Cotton is an international agricultural commodity and the main cash crop of Pakistan of which quality and quantity are subject to various whims of nature. Climate change, insect pest complex, and ...weeds are reducing its productivity. Here, we have developed triple gene cotton containing EPSPS gene along with two Bt toxin genes Cry1Ac and Cry2Ab using a strategy where all three genes are cloned in the same T-DNA, followed by successful cotton transformation via Agrobacterium-mediated transformation. This strategy has been developed to help cotton breeders in developing new cultivars by incorporating these genes into the non-transgenic or single Bt (Cry1Ac) gene cotton background where all three genes will inherit together. The expression of all three proteins was confirmed through immunostrips and was quantified through enzyme-linked immunosorbent assay (ELISA). The spatio-temporal expression of Bt protein in different parts of triple gene NIBGE cotton plants was determined. Maximum expression was found in leaves followed by seeds and boll rinds. Insect bioassays with cotton bollworms (Helicoverpa armigera), armyworms (Spodoptera litura), and pink bollworms (Pectinophora gossypiella) showed more than 90% mortality. The best performing line (NIBGE-E2) on the basis of spatiotemporal expression, glyphosate assays, and insect mortality data, was used for event characterization by using the genome sequencing approach. The event was successfully characterized and named NIBGE 20-01. A diagnostics test based on event-specific PCR was developed and its ability to distinguish NIBGE 20-01 event from other commercial transgenic cotton events was confirmed. To confirm stable expression of all three proteins in the field conditions, homozygous transgenic lines were grown in the field and the expression was confirmed through immunostrip assays. It was found that all three genes are expressed under field conditions. To show that all three genes are inherited together upon crossing with local elite cotton lines, the F
generation was grown under glasshouse and field conditions. The expression of all three genes was confirmed under field conditions. Our results showed that transgenic cotton with three genes cloned in the same T-DNA can express all genes and can be conveniently transferred into elite cotton lines through a single cross.
Cut-off operation is widely used in the manufacturing industry and is highly energy-intensive. Prediction of specific energy consumption (SEC) using data-driven models is a promising means to ...understand, analyze and reduce energy consumption for cut-off grinding. The present article aims to put forth a novel methodology to predict and validate the specific energy consumption for cut-off grinding of oxygen-free copper (OFC–C10100) using supervised machine learning techniques. State-of-the-art experimental setup was designed to perform the abrasive cutting of the material at various cutting conditions. First, energy consumption values were predicted on the bases of input process parameters of feed rate, cutting thickness, and cutting tool type using the three supervised learning techniques of Gaussian process regression, regression trees, and artificial neural network (ANN). Among the three algorithms, Gaussian process regression performance was found to be superior, with minimum errors during validation and testing. The predicted values of energy consumption were then exploited to evaluate the specific energy consumption (SEC), which turned out to be highly accurate, with a correlation coefficient of 0.98. The relationship of the predicted specific energy consumption (SEC) with material removal rate agrees well with the relationship depicted in physical models, which further validates the accuracy of the prediction models.
Pakistan experiences extreme flood events almost every year during the monsoon season. Recently, flood events have become more disastrous as their frequency and magnitude have increased due to ...climate change. This situation is further worsened due to the limited capacity of existing water reservoirs and their ability to absorb and mitigate peak floods. Thus, the simulation of stream flows using projected data from climate models is essential to assess flood events and proper water resource management in the country. This study investigates the future floods (in near future and far future periods) using the integrated flood analysis system (IFAS) model under the RCP2.6, RCP4.5, and RCP8.5 climate change scenarios. Downscaled and bias corrected climatic data of six general circulation models and their ensemble were used in this study. The IFAS model simulated the stream flow efficiently (R2 = 0.86–0.93 and Nash–Sutcliffe efficiency = 0.72–0.92) in the Jhelum River basin (JRB), Kabul River basin (KRB), and upper Indus River basin (UIRB) during the calibration and validation periods. The simulation results of the model showed significant impact of projected climate change on stream flows that will cause the mean monthly stream flow in the JRB to be lower, while that of the KRB and UIRB to be higher than that of the historical period. The highest flow months are expected to shift from May–June (Jhelum basin) and June–July (Kabul basin) to April–May with no changes in the UIRB. Higher frequencies of low to medium floods are projected in the KRB and UIRB, while the JRB expects fewer flood events. Based on the results from the IFAS model, it is concluded that stream flow in the study area will increase with several flood events.
Esophageal squamous cell carcinoma (ESCC) is one of the aggressive malignancies and mechanisms underlying its pathogenesis remain unclear. Cyclooxygenase-2 (COX-2) enzyme system plays a crucial role ...in many gastrointestinal malignancies and is an important regulator of cell growth, proliferation, apoptosis, differentiation and transformation. More precise outcome of COX-2 in ESCC is less investigated. In this study we investigated the risk factors of ESCC and expression of COX-2 in Carcinoma in situ (CIS) and ESCC compared to normal esophageal mucosa. ESCC relationship to clinico-pathological parameters using immunohistochemistry was also part of this investigation. Current study was conducted in the Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan. A total of 69 diagnosed patients of ESCC, both Pakistanis and Afghans were enrolled. Various risk factors associated with ESCC were recorded. Mean age at the time of diagnosis was 55 years. Out of 69 patients of ESCC 46 (67%) were users of dipping tobacco (Naswar). Expression of COX-2 was determined in normal esophageal mucosa, CIS and invasive ESCC using Immunohistochemistry (IHC). Differences of mean were computed using ANOVA followed by applying Post Hoc test. Patients were categorized as positive with high expression or negative with low to nil expression. ANOVA showed large differences in expression of COX-2 in normal healthy mucosa compared with CIS and ESCC with the mean difference of -9.529 and -7.370 respectively, p-value being <.05 at 95% confidence interval (CI). No significant difference was noticed in the expression of COX-2 in CIS compared with ESCC with p-value >.05 at 95% CI. Our complete cohort (23-85 years) showed statistically significant difference in the expression of COX-2 gene in ESCC and CIS tissue samples compared with normal healthy mucosa. Results of this study indicate that over-expression of COX-2 is positively associated with ESCC.
Extra-cellular glucoamylase of
Humicola sp. produced under submerged growth condition (10.44
U
mg
−1 protein) was purified to homogeneity level by using three-step purification procedure. Crude ...enzyme was subjected to ammonium sulphate precipitation, Hiload anion exchange chromatography and hydrophobic interaction column chromatography on FPLC purification system. The purified glucoamylase was monomeric in nature because native molecular mass on gel column chromatography and sub-unit mass on SDS-PAGE were the same, i.e. 72.8
kDa. Activation energy for soluble starch hydrolysis was 21.09
kJ
mol
−1, while temperature quotient (
Q
10) was 1.01. The enzyme was stable over a pH range of 3.5–5.9 and gave pH optimum of 4.7. Temperature optimum of the glucoamylase was 55
°C.
V
max for soluble starch hydrolysis was 56.63
U
mg
−1 protein and
K
m was 0.26
mg (soluble starch)
ml
−1. The turn over (
k
cat) was 69
s
−1. The p
Ka
1 and p
Ka
2 of ionizable groups of active site controlling
V
max were 3.65 and 6.3, respectively. Thermodynamic parameters for soluble starch hydrolysis were as follows: Δ
H*
=
18.36
kJ
mol
−1, Δ
G*
=
69.06
kJ
mol
−1, Δ
S*
=
−154.56
J
mol
−1
K
−1,
Δ
G
E
−
S
*
=
−
3.71
kJ
mo
l
−
1
and
Δ
G
E
−
T
*
=
−
26.41
kJ
mo
l
−
1
. Thermodynamic parameters (Δ
H*, Δ
G*, Δ
S*) for irreversible inactivation of glucoamylase at different temperatures (45–55
°C) were also determined. Current report has novelty as it explained for the first time kinetics and thermodynamics of soluble starch hydrolysis and irreversible inactivation of glucoamylase from
Humicola sp.
An Egyptian rhizobacterium Azospirillum sp. isolated from Sadat city was able to produce indole acetic acid (IAA) up to (30.59 µg/ml). The isolate was identified biochemically and by 16S rRNA ...sequencing which showed 99.9% similarity to Azospirillum brasilense. The new isolate has been registered in Genbank with accession number MH179119.1. Extracted IAA was used as reducing or stabilizing agent of sliver nanoparticles (AgNPs). Successful fabrication of biogenic IAA-AgNPs was confirmed by Fourier Transform Infrared Spectrophotometer (FTIR) analysis of IAA which showed absorbance peak at 3434.78 cm−1 due to the N-H stretch of primary amines. Highly resolution Transmission Electron Microscopy (HR-TEM) showed AgNPs coating or capping with IAA in spherical shaped with size ranged from 6.01 to 44.02 nm. Energy dispersive X-ray (EDX) analysis revealed that Ag+ ions were attached to the surface of IAA-AgNPs particles. HR-TEM examination showed cell wall damage of Citrobacter freundii cells after exposure to IAA-AgNPs leading to cell death. In vivo results showed that C. freundii infection of rats induced significant increase in liver and kidney functions and deleterious histopathological alteration in rat’s tissues. However, treatment by extracted IAA and IAA-AgNPs could normalize the biochemical and histopathological alterations occurred in infected rats. This is the first study to prove that IAA extracted from Azospirillum brasilense is a hopeful capping agent for NPs which has potential to protect against pathogenic infections, nontoxic and/or safe on rat’s metabolisms.
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•AgNPs were synthesized by indole acetic acid produced by rhizobacteril strain Azospirillum brasilense.•Biogenic IAA-AgNPs were characterized by UV-spectral, FTIR, EDX and HR-TEM examination.•Biogenic IAA-AgNPs were efficient against pathogenic Citrobacter freundii in vitro and in vivo.•Biogenic IAA-AgNPs proved safe/eco-friendly without any significant toxic effects on liver and kidney functions/tissues of rats.
Terminal irrigation drought stress is one of the most drastic abiotic stress to diminish the wheat crop development and grains yield in arid regions of the world. The use of moringa leaf extract ...(MLE30) via seed priming technique is investigated as an organic and sustainable approach for the mitigation of drought stress along with curtailed seed rate in wheat crop. The study investigated the interaction of organic seed priming: control (dry seeds), hydro-priming, MLE30-priming, seed rate: recommended @ 125 kg ha
, curtailed @ 25 kg ha
, and terminal irrigation drought (TID): normal irrigation, mild-TID, severe-TID in wheat crop at agronomic research station, Bahawalpur, Pakistan during the wheat winter season of 2021-2022 and 2022-2023. The application of organic MLE30-priming with curtailed seed rate enhanced antioxidant enzyme activity especially total soluble proteins by 15%, superoxide dismutase by 68%, peroxidase by 16%, catalase by 70%, ascorbic acid by 17% and total protein contents by 91% under severe-TID. Yield and yield-related morphological attributes performed better in MLE30-priming as compared to hydro-priming. An effective trend was observed in the plant's chlorophyll contents, K
, and water use efficiency after being treated with MLE30-priming followed by hydro-priming under curtailed seed rate. The higher benefit-cost ratio and net income return were observed with the application of MLE30-priming with curtailed seed rate under mild-TID and severe-TID. So, it is suggested to adopt the MLE30-priming technique along with a curtailed seed rate for improving the crop establishment, stress regulation, and economic return under limited availability of irrigation water. The project findings recommended that the application of exogenous application of organic MLE30-seed priming favored and compensated the maximum wheat grains production under curtailed seed rate @ 25 kg ha
and induced terminal drought stress at flowering and milking conditions.
Cellulose represents a major source of fermentable sugars in lignocellulosic biomass and a combined action of hydrolytic enzymes (exoglucanases , endoglucanases and β-glucosidases) is required to ...effectively convert cellulose to glucose that can be fermented to bio-ethanol. However, in-order to make the production of bio-ethanol an economically feasible process, the costs of the enzymes to be used for hydrolysis of the raw material need to be reduced and an increase in specific activity or production efficiency of cellulases is required. Among the cellulases, β-glucosidase not only hydrolyzes cellobiose to glucose but it also reduces the cellobiose inhibition, resulting in efficient functioning of endo- and exo-glucanases. Therefore, in the current study kinetic and thermodynamic characteristics of highly active β-glucosidase from randomly mutated Aspergillus niger NIBGE-06 have been evaluated for its industrial applications.
The main objective of this study was the identification of mutations and determination of their effect on the physiochemical, kinetic and thermodynamic characteristics of β-glucosidase activity and stability.
Pure cultures of Aspergillus niger NIBGE and its 2-Deoxy-D-glucose resistant γ-rays mutant Aspergillus niger NIBGE-06 were grown on Vogel's medium containing wheat bran (3% w/v), at 30±1 °C for 96-108 h. Crude enzymes from both strains were subjected to ammonium sulfate precipitation and column chromatography on Fast Protein Liquid Chromatography (FPLC) system. The purified β-glucosidases from both fungal sources were characterized for their native and subunit molecular mass through FPLC and SDS-PAGE, respectively. The purified enzymes were then comparatively characterized for their optimum temperature, activation energy (Ea), temperature quotient (Q10), Optimum pH, Heat of ionization (ΔHI) of active site residues , Michaelis-Menten constants (Vmax, Km, kcat and kcat/Km) and thermodynamics of irreversible inactivation through various enzyme assays. The genomic DNA from both fungal strains was also extracted by SDS-method and full length β- glucosidase genes (bgl) were amplified through PCR. The PCR products were cloned in TA cloning vector followed by the sequencing of potentially full length clones using the commercial services of Macrogen, Korea. The in silico analyses of the sequences thus obtained were also performed using various online tools such as blastn, blastp, GeneWise, SignalP, Inter- ProScan.
The extracellular β-glucosidases (BGL) from both fungal sources were purified to homogeneity level by ammonium sulfate precipitation and FPLC system. The BGLs from both strains were dimeric in nature, with subunit and native molecular masses of 130 kDa and 252 kDa, respectively. The comparative analysis of nucleotides of bgl genes revealed 8 point mutations. Significant improvement was observed in the kinetic properties of the mutant BGL relative to the wild type enzyme. Arrhenius plot for energy of activation (Ea) showed a biphasic trend and ES-complex formation required Ea of 50 and 42 kJ mol-1 by BGL from parent and mutant, respectively. The pKa1 and pKa2 of the active site residues were 3.4 & 5.5 and 3.2 & 5.6, respectively. The heat of ionization for the acidic limb (ΔHI-AL) and the basic limb (ΔHI-BL) of BGL from both strains were equal to 56 & 41 and 71 & 45 kJ mol-1, respectively. Kinetic constants of cellobiose hydrolysis for BGL from both strains were determined as follows: kcat = 2,589 and 4,135 s-1, Km = 0.24 and 0.26 mM cellobiose, kcat/Km = 10,872 and 15,712 s-1 mM-1 cellobiose, respectively. Thermodynamic parameters for cellobiose hydrolysis also suggested that mutant BGL is more efficient compared to the parent enzyme. Comparative analysis of Ea(d), ΔH* and ΔG* for irreversible thermostability indicated that the thermostabilization of mutant enzyme was due to higher functional energy (free energy), which enabled the enzyme to resist against unfolding of its transition state.
Physiochemical and thermodynamic characterization of extracellular β-glucosidases (BGL) from 2-Deoxy-Dglucose resistant mutant derivative of A. niger showed that mutagenesis did not greatly affect the physiochemical properties of the BGL enzyme, like temperature optima, pH optima and molecular mass, while the catalytic efficiency for cellobiose hydrolysis was significantly improved (High kcat and kcat/Km). Furthermore, the mutant BGL was more thermostable than the parent enzyme. This shows that random mutagenesis has changed the BGL structural gene, resulting in improvement within its stability- function characteristics. Hence, directed evolution or random mutagenesis with careful selection can result in the engineering of highly efficient enzymes for intended industrial applications.
Photosynthetic bacteria are beneficial to plants, but knowledge of photosynthetic bacterial community dynamics in field crops during different growth stages is scarce. The factors controlling the ...changes in the photosynthetic bacterial community during plant growth require further investigation. In this study, 35 microbial community samples were collected from the seedling, flowering, and mature stages of tomato, cucumber, and soybean plants. 35 microbial community samples were assessed using Illumina sequencing of the photosynthetic reaction center subunit M (pufM) gene. The results revealed significant alpha diversity and community structure differences among the three crops at the different growth stages. Proteobacteria was the dominant bacterial phylum, and Methylobacterium, Roseateles, and Thiorhodococcus were the dominant genera at all growth stages. PCoA revealed clear differences in the structure of the microbial populations isolated from leaf samples collected from different crops at different growth stages. In addition, a dissimilarity test revealed significant differences in the photosynthetic bacterial community among crops and growth stages (P<0.05). The photosynthetic bacterial communities changed during crop growth. OTUs assigned to Methylobacterium were present in varying abundances among different sample types, which we speculated was related to the function of different Methylobacterium species in promoting plant growth development and enhancing plant photosynthetic efficiency. In conclusion, the dynamics observed in this study provide new research ideas for the detailed assessments of the relationship between photosynthetic bacteria and different growth stages of plants.
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