Differential responses to double-stranded RNA injection and feeding in Mormon cricket (Orthoptera: Tettigoniidae).

The Mormon cricket, Anabrus simplex, is a flightless katydid, one of the major devastating rangeland pests in several states of the western United States. During the past few years, their sudden and periodic outbreaks into massive migratory bands caused significant economic losses to the rangeland forage and agricultural crops, particularly grain crops. Current population management methods rely heavily on broad-spectrum chemical insecticides, which could be toxic to nontargets, and even the targeted species might develop resistance in the long run. Therefore, we assessed the potential of RNA interference (RNAi)-based alternative management strategies that could supplement the current methods. In insects, RNAi efficiency varies with the method of double-stranded RNA (dsRNA) delivery. We tested 2 different methods of dsRNA delivery: injection and oral feeding of dsRNA. The results showed that Mormon crickets are sensitive to injection of dsRNA in a dose-dependent manner, but refractory to the oral feeding of dsRNA. Further, we confirmed the high nuclease activity in the insect midgut. In order to protect the dsRNA from the dsRNase activity and facilitate its uptake in the midgut, we encapsulated dsRNA inside poly lactic-co-glycolic acid (PLGA) nanoparticles and studied its release kinetics and RNAi efficiency by oral feeding. The release kinetics clearly suggested that the PLGA nanoparticle permeates from the insect digestive system to the hemolymph; however, it failed to induce an efficient RNAi response of the targeted genes. In conclusion, our findings suggest the different responses to dsRNA delivery methods in Mormon crickets, and further investigations involving dsRNA stability and its uptake mechanism are required to use RNAi as an alternative Mormon cricket population management strategy.

Development of RNAi Methods for the Mormon Cricket, Anabrus simplex (Orthoptera: Tettigoniidae).

Mormon crickets are a major rangeland pest in the western United States and are currently managed by targeted applications of non-specific chemical insecticides, which can potentially have negative effects on the environment. In this study, we took the first steps toward developing RNAi methods for Mormon crickets as a potential alternative to traditional broad-spectrum insecticides. To design an effective RNAi-based insecticide, we first generated a de novo transcriptome for the Mormon cricket and developed dsRNAs that could silence the expression of seven housekeeping genes. We then characterized the RNAi efficiencies and time-course of knockdown using these dsRNAs, and assessed their ability to induce mortality. We have demonstrated that it is possible to elicit RNAi responses in the Mormon cricket by injection, but knockdown efficiencies and the time course of RNAi response varied according to target genes and tissue types. We also show that one of the reasons for the poor knockdown efficiencies could be the presence of dsRNA-degrading enzymes in the hemolymph. RNAi silencing is possible in Mormon cricket, but more work needs to be done before it can be effectively used as a population management method.

Neurodevelopmental and transcriptomic effects of CRISPR/Cas9-induced somatic orco mutation in honey bees.

In insects, odorant receptors facilitate olfactory communication and require the functionality of the highly conserved co-receptor gene orco. Genome editing studies in a few species of ants and moths have revealed that orco can also have a neurodevelopmental function, in addition to its canonical role in adult olfaction, discovered first in Drosophila melanogaster. To extend this analysis, we determined whether orco mutations also affect the development of the adult brain of the honey bee Apis mellifera, an important model system for social behavior and chemical communication. We used CRISPR/Cas9 to knock out orco and examined anatomical and molecular consequences. To increase efficiency, we coupled embryo microinjection with a laboratory egg collection and in vitro rearing system. This new workflow advances genomic engineering technologies in honey bees by overcoming restrictions associated with field studies. We used Sanger sequencing to quickly select individuals with complete orco knockout for neuroanatomical analyses and later validated and described the mutations with amplicon sequencing. Mutant bees had significantly fewer glomeruli, smaller total volume of all the glomeruli, and higher mean individual glomerulus volume in the antennal lobe compared to wild-type controls. RNA-Sequencing revealed that orco knockout also caused differential expression of hundreds of genes in the antenna, including genes related to neural development and genes encoding odorant receptors. The expression of other types of chemoreceptor genes was generally unaffected, reflecting specificity of CRISPR activity in this study. These results suggest that neurodevelopmental effects of orco are related to specific insect life histories.

Comparative Analysis of Chitin SynthaseA dsRNA Mediated RNA Interference for Management of Crop Pests of Different Families of Lepidoptera.

RNA interference (RNAi) is a sequence-specific down-regulation in the expression of a particular gene, induced by double-stranded RNA (dsRNA). Feeding of dsRNA either directly or through transgenic plants expressing dsRNA of insect genes has been proven successful against lepidopteran and coleopteran pests, establishing an additional alternative to control insect pests. Lepidopteran crop pests including Spodoptera litura (Fabricius) (Noctuidae), Chilo partellus (Swinhoe) (Crambidae), Plutella xylostella (Linnaeus) (Plutellidae), and Maruca vitrata (Fabricius) (Pyralidae) are the devastating pests of a variety of crops. To tap the potential of RNAi against insect pests, a gene coding for the key enzyme in chitin biosynthesis in arthropods, the chitin synthaseA (CHSA), has been targeted through an exogenous delivery of dsRNA and plant-mediated RNAi. The introduction of dsCHSA caused "Half ecdysis" and "Black body" type lethal phenotypes and a significant reduction in larval body weight. Subsequent RT-qPCR analysis demonstrated the down-regulation of CHSA gene transcripts from 1.38- to 8.33-fold in the four target species. Meanwhile, when S. litura larvae fed with leaves of transgenic tobacco plants expressing dsSlCHSA, the mRNA abundance of CHSA gene was significantly decreased resulting in lethal phenotypes like "Double head formation," "Half ecdysis," and "Black body." In addition, abnormalities in pupal-adult and adult stage were also documented, strongly suggesting the RNAi effect of CHSA gene at late developmental stages. Overall, the results demonstrated that CHSA gene expression in Lepidopteran crop pests could be suppressed by application of dsRNA either as feeding or through transgenic crop plants.

Effect of sodium caseinate and vitamin A complexation on bioaccessibility and bioavailability of vitamin A in Caco-2 cells.

Native sodium caseinate-vitamin A (VA) complexes (Sodium caseinate-VA complex, NaCaS-VA) and modified sodium caseinate-VA complexes i.e. Succinylated sodium caseinate-VA complex (SNaCaS-VA), reassembled sodium caseinate-VA complex (RNaCaS-VA) and reassembled succinylated sodium caseinate-VA complex (RSNaCaS-VA) were prepared and evaluated for their in-vitro bioaccessibility and in-vitro bioavailability of VA through Caco-2 cell lines.VA degraded under acidic conditions as the physiological pH during digestion in stomach was highly acidic (1.2-1.8). During in-vitro gastric digestion, sodium caseinate provided protection to VA, hence, higher VA content was retained in digesta as compared to free VA (oily form). Vitamin uptake by Caco-2 cells was significantly different for digested sodium caseinate-VA complexes as compared to free VA. The peptide content of casein and various sodium caseinate-VA complexes was monitored throughout digestion process. Variation in the complex composition had an effect on protein digestibility and peptide distribution. The bioavailability of VA through sodium caseinate-VA complexes was evaluated by exposing Caco-2 cells to the digesta of milk fortified with various complexes. The total uptake of VA by Caco-2 cells was highest for milk fortified with RSNaCaS-VA followed by RNaCaS-VA, control milk, SNaCaS-VA, NaCaS-VA and free VA. During the formation of RNaCaS-VA and RSNaCaS-VA complexes more hydrophobic sites are exposed, leading to the attachment of VA on the interior hydrophobic regions of sodium caseinate molecule. This led to higher stability of VA during gastrointestinal digestion and further resulted in higher bioaccessibility and bioavailability of vitamin A in Caco-2 cells.

Validation of leaf enzymes in the detergent and textile industries: launching of a new platform technology.

Chemical catalysts are being replaced by biocatalysts in almost all industrial applications due to environmental concerns, thereby increasing their demand. Enzymes used in current industries are produced in microbial systems or plant seeds. We report here five newly launched leaf-enzyme products and their validation with 15 commercial microbial-enzyme products, for detergent or textile industries. Enzymes expressed in chloroplasts are functional at broad pH/temperature ranges as crude-leaf extracts, while most purified commercial enzymes showed significant loss at alkaline pH or higher temperature, required for broad range commercial applications. In contrast to commercial liquid enzymes requiring cold storage/transportation, chloroplast enzymes as a leaf powder can be stored up to 16 months at ambient temperature without loss of enzyme activity. Chloroplast-derived enzymes are stable in crude-leaf extracts without addition of protease inhibitors. Leaf lipase/mannanase crude extracts removed chocolate or mustard oil stains effectively at both low and high temperatures. Moreover, leaf lipase or mannanase crude-extracts removed stain more efficiently at 70 °C than commercial microbial enzymes (<10% activity). Endoglucanase and exoglucanase in crude leaf extracts removed dye efficiently from denim surface and depilled knitted fabric by removal of horizontal fibre strands. Due to an increased demand for enzymes in the food industry, marker-free lettuce plants expressing lipase or cellobiohydrolase were created for the first time and site-specific transgene integration/homoplasmy was confirmed by Southern blots. Thus, leaf-production platform offers a novel low-cost approach by the elimination of fermentation, purification, concentration, formulation and cold-chain storage/transportation. This is the first report of commercially launched protein products made in leaves and validated with current commercial products.

Comparative effect of different individual wrappings on shelf life of guava (Psidium guajava).

Being climacteric fruit the organoleptic quality of Guava deteriorates due to continuously occurring biochemical changes during the storage leading to heavy post harvest losses. The present research work was planned to depreciate the losses by assessing the effect of different individual packagings on the shelf life of the fruit. The individual wrapping of fruits were carried out in LDPE films by cling, shrink, vacuum and modified atmosphere packaging (MAP) and stored at room temperature (37 °C). Individual wrapping delayed the ripening process by slowing down the metabolic activities and reducing the magnitude of changes in physicochemical parameters. In control fruits significant compositional changes along with the total phenol content and ascorbic acid were observed with higher decay loss. However, wrapping of fruits maintained the natural freshness and helped in retaining the marketability of the fruits. Vacuum packing and MAP showed minimum PLW (3.5%), decay loss and ripening during storage. Cling wrapping and shrink wrapping were the best treatments that enhanced the shelf life of fruits by 4 days at room temperature.

Extension of the shelf life of guava by individual packaging with cling and shrink films.

Guava is a climacteric fruit so physico-chemical changes continuously occur after harvest till fruit become unfit for consumption and suffers from post harvest losses. The main objective of this work was to assess the effectiveness of individual film in form of Shrink and Cling wrap on shelf life of guava. Fruits were individually packed in polythene bags (LDPE) of 200 gauge thickness by Shrink and Cling wrapping and stored at 7 ± 3 °C. Individual wrapping reduced the magnitude of changes during storage i.e., ripening process drastically as evident from lower total soluble solids, higher ascorbic acid, polyphenol content with lower polyphenol oxidase activity and physiological loss of weight (PLW) was less than 3.5 %. Film wrapping preserved freshness of wrapped fruits as they remained acceptable for whole storage time in contrast to control fruits which turned unacceptable by 15(th) day of storage. Control fruits showed significant compositional changes as well as in polyphenol content, ascorbic acid and reduced number of marketable fruits while Cling and Shrink wrapping enhanced the shelf life by 10 days.

Infantile pericardial round cell tumor.

Cardiac malignancies presenting in infancy are rare. Desmoplastic small round cell tumor (DSRCT) is a rare occurrence in this age group. No case of intrapericardial DSRCT has been reported in the literature in infants.

Light chain proximal tubulopathy without crystals in a case of Burkitt lymphoma presenting with acute kidney injury.

Acute kidney injury is a common manifestation of malignancies, either directly by the tumor or indirectly from anticancer therapy. The pathologic correlates of acute kidney injury in malignancies are many and can be diagnosed conclusively using kidney biopsy. We report a rare case of a patient with light chain proximal tubulopathy without crystals who presented with acute kidney injury. Kidney biopsy showed proximal tubulopathy without crystals with λ light chain restriction. Detailed investigations lead to a diagnosis of Burkitt lymphoma with surface λ light chain restriction.

Heavy-chain deposition disease: a morphological, immunofluorescence and ultrastructural assessment.

Heavy-chain deposition disease (HCDD) is the least common of the monoclonal immunoglobulin deposition diseases with only 24 reported cases in English literature, including the present case. The rarity of this disease merits its documentation. We present a case of HCDD from our archival material, who presented with rapidly progressive renal failure and nephrotic syndrome and was found to have nodular glomerulosclerosis on renal biopsy which on immunofluorescence and electron microscopy confirmed HCDD of immunoglobulin G1 type without any light-chain deposition. We also present an in-depth literature review on HCDD.

Valvular vegetations? Cardiac amyloidosis is a possibility.

Cardiovascular system is a frequent target for the deposition of amyloid, specially in the primary type. Patients with this condition can present with myocardial dysfunction due to heavy interstitial myocardial deposits. We report a case of primary cardiovascular amyloidosis in a male with sudden cardiac death. The distribution of amyloid was remarkable for exclusive deposition in the pulmonary and systemic arterial adventitia and more strikingly over all valves, specially the mitral valve. These were large enough to simulate vegetations.