Advancements in Life Sciences (E-Journal, University of the Punjab)
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In vitro Micropropagation of Citrullus colocynthis (L.) Schrad: an endangered medicinal plant
Get PDFBackground: The experiment describes the establishment of in vitro conditions for seed germination, micropropagation, callogenesis, organogenesis and acclimatization of Citrullus colocynthis (L.) Schrad, of family Cucurbitaceae.Methods: In vitro grown seedlings from decontaminated seeds were micropropagated in basal MS medium at 23±2oC temperature and light intensity of 3000 Lux for 16 hours in culture room. In vitro grown nodal explants were supplemented with BAP (6-Benzylaminopurine) and NAA (Naphthalene acetic acid) with basal MS (Murashige and Skoog) medium to induce multiple shoots. Indole butyric acid (IBA; 0.1 to 2.0 mg/L) was supplemented to MS medium to develop roots of micropropagated shoots. Internodes and leaves of micropropagated shoots used to induce callus in MS medium enriched with varying concentration of 2, 4- dichlorophenoxyacetic acid (2, 4 D; 0 to 2.0mg/L) and kinetin (KIN; 0 to 1.0mg/L). Shoot initiation from callus was tested by adding 2, 4-D (0.1 to 2.0 mg/L) and BAP (1.0 to 1.5 mg/L) in basal MS medium. Conditions were carefully monitored during the experiment. After hardening, the micropropagated plantlets were placed in open filed environment in pots filled with sand and peat moss (3:1).Result: Surface sterilized seeds of Citrullus colocynthis (L.) showed 100% germination in regulator free medium. Significantly mature shoots (75%) from nodal explant recorded in BAP (2.0 mg/L) and NAA (1.0 mg/L) augmented MS medium. Highest number (90%) of roots per shoot explant were observed in IBA (2.0 mg/L). Leaf explants showed better response to form callus with a combination of 2, 4-D (1.0 mg/L) and KIN (1.0 mg/L) and further rise in 2, 4-D concentration caused a sharp decrease in callus formation. Shoot induction from callus cultures observed in MS medium containing 2, 4-D (2.0 mg/L) and BAP (1.5 mg/L), producing an average of 10 shoots per culture. Plants were effectively transplanted in open environment with survival rate of 85%.Conclusion: Results indicate the successful establishment of the growth room conditions for in vitro micropropagation of the endangered medicinal plant, Citrullus colocynthis.Keywords: In vitro; Indole butyric acid; callus induction; Citrullus colocynthis; organogenesi
CRISPR/Cas9 system: Current applications and future potential in rice breeding
Get PDFRice (Oryza sativa L.) plays a key role in human social and economic life. In order to meet the increasing needs of human food consumption, there is a constant requirement to develop rice cultivars with enhanced agricultural traits. The emerge of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (CRISPR/Cas9) system provides unprecedented opportunities in studying gene functions and creating new rice varieties with better characteristics, including improved tolerance to biotic and abiotic stresses, and increasing yield and quality. This review aims to provide details about the latest results of CRISPR/Cas9 system application on rice to obtain better adapted to environmental and commercial demands.Keywords: CRISPR/Cas9; Biotic stress; Abiotic stress; Yield; Quality
Single nucleotide polymorphism and phylogenetic analysis of the exon 2 of leptin gene in Lohi sheep
Get PDFBackground: Leptin hormone, encoded by leptin (LEP) gene is involved in many biological and physiological processes in the body. Polymorphism in LEP gene has been observed and correlated with a variety of reproductive and productive traits in several sheep breeds worldwide, but its role has not been much studied in local sheep breeds of Pakistan. The present study was conducted to analyze polymorphism in LEP gene in Lohi breed of sheep.Methods: Subsequent to statistical analysis (generalized linear model), 18 animals were selected randomly from the flock for blood samples collection followed by DNA extraction, amplification using PCR prior to sequencing. The amplified product of exon 2 and partial intron 2 regions of LEP gene was 268bp.Results: Molecular analysis showed a heterozygous condition i.e. C>Y at position 15 and 18 in exon 2. The data on average daily weight gain (ADG) from birthday to 90 days were used for association study, while environmental effects were minimized by means of generalized linear model. Association of polymorphisms in LEP gene with ADG did not yield any significant results.Conclusion: In conclusion, analysis of LEP gene sequence verified the existence of genetic changes in Lohi sheep. Further investigations are needed to find variations that might be linked with traits of economic importance for upcoming breeding program sand marker-assisted selection.Keywords: DNA; Exon 2; LEP; PCR; Loh
Evaluation of Antibiotics Pattern of Extended Spectrum Beta-Lactamase Producing Multi-Drug Resistant Pseudomonas aeruginosa
Get PDFBackground: Pseudomonas aeruginosa (Ps. aeruginosa) is considered as an opportunistic pathogen and the leading cause of morbidity and mortality in immunocompromised individuals. Globally, approximately 10-15% of the nosocomial infections are caused by Ps. aeruginosa. The Ps. aeruginosa can acquire resistance against broad-spectrum antibiotics. According to recent studies increased mortality has been observed due to infection with extended-spectrum-beta-lactamase (ESBL) producing Ps. aeruginosa strains. This study was designed to determined antibiogram of ESBL producing multi-drug resistant Ps. aeruginosa in Khyber Pakhtunkhwa.Methods: The clinical confirmed Ps. aeruginosa samples were collected according to the standard protocol, at Khyber Teaching Hospital (KTH), Peshawar. All collected samples were sub- cultured on appropriate culture media. After isolation and identification, the antibiotics susceptibility testing was performed. The detection of ESBL was carried out by the double-disc diffusion method. Carbapenemase-producing bacteria was confirmed by the modified Hodge test. Descriptive analysis was performed for statistical analysis of collected data.Results: A total of one hundred and sixty-two (n=162) Ps. aeruginosa confirmed isolates were collected, in which 59.3% were male and 40.7% were from female patients. The percentages of ESBL and carbapenemase producing Ps. aeruginosa isolates were 5.5% and 23.5%, respectively. The multidrug resistance was observed against 27.2% isolates. Among tested antibiotics highest percentages of resistance was observed against ciprofloxacin (43%) and ceftazidime (39.5%). Conclusion: We observed highest level of drug resistance in Ps. aeruginosa clinical isolates against tested antibiotics and majority of the isolates were Multi-drug resistant (MDR).Keywords: Pseudomonas; Multi-Drug Resistant; Extended Spectrum Beta Lactamases; Antibiotics Susceptibilit
Viral outbreaks: A real threat to the world
Get PDFGlobal public health is facing significant challenges in terms of emerging and re-emerging pathogens. The world is facing a new public health crisis emergence and spread of Coronaviruses outbreaks especially COVID-19 after nine deadliest viral outbreaks including Marburg virus, Ebola virus, Rabies, HIV, Smallpox, Hantavirus, Influenza, Dengue and Rotavirus. Coronaviruses (enveloped non-segmented positive-sense RNA viruses) belong to the Coronaviridae family, broadly distributed in humans as well as in other mammals. In December 2019, the COVID-19 outbreak was reported in the Wuhan, Hubei province of China. WHO confirmed that COVID-19 is associated with Huanan seafood (Wuhan). COVID-19 virus outbreak is more dangerous than its ancestors MERS-CoV and SARS-CoV. Although the case fatality rate is lower, it has alarmed the world because of its rapid spread during this era of the modern world where the whole world is connected through different channels of trade. As the world is already facing economic challenges, underdeveloped countries are not capable of facing such challenges, and this outbreak may become worse than ever before.Keywords: Coronaviruses; COVID-19; Marburg virus; Ebola virus; Rabies; HIV; Smallpox; Hantavirus; Influenza; Dengue; Rotaviru
Domestic animals’ identification using PCR-RFLP analysis of cytochrome b gene
Get PDFBackground: Species identification is an important process to identify the origin of meat, adulteration and for cooked and processed meat. The present study was conducted to identify cattle (Bos taurus) and buffalo (Bubalus bubalis) by using mitochondrial cytochrome-b (Cyt-b) gene. Size of the gene is 1140 bp, but we amplified 359 bp that is cleaved by specific restriction endonucleases. The aim of this study was species identification through Cyt-b gene by using PCR-RFLP analysis.Methods: For this study, 55 blood samples were collected from different species of domestic animals. The DNA was extracted from the whole blood through blood extraction kit. The DNA of these samples were amplified through PCR using universal Cyt-b primers. The amplified product was treated with restriction enzymes Alu I. The resultant fragments were viewed on 3.0 % agarose gel.Results: Cyt-b gene was amplified of all included animals. Different bands were observed as compared with 50 bp DNA ladder. Animals were identified on the base RFLP mediated by Alu1 restriction enzyme.Conclusion: We identified domestic animals on the basis of Mitochondrial Cyt-b gene by the process of PCR-RFLP. To identify specific animals through RFLP, a larger sample size and confirmation by gene sequence analysis may be helpful.Keywords: Domestic Animal Identification; Cytochrome b gene; AluI restriction enzyme; PCR-RFLP Analysi
Decreased Expression of Alpha Smooth Muscle Actin and Desmin Contributes to the Protection of Vitamin D3 against Diclofenac Induced Nephrotoxicity in Rats
Get PDFBackground: Diclofenac is widely prescribed for its analgesic and anti-inflammatory actions but it also has some harmful effects on the kidney. The current study was conducted to elucidate the possible mechanism of action of diclofenac sodium on kidney, and if it is affected by the addition of vitamin D.Methods: Rats were divided into 4 equal groups. G1 was the control group that received no treatment; G2 was treated with intramuscular injection of vitamin D (1,000 IU/kg, 3days/week); G3 was treated with intramuscular injection of diclofenac sodium (3.6 mg/kg, 3 days/week) and G4 treated simultaneously with both diclofenac (3.6 mg/kg, 3 days/week) and vitamin D (1,000 IU/kg, 3days/week) intramuscularly for four weeks. Kidneys sections were stained with H&E, Masson’s trichrome and immunohistochemical staining against α –SMA and desmin followed by the morphometric and statistical analysis.Results: Kidney sections from diclofenac sodium treated group showed degeneration and necrosis, small or atrophic glomeruli with dilated Bowman’s space and some of the renal tubular lining cells appeared vacuolated with small pyknotic nuclei. Renal fibrosis was confirmed by significant increase in collagen fibers, α –SMA and podocytes injury by significant increase of desmin. However, in diclofenac- vitamin D treated group significantly the expression of α –SMA and desmin were decreased.Conclusion: The current data suggested that vitamin D might play a protectant role against diclofenac induced kidney injury in rats through the preservation of the histological architecture of renal corpuscles, renal cortical tubules and down regulation of collagen, α – SMA and desmin.Keywords: α-SMA; Desmin; Diclofenac Sodium; Vitamin
Molecular cloning and expression of recombinant Trichoderma harzianum chitinase in Pichia pastoris
Get PDFBackground: The importance of chitinases over the years had attracted huge biotechnological attention because its usage cut across wide range of field. It plays a significant role in the defensive mechanism against fungal pathogens.Methods: In this study, an endochitinase gene was isolated from Trichoderma harzianum, and characterized in-silico by using various bioinformatics tools. Further, the gene was cloned in eukaryotic expression vector (pPICZA) under the control of AOX1 promoter for recombinant expression in Pichia pastoris GS115 host strain.Results: The chitinase cDNA was ~1000 bp long, while in-silico studies revealed an open reading frame of 888 bp encoding 295 amino acids with a calculated molecular mass of 37332.76 Da and an estimated isoelectric point of 4.07. Recombinant chitinase protein expressed intracellularly and revealed high expression in P. pastoris host. The 37 kDa recombinant chitinase protein developed with antigen antibody confirmed its expression in P. pastoris.Conclusion: Conclusively, T. harzianum derived chitinase gene was successfully over expressed in P. pastoris where recombinant protein was expressed intracellular in the form of inclusion bodies.Keywords: Trichoderma harzianum; Chitinase; Pichia pastori
Development of a diagnostic scar marker for Vibrio shilonii caused acute hepatopancreatic necrosis disease in whiteleg shrimp
Get PDFBackground: In a previous report, we showed that Vibrio shilonii was found on whiteleg shrimp (Litopenaeus vannamei) with acute hepatopancreatic necrosis disease in Thua Thien Hue province, Vietnam. This study was performed to develop a diagnostic molecular marker generated by random amplified polymorphic DNA (RAPD) for V. shilonii rapid detection.Methods: Pathogen Vibrio spp. were isolated from shrimps and fishes, and were identified by 16S rRNA sequencing. Genetic diversity of Vibrio strains was analysis by RAPD technique. Specific PCR product for V. shilonii was cloned and sequenced. SCAR marker was developed from specific PCR product.Result: Twenty random primers were evaluated for RAPD to identify DNA polymorphisms between Vibrio species. The random primer OPN-06 generated a 468-bp DNA fragment specific for V. shilonii. This was then converted into a sequence-characterized amplified region (SCAR) marker designated N6-441.Conclusion: Specific primers (Vshi-441F/Vshi-441R) amplified a unique DNA fragment in all V. shilonii isolates but not in the other Vibrio spp. This PCR assay showed significantly sensitive to the target DNA and reliably for the amplification the V. shilonii genome.Keywords: AHPND; RAPD; SCAR; Vibrio shilonii; Vietna
Study on significant changes in calcium, phosphorus and thyroid hormones level in hypothyroidism patients
Get PDFBackground: Thyroid hormones have a vital role in metabolism of lipids, cholesterol etc. They also have an important role in phosphorous and calcium homeostasis by their direct effect on bone turnover. The objective of present study was to find the significant differences of T3, T4, TSH, lipid profile (HDL, LDL, vLDL, TG, Cholesterol), electrolytes and minerals (sodium, potassium, chloride, phosphorus, calcium) between control group and hypothyroidism patients.Methods: Blood samples were collected from 72 patients and 12 control after their consent. Serum was used to determine biochemical parameters using standard protocol. Data obtained were statistically analyzed using “t” student test.Results: The level of TSH was significantly higher in female and male hypothyroidism patients (p < 0.01) as compared to control and it was inversely related to the level of T3 and T4. Patients with hypothyroidism have increased level of LFTs profile especially cholesterol and Triglycerides levels both in males and females. In hypothyroidism, the level of glomerulus filtrate rate decrease especially sodium level but thyroid hormonal disturbance does not affect serum electrolyte level. The serum phosphorus and calcium levels change significantly in thyroid disorder.Conclusion: Hypothyroidism is associated with increased TSH level and decreased T3 and T4 levels but does not appear to be associated with abnormalities in lipid profile. The serum phosphorus and calcium levels change significantly in thyroid disorder, Treatments of its primary causes should be given and if it is possible minerals can be added to avoid further bone complications.Keywords: Hypothyroid; T3; T4, TSH; Lipid profile; Minera