Inflammation in pathogenesis of chronic pain: Foe and friend.

Chronic pain is a refractory health disease worldwide causing an enormous economic burden on individuals and society. Accumulating evidence suggests that inflammation in the peripheral nervous system (PNS) and central nervous system (CNS) is the major factor in the pathogenesis of chronic pain. The inflammation in the early- and late phase may have distinctive effects on the initiation and resolution of pain, which can be viewed as friend or foe. On the one hand, painful injuries lead to the activation of glial cells and immune cells in the PNS, releasing pro-inflammatory mediators, which contribute to the sensitization of nociceptors, leading to chronic pain; neuroinflammation in the CNS drives central sensitization and promotes the development of chronic pain. On the other hand, macrophages and glial cells of PNS and CNS promote pain resolution via anti-inflammatory mediators and specialized pro-resolving mediators (SPMs). In this review, we provide an overview of the current understanding of inflammation in the deterioration and resolution of pain. Further, we summarize a number of novel strategies that can be used to prevent and treat chronic pain by controlling inflammation. This comprehensive view of the relationship between inflammation and chronic pain and its specific mechanism will provide novel targets for the treatment of chronic pain.

Ameliorating parkinsonian motor dysfunction by targeting histamine receptors in entopeduncular nucleus-thalamus circuitry.

In Parkinson's disease (PD), reduced dopamine levels in the basal ganglia have been associated with altered neuronal firing and motor dysfunction. It remains unclear whether the altered firing rate or pattern of basal ganglia neurons leads to parkinsonism-associated motor dysfunction. In the present study, we show that increased histaminergic innervation of the entopeduncular nucleus (EPN) in the mouse model of PD leads to activation of EPN parvalbumin (PV) neurons projecting to the thalamic motor nucleus via hyperpolarization-activated cyclic nucleotide-gated (HCN) channels coupled to postsynaptic H2R. Simultaneously, this effect is negatively regulated by presynaptic H3R activation in subthalamic nucleus (STN) glutamatergic neurons projecting to the EPN. Notably, the activation of both types of receptors ameliorates parkinsonism-associated motor dysfunction. Pharmacological activation of H2R or genetic upregulation of HCN2 in EPNPV neurons, which reduce neuronal burst firing, ameliorates parkinsonism-associated motor dysfunction independent of changes in the neuronal firing rate. In addition, optogenetic inhibition of EPNPV neurons and pharmacological activation or genetic upregulation of H3R in EPN-projecting STNGlu neurons ameliorate parkinsonism-associated motor dysfunction by reducing the firing rate rather than altering the firing pattern of EPNPV neurons. Thus, although a reduced firing rate and more regular firing pattern of EPNPV neurons correlate with amelioration in parkinsonism-associated motor dysfunction, the firing pattern appears to be more critical in this context. These results also confirm that targeting H2R and its downstream HCN2 channel in EPNPV neurons and H3R in EPN-projecting STNGlu neurons may represent potential therapeutic strategies for the clinical treatment of parkinsonism-associated motor dysfunction.

Histamine bidirectionally regulates the intrinsic excitability of parvalbumin-positive neurons in the lateral globus pallidus and promotes motor behaviour.

BACKGROUND AND PURPOSE: Parvalbumin (PV)-positive neurons are a type of neuron in the lateral globus pallidus (LGP) which plays an important role in motor control. The present study investigated the effect of histamine on LGPPV neurons and motor behaviour. EXPERIMENTAL APPROACH: Histamine levels in LGP as well as its histaminergic innervation were determined through brain stimulation, microdialysis, anterograde tracing and immunostaining. Mechanisms of histamine action were detected by immunostaining, single-cell qPCR, whole-cell patch-clamp recording, optogenetic stimulation and CRISPR/Cas9 gene-editing techniques. The effect of histamine on motor behaviour was detected by animal behavioural tests. KEY RESULTS: A direct histaminergic innervation in LGP from the tuberomammillary nucleus (TMN) and a histamine-induced increase in the intrinsic excitability of LGPPV neurons were determined by pharmacological blockade or by genetic knockout of the histamine H1 receptor (H1 R)-coupled TWIK-related potassium channel-1 (TREK-1) and the small-conductance calcium-activated potassium channel (SK3), as well as by activation or overexpression of the histamine H2 receptor (H2 R)-coupled hyperpolarization-activated cyclic nucleotide-gated channel (HCN2). Histamine negatively regulated the STN → LGPGlu transmission in LGPPV neurons via the histamine H3 receptor (H3 R), whereas blockage or knockout of H3 R increased the intrinsic excitability of LGPPV neurons. CONCLUSIONS AND IMPLICATIONS: Our results indicated that the endogenous histaminergic innervation in the LGP can bidirectionally promote motor control by increasing the intrinsic excitability of LGPPV neurons through postsynaptic H1 R and H2 R, albeit its action was negatively regulated by the presynaptic H3 R, thereby suggesting possible role of histamine in motor deficits manifested in Parkinson's disease (PD).

Receptor and Ionic Mechanism of Histamine on Mouse Dorsolateral Striatal Neurons.

The dorsolateral striatum (DLS) is the critical neural substrate that plays a role in motor control and motor learning. Our past study revealed a direct histaminergic projection from the tuberomammillary nucleus (TMN) of the hypothalamus to the rat striatum. However, the afferent of histaminergic fibers in the mouse DLS, the effect of histamine on DLS neurons, and the underlying receptor and ionic mechanisms remain unclear. Here, we demonstrated a direct histaminergic innervation from the TMN in the mouse DLS, and histamine excited both the direct-pathway spiny projection neurons (d-SPNs) and the indirect-pathway spiny projection neurons (i-SPNs) of DLS via activation of postsynaptic H1R and H2R, albeit activation of presynaptic H3R suppressed neuronal activity by inhibiting glutamatergic synaptic transmission on d-SPNs and i-SPNs in DLS. Moreover, sodium-calcium exchanger 3 (NCX3), potassium-leak channels linked to H1R, and hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) coupled to H2R co-mediated the excitatory effect induced by histamine on d-SPNs and i-SPNs in DLS. These results demonstrated the pre- and postsynaptic receptors and their downstream multiple ionic mechanisms underlying the inhibitory and excitatory effects of histamine on d-SPNs and i-SPNs in DLS, suggesting a potential modulatory effect of the central histaminergic system on the DLS as well as its related motor control and motor learning.

Neuroinflammation Involved in Diabetes-Related Pain and Itch.

Diabetes mellitus (DM) is a global epidemic with increasing incidence, which results in diverse complications, seriously affects the patient quality of life, and brings huge economic burdens to society. Diabetic neuropathy is the most common chronic complication of DM, resulting in neuropathic pain and chronic itch. The precise mechanisms of diabetic neuropathy have not been fully clarified, hindering the exploration of novel therapies for diabetic neuropathy and its terrible symptoms such as diabetic pain and itch. Accumulating evidence suggests that neuroinflammation plays a critical role in the pathophysiologic process of neuropathic pain and chronic itch. Indeed, researchers have currently made significant progress in knowing the role of glial cells and the pro-inflammatory mediators produced from glial cells in the modulation of chronic pain and itch signal processing. Here, we provide an overview of the current understanding of neuroinflammation in contributing to the sensitization of the peripheral nervous system (PNS) and central nervous system (CNS). In addition, we also summarize the inflammation mechanisms that contribute to the pathogenesis of diabetic itch, including activation of glial cells, oxidative stress, and pro-inflammatory factors. Targeting excessive neuroinflammation may provide potential and effective therapies for the treatment of chronic neuropathic pain and itch in DM.

Interleukin 17A deficiency alleviates neuroinflammation and cognitive impairment in an experimental model of diabetic encephalopathy.

Interleukin 17A (IL-17A) was previously shown to be a key pro-inflammatory factor in diabetes mellitus and associated complications. However, the role of IL-17A in diabetic encephalopathy remains poorly understood. In this study, we established a mouse model of diabetic encephalopathy that was deficient in IL-17A by crossing Il17a-/- mice with spontaneously diabetic Ins2Akita (Akita) mice. Blood glucose levels and body weights were monitored from 2-32 weeks of age. When mice were 32 weeks of age, behavioral tests were performed, including a novel object recognition test for assessing short-term memory and learning and a Morris water maze test for evaluating hippocampus-dependent spatial learning and memory. IL-17A levels in the serum, cerebrospinal fluid, and hippocampus were detected with enzyme-linked immunosorbent assays and real-time quantitative polymerase chain reaction. Moreover, proteins related to cognitive dysfunction (amyloid precursor protein, ß-amyloid cleavage enzyme 1, p-tau, and tau), apoptosis (caspase-3 and -9), inflammation (inducible nitric oxide synthase and cyclooxygenase 2), and occludin were detected by western blot assays. Pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1ß, and interferon-γ in serum and hippocampal tissues were measured by enzyme-linked immunosorbent assays. Microglial activation and hippocampal neuronal apoptosis were detected by immunofluorescent staining. Compared with that in wild-type mice, mice with diabetic encephalopathy had higher IL-17A levels in the serum, cerebrospinal fluid, and hippocampus; downregulation of occludin expression; lower cognitive ability; greater loss of hippocampal neurons; increased microglial activation; and higher expression of inflammatory factors in the serum and hippocampus. IL-17A knockout attenuated the abovementioned changes in mice with diabetic encephalopathy. These findings suggest that IL-17A participates in the pathological process of diabetic encephalopathy. Furthermore, IL-17A deficiency reduces diabetic encephalopathy-mediated neuroinflammation and cognitive defects. These results highlight a role for IL-17A as a mediator of diabetic encephalopathy and potential target for the treatment of cognitive impairment induced by diabetic encephalopathy.

Breaking through the Poisson Distribution: A compact high-efficiency droplet microfluidic system for single-bead encapsulation and digital immunoassay detection.

Droplet encapsulation of a single cell or bead is widely used in digital detection, single-cell sequencing, and drug screening. However, the encapsulation of particles is totally random restricted by the Poisson distribution. The theoretical possibility of single-particle encapsulation is usually only approximately 10%. In ultra-high multiplexed digital detection or other applications that needing to measure large numbers of particles, the number of the partitions required to be counted is extremely high, further result in great increase of statistical number of invalid droplets and the redundancy of detection data. Here, a bead ordered arrangement droplet (BOAD) system is proposed to break through the Poisson distribution. BOAD system tactfully combines sheath flow, Dean vortex, and compression flow channel to achieve orderly arrangement of particles for the first time, and could achieve the fastest orderly arrangement of particles in the shortest structure. The efficiency of single-bead encapsulation is improved to as high as 86%. Further application to encapsulate encoding beads and IL-10-targeted magnetic beads demonstrates the potential for bead-based ultra-high multiplexed digital detection. Thus, use of the BOAD system is very promising for many applications needing high single-particle encapsulation ratio in limited partitions, such as multiplexed digital bio-detection, single-cell analysis, drug screening, and single exosome detection.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos.

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

LIFGO: A modular laser-induced fluorescence detection system based on plug-in blocks.

In this work, a laser-induced fluorescence (LIF) detection system built in a modular assembling mode was developed based on commercial LEGO blocks and 3D printed blocks. We designed and fabricated a variety of 3D printed building blocks fixed with optical components, including laser light source, filters, lens, dichroic mirror, photodiode detector, and control circuits. Utilizing the relatively high positioning precision of the plug-in blocks, a modular construction strategy was adopted using the flexible plug-in combination of the blocks to build a highly sensitive laser-induced fluorescence detection system, LIFGO. The LIFGO system has a simple structure which could be constructed by inexperienced users within 3 h. We optimized the structure and tested the performance of the LIFGO system, and its detection limits for sodium fluorescein solution in 100 µm i.d. and 250 µm i.d. capillaries were 7 nM and 0.9 nM, respectively. Based on the LIFGO system, we also built a simple capillary electrophoresis (CE) system and applied it to the analysis of DNA fragments to demonstrate its application possibility in biochemical analysis. The separation of 7 fragments in DL500 DNA markers were completed in 600 s. Because of the features of low cost (less than $100) and easy-to-build construction, we introduced the LIFGO system to the experimental teaching of instrumental analysis for undergraduate students. The modular construction form of the LIF detection system greatly reduces the threshold of instrument construction, which is conducive to the popularization of the LIF detection technique in routine laboratories as well as the reform of experimental teaching mode.

[Effects of α1-adrenoreceptor on Treg cell function in collagen-induced arthritis].

Objective: An animal model of collagen-induced arthritis (CIA) was used to investigate the effects of norepinephrine (NE) and α1-adrenoreceptor (α1-AR) on Treg cells in CIA. Methods: Thirty-two male DBA/1 mice were randomly divided into control group and CIA model group. CIA was prepared by intradermal injection of collagen type II (CII, 100 µl) at the tail base of DBA/1 mice. On the 41th day following primary immunization, co-expression of CD4+T and α1-AR in mouse spleens was observed. Protein expressions of α1-AR in the ankle joints and the spleens of mice were measured by Western blot analysis. The CD4+ T cells were isolated from the mouse spleen tissues in CIA mice and treated with NE or α1-AR agonist phenylephrine. Percentage of Treg cells in mouse CD4+ T cells of CIA mice was determined by flow cytometry. Expressions of α1-AR, transforming growth factor-ß (TGF-ß) and IL-10 in CD4+T cells of CIA mice were assessed by Western blot. Results: Co-expression of CD4 and α1-AR was observed in spleens of both intact and CIA mice. Compared with intace mice, α1-AR expressions in the ankle joints and spleens were down-regulated in CIA mice. NE increased the function of Treg cells in CD4+ T cells of CIA mice compared with that of nothing-treated CD4+T cells of CIA mice. Moreover, the α1-AR agonist phenylephrine increased the Treg cell function in CD4+ T cells of CIA mice relative to that of nothing-treated CD4+T cells of CIA mice. Conclusion: Activating α1-AR on CD4+T cells of CIA mice enhances Treg cell function,facilitating a shift of CD4+T cells toward Treg polarization.

Ameliorated biomechanical properties of carotid arteries by puerarin in spontaneously hypertensive rats.

BACKGROUND: An emerging body of evidence indicates that puerarin (PUE) plays an important role in the treatment of angina pectoris, myocardial ischemia-reperfusion injury, hypertension and other cardiovascular diseases, but how PUE affects the vascular remodeling of hypertensive rats has not been reported yet. This study aimed to investigate the effect and mechanism of PUE on carotid arteries of spontaneously hypertensive rats (SHR) to provide the basis for the clinical application of PUE. METHODS: Thirty male SHR and six male Wistar Kyoto rats (WKY) aged 3 months were used in this study, SHR rats were randomly divided into 5 groups, PUE(40 or 80 mg/kg/d, ip) and telmisartan (TELMI) (30 mg/kg/d, ig) were administrated for 3 months. We use DMT myography pressure-diameter system to investigate biomechanical properties of carotid arteries, 10 µM pan-classical transient receptor potential channels (TRPCs) inhibitor SKF96365, 200 nM specific TRPC6 inhibitor SAR7334 and 100 µM Orai1 inhibitor ANCOA4 were used in the mechanical test. RESULTS: PUE can significantly decrease systolic and diastolic blood pressure, long-term administration of PUE resulted in a mild reduction of thickness and inner diameter of carotid artery. PUE ameliorate NE-response and vascular remodeling mainly through inhibiting TRPCs channel activities of VSMC. CONCLUSION: PUE can ameliorate biomechanical remodeling of carotid arteries through inhibiting TRPCs channel activities of VSMC in spontaneously hypertensive rats.

Precisely Encoded Barcodes through the Structure-Fluorescence Combinational Strategy: A Flexible, Robust, and Versatile Multiplexed Biodetection Platform with Ultrahigh Encoding Capacities.

With the rapid development of suspension array technology, microbeads-based barcodes as the core element with sufficient encoding capacity are urgently required for high-throughput multiplexed detection. Here, a novel structure-fluorescence combinational encoding strategy is proposed for the first time to establish a barcode library with ultrahigh encoding capacities. Based on the never revealed transformability of the structural parameters (e.g., porosity and matrix component) of mesoporous microbeads into scattering signals in flow cytometry, the enlargement of codes number has been successfully realized in combination with two other fluorescent elements of fluorescein isothiocyanate isomer I (FITC) and quantum dots (QDs). The barcodes are constructed with precise architectures including FITC encapsulated within mesopores and magnetic nanoparticles as well as QDs immobilized on the outer surface to achieve the ultrahigh encoding level of 300 accompanied with superparamagnetism. To the best of knowledge, it is the highest record of single excitation laser-based encoding capacity up to now. Moreover, a ten-plexed tumor markers bioassay based on the tailored-designed barcodes has been evaluated to confirm their feasibility and effectiveness, and the results indicate that the barcodes platform is a promising and robust tool for practical multiplexed biodetection.

Relationship between emotional intelligence and job well-being in Chinese Registered Nurses: Mediating effect of communication satisfaction.

AIM: To explore the relationship between emotional intelligence and job well-being among Chinese Registered Nurses, and the mediating role of communication satisfaction in the relationship. DESIGN: A descriptive cross-sectional study design was employed. METHODS: Random sampling was adopted. The study was conducted from September to December 2019, and 1,475 Registered Nurses from a Chinese hospital provided responses to Wong and Law's emotional intelligence scale, communication satisfaction scale, job well-being scale and general information questionnaire. SPSS25.0 software was used to calculate means, standard deviations and correlations, and AMOS 21.0 software was used to establish the structural equation model. RESULTS: The emotional intelligence, communication satisfaction and job well-being of Registered Nurses in China were related to positional rank, work department, monthly income, years of service, night shift work distribution and intensity of work. There were positive correlations among emotional intelligence, communication satisfaction and job well-being. Communication satisfaction partly mediated the relationship between emotional intelligence and job well-being. Improving the level of emotional intelligence and communication satisfaction should be an important strategy to improve nurses' job well-being. Therefore, nursing managers could carry out targeted training on emotional intelligence management and communication between nurses and patients and pay attention to the spiritual needs of nurses and provide psychological guidance on a regular basis.

Intervention of Tyrosine Hydroxylase Expression Alters Joint Inflammation and Th17/Treg Imbalance in Collagen-Induced Arthritis.

BACKGROUND/AIMS: Neuroendocrine dysregulation has been associated with rheumatoid arthritis (RA). Tyrosine hydroxylase (TH), a rate-limiting enzyme for synthesis of neuroendocrine hormones such as epinephrine, is also expressed in T lymphocytes and regulates balance between helper T (Th) 17 cells and regulatory T (Treg) cells. Herein, we aimed to show that TH expression in joints alleviates joint inflammation and Th17/Treg imbalance in collagen-induced arthritis (CIA), an animal model of RA, and these effects may be implemented by the mechanism of epinephrine action on α1-adrenoreceptor (α1-AR) in T cells. METHODS: CIA was prepared by intradermal injection of collagen type II in tail base of DBA1/J mice. On the 33rd day post-immunization, lentiviral vectors encoding TH or TH shRNA were injected into ankle joints of CIA mice. Limb inflammation of the mice was assessed beginning from day 21 until day 69 post-immunization by measurement of limb swelling, erythema and rigidity. Th17 and Treg differentiation and function in ankle joints were assessed on day 69 post-immunization by test of the expression of Th17 transcriptional factor ROR-γt and the levels of proinflammatory cytokines interleukin (IL)-17 and IL-22 as well as the expression of Treg transcriptional factor Foxp3 and the levels of antiinflammatory cytokines transforming growth factor (TGF)-ß1 and IL-10. T cells were obtained from the spleen of mice that had been immunized with collagen type II 41 day earlier and treated with epinephrine or α1-AR agonist phenylephrine in vitro. Flow cytometry was used to analyze the percentages of CD25-IL-17+ cells and CD25+Foxp3+ cells in CD4+ T cells. RESULTS: TH gene overexpression in ankle joints of CIA mice reduced limb inflammation and Th17-related transcription factor expression and inflammatory cytokine production but increased Treg-related antiinflammatory cytokine production in the joints. In contrast, TH gene silence in ankle joints of CIA mice enhanced limb inflammation and Th17 cell activity but decreased Treg cell function in the joints. Epinephrine upregulated α1-AR expression in T cells derived from CIA mice. Both epinephrine and phenylephrine reduced CIA-induced Th17 transcription factor expression and inflammatory cytokine production but enhanced Treg antiinflammatory cytokine production in vitro. CONCLUSION: Upregulating TH expression in joints alleviates joint inflammation and Th17/Treg imbalance in CIA at least partially by enhancing epinephrine action on α1-AR in T cells.

A magnetic bead-mediated selective adsorption strategy for extracellular vesicle separation and purification.

Extracellular vesicles (EVs) are membrane-encapsulated particles with critical biomedical functions, including mediating intercellular communication, assisting tumor metastasis, and carrying protein and microRNA biomarkers. The downstream applications of EVs are greatly influenced by the quality of the isolated EVs. However, almost none of the separation methods can simultaneously achieve both high yield and high purity of the isolated EVs, thus making the isolation of EVs an essential challenge in EV research. Here, we developed a magnetic bead-mediated selective adsorption strategy (MagExo) for easy-to-operate EV isolation. Benefited from the presence of an adsorption window between EVs and proteins under the effect of a hydrophilic polymer, EVs tend to adsorb on the surface of magnetic beads selectively and can be separated from biological fluids with high purity by simple magnetic separation. The proposed method was used for EV isolation from plasma and cell culture media (CCM), with two times higher yield and comparable purity of the harvested EVs to that obtained by ultracentrifugation (UC). Downstream applications in proteomics analysis showed 86.6% (plasma) and 86.5% (CCM) of the analyzed proteins were matched with the ExoCarta database, which indicates MagExo indeed enriches EVs efficiently. Furthermore, we found the target RNA amount of the isolated EVs by MagExo were almost dozens and hundred times higher than the gold standard DG-UC and ultracentrifugation (UC) methods, respectively. All the results show that MagExo is a reliable, easy, and efficient approach to harvest EVs for a wide variety of downstream applications with minimized sample usage. STATEMENT OF SIGNIFICANCE: Extracellular vesicles (EVs) are presently attracting increasing interest among clinical and scientific researchers. Although the downstream applications of EVs are recognized to be greatly affected by the quality of the isolated EVs, almost none of the separation methods can simultaneously achieve high yield and high purity of the isolated EVs; this makes the isolation of EVs an essential challenge in EV research. In the present work, we proposed a simple and easy-to-operate method (MagExo) for the separation and purification of EVs based on the phenomenon that EVs can be selectively adsorbed on the surface of magnetic microspheres in the presence of a hydrophilic polymer. The performance of MagExo was comparable to or even better than that of gold standard methods and commercial kits, with two times higher yield and comparable purity of the harvested EVs to that achieved with ultracentrifugation (UC); this could meet the requirements of various EV-associated downstream applications. In addition, MagExo can be easily automated by commercial liquid workstations, thus significantly improving the isolation throughput and paving a new way in clinical diagnosis and treatment.

A Selective TRPC3 Inhibitor Pyr3 Attenuates Myocardial Ischemia/Reperfusion Injury in Mice.

An emerging body of evidence indicates that transient receptor potential TRP channels act as important mediators for a wide variety of physiological functions and are potential targets for drug discovery. Our previous study has identified transient receptor potential channel 3 (TRPC3) and TRPC6 as cation channels through which most of the damaging calcium enters, aggravates pathological changes in vivo and increases ischemia/reperfusion (I/R) injury in mice. This study aimed to verify the effects of TRPC3 inhibitor Pyr3 on myocardial I/R injury in mice. C57BL/6J wild-type male mice (8 to 12 weeks old) were anesthetized with 3.3% chloral hydrate. A murine I (30 min)/R (24 h) injury model was established by temporary occlusion of the left anterior descending (LAD) coronary artery. Pyr3 was administered at concentrations of 0, 2.5, 5, or 10 mg/kg via the right jugular vein 5 min before reperfusion. We observed that the selective TRPC3 inhibitor, 10 mg/kg Pyr3, significantly decreased the infarct size of left ventricle, and reduced the myocardial cell apoptosis rate and inflammatory response in mice. In a conclusion, TRPC3 can function as a candidate target for I/R injury prevention, and Pyr3 may directly bind to TRPC3 channel protein, inhibit TRPC3 channel activity, and improve TRPC3-related myocardial I/R injury. Pyr3 may be used for clarification of TRPC3 functions and for treatments of TRPC3-mediated diseases.

Ultrabright dye-loaded spherical polyelectrolyte brushes and their fundamental structure-fluorescence tuning principles.

Ultrabright fluorescent particles (UFPs) have attracted increasing attention because of their outstanding signal amplification functions. However, there is still an urgent demand for designing novel UFPs with new components or structures as the existing ones can not satisfy the practical requirements due to their inherent disadvantages. Here we propose a novel ultrabright fluorescent particle platform by doping dyes of 5-aminofluorescein (5-AF) into silica core-based spherical poly (acrylic acid) brushes (SiO2@PAA@5-AF) and discuss their fundamental structure-fluorescence tuning principles. A series of brushes with different polymer chain lengths are successfully synthesized and then loaded with 5-AF through chemical binding. The high loading amount, suitable density or distribution, and enhanced quantum yield (QY) of 5-AF due to the amide bond formation with PAA chains on brushes are concluded as the three major reasons for the ultrabrightness of SiO2@PAA@5-AF. Therefore, a 2350 ± 445 times brighter brush particle in comparison to a single quantum dot (QD) is realized, and a 2.1 ± 0.4 times fluorescence improvement of a brush vs. a QD normalized by volume is also achieved when taking the hydrodynamic diameter into consideration (∼300 nm vs. ∼30 nm). Moreover, the excellent tolerance stabilities in normally applied environments and outstanding label effects to form 4-plexed encoded beads are demonstrated as well. The results in this work strongly indicate a promising potential of SiO2@PAA@5-AF as an ultrabright and stable signal amplification tool for biomedical related sensing, labeling, and biodetection.

A spherical poly(acrylic acid) brush-enzyme block with high catalytic capacity for signal amplification in digital biological assays.

Ultrasensitive determination of some ultra-low abundance biological molecules closely related to diseases is currently a wide concern and urgent issue to be addressed. Here, a spherical poly(acrylic acid)-alkaline phosphatase (SP-AKP) signal amplification block using spherical poly(acrylic acid) brush nanoparticles (SP) as the immobilized carriers was designed and synthesized optimally first. The results show that a single SP-AKP with high enzyme binding capacity and high catalytic ability (up to about 4800 effective free AKP per SP-AKP) has much greater fluorescence signal amplification ability than a single free AKP or SiO2-COOH-AKP. Then, a droplet generation microfluidic chip was prepared successfully, and the SP-AKP was loaded and confined in a 14 pL droplet by adjusting its concentration to ensure at most one SP-AKP was encapsulated in each droplet according to Poisson's theory. Finally, the fluorescence signals produced by 4-methylumbelliferyl phosphate (4-MUP) catalyzed via SP-AKP within 6 min were sufficient to be detected by a fluorescence microscope. Thus, the digital signal distribution of "1/0" (signal/background) was obtained, making this SP-AKP signal amplification block a promising enzyme label for potential high sensitivity digital biological detection applications.

A Low-Cost Palmtop High-Speed Capillary Electrophoresis Bioanalyzer with Laser Induced Fluorescence Detection.

In this work, we developed a miniaturized palmtop high-speed capillary electrophoresis (CE) system integrating whole modules, including picoliter-scale sample injection, short capillary-based fast CE, high-voltage power supply, orthogonal laser induced fluorescence (LIF) detection, battery, system control, on-line data acquisition, processing, storage, and display modules. A strategy of minimalist miniaturization combining minimal system design and low-cost system construction was adopted to achieve the instrument miniaturization with extremely low cost, which is differing from the current microfabrication strategy used in most reported miniaturized CE systems. With such a strategy, the total size of the bioanalyzer was minimized to 90 × 75 × 77 mm (length × width × height) and the instrument cost was reduced to ca. $500, which demonstrated the smallest and lowest-cost CE instrument with LIF detection in so far reported systems. The present bioanalyzer also exhibited comparable analytical performances to previously-reported high-speed CE systems. A limit of detection of 1.02 nM sodium fluorescein was obtained. Fast separations were achieved for multiple types of samples as amino acids, amino acid enantiomers, DNA fragments, and proteins with high efficiency. We applied this instrument in colorectal cancer diagnosis for detecting KRAS mutation status by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.

TGF-ß1 protection against Aß1-42-induced hippocampal neuronal inflammation and apoptosis by TßR-I.

Alzheimer's disease (AD), the most common chronic neurodegenerative disease, is pathologically characterized by the formation of neurofibrillary tangles because of hyperphosphorylation of tau protein and extracellular deposits of amyloid-ß (Aß) protein termed senile plaques. Recent studies indicate that neuronal apoptosis caused by chronic neuroinflammation is one of the important pathogenesis of AD. Transforming growth factor (TGF)-ß1 is a pleiotropic cytokine with immunosuppressive and anti-inflammatory properties. However, it is poorly known whether the anti-inflammatory property of TGF-ß1 is involved in a neuroprotection in AD. Here, an AD cell model of hippocampal neurons induced by Aß1-42 was used to show an anti-inflammatory and neuroprotective effect of TGF-ß1 through its receptor transforming growth factor-ß receptor type I (TßR-I). As expected, Aß1-42-induced an upregulation in neuronal expression of amyloid precursor protein (APP), tumor necrosis factor-α, cyclooxygenase-2, Bax, cleaved caspase-3, and cleaved caspase-9, and a downregulation in the expression of Bcl-2, as well as an increase in the number of NeuN/terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) double-positive cells. TGF-ß1 pretreatment reduced the Aß1-42-induced effects of upregulating APP, tumor necrosis factor-α, Bax, cleaved caspase-3 and cleaved caspase-9, and downregulating Bcl-2, in addition to increasing NeuNTUNEL cell number. TßR-I expression in hippocampal neurons was downregulated by Aß1-42 exposure, but upregulated by TGF-ß1 pretreatment. Silencing of the TßR-I gene in the neurons abolished the anti-inflammatory and antiapoptotic effects of TGF-ß1 in the Aß1-42-induced AD cell model. These findings suggest that TGF-ß1 protects neurons against Aß1-42-induced neuronal inflammation and apoptosis by activation of TßR-I.