Mapping of IgE-binding regions on recombinant Cyn d 1, a major allergen from Bermuda Grass Pollen (BGP)

<p>Abstract</p> <p>Background</p> <p>Bermuda grass (<it>Cynodon dactylon</it>; subfamily Chloridoideae) is an important source of seasonal aeroallergens in warm tropical and sub-tropical areas worldwide. Improved approaches to diagnosis and therapy of allergic diseases require a thorough understanding of the structure and epitopes on the allergen molecule that are crucial for the antigen-antibody interaction. This study describes the localization of the human IgE-binding regions of the major group 1 pollen allergen Cyn d 1 from Bermuda grass.</p> <p>Methods</p> <p>A cDNA library was constructed from Bermuda grass pollen (BGP) using a Lambda gt11 expression vector. The gene encoding the Cyn d 1 allergen was isolated by screening the library with a mouse monoclonal antibody raised against grass group 1 allergen. In order to characterize the IgE epitopes on Cyn d 1, seven overlapping fragments and three deletion mutants were cloned and over-expressed in E. coli. The recombinant fragments and deletion mutants were evaluated for their comparative IgE reactivity with sera of non atopic individuals and grass pollen allergic patients by ELISA and a dot-blot assay.</p> <p>Results</p> <p>Analysis of IgE binding regions by overlapping fragments and deletion mutants identified two major allergenic regions corresponding to amino acids 120–170 and 224–244. Deletion of either or both regions led to a significant reduction in IgE binding, emphasizing the importance of the C-terminal region on Cyn d 1 in epitope-IgE interaction.</p> <p>Conclusion</p> <p>Anti-Cyn d 1 IgE antibodies from allergic human sera recognize two epitopes located at the C-terminal end of the molecule. These data will enable the design of improved diagnostic and therapeutic approaches for BGP hypersensitivity.</p

Isolation and characterization of a flowering plant male gametic cell-specific promoter11GenBank accession number: AY207012.

AbstractFlowering plant male gametic cell-specific gene expression has been reported recently but the regulatory elements controlling specificity of such genes expressed in generative cell and sperm cells have not been identified and studied. Here, we report the 0.8 kb promoter sequence upstream of the start of the transcription site of the generative cell-specific gene, LGC1, sufficient to regulate the expression of reporter genes in a cell-specific manner. In addition, the diphtheria toxin A-chain- (DT-A)-coding region under the control of the LGC1 promoter sequence confirmed unequivocally the lack of LGC1 expression in vegetative tissues. Transgenic tobacco plants carrying the LGC1-DT/A construct showed normal phenotype except for anthers of these plants that contained sterile and aborted pollen. Truncation and internal deletion analysis of the LGC1 promoter identified −242 bp as the minimal sequence necessary for male gametic cell-specific expression. In addition, a regulatory sequence required for determining generative cell-specific expression of LGC1 was identified. Deletion of this regulatory sequence led to loss of the generative cell specificity resulting in activation of this promoter in other tissues where it is normally repressed. Therefore, male gametic cell specificity of the LGC1 gene seems to be regulated by factors that suppress its activation in other plant cells. This is the first report of a male gametic cell-specific promoter, hence can be used as a novel tool in molecular analyses and experimental manipulation of flowering plant spermatogenesis and fertilization

Biological parts for engineering abiotic stress tolerance in plants

It is vital to ramp up crop production dramatically by 2050 due to the increasing global population and demand for food. However, with the climate change projections showing that droughts and heatwaves becoming common in much of the globe, there is a severe threat of a sharp decline in crop yields. Thus, developing crop varieties with inbuilt genetic tolerance to environmental stresses is urgently needed. Selective breeding based on genetic diversity is not keeping up with the growing demand for food and feed. However, the emergence of contemporary plant genetic engineering, genome-editing, and synthetic biology offer precise tools for developing crops that can sustain productivity under stress conditions. Here, we summarize the systems biology-level understanding of regulatory pathways involved in perception, signalling, and protective processes activated in response to unfavourable environmental conditions. The potential role of noncoding RNAs in the regulation of abiotic stress responses has also been highlighted. Further, examples of imparting abiotic stress tolerance by genetic engineering are discussed. Additionally, we provide perspectives on the rational design of abiotic stress tolerance through synthetic biology and list various bioparts that can be used to design synthetic gene circuits whose stress-protective functions can be switched on/off in response to environmental cues

RNA-seq highlights molecular events associated with impaired pollen-pistil interactions following short-term heat stress in Brassica napus

The global climate change is leading to increased frequency of heatwaves with crops getting exposed to extreme temperature events. Such temperature spikes during the reproductive stage of plant development can harm crop fertility and productivity. Here we report the response of short-term heat stress events on the pollen and pistil tissues in a commercially grown cultivar of Brassica napus. Our data reveals that short-term temperature spikes not only affect pollen fitness but also impair the ability of the pistil to support pollen germination and pollen tube growth and that the heat stress sensitivity of pistil can have severe consequences for seed set and yield. Comparative transcriptome profiling of non-stressed and heat-stressed (40°C for 30 min) pollen and pistil (stigma + style) highlighted the underlying cellular mechanisms involved in heat stress response in these reproductive tissues. In pollen, cell wall organization and cellular transport-related genes possibly regulate pollen fitness under heat stress while the heat stress-induced repression of transcription factor encoding transcripts is a feature of the pistil response. Overall, high temperature altered the expression of genes involved in protein processing, regulation of transcription, pollen-pistil interactions, and misregulation of cellular organization, transport, and metabolism. Our results show that short episodes of high-temperature exposure in B. napus modulate key regulatory pathways disrupted reproductive processes, ultimately translating to yield loss. Further investigations on the genes and networks identified in the present study pave a way toward genetic improvement of the thermotolerance and reproductive performance of B. napus varieties

Rapid transcriptional reprogramming associated with heat stress-induced unfolded protein response in developing Brassica napus anthers

Climate change associated increases in the frequency and intensity of extreme temperature events negatively impact agricultural productivity and global food security. During the reproductive phase of a plant’s life cycle, such high temperatures hinder pollen development, preventing fertilization, and seed formation. At the molecular level, heat stress-induced accumulation of misfolded proteins activates a signaling pathway called unfolded protein response (UPR) in the endoplasmic reticulum (ER) and the cytoplasm to enhance the protein folding capacity of the cell. Here, we report transcriptional responses of Brassica napus anthers exposed to high temperature for 5, 15, and 30 min to decipher the rapid transcriptional reprogramming associated with the unfolded protein response. Functional classification of the upregulated transcripts highlighted rapid activation of the ER-UPR signaling pathway mediated by ER membrane-anchored transcription factor within 5 min of heat stress exposure. KEGG pathway enrichment analysis also identified “Protein processing in ER” as the most significantly enriched pathway, indicating that the unfolded protein response (UPR) is an immediate heat stress-responsive pathway during B. napus anther development. Five minutes of heat stress also led to robust induction of the cytosolic HSF-HSP heat response network. Our results present a perspective of the rapid and massive transcriptional reprogramming during heat stress in pollen development and highlight the need for investigating the nature and function of very early stress-responsive networks in plant cells. Research focusing on very early molecular responses of plant cells to external stresses has the potential to reveal new stress-responsive gene networks that can be explored further for developing climate change resilient crops

Uncommon presentation of a ganglionic cyst: a case study of intra muscular ganglion cyst of rectus femoris

Ganglionic cyst most commonly occurs in hand and wrist. When it presents in uncommon location like in lower limb, it causes a diagnostic dilemma. One such case is ours, an uncommon presentation of intra muscular cystic ganglion of rectus femoris. Due to its unlikely presentation in rectus femoris, diagnosis and management was delayed. A 12 years old boy presented with complaints of pain and swelling over left lower limb in suprapatellar region. On radiographic and ultrasound examination, swelling was found to be cystic lesion in rectus femoris. Histopathological examination of biopsied specimen was found to be intra muscular ganglion cyst of rectus femoris. The patient’s general condition improved with betterment in laboratory parameters, resolution of the lesion, without any sequelae, no residual deformity and excellent clinical outcome. To consider cystic ganglion as differential diagnosis, along with intra muscular myxoma, lipoma and synovial cyst in patients with lower limb intra muscular swelling

The role of endoplasmic reticulum stress response in pollen development and heat stress tolerance

Endoplasmic reticulum (ER) stress is defined by a protracted disruption in protein folding and accumulation of unfolded or misfolded proteins in the ER. This accumulation of unfolded proteins can result from excessive demands on the protein folding machinery triggered by environmental and cellular stresses such as nutrient deficiencies, oxidative stress, pathogens, and heat. The cell responds to ER stress by activating a protective pathway termed unfolded protein response (UPR), which comprises cellular mechanisms targeted to maintain cellular homeostasis by increasing the ER’s protein folding capacity. The UPR is especially significant for plants as being sessile requires them to adapt to multiple environmental stresses. While multiple stresses trigger the UPR at the vegetative stage, it appears to be active constitutively in the anthers of unstressed plants. Transcriptome analysis reveals significant upregulation of ER stress-related transcripts in diploid meiocytes and haploid microspores. Interestingly, several ER stress-related genes are specifically upregulated in the sperm cells. The analysis of gene knockout mutants in Arabidopsis has revealed that defects in ER stress response lead to the failure of normal pollen development and enhanced susceptibility of male gametophyte to heat stress conditions. In this mini-review, we provide an overview of the role of ER stress and UPR in pollen development and its protective roles in maintaining male fertility under heat stress conditions

Draft bills and research reports on: reducing judicial corruption and child labor in Nepal

These two draft bills and accompanying research report comprise the work of two teams of Nepali officials from Nepal's Ministry of Law and Justice who prepared them in the context of the Boston University School of Law Program on Legislative Drafting for Democratic Social Change. They attended that Program as part of a larger Ministry of Law and Justice Program, funded by the United Nations Development Program (UNDP), to strengthen Nepal's legal framework and the Rule of Law. Using the bills and reports as case studies, the four officials aimed to learn legislative theory, methodology and techniques. The Ministry had assigned them, on their return to Nepal, to play a significant role in institutionalizing an on-going learning process to strengthen Nepali drafters' capacity to prepare the effectively implementable legislation necessary to ensure good governance and development

Genome-wide in silico identification and comparative analysis of Dof gene family in Brassica napus

DNA binding with one finger (DOF) proteins are plant-specific transcription factors that play roles in diverse plant functions. However, little is known about the DOF protein repertoire of the allopolyploid crop, Brassica napus. This in silico study identified 117 Brassica napus Dof genes (BnaDofs) and classified them into nine groups (A, B1, B2, C1, C2.1, C2.2, C3, D1, and D2), based on phylogenetic analysis. Most members belonging to a particular group displayed conserved gene structural organisation and protein motif distribution. Evolutionary analysis exemplified that the divergence of the Brassica genus from Arabidopsis, the whole-genome triplication event, and the hybridisation of Brassica oleracea and Brassica rapa to form B. napus, followed by gene loss and rearrangements, led to the expansion and divergence of the Dof transcription factor (TF) gene family in B. napus. So far, this is the largest number of Dof genes reported in a single eudicot species. Functional annotation of BnaDof proteins, cis-element analysis of their promoters, and transcriptomic analysis suggested potential roles in organ development, the transition from the vegetative to the reproductive stage, light responsiveness, phytohormone responsiveness, as well as potential regulatory roles in abiotic stress. Overall, our results provide a comprehensive understanding of the molecular structure, evolution, and possible functional roles of Dof genes in plant development and abiotic stress response

BHUx: A Patent Polyherbal Formulation to Prevent Atherosclerosis

Ayurvedic medicine is a time-tested system of medicine which has been in clinical use for centuries in India. Being a time-tested system, it has an edge over other existing systems of health management, especially for dealing with chronic disorders such as coronary artery disease, which is of a complex multi-etiological nature. Recently, we have shown that BHUx, a patented polyherbal formulation consisting of the aqueous fraction of five medicinal plants of the ayurvedic system, has significant anti-inflammatory properties through inhibition of cyclooxygenase-2 and lipoxygenase-15. Here we have investigated its effect on diet-induced atherosclerosis in albino rabbits. BHUx was given orally for 3 months to rabbits pre-treated with an atherogenic diet for 3 months. After 6 months, the dorsal aorta was processed for histological studies for calcium and collagen content. The results demonstrated a remarkable reduction in intimal thickening in the treated animals. In addition, there was less calcification at the intima–medial interface and increased intensity of collagen cap on the surface along with an increase in survival, compared with the sham control. We suggest that BHUx is a potent, multi-factorial formulation against atherosclerosis