An Indigenous, Field-Deployable, Lateral Flow Immunochromatographic Assay Rapidly Detects Infectious Myonecrosis in Shrimp, Litopenaeus vannamei.

Shrimp farming is an important socioeconomic activity worldwide. Infectious myonecrosis virus (IMNV) is an important shrimp virus responsible for significant mortality (up to 70%) in Litopenaeus vannamei. We produced recombinant capsid protein (r-IMNV31) and obtained a highly specific antibody, anti-r-IMNV31, which was used in WOAH-approved ELISA and Western blot to detect IMNV. Further, anti-r-IMNV31 was employed in an indigenously developed lateral flow immunoassay (LFA) with gold nanoparticles as a visual label. Using LFA, IMNV could be detected rapidly (20 min) from tissue homogenate with high specificity, reproducibility, and sensitivity (LOD = 103 viral particles). LFA was validated with "gold standard" qRT-PCR using 60 samples with high sensitivity (100%), specificity (86%). A Cohen's kappa coefficient of 0.86 suggested "good agreement" between LFA and qRT-PCR. With a shelf-life of ~ 1 year at ambient temperature, the use of LFA in the on-site detection of IMNV by shrimp farmers will be a reality.

A concise review on implications of silver nanoparticles in bone tissue engineering.

Skeletal disorders represent a variety of degenerative diseases that affect bone and cartilage homeostasis. The regenerative capacity of bone is affected in osteoporosis, osteoarthritis, rheumatoid arthritis, bone fractures, congenital defects, and bone cancers. There is no viable, non-invasive treatment option and bone regeneration requires surgical intervention with the implantation of bone grafts. Incorporating nanoparticles in bone grafts have improved fracture healing by providing fine structures for bone tissue engineering. It is currently a revolutionary finding in the field of regenerative medicine. Silver nanoparticles (AgNPs) have garnered particular attention due to their well-known anti-microbial and potential osteoinductive properties. In addition, AgNPs have been demonstrated to regulate the proliferation and differentiation of mesenchymal stem cells (MSCs) involved in bone regeneration. Furthermore, AgNPs have shown toxicity towards cancer cells derived from bone. In the last decade, there have been multiple studies focusing on the effect of nanoparticles on the proliferation and/or differentiation of MSCs and bone cancer cells; however, the specific studies with AgNPs are limited. Although the reported investigations show promising in vitro and in vivo potential of AgNPs for application in bone regeneration, more studies are required to ensure their implications in bone tissue engineering. This review aims to highlight the current advances related to the production of AgNPs and their effect on MSCs and bone cancer cells, which will potentiate their possible implications in orthopedics. Moreover, this review article evaluates the future of AgNPs in bone tissue engineering.

Nanomaterials: new weapons in a crusade against phytopathogens.

Bacteria, fungi, viruses, and nematodes are the major causal agents of plant diseases. These phytopathogens are responsible for about 10-40% losses in productivity and quality of food crops and horticultural produce. Although eradication of pathogens is not possible, control of plant diseases has been an area of continuous improvement/research. Use of antimicrobials, bacteriophages, and biocontrol agents, natural and synthetic agrochemicals along with best farm management practices constitute integrated measures for disease control. However, the quest for new materials continues due to pesticide resistance in the pathogens, emergence of new serotypes, and accumulation of high quantities of agrochemical contaminants in the ecosystem and associated environmental hazards, specificity of biocontrol agents, succession of pathogens during the plant growth phase, etc. The emergence of "nanotechnology," a multidisciplinary field of research, has provided a plethora of nanomaterials for potential applications in the agricultural sector. Control of plant diseases requires agents that reduce the pathogen to manageable levels, tools for early-stage detection of pathogen, and compounds that elicit immune response in the host plants. Nanomaterials have in fact been assessed for their utility in all these approaches for disease control. The present review discusses nanomaterials for controlling phytopathogens, nanomaterials in plant disease diagnostics, and nanomaterials as elicitors of the plant immune system. These nanomaterials thus represent new weapons in the fight against the phytopathogens. Recent studies indicate that nanomaterials will be a crucial component in the agroecosystem.

Nanomaterials for the control of bacterial blight disease in pomegranate: quo vadis?

Bacterial blight, caused by Xanthomonas axonopodis pv. punicae, Xap is a serious threat to commercially successful pomegranate (Punica granatum L) crop. Owing to the non-availability of disease-resistant varieties of pomegranate, integrated disease management involving change of season, adequate nutrition, and preventive sprays of bactericides is used to control Xap. We undertook a systematic study to assess the efficacy of metal-based nanomaterials (Cu, CuO, ZnO, CaO, MgO) for the control of Xap. The antimicrobial effectiveness was in the order Cu > ZnO > MgO > CuO with MIC (minimum inhibitory concentration) 2.5, 20, 190, 200, and 1600 µg/ml. A time-to-kill curve indicated that Cu nanoparticles (CuNPs) killed Xap cells within 30 min at 2.5 µg/ml. Under controlled conditions (polyhouse), foliar application of CuNPs (400 µg/ml) resulted in ~ 90 and ~ 15% disease reduction in 6-month-old infected plants at early (disease severity 10%) and established (disease severity 40%) stages of infection, respectively. In a subsequent field study on severely infected 7-year-old plants, applications of nanoparticles reduced the disease incidence by ~ 20% as compared to untreated control. Microscopic observations revealed that CuNPs reduced the bacterial colonization of the leaf surface. Anti-Xap activity of foliar applied CuNPs was on par with conventionally used copper oxychloride (3000 µg/ml) albeit at 8-fold reduced copper concentration. Thus, early disease detection and application of effective dosage of copper nanoparticles can indeed help the farmer in achieving rapid infection control. Further studies on use of combinations of nanoparticles for management of bacterial blight are warranted.

Antioxidant-antibacterial containing bi-layer scaffolds as potential candidates for management of oxidative stress and infections in wound healing.

Tissue engineering techniques are continuously evolving towards providing better microenvironment along with therapeutic potential to address the skin tissue defects. Factors such as microbial infections, presence of excessive free radicals and depletion in antioxidant based scavenging systems pose serious challenges by prolonging inflammation and delaying the repair process. Incorporation of bioactive molecules in polymer based biomimetic scaffolds may present new vistas for handling chronic wounds. In this study, chitosan/collagen scaffolds incorporating 0.5, 1 and 2% (w/w) silymarin (CS-CO-SM) were synthesized and studied for their biocompatibility, in vitro release kinetics and anti-oxidant activity. The release kinetics of silymarin from the CS-CO-SM scaffold showed an initial burst followed by sustained release. The scaffolds were biocompatible and supported the recovery of COS-7 cells from UV induced oxidative stress. Further the CS-CO-SM(2) scaffolds were used to fabricate a bi-layer scaffold by layer upon layer arrangement with CS-Ag3 (3% Ag, w/w). The Ag was incorporated to impart antimicrobial property to the scaffold. The in vivo studies on bi-layer scaffolds were carried out in Wistar rat models at 3, 7 and 10 days post injury and the skin excisions were studied for wound contraction, histology (H&E staining), and lipid peroxidation. The bi-layer scaffold accelerated the process of wound healing with no inflammatory cells, proliferation of fibroblast, neovascularization and collagen deposition. By day 10 post transplantation of the scaffold, the skin had a structure similar to normal skin with complete re-epithelization. This bi-layer scaffold with antioxidant and antimicrobial properties promotes wound healing and is proposed as a potential tissue engineering material for managing chronic wounds.

Zinc use efficiency is enhanced in wheat through nanofertilization.

Ferti-fortification of wheat with zinc, an essential micronutrient is one of the strategies for combating 'hidden hunger' in a large proportion of people all over the world. During fertilization, application of large quantities of micronutrients often results in nutrient wastage and subsequent environmental pollution. Here, we report zinc complexed chitosan nanoparticles (Zn-CNP) for ferti-fortification of durum wheat in field-scale experiments. The efficacy of Zn-CNP was assessed vis-à-vis conventionally applied ZnSO4 (0.2%; 400 mgL-1 zinc) in two durum wheat genotypes (MACS 3125, an indigenous high yielding genotype and UC 1114, a genotype containing the Gpc-B1gene). The observed grain zinc enrichment using Zn-CNP nanocarrier (~36%) and conventional ZnSO4 (~50%) were comparable, despite 10 folds less zinc (40 mgL-1) used in the former. Nanofertilizer application increased grain zinc content without affecting grain yield, protein content, spikelets per spike, thousand kernel weight, etc. Grain zinc enrichment observed in the four-year field trials on plots with varying soil zinc content was consistent, proving the utility of Zn-CNP as a novel nanofertilizer which enhanced fertilizer use efficiency. Our work describes a new paradigm in micronutrient fortification, viz. 'use nanofertilizers at the right place, right time and in right doses'.

In vitro and in vivo studies of a novel bacterial cellulose-based acellular bilayer nanocomposite scaffold for the repair of osteochondral defects.

Bacterial cellulose (BC) is a naturally occurring nanofibrous biomaterial which exhibits unique physical properties and is amenable to chemical modifications. To explore whether this versatile material can be used in the treatment of osteochondral defects (OCD), we developed and characterized novel BC-based nanocomposite scaffolds, for example, BC-hydroxyapatite (BC-HA) and BC-glycosaminoglycans (BC-GAG) that mimic bone and cartilage, respectively. In vitro biocompatibility of BC-HA and BC-GAG scaffolds was established using osteosarcoma cells, human articular chondrocytes, and human adipose-derived mesenchymal stem cells. On subcutaneous implantation, the scaffolds allowed tissue ingrowth and induced no adverse immunological reactions suggesting excellent in vivo biocompatibility. Implantation of acellular bilayered scaffolds in OCD created in rat knees induced progressive regeneration of cartilage tissue, deposition of extracellular matrix, and regeneration of subchondral bone by the host cells. The results of micro-CT revealed that bone mineral density and ratio of bone volume to tissue volume were significantly higher in animals receiving bilayered scaffold as compared to the control animals. To the best of our knowledge, this study proves for the first time, the functional performance of acellular BC-based bilayered scaffolds. Thus, this strategy has great potential for clinical translation and can be used in repair of OCD.

Zinc complexed chitosan/TPP nanoparticles: A promising micronutrient nanocarrier suited for foliar application.

Cultivation of cereals in zinc deficient soils leads to declined nutritional quality of grain. Zinc deficiency in humans is a consequence of consumption of micronutrient deficient cereals as staple food. To achieve an increase in zinc density in grain, we evaluated zinc complexed chitosan nanoparticles (Zn-CNP) as a potential 'nanocarrier' suited for foliar fertilization. Zn-CNP were synthesized using tri-polyphosphate as a cross-linker. Spherical Zn-CNP (diameter 250-300nm) were positively charged (zeta potential, +42.34mV) and contained ∼20mg Zn/g (w/w). Plant growth in zinc deficient sand media, followed by foliar application of Zn-CNP (twice-a-week, for 5 weeks) after anthesis resulted in 27 and 42% increase in grain zinc content of MACS 3125 and UC1114 (durum wheat cultivars) respectively. Translocation of zinc ions from foliar applied Zn-CNP into the leaf and seed tissue was demonstrated using zinquin and dithizone stains, respectively. The study indicates the suitability of chitosan-based nanocarriers in agronomic biofortification.

Lateral flow assay for rapid detection of white spot syndrome virus (WSSV) using a phage-displayed peptide as bio-recognition probe.

White spot disease caused by the white spot syndrome virus (WSSV) has a major socio-economic impact on shrimp farming in India. It has been realized that a field-usable diagnostic capable of rapid detection of WSSV can prevent huge economic losses in disease outbreaks. In this work, we explored the possibility of using a peptide as bio-recognition probe in a field-usable device for the detection of WSSV from infected shrimps and prawns. A commercially available random phage-display library was screened against rVP28 (a major structural protein of WSSV, expressed as a recombinant protein in Escherichia coli). A bacteriophage clone VP28-4L was obtained, and its binding to purified rVP28 protein as well as WSSV from infected shrimp Litopaeneus vannamei tissue was confirmed by ELISA and western blot. The apparent equilibrium dissociation constant (Kd,app) was calculated to be 810 nM. VP28-4L did not show cross-reactivity with any other shrimp viruses. A 12-mer peptide (pep28, with the sequence 'TFQAFDLSPFPS') displayed on the VP28-4L was synthesized, and its diagnostic potential was evaluated in a lateral flow assay (LFA). Visual detection of WSSV could be achieved using biotinylated-pep28 and streptavidin-conjugated gold nanoparticles. In LFA, 12.5 µg/mL of the virus could be detected from L. vannamei gill tissue homogenate within 20 min. Pep28 thus becomes an attractive candidate in bio-recognition of WSSV in field-usable diagnostic platforms benefitting the aquaculture sector.

Field-Usable Lateral Flow Immunoassay for the Rapid Detection of White Spot Syndrome Virus (WSSV).

BACKGROUND: White spot disease (WSD), a major threat to sustainable aquaculture worldwide, is caused by White spot syndrome virus (WSSV). The diagnosis of WSD relies heavily on molecular detection of the virus by one-step PCR. These procedures are neither field-usable nor rapid enough considering the speed at which the virus spreads. Thus, development of a rapid, reliable and field-usable diagnostic method for the detection of WSSV infection is imperative to prevent huge economic losses. METHODS/PRINCIPAL FINDINGS: Here, we report on the development of a lateral flow immunoassay (LFIA) employing gold nanoparticles conjugated to a polyclonal antibody against VP28 (envelope protein of WSSV). The LFIA detected WSSV in ~20 min and showed no cross-reactivity with other shrimp viruses, viz. Monodon Baculovirus (MBV), Hepatopancreatic parvovirus (HPV) and Infectious Hypodermal and Hematopoietic Necrosis virus (IHHNV). The limit of detection (LOD) of the assay, as determined by real-time PCR, was 103 copies of WSSV. In a time course infectivity experiment, ~104 WSSV particles were injected in Litopenaeus vannamei. The LFIA could rapidly (~ 20 min) detect the virus in different tissues after 3 h (hemolymph), 6 h (gill tissue) and 12 h (head soft tissue, eye stalk, and pleopod) of infection. Based on these findings, a validation study was performed using 75 field samples collected from different geographical locations in India. The LFIA results obtained were compared with the conventional "gold standard test", viz. one-step PCR. The analysis of results in 2x2 matrix indicated very high sensitivity (100%) and specificity (96.77%) of LFIA. Similarly, Cohen's kappa coefficient of 0.983 suggested "very good agreement" between the developed LFIA and the conventional one-step PCR. CONCLUSION: The LFIA developed for the rapid detection of WSSV has an excellent potential for use in the field and could prove to be a boon to the aquaculture industry.

Anticancer activity of Indian stingless bee propolis: an in vitro study.

Indian stingless bee propolis has a complex chemical nature and is reported to possess various medicinal properties. In the present study, anticancer activity of the ethanolic extract of propolis (EEP) was explored by testing the cytotoxic and apoptotic effect in four different cancer cell lines, namely, MCF-7 (human breast cancer), HT-29 (human colon adenocarcinoma), Caco-2 (human epithelial colorectal adenocarcinoma), and B16F1 (murine melanoma), at different concentrations. Cytotoxicity was evaluated by MTT assay and Trypan blue dye exclusion assay. EEP at a concentration of 250 µg/mL exhibited ≥50% mortality in all cell lines tested (i.e., IC50 value). EEP revealed a concentration and time dependent cytotoxic effect. Apoptosis was estimated by differential staining (ethidium bromide/acridine orange) and TUNEL (deoxynucleotidyl transferase-dUTP nick end labeling) assay. Light microscopy and atomic force microscopy demonstrated morphological features of apoptosis in all the cell lines after treatment with 250 µg/mL EEP for 24 h. Thus, early onset of apoptosis is the reason for anticancer activity of Indian stingless bee propolis. Further, the antioxidant potential of Indian stingless bee propolis was demonstrated to substantiate its anticancer activity.

Nanotoxicology and in vitro studies: the need of the hour.

Nanotechnology is considered as one of the key technologies of the 21st century and promises revolution in our world. Objects at nano scale, take on novel properties and functions that differ markedly from those seen in the corresponding bulk counterpart primarily because of their small size and large surface area. Studies have revealed that the same properties that make nanoparticles so unique could also be responsible for their potential toxicity. Nanotechnology is rapidly advancing, with more than 1000 nanoproducts already on the market. Considering the fact that intended as well as unintended exposure to nanomaterials is increasing and presently no clear regulatory guideline(s) on the testing/evaluation of nanoparticulate materials are available, the in vitro toxicological studies become extremely relevant and important. This review presents a summary of nanotoxicology and a concise account of the in vitro toxicity data on nanomaterials. For nanomaterials to move into the applications arena, it is important that nanotoxicology research uncovers and understands how these multiple factors influence their toxicity so that the ensuing undesirable effects can be avoided.

Silver nanoparticles in therapeutics: development of an antimicrobial gel formulation for topical use.

Silver is an effective antimicrobial agent with low toxicity, which is important especially in the treatment of burn wounds where transient bacteremia is prevalent and its fast control is essential. Drugs releasing silver in ionic forms are known to get neutralized in biological fluids and upon long-term use may cause cosmetic abnormality, e.g., argyria and delayed wound healing. Given its broad spectrum activity, efficacy and lower costs, the search for newer and superior silver based antimicrobial agents is necessary. Among the various options available, silver nanoparticles have been the focus of increasing interest and are being heralded as an excellent candidate for therapeutic purposes. This report gives an account of our work on development of an antimicrobial gel formulation containing silver nanoparticles (SNP) in the size range of 7-20 nm synthesized by a proprietary biostabilization process. The typical minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against standard reference cultures as well as multidrug-resistant organisms were 0.78-6.25 microg/mL and 12.5 microg/mL, respectively. Gram-negative bacteria were killed more effectively (3 log(10) decrease in 5-9 h) than Gram-positive bacteria (3 log(10) decrease in 12 h). SNP also exhibited good antifungal activity (50% inhibition at 75 microg/mL with antifungal index 55.5% against Aspergillus niger and MIC of 25 microg/mL against Candida albicans). When the interaction of SNP with commonly used antibiotics was investigated, the observed effects were synergistic (ceftazidime), additive (streptomycin, kanamycin, ampiclox, polymyxin B) and antagonistic (chloramphenicol). Interestingly, SNP exhibited good anti-inflammatory properties as indicated by concentration-dependent inhibition of marker enzymes (matrix metalloproteinase 2 and 9). The post agent effect (a parameter measuring the length of time for which bacterial growth remains suppressed following brief exposure to the antimicrobial agent) varied with the type of organism (e.g., 10.5 h for P. aeruginosa, 1.3 h for Staphylococcus sp. and 1.6 h for Candida albicans) indicating that dose regimen of the SNP formulation should ensure sustained release of the drug. To meet this requirement, a gel formulation containing SNP (S-gel) was prepared. The antibacterial spectrum of S-gel was found to be comparable to that of a commercial formulation of silver sulfadiazine, albeit at a 30-fold less silver concentration. As part of toxicity studies, localization of SNP in Hep G2 cell line, cell viability, biochemical effects and apoptotic/necrotic potential were assessed. It was found that SNP get localized in the mitochondria and have an IC(50) value of 251 microg/mL. Even though they elicit an oxidative stress, cellular antioxidant systems (reduced glutathione content, superoxide dismutase, catalase) get triggered and prevent oxidative damage. Further, SNP induce apoptosis at concentrations up to 250 microg/mL, which could favor scarless wound healing. Acute dermal toxicity studies on SNP gel formulation (S-gel) in Sprague-Dawley rats showed complete safety for topical application. These results clearly indicate that silver nanoparticles could provide a safer alternative to conventional antimicrobial agents in the form of a topical antimicrobial formulation.