ABSTRACT
The choriogenin H - EGFP transgenic medaka (Oryzias melastigma) has been used to test estrogenic substances and quantify estrogenic activity into 17ß-estradiol (E2) equivalency (EEQ). The method uses 8 eleutheroembryos in 2 ml solution per well and 3 wells per treatment in 24-well plates at 26 ± 1 °C for 24 ± 2 h, with subsequent measurements of induced GFP signal intensity. EEQ measurements are calculated using a E2 probit regression model with a coefficient of determination (R2) > 0.90. The selectivity was confirmed evaluating 27 known estrogenic and 5 known non-estrogenic compounds. Limit of quantitation (LOQ), recovery rate and bias were calculated to be 1 ng/ml EEQ, 104% and 4% respectively. Robustness analysis revealed exposure temperature is a sensitive parameter that should be kept at 26 ± 1 °C. The repeatability of intra- and inter-laboratories achieved CV < 30% for most tested food and cosmetics samples. The lot-lot stability was confirmed by the stable EEQ qualitative control (QC, 1 ng/mL E2) and calibration curve results. The stability of standard reagents, samples and sample extracts was also investigated. These data demonstrated this method to be an accurate indicator of estrogenic activity for both chemicals and extracts.
Subject(s)
Animals, Genetically Modified/metabolism , Egg Proteins/analysis , Estradiol/chemistry , Oryzias/metabolism , Protein Precursors/analysis , Animals , Animals, Genetically Modified/embryology , Biosensing Techniques , Cell Extracts/chemistry , Estradiol/metabolism , Limit of Detection , Oryzias/embryology , Regression AnalysisABSTRACT
The ability to observe in situ 3D distribution and dynamics of endosymbionts in corals is crucial for gaining a mechanistic understanding of coral bleaching and reef degradation. Here, we report the development of a tissue clearing (TC) coupled with light sheet fluorescence microscopy (LSFM) method for 3D imaging of the coral holobiont at single-cell resolution. The initial applications have demonstrated the ability of this technique to provide high spatial resolution quantitative information of endosymbiont abundance and distribution within corals. With specific fluorescent probes or assays, TC-LSFM also revealed spatial distribution and dynamics of physiological conditions (such as cell proliferation, apoptosis, and hypoxia response) in both corals and their endosymbionts. This tool is highly promising for in situ and in-depth data acquisition to illuminate coral symbiosis and health conditions in the changing marine environment, providing fundamental information for coral reef conservation and restoration.
Subject(s)
Anthozoa/physiology , Coral Reefs , Symbiosis , Animals , Dinoflagellida/physiology , Microscopy, Fluorescence/methodsABSTRACT
Neutrophils are the first line defenders in the innate immune response, and rapidly migrate to an infected or injured area. Recently, bidirectional migration of neutrophils to the wound and the corresponding functions have become popular research pursuits. In zebrafish larvae, CXCR1/CXCL8 is the predominant chemoattractant pathway to recruit neutrophil to wound, while CXCR2/CXCL8 pathway mediate neutrophil dispersal in wound after injury. Here, we found that both CXCR1/CXCL8 and LTB4/BLT1 signals are activated in zebrafish heart after cryoinjury. And with a CXCR1/2 selective inhibitor (SB225002) treatment, the recruitment of neutrophils was not affected, but reverse migration of neutrophils was inhibited after cryoinjury of heart. We suggested that the neutrophil recruitment to cryoinjured area might be mediated by LTB4/BLT1 signals at the presence of SB225002. Therefore, SB225002 treatment resulted more accumulation and long retention of neutrophils in the injured heart. The long retention of neutrophils in the wound promoted revascularization in the injured heart; however, the AKT/mTOR pathway was inhibited and the regeneration was impaired. Our findings suggest that retention of neutrophils is a well-orchestrated process and might regulate regeneration by the AKT/mTOR pathway.
Subject(s)
Freezing/adverse effects , Heart/physiology , Regeneration , Signal Transduction/physiology , Zebrafish/physiology , Animals , Cryopreservation/veterinary , Heart Injuries/etiology , Heart Injuries/physiopathology , Neutrophils/immunologyABSTRACT
Inflammation plays a crucial role in cardiac regeneration. Numerous advantages, including a robust regenerative ability, make the zebrafish a popular model to study cardiovascular diseases. The zebrafish breakdance (bre) mutant shares several key features with human long QT syndrome that predisposes to ventricular arrhythmias and sudden death. However, how inflammatory response and tissue regeneration following cardiac damage occur in bre mutant is unknown. Here, we have found that inflammatory response related genes were markedly expressed in the injured heart and excessive leukocyte accumulation occurred in the injured area of the bre mutant zebrafish. Furthermore, bre mutant zebrafish exhibited aberrant apoptosis and impaired heart regenerative ability after ventricular cryoinjury. Mild dosages of anti-inflammatory or prokinetic drugs protected regenerative cells from undergoing aberrant apoptosis and promoted heart regeneration in bre mutant zebrafish. We propose that immune or prokinetic therapy could be a potential therapeutic regimen for patients with genetic long QT syndrome who suffers from myocardial infarction.
Subject(s)
Down-Regulation , Heart Injuries/physiopathology , Inflammation/physiopathology , Regeneration , Zebrafish/physiology , Animals , Cold Temperature/adverse effects , Disease Models, Animal , Heart , Heart Injuries/etiology , Inflammation/etiologyABSTRACT
Photon hormesis refers to the phenomenon where the biological effect of ionizing radiation with a high linear energy transfer (LET) value is diminished by photons with a low LET value. The present paper studied the effect of photon hormesis from X-rays on dose responses to alpha particles using embryos of the zebrafish (Danio rerio) as the in vivo vertebrate model. The toxicity of these ionizing radiations in the zebrafish embryos was assessed using the apoptotic counts at 20, 24, or 30 h post fertilization (hpf) revealed through acridine orange (AO) staining. For alpha-particle doses ≥ 4.4 mGy, the additional X-ray dose of 10 mGy significantly reduced the number of apoptotic cells at 24 hpf, which proved the presence of photon hormesis. Smaller alpha-particle doses might not have inflicted sufficient aggregate damages to trigger photon hormesis. The time gap T between the X-ray (10 mGy) and alpha-particle (4.4 mGy) exposures was also studied. Photon hormesis was present when T ≤ 30 min, but was absent when T = 60 min, at which time repair of damage induced by alpha particles would have completed to prevent their interactions with those induced by X-rays. Finally, the drop in the apoptotic counts at 24 hpf due to photon hormesis was explained by bringing the apoptotic events earlier to 20 hpf, which strongly supported the removal of aberrant cells through apoptosis as an underlying mechanism for photon hormesis.
Subject(s)
Alpha Particles , Embryo, Nonmammalian/radiation effects , Hormesis , Photons , Zebrafish , Animals , Apoptosis/radiation effects , Radiation Dosage , Radiation, Ionizing , X-RaysABSTRACT
BACKGROUND: Cell fusion is a fast and highly efficient technique for cells to acquire new properties. The fusion of somatic cells with stem cells can reprogram somatic cells to a pluripotent state. Our research on the fusion of stem cells and cancer cells demonstrates that the fused cells can exhibit stemness and cancer cell-like characteristics. Thus, tumor-initiating cell-like cells are generated. METHODS: We employed laser-induced single-cell fusion technique to fuse the hepatocellular carcinoma cells and human embryonic stem cells (hESC). Real-time RT-PCR, flow cytometry and in vivo tumorigenicity assay were adopted to identify the gene expression difference. RESULTS: We successfully produced a fused cell line that coalesces the gene expression information of hepatocellular carcinoma cells and stem cells. Experimental results showed that the fused cells expressed cancer and stemness markers as well as exhibited increased resistance to drug treatment and enhanced tumorigenesis. CONCLUSIONS: Fusion with stem cells transforms liver cancer cells into tumor initiating-like cells. Results indicate that fusion between cancer cell and stem cell may generate tumor initiating-like cells.
Subject(s)
Carcinoma, Hepatocellular/pathology , Cell Fusion , Liver Neoplasms/pathology , Neoplastic Stem Cells/pathology , Cell Line, Tumor , Flow Cytometry , Gene Expression Regulation, Neoplastic , Human Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/pathology , Humans , Hyaluronan Receptors/biosynthesis , Lasers , Liver/metabolism , Liver/pathology , Neoplasm Proteins/biosynthesis , Neoplastic Stem Cells/metabolismABSTRACT
Exposure to ionizing radiations (IRs) is ubiquitous in our environment and can be categorized into "targeted" effects and "non-targeted" effects. In addition to inducing deoxyribonucleic acid (DNA) damage, IR exposure leads to epigenetic alterations that do not alter DNA sequence. Using an appropriate model to study the biological effects of radiation is crucial to better understand IR responses as well as to develop new strategies to alleviate exposure to IR. Zebrafish, Danio rerio, is a scientific model organism that has yielded scientific advances in several fields and recent studies show the usefulness of this vertebrate model in radiation biology. This review briefly describes both "targeted" and "non-targeted" effects, describes the findings in radiation biology using zebrafish as a model and highlights the potential of zebrafish to assess the epigenetic effects of IR, including DNA methylation, histone modifications and miRNA expression. Other in vivo models are included to compare observations made with zebrafish, or to illustrate the feasibility of in vivo models when the use of zebrafish was unavailable. Finally, tools to study epigenetic modifications in zebrafish, including changes in genome-wide DNA methylation, histone modifications and miRNA expression, are also described in this review.
Subject(s)
Epigenesis, Genetic/radiation effects , Radiation, Ionizing , Zebrafish/genetics , Animals , DNA Methylation/radiation effects , Histones/metabolism , Models, AnimalABSTRACT
This study demonstrates a novel method of using silver nanoparticles for Achilles tendon injury healing. In vitro results indicated a stimulatory effect on cell proliferation and collagen synthesis with silver nanoparticles. Biomechanical test on the 42-day post operation Achilles tendon sample exhibited a significant improvement in tensile modulus when compared to the untreated group. Histology suggested that silver nanoparticles promoted cell alignment and proteoglycan synthesis. The collagen deposition was also improved. An alleviation of tumor necrosis factor α, and an increase in fibromodulin and proliferating cell nuclear antigen expression were seen in silver nanoparticles group by immunohistochemistry. This study further corroborates the finding of our previous study that silver nanoparticles help to restore the functionality of injured connective tissues. We believe that the anti-inflammatory nature of silver nanoparticles has an important role in accelerating the healing process and reducing scarring, leading to better functional outcome. From the clinical editor: Tendon healing after surgeries remains a slow and tedious process, typically requiring several weeks of recovery time and gradual introduction of physical therapy. There are no currently utilized methods that could promote tendon healing. In this study, silver nanoparticles are reported to facilitate Achilles tendon repair in a model system, through increased proteoglycan and collagen synthesis, paving the way to potential clinical applications in the future.
Subject(s)
Achilles Tendon/injuries , Metal Nanoparticles/therapeutic use , Proteoglycans/metabolism , Silver/chemistry , Tendon Injuries/therapy , Animals , Collagen/metabolism , Kidney/metabolism , Liver/metabolism , Rats , Rats, Sprague-Dawley , Tendon Injuries/physiopathologyABSTRACT
The efficient application of the newly developed gene-editing method CRISPR/Cas9 requires more accurate intracellular gene delivery. Traditional delivery approaches, such as lipotransfection and non-viral delivery methods, must contend with major problems to overcome the drawbacks of low efficiency, high toxicity, and cell-type dependency. The high-throughput microdroplet-based single-cell transfection method presented herein provides an alternative method for delivering genome-editing reagents into single living cells. By accurately controlling the number of exogenous plasmids in microdroplets, this method can achieve high-efficiency delivery of nucleic acids to different types of single cells. This paper presents a high-throughput quantitative DNA transfection method for single cells and explores the optimal DNA transfection conditions for specific cell lines. The transfection efficiency of cells at different concentrations of DNA in microdroplets is measured. Under the optimized transfection conditions, the method is used to construct gene-knockout cancer cell lines to determine specific gene functions through the CRISPR/Cas9 knockout system. In a case study, the migration ability of TRIM72 knockout cancer cells is inhibited, and the tumorigenicity of cells in a zebrafish tumor model is reduced. A single-cell microfluidic chip is designed to achieve CRISPR/Cas9 DNA transfection, dramatically improving the transfection efficiency of difficult-to-transfect cells. This research demonstrates that the microdroplet method developed herein has a unique advantage in CRISPR/Cas9 gene-editing applications.
Subject(s)
CRISPR-Cas Systems , Zebrafish , Animals , CRISPR-Cas Systems/genetics , Gene Knockout Techniques , Zebrafish/genetics , Transfection , DNAABSTRACT
Mapping brain activities is necessary for understanding brain physiology and discovering new treatments for neurological disorders. Such efforts have greatly benefited from the advancement in technologies for analyzing neural activity with improving temporal or spatial resolution. Here, we constructed a multielectrode array based brain activity mapping (BAM) system capable of stabilizing and orienting zebrafish larvae for recording electroencephalogram (EEG) like local field potential (LFP) signals and brain-wide calcium dynamics in awake zebrafish. Particularly, we designed a zebrafish trap chip that integrates with an eight-by-eight surface electrode array, so that brain electrophysiology can be noninvasively recorded in an agarose-free and anesthetic-free format with a high temporal resolution of 40 µs, matching the capability typically achieved by invasive LFP recording. Benefiting from the specially designed hybrid system, we can also conduct calcium imaging directly on immobilized awake larval zebrafish, which further supplies us with high spatial resolution brain-wide activity data. All of these innovations reconcile the limitations of sole LFP recording or calcium imaging, emphasizing a synergy of combining electrical and optical modalities within one unified device for activity mapping across a whole vertebrate brain with both improved spatial and temporal resolutions. The compatibility with in vivo drug treatment further makes it suitable for pharmacology studies based on multimodal measurement of brain-wide physiology.
Subject(s)
Brain , Electroencephalography , Zebrafish , Animals , Brain/drug effects , Brain/physiology , Electroencephalography/methods , Brain Mapping/methods , Calcium/metabolism , Larva , Optical Imaging/methodsABSTRACT
HEADINGS ETHNOPHARMACOLOGICAL RELEVANCE: Rehmanniae Radix Praeparata (RRP), a staple in traditional Chinese medicine, is derived from Rehmannia glutinosa Libosch and is renowned for its wound-healing properties. Despite its clinical prevalence, the molecular mechanisms underlying RRP's wound-healing effects have not been fully elucidated. AIM OF THE STUDY: This research endeavored to delineate the molecular and cellular mechanisms underlying the beneficial effects of RRP on wound healing, utilizing a zebrafish model. MATERIALS AND METHODS: Zebrafish larvae at 3 days post-fertilization were amputated at the fin and subsequently treated with RRP. The pro-wound healing and regenerative effects of RRP were evaluated through morphological analysis, assessment of cell proliferation and apoptosis, Additionally, mechanistic insights were gained through a comprehensive approach encompassing network pharmacology analysis, cell tracing, RNA-sequencing, CRISPR/Cas9 gene editing, and pharmacological inhibition. RESULTS: Our findings demonstrate that RRP significantly accelerates caudal fin regeneration in zebrafish following injury by suppressing cell apoptosis, promoting cell proliferation, and upregulating the expression of regenerative-related genes. Furthermore, RRP triggers autophagy signals during the regenerative process, which is attenuated by the autophagy inhibitor chloroquine (CQ). Notably, the administration of RRP enhances the expression of ahr1 and ahr2 in the regenerating fin. Genetic knockout of ahr1a, ahr1b, or ahr2 using CRISPR/Cas9, or pharmacological blockade of AHR signals with the antagonist CH-223191, diminishes the regenerative potential of RRP. Remarkably, zebrafish lacking ahr2 completely lose their fin regeneration ability. Additionally, inhibition of AHR signaling suppresses autophagy signaling during fin regeneration. CONCLUSIONS: This study uncovers that RRP stimulates fin regeneration in zebrafish by inducing AHR signals and, at least partially, activating the autophagy process. These findings provide novel insights into the molecular mechanisms underlying the wound-healing effects of RRP and may pave the way for the development of novel therapeutic strategies.
Subject(s)
Animal Fins , Autophagy , Cell Proliferation , Receptors, Aryl Hydrocarbon , Regeneration , Rehmannia , Zebrafish , Animals , Autophagy/drug effects , Animal Fins/drug effects , Animal Fins/physiology , Receptors, Aryl Hydrocarbon/metabolism , Receptors, Aryl Hydrocarbon/genetics , Rehmannia/chemistry , Regeneration/drug effects , Cell Proliferation/drug effects , Wound Healing/drug effects , Apoptosis/drug effects , Plant Extracts/pharmacology , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Plant RootsABSTRACT
Zebrafish is an important model organism in biological research. One of the least explored tissues of zebrafish is blood, because the existing methods for isolating blood from this organism are tedious and irreproducible. The small volume of blood collected by these methods also prohibits many biochemical and cytological analyses. This technical obstacle limits the utilization of zebrafish in many applications, particularly in hematological research and plasma biomarker discovery. To overcome this limitation, we have established a novel method of extracting blood from zebrafish, based on the use of low centrifugal force to collect blood from a wound. This method consistently recovers more blood than traditional methods. Gel electrophoresis and flow cytometry showed that composition of blood harvested by this method is indistinguishable from traditional methods. The increase in yield enables us to perform biochemical experiments on zebrafish blood. In particular, we have demonstrated that quantitative proteomics can be performed on plasma collected from single zebrafish. Here, we have compared, by using shotgun proteomic analysis, the plasma proteomes of adult male and female zebrafish. Twenty-seven gender-dependent plasma proteins are identified and their biochemical importance discussed. Taken together, this novel technique enables analyses that were previously difficult to perform on zebrafish blood.
Subject(s)
Blood Proteins/analysis , Blood Specimen Collection/methods , Zebrafish Proteins/blood , Zebrafish/blood , Animals , Female , Male , Proteomics/methods , Reproducibility of Results , Sex Factors , Tandem Mass SpectrometryABSTRACT
G-protein-coupled estrogen receptor 1 (Gper, formerly known as GPR30) is found to be a trophic and protective factor in mediating action of estrogen in adult brain, while its role in developing brain remains to be elucidated. Here we present the expression pattern of Gper and its functions during embryogenesis in zebrafish. Both the mRNA and protein of Gper were detected throughout embryogenesis. Whole mount in situ hybridization (WISH) revealed a wide distribution of gper mRNAs in various regions of the developing brain. Gper knockdown by specific morpholinos resulted in growth retardation in embryos and morphological defects in the developing brain. In addition, induced apoptosis, decreased proliferation of the brain cells and maldevelopment of sensory and motor neurons were also found in the morphants. Our results provide novel insights into Gper functions in the developing brain, revealing that Gper can maintain the survival of the brain cells, and formation and/or differentiation of the sensory and motor neurons.
Subject(s)
Brain/metabolism , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Receptors, G-Protein-Coupled/genetics , Zebrafish Proteins/genetics , Zebrafish/genetics , Animals , Apoptosis/genetics , Blotting, Western , Brain/cytology , Brain/embryology , Cell Differentiation/genetics , Cell Survival/genetics , Embryo, Nonmammalian/embryology , Gene Knockdown Techniques , In Situ Hybridization , Microscopy, Fluorescence , Models, Genetic , Models, Neurological , Motor Neurons/cytology , Motor Neurons/metabolism , Receptors, G-Protein-Coupled/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sensory Receptor Cells/cytology , Sensory Receptor Cells/metabolism , Zebrafish/embryology , Zebrafish/metabolism , Zebrafish Proteins/metabolismABSTRACT
Organelle-specific cell-permeable fluorescent dyes are invaluable tools in cell biology as they reveal intracellular dynamics in living cells. Mitrotracker is a family of dyes that strongly label the mitochondrion, a key organelle associated with many crucial cellular functions. Despite the popularity of these dyes, little is known about the molecular mechanism behind their staining specificity. Here, we aimed to identify the protein targets of one member of this dye family, mitotracker red (MTR), by 2DE and MS. MTR bound to cellular proteins covalently, and its fluorescence persisted even after cell lysis, protein solubilization, denaturation, and electrophoresis. This enabled us to display MTR-labeled proteins by 2DE. The MTR-specific fluorescent signals on the gel revealed the spots that contained MTR-conjugated proteins. These spots were analyzed by MS, resulting into the identification of ten proteins. We discovered that one major target is the mitochondrial protein HSP60 and that MTR staining could induce production of HSP60, predisposing cells to heat shock-like responses. The identification of the molecular targets of biological dyes, or "stainomics," can help correlate their intracellular staining properties with biochemical affinities. We believe this approach can be applied to a wide range of fluorescent probes.
Subject(s)
Fluorescent Dyes/chemistry , Mitochondrial Proteins/analysis , Proteomics/methods , Staining and Labeling/methods , Chaperonin 60/analysis , Chaperonin 60/chemistry , Chaperonin 60/metabolism , Electrophoresis, Gel, Two-Dimensional , Fluorescent Dyes/metabolism , HeLa Cells , Humans , Microscopy, Fluorescence , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolismABSTRACT
The protein corona of a nanomaterial is a complex layer of proteins spontaneously and stably formed when the material is exposed to body fluids or intracellular environments. In this study, we utilised stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to characterise the binding of human cellular proteins to two forms of carbon nanoparticles: namely multi-walled carbon nanotubes (MWCNTs) and carbon black (CB). The relative binding efficiency of over 750 proteins to these materials is measured. The data indicate that MWCNTs and CB bind to vastly different sets of proteins. The molecular basis of selectivity in protein binding is investigated. This study is the first large-scale characterisation of protein corona on CNT, providing the biochemical basis for the assessment of the suitability of CNTs as biomedical tools, and as an emerging pollutant. FROM THE CLINICAL EDITOR: This team of investigators performed the first large-scale characterization of protein corona on carbon nanotubes, studying 750 proteins and assessing the suitability of CNTs as biomedical tools and as an emerging pollutant.
Subject(s)
Amino Acids/chemistry , Carbon/chemistry , Nanotubes, Carbon/chemistry , Proteins/chemistry , Cell Line , Humans , Isotope Labeling , Nanoparticles/chemistry , Protein Binding , Proteomics , Soot/chemistryABSTRACT
BACKGROUND: Recent studies demonstrated that elevated osmolarity could induce adipocyte dedifferentiation, representing an appealing procedure to generate multipotent stem cells. Here we aim to elucidate the molecular mechanisms that underlie osmotic induction of adipocyte reprogramming. METHODS: To induce dedifferentiation, the 3T3-L1 or SVF adipocytes were cultured under the hypertonic pressure in 2% PEG 300 medium. Adipocyte dedifferentiation was monitored by aspect ratio measurement, Oil Red staining and qPCR to examine the morphology, lipid droplets, and specific genes of adipocytes, respectively. The osteogenic and chondrogenic re-differentiation capacities of dedifferentiated adipocytes were also examined. To investigate the mechanisms of the osmotic stress-induced dedifferentiation, extracellular vesicles (EVs) were collected from the reprograming cells, followed by proteomic and functional analyses. In addition, qPCR, ELISA, and TNF-α neutralizing antibody (20 ng/ml) was applied to examine the activation and effects of the TNF-α signaling. Furthermore, we also analyzed the Wnt signaling by assessing the activation of ß-catenin and applying BML-284, an agonist of ß-catenin. RESULTS: Hypertonic treatment induced dedifferentiation of both 3T3-L1 and the primary stromal vascular fraction (SVF) adipocytes, characterized by morphological and functional changes. Proteomic profiling revealed that hypertonicity induced extracellular vesicles (EVs) containing mitochondrial molecules including NDUFA9 and VDAC. Functionally, the mitochondrial EVs (MEVs) stimulated TNF-α signaling that activates Wnt-ß-catenin signaling and adipocyte dedifferentiation. Neutralizing TNF-α inhibited hypertonic dedifferentiation of adipocytes. In addition, direct activation of Wnt-ß-catenin signaling using BML-284 could efficiently induce adipocyte dedifferentiation while circumventing the apoptotic effect of the hypertonic treatment. CONCLUSIONS: Hypertonicity prompts the adipocytes to release MEVs, which in turn enhances the secretion of TNF-α as a pro-inflammatory cytokine during the stress response. Importantly, TNF-α is essential for the activation of the Wnt/ß-catenin signaling that drives adipocyte dedifferentiation. A caveat of the hypertonic treatment is apoptosis, which could be circumvented by direct activation of the Wnt/ß-catenin signaling using BML-284.
Subject(s)
Extracellular Vesicles , Tumor Necrosis Factor-alpha , Mice , Animals , Tumor Necrosis Factor-alpha/pharmacology , beta Catenin/metabolism , Proteomics , Adipocytes , Cell Differentiation , Wnt Signaling Pathway , Extracellular Vesicles/metabolism , 3T3-L1 Cells , AdipogenesisABSTRACT
Understanding health care resource utilisation and its associated costs are important for identifying areas of improvement regarding resource allocations. However, there is limited research exploring this issue in the setting of Brugada syndrome (BrS).This was a retrospective territory-wide study of BrS patients from Hong Kong. Healthcare resource utilisation for accident and emergency (A&E), inpatient and specialist outpatient attendances were analyzed over a 19-year period, with their associated costs presented in US dollars. A total of 507 BrS patients with a mean presentation age of 49.9 ± 16.3 years old were included. Of these, 384 patients displayed spontaneous type 1 electrocardiographic (ECG) Brugada pattern and 77 patients had presented with ventricular tachycardia/ventricular fibrillation (VT/VF). At the individual patient level, the median annualized costs were $110 (52-224) at the (A&E) setting, $6812 (1982-32414) at the inpatient setting and $557 (326-1001) for specialist outpatient attendances. Patients with initial VT/VF presentation had overall greater costs in inpatient ($20161 [9147-189215] vs $5290 [1613-24937],P < 0.0001) and specialist outpatient setting ($776 [438-1076] vs $542 [293-972],Pâ¯=â¯0.015) compared to those who did not present VT. In addition, patients without Type 1 ECG pattern had greater median costs in the specialist outpatient setting ($7036 [3136-14378] vs $4895 [2409-10554],p=0.019). There is a greater health care demand in the inpatient and specialist outpatient settings for BrS patients. The most expensive attendance type was inpatient setting stay at $6812 per year. The total median annualized cost of BrS patients without VT/VF presentation was 78% lower compared to patients with VT/VF presentation.
Subject(s)
Brugada Syndrome , Humans , Adult , Middle Aged , Aged , Brugada Syndrome/epidemiology , Brugada Syndrome/therapy , Retrospective Studies , Hong Kong/epidemiology , Ventricular Fibrillation/complications , Arrhythmias, Cardiac , Electrocardiography , Patient Acceptance of Health CareABSTRACT
We hypothesized that an interpretable gradient boosting machine (GBM) model considering comorbidities, P-wave and echocardiographic measurements, can better predict mortality and cerebrovascular events in mitral regurgitation (MR). Patients from a tertiary center were analyzed. The GBM model was used as an interpretable statistical approach to identify the leading indicators of high-risk patients with either outcome of CVAs and all-cause mortality. A total of 706 patients were included. GBM analysis showed that age, systolic blood pressure, diastolic blood pressure, plasma albumin levels, mean P-wave duration (PWD), MR regurgitant volume, left ventricular ejection fraction (LVEF), left atrial dimension at end-systole (LADs), velocity-time integral (VTI) and effective regurgitant orifice were significant predictors of TIA/stroke. Age, sodium, urea and albumin levels, platelet count, mean PWD, LVEF, LADs, left ventricular dimension at end systole (LVDs) and VTI were significant predictors of all-cause mortality. The GBM demonstrates the best predictive performance in terms of precision, sensitivity c-statistic and F1-score compared to logistic regression, decision tree, random forest, support vector machine, and artificial neural networks. Gradient boosting model incorporating clinical data from different investigative modalities significantly improves risk prediction performance and identify key indicators for outcome prediction in MR.
Subject(s)
Mitral Valve Insufficiency , Stroke , Humans , Mitral Valve Insufficiency/diagnostic imaging , Ventricular Function, Left , Stroke Volume/physiology , Systole/physiology , Stroke/epidemiology , Stroke/etiologyABSTRACT
We report the cellular properties of a luminescent cyclometalated iridium(III) complex, [Ir(pq)(2)(phen-ITC)](PF(6)) (Ir-ITC; Hpq=2-phenylquinoline, phen-ITC=5-isothiocyanate-1,10-phenanthroline), that efficiently and specifically labels mitochondria in living mammalian cells. Ir-ITC can be covalently conjugated to its protein targets, and its luminescence survived cell lysis, protein extraction, and gel electrophoresis under denaturing conditions. The conjugation of Ir-ITC with live-cell proteins is rapid and highly selective; the process requires active cellular metabolism, as the conjugation is abolished at nonphysiological temperature or in the presence of sodium azide. Based on measurements of the luminescence intensity, we have devised a biochemical fractionation procedure that allows the enrichment of the conjugated proteins, and their subsequent separation by two-dimensional gel electrophoresis (2DGE). Luminescent protein spots were picked from the gel and analyzed by mass spectrometry; this resulted in the identification of 46 proteins. Many of the strongly luminescently labeled proteins are mitochondrial proteins. One of the targets is VDAC1 (voltage-dependent anion channel 1). Consistent with known phenotypes of VDAC1 deregulation, prolonged exposure of cells to Ir-ITC led to significant mitochondrial shortening and fragmentation. As far as we know, this is the first report on the molecular characterization of the interactions of a luminescent dye with its biological targets. As many biological dyes exhibit specific intracellular staining patterns, the identification of their molecular targets can help elucidate the mechanisms behind their staining specificities and cytotoxicity. We believe our biochemical approach can be applied to identify the targets of a wide range of fluorescent and luminescent probes.
Subject(s)
Coordination Complexes/metabolism , Coordination Complexes/pharmacology , Iridium/chemistry , Luminescent Agents/chemistry , Mitochondria/drug effects , Mitochondria/metabolism , Organometallic Compounds/metabolism , Organometallic Compounds/pharmacology , Voltage-Dependent Anion Channel 1/metabolism , 3T3 Cells , Animals , Cell Survival/drug effects , Coordination Complexes/chemistry , Coordination Complexes/toxicity , Mice , Organometallic Compounds/chemistry , Organometallic Compounds/toxicity , Protein Binding , Proteomics , Substrate SpecificityABSTRACT
We report here a new class of biological reagents derived from luminescent rhenium(I) polypyridine complexes modified with a poly(ethylene glycol) (PEG) pendant. The PEG-amine complexes [Re(N(^)N)(CO)(3)(py-PEG-NH(2))](PF(6)) (py-PEG-NH(2) = 3-amino-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine, MW(PEG) = 5000 Da, PDI(PEG) < 1.08; N(^)N = 1,10-phenanthroline (phen) (1-PEG-NH(2)), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me(4)-phen) (2-PEG-NH(2)), 4,7-diphenyl-1,10-phenanthroline (Ph(2)-phen) (3-PEG-NH(2))) and [Re(bpy-PEG)(CO)(3)(py-NH(2))](PF(6)) (bpy-PEG = 4-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine; py-NH(2) = 3-aminopyridine) (4-PEG-NH(2)) have been synthesized and characterized. The photophysical properties, lipophilicity, water solubility, cytotoxic activity, and cellular uptake properties of these complexes have been compared to those of their PEG-free counterparts [Re(N(^)N)(CO)(3)(py-Et-NH(2))](PF(6)) (py-Et-NH(2) = 3-amino-5-(N-(ethyl)aminocarbonyl)pyridine; N(^)N = phen (1-Et-NH(2)), Me(4)-phen (2-Et-NH(2)), Ph(2)-phen (3-Et-NH(2))) and [Re(bpy-Et)(CO)(3)(py-NH(2))](PF(6)) (bpy-Et = 4-(N-(ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine) (4-Et-NH(2)). The PEG complexes exhibited significantly higher water solubility and lower cytotoxicity (IC(50) = 6.6 to 1152 µM) than their PEG-free counterparts (IC(50) = 3.6 to 159 µM), indicating that the covalent attachment of a PEG pendant to rhenium(I) polypyridine complexes is an effective way to increase their biocompatibility. The amine complexes 1-PEG-NH(2)-4-PEG-NH(2) have been activated with thiophosgene to yield the isothiocyanate complexes [Re(N(^)N)(CO)(3)(py-PEG-NCS)](PF(6)) (py-PEG-NCS = 3-isothiocyanato-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine; N(^)N = phen (1-PEG-NCS), Me(4)-phen (2-PEG-NCS), Ph(2)-phen (3-PEG-NCS)), and [Re(bpy-PEG)(CO)(3)(py-NCS)](PF(6)) (py-NCS = 3-isothiocyanatopyridine) (4-PEG-NCS) as a new class of luminescent PEGylation reagents. To examine their PEGylation properties, these isothiocyanate complexes have been reacted with a model substrate n-butylamine, resulting in the formation of the thiourea complexes [Re(N(^)N)(CO)(3)(py-PEG-Bu)](PF(6)) (py-PEG-Bu = 3-n-butylthioureidyl-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine; N(^)N = phen (1-PEG-Bu), Me(4)-phen (2-PEG-Bu), Ph(2)-phen (3-PEG-Bu)), and [Re(bpy-PEG)(CO)(3)(py-Bu)](PF(6)) (py-Bu = 3-n-butylthioureidylpyridine) (4-PEG-Bu). Additionally, bovine serum albumin (BSA) and poly(ethyleneimine) (PEI) have been PEGylated with the isothiocyanate complexes to yield bioconjugates 1-PEG-BSA-4-PEG-BSA and 1-PEG-PEI-4-PEG-PEI, respectively. Upon irradiation, all the PEGylated BSA and PEI conjugates exhibited intense and long-lived emission in aqueous buffer under ambient conditions. The DNA-binding and polyplex-formation properties of conjugate 3-PEG-PEI have been studied and compared with those of unmodified PEI. Furthermore, the in vivo toxicity of complex 3-PEG-NH(2) and its PEG-free counterpart 3-Et-NH(2) has been investigated using zebrafish embryos as an animal model. Embryos treated with the PEG complex at high concentrations revealed delayed hatching, which has been ascribed to hypoxia as a result of adhering of the complex to the external surface of the chorion.