Investigating the Protein-Based Therapeutic Relationship between Honey Protein SHP-60 and Bevacizumab on Angiogenesis: Exploring the Synergistic Effect through In Vitro and In Silico Analysis.

Honey is a natural product produced by honeybees, which has been used not only as food but also as a medicine by humans for thousands of years. In this study, 60 kDa protein was purified from Pakistani Sidr honey named as SHP-60 (Sidr Honey Protein-60), and its antioxidant potential and the effect of Bevacizumab with purified protein on in vitro angiogenesis using human umbilical vein endothelial cells (HUVEC) were investigated. We further validated the molecular protein-protein (SHP-60 with Bevacizumab) interactions through in silico analysis. It showed very promising antioxidant activity by reducing 2,2-diphenyl-1-picrylhydrazyl free radicals with a maximum of 83% inhibition at 50 µM and an IC50 of 26.45 µM statistically significant (**p < 0.01). Angiogenesis is considered a hallmark of cancer, and without it, the tumor cannot grow or metastasize. Bevacizumab, SHP-60, and both in combination were used to treat HUVEC, and the MTT assay was used to assess cell viability. To demonstrate in vitro angiogenesis, HUVEC was grown on Geltrex, and the formation of endotubes was examined using a tube formation assay. HUVEC viability was dose-dependently decreased by Bevacizumab, SHP-60, and both together. Bevacizumab and SHP-60 both inhibited angiogenesis in vitro, and their combination displayed levels of inhibition even higher than those of Bevacizumab alone. We investigated the protein-protein molecular docking interactions and molecular dynamics simulation analysis of MRJP3 (major royal jelly protein 3) similar to SHP-60 in molecular weight with both the heavy chain (HC) and light chain (LC) of Bevacizumab. We found significant interactions between the LC and MRJP3, indicating that ASN468, GLN470, and ASN473 of MRJP3 interact with SER156, SER159, and GLU161 of LC of Bevacizumab. The integration of experimental data and computational techniques is believed to improve the reliability of the findings and aid in future drug design.

Identification of specific codon 201 mutation of the DCC Gene in the colonoscopic specimen of colorectal cancer.

Objective: To identify the mutation in codon 201 of the deleted in colorectal cancer gene in colorectal cancer, and to correlate that mutation to the histopathological grading of colorectal cancer. METHODS: The cross-sectional study was conducted from February 2019 to February 2021 after approval from the ethics review board of the Dow University of Health Sciences, Karachi, and comprised biopsy-proven colorectal cancer patients regardless of age and gender. After histopathological reporting, formalin-fixed paraffin-embedded tissue blocks of colorectal cancer were used for deoxyribonucleic acid extraction, followed by polymerase chain reaction optimisation and deoxyribonucleic acid Sanger sequencing for mutational analysis. Data was analysed using SPSS 25. RESULTS: Of the 100 biopsy specimens assessed, 45(45%) were selected. Of them, 13(29%) samples failed to show any band on gel electrophoresis. The remaining 32(71%) samples were used for Sanger sequencing. Of these, 1(3%) sample did not sequence, while 31(97%) showed sequencing. All the sequenced samples identified a mutation in codon 201 of exon 3 in the deleted in colorectal cancer gene; 30(97%) showed homozygosity, and 1(3%) showed heterozygosity. No significant association of point mutation was noted with various demographic and clinicopathological parameters (p>0.05). Conclusion: The deleted in colorectal cancer gene's missense mutation in codon 201 was frequently observed in colorectal cancer patients.

Responses of ß-thalassemia and compound heterozygote of Sickle/ßthalassemia of BCL11A Gene Polymorphism in Pakistani Patients.

Background and Objective: Beta-thalassemia major (ß-Thal) and compound heterozygote of Sickle ß-thalassemia (S-ß Thal) are hereditary autosomal recessive disorders resulting from mutations or deletion in ß-globin gene cluster. Patients with increased HbF levels having polymorphism at BCL11A site loci have shown clinical significance. The present study aimed to assess the frequency of BCL11A gene polymorphism in a study population of ß-Thal, S-ß Thal & Controls using Sanger sequencing leading to plot the HbF response of polymorphism with reference to wild type. Methods: The sample size of the study is n=180, groups were divided in Controls, ß-thal & S-ß thal. One ml blood was drawn from patients and controls to extract DNA for PCR amplification and BCL11A locus genotyping using Sanger sequencing. This study was carried out at Dow Research Institute of Biotechnology and Biomedical Sciences, for one year from March 2021 to February 2022. Results: The HbF response of three groups is hyperbolic with 83 for ß-Thal, 16 for S-ß Thal and close to zero for controls. The frequency of heterozygous variant GA of BCL11A gene polymorphism is 51%. The frequency of homozygous variant GG is 49%. Complete absence of wild type AA in patient group. The frequency of BCL11A polymorphism in control group was 43% (with male 18% and female 21%) showing wild type status of 57%. Conclusions: The patient groups of SCD and Beta thalassemia are devoid of wild type status. The wild type status of BCL11A is 57% even in control population. Higher level of HbF in B-thalassemia and SCD and B Thalassemia is a cost-effective screening marker before switching to an expensive genotyping testing.

Optimisation of a Method for the Differentiation of Human Umbilical Cord-derived Mesenchymal Stem Cells Toward Renal Epithelial-like Cells.

Human umbilical cord-derived mesenchymal stem cells (hucMSCs) can differentiate into multiple cell lineages, but few methods have been developed to generate kidney lineage cells. Due to their human origin, pluripotent nature and immunomodulatory properties, these stem cells are attractive candidates for clinical applications such as the repair or regeneration of damaged organs. This study evaluated the renal differentiation potential of hucMSCs, when exposed for 10 days to optimised concentrations of retinoic acid, activin-A and bone morphogenetic protein-7 (BMP-7) in various combinations, with and without the priming of the cells with a Wnt signalling pathway activator (CHIR99021). The hucMSCs were isolated and characterised according to surface marker expression (CD73, CD90, CD44, CD146 and CD8) and tri-lineage differentiation potential. The expression of key marker genes (OSR1, TBXT, HOXA13, SIX2, PAX2, KRT18 and ZO1) was examined by qRT-PCR. Specific marker protein expression (E-cadherin, cytokeratin-8 and cytokeratin-19) was analysed by immunocytochemistry. CHIR99021-primed cells treated with the retinoic acid, activin-A and BMP-7 cocktail showed epithelial cell-like differentiation - i.e. distinct phenotypic changes, as well as upregulated gene and protein expression, were observed that were consistent with an epithelial cell phenotype. Thus, our results showed that hucMSCs can efficiently differentiate into renal epithelial-like cells. This work may help in the development of focused therapeutic strategies, in which lineage-defined human stem cells can be used for renal regeneration.

A novel experimental model to investigate fungal involvement shows expression of Dectin-1 in periapical lesion pathogenesis.

BACKGROUND: Candida albicans is linked to persistent endodontic lesions. However, the recognition receptor that identifies it is not explored previously. OBJECTIVES: The aim of this study was to (1) establish a zymosan-induced model of apical periodontitis in mouse, (2) observe the expression of Dectin-1 and its possible relationship with toll-like receptor (TLR) 2 and (3) observe relationship between Osteopontin (OPN) and inflammatory cytokines. METHODS: A total of 138 Naval Medical Research Institute (NMRI) mice were randomly divided into; Experimental Group n = 69 and Zymosan Group n = 69. Periapical periodontitis was developed in right maxillary molar. The animals were sacrificed at 7, 21 and 42 days. Bone blocks containing the mesial root (n = 15 for qRT-PCR, n = 45 for enzyme-linked immune sorbent assay (ELISA)) were collected for mRNA expression and ELISA. While whole maxilla (n = 3 from each time interval) were used for histology and immunohistochemical analysis. One way analysis of variance (ANOVA) and Tuckey's posthoc was used for statistical analysis at p ≤ .05. RESULTS: TLR-2, Dectin-1 and TLR4-positive cells was detected at all time intervals in both groups. A strong positive correlation was observed between TLR-2 and Dectin-1 in both lesions (regular r = .680, p = .015, zymosan (r = .861, p < .001)). A significant correlation was found between OPN and tumour necrosis factor-alpha (TNF-α) in zymosan lesion (r = .827, p = .001). CONCLUSIONS: Immune cells of inflamed periapical tissue expressed Dectin-1 receptor in response to the microbial challenge from infected root canals and showed positive correlation with TLR-2 and OPN suggesting a possible receptor collaboration mediated by OPN. The expression of OPN and TNF-α showed positive correlation in response to fungal antigen, indicating a possible relationship.

Differential Diagnosis of Hematologic and Solid Tumors Using Targeted Transcriptome and Artificial Intelligence.

Diagnosis and classification of tumors is increasingly dependent on biomarkers. RNA expression profiling using next-generation sequencing provides reliable and reproducible information on the biology of cancer. This study investigated targeted transcriptome and artificial intelligence for differential diagnosis of hematologic and solid tumors. RNA samples from hematologic neoplasms (N = 2606), solid tumors (N = 2038), normal bone marrow (N = 782), and lymph node control (N = 24) were sequenced using next-generation sequencing using a targeted 1408-gene panel. Twenty subtypes of hematologic neoplasms and 24 subtypes of solid tumors were identified. Machine learning was used for diagnosis between two classes. Geometric mean naïve Bayesian classifier was used for differential diagnosis across 45 diagnostic entities with assigned rankings. Machine learning showed high accuracy in distinguishing between two diagnoses, with area under the curve varying between 1 and 0.841. Geometric mean naïve Bayesian algorithm was trained using 3045 samples and tested on 1415 samples, and showed correct first-choice diagnosis in 100%, 88%, 85%, 82%, 88%, 72%, and 72% of acute lymphoblastic leukemia, acute myeloid leukemia, diffuse large B-cell lymphoma, colorectal cancer, lung cancer, chronic lymphocytic leukemia, and follicular lymphoma cases, respectively. The data indicate that targeted transcriptome combined with artificial intelligence are highly useful for diagnosis and classification of various cancers. Mutation profiles and clinical information can improve these algorithms and minimize errors in diagnoses.

Expression of Toll-like receptor 2, Dectin-1, and Osteopontin in murine model of pulpitis.

OBJECTIVES: This in vivo animal study aimed to develop a murine model of pulpitis induced by pulp exposure with or without application of zymosan in Naval Medical Research Institute (NMRI) mice and observe expressions of Toll-like receptor (TLR)-2, TLR-4, Dectin-1, Osteopontin (OPN), tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-1ß. MATERIAL AND METHODS: A total of 168 NMRI mice were divided into two groups, i.e., group A (n = 84) (pulpitis induced by pulp exposure only) and group B (n = 84) (pulpitis induced by pulp exposure and zymosan application). Right maxillary molar pulps were exposed with » round bur, and animals were sacrificed at 0, 6, 9, 12, 24, 48, and 72 h. The exposed teeth were obtained for real-time polymerase chain reaction (qRT-PCR) analysis and histological and immunohistochemistry (IHC) analysis. RESULTS: Histological evaluation revealed a time-dependent steady increase in inflammation. Similar time-dependent increase in the expression of inflammatory cytokines was noted. Group A exhibited an increase in TLR-4, Dectin-1, and OPN at 6 h, while TLR-2 was expressed at 24 h. Group B expressed TLR-2, Dectin-1, and OPN at 9, 48, and 72 h, respectively (p ≤ 0.05). Expression of OPN and TNF-α exhibited a similar pattern in both groups. IHC also detected expression of TLR-2, Dectin-1, TLR4, and CD68 in some cells at 6 and 9 h. CONCLUSIONS: NMRI mice provided for a stable pulp inflammation model. Zymosan may be used to develop pulp inflammation model and study inflammatory response towards fungal antigens. Dental pulp expressed Dectin-1 receptor. OPN and TNF-α exhibited a similar expression pattern. CLINICAL RELEVANCE: Innate immunity of dental pulp is capable of detecting fungal pathogens.

Gene expression analysis of toll like receptor 2 and 4, Dectin-1, Osteopontin and inflammatory cytokines in human dental pulp ex-vivo.

BACKGROUND: Toll like receptors (TLR) 2 and 4 present on innate immune cells of the dental pulp detect cariogenic bacteria. Along with bacteria, C. albicans may also be present in dental caries. The presence of C. albicans can be detected by Dectin-1 a C type Lectin receptor. Expression of Dectin-1 in human pulpits has not been reported. Similarly, cytokines are released as a consequence of dental pulp inflammation caused by cariogenic bacteria. The T helper (Th) 1 inflammatory response leads to exacerbation of inflammation and its relationship with Osteopontin (OPN) is not known in pulp inflammation. OBJECTIVE: The aim of this study was to observe the expression of Dectin-1, TLR-2, OPN and pro-inflammatory cytokines in irreversibly inflamed human dental pulp and to observe relationship between Dectin-1/TLR-2 and OPN/Pro-inflammatory cytokines in the presence of appropriate controls. METHODS: A total of 28 subjects diagnosed with irreversible pulpitis were included in this ex-vivo study. Fifteen samples were subjected to standard hematoxylin and Eosin (H&E) and immunohistochemistry staining. Whereas, gene expression analysis was performed on 13 samples to observe mRNA expression of pro-inflammatory cytokines; tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1 beta (ß), IL-6 Dectin-1, OPN, TLR-2 and TLR-4. SPSS version 21 was used for statistical analysis. One way analysis of variance (ANOVA), Pearson correlation and Chi-square test were used at p ≤ 0.05. RESULTS: Gene expressions of Dectin-1, TLR-2 and TLR-4 were observed in all samples. Dectin-1 and TLR-2 expressions were significantly correlated (r = 0.5587, p = 0.0002). Similarly, OPN and TNF-α expression showed a significant correlation (r = 0.5860, p = 0001). The agreement between histologic and clinical diagnosis was 69.2% in the cases of irreversible pulpitis. CONCLUSION: Dectin-1 was expressed by inflamed human dental pulp. Dectin-1 and TLR-2 expression pattern was suggestive of a collaborative receptor response in inflamed pulp environment. OPN and TNF-α expressions showed a positive correlation indicating a possible relationship.

Differentiation of CD117+ Amniotic Fluid Stem Cells towards Nephron Progenitors.

Objective: The study aimed at isolation of CD117+ stem cells from amniotic fluid samples followed by their invitro differentiation towards nephron progenitors that can be potentially used for regenerative medicine studies and to further understand pathways involved in renal pathogenesis. Methods: This experimental study was conducted at Dow Research Institute of Biotechnology and Biomedical Sciences (DRIBBS), Dow University of Health Sciences, OJHA Campus Karachi from November 2019 to December 2020. After patient consent, a Pfannenstiel incision was performed by the gynecologist through abdominal and uterine muscles without cutting into Amniotic Membrane. Using a needle of 5CC syringe connected to sterile Redivac bottle, a blunt end insertion was passed through the membrane and the amniotic fluid was aseptically sucked into Redivac bottle, the ice bag was used for transporting amniotic fluid from hospital to the lab and samples were processed within 60 minutes after collection. Amniotic fluid was centrifuged at 4º C for 20 minutes at 1400xg. After centrifugation the cell pellet was treated for analysis of CD117+ cells using flowcytometry, once small percentage of CD117+ cells were identified the cells were prepared for differentiation and that was carried out using specific growth factors including BMP4, BMP7, FGF2, and retinoic acid, providing the niche to the stem cells for differentiation towards nephron progenitors which was confirmed by protein expression of Wilms Tumor-1 (WT1) using immunofluorescence analysis. The sample size for this invitro work was n=3. Results: We successfully isolated small percentage of CD117+ cells in amniotic fluid followed by in vitro expansion and differentiation towards nephron progenitor cells (NPCs) using well defined media and growth factors, initially differentiated cells were spindle shaped and showed fibroblastic appearance later at stage of nephron progenitors it attained the shape of rounded big clusters, differentiated cells stained positive for WT1 and negative for cluster of differentiation (CD117). Therefore, confirming the successful isolation and differentiation of amniotic fluid stem cells towards nephron progenitors. Conclusion: To the best of our knowledge this is the first study from the country on the use of Amniotic fluid stem cells and their differentiation towards nephron progenitors that can be used as substitution source of cell therapy for exploration of renal diseases at cellular and molecular level and potential regenerative medicine applications.

Effect of glucose mediated oxidative stress on apoptotic gene expression in gingival mesenchymal stem cells.

BACKGROUND: Diabetes is a common disease that causes gingival and periodontal problems. Stem cells isolated from dental sources are an emerging area of research with a potential to facilitate regenerative medicine. The stem cells retain the property of self-renewal and the ones isolated from dental sources are mainly multipotent mesenchymal stem cells that have the ability to self-renew as well as differentiation towards multiple lineages. OBJECTIVES: We aimed to isolate and characterize gingival mesenchymal stem cells by pluripotency markers and investigated the effect of oxidative stress on growth kinetics and apoptotic gene expression of gingival cells exposed to glucose mediated oxidative stress. METHODS: In this study, we isolated gingival mesenchymal stem cells from gingiva. This was followed by morphologic analysis using inverted phase contrast microscopy and molecular profiling of these cells for the mRNA expression of specific genes. The isolated cells were cultured till passage 3 and then exposed to oxidative stress (high glucose concentration). We measured the apoptotic gene expression and compared their growth kinetics. RESULTS: The results showed that oxidative stress produced by glucose reduced growth kinetics and increased apoptotic gene expression in gingival mesenchymal stem cells. According to the genetic results, glucose activated TNF family to initiate apoptosis. CONCLUSION: In conclusion, the present study demonstrated that high glucose obliterated cellular proliferation testified by evaluating growth kinetics and induced apoptotic gene expression in gingival mesenchymal stem cells. This initiated extrinsic apoptotic pathway mediated by TNF family. Therefore, in diabetes oral health condition is compromised and periodontal disease is common.

Expression of miR-31 in saliva-liquid biopsy in patients with oral squamous cell carcinoma.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) is a commonly reported cancer in men and is second only to breast cancer in women in Pakistan.. Investigations for identifying biomarkers of OSCC are essential for diagnostic, therapeutic, or prognostic significance. This study aims to examine the miR-31 expression in the pre- and post-operative OSCC patients and correlate this expression with clinicopathological characteristics. METHODS: Patients with histopathologically confirmed OSCC who had undergone surgical resections of tumours were recruited. A total of 40 saliva samples (pre- and post-operative) were collected from 19 patients and two healthy individuals. Levels of salivary miR-31 expressions were examined through quantitative reverse transcription polymerase chain reaction. RESULTS: The salivary miR-31 expression was significantly higher in the preoperative patients than in postoperative patients (p < 0.001). However, no significant correlation had been found between the salivary miR-31 expression and clinicopathological characteristics (p > 0.05). CONCLUSION: Our data suggest that miR-31 can be used as an adjunct non-invasive marker to monitor surgery outcomes during postoperative follow-up in patients with OSCC.

Induced pluripotent stem cells derived cardiomyocytes from Duchenne Muscular Dystrophy patients in vitro.

OBJECTIVE: This study aimed at the in vitro generation of DMD-cardiomyocytes from patient-specific induced pluripotent stem cells derived from a Pakistani patient for future work on DMD in vitro disease modeling and drug testing for efficacy and toxicity. METHODS: This in vitro experimental study was carried out from December 2018 to January 2019 at Stem Cells and Regenerative Medicine Lab (SCRML) at Dow Research Institute of Biotechnology and Biomedical Sciences (DRIBBS), Dow University of Health Sciences (DUHS) Urine derived DMD-iPSCs were used which had been generated previously from a Pakistani DMD patient who had been selected through non-random purposive sampling. These were differentiated towards cardiomyocytes using Cardiomyocytes Differentiation media having specified growth factors and then the molecular characterization of the differentiated cells was done using immunofluorescence. RESULTS: Pakistani patient's DMD-Cardiomyocytes were generated and their identity was confirmed by positive immunofluorescence for the expression of cardiac markers NKX2-5 and TNNT-2. CONCLUSION: This study aimed for in vitro generation of DMD cardiomyocytes for future application in disease modeling, new drug testing for efficacy and toxicity, as well as for drug-testing for tailored personalized therapy. To the best of our knowledge, this was the first time DMD-Cardiomyocytes were generated from Pakistani DMD patients using their own induced pluripotent stem cells.

Genetic reprogramming of cord blood derived endothelial colony forming cells towards human induced pluripotent stem cells using episomal plasmids.

OBJECTIVE: This study aimed to isolate human umbilical cord blood derived endothelial colony forming cells (ECFCs) followed by their integration free reprogramming towards induced pluripotent stem cells (iPSCs) and molecular characterization of both cell types using multicolour flowcytometery and immunofluorescence respectively. METHODS: The cord blood was collected from 37-39 weeks of gestational ages after C-section ex-utero from Dow University Hospital. The ECFCs isolated after ficoll based separation of cord blood mononuclear cells (CBMNCs) which on emergence characterized through flow cytometry and reprogrammed towards induced pluripotent stem cells (iPSCs) using episomal vectors, the iPSCs were characterized using immunofluorescence. The study was conducted at Stem Cells and Regenerative lab, Dow Research Institute of Biotechnology and Biomedical Sciences, Dow University of health sciences OJHA campus. The study time duration was about one year (October 2017-October 2018); study design was in vitro experimental. The sample size of the study was n=3. RESULTS: The isolated ECFCs were evaluated using flowcytometery which showed positive expression for CD31, CD34, CD146 cell surface markers and negative for CD90. The successful reprogramming of ECFCs towards iPSCs was confirmed by immunofluorescence using OCT-4 which is considered to be a master regulator of pluripotency. CONCLUSIONS: To the best of our knowledge this study was the first attempt to integration free reprogramming of cord blood derived endothelial colony forming cells towards induced pluripotent stem using episomal plasmids. Cells that have been isolated from cord blood and those that have been reprogrammed both have potential therapeutic applications in regenerative medicine.

Episomal reprogramming of Duchenne muscular dystrophy patients derived CD3+ T cells towards induced pluripotent stem cells.

OBJECTIVE: To derive Duchenne muscular dystrophy patient specific induced pluripotent stem cells (iPSCs) from CD3+T cells of DMD patients using episomal reprogramming and characterization of these DMD-iPSCs using immunofluorescence to confirm their pluripotent state. METHODS: DMD patients were selected based upon their clinical history and examination. Peripheral blood mononuclear cells were isolated from peripheral blood of DMD patients (n=3) by density gradient centrifugation and were used to generate DMD patient specific T cells (DMD-T cells) using rhIL-2, plate bound anti CD3 antibody and T cell specific media along with specific culture conditions that promote T cell expansion. CD3+ T cells were characterized by flow cytometry and reprogrammed using episomal plasmid to generate DMD-iPSCs. These DMD-iPSCs were characterized using immunofluorescence. The study was carried out at Dow Research Institute of Biotechnology and Biomedical Sciences during August 2017- July 2018 for a period of approximately 12 months. RESULTS: The peripheral blood mononuclear cells (PBMNC) derived T cells appeared as suspended cell clumps morphologically were positive for the expression of CD3 and negative for CD31. The DMD patient specific iPSCs appeared as round, compact and flat colonies with well-defined edges morphologically and were positive for the expression of pluripotency markers OCT4, SSEA-4 and TRA-1-81 on immunofluorescence. CONCLUSION: CD3+ T cell derived DMD-iPSCs were obtained under feeder free and xeno-free culture conditions using episomal reprogramming.

Whole-Exome Sequencing Identifies Small Mutations in Pakistani Muscular Dystrophy Patients.

Background: Muscular dystrophies are a heterogeneous group of inherited disorders that cannot be diagnosed clinically due to overlapping clinical phenotypes. Whole-exome sequencing is considered as the diagnostic strategy of choice in these cases. In this study we aimed to determine the mutational spectrum of multiplex ligation-dependent probe amplification (MLPA)-negative muscular dystrophy patients in Pakistan using whole-exome sequencing. Subsequently the mutations identified via WES were used to screen additional dystrophinopathy patients by Sanger sequencing. Materials and Methods: DNA extracted from the peripheral blood of three MLPA-negative muscular dystrophy patients was sent for whole-exome sequencing. The identified variants in these 3 patients were then checked in 18 dystrophinopathy patients using Sanger sequencing. Results: Four missense variants and one nonsense variant in the Duchenne muscular dystrophy (DMD) gene were detected. WES diagnosed a DMD patient carrying a nonsense variant c.4375C>T (rs398123953) who can benefit from Ataluren therapy. The other two patients carried missense variant (c.572G>T) in the YARS2 gene (rs11539445) labeling them as patients of MLASA (myopathy, lactic acidosis, and sideroblastic anemia). The identified missense and nonsense variants in the DMD gene were detected in 18 clinically diagnosed dystrophinopathy patients using Sanger sequencing. Three missense variants were detected in our cohort of 18 dystrophinopathy patients. One missense variant c.3406A>T (rs3827462) and a nonsense variant c.4375C>T (rs398123953) were not detected in our cohort of 18 dystrophinopathy patients. Conclusions: Whole-exome sequencing identified a nonsense variant in Pakistani muscular dystrophy patients, which is amenable to treatment by Ataluren and a missense variant in YARS2 gene responsible for causing MLASA.

Induced pluripotent stem cells from urine of Duchenne muscular dystrophy patients.

BACKGROUND: The most common muscular dystrophy, Duchenne muscular dystrophy (DMD), is a lethal, X-linked disorder with no widespread cure. Worldwide, in vitro studies involving new, mutation-specific cures and regenerative therapies are employing disease-specific patient-specific cells. However, these may not be completely relevant for Pakistani children because of the human genome diversities and geographic variation in mutation type and frequency. Therefore, this study aimed to generate DMD induced pluripotent stem cells (iPSCs) from the urine of Pakistani children with DMD, to serve as a precious source of differentiated cells, such as Pakistani DMD-cardiomyocytes, for future disease-modelling, drug testing, and gene therapy. METHODS: Urine-derived cells (UDCs) isolated from mid-stream urine underwent molecular characterization and cellular reprogramming towards iPSCs using the episomal vector system followed by molecular profiling of the iPSCs. RESULTS: Colonies of elongated and spindle-shaped or rounded rice-grain like UDCs were spotted 4-7 days after plating and expanded rapidly with a second passage at 2-3 weeks. Multicolor flow cytometry confirmed the expression of mesenchymal stem-cell markers. The reprogramed iPSCs consisted of colonies of round, tightly-packed cells with large nuclei that were positively fluorescent for the pluripotency markers octamer binding transcription factor-4 (OCT-4), tumour resistance antigen 1-60 (TRA-1-60), and stage specific embryonic 4 antigen (SSEA-4), but not for the negative pluripotency marker SSEA-1. To the best of our knowledge, this was the first time DMD-iPSCs have been generated for Pakistani children. CONCLUSION: This integration-free, feeder-free, efficient, and reproducible reprogramming method employed UDCs. Urine is a low-cost, non-invasive, painless, and repeatable source of rapidly expandable cells from children and morbid individuals for obtaining autologous cells for drug-assays and disease-modelling, suitable for DMD and other debilitating diseases.

Synergistic effect of bevacizumab and celecoxib on angiogenesis in vitro using human umbilical vein endothelial cells.

OBJECTIVE: Angiogenesis is the underlying cause of a large number of neoplastic diseases. It is necessary for tumor metastasis, and without it the tumor cannot grow or metastasize. This study aimed to determine the synergistic effect of bevacizumab and celecoxib on angiogenesis using human umbilical vein endothelial cells (HUVEC) as an in vitro model. MATERIALS AND METHODS: HUVEC were isolated from the umbilical cord by enzymatic digestion using collagenase type IV. HUVEC characterization was done by flow cytometry using cell surface markers CD31, CD105, CD146, and CD45. HUVEC were treated with bevacizumab, celecoxib, and the combination of both drugs and the cell viability was assessed using MTT assay. The formation of capillary-like endotubes for angiogenesis was analyzed using a tube formation assay by measuring the total length of capillary tubes and branch points. RESULTS: Morphologically, HUVEC showed a typical cobblestone appearance using inverted-phase contrast microscopy and were further evaluated using flow cytometry, which showed positive expression for cell surface markers CD31, CD105, CD146, and negative for CD45. Celecoxib, bevacizumab, and the combination of both drugs showed a dose-dependent inhibition on HUVEC viability. Celecoxib inhibited total tube length by 15% and branch points by 16.5%. Bevacizumab inhibited total tube length by 34% and branch points by 49%. When the two drugs were combined, the total tube length was reduced due to synergism by 68% and branch points by 80%, and the difference was found to be statistically significant (p < 0.001). CONCLUSION: Bevacizumab and celecoxib have a synergistic effect in inhibiting in vitro angiogenesis and their combination achieved more strong inhibition than either drug alone.

Direct differentiation of cord blood derived mesenchymal stem cells into keratinocytes without feeder layers and cAMP inducers.

OBJECTIVES: The purpose of our study was isolation of umbilical cord blood derived mesenchymal stem (UCB-MSCs), their direct differentiation towards keratinocytes without using feeder layers, cAMP inducers and hormones known for morphological maintenance and proliferation of keratinocytes and characterization of UCB-MSCs through flowcytometry and keratinocytes through immunofluorescence. METHODS: We have isolated and cultured UCB-MSCs (n=4) following critical parameters for successful isolation like sample processing within an hour of collection, gestational age not more than 38 weeks, no co-morbid and blood volume at least 80 ml. Cord blood mononuclear cells were isolated through ficoll based density-gradient centrifugation then cultured to isolate MSCs, defined by minimum criteria of International Society for Cellular Therapy. UCB-MSCs were then differentiated directly into keratinocytes. Differentiation was confirmed by morphology and characterized through immunofluorescence staining. UCB samples were collected from gynae/obstetric ward of OJHA campus under sterile conditions and processed at Stem cells and Regenerative medicine Lab, Dow Research Institute of Biotechnology and Biomedical Sciences, Ojha campus. The total duration of study was approximately 12 months. RESULTS: We have successfully isolated UCB-MSCs that were plastic adherent, spindle shaped, showed trilineage mesodermal differentiation potential and were positive for CD90, CD73 and CD105 and negative for CD34 markers. UCB-MSCs were directly differentiated towards keratinocytes without using cAMP inducers, hormones or feeder layers. Differentiated keratinocytes attained typical honeycomb morphology and were stained positive on immunofluorescence for anti-pan cytokeratin antibody. CONCLUSION: Our study concludes possibility of direct differentiation of isolated and cultured UCB-MSCs into keratinocytes without using feeder layers and conventional keratinocyte culture media.

Magnetic resonance imaging (MRI) findings in white matter disease of brain.

Demyelinating and dysmyelinating white matter diseases are important components of neurological problems. Recently, Magnetic Resonance Imaging (MRI) has played a key role in diagnoses of white matter diseases. Therefore, the purpose of the current study is to evaluate the usefulness of MRI in determining the type and frequency of white matter disease. We studied 35 patients who visited the Radiology Department of the Aga Khan University Hospital (AKUH) for MRI with suspected demyelinating/dysmyelinating disorder from January 2003 to December 2005. Multiple Sclerosis (MS) (17; 48%) and leukodystrophies (10; 29%) were the most common diseases. The MRI helped identify the sites and types of the lesion precisely and thereby helped made clearer. distinction between various types of white matter diseases. The current study demonstrated the effective use of the imaging and clinical presentation for arriving at the correct diagnosis.