bluuebunny/crossref_metadata_2025 huggingface dataset:
Effectiveness of an Out-of-Pocket Cost Removal Intervention on Health Check Attendance in Japan
Authors: Murayama Hiroshi , Takahashi Yuta , Shimada Setaro
Published: May 2021

Annual health checks are important for identifying individuals at high risk for cardiometabolic diseases. However, there are socioeconomic disparities in health check attendance rates, and an intervention to lower financial barriers could be useful for increasing health check utilization. In this study, we aimed to evaluate the effectiveness of an out-of-pocket cost removal intervention on health check attendance in Japan. Data were obtained on beneficiaries of the National Health Insurance system of Yokohama City, Kanagawa Prefecture, Japan. In 2018, Yokohama started an intervention to remove out-of-pocket costs for specific health checks for all National Health Insurance beneficiaries. We analyzed data from 2015−2018 (131,295 people aged 40–74 years; 377,660 observations). A generalized estimating equation showed that people were more likely to receive specific health checks in 2018 (after the out-of-pocket cost removal intervention started) than in 2017 (immediately before the intervention; odds ratio [95% confidence interval] = 1.167 [1.149–1.185]), after adjusting for age, gender, tax exemption, and residential area. Stratified analyses revealed that the effectiveness of the out-of-pocket cost removal intervention was greater among the older age group and those who did not receive a tax exemption (i.e., those with relatively higher income). The present study showed that the out-of-pocket cost removal intervention could promote specific health check utilization. This indicates that removing financial barriers could motivate people’s behavior regarding health check attendance.
Examining the Effect of Squatter Settlements in the Evolution of Spatial Fragmentation in the Housing Market of the City of Buenos Aires by Using Geographical Weighted Regression
Authors: Ogas-Mendez A. Federico , Isoda Yuzuru
Published: May 2021

The spatial fragmentation in the housing market and the growth of squatter settlements are characteristic for the metropolitan areas in developing countries. Over the years, in large cities, these phenomena have been promoting an increase in the spatial concentration of poverty. Therefore, this study examined the relationship between the squatter settlement growth and spatial fragmentation in the housing market of Buenos Aires. By performing a spatiotemporal analysis using geographically weighted regression in the house prices for the years 2001, 2010, and 2018, the results showed that while squatter settlements had a strong negative effect on house prices, the affected areas shifted over time. Our findings indicate that it is not the growth of the squatter settlement that causes spatial fragmentation, but rather the widening income disparities and further segregation of low-income households. However, squatter settlements determined the spatial demarcation of fragmented housing market by attracting low-income households to surrounding low house price areas.
Possible Beneficial Actions of Caffeine in SARS-CoV-2
Authors: Romero-Martínez Bianca S. , Montaño Luis M. , Solís-Chagoyán Héctor , Sommer Bettina , Ramírez-Salinas Gemma Lizbeth , Pérez-Figueroa Gloria E. , Flores-Soto Edgar
Published: May 2021

The COVID-19 pandemic has established an unparalleled necessity to rapidly find effective treatments for the illness; unfortunately, no specific treatment has been found yet. As this is a new emerging chaotic situation, already existing drugs have been suggested to ameliorate the infection of SARS-CoV-2. The consumption of caffeine has been suggested primarily because it improves exercise performance, reduces fatigue, and increases wakefulness and awareness. Caffeine has been proven to be an effective anti-inflammatory and immunomodulator. In airway smooth muscle, it has bronchodilator effects mainly due to its activity as a phosphodiesterase inhibitor and adenosine receptor antagonist. In addition, a recent published document has suggested the potential antiviral activity of this drug using in silico molecular dynamics and molecular docking; in this regard, caffeine might block the viral entrance into host cells by inhibiting the formation of a receptor-binding domain and the angiotensin-converting enzyme complex and, additionally, might reduce viral replication by the inhibition of the activity of 3-chymotrypsin-like proteases. Here, we discuss how caffeine through certain mechanisms of action could be beneficial in SARS-CoV-2. Nevertheless, further studies are required for validation through in vitro and in vivo models.
Bacteriological Evaluation of Gingival Crevicular Fluid in Teeth Restored Using Fixed Dental Prostheses: An In Vivo Study
Authors: Heboyan Artak , Manrikyan Mikayel , Zafar Muhammad Sohail , Rokaya Dinesh , Nushikyan Ruzan , Vardanyan Izabella , Vardanyan Anna , Khurshid Zohaib
Published: May 2021

The present in vivo study determined the microbiological counts of the gingival crevicular fluid (GCF) among patients with fixed dental prostheses fabricated using three different techniques. A total of 129 subjects were divided into three study groups: first, cobalt-chrome-based, metal-ceramic prostheses fabricated by the conventional method (MC, n = 35); the second group consisted of cobalt-chrome-based, metal-ceramic prostheses fabricated by the computer-aided design and computer-aided manufacturing (CAD/CAM) technique (CC-MC, n = 35); the third group comprised zirconia-based ceramic prostheses fabricated using the CAD/CAM technique (CC-Zr, n = 35). The control consisted of 24 patients using prostheses fabricated with either MC, CC-MC, or CC-Zr. The GCF was obtained from the subjects before treatment, and 6 and 12 months after the prosthetic treatment. Bacteriological and bacterioscopic analysis of the GCF was performed to analyze the patients’ GCF. The data were analyzed using SPSS V20 (IBM Company, Chicago, IL, USA). The number of microorganisms of the gingival crevicular fluid in all groups at 12 months of prosthetic treatment reduced dramatically compared with the data obtained before prosthetic treatment. Inflammatory processes in the periodontium occurred slowly in the case of zirconium oxide-based ceramic constructions due to their biocompatibility with the mucous membranes and tissues of the oral cavity as well as a reduced risk of dental biofilm formation. This should be considered by dentists and prosthodontists when choosing restoration materials for subjects with periodontal pathology.
Urolithin A and B Alter Cellular Metabolism and Induce Metabolites Associated with Apoptosis in Leukemic Cells
Authors: Alzahrani Abdulaziz Musa , Shait Mohammed Mohammed Razeeth , Alghamdi Raed Ahmed , Ahmad Abrar , Zamzami Mazin A. , Choudhry Hani , Khan Mohammad Imran
Published: May 2021

Leukemia is persistently a significant cause of illness and mortality worldwide. Urolithins, metabolites of ellagic acid and ellagitannins produced by gut microbiota, showed better bioactive compounds liable for the health benefits exerted by ellagic acid and ellagitannins containing pomegranate and walnuts. Here, we assessed the potential antileukemic activities of both urolithin A and urolithin B. Results showed that both urolithin A and B significantly inhibited the proliferation of leukemic cell lines Jurkat and K562, among which urolithin A showed the more prominent antiproliferative capability. Further, urolithin treatment alters leukemic cell metabolism, as evidenced by increased metabolic rate and notable changes in glutamine metabolism, one-carbon metabolism, and lipid metabolism. Next, we evidenced that both urolithins equally promoted apoptosis in leukemic cell lines. Based on these observations, we concluded that both urolithin A and B alter leukemic cell metabolome, resulting in a halt of proliferation, followed by apoptosis. The data can be used for designing new combinational therapies to eradicate leukemic cells.
Structure, Dynamics, and Ligand Recognition of Human-Specific CHRFAM7A (Dupα7) Nicotinic Receptor Linked to Neuropsychiatric Disorders
Authors: Liu Danlin , de Souza João V. , Ahmad Ayaz , Bronowska Agnieszka K.
Published: May 2021

Cholinergic α7 nicotinic receptors encoded by the CHRNA7 gene are ligand-gated ion channels directly related to memory and immunomodulation. Exons 5–7 in CHRNA7 can be duplicated and fused to exons A-E of FAR7a, resulting in a hybrid gene known as CHRFAM7A, unique to humans. Its product, denoted herein as Dupα7, is a truncated subunit where the N-terminal 146 residues of the ligand binding domain of the α7 receptor have been replaced by 27 residues from FAM7. Dupα7 negatively affects the functioning of α7 receptors associated with neurological disorders, including Alzheimer’s diseases and schizophrenia. However, the stoichiometry for the α7 nicotinic receptor containing dupα7 monomers remains unknown. In this work, we developed computational models of all possible combinations of wild-type α7 and dupα7 pentamers and evaluated their stability via atomistic molecular dynamics and coarse-grain simulations. We assessed the effect of dupα7 subunits on the Ca2+ conductance using free energy calculations. We showed that receptors comprising of four or more dupα7 subunits are not stable enough to constitute a functional ion channel. We also showed that models with dupα7/α7 interfaces are more stable and are less detrimental for the ion conductance in comparison to dupα7/dupα7 interfaces. Based on these models, we used protein–protein docking to evaluate how such interfaces would interact with an antagonist, α-bungarotoxin, and amyloid Aβ42. Our findings show that the optimal stoichiometry of dupα7/α7 functional pentamers should be no more than three dupα7 monomers, in favour of a dupα7/α7 interface in comparison to a homodimer dupα7/dupα7 interface. We also showed that receptors bearing dupα7 subunits are less sensitive to Aβ42 effects, which may shed light on the translational gap reported for strategies focused on nicotinic receptors in ‘Alzheimer’s disease research.
Evaluation of Calyculin A Effect on γH2AX/53BP1 Focus Formation and Apoptosis in Human Umbilical Cord Blood Lymphocytes
Authors: Zastko Lucián , Račková Anna , Petrovičová Petra , Durdík Matúš , Míšek Jakub , Marková Eva , Belyaev Igor
Published: May 2021

Dephosphorylation inhibitor calyculin A (cal A) has been reported to inhibit the disappearance of radiation-induced γH2AX DNA repair foci in human lymphocytes. However, other studies reported no change in the kinetics of γH2AX focus induction and loss in irradiated cells. While apoptosis might interplay with the kinetics of focus formation, it was not followed in irradiated cells along with DNA repair foci. Thus, to validate plausible explanations for significant variability in outputs of these studies, we evaluated the effect of cal A (1 and 10 nM) on γH2AX/53BP1 DNA repair foci and apoptosis in irradiated (1, 5, 10, and 100 cGy) human umbilical cord blood lymphocytes (UCBL) using automated fluorescence microscopy and annexin V-FITC/propidium iodide assay/γH2AX pan-staining, respectively. No effect of cal A on γH2AX and colocalized γH2AX/53BP1 foci induced by low doses (≤10 cGy) of γ-rays was observed. Moreover, 10 nM cal A treatment decreased the number of all types of DNA repair foci induced by 100 cGy irradiation. 10 nM cal A treatment induced apoptosis already at 2 h of treatment, independently from the delivered dose. Apoptosis was also detected in UCBL treated with lower cal A concentration, 1 nM, at longer cell incubation, 20 and 44 h. Our data suggest that apoptosis triggered by cal A in UCBL may underlie the failure of cal A to maintain radiation-induced γH2AX foci. All DSB molecular markers used in this study responded linearly to low-dose irradiation. Therefore, their combination may represent a strong biodosimetry tool for estimation of radiation response to low doses. Assessment of colocalized γH2AX/53BP1 improved the threshold of low dose detection.
Fatty Acid Desaturases: Uncovering Their Involvement in Grapevine Defence against Downy Mildew
Authors: Laureano Gonçalo , Cavaco Ana Rita , Matos Ana Rita , Figueiredo Andreia
Published: May 2021

Grapevine downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most severe and devastating diseases in viticulture. Unravelling the grapevine defence mechanisms is crucial to develop sustainable disease control measures. Here we provide new insights concerning fatty acid’s (FA) desaturation, a fundamental process in lipid remodelling and signalling. Previously, we have provided evidence that lipid signalling is essential in the establishment of the incompatible interaction between grapevine and Plasmopara viticola. In the first hours after pathogen challenge, jasmonic acid (JA) accumulation, activation of its biosynthetic pathway and an accumulation of its precursor, the polyunsaturated α-linolenic acid (C18:3), were observed in the leaves of the tolerant genotype, Regent. This work was aimed at a better comprehension of the desaturation processes occurring after inoculation. We characterised, for the first time in Vitis vinifera, the gene family of the FA desaturases and evaluated their involvement in Regent response to Plasmopara viticola. Upon pathogen challenge, an up-regulation of the expression of plastidial FA desaturases genes was observed, resulting in a higher content of polyunsaturated fatty acids (PUFAs) of chloroplast lipids. This study highlights FA desaturases as key players in membrane remodelling and signalling in grapevine defence towards biotrophic pathogens.
Mechanical and Immunological Regulation in Wound Healing and Skin Reconstruction
Authors: Kimura Shun , Tsuji Takashi
Published: May 2021

In the past decade, a new frontier in scarless wound healing has arisen because of significant advances in the field of wound healing realised by incorporating emerging concepts from mechanobiology and immunology. The complete integumentary organ system (IOS) regeneration and scarless wound healing mechanism, which occurs in specific species, body sites and developmental stages, clearly shows that mechanical stress signals and immune responses play important roles in determining the wound healing mode. Advances in tissue engineering technology have led to the production of novel human skin equivalents and organoids that reproduce cell–cell interactions with tissue-scale tensional homeostasis, and enable us to evaluate skin tissue morphology, functionality, drug response and wound healing. This breakthrough in tissue engineering has the potential to accelerate the understanding of wound healing control mechanisms through complex mechanobiological and immunological interactions. In this review, we present an overview of recent studies of biomechanical and immunological wound healing and tissue remodelling mechanisms through comparisons of species- and developmental stage-dependent wound healing mechanisms. We also discuss the possibility of elucidating the control mechanism of wound healing involving mechanobiological and immunological interaction by using next-generation human skin equivalents.
Modeling the Formation of Gas Bubbles inside the Pores of Reactive Electrochemical Membranes in the Process of the Anodic Oxidation of Organic Compounds
Authors: Mareev Semyon , Skolotneva Ekaterina , Cretin Marc , Nikonenko Victor
Published: May 2021

The use of reactive electrochemical membranes (REM) in flow-through mode during the anodic oxidation of organic compounds makes it possible to overcome the limitations of plate anodes: in the case of REM, the area of the electrochemically active surface is several orders of magnitude larger, and the delivery of organic compounds to the reaction zone is controlled by convective flow rather than diffusion. The main problem with REM is the formation of fouling and gas bubbles in the pores, which leads to a decrease in the efficiency of the process because the hydraulic resistance increases and the electrochemically active surface is shielded. This work aims to study the processes underlying the reduction in the efficiency of anodic oxidation, and in particular the formation of gas bubbles and the recharge of the REM pore surface at a current density exceeding the limiting kinetic value. We propose a simple one-dimensional non-stationary model of the transport of diluted species during the anodic oxidation of paracetamol using REM to describe the above effects. The processing of the experimental data was carried out. It was found that the absolute value of the zeta potential of the pore surface decreases with time, which leads to a decrease in the permeate flux due to a reduction in the electroosmotic flow. It was shown that in the solution that does not contain organic components, gas bubbles form faster and occupy a larger pore fraction than in the case of the presence of paracetamol; with an increase in the paracetamol concentration, the gas fraction decreases. This behavior is due to a decrease in the generation of oxygen during the recombination reaction of the hydroxyl radicals, which are consumed in the oxidation reaction of the organic compounds. Because the presence of bubbles increases the hydraulic resistance, the residence time of paracetamol—and consequently its degradation degree—increases, but the productivity goes down. The model has predictive power and, after simple calibration, can be used to predict the performance of REM anodic oxidation systems.
Endocannabinoid System and Its Regulation by Polyunsaturated Fatty Acids and Full Spectrum Hemp Oils
Authors: Komarnytsky Slavko , Rathinasabapathy Thirumurugan , Wagner Charles , Metzger Brandon , Carlisle Carolina , Panda Chinmayee , Le Brun-Blashka Sara , Troup John P. , Varadharaj Saradhadevi
Published: May 2021

The endocannabinoid system (ECS) consists of endogenous cannabinoids, their receptors, and metabolic enzymes that play a critical homeostatic role in modulating polyunsaturated omega fatty acid (PUFA) signaling to maintain a balanced inflammatory and redox state. Whole food-based diets and dietary interventions linked to PUFAs of animal (fish, calamari, krill) or plant (hemp, flax, walnut, algae) origin, as well as full-spectrum hemp oils, are increasingly used to support the ECS tone, promote healthy metabolism, improve risk factors associated with cardiovascular disorders, encourage brain health and emotional well-being, and ameliorate inflammation. While hemp cannabinoids of THC and CBD groups show distinct but complementary actions through a variety of cannabinoid (CB1 and CB2), adenosine (A2A), and vanilloid (TRPV1) receptors, they also modulate PUFA metabolism within a wide variety of specialized lipid mediators that promote or resolve inflammation and oxidative stress. Clinical evidence reviewed in this study links PUFAs and cannabinoids to changes in ECS tone, immune function, metabolic and oxidative stress adaptation, and overall maintenance of a well-balanced systemic function of the body. Understanding how the body coordinates signals from the exogenous and endogenous ECS modulators is critical for discerning the underlying molecular mechanisms of the ECS tone in healthy and disease states. Nutritional and lifestyle interventions represent promising approaches to address chronic metabolic and inflammatory disorders that may overlap in the population at risk. Further investigation and validation of dietary interventions that modulate the ECS are required in order to devise clinically successful second-generation management strategies.
miR155 Deficiency Reduces Myofibroblast Density but Fails to Improve Cardiac Function after Myocardial Infarction in Dyslipidemic Mouse Model
Authors: Schumacher David , Curaj Adelina , Simsekyilmaz Sakine , Schober Andreas , Liehn Elisa A. , Mause Sebastian F.
Published: May 2021

Myocardial infarction remains the most common cause of heart failure with adverse remodeling. MicroRNA (miR)155 is upregulated following myocardial infarction and represents a relevant regulatory factor for cardiac remodeling by engagement in cardiac inflammation, fibrosis and cardiomyocyte hypertrophy. Here, we investigated the role of miR155 in cardiac remodeling and dysfunction following myocardial infarction in a dyslipidemic mouse model. Myocardial infarction was induced in dyslipidemic apolipoprotein E-deficient (ApoE−/−) mice with and without additional miR155 knockout by ligation of the LAD. Four weeks later, echocardiography was performed to assess left ventricular (LV) dimensions and function, and mice were subsequently sacrificed for histological analysis. Echocardiography revealed no difference in LV ejection fractions, LV mass and LV volumes between ApoE−/− and ApoE−/−/miR155−/− mice. Histology confirmed comparable infarction size and unaltered neoangiogenesis in the myocardial scar. Notably, myofibroblast density was significantly decreased in ApoE−/−/miR155−/− mice compared to the control, but no difference was observed for total collagen deposition. Our findings reveal that genetic depletion of miR155 in a dyslipidemic mouse model of myocardial infarction does not reduce infarction size and consecutive heart failure but does decrease myofibroblast density in the post-ischemic scar.
Melatonin for Neonatal Encephalopathy: From Bench to Bedside
Authors: Pang Raymand , Advic-Belltheus Adnan , Meehan Christopher , Fullen Daniel J. , Golay Xavier , Robertson Nicola J.
Published: May 2021

Neonatal encephalopathy is a leading cause of morbidity and mortality worldwide. Although therapeutic hypothermia (HT) is now standard practice in most neonatal intensive care units in high resource settings, some infants still develop long-term adverse neurological sequelae. In low resource settings, HT may not be safe or efficacious. Therefore, additional neuroprotective interventions are urgently needed. Melatonin’s diverse neuroprotective properties include antioxidant, anti-inflammatory, and anti-apoptotic effects. Its strong safety profile and compelling preclinical data suggests that melatonin is a promising agent to improve the outcomes of infants with NE. Over the past decade, the safety and efficacy of melatonin to augment HT has been studied in the neonatal piglet model of perinatal asphyxia. From this model, we have observed that the neuroprotective effects of melatonin are time-critical and dose dependent. Therapeutic melatonin levels are likely to be 15–30 mg/L and for optimal effect, these need to be achieved within the first 2–3 h after birth. This review summarises the neuroprotective properties of melatonin, the key findings from the piglet and other animal studies to date, and the challenges we face to translate melatonin from bench to bedside.
Modulation of Endocannabinoids by Caloric Restriction Is Conserved in Mice but Is Not Required for Protection from Acute Kidney Injury
Authors: Hoyer-Allo Karla Johanna Ruth , Späth Martin Richard , Hanssen Ruth , Johnsen Marc , Brodesser Susanne , Kaufmann Kathrin , Kiefer Katharina , Koehler Felix Carlo , Göbel Heike , Kubacki Torsten , Grundmann Franziska , Schermer Bernhard , Brüning Jens , Benzing Thomas , Burst Volker , Müller Roman-Ulrich
Published: May 2021

Acute kidney injury (AKI) is a frequent and critical complication in the clinical setting. In rodents, AKI can be effectively prevented through caloric restriction (CR), which has also been shown to increase lifespan in many species. In Caenorhabditis elegans (C. elegans), longevity studies revealed that a marked CR-induced reduction of endocannabinoids may be a key mechanism. Thus, we hypothesized that regulation of endocannabinoids, particularly arachidonoyl ethanolamide (AEA), might also play a role in CR-mediated protection from renal ischemia-reperfusion injury (IRI) in mammals including humans. In male C57Bl6J mice, CR significantly reduced renal IRI and led to a significant decrease of AEA. Supplementation of AEA to near-normal serum concentrations by repetitive intraperitoneal administration in CR mice, however, did not abrogate the protective effect of CR. We also analyzed serum samples taken before and after CR from patients of three different pilot trials of dietary interventions. In contrast to mice and C. elegans, we detected an increase of AEA. We conclude that endocannabinoid levels in mice are modulated by CR, but CR-mediated renal protection does not depend on this effect. Moreover, our results indicate that modulation of endocannabinoids by CR in humans may differ fundamentally from the effects in animal models.
Austalide K from the Fungus Penicillium rudallense Prevents LPS-Induced Bone Loss in Mice by Inhibiting Osteoclast Differentiation and Promoting Osteoblast Differentiation
Authors: Kim Kwang-Jin , Lee Jusung , Wang Weihong , Lee Yongjin , Oh Eunseok , Park Kyu-Hyung , Park Chanyoon , Woo Gee-Eun , Son Young-Jin , Kang Heonjoong
Published: May 2021

Osteoporosis is a chronic disease that has become a serious public health problem due to the associated reduction in quality of life and its increasing financial burden. It is known that inhibiting osteoclast differentiation and promoting osteoblast formation prevents osteoporosis. As there is no drug with this dual activity without clinical side effects, new alternatives are needed. Here, we demonstrate that austalide K, isolated from the marine fungus Penicillium rudallenes, has dual activities in bone remodeling. Austalide K inhibits the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and improves bone morphogenetic protein (BMP)-2-mediated osteoblast differentiation in vitro without cytotoxicity. The nuclear factor of activated T cells c1 (NFATc1), tartrate-resistant acid phosphatase (TRAP), dendritic cell-specific transmembrane protein (DC-STAMP), and cathepsin K (CTSK) osteoclast-formation-related genes were reduced and alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and osteopontin (OPN) (osteoblast activation-related genes) were simultaneously upregulated by treatment with austalide K. Furthermore, austalide K showed good efficacy in an LPS-induced bone loss in vivo model. Bone volume, trabecular separation, trabecular thickness, and bone mineral density were recovered by austalide K. On the basis of these results, austalide K may lead to new drug treatments for bone diseases such as osteoporosis.
TFEB Overexpression, Not mTOR Inhibition, Ameliorates RagCS75Y Cardiomyopathy
Authors: Kim Maengjo , Lu Linghui , Dvornikov Alexey V. , Ma Xiao , Ding Yonghe , Zhu Ping , Olson Timothy M. , Lin Xueying , Xu Xiaolei
Published: May 2021

A de novo missense variant in Rag GTPase protein C (RagCS75Y) was recently identified in a syndromic dilated cardiomyopathy (DCM) patient. However, its pathogenicity and the related therapeutic strategy remain unclear. We generated a zebrafish RragcS56Y (corresponding to human RagCS75Y) knock-in (KI) line via TALEN technology. The KI fish manifested cardiomyopathy-like phenotypes and poor survival. Overexpression of RagCS75Y via adenovirus infection also led to increased cell size and fetal gene reprogramming in neonatal rat ventricle cardiomyocytes (NRVCMs), indicating a conserved mechanism. Further characterization identified aberrant mammalian target of rapamycin complex 1 (mTORC1) and transcription factor EB (TFEB) signaling, as well as metabolic abnormalities including dysregulated autophagy. However, mTOR inhibition failed to ameliorate cardiac phenotypes in the RagCS75Y cardiomyopathy models, concomitant with a failure to promote TFEB nuclear translocation. This observation was at least partially explained by increased and mTOR-independent physical interaction between RagCS75Y and TFEB in the cytosol. Importantly, TFEB overexpression resulted in more nuclear TFEB and rescued cardiomyopathy phenotypes. These findings suggest that S75Y is a pathogenic gain-of-function mutation in RagC that leads to cardiomyopathy. A primary pathological step of RagCS75Y cardiomyopathy is defective mTOR–TFEB signaling, which can be corrected by TFEB overexpression, but not mTOR inhibition.
Insight into Bortezomib Focusing on Its Efficacy against P-gp-Positive MDR Leukemia Cells
Authors: Kyca Tomáš , Pavlíková Lucia , Boháčová Viera , Mišák Anton , Poturnayová Alexandra , Breier Albert , Sulová Zdena , Šereš Mário
Published: May 2021

In this paper, we compared the effects of bortezomib on L1210 (S) cells with its effects on P-glycoprotein (P-gp)-positive variant S cells, which expressed P-gp either after selection with vincristine (R cells) or after transfection with a human gene encoding P-gp (T cells). Bortezomib induced the death-related effects in the S, R, and T cells at concentrations not exceeding 10 nM. Bortezomib-induced cell cycle arrest in the G2/M phase was more pronounced in the S cells than in the R or T cells and was related to the expression levels of cyclins, cyclin-dependent kinases, and their inhibitors. We also observed an increase in the level of polyubiquitinated proteins (via K48-linkage) and a decrease in the gene expression of some deubiquitinases after treatment with bortezomib. Resistant cells expressed higher levels of genes encoding 26S proteasome components and the chaperone HSP90, which is involved in 26S proteasome assembly. After 4 h of preincubation, bortezomib induced a more pronounced depression of proteasome activity in S cells than in R or T cells. However, none of these changes alone or in combination sufficiently suppressed the sensitivity of R or T cells to bortezomib, which remained at a level similar to that of S cells.
High-Throughput Sequencing of Phage Display Libraries Reveals Parasitic Enrichment of Indel Mutants Caused by Amplification Bias
Authors: Plessers Sander , Van Deuren Vincent , Lavigne Rob , Robben Johan
Published: May 2021

The combination of phage display technology with high-throughput sequencing enables in-depth analysis of library diversity and selection-driven dynamics. We applied short-read sequencing of the mutagenized region on focused display libraries of two homologous nucleic acid modification eraser proteins—AlkB and FTO—biopanned against methylated DNA. This revealed enriched genotypes with small indels and concomitant doubtful amino acid motifs within the FTO library. Nanopore sequencing of the entire display vector showed additional enrichment of large deletions overlooked by region-specific sequencing, and further impacted the interpretation of the obtained amino acid motifs. We could attribute enrichment of these corrupted clones to amplification bias due to arduous FTO display slowing down host cell growth as well as phage production. This amplification bias appeared to be stronger than affinity-based target selection. Recommendations are provided for proper sequence analysis of phage display data, which can improve motive discovery in libraries of proteins that are difficult to display.
Both Specific Endothelial and Proximal Tubular Adam17 Deletion Protect against Diabetic Nephropathy
Authors: Palau Vanesa , Nugraha Bramasta , Benito David , Pascual Julio , Emmert Maximilian Y. , Hoerstrup Simon P. , Riera Marta , Soler Maria José
Published: May 2021

ADAM17 is a disintegrin and metalloproteinase capable of cleaving the ectodomains of a diverse variety of molecules including TNF-α, TGF-α, L-selectin, and ACE2. We have previously demonstrated that renal ADAM17 is upregulated in diabetic mice. The role of endothelial (eAdam17) and proximal tubular (tAdam17) Adam17 deletion in renal histology, modulation of the renin angiotensin system (RAS), renal inflammation, and fibrosis was studied in a mouse model of type 1 Diabetes Mellitus. Moreover, the effect of Adam17 deletion in an in vitro 3D cell culture from human proximal tubular cells under high glucose conditions was evaluated. eAdam17 deletion attenuates renal fibrosis and inflammation, whereas tAdam17 deletion decreases podocyte loss, attenuates the RAS, and decreases macrophage infiltration, α-SMA and collagen accumulation. The 3D in vitro cell culture reinforced the findings obtained in tAdam17KO mice with decreased fibrosis in the Adam17 knockout spheroids. In conclusion, Adam17 deletion either in the endothelial or the tubular cells mitigates kidney injury in the diabetic mice by targeting different pathways. The manipulation of Adam17 should be considered as a therapeutic strategy for treating DN.
Protein Kinase C as a Therapeutic Target in Non-Small Cell Lung Cancer
Authors: Sadeghi Mohammad Mojtaba , Salama Mohamed F. , Hannun Yusuf A.
Published: May 2021

Driver-directed therapeutics have revolutionized cancer treatment, presenting similar or better efficacy compared to traditional chemotherapy and substantially improving quality of life. Despite significant advances, targeted therapy is greatly limited by resistance acquisition, which emerges in nearly all patients receiving treatment. As a result, identifying the molecular modulators of resistance is of great interest. Recent work has implicated protein kinase C (PKC) isozymes as mediators of drug resistance in non-small cell lung cancer (NSCLC). Importantly, previous findings on PKC have implicated this family of enzymes in both tumor-promotive and tumor-suppressive biology in various tissues. Here, we review the biological role of PKC isozymes in NSCLC through extensive analysis of cell-line-based studies to better understand the rationale for PKC inhibition. PKC isoforms α, ε, η, ι, ζ upregulation has been reported in lung cancer, and overexpression correlates with worse prognosis in NSCLC patients. Most importantly, PKC isozymes have been established as mediators of resistance to tyrosine kinase inhibitors in NSCLC. Unfortunately, however, PKC-directed therapeutics have yielded unsatisfactory results, likely due to a lack of specific evaluation for PKC. To achieve satisfactory results in clinical trials, predictive biomarkers of PKC activity must be established and screened for prior to patient enrollment. Furthermore, tandem inhibition of PKC and molecular drivers may be a potential therapeutic strategy to prevent the emergence of resistance in NSCLC.
Orientated Immobilization of FAD-Dependent Glucose Dehydrogenase on Electrode by Carbohydrate-Binding Module Fusion for Efficient Glucose Assay
Authors: Han Qingye , Gong Weili , Zhang Zhenyu , Wang Lushan , Wang Binglian , Cai Lei , Meng Qingjun , Li Yiwei , Liu Qingai , Yang Yan , Zheng Lan , Ma Yaohong
Published: May 2021

The discovery or engineering of fungus-derived FAD-dependent glucose 1-dehydrogenase (FAD-GDH) is especially important in the fabrication and performance of glucose biosensors. In this study, a novel FAD-GDH gene, phylogenetically distantly with other FAD-GDHs from Aspergillus species, was identified. Additionally, the wild-type GDH enzyme, and its fusion enzyme (GDH-NL-CBM2) with a carbohydrate binding module family 2 (CBM2) tag attached by a natural linker (NL), were successfully heterogeneously expressed. In addition, while the GDH was randomly immobilized on the electrode by conventional methods, the GDH-NL-CBM2 was orientationally immobilized on the nanocellulose-modified electrode by the CBM2 affinity adsorption tag through a simple one-step approach. A comparison of the performance of the two electrodes demonstrated that both electrodes responded linearly to glucose in the range of 0.12 to 40.7 mM with a coefficient of determination R2 > 0.999, but the sensitivity of immobilized GDH-NL-CBM2 (2.1362 × 10−2 A/(M*cm2)) was about 1-fold higher than that of GDH (1.2067 × 10−2 A/(M*cm2)). Moreover, a lower detection limit (51 µM), better reproducibility (<5%) and stability, and shorter response time (≈18 s) and activation time were observed for the GDH-NL-CBM2-modified electrode. This facile and easy immobilization approach used in the preparation of a GDH biosensor may open up new avenues in the development of high-performance amperometric biosensors.
Bisphenol A Inhibits the Transporter Function of the Blood-Brain Barrier by Directly Interacting with the ABC Transporter Breast Cancer Resistance Protein (BCRP)
Authors: Engdahl Elin , van Schijndel Maarten D. M. , Voulgaris Dimitrios , Di Criscio Michela , Ramsbottom Kerry A. , Rigden Daniel J. , Herland Anna , Rüegg Joëlle
Published: May 2021

The breast cancer resistance protein (BCRP) is an important efflux transporter in the blood-brain barrier (BBB), protecting the brain from a wide range of substances. In this study, we investigated if BCRP function is affected by bisphenol A (BPA), a high production volume chemical used in common consumer products, as well as by bisphenol F (BPF) and bisphenol S (BPS), which are used to substitute BPA. We employed a transwell-based in vitro cell model of iPSC-derived brain microvascular endothelial cells, where BCRP function was assessed by measuring the intracellular accumulation of its substrate Hoechst 33342. Additionally, we used in silico modelling to predict if the bisphenols could directly interact with BCRP. Our results showed that BPA significantly inhibits the transport function of BCRP. Additionally, BPA was predicted to bind to the cavity that is targeted by known BCRP inhibitors. Taken together, our findings demonstrate that BPA inhibits BCRP function in vitro, probably by direct interaction with the transporter. This effect might contribute to BPA’s known impact on neurodevelopment.
Accurate Molecular Diagnosis of Gaucher Disease Using Clinical Exome Sequencing as a First-Tier Test
Authors: Zampieri Stefania , Cattarossi Silvia , Pavan Eleonora , Barbato Antonio , Fiumara Agata , Peruzzo Paolo , Scarpa Maurizio , Ciana Giovanni , Dardis Andrea
Published: May 2021

Gaucher disease (GD) is an autosomal recessive lysosomal disorder due to beta-glucosidase gene (GBA) mutations. The molecular diagnosis of GD is complicated by the presence of recombinant alleles originating from a highly homologous pseudogene. Clinical exome sequencing (CES) is a rapid genetic approach for identifying disease-causing mutations. However, copy number variation and recombination events are poorly detected, and further investigations are required to avoid mis-genotyping. The aim of this work was to set-up an integrated strategy for GD patients genotyping using CES as a first-line test. Eight patients diagnosed with GD were analyzed by CES. Five patients were fully genotyped, while three were revealed to be homozygous for mutations that were not confirmed in the parents. Therefore, MLPA (multiplex ligation-dependent probe amplification) and specific long-range PCR were performed, and two recombinant alleles, one of them novel, and one large deletion were identified. Furthermore, an MLPA assay performed in one family resulted in the identification of an additional novel mutation (p.M124V) in a relative, in trans with the known p.N409S mutation. In conclusion, even though CES has become extensively used in clinical practice, our study emphasizes the importance of a comprehensive molecular strategy to provide proper GBA genotyping and genetic counseling.
Latency Reversing Agents: Kick and Kill of HTLV-1?
Authors: Schnell Annika P. , Kohrt Stephan , Thoma-Kress Andrea K.
Published: May 2021

Human T-cell leukemia virus type 1 (HTLV-1), the cause of adult T-cell leukemia/lymphoma (ATLL), is a retrovirus, which integrates into the host genome and persistently infects CD4+ T-cells. Virus propagation is stimulated by (1) clonal expansion of infected cells and (2) de novo infection. Viral gene expression is induced by the transactivator protein Tax, which recruits host factors like positive transcription elongation factor b (P-TEFb) to the viral promoter. Since HTLV-1 gene expression is repressed in vivo by viral, cellular, and epigenetic mechanisms in late phases of infection, HTLV-1 avoids an efficient CD8+ cytotoxic T-cell (CTL) response directed against the immunodominant viral Tax antigen. Hence, therapeutic strategies using latency reversing agents (LRAs) sought to transiently activate viral gene expression and antigen presentation of Tax to enhance CTL responses towards HTLV-1, and thus, to expose the latent HTLV-1 reservoir to immune destruction. Here, we review strategies that aimed at enhancing Tax expression and Tax-specific CTL responses to interfere with HTLV-1 latency. Further, we provide an overview of LRAs including (1) histone deacetylase inhibitors (HDACi) and (2) activators of P-TEFb, that have mainly been studied in context of human immunodeficiency virus (HIV), but which may also be powerful in the context of HTLV-1.
Erythritol Ameliorates Small Intestinal Inflammation Induced by High-Fat Diets and Improves Glucose Tolerance
Authors: Kawano Rena , Okamura Takuro , Hashimoto Yoshitaka , Majima Saori , Senmaru Takafumi , Ushigome Emi , Asano Mai , Yamazaki Masahiro , Takakuwa Hiroshi , Sasano Ryoichi , Nakanishi Naoko , Hamaguchi Masahide , Fukui Michiaki
Published: May 2021

Background: Erythritol, a sugar alcohol, is widely used as a substitute for sugar in diets for patients with diabetes or obesity. Methods: In this study, we aimed to investigate the effects of erythritol on metabolic disorders induced by a high-fat diet in C57BL/6J mice, while focusing on changes in innate immunity. Results: Mice that were fed a high-fat diet and administered water containing 5% erythritol (Ery group) had markedly lower body weight, improved glucose tolerance, and markedly higher energy expenditure than the control mice (Ctrl group) (n = 6). Furthermore, compared with the Ctrl group, the Ery group had lesser fat deposition in the liver, smaller adipocytes, and significantly better inflammatory findings in the small intestine. The concentrations of short-chain fatty acids (SCFAs), such as acetic acid, propanoic acid, and butanoic acid, in the serum, feces, and white adipose tissue of the Ery group were markedly higher than those in the Ctrl group. In flow cytometry experiments, group 3 innate lymphoid cell (ILC3) counts in the lamina propria of the small intestine and ILC2 counts in the white adipose tissue of the Ery group were markedly higher than those in the Ctrl group. Quantitative real-time reverse transcription polymerase chain reaction analyses showed that the Il-22 expression in the small intestine of the Ery group was markedly higher than that in the Ctrl group. Conclusions: Erythritol markedly decreased metabolic disorders such as diet-induced obesity, glucose intolerance, dyslipidemia, and fat accumulation in the mouse liver by increasing SCFAs and modulating innate immunity.
Ganoderic Acid A Promotes Amyloid-β Clearance (In Vitro) and Ameliorates Cognitive Deficiency in Alzheimer’s Disease (Mouse Model) through Autophagy Induced by Activating Axl
Authors: Qi Li-Feng-Rong , Liu Shuai , Liu Yu-Ci , Li Ping , Xu Xiaojun
Published: May 2021

Alzheimer’s disease (AD) is thought to be caused by amyloid-β (Aβ) accumulation in the central nervous system due to deficient clearance. The aim of the present study was to investigate the effect of ganoderic acid A (GAA) on Aβ clearance in microglia and its anti-AD activity. Aβ degradation in BV2 microglial cells was determined using an intracellular Aβ clearance assay. GAA stimulated autophagosome formation via the Axl receptor tyrosine kinase (Axl)/RAC/CDC42-activated kinase 1 (Pak1) pathway was determined by Western blot analyses, and fluorescence-labeled Aβ42 was localized in lysosomes in confocal laser microscopy images. The in vivo anti-AD activity of GAA was evaluated by object recognition and Morris water maze (MWM) tests in an AD mouse model following intracerebroventricular injection of aggregated Aβ42. The autophagy level in the hippocampus was assayed by immunohistochemical assessment against microtubule-associated proteins 1A/1B light-chain 3B (LC3B). Intracellular Aβ42 levels were significantly reduced by GAA treatment in microglial cells. Additionally, GAA activated autophagy according to increased LC3B-II levels, with this increased autophagy stimulated by upregulating Axl and Pak1 phosphorylation. The effect of eliminating Aβ by GAA through autophagy was reversed by R428, an Axl inhibitor, or IPA-3, a Pak1 inhibitor. Consistent with the cell-based assay, GAA ameliorated cognitive deficiency and reduced Aβ42 levels in an AD mouse model. Furthermore, LC3B expression in the hippocampus was up-regulated by GAA treatment, with these GAA-specific effects abolished by R428. GAA promoted Aβ clearance by enhancing autophagy via the Axl/Pak1 signaling pathway in microglial cells and ameliorated cognitive deficiency in an AD mouse model.
Internal Determinants in the Field of RPA Technology Implementation on the Example of Selected Companies in the Context of Industry 4.0 Assumptions
Authors: Marciniak Piotr , Stanisławski Robert
Published: May 2021

As part of the assumptions of Industry 4.0. many modern technologies are developing. One of them is robotic process automation (RPA). It allows the replacement of human labor with robots, thus increasing the production capacity of enterprises. In practice, the implementation of RPA takes place through two solutions (models): using the services of an external entity offering RPA (service insourcing) or creating your own center of excellence (CoE). The use of both solutions is influenced by numerous conditions (determinants), among which we can mention benefits on the one hand and threats on the other. They are very different and depend on the model used. In this article, attempts were made to identify and determine their impact on the selection of the appropriate model from the point of view of the company (the purpose of the article). The research used two cases of entities implementing RPA on the basis of opposites, i.e., two different models, in which the discussed technology is treated as an important element of their automation. The achieved results indicate that there is no universalism, and their dominant feature is individualism concerning both enterprises and the solutions they implement. This is undoubtedly the effect of the currently too shallow RPA market, as well as the small number of entities using technologies based on intelligent systems. This is a serious research gap, which along with RPA growth will be reduced as a result of more and more intensive research in this field.
Circulating PTGS2, JAG1, GUCY2C and PGF mRNA in Peripheral Blood and Serum as Potential Biomarkers for Patients with Metastatic Colon Cancer
Authors: Jimenez-Luna Cristina , González-Flores Encarnación , Ortiz Raul , Martínez-González Luis J. , Antúnez-Rodríguez Alba , Expósito-Ruiz Manuela , Melguizo Consolación , Caba Octavio , Prados Jose
Published: May 2021

Genes involved in the angiogenic process have been proposed for the diagnosis and therapeutic response of metastatic colorectal cancer (CRC). This study aimed to investigate the value of PTGS2, JAG1, GUCY2C and PGF-circulating RNA as biomarkers in metastatic CRC. Blood cells and serum mRNA from 59 patients with metastatic CRC and 47 healthy controls were analyzed by digital PCR. The area under the receiver operating characteristic curve (AUC) was used to estimate the diagnostic value of each mRNA alone or mRNA combinations. A significant upregulation of the JAG1, PTGS2 and GUCY2C genes in blood cells and serum samples from metastatic CRC patients was detected. Circulating mRNA levels in the serum of all genes were significantly more abundant than in blood. The highest discrimination ability between metastatic CRC patients and healthy donors was obtained with PTGS2 (AUC of 0.984) and GUCY2C (AUC of 0.896) in serum samples. Biomarker combinations did not improve the discriminatory capacity of biomarkers separately. Analyzed biomarkers showed no correlation with overall survival or progression-free survival, but GUCY2C and GUCY2C/PTGS2 expression in serum correlated significantly with the response to antiangiogenic agents. These findings demonstrate that assessment of genes involved in the angiogenic process may be a potential non-invasive diagnostic tool for metastatic CRC and its response to antiangiogenic therapy.