Right Phrenic Nerve Palsy following Long-gap Esophageal Atresia and Tracheoesophageal Fistula Repair.

Esophageal atresia (EA) and tracheoesophageal fistula (TEF) are surgically correctable congenital anomalies with reported surgical common complications such as anastomotic leaks, recurrent TEF, and esophageal strictures; however, phrenic nerve injury (PNI) is a very rare but possible complication which we have highlighted in our case report. Here, we report a baby girl operated for long-gap EA and TEF having respiratory distress and failed attempts to wean off oxygen support. Serial chest X-rays showed elevated right hemidiaphragm, whereas ultrasound thorax confirmed our diagnosis of right PNI causing diaphragmatic palsy. Conservative management with the hope of spontaneous recovery failed, so diaphragmatic plication was done at 5 weeks from index surgery. Postplication, the baby was weaned off oxygen and pressure support the very 1st day and had improved respiratory physiology.

Influence of domestication on specialized metabolic pathways in fruit crops.

MAIN CONCLUSION: During the process of plant domestication, the selection and traditional breeding for desired characters such as flavor, juiciness and nutritional value of fruits, probably have resulted in gain or loss of specialized metabolites contributing to these traits. Their appearance in fruits is likely due to the acquisition of novel and specialized metabolic pathways and their regulation, driven by systematic molecular evolutionary events facilitated by traditional breeding. Plants change their armory of specialized metabolism to adapt and survive in diverse ecosystems. This may occur through molecular evolutionary events, such as single nucleotide polymorphism, gene duplication and transposition, leading to convergent or divergent evolution of biosynthetic pathways producing such specialized metabolites. Breeding and selection for improved specific and desired traits (fruit size, color, taste, flavor, etc.) in fruit crops through conventional breeding approaches may further alter content and profile of specialized metabolites. Biosynthetic routes of these metabolites have been studied in various plants. Here, we explore the influence of plant domestication and breeding processes on the selection of biosynthetic pathways of favorable specialized metabolites in fruit crops. An orderly clustered arrangement of genes associated with their production is observed in many fruit crops. We further analyzed selection-based acquisition of specialized metabolic pathways comparing first the metabolic profiles and genes involved in their biosynthesis, followed by the genomic organization of such genes between wild and domesticated horticultural crops. Domestication of crop plants favored the acquisition and retention of metabolic pathways that enhanced the fruit value while eliminated those which produced toxic or unfavorable metabolites. Interestingly, unintentional reorganization of complex metabolic pathways by selection and traditional breeding processes has endowed us with flavorful, juicy and nutritionally rich fruits.

Comparative Proteomics Unravels the Differences in Salt Stress Response of Own-Rooted and 110R-Grafted Thompson Seedless Grapevines.

Thompson Seedless, a commonly grown table grape variety, is sensitive to salinity when grown on its own roots, and therefore, it is frequently grafted onto salinity-tolerant wild grapevine rootstocks. Rising soil salinity is a growing concern in irrigated agricultural systems. The accumulation of salts near the root zone severely hampers plant growth, leading to a decrease in the productive lifespan of grapevine and causing heavy yield losses to the farmer. In the present study, we investigated the differences in response to salinity between own-rooted Thompson Seedless (TSOR) and 110R-grafted Thompson Seedless (TS110R) grapevines, wherein 110R is reported to be a salt-tolerant rootstock. The grapevines were subjected to salt stress by treating them with a 150 mM NaCl solution. The stress-induced changes in protein abundance were investigated using a label-free shotgun proteomics approach at three time-points viz. 6 h, 48 h, and 7 days of salt treatment. A total of 2793 proteins were identified, of which 246 were differentially abundant at various time-points in TSOR and TS110R vines. The abundance of proteins involved in several biological processes such as photosynthesis, amino acid metabolism, translation, chlorophyll biosynthesis, and generation of precursor metabolites was significantly affected by salt stress in both the vines but at different stages of stress. The results revealed that TSOR vines responded fervently to salt stress, while TS110R vines adopted a preventive approach. The findings of this study add to the knowledge of salinity response in woody and grafted plants and hence open the scope for further studies on salt stress-specific differences induced by grafting.

Integrated Transcriptomic and Proteomic Analyses Suggest the Participation of Endogenous Protease Inhibitors in the Regulation of Protease Gene Expression in Helicoverpa armigera.

Insects adapt to plant protease inhibitors (PIs) present in their diet by differentially regulating multiple digestive proteases. However, mechanisms regulating protease gene expression in insects are largely enigmatic. Ingestion of multi-domain recombinant Capsicum annuum protease inhibitor-7 (CanPI-7) arrests growth and development of Helicoverpa armigera (Lepidoptera: Noctuidae). Using de novo RNA sequencing and proteomic analysis, we examined the response of H. armigera larvae fed on recombinant CanPI-7 at different time intervals. Here, we present evidence supporting a dynamic transition in H. armigera protease expression on CanPI-7 feeding with general down-regulation of protease genes at early time points (0.5 to 6 h) and significant up-regulation of specific trypsin, chymotrypsin and aminopeptidase genes at later time points (12 to 48 h). Further, coexpression of H. armigera endogenous PIs with several digestive protease genes were apparent. In addition to the differential expression of endogenous H. armigera PIs, we also observed a distinct novel isoform of endogenous PI in CanPI-7 fed H. armigera larvae. Based on present and earlier studies, we propose potential mechanism of protease regulation in H. armigera and subsequent adaptation strategy to cope with anti-nutritional components of plants.

Time to Detection of Yeast Isolates in Pediatric and Adult Patients with Fungemia and its Relevance to Clinical Profile and Outcome.

CONTEXT: Time to detection (TTD) given by continuous monitoring automated blood culture systems (CMABS) have been found to be a predictor of clinical outcome, drug resistance and type of microorganism in cases of bacteremia but the studies evaluating TTD with respect to fungemia are scarce especially from India. AIMS: To evaluate TTD for yeast isolates in fungal bloodstream infections with respect to the type of yeast isolates, risk factors and outcome and to study yeast susceptibility and distribution of yeast isolates with respect to patient population. MATERIALS AND METHODS: All blood culture specimens were processed in CMABS. The TTD for yeast isolates were recorded. The identification of yeast and susceptibility testing was done by automated methods. A correlation of TTD was done with respect to prior/concurrent yeast isolates, use of antifungal, risk factors and clinical outcome. RESULTS: Out of 80 yeast isolates, the maximum was C. parapsilosis (26.25%) followed by C. albicans (16.25%) and C. tropicalis (13.75%). A statistically significant difference in the occurrence of yeasts with early TTD (TTD < = 48 hours) and late TTD (TTD > 48 hours) was found. TTD of C. glabrata was significantly longer (p = 0.002) while TTD of C. tropicalis was significantly shorter (p = 0.013). There was an observable favorable outcome in shorter TTD (< = 48 hours). C. albicans and C. tropicalis depicted 100% susceptibility for Azoles, Amphotericin B and Echinocandins. CONCLUSION: TTD may be used as both diagnostic and prognostic adjunct in fungal bloodstream infections. This study is a step towards this novel approach. We also emphasize on the importance of speciation of yeast isolates and susceptibility testing. HOW TO CITE THIS ARTICLE: Butta H, Sardana R, Mendiratta L, Sibal A, Gupta V, Chawla R, Jafri AA. Time to Detection of Yeast Isolates in Pediatric and Adult Patients with Fungemia and its Relevance to Clinical Profile and Outcome. Indian Journal of Critical Care Medicine, January 2019;23(1):27-30.

GA3 application in grapes (Vitis vinifera L.) modulates different sets of genes at cluster emergence, full bloom, and berry stage as revealed by RNA sequence-based transcriptome analysis.

In grapes (Vitis vinifera L.), exogenous gibberellic acid (GA3) is applied at different stages of bunch development to achieve desirable bunch shape and berry size in seedless grapes used for table purpose. RNA sequence-based transcriptome analysis was used to understand the mechanism of GA3 action at cluster emergence, full bloom, and berry stage in table grape variety Thompson Seedless. At cluster emergence, rachis samples were collected at 6 and 24 h after application of GA3, whereas flower clusters and berry samples were collected at 6, 24, and 48 h after application at full bloom and 3-4 mm berry stages. Seven hundred thirty-three genes were differentially expressed in GA3-treated samples. At rachis and flower cluster stage respectively, 126 and 264 genes were found to be significantly differentially expressed within 6 h of GA3 application. The number of DEG reduced considerably at 24 h. However, at berry stage, major changes occurred even at 24 h and a number of DEGs at 6 and 24 h were 174 and 191, respectively. As compared to upregulated genes, larger numbers of genes were downregulated. Stage-specific response to the GA3 application was observed as evident from the unique set of DEGs at each stage and only a few common genes among three stages. Among the DEGs, 67 were transcription factors. Functional categorization and enrichment analysis revealed that several transcripts involved in sucrose and hexose metabolism, hormone and secondary metabolism, and abiotic and biotic stimuli were enriched in response to application of GA3. A high correlation was recorded for real-time PCR and transcriptome data for selected DEGs, thus indicating the robustness of transcriptome data obtained in this study for understanding the GA3 response at different stages of berry development in grape. Chromosomal localization of DEGs and identification of polymorphic microsatellite markers in selected genes have potential for their use in breeding for varieties with improved bunch architecture.

Pharmaceutical Standardization and Physicochemical Characterization of Traditional Ayurvedic Marine Drug: Incinerated Conch Shell (Shankha Bhasma).

Natural resources such as plants, animals and minerals have always been used by mankind to develop drugs and marine world is no exception. Marine by-products like conches, pearls, mother of pearl shells, corals and so forth have been used by traditional Ayurvedic practitioners for centuries. The unique methods of these preparations are scientifically designed to eliminate unwanted impurities and convert them into bioavailable form. In this study, Conch (Xanchus pyrum) was used as a marine resource of calcium carbonate and was converted pharmaceutically from its aragonite form to calcite. All the steps of preparations and changes in the properties therein were documented and validated. Further, traditional as well as modern analytical tools were used to study its physical and chemical characters to develop a monograph. The physical characterization included particle size, X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Thermogravimetric Analysis (TGA) and Fourier Transform Infra-red (FTIR). Metal composition and heavy metal limits were determined using Inductively Coupled Plasma Optical Emission Spectrometry (ICPOES). This study revealed the rearrangement of aragonite crystals into calcite form by grinding, trituration with aloe vera juice and incineration under controlled conditions. Moreover, the finished product was found to be devoid of organic matrix that is nacre. This study creates a foundation for the development of a master formula for commonly used Shankha Bhasma in Ayurvedic medicines.

Identification and functionality prediction of pathogenesis-related protein 1 from legume family.

The production and accumulation of pathogenesis-related (PR) proteins in plants is one of the important responses to biotic and abiotic stress. Large number of identified PR proteins has been categorized into 17 functional families based on their structure, phylogenetics, and biological activities. However, they are not widely studied in legume crops. Using 29 PR1 proteins from Arabidopsis thaliana, as query, here we have predicted 92 candidate PR1 proteins through the PSI-BLAST and HMMER programs. These candidate proteins were comprehensively analyzed with, multiple sequence alignment, domain architecture studies, signal peptide, and motif extraction followed by phylogenetic analysis. Further, response of two candidate PR1 proteins from chickpea against Fusarium oxysporum f.sp.ciceri attack was validated using qRT-PCR followed by their 3D structure prediction. To decipher mode of action for PR1s, docking of pathogen extracellular matrix components along with fungal elicitors was performed with two chickpea PR1 proteins. Based on these findings, we propose carbohydrate to be the unique pathogen-recognition feature for PR1 proteins and ß-glucanase activity via ß-glucan binding or modification.

Genomic and functional characterization of coleopteran insect-specific α-amylase inhibitor gene from Amaranthus species.

The smallest 32 amino acid α-amylase inhibitor from Amaranthus hypochondriacus (AAI) is reported. The complete gene of pre-protein (AhAI) encoding a 26 amino acid (aa) signal peptide followed by the 43 aa region and the previously identified 32 aa peptide was cloned successfully. Three cysteine residues and one disulfide bond conserved within known α-amylase inhibitors were present in AhAI. Identical genomic and open reading frame was found to be present in close relatives of A. hypochondriacus namely Amaranthus paniculatus, Achyranthes aspera and Celosia argentea. Interestingly, the 3'UTR of AhAI varied in these species. The highest expression of AhAI was observed in A. hypochondriacus inflorescence; however, it was not detected in the seed. We hypothesized that the inhibitor expressed in leaves and inflorescence might be transported to the seeds. Sub-cellular localization studies clearly indicated the involvement of AhAI signal peptide in extracellular secretion. Full length rAhAI showed differential inhibition against α-amylases from human, insects, fungi and bacteria. Particularly, α-amylases from Helicoverpa armigera (Lepidoptera) were not inhibited by AhAI while Tribolium castaneum and Callosobruchus chinensis (Coleoptera) α-amylases were completely inhibited. Molecular docking of AhAI revealed tighter interactions with active site residues of T. castaneum α-amylase compared to C. chinensis α-amylase, which could be the rationale behind the disparity in their IC50. Normal growth, development and adult emergence of C. chinensis were hampered after feeding on rAhAI. Altogether, the ability of AhAI to affect the growth of C. chinensis demonstrated its potential as an efficient bio-control agent, especially against stored grain pests.

Effect of postharvest ethylene treatment on sugar content, glycosidase activity and its gene expression in mango fruit.

BACKGROUND: Ripening-associated softening is one of the important attributes that largely determines the shelf-life of mango (Mangifera indica Linn.) fruits. To reveal the effect of pre-climacteric ethylene treatment on ripening-related softening of Alphonso mango, ethylene treatment was given to mature, raw Alphonso fruits. Changes in the pool of reducing and non-reducing sugars, enzymatic activity of three glycosidases: ß-d-galactosidase, α-d-mannosidase and ß-d-glucosidase and their relative transcript abundance were analysed for control and ethylene treated fruits during ripening. RESULTS: Early activity of all the three glycosidases and accelerated accumulation of reducing and non-reducing sugars on ethylene treatment was evident. ß-d-Galactosidase showed the highest activity among three glycosidases in control fruits and marked increase in activity upon ethylene treatment. This was confirmed by the histochemical assay of its activity in control and ethylene treated ripe fruits. Relative transcript abundance revealed high transcript levels of ß-d-galactosidase in control fruits. Ethylene-treated fruits showed early and remarkable increase in the ß-d-galactosidase transcripts while α-d-mannosidase transcript variants displayed early accumulation. CONCLUSION: The findings suggest reduction in the shelf-life of Alphonso mango upon pre-climacteric ethylene treatment, a significant role of ß-d-galactosidase and α-d-mannosidase in the ripening related softening of Alphonso fruits and transcriptional regulation of their expression by ethylene. © 2016 Society of Chemical Industry.

Improved tolerance against Helicoverpa armigera in transgenic tomato over-expressing multi-domain proteinase inhibitor gene from Capsicum annuum.

Plant proteinase inhibitors (PIs) are plant defense proteins and considered as potential candidates for engineering plant resistances against herbivores. Capsicum annuum proteinase inhibitor (CanPI7) is a multi-domain potato type II inhibitor (Pin-II) containing four inhibitory repeat domains (IRD), which target major classes of digestive enzymes in the gut of Helicoverpa armigera larvae. Stable integration and expression of the transgene in T1 transgenic generation, were confirmed by established molecular techniques. Protein extract of transgenic tomato lines showed increased inhibitory activity against H. armigera gut proteinases, supporting those domains of CanPI7 protein to be effective and active. When T1 generation plants were analyzed, they exhibited antibiosis effect against first instar larvae of H. armigera. Further, larvae fed on transgenic tomato leaves showed delayed growth relative to larvae fed on control plants, but did not change mortality rates significantly. Thus, better crop protection can be achieved in transgenic tomato by overexpression of multi-domain proteinase inhibitor CanPI7 gene against H. armigera larvae.

Biochemical, structural and functional diversity between two digestive α-amylases from Helicoverpa armigera.

BACKGROUND: Helicoverpa armigera (Lepidoptera) feeds on various plants using diverse digestive enzymes as one of the survival tool-kit. The aim of the present study was to understand biochemical properties of recombinant α-amylases of H. armigera viz., HaAmy1 and HaAmy2. METHODS: The open reading frames of HaAmy1 and HaAmy2 were cloned in Pichia pastoris and expressed heterologously. Purified recombinant enzymes were characterized for their biochemical and biophysical attributes using established methods. RESULTS: Sequence alignment and homology modeling showed that HaAmy1 and HaAmy2 were conserved in their amino acid sequences and structures. HaAmy1 and HaAmy2 showed optimum activity at 60°C; however, they differed in their optimum pH. Furthermore, HaAmy2 showed higher affinity for starch and amylopectin whereas HaAmy1 had higher catalytic efficiency. HaAmy1 and HaAmy2 were inhibited to the same magnitude by a synthetic amylase inhibitor (acarbose) while wheat amylase inhibitor showed about 2-fold higher inhibition of HaAmy1 than HaAmy2 at pH7 while 6-fold difference at pH11. Interactions of HaAmy1 and HaAmy2 with wheat amylase inhibitor revealed 2:1 stoichiometric ratio and much more complex interaction with HaAmy1. CONCLUSIONS: The diversity of amylases in perspective of their biochemical and biophysical properties, and their differential interactions with amylase inhibitors signify the potential role of these enzymes in adaptation of H. armigera on diverse plant diets. GENERAL SIGNIFICANCE: Characterization of digestive enzymes of H. armigera provides the molecular basis for the polyphagous nature and thus could assist in designing future strategies for the insect control.

Fusarium oxysporum mediates systems metabolic reprogramming of chickpea roots as revealed by a combination of proteomics and metabolomics.

Molecular changes elicited by plants in response to fungal attack and how this affects plant-pathogen interaction, including susceptibility or resistance, remain elusive. We studied the dynamics in root metabolism during compatible and incompatible interactions between chickpea and Fusarium oxysporum f. sp. ciceri (Foc), using quantitative label-free proteomics and NMR-based metabolomics. Results demonstrated differential expression of proteins and metabolites upon Foc inoculations in the resistant plants compared with the susceptible ones. Additionally, expression analysis of candidate genes supported the proteomic and metabolic variations in the chickpea roots upon Foc inoculation. In particular, we found that the resistant plants revealed significant increase in the carbon and nitrogen metabolism; generation of reactive oxygen species (ROS), lignification and phytoalexins. The levels of some of the pathogenesis-related proteins were significantly higher upon Foc inoculation in the resistant plant. Interestingly, results also exhibited the crucial role of altered Yang cycle, which contributed in different methylation reactions and unfolded protein response in the chickpea roots against Foc. Overall, the observed modulations in the metabolic flux as outcome of several orchestrated molecular events are determinant of plant's role in chickpea-Foc interactions.

RNAi of selected candidate genes interrupts growth and development of Helicoverpa armigera.

Helicoverpa armigera is one of the major crop pests and is less amenable to current pest control approaches. RNA interference (RNAi) is emerging as a potent arsenal for the insect pest control over current methods. Here, we examined the effect on growth and development in H. armigera by targeting various enzymes/proteins such as proteases like trypsins (HaTry2, 3, 4 and 6), chymotrypsin (HaChy4) and cysteine protease like cathepsin (HaCATHL); glutathione S-transferases (HaGST1a, 6 and 8); esterases (HaAce4, HaJHE); catalase (HaCAT); super-oxide-dismutase (HaCu/ZnSOD); fatty acid binding protein (HaFabp) and chitin deacetylase (HaCda5b) through dsRNA approach. Significant downregulation of cognate mRNA expression and reduced activity of trypsin and GST-like enzyme were evident upon feeding candidate dsRNAs to the larvae. Among these, the highest mortality was observed in HaAce4 dsRNA fed larvae followed by HaJHE; HaCAT; HaCuZnSOD; HaFabp and HaTry3 whereas remaining ones showed relatively lower mortality. Furthermore, the dsRNA fed larvae showed significant reduction in the larval mass and abnormalities at the different stages of H. armigera development compared to their control diets. For example, malformed larvae, pupae and moth at a dose of 60µg/day were evident in high number of individual insects fed on dsRNA containing diets. Moreover, the growth and development of insects and moths were retarded in dsRNA fed larvae. These findings might provide potential new candidates for designing effective dsRNA as pesticide in crop protection.

Sequence characterization and in silico structure prediction of fatty acid desaturases in linseed varieties with differential fatty acid composition.

BACKGROUND: Linseed is the richest agricultural source of α-linolenic acid (ALA), an ω-3 fatty acid (FA) that offers several nutritional benefits. In the present study, sequence characterization of six desaturase genes (SAD1, SAD2, FAD2, FAD2-2, FAD3A and FAD3B) and 3D structure prediction of their proteins from ten Indian linseed varieties differing in ALA content were performed to determine whether the nucleotide and amino acid (AA) sequence variants have any functional implications in differential accumulation of ALA or other FAs in linseed. RESULTS: The SAD and FAD2 genes exhibited few sequence variations among the ten varieties, forming only one or two protein isoforms. In contrast, the FAD3A and FAD3B genes showed more sequence variations and three or four protein isoforms. Interestingly, the two high-ALA varieties NL260 and Padmini had the same FAD3B nucleotide and protein isoforms, which differed from all other varieties. Surprisingly, no AA changes altered the 3D structures of the desaturase proteins. CONCLUSION: Several nucleotide and AA sequence variations in desaturase genes were observed; however, they did not alter the 3D structure of any desaturase protein and were not correlated with FA levels among the ten linseed varieties, which had different ALA contents. This suggests a complex regulatory process of biosynthesis of FAs in linseed. © 2016 Society of Chemical Industry.

Ecological turmoil in evolutionary dynamics of plant-insect interactions: defense to offence.

MAIN CONCLUSION: Available history manifests contemporary diversity that exists in plant-insect interactions. A radical thinking is necessary for developing strategies that can co-opt natural insect-plant mutualism, ecology and environmental safety for crop protection since current agricultural practices can reduce species richness and evenness. The global environmental changes, such as increased temperature, CO2 and ozone levels, biological invasions, land-use change and habitat fragmentation together play a significant role in re-shaping the plant-insect multi-trophic interactions. Diverse natural products need to be studied and explored for their biological functions as insect pest control agents. In order to assure the success of an integrated pest management strategy, human activities need to be harmonized to minimize the global climate changes. Plant-insect interaction is one of the most primitive and co-evolved associations, often influenced by surrounding changes. In this review, we account the persistence and evolution of plant-insect interactions, with particular focus on the effect of climate change and human interference on these interactions. Plants and insects have been maintaining their existence through a mutual service-resource relationship while defending themselves. We provide a comprehensive catalog of various defense strategies employed by the plants and/or insects. Furthermore, several important factors such as accelerated diversification, imbalance in the mutualism, and chemical arms race between plants and insects as indirect consequences of human practices are highlighted. Inappropriate implementation of several modern agricultural practices has resulted in (i) endangered mutualisms, (ii) pest status and resistance in insects and (iii) ecological instability. Moreover, altered environmental conditions eventually triggered the resetting of plant-insect interactions. Hence, multitrophic approaches that can harmonize human activities and minimize their interference in native plant-insect interactions are needed to maintain natural balance between the existence of plants and insects.

Structural features of diverse Pin-II proteinase inhibitor genes from Capsicum annuum.

MAIN CONCLUSION: The proteinase inhibitor (PI) genes from Capsicum annuum were characterized with respect to their UTR, introns and promoter elements. The occurrence of PIs with circularly permuted domain organization was evident. Several potato inhibitor II (Pin-II) type proteinase inhibitor (PI) genes have been analyzed from Capsicum annuum (L.) with respect to their differential expression during plant defense response. However, complete gene characterization of any of these C. annuum PIs (CanPIs) has not been carried out so far. Complete gene architectures of a previously identified CanPI-7 (Beads-on-string, Type A) and a member of newly isolated Bracelet type B, CanPI-69 are reported in this study. The 5' UTR (untranslated region), 3'UTR, and intronic sequences of both the CanPI genes were obtained. The genomic sequence of CanPI-7 exhibited, exon 1 (49 base pair, bp) and exon 2 (740 bp) interrupted by a 294-bp long type I intron. We noted the occurrence of three multi-domain PIs (CanPI-69, 70, 71) with circularly permuted domain organization. CanPI-69 was found to possess exon 1 (49 bp), exon 2 (551 bp) and a 584-bp long type I intron. The upstream sequence analysis of CanPI-7 and CanPI-69 predicted various transcription factor-binding sites including TATA and CAAT boxes, hormone-responsive elements (ABRELATERD1, DOFCOREZM, ERELEE4), and a defense-responsive element (WRKY71OS). Binding of transcription factors such as zinc finger motif MADS-box and MYB to the promoter regions was confirmed using electrophoretic mobility shift assay followed by mass spectrometric identification. The 3' UTR analysis for 25 CanPI genes revealed unique/distinct 3' UTR sequence for each gene. Structures of three domain CanPIs of type A and B were predicted and further analyzed for their attributes. This investigation of CanPI gene architecture will enable the better understanding of the genetic elements present in CanPIs.

Plasticity of protease gene expression in Helicoverpa armigera upon exposure to multi-domain Capsicum annuum protease inhibitor.

BACKGROUND: A multi-domain Pin-II type protease inhibitor from Capsicum annuum (CanPI-7) is known to be effective against the insect pest, Helicoverpa armigera. The present study is an attempt to investigate the optimal dose of recombinant CanPI-7 (rCanPI-7) for effective antibiosis to H. armigera and further to characterize the responses of digestive proteases upon rCanPI-7 ingestion. METHODS: The gut protease activity was assessed biochemically and transcript accumulation pattern for selected trypsin and chymotrypsin genes was analyzed by quantitative Real-Time PCR. RESULTS: The growth retardation upon exposure to rCanPI-7 was more prominent in neonates as compared to third instar larvae. Influence of stage and dosage of rCanPI-7 was conspicuous on the expression and regulation of candidate trypsin and chymotrypsin genes in H. armigera. The transcript accumulation pattern correlated with the protease activity in rCanPI-7 exposed larvae. CONCLUSIONS: We conclude that early exposure and specific dose of protease inhibitor are essential for effective antibiosis despite the large diversity and plasticity in the expression of protease genes in H. armigera. Moreover, it is also evident that the regulation and expression of H. armigera gut proteases are specific to the stage of PI exposure. GENERAL SIGNIFICANCE: These results highlight the requirement of optimal PI concentration for effective growth retardation and for inhibiting the major gut proteases of H. armigera.

Complementation of intramolecular interactions for structural-functional stability of plant serine proteinase inhibitors.

BACKGROUND: Plant protease inhibitors (PIs) constitute a diverse group of proteins capable of inhibiting proteases. Among PIs, serine PIs (SPIs) display stability and conformational restrictions of the reactive site loop by virtue of their compact size, and by the presence of disulfide bonds, hydrogen bonds, and other weak interactions. SCOPE OF REVIEW: The significance of various intramolecular interactions contributing to protein folding mechanism and their role in overall stability and activity of SPIs is discussed here. Furthermore, we have reviewed the effect of variation or manipulation of these interactions on the activity/stability of SPIs. MAJOR CONCLUSIONS: The selective gain or loss of disulfide bond(s) in SPIs can be associated with their functional differentiation, which is likely to be compensated by non-covalent interactions (hydrogen bonding or electrostatic interactions). Thus, these intramolecular interactions are collectively responsible for the functional activity of SPIs, through the maintenance of scaffold framework, conformational rigidity and shape complementarities of reactive site loop. GENERAL SIGNIFICANCE: Structural insight of these interactions will provide an in-depth understanding of kinetic and thermodynamic parameters involved in the folding and stability mechanisms of SPIs. These features can be explored for engineering canonical SPIs for optimizing their overall stability and functionality for various applications.

Resistance through inhibition: ectopic expression of serine protease inhibitor offers stress tolerance via delayed senescence in yeast cell.

Protease inhibitors have been known to confer multiple stress tolerance in transgenic plants. We have assessed growth of yeast (Pichia pastoris GS115) strains expressing inhibitory repeat domains (PpIRD(+)) of previously characterized Capsicum annuum protease inhibitors under high salt, heavy metal and oxidative stress. PpIRD(+) strains exhibited multiple stress tolerance and showed differential molecular responses at transcriptional and translational level on exposure to stress inducing agents like heavy metal, high salt and H2O2. PpIRD(+) strains display significant reduction in metacaspase (Yca1) activity, the key enzyme in apoptosis, indicates the possibility of cross reactivity of IRDs (serine protease inhibitor) with cysteine proteases. PpIRD(+) and Saccharomyces cerevisiae knockout with Yca1 (ΔYca1) strain showed similar growth characteristics under stress, which indicated the delayed senescence due to cellular metacaspase inhibition. Molecular docking study showed a close proximity of IRDs reactive site and the active site of metacaspase in the complex that signified their strong interactions. Maintenance of GAPDH activity, primary target of metacaspase, in PpIRD(+) strain evidenced the inhibition of metacaspase activity and survival of these cells under stress. This report demonstrates a potential molecular mechanism of protease inhibitor-based multiple stress tolerance in yeast strains.