The human papillomavirus (HPV) E6 and E7 oncogenes have direct effects on host cell proliferation. The viral E2 protein regulates transcription of E6 and E7 and thereby has an indirect effect on cell proliferation. In HPV-induced tumours, misappropriate random integration of the viral genome into the host chromosome often leads to disruption of the E2 gene and the loss of E2 expression. This results in cessation of the virus life cycle and the deregulation of E6 and E7 and is an important step in tumourigenesis. However, prior to these integration events, E2 can interact directly with the E6 and E7 proteins and modulate their activities. E2 also interacts with a variety of host proteins, including the p53 tumour suppressor protein. Here we outline evidence that suggests a role for E2 in the regulation of cell proliferation, and we discuss the importance of this regulation in viral infection and cervical tumourigenesis.

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix.Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected. Protocol I. Preparation for fractionationThis section describes the preparation of poly-L-Lysine coated microscope coverslips and the attachment of cells prior to fractionation. If required the cells can be transiently transfected with protein expression vectors either before or after attachment. 2. Coat clean coverslips with poly-L-Lysine by incubating them in the solution for at least 1 hour on a rocking platform at 22°C. 3. Wash the coated coverslips with sterile distilled water twice and follow with a single wash with 96% ethanol. 4. Air dry the coated coverslips on a piece of filter paper and keep them in a dry container for future use (once dry they can be stacked). A. Preparation of poly-L-1. Place a poly-L-Lysine coated coverslip in a well in a 6-well plate with the coated surface facing up. 2. Seed K562 cells at a density of 7x10 5 cells/well in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and PS (penicillin 100units/ml, streptomycin 100mg/ml). In the case of K562 cells transient transfection can be performed using electroporation (0.4cm electroporation cuvettes with 1x10 7 cells in 200μl of media at 250V / 975μF) before seeding the cells. 3. Incubate the cells for 24 hours at 37°C in 5% CO2. 4. Pour off the medium and wash the cells twice with ice cold phosphate buffered saline (PBS). 5. Follow with the subcellular fractionation protocol.1. Place an uncoated coverslip in a well in a 6-well plate. 2. Wash twice in PBS prewarmed at 37°C. 3. Detach the cells by digesting with trypsin (0.03% EDTA, 0.25% trypsin) ...

Abstract. There is a lack of non-invasive screening modalities to diagnose chronic atrophic gastritis (CAG) and intestinal metaplasia (IM). Thus, the aim of the present study was to determine the sensitivity and specificity of serum pepsinogen I (PGI), PGI:II, the PGI:II ratio and gastrin-17 (G-17) in diagnosing CAG and IM, and the correlations between these serum biomarkers and pre-malignant gastric lesions. A cross-sectional study of 72 patients (82% of the calculated sample size) who underwent oesophageal-gastro-duodenoscopy for dyspepsia was performed in the present study. The mean age of the participants was 56.2±16.2 years. Serum PGI:I, PGI:II, G-17 and Helicobacter pylori antibody levels were measured by enzyme-linked immunosorbent assay. Median levels of PGI:I, PGI:II, the PGI:II ratio and G-17 for were 129.9 µg/l, 10.3 µg/l, 14.7 and 4.4 pmol/l, respectively. Subjects with corpus CAG/IM exhibited a significantly lower PGI:II ratio (7.2) compared with the control group (15.7; P<0.001). Histological CAG and IM correlated well with the serum PGI:II ratio (r=-0.417; P<0.001). The cut-off value of the PGI:II ratio of ≤10.0 demonstrated high sensitivity (83.3%), specificity (77.9%) and area under the receiver operating characteristic curve of 0.902 in detecting the two conditions. However, the sensitivity was particularly low at a ratio of ≤3.0. The serum PGI:II ratio is a sensitive and specific marker to diagnose corpus CAG/IM, but at a high cut-off value. This ratio may potentially be used as an outpatient, non-invasive biomarker for detecting corpus CAG/IM.

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Chronic myeloid leukaemia is blood cancer due to a reciprocal translocation, resulting in a BCR-ABL1 oncogene. Although tyrosine kinase inhibitors have been successfully used to treat CML, there are still cases of resistance. The resistance occurred mainly due to the mutation in the tyrosine kinase domain of the BCR-ABL1 gene. However, there are still many cases with unknown causes of resistance as the etiopathology of CML are not fully understood. Thus, it is crucial to figure out the complete pathogenesis of CML, and miRNA can be one of the essential pathogeneses. The objective of this study was to systematically review the literature on miRNAs that were differentially expressed in CML cases. Their target genes and downstream genes were also explored. An electronic search was performed via PubMed, Scopus, EBSCOhost MEDLINE, and Science Direct. The following MeSH (Medical Subject Heading) terms were used: chronic myeloid leukaemia, genes and microRNAs in the title or abstract. From 806 studies retrieved from the search, only clinical studies with in-vitro experimental evidence on the target genes of the studied miRNAs in CML cells were included. Two independent reviewers independently scrutinised the titles and abstracts before examining the eligibility of studies that met the inclusion criteria. Study design, sample size, sampling type, and the molecular method used were identified for each study. The pooled miRNAs were analysed using DIANA tools, and target genes were analysed with DAVID, STRING and Cytoscape MCODE. Fourteen original research articles on miRNAs in CML were included, 26 validated downstream genes and 187 predicted target genes were analysed and clustered into 7 clusters. Through GO analysis, miRNAs’ target genes were localised throughout the cells, including the extracellular region, cytosol, and nucleus. Those genes are involved in various pathways that regulate genomic instability, proliferation, apoptosis, cell cycle, differentiation, and migration of CML cells.

Cancer increases the global disease burden substantially, but it remains a challenge to manage it. The search for novel biomarkers is essential for risk assessment, diagnosis, prognosis, prediction of treatment response, and cancer monitoring. This paper examined NEDD8 ultimate buster-1 (NUB1) and F-adjacent transcript 10 (FAT10) proteins as novel biomarkers in cancer. This literature review is based on the search of the electronic database, PubMed. NUB1 is an interferon-inducible protein that mediates apoptotic and anti-proliferative actions in cancer, while FAT10 is a ubiquitin-like modifier that promotes cancer. The upregulated expression of both NUB1 and FAT10 has been observed in various cancers. NUB1 protein binds to FAT10 non-covalently to promote FAT10 degradation. An overexpressed FAT10 stimulates nuclear factor-kappa β, activates the inflammatory pathways, and induces the proliferation of cancer. The FAT10 protein interacts with the mitotic arrest deficient 2 protein, causing chromosomal instability and breast tumourigenesis. FAT10 binds to the proliferating cell nuclear antigen protein and inhibits the DNA damage repair response. In addition, FAT10 involves epithelial–mesenchymal transition, invasion, apoptosis, and multiplication in hepatocellular carcinoma. Our knowledge about them is still limited. There is a need to further develop NUB1 and FAT10 as novel biomarkers.

Intestinal fibrosis is a common complication of inflammatory bowel diseases. However, the possible involvement of epithelial-mesenchymal transition (EMT) has been scarcely investigated. This systematic review aims to search through research papers that are focusing on messenger RNA (mRNA) and protein expression profile in EMT in fistula or in intestinal fibrosis. Methods: Electronic exploration was performed until April 24, 2019 through PubMed, Ovid, Science Direct, and Scopus databases with the terms of "fistula" OR "intestinal fibrosis" AND "epithelial-mesenchymal transition". Two independent reviewers scrutinized the suitability of the title and abstract before examining the full text that met the inclusion criteria. For each study, the sample types that were used, methods for analysis, and genes expressed were identified. The list of genes was further analyzed using DAVID (Database for Annotation, Visualization, and Integrated Discovery) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway. Results: There were 896 citations found; however, only 3 studies fulfilled the requirements. Among the EMT-related genes, 5 were upregulated genes at mRNA level while 6 were at protein level. However, only 2 downregulated genes were found at each mRNA and protein level. Of the 4 inflammation-related genes found, 3 genes were upregulated at mRNA level and 1 at protein level. These genes were confirmed to be involved in the development of inflammatory induced fibrosis and fistula through EMT. Results from quantitative real-time polymerase chain reaction analysis were consistent with the process of EMT, confirmed by the western blot protein analysis. Conclusion: Many significant genes which are involved in the process of EMT in fistula and intestinal fibrosis have been identified. With high-end technology many more genes could be identified. These genes will be good molecular targets in the development of biomarkers for precision drug targeting in the future treatment of intestinal fibrosis and fistula.

Background: Chronic myeloid leukemia (CML) is associated with BCR-ABL1 gene that plays a central role in the pathogenesis of CML. Thus, it is crucial to supress the expression of BCR-ABL1 in the treatment of CML. MiRNA is known to be a gene expression regulator, thus it is a good candidate for molecularly targeted therapy for CML. Objective: This study aims to identify the miRNAs from edible plants that targeted the 3’ untranslated region (3’UTR) of BCR-ABL1. Method: In this in silico analysis, the sequence of 3’UTR of BCR-ABL1 was obtained from Ensembl Genome Browser. PsRNATarget Analysis Server and MicroRNA Target Prediction (miRTar) Server were utilised to identify miRNAs that have binding conformity with 3’UTR of BCR-ABL1. MiRBase database was used to validate the species of plants expressing the miRNAs. RNAfold web server and RNA COMPOSER were used for secondary and tertiary structure prediction respectively. Results: In silico analyses reveal cpa-miR8154, csi-miR3952, gma-miR4414-5p, mdm-miR482c, osa-miR1858a and osa-miR1858b are having binding conformity with strong molecular interaction toward 3’UTR region of BCR-ABL1. However only cpa-miR-8154, osa-miR-1858a and osa-miR-1858b showed good target site accessibility. Conclusion: It is predicted that these miRNAs post-transcriptionally inhibit BCR-ABL1 gene via cleavage and thus, they could be a potential molecular targeted therapy for CML. However, further studies involving in vitro, in vivo and functional analyses need to be carried out in order to determine the ability of these miRNAs as a targeted therapy for CML.