2020

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Anti-cancer potential of R. STRICTA and its effect on DE NOVO lipid biosynthesis pathway in MCF-7 breast cancer cell line
(UMT Lahore, 2020) MUQADAS
Breast cancer has become the major issue and the most common type of invasive cancer worldwide among the women. Plant based treatment of breast cancer is considered safe because plants do not affect surrounding part of body. Rhazya stricta is a plant that is seem to have many alkaloids, flavonoids and many other phytochemical contributing for its potential to cure many diseases. Aim of this study is to evaluate the anticancer potential of R.stricta and its effect on de novo lipid biosynthesis pathway in MCF-7 breast cancer cell line. In current study, methanolic extract of R.stricta was used. A range of plant extract dilutions (0µg/ml-300µg/ml) applied on MCF-7 cells. MTT assay performed to check the cell viability and to determine IC50 value of extract. Then MCF-7 was treated with IC50 value of dose i.e. 25µg/ml for 72 hours’ incubation. Furthermore, expression analysis of FASN and ELOVL6 genes evaluated in the treated cells that were seem to down-regulate after treatment with methanolic extract of plant.
Children hospital, Lahore
(UMT Lahore, 2020) AQSA AZIZ QURESHI
City pharmaceuticals PVT, Limited
(UMT Lahore, 2019-10-15) SAQIB ABBAS
City pharmaceuticals PVT, Limited
(UMT Lahore, 2020) SAQIB ABBAS
Cloning of fused gene of Thymosin alpha-1-p28 domain of Azurin in E. Coli
(UMT Lahore, 2020) MAIRA NAEEM
Thymosin alpha 1 is a hormone consists of 28 amino acid and play role in treatment of diseases because of its immune stimulation, anti-viral and anti-cancer properties. p28 is a peptide derived from bacterial azurin protein and have anti-tumor activity. T-1 and p28 are both fused with the help of GGGGS linker. The objectives are to clone the fusion protein in E. coli DH5 cells. TA cloning is specifically used for the process cloning and other steps of cloning have been followed by using kit method and plasmid pTZ57R/T was used. The fusion protein expects to be a strong candidate for cancer therapy because it is not only beneficial in stimulating immune response but it also targets tumor cells and block its way to escape from immune surveillance.
Comparison of different risk factors associated with polycystic ovarian syndrome in healthy and diseased women
(UMT Lahore, 2020) Abdul Ahad
Polycystic Ovarian Syndrome is one of the most frequent disorder and one of the main leading cause of infertility among women of reproductive period. There are three main symptoms linked with PCOS as stated by Rotterdam Criteria, such as Irregular menstruations, hyperandrogenism and polycystic ovaries. There are other factors as well that are linked with PCOS such as infertility, insulin resistance, obesity, infertility, diabetes mellitus type 2, endometrial cancer, cardiovascular diseases, dyslipidemia, hirsutism and acne. The purpose of this research was to analyze and study the different factors associate with PCOS by comparing the data of healthy women and women with PCOS. By analyzing and comparing different factors, our study concludes that there are factors that are very different between women with PCOS and healthy women, such as hirsutism, acne, irregular menstruations and obesity. In our study, one of the factors that we did not found very significant was the prevalence of Diabetes mellitus type 2 (insulin resistance). Moreover, DNA extraction was done from collected blood samples of healthy and diseased women and our further aim was to study the variance elements between healthy and diseased women more deeply on a DNA sequencing level.
Comparison of three different DNA extraction methods for betel quid spits and gutka spits collected from ten different surfaces
(UMT Lahore, 2020) Ameer Hamza
Betel quid and Gutka chewing is habit of a mass population mainly consist of Asian countries and is also spread to other world through immigrants. Chewing of Betel quid and Gutka induce some chemical changes in the saliva of consumers due to the presence of a number of carcinogenic and other substances present in them which cause inhibition in the isolation of DNA from their chewer’s saliva. Saliva is an important examined biological fluid in forensic analysis. It is a significant evidence that can help to investigate a rape case, assault and homicide cases. This study was designed to make an evaluation and compare three manual methods consisting of Phenol Chloroform isoamyl alcohol (PCI) test, Trehalose test and Alkaline lysis test for DNA isolation from Betel quid and Gutka samples collected from ten different surfaces. The aim of this study was to find a most suitable method for DNA isolation to face these kind of challenging samples in the field of Forensic science. The surfaces which were supposed in this study were more susceptible as a part of crime scene where evidential saliva might be found. soil, marble, glass, cloth, wood, plastic, cardboard, paper, metal and plant material were the surfaces selected in this study. The isolated DNA was checked for concentration and purity through spectrophotometer method in order to evaluate the most favorable method for DNA isolation. In this study Phenol Chloroform Isoamyl Alcohol was found to be the most favorable and efficient method from two other methods for both Betel quid and Gutka spit samples. However, it was observed in this study that it is difficult to recover DNA from these kind of Forensic samples.
Computational sequence analysis of DNA-A&B segments of Begomoviruses infecting vegetables.
(UMT Lahore, 2020) Khizar Hayat
Begomoviruses are the members of Geminiviridae, which mostly infects dicots. Vector which is the cause transmission of this virus are whiteflies Bemisiatabaci. They have been characterized into monopartite and bipartite genome associated with DNA A&B. including some devastating strains which are linked with monopartite genome. They infect a broad range of horticulture and vegetable crops resulting into a huge economic loss every year. It has been a nutshell for scientists since the twentieth century and till now there are regular updates in its strains. Total number of DNA-A sequences infecting in vegetables were 151. 30 Species including the extract from phylogenetic tree of maximum likelihood. Species from tree were 15 (Based on 91% identities Brown et al. 2015) Pair wise sequence comparison revealed 86% cutoff value and beyond this point new Species are predicted DNA -B consisted on total number of 17 sequences from which 13 Species were included ( Based on 69% sequence identities) Pair wise sequence Comparison for DNA-B segment depicted 69% Computational sequence analysis of DNA-A&B segments of Begomoviruses infecting vegetables.
Effect of water and detergent washings on detection of saliva stains from commonly used natural and synthetic clothes
(UMT Lahore, 2020) FATIMA ARSHAD
Forensic science plays main role in criminal justice system. Mainly, homicide, sexual assault and burglary, permit the assembly of biological evidence. Blood, semen and saliva commonly obtain from the crime scene. Culprit often tries to remove these biological stains through washing out the crime scene or the items but previously it has demonstrated that DNA can still recover from laundered clothes. The purpose of this study was to observe the effect of water and detergent wash on clothes and to extract, detect and quantify the amount DNA from these clothes. In this study, natural and synthetic clothes gave positive results under various presumptive tests. Both types of clothes gave different ranges of mean diameter after water wash till three washings. Saliva was found retain from laundered natural clothes till ten washings after processing with detergent while the laundered synthetic clothes could not efficiently retain saliva after treatment with detergent. DNA extracted from clothes analyzed under UV spectrophotometer demonstrated that both natural and synthetic clothes could give the good amount of DNA that was enough to generate the DNA profile. Overall terry, cotton, linen, khaddar, denim and spandex clothes showed the efficient results for saliva detection while wool, polyester and nylon exhibited the poor results.
Immunoinformatic-based screening of immunogenic fragments from the pathogenic proteins of NAEGLERIA FOWLERI against the HLA-alleles of majorly affected population
(UMT Lahore, 2020) Mehwish Andleeb
Naegleria fowleri is a unicellular pathogenic amoeboid flagellate. The trophozoite phase is responsible for causing primary amoebic meningoencephalitis, with the 98% fatality rate. Considering the complex pathogenicity, as well as the resistance towards most of the antifungal drugs, present study aimed to propose a multi-epitope ensemble against N. fowleri restricted to targeted populations. The significant B-cell and T-cell epitopes from all five immunogenic surface proteins, such as Mp2CL5 (181), HSP70 (659), Naegleria Pore A (307), Naegleria Pore B (484), and NF23 (229), were selected. The immunogenicity and allergenicity of selected epitopes were analyzed using VaxiJen and AllerTop v. 2.0. The binding affinities of selected T-cell epitopes with the South Asian, USA, and Australian specific HLA alleles, retrieved through AFND, were evaluated through the IEDB server. The antigenic fragments with the highest binding affinity towards these alleles were analyzed. The nominated epitopes were covering 94.53% for the USA, 84.27% for Australia, and 73.77% for the South Asian region. The structural modeling through I-TASSER and validation via SAVES server v.5.0 suggested all these proteins were stable. The common T-cell epitopes from all these populations were labeled using the PyMol graphic tool. The immunogenicity, non-allergenicity, and maximum population coverage of short-listed epitopes marked them as efficient candidates for vaccine designing.
In silico analysis of functional nonsynonymous SNP in human INSR gene associated with polycystic ovarian syndrome
(UMT Lahore, 2020) KHIZRA BANO
Polycystic ovarian syndrome (PCOS) is a very complicated condition having many risk factors behind it, which in turn lead to other diseases. According to Rotterdam criteria, three factors are necessary for its diagnosis such as hyperandrogenism, irregular menstruation and polycystic ovaries on ultrasound. It also results in increased chances of obesity, type two diabetes and cardiovascular diseases. We compared various factors between PCOS women and healthy women after signing written consent by them, which revealed that PCOS women were more prone to Obesity, diabetes and irregular menstruation as compared to healthy women. In order to study genetic factors behind PCOS, we analyzed role of INSR gene polymorphism with the help of various bioinformatics tools showing role of non-synonymous SNP in causing damage to protein function. Structural comparison between wild type and mutant strain of protein showed no interaction between protein and ligand but having impact on protein stability predicted by I-mutant server showed that SNP at position 1161 where alanine changes to threonine resulting in decreased protein stability. The protein 3D structure was further validate by Ramachandran plot.
In silico analysis of LR34 gene resistance to leaf rust in wheat TRITICUM AESTIVUM L
(UMT Lahore, 2020) Muhammad Usama Yaqoob
Lr34 is a leaf rust resistance gene of hexaploid wheat Triticum aestivum L. This wheat is also known as the bread wheat as it is the source of food for humans and also feeding stuff for the animals. 95% of the world wheat cultivated constitutes hexaploid wheat which is Triticum aestivum L. It is the main host of leaf rust pathogen called Puccinia triticina. Lr34 gene provides resistance against the urediniospore's life stage of Pt. Some studies show that the locus of LR34 is on 7D chromosomes while other homologs are present on other chromosomes. This study involves various in silico techniques to carry out the analysis, characterization, structural and functional predictions, and the phylogenetic and evolutionary relationship of the LR34 protein encoded by the Lr34 gene. Various computational tools were used for the predictions and verification of the results. NCBI was used for the data and the sequence retrieval which showed that the protein contains 1402 amino acids and has an accession ID ADK62371. Other tools that were used in this study include Scanprosite, InterPro, NCBI CDD, SOPMA, DNAssist, I-TASSER, SWISS MODEL, SAVES 5.0, and MEGAX. PyMOL and Chimera were used for visualization. From different domain search tools, it was found that LR34 contains the domains of the ABC transporter family. Out of three NCBI, CDD predicted the conserved domain region from amino acod#43 onward. While the sequence homology and the phylogenetic analysis confirmed hypothetical protein CFC21_054641 [Triticum aestivum] as the nearest neighbor and homolog of LR34 in the evolutionary history. I- TASSER predicted the model which had only 6% residues in the disallowed region in the Ramachandran plot while the rest were in the most favored, additional allowed, and generously allowed regions. To make the model more favorable the model was further energy minimized. It was found that the protein is involved in oxidoreductase reaction more specifically in fatty acid catalysis and is an obsolete intracellular part.
In silico analysis of NF1 network in cutaneous melanoma
(UMT Lahore, 2020) HAFSA AHMAD
Cutaneous melanoma is a type of cancer formed by transformation of melanocytes in malignant manner. This skin cancer varies among same age of people with same stage. This disease is increasing day by day in all over the world. Mutations in NF1 causes major dysregulation of RAS/RAF/MAPK pathways which inhibits apoptosis and proliferation of melanocytes. For understanding major onco-proteins role in cancer, it is possible by network biology approach. NF1 protein-protein interaction network was extracted and its cluster was formed on STRING database version 11.0. Enrichment analysis were done on ShinyGo version 0.61. Hub proteins were identified on CytoHubba of Cytoscape plugin version 3.4. Mutations of hub proteins were analyzed on cbioportal. Thirty hub proteins showed novel mutations in 181 samples of cutaneous melanoma. Gene alteration frequency was 50 %. These hub proteins showed their role in cutaneous melanoma.
In silico analysis of uncharacterized gene of cotton cotad_59296 putatively involved in resistance to cotton leaf curl virus disease
(UMT Lahore, 2020) TAYYABA ALI
Gossypium is a major non vegetative crop that is cultivated across the globe to get fiber. Beside textile industry it has immense uses in different pharmaceutical and chemical products, paper and pulp industry, food industry and cotton-seed oil industries. Pakistan is 3rd position as exporter, 4th rank as yarn producer and 7th as in cloth production. Cotton adds about 11.5 % value in agriculture sector and contributes to about 2.7% GDP and about 60% in earning foreign currency. But the lamenting fact is that our cotton is decreasing day by day due to various biotic and abiotic factor. One such biotic factor is Cotton Leave Curl Disease caused by a Begomovirus. To combat against biotic factor Effector-triggered immune (ETI) of plants is activated via intracellular receptors. These receptors activations depend directly or indirectly on resistance (R) genes. The resistance genes can be broadly classified into eight domains. Among these R gene one is COTAD_59296 belong to XII group. The protein putatively involved in Cotton Leaf Curl Disease. The protein have maximum homology with cysteine rich repeat secondary protein of G.hirsutum having accession no XP_0166034 and it is related to same protein in evolutionary relation also. The motif and domain analysis of protein are involved in stress response, antiviral and antifungal activity. The protein resembles the most with CotAd_59296 in PDB database is 6GRF which is also a stress response anti-fungal protein and it is responsible for symbiotic process, viral process and in interaction between organisms. In HoPItor: Host-pathogen protein-protein interaction prediction server the highest value was obtained was 0.926029 with AV2. AV2 is involved in the movement of bipartite Gemini viruses. Thus CotAD_59296 is stable and interacting protein with AV2 gene of Begomovirus.
In silico characterization of hypothetical proteins of vibrio parahaemolyticus
(UMT Lahore, 2020) Rabia Akhtar
Vibrio parahaemolyticus is a gram-negative, rod-shaped, halophilic bacterium that lives in warm aquatic environments and is involved in causing acute gastrointestinal infections among humans, due to the consumption of contaminated, raw, or uncooked seafood. It was first reported in 1950, in Japan, and in the early 1996 it disseminated throughout the world and was declared as a pandemic. This seafood-borne pathogen contains several virulence factors, including adhesion molecules, hemolysins, and secretion systems, however, many of its proteins are still uncharacterized. The pandemic of this pathogen demands the characterization of all of its proteins to trace the virulent factors and their effect on humans. In this study, 23 hypothetical proteins of V. parahaemolyticus have been targeted for functional and structural annotation. The selected proteins were first subjected to homology screening to check the templates accessibility. After filtering, the 11 hypothetical proteins were evaluated further for the conserved domain analysis via CDD, Pfam, Interproscan, and scanprosite, physicochemical characterization via ExPASy ProtParam, subcellular localization via CELLO v2.5 and PSORTb v3.0, and structural modeling via I-TASSER and SAVES server (v.5.0). The pathogenicity assessment, using VirulentPred and functional annotation through CATH v4.2 and Argot v2.5 revealed that 7 out of 11 hypothetical proteins, could act as the potential virulent factors. The finding of this study could assist in confirming the functions and pathogenic involvement of these proteins and may, therefore, help in target identification for drug discovery and vaccine designing for V. parahaemolyticus.
In silico designing of a multi-epitope vaccine candidate against denguevirus specific to HLA alleles of the USA populations
(UMT Lahore, 2020) Anila Ilyas
Dengue is a single-stranded RNA virus, belonging to the genus Flavivirus, and causes systematic viral infection among humans. There are four different serotypes of this virus reported in the literature, including DENV1, DENV2, DENV3, and DENV4. Its outbreak started in Africa, Asia, and America then spread to the whole world. Currently, there is no authorized vaccine against dengue, and only one vaccine named dengvaxia was approved, in 2015, which was also proved to be less effective for the USA populations. Therefore, it is the need of the time to develop a tetravalent vaccine, which would be effective for all the serotypes of the dengue virus in this population. The present study involves the screening of highly immunogenic and non-allergenic B and T-cell epitopes, from the three proteins of the dengue virus, NS1, NS3, and E, through IEDB. The binding affinities of all selected T-cell epitopes with the USA-specific HLA alleles, retrieved via AFND, were further checked using IEDB binding affinity tools. Three epitopes, having the strongest binding affinities with the HLA alleles, immunogenic, and non-allergenic natures, were considered efficient for designing a multi-epitope construct, with the GSGSG linker among the adjacent epitopes. The structural modeling and validation of all possible multi-epitope constructs revealed that variant 1 was the most stable among all with the proper folding and structural integrity. Moreover, the population coverage of this construct was 67.67% for the USA populations, suggesting this construct as the favorable candidate for designing a tetravalent vaccine, against the four serotypes of dengue virus, against these populations. Still, additional experimental studies are required to confirm its soluble expression, non-allergenic nature, and immunogenic effects on diseased subjects.
In silico designing of a multi-epitope vaccine candidate against mycobacterium tuberculosis specific to HLA alleles of USA
(UMT Lahore, 2020) Amara Saif
Mycobacterium tuberculosis is a remarkable human pathogen that has infected about one-third of the world’s population, along with the increment of more than 10 million new cases and 1.5 billion deaths every year. Altogether, its complex pathogenicity, dormancy, antibiotic resistance has driven the research towards the designing of a viable vaccine against this deadliest infectious disease. Considering this fact, the current research was aimed to design a population-specific, multi-epitope ensemble vaccine against MTB by employing various computational approaches. The substantial B and T-cell epitopes were screened from the three immunogenic surface proteins of MTB, including PEPGRS33 (501 aa), Mpt 83 (239 aa), and LipC, (403 aa). The epitope prediction was followed by antigenic and allergenic assessment via VexiJen and AllerTop v.2.0, respectively. Finally, the binding affinities of selected T-cell epitopes with the USA specific HLA alleles, retrieved through AFND, was evaluated using IEDB, and the antigenic fragments, with the maximum binding affinity towards these alleles, were screened. The selected epitopes were covering 83.1% of the USA populations and therefore, were considered efficient candidates for the designing of multi-epitope constructs with the GPGPG linker region between the adjacent epitopes. The structural modeling and validation through I-TASSER and SAVES server 5.0, suggested variant 4 as the most stable construct with the proper folding and structural integrity. Moreover, this variant is likely to give a soluble expression based on the Protein-Sol score of 0.65. Thus, the designed chimera can act as a potential vaccine candidate against TB. Still, a detailed experimental analysis is required to develop an effective vaccine against tuberculosis.
In silico screening of the putative epitopes from the surface proteins of streptococcus pneumoniae against south asians specific human leukocyte antigens
(UMT Lahore, 2020) Anosha Liaqat
Streptococcus pneumoniae is a causative agent of sinusitis, bacteremia, pneumococcal pneumonia, and other pneumococcal infections, most commonly in children under five years. Two serotype-based vaccines, polysaccharide vaccines (PPV) and protein conjugated polysaccharide vaccine (PCV), have provided some protection against this organism, but serotype specificity, and low immunogenicity of these conventional vaccines, have driven the research toward the designing of more efficient treatment strategy against this organism. This study attempted to design the population-specific and highly immunogenic vaccine candidates that can evoke both cell-mediated and humoral immune responses against S. pneumoniae. Consequently, both B and T-cell epitopes from the four pathogenic surface proteins of S. pneumoniae, including PspC, PvaA, PhtD, and Pneumolysin, were identified. Following the epitope prediction, the antigenicity and allergenicity assessments of these epitopes were done by using VaxiJen and AllerTOP. Binding restrictions of selected T-cell epitopes with South Asian specific alleles, retrieved through AFND, were determined using IEDB MHC-binding prediction tools, with the selection of best binder epitopes based on IC50 values of less than 50nM. Population coverage analysis revealed that these epitopes were covering 83.75% of the South Asian population. Finally, after the structural modeling and validations through I-TASSER and SAVES v5.0, the identified epitopes were labeled on their respective protein models using PyMOL. The identified epitopes were not only immunogenic and non-allergenic but also show restriction to the majority of the HLA alleles of the South Asian populations, the identified epitopes might therefore be the probable vaccine candidates for these populations.
In silico structural and functionalcharacterization of hypothetical proteinsof candida auris
(UMT Lahore, 2020) Qamar Abbas
Candida auris is a fungal pathogen, which provokes a wide array of infections in humans including, bloodstream, wound, and ear infection. Due to its resistivity to all types of antifungal drugs, the treatment of infections caused by this pathogen has become very challenging. Previous studies revealed that some of its proteins are involved in causing the pathogenicity in the host but these proteins are not structurally and functionally annotated, and, therefore, are hypothetical. So, the present study is designed to use multiple in silico strategies to characterize the hypothetical proteins of C.auris to get a structural and functional insight about this deadly microorganism. The study started initially with the 50 proteins, selected randomly from NCBI. The homolog screening via BLAST based on percentage identity and E-value reduced the number of proteins from 50 to 12. All these 12 hypothetical proteins were then characterized using multiple computational tools, including physicochemical characterization via ExPASY ProtParam, conserved domain search through CDD, Pfam and, InterProScan. The analysis of the subcellular localization of these proteins via CELLO and SecretomeP v 2.0 marked all these proteins to be present in the cytoplasm. The structural modeling of these hypothetical proteins was performed using I-TASSER, followed by the model validations via SAVES V.5.0. Finally, the functional annotations revealed that these proteins are likely to have diverse functions, including, transposition, transmembrane transporter activity, DNA directed DNA polymerase activity, and hydrolase and peptidase activity. The results of this study may help in understanding the function of uncharacterized proteins from C.auris. After the screening of virulent factors from this pathogen, it would be easy to identify ligand-binding sites and drug targets through docking analysis.
Isolaion and optimization of efficient bacteria during thermophillic phase of municipal solid waste composting
(UMT Lahore, 2020) KHADIJA TARIQ
Composting is biological decomposition of organic waste into more stable humus like compound carried by microbial population. The complex organic matter into simpler form by the help of microbial activity of bacteria and fungi. Different physical and chemical parameter like pH, moisture content of substrate, oxygen concentration, carbon-nitrogen ratio, particle size play important factors in supporting stability of the final product. Thermophilic stage is the most active stage of composting starts by replacing microphillic flora and continues until 62℃ is reached. The optimum temperature for thermophilic stage is 35 to 55 °C. Thermophiles plays an important role and have a huge diversity, thermophilic phase was found to be dominant by Bacillus. On the other hand degree of humification and maturation process in composting is improved by microbial inoculants. This study is designed to isolate and identify most efficient degrading bacteria at thermophilic stage of composting. Growth of strain ‘X’ on different carbohydrates media and blood agar media was observed and evaluated. Optimization of Bacterial ‘X’ strain at different temperature and pH was also evaluated by spectrophotometry and electrical impedance. The most suitable temperature for better growth of bacteria ‘X’ was 450C and 5.5 pH.

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