Spatial and temporal expression analysis of D-myo-inositol 3-phosphate synthase (MIPS) gene family in Glycine max

Phytic acid, the principal storage form of phosphorus in plant seeds accounts for up to 60 to 80% of the total seed phosphorus content in soybean. Its accumulation increases linearly throughout seed  development and it strongly chelates essential mineral cations and charged proteins decreasing their bioavailability. D-Myo-inositol 3-phosphate synthase (MIPS; EC 5.5.1.4), the evolutionarily conserved enzyme in plants, catalyzes the first and the rate limiting step in phytic acid biosynthetic pathway. Aiming at controlling the level of phytate, we monitored the differential expression profile of four, previously reported, members of the MIPS gene family in developing seeds and vegetative tissues of soybean by quantitative real-time PCR (qRT-PCR). Transcript levels were measured relative to the endogenous reference gene eEF-1á (eukaryotic elongation factor 1-alpha) using SYBER-Green. The qRT-PCR data analysis indicated that the expression of the four highly conserved MIPS genes is both temporally and spatially regulated, information much needed for reverse genetic applications. MIPS1 exhibited high transcript levels in the early developing cotyledons with the levels peaking at around 4 to 6 mm seed size stage. Despite of high level of nucleotide sequence conservation amongst the MIPS gene family members, MIPS2, MIPS3 and MIPS4 were poorly expressed in developing seed tissues, although their transcript levels were relatively high in the other organ tissues. MIPS1 was however moderately expressed in seedlings where MIPS2 showed relatively higher expression levels. Among the four isoforms, MIPS4 had the highest transcript levels in the leaf tissue. The data was clearly indicative of the fact that the four isoforms had diverged regulatory elements resulting in their differential expression. Of the four members of the MIPS gene family, MIPS1 is thus the major isoform that had high expression in the developing seed tissues and can be targeted using the dsRNA induced sequence specific RNA degradation mechanism for reduction of phytate levels without affecting the critical aspects of inositol metabolism in other tissues of the plant.Key words: Soybean, MIPS isoforms, differential expression, endogenous reference gene, qRT-PCR

Hodgkin Lymphoma in a Case of Chronic Myeloid Leukemia Treated with Tyrosine Kinase Inhibitors

Chronic myeloid leukemia (CML) is characterized by increased and unregulated proliferation of granulocytic lineage in the bone marrow and presence of these immature myeloid cells in the peripheral blood with presence of Philadelphia (Ph) chromosome. Tyrosine kinase inhibitors are the most important drugs in the CML therapy and provide long disease-free survival. Due to the increased survival of CML patients with continual administration of these drugs, the chance of development of secondary malignancies may increase. The most common secondary malignancies are prostate, colorectal and lung cancer, non-Hodgkin lymphoma, malignant melanoma, non-melanoma skin tumors and breast cancer. Herein, we are describing a rare case of Hodgkin lymphoma in a patient of CML after ten year of primary disease presentation. Hodgkin lymphoma in a known case of CML is very rare and further studies are also needed to know the pathogenic relationship between the two entities and to assess the risk of secondary Hodgkin lymphoma in CML patients treated with tyrosine kinase inhibitors. CML itself is a risk factor for development of solid cancers and hematologic malignancies. In addition, patients on chemotherapy are immune-compromised and may be at greater risk of neoplasm driven by infectious agents such as Epstein-Barr virus

Valorisation of black carrot pomace: microwave assisted extraction of bioactive phytoceuticals and antioxidant activity using Box–Behnken design

The present study compares three methods viz. microwave assisted extraction (MAE), ultrasonic-assisted extraction (UAE) and conventional solvent extraction (CSE) for extraction of phenolic compounds from black carrot pomace (BCP). BCP is the major by-product generated during processing and poses big disposal problem. Box–Behnken design using response surface methodology was employed to investigate and optimize the MAE of phenolics, antioxidant activity and colour density from BCP. The conditions for maximum recovery of polyphenolics were: microwave power (348.07 W), extraction time (9.8 min), solvent–solid ratio (19.3 mL/g) and ethanol concentration (19.8%). Under these conditions, the extract contained total phenolic content of 264.9 ± 10.02 mg gallic acid equivalents (GAE)/100 mL, antioxidant capacity (AOC) of 13.14 ± 1.05 lmol Trolox equivalents (TE)/mL and colour density of 68.63 ± 5.40 units. The total anthocyanin content at optimized condition was 753.40 ± 31.6 mg/L with low % polymeric colour of 7.40 ± 0.42. At optimized conditions, MAE yielded higher colour density (68.63 ± 5.40), polyphenolic content (264.9 ± 10.025 mg GAE/100 mL) and AOC (13.14 ± 1.05 lmol TE/mL) in a short time as compared to UAE and CSE. Overall results clearly indicate that MAE is the best suited method for extraction in comparison to UAE and CSE. The phenolic rich extract can be used as an effective functional ingredient in foods

Optimization of the use of cellulolytic enzyme preparation for the extraction of health promoting anthocyanins from black carrot using response surface methodology

Anthocyanin-rich extracts from black carrots are being considered as a candidate replacer for the red colour in processed foods. The present investigation optimizes the extraction of anthocyanins with high phenolic content and low degradation parameters from black carrots using a cellulolytic multi-enzyme preparation known as Viscozyme. The optimized conditions for Viscozyme using a Box-Behnken design (BBD) of response surface methodology (RSM) were as follow: temperature (50.2 ◦C), extraction time (58.4 min) and enzyme concentration (0.20%). The predicted value of anthocyanins content was 1380 mg/L, which was near to the optimized experimental value of 1375 mg/L. The extracted anthocyanins based on above mentioned conditions exhibited the lowest degradation parameters such as degradation index (DI) of (0.86), browning index of (BI) (1.31) and were characterized with cyanidin 3-sinapoylxylosylglucosylgalactoside as being the most abundant. The findings clearly reveal that Viscozyme-assisted extraction (VAE) is the best approach for extracting superior quality extracts from black carrots with high anthocyanin and other phenolic component concentrations

Targeting the MAPK7/MMP9 axis for metastasis in primary bone cancer

Metastasis is the leading cause of cancer related death. This multistage process involves contribution from both tumour cells and the tumour stroma to release metastatic cells into the circulation. Circulating tumour cells (CTCs) survive circulatory cytotoxicity, extravasate and colonise secondary sites effecting metastatic outcome. Reprogramming the transcriptomic landscape is a metastatic hallmark but detecting underlying master regulators that drive pathological gene expression is a key challenge, especially in childhood cancer. Here we used whole tumour plus single cell RNA sequencing in primary bone cancer and CTCs to perform weighted gene co-expression network analysis to systematically detect coordinated changes in metastatic transcript expression. This approach with comparisons applied to data collected from cell line models, clinical samples and xenograft mouse models revealed MAPK7/MMP9 signalling as a driver for primary bone cancer metastasis. RNAi knockdown of MAPK7 reduces proliferation, colony formation, migration, tumour growth, macrophage residency/polarisation and lung metastasis. Parallel to these observations were reduction of activated interleukins IL1B, IL6, IL8 plus mesenchymal markers VIM and VEGF in response to MAPK7 loss. Our results implicate a newly discovered, multidimensional MAPK7/MMP9 signalling hub in primary bone cancer metastasis that is clinically actionable

Interview with the Master Organelle – ‘Ribosome’

41-42An article that unveils some possible queries addressed in recent years in ribosome research

Moguća uloga izoflavona u uklanjanju nepoželjnog okusa soje, te ključna uloga IFS2 u akumulaciji izoflavona u sjemenkama soje

Research background. Soybean (Glycine max (L.) Merr) is a nutrient-rich crop with a high protein content and various bioactive compounds with health-promoting properties. Nevertheless, it is poorly accepted as a food by consumers due to its off-flavour. Due to the ubiquitous presence of isoflavones in soybeans, their inherent antioxidant potential and inhibitory effect on lipoxygenase activity, their sensory properties are currently being considered to mitigate the off-flavour. Experimental approach. In the present study, the content and composition of isoflavones in 17 soybean cultivars are determined. The correlation between the isoflavone mass fraction and lipid peroxidation was also established, using thiobarbituric acid (TBA) value and carbonyl compound concentration as indices for the development of off-flavour. Cloning, gene expression analysis and in silico analysis of isoflavone synthase isoforms (IFS1 and IFS2) were also performed. Results and conclusions. The total isoflavone mass fraction in soybean genotypes ranged from (153.5±7.2) µg/g for PUSA 40 to (1146±43) µg/g for Bragg. There was a moderately negative correlation between the indices of off-flavour formation and the genistein/daidzein ratio (p<0.1). However, the correlation with total isoflavone mass fraction was found to be insignificant, indicating complex interactions. Higher protein-protein interactions for the predicted structure of IFS2 with other biosynthesis enzymes and its comparatively higher expression in the Bragg than that of IFS1 indicated its more important role in isoflavone synthesis. Novelty and scientific contribution. The genistein/daidzein mass ratio was found to be an important factor in controlling off-flavour. IFS2 was identified as key to produce soybeans with high isoflavone mass fraction and potentially lower off-flavour formation.Pozadina istraživanja. Soja (Glycine max (L.) Merr) je žitarica bogata hranivima, s velikim udjelom proteina i različitih biološki aktivnih spojeva koji imaju pozitivan učinak na zdravlje. Usprkos tome, nije prihvaćena među potrošačima zbog svog nepoželjnog okusa. Budući da su izoflavoni prisutni u soji u velikoj količini, te imaju antioksidacijska svojstva i inhibiraju aktivnost lipoksigenaze, ispituju se njihova senzorska svojstva u svrhu uklanjanja nepoželjnog okusa soje. Eksperimentalni pristup. U ovom su radu određeni udjel i sastav izoflavona u 17 kultivara soje. Korelacija između masenog udjela izoflavona i lipidne peroksidacije utvrđena je testom tiobarbiturne kiseline i mjerenjem koncentracije karbonila kao pokazateljima razvoja nepoželjnog okusa. Osim toga, klonirane su izoforme izoflavon sintaze (IFS1 i IFS2), te je ispitana ekspresija gena i provedena analiza in silico. Rezultati i zaključci. Ukupni maseni udjel izoflavona u soji iznosio je od (153,5±7,2) µg/g u genotipu PUSA 40 do (1146±43) µg/g u genotipu Bragg. Opažena je blago negativna korelacija između indikatora nastanka nepoželjnog okusa i omjera genisteina i daidzeina (p<0,1). Međutim, korelacija s ukupnim masenim udjelom izoflavona nije bila značajna, što potvrđuje kompleksnost interakcija. Čini se da IFS2 ima veću ulogu od IFS1 u sintezi izoflavona, što je vidljivo iz veće interakcije između proteina u predviđenoj strukturi IFS2 i drugih proteina odgovornih za biosintezu izoflavona, te njegove bitno veće ekspresije u genotipu Bragg nego IFS1. Novina i znanstveni doprinos. Maseni omjer genisteina i daidzeina bitno je utjecao na smanjenje nepoželjnog okusa soje. Utvrđeno je da IFS2 ima ključnu ulogu u proizvodnji soje s velikim masenim udjelom izoflavona, što bi trebalo smanjiti nastanak nepoželjnog okusa

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Not AvailablePhytic acid, the major storage form of phosphorus in plant seeds is degraded by the phytases to yield inositol and free phosphate, contributing thereby to the improved bioavailability of phytate phosphorus and essential minerals in plant foods and simultaneous reduction in phosphorus pollution of the terrestrial and aquatic ecosystems. As a possible strategy for altering seed phytate levels, the approach involving reduction of phytate content by ectopically expressing endogenous phytase gene during seed development of soybean (Glycine max L. cv. Pusa - 20) was attempted in the present study. Semi-quantitative RT-PCR revealed the maximum expression of phytase gene transcripts in germinating cotyledons (approximately 10 days after germinations), compared to other vegetative tissues. A full-length phytase cDNA was amplified from the germinating seedlings by splicing by overlap extension (SOE)-PCR and its sequence analysis revealed an open-reading-frame of 1644 bp, including an N terminal signal peptide of 28 amino acids. Predicted amino acid sequence (547 - aa) of molecular mass 62 kDa on alignment with related purple acid phosphatases in other plants shared five conserved domains and seven invariant amino acids involved in coordination of the metals in the binuclear center of purple acid phosphatases. Owing to a large number of E. coli low-usage codons in soybean phytase gene, the modified gene was cloned into a prokaryotic expression vector pET - 28a ( + ) and its expression in E. coli was confirmed by SDS-PAGE and Western blot analysis. Bioassay of the crude expression product in E. coli revealed a functional phytase gene, showing a great potential for developing low phytate transgenic soybean through its seed-specific overexpression in the early stages of seed development.Not Availabl

Targeted genome editing with CRISPR/Cas9: a step towards generating nutritionally rich soybean

63-68Phytic acid is 75-80% of the total phosphorus in cereal and legume seeds but maximum portion of it is not accessible as the monogastric animals don't have phytase in their digestive tracts which is important for hydrolysis of the phytate molecule. This leads to eutrophication due to the influx of phosphorus from undigested animal waste which is the major source of agricultural phosphorus runoff. Also the polyanionic antinutrient, phytic acid binds to mineral cations and makes them unavailable for absorption by animals leading to mineral deficiencies. Low phytate soybean is thus desirable not only for improving human health but also for reducing the environmental phosphorus load. The implications of phytic acid on the environment, human nutrition and livestock industry have sparked numerous nutritional studies focussing on reduction of its levels. Unfortunately, the negative and altered phenotypes obtained by classical breeding mutations and reverse genetics approaches necessitate the usage of a more targeted and efficient biotechnological intervention for tissue-specific knockdown of phytic acid, obliterating the negative impacts on basal metabolic functions. The steps undertaken in the present study are for developing low-phytate soybean using the latest developed, targeted genome editing tool, CRISPR/Cas9 system for precise and efficient editing of the IPK2 gene of phytic acid biosynthesis pathway. A CRISPR/Cas9 construct for editing the IPK2 gene, involved in phytic acid biosynthesis was successfully designed and generated