Current Issue

Advancements in Life Sciences, volume 8, issue 3Cover; Volume 8, Issue 3
Published: 25 July 2021
ISSN 2310-5380 

Important Note: This issue was due to be published in May 2021 but appeared online in July 2021. We were adapting to changing working dynamics due to COVID-19 pandemic. It took us some time to complete the due processing. We are sorry for this delay and appreciate the understanding of our readers and esteemed authors.


IN THIS ISSUE

 

Review Articles


Contribution of GJB2 gene mutations to hearing loss in Pakistani population – A Narrative Review
Ejaz Ali, Nageen Hussain, pages 217-220
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 Pakistan has a unique population for the study of recessive genetic diseases due to a higher consanguinity rate. Hearing impairment is the loss of hearing normal sounds, and it is a common sensory disorder that affects more than 466 million people worldwide. Immuno-genetic and other environmental factors like loud noises, drug usage, and viral infections are the causes of hearing loss. Hearing loss is categorized into a syndromic hearing loss (70%) and non-syndromic hearing loss (30%). GJB2 mutations are one of the main causes of hearing loss in different populations, including Pakistan. The GJB2 gene encodes a gap junction protein involved in the homeostasis of the inner ear through the recycling of potassium ions. The prevalence of GJB2 mutation in the Pakistani population varies from 6.1 to 9.2%. The most common mutations found in the Pakistani population are 71G>A (p.(Trp24*), 231G > A (p. Trp77*), c.35delG (p. Gly11Leufs24*),c.355G>T (p. Glu119*) 457G > A (p.Val153Ile), 598G > A (p.Gly200Arg), 439G > A (p.Glu147Lys), c.377_378insATGCGGA (p.Arg127Cysfs*85). c.1055C>T (p. Pro352Leu), c.6202A>C p.(Thr2068Pro), c.2496_2496delC p.(Tyr832*) and c.355G>T p.(Glu119*).  

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Clinical Guidelines for Management of Acute Painful Episodes of Vaso-Occlusive Crisis in Children with Sickle Cell Disease
Nawaf Alanazi, pages 221-227
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 Sickle cell disease (SCD) is an inherited disease resulted due to a single mutation in beta globin gene. It changes normal red blood cells into sickle-shaped cells, just rendering their ability to carry oxygen to tissues and cells. SCD involves many fatal clinical complications including very painful vaso-occlusive crisis (VOC), the later caused by sickled erythrocytes-mediated ischemic tissue injury and obstruction of blood flow that leads. Pain in VOC is caused by hypoxia, acidosis, fever, infections, dehydration, obstructive sleep apnea, abrupt changes in weather (hot / cold), menstruation and pregnancy etc. Pain in VOC is very severe and is associated with many other life-threatening clinical complications. Therefore, it is needed be managed as early as possible, otherwise persistent pain can weaken patients physically and psychologically. There are no specific laboratory indicators for VOC and diagnosis is made on the basis of history and physical examination. We formulated clinical guidelines for the management of acute painful crisis (VOC) in sickle cell patients based upon updated published medical literature, in addition to our experience of treating sickle cell patients King Abdulaziz National Guards Hospital, Al-Ahsa, Saudi Arabia, which is its application to an Asian publication. This report describes details of these clinical guidelines for management of acute painful vaso-occlusive crisis in sickle cell patients.  

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A Review on the impacts of Azadirachta indica on Multi-drug Resistant Extended Spectrum Beta Lactamase-positive of Escherichia coli and Klebsiella pneumonia
Faisal MB. Al-Sarraj, pages 228-232
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 Neem trees have long been considered the holy grail of holistic and nature-based treatments. The medicinal properties that constituents of the trees possess to range from traits that are immunomodulatory to traits combat different disease and infections such as anti-inflammatory and antibacterial effects, to insulating properties that include cardioprotective and hepatoprotective effects. The role of Extended Spectrum Beta-Lactamase positive (ESBL+) bacteria in the occurrence and recurrence of Urinary Tract Infection (UTI) infections, particularly in the Gulf region has been studied extensively. However, suggested treatment methods have had little success due to a variety of factors which include drug resistance, the inability of doctors to calculate the optimal duration for treatment, which has resulted in wrong antibiotic prescriptions, as well as lack of understanding of how UTIs work and thrive in different demographics/ populations which again, results in inappropriate antibiotic treatment of the disease. These discoveries raised the issue of professionals needing better training and education in issues to do with UTIs amongst different demographics of people. In this investigation, the medicinal and pharmacological properties of A. indica from neem leaves were assessed by studying how they affected the activity of ESBL+ bacteria, based on literature from similar studies. The goal and objective of this study were to see if ESBL+ bacteria persisted in the presence of A. Indica from leaf extract, as well as gaining an understanding of the factors that affected the persistence and subsequent treatment of the bacteria using A. indica.  

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Full Length Research Articles


Efficiency of Virgin's Mantle (Fagonia cretica L.) as an Antibacterial and Antifungal Agent
Khushnood ur Rehman, Muhammad Hamayun, Shahab Saeed Khan, Nadeem Ahmad, Sher Wali, pages 233-237
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 Background: For medicinal purposes, biological activities are carried out on plant secondary metabolites in which common but very significant antimicrobial activities are focused. To evaluate the antimicrobial potential of Fagonia cretica L., different pathogenic microbial strains were obtained from KP, hospitals (already identified) to resolve the objectives of the current study.

Methods: In the agar, well diffusion method, a total of eight strains (4 bacterial 4 fungal) Streptococcus mutans, MRSA (Methicillin resistance staphylococcus aureus), Serratia marcescens, and Staphylococcus aureus are the bacterial strains while the fungal strains are Alternaria alternate Aspergillus flavus, Fusarium oxysporum, and Polysphondylium pallidum pre-identified and isolated in hospitals, were used respectively to evaluate the potentiality of n-hexane, ethyl acetate, chloroform, aqueous, and crude methanolic fractions against these strains.

Results: The highest significant (46-57% & 39-60%) antibacterial and antifungal activities were shown by the methanolic fraction while the lowest (28-35% & 25-35%) antibacterial and antifungal was shown by aqueous fraction against the selected microbial strains. Other fractions were also exhibited reasonable antimicrobial activities.

Conclusion: The current study concluded that different fractions of F. cretica have significant antimicrobial potential and might be a source of antibiotics in future studies of that plant.  

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Prevalence of Colibacillosis in Young Broiler Chicks and Antibiogram of Escherichia coli in Different Areas of Hazara Region
Aysha Tahir, Muhammad Azam Khan, Kayanat Bibi, Sana Bibi, Fatima Rauf, Farwa Ayaz, pages 238-240

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 Background: Poultry sector is a vital zone of Pakistan economy and is a feasible source of animal derivative protein.  In term of mortality and morbidity the development of this segment is significantly affected by a lot of problems. Hazara region is well known for poultry in Pakistan. Colibacillosis, caused by Escherichia coli is vital disease among poultry of all ages resulting in huge economic losses. The aim of this study was to evaluate the prevalence of colibacillosis in young broiler chickens and antibiogram of E. coli in different areas of Hazara region.

Methods: The current study was conducted to evaluate the prevalence of colibacillosis in young broiler chickens and antibiogram of E. coli. This study was done during February 2019 to June 2019; a total of 200 (n=200) liver samples of freshly dead young broiler chicks were randomly collected from the postmortem section of Veterinary research and disease investigation center Abbottabad (VRDICA). The identification of E. coli was confirmed by gram staining and biochemical tests. Susceptibility pattern to 13 antibiotics was also checked.

Results: Total of 200 (n=200) samples were brought to the VRDICA for the diagnosis of disease and antibiotic susceptibility testing. Out of these 130 were positive (Overall prevalence =65%).Area wise prevalence varied from area to area and highest prevalence was recorded in Abbottabad (81.11%) followed by Havelian (75%), Manghal (72.22%), Mansehra (60%), Qalandarabad (50%) and Haripur (28.57%). About age group, highest mortality was found in 11-15 days old chicks (93%) as compared to 6-10days (83.33%) and 1-5 days old chicks (21.42%). Antibiogram showed highest sensitivity to Gentamicin (100%) afterward Colistin Sulphate (92.30%). However, lincomycin (92%) and streptomycin (96%) display highest resistance.

Conclusion:  In this study, the highest prevalence of infection was observed in young chicks that play a crucial role in their mortality. E. coli had showed resistance to commonly recommended antibiotic so care should be taken while prescribing the drugs.  

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Characterization of Bacterial Strains from Rotten Fruits Treated with Harmful Preservatives
Roheen, Zakia Latif, pages 241-245

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 Background: Fruits are beneficial to maintain good health, but microbes can spoil them. To avoid spoilage of fruits different harmful preservatives are being used that pose danger to human health and environment. This study was designed to isolate bacterial strains from rotten fruits preserved by using different preservatives.

Methods: Different rotten fruit samples were collected from different shops of Moon Market and Neelam block, Allama Iqbal Town, Lahore and used to purify bacterial cultures by growing on simple N-agar medium. Biochemical characterization was performed by different tests including gram staining, catalase, mannitol salt agar, glucose and fructose fermentation and nitrate reduction. Bacterial strains were further subjected to additional tests like HCN, H2S production, metal resistance and antibiotic sensitivity.

Results: Nineteen bacterial strains were found positive for different tests and were characterized as Bacillus sp, Staphylococcus aureus, Micrococcus varians, Staphylococcus epidermidis and Staphylococcus saprophyticus. Many bacterial strains were resistant to antibiotics and high doses of most metals specially mercury. 

Conclusion: Due to the use of high doses of mercury for the storage of fruits, microorganisms have evolved resistance. It is an urgent need to take alternative measures for the storage of fruits for the safe lives.  

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Extraction and Amplification of mtDNA HVR1 from Old and Degraded Samples of Human Skeletal Collected from Khyber Pakhtunkhwa province of Pakistan
Nasir Ali, Muhammad Ilyas, Habib Ahmad, Shaker Khan, Wajiha Shafique, Ghani ur Rehma, Muhammad Jamil, Nazia Akbar, Shkair ul llah, Ikram Qayum, Gohar Rahman, pages 246-250

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 Background: The study of ancient DNA enables the examination of genetic associations between past and present individuals and populations. The recovery and analysis of human DNA from degraded samples precisely has become a central research tool in various scientific fields’ ranges from ancient DNA to forensics and medical sciences. 

Methods: In the current study, remains of human bones are analyzed that dates back to 1000-1200 BC from different burial sites of Khyber Pakhtunkhwa province of Pakistan. Hypervariable region 1 (HVR1) as a genetic marker was selected. Multiple extractions, qubit assays quantification and cloning of PCR products were performed to get the sequences from the hypervariable region 1 of mtDNA. In this study, we also evaluate the possibility of extracting DNA from degraded human tissues (bones and teeth) such as remains buried in archaeological sites and remains of dead bodies buried for many decades. 

Results: Mitochondrial DNA HVR1 of 2000 years old degraded bone specimens were successfully determined by analyzing haplotype, defining polymorphisms in the hypervariable region 1. The prominent haplogroup found was eastern European R  (63%) that was also found in present day population by previous studies.

Conclusion: The protocol developed in this study could be used for the extraction of DNA from old degraded and ancient bone samples.  

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Biofilm Forming Bacteria Isolated from Medical Implants
Aimen Fatima, Mahwish Batool Kazmi, Humaira Yasmeen, pages 251-256

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 Background: Biofilm formation in indwelling medical devices poses serious risk of infection and increases the likelihood of  recurrence of infections. The study was carried out to identify the microbes which form biofilms on medical implants and are thus involved in nosocomial infections, to assess the potential of biofilm producing ability of these isolated microbes and to determine antibiotic resistance towards ampicillin, vancomycin ceftazidime, streptomycin and tetracycline.

Methods: For this, 11 samples of 5 different implants were taken from Tertiary Care Hospital Multan, Pakistan. Bacteria were isolated and identified by culture plate method. Tryptone soy broth (TBS) media was used for biofilm development by microbes in plastic tubes. Developed biofilm in tubes was visualized with crystal violet staining method and then biofilm forming potential was estimated by measuring the optical density through spectrophotometer. Antibiotic susceptibility was done by Kirby Bauer disk diffusion method to determine the resistance and susceptibility pattern of biofilm producers.

Results: Out of 11 different samples of indwelling medical devices, a total of 131 bacterial strains were isolated. The percentage of bacterial isolates which produced biofilms were Staphylococcus spp. (41%) followed by Escherichia coli (18%), Pseudomonas spp. (4%), Proteus spp. (7.2%), Klebsiella spp. (8.6%), Bacillus spp. (8.6%), Fusobacterium spp. (1.4%) Clostridium spp.(1.4%), Enterococcus spp. (7.2%) and Neisseria spp. (1.4%). Sixty-nine isolates were considered positive for biofilm formation while 58 were considered negative. The resistance was maximum against ampicillin (42%) followed by ceftazidime (17.1%), tetracycline (34%) and streptomycin (30%) while against vancomycin no resistance was observed.

Conclusion: Biofilms produced on medical implants by different bacteria are considered to be the major cause of hospital acquired infections and are very difficult to eradicate. These biofilms associated implant infections are challenging to treat because of their resistance towards various antibiotic therapies. Therefore, some efficient methods of prevention of biofilm formation should be introduced. 

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Response of Starter Broiler Chickens to Feed Diets Treated with Organic Acids
Dozie Ndubisi Onunkwo, Abdul Jabbar, Muhammad Talha, Areej Rauf, Hamza Javaid, Muhammad Usama Munir, Nimra Irm, Muhammad Hassan Saleem, pages 257-261

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 Background: Organic acids contain one or more carboxylic acid groups which are linked with covalent bond and  have acidic properties that can enhance the reservation of protein and some other nutrients in birds. Antibiotic growth promoters have been banned due to their residues that remain in the meat and effects the human beings. Therefore, the organic acids are used as their alternatives. The present study is aimed to inspect the outcome of organic acids on uptake of feed, feed gain ratio, and live weight gain in broiler chickens.

Methods: A total of 150 unsexed broiler chickens were used for this experiment which were having five categories of treatment as T1, T2, T3, T4, and T5. Each category had thirty birds. Twas treated with a standard diet, T2was treated with acetic acid, T3 with butyric acid, T4 with citric acid and T5 with formic acid.  The duration of this experiment was 28 days. After the specified time of this experiment, the data of uptake of feed and body weight was gathered on weekly basis.  Comparison of all the five treatments was done by using the Duncan's multiple range test.

Results: Feed Conversion Ratio (FCR) was found lower in Tthan other treatments. Tgroup showed the highest average value of final body weight of broilers in contrast to the T3 group which showed the lowest final body weight.  Feed intake was found significantly different within the treatments. T3 showed significantly lower value as compared to other treatments. The lowest value of average regular uptake of grains was found in Ttreatment group. Broilers fed on formic acid have shown a better protein efficiency ratio than that of butyric acid and citric acid. Broilers treated with citric acid have a significant difference which indicates more water consumption as compared to other treatments.

Conclusion: Organic acids have a productive effect on the growth of animals and broiler chickens. Organic acids including butyric acid, acetic, citric, formic, fumaric, and propionic acid vary in their biochemical actions in the system of animals. Organic acid affects the final weight gain, average regular gain in weight, total uptake of feed, and feed to gain ratio, daily intake of protein, protein efficiency ratio, total water uptake, average water intake, and water feed ratio. Based on the present study, further analysis is required to look over the impacts of addition of organic acid on the growth accomplishment of broiler chicks. 

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Empirical Analysis of Farmers Preferences and Willingness towards Organic Farming in Gilgit-Baltistan, Pakistan
Adil Daniel, Zoia Arshad Awan, Asad Imran, Rida Munir Khan, pages 262-266

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 Background: Organic farming is an eco-friendly approach in the agricultural production system that ensures food safety, reduces health problems, promotes environmental conservation, and generates employment opportunities for local communities.

Methods: The survey was conducted using a well-structured questionnaire that focused on the scope and potential of organic farming in Gilgit-Baltistan (GB), Pakistan. The data were collected from randomly selected 150 respondents in nine districts of GB between May and June (2019). This survey analyzed the factors influencing the willingness of farmers to pay attention to organic farming and evaluated the possibilities of espousal organic farming as an effective approach.

Results: In general, mixed perceptions were revealed by the behavior and attitudes of the farmers, however, it is necessary to notice the intentions of the farmers for substitute methods to make organic farming feasible. Based on the survey, people were not reluctant to adopt organic farming to ensure better market value for their products. As a result, they will sustainably get a better livelihood.

Conclusion: The current study is concluded that the espousal of organic farming could have a positive and noteworthy outcome on a farmer’s life in terms of profitability, compatibility, productivity, and sustainability in GB. There is a dire need for organic certification to increase the market value both at local and international levels. 

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Probiotic potential of encapsulated Lactobacillus species in yogurt formation indigenously isolated from dairy source
Atia Iqbal, Saba Irshad, Saira Saeed, Aatikah Tareen, pages 267-274

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 Background: Exploration of beneficial bacteria as probiotics inputs are drawing interest in dairy industry but their long-term survival and viability is an important consideration.

Methods: The current work focused on the exploration of probiotic potential of indigenously isolated Lactobacillus strains from dairy products and their encapsulation and utilization in yogurt formation.

Results: These Lactobacillus strains were identified as Lactobacillus fermentum MGA23-1 and Lactobacillus fermentum LMEM19 and found resistant to inhibitory substances like phenol (0.2%), bile salts (0.3%), pancreatin (0.5%) and pepsin (0.3%). The highest antibacterial activity was observed by Lactobacillus fermentum MGA23-1 against Pseudomonas aeruginosa (13mm). Encapsulation experiment showed that the number of bacterial CFU/g increased significantly (p < 0.05) in beads during storage up to 7 days. Chemical characterization of microcapsules was assessed using FTIR and showed characteristics wavelength major at 1541 – 1716 cm-1 and 3336 cm-1. Yogurt was prepared using a single probiotic strain, in a consortium of Lactobacillus fermentum MGA23-1 and Lactobacillus fermentum LMEM19 and in the form of beads. Best results were observed in the case of microencapsulation.

Conclusion: It was concluded that both strains had the potential to be used as a probiotic in the dairy industry. 

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Cloning and expression of hepatitis B surface gene in E. coli
Krishma Gulzar, Maria Sharif, Arif M Khan, Muhammad Rizwan Javed, Imran Riaz Malik, pages 275-280

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 Background: Hepatitis B virus (HBV) is among the smallest DNA viruses resulting in ~800,000 deaths each year. Pakistan is considered a country affected by HBV. In Pakistan, the most dominant genotype is D. HBV is an enveloped virus of 3.2 kb. The study's goal was to express hepatitis B surface antigen in a bacterial host to produce a recombinant protein.

Method: Blood samples were collected in EDTA coated vacutainer from patients after their consent. DNA was extracted from serum through the phenol-chloroform method; Hepatitis B surface gene was cloned in TA cloning vector, subclone in pET 28a expression vector. An expression vector containing the Surface gene was then transformed into a competent bacterial host BL21 and inducted with IPTG at 0.1-0.2mM concentration for expression. The expressed proteins (soluble and pellet form) were analyzed on SDS PAGE.

Results: Hepatitis B Surface gene of 681bp after PCR were detected under UV light then successfully cloned and subcloned in pET 28 expression vector. The restricted fragment indicating the gene of interest was 681bp when analyzed on 1.2% Agarose gel under UV light. The required protein of 25kDa was obtained in soluble form when detected on 12% SDS PAGE after staining with Coomassie Blue dye.

Conclusion: Hepatitis B surface gene was successfully expressed in both insoluble and pellet forms using E.coli. The expression of surface protein needs to maximize through optimizing conditions to be used as potent candidate for vaccine production to prevent hepatitis B infection.  

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Knowledge, Attitude, Practices (KAP) and Psychological Impact of COVID-19 among Pakistani Population: A Quick Cross-Sectional Online Study
Saadia Ijaz, Masood Afzal, Tahir Naeem, Nayab Zaid, Syed Faraz Gillani, Aqib Saeed, Kashif Ur Rehman, Shafay Haseeb, Maria Najeeb, pages 287-292

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 Background: Coronavirus causes extremely contagious infection and currently is a major threat for the public health globally. The aim of this study was to evaluate the current level of awareness towards Corona Virus Disease 2019 (COVID-19) among Pakistani residents, their attitude, practices and psychological impact towards COVID-19 those contribute in an important way to control this pandemic.

Methods: A cross sectional online study was conducted. A self-developed online questionnaire was made by Google forms and sent to the author’s network with local residents in Pakistan. The questionnaire contained 35 questions among which 6 questions were related with the demographic features, 14 questions assess knowledge of participants, 4 questions evaluate the attitude, 7 questions assess the practices of the participants towards COVID-19 and 4 questions assess the psychological impact towards COVID-19.

Results: Among the participants (n=528) 66.7% were female and 33.3% were male. Among them 49.8% of the participants were from the age group of 18-24 and the percentage of students was higher i.e. 39.8%. The overall range for correct answers for the knowledge questionnaire was 97.5%-46.8%, for attitude 75.2%-91.9% and for practices 39%-95%. 79% of the participants were confident that Pakistan will overcome the disaster situation of COVID-19.

Conclusion: The findings of the current study indicated that Knowledge among the Pakistani residents about COVID-19 was found satisfactory. A significant number of the participants still lacking confidence when compared with the other countries. However, to effectively control the infection spread and improving COVID-19 knowledge the well-structured awareness programs must be launched by the government those could be helpful for Pakistani residents to hold optimistic attitudes and maintain appropriate practices. 

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DNA methylation profile of multiple genes involved in bladder cancer among Saudi population – A pilot study
Ahmed Yaqinuddin, Amna Shoaib Siddiqui, Ayesha Rahman Ambia, pages 293-299

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 Background: To identify bladder cancer specific methylated DNA sequences for the Saudi population in order to detect and predict bladder cancer progression.

Methods: In this study, we analysed DNA methylation levels of 48 tumour suppressor genes loci in 24 bladder tissues (19 bladder cancer samples and 5 control samples taken from histologically normal bladders).  DNA Methylation analysis was done using Human Tumour Suppressor Genes EpiTect Methyl II Complete PCR Array from Qiagen TM.

Results: We identified significant difference in DNA hypermethylation levels at APC, BRCA1, CDH1, CDH13, CDKN2A, DAPK1, ESR1, FHIT, MGMT, RASSF1, SOCS1, TIMP3, TP73, VHL, WIF1 between controls and cancerous samples. It was also observed that CADM1 and DKK3 were differentially methylated in non-muscle invasive versus muscle invasive bladder cancer samples. Additionally, DNA hypermethylation of ESR1 was notified as the novel tumour suppressor gene specific for the Saudi population in bladder cancer.

Conclusion: Our findings suggest that these aberrant DNA methylation patterns in bladder cancer are disease and population specific and have a potential to develop as distinct DNA methylation-based biomarkers in future. 

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Reconnoitering Mycobacterium tuberculosis lipoproteins to design subunit vaccine by immunoinformatics approach
Muhammad Ahsan Naeem, Muhammad Muzammal Adeel, Ayesha Kanwal, Sajjad Ahmad, Waqas Ahmad, Qaiser Akram, Asif Saleh, Waqas Ahmed, pages 300-306

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 Background: Tuberculosis is an aerosol transmitted disease of human beings caused by Mycobacterium tuberculosis (Mtb). The only available vaccine for Mtb is Bacillus Calmette-Guérin (BCG). Currently no alternative or booster is available for BCG. The objective of this predictive approach was based on binding of MHC-I and MHC-II and B cell epitopes of Mtb for mouse host.

Methods: Immunoinformatics approach was used to design subunit vaccine (SV) by joining 8 MHC-I bindings, 6 MHC-II bindings, and 8 B-Cell epitopes with AAV, GPGPG, and KK amino acid linkers, respectively. The efficacy of the SV was enhanced through Mtb protein Rv3763 (LpqH, PDB ID= 4ZJM) as an adjuvant at the N-terminal of SV. The in silico analyses evaluated the SV to predict allergenicity, antigenicity, and physico-chemical properties.

Results: Predictions revealed that SV is non-allergic and highly antigenic. The physico-chemical analysis showed that the SV was stable and basic in nature. The three-dimensional structure of SV was stable with a high binding affinity against the mouse TLR2 receptor. In silico cloning suggested the effective transformation of SV into the eukaryotic expression vector.

Conclusion: This study permits preclinical validation of the designed SV in mouse host to confirm its immunogenic potential and efficacy, which will help in controlling tuberculosis.  

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