Search

The study evaluated 297 carrot germplasm lines, focusing on 52 cultivars to explore their therapeutic potential and address challenges related to the accessibility and affordability of nutraceuticals and health promoting foods. The investigation explores the application of DNA barcoding using the ITS region for precise species identification, highlighting genetic diversity among the examined cultivars. Through ITS sequence-based analysis and phylogenetic examination, six diverse Daucus spp. genotypes were differentiated and classified into distinct groups, indicating the presence of vast genetic variation. Evaluation of antioxidant activities using the DPPH radical scavenging assay revealed varying degrees of scavenging ability among genotypes with SKAU-C-15, SKAU-C-17, and SKAU-C-16 exhibiting the highest activity, suggesting their potential for antioxidant-rich products. Thin Layer Chromatography (TLC) bioautography confirmed the presence of bioactive compounds in carrot extracts responsible for their antioxidant properties. In cell culture studies, specific carrot genotype extracts demonstrated potential anti-proliferative and anti-invasive effects on recurrent prostate cancer cell line - C4-2 (SKAU-C-30, SKAU-C-10, and SKAU-C-42) and non-small cell lung cancer cell line - A549 (SKAU-C-18 and SKAU-C-11) cancer cells, as indicated by MTT assay, wound healing assay, and Colony Forming Unit assay. These findings suggest the promising therapeutic potential of carrot genotypes for developing anti-cancer functional foods, nutraceuticals and health supplements.Therefore, the study contributes to the nutrition security, paving the way for advancements in functional foods and health applications, particularly in cancer treatment and prevention., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The cold stress susceptibility of tomato (Solanum lycopersicum) curtails its cultivation, with significant impact in temperate regions and on cropping seasons. To unravel genomic regions responsible for cold stress resilience, a diverse set of fifty genotypes encompassing cultivated, wild species, and landraces were genotyped using genotyping-by-sequencing. Over two years and six trials employing both early and late sowing, these lines were evaluated. Illumina-based next-generation sequencing produced up to 3 million reads per sample from individually sequenced library pools. The Tassel pipeline yielded 10,802 variants, subsequently filtered to 3,854 SNPs for genome-wide association analysis (GWAS). Employing clustering methods (population structure) via TASSEL, SNPhylo, and Kinship matrix, the fifty genotypes clustered into four distinct gene pools. The GWAS for cold tolerance in tomato integrated key traits including yield. Using six independent phenotypic datasets representing various environments, the study identified 4,517 significant marker-trait associations for cold tolerance traits. Notably, pivotal variations (> 10%) in cold stress tolerance, particularly proline content, were linked to marker-trait associations. Additionally, 5,727 significant marker-trait associations for yield and yield-related traits were unveiled, shedding light on fruit yield and directly associated attributes. The investigation pinpointed 685 candidate genes across all examined traits, including 60 genes associated with biological processes within these genomic regions. Remarkably, 7 out of the 60 genes were directly linked to abiotic stress tolerance, functioning as stress-responsive genes either directly or indirectly. The identified genes, particularly those associated with stress response, could hold the key to enhancing cold tolerance and overall crop productivity in tomato cultivation., (© 2024. The Author(s).)

In August 2020 chili (Capsicum annuum L.) showing wilt symptoms were collected from different districts of the Kashmir: Pulwama, Srinagar, Baramulla, and Anantnag. From each district one location was selected for sample collection and a total of 23 chili isolates were isolated. The tissue bit technique was used to isolate fungus from the infected samples on potato dextrose agar (PDA) medium, purified using the single spore technique, maintained at 25⁰±1℃ and then stored at 4° C (Ferniah et al. 2014) . Initially cultural characteristics appeared as white colonies which gradually turned to pale white colored and attained a growth of 90 mm in 18 days of incubation at 25 ± 1°C. Microscopic observations revealed that mycelium was branched and cylindrical, 3.53-4.98 µm in width. Microconidia were ellipsoidal, hyaline, 0-1 septa werepresent, and 6-7 x 3-4 µm in size. Macroconidia were cylindrical, hyaline, 2-6 septa, measuring 20-60 x 40-45 µm in size. Molecular identification of the pathogens with ITS, TEF, and RPB2 was successfully carried out and the fungi was confirmed as Fusarium flocciferum infecting chili. Amplified PCR products were sequenced and were successfully submitted and accessioned in GenBank with accession number OM189458, OM441199, OR484037 for ITS, TEF, and RPB2 gene. To confirm Koch's postulates pathogenicity test was carried out using rhizosphere inoculation technique (Najar et al. 2011, Parihar et al. 2022). In total 7 replications for sand maize meal medium (potting mixture) was prepared by autoclaving 90 g of sand and 10 g of maize meal in 250 ml of erlenmeyer flask comprising 40 ml of distilled water. The spore suspension at 100 µl per pot was inoculated and was mixed with the sterilized potting mixture in a ratio of (2:1) and up to seven days pathogen was allowed to infect the soil (Davey and Papavizas 1962; Hami et al. 2021). Then chili seeds (cv. Kashmir long-1) were sown in infected potting mixture and grown for three weeks to allow the pathogen to infect the host plants. F. flocciferum took six weeks for appearance of symptoms in the infected potted plants. Control mock inoculation of the potting mixture was carried out using water droplets instead of spore suspension at 100 µl per pot. Seven replications were kept for both inoculated and un-inoculated / control mock pots. The plants developed initial symptoms from light green to yellowish discoloration of leaves followed by the drooping, shriveling, and ultimately leading to death. The collar region of the plant was cut vertically and observed that vascular bundles showed brownish spots and discoloration, indicating wilt as the cause of death. The pathogens were re-isolated and inoculated from all infected plants, then compared with their original pure culture inoculated first, which completely resembled based on morphological, cultural, and pathogenic characteristics. No symptoms were observed on control plants. A phylogenetic analysis was also carried out using ClustalW software that grouped the species identified by different genes into different clades. F. flocciferum has been reported earlier in pea, faba bean and bamboo (Kainthola et al. 2022; Šišić et al. 2020) . In solanaceous crops, this species have been explored as wilt pathogens for the first time from India, indicating diversifying nature of Fusarium flocciferum across various hosts including solanaceous crops.

Background: To decipher the capability of Methyl Jasmonate (MeJA) in resisting cold stress in Solanum lycopersicum assessment regarding various physiological parameters in response to diverse doses of MeJA was done. Low temperature (LT) were given to the plants with MeJA (J1C, J2C, J3C) or without MeJA (LT) application. MeJA in the form of foliar spray was given before stress, during stress and after stress. Three concentrations of MeJA were used under normal and LT stress conditions that includes of J1 (0.5 µM), J2 (10 µM), and J3 (15 µM)., Results: Oxidative stress, growth characteristics, stress tolerance parameters, antioxidant response and photosynthetic parameters were investigated. In our current study we observed that oxidative stress markers declined by MeJA supplementation under cold stress conditions. MeJA boosted antioxidant enzyme activity along with photosynthetic parameters. The best concentration of MeJA was J2 based on results obtained. This is the first study related to MeJA best dose screening in Solanum lycopersicum under LT stress conditions., Conclusion: The LT stress in the Solanum lycopersicum plant was reduced by MeJA. The adverse consequences of LT stress can be significantly attenuated by the J2 concentration of MeJA. So, the optimal concentration of MeJA supplied exogenously to LT stressed Solanum lycopersicum can be a smart strategy to mitigate harmful impact of LT stress on detox system and overall growth of plant., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Kashmir valley, India is a homeland to rice landraces like Zag, Nunbeoul, Qadirbeigh, Kawkadur, Kamad, Mushk Budji, etc., generally characterized by short grains, aroma, earliness and cold tolerance. Mushk Budji is a commercially important speciality rice known for its taste and aroma, nonetheless, is extremely vulnerable to blast disease. Through the use of the marker-assisted backcrossing (MABC) approach, a set of 24 Near-isogenic lines (NILs) was created, and the lines with the highest background genome recovery were chosen. The expression analysis was carried out for the component genes and other eight pathway genes related to blast resistance., Results: The major blast resistance genes Pi9 (from IRBL-9W) and Pi54 (from DHMAS 70Q 164-1b) were incorporated following simultaneous-but-step-wise MABC. The NILs harbouring genes Pi9 + Pi54, Pi9 and Pi54 expressed resistance to isolate (Mo-nwi-kash-32) under controlled and natural field conditions. The loci controlling ETI (effector triggered immunity) included the gene Pi9 and showed 61.18 and 60.27 fold change in relative gene expression in Pi54 + Pi9 and Pi9 carrying NILs against RP Mushk Budji. Pi54 was up regulated and showed 41 and 21 fold change in relative gene expression for NIL-Pi54 + Pi9 and NIL-Pi54, respectively. Among the pathway genes, LOC_Os01g60600 (WRKY 108) recorded 8 and 7.5 fold up regulation in Pi9 and Pi54 NILs., Conclusion: The NILs showed recurrent parent genome recovery (RPG) per cent of 81.67 to 92.54 and were on par in performance to recurrent parent Mushk Budji. The lines were utilized to study the expression of the loci controlling WRKYs, peroxidases and chitinases that confer overall ETI response., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Shot hole is one of the important fungal diseases in stone fruits viz., peach, plum, apricot and cherry caused by Wilsonomyces carpophilus and almond among nut crops. Fungicides significantly decrease the disease. Pathogenicity studies proved a wide host range of the pathogen infecting all stone fruits and almond among the nut crops, however, the mechanism underlying host-pathogen interaction is still unknown. Molecular detection of the pathogen using polymerase chain reaction (PCR) based simple sequence repeat (SSR) markers is also unknown due to the unavailability of the pathogen genome., Methods and Results: We examined the morphology, pathology and genomics of the Wilsonomyces carpophilus. Whole genome sequencing of the W. carpophilus was carried out by Illumina HiSeq and PacBio high throughput sequencing plate-forms through hybrid assembly. Constant selection pressure alters the molecular mechanism of the pathogen causing disease. The studies revealed that the necrotrophs are more lethal with a complex pathogenicity mechanism and little-understood effector repositories. The different isolates of necrotrophic fungus W. carpophilus causing shot hole in stone fruits namely peach, plum, apricot and cherry, and almonds among the nut crops showed a significant variation in their morphology, however, the probability value (p = 0.29) suggests in-significant difference in the pathogenicity. Here, we reported draft genome of W. carpophilus of size 29.9 Mb (Accession number: PRJNA791904). A total of 10,901 protein-coding genes were predicted, including heterokaryon incompatibility genes, cytochrome-p450 genes, kinases, sugar transporters among others. We found 2851 simple sequence repeats (SSRs), tRNAs, rRNAs and pseudogenes in the genome. The most prominent proteins showing necrotrophic lifestyle of the pathogen were hydrolases, polysaccharide-degrading enzymes, esterolytic, lipolytic, and proteolytic enzymes accounted for 225 released proteins. Among the 223 fungal species, top-hit species distribution revealed the majority of hits against the Pyrenochaeta species followed by Ascochyta rabiei and Alternaria alternata., Conclusion: Draft genome of W. carpophilus is 29.9 Mb based on Illumina HiSeq and PacBio hybrid assembly. The necrotrophs are more lethal with a complex pathogenicity mechanism. A significant variation in morphology was observed in different pathogen isolates. A total of 10,901 protein-coding genes were predicted in the pathogen genome including heterokaryon incompatibility, cytochrome-p450 genes, kinases and sugar transporters. We found 2851 SSRs, tRNAs, rRNAs and pseudogenes, and prominent proteins showing necrotrophic lifestyle such as hydrolases, polysaccharide-degrading enzymes, esterolytic, lipolytic and proteolytic enzymes. The top-hit species distribution were against the Pyrenochaeta spp. followed by Ascochyta rabiei., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Mineral stress is one of the dominating abiotic stresses, which leads to decrease in crop production. Selenium (Se) seed priming is a recent approach to mitigate the plant's mineral deficiency stress. Although not an essential element, Se has beneficial effects on the plants in terms of growth, quality, yield and plant defense system thus, enhancing plant tolerance to mineral deficiency., Methods and Results: The present research was accomplished to find out the effect of Se priming on common bean plant (SFB-1 variety) under phosphorus (P) stress. The seeds were grown invitro on four different MGRL media which are normal MGRL media as control with non-Se primed seeds (Se -  P + ), non -Se primed seeds grown on P deficient MGRL media (Se -  P - ), Se primed seeds grown on normal MGRL media (Se + P + ) and Se primed seeds grown on P deficient MGRL media (Se + P  - ). The various morphological and biochemical parameters such as proline content, total sugar content, polyphenols and expression of proteins were analyzed under P stress. The results showed that Se priming has significantly (p ≤ 0.05) affected the morphological as well as biochemical parameters under normal and P stress conditions. The morphological parameters-length, weight, number of nodes and leaves of Se + P + , Se + P - root and shoot tissue showed significant increase as compared to Se - P + , Se - P - . Similarly various biochemical parameters such as total chlorophyll content, proline, total sugar content and polyphenols of Se + P + , Se + P -  increased significantly as compared to Se - P + , Se - P - . The differential protein expression in both Se + P + , Se + P - and Se - P + , Se - P -  plants were determined using MALDI-MS/MS. The differentially expressed proteins in Se + P + , Se + P -  plants were identified as caffeic acid-3-O-methyltransferase (COMT) and SecA protein (a subunit of Protein Translocan transporter), and are found responsible for lignin synthesis in root cell walls and ATP dependent movement of thylakoid proteins across the membranes in shoot respectively. The differential expression of proteins in plant tissues, validated morphological and biochemical responses such as maintaining membrane integrity, enhanced modifications in cellular metabolism, improved polyphenol activities and expression of defensive proteins against mineral deficiency., Conclusions: The study provided an understanding of Se application as a potential approach increasing tolerance and yield in crop plants against mineral deficiency., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Cancer is the major challenge across world and the adenocarcinoma of prostate malignancy is the second most prevalent male cancer. Various medicinal plants are used for the treatment and management of various cancers. Matricaria chamomilla L., is one of the extensively used Unani medicament for the treatment of various type of diseases. In the current study we evaluated most of the parameters prescribed for drug standardization using pharmacognostic approaches. The 2,2 Diphenyl-1-picryl hydrazyl (DPPH) method was utilized for the analysis of antioxidant activity in the flower extracts of M. chamomilla . Moreover, we analyzed the antioxidant and cytotoxic activity of M. chamomilla (Gul-e Babuna ) through in-vitro method. DPPH (2,2-diphenyl-1-picryl-hydrazl-hydrate) method was utilized for the analysis of antioxidant activity in the flower extracts of M. chamomilla . CFU and wound healing assay were performed to determine the anti-cancer activity. The results demonstrated that various extracts of M. chamomilla fulfilled most of the parameters of drug standardization and contained good antioxidant and anticancer activities. The ethyl acetate showed higher anticancer activity followed by aqueous, hydroalcoholic, petroleum benzene and methanol by CFU method. Also, the wound healing assay demonstrated that ethyl acetate extract has more significant effect followed by methanol and petroleum benzene extract on prostate cancer cell line (C4-2). The current study concluded that the extract of M. chamomilla flowers could act as good source of natural anti-cancer compounds., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Apple scab is caused by an ascomycete fungus, Venturia inaequalis (Cke.) Wint., which is one of the most severe disease of apple (Malus × Domestica Borkh.) worldwide. The disease results in 30-40% fruit loss annually and even complete loss in some places. Owing to the evolving susceptibility of resistant apple genotypes harboring R-genes to new variants of V. inaequalis, a comparative transcriptome analysis using Illumina (HiSeq) platform of three scab-resistant (Florina, Prima, and White Dotted Red) and three susceptible (Ambri, Vista Bella, and Red Delicious) apple genotypes was carried out to mine new scab resistance genes. The study led to the identification of 822 differentially expressed genes in the tested scab-resistant and scab-susceptible apple genotypes. The most upregulated genes uniformly expressed in resistant varieties compared to susceptible ones were those coding for 17.3 kDa class II heat shock protein-like, chaperone protein ClpB1, glutathione S-transferase L3-like protein, B3 domain-containing protein At3g18960-like, transcription factor bHLH7, zinc finger MYM-type protein 1-like, and nine uncharacterized proteins, besides three lncRNAs. The genes that were downregulated in susceptible and upregulated in resistant cultivars were those coding for non-specific lipid transfer protein GPI-anchored 1, rust resistance kinase Lr10-like, disease resistance protein RPS6-like, and many uncharacterized proteins. DESeq2 analysis too revealed 20 DEGs that were upregulated in scab-resistant cultivars. Furthermore, a total of 361 genes were significantly upregulated in scab-susceptible variety, while 461 were found downregulated (P value < 0.05 and Log2 (FC) > 1). The differentially expressed genes (DEGs) were related to various pathways, i.e., metabolic, protein processing, biosynthesis of secondary metabolites, plant hormone signal transduction, autophagy, ubiquitin-mediated proteolysis, plant-pathogen interaction, lipid metabolism, and protein modification pathways. Real-time expression of a set of selected twelve DEGs further validated the results obtained from RNA-seq. Overall, these findings lay the foundation for investigating the genetic basis of apple scab resistance and defense pathways that might have a plausible role in governing scab resistance in apple against V. inaequalis., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Chili (Capsicum annuum L.) and brinjal (Solanum melongena L.) are the most widely grown solanaceous crops in the world. However, their production has reduced over several years due to the attack of various fungal and bacterial pathogens and various abiotic factors. Still, the major constrain in their production are pathogens with fungal etiology, especially the fungal wilt of solanaceous crops. Fusarium oxysporum and Fusarium solani have been previously identified as the pathogens causing wilt disease in chili and brinjal. Recently, a new fungal pathogen F. equiseti has been reported as the causal agent of wilt disease infecting chili. The current study focused on identifying fungal pathogens associated with the wilted plants of chili and brinjal, collected from different parts of the Himalayan region of Kashmir valley, through morpho-cultural and molecular characterization. DNA extraction, PCR amplification, and sequencing were performed on various isolates. DNA barcoding using the internal transcribed spacer region (ITS) was used to identify the pathogen followed by the pathogenicity test. Further confirmation of the pathogen was done by sequencing of transcription elongation factor (TEF) and Calmodulin (CAL2). In current study Fusarium chlamydosporum has been reported as the wilt causing pathogen of chili and brinjal for the first time in Kashmir Himalayas., (© 2022. The Author(s).)

The prevalence of multidrug resistance has been increasingly witnessed during the past few decades. Resistance of human pathogenic fungi against the currently available antifungal agents has increased the frequency of fungal infections and associated mortality rates. The discovery of novel lead antifungal agents is important to challenge multidrug resistance. The present study examined the antifungal potential of chemically synthesized β-Nitrostyrene derivatives. Among the eight β-Nitrostyrene derivatives used in this study, SS45, SS46 and SS47 showed strong antifungal potential. The results show that β-Nitrostyrene derivatives inhibited the growth of different species of human pathogenic Candida, particularly the highly prevalent C. albicans, C. glabrata and the emerging pathogenic C. auris species. Moreover, β-Nitrostyrene derivatives also show strong antifungal activities against drug-resistant clinical isolates and drug transporter overexpressing fungal species. The drug susceptibility assays revealed that β-Nitrostyrene derivatives are fungicidal and show the synergy of action when combined with antifungal drugs caspofungin and fluconazole. The transcriptomic study performed on C. albicans in the presence of β-Nitrostyrene derivatives revealed the differential expression of genes related to cell wall metabolism. Mechanistically, β-Nitrostyrene derivatives impact cell wall morphology, enhance ROS generation and modulate drug efflux. Collectively this study reveals that β-Nitrostyrene derivatives have strong antifungal potential with a particular mode of activity similar to known cell wall perturbing antifungal agents and thus can be exploited as promising potential antifungal agents for further studies., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Colletotrichum lindemuthianum is a hemibiotrophic fungal pathogen that causes bean anthracnose and it is rated among the top 10 important diseases infecting beans. Currently our knowledge on molecular mechanisms underlying C. lindemuthianum pathogenesis is limited. About five pathogenicity genes have been identified in C. lindemuthianum using Restricted Enzyme Mediated Integration and the transformation using Agroinfection has not been optimized. In this study, a series of experiments were conducted to optimize the key parameters affecting the Agrobacterium tumefaciens-mediated transformation for C. lindemuthianum. The transformation efficiency increased with increase in spore concentration and co-cultivation time. However, the optimum conditions that yielded significant number of transformants were 10 6  ml -1 spore concentration, co-cultivation time of 72 h, incubation at 25°C and using a cellulose membrane filter for the co-cultivation. The optimized protocol resulted in establishment of large mutant library (2400). A few mutants were melanin deficient and a few were unable to produce conidia. To determine the altered pathogenicity, two new approaches such as detached leaf and twig techniques proved reliable and require fewer resources to screen the large mutant libraries in a short time. Among the 1200 transformants tested for virulence, 90% transformants were pathogenically similar to wild type (race 2047), 96 and 24 were reduced and impaired, respectively. The altered avirulent transformants can prove vital for understanding the missing link between growth and developmental stages of pathogen with virulence. This platform will help to develop strategies to determine the potential pathogenicity genes and to decipher molecular mechanisms of host-pathogen interactions in more detail., (© 2022 The Society for Applied Microbiology.)

Buckwheat ( Fagopyrum spp . ) has immense nutritional and nutraceutical potential. All the plant parts of buckwheat possess various metabolites, such as rutin, quercetin, vitexin etc. The high content of rutin in this pseudo cereal crop strongly adapts it to grow under adverse environments. In the present study 50 germplasm lines of Fagopyrum tataricum were used for estimation of seed endosperm rutin content through HPLC. Furthermore, molecular analysis of PAL gene (Phenylalanine Ammonia Lyase), an upstream gene in rutin biosynthesis pathway was targeted for detection of SNPs to understand the variations in the concentrations of seed endosperm rutin content, among tartary buckwheat genotypes with highest and lowest seed endosperm rutin content. Three primer pairs were employed for amplification of PAL gene for F. tartaricum (covering whole gene) followed by sequencing. Rutin concentration in seed endosperm of F. tartaricum ranged from 194.86 to 1403.22 ppm with an average of 617.06 ppm. Highest rutin concentration was found in genotype BWZ90 and lowest in BWZ16. Significant variations were observed in the seed endosperm rutin content among the genotypes of tartary buckwheat. Furthermore, alignment of PAL gene sequences of genotypes with high seed endosperm rutin content and low seed endosperm rutin content revealed variations at 21 polymorphic sites. The amino acid sequences obtained from the nucleotide sequences were also aligned and the variations were detected at 19 positions . The putative protein structure showed conformational changes among predicted proteins from two contrasting genotypes for endosperm rutin content. We here established an inventory of seed endosperm rutin content of tartary buckwheat. This study also provided insights about role of these SNPs in rutin biosynthesis. Furthermore, this information can be used for breeding buckwheat for high metabolite contents., Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03218-y., Competing Interests: Conflict of interestThe Authors declare that they have no conflict of interests., (© King Abdulaziz City for Science and Technology 2022.)

Background: Sea buckthorn (Hippophae) is in the focus of interest mainly for its positive effects on health of both human and animal organisms. Due to the similarities in vegetative morphology, Hippophae species are often misidentified. Therefore, current study was focused on ITS based sequence characterization of sea buckthorn species and comparative biochemical evaluation for its antioxidant properties., Methods and Results: DNA was extracted from leaf samples. Primer pairs K-Lab-SeaBukRhm-ITS1F1- K-Lab-SeaBukRhm-ITS1R1 and K-LabSeaBukTib- ITSF1- K-LabSeaBukTib-ITSR1 were used for PCR amplification. The purified PCR products were outsourced for sequencing. Phylogenetic tree was constructed based on neighbor-joining (NJ) method. Moreover, comparison of antioxidant potential of leaves of two sea buckthorn species (Hippophae rhamnoides and Hippophae tibetana) collected from different regions of Ladakh viz., Stakna, Nubra, DRDO Leh and Zanskar was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis (3- ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), and Total antioxidant capacity (TAC) by phosphomolybdenum assays. The present investigation led to the differentiation of two sea buckthorn species viz., H. rhamnoides and H. tibetana based on Internal Transcribed Spacer (ITS) region. Moreover, significant variation was observed in antioxidant potential of leaf extracts collected from different regions., Conclusions: Primary ITS sequence analysis was found to be powerful tool for identification and genetic diversity studies in sea buckthorn. Leaves of sea buckthorn have pronounced antioxidant properties and can be used in food, neutraceuticals and pharmaceutical industries etc. The current study will pave the way to discover small bioactive molecules responsible for antioxidant and anticancer properties in sea buckthorn., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Aim: Anthocyanin, an essential ingredient of functional foods, is present in a wide range of plants, including black carrots. The current investigation was carried out to analyse the effect of cold stress on the expression of major anthocyanins and anthocyanin biosynthetic pathway genes, MYB6 and LDOX-1., Methods and Results: Five cultivated carrot genotypes belonging to the eastern group, having anthocyanin pigment, were used in the current study. The qRT-PCR analysis revealed that relative gene expression of transcription factor MYB-6 and LDOX1gene was highly expressed upon cold stress compared to non-stress samples. High-performance liquid chromatography-based quantification of Cyanidin 3-O-glucoside (Kuromanin chloride), Ferulic acid, 3,5-Dimethoxy-4-hydroxycinnamic acid (Sinapic acid), and Rutin revealed a significant increase in these major anthocyanins in response to cold stress when compared to control plants., Conclusion: We conclude that MYB6 and LDOX1 gene expression increases upon cold stress, which induces accumulation of major anthocyanins in purple black carrot and suggests a possible cross-link between cold stress and anthocyanin biosynthesis in purple black carrot., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Scab caused by Venturia inaequalis (Cke.) Wint. is the most important fungal disease of apple. Fungicide application is a widely practiced method of disease control. However, the use of chemicals is costintensive, tedious, and ecologically unsafe. The development of genetic resistance and the breeding of resistant cultivars is the most reliable and safest option. One such source of scab resistance happens to be the variety 'Shireen', released from SKUAST-Kashmir. However, to date, the nature of resistance and its genetic control have not been characterized. Objective This research aimed to elucidate the genetic basis of scab resistance in Shireen., Methods: Genetic mapping of quantitative trait loci (QTL) for resistance to apple scab disease was performed using an F1 cross developed between the susceptible cultivar 'StarKrimson' and the resistant cultivar 'Shireen'. The population was evaluated for two consecutive years. Further, six candidate genes were analyzed via quantitative real-time PCR, to determine their expression level in response to the pathogen infestation., Results: Genotyping and disease phenotyping of populations led us to identify two quantitative trait loci (QTLs), namely qRVI.SS-LG2.2019 and qRVI.SS-LG8.2019 on chromosomes 2 and 8 with LOD-values of 7.67 and 4.99 respectively, and six potential CDGs for the polygenic resistance in 'Shireen'. The genomic region corresponding to the mapped QTLs in LG 2 and LG 8 of 'Shireen' was examined for candidate genes possibly related to scab resistance using in silico analysis., Conclusion: The QTLs mapped in the genetic background of Shireen are the novel QTLs and may be transferred to desirable genetic backgrounds and provide opportunities for isolation and cloning of genes apart from their utility to achieve durable resistance to scab., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

In August 2020 powdery mildew was observed on pear cv. Fertility at the University research field in Shalimar, Srinagar (J&K), India (34° 08' 30.5'' N and 74° 51' 42.0'' E) with a disease incidence up to 30% (100 leaves observed from ten trees). White irregularly shaped fungal colonies were observed on the abaxial leaf surface which latter covered the whole leaf surface and developed black chasmothecia. The affected leaves appeared brittle, slightly curved upwards and dropped prematurely. Mycelium was hypophyllous, septate and measured 2.0 to 5.0 μm in width. Appressoria were nipple shaped, solitary or present in opposite pairs. Conidiophores were erect, up to 440.0 µm long (n=50), mostly centrally on upper surface of mother cells. Conidiophore foot-cells were filiform, followed by 1 to 3 shorter cells, producing single conidia at the tip. Conidia were hyaline, lanceolate, with a non-papillate rounded apex, measuring55.5 to 81.4 × 14.8 to 22.5 µm (n=50) and devoid of any conspicuous fibrosin bodies. Germ tube was, filiform, twisted, arose basally and measured 2.0 to 5.0 µm in width. Chasmothecia were hypophyllous, black, scattered, globose and measured 195.0 to 255.0 µm in diameter (n=50) having 8 to 12 equatorial, acicular, up to 270.0 µm length appendages with 25.9 to 44.4 µm diameter bulbous base (n=50) and obtuse or subacute apex. Asci in a chasmothecium were clavate to saccate, 62.9 to 81.4 × 18.5 to 22.2 µm (n=50), stalked, and two- spored. Ascospores were 33.3 to 40.7 × 12.9 to 18.5 µm (n=50), pale yellowish or golden brown in color. All morphological features were consistent with Phyllactinia pyri-serotinae (Braun and Cook 2012). To confirm the fungus identity at molecular level, DNA of two isolates was extracted from chasmothecia. The internal transcribed spacer (ITS) sequence of ribosomal DNA was amplified with the primers ITS1 and ITS4 (White et al. 1990) and sequenced. The ITS sequences submitted to NCBI GenBank under Accession No. MZ505441 and MZ505442 have 97 (416/427) & 96 (424/440) per cent and 99 (424/430) & 98 (428/438) per cent base pair matching, with that of P. pyri-serotinae isolates from Japan (AB080521 and AB985507), respectively. Thus, the pathogen was identified as Phyllactinia pyri-serotinae Sawada based on morphological and molecular sequence analyses. The pathogenicity tests of both the isolates were carried out on one year old pear saplings (cv. Fertility) and repeated twice. The inoculum was prepared by collecting P. pyri-serotinae conidia in sterile distilled water from infected pear leaves. Three saplings were inoculated by spraying (15ml per sapling) the inoculum (3 x 105 spores ml-1) on leaf surfaces, while same number of saplings sprayed with sterile distilled water served as non-inoculated controls. After 15 days of incubation at 25oC in a green house, similar symptoms as observed on naturally infected plants were observed on inoculated plants and uninoculated plants remained symptomless. The pathogen of interest observed on inoculated plants was morphologically characterized and found to be similar to P. pyri-serotinae. The voucher specimen was deposited in the Herbarium Crytogamae Indiae Orientalis (HCIO), IARI, New Delhi under accession number 52213. Pear is the third most important temperate fruit grown in India (Chattopadhyay 2009) and our study reveal P. pyri-serotinae as the new causal agent of powdery mildew in addition to P. guttata (Dhar and Shah 1982) under Indian conditions.

Scavenger receptors belong to a superfamily of proteins that are structurally heterogeneous and encompass the miscellaneous group of transmembrane proteins and soluble secretory extracellular domain. They are functionally diverse as they are involved in various disorders and biological pathways and their major function in innate immunity and homeostasis. Numerous scavenger receptors have been discovered so far and are apportioned in various classes (A-L). Scavenger receptors are documented as pattern recognition receptors and known to act in coordination with other co-receptors such as Toll-like receptors in generating the immune responses against a repertoire of ligands such as microbial pathogens, non-self, intracellular and modified self-molecules through various diverse mechanisms like adhesion, endocytosis and phagocytosis etc. Unlike, most of the scavenger receptors discussed below have both membrane and soluble forms that participate in scavenging; the role of a potential scavenging receptor Angiotensin-Converting Enzyme-2 has also been discussed whereby only its soluble form might participate in preventing the pathogen entry and replication, unlike its membrane-bound form. This review majorly gives an insight on the functional aspect of scavenger receptors in host defence and describes their mode of action extensively in various immune pathways involved with each receptor type. Video abstract., (© 2021. The Author(s).)

Aim: There is an urgent need to set up a useful biomarker for ovarian cancer. Galectin-1 is a promising carbohydrate-binding protein which plays a remarkable role in various malignancies yet its clinical significance is questionable. In this study, we have tested the clinical implications of serum Galectin-1 levels in patients with ovarian tumours., Main Methods: Serum Galectin-1 levels were quantified in 84 newly diagnosed ovarian tumour patients and 20 healthy controls by Enzyme Linked Immuno Sorbent Assay during the course of the disease. Therefore the samples were taken at diagnosis, after surgery and after chemotherapy., Key Findings: The Galectin-1 levels were found to be associated with various variables of Ovarian Cancer patients. The levels were found to be prominently high in postmenopausal patients. Galectin-1 levels were raised in epithelial ovarian tumours with significantly high levels in serous subtype. A decrease in Galectin-1 levels post-surgical intervention and after receiving chemotherapy was found. Galectin-1 levels evidently distinguished between normal, benign, malignant and metastatic cases as compared to CA125 levels. Galectin-1 demonstrated to be a better biomarker than CA125 according to the Receiver Operating Characteristic (ROC) curve analysis., Significance: The study emphasizes that serum Galectin-1 may serve as a better surrogate biomarker in Ovarian Cancer for early detection, discriminating between malignant and benign abdominal masses and monitoring the progression of the disease and response to treatment., (© 2021. The Author(s).)

Tulip is an ornamental bulbous flowering crop belonging to the Genus Tulipa and family Liliaceae . It is the first ranking bulbous ornamental plant in the world (Nayeem and Qayoom 2015). They are often the first flowers to witness the bloom in the spring. Kashmir valley is located in northern Himalayas in northwestern region of Indian subcontinent. It is the most alluring and fascinating place all over India and the home of famous "Indhra Gandhi Memorial Tulip garden", the largest tulip garden in the entire Asia. However there are number of constraints in tulip cultivation among which bulb rot occupy a prominent place (Piwoni 2000). Bulb rot is posing problem to all the tulip growers throughout the world (De Hertogh et al. 1983). Rot symptoms were observed on tulip bulbs in field as well as in storage conditions (20-22◦C temperature with a relative humidity of 65%) in the summers of 2018 and 2019 in Shalimar fields of Kashmir. The main disease symptoms are yellow sunken spots on bulbs, purple-yellow coloration of leaves. Causal agent was isolated using tissue bit technique (Pathak 1972) on potato dextrose agar plates which where incubated at 24±2◦C . Single spore technique was used to obtain the pure isolate (Johnston and Booth 1983). The isolate covered the full plate (90mm) in ten days. The colony was dull whitish in color, flat and smooth with concentric ring formation in the culture plate with inner ring having a creamy exudation. The mycelium was septate, branched and hyaline in color and measured 3.50-5.20 µm in width with an average of 4.4 µm. Micro-conidia were hyaline, cylindrical to oval, 0-1 septa and measured 7.50-11.00×2.80-3.75 µm in size. Macro conidia were hyaline with 3-4 septa, fusiform, moderately curved which measured 21.15- 32.00×3.80-4.75 µm in size with an average of 28.50±0.21× 4.30±0.2 µm. On the basis of these morphological and cultural characteristics of the fungus, it was identified as Fusarium solani (Mar.) Sacc.,. To confirm the identity the PCR amplification was carried out for two genes Internal Transcribed Spacer (ITS 1, ITS 4)and Translation Elongation factor1-alpha gene (tef1- alpha) (O'Donnell et al. 1998; White et al. 1990). BLAST analysis of the sequence obtained for both the genes showed 99% homology with F. solani sequences in GenBank and Fusarium -ID databases. The sequences were deposited in the GenBank (Accession No MN611433, MW995477). Pathogenicity test was conducted on variety orange emperor both in laboratory and polyhouse. Bulbs were divided into three sets, (three bulbs per set) one set was given injury and dipped in conidial suspension (10 6 conidia/ml) for 30 min, another set was kept uninjured and dipped in spore suspension of same concentration, the third set was served as control and dipped in sterilized distilled water. All the respective sets were incubated in a moist chamber maintained at a temperature of 22 ◦C to observe symptoms. The injured ones showed symptoms after 7-8 days of inoculation, whereas the uninjured bulbs showed symptoms after 11-12 days. No symptoms were observed in controlled set. A pot experiment was also conducted to carry the pathogenicity tests. Bulbs were injured with the help of sterile needle and were dipped in conidial suspension (10 6 conidia/ml) for 30 min (Pastrana et al. 2014). The bulbs kept for control were dipped in sterilized distilled water. Bulbs were then planted in pots maintained at 18◦C. The above ground parts of the inoculated bulbs showed symptoms like stunted growth which gradually turned yellow and did not produced flowers. The bulbs after harvesting were rotten .No symptoms were observed in controlled plants. To fulfill the Koch's postulates the fungal pathogen was re-isolated which was identified as F. solani . The pathogen is reported to cause disease in other crops (Gupta et al. 2012) but to our knowledge and on the basis of literature, this is the first report of F. solani causing bulb rot of tulip in India.

An efficient protocol for in-vitro propagation of an important ornamental crop, Petunia hybrida Vilm. Cv. "Bravo" was developed. The explants that were used to carry out the experiment were Leaf segments, nodal segments and shoot tips. Nodal segments recorded highest per cent asepsis followed by shoot tips and leaf segments. Asepsis was found to be highest when the explants were sterilized with Fungicide (Carbendazim) 0.02% for the duration of 30 min followed by 0.1% HgCl 2 for duration of 10 min and then ethanol 70% for 10 s. Longer duration of the sterilant treatment showed more necrotic effects on the explants, thus mercuric chloride treatment when given for 5 min proved to be more effective in terms of survival of the explants. Maximum establishment per cent was recorded in Murashige and Skoog (MS) media fortified with BAP (1.5 mg L -1 ) and IBA (0.5 mg L -1 ) in shoot tips and nodal segments, i.e. 97.90 and 95.74% respectively. Callus was efficiently induced and developed when PGR amalgamation of BAP (0.1 mg L -1 ) and 2,4-D (1.5mg L -1 ) was used. Kinetin at the concentration of 2.0 mg L -1 along with IBA at 0.5mg L -1 recorded highest callus regeneration in both leaf and internodal segment derived callus. Maximum proliferation percent of shoots (97.90%), highest number of shoots (20.50 explant -1 ) and maximum length of shoot (2.70 cm) was recorded in PGR combination of IBA and BAP both at 0.5 mg L - 1 concentration level. Rhizogenesis was recorded to be highest in the MS media containing IBA 1.00 mg L -1 . Best hardening media which recorded maximum survival per cent 92.50% was noticed on the media formulation comprised of equal ratio of perlite and vermiculite mix, under poly house conditions., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Soil salinity is one of the major environmental stresses faced by the plants. Sodium chloride is the most important salt responsible for inducing salt stress by disrupting the osmotic potential. Due to various innate mechanisms, plants adapt to the sodic niche around them. Genes and transcription factors regulating ion transport and exclusion such as salt overly sensitive (SOS), Na + /H + exchangers (NHXs), high sodium affinity transporter (HKT) and plasma membrane protein (PMP) are activated during salinity stress and help in alleviating cells of ion toxicity. For salt tolerance in plants signal transduction and gene expression is regulated via transcription factors such as NAM (no apical meristem), ATAF (Arabidopsis transcription activation factor), CUC (cup-shaped cotyledon), Apetala 2/ethylene responsive factor (AP2/ERF), W-box binding factor (WRKY) and basic leucine zipper domain (bZIP). Cross-talk between all these transcription factors and genes aid in developing the tolerance mechanisms adopted by plants against salt stress. These genes and transcription factors regulate the movement of ions out of the cells by opening various membrane ion channels. Mutants or knockouts of all these genes are known to be less salt-tolerant compared to wild-types. Using novel molecular techniques such as analysis of genome, transcriptome, ionome and metabolome of a plant, can help in expanding the understanding of salt tolerance mechanism in plants. In this review, we discuss the genes responsible for imparting salt tolerance under salinity stress through transport dynamics of ion balance and need to integrate high-throughput molecular biology techniques to delineate the issue., (© 2020 Scandinavian Plant Physiology Society.)

Chilli (Capsicum annuum L.) is one of the most significant vegetable and spice crop. Wilt caused by Fusarium Sp. has emerged as a serious problem in chilli production. Internal transcribed spacer (ITS) region is widely used as a DNA barcoding marker to characterize the diversity and composition of Fusarium communities. ITS regions are heavily used in both molecular methods and ecological studies of fungi, because of its high degree of interspecific variability, conserved primer sites and multiple copy nature in the genome. In the present study we focused on morphological and molecular characterization of pathogen causing chilli wilt. Chilli plants were collected from four districts of Kashmir valley of Himalayan region. Pathogens were isolated from infected root and stem of the plants. Isolated pathogens were subjected to DNA extraction and PCR amplification. The amplified product was sequenced and three different wilt causing fungal isolates were obtained which are reported in the current investigation. In addition to Fusarium oxysporum and Fusarium solani, a new fungal species was found in association with the chilli wilt in Kashmir valley viz., Fusarium equiseti that has never been reported before from this region. The studies were confirmed by pathogenicity test and re-confirmation by DNA barcoding.

Apple ( Malus domestica Borkh) is rich in phenolic compounds, which may enhance resistance to scab disease caused by Venturia inaequalis . In present study, apple cv. Golden Delicious was used for estimation of total phenols, flavonoids and quantification of six individual phenolic compounds. between control vs inoculated samples at different inoculation stages. The relative gene expression of phenylalanine ammonia lyase, chalcone synthase and flavanone 3 hydroxylase increased and polyphenolic compounds were constitutively upregulated at different post-inoculation stages. Data suggest that synthesis and accumulation of polyphenols is closely related with disease resistance against Venturia inaequalis. This study may play a vital role in understanding and finding out the governing mechanisms of scab resistance., Competing Interests: No conflict of interest exists neither in the conduct of this research nor in preparation of this manuscript., (© 2020 The Author(s).)

Codling moth ( Cydia pomonella , Lepidoptera: Tortricidae) is a quarantine pest of apple in Ladakh, India. We report Cydia pomonella granulovirus from infected larvae of codling moth for the first time in India. The two CpGV isolates were identified as (CpGV SKUAST-1 and CpGV SKUAST-2) and published in Genbank under accession number, MK801791 and MK801792, respectively. The mortality of CpGV was evaluated against 3rd instar larvae of codling moth at various concentrations viz., 10 2 , 10 4 , 10 6 , 10 8 , 10 10 , 10 12 and 10 14 OB S /ml. The median lethal concentrations (LC 50 and LC 90 ) were observed at 7.08 and 28.56 OB S /ml, respectively. In field, the infection rate by CpGV was 5.95 to 15.65%. Based on typical infection symptoms on the larvae, morphological features under the microscope and sequence results of the amplified product confirmed the first occurrence of CpGV from India. Thus, CpGV will form an important non-chemical strategy for managing this pest., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© Indian Virological Society 2020.)

Thyrostroma carpophilum, a causal agent of shot hole disease of stone fruits, cause severe loss in economically important fruit crops of Kashmir. Understanding its pathogenesis at molecular level will aid in devising a better management strategy. In this study, we optimized Agrobacterium tumefaciens mediated transformation (ATMT) conditions for T. carpophilum using PBIF2-EGFP construct. Using this protocol, we obtained 328 positive transformants per 10 4 spores and subsequent sub-culturing of transformants on selective and non-selective media resulted in stable T-DNA integration. Southern blot analysis revealed that most of the transformants embodied single T-DNA integration. Using this method, we obtained a small-scale transformant library (2050 transformants). Among this pool, we tested 1005 transformants for their pathogenicity; out of which 185 showed complete pathogenicity loss, 35 displayed reduced virulence and 785 were pathogenically similar to wild type. Out of this experimental stock, three transformants from each category were randomly selected to dissect the infection assay. The findings deciphered that transformants with complete pathogenicity loss failed to penetrate the host tissue and a few transformants failed to sporulate in laboratory. Transformants from reduced category could not form appressorium and occasionally sporulated. Transformants similar to wild type were morphologically and pathogenically similar to wild type because of un-alteration in their modus operandi. Our work provides a new platform to understand the pathogenicity mechanism of T. carpophilum. The optimized ATMT protocol will help in developing large transformant library that can help to identify the virulence arsenals necessary for the pathogen to cause disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Saffron plant ( Crocus sativus L.) is being used as a source of saffron spice and medicine to cure or prevent different types of diseases including cancers. We report the isolation, characterization of bioactive small molecule ([crocetin (β-d-glucosyl) ester] from the leaf biowastes of saffron plant of Kashmir, India. MTTC assay and Bio-autography aided approach were used to assess anti-oxidant activity and anti-cancer properties of crocin (s) against DPPH free radical and breast cancer cell line respectively. Crocetin beta-d-glucosyl ester restrained proliferation of human breast adeno-carcinoma cell model (MCF-7) without significantly affecting normal cell line (L-6). Further studies involving molecular mechanics generalized born surface area and molecular docking showed that crocetin beta-d-glucosyl ester exhibits strong affinity for estrogen receptor alpha and histone deacetylase 2 (crucial receptors involved in breast cancer signalling) as evidenced by the negative docking score and binding free energy (BFE) values. Therefore, crocetin beta-d-glucosyl ester from Crocus sativus biowastes showed antiproliferative effect possibly by inhibiting estrogen receptor alpha and HDAC2 mediated signalling cascade., (© 2020 The Author(s).)

Apple scab caused by Venturia inaequalis Cooke (Wint.) is one the important diseases of trade and industrial significance in apple. In present study variability studies in pathogen isolates were studied, which is one of the most important factors for devising management studies of scab disease in apple. Genetic diversity of 30 Venturia inaequalis isolates from 12 districts of two geographical distinct regions of Jammu and Kashmir was calculated based on the allele frequencies of 28 SSR markers and the internal transcribed spacer (ITS) region of the ribosomal DNA. The ITS based characterized sequences were submitted to NCBI GenBank and accession numbers were sanctioned. Dendrogram showed that all the accessions formed 2 main clusters with various degree of sub clustering within the clusters. Analysis based on SSR study reveals that the heterozygosity ranged from 0.0 and 0.5, with an average value of 0.39. The expected heterozygosis or gene diversity (He) ranged from 0.0 to 0.50 with an average of 0.40. The Fst value ranges from 0 to 0.6 with an average of 0.194. Diversity within each population (HS) values ranging from 0.26 to 0.33. Average differentiation among populations (GST) was 0.11 and populations were isolated by significant distance (r 2 = 0.50, P < 0.01). From the AMOVA analysis, 25% of variation was observed among population, 9% among individuals and 66% within individuals observed in the population. Structure analysis grouped isolates into two populations. Principle coordinate analysis explained variation of 36.6% in population 1, 14.30% in population 2 and 13.10% in population 3(Admixture) with 64.07% as overall cumulative percentage of variation. This indicates that extensive short-distance gene flow occurs in Kashmir region that dispersal over longer distances also appears to occur frequently enough to prevent differentiation due to genetic drift. Also it is evident that Jammu and Kashmir most likely has V. inaequalis subpopulations linked to diverse climatic conditions of the Jammu region compared to the mountainous inland Kashmir region. The results of present study would help to understand the genetic diversity of V. inaequalis from Jammu and Kashmir that would lead in the development of more effective management strategies and development of new resistant cultivars through marker-assisted selection., Competing Interests: The authors have declared that no competing interests exist.

Elongation factor for RNA polymerase II 2 (ELL2) and ELL-associated factor 2 (EAF2) are two functionally related androgen responsive gene-encoded proteins with prostate tumor suppressor characteristics. EAF2 and ELL2 have both been shown to be down-regulated in advanced prostate cancer, and mice with either Eaf2 or Ell2 deficiency developed murine prostatic intraepithelial neoplasia (mPIN), increased cellular proliferation and increased vascularity. Functional studies have revealed that EAF2 and ELL2 can bind to each other and have similar roles in regulating cell proliferation, angiogenesis and prostate homeostasis. Here, cell line experiments showed that knockdown of EAF2 or ELL2 induced an increase in proliferation and migration in C4-2 and 22Rv1 prostate cancer cells. Concurrent knockdown of EAF2 and ELL2 increased proliferation and migration similarly to the loss of EAF2 or ELL2 alone. Mice with homozygous deletion of Ell2 or heterozygous deletion of Eaf2 developed mPIN lesions characterized by increased epithelial proliferation, intraductal microvessel density, and infiltrating intraductal CD3-positive T-cells compared to wild-type controls. Mice with combined heterozygous deletion of Eaf2 and Ell2 developed mPIN lesions that were similar to those observed in mice with deficiency in Eaf2 or Ell2 alone. These results suggest that EAF2 and ELL2 have similar functions and are likely to require each other in their regulation of prostate epithelial cell proliferation and migration in prostate cancer cells as well as their tumor suppressive properties in the murine prostate., Competing Interests: None.

Androgen receptor (AR) activation is critical for prostate cancer (PCa) development and progression, including castration resistance. The nuclear export signal of AR (NES AR ) has an important role in AR intracellular trafficking and proteasome-dependent degradation. Here, we identified the RNA helicase DHX15 as a novel AR co-activator using a yeast mutagenesis screen and revealed that DHX15 regulates AR activity by modulating E3 ligase Siah2-mediated AR ubiquitination independent of its ATPase activity. DHX15 and Siah2 form a complex with AR, through NES AR . DHX15 stabilized Siah2 and enhanced its E3 ubiquitin-ligase activity, resulting in AR activation. Importantly, DHX15 was upregulated in PCa specimens and its expression was correlated with Gleason scores and prostate-specific antigen recurrence. Furthermore, DHX15 immunostaining correlated with Siah2. Finally, DHX15 knockdown inhibited the growth of C4-2 prostate tumor xenografts in mice. Collectively, our data argue that DHX15 enhances AR transcriptional activity and contributes to PCa progression through Siah2.

Elongation factor, RNA polymerase II, 2 (ELL2) is an RNA Pol II elongation factor with functional properties similar to ELL that can interact with the prostate tumor suppressor EAF2. In the prostate, ELL2 is an androgen response gene that is upregulated in benign prostatic hyperplasia (BPH). We recently showed that ELL2 loss could enhance prostate cancer cell proliferation and migration, and that ELL2 gene expression was downregulated in high Gleason score prostate cancer specimens. Here, prostate-specific deletion of ELL2 in a mouse model revealed a potential role for ELL2 as a prostate tumor suppressor in vivo Ell2 -knockout mice exhibited prostatic defects including increased epithelial proliferation, vascularity and PIN lesions similar to the previously determined prostate phenotype in Eaf2 -knockout mice. Microarray analysis of prostates from Ell2 -knockout and wild-type mice on a C57BL/6J background at age 3 months and qPCR validation at 17 months of age revealed a number of differentially expressed genes associated with proliferation, cellular motility and epithelial and neural differentiation. OncoPrint analysis identified combined downregulation or deletion in prostate adenocarcinoma cases from the Cancer Genome Atlas (TCGA) data portal. These results suggest that ELL2 and its pathway genes likely play an important role in the development and progression of prostate cancer., (© 2017 Society for Endocrinology.)

Aquaporins (AQPs) are primarily involved in maintaining cellular water homeostasis. Their role in diverse physiological processes has fascinated plant scientists for more than a decade, particularly concerning abiotic stresses. Increasing examples of evidence in various crop plants indicate that the AQPs are responsible for precise regulation of water movement and consequently play a crucial role in the drought stress tolerance. Since drought is one of the major abiotic stresses affecting agricultural production worldwide, it has become a critical agenda to focus research on the development of drought tolerant crop plants. AQPs can act as key candidate molecules to confront this issue. Hence, there is an important need to explore the potential of AQPs by understanding the molecular mechanisms and pathways through which they induce drought tolerance. Moreover, the signalling network/s involved in such pathways needs to be mined and understood correctly, and that may lead to the development of drought tolerance in crop plants. In the present review, opportunity and challenges regarding the efficient utilization of AQP-related information is presented and discussed. The complied information and the discussion will be helpful for designing future experiments and to set the specific goals for the enhancement of drought tolerance in crop plants. Biological Significance Knowledge on the role of AQPs in maintaining cellular water homeostasis has given new hope for developing drought tolerance in crop plants. Since drought is one of the major abiotic stresses affecting agricultural production worldwide, it has become a critical agenda to focus research on the development of drought-tolerant crop plants. AQPs can act as key candidate molecules to solve this problem through genetic engineering. For this, it is important to understand the molecular mechanisms and inter-related pathways through which AQPs induce drought tolerance and to explore the signaling network/s involved in such pathways., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The androgen receptor (AR) plays a critical role in the development of castration-resistant prostate cancer (CRPC) as well as in the resistance to the second-generation AR antagonist enzalutamide and the selective inhibitor of cytochrome P450 17A1 (CYP17A1) abiraterone. Novel agents targeting AR may inhibit the growth of prostate cancer cells resistant to enzalutamide and/or abiraterone. Through a high-throughput/high-content screening of a 220,000-member small molecule library, we have previously identified 2-[(isoxazol-4-ylmethyl)thio]-1-(4-phenylpiperazin-1-yl)ethanone (IMTPPE) (SID 3712502) as a novel small molecule capable of inhibiting AR transcriptional activity and protein level in C4-2 prostate cancer cells. In this study, we show that IMTPPE inhibits AR-target gene expression using real-time polymerase chain reaction, Western blot, and luciferase assays. IMTPPE inhibited proliferation of AR-positive, but not AR-negative, prostate cancer cells in culture. IMTPPE inhibited the transcriptional activity of a mutant AR lacking the ligand-binding domain (LBD), indicating that IMTPPE inhibition of AR is independent of the LBD. Furthermore, animal studies showed that IMTPPE inhibited the growth of 22Rv1 xenograft tumor, a model for enzalutamide-resistant prostate cancer. These findings suggest that IMTPPE is a potential lead compound for developing clinical candidates for the treatment of CRPC, including those resistant to enzalutamide., (Copyright © 2017 Endocrine Society.)

The androgen receptor (AR) is a ligand-dependent transcription factor that controls the expression of androgen-responsive genes. A key step in androgen action, which is amplified in castration-resistant prostate cancer (CRPC), is AR nuclear translocation. Small molecules capable of inhibiting AR nuclear localization could be developed as novel therapeutics for CRPC. We developed a high-throughput screen and identified two structurally-related pyrroloimidazoles that could block AR nuclear localization in CRPC cells. We show that these two small molecules, 3-(4-ethoxyphenyl)-6,7-dihydro-5 H -pyrrolo[1,2- a ]imidazole (EPPI) and 3-(4-chlorophenyl)-6,7-dihydro-5 H -pyrrolo[1,2- a ]imidazole (CPPI) can inhibit the nuclear localization and transcriptional activity of AR and reduce the proliferation of AR-positive but not AR-negative prostate cancer cell lines. EPPI and CPPI did not inhibit nuclear localization of the glucocorticoid receptor or the estrogen receptor, suggesting they selectively target AR. In LNCaP tumor xenografts, CPPI inhibited the proliferation of relapsed LNCaP tumors. These findings suggest that EPPI and CPPI could serve as lead structures for the development of therapeutic agents for CRPC. Mol Cancer Ther; 16(10); 2120-9. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Patients with castration-resistant prostate cancer (CRPC) can be treated with abiraterone, a potent inhibitor of androgen synthesis, or enzalutamide, a second-generation androgen receptor (AR) antagonist, both targeting AR signaling. However, most patients relapse after several months of therapy and a majority of patients with relapsed CRPC tumors express the AR target gene prostate-specific antigen (PSA), suggesting that AR signaling is reactivated and can be targeted again to inhibit the relapsed tumors. Novel small molecules capable of inhibiting AR function may lead to urgently needed therapies for patients resistant to abiraterone, enzalutamide, and/or other previously approved antiandrogen therapies. Here, we describe a high-throughput high-content screening (HCS) campaign to identify small-molecule inhibitors of AR nuclear localization in the C4-2 CRPC cell line stably transfected with GFP-AR-GFP (2GFP-AR). The implementation of this HCS assay to screen a National Institutes of Health library of 219,055 compounds led to the discovery of 3 small molecules capable of inhibiting AR nuclear localization and function in C4-2 cells, demonstrating the feasibility of using this cell-based phenotypic assay to identify small molecules targeting the subcellular localization of AR. Furthermore, the three hit compounds provide opportunities to develop novel AR drugs with potential for therapeutic intervention in CRPC patients who have relapsed after treatment with antiandrogens, such as abiraterone and/or enzalutamide.

Androgen receptor (AR) plays a pivotal role in the development of primary as well as advanced castration-resistant prostate cancer. Previous work in our lab identified a novel nuclear export signal (NES) (NES(AR)) in AR ligand-binding domain essential for AR nucleocytoplasmic trafficking. By characterizing the localization of green fluorescence protein (GFP)-tagged NES(AR), we designed and executed a yeast mutagenesis screen and isolated 7 yeast mutants that failed to display the NES(AR) export function. One of those mutants was identified as the splicing factor pre-mRNA processing factor 8 (Prp8). We further showed that Prp8 could regulate NES(AR) function using short hairpin RNA knockdown of Prp8 coupled with a rapamycin export assay in mammalian cells and knockdown of Prp8 could induce nuclear accumulation of GFP-tagged AR in PC3 cells. Prp8 expression was decreased in castration-resistant LuCaP35 xenograft tumors as compared with androgen-sensitive xenografts. Laser capture microdissection and quantitative PCR showed Prp8 mRNA levels were decreased in human prostate cancer specimens with high Gleason scores. In prostate cancer cells, coimmunoprecipitation and deletion mutagenesis revealed a physical interaction between Prp8 and AR mainly mediated by NES(AR). Luciferase assay with prostate specific antigen promoter-driven reporter demonstrated that Prp8 regulated AR transcription activity in prostate cancer cells. Interestingly, Prp8 knockdown also increased polyubiquitination of endogenous AR. This may be 1 possible mechanism by which it modulates AR activity. These results show that Prp8 is a novel AR cofactor that interacts with NES(AR) and regulates AR function in prostate cancer cells.

The androgen receptor (AR) is a member of the steroid receptor superfamily that regulates gene expression in a ligand-dependent manner. The NTD of the AR plays a key role in AR transactivation including androgen-independent activation of the AR in castration-resistant prostate cancer (CRPC) cells. We recently reported that amino acids (a.a.) 50-250 of the NTD are capable of modulating AR nucleocytoplasmic trafficking. To further explore the mechanism associated with a.a. 50-250, GFP pull-down assays were performed in C4-2 CRPC cells transfected with GFP tagged a.a. 50-250 of the AR. Mass spectrometry analysis of the pulled down proteins identified poly (A) binding protein cytoplasmic 1 (PABPC1) interaction with this region of the AR. In silico analysis of gene expression data revealed PABPC1 up-regulation in prostate cancer tissue specimens and this up-regulation correlates to increased disease recurrence. Co-immunoprecipitation assays confirmed the association of PABPC1 with a.a. 50-250 of the NTD of the AR. Knockdown of PABPC1 decreased nuclear AR protein levels and inhibited androgen activation of the AR target PSA in LNCaP and C4-2 cells. Additionally, knockdown of PABPC1 inhibited transactivation of the PSA promoter by NAR (AR lacking the LBD) and attenuated proliferation of AR-positive prostate cancer cells. These findings suggest that PABPC1 is a novel co-regulator of the AR and may be a potential target for blocking activation of the AR in CRPC.

Background: Several studies suggest that Vitamin D (Vit-D3) supplementation reduces Chronic Urticaria (CU) symptoms., Objectives: Evaluation of serum 25-hydroxyvitamin-D (25 (OH)2D) level and assessment of therapeutic effect of VitD3in CU patients., Methods: 192 subjects were stratified according to the baseline 25(OH)2D levels and subsequently randomized into three subgroups to receive Vit-D3 alone (VD) or antihistamine and systemic corticosteroid (H+S) or VitD3 with antihistamine and systemic corticosteroid (VD+H+S) for 6 weeks between July 2012 to Oct 2014. 130 healthy controls (HC) were followed without any intervention. The patients were evaluated for reduction in urticarial symptoms using visual analogue scale (VAS) and 5-D itch score., Results: Low serum levels of 25 (OH)2D was observed in 91% of CU patients and 64% of the healthy controls (P < 0.0001). VAS and 5-D Score in subgroups VD, H + S and VD + H + S decreased significantly from 6 · 7 ± 0 · 043, 6 · 6 ± 0 · 42 and 6 · 68 ± 0 · 40 at baseline to 5 · 2 ± 0 · 70 (P = 0 · 0088), 3 · 3 ± 0 · 50 (P < 0 · 0001) and1 · 86 ± 0 · 39 (P < 0 · 0001) after treatment and from 14 · 5 ± 0 · 72, 13 · 9 ± 0 · 77 and 13 · 9 ± 0 · 221 to 12 · 06 ± 1 · 10 (P = 0 · 0072), 8 · 1 ± 1 · 13 (P < 0 · 0001) and 5 · 01 ± 0 · 94 (P < 0 · 0001) respectively., Conclusions: CU patients have low serum 25(OH)2D levels and Vit-D3 supplementation in combination with antihistamine and systemic corticosteroid show elevated response in resolving the symptoms of CU. This study also warrants that each subject with CU should be screened for serum 25 (OH)2D levels before starting a treatment.

Purpose: Intermittent androgen deprivation therapy in patients with prostate specific antigen progression after localized prostate cancer treatment is an alternative to standard continuous androgen deprivation therapy. Intermittent androgen deprivation therapy allows for testosterone recovery during off cycles. This stimulates regrowth and differentiation of the regressed prostate tumor, lessens the side effects of continuous androgen deprivation therapy and potentially prolongs survival. Previously intermittent androgen deprivation therapy coupled with finasteride was shown to prolong survival in animals bearing androgen sensitive prostate tumors when the off cycle duration was not prolonged but rather fixed at 10 to 14 days. Regressed prostate tumor xenografts with testosterone replacement were initially responsive to 5α-reductase inhibition but growth resumed after several days. In shorter off cycles of testosterone recovery 5α-reductase inhibition might maximize tumor growth inhibition during intermittent androgen deprivation therapy and perhaps increase survival., Materials and Methods: We used the LNCaP xenograft tumor model to evaluate the effectiveness of short off cycles of 4 days coupled with 5α-reductase inhibition on survival and tumor regrowth while on intermittent androgen deprivation therapy., Results: Dutasteride inhibited initial testosterone induced tumor regrowth off cycles 1 and 2, and significantly increased survival., Conclusions: These results further support the potential for intermittent androgen deprivation therapy combined with 5α-reductase inhibition to improve survival in patients with prostate cancer when off cycle duration is short or very short., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Even after adequate immunosuppression therapy, acute rejection continues to be the single most important cause of graft dysfunction after renal transplantation. Renal allograft biopsy continues to be the reference standard, though certain clinical and biochemical parameters are helpful in assessment of these patients. Renal allograft rejection is mediated by T lymphocytes, expressing cell surface interleukin-2 receptors (IL-2R) which has been suggested as a marker of acute rejection episodes after organ transplantation., Objective: To determine the pre- and post-transplantation serum soluble IL-2R levels in live related kidney transplant patients to predict acute rejection episodes., Methods: Serial serum samples from 75 recipients and 41 healthy controls were assessed for soluble IL-2R levels by ELISA. The outcome of the graft was also determined for each recipient., Results: The mean±SD serum soluble IL-2R levels in renal allograft recipients with rejection were significantly (p<0.001) higher than those without rejection (329.85±59.22 vs 18.12±11.22 pg/mL). The elevation of serum soluble IL-2R was evident in acute rejection episodes and found before elevation of serum creatinine. The higher values of serum soluble IL-2R in the rejection group were significantly reduced after recovery of allograft function by adequate anti-rejection therapy. 36.4% of patients in the rejection group had proven positive biopsies for the rejection and higher creatinine values, which was found to be statistically significant (p<0.001). A cohort of 41 healthy controls showed significantly (p<0.05) lower serum soluble IL-2R concentrations (15.27±7.79 pg/mL) when compared with the rejection group., Conclusion: Serum soluble IL-2R concentrations showed significant correlation with the acute rejection episodes in the renal allograft recipients. Prediction of soluble IL-2R levels might help the early detection of rejection episodes, which may pave way for the management of immunosuppression regimes and better graft functioning.

To study the possible role of proinflammatory interleukin 6 -174 G>C (rs 1800795) and -634 C>G (rs 1800796) polymorphism in the pathogenesis of non-small cell lung cancer (NSCLC). A total of 190 NSCLC patients and 200 healthy controls were evaluated for polymorphic analysis of -174 G/C and -634 C/G by PCR-RFLP followed by DNA sequencing. A significant association was observed in the genotypic and allelic distribution of IL-6 -174 G/C in the NSCLC group as compared to control group [OR = 2.7 (1.77-4.11), p < 0.0001]. Smokers with the -174C allele were found to be significantly associated with NSCLC (p = 0.01), while 634C/G SNP showed an inverse relation [OR-0.4, p < 0.0001]. The present investigation revealed a significant association of the IL6 -174 G/C gene promoter polymorphism with NSCLC, and thus, the IL-6 -174G/C genotype can be considered as one of the biological markers in the etiology of NSCLC.

Background: The gene for the vascular endothelial growth factor (VEGF), which promotes angiogenesis and permeability, is polymorphic. The aim of the present study was to evaluate the relationship between +936C>T and +404C>G polymorphism of VEGF with risk of esophageal cancer in the Kashmiri population in India., Materials and Methods: 150 esophageal cancer patients and 150 unrelated healthy controls were genotyped for two VGEF SNPs (+405C/G, and +936C/T) using DNA extracted from prospectively collected blood samples by the PCR-RFLP method., Results: For the VEGF +936C>T polymorphism a significant association of CT and combined CT+TT genotypes was observed with increased risk of esophageal cancer (p=0.021; 0.024). For the +405C>G polymorphism we observed significantly increased frequency of GG genotype in cases as compared to controls and also the +405 GG Genotype was observed to have a two fold risk(OR=2.7356; 95%CI=1.1409- 6.5593; p=0.020). The combined genotypes of GG-CC and GG-CT of +405C>G and +936C>T were found to be significantly associated with increased risk of esophageal cancer (p=0.0376; 0.0099)., Conclusions: From the results of the present study a significant association of +936C>T and +405C>G polymorphisms with increased esophageal cancer risk exists in the Kashmiri population.

A new class of compounds targeting cyclooxygenase 2 (COX-2) together with other different clinically used therapeutic strategies has recently shown a promise for the chemoprevention of several solid tumors including lung cancer. The aim was to study the possible role of COX-2 -8473 T/C NP and its expression in the pathogenesis of non-small cell lung cancer. One hundred ninety non-small cell lung cancer (NSCLC) patients and 200 healthy age-, sex-, and smoking-matched controls were used for polymorphic analysis, and 48 histopathologically confirmed NSCLC patients were analyzed for COX-2 messenger RNA (mRNA) and protein expression. Our results showed that the frequencies of variant genotypes 8473 CT/CC were significantly less common in the cases (30.0%) than in the controls (36%), suggesting that the 8473 C variant allele is related with lower susceptibility in NSCLC (OR = 0.79, 95% CI 0.54-1.4). However, the frequency of COX-2 -8473 TC and CC genotypes were significantly associated with age in NSCLC (P = 0.02). Quantitative real-time expression analysis showed a significant increase in the COX-2 mRNA in tumor tissues as compared to their adjacent normal tissues [delta cycle threshold (ΔCT) = 9.25 ± 4.67 vs 5.63 ± 3.85, P = 0.0001]. Multivariate logistic regression analyses revealed that the COX-2 expression was associated significantly with age (P = 0.044). Also, an increasing trend was observed in stages I and II and in female patients compared to stages III and IV and male patients, respectively, but no statistical significance was observed. However, COX-2 mRNA expression shown no association with the -8473 C variant allele. Our findings indicate that the COX-2 T8473C polymorphism may contribute to NSCLC cancer susceptibility in the Kashmiri population, while our expression analysis revealed a significant increase of COX-2 in tumor tissues as compared to their adjacent normal tissues, suggesting that it could become an important therapeutic marker in NSCLC in the future.

Background: Stroke leads to transient immunedepression, which leads to increased incidence of poststroke infections. Because infection is one of the most common causes of increased mortality in patients with stroke, this study was undertaken to document immunedepression after stroke in our population., Methods: A case-controlled study wherein 39 patients with acute ischemic stroke in the age group of 18 and 60 years without any evidence of previous immunedepression were included. Interleukin 6 (IL-6) and interleukin 10 (IL-10) levels were checked in plasma in both the groups on day 3 and day 45. Also Cortisol and epinephrine levels were checked in the urine samples collected on day 3 and day 8., Results: No significant difference was seen between the IL-6 and the IL-10 levels in samples collected on day 3 between the controls and cases, whereas Cortisol and norepinephrine were significantly raised in samples collected on day 3 in cases who developed infection as compared with controls., Conclusions: The higher levels of urinary cortisol and norepinephrine were observed in patients with stroke who developed infections, which indirectly reflected increased amount of stroke related stress. Furthermore, the levels of plasma IL-6 and IL-10 were also elevated in the same group of patients, which means transformation of immunecompetence to immunedepression, which is responsible for higher mortality. Subsequently on recovery from infection the plasma levels of interleukins and urinary cortisol and norepinephrine did not show any difference, which indirectly means recovery of the immune system on recovery from acute stage of stroke. Mortality in the patients with infection was increased than controls., (Copyright © 2014 National Stroke Association. All rights reserved.)

Androgen-independent nuclear localization is required for androgen receptor (AR) transactivation in castration-resistant prostate cancer (CRPC) and should be a key step leading to castration resistance. However, mechanism(s) leading to androgen-independent AR nuclear localization are poorly understood. Since the N-terminal domain (NTD) of AR plays a role in transactivation under androgen-depleted conditions, we investigated the role of the NTD in AR nuclear localization in CRPC. Deletion mutagenesis was used to identify amino acid sequences in the NTD essential for its androgen-independent nuclear localization in C4-2, a widely used CRPC cell line. Deletion mutants of AR tagged with green fluorescent protein (GFP) at the 5'-end were generated and their signal distribution was investigated in C4-2 cells by fluorescent microscopy. Our results showed that the region of a.a. 294-556 was required for androgen-independent AR nuclear localization whereas a.a. 1-293 mediates Hsp90 regulation of AR nuclear localization in CRPC cells. Although the region of a.a. 294-556 does not contain a nuclear import signal, it was able to enhance DHT-induced import of the ligand binding domain (LBD). Also, transactivation of the NTD could be uncoupled from its modulation of AR nuclear localization in C4-2 cells. These observations suggest an important role of the NTD in AR intracellular trafficking and androgen-independent AR nuclear localization in CRPC cells., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Intermittent androgen deprivation therapy (IADT) allows prostate cancer patients a break from the side-effects of continuous androgen deprivation therapy (ADT). Although clinical studies suggest that IADT can significantly improve patient quality of life over ADT, it has not been demonstrated to improve patient survival. Recently, increased survival has been demonstrated when 5α-reductase inhibitors have been used during the off-cycle of IADT in animal xenograft tumor models LNCaP and LuCaP35. In the current study, the sensitivity of LAPC4 xenograft tumor regrowth to the 5ARI dutasteride was determined. Tumor regrowth and gene expression changes in LAPC4 tumors were compared to the previously determined response of LNCaP and LuCaP35 xenograft tumors to 5ARI treatment during the off-cycle of IADT, LAPC4, LNCaP and LuCaP35 tumors were sensitive to androgen manipulation. However, in contrast to LNCaP and LuCaP35, dutasteride treatment during testosterone-stimulated prostate regrowth did not affect tumor regrowth or the expression of androgen responsive genes. Tumor response to dutasteride during the off-cycle of IADT is variable in xenograft prostate tumor models. Future studies will be required to elucidate the mechanisms contributing to the dutasteride resistance observed in the LAPC4 model during the off-cycle.

Nucleocytoplasmic trafficking of the androgen receptor (AR) represents an essential step in androgen action. To determine whether the amino-terminal domain (NTD) contains potential nuclear import and/or export signals, deletion mutants of the NTD tagged with green fluorescent protein (GFP) were generated and tested for their intracellular localization in both AR-negative and AR-positive cell lines. Subcellular localization analysis suggested a role of the NTD in regulating AR subcellular localization and revealed that the region of a.a. 50-250 of the NTD of AR (AR(50-250)) could promote cytoplasmic localization. Leptomycin B inhibited the activity of AR(50-250), suggesting that AR(50-250) export is mediated through exportin 1, either directly or indirectly. These observations argue for an important role of the NTD in regulating AR nucleocytoplasmic trafficking and will facilitate further investigation of interactions among different signals in regulating AR nucleocytoplasmic trafficking, which may lead to new approaches to inhibit AR nuclear localization., (Copyright © 2013 Elsevier Ltd. All rights reserved.)