Single-cell analysis of proteins is critical to gain precise information regarding the mechanisms that dictate the heterogeneity in cellular phenotypes and their differential response to internal and ...external stimuli. However, tools that allow sensitive and easy measurement of proteins in individual cells are still limited. The emerging semiconductor-based bioelectronics may provide a new approach to overcome the challenges in this field, however its utility in single-cell protein analysis has not been explored. In this study, we investigated multiple protein detection in single cells by MoS2 field effect transistors (MoS2-FETs) modified with specific biological probes. First, β-actin antibody was connected to the surface of MoS2-FETs by covalent bonds, and the fabricated device was tested using β-actin solution with concentrations from 10−9 to 10−3 μg/μL. Next, we examined the application of MoS2-FET for protein analysis in complex biological samples, and the device showed electrical signal response to human embryonic kidney cell line HEK293T in a dose-dependent manner. Furthermore, we applied this method to analyze individual liver cancer MHCC-97L cells, targeting four cellular proteins, including β−actin, epidermal growth factor receptor, sirtuin-2, and glyceraldehyde-3-phosphate dehydrogenase. The devices modified with corresponding probes could identify the target proteins and showed cell number-dependent responses. As a proof of principle, we demonstrated sensitive and multiplexed detection of proteins in single cells using MoS2-FETs. The biosensor and this detection method are cost-efficient and user-friendly with broad application prospects in biological studies and clinical diagnosis.
Display omitted
•This study demonstrates the use of semiconductor device for single-cell protein analysis for the first time.•Antibody-functionalized MoS2-FETs are developed for protein detection.•The fabricated MoS2-FETs exhibit high sensitivity with detection limit as low as 10−9 μg/μL for β-actin.•The MoS2-FETs enable detection of four cellular proteins in single HEK293T and MHCC-97L cells.
Abstract
The non-selective cation channel transient receptor potential vanilloid 1 (TRPV1) is expressed throughout the cardiovascular system. Recent evidence shows a role for TRPV1 in inflammatory ...processes. The role of TRPV1 for myocardial inflammation has not been established yet. Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (hiPSC-CM) from 4 healthy donors were incubated with lipopolysaccharides (LPS, 6 h), TRPV1 agonist capsaicin (CAP, 20 min) or the antagonist capsazepine (CPZ, 20 min). TRPV1 expression was studied by PCR and western blotting. TRPV1 internalization was analyzed by immunofluorescence. Interleukin-6 (IL-6) secretion and phosphorylation of JNK, p38 and ERK were determined by ELISA. TRPV1-associated ion channel current was measured by patch clamp. TRPV1-mRNA and -protein were expressed in hiPSC-CM. TRPV1 was localized in the plasma membrane. LPS significantly increased secretion of IL-6 by 2.3-fold, which was prevented by pre-incubation with CPZ. LPS induced TRPV1 internalization. Phosphorylation levels of ERK, p38 or JNK were not altered by TRPV1 stimulation or inhibition. LPS and IL-6 significantly lowered TRPV1-mediated ion channel current. TRPV1 mediates the LPS-induced inflammation in cardiomyocytes, associated with changes of cellular electrophysiology. LPS-induced inflammation results in TRPV1 internalization. Further studies have to examine the underlying pathways and the clinical relevance of these findings.
Severe infections like sepsis lead frequently to cardiomyopathy. The mechanisms are unclear and an optimal therapy for septic cardiomyopathy still lacks. The aim of this study is to establish an ...endotoxin-induced inflammatory model using human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (hiPSC-CMs) for mechanistic and therapeutic studies. hiPSC-CMs were treated by lipopolysaccharide (LPS) in different concentrations for different times. ELISA, FACS, qPCR, and patch-clamp techniques were used for the study. TLR4 (Toll-like receptor 4) and its associated proteins, CD14, LBP (lipopolysaccharide binding protein), TIRAP (toll-interleukin 1 receptor domain containing adaptor protein), Ly96 (lymphocyte antigen 96) and nuclear factor kappa B as well as some pro-and anti-inflammatory factors are expressed in hiPSC-CMs. LPS-treatment for 6 hours increased the expression levels of pro-inflammatory and chemotactic cytokines (TNF-a, IL-1ß, IL-6, CCL2, CCL5, IL-8), whereas 48 hour-treatment elevated the expression of anti-inflammatory factors (IL-10 and IL-6). LPS led to cell injury resulting from exaggerated cell apoptosis and necrosis. Finally, LPS inhibited small conductance Ca
-activated K
channel currents, enhanced Na
/Ca
-exchanger currents, prolonged action potential duration, suggesting cellular electrical dysfunctions. Our data demonstrate that hiPSC-CMs possess the functional reaction system involved in endotoxin-induced inflammation and can model some bacterium-induced inflammatory responses in cardiac myocytes.
DNA hydroxymethylation (5-hmC) is a kind of new epigenetic modification, which plays key roles in DNA demethylation, genomic reprogramming, and the gene expression in mammals. For further exploring ...the functions of 5-hmC, it is necessary to develop sensitive and selective methods for detecting 5-hmC. Herein, we developed a novel multiplexing electrochemical (MEC) biosensor for 5-hmC detection based on the glycosylation modification of 5-hmC and enzymatic signal amplification. The 5-hmC was first glycosylated by T4 β-glucosyltransferase and then oxidated by sodium periodate. The resulting glucosyl-modified 5-hmC (5-ghmC) was incubated with ARP-biotin and was bound to avidin-HRP. The 5-hmC can be detected at the subnanogram level. Finally, we performed 5-hmC detection for mouse tissue samples and cancer cell lines. The limit of detection of the MEC biosensor is 20 times lower than that of commercial kits based on optical meaurement. Also, the biosensor presented high detection specificity because the chemical reaction for 5-hmC modification can not happen at any other unhydroxymethylated nucleic acid bases. Importantly, benefited by its multiplexing capacity, the developed MEC biosensor showed excellent high efficiency, which was time-saving and cost less.
In December 2019, the novel coronavirus disease (COVID-19) due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection broke. With the gradual deepening understanding of SARS-CoV-2 ...and COVID-19, researchers and clinicians noticed that this disease is closely related to the nervous system and has complex effects on the central nervous system (CNS) and peripheral nervous system (PNS). In this review, we summarize the effects and mechanisms of SARS-CoV-2 on the nervous system, including the pathways of invasion, direct and indirect effects, and associated neuropsychiatric diseases, to deepen our knowledge and understanding of the relationship between COVID-19 and the nervous system.
•About 1/3 of people with COVID-19 will show some kind of neurological symptoms.•These symptoms will be produced through direct infection and indirect effects.•These effects will relate to almost all common types of nervous system disease.
Origin of the Chordate Notochord Sui, Zihao; Zhao, Zhihan; Dong, Bo
Diversity (Basel),
10/2021, Letnik:
13, Številka:
10
Journal Article
Recenzirano
Odprti dostop
The phylum of Chordata is defined based on the discovery of a coelom-like dorsal notochord in ascidian and amphioxus embryos. Chordata can be classified into three subphylums, Cephalochordata, ...Urochordata, and Vertebrata, united by the presence of a notochord at some point during development. The origin of the notochord, the signature anatomical structure of chordates, has been under debate since the publication of Alexander Kovalevsky’s work in the mid-19th century that placed ascidians close to the vertebrates on the phylogenetic tree. During the late 20th century, the development of molecular and genetic tools in biology brought about a revival of studies on the evolutionary path of notochord development. Two main hypotheses for the origin of the notochord were proposed, the de novo theory and the axochord theory. The former states that notochord has developed de novo from the mid-dorsal archenteron of a chordate ancestor with simple morphology and no central nervous system nor notochord homolog. The putative notochord along the dorsal side of the animal is proposed to take on the signal functions later from the endoderm and ectoderm. An alternative hypothesis, the axochord theory, proposes that notochord has evolved from the mid-line muscle tissue, the so-called axochord, in annelids. Structural and molecular evidence point to the midline muscle of annelids as a distant homolog of the notochord. This hypothesis thus suggests a notochord-like structure in the urbilaterian ancestor, opposed to the consensus that notochord is a chordate-specific feature. In this review, we introduce the history of the formation of these views and summarize the current understandings of embryonic development, molecular profile, and gene regulatory networks of notochord and notochord-like structures.
Background
Short QT syndrome (SQTS), a disorder associated with characteristic ECG QT‐segment abbreviation, predisposes affected patients to sudden cardiac death. Despite some progress in assessing ...the organ‐level pathophysiology and genetic changes of the disorder, the understanding of the human cellular phenotype and discovering of an optimal therapy has lagged because of a lack of appropriate human cellular models of the disorder. The objective of this study was to establish a cellular model of SQTS using human‐induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs).
Methods and Results
This study recruited 1 patient with short QT syndrome type 1 carrying a mutation (N588K) in KCNH2 as well as 2 healthy control subjects. We generated hiPSCs from their skin fibroblasts, and differentiated hiPSCs into cardiomyocytes (hiPSC‐CMs) for physiological and pharmacological studies. The hiPSC‐CMs from the patient showed increased rapidly activating delayed rectifier potassium channel current (IKr) density and shortened action potential duration compared with healthy control hiPSC‐CMs. Furthermore, they demonstrated abnormal calcium transients and rhythmic activities. Carbachol increased the arrhythmic events in SQTS but not in control cells. Gene and protein expression profiling showed increased KCNH2 expression in SQTS cells. Quinidine but not sotalol or metoprolol prolonged the action potential duration and abolished arrhythmic activity induced by carbachol.
Conclusions
Patient‐specific hiPSC‐CMs are able to recapitulate single‐cell phenotype features of SQTS and provide novel opportunities to further elucidate the cellular disease mechanism and test drug effects.
The purpose of this study was to verify the veracity and reliability of the INCNS score for prediction of neurological ICU (NICU) mortality and 3-month functional outcome and mortality in comatose ...patients.
In this prospective study, data of the patients admitted to NICU from January 2013 to January 2019 were collected for validation. The 3-month functional outcomes were evaluated using modified Rankin Scale (mRS). By using the receiver operating characteristics curve (ROC) analysis, we compared the INCNS score with Glasgow Coma Scale (GCS), Full Outline of Un-Responsiveness Score (FOUR) and Acute Physiology and Chronic Health Evaluation II (APACHE II) for assessment of the predictive performance of these scales for 3-month functional outcome and mortality and NICU mortality performed at 24- and 72-h after admission to the NICU.
Totally 271 patients were used for evaluation; the INCNS score achieved an AUC (area under the receiver operating characteristic curve) of 0.766 (95% CI: 0.711-0.815) and 0.824 (95% CI: 0.774-0.868) for unfavorable functional outcomes, an AUC of 0.848 (95% CI: 0.800-0.889) and 0.892 (95% CI: 0.848-0.926) for NICU mortality, and an AUC of 0.811 (95% CI: 0.760-0.856) and 0.832 (95% CI: 0.782-0.874) for the 3-month mortality after discharge from the NICU at 24- and 72-h. The INCNS score exhibited a significantly better predictive performance of mortality and 3-month functional outcomes than FOUR and GCS. There was no significant difference in predicting NICU mortality and 3-month functional outcomes between INCNS and APACHE II, but INCNS had better predictive performance of 3-month mortality than APACHE II.
The INCNS score could be used for predicting the functional outcomes and mortality rate of comatose patients.
Ferroptosis is a special form of regulated cell death, which is reported to play an important role in a variety of traumatic diseases by promoting lipid peroxidation and devastating cell membrane ...structure. Pelvic floor dysfunction (PFD) is a kind of disease affecting the quality and health of many women's lives, which is closely related to the injury of the pelvic floor muscle. Clinical findings have discovered that there is anomalous oxidative damage to the pelvic floor muscle in women with PFD caused by mechanical trauma, but the specific mechanism is still unclear. In this study, we explored the role of ferroptosis-associated oxidative mechanisms in mechanical stretching-induced pelvic floor muscle injury, and whether obesity predisposed pelvic floor muscle to ferroptosis from mechanical injury. Our results, in vitro, showed that mechanical stretch could induce oxidative damage to myoblasts and trigger ferroptosis. In addition, glutathione peroxidase 4 (GPX4) down-regulation and 15-lipoxygenase 1(15LOX-1) up-regulation exhibited the same variational characteristics as ferroptosis, which was much more pronounced in palmitic acid (PA)-treated myoblasts. Furthermore, ferroptosis induced by mechanical stretch could be rescued by ferroptosis inhibitor (ferrostatin-1). More importantly, in vivo, we found that the mitochondria of pelvic floor muscle shrank, which were consistent with the mitochondrial morphology of ferroptosis, and GPX4 and 15LOX-1 showed the same change observed in cells. In conclusion, our data suggest ferroptosis is involved in the injury of the pelvic floor muscle caused by mechanical stretching, and provide a novel insight for PFD therapy.
PDF
