Genetic modification of a baculovirus vector for increased expression in insect cells

Generating large amounts of recombinant protein in transgenic animals is often challenging and has a number of drawbacks compared to cell culture systems. The baculovirus expression vector system (BEVS) uses virus-infected insect cells to produce recombinant proteins to high levels, and these are usually processed in a similar way to the native protein. Interestingly, since the development of the BEVS, the virus most often used (Autographa californica multi-nucleopolyhedovirus; AcMNPV) has been little altered genetically from its wild-type parental virus. In this study, we modified the AcMNPV genome in an attempt to improve recombinant protein yield, by deleting genes that are non-essential in cell culture. We deleted the p26, p10 and p74 genes from the virus genome, replacing them with an antibiotic selection cassette, allowing us to isolate recombinants. We screened and identified recombinant viruses by restriction enzyme analysis, PCR and Western blot. Cell viability analysis showed that the deletions did not improve the viability of infected cells, compared to non-deletion viruses. However, expression studies showed that recombinant protein levels for the deletion viruses were significantly higher than the expression levels of non-deletion viruses. These results confirm that there is still great potential for improving the BEVS, further increasing recombinant protein expression yields and stability in insect cells.     

Calcium control of waveform in isolated flagellar axonemes of chlamydomonas

The effect of Ca(++) on the waveform of reactivated, isolated axonemes of chlamydomonas flagella was investigated. Flagella were detached and isolated by the dibucaine procedure and demembranated by treatment with the detergent Nonidet; the resulting axomenes lack the flagellar membrane and basal bodies. In Ca(++)-buffered reactivation solutions containing 10(-6) M or less free Ca(++), the axonemes beat with a highly asymmetrical, predominantly planar waveform that closely resembled that of in situ flagella of forward swimming cells. In solutions containing 10(-4) M Ca(++), the axonemes beat with a symmetrical waveform that was very similar to that of in situ flagella during backward swimming. In 10(-5) M Ca(++), the axonemes were predominantly quiescent, a state that appears to be closely associated with changes in axomenal waveform or direction of beat in many organisms. Experiments in which the concentrations of free Ca(++), not CaATP(--) complex were independently varied suggested that free Ca(++), not CaATP(--), was responsible for the observed changes. Analysis of the flagellar ATPases associated with the isolated axonemes and the nonidet- soluble membrane-matrix fraction obtained during preparation of the axonemes showed that the axonemes lacked the 3.0S Ca(++)-activated ATPase, almost all of which was recovered in the membrane-matrix fraction. These results indicate that free Ca(++) binds directly to an axonemal component to alter flagellar waveform, and that neither the 3.0S CaATPase nor the basal bodies are directly involved in this change.     

Histiostoma papillata sp. n. (Acari: Histiostomatidae), a mite attacking fish in Australia

Histiostoma papillata sp. n. (Acari: Histiostomatidae) is described from Victoria, Australia. It was found in the fins and gills of juvenile Murray cod, Maccullochella peelii peelii (Mitchell), where it appeared to inflict injuries thought to be responsible for the mortality of fish in a diet trial.     

Optimizing the baculovirus expression vector system

Baculoviruses have a unique bi-phasic life cycle and powerful promoters, which greatly facilitates their use for recombinant protein expression in insect cells. We have developed an expression system that utilizes homologous recombination in insect cells between a transfer plasmid containing a gene to be expressed and a replication-deficient virus (bacmid). Only recombinant virus can replicate facilitating the rapid production of multiple recombinant viruses using robotic liquid handlers. The bacmid has also been genetically optimized for improved protein expression and stability. We describe the application of this system for high level production of recombinant proteins.     

Intestinal calcium-binding protein in animals fed normal and rachitogenic diets: I. Rat studies.

Measurements were made of duodenal calcium-binding protein (CaBP) on rats during development of rickets and, subsequently, following vitamin-D2 treatment. Results showed a poor inverse correlation between duodenal CaBP and rickets. In rats fed a phosphate-deficient rachitogenic diet, duodenal CaBP concentration finally fell below detectable limits, but CaBP was still readily measurable 2 weeks after rickets was clearly established. Following a massive dose of vitamin D2 (50 000 I.U.) to rachitic animals, CaBP was formed. However, a small dose of vitamin D2 (500 I.U. daily for 4 days) was insufficient to demonstrate CaBP synthesis than vitamin-D treatment alone. The rachitogenic diet supplemented with phosphate, which caused osteoporosis but not rickets, inhibited CaBP synthesis. The results suggest that nutritional deficiencies from the rachitogenic diet, in addition to vitamin-D deficiency, inhibited CaBP synthesis.     

The effect of diet calcium on fluoride toxicity in growing rats

The effect of dietary Ca in response to fluoride (F) treatment was investigated in rats. Rats were maintained on either adequate (0.5%) or high (2.0%) dietary Ca and given for 5 weeks, NaF in drinking water. The minimum NaF levels that inhibited body growth and reduced survival were 300 mg/L with 0.5% diet Ca and 550 mg/L with 2.0% diet Ca. With these toxic F doses, bone histology showed increased formation surfaces and thickened osteoid seams (osteoid index 6-7%). Fluoride doses 30% below toxic levels (200 and 350 mg/L for 0.5 and 2.0% diet Ca, respectively) had no demonstrable effect on bone. Additional diet Ca reduced F absorption from 76 +/- 3 to 47 +/- 3% for 0.5 and 2.0% diet Ca, respectively. Comparable absorbed doses of F produced comparable effects on bone and body growth but, with additional dietary Ca, these effects were observed with 50% lower serum and bone F levels. Variable response to NaF therapy can be produced in rats by alterations in dietary Ca alone. Results indicate that for clinical treatment the NaF dose needs to be adjusted on an individual basis but neither serum nor bone F levels can be used reliably to establish optimal doses.     

Potentialities of fluorescent carbon nanomaterials as sensor for food analysis

Food safety and quality are among the most significant and prevalent research areas worldwide. The fabrication of appropriate technical procedures or devices for the recognition of hazardous features in foods is essential to safeguard food materials. In the recent era, developing high-performance sensors based on carbon nanomaterial for food safety investigation has made noteworthy progress. Hence this review briefly highlights the different detection approaches (colorimetric sensor, fluorescence sensor, surface-enhanced Raman scattering, surface plasmon resonance, chemiluminescence, and electroluminescence), functional carbon nanomaterials with various dimensions (quantum dots, graphene quantum dots) and detection mechanisms. Further, this review emphasizes the assimilation of carbon nanomaterials with optical sensors to identify multiple contaminants in food products. The insights of carbon-based nanomaterials optical sensors for pesticides and insecticides, toxic metals, antibiotics, microorganisms, and mycotoxins detection are described in detail. Finally, the opportunities and future perspectives of nanomaterials-based optical analytical approaches for detecting various food contaminants are discussed.