Phylum-level studies of bacterial cutinases for unravelling enzymatic specificity toward PET degradation: an in silico approach.

The overwhelming use of PET plastic in various day-to-day activities led to the voluminous increase in PET waste and growing environmental hazards. A plethora of methods have been used that are associated with secondary pollutants. Therefore, microbial degradation of PET provides a sustainable approach due to its versatile metabolic diversity and capacity. The present work highlights the cutinase enzyme's role in PET degradation. This study focuses on the bacterial cutinases homologs screened from 43 reported phylum of bacteria. The reported bacterial cutinases for plastic degradation have been chosen as reference sequences, and 917 sequences have shown homology across the bacterial phyla. The dienelactone hydrolase (DLH) domain was identified for attaining specificity towards PET binding in 196 of 917 sequences. Various computational tools have been used for the physicochemical characterization of 196 sequences. The analysis revealed that most selected sequences are hydrophilic, extracellular, and thermally stable. Based on this analysis, 17 sequences have been further pursued for three-dimensional structure prediction and validation. The molecular docking studies of 17 selected sequences revealed efficient PET binding with the three sequences derived from the phylum Bacteroidota, the lowest binding energy of -5.9 kcal/mol, Armatimonadota, and Nitrososphaerota with -5.8 kcal/mol. The two enzyme sequences retrieved from the phylum Bacteroidota and Armatimonadota are metagenomically derived. Therefore, the present studies concluded that there is a high probability of finding cutinase homologs in various environmental resources that can be further explored for PET degradation.

Evolutionary dynamics of the cytoskeletal profilin gene family in Brassica juncea L. reveal its roles in silique development and stress resilience.

Essential to plant adaptation, cell wall (CW) integrity is maintained by CW-biosynthesis genes. Cytoskeletal actin-(de)polymerizing, phospholipid-binding profilin (PRF) proteins play important roles in maintaining cellular homeostasis across kingdoms. However, evolutionary selection of PRF genes and their systematic characterization in family Brassicaceae, especially in Brassica juncea remain unexplored. Here, a comprehensive analysis of genome-wide identification of BjPRFs, their phylogenetic association, genomic localization, gene structure, and transcriptional profiling were performed in an evolutionary framework. Identification of 23 BjPRFs in B. juncea indicated an evolutionary conservation within Brassicaceae. The BjPRFs evolved through paralogous and orthologous gene formation in Brassica genomes. Evolutionary divergence of BjPRFs indicated purifying selection, with nonsynonymous (dN)/synonymous (dS) value of 0.090 for orthologous gene-pairs. Hybrid homology-modeling identified evolutionary distinct and conserved domains in BjPRFs which suggested that these proteins evolved following the divergence of monocot and eudicot plants. RNA-seq profiles of BjPRFs revealed their functional evolution in spatiotemporal manner during plant-development and stress-conditions in diploid/amphidiploid Brassica species. Real-Time PCR experiments in seedling, vegetative, floral and silique tissues of B. juncea suggested their essential roles in systematic plant development. These observations underscore the expansion of BjPRFs in B. juncea, and offer valuable evolutionary insights for exploring cellular mechanisms, and stress resilience.

Neurological, Psychiatric, and Multisystemic Involvement of Fragile X Syndrome Along With Its Pathophysiology, Methods of Screening, and Current Treatment Modalities.

Fragile X syndrome (FXS) is a hereditary disease that predominantly leads to intellectual disability (ID) in boys. It is the second prominent cause of ID, which manifests as a result of the atypical development of the cytosine-guanine-guanine (CGG) region. This irregular extension of the CGG region gives rise to methylation and silencing of the fragile X mental retardation 1 (FMR1) gene, causing a loss of the fragile X mental retardation 1 protein (FMRP). This reduction or loss of FMRP is the main cause of ID. It has a multisystemic involvement showing neuropsychiatric features such as ID, speech and language delay, autism spectrum disorder, sensory hyperarousal, social anxiety, abnormal eye contact, shyness, and aggressive behaviour. It is also known to cause musculoskeletal symptoms, ocular symptoms, cardiac abnormalities, and gastrointestinal symptoms. The management is challenging, and there is no known cure for the disease; hence an early diagnosis of the condition is needed through prenatal screening offered to couples with familial history of ID before conception. The management rests on non-pharmacological modalities, including applied behaviour analysis, physical therapy, occupational therapy, speech-language therapy, and pharmacologic management through symptomatic treatment of comorbid behaviours and psychiatric problems and some forms of targeted therapy.

Coordination of floral and fiber development in cotton (Gossypium) by hormone- and flavonoid-signalling associated regulatory miRNAs.

Various plant development activities and stress responses are tightly regulated by various microRNAs (miRNA) and their target genes, or transcription factors in a spatiotemporal manner. Here, to exemplify how flowering-associated regulatory miRNAs synchronize their expression dynamics during floral and fiber development in cotton, constitutive expression diminution transgenic lines of auxin-signaling regulatory Gh-miR167 (35S-MIM167) were developed through target mimicry approach. 'Moderate' (58% to 80%)- and 'high' (> 80%)-Gh-miR167 diminution mimic lines showed dosage-dependent developmental deformities in anther development, pollen maturation, and fruit (= boll) formation. Cross pollination of 'moderate' 35S-MIM167 mimic lines with wild type (WT) plant partially restored boll formation and emergence of fiber initials on the ovule surface. Gh-miR167 diminution favored organ-specific transcription biases in miR159, miR166 as well as miR160, miR164, and miR172 along with their target genes during anther and petal development, respectively. Similarly, accumulative effect of percent Gh-miR167 diminution, cross regulation of its target ARF6/8 genes, and temporal mis-expression of hormone signaling- and flavonoid biosynthesis-associated regulatory miRNAs at early fiber initiation stage caused irregular fiber formation. Spatial and temporal transcription proportions of regulatory miRNAs were also found crucial for the execution of hormone- and flavonoid-dependent progression of floral and fiber development. These observations discover how assorted regulatory genetic circuits get organized in response to Gh-miR167 diminution and converge upon ensuing episodes of floral and fiber development in cotton.

Evolutionary expansion and expression dynamics of cytokinin-catabolizing CKX gene family in the modern amphidiploid mustard (Brassica sp.).

Plant cytokinins (CKs) promote development and physiological processes, drought tolerance, root architecture, and ultimately crop productivity. Biologically active CKs (iP, tZ, and cZ) are precisely maintained in the vegetative and floral tissues through their irreversible degradation by developmentally regulated CK-catabolizing cytokinin oxidase/dehydrogenase (CKX) enzyme. A meta-analysis of CKX proteins was performed through an exhaustive exploration of multiple genome databases of cyanobacteria, bryophyte, monocot and eudicot plants to reveal the intricate evolutionary profiles of CKX enzymes specific to the family Brassicaceae. At least 175 unique paralogous/orthologous CKX sequences were successfully retrieved and phylogenetically clustered into distinct groups. Observations of structural divergences among paralogous sequences compared to their orthologs indicated that the progenitor CKX sequence had been subjected to massive structural modifications, possibly as a result of the evolutionary split between monocots and eudicots. An analysis of dN/dS comparisons of orthologous genes revealed that segmental CKX gene duplications have evolved primarily under purifying selection. Further, 24 CKX genes with conserved signature domain were identified in the amphidiploid Brassica juncea genome (AABB; 2n = 36). Genetic evolution of paralogous and orthologous genes was largely responsible for the expansion of CKX homoeologs in the amphidiploid Brassica genomes. Also, comparative analyses of 1.5 kb-long upstream regulatory regions of BjCKX genes identified various development- and stress-responsive elements. Spatial and temporal expression profiles of CKX genes were primarily attributed to their structural diversity observed in the 5'-regulatory regions along with species evolution. This data suggested that CKX duplicate genes had partitioned their spatial expression (= function) during evolution. These findings illustrated the evolutionary importance of CKX genes during plant development, and also suggested their deployment for future crop improvement programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03294-0.

Transcriptional loss of domestication-driven cytoskeletal GhPRF1 gene causes defective floral and fiber development in cotton (Gossypium).

KEY MESSAGE: Constitutive- and fiber-specific RNAi of GhPRF1 gene illustrated strong correlation between domestication-driven profilin genes and floral/fiber architecture in cotton. During morpho-transformation of short-fuzz of wild cotton into the elongating spinnable fibers under the millennia of human selection, actin-polymerizing cytoskeletal profilin genes had undergone significant sequence alterations and spatiotemporal shift in their transcription levels. To comprehend the expression dynamics of profilin genes with their phenotypic implications, transgenic expression modulation of cotton profilin 1 (GhPRF1) gene was performed in the constitutive- and fiber-specific manner in Coker 310FR cotton cultivar. The constitutive GhPRF1-RNAi lines (35S:GhPRF1-RNAi) exhibited distorted 'monadelphous' staminal-tube, reduced pollen-viability and poorly developed fibers, whereas floral and fiber development of fiber-specific GhPRF1-RNAi lines showed no abnormalities. Moreover, the fiber-specific GhPRF1 overexpression lines (FBP7:GhPRF1-Ox) showed increased emergence of fiber-initials on the ovule surface, on the contrary to no fiber-initials in fiber-specific RNAi lines (FBP7:GhPRF1-RNAi). Interestingly, the average seed weight and fiber weight of FBP7:GhPRF1-Ox lines increased > 60% and > 38%, respectively, compared with FBP7:GhPRF1-RNAi lines and untransformed control seeds. On a molecular basis, the aberrant floral and fiber development of 35S:GhPRF1-RNAi lines was largely associated with sugar metabolism and hormone-signaling mechanisms. These observations illustrated the strong correlation between domestication-driven GhPRF genes, and floral/fiber development in cotton. Also, the enhanced agronomic traits in GhPRF1-Ox lines of cotton empowered us to recognize their imperative roles, and their future deployment for the sustainable cotton crop improvement.

Global expression dynamics and miRNA evolution profile govern floral/fiber architecture in the modern cotton (Gossypium).

MAIN CONCLUSION: Majority of differentially expressed miRNAs with functional attributes have been recruited independently and parallelly during allopolyploidy followed by the millennia of human selection of both domesticated G. hirsutum and G. barbadense. The genus Gossypium is a marvelous evolutionary model for studying allopolyploidy and morpho-evolution of long-spinnable fibers from the ancestral wild-fuzz. Many genes, transcription factors, and notably, the regulatory miRNAs essentially govern such remarkable modern fiber phenotypes. To comprehend the impact of allopolyploidy on the evolutionary selection of transcriptional dynamicity of key miRNAs, comparative transcriptome profiling of vegetative and fiber tissues of domesticated diploid G. arboreum (A2) and allopolyploid cotton species G. hirsutum (AD1), and G. barbadense (AD2) identified > 300 differentially expressed miRNAs (DEmiRs) within or between corresponding tissues of A2, AD1 and AD2 species. Up to 49% and 32% DEmiRs were up- and down-regulated at fiber initiation stage of AD1 and AD2 species, respectively, whereas 50% and 18% DEmiRs were up- and down-regulated at fiber elongation stage of both the allopolyploid species. Interestingly, A-subgenome-specific DEmiRs exhibit expression dominance in the allopolyploid genetic backgrounds. Comparative spatio-temporal expression analyses of AD1 and AD2 species discovered that a majority of DEmiRs were recruited independently under millennia of human selection during domestication. Functional annotations of these DEmiRs revealed selection of associated molecular functions such as hormone-signaling, calcium-signaling and reactive oxygen species (ROS) signaling during fiber initiation and elongation. To validate the functional attributes of annotated DEmiRs, we demonstrated for the first time that the target-mimicry-based constitutive diminution of auxin-signaling associated miR167 directly affected the differentiation of floral and fiber tissues of transgenic cotton. These results strongly suggested that the evolutionarily favored DEmiRs including miR167 were involved in the transcriptional regulation of numerous genes during cotton evolution for enhanced fiber-associated agronomic traits.

Genome-wide identification and characterization of ABA receptor PYL gene family in rice.

BACKGROUND: Abscisic acid (ABA), a key phytohormone that controls plant growth and stress responses, is sensed by the pyrabactin resistance 1(PYR1)/PYR1-like (PYL)/regulatory components of the ABA receptor (RCAR) family of proteins. Comprehensive information on evolution and function of PYL gene family in rice (Oryza sativa) needs further investigation. This study made detailed analysis on evolutionary relationship between PYL family members, collinearity, synteny, gene structure, protein motifs, cis-regulatory elements (CREs), SNP variations, miRNAs targeting PYLs and expression profiles in different tissues and stress responses. RESULTS: Based on sequence homology with Arabidopsis PYL proteins, we identified a total of 13 PYLs in rice (BOP clade) and maize (PACCMAD clade), while other members of BOP (wheat - each diploid genome, barley and Brachypodium) and PACCMAD (sorghum and foxtail millet) have 8-9 PYLs. The phylogenetic analysis divided PYLs into three subfamilies that are structurally and functionally conserved across species. Gene structure and motif analysis of OsPYLs revealed that members of each subfamily have similar gene and motif structure. Segmental duplication appears be the driving force for the expansion of PYLs, and the majority of the PYLs underwent evolution under purifying selection in rice. 32 unique potential miRNAs that might target PYLs were identified in rice. Thus, the predicted regulation of PYLs through miRNAs in rice is more elaborate as compared with B. napus. Further, the miRNAs identified to in this study were also regulated by stresses, which adds additional layer of regulation of PYLs. The frequency of SAPs identified was higher in indica cultivars and were predominantly located in START domain that participate in ABA binding. The promoters of most of the OsPYLs have cis-regulatory elements involved in imparting abiotic stress responsive expression. In silico and q-RT-PCR expression analyses of PYL genes revealed multifaceted role of ABARs in shaping plant development as well as abiotic stress responses. CONCLUSION: The predicted miRNA mediated regulation of OsPYLs and stress regulated expression of all OsPYLs, at least, under one stress, lays foundation for further validation and fine tuning ABA receptors for stress tolerance without yield penalty in rice.

De novo transcriptome assembly and protein profiling of copper-induced lignocellulolytic fungus Ganoderma lucidum MDU-7 reveals genes involved in lignocellulose degradation and terpenoid biosynthetic pathways.

Ganoderma lucidum is an important medicinal fungus that possesses exceedingly high lignocellulose degrading ability. Evidently, Cu2+ has decisive roles on the mycelial growth and enzyme production. To reveal the effect of Cu2+ on G. lucidum transcriptome, predominantly associated with lignocellulolytic progression, we conducted comparative NGS based de novo transcriptome assembly using Illumina Hi SeqTM sequencing platform. We obtained 26,083,372 and 35,713,076 high-quality reads from induced and uninduced cultures. For wood degrading activity, 194 transcript coding for oxidoreductases and 402 transcripts for CAZymes were predicted. Further, secretome studies revealed high score GO terms related to oxidoreductases, glycosyl hydrolases, and chitinases from Cu-induced mycelia. The increased Cu2+ concentrations showed higher secretion of lignocellulases such as laccases, cellulases, and xylanases along with increased production of phenolics and antioxidants. Several differences in the transcriptomic and proteomic signatures for lignocellulolytic enzymes provide vital clues about Cu2+ mediated gene regulation and metabolic pathways in basidiomycetous fungi.

Interspersed 5'cis-regulatory elements ascertain the spatio-temporal transcription of cytoskeletal profilin gene family in Arabidopsis.

Spatio-temporal expression patterns of cytoskeleton-associated profilin (PRF) family proteins in response to varied environmental stimuli are tightly regulated. Functional analyses of PRFs have revealed their crucial roles in varied developmental and stress related traits, but very little is implicit pertaining to cis-acting regulatory elements that regulate such intricate expression patterns. Here, we identified cis-elements with their varying distribution frequencies by scanning 1.5kbp upstream sequences of 5'regulatory regions of PRFs of dicot and monocot plant species. Predicted cis-elements in the regulatory sub-regions of Arabidopsis PRFs (AtPRFs) were predominantly associated with development-responsive motifs (DREs), light responsive elements (LREs), hormonal responsive elements (HREs), core motifs and stress-responsive elements (SREs). Interestingly, DREs, LREs and core promoter motifs, were extensively distributed up to the distal end of 5'regulatory regions on contrary to HREs present closer to the translational start site in Arabidopsis. The evolutionary footprints of predicted orthologous cis-elements were conserved, and preferably located in the proximal regions of 5'regulatory regions of evolutionarily diverged plant species. We also explored comprehensive tissue-specific global gene expression levels of PRFs under diverse hormonal and abiotic stress regimes. In response, the PRFs exhibited large transcriptional biases in a time- and organ-dependent manner. Further, the methodical elucidation of spatial expression analysis of predicted cis-elements binding transcription factors and relevant PRFs showed notable correlation. Results indicate that binding transcription factors' expression data is largely informative for envisaging their precise roles in the spatial regulation of target PRFs. These results highlight the importance of PRFs during plant development; and establish a relationship between their spatial expression patterns and presence of respective regulatory motifs in their promoter sequences. This information could be employed in future studies and field-utilization of cell wall structural genes.

Target-mimicry based diminution of miRNA167 reinforced flowering-time phenotypes in tobacco via spatial-transcriptional biases of flowering-associated miRNAs.

Evolutionarily conserved microRNAs such as miR156, miR159, miR167 and miR172 tightly regulate the extensive array of gene expression during flowering in plants, through instant and long-term alterations in the expression of their target genes. Here we employed a novel target-mimicry approach for the diminution of auxin signalling regulator miRNA167 by developing mimic-transgenic lines in tobacco, to investigate the transcriptional biases of flowering-associated miRNAs in apical and floral meristematic tissues and their phenotypic implications. Recorded morpho-alterations such as uneven flowering-time phenotypes, anomalous floral organ formation, and large variations in the seed forming characteristics permitted us to determine the consequence of the extent of miR167 expression diminution accompanying the transcriptional biases of interrelated miRNAs. We demonstrate that percent diminution of miR167 gene expression is proportionally associated with both early and late flowering-time phenotypes in mimic lines. Also, the associated miRNAs, miR156, miR159, and miR172 showed >90% transcriptional diminution in at least 'early-flowering' miR167 mimic lines. On contrary, low percentages of their respective diminution were recorded in 'late-flowering' lines. Evidently, the misexpression of miR156, miR159, and miR172 led to the over-expression of their respective target genes SPL9, AtMYB33-like and AP2 genes in mimic lines which resulted in assorted phenotypes. We describe the scope of spatial regulation of these microRNAs in floral bud tissues of mimic lines which showed negative- or very low (<25%) misexpression levels in early/late-flowering lines highlighting their roles in the acquisition of flowering mechanism. To our knowledge, this study represents the first characterization of transcriptional biases of flowering associated miRNAs in miR167-mimic lines and certainly augments our understanding of the importance of microRNA-mediated regulation of flowering in plants.

Bioengineering of crop plants for improved tetrahydrofolate production.

De novo synthesis of folates in plants is tightly regulated through feedback-regulation of certain pathway catalysts. Recently, we investigated the prospects of incessant production of folates in an evolutionary conjunction, through the overexpression of feedback targeted and evolutionarily conserved heterologous E.coli dihydroneopterin aldolase (EcDHNA) in tobacco. 1 The enhanced production of folates in the transgenic lines was associated with differential allosteric regulatory cavities accessible at EcDHNA surface having critical amino-acid differences as Ile 64 (His_63), Val 70 (Phe_69), His 75 (Arg_78) and Arg 79 (Glu_72). These structural characteristics are indicative of evolutionary signatures of the catalytic feedback-regulation of folate manufacturing. We exploited the biotechnological potential of such allosterically diverged trans-DHNA for improved folate production in plants. Nonetheless, genetic manipulation of single enzymes modulating complex pathways such as folate biosynthesis is often inadequate to achieve desired phenotypes; therefore, multi-gene integration with explicit genic-combination for folate enrichment in plants has also been projected for future folate agri-biofortification schemes.

Evolutionary expansion and structural functionalism of the ancient family of profilin proteins.

Structure and functional similarities of a recent protein's orthologs with its ancient counterpart are largely determined by the configuration of evolutionary preservation of amino acids. The emergence of genome sequencing databases allowed dissecting the evolutionarily important gene families at a comprehensive and genome-wide scale. The profilin multi-gene family is an ancient, universal, and functionally diverged across kingdoms, which regulates various aspects of cellular development in both prokarya and eukarya, especially cell-wall maintenance through actin sequestering, nucleation and cytokinesis. We performed a meta-analysis of the evolutionary expansion, structural conservation, evolution of function motifs, and transcriptional biases of profilin proteins across kingdoms. An exhaustive search of various genome databases of cyanobacteria, fungi, animalia and plantae kingdoms revealed 172 paralogous/orthologous profilins those were phylogenetically clustered in various groups. Orthologous gene comparisons indicated that segmental and tandem duplication events under strong purifying selection are predominantly responsible for their convoluted structural divergences. Evidently, structural divergences were more prevalent in the paralogs than orthologs, and evolutionary variations in the exon/intron architecture were accomplished by 'exon/intron-gain' and insertion/deletion during sequence-exonization. Remarkably, temporal expression evolution of profilin paralogs/homeologs during cotton fiber domestication provides evolutionary impressions of the selection of highly diverged transcript abundance notably in the fiber morpho-evolution. These results provide global insights into the profilin evolution, their structural design across taxa; and their future utilization in translational research.

Recombinant overexpression of dihydroneopterin aldolase catalyst potentially regulates folate-biofortification.

We aim to investigate the prospects of increased production of folate through the overexpression of heterologous dihydroneopterin aldolase catalyst. The gene encoding aldolase catalyst was cloned into an expression vector and the induced recombinant protein was purified through metal-affinity chromatography which appeared at 14 kDa position on polyacrylamide-gel. Remarkably, a periodic increase in the extracellular and intracellular folic acid concentration was observed at 4 h growth of induced recombinant DHNA samples than control in a pH-dependent manner. Maximum folate concentration was observed with at least twofold increase in induced recombinant samples at pH8.0 compared to the significant decline at 6 h growth. Consistently, heterologous overexpression of bacterial aldolase through Agrobacterium-mediated genetic transformation of tobacco led to more than 2.5-fold increase in the folate concentration in the transgenic leaves than control tissues. These data are veritable inspecting metabolic flux in both bacterial and plant systems, thus providing directions for future research on folate agri-fortification.

Domestication-driven Gossypium profilin 1 (GhPRF1) gene transduces early flowering phenotype in tobacco by spatial alteration of apical/floral-meristem related gene expression.

BACKGROUND: Plant profilin genes encode core cell-wall structural proteins and are evidenced for their up-regulation under cotton domestication. Notwithstanding striking discoveries in the genetics of cell-wall organization in plants, little is explicit about the manner in which profilin-mediated molecular interplay and corresponding networks are altered, especially during cellular signalling of apical meristem determinacy and flower development. RESULTS: Here we show that the ectopic expression of GhPRF1 gene in tobacco resulted in the hyperactivation of apical meristem and early flowering phenotype with increased flower number in comparison to the control plants. Spatial expression alteration in CLV1, a key meristem-determinacy gene, is induced by the GhPRF1 overexpression in a WUS-dependent manner and mediates cell signalling to promote flowering. But no such expression alterations are recorded in the GhPRF1-RNAi lines. The GhPRF1 transduces key positive flowering regulator AP1 gene via coordinated expression of FT4, SOC1, FLC1 and FT1 genes involved in the apical-to-floral meristem signalling cascade which is consistent with our in silico profilin interaction data. Remarkably, these positive and negative flowering regulators are spatially controlled by the Actin-Related Protein (ARP) genes, specifically ARP4 and ARP6 in proximate association with profilins. This study provides a novel and systematic link between GhPRF1 gene expression and the flower primordium initiation via up-regulation of the ARP genes, and an insight into the functional characterization of GhPRF1 gene acting upstream to the flowering mechanism. Also, the transgenic plants expressing GhPRF1 gene show an increase in the plant height, internode length, leaf size and plant vigor. CONCLUSIONS: Overexpression of GhPRF1 gene induced early and increased flowering in tobacco with enhanced plant vigor. During apical meristem determinacy and flower development, the GhPRF1 gene directly influences key flowering regulators through ARP-genes, indicating for its role upstream in the apical-to-floral meristem signalling cascade.

Thoracic Endometriosis-A Rare Cause of Haemoptysis.

Thoracic endometriosis is a rare condition and occurs in females of reproductive age due to the presence of active endometrial tissue in tracheobronchial tree, lung parenchyma and lung pleura. A typical history of haemoptysis during menstrual periods and strong suspicion of the disease entity is important for the diagnosis and management of the case. Diagnosis of the disease is usually delayed. Serial CT thorax during menstrual period and in non-menstrual period supports the diagnosis. We present here a case of catamenial haemoptysis. The diagnosis was missed initially but later a detailed clinical history revealed the same. Serial computed tomography of thorax taken during menstrual and after menstrual period supported the diagnosis. Though bronchoscopy was able to reveal hyperemic tissue in the tracheobronchial tree, bronchial washing was inconclusive. The patient was treated successfully with danazol.

Plant domestication and resistance to herbivory.

Transformation of wild species into elite cultivars through "domestication" entails evolutionary responses in which plant populations adapt to selection. Domestication is a process characterized by the occurrence of key mutations in morphological, phenological, or utility genes, which leads to the increased adaptation and use of the plant; however, this process followed by modern plant breeding practices has presumably narrowed the genetic diversity in crop plants. The reduction of genetic diversity could result in "broad susceptibility" to newly emerging herbivores and pathogens, thereby threatening long-term crop retention. Different QTLs influencing herbivore resistance have also been identified, which overlap with other genes of small effect regulating resistance indicating the presence of pleiotropism or linkage between such genes. However, this reduction in genetic variability could be remunerated by introgression of novel traits from wild perhaps with antifeedant and antinutritional toxic components. Thus it is strongly believed that transgenic technologies may provide a radical and promising solution to combat herbivory as these avoid linkage drag and also the antifeedant angle. Here, important questions related to the temporal dynamics of resistance to herbivory and intricate genetic phenomenon with their impact on crop evolution are addressed and at times hypothesized for future validation.

Detrimental effect of expression of Bt endotoxin Cry1Ac on in vitro regeneration, in vivo growth and development of tobacco and cotton transgenics.

High levels of expression of the cry1Ac gene from Bacillus thuringiensis cannot be routinely achieved in transgenic plants despite modifications made in the gene to improve its expression. This has been attributed to the instability of the transcript in a few reports. In the present study, based on the genetic transformation of cotton and tobacco, we show that the expression of the Cry1Ac endotoxin has detrimental effects on both the in vitro and in vivo growth and development of transgenic plants. A number of experiments on developing transgenics in cotton with different versions of cry1Ac gene showed that the majority of the plants did not express any Cry1Ac protein. Based on Southern blot analysis, it was also observed that a substantial number of lines did not contain the cry1Ac gene cassette although they contained the marker gene nptII. More significantly, all the lines that showed appreciable levels of expression were found to be phenotypically abnormal. Experiments on transformation of tobacco with different constructs expressing the cry1Ac gene showed that in vitro regeneration was inhibited by the encoded protein. Further, out of a total of 145 independent events generated with the different cry1Ac gene constructs in tobacco, only 21 showed expression of the Cry1Ac protein, confirming observations made in cotton that regenerants that express high levels of the Cry1Ac protein are selected against during regeneration of transformed events. This problem was circumvented by targeting the Cry1Ac protein to the chloroplast, which also significantly improved the expression of the protein.