Effect of ABO blood group on the severity and clinico-pathological parameters of COVID-19.

Background and Objective: The COVID-19 pandemic has highlighted the need to understand the factors affecting disease severity. Prior research has indicated the potential roles of the ABO blood group system in disease susceptibility and progression. Our objective was to investigate the association between ABO Blood groups and the severity of COVID-19 and clinicopathological parameters. Methods: An analytical cross-sectional study was conducted across three locations of Ziauddin University Hospital, including COVID-19 outpatient departments (OPDs), wards, and intensive care units (ICUs) from May 2020 to December 2020.The study utilized a non-probability convenient sampling technique with a sample size of 120 PCR-positive adult patients, as calculated by OpenEpi with a 95% confidence interval. Patients were excluded if they were under 14, intellectually impaired, post-chemotherapy or radiotherapy, or had a malignant condition. Disease severity was determined based on clinicopathological parameters and associated with blood group data using ANOVA and Chi-square tests in SPSS version 21. Results: A significant association was found between the ABO blood groups and COVID-19 severity. Blood group-A was notably overrepresented in patients with severe COVID-19 and correlated with higher inflammatory markers and coagulation parameters. Conclusion: ABO blood group, particularly Blood Group-A significantly associates with the severity of COVID-19. This finding suggests the potential utility of ABO blood group typing as a predictive marker for disease severity, which could contribute to personalized patient management strategies. Further research is necessary to understand the mechanisms underlying this association.

Human amniotic epithelial cells (HAECS); a potential cell type for the hepatic disorders.

OBJECTIVE: To isolate a homogenous population of human amniotic epithelial cells (hAECs) from the amniotic membrane of the human placenta and differentiate them into hepatic-like cells with the help of small molecules. METHODS: hAECs were isolated by using the enzymatic digestion method and characterized for the presence of specific stem cell markers. In-vitro, hepatic differentiation of hAECs was carried out by using a combination of small molecules. Differentiated cells were observed under a live cell imaging microscope for morphological changes followed by gene and protein expression analysis by qPCR and immunocytochemistry respectively. RESULTS: The isolated hAECs attained characteristic cuboid epithelial shape and express stem cells marker. The hepatic differentiation method was optimized based on soluble chemical compounds supplied in the culture medium. The differentiated hAECs phenotypically acquire hepatic-like cell features and expressed hepatic markers as well as hepatic protein albumin at immature levels. CONCLUSIONS: The isolated population of hAECs is highly proliferative. Moreover, hepatic markers expression in the isolated hAECs makes them an exclusive source for the treatment of chronic liver diseases.

Investigating the Association between the Autophagy Markers LC3B, SQSTM1/p62, and DRAM and Autophagy-Related Genes in Glioma.

High-grade gliomas are extremely fatal tumors, marked by severe hypoxia and therapeutic resistance. Autophagy is a cellular degradative process that can be activated by hypoxia, ultimately resulting in tumor advancement and chemo-resistance. Our study aimed to examine the link between autophagy markers' expression in low-grade gliomas (LGGs) and high-grade gliomas (HGGs). In 39 glioma cases, we assessed the protein expression of autophagy markers LC3B, SQSTM1/p62, and DRAM by immunohistochemistry (IHC) and the mRNA expression of the autophagy genes PTEN, PI3K, AKT, mTOR, ULK1, ULK2, UVRAG, Beclin 1, and VPS34 using RT-qPCR. LC3B, SQSTM1/p62, and DRAM expression were positive in 64.1%, 51.3%, and 28.2% of glioma cases, respectively. The expression of LC3B and SQSTM1/p62 was notably higher in HGGs compared to LGGs. VPS34 exhibited a significant differential expression, displaying increased fold change in HGGs compared to LGGs. Additionally, it exhibited robust positive associations with Beclin1 (rs = 0.768), UVRAG (rs = 0.802), and ULK2 (rs = 0.786) in HGGs. This underscores a potential association between autophagy and the progression of gliomas. We provide preliminary data for the functional analysis of autophagy using a cell culture model and to identify potential targets for therapeutic interventions.

Role of Alanine Transaminase and Transient Elastography in Categorising Nonalcoholic Fatty Liver Disease Subgroups.

OBJECTIVE: To investigate the association of alanine transaminase (ALT) with transient elastography grades to define various nonalcoholic fatty liver disease (NAFLD) groups for disease status. STUDY DESIGN: Cross-sectional, descriptive study. Place and Duration of the Study: Gastroenterology Outpatient Clinic, Ziauddin Hospital, from January to December 2022. METHODOLOGY: This study included 194 NAFLD patients. Demographic data, body mass index, enzymes, and transient elastography (TE) findings were recorded. NAFLD patients were categorised as nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), steatofibrosis (significant fibrosis F2-F3 with normal ALT), and cirrhosis using TE grades. RESULTS: The median age of the patients was 44 [IQR 18.25] years; 146 (75.3%) were males. Out of 194 NAFLD patients, 21 (10.8%) were NAFL, 116 (59.8%) were NASH, 14 (7.2%) showed steatofibrosis, and 43 (22.2%) were cirrhotic. On transient elastography, the majority were with S3 steatosis (n=107, 55.2%) and 59 (30.2%) had F0-F1 fibrosis. There was a statistically significant difference in the mean rank of age, ALT, AST, and GGT levels within 4 groups of NAFLD (p <0.001). Most of the patients with all the stages of fibrosis had increased ALT levels (p=0.034). CONCLUSION: This study concluded that a combination of ALT levels and transient elastography findings could be considered for differentiating uncomplicated steatosis from NASH, steatofibrosis, and cirrhosis, hence limiting the use of liver biopsy. This may prove a reliable way to measure the severity of the disease. KEY WORDS: Nonalcoholic fatty liver disease, Cirrhosis, Transient elastography.

Role of Human Papilloma Virus 16/18 In Laryngeal Carcinoma with Correlation to the Expression of Cyclin D1, p53, p16 and EGFR.

Objective: To investigate the role of Human Papilloma Virus (HPV) (16/18) in relation to the molecular genetic mechanisms of Cyclin D1, p53, p16, and Epidermal growth factor receptor (EGFR) in Laryngeal Squamous Cell Carcinoma (LSSC). Methods: A cross-sectional study of 88 (Formalin-fixed Paraffin Embedded) FFPE laryngeal biopsies were done at Basic Medical Sciences Institute, Jinnah Postgraduate Centre, Karachi from 2010 to 2019 with the application of Polymerase chain reaction (PCR) for HPV 16/18and Immuno-histochemical staining for molecular genetic expression of proteins, Cyclin D1, p53, p16, and EGFR. Results: Out of 88 cases of Laryngeal Squamous Cell Carcinoma (LSSC) there was female preponderance. Mean age of the participants was found as 50.7±12.8 years. High risk HPV 16/18 was positive in 28 cases (31.8%), largely related to Grade-II and Grade-III. Immunohistochemically, Cyclin D1 (87.5%) appeared as the most important driver mutation followed by p16 (86.4%), EGFR (65.9%), and, p53 was positive in (61.4%) of cases. Conclusion: The role of high-risk HPV 16/18 is concurred in the present study strongly in correlation to p16 as a surrogate marker. Moreover, the other driver mutations of Cyclin D1, p53, and EGFR are also implicated as cumulative molecular events in tumor progression as mostly seen in higher Grades.

Inflammatory markers and COVID-19 disease progression.

BACKGROUND: The COVID-19 pandemic has resulted in a global humanitarian crisis. Despite ongoing research, transmission risks and many disease characteristics remained unclear. Most patients have displayed elevated levels of certain inflammatory markers, which we sought to investigate further in relation to disease severity. The aim of this study was to examine the correlation between inflammatory markers and the severity of COVID-19 among patients. METHODS: We conducted a cross-sectional study from April to September 2020, involving 143 COVID-19 PCR-positive patients from Ziauddin Hospital. Electronic patient records provided data on demographics, clinical status, and laboratory results. RESULTS: The majority of PCR-positive patients were elderly males with comorbidities such as diabetes and hypertension. Almost all patients exhibited increased levels of various inflammatory markers, with procalcitonin (97.2%) being the most common. Statistically significant differences were observed in the levels of TLC (p = 0.005), CRP (p = 0.001), LDH (p = 0.001), Ferritin (p = 0.001), D-dimer (p = 0.001), and procalcitonin (p = 0.028), in relation to COVID-19 severity. CONCLUSIONS: The data suggest a significant association between levels of inflammatory markers and COVID-19 severity. All markers, except procalcitonin, demonstrated a significant correlation with disease severity. These results could enhance our understanding of COVID-19 pathogenesis and help predict and manage severe cases.

Angiotensin-converting enzyme 2 (ACE2) polymorphisms and susceptibility of severe SARS-CoV-2 in a subset of Pakistani population.

Science is digging for the varied presentation of COVID-19 patients exposed to the same risk factors, and medical conditions may be influenced by the presence of polymorphic genetic variants. This study investigated the link between ACE2 gene polymorphisms and the severity of SARS-CoV-2. This cross-sectional study recruited COVID-19 PCR-positive patients by consecutive sampling from Ziauddin Hospital from April to September 2020. DNA was extracted from whole blood, followed by gene amplification and Sanger's sequencing. Most of the patients, 77: 53.8%, were serious. Males were higher (80; 55.9%) with age more than 50 years (106: 74.1%). We found 22 ACE2 SNPs. rs2285666 SNP was most prevalent with 49.2% CC, 45.2% TT, 4.8% CT heterozygosity, and 0.8% AA genotypes. Variants with multiple genotypes were also insignificantly associated with the severity of COVID-19 in the analysis of the dominant model. Only rs2285666 had a significant statistical link with gender (p-value 0.034, OR; 1.438, CI; 1.028-2.011) while rs768883316 with age groups (p-value 0.026, OR; 1.953, CI; 1.085-3.514). Haplotypes ATC of three polymorphisms (rs560997634, rs201159862, and rs751170930) commonly found in 120 (69.77%) and TTTGTAGTTAGTA haplotype consisting of 13 polymorphisms (rs756737634, rs146991645, rs1601703288, rs1927830489, rs1927831624, rs764947941, rs752242172, rs73195521, rs781378335, rs756597390, rs780478736, rs148006212, rs768583671) in 112 (90.32%) had statistically significant association with the severity having p = value 0.029 and 0.001 respectively. Males of old age and diabetics are found to have more severe COVID-19 infection in the current study. We also found that common ACE2 polymorphism rs2285666 influences the susceptibility of acquiring the severe SARS-CoV-2 infection.

RAS gene mutations and histomorphometric measurements in oral squamous cell carcinoma.

Members of the RAS gene family frequently are mutated in cancers including oral squamous cell carcinoma (OSCC). We investigated the correlation of histological characteristics of OSCC with RAS gene mutations. We graded tumors and extracted genomic DNA from OSCC. The first two exons of KRAS, HRAS and NRAS genes were subjected to PCR amplification and DNA sequencing followed by bioinformatic analysis to explore the structural and functional impact of the mutations on encoding of proteins. Cellular and nuclear diameters in histological sections were varied in all grades of cancer. Using sequence analysis, we identified nonsynonymous mutations in both HRAS (G12S, G15C, D54H, Q61H, Q61L, E62D, E63D, Q70E, Q70V) and NRAS (Q22P, K88R). Stop codon mutations, however, were observed in KRAS. Spatial orientation of substituted amino acids was observed despite conservation of overall structure of variant proteins. Our findings suggest that KRAS may be mutated more frequently in OSCC compared to HRAS and NRAS. Also, the histological features of nuclear and cellular diameter differed significantly between the KRAS mutated and unmutated cases.

Investigating In Situ Expression of c-MYC and Candidate Ubiquitin-Specific Proteases in DLBCL and Assessment for Peptidyl Disruptor Molecule against c-MYC-USP37 Complex.

Diffuse Large B-Cell Lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma (NHL). Elevated expression of c-MYC in DLBCL is associated with poor prognosis of the disease. In different cancers, c-MYC has been found regulated by different ubiquitin-specific proteases (USPs), but to date, the role of USPs in c-MYC regulation has not been investigated in DLBCL. In this study, in situ co expression of c-MYC and three candidates USPs, USP28, USP36 and USP37, have been investigated in both the ABC and GCB subtypes of DLBCL. This shows that USP37 expression is positively correlated with the c-MYC expression in the ABC subtype of DLBCL. Structurally, both c-MYC and USP37 has shown large proportion of intrinsically disordered regions, minimizing their chances for full structure crystallization. Peptide array and docking simulations has shown that N-terminal region of c-MYC interacts directly with residues within and in proximity of catalytically active C19 domain of the USP37. Given the structural properties of the interaction sites in the c-MYC-USP37 complex, a peptidyl inhibitor has been designed. Molecular docking has shown that the peptide fits well in the targeted site of c-MYC, masking most of its residues involved in the binding with USP37. The findings could further be exploited to develop therapeutic interventions against the ABC subtype of DLBCL.

Analysis Of Common Somatic Mutations In Colorectal Carcinoma And Associated Dysregulated Pathwaysarts.

BACKGROUND: Identification of gene targets and biological pathways involved in colorectal carcinoma (CRC) is essential for better management of patients. Our study aims to highlight common somatic mutations in colorectal carcinoma and to identify dysregulated pathways and gene enrichment based on KRAS and BRAF interaction network analysis. METHODS: By using cancer browser tool in COSMIC database, mutation frequencies of the top 20 mutated genes listed for colorectal adenocarcinoma were identified. The most frequent variants of selected genes were explored with ClinVar database which led to identification of protein change along with its cytogenic location, variant type, variant length and the associated single nucleotide polymorphism (SNP). These identified SNPs were searched in Pakistani database using 1000genome in an attempt to identify common polymorphisms. Using the database ClinicalTrial.gov the number of clinical trials based upon these selected mutations was explored. Enrichment and protein interaction (PI) analysis of KRAS and BRAF was carried out to reveal significant biological pathways associated with these genes. RESULTS: In cumulative data, among all variants about 57% of substitution mutations are observed to be G>A including mutations in KRAS, Tp53, SMAD4, PI3K and NRAS. The mutations of KRAS (c.35G>A), TP53 (c.524G>A) and APC (c.4348C>T) were found to be pathogenic with single nucleotide variation and variant length of 1bp. Searching 1000genome database revealed that 100 % of alleles found in East Asian population studied are 'C'(frequency=1). Significant biological pathways (<0.05) identified by our search include Trk receptor signalling mediated by the MAPK pathway, signalling to p38 via RIT and RIN, signalling to ERKs, Frs2-mediated activation, ARMS-mediated activation and prolonged ERK activation events. CONCLUSIONS: Our study highlights the role of genetic profiling in CRC, with emphasis on mutations which may define treatment outcome. Targeting several collateral pathways simultaneously may be further explored to improve colorectal cancer therapeutics.

The Effects of Ficus carica on Male and Female Reproductive Capabilities in Rats.

The basic purpose of pharmacology is to look into the benefits of natural remedies and make them available to the general populace. Herbal medicines are now considered to be the future of humanity. The current study explored the effects of Ficus carica (FC) extract in rats of two-generation F0 (parents) and F1 (offspring) in either sex. The F. carica extract was initially tested for acute and sub-chronic toxicity. Extracts were also tested for fertility assessment and effects on reproductive hormones like follicle-stimulating hormone (FSH), luteinizing hormone (LH), gonadotropin-releasing hormone (GnRH), estradiol, testosterone, dihydrotestosterone (DHT), dehydroepiandrosterone-sulfate (DHEAS), insulin, progesterone, and prolactin. The antioxidant activity of the extract was also evaluated by testing mRNA expressions of SOD2, GPX1, CAT, and GR in male testes and female ovaries. The animals treated with 100 mg/kg FC extract produced a more pronounced fertility effect in both genders. Expression of CAT, SOD2, GPX1, and GR was found to be increased in the reproductive organs of both sexes. Histology of the testes reveals increased spermatogenesis and increased folliculogenesis in ovaries. The hormone profile showed an increase in FSH, DHT, estradiol, and DHEAS levels in males and FSH, LH, estrogen, and DHEAS in females. The results of the study establish the effectiveness of natural products in improving fertility issues in either sex.

Genetic profiles of different ethnicities living in Karachi as regards to tobacco-metabolising enzyme systems and the risk of oral cancer.

OBJECTIVE: To detect polymorphisms of CYP1A1, GSTM1 and GSTT1 gene loci among various tobacco-consuming ethnicities in an urban centre, and to relate these with susceptibility to oral cancer. METHODS: The cross-sectional, case-control study was conducted at Ziauddin University, Karachi, and the Dow University of Health Sciences, Karachi, from 2011 to 2016, and comprised patients having oral squamous cell carcinoma in group A, with oral precancerous lesions in group B, and tobacco habit-matched controls in group C. Routine histopathology was followed by molecular analysis through polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism techniques. Data was analysed using SPSS 20. RESULTS: Of the 358 subjects, 150(42%) were in group A, 100(28%) in group B, and 108(30%) in group C. There were 190(53.1%) Urdu-speaking subjects, 42(11.7%) Memoni-speaking, 37(10.3%) Sindhi-speaking, 34(9.5%) Balochi-speaking, 25(7%) Pashto-speaking, 15(4.2%) Punjabi-speaking, and 15(4.2) of other ethnicities. Among the Urdu-speaking ethnicity, CYP1A1 MspI heterozygous variant was the most prevalent genotype ingroup A 50(66.7%), group B 37(62.7%) and group C 36(64.3%). The homozygous variant was equally distributed in group A 8(13.5%) and group B 10(13.3%), while it remained quite low in group C 4(7.1%). Homozygous genotype was most common in Pashto-speaking subjects in group A 4(57.1%). In Urdu-speaking subjects, GSTM1-null genotype was mostly found in group B 19(32.2%), while GSTT1-null genotype was most common in group A 12(16%). Other than Urdu-speaking, GSTM1-null variant was most frequent in Sindhi-speaking subjects in group B 8(80%). CONCLUSIONS: Intra-ethnic distribution of tobacco-metabolising enzyme genes can be considered an important contributor to oral cancer risk in the population of Karachi.

Gene-gene and gene-environment interaction: An important predictor of oral cancer among smokeless tobacco users in Karachi.

OBJECTIVE: To determine the risk for oral cancer caused by simultaneous occurrence of more than one of the tested cytochrome P450 CYP1A1 MspI, glutathione S-transferase M1 null gnd Glutathione S-transferases T1 null gene polymorphisms. METHODS: The cross-sectional case-control study was conducted from December 2011 to October 2016 at the Ziauddin University, Karachi, in collaboration with Dow University of Health Sciences, Karachi, and comprised oral squamous cell carcinoma cases in group A and healthy tobacco habit-matched controls in group B. All investigations were done using standardised laboratory protocols. The outcomes were determined in terms of association of various combinations of cytochrome P450 1A1MspI, glutathione S-transferasesM1 null and glutathione S-transferases T1 null polymorphisms with oral cancer. Data was analysed using SPSS 20. RESULTS: Of the 238 subjects, 140(58.8%) were in group A and 98(41.2%) were in group B. Mean ages in group A and B were 47.1±12.22 and 41.6±14.58 years, respectively. Male/Female ratio in group A was 1.88:1 while 83.4% were using tobacco. When cytochrome P450 1A1MspI homozygous (m2/m2) and glutathione S-transferases M1 null variants occured simultaneously in an individual, an odds ratio of 12.8 (95% confidence interval: 1.20-135.5; p=0.03) among overall tobacco chewers was observed. For glutathione S-transferases M1 not null and glutathione S-transferases T1 null variant combination among overall tobacco users, the conferred odds ratio was 4.58 (95% confidence interval: 0.99-21.2; p=0.05). The other studied gene combinations did not reveal significant associations (p>0.05). CONCLUSIONS: A higher risk of oral squamous cell carcinoma was found to be associated with combined-gene polymorphisms of phase I and phase II enzymes than that attributed to a single-gene polymorphism.

Intensification in Genetic Information and Acquisition of Resistant Genes in Genome of Acinetobacter baumannii: A Pan-Genomic Analysis.

Acinetobacter baumannii (A. baumannii) attributes 26% of the mortality rate in hospitalized patients, and the percentage can rise to 46 in patients admitted to ICU as it is a major cause of ventilator-associated pneumonia. It has been nominated as the critical priority organism by WHO for which new therapeutic drugs are urgently required. To understand the genomic identification of different strains, antimicrobial resistance patterns, and epidemiological typing of organisms, whole-genome sequencing (WGS) analysis provides insight to explore new epitopes to develop new drugs against the organism. Therefore, the study is aimed at investigating the whole genome sequence of A. baumannii strains to report the new intensifications in its genomic profile. The genome sequences were retrieved from the NCBI database system. Pan-genome BPGA (Bacterial Pan-genome Analysis Tool) was used to analyze the core, pan, and species-specific genome analysis. The pan and core genome curves were extrapolated using the empirical power law equation f(x) = a.xb and the exponential equation f1(x) = c.e (d.x). To identify the resistant genes with resistant mutations against antibiotics, ResFinder and Galaxy Community hub bioinformatics tools were used. According to pan-genome analysis, there were 2227 core genes present in each species of the A. baumannii genome. Furthermore, the number of accessory genes ranged from 1182 to 1460, and the unique genes in the genome were 931. There were 325 exclusively absent genes in the genome of Acinetobacter baumannii. The pan-genome analysis showed that there is a 5-fold increase in the genome of A. baumannii in 5 years, and the genome is still open. There is the addition of multiple unique genes; among them, genes participating in the function of information and processing are increased.

Opuntiol Inhibits Growth and Induces Apoptosis in Human Glioblastoma Cells by Upregulating Active Caspase 3 Expression.

BACKGROUND: Glioblastoma Multiforme (GBM) is a deadly tumor with poor prognosis. Resistance to apoptosis considered as an important factor in treatment failure. Therefore, identification of new compounds that facilitates apoptosis is crucial. Natural Anti-inflammatory compounds have emerged as potential anti-cancer agents and should be explored for their apoptotic activity against GBM. Therefore, the present study aims to evaluate growth inhibitory and apoptotic activity of a natural anti-inflammatory compound "Opuntiol" against GBM cell line U87. METHODS: MTT assay was performed to determine the effect of Temozolomide and Opuntiol on growth inhibition of U87 cell. While, TUNEL assay was used to assess their apoptotic activity. To further assess apoptosis, nuclear condensation and nuclear area factor (NAF) was evaluated through DAPI staining. Whereas, active caspase-3 protein expression determined using immunocytochemistry. RESULTS: Significant growth inhibition was observed in U87 cells treated with Temozolomide (IC50 380 µM) and Opuntiol (IC50 357 µM). Temozolomide (p<0.001) and Opuntiol (p<0.001) significantly improved rate of apoptosis when compared to control group. A significant decrease in NAF was also observed in Temozolomide (p < 0.05) and Opuntiol (p < 0.05) treated cells. There was a significant increase in active caspase-3 expression when observed in Temozolomide (p<0.001) and Opuntiol (p<0.05) treated groups as compared to control. CONCLUSION: In conclusion our findings suggests, Opuntiol repress cell viability and possess strong apoptotic activity against GBM cell line U-87. However, further mechanistic studies will be required to confirm whether it can be develop as a potential drug against GBM.

Differential Immune Landscape of Hepatocellular Carcinoma Suggests Potential role of Macrophages in Hepatocarcinogenesis.

OBJECTIVES: To investigate immune cell densities in tumor microenvironment of hepatocellular carcinoma. METHODS: This cross-sectional study was conducted during 2017-2019 at the Dow University of Health Sciences Karachi. A total of 42 subsequent patients undergoing liver biopsy/resection and diagnosed with hepatocellular carcinoma were included in the study. Moreover, a total of 10 control tissues were also included. In order to investigate immune cells densities in hepatocellular carcinoma, immunohistochemistry was performed using antibodies including α-MPO(neutrophils), α-CD-68(macrophages), α-CD-3(T-cells), α-CD-20(B-cells), α-CD-4(CD4+ T-cells) and α-CD-8(CD8+ T-cells). Quantification of immune cells/mm2 was performed as per the College of American Pathologists' guidelines. Data were analyzed using SPSS version 21. A p-value of 0.05 was considered significant at all times. RESULTS: We report significantly increased infiltration of macrophages (mean macrophages= 306.57/mm2, p-value <0.05), moderately significant infiltration of neutrophils (p-value=0.06) and B-cells (p-value=0.07) while no significant infiltration of CD4+T-cells (p- value=0.31), and CD8+T-cells (p-value=0.39) in tumour microenvironment of patients with hepatocellular carcinoma. CONCLUSION: We provide evidence for increased macrophage infiltration in liver cancer microenvironment suggesting a potential role of these cells in hepatocarcinogenesis.

Attenuation of cisplatin-induced acute kidney injury by N-(2-Hydroxyphenyl) acetamide and its gold conjugated nano-formulations in mice.

The attenuation of cisplatin-induced acute kidney injury (AKI) in mice by N-(2-hydroxyphenyl) acetamide (NA-2) and NA-2-conjugated gold nanoparticles (NA2-AuNPs) was investigated. Male BALB/c mice (n = 54) were divided into nine groups having six animals in each group. Animals in groups 3-9 were pre-treated for 5 days with test compounds, whereas, animals in group 1 and 2 received normal saline. On day 4, animals in groups 2, 3, 4, 5, 6 and 9 were given single intra-peritoneal injection of CP at the dose of 5 mg/kg. After 72 hours of CP injection, all animals were sacrificed. Blood was collected for serum urea and creatinine estimation, and kidneys were harvested for histo-pathological examinations and qPCR studies for nuclear factor-κB p50, (NFκB) ; inducible nitric oxide synthase (iNOS); hemeoxygenase-1 (HO-1); and interleukin-6 (IL-6).NA-2 and NA2-AuNPs was observed to decrease the serum urea and creatinine levels. Both the test compounds reduced kidney injury damage score and improved histological architecture in the treated animals in dose dependent manner. Furthermore, the mRNA expressions of NFkB p50, iNOS and IL-6 genes were down-regulated, and HO-1 gene was up-regulated in the animals treated with the test compounds. It is concluded that NA-2 and NA2-AuNPs attenuates CP-induced AKI in mice models through anti-inflammatory and anti-oxidant mechanisms.

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.

Spectrum of Preneoplastic and Neoplastic lesions of intestine in a Tertiary Care Hospital of Karachi, Pakistan.

OBJECTIVES: To present 7 years data mentioning the spectrum of preneoplastic & neoplastic cases of intestine received at Dow Diagnostic Research and Reference Laboratory. METHODS: All the cases of preneoplastic & neoplastic lesions of intestine received during 2009 - 2015 were reviewed. The data obtained were subjected to descriptive statistical analysis using SPSS version 22. Furthermore, the association of diagnosis was seen with various other variables including age, gender & site of the lesion. A p-value of < 0.05 was considered as significant. RESULTS: The total samples were 486, out of which 33 cases were of premalignant and 453 were of malignant lesions. Out of total 33 cases of premalignant lesions of intestine, it consisted adenomatous polyp = 39.4% (n=13), dysplasia = 36.4% (n=12) and adenoma = 24.2% (n=8). From the total of 453 cases diagnosed as malignant lesions; adenocarcinoma as Grade-I were 14.2% (n=64), Grade-II were 7.6% (n=260) and Grade-III were 22% (n=99). Squamous cell carcinoma Grade-I were 0.4% (n=2), Grade-II 1.6% (n=7) and Grade-III 0.9% (n=4). 2.4% (n=11) cases were of metastatic adoncarcinoma, 0.9% (n=4) were diagnosed as neuroendocrine tumors and 0.4% (n=2) as lymphoma. A significant association was seen between site of the tumor and diagnosis, rectum was the commonest site for adenocarcinomas (p=0.001). Moderately differentiated adenocarcinoma was predominantly present in young age (p=0.001). CONCLUSION: Colorectal carcinoma is on rise in Pakistan, predominantly in young males, and rectum being the commonest site. In our study, all the lesions showed male predominance with adenomatous polyp as the commonest premalignant lesion & Grade-II adenocarcinoma the most common malignancy of intestine.

Neuroprotective effect of diclofenac on chlorpromazine induced catalepsy in rats.

Neuroinflammation plays a key role in progressive degeneration of dopaminergic cells. Upregulation of prostaglandins and free radicals formation are involved in the mechanisms of cell death in Parkinson's disease (PD). The present study aimed to investigate the neuroprotective effect of diclofenac against chlorpromazine (CPZ) induced catalepsy and motor impairment in mice. Adult Wistar rats treated with CPZ (3 mg/kg/day, IP) were orally dosed with diclofenac and L-dopa/carbidopa for 21 days. Catalepsy was measured after 21 days of dosing by using standard bar test at 30, 60, 90, 120 and 180 min then motor performances were assessed via open field test and wire hanging test. Histopathological investigation and determination of dopamine (DA) and 3,4-Dihydroxyphenylacetic acid (DOPAC) levels of rat's brain was also carried out. We found that CPZ treated group exhibited reduced motor impairment after 21 days of treatment in open field and wire hanging test (P < 0.01) as compared to control group. The cataleptic scores of CPZ treated rats were also significantly increased (P < 0.01) after 21 days of chronic dosing, however diclofenac treated groups showed significant reduction in cataleptic scores with improved motor performances. Histopathology of CPZ treated rats showed marked degeneration with architecture distortion in the mid brain region. Dopaminergic degeneration is confirmed by neurochemical results that showed reduced amount of dopamine and DOPAC levels in mid brain. Moreover, histopathological slides of diclofenac treated rats showed improved architecture with reduced gliosis of mid brain region as well as improved dopamine and DOPAC levels were achieved after 21 days dosing of diclofenac. Taken together, the present work provide an evidence that diclofenac ameliorated behavioral performances by mediating neuroprotection against CPZ induced PD via preventing dopaminergic neuronal cell death.