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Cenchrus ciliaris is a potential forage grass that widely grows in saline desert environment. To examine root, stem and leaf anatomical modifications, population of Cenchrus ciliaris L. (Buffal grass) was collected from the naturally saline patches of the Cholistan desert, Pakistan. Four salinity treatments 0 mM of NaCl (control), 100, 200 and 300 mM of NaCl were maintained in half strength Hoagland nutrient solution by using hydroponic system. After 16 weeks of growth in nutrient solution root, stem and leaf anatomical characteristics were studied. Salt stress induced anatomical modification in root, stem and leaf that help to survive species in adverse environmental condition. At root level, salinity increased the thickness of endodermis and sclerenchyma tissues both prevent the water loss from root surface and also increased in number of parenchyma cells in pith and cortex region that improved the water storage capacity of root. At stem level, salinity contributed in epidermis and sclerenchyma thickness along with increase in number of vascular bundles and its area that might be improved conduction of water and solute, increase succulence ability and preventing water loss. In case of leaf and leaf sheath, decreased in stomatal density and its area, increased bulliform cell, and rich density of vesicular hairs and trichomes might be essential for water conservation and salt excretion. On the basis of anatomical modifications and its correlation with biomass it was concluded that C. ciliaris can easily be grow at salt affected areas and such anatomical modifications seemed to be critical for the better survival of species under harsh sandy and salty environment. [ABSTRACT FROM AUTHOR]

Filamentous fungi is the basic source of hydrolytic enzymes and widely used for industrial applications. Aspergillus fumigatus have been widely used for the production of hydrolytic enzymes. The present study was undertaken to demonstrate the production of glucoamylase from Aspergillus fumigatus using wheat bran as substrate. A. fumigatus was isolated from organic waste samples from river Ravi, Lahore, Punjab, Pakistan and the conditions were optimized for its growth. Various carbon and nitrogen sources were utilized for the supplementation of growth medium in order to produce higher level of glucoamylase. The produced enzyme was partially purified by fractional ammonium sulphate precipitation followed by dialysis for the removal of salt. The dialyzed sample was further purified by ion exchange and gel filtration chromatography. The purified protein was utilized for the kinetic studies. Purification studies resulted in 12 fold purification with 45% yield of the glucoamylase. SDS-PAGE analysis of the purified enzyme revealed an approximate size of 60 kDa. Under optimal conditions, we were able to produce 28 U/mL of glucoamylase. Highest enzyme activity was observed when A. fumigatus was grown on 3% wheat bran in the presence of 0.7% Tween-80 at 40°C for 72 hours at pH 5.0. Kinetic studies demonstrated the Km and Vmax values 9.1 mg/mL and 40 µM min-1 mg-1 respectively when starch was utilized as substrate. Present study revealed a new source of glucoamylase, which have ability to utilize a variety of agriculture products as carbon sources that can be useful for enzyme industry. This study discovered a new strain, which is a unique machine that can act as an attractive alternative source for the production of industrial enzymes for starch hydrolysis. To our knowledge this is the highest glucoamylase activity from Aspergillus fumigatus sp. so far. [ABSTRACT FROM AUTHOR]

p21 is a cyclin dependent kinase inhibitor, that plays an important role in cell growth, differentiation and apoptosis. The aim of present study was to analyze the mutation and expression of tumor suppressor gene ($21) in canine tumors. A total of 26 tumors (mammary tumors and mast cell tumors) and normal samples from different breed of dogs were analyzed by using direct DNA sequencing and quantitative RT-PCR for mutation and expression profiling of $21gene respectively. The coding exon of $21 gene (Exon2 and 3) did not show any polymorphism in studied samples while 69% tumors showed up-regulation of gene. Seventy five percent mammary tumors showed up-regulation of gene with highest fold change 8.69, 7.6, 5.63, 5.21 and 60% mast cell tumor showed up-regulation of gene with highest fold change 7.31. These results indicate that altered expression of tumor suppressor gene may be involved in malignant progression of tumors and might be helpful in the diagnosis and prognosis of canine tumors. [ABSTRACT FROM AUTHOR]

Current study deals with the production of locally characterized recombinant thermostable phytase from Thermotoga naphthophila (PHYTN) and its biological evaluation in poultry broiler chicks. The PHYTN was produced from 16-liter LB medium using BL21 CodonPlus cells having pET-21a containing phytase gene from Thermotoga naphthophila. A total of 135 birds were divided into 5 groups each having 27 birds. Group A served as negative control, group B, C and D were given feed supplemented with 500, 1000 and 1500 IU/kg of locally produced recombinant phytase while group E served as positive control. The results depicted that locally produced recombinant phytase showed significant effect on bird's weight gain, feed intake and feed efficiency ratio. Presence of 1000 IU/kg of phytase resulted in the weight gain in 3rd, 4th and 5th week of trial from 504.77±1 to 533.53±1.4, 767.9±2 to 823.73±2.84 and 999.8±7.66 to 1122.8±2g respectively when compared with the control. The group B, C and D showed an average feed intake of 2710, 2780 and 2790 g as compared to 2669 g for group A. The supplementation of 1000 IU/kg of locally produced phytase in feed resulted in the improvement of feed conversion ratio from 2.6 to 2.4. The study demonstrated that PHYTN is suitable for poultry feed industry and its domestic production will contribute in the economic availability of locally produced phytase for the poultry feed industry. [ABSTRACT FROM AUTHOR]

Newcastle disease (ND) is one of the most significant OIE listed disease that causes a huge economic losses in the poultry sector worldwide. ND is endemic in Pakistan and continues outbreaks have been reported in vaccinated and non-vaccinated birds. Sequence analysis revealed several amino acids substitutions at the functional domains of fusion (F) and hemagglutinin-neuroaminidase (HN) protein. The current study proposed that the vaccination is leading to pressure on the NDV to mutate into more virulent strains. We noticed that the host range of NDV is extending to pet and other species and suggest it could be due more virulent strains emerging. The NDV strain Chicken/NDV/Pak/AW-14 was isolated from the vaccinated chicken from a poultry farm located near Lahore, Punjab. The isolated was biologically and genetically characterized through intra cerebral pathogenicity index (ICPI) and real time PCR (rRT-PCR). To better understand the epidemiology of ND outbreak, the full genome was sequenced and phylogenetic analysis was performed on the basis of full F and genome sequencing. AW-14 isolate was categorized as a velogenicNDV strain assessed biologically by the ICPI (1.8) and polybasic amino acid residues at the F protein cleavage site. The complete genome was 15,192 nucleotides (nt) in length with six genes in order of 3�€�-NP-P-M-F-HN-L-5�€�. Several polymorphisms were identified at the functional domains of surface glycoprotein F and HN, including signal peptide, transmembrane domain, he pad repeat region and N-glycosylation site, transmembrane domain, neutralizing epitope respectively. Phylogenetic analysis showed that the AW-14 belongs to sub-genotype VlIi, the newly emerging NDV strain has been implicated in the recent outbreaks in Pakistan, Indonesia and Israel and becoming established in poultry sector through Asia and Middle East. Here, we provide a summary of the genetic evolution and molecular epidemiology of the vNDV strain suggesting responsible of a fifth panzootic. [ABSTRACT FROM AUTHOR]

Francisella tularensis (FT) is a Tier 1 select agent due to its low infectious dose and potential threat of being a bio-weapon while causing zoonotic disease in more than 150 mammalian species. Since there are concerns in its culturing at bio-containment deficient labs in developing countries and its subsequent diagnostics and surveillance accordingly, here we proposed a conventional PCR based test of choice that have been developed and optimized for detection of FT using tul4 gene specific primers. Annealing temperature and genomic DNA concentration was optimized using a live vaccine strain. Specificity of primer showed FT detection in soil DNA in the presence of other cross-reactive organism. Sensitivity was determined in two fold dilutions with detection limit of up to 320 pg/μL. Utilizing pET28a vector, a construct was prepared containing transformed tul4 gene (450bp) showing 100% sequence homology to query gene sequence. These results suggest tul4 gene based constructs in vector could be shared to developing countries while developing diagnostic assays as positive control and could potentially be used for lipoprotein purification for ELISA kit development. [ABSTRACT FROM AUTHOR]

Francisella tularensis (FT) is gram negative, non-motile, capsulated and intracellular zoonotic fastidious bacteria, causing tularemia in both animals and human. Soil texture and heavy metals play an important role in survival and persistence of microbes in environment. In the present study, association of physical and chemical composition of soil was determined in 74 FT positive and negative sites in seven districts of Punjab, Pakistan. Soil samples were analyzed for macro and micronutrients including magnesium, copper, chromium, nickel, manganese, cobalt, lead, cadmium, sodium, iron, calcium, nitrogen, phosphorus and potassium by ammonium bicarbonate-diethylenetriaminepenta acetic acid (DTPA) method using atomic absorption spectrophotometer. Soil pH, moisture, clay, silt, sand, soluble salt and organic matters were determined using standard protocols. A significant difference was found in silt (21.49±14.36), clay (69.68±19.34), soluble salt (3.96±2.89), organic matters (7.32±3.95), nitrogen (0.37±0.20), nickel (1.06±0.23), cadmium (0.37±0.18), magnesium (1.51±3.44), lead (3.51±0.85) and zinc (0.56±0.19) in soil collected from both FT positive and negative sites. There was a non-significant difference in pH (8.21±1.93), sand (10.75±7.73), moisture (11.34±6.94), phosphorus (15.98±16.07), copper (0.23±0.18), chromium (0.38±0.97), manganese (1.99±9.15), iron (7.95±80.42), calcium (75.53±720.29), sodium (49.66±48.06) and potassium (43.59±37.66) in soil samples collected from all districts. In conclusion, physicochemical factors of soil play some role in persistence of FT in environment. [ABSTRACT FROM AUTHOR]

Cancer originates from the unnatural divisions of cells that have constant mutations in the genes which are involved in cell propagation and endurance. Dogs are beloved pet animals and they suffer from many cancers like humans. Mutated BRCA1 and COX-2 abnormal gene expression have been seen in a large number of cancers previously. In this research gene sequencing and gene expression of COX-2 and BRCA1 genes in 22 different canine tumors and 25 normal canine samples were performed. All samples were collected from Punjab Province, Pakistan. The whole coding region of COX- 2, comprised of almost 1812bp with ten exons in dog and the hot spot N (1, 2 and 12 exons) and C- terminal (18, 19, 20, 21 and 22 exons) of BRCA1 gene which include 2024bp were included in mutational studies. There was no mutation in the coding region of COX-2 and 5'- and -3' region of BRCA1. The gene expression of COX-2 and BRCA1 in all aforementioned tumors was measured by real time qPCR. BRCA1 high grade negative expression and COX-2 positive gene expression were observed in this study. About 45% tumors showed up-regulation of COX-2 and four tumors showed highest gene expression, two CTVTs, one head tumor and one mammary tumor with fold changes 942.93, 122.79, 85.95 and 44.49 respectively. All granulomas showed down-regulation of COX-2 gene. It has been observed during this study that the loss of BRCA1 gene expression and up-regulation of the COX-2 gene may have a role in canine tumors. Altered expression of COX-2 gene in tumors may have link with the extent of aggressive tumor types. The mechanism behind the abnormal function of these genes is not clear yet, however, abnormal expression of COX-2 and BRCA1 in tumors may be associated with the most aggressive and poor prognosis of cancer types and further studies are required. [ABSTRACT FROM AUTHOR]

This study was conducted to analyze the genetic diversity and variability of two sheep breeds (Balochi and Rakhshani), from Balochistan province of Pakistan, through the use of 11 microsatellite markers recommended by FAO. All the screened loci were polymorphic and 70 alleles in total were observed in all studied loci with average polymorphic information content equal to 0.57, showing that the microsatellite panel used was highly informative. The result divulged high level of genetic variability in each of the two investigated sheep breeds, allele diversity in Balochi 4.5455 and Rakhshani was 4.0909; gene diversity in Balochi 0.5927 and Rakhshani was 0.6182. High heterozygosity value indicated low level of inbreeding, low or no selection pressure and large number of alleles. Further support in this regard was observed by inbreeding estimate (Balochi F[sub IS] = 0.0292 and Rakhshani F[sub IS] = 0.0084) in our sheep population. High level of genetic differentiation between Balochi and Rakhshani sheep breeds was evident from high genetic differentiation estimates (F[sub ST] = 0.1884). The pair wise comparison between both breeds at each locus in term of number of alleles shared (36%, 25/70) reflected the variation between them. The Nei's genetic distances (D[sub S] = 1.3001 and D[sub A] = 0.2725) and gene flow (N[sub m] = 1.0767) further indicating the genetic variation between Balochi and Rakhshani sheep breeds. These data depicts the effectiveness of FAO recommended microsatellite markers for estimation of genetic diversity in Balochi and Rakhshani sheep breeds of Pakistan and may be helpful for comparison with other reported data and for better understanding and breed conservation efforts locally and worldwide. [ABSTRACT FROM AUTHOR]

Minimum inhibitory concentrations (MICs) of methanol extracts of three herbal plants namely garlic, Glycyrrhiza and Neem against three local Mycoplasma gallisepticum (MG) isolates were determined using micro dilution method. A constant number (108CFU/ml) of three MG isolates were used after confirmation through polymerase chain reaction (PCR). A total of 50 mg/ml concentration of each extract was maintained in first well and then two folds serial dilutions were made. Mean values of MICs of glycyrrhiza and neem extracts were 6.25±0.000 against all of the three isolates showing non-significant differences statistically. While, garlic extract showed MIC 3.125±0.000 against two isolates and 6.25±0.000 for third isolate showing significant difference (p<0.05). It was concluded that garlic is more effective than glycyrrhiza and neem in controlling the growth of MG and can be used as therapeutic agent in poultry to control MG infection, although field trials are needed. [ABSTRACT FROM AUTHOR]