A naked-eye detection of cholesterol using enzyme cascade reactions on chitosan beads

Cholesterol is an important biomarker that is involved in the implication of many diseases including coronary heart disease, hypertension, arteriosclerosis and etc. Faster assessment of the cholesterol level in the body may help the users to monitor their health status and allow faster action for the appropriate treatments. A hydrogel based enzyme cascade system using multiple enzymes was developed to detect the cholesterol levels with a naked-eye. Cholesterol esterase, cholesterol oxidase, and horseradish peroxidase were immobilized onto chitosan beads through a glutaraldehyde cross-linkage. The cholesterol level was monitored through the oxidation of ABTS in the assay system. The optimum temperature of the immobilized chitosan beads was 30ºC and the beads retained its activity up to 9 successive operations. We found that glycerol coated immobilized chitosan beads to be more stable in retaining its structure and activity at room temperature over time. Because enzyme has great specificity against its substrate molecules, we believe that the chitosan beads provide an excellent platform for the enzyme cascade system that may be applied in detecting the small molecule biomarkers

Detecting Anomalous Transactions via an IoT Based Application: A Machine Learning Approach for Horse Racing Betting

During the past decade, the technological advancement have allowed the gambling industry worldwide to deploy various platforms such as the web and mobile applications. Government agencies and local authorities have placed strict regulations regarding the location and amount allowed for gambling. These efforts are made to prevent gambling addictions and monitor fraudulent activities. The revenue earned from gambling provides a considerable amount of tax revenue. The inception of internet gambling have allowed professional gamblers to par take in unlawful acts. However, the lack of studies on the technical inspections and systems to prohibit unlawful internet gambling has caused incidents such as the Walkerhill Hotel incident in 2016, where fraudsters placed bets abnormally by modifying an Internet of Things (IoT)-based application called “MyCard”. This paper investigates the logic used by smartphone IoT applications to validate the location of users and then confirm continuous threats. Hence, our research analyzed transactions made on applications that operated using location authentication through IoT devices. Drawing on gambling transaction data from the Korea Racing Authority, this research used time series machine learning algorithms to identify anomalous activities and transactions. In our research, we propose a method to detect and prevent these anomalies by conducting a comparative analysis of the results of existing anomaly detection techniques and novel techniques

<i>Aster glehni</i> F. Schmidt Extract Modulates the Activities of HMG-CoA Reductase and Fatty Acid Synthase

Aster glehni F. Schmidt (AG), is a natural product known to have anti-obesity effects, but the mechanism underlying these effects is not well documented. We hypothesized that AG may have inhibitory effects on enzymes related to lipid accumulation. Herein, AG fractions were tested against HMG-CoA reductase (HMGR) and fatty acid synthase (FAS), two important enzymes involved in cholesterol and fatty acid synthesis, respectively. We found that dicaffeoylquinic acid (DCQA) methyl esters present in AG are largely responsible for the inhibition of HMGR and FAS. Since free DCQA is a major form present in AG, we demonstrated that a simple methylation of the AG extract could increase the overall inhibitory effects against those enzymes. Through this simple process, we were able to increase the inhibitory effect by 150%. We believe that our processed AG effectively modulates the HMGR and FAS activities, providing promising therapeutic potential for cholesterol- and lipid-lowering effects

USP14 Regulates Cancer Cell Growth in a Fatty Acid Synthase-Independent Manner

Fatty acid synthase (FASN) plays an important role in cancer development, providing excess lipid sources for cancer growth by participating in de novo lipogenesis. Although several inhibitors of FASN have been developed, there are many limitations to using FASN inhibitors alone as cancer therapeutics. We therefore attempted to effectively inhibit cancer cell growth by using a FASN inhibitor in combination with an inhibitor of a deubiquitinating enzyme USP14, which is known to maintain FASN protein levels in hepatocytes. However, when FASN and USP14 were inhibited together, there were no synergistic effects on cancer cell death compared to inhibition of FASN alone. Surprisingly, USP14 rather reduced the protein levels and activity of FASN in cancer cells, although it slightly inhibited the ubiquitination of FASN. Indeed, treatment of an USP14 inhibitor IU1 did not significantly affect FASN levels in cancer cells. Furthermore, from an analysis of metabolites involved in lipid metabolism, metabolite changes in IU1-treated cells were significantly different from those in cells treated with a FASN inhibitor, Fasnall. These results suggest that FASN may not be a direct substrate of USP14 in the cancer cells. Consequently, we demonstrate that USP14 regulates proliferation of the cancer cells in a fatty acid synthase-independent manner, and targeting USP14 in combination with FASN may not be a viable method for effective cancer treatment

Aster glehni F. Schmidt Extract Modulates the Activities of HMG-CoA Reductase and Fatty Acid Synthase

Aster glehni F. Schmidt (AG), is a natural product known to have anti-obesity effects, but the mechanism underlying these effects is not well documented. We hypothesized that AG may have inhibitory effects on enzymes related to lipid accumulation. Herein, AG fractions were tested against HMG-CoA reductase (HMGR) and fatty acid synthase (FAS), two important enzymes involved in cholesterol and fatty acid synthesis, respectively. We found that dicaffeoylquinic acid (DCQA) methyl esters present in AG are largely responsible for the inhibition of HMGR and FAS. Since free DCQA is a major form present in AG, we demonstrated that a simple methylation of the AG extract could increase the overall inhibitory effects against those enzymes. Through this simple process, we were able to increase the inhibitory effect by 150%. We believe that our processed AG effectively modulates the HMGR and FAS activities, providing promising therapeutic potential for cholesterol- and lipid-lowering effects

Polyamine and EIF5A hypusination downstream of c-Myc confers targeted therapy resistance in BRAF mutant melanoma

Abstract Background BRAF inhibitors are widely employed in the treatment of melanoma with the BRAF V600E mutation. However, the development of resistance compromises their therapeutic efficacy. Diverse genomic and transcriptomic alterations are found in BRAF inhibitor resistant melanoma, posing a pressing need for convergent, druggable target that reverse therapy resistant tumor with different resistance mechanisms. Methods CRISPR-Cas9 screens were performed to identify novel target gene whose inhibition selectively targets A375VR, a BRAF V600E mutant cell line with acquired resistance to vemurafenib. Various in vitro and in vivo assays, including cell competition assay, water soluble tetrazolium (WST) assay, live-dead assay and xenograft assay were performed to confirm synergistic cell death. Liquid Chromatography-Mass Spectrometry analyses quantified polyamine biosynthesis and changes in proteome in vemurafenib resistant melanoma. EIF5A hypusination dependent protein translation and subsequent changes in mitochondrial biogenesis and activity were assayed by O-propargyl-puromycin labeling assay, mitotracker, mitoSOX labeling and seahorse assay. Bioinformatics analyses were used to identify the association of polyamine biosynthesis with BRAF inhibitor resistance and poor prognosis in melanoma patient cohorts. Results We elucidate the role of polyamine biosynthesis and its regulatory mechanisms in promoting BRAF inhibitor resistance. Leveraging CRISPR-Cas9 screens, we identify AMD1 (S-adenosylmethionine decarboxylase 1), a critical enzyme for polyamine biosynthesis, as a druggable target whose inhibition reduces vemurafenib resistance. Metabolomic and proteomic analyses reveal that polyamine biosynthesis is upregulated in vemurafenib-resistant cancer, resulting in enhanced EIF5A hypusination, translation of mitochondrial proteins and oxidative phosphorylation. We also identify that sustained c-Myc levels in vemurafenib-resistant cancer are responsible for elevated polyamine biosynthesis. Inhibition of polyamine biosynthesis or c-Myc reversed vemurafenib resistance both in vitro cell line models and in vivo in a xenograft model. Polyamine biosynthesis signature is associated with poor prognosis and shorter progression free survival after BRAF/MAPK inhibitor treatment in melanoma cohorts, highlighting the clinical relevance of our findings. Conclusions Our findings delineate the molecular mechanisms involving polyamine-EIF5A hypusination-mitochondrial respiration pathway conferring BRAF inhibitor resistance in melanoma. These targets will serve as effective therapeutic targets that can maximize the therapeutic efficacy of existing BRAF inhibitors

Inhibiting peripheral and central MAO-B ameliorates joint inflammation and cognitive impairment in rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic inflammation and the destruction of joints and systemic organs. RA is commonly accompanied by neuropsychiatric complications, such as cognitive impairment and depression. However, the role of monoamine oxidase (MAO) and its inhibitors in controlling neurotransmitters associated with these complications in RA have not been clearly identified. Here, we report that peripheral and central MAO-B are highly associated with joint inflammation and cognitive impairment in RA, respectively. Ribonucleic acid (RNA) sequencing and protein expression quantification were used to show that MAO-B and related molecules, such as gamma aminobutyric acid (GABA), were elevated in the inflamed synovium of RA patients. In primary cultured fibroblast-like synoviocytes in the RA synovium, MAO-B expression was significantly increased by tumor necrosis factor (TNF)-alpha-induced autophagy, which produces putrescine, the polyamine substrate for GABA synthesis. We also observed that MAO-B-mediated aberrant astrocytic production of GABA was augmented by interleukin (IL)-1 beta and inhibited CA1-hippocampal pyramidal neurons, which are responsible for memory storage, in an animal model of RA. Moreover, a newly developed reversible inhibitor of MAO-B ameliorated joint inflammation by inhibiting cyclooxygenase (Cox)-2. Therefore, MAO-B can be an effective therapeutic target for joint inflammation and cognitive impairment in patients with RA. Rheumatoid arthritis: Potential therapeutic target identified Inhibiting an enzyme that is upregulated during joint inflammation may prove a valuable therapy for rheumatoid arthritis (RA). As well as causing considerable pain and discomfort in the joints, RA can also trigger neuropsychiatric problems including depression and memory impairment. The monoamine oxidase (MAO) enzyme family is involved in the control of neurotransmitters, and there is evidence that links MAO-B levels with systemic inflammation. C. Justin Lee at Center for Cognition and Sociality, Institute for Basic Science,, Daejeon, South Korea, and co-workers examined the role of MAO-B in RA using patient tissue samples and mouse models. MAO-B and related molecules were upregulated in patients&apos; inflamed joint tissues. In mice, elevated MAO-B triggered the inhibition of nerve cell activity related to memory storage. A novel drug that inhibits MAO-B reduced RA-related inflammation and cognitive impairment in mice, suggesting a promising approach to treatment.11Nsciescopuskc

Astrocytic urea cycle detoxifies A beta-derived ammonia while impairing memory in Alzheimer&apos;s disease

Alzheimer&apos;s disease (AD) is one of the foremost neurodegenerative diseases, characterized by beta-amyloid (A beta) plaques and significant progressive memory loss. In AD, astrocytes are proposed to take up and clear A beta plaques. However, how A beta induces pathogenesis and memory impairment in AD remains elusive. We report that normal astrocytes show non-cyclic urea metabolism, whereas A beta-treated astrocytes show switched-on urea cycle with upregulated enzymes and accumulated entering-metabolite aspartate, starting-substrate ammonia, end-product urea, and side-product putrescine. Gene silencing of astrocytic ornithine decarboxylase-1 (ODC1), facilitating ornithine-to-putrescine conversion, boosts urea cycle and eliminates aberrant putrescine and its toxic byproducts ammonia and H2O2 and its end product GABA to recover from reactive astrogliosis and memory impairment in AD. Our findings implicate that astrocytic urea cycle exerts opposing roles of beneficial A beta detoxification and detrimental memory impairment in AD. We propose ODC1 inhibition as a promising therapeutic strategy for AD to facilitate removal of toxic molecules and prevent memory loss.11Nsciescopu

Astrocytic autophagy plasticity modulates Aβ clearance and cognitive function in Alzheimer’s disease

Abstract Background Astrocytes, one of the most resilient cells in the brain, transform into reactive astrocytes in response to toxic proteins such as amyloid beta (Aβ) in Alzheimer’s disease (AD). However, reactive astrocyte-mediated non-cell autonomous neuropathological mechanism is not fully understood yet. We aimed our study to find out whether Aβ-induced proteotoxic stress affects the expression of autophagy genes and the modulation of autophagic flux in astrocytes, and if yes, how Aβ-induced autophagy-associated genes are involved Aβ clearance in astrocytes of animal model of AD. Methods Whole RNA sequencing (RNA-seq) was performed to detect gene expression patterns in Aβ-treated human astrocytes in a time-dependent manner. To verify the role of astrocytic autophagy in an AD mouse model, we developed AAVs expressing shRNAs for MAP1LC3B/LC3B (LC3B) and Sequestosome1 (SQSTM1) based on AAV-R-CREon vector, which is a Cre recombinase-dependent gene-silencing system. Also, the effect of astrocyte-specific overexpression of LC3B on the neuropathology in AD (APP/PS1) mice was determined. Neuropathological alterations of AD mice with astrocytic autophagy dysfunction were observed by confocal microscopy and transmission electron microscope (TEM). Behavioral changes of mice were examined through novel object recognition test (NOR) and novel object place recognition test (NOPR). Results Here, we show that astrocytes, unlike neurons, undergo plastic changes in autophagic processes to remove Aβ. Aβ transiently induces expression of LC3B gene and turns on a prolonged transcription of SQSTM1 gene. The Aβ-induced astrocytic autophagy accelerates urea cycle and putrescine degradation pathway. Pharmacological inhibition of autophagy exacerbates mitochondrial dysfunction and oxidative stress in astrocytes. Astrocyte-specific knockdown of LC3B and SQSTM1 significantly increases Aβ plaque formation and GFAP-positive astrocytes in APP/PS1 mice, along with a significant reduction of neuronal marker and cognitive function. In contrast, astrocyte-specific overexpression of LC3B reduced Aβ aggregates in the brain of APP/PS1 mice. An increase of LC3B and SQSTM1 protein is found in astrocytes of the hippocampus in AD patients. Conclusions Taken together, our data indicates that Aβ-induced astrocytic autophagic plasticity is an important cellular event to modulate Aβ clearance and maintain cognitive function in AD mice