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.