An integral blood-brain barrier in adulthood relies on microglia-derived PDGFB.

Pericyte is an indispensable cellular constituent of blood-brain barrier (BBB) and its homeostasis heavily rely on PDGFB-PDGFRß signaling. However, the primary cellular sources of PDGFB in the central nervous system (CNS) are unclear. Microglia is not considered a component of BBB and its role in maintaining BBB integrity in steady state is controversial. In this study, by analyzing transcriptomic data and performing in situ hybridization, we revealed a transition of the primary central PDGFB producers from endothelial cells in newborns to microglia in adults. Acute loss of microglial PDGFB profoundly impaired BBB integrity in adult but not newborn mice, and thus, adult mice deficient of microglial PDGFB could not survive from a sublethal endotoxin challenge due to rampant microhemorrhages in the CNS. In contrast, acute abrogation of endothelial PDGFB had minimal effects on the BBB of adult mice but led to a severe impairment of CNS vasculature in the neonates. Moreover, we found that microglia would respond to a variety of BBB insults by upregulating PDGFB expression. These findings underscore the physiological importance of the microglia-derived PDGFB to the BBB integrity of adult mice both in steady state and under injury.

Striatal CDK5 Regulates Cholinergic Neuron Activation and Dyskinesia-like Behaviors through BK Channels.

Disturbance of the cholinergic system plays a crucial role in the pathological progression of neurological diseases that cause dyskinesia-like behaviors. However, the molecular mechanisms underlying this disturbance remain elusive. Here, we showed that cyclin-dependent kinase 5 (Cdk5) was reduced in cholinergic neurons of midbrain according to the single-nucleus RNA sequencing analysis. Serum levels of CDK5 also decreased in patients with Parkinson's disease accompanied by motor symptoms. Moreover, Cdk5 deficiency in cholinergic neurons triggered paw tremors, abnormal motor coordination, and motor balance deficits in mice. These symptoms occurred along with cholinergic neuron hyperexcitability and increases in the current density of large-conductance Ca2+-activated K+ channels (BK channels). Pharmacological inhibition of BK channels restrained the excessive intrinsic excitability of striatal cholinergic neurons in Cdk5-deficient mice. Furthermore, CDK5 interacted with BK channels and negatively regulated BK channel activity via phosphorylation of threonine-908. Restoration of CDK5 expression in striatal cholinergic neurons reduced dyskinesia-like behaviors in ChAT-Cre;Cdk5f/f mice. Together, these findings indicate that CDK5-induced phosphorylation of BK channels involves in cholinergic-neuron-mediated motor function, providing a potential new therapeutic target for treating dyskinesia-like behaviors arising from neurological diseases.

Endothelial TFEB signaling-mediated autophagic disturbance initiates microglial activation and cognitive dysfunction.

Cognitive impairment caused by systemic chemotherapy is a critical question that perplexes the effective implementation of clinical treatment, but related molecular events are poorly understood. Herein, we show that bortezomib exposure leads to microglia activation and cognitive impairment, this occurs along with decreased nuclear translocation of TFEB (transcription factor EB), which is linked to macroautophagy/autophagy disorder, STAT3 (signal transducer and activator of transcription 3) phosphorylation and IL23A (interleukin 23 subunit alpha) expression. Pharmacological enhancement of TFEB nuclear translocation by digoxin restores lysosomal function and reduces STAT3-dependent endothelial IL23A secretion. As a consequence, we found that brain endothelial-specific ablation of Il23a ameliorated both microglia activation and cognitive dysfunction. Thus, the endothelial TFEB-STAT3-IL23A axis in the brain represents a critical cellular event for initiating bortezomib-mediated aberrant microglial activation and synapse engulfment. Our results suggest the reversal of TFEB nuclear translocation may provide a novel therapeutic approach to prevent symptoms of cognitive dysfunction during clinical use of bortezomib.Abbreviations: AAV: adeno-associated virus; BBB: blood-brain barrier; BTZ: bortezomib; DG: digoxin; DGs: dentate gyrus; DLG4/PSD95: discs large MAGUK scaffold protein 4; HBMECs: human brain microvascular endothelial cells; HP: hippocampus; IL23A: interleukin 23 subunit alpha; MBVECs: mouse brain vascular endothelial cells; mPFC: medial prefrontal cortex; NORT: novel object recognition test; OLT: object location test; PLX5622: 6-fluoro-N-([5-fluoro-2-methoxypyridin-3-yl]methyl)-5-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3- yl)methyl; PPP3/calcineurin: protein phosphatase 3; SBEs: STAT3 binding elements; shRNA: small hairpin RNA; SLC17A7/VGLUT1: solute carrier family 17 member 7; SLC32A1/VGAT: solute carrier family 32 member 1; STAT3: signal transducer and activator of transcription 3, TFEB: transcription factor EB; Ub: ubiquitin.

Divergent projections of the prelimbic cortex mediate autism- and anxiety-like behaviors.

The comorbidity of autism spectrum disorder and anxiety is common, but the underlying circuitry is poorly understood. Here, Tmem74-/- mice showed autism- and anxiety-like behaviors along with increased excitability of pyramidal neurons (PNs) in the prelimbic cortex (PL), which were reversed by Tmem74 re-expression and chemogenetic inhibition in PNs of the PL. To determine the underlying circuitry, we performed conditional deletion of Tmem74 in the PNs of PL of mice, and we found that alterations in the PL projections to fast-spiking interneurons (FSIs) in the dorsal striatum (dSTR) (PLPNs-dSTRFSIs) mediated the hyperexcitability of FSIs and autism-like behaviors and that alterations in the PL projections to the PNs of the basolateral amygdaloid nucleus (BLA) (PLPNs-BLAPNs) mediated the hyperexcitability of PNs and anxiety-like behaviors. However, the two populations of PNs in the PL had different spatial locations, optogenetic manipulations revealed that alterations in the activity in the PL-dSTR or PL-BLA circuits led to autism- or anxiety-like behaviors, respectively. Collectively, these findings highlight that the hyperactivity of the two populations of PNs in the PL mediates autism and anxiety comorbidity through the PL-dSTR and PL-BLA circuits, which may lead to the development of new therapeutics for the autism and anxiety comorbidity.

Glioblastoma multiforme: Diagnosis, treatment, and invasion.

Glioblastoma multiforme (GBM) is an essentially incurable brain tumor, which has been explored for approximately a century. Nowadays, surgical resection, chemotherapy, and radiation therapy are still the standardized therapeutic options. However, due to the intrinsic invasion and metastasis features and the resistance to chemotherapy, the survival rate of glioblastoma patients remains unsatisfactory. To improve the current situation, much more research is needed to provide comprehensive knowledge of GBM. In this review, we summarize the latest updates on GBM treatment and invasion. Firstly, we review the traditional and emerging therapies that have been used for GBM treatment. Given the limited efficiency of these therapies, we further discuss the role of invasion in GBM recurrence and progression, and present current research progress on the mode and mechanisms of GBM invasion.

Signalling pathways in autism spectrum disorder: mechanisms and therapeutic implications.

Autism spectrum disorder (ASD) is a prevalent and complex neurodevelopmental disorder which has strong genetic basis. Despite the rapidly rising incidence of autism, little is known about its aetiology, risk factors, and disease progression. There are currently neither validated biomarkers for diagnostic screening nor specific medication for autism. Over the last two decades, there have been remarkable advances in genetics, with hundreds of genes identified and validated as being associated with a high risk for autism. The convergence of neuroscience methods is becoming more widely recognized for its significance in elucidating the pathological mechanisms of autism. Efforts have been devoted to exploring the behavioural functions, key pathological mechanisms and potential treatments of autism. Here, as we highlight in this review, emerging evidence shows that signal transduction molecular events are involved in pathological processes such as transcription, translation, synaptic transmission, epigenetics and immunoinflammatory responses. This involvement has important implications for the discovery of precise molecular targets for autism. Moreover, we review recent insights into the mechanisms and clinical implications of signal transduction in autism from molecular, cellular, neural circuit, and neurobehavioural aspects. Finally, the challenges and future perspectives are discussed with regard to novel strategies predicated on the biological features of autism.

Decreased synapse-associated proteins are associated with the onset of epileptic memory impairment in endothelial CDK5-deficient mice.

Accumulating evidence indicates that epilepsy has a higher risk of inducing memory impairment and dementia. However, the underlying onset mechanism remains unclear. Here, we found that mice with spontaneous epilepsy induced by endothelial CDK5 deficiency exhibited hippocampal-dependent memory impairment at 6 months of age, but not at 2 months of age. Moreover, the persistent epileptic seizures induce aberrant changes in phosphorylation of CaMKII protein in the hippocampus of spontaneous epileptic mice. Using genome-wide RNA sequencing and intergenic interaction analysis of STRING, we found that in addition to epilepsy-related genes, there are changes in synaptic organization pathway node genes, such as Bdnf and Grin1. The synapse-related proteins by Western blot analysis, such as NMDA receptors (NR1 and NR2B), PSD95, and the phosphorylation of synapsin1, are progressively decreased during epileptic seizures in Cdh5-CreERT2;CDK5f/f mice. Notably, we found that valproate (VPA) and phenytoin (PHT) augment mRNA expression and protein levels of synapse-related genes and ameliorate memory impairment in Cdh5-CreERT2;CDK5f/f mice. Our study elucidates a potential mechanism of memory deficits in epilepsy, and pharmacological reversal of synaptic pathology targeting might provide a new therapeutic intervention for epileptic memory deficits.

BOD1 regulates the cerebellar IV/V lobe-fastigial nucleus circuit associated with motor coordination.

Cerebellar ataxias are characterized by a progressive decline in motor coordination, but the specific output circuits and underlying pathological mechanism remain poorly understood. Through cell-type-specific manipulations, we discovered a novel GABAergic Purkinje cell (PC) circuit in the cerebellar IV/V lobe that projected to CaMKIIα+ neurons in the fastigial nucleus (FN), which regulated sensorimotor coordination. Furthermore, transcriptomics profiling analysis revealed various cerebellar neuronal identities, and we validated that biorientation defective 1 (BOD1) played an important role in the circuit of IV/V lobe to FN. BOD1 deficit in PCs of IV/V lobe attenuated the excitability and spine density of PCs, accompany with ataxia behaviors. Instead, BOD1 enrichment in PCs of IV/V lobe reversed the hyperexcitability of CaMKIIα+ neurons in the FN and ameliorated ataxia behaviors in L7-Cre; BOD1f/f mice. Together, these findings further suggest that specific regulation of the cerebellar IV/V lobePCs → FNCaMKIIα+ circuit might provide neuromodulatory targets for the treatment of ataxia behaviors.

Carbamate-based N-Substituted tryptamine derivatives as novel pleiotropic molecules for Alzheimer's disease.

A novel series of carbamate-based N-substituted tryptamine derivatives were designed and synthesized based on functional group combination strategy, and possessed both cholinesterase inhibition and neuroprotective effects. After systematically evaluating the cholinesterase inhibitory activity of 24 synthesized compounds, compound 6H6, bearing n-heptyl residue as carbamate moiety, was highlighted due to its great BChE-selective inhibition (eeAChE IC50 > 100 µM; eqBChE IC50 = 7 nM), neuronal protection, antioxidation and anti-neuroinflammation efficacy. Cytotoxicity and acute toxicity assays confirmed the safety-efficacy profiles of compound 6H6. Besides, pharmacokinetic properties and blood-brain barrier (BBB) permeability of compound 6H6 were favorable and suitable for further study in vivo. The behavioral tests revealed that compound 6H6 could remarkably improve the scop-induced ethological changes and memory impairment, suggesting compound 6H6, as an attractive pleiotropic molecule, had great promise in treating Alzheimer's disease.

Design, synthesis and biological evaluation of novel o-aminobenzamide derivatives as potential anti-gastric cancer agents in vitro and in vivo.

Although gastric cancer has become a major public health problem, oral agents applied in clinics for gastric cancer therapy are scarce. Therefore, to explore new oral chemical entities with high efficiency and low toxicity, 41 o-aminobenzamide derivatives based on the scaffolds of MS-275 and SAHA were designed, synthesized, and evaluated for their anti-gastric cancer abilities in vitro and in vivo. Structure-activity relationships were discussed, leading to the identification of compounds F8 (IC50 = 0.28 µM against HGC-27 cell) and T9 (IC50 = 1.84 µM against HGC-27 cell) with improved cytotoxicity, anti-gastric cancer proliferation potency, induction of cell apoptosis and cell cycle arrest ability, inhibition of cell migration and invasion. What is worth mentioning is that compound F8 was more efficient and less toxic than the positive drug capecitabine in vivo on the HGC-27-xenograft model. Meanwhile, compound F8 exhibited suitable pharmacokinetic properties and less acute toxicity (LD50 > 1000 mg/kg). Besides, western blotting analysis, IHC analysis, differentially expressed proteins analysis and ABPP experiment indicated that compound F8 could modulate molecular pathways involved in apoptosis and cell cycle progression. Consequently, compound F8 is a strong candidate for the development of human gastric cancer therapy.

Novel piperazine based benzamide derivatives as potential anti-glioblastoma agents inhibiting cell proliferation and cell cycle progression.

Highly efficacious and tolerable agents for the treatment of glioblastoma (GBM), the most common and aggressive primary brain tumor, are urgently needed. Herein, we reveal the design, synthesis and biological evaluation of several piperazine based benzamide derivatives, which are based on the non-classical isostere principle and combination principle for GBM therapy. After structure-activity relationship (SAR) study, compound L19 was demonstrated as the most promising compound with IC50 values of 0.15 µM, 0.29 µM, 1.25 µM against GBM C6, U87-MG, U251 cells, respectively. Moreover, compound L19 could inhibit the proliferation, migration and invasion, as well as induce apoptosis and cell cycle arrest of GBM cell lines in vitro. From mechanism perspective, compound L19 could regulate the cell cycle-related proteins and influence the p16INK4a-CDK4/6-pRb pathway by western blotting experiment. What is worth mentioning is that compound L19 could penetrate the blood-brain barrier (BBB) with an exceptional brain-to-plasma ratio of 1.07 in vivo. Besides, the superior anti-glioblastoma potency in vivo of compound L19 was identified on U87-MG-xenograft model without any apparent host toxicity. Overall, the potential of compound L19 warrants further pre-clinical investigation for GBM therapy.

Endothelial peroxynitrite causes disturbance of neuronal oscillations by targeting caspase-1 in the arcuate nucleus.

Severe anorexia limits the clinical application of cisplatin, and even leads to the discontinuation of treatment. However, the mechanisms underlying cisplatin-induced anorexia are unknown. Herein, we demonstrated that cisplatin could affect neuronal gamma oscillations and induce abnormal neuronal theta-gamma phase-amplitude coupling in the arcuate nucleus (Arc) of the hypothalamus, and these findings were associated with significantly decreased food intake and weight loss in mice. Chemogenetic activation of AgRP neurons in the Arc reversed the cisplatin-induced food intake reduction in mice. We further demonstrated that endothelial peroxynitrite (ONOO-) formation in the Arc induced nitrosative stress following cisplatin treatment via a previously uncharacterized pathway involving neuronal caspase-1 activation. Strikingly, treatment with the ONOO- scavenger uric acid (UA) reversed the reduced action potential (AP) frequency of AgRP neurons and increased the AP frequency of POMC neurons induced by SIN1, a donor of ONOO-, in the Arc, as determined by whole-cell patch-clamp electrophysiological recording. Consistent with these findings, UA treatment effectively alleviated cisplatin-induced dysfunction of neuronal oscillations and neuronal theta-gamma phase-amplitude coupling in the Arc of mice. Taken together, these results suggest, for the first time, that targeting the overproduction of endothelial ONOO- can regulate cisplatin-induced neurotoxicity through neuronal caspase-1, and thereby serve as a potential therapeutic approach to alleviate chemotherapy-induced anorexia and weight loss.

N-2-(phenylamino) benzamide derivatives as novel anti-glioblastoma agents: Synthesis and biological evaluation.

Glioblastoma is one of the most lethal brain tumors. The crucial chemotherapy is mainly alkylating agents with modest clinical success. Given this desperate need and inspired by the encouraging results of a phase II trial via concomitant Topo I inhibitor plus COX-2 inhibitor, we designed a series of N-2-(phenylamino) benzamide derivatives as novel anti-glioblastoma agents based on structure modification on 1,5-naphthyridine derivatives (Topo I inhibitors). Notably, the target compounds I-1 (33.61 ± 1.15 µM) and I-8 (45.01 ± 2.37 µM) were confirmed to inhibit COX-2, while a previous reported compound (1,5-naphthyridine derivative) resulted nearly inactive towards COX-2 (IC50 > 150 µM). Besides, I-1 and I-8 exhibited higher anti-proliferation, anti-migration, anti-invasion effects than the parent compound 1,5-naphthyridine derivative, suggesting the success of modification based on the parent. Moreover, I-1 obviously repressed tumor growth in the C6 glioma orthotopic model (TGI = 66.7%) and U87MG xenograft model (TGI = 69.4%). Besides, I-1 downregulated PGE2, VEGF, MMP-9, and STAT3 activation, upregulated E-cadherin in the orthotopic model. More importantly, I-1 showed higher safety than temozolomide and different mechanism from temozolomide in the C6 glioma orthotopic model. All the evidence demonstrated that N-2-(phenylamino) benzamide derivatives as novel anti-glioblastoma agents could be promising for the glioma management.

Characterization of porcine sapelovirus prevalent in western Jiangxi, China.

BACKGROUND: Porcine sapelovirus (PSV) infection can lead severe polioencephalomyelitis with high morbidity and mortality, which result in significant economic losses. Infection with the PSV is believed to be common yet limited information is available on the prevalence and molecular characterization of PSV in China. Therefore, the objective of this study was to characterize the prevalence and genome of PSV strains identified in the western Jiangxi province of China. RESULTS: A high specificity and sensitivity SYBR Green I-based RT-PCR method for PSV detection was developed. Two hundred and ninety four fecal samples were collected from December 2018 to March 2019 in 4 farms. An overall PSV-positivity rate of 11.22% (33/294) was detected with the real-time RT-PCR method, and a high infection rate and viral load of PSV were found in nursery pigs. In total, complete VP1 gene sequences of 11 PSV strains (PSV-YCs) were obtained. Homology comparisons of the VP1 gene of the 11 PSV-YCs with previously reported PSVs revealed nucleotide sequence identities ranging from 63% to 96.8%, and deduced amino acid sequence identities from 61.4% to 99.7%. Phylogenetic analyses based on the VP1 gene exhibited 2 main clades corresponding to PSV-1 and PSV-2, and all PSV-YCs prevalent in western Jiangxi belonged to the traditional genotype (PSV-1). In addition, the pairwise distances of VP1 gene sequences between PSV-YCs ranged from 0.009 to 0.198, which indicating that substantial genetic diversity among the PSVs in western Jiangxi. CONCLUSIONS: To the authors' knowledge, this is the first description of PSV in the Jiangxi province pig herds in China, and it is crucial to understand the epidemiology of the viruses in China. The results also provide an important theoretical foundation for diagnosis and early warning of epidemic diseases caused by PSVs prevailing in this region.

Endothelium-derived Cdk5 deficit aggravates air pollution-induced peripheral vasoconstriction through AT1R upregulation.

PM2.5 infiltrates into circulation and increases the risk of systemic vascular dysfunction. As the first-line barrier against external stimuli, the molecular mechanism of the biological response of vascular endothelial cells to PM2.5 exposure remains unclear. In this study, 4-week-old mice were exposed to Hangzhou 'real' airborne PM2.5 for 2 months and were found to display bronchial and alveolar damage. Importantly, in the present study, we have demonstrated that Cdk5 deficit induced peripheral vasoconstriction through angiotensin II type 1 receptor under angiotensin II stimulation in Cdh5-cre;Cdk5f/n mice. In the brain, Cdk5 deficit increased the myogenic activity in the medullary arterioles under external pressure. On the other hand, no changes in cerebral blood flow and behavior patterns were observed in the Cdh5-cre;Cdk5f/n mice exposed to PM2.5. Therefore, our current findings indicate that CDK5 plays an important role in endothelium cell growth, migration, and molecular transduction, which is also a sensor for the response of vascular endothelial cells to PM2.5.

Targeted Regulation of Blood-Brain Barrier for Enhanced Therapeutic Efficiency of Hypoxia-Modifier Nanoparticles and Immune Checkpoint Blockade Antibodies for Glioblastoma.

Glioblastoma is the most destructive type of brain cancer. The blood-brain barrier (BBB) is a tremendous obstacle that hinders therapeutic agents, such as chemical drugs and antibodies, from reaching glioblastoma tissues. Meanwhile, the abnormal microenvironment of glioblastoma extremely restricts the expected therapeutic effects of accumulated drugs. Therefore, in the present study, BBB-regulating nanovesicles (BRN) are developed to achieve targeted and controlled BBB regulation, carrying adenosine 2A receptor (A2AR) agonists and perfluorocarbon (PF). The red-blood-cell membrane (RBCM) is included on the outside to avoid the premature release of therapeutic agents. In the presence of ultrasonication (US), A2AR agonists are released and induce effects on both F-actin and tight junctions of endothelial cells. Subsequently, BBB permeability is temporarily increased and enables small molecules and nanoparticles to enter brain parenchymal tissues. The high affinity between manganese dioxide and temozolomide (TMZ) is utilized to form multifunctional nanoparticles to ameliorate the hypoxic microenvironment, which yields improved glioblastoma inhibition combined with radiotherapy. Moreover, with the aid of targeted BBB regulation, programmed death ligand-1 (PD-L1) antibody induces a tumor-specific immune response. Taken together, the findings suggest that synergistic combination may have the potential in amplifying the therapeutic efficacies of clinical drugs and immune checkpoint blockade antibodies to overcome the therapeutic resistance of glioblastoma.

Endothelium-derived semaphorin 3G attenuates ischemic retinopathy by coordinating ß-catenin-dependent vascular remodeling.

Abnormal angiogenesis and regression of the diseased retinal vasculature are key processes associated with ischemic retinopathies, but the underlying mechanisms that regulate vascular remodeling remain poorly understood. Here, we confirmed the specific expression of semaphorin 3G (Sema3G) in retinal endothelial cells (ECs), which was required for vascular remodeling and the amelioration of ischemic retinopathy. We found that Sema3G was elevated in the vitreous fluid of patients with proliferative diabetic retinopathy (PDR) and in the neovascularization regression phase of oxygen-induced retinopathy (OIR). Endothelial-specific Sema3G knockout mice exhibited decreased vessel density and excessive matrix deposition in the retinal vasculature. Moreover, loss of Sema3G aggravated pathological angiogenesis in mice with OIR. Mechanistically, we demonstrated that HIF-2α directly regulated Sema3G transcription in ECs under hypoxia. Sema3G coordinated the functional interaction between ß-catenin and VE-cadherin by increasing ß-catenin stability in the endothelium through the neuropilin-2 (Nrp2)/PlexinD1 receptor. Furthermore, Sema3G supplementation enhanced healthy vascular network formation and promoted diseased vasculature regression during blood vessel remodeling. Overall, we deciphered the endothelium-derived Sema3G-dependent events involved in modulating physiological vascular remodeling and regression of pathological blood vessels for reparative vascular regeneration. Our findings shed light on the protective effect of Sema3G in ischemic retinopathies.

CRISPR-Sunspot: Imaging of endogenous low-abundance RNA at the single-molecule level in live cells.

CRISPR/Cas-based mRNA imaging has been developed to labeling of high-abundance mRNAs. A lack of non-genetically encoded mRNA-tagged imaging tools has limited our ability to explore the functional distributions of endogenous low-abundance mRNAs in cells. Here, we developed a CRISPR-Sunspot method based on the SunTag signal amplification system that allows efficient imaging of low-abundance mRNAs with CRISPR/Cas9. Methods: We created a stable TRE3G-dCas9-EGFP cell line and generated an Inducible dCas9-EGFP imaging system for assessment of two factors, sgRNA and dCas9, which influence imaging quality. Based on SunTag system, we established a CRISPR-Sunspot imaging system for amplifying signals from single-molecule mRNA in live cells. CRISPR-Sunspot was used to track co-localization of Camk2a mRNA with regulatory protein Xlr3b in neurons. CRISPR-Sunspot combined with CRISPRa was used to determine elevated mRNA molecules. Results: Our results showed that manipulating the expression of fluorescent proteins and sgRNA increased the efficiency of RNA imaging in cells. CRISPR-Sunspot could target endogenous mRNAs in the cytoplasm and amplified signals from single-molecule mRNA. Furthermore, CRISPR-Sunspot was also applied to visualize mRNA distributions with its regulating proteins in neurons. CRISPR-Sunspot detected the co-localization of Camk2a mRNA with overexpressed Xlr3b proteins in the neuronal dendrites. Moreover, we also manipulated CRISPR-Sunspot to detect transcriptional activation of target gene such as HBG1 in live cells. Conclusion: Our findings suggest that CRISPR-Sunspot is a novel applicable imaging tool for visualizing the distributions of low-abundance mRNAs in cells. This study provides a novel strategy to unravel the molecular mechanisms of diseases caused by aberrant mRNA molecules.

Role of pericyte-derived SENP1 in neuronal injury after brain ischemia.

AIMS: SUMOylation is a posttranslational modification related to multiple human diseases. SUMOylation can be reversed by classes of proteases known as the sentrin/SUMO-specific proteases (SENPs). In the present study, we investigate the potential role of SENP1 in pericytes in the brain ischemia. METHODS: Pericyte-specific deletion of senp1 mice (Cspg4-Cre; senp1f/f ) were used for brain function and neuronal damage evaluation following brain ischemia. The cerebral blood vessels of diameter, velocity, and flux were performed in living mice by two-photon laser scanning microscopy (TPLSM). Biochemical analysis and immunohistochemistry methods were used to address the role and mechanism of pericyte-specific SENP1 in the pathological process of brain ischemia. A coculture model of HBVPs and HBMECs mimicked the BBB in vitro and was used to evaluate BBB integrity after glucose deprivation. RESULTS: Our results showed that senp1-specific deletion in pericytes did not affect the motor function and cognitive function of mice. However, the pericyte-specific deletion of senp1 aggravated the infarct size and motor deficit following focal brain ischemia. Consistently, the TPLSM data demonstrated that SENP1 deletion in pericytes accelerated thrombosis formation in brain microvessels. We also found that pericyte-specific deletion of senp1 exaggerated the neuronal damage significantly following brain ischemia in mice. Moreover, SENP1 knockdown in pericytes could activate the apoptosis signaling and disrupt the barrier integrity in vitro coculture model. CONCLUSIONS: Our findings revealed that targeting SENP1 in pericytes may represent a novel therapeutic strategy for neurovascular protection in stroke.

Design and synthesis of 1,3-benzothiazinone derivatives as potential anti-inflammatory agents.

A series of 1,3-benzothiazinone derivatives were designed and synthesized for pharmacological assessments. Among the synthesized 19 compounds, some compounds showed high activities on inhibiting LPS-induced nitrite oxide and TNF-α production, down-regulating COX-2 and increasing IL-10 production in RAW264.7 cells. All the compounds had no obvious cytotoxicity in in vitro assay. LD50 value of compound 25 was greater than 2000 mg/kg, which was safer than meloxicam. Compound 25 significantly inhibited phosphorylation of NF-κB and STAT3 in LPS-induced RAW264.7 cells. Inhibition of synthesized compounds on COX activity was weaker than meloxicam. Compound 25 displayed lower gastrointestinal toxicity than meloxicam. Besides, compound 25 decreased the swelling in carrageenan-induced paw edema models of inflammation and reduced PGE2 level significantly. In summary, 1,3-benzothiazinone derivatives are unique scaffolds with anti-inflammatory activity and low toxicity.