The Role of Hedgehog Acyltransferase & Heparan Sulphate Proteoglycans in Human Sonic Hedgehog Signalling

Hedgehog proteins (Hh) are morphogens and major mediators in many developmental processes. Hh signalling is significant for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancers. Hh proteins require dual lipidation and Heparan Sulfate Proteoglycans (HSPGs) for their proper distribution and signalling activity. My first aim was to study the role of HSPGs in human (h) Sonic Hedgehog (Shh) signalling and clarify the biological function of hShh/HSPGs complexes in hShh signalling, by investigating the interaction between human hShh and HSPGs. I used DNA mutagenesis and heparin affinity chromatography to determine key residues in hShh involved in heparin binding (K37/38 and K178). The activity of these mutants was tested by detecting induced Alkaline Phosphatase activity in C3H10T1/2 cells and hShh-inducible gene expression in PANC1 human pancreatic carcinoma cells. I examined the biological function of mutated hShhs (K37/38S, K178S and K37/38/178S) that cannot interact with heparin efficiently and showed that they had reduced signalling activity compared to wild type hShh and a control mutation (K74S). Also, I showed that mutant hShh proteins mediate reduced proliferation and invasion of PANC1 cells following hShh RNAi knockdown (KD), and this correlated with reduced Shh multimeric complex formation. Structurally, Shh proteins are unusual in being dually lipid-modified to be fully active. During the post-translational modifications of Shh, N-terminal palmitoylation is facilitated by the product (Hhat) of the hedgehog acyltransferase gene. I have carried out a thorough analysis of Hhat in PANC1 cells. First, I characterised an antibody prepared in the lab to hHhat. I confirmed the specificity of the antibody by immunoblotting using a self-constructed hHhat-EGFP clone, and a control mGup1-EGFP clone. By subcellular fractionation and Western blotting I found Hhat to be a membrane protein. In addition, I used the hHhat antibody to determine the intracellular localisation of hHhat in PANC1 cells by confocal microscopy and showed that hHhat localised in ER mainly but not in Golgi apparatus. I confirmed this using the hHhat-EGFP clone for fluorescence microscopy in transfected cells. To illuminate the biological function of palmitoylation of hShh in production of active hShh and in the formation of hShh multimeric complex I optimised hHhat RNAi knockdown (KD) in PANC1 cells and confirmed this by a cell-based palmitoylation assay. Using semi-quantitative RT-PCR and immunoblot analyses, I showed that hHhat KD caused decreased signalling through the Shh pathway due to reduced production of active hShh. In addition, I investigated the effect of the addition of palmitate to hShh on its association with cells by comparing hHhat KD cells with control cells. Immunoblotting suggests that palmitoylation of Shh improves its ability to associate to cell membranes. Using hHhat KD, gel filtration of high molecular weight complexes of hShh and immunoblotting of hShh I characterised the role of palmitoylation of hShh in multimeric complex formation. Lastly, I investigated the effect of hHhat KD on PANC1 proliferation and invasion, showing that it represses PANC1 proliferation and invasion. These studies provide a firm basis for understanding the functional roles of hShh palmitoylation and its interactions with HSPGs, and provide proof-of-principle for targeting these aspects of hShh biology in tumour cell therapeutics, specifically in the pancreatic carcinoma context

Productivity and Territorial Specialisation

On the basis of a relatively free trade world and global economy, specialisation tends to increase the comparative advantages of a country. But in this scenario, not countries but cities are the territorial organization that play the main role. Cities concentrate the economic activity and political influential household that reflects economic changes. The purpose of this study is to explore the principles underlie the distribution of population on a territory and the likely effect of economic development on redistributing population geographically

Gender-related issues in a Taiwanese university medical science laboratory setting: a qualitative analysis

IntroductionThis paper provides a glimpse into gender issues in a university-based medical science laboratory setting in northern Taiwan. In this study, gender issues with respect to perceptions regarding gender, the degree of gender neutrality in the work environment, and the influence of gender on researchers’ academic careers were analyzed.MethodsFrom July to August 2021, semistructured interviews to understand the perspectives of five faculty members at Chang Gung University School of Medicine regarding gender issues were conducted. The data were transcribed verbatim and analyzed thematically. Subsequently, coding was performed using ATLAS.ti Web (Version 4.0.10).Results and discussionIt was found that gender is not perceived to correlate with performance in the medical sciences. Although the medical science laboratories in the study institution are mostly gender-neutral, instances of discrimination might have been concealed elsewhere because of underreporting. Nevertheless, medical science research culture in Chang Gung University appears to promote respect and equality owing to increased general awareness regarding such issues as well as robust policies that protect women’s rights and promote gender equality. Marriage, motherhood, and family obligations remain key challenges to the academic career of female scientists in the institution. To achieve more equitable representation of male and female scientists as well as to prevent female scientists from leaving medical science laboratories in Taiwan, institutional and national policies that provide tailored support for female scientists intending to start families must continue to be implemented

Technology computer-aided design simulation study for a strained InGaAs channel n-type metal-oxide-semiconductor field-effect transistor with a high-k dielectric oxide layer and a metal gate electrode

The stress distributions in the InGaAs channel regions of strained InGaAs metal-oxide-semiconductor (MOS) field-effect transistors with high-k dielectric layer, metal gate, and InGaAs alloy souce/drain (S/D) stressors were studied with three-dimensional process simulations. It was shown that the geometric effects, such as channel width and length, could impact the achievable transistor performance gains. In this work, high-performance III-V MOS devices were achieved by stressors, such as S/D stressors, with the InGaAs alloy material. The resulting mobility improvement was analyzed by the Monte Carlo simulations. Tensile stress along the transport direction was found to dominate mobility gain while narrower devices (<1 mu m), and a decrease of tensile stress along the channel direction contributed to a decrease in mobility gain owing to the decreasing width. This work helps the future III-V-based MOS device design and demonstrates that strain engineering is important for future nanoscale device technology

Stateless Two-Stage Multiple Criteria Scheduling in Nuclear Medicine

Examination in nuclear medicine exhibits scheduling difficulties due to its intricate clinical issues, such as varied radiopharmaceuticals for different diseases, machine preparation and length of scan, and patients’ and hospital’s criteria and/or limitations. Many scheduling methods exist but are limited for nuclear medicine. In this paper, we present stateless two-stage scheduling to cope with multiple criteria decision making. The first stage mostly deals with patients’ conditions. The second stage concerns more the clinical condition and its correlations with patients’ preference which presents more complicated intertwined configurations. A greedy algorithm is proposed in the second stage to determine the (time slot and patient) pair in linear time. The result shows practical and efficient scheduling for nuclear medicine

Discovery of serum biomarkers of alcoholic fatty liver in a rodent model: C-reactive protein

<p>Abstract</p> <p>Background</p> <p>Excessive consumption of alcohol contributes to alcoholic liver disease. Fatty liver is the early stage of alcohol-related liver disease. The aim of this study was to search for specific serological biomarkers of alcoholic fatty liver (AFL) compared to healthy controls, non-alcoholic fatty liver (NAFL) and liver fibrosis in a rodent model.</p> <p>Methods</p> <p>Serum samples derived from animals with AFL, NAFL, or liver fibrosis were characterized and compared using two-dimensional differential gel electrophoresis. A matrix-assisted laser desorption ionization-time of flight tandem mass spectrometer in conjunction with mascot software was used for protein identification. Subsequently, Western blotting and flexible multi-analyte profiling were used to measure the expressions of the putative biomarkers present in the serum of animals and clinical patients.</p> <p>Results</p> <p>Eight differential putative biomarkers were identified, and the two most differentiated proteins, including upregulated C-reactive protein (CRP) and downregulated haptoglobin (Hp), were further investigated. Western blotting validated that CRP was dramatically higher in the serum of AFL compared to healthy controls and other animals with liver disease of NAFL or liver fibrosis (<it>p </it>< 0.05). Moreover, we found that CRP and Hp were both lower in liver fibrosis of TAA-induced rats and clinical hepatitis C virus-infected patients.</p> <p>Conclusion</p> <p>The results suggest that increased levels of CRP are an early sign of AFL in rats. The abnormally elevated CRP induced by ethanol can be used as a biomarker to distinguish AFL from normal or otherwise diseased livers.</p

Recruitment of Rad51 and Rad52 to Short Telomeres Triggers a Mec1-Mediated Hypersensitivity to Double-Stranded DNA Breaks in Senescent Budding Yeast

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of telomerase. In both mammalian tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative recombination mechanism. Here we demonstrated that the budding yeast Saccharomyces cerevisiae type I survivors derived from telomerase-deficient cells were hypersensitive to DNA damaging agents. Assays to track telomere lengths and drug sensitivity of telomerase-deficient cells from spore colonies to survivors suggested a correlation between telomere shortening and bleomycin sensitivity. Our genetic studies demonstrated that this sensitivity depends on Mec1, which signals checkpoint activation, leading to prolonged cell-cycle arrest in senescent budding yeasts. Moreover, we also observed that when cells equipped with short telomeres, recruitments of homologous recombination proteins, Rad51 and Rad52, were reduced at an HO-endonuclease-catalyzed double-strand break (DSB), while their associations were increased at chromosome ends. These results suggested that the sensitive phenotype may be attributed to the sequestration of repair proteins to compromised telomeres, thus limiting the repair capacity at bona fide DSB sites