Continuous monitoring of IgG using immobilized fluorescent reporters

In the manufacture of therapeutic monoclonal antibodies, the clarified cell culture fluid (CCF) is typically loaded onto an initial protein A affinity capture column. Imperfect mass transfer and loading to maximum capacity can risk antibody breakthrough and loss of valuable product, but conservative underloading wastes expensive protein A resin. In addition, the effects of column fouling and ligand degradation require the frequent optimization of immunoglobulin G (IgG) loading to avoid wastage. Continuous real-time monitoring of IgG flowthrough is of great interest, therefore. We previously developed a fluorescence-based monitoring technology that allows batch mix-and-read mAb detection in the CCF. Here, we report the use of reporters immobilized on cyanogenbromide-activated Sepharose 4B resin for continuous detection of IgG in column breakthrough. The column effluent is continuously contacted with immobilized fluorescein-labeled Fc-binding ligands in a small monitoring column to produce an immediately-detectable change in fluorescence intensity. The technology allows rapid and reliable monitoring of IgG in a flowing stream of clarified CCF emerging from a protein A column, without prior sample preparation. We observed a significant change in fluorescence intensity at 0.5 g/L human IgG, sufficient to detect a 5% breakthrough of a 10 g/L load, within 18 s at a flow rate of 0.5 ml/min. The current small-scale technology is suitable for use in process development, but the chemistry should be readily adaptable to larger scale applications using fiber-optic sensors, and continuous IgG monitoring could be applicable in a variety of upstream and downstream process settings.     

Rapid in vitro propagation of cauliflower

A rapid single step method for complete plantlet regeneration in cauliflower is described. Curd explants cultured on Murashige and Skoog (MS) medium supplemented with 1 mg⁻¹ indoleacetic acid (IAA) developed complete plantlets in 25 days. Regeneration potential of curd explants was drastically reduced with increasing storage of cauliflower curds at room temperature beyond 8 days of harvesting. Light intensity of 3000 lux supplied by white fluorescent tubes was optimum to evoke the best response. It was possible to field transfer the hardened plantlets within 35 days of culture initiation.     

Effect of 24-epibrassinolide on oxidative stress markers induced by nickel-ion in <Emphasis Type="Italic">Raphanus sativus</Emphasis> L.

The present study illustrates the effect of 24-epibrassinolide (24-EBL) on morphological and biochemical parameters in radish (Raphanus sativus L.) seedlings grown under nickel (Ni) ion stress. The radish seeds pre-soaked in different concentrations of 24-EBL were sown in petridishes containing various concentrations of heavy metal (Ni).Observations were made on root/shoot length, fresh biomass, activities of antioxidant enzymes (ascorbate peroxidase, superoxide dismutase, catalase, monodehydroascorbate reductase, dehydroascorbate reductase, guaiacol peroxidase and glutathione reductase), lipid peroxidation, proline and protein content in 7-day-old Ni-stressed radish seedlings. Results indicate that seeds presoaked with 24-EBL reduced the impact of Ni-stress which was evident by assessing the morphological parameters, protein content and antioxidant enzyme activities. It was also observed that 24-EBL reduced the toxicity of heavy metal by influencing proline and malondialdehyde (MDA) content. The present study lays a foundation for understanding the role of 24-EBL in heavy metal stress amelioration, particularly in food crop. Analysis of behaviour of antioxidant enzymes will play a critical role in understanding the stress networking, further filling the knowledge gap on the subject.     

Oestrone 3-O-(N-acetyl)sulphamate, a potential molecular probe of the active site of oestrone sulphatase

N,N-Dialkylated derivatives of the steroid sulphatase inhibitor, oestrone 3-O-sulphamate (EMATE) are weak reversible inhibitors of the enzyme. N-Acetylated-EMATE (8), but not the benzoyl derivative, inhibits the enzyme irreversibly, albeit less potently than EMATE and will allow hitherto difficult radiolabelling on the sulphamate group to facilitate investigation of the enzyme inactivation mechanism.     

Schistosome apyrase SmATPDase1, but not SmATPDase2, hydrolyses exogenous ATP and ADP

Schistosomes are parasitic worms that can live in the bloodstream of their vertebrate hosts for many years. It has been proposed that the worms impinge on host purinergic signalling by degrading proinflammatory molecules like ATP as well as prothrombotic mediators like ADP. This capability may help explain the apparent refractoriness of the worms to both immune elimination and thrombus formation. Three distinct ectoenzymes, expressed at the host-exposed surface of the worm’s tegument, are proposed to be involved in the catabolism of ATP and ADP. These are alkaline phosphatase (SmAP), phosphodiesterase (SmNPP-5), and ATP diphosphohydrolase (SmATPDase1). It has recently been shown that only one of these enzymes—SmATPDase1—actually degrades exogenous ATP and ADP. However, a second ATP diphosphohydrolase homolog (SmATPDase2) is located in the tegument and has been reported to be released by the worms. It is possible that this enzyme too participates in the cleavage of exogenous nucleotide tri- and di-phosphates. To test this hypothesis, we employed RNA interference (RNAi) to suppress the expression of the schistosome SmATPDase1 and SmATPDase2 genes. We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade exogenously added ATP or ADP. Suppression of SmATPDase2 does not appreciably affect the worms’ ability to catabolize ATP or ADP. Furthermore, we detect no evidence for the secretion or release of an ATP-hydrolyzing activity by cultured parasites. The results confirm the role of tegumental SmATPDase1, but not SmADTPDase2, in the degradation of the exogenous proinflammatory and prothrombotic nucleotides ATP and ADP by live intravascular stages of the parasite.     

A substitution mutation in OsPELOTA confers bacterial blight resistance by activating the salicylic acid pathway

We previously reported a spotted-leaf mutant pelota(originally termed HM_(47)) in rice displaying arrested growth and enhanced resistance to multiple races of Xanthomonas oryzae pv. oryzae. Here, we report the mapbased cloning of the causal gene OsPELOTA(originally termed spl~(HM47)). We identified a single base substitution from T to A at position 556 in the coding sequence of OsPELOTA, effectively mutating phenylalanine to isoleucine at position 186 in the translated protein sequence. Both functional complementation and over-expression could rescue the spotted-leaf phenotype. OsPELOTA, a paralogue to eukaryotic release factor 1(eRF_1), shows high sequence similarity to Drosophila Pelota and also localizes to the endoplasmic reticulum and plasma membrane.OsPELOTA is constitutively expressed in roots, leaves,sheaths, stems, and panicles. Elevated levels of salicylic acid and decreased level of jasmonate were detected in the pelota mutant. RNA-seq analysis confirmed that genes responding to salicylic acid were upregulated in the mutant. Our results indicate that the rice PELOTA protein is involved in bacterial leaf blight resistance by activating the salicylic acid metabolic pathway.     

Multivariate analysis on the distribution of elements in plants

The present review accentuates the elemental composition of plants as investigated by various researchers utilizing sophisticated techniques. Plants contain plethora of elements which extend their benefits to humans both in terms of medicine as well as nutrition. Therefore, there is a need to explore the elemental composition of plants to investigate their nutraceutical importance. In this review, the elemental data was analyzed using different multivariate techniques. The average values of elements used in this review are sodium (Na) (241.1 µg/g dw), magnesium (Mg) (1202.4 µg/g dw), aluminum (Al) (159.6 µg/g dw), silicon (Si) (133.7 µg/g dw), phosphorus (P) (1947 µg/g dw), sulfur (S) (1483.1 µg/g dw), chloride (Cl) (624.5 µg/g dw), potassium (K) (3008.5 µg/g dw), calcium (Ca) (1663.1 µg/g dw) and iron (Fe) (143.6 µg/g dw) respectively. Maximum accumulation of Na, K, Ca, Mg, Fe, P and S occurs in Poaceae, Lamiaceae, Lauraceae, Sapotaceae, Apiaceae, Pedaliaceae and Brassicaceae, respectively.