Role of in vitro two-dimensional (2D) and three-dimensional (3D) cell culture systems for ADME-Tox screening in drug discovery and development: a comprehensive review.

Drug discovery and development have become a very time-consuming and expensive process. Preclinical animal models have become the gold standard for studying drug pharmacokinetic and toxicity parameters. However, the involvement of a huge number of animal subjects and inter-species pathophysiological variations between animals and humans has provoked a lot of debate, particularly because of ethical concerns. Although many efforts are being established by biotech and pharmaceutical companies for screening new chemical entities in vitro before preclinical trials, failures during clinical trials are still involved. Currently, a large number of two- dimensional (2D) in vitro assays have been developed and are being developed by researchers for the screening of compounds. Although these assays are helpful in screening a huge library of compounds and have shown perception, there is a significant lack in predicting human Absorption, Distribution, Metabolism, Excretion and Toxicology (ADME-Tox). As a result, these assays cannot completely replace animal models. The recent inventions in three-dimensional (3D) cell culture-based assays like organoids and micro-physiological systems have shown great potential alternative tools for predicting the compound pharmacokinetic and pharmacodynamic fate in humans. In this comprehensive review, we have summarized some of the most commonly used 2D in vitro assays and emphasized the achievements in next-generation 3D cell culture-based systems for predicting the compound ADME-Tox.

Gene Expression and Proteomics Studies Suggest an Involvement of Multiple Pathways Under Day and Day-Night Combined Heat Stresses During Grain Filling in Wheat.

Recent weather fluctuations imposing heat stress at the time of wheat grain filling cause frequent losses in grain yield and quality. Field-based studies for understanding the effect of terminal heat stress on wheat are complicated by the effect of multiple confounding variables. In the present study, the effect of day and day-night combined heat stresses during the grain-filling stage was studied using gene expression and proteomics approaches. The gene expression analysis was performed by using real-time quantitative PCR (RT-qPCR). The expression of genes related to the starch biosynthetic pathway, starch transporters, transcription factors, and stress-responsive and storage proteins, at four different grain developmental stages, indicated the involvement of multiple pathways. Under the controlled conditions, their expression was observed until 28 days after anthesis (DAA). However, under the day stress and day-night stress, the expression of genes was initiated earlier and was observed until 14 DAA and 7 DAA, respectively. The protein profiles generated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS/MS) showed a differential expression of the proteins belonging to multiple pathways that included the upregulation of proteins related to the translation, gliadins, and low-molecular-weight (LMW) glutenins and the downregulation of proteins related to the glycolysis, photosynthesis, defense, and high-molecular-weight (HMW) glutenins. Overall, the defense response to the day heat stress caused early gene expression and day-night heat stress caused suppression of gene expression by activating multiple pathways, which ultimately led to the reduction in grain-filling duration, grain weight, yield, and processing quality.

Spotlight on the overlapping routes and partners for anthocyanin transport in plants.

Secondary metabolites are produced by plants and are classified based on their chemical structure or the biosynthetic routes through which they are synthesized. Among them, flavonoids, including anthocyanins and pro-anthocyanidins (PAs), are abundant in leaves, flowers, fruits, and seed coats in plants. The anthocyanin biosynthetic pathway has been intensively studied, but the molecular mechanism of anthocyanin transport from the synthesis site to the storage site needs attention. Although the major transporters are well defined yet, the redundancy of these transporters for structurally similar or dis-similar anthocyanins motivates additional research. Herein, we reviewed the role of membrane transporters involved in anthocyanin transport, including ATP-binding cassette, multidrug and toxic compound extrusion (MATE), Bilitranslocase-homolog (BTL), and vesicle-mediated transport. We also highlight the ability of transporters to cater distinct anthocyanins or their chemically-modified forms with overlapping transport mechanisms and sequestration into the vacuoles. Our understanding of the anthocyanin transporters could provide anthocyanin-rich crops and fruits with a benefit on human health at a large scale.

A null allele of granule bound starch synthase (Wx-B1) may be one of the major genes controlling chapatti softness.

Chapatti (unleavened flatbread) is a staple food in northern India and neighboring countries but the genetics behind its processing quality are poorly understood. To understand the genes determining chapatti quality, differentially expressed genes were selected from microarray data of contrasting chapatti cultivars. From the gene and trait association studies, a null allele of granule bound starch synthase (GBSS; Wx-B1) was found to be associated with low amylose content and good chapatti quality. For validation, near-isogenic lines (NILs) of this allele were created by marker assisted backcross (MAB) breeding. Background screening indicated 88.2 to 96.7% background recovery in 16 selected BC3F5 NILs. Processing quality and sensory evaluation of selected NILs indicated improvement in chapatti making quality. Traits that showed improvement were mouthfeel, tearing strength and softness indicating that the Wx-B1 may be one of the major genes controlling chapatti softness.

Evaluation of Anthocyanin Content, Antioxidant Potential and Antimicrobial Activity of Black, Purple and Blue Colored Wheat Flour and Wheat-Grass Juice against Common Human Pathogens.

The present study aimed to analyze the antioxidant and antimicrobial activity of anthocyanins extracted from colored wheat flour and wheat-grass juice against human pathogens. The total anthocyanin content and antioxidant potential in colored wheat flour and wheat-grass juice extracts were significantly higher than white flour and wheat-grass juice extracts. Ultra-performance liquid chromatography showed the maximum number of anthocyanin peaks in black wheat, with delphinidin-3-o-galactoside chloride, delphinidin-3-o-glucoside chloride, and cyanindin-3-o-glucoside chloride as the major contributors. Among flour extracts, maximum zones of inhibition against Staphylococcus aureus (MTCC 1934), Pseudomonas aeruginosa (MTCC 1434), Escherichia coli, and Candida albicans (MTCC 227) were produced by black flour extract, having the highest anthocyanin content. It exhibited a minimum microbicidal concentration (MMC) of 200 mg/mL against E. coli and C. albicans; and 100 and 150 mg/mL against S. aureus and P. aeruginosa, respectively. Black and purple flour extracts exhibited a minimum inhibitory concentration (MIC) of 50 mg/mL against S. aureus and P. aeruginosa. White flour extracts did not show MMC against E. coli and C. albicans. Among wheat-grass juice extracts, black wheat-grass was most effective and showed an MIC of 100-150 mg/mL against all pathogens. It exhibited an MMC of 200 mg/mL against S. aureus and P. aeruginosa. Hence, anthocyanin-rich colored wheat could be of nutraceutical importance.

Influence of Biofortified Colored Wheats (Purple, Blue, Black) on Physicochemical, Antioxidant and Sensory Characteristics of Chapatti (Indian Flatbread).

Biofortified colored wheat (black, blue, and purple) is rich in anthocyanins and phenolic acid compounds that impart positive physiological effects in humans. A large proportion of wheat is consumed in the form of Chapatti in Asian countries. The effect of chapatti cooking on the proximate composition, bioactive compounds (anthocyanins and phenolics), and antioxidant activities of these wheat varieties were checked in this study. Apart from acceptable sensory parameters, good taste, and soft texture of chapatti, biofortified colored wheat chapatti and flour had higher dietary fibers, protein content, and lower carbohydrate content. Higher soluble and insoluble phenolic compounds, anthocyanin content, and antioxidant activity were in the order of black > blue > purple > white. Chapatti making has reduced their antioxidant activity and anthocyanin content in comparison to flour. Moreover, the reduction in antioxidant activity is less as compared to the decrease in anthocyanin content. Our results suggest that colored wheat can be a better alternative to normal wheat for preparing chapatti as it would have additional health-promoting activities.

Genome-wide Identification and Characterization of Heat Shock Protein Family Reveals Role in Development and Stress Conditions in Triticum aestivum L.

Heat shock proteins (HSPs) have a significant role in protein folding and are considered as prominent candidates for development of heat-tolerant crops. Understanding of wheat HSPs has great importance since wheat is severely affected by heat stress, particularly during the grain filling stage. In the present study, efforts were made to identify HSPs in wheat and to understand their role during plant development and under different stress conditions. HSPs in wheat genome were first identified by using Position-Specific Scoring Matrix (PSSMs) of known HSP domains and then also confirmed by sequence homology with already known HSPs. Collectively, 753 TaHSPs including 169 TaSHSP, 273 TaHSP40, 95 TaHSP60, 114 TaHSP70, 18 TaHSP90 and 84 TaHSP100 were identified in the wheat genome. Compared with other grass species, number of HSPs in wheat was relatively high probably due to the higher ploidy level. Large number of tandem duplication was identified in TaHSPs, especially TaSHSPs. The TaHSP genes showed random distribution on chromosomes, however, there were more TaHSPs in B and D sub-genomes as compared to the A sub-genome. Extensive computational analysis was performed using the available genomic resources to understand gene structure, gene expression and phylogentic relationship of TaHSPs. Interestingly, apart from high expression under heat stress, high expression of TaSHSP was also observed during seed development. The study provided a list of candidate HSP genes for improving thermo tolerance during developmental stages and also for understanding the seed development process in bread wheat.

Anthocyanin-Biofortified Colored Wheat Prevents High Fat Diet-Induced Alterations in Mice: Nutrigenomics Studies.

SCOPE: Effective health-promoting results of either anthocyanins or whole wheat against chronic diseases are well reported. The current study is designed to understand the effect and underlying mechanism of anthocyanins-biofortified whole wheat on high-fat diet (HF)-induced obesity and its comorbidities. METHOD AND RESULTS: Mice are fed a HFD supplemented with isoenergetic white, purple, or black whole wheat for 12 weeks and analyzed by physiological, biochemical, and nutrigenomics studies (qRT-PCR and RNA-Seq analysis). Black wheat significantly reduces body weight gain and fat pad. Both black and purple wheats reduce total cholesterol, triglyceride, and free fatty acid levels in serum, with the restoration of blood glucose and insulin resistance. Black wheat significantly elevates the expression of enzymes related to fatty acid balancing, ß-oxidation, and oxidative stress that supported the biochemical and physiological positive outcomes. Moreover, the transcriptome analysis of adipose and liver tissue reveals activation of multiple pathways and genes related to fatty acid-ß oxidation (crat, acca2, lonp2 etc.), antioxidative enzymes (gpx1, sod1, nxnl1 etc.), along with balancing of fatty acid metabolism specifically in black wheat supplemented mice. CONCLUSION: Taken together, the results suggest that the incorporation of colored wheat (especially black wheat) in the diet can prevent obesity and related metabolic complications.

Pathogenesis of Celiac Disease and Other Gluten Related Disorders in Wheat and Strategies for Mitigating Them.

Wheat is a major cereal crop providing energy and nutrients to the billions of people around the world. Gluten is a structural protein in wheat, that is necessary for its dough making properties, but it is responsible for imparting certain intolerances among some individuals, which are part of this review. Most important among these intolerances is celiac disease, that is gluten triggered T-cell mediated autoimmune enteropathy and results in villous atrophy, inflammation and damage to intestinal lining in genetically liable individuals containing human leukocyte antigen DQ2/DQ8 molecules on antigen presenting cells. Celiac disease occurs due to presence of celiac disease eliciting epitopes in gluten, particularly highly immunogenic alpha-gliadins. Another gluten related disorder is non-celiac gluten-sensitivity in which innate immune-response occurs in patients along with gastrointestinal and non-gastrointestinal symptoms, that disappear upon removal of gluten from the diet. In wheat allergy, either IgE or non-IgE mediated immune response occurs in individuals after inhalation or ingestion of wheat. Following a life-long gluten-free diet by celiac disease and non-celiac gluten-sensitivity patients is very challenging as none of wheat cultivar or related species stands safe for consumption. Hence, different molecular biology, genetic engineering, breeding, microbial, enzymatic, and chemical strategies have been worked upon to reduce the celiac disease epitopes and the gluten content in wheat. Currently, only 8.4% of total population is affected by wheat-related issues, while rest of population remains safe and should not remove wheat from the diet, based on false media coverage.

Potential of Aegilops sp. for Improvement of Grain Processing and Nutritional Quality in Wheat (Triticum aestivum).

Wheat is one of the most important staple crops in the world and good source of calories and nutrition. Its flour and dough have unique physical properties and can be processed to make unique products like bread, cakes, biscuits, pasta, noodles etc., which is not possible from other staple crops. Due to domestication, the genetic variability of the genes coding for different economically important traits in wheat is narrow. This genetic variability can be increased by utilizing its wild relatives. Its closest relative, genus Aegilops can be an important source of new alleles. Aegilops has played a very important role in evolution of tetraploid and hexaploid wheat. It consists of 22 species with C, D, M, N, S, T and U genomes with high allelic diversity relative to wheat. Its utilization for wheat improvement for various abiotic and biotic stresses has been reported by various scientific publications. Here in, for the first time, we review the potential of Aegilops for improvement of processing and nutritional traits in wheat. Among processing quality related gluten proteins; high molecular weight glutenins (HMW GS), being easiest to study have been explored in highest number of accessions or lines i.e., 681 belonging to 13 species and selected ones like Ae. searsii, Ae. geniculata and Ae. longissima have been linked with improved bread making quality of wheat. Gliadins and low molecular weight glutenins (LMW GS) have also been extensively explored for wheat improvement and Ae. umbellulata specific LMW GS have been linked with wheat bread making quality improvement. Aegilops has been explored for seed texture diversity and proteins like puroindolins (Pin) and grain softness proteins (GSP). For nutrition quality improvement, it has been screened for essential micronutrients like Fe, Zn, phytochemicals like carotenoids and dietary fibers like arabinoxylan and ß-glucan. Ae. kotschyi and Ae. biuncialis transfer in wheat have been associated with higher Fe, Zn content. In this article we have tried to compile information available on exploration of nutritional and processing quality related traits in Aegilops section and their utilization for wheat improvement by different approaches.

Anthocyanin bio-fortified colored wheat: Nutritional and functional characterization.

Colored wheat, rich in anthocyanins, has created interest among the breeders and baking industry. This study was aimed at understanding the nutritional and product making potential of our advanced, high yielding and regionally adapted colored wheat lines. Our results indicated that our advanced colored wheat lines exhibited higher anthocyanin content and antioxidant activity than donor wheat lines and it varied in the order of white

Biofortified Crops Generated by Breeding, Agronomy, and Transgenic Approaches Are Improving Lives of Millions of People around the World.

Biofortification is an upcoming, promising, cost-effective, and sustainable technique of delivering micronutrients to a population that has limited access to diverse diets and other micronutrient interventions. Unfortunately, major food crops are poor sources of micronutrients required for normal human growth. The manuscript deals in all aspects of crop biofortification which includes-breeding, agronomy, and genetic modification. It tries to summarize all the biofortification research that has been conducted on different crops. Success stories of biofortification include lysine and tryptophan rich quality protein maize (World food prize 2000), Vitamin A rich orange sweet potato (World food prize 2016); generated by crop breeding, oleic acid, and stearidonic acid soybean enrichment; through genetic transformation and selenium, iodine, and zinc supplementation. The biofortified food crops, especially cereals, legumes, vegetables, and fruits, are providing sufficient levels of micronutrients to targeted populations. Although a greater emphasis is being laid on transgenic research, the success rate and acceptability of breeding is much higher. Besides the challenges biofortified crops hold a bright future to address the malnutrition challenge.

Rapid Development and Characterization of Chromosome Specific Translocation Line of Thinopyrum elongatum with Improved Dough Strength.

The protein content and its type are principal factors affecting wheat (Triticum aestivum) end product quality. Among the wheat proteins, glutenin proteins, especially, high molecular weight glutenin subunits (HMW-GS) are major determinants of processing quality. Wheat and its primary gene pool have limited variation in terms of HMW-GS alleles. Wild relatives of wheat are an important source of genetic variation. For improvement of wheat processing quality its wild relative Thinopyrum elongatum with significant potential was utilized. An attempt was made to replace Th. elongatum chromosome long arm (1EL) carrying HMW-GS genes related to high dough strength with chromosome 1AL of wheat with least or negative effect on dough strength while retaining the chromosomes 1DL and 1BL with a positive effect on bread making quality. To create chromosome specific translocation line [1EL(1AS)], double monosomic of chromosomes 1E and 1A were created and further crossed with different cultivars and homoeologous pairing suppressor mutant line PhI . The primary selection was based upon glutenin and gliadin protein profiles, followed by sequential genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH). These steps significantly reduced time, efforts, and economic cost in the generation of translocation line. In order to assess the effect of translocation on wheat quality, background recovery was carried out by backcrossing with recurrent parent for several generations and then selfing while selecting in each generation. Good recovery of parent background indicated the development of almost near isogenic line (NIL). Morphologically also translocation line was similar to recipient cultivar N61 that was further confirmed by seed storage protein profiles, RP-HPLC and scanning electron microscopy. The processing quality characteristics of translocation line (BC4F6) indicated significant improvement in the gluten performance index (GPI), dough mixing properties, dough strength, and extensibility. Our work aims to address the challenge of limited genetic diversity especially at chromosome 1A HMW-GS locus. We report successful development of chromosome 1A specific translocation line of Th. elongatum in wheat with improved dough strength.

Chromosome Specific Substitution Lines of Aegilops geniculata Alter Parameters of Bread Making Quality of Wheat.

Wheat cultivars with wide introgression have strongly impacted global wheat production. Aegilops geniculata (MgUg) is an important wild relative with several useful traits that can be exploited for wheat improvement. Screening of Ae. geniculata addition lines indicated a negative effect of 1Ug and the positive effect of 1Mg chromosome on wheat dough strength. Negative effect of 1Ug is probably associated with variation in number and position of the tripeptide repeat motif in the high molecular weight glutenin (HMW-G) gene. To utilize the positive potential of 1Mg chromosome, three disomic substitution lines (DSLs) 1Mg(1A), 1Mg(1B) and 1Mg(1D) were created. These lines were characterized for morphological, cytogenetic properties and biochemical signatures using FISH, 1D-, 2D-PAGE and RP-HPLC. Contribution of wheat 1A, 1B and 1D chromosomes towards dough mixing and baking parameters, chapatti quality, Fe/Zn content and glume color were identified. Observed order of variation in the dough mixing and baking parameters {1Mg(1D) ≤wheat ≤1Mg(1B) ≤1Mg(1A)} indicated that chromosome specific introgression is desirable for best utilization of wild species' potential.

Soft and Hard Textured Wheat Differ in Starch Properties as Indicated by Trimodal Distribution, Morphology, Thermal and Crystalline Properties.

Starch and proteins are major components in the wheat endosperm that affect its end product quality. Between the two textural classes of wheat i.e. hard and soft, starch granules are loosely bound with the lipids and proteins in soft wheat due to higher expression of interfering grain softness proteins. It might have impact on starch granules properties. In this work for the first time the physiochemical and structural properties of different sized starch granules (A-, B- and C-granules) were studied to understand the differences in starches with respect to soft and hard wheat. A-, B- and C-type granules were separated with >95% purity. Average number and proportion of A-, B-, and C-type granules was 18%, 56%, 26% and 76%, 19%, 5% respectively. All had symmetrical birefringence pattern with varied intensity. All displayed typical A-type crystallites. A-type granules also showed V-type crystallinity that is indicative of starch complexes with lipids and proteins. Granules differing in gelatinization temperature (ΔH) and transition temperature (ΔT), showed different enthalpy changes during heating. Substitution analysis indicated differences in relative substitution pattern of different starch granules. Birefringence, percentage crystallinity, transmittance, gelatinization enthalpy and substitution decreased in order of A>B>C being higher in hard wheat than soft wheat. Amylose content decreased in order of A>B>C being higher in soft wheat than hard wheat. Reconstitution experiment showed that starch properties could be manipulated by changing the composition of starch granules. Addition of A-granules to total starch significantly affected its thermal properties. Effect of A-granule addition was higher than B- and C-granules. Transmittance of the starch granules paste showed that starch granules of hard wheat formed clear paste. These results suggested that in addition to differences in protein concentration, hard and soft wheat lines have differences in starch composition also.