Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes

<p>Abstract</p> <p>Background</p> <p>Infection with high-risk type human papilloma viruses (HPVs) is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features.</p> <p>Results</p> <p>Six replicate measurements were performed for each group using two-dimensional difference gel electrophoresis (2D-DIGE). The median within-group coefficient of variation was 19–21%. Significance of between-group differences was tested based on Significance Analysis of Microarray and fold change. Expression of 170 (23%) of the protein features changed significantly in immortalized cells compared to primary keratinocytes. Most of these changes were qualitatively similar in cells immortalized by E6, E7, or E6/7 expression, indicating convergence on a common phenotype, but fifteen proteins (~2%) were outliers in this regulatory pattern. Ten demonstrated opposite regulation in E6- and E7-expressing cells, including the cell cycle regulator p16^INK4a; the carbohydrate binding protein Galectin-7; two differentially migrating forms of the intermediate filament protein Cytokeratin-7; HSPA1A (Hsp70-1); and five unidentified proteins. Five others had a pattern of expression that suggested cooperativity between the co-expressed oncoproteins. Two of these were identified as forms of the small heat shock protein HSPB1 (Hsp27).</p> <p>Conclusion</p> <p>This large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.</p

Functional Changes Within the Rod Inner Segment Ellipsoid in Wildtype Mice: An Optical Coherence Tomography and Electron Microscopy Study

Purpose: To test the hypothesis that changing energy needs alter mitochondria distribution within the rod inner segment ellipsoid. Methods: In mice with relatively smaller (C57BL/6J [B6J]) or greater (129S6/ev [S6]) retina mitochondria maximum reserve capacity, the profile shape of the rod inner segment ellipsoid zone (ISez) was measured with optical coherence tomography (OCT) under higher (dark) or lower (light) energy demand conditions. ISez profile shape was characterized using an unbiased ellipse descriptor (minor/major aspect ratio). Other bioenergy indexes evaluated include the external limiting membrane–retinal pigment epithelium (ELM-RPE) thickness and the magnitude of the signal intensity of a hyporeflective band located between the photoreceptor tips and apical RPE. The spatial distribution of rod ellipsoid mitochondria were also examined with electron microscopy. Results: In B6J mice, darkness produced a greater ISez aspect ratio, thinner ELM-RPE, and a smaller hyporeflective band intensity than in light. In S6 mice, dark and light ISez aspect ratio values were not different and were greater than in light-adapted B6J mice; dark-adapted S6 mice showed smaller ELM-RPE thinning versus light, and negligible hyporeflective band intensity in the light. In B6J mice, mitochondria number in light increased in the distal inner segment ellipsoid and decreased proximally. In S6 mice, mitochondria number in the inner segment ellipsoid were not different between light and dark, and were greater than in B6J mice. Conclusions: These data raise the possibility that rod mitochondria activity in mice can be noninvasively evaluated based on the ISez profile shape, a new OCT index that complements OCT energy biomarkers measured outside of the ISez region

QUEnch assiSTed (QUEST) MRI Used as a Novel Approach to Identify Reactive Oxygen Species as a Result of Experimental TBI

Introduction: Traumatic brain injury (TBI) generates reactive oxygen species (ROS), promoting inflammatory processes and impeding TBI recovery. Within the VA population, over 70% of military personnel that sustain a TBI receive opioid-based pain relief, however, opiates may actually exacerbate post-TBI complications through its documented recruitment of oxidative and inflammatory systems. Thus, we hypothesize that TBI and opioid treatment act synergistically to worsen post-TBI oxidative stress. Methods: Mice were exposed to either TBI or sham injury and administered morphine or saline in the acute post-injury period. Afterwards, neuroimaging was conducted using a novel technique, QUEnch assiSTed (QUEST) MRI, which compares standard MRI signals across mice that acutely receive an antioxidant “quench” therapy and those receiving saline as control. Therefore, differential MRI signals between these groups are an index of ROS generation. Changes in hippocampus and cortex signals were measured, as these structures are most commonly affected by TBI. Methylene blue and α-lipoic acid were used as antioxidants in the quenching step as they halt mitochondrial ROS production and scavenge excess ROS, respectively. Results: No significant changes in ROS levels were detected as a result of TBI, opioid exposure or their combination using QUEST MRI in either the cortex or hippocampus. Conclusions and Future Directions: While QUEST imaging did not yield significant changes between experimental groups, future work will include ex-vivo biochemical ROS analyses from harvested tissues, which will provide higher resolution quantification of oxidative processes than that of QUEST MRI

In vivo imaging of prodromal hippocampus CA1 subfield oxidative stress in models of Alzheimer disease and Angelman syndrome

Hippocampus oxidative stress is considered pathogenic in neurodegenerative diseases, such as Alzheimer disease (AD), and in neurodevelopmental disorders, such as Angelman syndrome (AS). Yet clinical benefits of antioxidant treatment for these diseases remain unclear because conventional imaging methods are unable to guide management of therapies in specific hippocampus subfields in vivo that underlie abnormal behavior. Excessive production of paramagnetic free radicals in nonhippocampus brain tissue can be measured in vivo as a greaterâ thanâ normal 1/T1 that is quenchable with antioxidant as measured by quenchâ assisted (Quest) MRI. Here, we further test this approach in phantoms, and we present proofâ ofâ concept data in models of ADâ like and AS hippocampus oxidative stress that also exhibit impaired spatial learning and memory. ADâ like models showed an abnormal gradient along the CA1 dorsalâ ventral axis of excessive free radical production as measured by Quest MRI, and redoxâ sensitive calcium dysregulation as measured by manganeseâ enhanced MRI and electrophysiology. In the AS model, abnormally high free radical levels were observed in dorsal and ventral CA1. Quest MRI is a promising in vivo paradigm for bridging brain subâ field oxidative stress and behavior in animal models and in human patients to better manage antioxidant therapy in devastating neurodegenerative and neurodevelopmental diseases.â Berkowitz, B. A., Lenning J., Khetarpal, N., Tran, C., Wu, J. Y., Berri, A. M., Dernay, K., Haacke, E. M., Shafieâ Khorassani, F., Podolsky, R. H., Gant, J. C., Maimaiti, S., Thibault, O., Murphy, G. G., Bennett, B. M., Roberts, R. In vivo imaging of prodromal hippocampus CA1 subfield oxidative stress in models of Alzheimer disease and Angelman syndrome. FASEB J. 31, 4179â 4186 (2017). www.fasebj.orgâ Berkowitz, Bruce A., Lenning, Jacob, Khetarpal, Nikita, Tran, Catherine, Wu, Johnny Y., Berri, Ali M., Dernay, Kristin, Haacke, E. Mark, Shafieâ Khorassani, Fatema, Podolsky, Robert H., Gant, John C., Maimaiti, Shaniya, Thibault, Olivier, Murphy, Geoffrey G., Bennett, Brian M., Roberts, Robin, In vivo imaging of prodromal hippocampus CA1 subfield oxidative stress in models of Alzheimer disease and Angelman syndrome. FASEB J. 31, 4179â 4186 (2017)Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154241/1/fsb2fj201700229r.pd

Dissection of the Inflammatory Bowel Disease Transcriptome Using Genome-Wide cDNA Microarrays

BACKGROUND: The differential pathophysiologic mechanisms that trigger and maintain the two forms of inflammatory bowel disease (IBD), Crohn disease (CD), and ulcerative colitis (UC) are only partially understood. cDNA microarrays can be used to decipher gene regulation events at a genome-wide level and to identify novel unknown genes that might be involved in perpetuating inflammatory disease progression. METHODS AND FINDINGS: High-density cDNA microarrays representing 33,792 UniGene clusters were prepared. Biopsies were taken from the sigmoid colon of normal controls (n = 11), CD patients (n = 10) and UC patients (n = 10). (33)P-radiolabeled cDNA from purified poly(A)(+) RNA extracted from biopsies (unpooled) was hybridized to the arrays. We identified 500 and 272 transcripts differentially regulated in CD and UC, respectively. Interesting hits were independently verified by real-time PCR in a second sample of 100 individuals, and immunohistochemistry was used for exemplary localization. The main findings point to novel molecules important in abnormal immune regulation and the highly disturbed cell biology of colonic epithelial cells in IBD pathogenesis, e.g., CYLD (cylindromatosis, turban tumor syndrome) and CDH11 (cadherin 11, type 2). By the nature of the array setup, many of the genes identified were to our knowledge previously uncharacterized, and prediction of the putative function of a subsection of these genes indicate that some could be involved in early events in disease pathophysiology. CONCLUSION: A comprehensive set of candidate genes not previously associated with IBD was revealed, which underlines the polygenic and complex nature of the disease. It points out substantial differences in pathophysiology between CD and UC. The multiple unknown genes identified may stimulate new research in the fields of barrier mechanisms and cell signalling in the context of IBD, and ultimately new therapeutic approaches

Microbial Patterns Signaling via Toll-Like Receptors 2 and 5 Contribute to Epithelial Repair, Growth and Survival

Epithelial cells (ECs) continuously interact with microorganisms and detect their presence via different pattern-recognition receptors (PRRs) including Toll-like receptors (TLRs). Ligation of epithelial TLRs by pathogens is usually associated with the induction of pro-inflammatory mediators and antimicrobial factors. In this study, using human airway ECs as a model, we found that detection of microbial patterns via epithelial TLRs directly regulates tissue homeostasis. Staphylococcus aureus (S. aureus) and microbial patterns signaling via TLR2 and TLR5 induce a set of non-immune epithelial responses including cell migration, wound repair, proliferation, and survival of primary and cancerous ECs. Using small interfering RNA (siRNA) gene targeting, receptor-tyrosine kinase microarray and inhibition studies, we determined that TLR and the epidermal growth factor receptor (EGFR) mediate the stimulating effect of microbial patterns on epithelial repair. Microbial patterns signaling via Toll-like receptors 2 and 5 contribute to epithelial repair, growth and survival. This effect is independent of hematopoietic and other cells as well as inflammatory cytokines suggesting that epithelia are able to regulate their integrity in an autonomous non-inflammatory manner by sensing microbes directly via TLRs

Activation of Aryl Hydrocarbon Receptor (AhR) Leads to Reciprocal Epigenetic Regulation of FoxP3 and IL-17 Expression and Amelioration of Experimental Colitis

Aryl hydrocarbon receptor (AhR), a transcription factor of the bHLH/PAS family, is well characterized to regulate the biochemical and toxic effects of environmental chemicals. More recently, AhR activation has been shown to regulate the differentiation of Foxp3(+) Tregs as well as Th17 cells. However, the precise mechanisms are unclear. In the current study, we investigated the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AhR ligand, on epigenetic regulation leading to altered Treg/Th17 differentiation, and consequent suppression of colitis.Dextran sodium sulphate (DSS) administration induced acute colitis in C57BL/6 mice, as shown by significant weight loss, shortening of colon, mucosal ulceration, and increased presence of CXCR3(+) T cells as well as inflammatory cytokines. Interestingly, a single dose of TCDD (25 µg/kg body weight) was able to attenuate all of the clinical and inflammatory markers of colitis. Analysis of T cells in the lamina propria (LP) and mesenteric lymph nodes (MLN), during colitis, revealed decreased presence of Tregs and increased induction of Th17 cells, which was reversed following TCDD treatment. Activation of T cells from AhR(+/+) but not AhR (-/-) mice, in the presence of TCDD, promoted increased differentiation of Tregs while inhibiting Th17 cells. Analysis of MLN or LP cells during colitis revealed increased methylation of CpG islands of Foxp3 and demethylation of IL-17 promoters, which was reversed following TCDD treatment.These studies demonstrate for the first time that AhR activation promotes epigenetic regulation thereby influencing reciprocal differentiation of Tregs and Th17 cells, and amelioration of inflammation

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research