Author Correction: Association of Cyclin Dependent Kinase 10 and Transcription Factor 2 during Human Corneal Epithelial Wound Healing in vitro model.

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Increased NID1 Expression among Breast Cancer Lung Metastatic Women; A Comparative Analysis between Naive and Treated Cases.

BACKGROUND: Lungs are the second most common reported site of distant metastasis in Breast cancer after bone. Mostly the studies were conducted in cell lines and animal model. To date, there is no blood biomarker reported that could determine the breast cancer progression in terms of lung metastasis. OBJECTIVE: The aim of this study is to determine Nidogen-1 (NID1)'s mRNA and protein expressions in non-invasive blood samples of breast cancer, in early (II) and lung metastasis advanced stages (III & IV) of naive and treated groups. To determine the functional association of NID1, we employed an in silico analysis, STRING database version 11. METHODS: A total of n = 175 cases of breast cancer were recruited in our study. Real time quantitative PCR and ELISA were performed to analyze the mRNA and protein expressions of NID1 respectively. An in silico method is also used to assess NID1's interactome. Some significant patents related to this topic were also studied and discussed in this research paper. RESULTS: The results show high levels of NID1's mRNA in the naive group (Group A) as compared to treated group (Group B). Similar trend of increased NID1's protein expressions was also observed among naive and treated groups, respectively. Our results also show the significant impact of treatment on NID1's gene and protein expressions. In silico analysis has revealed the functional association of NID1 with its different interactome protein partners. CONCLUSION: The increased expression of NID1 in early to advanced naive as compared to the treated groups with lung metastasis makes it a promising marker which has pro-metastatic role in breast cancer.

Association of Cyclin Dependent Kinase 10 and Transcription Factor 2 during Human Corneal Epithelial Wound Healing in vitro model.

Proper wound healing is dynamic in order to maintain the corneal integrity and transparency. Impaired or delayed corneal epithelial wound healing is one of the most frequently observed ocular defect and difficult to treat. Cyclin dependen kinase (cdk), a known cell cycle regulator, required for proper proliferating and migration of cell. We therefore investigated the role of cell cycle regulator cdk10, member of cdk family and its functional association with transcriptional factor (ETS2) at active phase of corneal epithelial cell migration. Our data showed that cdk10 was associated with ETS2, while its expression was upregulated at the active phase (18 hours) of cell migration and gradually decrease as the wound was completely closed. Topical treatment with anti-cdk10 and ETS2 antibodies delayed the wound closure time at higest concentration (10 µg/ml) compared to control. Further, our results also showed increased mRNA expression of cdk10 and ETS2 at active phase of migration at approximately 2 fold. Collectively, our data reveals that cdk10 and ETS2 efficiently involved during corneal wound healing. Further studies are warranted to better understand the mechanism and safety of topical cdk10 and ETS2 proteins in corneal epithelial wound-healing and its potential role for human disease treatment.

Identification of Aspergillus flavus and aflatoxin in home mix layer poultry feed in relation to seasons in Karachi, Pakistan.

Fungal toxins in feed are leading issue in poultry industry causing a detrimental effect on the performance and health of poultry. The study was carried out to determine the incidence and concentration of the aflatoxins and their major producer Aspergillus flavus in home mix layer poultry feed in respect of seasonal variation throughout the year. A total of (n = 204) home mix poultry layer feed samples were analyzed for the isolation of fungi. The isolates were initially screened through colony morphology and microscopic examination. However, aflatoxin concentration was determined by ELISA. Revealed results indicated that, the highest percentage of A. flavus was found during the months of June to August 50/54 (92.5%) followed by September to November 43/65 (66.1%), March to May 21/40 (52.5%), and December to February 18/45 (40%). As a whole, the incidence was recorded 132/204 (64.7%). Moreover, of the 132 samples, 41 (31%) were exceeded in respect of aflatoxin contamination from the legal limit (20 µg/kg) imposed by Food Drug Association (FDA). Statistically, the growth of A. flavus and aflatoxin production was found significantly different in respect of seasonal variation. As highest total viable fungal count (9.9 × 104 CFU/g) and aflatoxin level (72.27 µg/kg) were recorded during the months of June to August and lowest in December to February. Consequently, instantaneous essential control measures are demanded regarding appropriate storage and adequate drying in post-harvesting season. Along with surveillance plans and austere regulations for monitoring the aflatoxin contents for the wellbeing of consumers.

Molecular basi of superoxide dismutase alterations in Spirometry proven bronchial asthma.

OBJECTIVE: To study alterations in superoxide dismutase at molecular level in spirometry-proven bronchial asthma. METHODS: This pilot study was conducted at Baqai Medical University Hospital, Karachi, from June to December 2013, and comprised spirometry-proven asthmatics. The allele frequencies of missense polymorphisms of the exon-intron of a superoxide dismutase, copper-zinc superoxide dismutase were included in the analysis and compared with their age- and gender-matched healthy controls. RESULTS: Of the 45 participants, 30(66.7%) were cases and 15(33.3%) were controls. The mean age of cases and controls was 37.77±11.95 and 37.27±11.81 years, respectively. The case population showed significant mean baseline and predicted spirometric values (p<0.05). The mean serum superoxide oxide dismutase in cases and controls was 62.53± 15.23 and 55.65± 15.87, respectively. The superoxide dismutase genetic variants studied for the intronic polymorphism in copper-zinc superoxide dismutase showed increased risk of asthma compared with non-asthmatic controls. CONCLUSIONS: Levels of serum superoxide dismutase were elevated with concomitant amplification of copper-zinc superoxide dismutase gene.

Single step PCR for the identification of Low Density Lipoprotein Receptor (LDL-R) gene mutations.

BACKGROUND AND OBJECTIVE: This study was conducted to determine the common mutation of low density lipoprotein receptor in patients with familial hypercholesterolemia (FH) in our population and identify the different point mutation in the LDL-receptor gene. The main aim of this study was to reduce the cost of PCR without extracting DNA and do the diagnosis at single step. METHODS: This study was carried out in the period of one year, from 2009- 2011. All the patients selected for this study were from Dr. Ziauddin Hospital, National Institute of Cardiovascular Diseases, and Dr. Rubina Ghani's Pathological & Molecular Laboratories. While collecting the blood sample, the patients were in overnight fasting condition. The clinical and biochemical analysis was performed on hyperlipidemic patients (n=120) to determine the frequency of familial hypercholesterolemia in our population. After lipid profile the patients were selected and direct multiplex PCR (Polymerase chain reaction) was performed from whole blood collected in a single tube using forward and reverse primers of exons 3, 4, 9 and 14 of without extracting DNA. RESULTS: Genomic DNA was extracted from blood samples as well as direct whole ETDA blood of healthy control group and hypercholesterolemia patients to detect mutations in exons 3, 4, 9, and 14 of the LDLR gene, with modification in the technique by using type-specific primers. These results for exon 4 mutation were confirmed by DNA sequencing. CONCLUSION: Screening method based on PCR by using Kappa direct PCR could be a faster and cheaper method with least contamination for screening a large number of FH patients for mutation of LDLR gene.

Heterogeneity of BCR-ABL rearrangement in patients with chronic myeloid leukemia in Pakistan.

BACKGROUND AND OBJECTIVE: Breakpoint cluster region-Abelson (BCR-ABL) rearrangement or Philadelphia (Ph) chromosome in Chronic Myeloid Leukemia (CML) is derived from a reciprocal chromosomal translocation between ABL gene on chromosome 9 and BCR gene on chromosome 22. This chimeric protein has various sizes and therefore different clinical behaviour. The purpose of this study was to determine the heterogeneity of BCR-ABL rearrangement in patients with Ph(+)CML in Pakistan. METHODS: The study was conducted at Civil Hospital and Baqai Institute of Hematology (BIH) Karachi. Blood samples from 25 patients with CML were collected. Multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed to identify various BCR-ABL transcripts. RESULTS: All 25 samples showed BCR-ABL rearrangements. Out of these, 24 (96%) patients expressed p210 BCR-ABL rearrangements i.e. 60% (n=15) had b3a2 and 32% (n=8) had b2a2 rearrangements. Co-expression of b3a2 /b2a2 rearrangement and p190 (e1a3) rearrangement was also identified in two patients. CONCLUSION: It is apparent that majority of the patients had p210 BCR-ABL rearrangements. Frequency of co-expression and rare fusion transcripts was very low.

Frequency of JAK2 V617F mutation in patients with Philadelphia positive Chronic Myeloid Leukemia in Pakistan.

BACKGROUND AND OBJECTIVE: Co-existence of myeloproliferative disorders (MPD) and Janus associated kinase 2 mutation (JAK2 V617F) is a well-established fact. Only few case reports are available showing presence of JAK2 V617F mutation in chronic myeloid leukemia (CML). Purpose of this study was to determine the frequency of JAK2 V617F mutation in Philadelphia Chromosome positive (Ph (+)) CML patients in Pakistan. METHODS: The study was conducted from August 2009 to July 2010 at Civil Hospital and Baqai Institute of Hematology (BIH) Karachi. Blood samples from 25 patients with CML were collected. Multiplex reverse transcription polymerase chain reaction (RT-PCR) was performed for Breakpoint Cluster Region - Abelson (BCR-ABL) rearrangement. Conventional PCR was performed for JAK2 V617F mutation on BCR-ABL positive samples. RESULTS: All 25 samples showed BCR-ABL rearrangement. Out of these 11 samples (44%) had JAK2 V617F mutation; the remaining 14 (56%) cases showed JAK2 617V wild type. CONCLUSION: It is concluded that the co-existence of Ph (+)CML and JAK2 V617F mutation is possible.

Two novel mutations in exon 3 and 4 of low density lipoprotein (LDL) receptor gene in patients with heterozygous familial hypercholesterolemia.

OBJECTIVE: To determine the common mutation of low density lipoprotein receptor in hypercholesterolemia patients requiring screening for heterozygous familial hypercholesterolemia (HeFH) in Karachi. STUDY DESIGN: Case-series. PLACE AND DURATION OF STUDY: Dr. Ziauddin Hospital Laboratory and Dr. Rubina Ghani's Pathological and Molecular Laboratories, Karachi, for the PCR bench work from June 2008 to October 2009. METHODOLOGY: All the patients selected for this study were from Dr. Ziauddin Hospital and National Institute of Cardiovascular Diseases. All the patients having high total cholesterol and LDL-cholesterol were included in this study with premature coronary artery diseases or a family history of hypercholesterolemia. Exclusion criteria included Diabetes mellitus, hypertension, renal disease, hypothyroidism and steroid therapy. After lipid profile with overnight fasting, DNA was extracted from whole blood collected in EDTA (ethylenediamine tetra acetic acid) tube and multiplex PCR (polymerase chain reaction) using forward and reverse primers of exons 3, 4, 9 and 14 of base pairs 162, 431, 550 and 496 respectively. RESULTS: Out of total of 120 hypercholesterolemia cases, 42 patients were classical cases of HeFH (heterozygous familial hypercholesterolemia) with xanthomas, xanthelasmas and LDL-C > 160 mg/dl. The total cholesterol (260± 57 mg/dL) and LDL-C (192 ± 39 mg/dL ) of cases was significantly high as compared to, controls having total cholesterol (184 ± 27 mg/dL) and LDL-C (105 ± 22 mg/dL), p > 0.001. Two novel point mutations were noted in exon 3 and exon 4. The other 78 cases were probable with raised LDL-C (low density lipoprotein cholesterol) and family history of premature coronary heart diseases. CONCLUSION: The frequency of HeFH was 35% classical and 65% probable cases out of total 120 hypercholesterolemia patients from two tertiary care hospitals in Karachi. The point mutation on exon 3 and exon 4 of LDLR gene was the most common. PCR is useful for the detection of large re-arrangements in the LDL-receptor gene and is a rapid and reliable method for diagnosis of familial hypercholesterolemia.

Carotid intima-media thickness correlation with lipid profile in patients with familial hypercholesterolemia versus controls.

OBJECTIVE: To determine the variations in carotid intima-media thickness (CIMT) in familial hypercholesterolemia (FH) patients and its use as predictive marker for premature cardiovascular diseases. STUDY DESIGN: Comparative study. PLACE AND DURATION OF STUDY: National Institute of Cardiovascular Diseases and Dr. Ziauddin Hospital, Karachi, from June 2008 to October 2009. METHODOLOGY: Familial hypercholesterolemia was clinically diagnosed by premature coronary diseases, xanthomas, arcus cornealis and family history of premature coronary heart diseases. Controls were age matched normal individuals without hypercholesterolemia. Their lipid profile was tested after overnight fasting. CIMT was measured in mm using B-mode ultrasonography using linear probe. Student t-test was applied to compare mean CIMT of cases and the control. The mean CIMT values of the FH cases were correlated with LDL using Pearson's correlation test. RESULTS: Forty cases with hypercholesterolemia gave consent to participate in the study. These patients had total cholesterol ≥200 mg/dL and LDL ≥160 mg/dL as compared to twenty controls of similar age with total cholesterol ≤200 mg/dL and LDL ≤130 mg/dL. Mean CIMT for the cases was 0.77 ± 0.18 mm while mean CIMT for control was 0.59 ± 0.08 mm. The mean CIMT for the cases ranged from 0.7-1.83 mm and 0.48-0.73 mm for controls. Among the FH cases, 25% (n=11) had arterial plaques. Mean CIMT was significantly correlated to LDL-cholesterol (r = 0.725**, p < 0.001). CONCLUSION: In this study, CIMT was found to be significantly increased in familial hypercholesterolemia and it correlated with raised LDL-cholesterol. Both are predictive of premature cardiovascular diseases.

Elevated frequency of p53 genetic mutations and AgNOR values in squamous cell carcinoma.

BACKGROUND: Epidermal squamous cell carcinoma (SCC) isa common malignancy in Pakistan. We hypothesize that it is characterized by higher frequency of p53 genetic mutations and increased AgNOR values compared with squamous cell papilloma(SCP) and basal cell carcinoma (BCC). EXPERIMENTAL DESIGN: To test our hypothesis, 140 skin biopsies(including 20 normal skin, 20 SCP, 20 BCC and 80 SCC samples of various grades) were examined for p53 mutations using immunohistochemistry (IHC) and polymerase chain reaction (PCR). AgNOR staining was used for histological determination of AgNORindex. RESULTS: Both markers were undetectable in normal skin and were low in SCP. They were upregulated in BCC and SCC. PCR experiments revealed p53 mutations in 70% and 96.25% of BCC and SCC, respectively. Higher AgNOR values were seen in SCC than in BCC (mean AgNOR count = 5.81 +/- 31 and 8.36 +/- 19; percentage of AgNOR was 43.5% and 53% in BCC and SCC, respectively). Finally, p53 IHC score was found to be related to the AgNOR index in the histological grading of BCC and SCC (r = 10.983, p < 0.0001). CONCLUSION: Our results suggest that a higher frequency of p53 genetic mutations and increased AgNOR values exist in SCC compared with BCC and SCP. 'Consequently, SCC patients may have poorer prognosis'.

Screening of Five Common Beta Thalassemia Mutations in the Pakistani Population: A basis for prenatal diagnosis.

OBJECTIVES: Thalassemia is one of the most common autosomal single-gene disorder worldwide. The highest prevalence of the disease is in the "thalassemia belt" which includes the Mediterranean region, parts of the Middle East, the Indian subcontinent, the southern parts of the Far East, Pakistan and South-East Asia. This study aimed to detect the common molecular abnormalities of the beta thalassemia syndrome in Pakistan. METHODS: The study was conducted at the Institute of Hematology, Baqai Medical University, Karachi, Pakistan from August 2004 to November 2007. Blood samples of patients with beta thalassemia major (n = 400) were collected from hospital transfusion centres and diagnostic laboratories in different districts of Karachi representing five major ethnic groups including Punjabi, Pathan, Sindhi, Baluchi and Urdu speaking. All the samples were analysed for five common mutations by using the polymerase chain reaction technique ARMS (amplification of refractory mutation system). RESULTS: The data revealed five common mutations including IVS 1-5(G→C), Fr 41/42(-CTTT), Fr 8/9 (+G), IVS 1-1 and Del 619. These accounted for 90% of the total beta thalassemia genes in Pakistan. The IVS 1-5(G→C) was found to be the most common beta thalassemia gene in the Pakistani population with a frequency of 44.4% present in all major ethnic groups. CONCLUSION: The results of this study will be helpful in the establishment of a large scale prenatal diagnosis programme in Pakistan.

Clozapine induced neutrophil cytotoxicity in rats.

OBJECTIVE: The study was carried out to understand the pathogenesis of hematological dyscrasias and cytotoxicity following administration of both purified and commercially available form of Clozapine in an animal model. METHODS: The Albino Sprague Dawley rats (n=30) with an average weight of 180g were taken and divided into three groups. Haematological parameters including haemoglobin, haematocrit, RBC and differential counts, absolute indices, Red cell Distribution Width (RDW) and morphological features of RBCs by peripheral blood smear were performed by standard laboratory methods. Additionally Serum Iron Concentration (SIC), Total Iron Binding Capacity (TIBC) (Roche Ltd.) and the serum ferritin level (Randox Ltd.) were also determined in each group. All statistical analysis was performed using graph pad prism. RESULTS: Clozapine induced neutrophil toxicity was manifested in both experimental groups, with condensation and subsequent breakdown of chromatin material. CONCLUSION: Our data, raised concerns about haematological safety and the potential mechanisms of neutrophil cytotoxicity related to the use of this drug.

Hemoglobinopathies among five major ethnic groups in Karachi, Pakistan.

A brief survey of abnormal hemoglobin variants among the major ethnic groups of Karachi was conducted; 202,600 subjects were studied. Patients with low hemoglobin (Hb), low mean cell volume (MCV) and mean cell hemoglobin (MCH) including anemia, microcytosis, hypochromic hemolysis and target cells, were refered for the identification of hemoglobinopathy by molecular methods. Population screening showed that 60% had iron-deficiency anemia and 40% had hemolytic anemia, of which 20.6% was due to beta-thalassemia major, 13% beta-thalassemia trait, 5.1% sickle cell disease, 0.76% hemoglobin D Punjab (HbD Punjab), 0.32% hemoglobin C (HbC), and 0.22% hereditary persistence of fetal hemoglobin (HPFH).