Ethanolic extract of Euphorbia royleana Boiss. reduces metastasis of breast cancer cells and inhibits tumor progression in vivo.

Metastasis is the most devastating attribute of breast cancer (BC) that leads to high mortality. It is a complex process of tumor cell migration, invasion, and angiogenesis. In this study, we evaluated the effect of ERA on BC metastasis and BC progression in vivo. The transwell invasion/migration and wound healing assays showed that ERA treatment significantly reduced the invasion and migration of BC cell lines. The expression of mesenchymal (E-cadherin and N-cadherin), matrix metalloproteinases (MMP2, MMP9), and stemness markers (Oct3) were down-regulated by ERA. Furthermore, ERA down-regulated angiogenic chemokines (CXCL1/2/3, CXCL5, and CXCL12) expression in the highly metastatic MDA-MB-231 cell line. The clonogenic survival of BC cells was also reduced by ERA treatment. Strikingly, ERA prevented DMBA-induced tumor growth in Swiss albino mice as depicted by a high animal survival rate (84%) in the ERA group and histopathological analysis. Conclusively, this study revealed that ERA possesses anti-metastatic potential and also reduces the growth of BC in vivo. Moreover, the GC-MS data revealed the presence of biologically active compounds (Lupeol, Phytol, phytosterol) and some rare (9, 19-Cyclolanost) phyto metabolites in ERA extract. However, further studies are suggestive to identify and isolate the therapeutic agents from ERA to combat BC and metastasis.

Detection of BK and JC polyomaviruses in sewage water of the urban areas of Lahore, Pakistan.

The surveillance of sewage water has become an extremely essential tool to trace the circulation of viruses in a population and to predict the outbreak of viral diseases. Sewage monitoring is important for those viruses which cause subclinical infections since it is difficult to determine their prevalence. Polyomaviruses are ubiquitously present, circular double-stranded DNA viruses that can infect humans as well. Among all human polyomaviruses, BK polyomavirus and JC polyomavirus associated with the development of aggressive diseases in immunocompromised individuals, are highly prevalent. This study aimed to investigate the presence and the quantitative prevalence of these two disease-associated human polyomaviruses in sewage water collected from six drains of Lahore, Pakistan. The viruses present in the environmental samples were concentrated by PEG method before isolating viral nucleic acids. Conventional PCR amplifications were performed for molecular detection of BK polyomavirus and JC polyomavirus targeting their large tumor antigen genetic region. The presence of BK polyomavirus and JC polyomavirus was confirmed in the DNA extracted from concentrated sewage samples of each drain by performing both qualitative and quantitative PCR. Our data shows that the viral load ranged from 1278 to 178368 copies per µg of environmental DNA for BK polyomavirus and 5173 to 79129 copies per µg of environmental DNA for JC polyomavirus. In conclusion, here we report first time the detection of BK polyomavirus and JC polyomavirus in sewage water collected from six main drains in urban areas of Lahore, Pakistan showing the high prevalence of these viruses in the Pakistani population. This assay could be used as a proxy to determine the prevalence of these viruses in the Pakistani population.

Ethanolic extracts of Pakistani euphorbiaceous plants induce apoptosis in breast cancer cells through induction of DNA damage and caspase-dependent pathway.

BACKGROUND: Numerous plants of Euphorbiaceae, thespurgefamily are traditionally used for the treatment of different diseases and recent studies also reported anti-oxidant, anti-inflammatory, and anti-tumor activities of these plants. However, the medicinal potential of several indigenous euphorbiaceous plants of Pakistan is not described yet. Therefore, we intended to evaluate the in vitro anti-breast cancer potential of 10 euphorbiaceous plants of Pakistan. METHODS: Cytotoxic screening of ethanolic extracts of selected plants was performed by MTT assay. The qualitative phytochemical analysis was performed to find the major groups of chemicals responsible for cytotoxic activity. To determine the genotoxic effect of plant extracts, microscopic analysis was carried out. Flow cytometry and fluorescent microscopic analysis were done to detect apoptosis. To find out the expression analysis of cell cycle and cell death regulatory genes, quantitative real-time polymerase reaction (qRT-PCR) was performed. RESULTS: Among the 10 tested plants, ethanolic extracts of Croton tiglium (CTL) and Euphorbia royleana (ERA) were found to possess the highest anti-proliferative activity against breast cancer cells (MDA-MB-231, MCF-7), with IC50 values 100 and 80 µg/mL respectively. The phytochemical analysis confirmed the presence of phenols, flavonoids, and steroids in both plant extracts, whereas, glycosides and saponins were found only in CTL and ERA, respectively. The cellular aberrations and nuclear morphologies with a distinct DNA laddering pattern substantiated the genotoxic effects. Furthermore, our data showed that CTL and ERA induce cell cycle arrest at the G1/S phase by down-regulating the CDK4 and Cyclin D1 expression followed by caspase-dependent induction of apoptosis in both MCF-7 and MDA-MB-231 cells. However, based on the activation of initiator and executioner caspases, two distinct types of apoptotic pathways are proposed for these plants. The CTL prompted extrinsic while ERA triggered the intrinsic pathways of apoptosis. CONCLUSION: Our data demonstrate the strong anti-proliferative and caspase-dependent apoptotic potential of CTL and ERA against breast cancer cells. Further studies are suggested to find clinical implications of these plants in breast cancer therapeutic.

A novel RdRp-based colorimetric RT-LAMP assay for rapid and sensitive detection of SARS-CoV-2 in clinical and sewage samples from Pakistan.

Novel corona virus SARS-CoV-2, causing coronavirus disease 2019 (COVID-19), has become a global health challenge particularly for developing countries like Pakistan where overcrowded cities, inadequate sanitation, little health awareness and poor socioeconomic conditions exist. The SARS-CoV-2 has been known to spread primarily through direct contact and respiratory droplets. However, detection of SARS-CoV-2 in stool and sewage have raised the possibility of fecal-oral mode of transmission. Currently, quantitative reverse-transcriptase PCR (qRT-PCR) is the only method being used for SARS-CoV-2 detection, which requires expensive instrumentation, dedicated laboratory setup, highly skilled staff, and several hours to report results. Considering the high transmissibility and rapid spread, a robust, sensitive, specific and cheaper assay for rapid SARS-CoV-2 detection is highly needed. Herein, we report a novel colorimetric RT-LAMP assay for naked-eye detection of SARS-COV-2 in clinical as well as sewage samples. Our SARS-CoV-2 RdRp-based LAMP assay could successfully detect the virus RNA in 26/28 (93%) of RT-PCR positive COVID-19 clinical samples with 100% specificity (n = 7) within 20 min. We also tested the effect of various additives on the performance of LAMP assay and found that addition of 1 mg/ml bovine serum albumin (BSA) could increase the sensitivity of assay up to 101 copies of target sequence. Moreover, we also successfully applied this assay to detect SARS-CoV-2 in sewage waters collected from those areas of Lahore, a city of Punjab province of Pakistan, declared as virus hotspots by local government. Our optimized LAMP assay could provide a sensitive first tier strategy for SARS-CoV-2 screening and can potentially help diagnostic laboratories in better handling of high sample turnout during pandemic situation. By providing rapid naked-eye SARS-CoV-2 detection in sewage samples, this assay may support pandemic readiness and emergency response to any possible virus outbreaks in future.

Novel coronavirus disease (COVID-19) pandemic: A recent mini review.

The COVID-19, caused by a novel coronavirus, was declared as a global pandemic by WHO more than five months ago, and we are still experiencing a state of global emergency. More than 74.30 million confirmed cases of the COVID-19 have been reported globally so far, with an average fatality rate of almost 3.0%. Seven different types of coronaviruses had been detected from humans; three of them have resulted in severe outbreaks, i.e., MERS-CoV, SARS-CoV, and SARS-CoV-2. Phylogenetic analysis of the genomes suggests that the possible occurrence of recombination between SARS-like-CoVs from pangolin and bat might have led to the origin of SARS-CoV-2 and the COVID-19 outbreak. Coronaviruses are positive-sense, single-stranded RNA viruses and harbour a genome (30 kb) consisting of two terminal untranslated regions and twelve putative functional open reading frames (ORFs), encoding for non-structural and structural proteins. There are sixteen putative non-structural proteins, including proteases, RNA-dependent RNA polymerase, helicase, other proteins involved in the transcription and replication of SARS-CoV-2, and four structural proteins, including spike protein (S), envelope (E), membrane (M), and nucleocapsid (N). SARS-CoV-2 infection, with a heavy viral load in the body, destroys the human lungs through cytokine storm, especially in elderly persons and people with immunosuppressed disorders. A number of drugs have been repurposed and employed, but still, no specific antiviral medicine has been approved by the FDA to treat this disease. This review provides a current status of the COVID-19, epidemiology, an overview of phylogeny, mode of action, diagnosis, and possible treatment methods and vaccines.

Isocitrate dehydrogenase 1 gene variants analysis of glioma patients from Pakistan.

Various somatic isocitrate dehydrogenase 1 (IDH1) gene variants have been reported to drive lower-grade gliomas and secondary glioblastomas. In the current study, we explored the IDH1 variants in the glioma biopsy samples of patients from Pakistan. We explored the incidence of isocitrate dehydrogenase 1 gene variants by hotspot sequencing in 80 formalin-fixed paraffin-embedded tissues of different types of glioma biopsy samples. Structural modeling of the identified variants in isocitrate dehydrogenase 1 protein was done to see their possible consequences. The frequently described p.Arg132 variants were not found in any of the glioma types. However, in our study, we identified nonsynonymous variants at the residues p.R109 and p.G136 in astrocytomas and p.R100 in oligodendroglioma. These variants are affecting a part of the conserved domain in isocitrate dehydrogenase 1. Both of p.R100 and p.R109 variants are rare and described before, whereas the p.G136 variant identified in this study has never been described previously. Structural modeling showed that variants of these residues would directly affect the substrate binding and hence the enzyme activity.

SHISA3, an antagonist of the Wnt/ß-catenin signaling, is epigenetically silenced and its ectopic expression suppresses growth in breast cancer.

Breast cancer (BC) is the foremost cause of cancer related deaths in women globally. Currently there is a scarcity of reliable biomarkers for its early stage diagnosis and theranostics monitoring. Altered DNA methylation patterns leading to the silencing of tumor suppressor genes are considered as an important mechanism underlying tumor development and progression in various cancer types, including BC. Very recently, epigenetic silencing of SHISA3, an antagonist of ß-catenin, has been reported in various types of tumor. However, the role of SHISA3 in BC has not been investigated yet. Therefore, we aimed at evaluating the contribution of SHISA3 in BC causation by analyzing its expression and methylation levels in BC cell lines (MDA-MB231, MCF-7 and BT-474) and in 103 paired BC tissue samples. The SHISA3 expression and methylation status was determined by qPCR and methylation specific PCR (MSP) respectively. The role of SHISA3 in BC tumorigenesis was evaluated by proliferation and migration assays after ectopic expression of SHISA3. The association between SHISA3 hypermethylation and clinicopathological parameters of BC patients was also studied. The downregulation of SHISA3 expression was found in three BC cell lines used and in all BC tissue samples. However, SHISA3 promoter region was hypermethylated in 61% (63/103) tumorous tissues in comparison to the 18% of their matched normal tissues. The 5-aza-2'-deoxycytidine treatment restored SHISA3 expression by reversing promoter hypermethylation in both MDA-MB231 and MCF-7 cells. Furthermore, ectopic expression of SHISA3 significantly reduced the proliferation and migration ability of these cells. Taken together, our findings for the first time reveal epigenetic silencing and tumor suppressing role of SHISA3 in BC. Henceforth, this study has identified SHISA3 as potentially powerful target for the development of new therapies against BC, as well as novel diagnostic and therapy response monitoring approaches.

The First blaKPC Harboring Klebsiella pneumoniae ST258 Strain Isolated in Pakistan.

Klebsiella pneumoniae is an important pathogen that causes pneumonia and bloodstream infections, especially in neonates and intensive care patients. The carbapenems remain an important therapeutic option for clinicians, particularly against cephalosporin-resistant Gram-negative pathogens. This increased use of carbapenems at clinics has resulted in the evolution and spread of carbapenem-resistant K. pneumoniae. In this study, we isolated six bla K. pneumoniae carbapenemase (KPC)-producing strains belonging to sequence type 258 (ST258) from clinical, environmental, and veterinary sources. Antibiotic susceptibility was performed on these isolates and the genes responsible for extended-spectrum beta-lactamases and metallo-beta-lactamase production were screened. The molecular typing was done using multilocus sequence typing. Isolated strains were resistant to various antibiotic classes, including carbapenems, and carried the carbapenem-resistant gene, blaKPC. All strains were susceptible to tigecycline and colistin. This is the first report detecting K. pneumoniae ST258 strains in Pakistan.

Human BK and JC polyomaviruses: Molecular insights and prevalence in Asia.

Polyomaviridae family consists of small circular dsDNA viruses. Out of the 14 human polyomaviruses described so far, BKPyV and JCPyV have been studied extensively since their discovery in 1971. Reportedly, both BKPyV and JCPyV are widely distributed across the globe with the frequency of 80-90 % in different populations. The primary infection of these viruses is usually asymptomatic and latent which is activated as a consequence of immunosuppression. Activated BKPyV and JCPyV viruses lead to the development of BK Virus Associated Nephropathy and Progressive Multifocal Leukoencephalopathy, respectively. Immense progress has been made during the last few decades regarding the molecular understanding of polyomaviruses. Epidemiology of polyomaviruses has also been studied extensively. However, most of the epidemiological studies have focused on European and American populations. Therefore, limited data is available regarding the geographical distribution of these potentially oncogenic viruses in Asian countries. In this article, we have presented a compendium of latest advances in the molecular understanding of polyomaviruses and their pathobiology. We also present a comprehensive review of published literature regarding the epidemiology and prevalence of BKPyV and JCPyV in Asian regions. For this purpose, a thorough search of available online resources was performed. As a result, we retrieved 24 studies for BKPyV and 22 studies for JCPyV, that describe their prevalence in Asia. These studies unanimously report high occurrence of both BKPyV and JCPyV in Asian populations. The available data from these studies was categorized into two groups: on the basis of prevalence (low, medium and high) and disease development (healthy and diseased). Altogether, Korean population hasbeen evidenced to possess highest frequency of BKPyV (66.7 %), while JCPyV was found to be most prevalent in Taiwan (88 %). Due to high and ubiquitous distribution of these viruses, frequent studies are required to develop a better understanding regarding the epidemiology and pathobiology of these viruses in Asia.

Genetic diversity of Paramecium species on the basis of multiple loci analysis and ITS secondary structure models.

Among ciliates, Paramecium has become a privileged model for the study of "species problem" particularly in the case of the "Paramecium aurelia complex" that has been intensely investigated. Despite extensive studies, the taxonomy of Paramecium is still challenging. The major problem is an uneven sampling of Paramecium with relatively few representatives of each species. To investigate species from the less discovered region (Pakistan), 10 isolates of Paramecium species including a standing-alone FT8 strain previously isolated by some of us were subjected to molecular characterization. Fragments of 18S recombinant DNA (rDNA), ITS1-5.8S-ITS2-5'LSU rDNA, cytochrome c oxidase subunit II, and hsp70 genes were used as molecular markers for phylogenetic analysis of particular isolates. The nucleotide sequences of polymerase chain reaction products of all markers were compared with the available sequences of relevant markers of other Paramecium species from GenBank. Phylogenetic trees based on all molecular markers showed that all the nine strains had a very close relationship with Paramecium primaurelia except for the FT8 strain. FT8 consistently showed its unique position in comparison to all other species in the phylogenetic trees. Available sequences of internal transcribed spacer 1 (ITS1) and ITS2 and some other ciliate sequences from GenBank were used for the construction of secondary models. Two highly conserved helices supported by compensatory base changes among all ciliates of ITS2 secondary structures were found similar to other eukaryotes. Therefore, the most conserved 120 to 180 base pairs regions were identified for their comparative studies. We found that out of the three helices in ITS1 structure, helix B was more conserved in Paramecium species. Despite various substitutions in the primary sequence, it was observed that secondary structures of ITS1 and ITS2 could be helpful in interpreting the phylogenetic relationships both at species as well as at generic level.

Merkel Cell Polyomavirus Downregulates N-myc Downstream-Regulated Gene 1, Leading to Cellular Proliferation and Migration.

Merkel cell polyomavirus (MCPyV) is the first human polyomavirus etiologically associated with Merkel cell carcinoma (MCC), a rare and aggressive form of skin cancer. Similar to other polyomaviruses, MCPyV encodes early T antigen genes, viral oncogenes required for MCC tumor growth. To identify the unique oncogenic properties of MCPyV, we analyzed the gene expression profiles in human spontaneously immortalized keratinocytes (NIKs) expressing the early genes from six distinct human polyomaviruses (PyVs), including MCPyV. A comparison of the gene expression profiles revealed 28 genes specifically deregulated by MCPyV. In particular, the MCPyV early gene downregulated the expression of the tumor suppressor gene N-myc downstream-regulated gene 1 (NDRG1) in MCPyV gene-expressing NIKs and hTERT-MCPyV gene-expressing human keratinocytes (HK) compared to their expression in the controls. In MCPyV-positive MCC cells, the expression of NDRG1 was downregulated by the MCPyV early gene, as T antigen knockdown rescued the level of NDRG1. In addition, NDRG1 overexpression in hTERT-MCPyV gene-expressing HK or MCC cells resulted in a decrease in the number of cells in S phase and cell proliferation inhibition. Moreover, a decrease in wound healing capacity in hTERT-MCPyV gene-expressing HK was observed. Further analysis revealed that NDRG1 exerts its biological effect in Merkel cell lines by regulating the expression of the cyclin-dependent kinase 2 (CDK2) and cyclin D1 proteins. Overall, NDRG1 plays an important role in MCPyV-induced cellular proliferation.IMPORTANCE Merkel cell carcinoma was first described in 1972 as a neuroendocrine tumor of skin, most cases of which were reported in 2008 to be caused by a PyV named Merkel cell polyomavirus (MCPyV), the first PyV linked to human cancer. Thereafter, numerous studies have been conducted to understand the etiology of this virus-induced carcinogenesis. However, it is still a new field, and much work is needed to understand the molecular pathogenesis of MCC. In the current work, we sought to identify the host genes specifically deregulated by MCPyV, as opposed to other PyVs, in order to better understand the relevance of the genes analyzed on the biological impact and progression of the disease. These findings open newer avenues for targeted drug therapies, thereby providing hope for the management of patients suffering from this highly aggressive cancer.

A cross-sectional study of methicillin-resistant Staphylococcus aureus at the equine-human interface.

The present study aimed at investigating the percent prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in equines and associated personnel. A total of 150 swabs of equines and 50 nasal swab samples of associated personnel were collected. These samples were processed in mannitol salt broth for enrichment. A total of 175 nasal swab samples changed the broth color from pink to yellow which were detected as samples containing S. aureus. These samples were processed further on specific media, namely mannitol salt agar, Staph-110, and blood agar, for phenotypic and Gram's staining-based confirmation of S. aureus isolates. Out of these 175 S. aureus-positive samples, 150 were of equine and 25 were of human origin. Identification of MRSA isolates in 175 S. aureus-positive samples was carried out by antimicrobial susceptibility testing by disc diffusion method. Results showed the presence of MRSA in 87 samples, out of which 81 samples were collected from equines and six samples from humans. Results of antibiotic testing revealed that percentage positivity of MRSA was higher (54%) in equines as compared with the associated personnel (24%). Most resistant antibiotics against MRSA isolates were oxacillin and methicillin while linezolid was found to be the most sensitive antibiotic against MRSA. In conclusion, our findings indicated prevalence of MRSA in equines and associated personnel evidencing an occupational risk of contracting MRSA from horses.

Merkel cell polyomavirus DNA sequences in the blood of healthy population of Pakistan.

Aim: This study aimed at detecting and quantifying Merkel cell polyomavirus (MCPyV) viral loads in the peripheral blood of healthy Pakistani individuals. Patients & methods: A total of 221 whole blood samples obtained from healthy individuals were examined by qPCR. Results & conclusion: MCPyV was detected in the peripheral blood of 31.2% healthy individuals. The rate of MCPyV positivity decreased from young (36%), to middle (33.7%) and elder (25.3%) age groups. Our data revealed higher prevalence of MCPyV in women (43.93%) than men (25.80%). The MCPyV viral load was calculated in the range of 0.06 -11 copies/ng of isolated DNA. The MCPyV viral load increased from young (median = 3.35) to elder (median = 5.66) age groups. The MCPyV circulate at a higher frequency by residing dormant in certain blood cells, which might act as potential vehicles for the spread of MCPyV infection among general population.

The implication of CRISPR/Cas9 genome editing technology in combating human oncoviruses.

It is evidenced that 20% of all tumors in humans are caused by oncoviruses, including human papilloma viruses, Epstein-Barr virus, Kaposi sarcoma virus, human polyomaviruses, human T-lymphotrophic virus-1, and hepatitis B and C viruses. Human immunodeficiency virus is also involved in carcinogenesis, although not directly, but by facilitating the infection of many oncoviruses through compromising the immune system. Being intracellular parasites with the property of establishing latency and integrating into the host genome, these viruses are a therapeutic challenge for biomedical researchers. Therefore, strategies able to target nucleotide sequences within episomal or integrated viral genomes are of prime importance in antiviral or anticancerous armamentarium. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has emerged as a powerful genome editing tool. Standing out as a precise and efficient oncoviruses method, it has been extensively applied in recent experimental ventures in the field of molecular medicine, particularly in combating infections including tumor inducing viruses. This review is aimed at collating the experimental and clinical advances in CRISPR/Cas9 technology in terms of its applications against oncoviruses. Primarily, it will focus on the application of CRISPR/Cas9 in combating tumor viruses, types of mechanisms targeted, and the significant outcomes till date. The technical pitfalls of the CRISPR/Cas9 and the comparative approaches in evaluating this technique with respect to other available alternatives are also described briefly. Furthermore, the review also discussed the clinical aspects and the ethical, legal, and social issues associated with the use of CRISPR/Cas9.

Circulating liver-specific microRNAs as noninvasive diagnostic biomarkers of hepatic diseases in human.

CONTEXT: Hepatitis is an endemic disease worldwide leading to chronic and debilitating cancers. The viral agents and hepatotoxic substances lead to damage of hepatocytes and release of damage associated molecules in circulation. The lack of timely and rapid diagnosis of hepatitis results in chronic disease. OBJECTIVE: The present review aimed to describe regulation, release and functions of microRNAs (miR) during human liver pathology and insights into their promising use as noninvasive biomarkers of hepatitis. METHODS: Comprehensive data were collected from PubMed, ScienceDirect and the Web of Science databases utilizing the keywords "biomarkers", "microRNAs" and "hepatic diseases". RESULTS: The miRs are readily released in the body fluids and blood during HBV/HCV associated hepatitis as well as metabolic, alcoholic, drug induced and autoimmune hepatitis. The liver-specific microRNAs including miR-122, miR-130, miR-183, miR-196, miR-209 and miR-96 are potential indicators of liver injury (mainly via apoptosis, necrosis and necroptosis) or hepatitis with their varied expression during acute/fulminant, chronic, liver fibrosis/cirrhosis and hepato-cellular carcinoma. CONCLUSIONS: The liver-specific miRs can be used as rapid and noninvasive biomarkers of hepatitis to discern different stages of hepatitis. Blocking or stimulating pathways associated with miR regulation in liver could unveil novel therapeutic strategies in the management of liver diseases. Clinical significance Liver specific microRNAs interact with cellular proteins and signaling molecules to regulate the expression of various genes controlling biological processes. The circulatory level of liver specific microRNAs is indicator of severity of HBV and HCV infections as well as prognostic and therapeutic candidates. The expression of liver specific microRNAs is strongly associated with infectious, drug-induced, hepatotoxic, nonalcoholic steatohepatitis and nonalcoholic fatty liver diseases.

Molecular basis of quinolone resistance in clinical isolates of Klebsiella pneumoniae from Pakistan.

Antibiotic resistant Klebsiella pneumoniae, is associated with various nosocomial infections that are difficult to treat. This study is designed to find out the patterns of resistance against commonly used antibiotics in K. pneumoniae clinical isolates with special attention to fluoroquinolones. A total number of 200 K. pneumoniae clinical isolates collected from various tertiary care hospitals of Punjab, Pakistan for a span of 1 year were investigated. Isolates were identified biochemically and genetically using VITEK® system and species-specific PCR, respectively. Antibiogram of isolates was studied by using disc diffusion and broth micro-dilution assays. Highest infection of K. pneumoniae detected in urinary tract (43%) followed by respiratory tract (25.5%). Most of the isolates displayed strong resistance against ampicillin, cefotetan, tazobactam, cefuroxime, cefixime, ceftriaxone, ampicillin-sulbactam imipenem, meropenem, ciprofloxacin and moxifloxacin, while sensetive to cefotaxime. Chromosoaml mutation was deteted in gyrA gene, gyrA harbors a strong mutation which provides resistance against ciprofloxacin by substituting Ser83→Ile. However, no mutation was detected in gyrB gene. Moreover, qnrB1 plasmid born resistant gene was only detected among qnrA, qnrB and qnrS. The story depicts an alarming situation of antibiotic resistance among K. pneumoniae associated with various nosocomial infections.

Differentiation of Mesenchymal Stem Cells Towards Nephrogenic Lineage and Their Enhanced Resistance to Oxygen Peroxide-induced Oxidative Stress.

INTRODUCTION: Mesenchymal stem cells (MSCs) have been publicized to ameliorate kidney injury both in vitro and in vivo. However, very less is known if MSCs can be differentiated towards renal lineages and their further application potential in kidney injuries. MATERIALS AND METHODS: The present study developed a conditioning system of growth factors fibroblast growth factor 2, transforming growth factor-ß2, and leukemia inhibitory factor for in vitro differentiation of MSCs isolated from different sources towards nephrogenic lineage. Less invasively isolated adipose-derived MSCs were also compared to bone marrow-derived MSCs for their differentiation potential to induce renal cell. Differentiated MSCs were further evaluated for their resistance to oxidative stress induced by oxygen peroxide. RESULTS: A combination of growth factors successfully induced differentiation of MSCs. Both types of differentiated cells showed significant expression of pronephrogenic markers (Wnt4, Wt1, and Pax2) and renal epithelial markers (Ecad and ZO1). In contrast, expression of mesenchymal stem cells marker Oct4 and Vim were downregulated. Furthermore, differentiated adipose-derived MSCs and bone marrow-derived MSCs showed enhanced and comparable resistance to oxygen peroxide-induced oxidative stress. CONCLUSIONS: Adipose-derived MSC provides a promising alternative to bone marrow-derived MSC as a source of autologous stem cells in human kidney injuries. In addition, differentiated MSCs with further in vivo investigations may serve as a cell source for tissue engineering or cell therapy in different renal ailments.

Specific and quantitative detection of Human polyomaviruses BKPyV and JCPyV in the healthy Pakistani population.

BACKGROUND: The BK Polyomavirus (BKPyV) and JC polyomavirus (JCPyV) infections are widespread in human population and have been associated with severe kidney and brain disorders, respectively. The viruses remain latent primarily in reno-urinary tract, reactivating only in case of a compromised immune system. The seroepidemiology and molecular prevalence of BKPyV and JCPyV have been widely studied both in healthy and immunocompromised patients worldwide. However, data regarding the prevalence of these viruses in the immunocompetent or apparently healthy Pakistani population is lacking. Herein, we present the first ever report on quantitative prevalence of BKPyV and JCPyV in the peripheral blood of a randomly selected cohort of healthy Pakistani population. METHODS: A total of 266 whole blood samples were examined. The subjects were divided into three age groups: ≤ 25 years (young), 26-50 years (middle) and ≥ 51 years (elder). Absolute real time PCR assay was designed to quantify the BKPyV and JCPyV viral copy numbers in the range of 106 to 100 copies/mL. RESULTS: Overall, BKPyV was detected in 27.1% (72/266) individuals while JCPyV in 11.6% (31/266) indicating significant difference (p < 0.005) in the distribution of these two viruses. The prevalence of BKPyV significantly decreased from 51% (49/96) in young age group to 8.2% (7/85) in eldest age group. Whereas, JCPyV positivity rate slightly increased from 8.3% (8/96) in young age group to 11.8% (10/85) in elder age group. The median viral load was calculated as 6.2 log and 3.38 log copies/mL of blood for BKPyV and JCPyV, respectively. Notably, no significant difference in viral load of either of the subtypes was found between different age groups. CONCLUSION: The current study provides an important baseline data on the prevalence and viral load of circulating BKPyV and JCPyV in Pakistani population. The prevalence and viral load of BKPyV was comparatively higher than JCPyV. The prevalence of BKPyV significantly decreased with increase in age while JCPyV positivity rate slightly increased with increasing age. Viral load of both BKPyV and JCPyV was not correlated with the individual ages.

Incidence of pediatric acute kidney injury in hospitalized patients.

Pediatric acute kidney injury (pAKI) is a common complication associated with high mortality in children. The objective of this study was to determine the incidence of acute kidney injury (AKI) and mortality in hospitalized (critically ill and non-critically ill) patients. This was a retrospective study conducted during the period of June 1, 2013, to May 31, 2014, at the Postgraduate Department of Pediatrics, G. B. Pant Hospital, an Associated Hospital of Government Medical College, Srinagar, Jammu and Kashmir, India. All patients between the ages of one month and 18 years were included in the study, who had AKI. In general, out of 23,794 patients, 197 developed AKI (0.8%). On subgroup analysis, 2460 were critically ill and had Intensive Care Unit (ICU) admission among whom 99 developed AKI (4%), whereas 21,334 had general pediatric ward admissions and 98 developed AKI (0.5%). Infantile age group was the most commonly 91 (46.2%) affected. The common causes of AKI were renal in 73 (37%), neurologic in 38 (19%), septicemia in 35 (18%), and inborn errors of metabolism in 30 (15.2%). Out of 197 pAKI patients, 42 (21.3%) died and all of them were critically sick (ICU admissions). The incidence of pAKI in general was 0.8%, whereas it was 4% in critically ill children and 0.5% in general ward admissions implying an eight-fold increased risk of pAKI in critically ill patients.

A cornucopia of screening and diagnostic techniques for human papillomavirus associated cervical carcinomas.

Cervical carcinoma is one of the major consequences of human papillomavirus (HPV) infections. Although HPV infections of cervix do not always progress to cancer, 90% cases of cervical cancer have been found associated with high risk HPV (hrHPV) infection. Usually, HPV infection is asymptomatic; however, this asymptomatic infection can cause abnormal changes in cervix ultimately leading to cancer development. These changes can be detected by the application of screening tests at regular time intervals. For this purpose, morphological, cytological, and DNA based techniques are available. Nevertheless, abnormal screening tests have only the predictive value for precancerous lesions and thus require further evaluation which is usually done by using diagnostic techniques. So far, colposcopy and histological examination alone were considered as the gold standards for cervical cancer diagnosis. Currently, some tests based on expression level of host cell biomarkers are also being used along with histology for diagnostic purpose. Albeit, these tests have significant specificity and sensitivity values but they are unable to suggest a particular viral genotype involved in infection. Diagnostic methods such as PCR, HPV genotyping assays, microarray, and mRNA based assays are useful to predict the genotypes as well as the quantity of viral load in a host cell. Similarly, these diagnostic procedures have high specificity and sensitivity ranges. However, only few of them are practiced commonly, as approval of these tests as routine diagnostic tests requires clinical validation and cost effectiveness.