Intraperitoneal injections of copper ferrite nanoparticles disturb blood, plasma, and antioxidant parameters of Wistar rats in a sex-specific manner.

The present study was designed to report the synthesis and characterization of copper ferrite nanoparticles (CF NPs) and their biocompatibility in Wistar rats. Coprecipitation method was used to generate CF NPs having average diameter of 14.06 nm. NPs were characterized by scanning electron microscopy and X-ray diffraction. Six-week-old Wistar rats of both sexes intraperitoneally received 10 mg/ml saline/Kg body weight of CF NPs for 14 days. Control groups were maintained in parallel that received saline solution for 14 days through same route. Open field and novel object recognition tests, complete blood count, selected plasma parameters, antioxidants, and copper concentration in vital organs were determined in all treatments. Female rats treated with CF NPs had significantly higher platelet count (P = 0.05) and platelet crit (P = 0.05) and decreased plasma triglyceride concentration levels (P = 0.02) than control group. Female rats had significantly increased levels of superoxide dismutase (P = 0.01), catalase (P = 0.05), and malonaldehyde (P = 0.05) in the kidney, while male rats had significantly elevated levels of superoxide dismutase in the lungs (P = 0.01) as compared with respective control groups. Copper concentrations in the liver were significantly higher in both female (P = 0.04) and male (P = 0.05) rats exposed to CF NPs than control group. All other studied parameters of behavioral tests, blood biochemistry, antioxidant, and copper concentrations in the brain varied nonsignificantly (P > 0.05) when compared between CF NPs treated and untreated rats of both sexes. Intraperitoneal supplementation of CF NPs for 14 days disturbed the platelet count, plasma triglyceride concentration, copper levels in the liver, and antioxidant concentrations in the kidney of female Wistar rats. These parameters remained unaffected in male subjects.

Forensic SNP Genotyping with SNaPshot: Development of a Novel In-house SBE Multiplex SNP Assay.

This study introduces a newly developed in-house SNaPshot single-base extension (SBE) multiplex assay for forensic single nucleotide polymorphism (SNP) genotyping of fresh and degraded samples. The assay was validated with fresh blood samples from four different populations. In addition, altogether 24 samples from skeletal remains were analyzed with the multiplex. Full SNP profiles could be obtained from 14 specimens, while ten remains showed partial SNP profiles. Minor allele frequencies (MAF) of bone samples and different populations were compared and used for association of skeletal remains with a certain population. The results reveal that the SNPs of the bone samples are genetically close to the Pathan population. The findings show that the new multiplex system can be utilized for SNP genotyping of degraded and forensic relevant skeletal material, enabling to provide additional investigative leads in criminal cases.

Development of new PCR multiplex system by the simultaneous detection of 10 miniSTRs, SE33, Penta E, Penta D, and four Y-STRs.

The 18 loci multiplex system has been instigated for co-amplification and fluorescent detection of Amelogenin and 17 STRs, including 10 MiniSTRs (CSF1PO, D18S51, D7S820, D2S1338, TPOX, D13S317, FGA, D5S818, D21S11, D16S539), SE33, Penta E, Penta D, and four Y-STRs (DYS385a/b, DYS438, DYS392). This multiplex system was developed for the simultaneous analysis of compromised DNA samples, Y-amelogenin marker mutation, motherless paternity issues where single allele sharing occurs at autosomal STRs in unrelated individuals, and other complex forensic cases. Selection of loci, primers, and allelic ladders were designed and created in-house with a design strategy to work in this multiplex. The multiplex system was evaluated by sensitivity, specificity, stability, precision and accuracy, case-type samples, mixture studies, PCR-based and population distribution studies to establish the robustness and reliability of the system as the current requirements of the forensic case work. Among all the markers evaluated for this study, 209 alleles including 44 variants were observed with combined power of discrimination, combined power of exclusion, and the combined probability of matching calculated as 0.999999999999999999893916339344, 0.999993816173890, and 5.90019 × 10-19, respectively. Due to highly polymorphic characteristics of these loci particularly SE33 and Penta E which are most discriminatory (PD = 0.991 and 0.983, respectively) in the Pakistani population, this multiplex would be highly valuable for individual identification in complex forensic cases and paternity issues as well as population database.

Baluchi and Pakhtun population data of 9 X-chromosomal short tandem repeat loci.

Baluchistan is the largest province of Pakistan in terms of area, constituting approximately 44% of the country's total land mass, and the smallest in terms of population, being home to less than 5% of the country's population. Khyber Pakhtunkhwa (KPK) formerly called North-West Frontier Province is located in the north-west of Pakistan having an estimated 13.4% of total population of Pakistan in which Pakhtuns are the major ethnic group. A total of 250 samples from Baluchi population and 250 samples from Pakhtun population were typed for 9 X-chromosomal STR markers: DXS101, DXS6789, DXS7132, DXS7423, DXS7424, DXS8378, GATA31E08, GATA172D05 and HPRTB along with sex typing locus, Amelogenin. A total of 59 alleles were found in Baluchi population while 61 alleles were found in Pakhtun population. This is the first study of the two populations based on these markers and the population data can be used as reference database for Baluchi and Pakhtun populations.

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.

Allele frequency distribution of 10 MiniSTRs in the Pashtun population of Pakistan.

Two hundred individual samples of Pashtun population from Khyber Pakhtunkhwa province of Pakistan were randomly evaluated through 10 MiniSTR loci (CSF1PO, D7S820, TPOX, D18S51, D2S1338, D13S317, FGA, D5S818, D21S11, and D16S539). The PCR product size was reduced in the range of 65 to 280 bp. A total of 112 alleles were observed containing allelic frequency ranging from 0.0025 to 0.4325. Statistical values for forensic and parentage analysis were calculated including combined power of discrimination (PD), combined power of exclusion (PE), and cumulative probability of matching (PM) and equaled to 0.99999999999768, 0.99984944, and 2.33 × 10(-12), respectively. These MiniSTRs show a high degree of polymorphism information content and discriminatory power which would be helpful to resolve forensic cases and establish DNA database for major population groups of Pakistan. In contrast to different populations, significant differences were also observed on these loci.

Genetic polymorphism of ten MiniSTR loci in the population of Punjab Pakistan for forensic application.

Ten MiniSTR loci were analyzed on 250 unrelated individuals of Punjabi population from Punjab province of Pakistan. The product amplification range is from 65 to 280 bp. Allele frequency for a total of 98 observed alleles is from 0.002 to 0.41. Forensic and paternity statistical parameters were investigated including combined power of discrimination (PD), combined power of exclusion (PE), and cumulative probability of matching (PM) equaled to 0.99999999999824, 0.999824, and 1.75931 × 10(-12), respectively. These MiniSTRs on the basis of high degree polymorphism and fragment size reduction will be highly informative in most of the forensic cases where DNA of the samples is degraded, mass disasters, and dead body identification. Significant differences were observed in all loci when comparing with same STR loci in previously published different world populations.

Development and characterization of a new 12-plex ChrX miniSTR system.

Short tandem repeat (STR) markers are extensively used for human identification as well as paternity and forensic casework. X-chromosome STR (X-STR) markers are a powerful complementary system especially in deficiency paternity testing. This study presents the development and characterization of a new X-chromosomal short tandem repeat (STR) multiplex using short amplicon (<200 bp). A total of 366 samples from Punjabi population and 346 samples from Sindhi population were typed for 11 X-chromosomal STR markers: DXS101, DXS6789, DXS6793, DXS7132, DXS7423, DXS7424, DXS8378, DXS9902, GATA31E08, GATA172D05, and HPRTB along with sex-typing locus, amelogenin. Each marker showed a high degree of polymorphism, and the multiplex was sensitive down to 250 pg of human DNA. A total of 78 alleles were found with 5-11 alleles for each marker. The population data can be used as reference database for Sindhi and Punjabi populations.