Effects of selenium depletion and selenoprotein knockdown on inflammatory signalling in a gut epithelial cell line

Selenium is a micronutrient essential for human health. Low Se intake versus Se supplementation have been reported to elevate and lower mortality from colorectal cancer. Se is present in selenoproteins including glutathione peroxidases (GPx) 1-4. GPxs are antioxidant enzymes that protect cells from excessive oxidative stress and they may have a role in maintaining innate immunity homeostasis against inflammatory stimulation from exposure to luminal bacteria. This thesis describes studies of the regulatory effects of Se depletion and selenoprotein knockdown in the gastrointestinal cell line Caco-2 in relation to inflammatory responses. A luciferase reporter model was developed in which Caco-2 cells were stably transfected with gene constructs in which luciferase expression was under the control of regulatory elements that bind the Nuclear Factor-kappa B (NF B), a transcription factor central to inflammatory signaling pathways (Chapter 3). As a control reporter luciferase coding sequences were linked to a TATA box. When Caco-2 cells expressing these constructs were grown in Se-deficient conditions, a 30% increase of reporter activity and a 50% increase in endogenous interleukin 8 mRNA levels were observed after stimulation with TNF . In comparison, no changes in reporter activity were found in Se-deficient cells after stimulation with flagellin (Chapter 4). Small interfering RNA was used to knockdown expression of individual selenoproteins including GPx1, GPx4, SelW and SelH (Chapter 5). Knockdown of GPx1 expression by ~55% led to a 25% decrease of reporter activity and a 17% decrease of IL8 mRNA level after stimulation with TNF ✁ . Knockdown of SelW or SelH expression had no observable effect on reporter activity. In addition, Se depletion elevated cellular ROS production as assessed by Carboxy-2’7’-dichlorodihydrofluorescein diacetate staining whereas GPx1 knockdown had no significant effect (Chapter 5). Knockdown of GPx4 by 85%, as assessed by RT-PCR and Western blotting, had little effect on TNF -driven luciferase activity. However, GPx4 knockdown lowered flagellin-induced reporter activity by 20% and interleukin 8 mRNA levels by 40% (Chapter 6). It is concluded that 1] low Se supply affects NF B inflammatory response in Caco-2 cells, 2] the exact role of different selenoproteins in this effect remains to be elucidated, and 3] the endogenous and exogenous inflammatory responses in Caco-2 cells are differentially regulated by Se supply and by antioxidant selenoproteins.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Inflammation-Induced Cell Proliferation Potentiates DNA Damage-Induced Mutations In Vivo

Mutations are a critical driver of cancer initiation. While extensive studies have focused on exposure-induced mutations, few studies have explored the importance of tissue physiology as a modulator of mutation susceptibility in vivo. Of particular interest is inflammation, a known cancer risk factor relevant to chronic inflammatory diseases and pathogen-induced inflammation. Here, we used the fluorescent yellow direct repeat (FYDR) mice that harbor a reporter to detect misalignments during homologous recombination (HR), an important class of mutations. FYDR mice were exposed to cerulein, a potent inducer of pancreatic inflammation. We show that inflammation induces DSBs (γH2AX foci) and that several days later there is an increase in cell proliferation. While isolated bouts of inflammation did not induce HR, overlap between inflammation-induced DNA damage and inflammation-induced cell proliferation induced HR significantly. To study exogenously-induced DNA damage, animals were exposed to methylnitrosourea, a model alkylating agent that creates DNA lesions relevant to both environmental exposures and cancer chemotherapy. We found that exposure to alkylation damage induces HR, and importantly, that inflammation-induced cell proliferation and alkylation induce HR in a synergistic fashion. Taken together, these results show that, during an acute bout of inflammation, there is a kinetic barrier separating DNA damage from cell proliferation that protects against mutations, and that inflammation-induced cell proliferation greatly potentiates exposure-induced mutations. These studies demonstrate a fundamental mechanism by which inflammation can act synergistically with DNA damage to induce mutations that drive cancer and cancer recurrence.Austrian Academy of Sciences (APART Fellowship)Singapore-MIT Alliance for Research and TechnologySingapore. National Research FoundationNational Institutes of Health (U.S.) (NIH R33-CA112151)National Institutes of Health (U.S.) (grant R01-CA079827

High-Performance Non-enzymatic Glucose Sensors Based on CoNiCu Alloy Nanotubes Arrays Prepared by Electrodeposition

Transition metal alloys are good candidate electrodes for non-enzymatic glucose sensors due to their low cost and high performance. In this work, we reported the controllable electrodeposition of CoNiCu alloy nanotubes electrodes using anodic aluminum oxide (AAO) as template. Uniform CoNiCu alloy arrays of nanotubes about 2 μm in length and 280 nm in diameter were obtained by optimizing the electrodeposition parameters. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) measurements indicated that the as-prepared alloy nanotubes arrays are composed of 64.7 wt% Co-19.4 wt% Ni-15.9 wt% Cu. Non-enzymatic glucose sensing measurements indicated that the CoNiCu nanotubes arrays possessed a low detection limit of 0.5 μM, a high sensitivity of 791 μA mM−1 cm−2 from 50 to 1,551 μM and 322 μA mM−1 cm−2 from 1,551 to 4,050 μM. Besides, they showed high reliability with the capacity of anti-jamming. Tafel plots showed that alloying brought higher exchange current density and faster reaction speed. The high performance should be due to the synergistic effect of Co, Ni, and Cu metal elements and high surface area of nanotubes arrays

Bevacizumab Combined With Oxaliplatin/Capecitabine in Patient With Refractory and Recurrent Mucinous Adenocarcinoma of the Appendix: A Case Report

Primary appendiceal adenocarcinoma with peritoneal pseudomyxoma (PPM) has a high recurrence rate and refractory to medical interventions such as repetitive debulking surgery and systemic chemotherapy. Genome-based targeted therapy for such cases has not been well-documented. Here we present a 63-years-old women, who was diagnosed with recurrent mucinous adenocarcinoma of the appendix with local invasions and peritoneal carcinomatosis, was refractory to systemic chemotherapy after surgery. We used a regime developed using whole exome sequencing. Somatic mutations in the genes encoding VEGFR2, FGFR1, FGFR2, FGFR3, and KRAS were identified in the patient's tumor tissue. The patient was then treated with bevacizumab plus oxaliplatin. After 4 months of treatment, pelvic CT showed dramatic reduction of pseudomyoma and a decline of CA199 level from 5436.7 to 1121.4 U/ml. Continual treatment with bevacizumab-capecitabine remained effective and the patient's CA199 level further decreased to 401.26 U/ml according to the follow-up examination on Aug 15th, 2018. Results from this study show the evidence of gene mutations involving VEGF signal activation in the recurrence of appendiceal adenocarcinoma. Our results also suggest the association of these mutations with the effectiveness of anti-VEGF treatment using bevacizumab. Therefore, the screening of gene mutations involved in VEGF signaling and targeted therapy with anti-VEGF drugs may provide a new option to manage refractory/recurrent advanced-stage appendiceal adenocarcinoma

Lamellipodin-Deficient Mice: A Model of Rectal Carcinoma

During a survey of clinical rectal prolapse (RP) cases in the mouse population at MIT animal research facilities, a high incidence of RP in the lamellipodin knock-out strain, C57BL/6-Raph1[superscript tm1Fbg] (Lpd[superscript -/-]) was documented. Upon further investigation, the Lpd[superscript -/-] colony was found to be infected with multiple endemic enterohepatic Helicobacter species (EHS). Lpd[superscript -/-] mice, a transgenic mouse strain produced at MIT, have not previously shown a distinct immune phenotype and are not highly susceptible to other opportunistic infections. Predominantly male Lpd[superscript -/-] mice with RP exhibited lesions consistent with invasive rectal carcinoma concomitant to clinically evident RP. Multiple inflammatory cytokines, CD11b+Gr1+ myeloid-derived suppressor cell (MDSC) populations, and epithelial cells positive for a DNA damage biomarker, H2AX, were elevated in affected tissue, supporting their role in the neoplastic process. An evaluation of Lpd[superscript -/-] mice with RP compared to EHS-infected, but clinically normal (CN) Lpd[superscript -/-] animals indicated that all of these mice exhibit some degree of lower bowel inflammation; however, mice with prolapses had significantly higher degree of focal lesions at the colo-rectal junction. When Helicobacter spp. infections were eliminated in Lpd[superscript -/-] mice by embryo transfer rederivation, the disease phenotype was abrogated, implicating EHS as a contributing factor in the development of rectal carcinoma. Here we describe lesions in Lpd[superscript -/-] male mice consistent with a focal inflammation-induced neoplastic transformation and propose this strain as a mouse model of rectal carcinoma.United States. National Institutes of Health (T32-OD010978)United States. National Institutes of Health (R01-OD011141)United States. National Institutes of Health (P30-ES002109)Massachusetts Institute of Technology. Ludwig Center for Molecular Oncology (U54- CA114462)National Cancer Institute (U.S.) (P30-CA14051

Measuring the missing: Knowledge, risk perceptions and self-protection practices of COVID-19 among the Asian population in New Zealand: An online survey

AimAsians are the second largest and fastest growing non-European population in New Zealand but are under-researched in terms of their COVID-19 pandemic response. The paper aims to illustrates Asians’ risk perceptions and knowledge of COVID-19, and self-protection practices to avoid infection and prevent community transmission.Subject and methodsAn online survey was used to collect data and received 402 valid responses. Data analyses included: 1) a descriptive analysis by using Chi-square tests and a Kruskal-Wallis rank sum tests to explore associations between responses and the four demographic variables (i.e. age, gender, country of origin/ethnicity, and region); and 2) a correlation analysis between different survey objectives.ResultsThe descriptive analysis of the survey found that while ethnicity (within the Asian category) was the most influential variable that resulted in varying responses to many questions, gender and age were other two important variables in influencing the answering patterns. The correlation analysis found a positive correlation between the perceived ‘dangerousness’ of COVID-19 and respondents’ overall compliance behaviour to New Zealand authorities’ recommendations to prevent spread of COVID-19.ConclusionThe majority of the respondents provided correct answers to the questions about the vulnerable populations, symptoms, asymptomatic transmission and potential sequelae of COVID-19; however, their understanding of the availability of a cure for, and the incubation period of COVID-19 was not consistent with the official information. The research also found that the higher perceived dangerousness of COVID-19, the better compliance to self-protection practices among the surveyed population

Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies

Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction-limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (~70 nm) imaging of cells and mammalian tissues on conventional microscopes.United States. National Institutes of Health (1R01GM104948)United States. National Institutes of Health (1DP1NS087724)United States. National Institutes of Health ( NIH 1R01EY023173)United States. National Institutes of Health (1U01MH106011