The interplay of PTEN and AKT nexus in breast cancer: a molecular perspective.

BACKGROUND: Breast cancer is a highly prevalent and life-threatening ailment that is commonly detected among the females. The downregulation of PTEN in breast cancer is associated with a poor prognosis, aggressive tumor type, and metastasis to lymph nodes, as it activates the pro-survival pathway PI3K/AKT, which is considered the ultimate proliferative pathway. MATERIAL AND METHODS: The mRNA expression of PTEN and AKT genes was investigated using RT-qPCR and TaqMan primer probe chemistry. Moreover DNA was also isolated from the same tissue samples and exonic regions of both genes were amplified for mutational analysis. The proteins expression of PTEN and AKT from seven human breast cancer cell lines was checked through western blot experiments. RESULT: The study revealed a decrease in PTEN expression in 73.3% of the samples, whereas an increase in AKT expression in 40% of samples was observed when compared to the distant normal breast tissue. Conversely, the remaining 60% of samples exhibited a decrease in AKT mRNA expression. There was no observed alteration in the genetic sequence of AKT and PTEN within the targeted amplified regions of breast cancer samples. The high levels of PTEN protein in T-47D and MDA-MB-453 resulted in a lower p-AKT. Two cell lines ZR-75-1 and MDA-MB-468 appeared to be PTEN negative on western blot but mRNA was detected on RT-qPCR. CONCLUSION: In breast cancer the status/expression of PTEN & AKT at mRNA and protein level might be obliging in forecasting the path of disease progression, treatment and prognosis.

Alterations in the RB1 gene in Pakistani patients with retinoblastoma using direct sequencing analysis.

PURPOSE: Retinoblastoma (RB) is a rare intraocular malignant tumor of the developing retina with an estimated incidence of 1:20,000 live births in children under the age of 5 years. In addition to the abnormal whitish appearance of the pupil or leukocoria, strabismus has also been reported as a clinical symptom of the disease. RB1 is the first cloned tumor suppressor gene, and mutational inactivation of this gene is responsible for the development of RB during early childhood. The purpose of this study was to identify mutational alterations in the RB1 gene in Pakistani patients with RB. METHODS: During this study, 70 clinically evaluated patients with RB were recruited from different regions of Pakistan. The cases included 23 sporadic bilateral (32.9%), 34 sporadic unilateral (48.6%), nine familial bilateral (12.8%), and four familial unilateral (5.7%) cases. Constitutional causative mutations in the RB1 gene were screened via direct sequencing of all RB1 exons and their flanking regions. RESULTS: In this report, genetic testing resulted in the identification of 18 mutations in 25 patients with RB including six novel RB1 mutations. Of the total mutations identified, 13 (72.22%) were found to be null mutations caused by nine nonsense, three deletions, and one insertion. Two (11.11%) missense, two (11.11%) splice site mutations, and one (5.55%) base substitution in the promoter region were also found. Moreover, ten intronic variants were identified, one of which is novel. CONCLUSIONS: Molecular screening and identification of these mutations in Pakistani patients with RB provide the mutational variants of the RB1 gene in the Pakistani population. The detection of oncogenic mutations in patients with RB and genetically predisposed individuals is a major step in clinical management, prognosis, follow-up care, accurate genetic counseling, and presymptomatic diagnosis of RB.

Molecular classification of Pakistani collared dove through DNA barcoding.

Pakistan is bestowed by a diversified array of wild bird species including collared doves of which the taxonomy has been least studied and reported. DNA barcoding is a geno-taxonomic tool that has been used for characterization of bird species using mitochondrial cytochrome c oxidase I gene (COI). This study aimed to identify taxonomic order of Pakistani collared dove using DNA barcoding. Purposely herein, we present a phylogenetic analysis of Pakistani collared dove based on 650 base pairs of COI gene sequences. Analysis of phylogenetic tree revealed that Pakistani collared dove shared a common clade with Eurasian collared dove (Streptopelia decaocto) and African collared dove (Streptopelia roseogrisea) which indicated a super-species group in Streptopelia genus. This is the first report of molecular classification of Pakistani collared dove using DNA barcoding.

Quantification and characterization of microplastics (MPs) pollution in peri-uburban agricultural lands of Lahore, Pakistan.

Microplastics (MPs) contaminate every conceivable terrestrial and aquatic environment including high peaks and deep marine trenches. Agricultural lands alone are expected to receive plastic up to 23 times more than ocean basins. In this study, soil samples were collected from peri-urban agricultural lands of Lahore on four sides including Kala Shah Kaku (KSK), Punjab University (PU), Dera Gujran (DG), and Sagian (SG). National Oceanic and Atmospheric Administration (NOAA) protocol was used for MPs extraction and analysis. Extracted MPs were analyzed under microscope at 40X magnification and their composition was analyzed using Fourier Transform Infrared (FTIR) spectroscopy. A considerable concentration of MPs was recorded at all sites. The highest contamination was found at SG with 876 ±194 MPs/kg of soil, and the lowest contamination was recorded at PU with 672 ±235 MPs/kg of soil. However, these differences among the sites were not statistically significant (p = 0.29). The overall predominant shape of MPs was fibers (613±71, 79.73%) followed by sheets (125±55, 16.28%), fragments (30±5, 3.9%) and foam particles (1±2, .09%). The differences in the distribution of MPs in various types were statistically significant (p = 0), while differences between sites were insignificant (p = 0.13). About 95% of MPs were less than 2 mm and 85% were less than 1 mm size. The distribution of MPs in various sizes (p = 0) and differences of this distribution between sites (p = 0.037) were both statistically significant. A good diversity of nine colored MPs was recorded, however majority of the MPs were transparent (89.57%). Six polymer including Polyethylene (PE), Polyethylene terephthalate (PET), Polypropylene (PP), Polystyrene (PS), Polycarbonate (PC), and Polyvinyl Chloride (PVC) were identified by FTIR. The current levels of MPs pollution are higher than in many other parts of the world. Composition of MPs (types, colors, sizes, and polymer types) indicates the diversity of their sources and their possible implications on agricultural ecosystem.

Novel mutations of endothelin-B receptor gene in Pakistani patients with Waardenburg syndrome.

Mutations in EDNRB gene have been reported to cause Waardenburg-Shah syndrome (WS4) in humans. We investigated 17 patients with WS4 for identification of mutations in EDNRB gene using PCR and direct sequencing technique. Four genomic mutations were detected in four patients; a G to C transversion in codon 335 (S335C) in exon 5 and a transition of T to C in codon (S361L) in exon 5, a transition of A to G in codon 277 (L277L) in exon 4, a non coding transversion of T to A at -30 nucleotide position of exon 5. None of these mutations were found in controls. One of the patients harbored two novel mutations (S335C, S361L) in exon 5 and one in Intronic region (-30exon5 A>G). All of the mutations were homozygous and novel except the mutation observed in exon 4. In this study, we have identified 3 novel mutations in EDNRB gene associated with WS4 in Pakistani patients.

DNA fingerprinting of Pakistani buffalo breeds (Nili-Ravi, Kundi) using microsatellite and cytochrome b gene markers.

Cytochrome b gene markers have been proved as an efficient and powerful tool for breed characterization and species identification of buffaloes. This study represents the substantial analysis of mitochondrial DNA variation in Pakistani buffalo breeds and provides information about their genetic diversity. In this study partial amplification of cytochrome b gene of 1,061 bp was done and sequencing results showed ten haplotypes. Comparing all fifty samples from two buffalo breeds of Pakistan, fifteen polymorphic sites were observed out of which, twelve codons 42, 71, 118, 120, 199, 235, 269, 297, 318, 327, 350, 355 of mitochondrial cytochrome b gene are monomorphic which translate same amino acids as in the reference protein sequence due to silent mutation while different in DNA sequence. Similarly three codons 163, 246, 337 of mitochondrial cytochrome b are polymorphic and different from the reference sequence with respect to DNA as well as protein sequence. For the further confirmation a panel of nine microsatellite markers was used with high polymorphism information content (PIC). The frequency distribution of these alleles varies from three to eight allele at locus CSSM66 and ILST029 respectively. The results obtained from this study may contribute to the establishment of routine genotyping service of buffalo breeds for buffalo farmers for animal forensic application in case of any dispute. Additionally this study may help for breed characterization and phylogeny of aforementioned breeds of buffalo.

Promyelocytic leukemia zinc finger protein (PLZF) enhances glucocorticoid-induced apoptosis in leukemic cell line NALM6.

Glucocorticoids (GC) actuate apoptosis as well as cell cycle arrest in lymphocytes, and included as core element in the lymphoid malignancy treatment. Despite clinical significance of GC and considerable efforts to understand it, the molecular basis of GC regulated cell death and the resistance phenomenon remains, however, poorly understood. Using Affymetrix-based whole genome expression profiling our group has previously identified a number of prominent glucocorticoid-response genes (Blood 107: 2061, 2006). Promyelocytic leukemia zinc finger (PLZF) was one of the best candidate genes. This study was proposed to investigate the possible role of PLZF in GC regulated cell death in leukemic model cell line NALM6. To this end, we generated NALM6 cell line (bulk) transduced with a retroviral expression vectors, pHR-SFFV-PLZF-IRES-Puro (U426) and pHR-SFFV-Venus-IRES-Puro (U417), as control, for constitutive gene-expression. HEK293T cells were transfected transiently to generate viral particles. These cell lines were characterized by Western blotting and used to assay the effect of constitutive PLZF expression. In conclusion, we report that bona fide transcription repressor PLZF, which turned out as prominent GC-regulated gene both in vivo and in vitro situations was found to enhance the GC-induced cell death (basal) in leukemic model cell line NALM6 after 48 and 72h time points.

Novel single nucleotide polymorphism identification in interleukin-6 gene of Pakistani sheep.

The present study aimed to identify single-nucleotide polymorphism (SNP) in coding and non-coding regions of interleukin-6 (IL-6) gene of Pakistani sheep. The IL-6 gene of 205 animals from nine sheep breeds were sequenced for screening of SNP. Characterizing the IL-6 gene revealed thirteen SNP sites within the intronic region of IL-6 gene. The novel SNPs found in the present study can serve as genetic marker for association studies with susceptibility/resistance to parasite infection in sheep. This is first report of SNP polymorphism of IL-6 gene of Pakistani sheep.

Expression Profiling of Hspb1 and Tp53 Genes through RT-qPCR in Different Cancer Types of Canis familiaris.

Background: Diagnostic molecular marker studies are in vogue to have insight of most prevalent animal diseases including cancer. Objectives: Gene expression profiling of pro and anti-apoptotic genes was conducted in dog Lymphoma, CTVT, SCC, granuloma, perianal adenocarcinoma and mammary tumors. Materials and Methods: Cancerous tissues of 21 affected animals were obtained. Total RNA was extracted followed by cDNA synthesis. Comparative Ct method via Taqman assay (RT-qPCR) was used to quantify corresponding mRNA molecules, Tp53 and Hspb1, as normalized by GAPDH as the reference gene . Results:Hspb1 showed ectopic expression in lymphoma, CTVT and mammary tumors; its down-regulation was observed in granuloma and oral SCC with fold difference (FD) of ±35. Similarly, Tp53 as the tumor suppressor gene with pro-apoptotic properties, showed up-regulation in all tumor types, notably 80% of mammary tumors and 60% of CTVT. The FD values were 33.31 and 2.27, respectively. Conclusion: Altered transcriptomic response of Hspb1 and Tp53 was observed in all cancer types of Canis familiaris. The resulting profile depicts the involvement of the genes in cancer pathways. Thus, the data might be helpful for diagnosis, prognosis, identification and classification of these widespread neoplasms in this species.

Hspb1 and Tp53 Mutation and Expression Analysis in Cat Mammary Tumors.

BACKGROUND: Molecular marker based cancer diagnosis gaining more attention in the current genomics era. So, Hspb1 and Tp53 gene characterization and their mRNA expression might be helpful in diagnosis and prognosis of cat mammary adenocarcinoma. It will also add information in comparative cancer genetics and genomics. OBJECTIVES: Eight tumors of Siamese cats were analyzed to ascertain germ-line and tissue-specific somatic DNA variations of Hspb1 and Tp53 genes along with the ectopic differential expression in tumorous and normal tissues were also analyzed. MATERIALS AND METHODS: Tumorous tissues and peripheral blood from mammary adenocarcinoma affected Siamese cats were collected from the Pet center-UVAS. DNA and RNA were extracted from these tissues to analyze the Hspb1 and Tp53 DNA variants and ectopic expression of their mRNA within cancerous and normal tissues. RESULTS: Exon 1 and 3 revealed as hotspots in Hspb1 gene. The 5´UTR region of the exon1 bear six mutation including 3 transitions, 2 transversion and one heterozygous synonymous transversion in two samples at locus c.34C>C/A. Exon 3 has 1 transversion at c.773A>A/T, 3´UTR of this exon harbor two point mutations at 1868A>T and 2193C>T loci. Intron 2 has two alterations at 1490C>C/T and GTCT4del at 1514. Overall up-regulation of Hspb1 gene was observed. While exons 3, 4 and 7 of Tp53 harbor a single variationat c.105A>A/G, c.465T>T/C and c.859G>T respectively. The locus c.1050G>G/A in exon 9 is a heterozygous (G/A) in 3 samples and homozygous (G) in 2 other tumours. Introns 3, 5, 7 and 9 harbor 3, 4, 2 and 7 altered loci respectively. Sixty percent of cancers showed up-regulated trend of Tp53 gene. CONCLUSIONS: Tumor specific mutations and ectopic expression of Hspb1 and Tp53 genes might be helpful in the diagnosis of the mammary lesions and endorse their involvement in cat mammary neoplasm.

Evaluating the accuracy and efficiency of multiple sequence alignment methods.

A comparison of 10 most popular Multiple Sequence Alignment (MSA) tools, namely, MUSCLE, MAFFT(L-INS-i), MAFFT (FFT-NS-2), T-Coffee, ProbCons, SATe, Clustal Omega, Kalign, Multalin, and Dialign-TX is presented. We also focused on the significance of some implementations embedded in algorithm of each tool. Based on 10 simulated trees of different number of taxa generated by R, 400 known alignments and sequence files were constructed using indel-Seq-Gen. A total of 4000 test alignments were generated to study the effect of sequence length, indel size, deletion rate, and insertion rate. Results showed that alignment quality was highly dependent on the number of deletions and insertions in the sequences and that the sequence length and indel size had a weaker effect. Overall, ProbCons was consistently on the top of list of the evaluated MSA tools. SATe, being little less accurate, was 529.10% faster than ProbCons and 236.72% faster than MAFFT(L-INS-i). Among other tools, Kalign and MUSCLE achieved the highest sum of pairs. We also considered BALiBASE benchmark datasets and the results relative to BAliBASE- and indel-Seq-Gen-generated alignments were consistent in the most cases.