Correlation of serum kisspeptin and leptin with non-obstructive azoospermia: A cross-sectional study in a subset of Karachi population.

Objective: To determine the association of serum kisspeptin, leptin, and other hormonal profile with non-obstructive azoospermia (NOA) in infertile male subjects. Methods: This cross-sectional study was conducted at Australian Concept Infertility Medical Center, and Ziauddin University, Karachi from March 2018 to March 2020. The duration of the study was two years. Serum samples of 106 azoospermic participants were taken. Division of the subjects was done on a histological basis into obstructive azoospermia (OA) n=36, NOA n=70 which were further divided into spermatid maturation arrest (SMA), n=41, and sertoli cell-only syndrome (SCOS) n=29. Serum kisspeptin and leptin were measured by ELISA (Cloud-Clone Corp). Results: The follicle-stimulating hormone (FSH) (p<0.01), luteinizing hormone (LH) (p<0.01), thyroid-stimulating hormone (TSH) (p<0.01), and estradiol (p<0.01) was significantly high in the NOA group. However, kisspeptin was significantly decreased (p<0.01) in the NOA group. In the multivariate analysis after adjusting for other variables, the results showed that with the decrease in kisspeptin, the chances of being NOA were increased. Moreover, with the increase in Leptin, FSH, LH, and TSH the chances of being NOA were significantly enhanced. Conclusion: Serum kisspeptin, leptin, FSH, LH, TSH, and estradiol can be a potential marker for NOA in terms of better diagnosis, targeted therapeutic management, and the decision to proceed with surgical intervention.

Comparison of KRAS gene in circulating tumor DNA levels vs histological grading of colorectal cancer patients through liquid biopsy.

Background: To determine KRAS gene in circulating tumor DNA in comparison with histological grading through liquid biopsy in colorectal cancer patients. Methods: This dual-centered cross-sectional study included 73 diagnosed patients of colorectal cancer at different grading levels [Grade I, well differentiated (n = 7, 9.5%); Grade II, moderately differentiated (n = 14,18.9%); and Grade III, poorly differentiated (n = 52, 70%)]. Blood was collected, and plasma was separated. ctDNA was extracted, using magnetic bead-based technique (MagMAX Cell-Free DNA kit). KRAS gene was quantified through qPCR. STRING database was used to find KRAS interactomes. Results: Mean threshold cycle (CT value) of KRAS gene in Grade III samples showed significantly higher (P = 0.001) levels of ctDNA (2.7 ± 1.14) compared with Grade II and Grade I (3.1 ± 0.68, 2.3 ± 0.60), respectively. Grading characterization showed that rectal cancer (n = 22, 42.3%) with Grade III (68.8%) was more prevalent than colon and sigmoid cancer (n = 19, 36.5%, n = 11, 21%, respectively). STRING database showed 10 functional genes interacting with KRAS expressed as gene/proteins. Conclusion: Liquid biopsy can be used to detect ctDNA in plasma of CRC patients and enabled to detect the KRAS gene by qPCR. The technique being less invasive and cost-effective is convenient for multiple biopsies in different cancers.

Detection of Kras Gene in Colorectal Cancer Patients through Liquid Biopsy: A Cost-effective Method.

OBJECTIVE: To detect the Kras gene through liquid biopsy, a less invasive technique in diagnosed colorectal cancer patients. STUDY DESIGN: Cross-sectional study. PLACE AND DURATION OF STUDY: Department of Oncology, Dr. Ziauddin Hospital and Bait-us-Sukoon Cancer Hospital, Karachi, from 2019 to 2020. METHODOLOGY: Circulating tumor DNA (ctDNA) in colorectal cancer patients was extracted through magnetic bead technique using MagMAX cell free DNA kit (Thermofisher, Uk). The frequency of Kras gene was quantified using a real-time polymerase chain reaction (RT-PCR) assay (qPCR). ANOVA and Chi-square tests were utilised for statistical analysis. RESULTS: Mean threshold cycle (CT) of Kras gene showed significantly higher expression 15.6 ± 1.82 (p=0.001) in stage IV CRC cases compared to early stages (19.53 ± 18.223.7 ± 2.9 and 19.8 ± 2.69 of stage 1, 2 and 3, respectively. Similarly, ΔCT mean of Kras gene at stage IV showed significantly higher expression of 2.48 ± 1.40 (0.048), compared to 2.39 ± 0.6, 3.12 ± 0.68 and 3.15 ± 0.41 of stage 1, 2 and 3, respectively. Males (n=40, 55%) showed significant association (p=0.001) with CRC compared to females (n=33, 45%). Categorisation of tumor types within different age groups revealed that colon cancer was more frequent (n=11, 15.1%) in the 41-50 age group, while rectal cancer was more frequent (n= 11, 15.1%) in the 41-50 age group, while rectal cancer was more in the 51-60 age group (n=11, 15.1%). CONCLUSION: Kras gene was detected with significantly increased levels in plasma of CRC patients at advanced stages. This confirms that liquid biopsy can be used to detect Kras gene in ctDNA of CRC patients through a magnetic bead based technique. Key Words: Liquid biopsy, Circulating tumor DNA, KRAS, Colorectal cancer, Real-time polymerase chain reaction.

Spermatozoa retrieval in azoospermia and expression profile of JMJD1A, TNP2, and PRM2 in a subset of the Karachi population.

BACKGROUND: The difficulties encountered in surgical spermatozoa retrieval for intracytoplasmic sperm injection procedure in azoospermic men have stressed the dire need for a robust biomarker for the prediction of spermatozoa retrieval. Data have highlighted the role of JMJD1A (Jumonji domain-containing 1A), a histone H3K9 demethylase, and other nuclear proteins, protamines (PRM) and transition nuclear proteins (TNP), as biomarkers in male infertility. OBJECTIVE: To access successful spermatozoa retrieval at the time of intracytoplasmic sperm injection by evaluating the mRNA expression profile of JMJD1A, TNP, and PRM in testicular tissue. MATERIALS/METHODS: About 100 azoospermic patients, who visited the Australian Concept Infertility Medical Center, Karachi for spermatozoa retrieval by testicular sperm extraction or microsurgical testicular sperm extraction participated in the study. mRNA expression of the JMJD1A, TNP1, TNP2, PRM1, and PRM2 genes was determined. Patients were categorized into successful spermatozoa retrieval (n = 42) group and unsuccessful spermatozoa retrieval (n = 58) group. RESULTS: Azoospermic men in successful spermatozoa retrieval had significantly increased expression of JMJD1A, TNP2, and PRM2. The hormonal parameters - follicle-stimulating hormone, luteinizing hormone, and thyroid-stimulating hormone were significantly higher in unsuccessful spermatozoa retrieval. Multivariate analysis showed a significant association between JMJD1A, TNP2, PRM2, and successful spermatozoa retrieval. The area under the receiver operating characteristics curve showed a significant discriminatory ability to predict the spermatozoa retrieval outcome in azoospermic patients for mRNA expression of JMJD1A, TNP2, and PRM2 was 71, 72, and 73%, respectively. The area under the curve for follicle-stimulating hormone, luteinizing hormone, and thyroid-stimulating hormone was 0.67, 0.81, and 0.65, respectively. DISCUSSION: Our study demonstrates that the mRNA expression profile of JMJD1A, TNP2, and PRM2 along with hormonal parameters, is a useful marker to assess the probability of spermatozoa retrieval before intracytoplasmic sperm injection intervention. CONCLUSION: The probability of spermatozoa retrieval in azoospermic patients is increased when the mRNA expression profile of JMJD1A, TNP2, and PRM2 in testicular tissue is increased.

Potential Utility of Cell Free High Mobility Group AT-hook 2 (HMGA2) as a Prognostic Biomarker in Liquid Biopsies of Oral Squamous Cell Carcinoma.

BACKGROUND: Locoregional spread is a frequent finding in oral cancer which dictates poor prognosis. HMGA2 expression has been linked to malignant traits of oral cancer in tissue biopsies however, data on HMGA2 expression in liquid biopsies in oral cancer is sparse. Purpose of this study was to explore prognostic relevance of HMGA2 in liquid biopsies of oral cancer patients. PATIENTS AND METHODS: After obtaining approval from Institutional Review Board of Ziauddin University and informed written consent from study subjects, expression of circulating HMGA2 was evaluated in 96 OSCC cases and 100 age and sex matched controls via real time PCR using specific set of primers. We further analyzed relationship of various sociodemographic and clinicopathological variables with HMGA2expression and explored its prognostic potential. RESULTS: Expression was seen in 22 (23%) cases. A higher expression was observed among subjects with local invasion (52.6% vs 47.4 %), distant metastasis (71.4% vs 28.6%) and tumor recurrence (57.1% vs 42.9%) p.

Protamine 1/Protamine 2 mRNA ratio in nonobstructive azoospermic patients.

Altered protamine 1 (PRM1)/ protamine 2 (PRM2) mRNA ratio in testicular biopsy samples correlates with sperm quality and its fertilising ability. This study is planned to assess PRM1/ PRM2 mRNA ratio in subgroups of azoospermia to suggest a more reliable and accurate marker for assessing sperm quality in nonobstructive azoospermia (NOA). A cross-sectional study was done on testicular biopsy samples, taken from 106 azoospermic patients. Samples were histologically classified into subgroups: 36 obstructive azoospermia (OA), and two groups of NOA: 41 round spermatid maturation arrest (SMA) and 29 Sertoli cell-only syndrome (SCOS). OA samples showed histologically normal spermatogenesis and serve as a positive control. mRNA expression of jumonji domain-containing 1A (JMJD1A), PRM1, PRM2 and transition nuclear proteins (TNP1, TNP2) genes was determined, by RT-qPCR. Significantly lower expression of JMJD1A (p < .001), PRM1 (p = .0265) and PRM2 (p = .0032) has been seen in the SCOS group of NOA. We found significant (p < .001) increase in PRM1/PRM2 mRNA ratio in testicular biopsy samples of SCOS group of NOA patients and significant negative correlation of PRM1/PRM2 mRNA ratio with JMJD1A. Hence, PRM1/PRM2 mRNA ratio may represent a more reliable and accurate marker to assess sperm quality in NOA in addition to standard semen parameters.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Cancer Cell-derived Secretory Factors in Breast Cancer-associated Lung Metastasis: Their Mechanism and Future Prospects.

In Breast cancer, Lung is the second most common site of metastasis after the bone. Various factors are responsible for Lung metastasis occurring secondary to Breast cancer. Cancer cellderived secretory factors are commonly known as 'Cancer Secretomes'. They exhibit a prompt role in the mechanism of Breast cancer lung metastasis. They are also major constituents of hostassociated tumor microenvironment. Through cross-talk between cancer cells and the extracellular matrix components, cancer cell-derived extracellular matrix components (CCECs) such as hyaluronan, collagens, laminin and fibronectin cause ECM remodeling at the primary site (breast) of cancer. However, at the secondary site (lung), tenascin C, periostin and lysyl oxidase, along with pro-metastatic molecules Coco and GALNT14, contribute to the formation of pre-metastatic niche (PMN) by promoting ECM remodeling and lung metastatic cells colonization. Cancer cell-derived secretory factors by inducing cancer cell proliferation at the primary site, their invasion through the tissues and vessels and early colonization of metastatic cells in the PMN, potentiate the mechanism of Lung metastasis in Breast cancer. On the basis of biochemical structure, these secretory factors are broadly classified into proteins and non-proteins. This is the first review that has highlighted the role of cancer cell-derived secretory factors in Breast cancer Lung metastasis (BCLM). It also enumerates various researches that have been conducted to date in breast cancer cell lines and animal models that depict the prompt role of various types of cancer cell-derived secretory factors involved in the process of Breast cancer lung metastasis. In the future, by therapeutically targeting these cancer driven molecules, this specific type of organ-tropic metastasis in breast cancer can be successfully treated.

Circulating miR-21 as a prognostic and predictive biomarker in oral squamous cell carcinoma.

BACKGROUND & OBJECTIVE: The high-throughput analysis of circulating microRNAs (miRNAs) is an active area of biomarker research. The oral cancer remains a common cancer among Pakistani males that continues to present at an advance stage, thus exhibiting poor survival. MiRNA 21 (miR-21) is the most consistently over-expressed miRNA in different types of tumor tissues. However, information regarding expression of miR-21 in plasma remains to be resolved. Therefore, present study was designed to investigate if miR-21 was expressed in plasma of patients with oral cancer, and further explore its diagnostic and prognostic potential. METHODS: Present study was conducted at Ziauddin University and Karachi Institute of Radiotherapy and Nuclear Medicine (KIRAN). Histologically confirmed cases of oral squamous cell carcinoma were recruited from Oncology Department of Ziauddin Hospital between 2013 and 2017. Controls were carefully selected after considering age, gender and socioeconomic condition. MiRNA was extracted and immediately reverse transcribed to cDNA. MiR-21 expression was evaluated using probes specifically designed for Real time quantitative polymerase chain reaction. RESULTS: A significant over expression of miRNA 21 was observed in histologically confirmed cases as compared to controls. The increased expression of miRNA 21 was also reported to be associated with tumor size, metastasis and local invasion (p<0.05). CONCLUSION: The expression of circulating miR-21 in plasma samples of oral cancer patients makes it a promising diagnostic and prognostic marker.

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.

A study of genotypes, mutants and nucleotide sequence of hepatitis B virus in Pakistan: HBV genotypes in pakistan.

BACKGROUND: Hepatitis B virus (HBV) genotypes and mutations are gaining importance in determining the clinical course of chronic liver disease. OBJECTIVES: To determine and compare the distribution of HBV genotypes and genomic variations in Pakistan to other parts of the world. PATIENTS AND METHODS: We conducted a prospective study at Aga Khan University Hospital from December 2006 to December 2008. HBV genotype was determined in 257 HBV DNA-positive patients. Patients were divided into two groups according to HBeAg positivity. Mutations in the pre-core and core promoter regions of HBV were determined in HBeAg-negative patients by line probe INNOLIPA assay. RESULTS: The mean±SD age of patients was 28±5 years; there were 201 (78%) men. HBeAg was positive in 219 (85%) patients and negative in 38 (15%). HBeAg-positive patients were younger than HBeAg-negative patients (95% vs 21% in ≤30 years, p<0.001). HBV genotype D found in 247 (96.2 %) patients followed by a combined infection with HBV genotype B+D in 9 (3.3%) and 1 (0.5%) with genotype A. The mutations identified in 38 HBeAg-negative patients were T1762/A1764 in 21 (55.2%), PC mutant in 7 (18.4%), T1762/A1764/PC mutant in 2 (5%) and T1762/A1764/PC wild mutation in 1 (2%); no mutation identified in 7 (18.4%). Phylogenetic analysis did not show any significant differences between HBV genotype D isolated from Pakistan and those isolated from other parts of the world. CONCLUSIONS: HBV genotype D is predominant in Pakistan, irrespective of HBeAg status. PC and BCP mutations were found in significant numbers of patients infected with genotype D. The HBV genotype D isolates from Pakistan are identical to the sequences isolated from other parts of the world.

Comparison of high performance liquid chromatography, radio immunoassay and electrochemiluminescence immunoassay for quantification of serum 25 hydroxy vitamin D.

OBJECTIVE: To compare the performance of radioimmunoassay (RIA) with high-performance liquid chromatography (HPLC) and electrochemiluminescence (ECLIA) for the quantification of vitamin D (25OHD). METHODS: HPLC method for the determination of 25OHD in human biological samples was developed and compared in terms of accuracy and precision with a commercially available RIA assay. Performance of RIA assay with ECLIA technology for 25OHD analysis was further compared. RESULTS: Median 25OHD levels with HPLC vs. RIA were 50.1nmol/L (IQ=17.7-199.4nmol/L) and 51.1nmol/L (IQ=12.5-187.2nmol/L) respectively, whereas median 25OHD concentration with RIA vs. ECLIA was 32.4nmol/L (9.98-199.7nmol/L) and 29.9nmol/L (4.9-214.6nmol/L), respectively. Comparison data for HPLC vs. RIA showed RIA=-1.13+1.01 (HPLC) (RMSE=11.2nmol/L) and for RIA vs. ECLIA revealed, ECLIA=3.21+0.9 (RIA) (RMSE9.6nmol/L). CONCLUSION: Acceptable correlation was observed among HPLC and RIA and also with RIA and ECLIA in quantification of 25OHD.

Albumin precursor and Hsp70 modulate corneal wound healing in an organ culture model.

In order to investigate the role of albumin precursor and Hsp70 in corneal wound healing, we have analyzed the distribution of these proteins in wounded and non-wounded corneas of rabbits and the effects of topical applications of anti-albumin precursor and anti-Hsp70 antibodies on wound healing. Anti-albumin precursor and anti-Hsp70 antibodies were topically applied in healing corneal epithelium of rabbit eyes in organ culture. Corneas were allowed to heal in vitro for up to 120 h in serum-free medium with 5 and 10 µg/ml or without (migrating control) anti-albumin precursor/ or anti-Hsp70 antibodies. Fibronectin (Fb) (5 µg/ml) was used as a positive control. Immunofluorescence labelling was used to detect proteins in corneal epithelium at various time intervals following an epithelial defect. Delay in wound healing (p<0.005) was observed with 10 µg/ml anti-albumin antibody labelling. A similar pattern was observed when anti-fibronectin antibody (5 µg/ml) alone and in combination with anti-albumin (10 µg/ml) was ectopically added with wound closure occurring at 120 h. However with anti-Hsp70 antibody (5 µg/ml) slightly delayed (p<0.005) wound closure was observed at 96 h and considerable retardation >120 h with 10 µg/ml. Additionally, immunofluoresence showed a strong co-localization of Hsp70 and albumin precursor during the active phase of wound healing. The presence of albumin precursor and Hsp70 in the epithelial compartment of the cornea indicates a role for these proteins in modulating cell behavior such as epithelial growth, adhesion or regeneration, thus contributing to corneal epithelial wound healing.

Identification of myeloperoxidase, alpha-defensin and calgranulin in calcium oxalate renal stones.

BACKGROUND: In order to understand the mechanism of stone genesis, it is essential to determine the characteristics of macromolecules constituting the urinary stones. We characterized proteins from the inner core and outer matrix of calcium oxalate (CaOx) renal stones. METHODS: Inner core and outer matrix of CaOx renal stones were separated and proteins were extracted with a buffer containing SDS and beta-mercaptoethanol. Proteins were analyzed and purified by SDS-PAGE and RP-HPLC respectively. The protein bands from gel and protein fractions were sequenced by MALDI TOF mass spectrometry. ELISA, western and slot blot immunoassays were performed to confirm the identity of the proteins in stones and urine of the stone formers. The potential of the identified protein as an effective promoter or inhibitor was assessed by observing their effects on CaOx crystallization using aggregometer. RESULTS: The inner core extract predominantly exhibited protein species in the molecular weight range of 12-14 kDa. However, a 66 kDa band, identified as osteopontin was also detected in the inner core along with outer matrix and in the urine of stone formers and non stone formers. Purification of low molecular weight proteins was carried out by reversed phase HPLC. Tandem mass spectrometry analysis identified them as myeloperoxidase chain A (MPO-A), alpha-defensin, and calgranulin. ELISA, western blot and slot-blot immuno-assays further confirmed their presence restricted to the inner core and not in the outer matrix. Turbidity assays showed that low molecular weight renal stone proteins promoted the aggregation of CaOx crystals. CONCLUSIONS: Persistent hyperoxaluria leads to tubular epithelial injury, resulting in the release of these anti-inflammatory proteins. These proteins could have been first adsorbed on CaOx crystals thereby become a part of nucleation process leading to inner matrix formation.

Changes in albumin precursor and heat shock protein 70 expression and their potential role in response to corneal epithelial wound repair.

Many proteins displayed differential expression (either up- or down-regulation) when proteome of migrating and non-migrating epithelium was assessed using 2-DE and ESI-Q-TOF MS/MS. From the up-regulated set, we have identified for the first time a 69-kDa albumin precursor protein with four peptides sequences and 70-kDa heat shock protein (hsp70) with one peptide in the active phase of cell migration (48 h) during the healing process. Western blot analysis was used to further characterize these proteins at different phases (24, 48 and 72 h) of healing. An increase in the mRNA expression (measured using RT-PCR) in the active migration phase (48 h) for albumin precursor and hsp70 was also observed. Furthermore, co-immunoprecipitation studies with anti-albumin precursor and anti-hsp70 antibodies, followed by immunoblotting with anti-fibronectin antibody demonstrated a novel and biologically relevant interaction between albumin precursor protein and fibronectin in corneal epithelial wound healing but not with hsp70. The increased gene and protein expression of albumin and hsp70 during the active phase of cell migration (48 h) in the corneal epithelium suggests their possible role in corneal wound healing. These findings may have broader implications for developing therapeutic strategies for treating wound healing disorders.