the potency of cytotoxic and apoptosis inducing effects of dietary agents on CAL-27 cells was in the order P-E and bLF combination (1:2 ratio)>P-E>P-B. These results suggest that a "designer" approach may be useful for oral cancer prevention strategies.

Modulatory effects of black tea polyphenols on rat forestomach carcinogenesis.

ABSTRACT The present study was designed to evaluate the chemopreventive effects of black tea polyphenols (Polyphenon-B) on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis in Wistar rats. Intragastric administration of MNNG induced well-differentiated squamous cell carcinomas that showed diminished mitochondrial lipid and protein oxidation and an increase in antioxidants. In contrast to tumor tissue, the liver mitochondria of tumor-bearing animals showed elevated lipid and protein oxidation with compromised antioxidant defenses. Dietary administration of Polyphenon-B effectively suppressed MNNG-induced stomach tumors, modulated mitochondrial lipid and protein oxidation, and enhanced antioxidant enzyme activities in the stomach and liver. Our results suggest that Polyphenon-B may exert its chemopreventive effects by modulating mitochondrial cellular redox status in the tumor as well as in the host liver.

Black tea polyphenols protect against 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis.

Dietary chemoprevention has emerged as a cost-effective approach for cancer control. We evaluated the chemopreventive effects of black tea polyphenols (Polyphenon-B) administration during the preinitiation phase of 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. The expression of proliferating cell nuclear antigen (PCNA) in the buccal pouch and the concentration of lipid peroxides, protein carbonyl, and the antioxidant status in the buccal pouch, liver and erythrocytes were used as biomarkers of chemoprevention. All the hamsters painted with DMBA alone for 14 weeks developed buccal pouch carcinomas associated with increased expression of PCNA, diminished lipid and protein oxidation, and enhanced antioxidant status. In the liver and erythrocytes of tumor-bearing animals, enhanced oxidation of lipids and proteins was accompanied by compromised antioxidant defenses. Dietary administration of Polyphenon-B effectively suppressed DMBA-induced HBP carcinogenesis as revealed by decreased incidence of tumours and PCNA expression. In addition, Polyphenon-B modulated lipid and protein oxidation and enhanced the antioxidant status in the pouch, liver, and erythrocytes. We suggest that Polyphenon-B exerts its chemopreventive effects by inhibiting cell proliferation in the target tissue and modulating the oxidant-antioxidant status in the target as well as in host tissues.

Enhancement of erythrocyte antioxidants by green and black tea polyphenols during 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis.

We evaluated the comparative chemopreventive efficacy of green tea polyphenols (polyphenon-E) and black tea polyphenols (polyphenon-B) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Lipid peroxidation, reduced and oxidized glutathione (GSH and GSSG, respectively), and the GSH-dependent enzymes glutathione peroxidase and glutathione S-transferase in the erythrocytes were used as biomarkers of chemoprevention. Enhanced lipid peroxidation in erythrocytes of DMBA-treated animals was accompanied by a significant decrease in the antioxidant status. Dietary administration of polyphenon-E and -B to DMBA-treated animals significantly decreased the extent of lipid peroxidation and enhanced the levels of GSH, GSH/GSSG ratio, and activities of GSH-dependent enzymes. Our study provides evidence that polyphenon-B is more effective in inhibiting HBP carcinogenesis than polyphenon-E by enhancing the antioxidant status, suggesting that polyphenon-B may have a major impact in the chemoprevention of oral cancer.

Protein disulfide isomerase assisted protein folding in malaria parasites.

In eukaryotes, the formation of protein disulfide bonds among cysteine residues is mediated by protein disulfide isomerases and occurs in the highly oxidised environment of the endoplasmic reticulum. This process is poorly understood in malaria parasites. In this paper, we report the gene isolation, sequence and phylogenetic comparisons, protein structure and thioredoxin-domain analyses of nine protein disulfide isomerases-like molecules from five species of malaria parasites including Plasmodium falciparum and Plasmodium vivax (human), Plasmodium knowlesi (simian) and Plasmodium berghei and Plasmodium yoelii (murine). Four of the studied protein disulfide isomerases belong to P. falciparum malaria and have been named PfPDI-8, PfPDI-9, PfPDI-11 and PfPDI-14, based on their chromosomal location. Among these, PfPDI-8 bears the closest similarity to a prototype PDI molecule with two thioredoxin domains (containing CGHC active sites) and a C-terminal Endoplasmic reticulum retrieval signal, SEEL. PfPDI-8 is expressed during all stages of parasite life cycle and is highly conserved (82-96% identity at amino acid level) in the other four Plasmodium species studied. Detailed biochemical analysis of PfPDI-8 revealed that this molecule is a potent oxido-reductase enzyme that facilitated the disulfide-dependent conformational folding of EBA-175, a leading malaria vaccine candidate. These studies open the avenues to understand the process of protein folding and secretory pathway in malaria parasites that in turn might aid in the production of superior recombinant vaccines and provide novel drug targets.

Comparative molecular characterization of gene segment 11-derived NSP6 from lamb rotavirus LLR strain used as a human vaccine in China.

Sequence-length polymorphism is known for rotavirus genetic segment 11 (encodes non-structural protein, NSP6). With the exception of 11 strains that have the coding potential for a 98-residue NSP6, majority of the strains have the potential for a 92-residue NSP6. In nine strains, the coding potential for this protein is even shorter. This report focuses on the NSP6 gene nucleotide sequence of Lanzhou Lamb Rotavirus (LLR) strain and its comparative molecular characterization. The LLR strain is a G10 P12 type, which is in use as a licensed human vaccine in China. The LLR NSP6 was compared with 56 other rotaviral NSP6 sequences including a rhesus strain (RRV) available in the database. Analyses indicate that while RRV-NSP6 belongs to the majority (92-residue) group, the LLR NSP6 belongs to the 98-residue group. When the rotavirus NSP6 protein was expressed in cells as GFP fusion protein from human, simian and the LLR strains, they all demonstrated punctate cytoplasmic distribution and, contrary to the computer-aided prediction, the NSP6 did not undergo phosphorylation, which in itself is a novel observation for the rotavirus NSP6.

Comparative evaluation of the chemopreventive efficacy of green and black tea polyphenols in the hamster buccal pouch carcinogenesis model.

OBJECTIVES: To evaluate the comparative chemopreventive efficacy of green tea polyphenols (Polyphenon-E) and black tea polyphenols (Polyphenon-B) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. DESIGN AND METHODS: Hamsters were divided into 6 groups. Animals in group 1 served as controls. Animals in groups 2 and 3 were administered 0.05% Polyphenon-E and B, respectively, in the diet. The right buccal pouches of animals in groups 4-6 were painted with 0.5% DMBA three times a week for 14 weeks. While group 4 received no further treatment, hamsters in groups 5 and 6 received diet containing 0.05% Polyphenon-E and B, respectively. The status of carcinogen-metabolising enzymes, lipid peroxidation and glutathione-dependent antioxidants in the buccal pouch and liver, as well as the frequency of bone marrow micronuclei were used as biomarkers. RESULTS: Application of DMBA induced HBP carcinomas, increased genotoxicity with an imbalance in carcinogen-metabolising enzymes and the cellular redox status. Inhibition of HBP carcinomas by Polyphenon-E and B was associated with a significant decrease in phase I enzymes, modulation of lipid peroxidation and enhanced antioxidant and phase II enzyme activities. CONCLUSION: The greater efficacy of Polyphenon-B in inhibiting HBP carcinogenesis suggests that it may have a major impact in the chemoprevention of oral cancer.

Attenuation of N-methyl-N'-nitro-N-nitrosoguanidine induced genotoxicity and oxidative stress by tomato and garlic combination.

The protective effect of pretreatment with tomato and garlic against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced genotoxicity and oxidative stress was investigated in male Swiss mice. In vivo bone marrow micronucleus test was performed to assess the antigenotoxic effect of tomato and garlic. Oxidative stress was monitored by estimating the extent of lipid peroxidation and the status of the glutathione redox cycle antioxidants. Increased frequency of bone marrow micronuclei with enhanced lipid peroxidation was associated with compromised antioxidant defenses in MNNG treated animals. Although pretreatment with tomato and garlic significantly reduced the frequencies of MNNG-induced bone marrow micronuclei, the combination of tomato and garlic exerted a greater protective effect. This was associated with modulation of lipid peroxidation as well as reduced glutathione (GSH) and the GSH-dependent enzymes glutathione peroxidase (GPx) and glutathione-S-transferase (GST). These findings suggest that a diet containing even low levels of different naturally occurring compounds is effective in exerting antigenotoxic effects by modulating oxidative stress.

Rubella virus and birth defects: molecular insights into the viral teratogenesis at the cellular level.

BACKGROUND: In utero rubella virus (RV) infection of a fetus can result in birth defects that are often collectively referred to as congenital rubella syndrome (CRS). In extreme cases, fetal death can occur. In spite of the availability of a safe and effective vaccine against rubella, recent worldwide estimates are that more than 100,000 infants are born with CRS annually. RECENT PROGRESS: Recently, several significant findings in the field of cell biology, as well as in the RV replication and virus-cell interactions, have originated from the authors' laboratory, and other researchers have provided insights into RV teratogenesis. It has been shown that 1) an RV protein induces cell-cycle arrest by generating a subpopulation of tetraploid nuclei (i.e., 4N DNA) cells, perhaps representative of the tetraploid state following S phase in the cell cycle, due to its interaction with citron-K kinase (CK); 2) RV infection induces apoptosis in cell culture, and 3) CK functional perturbations lead to tetraploidy, followed by apoptosis, in specific cell types. CONCLUSIONS: Based on several similarities between known RV-associated fetal and cellular manifestations and CK deficiency-associated phenotypes, it is reasonable to postulate that P90-CK interaction in RV-infected cells interferes with CK function and induces cell-cycle arrest following S phase in a subpopulation, perhaps representative of tetraploid stage, which could lead to subsequent apoptosis in RV infection. Taking all these observations to the fetal organogenesis level, it is plausible that P90-CK interaction could perhaps be one of the initial steps in RV infection-induced apoptosis-associated fetal birth defects in utero.

Protective effects of a mixture of dietary agents against 7,12-dimethylbenz[a]anthracene-induced genotoxicity and oxidative stress in mice.

We investigated the effects of pretreatment with tomato, garlic, and turmeric, alone and in combination, against 7,12-dimethylbenz[a]anthracene (DMBA)-induced genetic damage and oxidative stress in male Swiss mice. Measurement of the incidence of bone marrow micronuclei as well as the extent of lipid peroxidation and the status of the antioxidants reduced glutathione, glutathione peroxidase, and glutathione-S-transferase in the liver and erythrocytes were used as biomarkers of chemoprotection. In DMBA-treated animals, increased frequency of bone marrow micronuclei was accompanied by enhanced lipid peroxidation and antioxidant depletion. Pretreatment with tomato, garlic, and turmeric alone and a combination of these agents significantly reduced the frequencies of DMBA-induced bone marrow micronuclei as well as the extent of lipid peroxidation. These changes may be mediated by the antioxidant-enhancing effects of the dietary agents. The results of the present study suggest that a diet containing even low levels of different naturally occurring compounds is effective in exerting antigenotoxic effects by inhibiting DMBA-induced oxidative stress.

Rubella virus P90 associates with the cytokinesis regulatory protein Citron-K kinase and the viral infection and constitutive expression of P90 protein both induce cell cycle arrest following S phase in cell culture.

In utero infection of developing fetus by Rubella virus (RV) causes cell division inhibition of critical precursor cells in organogenesis, CNS-associated birth defects and induction of apoptosis in cell culture. The underlying mechanisms of RV-induced congenital abnormalities are not known. Here, we identified a novel interaction between RV replicase P90 protein and a cytokinesis-regulatory protein, the Citron-K kinase (CK), in a yeast two-hybrid cDNA library screen. Aberrations in cytokinesis and subsequent apoptosis do occur in specific cell types when the CK gene is knocked out or, its regulatory function is perturbed. Our analysis found that full-length P90 binds CK and in RV-infected cells P90 colocalizes with CK in the cytoplasm. Furthermore, during RV infection as well as cellular expression of P90 alone, we identified a discrete subpopulation of cells containing 4N DNA content, indicating that these cells are arrested in the cell cycle following S phase, suggesting that cellular expression of viral P90 during RV infection perturbs cytokinesis. Previous reports by others established that RV infection leads to apoptosis in cell culture. These observations together taken to the fetal organogenesis level, favor the idea that RV P90, by binding to cellular CK, invokes cell cycle aberrations resulting in the cell- and organ-specific growth inhibition and programmed cell death during RV infection in utero, which commonly is referred to as RV-induced teratogenesis.

The N- and C-terminal regions of rotavirus NSP5 are the critical determinants for the formation of viroplasm-like structures independent of NSP2.

Molecular events and the interdependence of the two rotavirus nonstructural proteins, NSP5 and NSP2, in producing viroplasm-like structures (VLS) were previously evaluated by using transient cellular coexpression of the genes for the two proteins, and VLS domains as well as the NSP2-binding region of NSP5 were mapped in the context of NSP2. Review of the previous studies led us to postulate that NSP2 binding of NSP5 may block the N terminus of NSP5 or render it inaccessible and that any similar N-terminal blockage may render NSP5 alone capable of producing VLS independent of NSP2. This possibility was addressed in this report by using two forms of NSP5-green fluorescent protein (GFP) chimeras wherein GFP is fused at either the N or the C terminus of NSP5 (GFP-NSP5 and NSP5-GFP) and evaluating their VLS-forming capability (by light and electron microscopy) and phosphorylation and multimerization potential independent of NSP2. Our results demonstrate that NSP5 alone can form VLS when the N terminus is blocked by fusion with a nonrotavirus protein (GFP-NSP5) but the C terminus is unmodified. Only GFP-NSP5 was able to undergo hyperphosphorylation and multimerization with the native form of NSP5, emphasizing the importance of an unmodified C terminus for these events. Deletion analysis of NSP5 mapped the essential signals for VLS formation to the C terminus and clearly suggested that hyperphosphorylation of NSP5 is not required for VLS formation. The present study emphasizes in general that when fusion proteins are used for functional studies, constructs that represent fusions at both the N and the C termini of the protein should be evaluated.