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Transgenic Arabidopsis thaliana (ecotype Columbia) was successfully transformed with the gene fructose-1,6-bisphosphatase (FBPase) and named as AtFBPase plants. Transgenic plants exhibited stable transformation, integration and significantly higher expressions for the transformed gene. Morphological evaluation of transgenic plants showed increased plant height (35 cm), number of leaves 25), chlorophyll contents (28%), water use efficiency (increased from 1.5 to 2.6 µmol CO2 µmol-1 H2O) and stomatal conductance (20%), which all resulted in an enhanced photosynthetic rate 2.7 µmol m-2 s-1) compared to wild type plants. This study suggests the vital role of FBPase gene in the modification of regulatory pathways to enhance the photosynthetic rate, which can also be utilised for economic crops in future. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surfaced on 31 December, 2019, and was identified as the causative agent of the global COVID-19 pandemic, leading to a pneumonia-like disease. One of its accessory proteins, ORF6, has been found to play a critical role in immune evasion by interacting with KPNA2 to antagonize IFN signaling and production pathways, resulting in the inhibition of IRF3 and STAT1 nuclear translocation. Since various mutations have been observed in ORF6, therefore, a comparative binding, biophysical, and structural analysis was used to reveal how these mutations affect the virus's ability to evade the human immune system. Among the identifiedmutations, the V9F, V24A,W27L, and I33T, were found to have a highly destabilizing effect on the protein structure of ORF6. Additionally, the molecular docking analysis of wildtype andmutant ORF6 and KPNA2 revealed the docking score of - 53.72 kcal/mol for wildtype while, -267.90 kcal/mol, -258.41kcal/mol, -254.51 kcal/mol and -268.79 kcal/mol for V9F, V24A, W27L, and I33T respectively. As compared to the wildtype the V9F showed a stronger binding affinity with KPNA2 which is further verified by the binding free energy (-42.28 kcal/mol) calculation. Furthermore, to halt the binding interface of the ORF6-KPNA2 complex, we used a computational molecular search of potential natural products. A multi-step virtual screening of the African natural database identified the top 5 compounds with best docking scores of -6.40 kcal/mol, -6.10 kcal/mol, -6.09 kcal/mol, -6.06 kcal/mol, and -6.03 kcal/mol for tophit1-5 respectively. Subsequent all-atoms simulations of these top hits revealed consistent dynamics, indicating their stability and their potential to interact effectively with the interface residues. In conclusion, our study represents the first attempt to establish a foundation for understanding the heightened infectivity of new SARS-CoV-2 variants and provides a strong impetus for the development of novel drugs against them. [ABSTRACT FROM AUTHOR]

The cellular response to stress is mediated by intracellular proteins, called heat shock proteins (HSPs). Among various known stressors, heat is a major factor that induces the production of HSP70. Keeping in view the very hot conditions of Dera Ismail Khan (D.I. Khan) division where the temperature remains at 45-50°C during the months of June to September, it was hypothesized that heat stress conditions do induce the overexpression of HSPs, especially Hsp70. It was thus attempted to find out the possible role of Hsp70 in those human being having the age of 90 years or above, called longevity people, against heat stress conditions. Whole blood samples of 45 longevity individuals and 20 samples of control people were collected in D. I. Khan during September 2018 to October, 2019 after proper approval from Gomal University ethical review board and written consent of each individual was taken prior to collection of blood sample. For serum collection, blood samples were centrifuged at 1000xg for 15 minutes. Quantitative measurement of Hsp70 protein was done using sandwich ELISA technique. The maximum serum Hsp70 level observed was 42ng/ml and minimum serum Hsp70 value was 13ng/ml, with median value of 28ng/ml. In control group, maximum concentration observed was 38ng/ml while the minimum serum level was 18ng/ml with median value of 13ng/ml. In longevity males, serum Hsp70 levels increased in individuals between 89 to 91 years of age, peaked between 92 to 97 years but comparatively lower having the age of above 98 years. On the other hand, serum concentration of Hsp70 in longevity females were highest in those having 89 to 92 years, lower in 93 to 97 years and, like males, lowest having age above 98 years. The study hence, showed that longevity individuals had higher quantities of serum Hsp70 compared to control group, and again, males contained higher concentration than longevity females, showing that thermal stress was the agent leading to over-expression of Hsp70 especially in longevity individuals but in longevity individuals. Whether this increased expression had any impact on longevity is still not clear that needs to be deciphered. [ABSTRACT FROM AUTHOR]

The fibrillin (FBN) gene family is widely distributed in all photosynthetic organisms. Members of this gene family are involved in plant growth and development and their response to various biotic and abiotic stress factors. In this study, 16 members of FBN were identified in Glycine max and characterized by using different bioinformatics tools. Phylogenetic analysis classified FBN genes into seven groups. The presence of stress-related cis-elements in the upstream region of GmFBN highlighted their role in tolerance against abiotic stresses. To further decipher the function, physiochemical properties, conserved motifs, chromosomal localization, subcellular localization, and cis-acting regulatory elements were also analyzed. Gene expression analysis based on FPKM values revealed that GmFBNs greatly enhanced soybean drought tolerance and controlled the expression of several genes involved in drought response, except for GmFBN-4, GmFBN-5, GmFBN-6, GmFBN-7 and GmFBN-9. For high throughput genotyping, an SNP-based CAPS marker was also developed for the GmFBN-15 gene. The CAPS marker differentiated soybean genotypes based on the presence of either the GmFBN-15-G or GmFBN-15-A alleles in the CDS region. Association analysis showed that G. max accessions containing the GmFBN-15-A allele at the respective locus showed higher thousand seed weight compared to accessions containing the GmFBN-15-G allele. This research has provided the basic information to further decipher the function of FBN in soybean. [ABSTRACT FROM AUTHOR]

Cysteine-rich poly comb-like protein (CPP) is a member of cysteine-rich transcription factors that regulates plant growth and development. In the present work, we characterized twelve CPP transcription factors encoding genes in soybean (Glycine max). Phylogenetic analyses classified CPP genes into six clades. Sequence logos analyses between G. max and G. soja amino acid residues exhibited high conservation. The presence of growth and stress-related cis-acting elements in the upstream regions of GmCPPs highlight their role in plant development and tolerance against abiotic stress. Ka/Ks levels showed that GmCPPs experienced limited selection pressure with limited functional divergence arising from segmental or whole genome duplication events. By using the PAN-genome of soybean, a single nucleotide polymorphism was identified in GmCPP-6. To perform high throughput genotyping, a kompetitive allele-specific PCR (KASP) marker was developed. Association analyses indicated that GmCPP-6-T allele of GmCPP-6 (in exon region) was associated with higher thousand seed weight under both water regimes (well-water and water-limited). Taken together, these results provide vital information to further decipher the biological functions of CPP genes in soybean molecular breeding. [ABSTRACT FROM AUTHOR]

Several health benefits are obtained from resistant starch, also known as healthy starch. Enhancing resistant starch with genetic modification has huge commercial importance. The variation of resistant starch content is narrow in wheat, in relation to which limited improvement has been attained. Hence, there is a need to produce a wheat population that has a wide range of variations in resistant starch content. In the present study, stable mutants were screened that showed significant variation in the resistant starch content. A megazyme kit was used for measuring the resistant starch content, digestible starch, and total starch. The analysis of variance showed a significant difference in the mutant population for resistant starch. Furthermore, four diverse mutant lines for resistant starch content were used to study the quantitative expression patterns of 21 starch metabolic pathway genes; and to evaluate the candidate genes for resistant starch biosynthesis. The expression pattern of 21 starch metabolic pathway genes in two diverse mutant lines showed a higher expression of key genes regulating resistant starch biosynthesis (GBSSI and their isoforms) in the high resistant starch mutant lines, in comparison to the parent variety (J411). The expression of SBEs genes was higher in the low resistant starch mutants. The other three candidate genes showed overexpression (BMY, Pho1, Pho2) and four had reduced (SSIII, SBEI, SBEIII, ISA3) expression in high resistant starch mutants. The overexpression of AMY and ISA1 in the high resistant starch mutant line JE0146 may be due to missense mutations in these genes. Similarly, there was a stop_gained mutation for PHO2; it also showed overexpression. In addition, the gene expression analysis of 21 starch metabolizing genes in four different mutants (low and high resistant starch mutants) shows that in addition to the important genes, several other genes (phosphorylase, isoamylases) may be involved and contribute to the biosynthesis of resistant starch. There is a need to do further study about these new genes, which are responsible for the fluctuation of resistant starch in the mutants. [ABSTRACT FROM AUTHOR]

Food security is facing a major threat from salinity and there is a need to develop salt tolerant crop varieties to ensure that the demand for food from the world's increasing population is met. Salinity mostly occurs in arid and semi-arid regions. It may cause many adverse physiological effects on plants, i.e., toxic ion accumulation, disturbed osmotic potential, and decreased crop yield. The present study aimed to investigate the morphological, physiological, biochemical, and genetic parameters of wheat genotypes under salt stress. Six wheat genotypes were screened for salt tolerance at the seedling and maturity stage. Seeds were sown at 0 and 150 mM of salinity level. Biochemical traits, i.e., shoot/root fresh and dry weight, chlorophyll a/b and total chlorophyll contents, shoot nitrogen, shoot phosphorus, proline, and carbohydrates were measured. Wheat genotypes showed a significant increase in free amino acids, shoot nitrogen, and total soluble proteins under saline conditions. Higher Na+/K+ ratio and free amino acids were estimated under 150 mM NaCl treatment in Pasban-90 and found to be the most salt-tolerant genotype. By contrast, reduced proline, total chlorophyll, and Na+/K+ ratio were found in Kohistan-97 marking it to be sensitive to stress. Expression analysis of HKTs genes was performed to validate the results of two contrasting genotypes. The differential expression of HKT2; 1 and HKT2; 3 explained the tissue and genotype specific epigenetic variations. Our findings indicated that these selected genotypes can be further used for molecular studies to find out QTLs/genes related to salinity. This suggests that, in contrasting wheat genotypes, there is a differentially induced defense response to salt stress, indicating a functional correlation between salt stress tolerance and differential expression pattern in wheat. [ABSTRACT FROM AUTHOR]

Multi-ovary wheat (three pistil) is a unique germplasm for the seed production of hybrid wheat. The purpose of the present study was to transfer the multi-ovary trait to semi-dwarf plants to increase the production of grains in wheat crops. Therefore, tall, semi-dwarf, and dwarf plants were crossed with plants with the three-pistil trait. A three-pistil tall plant was used as the female parent, while tall (Synthetic hexaploid), semi-dwarf, and dwarf plants were used as male parents. F1 and F2 progenies with parents were planted in 2015-16 using RCBD. The outcome of the crosses showed that multi-ovary tall plants gave significant difference for all five traits (days to maturity, plant height, number of seeds per spike, grain weight per spike, and grain yield per unit area) in both generations. The greatest number of grains per spike and grain yield per unit area were obtained from the cross of three-pistil tall and dwarf parent (P1/P6) in the F1 and F2 generations. The cross also resulted in a significant reduction in height (96 cm). Further heterosis studies conducted with crosses between three-pistil tall and dwarf parent (P1/P6) showed the greatest heterosis and heterobeltiosis for the number of grains per spike (60.0 and 26.19%, respectively) and grain yield per m2 (27.68 and 2.85%, respectively). In the case of grain weight per spike, the heterosis value was also positive and significant (17.7). Meanwhile, for other traits, their values were negative for heterosis and heterobeltiosis. High numbers of grains and grain weight were found to be associated with positive heterobeltiosis and in turn the grain yield per m2, but plant height and maturity had negative affirmation with heterobeltiosis. Heterosis studies also indicated the dominant gene action for the three-pistil trait. Thus, the study clearly signified that grain yield can be increased by using the multi-ovary genotype with the semi-dwarf height. This new germplasm will be helpful for breeders to increase the production of wheat crops in the southern climate of Pakistan. [ABSTRACT FROM AUTHOR]

Manipulation of genes involved in starch synthesis could significantly affect wheat grain weight and yield. The starch-branching enzyme (SBE) catalyzes the formation of branch points by cleaving the α-1,4 linkage in polyglucans and reattaching the chain via an α-1,6 linkage. Three types of SBE isoforms (SBEI, SBEII, and SBEIII) exist in higher plants, with the number of SBE isoforms being species-specific. In this study, the coding sequence of the wheat TaSBEIII gene was amplified. After the multiple sequence alignment of TaSBEIII genome from 20 accessions in a wheat diversity panel, one SNP was observed in TaSBEIII-A , which formed the allelic marker allele-T. Based on this SNP at 294 bp (C/T), a KASP molecular marker was developed to distinguish allelic variation among the wheat genotypes for thousand grain weight (TGW). The results were validated using 262 accessions of mini core collection (MCC) from China, 153 from Pakistan, 53 from CIMMYT, and 17 diploid and 18 tetraploid genotypes. Association analysis between TaSBEIII-A allelic variation and agronomic traits found that TaSBEIII-A was associated with TGW in mini core collection of China (MCC). The accessions possessing Allele-T had higher TGW than those possessing Allele-C ; thus, Allele-T was a favorable allelic variation. By analyzing the frequency of the favorable allelic variation Allele-T in MCC, it increased from pre-1950 (25%) to the 1960s (45%) and increased continuously from 1960 to 1990 (80%). The results suggested that the KASP markers can be utilized in grain weight improvement, which ultimately improves wheat yield by marker-assisted selection in wheat breeding. The favorable allelic variation allele-T should be valuable in enhancing grain yield by improving the source and sink simultaneously. Furthermore, the newly developed KASP marker validated in different genetic backgrounds could be integrated into a breeding kit for screening high TGW wheat. [ABSTRACT FROM AUTHOR]

High-throughput genotyping for functional markers offers an excellent opportunity to effectively practice marker-assisted selection (MAS) while breeding cultivars. We developed kompetitive allele-specific PCR (KASP) assays for genes conferring drought tolerance in common wheat (Triticum aestivum L.). In total, 11 KASP assays developed in this study and five already reported assays were used for their application in wheat breeding. We investigated alleles at 16 loci associated with drought tolerance among 153 Pakistani hexaploid wheat cultivars released during 1953–2016; 28 diploid wheat accessions (16 for AA and 12 for BB) and 19 tetraploid wheat (AABB) were used to study the evolutionary history of the studied genes. Superior allelic variations of the studied genes were significantly associated with higher grain yield. Favored haplotypes of TaSnRK2.3 -1A, TaSnRK2.3 -1B, TaSnRK2.9 -5A, TaSAP -7B, and TaLTPs -1A predominated in Pakistani wheat germplasm indicating unconscious pyramiding and selection pressure on favorable haplotypes during selection breeding. TaSnRK2.8 -5A, TaDreb -B1, 1-feh w3 , TaPPH -7A, TaMOC -7A, and TaPARG -2A had moderate to low frequencies of favorable haplotype among Pakistani wheat germplasm pointing toward introgression of favorable haplotypes by deploying functional markers in marker-assisted breeding. The KASP assays were compared with gel-based markers for reliability and phenotypically validated among 62 Pakistani wheat cultivars. Association analyses showed that the favorable allelic variations were significantly associated with grain yield-contributing traits. The developed molecular marker toolkit of the genes can be instrumental for the wheat breeding in Pakistan. [ABSTRACT FROM AUTHOR]

Heat and drought are among the major obstacles confronting crop production under climate change. The present study was conducted to evaluate 50 diverse wheat genotypes for cell membrane stability (CMS) and chlorophyll content at seedling and anthesis stages under heat and drought stress conditions, to understand the effect of the two abiotic factors and to find promising genotypes for future breeding. Experiments were conducted in the glasshouse (seedling stage) and the field (anthesis stage). Analysis of variance showed significant variation (P≤0.05) for all of the traits at seedling and anthesis stages. High levels of broad-sense heritability and genetic advance at 5% selection intensity indicated the presence of a high genetic component of variation and potential for genetic improvement through selection among the existing genetic variation. CMS showed a significant positive correlation with 1000-grain weight (TGW) under heat and drought conditions at both seedling and anthesis stages. Chlorophyll a/b ratio at seedling stage exhibited a significant negative correlation (r = -0.39, P < 0.05) with TGW under heat stress. Total chlorophyll content was significantly (r = 0.42, P < 0.05) correlated with TGW under heat stress at anthesis. Genotypes ETAD248 and ETAD7 showed the highest CMS and TGW values, whereas their chlorophyll a/b values were lowest, at both seedling and anthesis stages under heat and drought stress conditions. Higher CMS and total chlorophyll content, and lower chlorophyll a/b, were found to be useful indicators to identify genotypes with high TGW under heat and drought stress conditions. This study indicated the possibility of using seedling resistance as an indicator for later stage response in breeding for heat and drought resistance. The resistant genotypes identified can be used as potential germplasm in breeding programs. [ABSTRACT FROM AUTHOR]

Objective: Thalassemia is blood related disease which arises from the reduced level of hemoglobin in red blood cells (RBC), a protein responsible for carrying oxygen inside the body. Considering its widespread occurrence in developing countries like Pakistan, this study aims to investigate the common molecular anomalies of the beta thalassemia disease in district Charsadda, Khyber Pakhtunkhwa. Methods: This work was done at Abdul Wali Khan University (AWKU) Mardan, Khyber Pakhtunkhwa, Pakistan. The work was performed on the blood samples collected from the patients and their families with beta thalassemia major (n = 13 families) belonged to District Charsadda. The collected blood samples were analyzed for presence of six known mutations with the help of polymerase chain reaction technique i.e. amplification of refractory mutation system. Results: Our Study reports six known mutations (IVS-1-5, FSC 8/9, CD 41/42, IVS-1-1, CD 15 and FSC-5) accounting for about 90% of total beta thalassemia genes in this country. Among the reported mutations, IVS 1-5 was the most prevalent beta thalassemia gene in patients belonging to District Charsadda. Conclusion: The results and findings of the current study may help in accessing the frequency of these common mutations and in initiating pre-natal diagnosis programme in Pakistan. [ABSTRACT FROM AUTHOR]

Objective: To characterize the disease causing mutation in a large consanguineous Pakistani family with severe Mal de Meleda (MDM) or keratosis palmoplantaris transgrediens, a rare autosomal recessive skin disorder. Methodology: Single nucleotide polymorphism (SNPs) genotyping was performed using the GeneChip Mapping 250K array (Affymetrix). Homozygosity mapping and sorting of genomic regions were performed with dedicated software called AutoSNPa. Selected regions were further investigated by genotyping with microsatellite markers derived from known and novel polymorphic repeats. Two-point LOD score calculation was performed by using the MLINK of Fastlink computer package. All three coding exons of ARS (component B) gene were amplified by PCR and sequenced. Conclusion: Sequencing of all the coding exons of ARS (component B) gene in the affected individuals revealed a recurrent missense mutation in exon 3 at base pair 256 from Guanine to Alanine (256G>A) and as a result the amino acid Glycine is replaced by Arginine at position 86 (G86R). This finding will facilitate control of affected MDM births in the Pakistani families. [ABSTRACT FROM AUTHOR]

Interrogations of local germplasm and landraces can offer a foundation and genetic basis for drought tolerance in wheat. Potential of drought tolerance in a panel of 30 wheat genotypes including varieties, local landraces, and wild crosses were explored under drought stress (DS) and well-watered (WW) conditions. Considerable variation for an osmotic adjustment (OA) and yield components, coupled with genotype and environment interaction was observed, which indicates the differential potential of wheat genotypes under both conditions. Reduction in yield per plant (YP), thousand kernel weight (TKW), and induction of OA was detected. Correlation analysis revealed a strong positive association of YP with directly contributing yield components under both environments, indicating the impotence of these traits as a selection-criteria for the screening of drought-tolerant genotypes for drylands worldwide. Subsequently, the association of OA with TKW which contributes directly to YP, indicates that wheat attains OA to extract more water from the soil under low water-potential. Genotypes including WC-4, WC-8 and LLR-29 showed more TKW under both conditions, among them; LLR-29 also has maximum OA and batter yield comparatively. Result provides insight into the role of OA in plant yield sustainability under DS. In this study, we figure out the concept of OA and its incredible role in sustainable plant yield in wheat. [ABSTRACT FROM AUTHOR]

PGAP2 encodes a protein involved in remodeling the glycosylphosphatidylinositol (GPI) anchor in the Golgi apparatus. After synthesis in the endoplasmic reticulum (ER), GPI anchors are transferred to the proteins and are remodeled while transported through the Golgi to the cell membrane. Germline mutations in six genes (PIGA, PIGL, PIGM, PIGV, PIGN, and PIGO) in the ER-located part of the GPI-anchor-biosynthesis pathway have been reported, and all are associated with phenotypes extending from malformation and lethality to severe intellectual disability, epilepsy, minor dysmorphisms, and elevated alkaline phosphatase (ALP). We performed autozygosity mapping and ultra-deep sequencing followed by stringent filtering and identified two homozygous PGAP2 alterations, p.Tyr99Cys and p.Arg177Pro, in seven offspring with nonspecific autosomal-recessive intellectual disability from two consanguineous families. Rescue experiments with the altered proteins in PGAP2-deficient Chinese hamster ovary cell lines showed less expression of cell-surface GPI-anchored proteins DAF and CD59 than of the wild-type protein, substantiating the pathogenicity of the identified alterations. Furthermore, we observed a full rescue when we used strong promoters before the mutant cDNAs, suggesting a hypomorphic effect of the mutations. We report on alterations in the Golgi-located part of the GPI-anchor-biosynthesis pathway and extend the phenotypic spectrum of the GPI-anchor deficiencies to isolated intellectual disability with elevated ALP. GPI-anchor deficiencies can be interpreted within the concept of a disease family, and we propose that the severity of the phenotype is dependent on the location of the altered protein in the biosynthesis chain. [ABSTRACT FROM AUTHOR]

Wheat is one of the world's major staple food crops, and breeding for improvement of grain yield is a priority under the scenarios of climate change and population growth. WRKY transcription factors are multifaceted regulators in plant growth, development, and responses to environmental stimuli. In this study, we identify the WRKY gene TaGSNE (Grain Size and Number Enhancer) in common wheat, and find that it has relatively high expression in leaves and roots, and is induced by multiple abiotic stresses. Eleven single-nucleotide polymorphisms were identified in TaGSNE, forming two haplotypes in multiple germplasm collections, named as TaGSNE-Hap-1 and TaGSNE-Hap-2. In a range of different environments, TaGSNE-Hap-2 was significantly associated with increases in thousand-grain weight (TGW; 3.0%) and spikelet number per spike (4.1%), as well as with deeper roots (10.1%) and increased root dry weight (8.3%) at the mid-grain-filling stage, and these were confirmed in backcross introgression populations. Furthermore, transgenic rice lines overexpressing TaGSNE had larger panicles, more grains, increased grain size, and increased grain yield relative to the wild-type control. Analysis of geographic and temporal distributions revealed that TaGSNE-Hap-2 is positively selected in China and Pakistan, and TaGSNE-Hap-1 in Europe. Our findings demonstrate that TaGSNE overcomes the trade-off between TGW/grain size and grain number, leading us to conclude that these elite haplotypes and their functional markers could be utilized in marker-assisted selection for breeding high-yielding varieties., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The main function of histone protein is to provide support to the structure of chromosomes. It helps in binding a long thread of DNA into a more condensed shape to fit into the nucleus. From histone variants, histone H3 ( HH3 ) plays a crucial role in plant growth and development. Characterization of histones has not been reported in Glycine max till now. The objective of this study was to characterize the HH3 gene family for molecular breeding of G. max . In this study, 17 HH3 members in G. max were identified by performing local BLASTp using HH3 members from Arabidopsis as a query. Phylogenetic analysis classified HH3 genes in seven clades. Sequence logo analysis among Arabidopsis thaliana , Oryza sativa , and Glycine max showed a higher level of similarity in amino acids. Furthermore, conserveness of G. max HH3 genes was also confirmed by Gene Structure Display. Ten paralogous gene pairs were identified in GmHH3 genes in the Glycine max genome by conducting collinearity analysis. G. max HH3 genes have experienced strong purifying selection pressure, with limited functional divergence originating from the segmental and whole-genome duplication, as evidenced by the Ka/Ks ratio. The KASP marker was developed for GmHH3-3 gene. Genotyping was performed on 46 G. max genotypes. This differentiation was based upon the presence of either GmHH3-3-C or GmHH3-3-T allele in the CDS region. The results showed that G. max accessions containing the GmHH3-3-T allele at respective locus showed higher thousand seed weight than that of those accessions that contain the GmHH3-3-C allele. This research provides the basic information to further decipher the function of HH3 in soybean., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fatima, Tahir, Ikram, Khan, Sajjad, Qanmber, Darwish, Geng, Xiangkuo and Ur Rehman.)