Characterization, antioxidant activity, and mineral profiling of Auricularia cornea mushroom strains.

Background: Mushrooms are considered as next-generation healthy food components. Owing to their low-fat content, high-quality proteins, dietary fiber, and rich source of nutraceuticals. They are ideally preferred in formulation of low-caloric functional foods. In this view, the breeding strategies of mushroom Auricularia cornea (A. cornea) focusing on high yield and higher quality with rich nutritional values and health benefits are still needed. Materials and methods: A total of 50 strains of A. cornea were used to analyze the bio efficiency and the time required for fruiting body formation following the cultivation experiment. The calorimetric method was used to evaluate the antioxidant activity and quantify the crude polysaccharides and minerals content thereafter. Results: The results showed that the time required for fruiting body formation and biological efficiency varied significantly among the selected strains. Noticeably, the wild domesticated strain Ac13 of A. cornea mushroom showed the shortest fruit development time (80 days). Similarly, the hybrid strains including Ac3 and Ac15 possessed the highest biological efficiency (82.40 and 94.84%). Hybrid strains Ac18 (15.2%) and cultivated strains Ac33 (15.6%) showed the highest content of crude polysaccharides, while cultivated strains Ac1 and Ac33, demonstrated the highest content of total polysaccharides in the fruiting body (216 mg. g-1 and 200 mg. g-1). In the case of mineral content, the highest zinc contents were observed from the cultivated strain Ac46 (486.33 mg·kg-1). The maximum iron content was detected from the hybrid strain Ac3 (788 mg·kg-1), and the wild domesticated strain Ac28 (350 mg·kg-1). The crude polysaccharides of the A. cornea strain showed significant antioxidant potential, and the ability of Ac33 and Ac24 to scavenge DPPH radicals and ABTS, which was significantly improved compared to other strains, respectively. Principal component analysis was applied to examine the agronomic traits and chemical compounds of various strains of A. cornea mushrooms. The results revealed that cultivated, wild domesticated, and hybrid strains of A. cornea exhibited distinct characteristics in terms of growth, yield, and nutritional properties. Conclusion: The crude polysaccharides from A. cornea mushroom strains act as natural antioxidants, the wild, hybrid, and commercial A. cornea mushroom strains can achieve rapid growth, early maturation, and high yields. The evaluation of biochemical indexes and nutritional characteristics of strains with excellent traits provided a scientific basis for initiating high-quality breeding, provided germplasm resources for the production of "functional food" with real nutritional and health value.

TPD1-like Gene as a Suitable Marker for Early Sex Determination in Date Palm (Phoenix dactylifera L.).

Date palm (Phoenix dactylifera L.) is a considerably beneficial and economically profitable fruit crop. Female date palm plants produce fruit that is rich in fiber and sugar. Date palm is propagated by two means: suckers and seed. The propagation of date palm through seeds is very necessary for germplasm conservation and breeding. The late reproductive age (4-5 years) and dioecious nature of date palm make genetic improvement and breeding difficult. Early sex determination is the only way to improve breeding by selecting experimental male and female plants at the seedling stage. The primers for Tapetum Determinant 1 (TPD1-like) were designed using Amplify software. The DNA amplification of selected date palm suckers of three genotypes (Ajwa, Amber, and Medjool) was observed through PCR. Expression profiling of selected genotypes was carried out through semi-q PCR and RT-PCR by using the cDNA of suckers and unknown seedlings. Different in silico analyses were performed for the gene and protein characterization and identification of cis-acting elements in the promoter region. The promoter was identified along with the protein's properties and functionality. The expression of TPD1-like gene was found in the leaves of three selected genotypes of male sucker and in some plants of selected unknown seedlings that are considered male plants, and no expression was observed in female suckers and unknown seedlings that are considered female plants. The findings suggested that the TPD1-like gene has the potential for sex differentiation at the seedling stage, as the TPD1-like gene is essential to the specialization of tapetal cells and plays a critical role in plant reproduction.

Pharmacological activities of Artemisia absinthium and control of hepatic cancer by expression regulation of TGFß1 and MYC genes.

Plant derived compounds have always been an important source of medicines and have received significant attention in recent years due to their diverse pharmacological properties. Millions of plant-based herbal or traditional medicines are used to cure various types of cancers especially due to activation of proliferative genes. The aim of the present study was to characterize the altered and attenuated gene expression of the selected growth factor namely Transforming growth factor Beta -1 (TGFß1) and MYC in human hepatoma-derived (Huh7) liver cancer cell lines in response to extracts of Artemisia absinthium dissolved in selected organic solvents. Ethanolic, methanolic and acetone extract of different plant parts (leaf, stem and flowers) was used to access the antiproliferative activity by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Quantitative Real-Time RT-PCR revealed that the transcript levels of TGFß1 are induced in the samples treated with methanolic extract of Artemisia absinthium. Furthermore, reduced expression levels of MYC gene was noticed in cancerous cells suggesting antiproliferative properties of the plant. This study further highlights the resistance profile of various microbes by antimicrobial susceptibility test with plant extracts. In addition, antidiabetic effect of Artemisia absinthium have also shown positive results. Our study elucidates the potentials of Artemisia absinthium as a medicinal plant, and highlights the differential expression of genes involved in its mitogenic and anti-proliferative activity with a brief account of its pharmacological action.

In-situ stabilization of potentially toxic elements in two industrial polluted soils ameliorated with rock phosphate-modified biochars.

The present study was aimed at determining the efficacy of rock phosphate (RP) 3% loaded in a green coconut shell, chicken manure, and vegetable waste to make green coconut-modified biochar (GMB), chicken manure modified-biochar (CMB), and vegetable waste-modified biochar (VMB) in the fixation of Cr, Pb, Cu, Zn, Ni, and Cd in Sharafi goth and Malir polluted soils. The impact of RP impregnated with organic waste material to produce modified biochars (MBs) on stabilizing PTEs from polluted soils and reducing their uptake by mustard plant has not yet been thoroughly investigated. All modified BCs in 0.5, 1, and 2% doses were used to stabilize Cr, Pb, Cu, Zn, Ni, and Cd in two polluted soils and to reduce their uptake by the mustard plant. The obtained results revealed that the maximum mustard fresh biomass was 17.8% higher with GMB 1% in Sharafi goth polluted soil and 25% higher with VMB 0.5% in Malir polluted soil than in the control treatment. After applying modified BCs, immobilization of Cr, Pb, Cu, Ni, and Cd was observed in both soils and it reduced the uptake of these elements by mustard plants. On the other hand, although Zn mobilization increased by 0.38% for CMB 0.5% and by 5.9% for VMB 0.5% in Sharafi goth polluted soil, as well as by 3.15% for GMB 1%, 6.34% for GMB 2%, and 4.78% for VMB 0.5% in Malir polluted soil, this was due to changes in soil pH and OM. It was found that GMB 1%, CMB 0.5%, and VMB 0.5% have the potential to increase Zn uptake by mustard, while VMB 2% can reduce the element uptake by the plant. Redundancy analysis showed that soil chemical parameters were negatively correlated with PTEs in both soils and reduced their uptake by mustard. The present study revealed that MBs can stabilize PTEs in industrial and wastewater soils polluted with multiple metals and reduce their uptake by plants.

In silico analysis and expression profiling of Expansin A4, BURP domain protein RD22-like and E6-like genes associated with fiber quality in cotton.

BACKGROUND: To supply high-quality cotton fibre for the textile industry, the development of long, strong and fine fibre cotton varieties is imperative. An interlinked approach was used to comprehend the role of fibre genes by analyzing interspecific progenies of cotton species. Wild Gossypium species and races are rich source of genetic polymorphism due to environmental dispersal and continuous natural selection. These genetic resources hold mass of outclass genes that can be used in cotton improvement breeding programs to exploit possible traits such as fibre quality, abiotic stress tolerance, and disease and insect resistance. Therefore, use of new molecular techniques such as genomics, transcriptomics and bioinformatics is very important to utilize the genetic potential of wild species in cotton improvement programs. METHODS: Interspecific lines and Gossypium species used in the study were grown at Central Cotton Research Institute (CCRI), Multan. After retrieving DNA sequence of the genes from NCBI, the primers for gene expression and full-length gene sequence were designed. Expression profiling of Expansin A4, BURP Domain protein RD22-like and E6-like fibre genes was performed through Real Time PCR. BLAST and DNA sequence alignment was conducted for sequence comparison of interspecific lines and Gossypium species. Different in silico analysis were used for characterization of fibre genes and identification of cis acting promoter elements in promoter region. RESULTS: Variable expression of genes related to fibre development was observed at different stages. BLAST and DNA sequence alignment demonstrated resemblance of interspecific lines with G. hirsutum. In silico analysis on the sequence data also confirmed the role of Expansin A4, BURP Domain protein RD22-like and E6-like fibre genes in fibre development. Genetic engineering is also recommended by transferring E6-like, Expansin A4 and BURP Domain RD22-like genes in local cotton cultivars. Similarly, several stress tolerant and light responsive cis acting elements were identified through promotor analysis, which may contribute for fibre development in the breeding programs. CONCLUSION: Expansin A4, BURP Domain RD22-like and E6-like have positive role in fibre development with variable expression at fiber length and strength associated stages.

Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus.

In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome editing through CRISPR/Cas9 has revolutionized plant biology, especially to develop immunity in plants against viral diseases. Here we demonstrate multiplex CRISPR/Cas-mediated genome editing against CLCuD using transient transformation in N. benthamiana plants and cotton seedlings. The genomic sequences of cotton leaf curl viruses (CLCuVs) were obtained from NCBI and the guide RNA (gRNA) were designed to target three regions in the viral genome using CRISPR MultiTargeter. The gRNAs were cloned in pHSE401/pKSE401 containing Cas9 and confirmed through colony PCR, restriction analysis, and sequencing. Confirmed constructs were moved into Agrobacterium and subsequently used for transformation. Agroinfilteration in N. benthamiana revealed delayed symptoms (3-5 days) with improved resistance against CLCuD. In addition, viral titer was also low (20-40%) in infected plants co-infiltrated with Cas9-gRNA, compared to control plants (infected with virus only). Similar results were obtained in cotton seedlings. The results of transient expression in N. benthamiana and cotton seedlings demonstrate the potential of multiplex CRISPR/Cas to develop resistance against CLCuD. Five transgenic plants developed from three experiments showed resistance (60-70%) to CLCuV, out of which two were selected best during evaluation and screening. The technology will help breeding CLCuD-resistant cotton varieties for sustainable cotton production.

Analysis of the Genome Sequence of Strain GiC-126 of Gloeostereum incarnatum with Genetic Linkage Map.

Gloeostereum incarnatum has edible and medicinal value and was first cultivated and domesticated in China. We sequenced the G. incarnatum monokaryotic strain GiC-126 on an Illumina HiSeq X Ten system and obtained a 34.52-Mb genome assembly sequence that encoded 16,895 predicted genes. We combined the GiC-126 genome with the published genome of G. incarnatum strain CCMJ2665 to construct a genetic linkage map (GiC-126 genome) that had 10 linkage groups (LGs), and the 15 assembly sequences of CCMJ2665 were integrated into 8 LGs. We identified 1912 simple sequence repeat (SSR) loci and detected 700 genes containing 768 SSRs in the genome; 65 and 100 of them were annotated with gene ontology (GO) terms and KEGG pathways, respectively. Carbohydrate-active enzymes (CAZymes) were identified in 20 fungal genomes and annotated; among them, 144 CAZymes were annotated in the GiC-126 genome. The A mating-type locus (MAT-A) of G. incarnatum was located on scaffold885 at 38.9 cM of LG1 and was flanked by two homeodomain (HD1) genes, mip and beta-fg. Fourteen segregation distortion markers were detected in the genetic linkage map, all of which were skewed toward the parent GiC-126. They formed three segregation distortion regions (SDR1-SDR3), and 22 predictive genes were found in scaffold1920 where three segregation distortion markers were located in SDR1. In this study, we corrected and updated the genomic information of G. incarnatum. Our results will provide a theoretical basis for fine gene mapping, functional gene cloning, and genetic breeding the follow-up of G. incarnatum.

Comparative Profiling of Button Mushroom, Agaricus bisporus (Agaricomycetes), Possessing Textural, Antibacterial, Elemental, and Antioxidant Properties in Diverse Collections from China.

Button mushroom (Agaricus bisporus) is cultured commercially and consumed worldwide for its unique flavor, texture, and culinary qualities. The objective of this study was to assess the textural profile together with the phenolic content and antioxidant, antibacterial, and elemental composition of wild and commercially cultivated A. bisporus from China. Of the six mushroom strains studied, both cultivated strains CCMJ1013 (fresh: 85.8 N/m2; stored: 57.9 N/m2) and CCMJ1343 (fresh: 94.7 N/m2; stored: 52.9 N/m2) recorded elevated hardness. Our results revealed that wild A. bisporus strain CCMJ1363 contained the highest phenolic content (7.840 mg gallic acid equivalents [GAE]/g), followed by CCMJ1361 (7.125 mg GAE/g) and CCMJ1351 (6.709 mg GAE/g). Antioxidant activity was elevated in CCMJ1351 (inhibition concentration at 50% inhibition [IC50] = 1.04 mg/mL) followed by CCMJ1361 (IC50 = 1.67 mg/mL) in the 1,1-diphenyl-2-picrylhydrazyl assay, whereas CCMJ1351 showed the maximum antioxidant activity (353.54 mg Trolox equivalents/g) in the ferric reducing antioxidant power assay. The maximum 2,2'-azobis(2-amidinopropane) di-hydrochloride-induced erythrocyte hemolysis inhibition was found in wild A. bisporus CCMJ1363 (86.09% inhibition) followed by CCMJ1351 (84.45% inhibition). In terms of antibacterial activity, only a wild A. bisporus strain (CCMJ1361) showed antibacterial activity against pathogenic bacteria (Bacillus coli and Staphylococcus aureus). Wild strain CCMJ1110 contained the highest level of zinc and magnesium; CCMJ1361 showed higher levels of calcium and iron, and CCMJ1363 contained a comparatively higher composition of nitrogen, sodium, and aluminum. Therefore, the current study lays a foundation for creating high-performance, culinary-quality, and stress-resistant germplasms in breeding for A. bisporus strains.

Fabrication of a lead ion selective membrane based on a polycarbazole Sn(iv) arsenotungstate nanocomposite and its ion exchange membrane (IEM) kinetic studies.

A polycarbazole-Sn(iv) arsenotungstate (Pcz-SnAT) nanocomposite cation exchanger membrane (CEM) was prepared via the casting solution technique utilizing polycarbazole-Sn(iv) arsenotungstate and PVC (polyvinyl chloride) as a binder. The synthesis of the Pcz-SnAT membrane was confirmed via various characterization methods such as EDX, SEM, TGA, XRD, and FTIR spectroscopy. This membrane having a 4.5 : 1 composition ratio of composite by PVC exhibited the most effective outcomes for swelling, thickness, porosity, and water content. Our research indicates that the present ion selective membrane electrode is selective towards Pb(ii) ions, with the detection limit ranging from 1 × 10-7 mol L-1 to 1 × 10-1 mol L-1 where 20 s is the response time and 3-7 is the working value pH. The mechanism of the Pcz SnAT ion exchange membrane was obtained by kinetic studies by utilizing the equation given by Nernst Planck at 40-80 °C. As a result, activation energy and thermodynamic studies were done. The analytical utility of this electrode is conventional by utilizing it as an electrode indicator within the potentiometric titration.

Novel mutant camelina and jatropha as valuable feedstocks for biodiesel production.

Novel mutant camelina has become a crop of interest inspired by its short growing season, low harvesting costs and high oil composition. Despite those advantages, limited research has been done on novel mutant lines to determine applicability for biodiesel production. Jatropha is an extremely hardy, frugal and high oil yielding plant species. The major aim of the present study was not only to compare biodiesel production from jatropha and camelina but was also to test the efficacy of camelina mutant lines (M6 progenies) as superior feedstock. The biodiesel yield from camelina oil and jatropha oil was 96% and 92%, respectively. The gas chromatographic analysis using flame ionization detector (GC-FID) showed that mutant camelina oil biodiesel sample contain major amount of oleic acid (46.54 wt%) followed by linolenic acid (20.41 wt%) and linoleic acid (16.55 wt%). Jatropha biodiesel found to contain major amount of oleic acid (45.03 wt%) followed by linoleic acid (25.07 wt%) and palmitic acid (19.31 wt%). The fuel properties of produced biodiesel were found in good agreement with EN14214 and ASTM D6751 standards. The mutant camelina lines biodiesel have shown comparatively better fuel properties than jatropha. It has shown low saponification value (120.87-149.35), high iodine value (130.2-157.9) and better cetane number (48.53-59.35) compared to jatropha biodiesel which have high saponification value (177.39-198.9), low iodine value (109.7-123.1) and lesser cetane number (47.76-51.26). The results of the present student of utilizing novel mutant camelina lines for biodiesel production are quite promising and are helpful in turning out the outcomes of the previous studies suggesting that C. sativa biodiesel presents serious drawbacks for biodiesel applications.

A Comparison of the Physical, Chemical, and Structural Properties of Wild and Commercial Strains of Button Mushroom, Agaricus bisporus (Agaricomycetes).

Agaricus bisporus is well known for its nutraceutical properties. To meet consumer market demand, there is an urgent need for new strains with disease resistance and a diverse nutrient profile for commercial cultivation. Wild germplasm resources provide a good source for the breeding of new strains for this purpose. In this study, we evaluated the physical, chemical, and structural properties of wild domesticated (CCMJ1351) and major commercially cultivated strains (CCMJ1013, CCMJ1028, and CCMJ1343) of A. bisporus from China. The results showed significant differences among the strains for all parameters measured. In terms of morphological characteristics, CCMJ1351 possessed the highest stipe thickness, fruiting body individual weight, cohesiveness, and springiness; CCMJ1013 demonstrated maximum pileus diameter and thickness; CCMJ1028 exhibited the highest textural hardness and color characteristics; and strain CCMJ1343 had the highest yield. CCMJ1351 ranked top among all the strains for proximate composition, rheological profile, and structural and mechanical properties, containing 21.93% crude protein and the highest dry matter, crude fat, and fiber contents. However, the bioactive chemical constituents present in the four strains were very similar, especially ß-(1→3)-glucan, according to Fourier transform infrared spectroscopy analysis, while some minimal peaks varied among the different strains. Therefore, in combination with previously identified high disease-resistance traits, the wild domesticated strain CCMJ1351 constitutes a good candidate for further exploitation in breeding programs and is suitable for fresh consumption as well as incorporation into various food products.

Map-based cloning of genes encoding key enzymes for pigment synthesis in Auricularia cornea.

Color is an important quality attribute of fungi, and a useful marker for classification, genetic, and molecular research. However, there is much debate over which enzymes play key regulatory roles in pigment synthesis pathways among different fungi and even within the same species. Auricularia cornea is the most widely cultivated mushroom in the genus Auricularia; 1.834 million tons of this mushroom were produced in 2016 in China. Thus, systematic studies on its color inheritance and the genes encoding key enzymes for pigment synthesis have high scientific and economic value. In this study, the white strain ACW001 and the purple strain ACP004 of A. cornea were used as dikaryotic parents. Selfing populations of ACW001 and ACP004 were constructed with their monokaryotic strains. The fruiting body color of the two populations was consistent with that of their parents, confirming that the two parents were color homozygotes. All strains in the hybrid population of the two parents produced purple fruiting bodies. A robust hybrid strain (ACW001-33×ACP004-33) was selected from the hybrid population, and 87 monokaryotic strains of ACW001-33×ACP004-33 were obtained as a mapping population. Finally, a testcross population was constructed by crossing the mapping population with the test strain ACW001-9. The color genotype of each monokaryotic strain in the mapping population was identified by a fruiting test. The genomes of the two monokaryotic strains ACW001-33 and ACP004-33 were sequenced, and then simple sequence repeat (SSR) and sequence-related amplified polymorphism (SRAP) molecular marker primers were developed. Then, 88 pairs of primers that could distinguish the genotypes of the mapping population were used to construct a genetic linkage map. The genetic linkage map consisted of 12 linkage groups (LGs) spanning 1315.2 cM. The color control locus was preliminarily located at 24.5 cM of the 11th LG. Fine-mapping primers were designed based on sequence differences between ACW001-33 and ACP004-33 in the primary location region. Four color control candidate genes were located in an 8.2-kb region of ACW001-33_contig733 and a 9.2-kb region of ACP004-33_contig802. Homologous alignment and prediction of conserved domain analyses indicated that two of the color control candidate genes encoded proteins with unknown function, and the other two, ACP004_g11815 and ACP004_g11816, encoded glutamyl aminotransferases. These two genes were consecutively arranged on ACP004-33_contig802, and were likely to encode key enzymes in the γ-glutamine-4-hydroxy-benzoate (GHB) pigment synthesis pathway. Primers were designed from the flanking sequences of the two genes and used to analyze the testcross population. Products were amplified only from the 30 testcross strains with purple fruiting bodies, confirming the accuracy of the localization results. We discuss the deficiencies and advantages of map-based cloning in fungi vs. plants, and summarize the steps and requirements of the map-based cloning method for fungi. This study has provided novel ideas and methods for locating functional genes in fungi.

Modulation of agronomic and nutritional response of Pleurotus eryngii strains by utilizing glycine betaine enriched cotton waste.

BACKGROUND: This study aimed to evaluate the possibility of cotton waste enrichment with glycine betaine (GB) for production of two strains (P9, P10) of king oyster (Pleurotus eryngii). Cotton waste was used as (100%) control (T0 = cotton waste) and augmented with various combinations of GB, (T1 = 2 mmol L-1 , T2 = 4 mmol L-1 , T3 = 6 mmol L-1 , T4 = 8 mmol L-1 and T5 = 10 mmol L-1 ). The response of king oyster to GB was evaluated by earliness, yield, biological efficiency (BE), minerals (nitrogen, phosphorus, potassium, zinc (Zn), copper (Cu), magnesium (Mg), manganese (Mn), iron (Fe), sodium (Na), calcium (Ca)), total sugars, total soluble solids, reducing sugars, non-reducing sugars, ascorbic acid, proximate (crude protein, carbohydrates, crude fibers, ash, fats) content of fruiting body and Fourier-transform infrared (FTIR) spectroscopy analysis compared with the control substrate (cotton waste). RESULTS: The earliness, yield, and BE were higher as compared to control substrate and increased with an augmentation in the concentration of GB within the cotton waste. Two strains showed (on dry weight basis) 33.9-54.9 mg g-1 nitrogen, 6.8-12.5 mg g-1 phosphorus, 16.9-25.1 mg g-1 potassium, 40.5-64.2 mg kg-1 Zn, 17.1-37.3 mg kg-1 Cu, 1174-1325 mg kg-1 Mg, 20.1-29.1 mg kg-1 Mn, 129-265 mg kg-1 Fe, 779-835 mg kg-1 Ca), 6.3%-11.3% total sugars, 7.3-14.9 °Brix total soluble solids, 2.1-7.3% reducing sugars, 10.4-18.1% crude protein, 3.6-4.4% crude fiber and 5.6-16.7 mg (100 g)-1 on various concentration of GB enrich cotton waste. Cotton waste enriched with GB significantly affected nutritional profile of king oyster mushroom. CONCLUSION: The results revealed that GB enriched cotton waste can be used as an innovative substrate to enhance the yield and quality of king oyster mushroom. © 2019 Society of Chemical Industry.

Resistance status of Helicoverpa armigera against Bt cotton in Pakistan.

Transgenic cotton expressing the toxin Cry1Ac from Bacillus thuringiensis L. (Bt) is widely cultivated in Pakistan after its formal approval in 2010. The exposure of the local target pests to the Cry1Ac endotoxin for this duration might have changed the baseline susceptibility. To probe the status of resistance in one of the main target pests, Helicoverpa armigera, field-collected larvae were reared in the lab for conducting leaf fed bioassays. Twenty-six cotton accessions collected from farmers, including 25 Bt-cotton and one non-Bt, were tested to quantify the level of Cry1Ac, an insecticidal crystalline protein (ICP), in leaves of lower, middle and upper canopies of plants. The concentration of ICP was tested through Enzyme-linked Immunosorbent Assay and found significantly variable (P < 0.01) between and within accessions. The highest mean expression was observed in Accession-2 and Accession-4, while the lowest in Accession-21 and Accession-19. Among fresh leaf tissues from different parts of the plant, the highest mean expression was recorded at 60 days after sowing in upper canopy leaves of cotton accessions, which decreased in lower parts of the plant with the lowest mean expression in lower canopy leaves. Laboratory bioassays, to calculate lethal dose, for H. armigera showed that LD50 and LD95 were 0.62 µg/g and 1.59 µg/g of fresh tissue weight, respectively. A strong positive correlation also exists between the levels of Cry1Ac protein and insect mortality (r = 0.84). These findings suggested the future risk of cultivation of Bt cotton, carrying single Cry1Ac gene, in Pakistan, as resistance surging in H. armigera against Cry protein. These results may also have significant implications for the resistance management in Bt crops, especially cotton, in future.

Effect of Early ≤ 3 Mets (Metabolic Equivalent of Tasks) of Physical Activity on Patient's Outcome after Cardiac Surgery.

OBJECTIVE: To determine the effect of <3 Mets (Metabolic Equivalent of Tasks) of physical activity on zero postoperative days for improving hemodynamic and respiratory parameters of patients after cardiac surgeries. STUDY DESIGN: Randomized control trial. PLACE AND DURATION OF STUDY: BARMWTHospital, Rawalpindi, from March to August 2015. METHODOLOGY: Arandomized controlled trial was conducted on 174 CABG and valvular heart disease patients undergoing cardiac surgical procedures. After selection of sample via non-probability purposive sampling, they were randomly allocated into interventional group (n=87) and control group (n=87). Treatment protocol for experimental group was ≤3 Mets of physical activity, i.e. chest physiotherapy, sitting over edge of bed, standing and sitting on chair at bedside, on zero postoperative day but the control group was treated with conventional treatment on first postoperative day. Pre- and post-treatment assessment was done in control and interventional groups on both zero and first postoperative days. Data was analyzed on SPSS version 21. RESULTS: The patients' mean age was 51.86 ±13.76 years. Male to female ratio was 132:42. Statistically significant differences in respiratory rate and SpO2 (p=0.000 and 0.000, respectively) were found between both groups. Among ABG's, PCO2 and pH showed significant differences with p values of 0.039 and <0.001, respectively. No significant differences were observed between both groups regarding electrolytes (Na+, K+, Cl-, p-values of 0.361, 0.575 and 0.120 respectively) and creatinine (p=0.783). Marked improvement in oxygen saturation, dyspnea and a fall in systolic BPwas seen in interventional group. There was also observed to be a reduction in the length of ICU stay among interventional group patients as frequency with percentage of total stay was compared to control group. CONCLUSION: Early physical activity (≤3 METS) post-cardiac surgeries prevent respiratory complications through improvement in dyspnea, respiratory rate, and oxygen saturation.

What are farmers really planting? Measuring the presence and effectiveness of Bt cotton in Pakistan.

Genetically modified, insect-resistant Bacillus thuringiensis (Bt) cotton is cultivated extensively in Pakistan. Past studies, however, have raised concerns about the prevalence of Bt cotton varieties possessing weak or nonperforming insect-resistance traits conferred by the cry gene. We examine this issue using data drawn from a representative sample of cotton-growing households that were surveyed in six agroclimatic zones spanning 28 districts in Pakistan in 2013, as well as measurements of Cry protein levels in cotton tissue samples collected from the sampled households' main fields. The resultant dataset combines information from 593 sampled households with corresponding plant tissue diagnostics from 70 days after sowing, as well as information from 589 sampled households with corresponding diagnostics from 120 days after sowing. Our analysis indicates that 11 percent of farmers believed they were cultivating Bt cotton when, in fact, the Cry toxin was not present in the tested tissue at 70 days after sowing (i.e., a Type I error). The analysis further indicates that 5 percent of farmers believed they were cultivating non-Bt cotton when, in fact, the Cry toxin was present in the tested tissue (i.e., a Type II error). In addition, 17 percent of all sampled farmers were uncertain whether or not they were cultivating Bt cotton. Overall, 33 percent of farmers either did not know or were mistaken in their beliefs about the presence of the cry gene in the cotton they cultivated. Results also indicate that toxic protein levels in the plant tissue samples occurred below threshold levels for lethality in a significant percentage of cases, although these measurements may also be affected by factors related to tissue sample collection, handling, storage, and testing procedures. Nonetheless, results strongly suggest wide variability both in farmers' beliefs and in gene expression. Such variability has implications for policy and regulation in Pakistan's transgenic cotton seed market.

Diversity, Mutation and Recombination Analysis of Cotton Leaf Curl Geminiviruses.

The spread of cotton leaf curl disease in China, India and Pakistan is a recent phenomenon. Analysis of available sequence data determined that there is a substantial diversity of cotton-infecting geminiviruses in Pakistan. Phylogenetic analyses indicated that recombination between two major groups of viruses, cotton leaf curl Multan virus (CLCuMuV) and cotton leaf curl Kokhran virus (CLCuKoV), led to the emergence of several new viruses. Recombination detection programs and phylogenetic analyses showed that CLCuMuV and CLCuKoV are highly recombinant viruses. Indeed, CLCuKoV appeared to be a major donor virus for the coat protein (CP) gene, while CLCuMuV donated the Rep gene in the majority of recombination events. Using recombination free nucleotide datasets the substitution rates for CP and Rep genes were determined. We inferred similar nucleotide substitution rates for the CLCuMuV-Rep gene (4.96X10-4) and CLCuKoV-CP gene (2.706X10-4), whereas relatively higher substitution rates were observed for CLCuMuV-CP and CLCuKoV-Rep genes. The combination of sequences with equal and relatively low substitution rates, seemed to result in the emergence of viral isolates that caused epidemics in Pakistan and India. Our findings also suggest that CLCuMuV is spreading at an alarming rate, which can potentially be a threat to cotton production in the Indian subcontinent.

Comparative analysis of resistance gene analogues encoding NBS-LRR domains in cotton.

BACKGROUND: Plant production is severely affected by biotic and abiotic stresses R-genes exhibit resistance against a range of diseases and pathogens in plants. The nucleotide binding site and leucine rich repeat (NBS-LRR) class of R-genes is the most comprehensively studied in terms of sequence evolution and genome distribution. The differential response for resistance against biotic and abiotic stress has been observed in cultivated and wild relatives of the genus Gossypium. RESULTS: Efforts have been made to address the recent evolution of NBS-LRR sequences within Gossypium hirsutum and resistance gene analogue (RGA) sequences derived from G. arboreum and G. raimondii. The % identity and phylogenetic analysis of NBS-LRR-encoded RGAs from tetraploid New World cotton and its diploid ancestors G. raimondii and G. arboreum suggest that the evolution of NBS-LRR-encoding sequences in G. hirsutum occurred by gradual accumulation of mutants that led to positive selection and a slow rate of divergence within distinct R-gene families. CONCLUSION: The allotetraploid genome of cotton, after separating from its diploid parents, experienced polyploidisation, natural and artificial selection, hybrid necrosis, duplication and recombination which became the reason to shed off and evolve new genes for its survival. These driving forces influenced the development of genomic architecture that make it susceptible to diseases and pathogens as compared to donor parents.

Molecular markers and cotton genetic improvement: current status and future prospects.

Narrow genetic base and complex allotetraploid genome of cotton (Gossypium hirsutum L.) is stimulating efforts to avail required polymorphism for marker based breeding. The availability of draft genome sequence of G. raimondii and G. arboreum and next generation sequencing (NGS) technologies facilitated the development of high-throughput marker technologies in cotton. The concepts of genetic diversity, QTL mapping, and marker assisted selection (MAS) are evolving into more efficient concepts of linkage disequilibrium, association mapping, and genomic selection, respectively. The objective of the current review is to analyze the pace of evolution in the molecular marker technologies in cotton during the last ten years into the following four areas: (i) comparative analysis of low- and high-throughput marker technologies available in cotton, (ii) genetic diversity in the available wild and improved gene pools of cotton, (iii) identification of the genomic regions within cotton genome underlying economic traits, and (iv) marker based selection methodologies. Moreover, the applications of marker technologies to enhance the breeding efficiency in cotton are also summarized. Aforementioned genomic technologies and the integration of several other omics resources are expected to enhance the cotton productivity and meet the global fiber quantity and quality demands.

Aortic aneurysm: a rare cause of Ortner's syndrome.

A young man presented with hoarseness of voice and was found to have left vocal cord paralysis and a large opacity on chest X-ray in the left upper zone. CT angiography showed a giant aneurysm of the aortic arch involving the left subclavian artery. Using a dual perfusion system, with the femoral bypass circuit taking care of the spinal protection and the aortic bypass circuit providing the cerebral protection, the aneurysm was excised and a 16 mm Dacron graft was anastomosed to the aortic arch and the left subclavian artery was anastomosed to the interposition graft. He had a smooth postoperative course and his hoarseness subsided in next 6 months.