Centre of Excellence in Molecular Biology
Recent publications
Tomato leaf curl New Delhi virus (ToLCNDV), a begomovirus, that causes severe leaf curling, stunting, and reduced yield in tomato plants is consistently threatening its production worldwide. CRISPR/Cas9‐mediated genome editing has shown immense potential in developing disease‐resistant crops. This study successfully focuses on designing a precise and efficient strategy for in planta defence against ToLCNDV. Five key targets within the viral genome, essential for its replication and pathogenicity, were selected. Five Cas9‐expressing constructs, along with the ToLCNDV infectious clone, were agroinfiltrated into tomato plants. Three constructs effectively disrupted the ToLCNDV genome. These three constructs, 1T, 2T, and 4T, were shortlisted based on symptom severity level. They showed a relatively low viral titre of 0.5, 0.42, and 0.25 through quantitative real time PCR (qPCR) after 3, 6, and 9 days of post‐co‐infiltration, respectively. Positive control plants showed significant signs of infection like yellowing of leaves, thickening of veins, and majorly upward curling of leaves. In comparison, plants infiltrated with three Cas9 constructs had mild yellowing of leaves that recovered after approximately 21‐dpi. Furthermore, we assessed the agronomic performance of Cas9‐mediated tomato plants through in planta Agrobacterium‐ mediated transformation with three short‐listed guided RNA (gRNA) constructs under greenhouse conditions. Also, qPCR analysis of Cas9 protein in 7, 14, and 21‐day of intervals gave a relative expression of 0.85, 0.76 and 0.51 respectively in genetically engineered (GE) plants through in planta transformation. In conclusion, this research contributes to CRISPR‐Cas9‐mediated plant genome editing. Our findings substantiate the efficacy of the CRISPR/Cas9 system in achieving durable engineering of resistance against ToLCNDV with 30% transformation efficiency in tomato plant. Furthermore, this study illuminates potential avenues for extending the application of this technology to confer resistance against singular and multiple infectious viruses in diverse crop species.
The red palm weevil, Rhynchophorus ferrugineus (Oliver, 1970) (Coleoptera: Dryophthoridae) is the most devastating insect-pest of palm trees worldwide. Synthetic insecticides are the most preferred tool for the management of RPW. Alternatively, RNA interference (RNAi) mediated silencing of crucial genes provides reasonable control of insect pests. Recently, we have targeted four important genes; ecdysone receptor (EcR), serine carboxypeptidase (SCP), actin and chitin-binding peritrophin (CBP) in the 3rd and 5th instar larvae RPW. The results from 20 days trial showed that the survival rate of 3rd instar larvae fed on SCP and actin dsRNAs exhibited the lowest survival (12–68%). While, in the 5th instar larvae, the lowest survival rate (24%) was recorded for SCP after 20 days of incubation. Similarly, the weight of the 3rd and 5th instar larvae treated with SCP and actin was significantly reduced to 2.30–2.36 g and 4.64–4.78 g after 6 days of dsRNA exposure. The larval duration was also decreased significantly in the larvae treated with all the dsRNA treatments. The qRT-PCR results confirmed a significant suppression of the targeted genes as 90–97% and 85–93% in the 3rd and 5th instar larvae, respectively. The results suggest that the SCP and the actin genes can be promising targets to mediate RNAi-based control of RPW.
Long non-coding RNAs (lncRNAs) have been in the spotlight for the past two decades due to their extensive role in regulating a wide range of cellular processes. Development, differentiation, regulation, and modulation are some of the vital cellular cascades coordinated by these molecules. Despite their importance, there has been limited literature on their practical implications in cancer prevention. Advancements in lncRNA biology have enabled the characterization of numerous secondary structures and sequence motifs, which could serve as potential targets for cellular therapies. Several studies have highlighted the involvement of lncRNAs in human pathologies, where they can be targeted by small molecules or antisense oligonucleotides to prevent diseases. However, progress has been hindered by the challenge of developing specific delivery vehicles for targeted delivery. Recent improvements in sequence optimization and nucleotide modification have enhanced drug stability and reduced the immunogenicity of lncRNA-based therapies, yet further advances are needed to fully realize their potential in treating complex diseases like cancer. This review aims to explore current lncRNA biology, their mechanisms of action, nanoformulation strategies, and the clinical trials focused on lncRNA delivery systems.
Interleukin-37 is a cytokine with potent immunosuppressive properties that has been shown to have potential to treat autoimmune and chronic inflammatory diseases, as well as certain types of cancer. IL-37 is a 19 kDa protein which interacts with proteins in receptor-dependent and receptor-independent pathways. The expression of the IL-37 protein cloned into the pET-28a vector was optimized in Rosetta 2(DE3) after comparing its expression with Rosetta-gami 2(DE3) and Rosetta 2(DE3) pLysS, which was then used for the large-scale production of IL-37. IMAC purification of IL-37 yielded > 97% pure 0.9 mg/mL protein from auto-induced fermentation. The IC50 value of IL-37 was < 1 µM, which was similar to that of doxorubicin, and proliferation of > 80% of all cancer cells was inhibited by 100 µg/mL of IL-37 protein. IL-37 may be a promising theragnostic target for cancer due to its comparable IC50 value with that of doxorubicin.
The search for novel therapeutic agents to treat breast cancer has compelled the development of fusion proteins that synergize the functional benefits of different bioactive peptides. Leptulipin, derived from scorpion venom, exhibits antitumor properties. On the other hand, p28, a peptide from the bacterial protein azurin, enhances cell penetration. The current study investigated the design and computational evaluation of a Leptulipin-p28 fusion protein for breast cancer treatment. The amino acid sequences of Leptulipin and p28 were joined via a rigid linker to maintain structural and functional integrity. Secondary and tertiary structure predictions were performed using online servers of GOR-IV and I-TASSER. Physicochemical properties and solubility were analyzed using ProtParam and Protein-Sol. Validation and quality assessment of the fusion protein were confirmed through Rampage and ERRAT2. Finally, the fusion protein was docked with 2 receptors (VEGFR and Cadherin) and docked complexes were simulated on GROMACS. The Leptulipin-p28 fusion protein exhibited a stable structure exhibiting a high quality score of 92 on ERRAT and Ramachandran plot analysis highlighting 76.3% of residues in the favorable region. Docking studies with VEGFR and Cadherin receptors followed by 100 ns simulations on GROMACS showed stable complex formation. Molecular dynamics simulations confirmed the stability and robust interaction of the fusion protein-receptor complexes over time. The computational analysis indicates that the Leptulipin-p28 fusion protein holds promise as a multitarget therapeutic agent in breast cancer. The current findings warrant further investigation through in vitro and in vivo studies to validate the current outcomes.
Pathogen infections that affect potato yield cause severe economic losses every year. Many studies point to the role of apoplastic (cell wall) invertase (CWIN) enzyme in plant defence mechanisms. Apoplastic invertase inhibitor (INVINH1) post-translationally regulates the CWIN enzyme activity. Nevertheless, the role of INVINH1 needs to be elucidated for several effects in plant transformation parameters and its gene expression which we sought to explore using CRISPR/Cas9 technology. In this study, we sequenced the first exon of INVINH1 gene in cv. Desiree and Solanum chacoense M6. We identified in the first exon two alleles for StINVINH1 gene in cv. Desiree and one allele for ScINVINH1 gene in S. chacoense M6. We designed two single-guided RNAs (sgRNAs) to target INVINH1 gene from diploid S. chacoense M6 and tetraploid S. tuberosum cv. Desiree using CRISPR/Cas9-based technology. In our earlier study, we have already optimised the transformation protocol for M6 and cv. Desiree using Agrobacterium strains, based on which Agrobacterium strain AGL1 was chosen for the CRISPR/Cas9 experiment. Our experimentation showed that heat stress at 37 °C could increase the mutagenesis capability and CRISPR/Cas9 targeting affected the plant transformation parameters. It was found from the knockout experiment that the indels were present in the calli and the candidate regenerated plants showed reduced gene expression level conducted via RT-qPCR. Our study demonstrated that INVINH1 targeting affected the calli induction and regeneration rates, was effective under heat stress and reduced its gene expression level. More studies are required to comprehend the function of the INVINH1 enzyme in potato stress response and defence mechanism.
Cystic fibrosis (CF) is a genetic monogenic disorder inherited in an autosomal recessive manner, marked by persistent airway infections in the endobronchial region. This condition leads to the gradual development of bronchiectasis and, ultimately, respiratory failure, emerging as the primary cause of mortality in individuals diagnosed with CF. Diagnosis is done depending on the patient’s symptoms and lung radiological findings like chest X-rays and CTs. In younger patients and children, diagnosis becomes difficult due to overlapping symptoms with other diseases such as CF which is a rare genetic disease in our population. Diagnosis of CF usually relies on characteristic symptoms, a family history of CF, and an abnormal sweat chloride test, but in children, low sweat production during testing leads to false negative results. In this case report, a suspected patient with ambiguous respiratory symptoms underwent a comprehensive investigation revealing elevated CRP levels, TLC, and characteristic pulmonary manifestations on chest X-ray, suggesting cystic fibrosis. Despite negative sweat chloride tests, the patient was analysed for potential candidate SNPs and was also tested for potential CFTR mutations to rule out CF, genetic analysis confirmed the diagnosis. Genetic testing plays a crucial role in diagnosing cystic fibrosis, even when traditional tests are inconclusive. Specific mutations like Δ508 deletion and rs213950 guide personalized treatment. Consanguinity and family history highlight genetic predisposition, while environmental factors may influence symptom onset. Further research is needed to understand these complexities and improve diagnostic and treatment approaches.
The tomato leaf miner Tuta absoluta Meyrick is the most serious pest of potato worldwide. In the present study, we explored an alternate approach to induce resistance in potatoes against T. absoluta by engineering Jaburetox-2Ec, an insecticidal peptide derived from an isoform of urease from Jack bean (Canavalia ensiformis L.). The construct was developed by cloning a PCR-amplified fragment of Jaburetox-2Ec (270 bp) in a pBIN61 vector under the control of a 35S promoter and was named pBIN-JBTX. Furthermore, the recombinant plasmid expressing Jaburetox-2Ec was transformed into potato cv. Lady Olympia using Agrobacterium tumefaciens strain EHA105. The results showed the proper integration of the T-DNA region in primary transformants. Quantitative PCR assays showed the independent transgenic lines had enhanced transcript levels of Jaburetox. The leaf biotoxicity assays were performed by releasing pre-starved 2nd, 3rd, and 4th instar larvae of T. absoluta on transgenic potato plants. As a result, depending on the larval stage, the mortality rate of larvae feeding on transgenic plants ranged from 11 to 33%. Additionally, it was found that larvae feeding on control plants gained more weight than those feeding on transgenic plants. Besides that, deformities in larvae feeding on transgenic plants were also recorded. Overall, the results exhibited the insecticidal activity of Jaburetox 2-Ec against T. absoluta. The transgenic lines can be used as a source of germplasm for an efficient potato breeding programme.
The cotton aphid Aphis gossypii is a major agricultural pest of cotton that causes substantial damage to the crop not only by sucking sap but also through virus transmission. Globally adopted traditional and contemporary approaches to control aphid infestation have certain limitations and are hazardous to human health. RNA interference (RNAi) technology has unfolded its potential as an effective crop protection strategy against various pests. In this study, we adopted plant-mediated RNAi strategy to enhance aphid resistance in cotton by targeting arginine kinase (AK), which is a crucial enzyme responsible for energy homeosta-sis in insects. We selected a 312bp dsRNA fragment containing eight siRNAs and showing optimum GC content, Hb index, and stable secondary structure based on computational prediction studies. The binary construct expressing dsRNA was used to transform local cotton variety MNH886 and four transgenic lines were obtained in the T 1 generation. Out of the four T 1 transgenic cotton lines, dsA-7 exhibited the highest aphid mortality (73.3%), whereas, dsA-1, dsA-3 and dsA-6 revealed 60%, 61%, and 66.6% aphid mortality, respectively , in comparison to 13.3% mortality in the mock control cotton line. Moreover, significant knockdown in mRNA expression of AK was observed in aphids fed dsA-7 which was 79%. In comparison, 54%, 47%, and 45% downregulation was recorded in aphids which fed on dsA-6, dsA-3, and dsA-1 transgenic cotton lines, respectively. These results revealed that plant-mediated downregulation of aphid RNA induced significant RNA interference in A. gossypii which resulted in considerable aphid mortality and led to plant protection against aphids.
The whitefly, a polyphagous insect pest feeding on nearly 1328 plant species, is a major threat to global cotton production and incurs up to 50% yield losses in cotton production in Pakistan. We investigated whether increased aspartate in phloem sap imparts whitefly toxicity and protects cotton plants from intense damage. The enzymatic step for aspartate production is carried through aspartate aminotransferase (AAT). In this study, we constitutively overexpressed the Oryza sativa cytoplasmic AAT ( OsAAT2) under the CaMV35S promoter in Gossypium hirsutum cv. CIM‐482. Real‐time PCR analysis of the AAT transcripts revealed a 2.85‐ to 31.7‐fold increase in mRNA levels between the different cotton lines. A substantial increase in the free‐amino acid content of the major N‐assimilation and transport amino acids (aspartate, glutamate, asparagine, and glutamine) was seen in the phloem sap of the transgenic cotton lines. The bioassay revealed that the two transgenic cotton lines with the highest free aspartate content in the phloem sap exhibited 97 and 94% mortality in the adult whitefly population and a 98 and 96% decline in subsequent nymph populations, respectively. There was also a significant change in the physiological behaviour of the transgenic cotton lines, with an increased net assimilation ( A ), gaseous exchange ( Gs ) and rate of transpiration ( E ). Improved morphological characteristics like plant height, total number of bolls and fiber yield were recorded in transgenic cotton lines. The AAT gene shows promise in mitigating whitefly infestations and enhancing the overall health and yield of cotton plants.
The assessment of genetic diversity within and between grapevine populations is routinely performed at the molecular level using various laboratory-based techniques. Inter Simple Sequence Repeats (ISSR) markers have been widely used for plant varietal fingerprinting analysis, assessment of hybridization as well as detection of clonal variations and identification of cultivars. The objective of the present study was to reveal the ISSR-based genetic and morphological diversity among different grapevine genotypes, analyse polymorphism level of ISSR primers used, and determine genetic relationship of some Azerbaijani, introduced cultivars and hybrids. Twenty-one cultivars were analysed via ten polymorphic ISSR primers.. Morphological and ampelography data of genotypes studied according OIV descriptors to provide complete information of accessions. ISSR primers produced a total of 56 bands of which 44 were polymorphic. Resolving power of the ISSR primers ranged between 1.61 (UBC 826) and 4.09 (UBC 840). PIC (Polymorphism Information Content) values varied from 0.211(UBC 842) to 0.401 (UBC 810) respectively. The grapevine genotypes were grouped into five major kinship groups. Considering morphological and high genetic similarity value Khalbasar and Apoi Khagog genotypes showed highest genetic similarity index (0.923) and could be theoretically identical. Our results revealed that ISSR genetic markers combined with morphological and ampleography data could be a better tool for evaluation of genetic diversity among the grapevine genotypes.
Background/Objectives This study aims to elucidate the genetic causes of congenital hypogonadotropic hypogonadism (CHH), a rare genetic disorder resulting in GnRH deficiency, in six families from Pakistan. Methods Eighteen DNA samples from six families underwent genome sequencing followed by standard evaluation for pathogenic single nucleotide variants (SNVs) and small indels. All families were subsequently analyzed for pathogenic copy number variants (CNVs) using CoverageMaster. Results Novel pathogenic homozygous SNVs in known CHH genes were identified in four families: two families with variants in GNRHR, and two others harboring KISS1R variants. Subsequent investigation of CNVs in the remaining two families identified novel unique large deletions in ANOS1. Conclusion A combined, systematic analysis of single nucleotide and CNVs helps to improve the diagnostic yield for variants in patients with CHH.
Multiple abiotic stresses like extreme temperatures, water shortage, flooding, salinity, and exposure to heavy metals are confronted by crop plants with changing climatic patterns. Prolonged exposure to these adverse environmental conditions leads to stunted plant growth and development with significant yield loss in crops. CRISPR-Cas9 genome editing tool is being frequently employed to understand abiotic stress-responsive genes. Noteworthy improvements in CRISPR-Cas technology have been made over the years, including upgradation of Cas proteins fidelity and efficiency, optimization of transformation protocols for different crop species, base and prime editing, multiplex gene-targeting, transgene-free editing, and graft-based heritable CRISPR-Cas9 approaches. These developments helped to improve the knowledge of abiotic stress tolerance in crops that could potentially be utilized to develop knock-out varieties and over-expressed lines to tackle the adverse effects of altered climatic patterns. This review summarizes the mechanistic understanding of heat, drought, salinity, and metal stress-responsive genes characterized so far using CRISPR-Cas9 and provides data on potential candidate genes that can be exploited by modern-day biotechnological tools to develop transgene-free genome-edited crops with better climate adaptability. Furthermore, the importance of early-maturing crop varieties to withstand abiotic stresses is also discussed in this review.
Cystic Fibrosis Transmembrane Regulator (CFTR) is a significant protein that is responsible for the movement of ions across cell membranes. The cystic fibrosis (CF) occur due to the mutations in the CFTR gene as it produces the dysfunctional CFTR protein. The sequence of CFTR protein as a target structure was retrieved from UniProt and PDB database. The ligands selection was performed through virtual screening and top 3 ligands choose out of 65 ligands silibinins, curcumin, demethoxycurcumin were selected with a reference drug Trikafta (R*). According to docking, ADMET analyses, the natural ligands (Silibinins and Curcumin) displayed best binding energy, pharmacokinetic and free toxicity than other natural compounds and reference drug (R*). An MD simulation for 200 ns was also established to ensure that natural ligands (Silibinins and Curcumin) attached to the target protein favorably and dynamically, and that protein–ligand complex stability was maintained. It is concluded that silibinins and curcumins have a better capacity to decrease the effect of mutant CFTR protein through improved trafficking and the restoration of original function. In conclusion, in silico studies demonstrate the potential of silibinins and curcumin as therapeutic agents for cystic fibrosis, particularly for the D614G mutated protein. Their ability to increase CFTR function while reducing cellular stress and inflammation, together with their favorable safety profile and accessibility could make them valuable additions to cystic fibrosis treatment options. Further experimental and clinical validation will be required to fully realize their potential and include them into effective therapy regimens.
The potato tuber moth, Phthorimaea operculella (Zeller), is a notorious insect pest of potato incurring substantial yield losses in the field as well as in storage. Chemical control is difficult to exercise due to the latent feeding of the caterpillars and their ability to develop resistance against insecticides. One of the essential components of efficient insect-resistant management is using two or more different insecticidal genes in transgenic crops to effectively avoid and delay the resistance development in insect pests. Two constructs, namely DS-1 (cry3A + SN-19 genes) and DS-2 (OCII + SN-19 genes) in pCAMBIA1301 binary vector, were developed and were transformed in potato cultivars (Agria and Lady Olympia) via Agrobacterium-mediated transformation. The molecular analysis confirmed gene integration and expression of the introduced genes in transgenic plants. The insecticidal effects of incorporated genes in transgenic plants were assessed against 1st, 2nd, 3rd, and 4th instar potato tuber moth (PTM) larvae. The transgenic plants endured significantly high mortalities (100%) of larval stages of PTM within 72 h. Our results show that these transgenic potato plants have the potential to control populations of PTM and are also useful tools in managing PTM that would ultimately reduce the dependency on conventional chemical pesticides with potentially less or minimal hazards. These lines can also serve as an excellent source of germplasm for potato breeding program.
Microtubule associated proteins (MAPs) are widely expressed in the central nervous system, and have established roles in cell proliferation, myelination, neurite formation, axon specification, outgrowth, dendrite, and synapse formation. We report eleven individuals from seven families harboring predicted pathogenic biallelic, de novo, and heterozygous variants in the NAV3 gene, which encodes the microtubule positive tip protein neuron navigator 3 (NAV3). All affected individuals have intellectual disability (ID), microcephaly, skeletal deformities, ocular anomalies, and behavioral issues. In mouse brain, Nav3 is expressed throughout the nervous system, with more prominent signatures in postmitotic, excitatory, inhibiting, and sensory neurons. When overexpressed in HEK293T and COS7 cells, pathogenic variants impaired NAV3 ability to stabilize microtubules. Further, knocking-down nav3 in zebrafish led to severe morphological defects, microcephaly, impaired neuronal growth, and behavioral impairment, which were rescued with co-injection of WT NAV3 mRNA and not by transcripts encoding the pathogenic variants. Our findings establish the role of NAV3 in neurodevelopmental disorders, and reveal its involvement in neuronal morphogenesis, and neuromuscular responses.
Epigenetic machinery is a cornerstone in normal cell development, orchestrating tissue-specific gene expression in mammalian cells. Aberrations in this intricate landscape drive substantial changes in gene function, emerging as a linchpin in cancer etiology and progression. While cancer was conventionally perceived as solely a genetic disorder, its contemporary definition encompasses genetic alterations intertwined with disruptive epigenetic abnormalities. This review explores the profound impact of DNA methylation, histone modifications, and noncoding RNAs on fundamental cellular processes. When these pivotal epigenetic mechanisms undergo disruption, they intricately guide the acquisition of the 6 hallmark characteristics of cancer within seemingly normal cells. Leveraging the latest advancements in decoding these epigenetic intricacies holds immense promise, heralding a new era in developing targeted and more efficacious treatment modalities against cancers driven by aberrant epigenetic modifications.
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107 members
Aneela Yasmeen
  • Plant Biotechnology
Ayesha Latif
  • PLANT BIOTECHNOLOGY
Muhammad Islamkhan
  • BIOPHARMACUETICAL
Ayesha Malik
  • Molecular Biology
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Lahore, Pakistan