Recent Advances in and Application of Fluorescent Microspheres for Multiple Nucleic Acid Detection.

Traditional single nucleic acid assays can only detect one target while multiple nucleic acid assays can detect multiple targets simultaneously, providing comprehensive and accurate information. Fluorescent microspheres in multiplexed nucleic acid detection offer high sensitivity, specificity, multiplexing, flexibility, and scalability advantages, enabling precise, real-time results and supporting clinical diagnosis and research. However, multiplexed assays face challenges like complexity, costs, and sample handling issues. The review explores the recent advancements and applications of fluorescent microspheres in multiple nucleic acid detection. It discusses the versatility of fluorescent microspheres in various fields, such as disease diagnosis, drug screening, and personalized medicine. The review highlights the possibility of adjusting the performance of fluorescent microspheres by modifying concentrations and carrier forms, allowing for tailored applications. It emphasizes the potential of fluorescent microsphere technology in revolutionizing nucleic acid detection and advancing health, disease treatment, and medical research.

Selenium nanoparticles improve nickel-induced testosterone synthesis disturbance by down-regulating miR-708-5p/p38 MAPK pathway in Leydig cells.

The present study was designed to investigate the role of miR-708-5p/p38 mitogen-activated protein kinase (MAPK) pathway during the mechanism of selenium nanoparticles (Nano-Se) against nickel (Ni)-induced testosterone synthesis disorder in rat Leydig cells. We conducted all procedures based on in vitro culture of rat primary Leydig cells. After treating Leydig cells with Nano-Se and NiSO4 alone or in combination for 24 h, we determined the cell viability, reactive oxygen species (ROS) levels, testosterone production, and the protein expression of key enzymes involved in testosterone biosynthesis: steroidogenic acute regulatory (StAR) and cytochrome P450 cholesterol side chain cleavage enzyme (CYP11A1). The results indicated that Nano-Se antagonized cytotoxicity and eliminated ROS generation induced by NiSO4 , suppressed p38 MAPK protein phosphorylation and reduced miR-708-5p expression. Importantly, we found that Nano-Se upregulated the expression of testosterone synthase and increased testosterone production in Leydig cells. Furthermore, we investigated the effects of p38 MAPK and miR-708-5p using their specific inhibitor during Nano-Se against Ni-induced testosterone synthesis disorder. The results showed that Ni-inhibited testosterone secretion was alleviated by Nano-Se co-treatment with p38 MAPK specific inhibitor SB203580 and miR-708-5p inhibitor, respectively. In conclusion, these findings suggested Nano-Se could inhibit miR-708-5p/p38 MAPK pathway, and up-regulate the key enzymes protein expression for testosterone synthesis, thereby antagonizing Ni-induced disorder of testosterone synthesis in Leydig cells.

Leveraging the ATP-P2X7 receptor signalling axis to alleviate traumatic CNS damage and related complications.

The P2X7 receptor is an exceptional member of the P2X purinergic receptor family, with its activation requiring high concentrations of extracellular adenosine 5'-triphosphate (ATP) that are often associated with tissue damage and inflammation. In the central nervous system (CNS), it is highly expressed in glial cells, particularly in microglia. In this review, we discuss the role and mechanisms of the P2X7 receptor in mediating neuroinflammation and other pathogenic events in a variety of traumatic CNS damage conditions, which lead to loss of neurological and cognitive functions. We raise the perspective on the steady progress in developing CNS-penetrant P2X7 receptor-specific antagonists that leverage the ATP-P2X7 receptor signaling axis as a potential therapeutic strategy to alleviate traumatic CNS damage and related complications.

A novel dephosphorylation targeting chimera selectively promoting tau removal in tauopathies.

Intraneuronal accumulation of hyperphosphorylated tau is a hallmark pathology shown in over twenty neurodegenerative disorders, collectively termed as tauopathies, including the most common Alzheimer's disease (AD). Therefore, selectively removing or reducing hyperphosphorylated tau is promising for therapies of AD and other tauopathies. Here, we designed and synthesized a novel DEPhosphorylation TArgeting Chimera (DEPTAC) to specifically facilitate the binding of tau to Bα-subunit-containing protein phosphatase 2A (PP2A-Bα), the most active tau phosphatase in the brain. The DEPTAC exhibited high efficiency in dephosphorylating tau at multiple AD-associated sites and preventing tau accumulation both in vitro and in vivo. Further studies revealed that DEPTAC significantly improved microtubule assembly, neurite plasticity, and hippocampus-dependent learning and memory in transgenic mice with inducible overexpression of truncated and neurotoxic human tau N368. Our data provide a strategy for selective removal of the hyperphosphorylated tau, which sheds new light for the targeted therapy of AD and related-tauopathies.

Tau inhibits PKA by nuclear proteasome-dependent PKAR2α elevation with suppressed CREB/GluA1 phosphorylation.

Intraneuronal accumulation of wild-type tau plays a key role in Alzheimer's disease, while the mechanisms underlying tauopathy and memory impairment remain unclear. Here, we report that overexpressing full-length wild-type human tau (hTau) in mouse hippocampus induces learning and memory deficits with remarkably reduced levels of multiple synapse- and memory-associated proteins. Overexpressing hTau inhibits the activity of protein kinase A (PKA) and decreases the phosphorylation level of cAMP-response element binding protein (CREB), GluA1, and TrkB with reduced BDNF mRNA and protein levels both in vitro and in vivo. Simultaneously, overexpressing hTau increased PKAR2α (an inhibitory subunit of PKA) in nuclear fraction and inactivated proteasome activity. With an increased association of PKAR2α with PA28γ (a nuclear proteasome activator), the formation of PA28γ-20S proteasome complex remarkably decreased in the nuclear fraction, followed by a reduced interaction of PKAR2α with 20S proteasome. Both downregulating PKAR2α by shRNA and upregulating proteasome by expressing PA28γ rescued hTau-induced PKA inhibition and CREB dephosphorylation, and upregulating PKA improved hTau-induced cognitive deficits in mice. Together, these data reveal that intracellular tau accumulation induces synapse and memory impairments by inhibiting PKA/CREB/BDNF/TrkB and PKA/GluA1 signaling, and deficit of PA28γ-20S proteasome complex formation contributes to PKAR2α elevation and PKA inhibition.

Adenosine Triphosphate Release and P2 Receptor Signaling in Piezo1 Channel-Dependent Mechanoregulation.

Organs and tissues and their constituent cells are physiologically submitted to diverse types of mechanical forces or stress, one common sequence of which is release of intracellular ATP into extracellular space. Extracellular ATP is a well-established autocrine or paracrine signaling molecule that regulates multiple cell functions and mediates cell-to-cell communications via activating the purinergic P2 receptors, more specifically, ligand-gated ion channel P2X receptors and some of the G-protein-coupled P2Y receptors. The molecular mechanisms that sense mechanical and transduce forces to trigger ATP release are poorly understood. The Piezo1, a newly identified mechanosensing ion channel, shows widespread expression and confers mechanosensitivity in many different types of cells. In this mini-review, we briefly introduce the Piezo1 channel and discuss the evidence that supports its important role in the mechanoregulation of diverse cell functions and, more specifically, critical engagement of ATP release and subsequent P2 receptor activation in Piezo1 channel-dependent mechanoregulation. Such ATP release-mediated coupling of the Piezo1 channel and P2 receptors may serve a signaling mechanism that is more common than we currently understand in transducing mechanical information to regulation of the attendant cell functions in various organs and tissues.

Increasing the TRPM2 Channel Expression in Human Neuroblastoma SH-SY5Y Cells Augments the Susceptibility to ROS-Induced Cell Death.

Human neuroblastoma SH-SY5Y cells are a widely-used human neuronal cell model in the study of neurodegeneration. A recent study shows that, 1-methyl-4-phenylpyridine ion (MPP), which selectively causes dopaminergic neuronal death leading to Parkinson's disease-like symptoms, can reduce SH-SY5Y cell viability by inducing H2O2 generation and subsequent TRPM2 channel activation. MPP-induced cell death is enhanced by increasing the TRPM2 expression. By contrast, increasing the TRPM2 expression has also been reported to support SH-SY5Y cell survival after exposure to H2O2, leading to the suggestion of a protective role for the TRPM2 channel. To clarify the role of reactive oxygen species (ROS)-induced TRPM2 channel activation in SH-SY5Y cells, we generated a stable SH-SY5Y cell line overexpressing the human TRPM2 channel and examined cell death and cell viability after exposure to H2O2 in the wild-type and TRPM2-overexpressing SH-SY5Y cells. Exposure to H2O2 resulted in concentration-dependent cell death and reduction in cell viability in both cell types. TRPM2 overexpression remarkably augmented H2O2-induced cell death and reduction in cell viability. Furthermore, H2O2-induced cell death in both the wild-type and TRPM2-overexpressing cells was prevented by 2-APB, a TRPM2 inhibitor, and also by PJ34 and DPQ, poly(ADP-ribose) polymerase (PARP) inhibitors. Collectively, our results show that increasing the TRPM2 expression renders SH-SY5Y cells to be more susceptible to ROS-induced cell death and reinforce the notion that the TRPM2 channel plays a critical role in conferring ROS-induced cell death. It is anticipated that SH-SY5Y cells can be useful for better understanding the molecular and signaling mechanisms for ROS-induced TRPM2-mediated neurodegeneration in the pathogenesis of neurodegenerative diseases.

ATP-activated P2X7 receptor in the pathophysiology of mood disorders and as an emerging target for the development of novel antidepressant therapeutics.

Mood disorders are a group of psychiatric conditions that represent leading global disease burdens. Increasing evidence from clinical and preclinical studies supports that innate immune system dysfunction plays an important part in the pathophysiology of mood disorders. P2X7 receptor, belonging to the ligand-gated ion channel P2X subfamily of purinergic P2 receptors for extracellular ATP, is highly expressed in immune cells including microglia in the central nervous system (CNS) and has a vital role in mediating innate immune response. The P2X7 receptor is also important in neuron-glia signalling in the CNS. The gene encoding human P2X7 receptor is located in a locus of susceptibility to mood disorders. In this review, we will discuss the recent progress in understanding the role of the P2X7 receptor in the pathogenesis and development of mood disorders and in discovering CNS-penetrable P2X7 antagonists for potential uses in in vivo imaging to monitor brain inflammation and antidepressant therapeutics.

[Effect of NaHS on ATP-induced P2X receptor expression in rat microglia].

OBJECTIVE: To observed the effect of sodium hydrosulphide (NaHS), a donor of H2S on the cell viability,the membrane permeability and the expression of P2X7 receptor induced by adenosine triphosphate(ATP) in rat microglia. METHODS: Rat microglia in logarithmic growth phase was randomly divided into 4 groups. In control group, the cells were cultured without ATP treatment. In ATP group, the cells were treatment with ATP after cultured for 24 hours. In NaHS+ATP group, the cells were incubated with NaHS for 30 min before ATP, and NaHS always existed in the reaction system. In KN-62+ATP group, the cells were pretreated with KN-62 for 30 min, the others were as the same as NaHS+ATP group. The cell viability was detected by MTT. Fluorescent dyes YO-PRO-1 was used to observe the membrane permeability. The expression of P2X7 receptor was examined by immunofluorescence staining. RESULTS: ① Compared with control group, the cell viability dropped after treatment with ATP (1、3、5、10 mmol/L) for 3 hours. When pre-incubation with NaHS(200 µmol/L), the cell viability was apparently higher than that of ATP alone group(P<0.01), while 400 µmol/L had no further beneficial.②The YO-PRO-1 fluorescence intensity was obviously elevated by ATP in rat microglia, but this effect was counteracted by NaHS pretreatment (P<0.01). ③ The expression of P2X7 receptor protein was significantly increased after ATP(3 mmol/L) for 3 h. While the expression upregulation of P2X7 receptor protein induced by ATP was significantly counteracted by pretreating with NaHS(200 µmol/L) (P<0.01). CONCLUSIONS: NaHS could reduce the expression of P2X7 receptor, decrease membrane permeability, and increase the cell viability in rat microglia injured by ATP. So the cytoprotection of hydrogen sulfide may be related to the expression and function of P2X7 receptor.

[Neuroprotective effect of progesterone on focal cerebral ischemia/reperfusion injury in rats and its mechanism].

OBJECTIVE: To observe the neurological protective effects of progesterone (PROG) on focal cerebral ischemia/reperfusion injury in rats and to explore its possible mechanism. METHODS: One handred and twenty male SD rats were divided into three groups randomly: sham-operated group, middle cerebral artery occlusion ( MCAO ) group and PROG + MCAO group( n = 40). The right temporary MCAO model was established by the line-embolism method. The PROG + MCAO group rats were according to 8 mg/kg intraperitoneal injection PROG, after that 30 min, the rats were suffered ischemia/reperfusion. After rats were suffered ischemia for 2 h and reperfusion 0, 24, 48, 72 h stress, the nervous functional defect degree were evaluated by longe scoring, and the expression of two-pore domain K channel 3 (TASK3) mRNA in brain tissue were detected by the real-time PCR. RESULTS: PROG (8 mg/kg) could significantly reduced the nervous functional defect degree in rats after ischemia/reperfusion 24, 48, 72 h (P < 0.05). The results of real-time PCR showed that the TASK3 mRNA expression in the brain tissue at all time points significantly decreased in MCAO group compared with sham-operated group (P < 0.05). However, compared with MCAO group, the expression of TASK3 mRNA in brain tissue at all time points dramatically increased in PROG + MCAO group (P < 0.05). CONCLUSION: PROG can improve the nervous functional defect degree after focal cerebral ischemia/reperfusion injury in rats, and the mechanism might be associated with up-regulating the expression of TASK3 mRNA in brain tissue.

Stable cell line of human SH-SY5Y uniformly expressing TWIK-related acid-sensitive potassium channel and eGFP fusion.

TWIK-related acid-sensitive potassium channels (TASK3) are pharmacological targets of CNS inflammation induced by acidification. They function as molecular switches between survival and death of neurons. In this report, TASK3 cloned from human brain cDNA was tagged with enhanced green fluorescent protein (eGFP), and the fusion gene was transiently expressed in human neuroblastoma SH-SY5Y cells. A cell line stably expressing TASK-eGFP fusion proteins was generated from transient expression cells by using fluorescence-activated cell sorting followed by antibiotic selection. The uniform expression of TASK3 fusion proteins was further confirmed by flow cytometry. Moreover, the localization of TASK3 tagged with eGFP was checked by confocal microcopy. TASK3-eGFP fusion proteins are observed on the SH-SY5Y cell membrane. The strategies using eGFP as a fusion tag facilitate the monitoring of the TASK3 expression and enable the successful employment of FACS for screening and construction of cell lines stably expressing TASK3. The TASK3 overexpression cell line will lay a fundamental for the in vitro evaluation of TASK3 function during hypoxic/ischemic injury.

Progesterone treatment before experimental hypoxia-ischemia enhances the expression of glucose transporter proteins GLUT1 and GLUT3 in neonatal rats.

Progesterone is an efficient candidate for treating stroke and traumatic brain damage. The current study was designed to investigate the effects of progesterone on glucose transporter proteins (GLUT1 and GLUT3) during hypoxic-ischemic injury in a neonatal rat model. We demonstrated strong staining for GLUT1 in the walls of blood vessels and GLUT3 immunoreactivity in hippocampal neurons after hypoxiaischemia. Hypoxia-ischemia elevated GLUT1 and GLUT3 at both the mRNA and protein levels in the hippocampus, and pre-treatment with progesterone (8 mg/kg) further enhanced their accumulation until 24 h after hypoxic-ischemic injury. These results showed that progesterone treatment induced the accumulation of both GLUT1 and GLUT3 transporters, and an energy-compensation mechanism may be involved in the neuroprotective effect of progesterone during hypoxic-ischemic injury after cerebral ischemic attacks.