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BACKGROUND: Type 2 diabetes is a chronic disease with a significant medical burden. eHealth care integrates medicine and technology to enhance the outcomes of such patients; however, adequate eHealth literacy (eHL) is necessary for that to happen. Fostering eHL is crucial for patients with diabetes to engage with eHealth care and receive quality care and timely support. Experiential learning theory can enhance patients' eHL and skills to use eHealth care technology in their daily care. OBJECTIVE: This study explored the effectiveness of an eHealth care experiential learning program in improving eHL, patient health engagement, and eHealth care use status among patients with type 2 diabetes in 3 months. METHODS: In this randomized controlled trial, patients under case management services from various clinics in Taiwan were randomly assigned to either the intervention group receiving the 6-session eHealth care experiential learning program or the control group receiving the usual care. Data were collected using structured questionnaires at 3 time points: pretest, postintervention, and 3 months after the intervention. Descriptive data were presented using frequency distribution, percentage, mean, and SD. The outcomes were analyzed using a generalized estimating equation method by intention-to-treat analysis. RESULTS: A total of 92 participants (46 in each group) were recruited in this study. Of these, 86 completed the course and follow-up evaluations with a mean age of 62.38 (SD 12.91) years. After completing the intervention, the intervention group had significantly higher posttest scores in eHL (ß=19.94, SE 3.52; P<.001), patient health engagement (ß=.28, SE 0.13; P=.04), and eHealth use (ß=3.96, SE 0.42; P<.001) than the control group. Furthermore, the intervention group maintained these significant improvements in eHL (ß=18.19, SE 3.82; P<.001) and eHealth use (ß=3.87, SE 0.49; P<.001) after 3 months. CONCLUSIONS: Participating in the eHealth care experiential learning program resulted in significant improvements in eHL, patient health engagement, and eHealth use among patients with type 2 diabetes. Our interventional program can inform future clinical practice and policies to strengthen self-management skills and facilitate the use of health technology in caring for patients with chronic diseases. TRIAL REGISTRATION: ClinicalTrials.gov NCT05180604; https://clinicaltrials.gov/ct2/show/NCT05180604.
Sujet(s)
Diabète de type 2 , Apprentissage par problèmes , Télémédecine , Humains , Diabète de type 2/thérapie , Adulte d'âge moyen , Femelle , Mâle , Apprentissage par problèmes/méthodes , Taïwan , Sujet âgé , Adulte , Enquêtes et questionnairesRÉSUMÉ
Cesium carbonate promoted direct amidation of unactivated esters with amino alcohols was developed without the use of transition-metal catalysts and coupling reagents. This method enabled the synthesis of several serine-containing oligopeptides and benzamide derivatives with yields up to 90%. The methodology proceeds under mild reaction conditions and exhibits no racemization for most naturally occurring amino acid substrates. The reaction demonstrates good compatibility with primary alkyl and benzyl esters and broad tolerance for a range of amino acid substrates with nonpolar and protected side chains. The hydroxy group on the amine nucleophile was found to be critical for the reaction to be successful. A likely mechanism involving cesium coordination to the substrates enabling the subsequent proximity-driven acyl transfer was proposed. The practicality of this approach was demonstrated in the preparation of a biologically active nicotinamide derivative in a reasonable yield.
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Human induced pluripotent stem cells (hiPSCs) are characterized by unlimited self-renewal and the capability to differentiate into all three germ layers, with the potential to further differentiate into all types of cells and tissues. Human iPSCs retain all genetic information from their original donors and can be developed into disease models to study disease pathophysiology, identify disease phenotypes and biomarkers, and evaluate therapeutic efficacy and toxicity for drug development. Human iPSCs can also be used to develop cell therapies and regenerative medicine. In the last decade, the technologies for hiPSC generation and differentiation have advanced rapidly. Human iPSC culture and propagation are tedious and require careful handling. High-quality hiPSCs are necessary for downstream applications. The methods, techniques, and skills for hiPSC maintenance and characterization are very different from those for immortalized cell lines. It can be a challenge for new laboratory staff, and sometimes even for experienced staff, to properly culture and maintain the high quality of these cells. Here, we describe a comprehensive set of protocols for hiPSC propagation under chemically defined and feeder-free culture conditions. These step-by-step protocols describe in detail all the reagents and experimental procedures needed to culture hiPSCs. The protocols also describe experimental methods for hiPSC characterization, including immunofluorescence staining and flow cytometric analysis with a panel of pluripotency markers, a teratoma formation assay for validation of in vivo pluripotency, and detection of Sendai virus to ensure elimination of the viral vectors. These protocols have been successfully used in our laboratory for hiPSC expansion and propagation, and this article provide a useful reference guide for laboratory staff to work on hiPSC culture. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Propagation and cryopreservation of hiPSC cultures Basic Protocol 2: Recovery of cryopreserved hiPSCs Basic Protocol 3: Validation of pluripotency markers via immunocytochemical analysis Alternate Protocol: Determination of the expression of pluripotency markers via flow cytometry analysis Basic Protocol 4: Assessment of pluripotency via in vivo teratoma formation assay Basic Protocol 5: Confirmation of Sendai viral vector clearance via RT-PCR.
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Cellules souches pluripotentes induites , Humains , Dosage biologique , Différenciation cellulaire , Lignée cellulaire , Thérapie cellulaire et tissulaireRÉSUMÉ
Ovarian cancer is one of the most lethal gynecological malignancies. Recurrence or acquired chemoresistance is the leading cause of ovarian cancer therapy failure. Overexpression of ATP-binding cassette subfamily B member 1 (ABCB1), commonly known as P-glycoprotein, correlates closely with multidrug resistance (MDR). However, the mechanism underlying aberrant ABCB1 expression remains unknown. Using a quantitative high-throughput combinational screen, we identified that terfenadine restored doxorubicin sensitivity in an MDR ovarian cancer cell line. In addition, RNA-seq data revealed that the Ca2+-mediated signaling pathway in the MDR cells was abnormally regulated. Moreover, our research demonstrated that terfenadine directly bound to CAMKIID to prevent its autophosphorylation and inhibit the activation of the cAMP-responsive element-binding protein 1 (CREB1)-mediated pathway. Direct inhibition of CAMKII or CREB1 had the same phenotypic effects as terfenadine in the combined treatment, including lower expression of ABCB1 and baculoviral IAP repeat-containing 5 (BIRC5, also known as survivin) and increased doxorubicin-induced apoptosis. In this study, we demonstrate that aberrant regulation of the Ca2+-mediated CAMKIID/CREB1 pathway contributes to ABCB1 over-expression and MDR creation and that CAMKIID and CREB1 are attractive targets for restoring doxorubicin efficacy in ABCB1-mediated MDR ovarian cancer.
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BACKGROUND AND PURPOSE: A high plasma concentration of proprotein convertase subtilisin/kexin type 9 is characteristic of a prothrombotic state in cardiovascular diseases. Elevated inflammatory markers, such as interleukin-6, are associated with worse outcomes after ischemic stroke. We aimed to study the role of plasma PCSK9 and IL-6 in acute ischemic stroke with dyslipidemia. METHODS: We divided 123 enrolled patients with first-ever acute ischemic stroke into normotensive and high blood pressure groups and further into high and low pulse pressure subgroups. Clinical characteristics and inflammatory and metabolic parameters, including plasma PCSK9 and IL-6, were recorded. RESULTS: After the analysis of the normotensive and BP groups, there were positive correlations between PP and carotid stenosis (P = 0.031) and plaque numbers (P = 0.013) and between National Institute of Health Stroke Scale scores (P = 0.019) and carotid stenosis severity (P = 0.021) and resistance index (P = 0.04). There was a significant association between plasma cholesterol and PCSK9 (P = 0.044) in the low PP subgroup and IL-6 (P = 0.042) in the high PP subgroup. CONCLUSIONS: Our findings indicated that plasma PCSK9 levels were associated with the low PP subgroup, while IL-6 was associated with the high PP subgroup. Dyslipidemia control is also necessary for those who had a stroke and who have high PP. Further investigation to assess the role of PCSK9 and IL-6 in patients with stroke is required for early treatment and secondary prevention.
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Sténose carotidienne , Dyslipidémies , Accident vasculaire cérébral ischémique , Accident vasculaire cérébral , Pression sanguine , Cholestérol , Humains , Interleukine-6 , Proprotéine convertase 9 , Accident vasculaire cérébral/métabolisme , SubtilisinesRÉSUMÉ
Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome, is a rare, lysosomal disorder caused by mutations in a gene encoding iduronate-2-sulfatase (IDS). IDS deficiency results in an accumulation of glycosaminoglycans (GAGs) and secondary accumulations of other lipids in lysosomes. Symptoms of MPS II include a variety of soft and hard tissue problems, developmental delay, and deterioration of multiple organs. Enzyme replacement therapy is an approved treatment for MPS II, but fails to improve neuronal symptoms. Cell-based neuronal models of MPS II disease are needed for compound screening and drug development for the treatment of the neuronal symptoms in MPS II. In this study, three induced pluripotent stem cell (iPSC) lines were generated from three MPS II patient-derived dermal fibroblast cell lines that were differentiated into neural stem cells and neurons. The disease phenotypes were measured using immunofluorescence staining and Nile red dye staining. In addition, the therapeutic effects of recombinant human IDS enzyme, delta-tocopherol (DT), and hydroxypropyl-beta-cyclodextrin (HPBCD) were determined in the MPS II disease cells. Finally, the neural stem cells from two of the MPS II iPSC lines exhibited typical disease features including a deficiency of IDS activity, abnormal glycosaminoglycan storage, and secondary lipid accumulation. Enzyme replacement therapy partially rescued the disease phenotypes in these cells. DT showed a significant effect in reducing the secondary accumulation of lipids in the MPS II neural stem cells. In contrast, HPBCD displayed limited or no effect in these cells. Our data indicate that these MPS II cells can be used as a cell-based disease model to study disease pathogenesis, evaluate drug efficacy, and screen compounds for drug development.
Sujet(s)
Cellules souches pluripotentes induites/effets des médicaments et des substances chimiques , Cellules souches pluripotentes induites/métabolisme , Mucopolysaccharidose de type II/traitement médicamenteux , Mucopolysaccharidose de type II/métabolisme , Cellules souches neurales/effets des médicaments et des substances chimiques , Cellules souches neurales/métabolisme , 2-Hydroxypropyl-beta-cyclodextrin/usage thérapeutique , Lignée cellulaire , Thérapie enzymatique substitutive , Glycosaminoglycanes/métabolisme , Humains , Iduronate 2-sulfatase/usage thérapeutique , Cellules souches pluripotentes induites/anatomopathologie , Métabolisme lipidique/effets des médicaments et des substances chimiques , Modèles neurologiques , Mucopolysaccharidose de type II/anatomopathologie , Cellules souches neurales/anatomopathologie , Phénotype , Protéines recombinantes/usage thérapeutique , Tocophérols/usage thérapeutiqueRÉSUMÉ
NGLY1 deficiency is a rare recessive genetic disease caused by mutations in the NGLY1 gene which codes for N-glycanase 1 (NGLY1). Here, we report the generation of two gene corrected iPSC lines using a patient-derived iPSC line (NCATS-CL6103) that carried a homozygous p.R401X mutation in the NGLY1 gene. These lines contain either one (NCATS-CL6104) or two (NCATS-CL6105) CRISPR/Cas9 corrected alleles of NGLY1. This pair of NGLY1 mutation corrected iPSC lines can be used as a control for the NCATS-CL6103 which serves as a cell-based NGLY1 disease model for the study of the disease pathophysiology and evaluation of therapeutics under development.
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Troubles congénitaux de la glycosylation , Cellules souches pluripotentes induites , Peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase/déficit , Systèmes CRISPR-Cas/génétique , Troubles congénitaux de la glycosylation/génétique , Homozygote , Humains , Mutation/génétique , National Center for Advancing Translational Sciences (USA) , Peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase/génétique , États-UnisRÉSUMÉ
Mucopolysaccharidosis type IIIB (MPS IIIB) is a lysosomal disease caused by mutations in the NAGLU gene encoding α-N-acetylglucosaminidase (NAGLU) which degrades heparan sulfate in lysosomes. Deficiency in NAGLU results in lysosomal accumulation of glycosaminoglycans (GAGs) and neurological symptoms. Currently, there is no effective treatment or cure for this disease. In this study, induced pluripotent stem cell lines were established from two MPS IIIB patient fibroblast lines and differentiated into neural stem cells and neurons. MPS IIIB neural stem cells exhibited NAGLU deficiency accompanied with GAG accumulation, as well as lysosomal enlargement and secondary lipid accumulation. Treatments with recombinant NAGLU, δ-tocopherol, and 2-hydroxypropyl-b-cyclodextrin significantly reduced the disease phenotypes in these cells. These results indicate the MPS IIIB neural stem cells and neurons have the disease relevant phenotype and can be used as a cell-based disease model system for evaluation of drug efficacy and compound screening for drug development.
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Acetylglucosaminidase/métabolisme , Cellules souches pluripotentes induites/métabolisme , Mucopolysaccharidose de type III/métabolisme , Cellules souches neurales/métabolisme , Acetylglucosaminidase/génétique , Différenciation cellulaire/physiologie , Héparitine sulfate/métabolisme , Humains , Lysosomes/métabolisme , Mucopolysaccharidose de type III/génétique , Neurones/métabolisme , PhénotypeRÉSUMÉ
Alagille syndrome (ALGS) is a rare autosomal dominant disorder caused by disruption of the Notch signaling pathway due to mutations in either JAGGED1 (JAG1) (ALGS type 1) or NOTCH2 (ALGS type 2). Loss of this signaling interferes with the development of many organs, but especially the liver. A human induced pluripotent stem cell (iPSC) line was generated from the fibroblasts of a patient with a p. C312X (c. 936 T > A) variant in JAG1. This iPSC line offers a valuable resource to study the disease pathophysiology and develop therapeutics to treat patients with ALGS.
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Syndrome d'Alagille , Cellules souches pluripotentes induites , Syndrome d'Alagille/génétique , Hétérozygote , Humains , Cellules souches pluripotentes induites/métabolisme , Protéine jagged-1/génétique , Protéine jagged-1/métabolisme , Mutation/génétiqueRÉSUMÉ
Alagille syndrome (ALGS) is a multisystem autosomal dominant disorder caused by defects in the Notch signaling pathway, including the mutation in JAGGED1 (JAG1) (ALGS type 1) or NOTCH2 (ALGS type 2). An induced pluripotent stem cell (iPSC) line was generated from the dermal fibroblasts of a 3-month-old patient with heterozygous mutation at JAG1 splicing site (Chr20: 10,629,709C>A) before exon 11. This iPSC model offers a useful resource for disease modeling to study the disease pathophysiology and to develop therapeutics for treatment of ALGS.
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Syndrome d'Alagille , Cellules souches pluripotentes induites , Syndrome d'Alagille/génétique , Exons/génétique , Humains , Cellules souches pluripotentes induites/métabolisme , Nourrisson , Protéine jagged-1/génétique , Protéine jagged-1/métabolisme , MutationRÉSUMÉ
Farber disease is an ultra-rare lysosomal storage disease. Mutations in the N-acylsphingosine amidohydrolase (ASAH1) gene, which encodes for the enzyme acid ceramidase (ACDase), cause ceramides to accumulate in the body. A human induced pluripotent stem cell (iPSC) line TRNDi030-A was generated from fibroblasts of a male patient with a homozygous p. Y36C (c.107 A>G) variant in the second exon of the ASAH1 producing the alpha subunit of ACDase. This Farber disease iPSC line is a useful resource to study disease pathophysiology and to develop therapeutics for treatment of patients with Farber disease.
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Lipogranulomatose de Farber , Cellules souches pluripotentes induites , Acid Ceramidase/génétique , Homozygote , Humains , Mâle , Mutation/génétiqueRÉSUMÉ
Effective therapeutics to combat emerging viral infections are an unmet need. Historically, treatments for chronic viral infections with single drugs have not been successful, as exemplified by human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections. Combination therapy for these diseases has led to improved clinical outcomes with dramatic reductions in viral load, morbidity, and mortality. Drug combinations can enhance therapeutic efficacy through additive, and ideally synergistic, effects for emerging and re-emerging viruses, such as influenza, severe acute respiratory syndrome-coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS)-CoV, Ebola, Zika, and SARS-coronavirus 2 (CoV-2). Although novel drug development through traditional pipelines remains a priority, in the interim, effective synergistic drug candidates could be rapidly identified by drug-repurposing screens, facilitating accelerated paths to clinical testing and potential emergency use authorizations.
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Antiviraux/usage thérapeutique , Maladies transmissibles émergentes/traitement médicamenteux , Association médicamenteuse , Association de médicaments/tendances , Maladies virales/traitement médicamenteux , Repositionnement des médicaments , Humains , Traitements médicamenteux de la COVID-19RÉSUMÉ
NGLY1 deficiency is a rare disorder caused by mutations in the NGLY1 gene which codes for the highly conserved N-glycanase1 (NGLY1). This enzyme functions in cytosolic deglycosylation of N- linked glycoproteins. An induced pluripotent stem cell (iPSC) line was generated from the dermal fibroblasts of a 2-year-old patient carrying compound heterozygous mutations, p.R390P and p.L318P in the NGLY1 gene. This cell-based iPSC disease model provides a resource to study disease pathophysiology and to develop a cell-based disease model for drug development for NGLY1 patients.
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Cellules souches pluripotentes induites , Peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase/génétique , Lignée cellulaire , Enfant d'âge préscolaire , Glycoprotéines , Hétérozygote , Humains , MutationRÉSUMÉ
Candida auris is an emerging fatal fungal infection that has resulted in several outbreaks in hospitals and care facilities. Current treatment options are limited by the development of drug resistance. Identification of new pharmaceuticals to combat these drug-resistant infections will thus be required to overcome this unmet medical need. We have established a bioluminescent ATP-based assay to identify new compounds and potential drug combinations showing effective growth inhibition against multiple strains of multidrug-resistant Candida auris The assay is robust and suitable for assessing large compound collections by high-throughput screening (HTS). Utilizing this assay, we conducted a screen of 4,314 approved drugs and pharmacologically active compounds that yielded 25 compounds, including 6 novel anti-Candida auris compounds and 13 sets of potential two-drug combinations. Among the drug combinations, the serine palmitoyltransferase inhibitor myriocin demonstrated a combinational effect with flucytosine against all tested isolates during screening. This combinational effect was confirmed in 13 clinical isolates of Candida auris.
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Candida , Préparations pharmaceutiques , Antifongiques/pharmacologie , Antifongiques/usage thérapeutique , Repositionnement des médicaments , Tests de sensibilité microbienneRÉSUMÉ
Understanding the SARS-CoV-2 virus' pathways of infection, virus-host-protein interactions, and mechanisms of virus-induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID-19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose-limiting toxicities in patients. Therefore, we evaluated additional lysosomotropic compounds to identify an alternative lysosome-based drug repurposing opportunity. We found that six of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero E6 cells with half-maximal effective concentration (EC50) values ranging from 2.0 to 13 µM and selectivity indices (SIs; SI = CC50/EC50) ranging from 1.5- to >10-fold. The compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) reduced (ROC-325) viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV-NL63 cytopathic effect in LLC-MK2 cells. Moreover, an analysis of SARS-CoV-2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS-CoV-2. Our findings suggest the lysosome as a potential host cell target to combat SARS-CoV-2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID-19.
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COVID-19 , SARS-CoV-2 , Antiviraux/pharmacologie , Antiviraux/usage thérapeutique , Repositionnement des médicaments , Humains , LysosomesRÉSUMÉ
Pompe disease is a lysosomal storage disorder caused by autosomal recessive mutations in the acid alpha-glucosidase (GAA) gene. Acid alpha-glucosidase deficiency leads to abnormal glycogen accumulation in patient cells. Given the increasing evidence of central nervous system (CNS) involvement in classic infantile Pompe disease, we used neural stem cells, differentiated from patient induced pluripotent stem cells, to model the neuronal phenotype of Pompe disease. These Pompe neural stem cells exhibited disease-related phenotypes including glycogen accumulation, increased lysosomal staining, and secondary lipid buildup. These morphological phenotypes in patient neural stem cells provided a tool for drug efficacy evaluation. Two potential therapeutic agents, hydroxypropyl-ß-cyclodextrin and δ-tocopherol, were tested along with recombinant human acid alpha-glucosidase (rhGAA) in this cell-based Pompe model. Treatment with rhGAA reduced LysoTracker staining in Pompe neural stem cells, indicating reduced lysosome size. Additionally, treatment of diseased neural stem cells with the combination of hydroxypropyl-ß-cyclodextrin and δ-tocopherol significantly reduced the disease phenotypes. These results demonstrated patient-derived Pompe neural stem cells could be used as a model to study disease pathogenesis, to evaluate drug efficacy, and to screen compounds for drug discovery in the context of correcting CNS defects.
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2-Hydroxypropyl-beta-cyclodextrin/pharmacologie , Glycogénose de type II , alpha-Glucosidase/pharmacologie , gamma-Tocophérol/pharmacologie , Lignée cellulaire , Fibroblastes , Glycogénose de type II/traitement médicamenteux , Glycogénose de type II/métabolisme , Humains , Cellules souches pluripotentes induites , Cellules souches neuralesRÉSUMÉ
Expanded human skin fibroblast cells from four different aged healthy individuals, 11-year-old female, 1-year-old male, 2-month-old male, and 8-year-old male, were used to generate integration-free induced pluripotent stem cell (iPSC) lines TRNDi021-C, TRNDi023-D, TRNDi024-D, and TRNDi025-A, respectively, by exogenous expression of four reprogramming factors, human SXO2, OCT3/4, C-MYC, KLF4. The authenticity of established iPSC lines was confirmed by the expressions of stem cell markers, karyotype analysis, and teratoma formation. These iPSC lines could serve as young healthy controls for the studies involving patient-specific iPSCs.
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Cellules souches pluripotentes induites , Tératome , Sujet âgé , Différenciation cellulaire , Reprogrammation cellulaire , Enfant , Femelle , Fibroblastes , Humains , Nourrisson , Caryotype , Caryotypage , Facteur-4 de type Kruppel , MâleRÉSUMÉ
The 2015 to 2016 outbreak of Zika virus (ZIKV) infections in the Americas coincided with a dramatic increase in neurodevelopmental abnormalities, including fetal microcephaly, in newborns born to infected women. In this study, we observed mitochondrial fragmentation and disrupted mitochondrial membrane potential after 24 h of ZIKV infection in human neural stem cells and the SNB-19 glioblastoma cell line. The severity of these changes correlated with the amount of ZIKV proteins expressed in infected cells. ZIKV infection also decreased the levels of mitofusin 2, which modulates mitochondria fusion. Mitochondrial division inhibitor 1 (Mdivi-1), a small molecule inhibiting mitochondria fission, ameliorated mitochondria disruptions and reduced cell death in ZIKV-infected cells. Collectively, this study suggests that abnormal mitochondrial fragmentation contributes to ZIKV-induced neuronal cell death; rebalancing mitochondrial dynamics of fission-fusion could be a therapeutic strategy for drug development to treat ZIKV-mediated neuronal apoptosis.