Use superb microvascular imaging to diagnose and predict metastatic cervical lymph nodes in patients with papillary thyroid carcinoma.

PURPOSE: Papillary thyroid carcinoma (PTC) with metastatic lymph nodes (LNs) is closely associated with disease recurrence. This study accessed the value of superb microvascular imaging (SMI) in the diagnosis and prediction of metastatic cervical LNs in patients with PTC. METHODS: A total of 183 cervical LNs (103 metastatic and 80 reactive) from 116 patients with PTC were analysed. Metastatic cervical LNs were confirmed by pathology or/and cytology; reactive cervical LNs were confirmed by pathology or clinical features. The characteristic of conventional ultrasound (US) was extracted using univariate and multivariate analyses. The diagnostic performance of US and SMI were compared using the area under the receiver operating curve (AUC) with corresponding sensitivity and specificity. A nomogram was developed to predict metastatic LNs in patients with PTC, based on multivariate analyses. RESULTS: L/S < 2, ill-defined border, absence of hilum, isoechoic or hyperechoic, heterogeneous internal echo, peripheral or mixed vascular pattern on color Doppler flow imaging (CDFI) and SMI, and a larger SMI vascular index appeared more frequently in metastatic LNs in the training datasets than in reactive LNs (P < 0.05). The diagnostic sensitivity, specificity and accuracy of SMI vs US are 94.4% and 87.3%, 79.3% and 69.3%, and 87.6% and 79.1%, respectively; SMI combined with US exhibited a higher AUC [0.926 (0.877-0.975)] than US only [0.829 (0.759-0.900)]. L/S < 2, peripheral or mixed vascular type on CDFI, and peripheral or mixed vascular types on SMI were independent predictors of metastatic LNs with PTC. The nomogram based on these three parameters exhibited excellent discrimination, with an AUC of 0.926. CONCLUSION: SMI was superior to US in diagnosing metastatic LNs in PTC. US combined with SMI significantly improved the diagnostic accuracy of metastatic cervical LNs with PTC. SMI is efficacious for differentiating and predicting metastatic cervical LNs.

Prostaglandin E2 accumulation is closely associated with S. aureus-infected bovine endometritis.

S. aureus isolated from bacterial bovine endometritis is common in epidemiological reports, but is often ignored as a subclinical pathogenic microorganism. In a previous study, we showed that live S. aureus (LSA) and heat killed S. aureus (HK-SA) induce different inflammatory responses in bovine endometrial tissue, and possibly being associated with the accumulation of prostaglandin E2 (PGE2). Thus, in this study, we varied PGE2 concentrations using inhibitors or agonists in HK-SA-treated bovine endometrial tissues. The results demonstrated that PGE2 has a positive relationship with IL-6, TNF-α, and damage-associated molecular patterns (DAMPs; e.g., HMGB-1 and HABP-1) expression and tissues damage, and is regulated by the EP4-p38 MAPK pathway. We concluded that lipoproteins of S. aureus are associated with PGE2 generation. To further explore the relationship between LSA and PGE2 accumulation, we used the S. aureus strain SA113 lipoprotein knockout (SA113Δlpl) to infect bovine endometrial epithelial cells (BECs). LSA decreased PGE2, cAMP, EP4, IL-6, IL-8, cAMP secretion, and the MAPK and PKA signaling pathways when infected with SA113Δlpl, as compared with SA113-infected groups. Moreover, the adhesion and invasion of BECs were similarly downregulated when lipoproteins in S. aureus were knocked out. The results of this study show that PGE2 is involved in both HK-SA- and LSA-induced inflammatory responses in the bovine endometrium. We suggest that S. aureus infection is associated with bovine endometritis, and although HK-SA and LSA induce different inflammatory responses, the strategy of decreasing PGE2 accumulation is helpful in reducing the inflammation stage caused by S. aureus.

Equine ß-defensin 1 regulates cytokine expression and phagocytosis in S. aureus-infected mouse monocyte macrophages via the Paxillin-FAK-PI3K pathway.

ß-defensin-1 (BD-1) is a rich source of disulfide bonds and antibacterial peptides that exhibit direct bactericidal function. The expression of BD-1 is primarily induced by external stimulation and is known to correlate with TLR-mediated inflammation, suggesting its association with innate immune responses. Equine ß-defensin-1 (eBD-1) belongs to the BD-1 family. Our previous study demonstrated that eBD-1 enhances cytokine expression and promotes macrophage phagocytosis of S. aureus, although the underlying mechanism remains unknown. In this study, we utilized a PI-3K inhibitor (PKI-402) to treat eBD-1 -treated S. aureus-infected macrophages in vitro. Our results revealed that PKI-402 decreased the expression of eBD-1-promoted TNF-α, IL-6, CXCL10, CD40, RANTES, and p65 mRNA. To further investigate the relationship between eBD-1 and phagocytosis, we examined the expression of paxillin and FcγRIII (CD16 receptor) using western blot and immunofluorescence techniques. Our findings demonstrated that eBD-1 enhanced CD16 and paxillin expression in S. aureus -infected macrophages. Considering the correlation between paxillin expression and focal adhesion kinase (FAK), we transfected FAK siRNA into macrophages and evaluated paxillin expression using western blot analysis. Additionally, we quantified the number of S. aureus phagocytosed by macrophages. The results indicated a reduction in both paxillin expression and the number of S. aureus phagocytosed by macrophages upon FAK siRNA treatment. Our study showed the eBD-1 promotes cytokine mRNA expression in S. aureus-infected macrophages regulated by PI-3K-NF-κB pathway, and it increases macrophage phagocytosis of S. aureus associated with the FAK-paxillin signaling pathway.

Size-matched hydrogen bonded hydroxylammonium frameworks for regulation of energetic materials.

Size matching molecular design utilizing host-guest chemistry is a general, promising strategy for seeking new functional materials. With the growing trend of multidisciplinary investigations, taming the metastable high-energy guest moiety in well-matched frameworks is a new pathway leading to innovative energetic materials. Presented is a selective encapsulation in hydrogen-bonded hydroxylammonium frameworks (HHF) by screening different sized nitrogen-rich azoles. The size-match between a sensitive high-energy guest and an HHF not only gives rise to higher energetic performance by dense packing, but also reinforces the layer-by-layer structure which can stabilize the resulting materials towards external mechanic stimuli. Preliminary assessment based on calculated detonation properties and mechanical sensitivity indicates that HHF competed well with the energetic performance and molecular stability (detonation velocity = 9286 m s-1, impact sensitivity = 50 J). This work highlights the size-matched phenomenon of HHF and may serve as an alternative strategy for exploring next generation advanced energetic materials.

Equus ß-Defensin-1 Regulates Innate IMMUNE Response in S. aureus-Infected Mouse Monocyte Macrophage.

Beta-defensin-1 (BD-1) is among the class of antibacterial peptides that are rich in disulfide bonds, have direct antibacterial activity and showed enhanced expression following external stimulation. However, existing research studies only treated BD-1 to cell models without stimulation from pathogen-associated molecular patterns (PAMPs), which will further influence our understanding of the role of BD-1. In this study, we map the tissue distribution of Equus BD-1 (i.e., eBD-1, ass BD-1, and mule BD-1) and compare their expression levels in various tissues. We further characterize the three kinds of Equus BD-1 by analyzing their full-length cDNA. We showed that eBD-1, ass BD-1, and mule BD-1 have an identical (100%) open reading frame (ORF). The ORF encoding OEBD-1 expressed the ORF in the E. coli Top10 expression system. This expression system was combined with an S. aureus-infected J774A.1 macrophage cell line to determine the influence on innate immune mediator expression. Using this expression model system, it was determined that the OEBD-1 protein enhanced IL-6 and TNF-α secretion. It can also promote TLR2, IL-1ß, CCL2, CCL7, CXCL10 and NF-κB p65 mRNA expression. Moreover, OEBD-1 upregulates phosphorylation of ATK, Syk and IκB-α. In addition, OEBD-1 enhances the macrophage's ability to phagocytose S. aureus. In conclusion, Equus BD-1 was shown to play an essential role in macrophage-involved innate immune responses in an in vitro system.

Imidazole-Based Energetic Materials: A Promising Family of N-Heterocyclic Framework.

Imidazole represents a fascinating class of explosophoric units with exciting structures and unique properties. As compared to other nitrogen-rich heterocycles, imidazole demonstrates great potential applications due to economic effectiveness and superior energetic performances. The field of traditional chemistry has been extensively explored for imidazole, and thus established bond-building methods and functionalization strategies promote further development as high-energy density materials (HEDMs). This review addresses the development of energetic imidazole compounds in the past decade, summarizes their physiochemical properties, and is divided into three parts (explosives, propellants, and energetic biocides) according to application requirements. Various synthetic strategies for these energetic molecules are highlighted, including the construction of heterocyclic frameworks and following functionalization. The selected and discussed reactions illustrate the versatility of imidazole in energetic applications as building blocks for the future design of new HEDMs.

Population pharmacokinetics and clinical outcomes of polymyxin B in paediatric patients with multidrug-resistant Gram-negative bacterial infections.

BACKGROUND: Current polymyxin B dosing in children relies on scant data. OBJECTIVES: To build a population pharmacokinetic (PK) model for polymyxin B in paediatric patients and assess the likely appropriateness of different dosages. METHODS: A total of 19 paediatric patients were enrolled to receive intravenous polymyxin B (1.33-2.53 mg/kg/day), and the median age was 12.5 (range 3.2-17.8) years. Serial plasma samples were collected at steady-state and modelled by population PK analysis. Clinical efficacy and nephrotoxicity of polymyxin B treatment were also assessed. RESULTS: PK data were adequately described by a two-compartment model with first-order elimination, and weight was a significant covariate of polymyxin B clearance. Clinical success occurred in 14 of 19 patients (73.7%) and only one patient developed acute kidney injury. The 28 day mortality was 10.5% (2/19). The steady-state polymyxin B exposure was 36.97 ±â€Š9.84 mg·h/L, lower than the therapeutic exposure of 50-100 mg·h/L. With the AUC24h/MIC target of 50, the dosage of 1.5-3.0 mg/kg/day had a probability of target attainments over 90% when MICs were <0.5 mg/L. CONCLUSIONS: Dose adjustment of polymyxin B needs to consider the MIC of infecting pathogens. Current polymyxin B dosing for paediatric patients may be acceptable when MICs are <0.5 mg/L.

The Functions of PCNA in Tumor Stemness and Invasion.

Invasion is the most prominent lethal feature of malignant cancer. However, how cell proliferation, another important feature of tumor development, is integrated with tumor invasion and the subsequent cell dissemination from primary tumors is not well understood. Proliferating cell nuclear antigen (PCNA) is essential for DNA replication in cancer cells. Loss of phosphorylation at tyrosine 211 (Y211) in PCNA (pY211-PCNA) mitigates PCNA function in proliferation, triggers replication fork arrest/collapse, which in turn sets off an anti-tumor inflammatory response, and suppresses distant metastasis. Here, we show that pY211-PCNA is important in stromal activation in tumor tissues. Loss of the phosphorylation resulted in reduced expression of mesenchymal proteins as well as tumor progenitor markers, and of the ability of invasion. Spontaneous mammary tumors that developed in mice lacking Y211 phosphorylation contained fewer tumor-initiating cells compared to tumors in wild-type mice. Our study demonstrates a novel function of PCNA as an essential factor for maintaining cancer stemness through Y211 phosphorylation.

miR-4759 suppresses breast cancer through immune checkpoint blockade.

Programmed cell death protein 1 (PD-1)/ programmed cell death protein ligand 1 (PD-L1) is the key immune checkpoint governing evasion of advanced cancer from immune surveillance. Immuno-oncology (IO) therapy targeting PD-1/PD-L1 with traditional antibodies is a promising approach to multiple cancer types but to which the response rate remains moderate in breast cancer, calling for the need of exploring alternative IO targeting approaches. A miRNA-gene network was integrated by a bioinformatics approach and corroborated with The Cancer Genome Atlas (TCGA) to screen miRNAs regulating immune checkpoint genes and associated with patient survival. Here we show the identification of a novel microRNA miR-4759 which repressed RNA expression of the PD-L1 gene. miR-4759 targeted the PD-L1 gene through two binding motifs in the 3' untranslated region (3'-UTR) of PD-L1. Reconstitution of miR-4759 inhibited PD-L1 expression and sensitized breast cancer cells to killing by immune cells. Treatment with miR-4759 suppressed tumor growth of orthotopic xenografts and promoted tumor infiltration of CD8+ T lymphocytes in immunocompetent mice. In contrast, miR-4759 had no effect to tumor growth in immunodeficient mice. In patients with breast cancer, expression of miR-4759 was preferentially downregulated in tumors compared to normal tissues and was associated with poor overall survival. Together, our results demonstrated miR-4759 as a novel non-coding RNA which promotes anti-tumor immunity of breast cancer.

Live S. aureus and heat-killed S. aureus induce different inflammation-associated factors in bovine endometrial tissue in vitro.

Staphylococcus aureus is majorly involved in bovine mastitis; however, it weakly induces pro-inflammatory factors in mammary gland epithelial cells. We aimed to clarify the involvement of S. aureus in other inflammation types and its relationship with inflammatory factor secretion in bovine endometritis. We used live S. aureus (LSA)- and heat-killed S. aureus (HK-SA)-treated bovine endometrial tissue in vitro. The HK-SA-treated group showed significantly higher IL-6, IL-1ß, TNF-α, CXCL1/2 and TLR2 expression than the LSA-infected group. Contrastingly, the LSA-infected group showed significantly higher PTGS2, mPGES-1, and EP4 expression than the HK-SA treated group. There was no significant between-group difference in hyaluronan-binding protein 1 expression, which suggested similar inflammatory responses. H&E results indicated that LSA and HK-SA induced shedding of endometrial gland epithelial cells. The LSA-infected group showed higher high-mobility group box 1 protein expression than the HK-SA treated groups, which indicated differences in signaling pathway activation. Further, the LSA-treated group had higher JNK and p38 MAPK levels while the HK-SA-treated group had higher IκB-α levels. There was no significant between-group difference in the ERK signaling pathway. Our findings indicate that the pathogen-associated molecular patterns (PAMPs) of S. aureus activate pro-inflammatory factor expression via the TLR2-ERK-NF-κB signaling pathway. Contrastingly, LSA induced PGE2 accumulation via the TLR2/MAPKs signaling pathway. This is the first report that S. aureus and the PAMPs of S. aureus activate different signaling pathways and that LSA mainly induce PGE2 accumulation rather than cytokine secretion.

Evading immune surveillance via tyrosine phosphorylation of nuclear PCNA.

Increased DNA replication and metastasis are hallmarks of cancer progression, while deregulated proliferation often triggers sustained replication stresses in cancer cells. How cancer cells overcome the growth stress and proceed to metastasis remains largely elusive. Proliferating cell nuclear antigen (PCNA) is an indispensable component of the DNA replication machinery. Here, we show that phosphorylation of PCNA on tyrosine 211 (pY211-PCNA) regulates DNA metabolism and tumor microenvironment. Abrogation of pY211-PCNA blocks fork processivity, resulting in biogenesis of single-stranded DNA (ssDNA) through a MRE11-dependent mechanism. The cytosolic ssDNA subsequently induces inflammatory cytokines through a cyclic GMP-AMP synthetase (cGAS)-dependent cascade, triggering an anti-tumor immunity by natural killer (NK) cells to suppress distant metastasis. Expression of pY211-PCNA is inversely correlated with cytosolic ssDNA and associated with poor survival in patients with cancer. Our results pave the way to biomarkers and therapies exploiting immune responsiveness to target metastatic cancer.

[Study on metabolic dynamics,metabolic enzyme phenotype and species difference of hepatic and intestinal microsome of psoralidin].

This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5'-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 µL·min~(-1)·mg~(-1),respectively,while those for G1 were 543. 3,and 75. 9 µL·min~(-1)·mg~(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 µL·min~(-1)·mg~(-1)),CYP2 C8( 88. 0 µL·min~(-1)·mg~(-1)),CYP2 C19( 166. 7 µL·min~(-1)·mg~(-1)),and CYP2 D6( 9. 1 µL·min~(-1)·mg~(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 µL·min~(-1)·mg~(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 µL·min~(-1)·mg~(-1)),UGT1 A7( 922. 8 µL·min~(-1)·mg~(-1)),UGT1 A8( 133. 0 µL·min~(-1)·mg~(-1)),UGT1 A9( 348. 6 µL·min~(-1)·mg~(-1)) and UGT2 B7( 118. 7 µL·min~(-1)·mg~(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 µL·min~(-1)·mg~(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.

Prostaglandin E2 promotes Staphylococcus aureus infection via EP4 receptor in bovine endometrium.

Prostaglandin E2 (PGE2) enhances Staphylococcus aureus infection but its mechanism is not well understood. Here, we examined the effect of PGE2 on Staphylococcal Protein A (SPA) expression in bovine endometrium and determined the role of select PGE2 receptors (i.e., EP2 and EP4) in adhesion and internalization of S. aureus. S. aureus isolate SA113 was used for in vitro infection of bovine endometrial tissues and epithelial cells, with treatment conditions consisting of untreated control, SA113 treatment, SA113 + PGE2, SA113 + PGE2 + EP2 receptor antagonist (AH-6809), and SA113 + PGE2 + EP4 receptor antagonist (AH-23848). Immunofluorescence assay revealed that PGE2 could promote SPA expression in S. aureus-infected bovine endometrial tissues. PGE2 also enhanced the adhesion and internalization of S. aureus in bovine endometrial cells. The addition of EP4 antagonist, but not the EP2 antagonist, abrogated the ability of PGE2 to promote S. aureus SPA expression, adhesion, and internalization in endometrial cells. Our findings suggest that S. aureus infection in the endometrium is enhanced by PGE2 through the EP4 receptor. This result is essential for the development of new approach to treating S. aureus infection, such as the application of EP4 antagonist as an adjunct drug treatment.

Constraint-induced movement therapy enhances AMPA receptor-dependent synaptic plasticity in the ipsilateral hemisphere following ischemic stroke.

Constraint-induced movement therapy (CIMT) can promote the recovery of motor function in injured upper limbs following stroke, which may be associated with upregulation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) at synapses in the ipsilateral sensorimotor cortex in our previous study. However, AMPAR distribution is tightly regulated, and only AMPARs on the postsynaptic membrane can mediate synaptic transmission. We speculated that synaptic remodeling induced by movement-associated synaptic activity can promote functional recovery from stroke. To test this hypothesis, we compared AMPAR expression on the postsynaptic membrane surface in a rat model of ischemic stroke induced by middle cerebral artery occlusion (MCAO) with versus without CIMT, which consisted of daily running wheel training for 2 weeks starting on day 7 after MCAO. The results showed that CIMT increased the number of glutamate receptor (GluR)2-containing functional synapses in the ipsilateral sensorimotor cortex, and reduced non-GluR2 AMPARs in the ipsilateral sensorimotor cortex and hippocampal CA3 region. In addition, CIMT enhanced AMPAR expression on the surface of post-synaptic membrane in the ipsilateral sensorimotor cortex and hippocampus. Thus, CIMT promotes the recovery of motor function of injured upper limbs following stroke by enhancing AMPAR-mediated synaptic transmission in the ischemic hemisphere. These findings provide supporting evidence for the clinical value of CIMT for restoring limb movement in stroke patients. All experimental procedures and protocols were approved by the Department of Laboratory Animal Science of Fudan University, China (approval No. 201802173S) on March 3, 2018.

Study on metabolic dynamics,metabolic enzyme phenotype and species difference of hepatic and intestinal microsome of psoralidin / 中国中药杂志

This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5'-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 μL·min~(-1)·mg~(-1),respectively,while those for G1 were 543. 3,and 75. 9 μL·min~(-1)·mg~(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 μL·min~(-1)·mg~(-1)),CYP2 C8( 88. 0 μL·min~(-1)·mg~(-1)),CYP2 C19( 166. 7 μL·min~(-1)·mg~(-1)),and CYP2 D6( 9. 1 μL·min~(-1)·mg~(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 μL·min~(-1)·mg~(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 μL·min~(-1)·mg~(-1)),UGT1 A7( 922. 8 μL·min~(-1)·mg~(-1)),UGT1 A8( 133. 0 μL·min~(-1)·mg~(-1)),UGT1 A9( 348. 6 μL·min~(-1)·mg~(-1)) and UGT2 B7( 118. 7 μL·min~(-1)·mg~(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 μL·min~(-1)·mg~(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.

[Effect of Modified Constraint-induced Movement Therapy on Neurotransmitter Levels of Motor Cortex in Cerebral Ischemia-Reperfusion Injured Rats].

OBJECTIVE: To study the effect and mechanism of modified constraint-induced movement therapy (mCIMT) on motor function recovery in cerebral ischemia-reperfusion rats. METHODS: The rats were randomly divided into the control group and the mCIMT group, with 12 rats in each group. The left middle cerebral artery occlusion (MCAO) model was established by the Longa suture method. In the mCIMT group, the rats started continuous training for 14 d on the 7 th day after modeling. The unaffected limb was tied to the chest with elastic bandages, and the affected limb was trained in the compulsory runner equipment. In the control group, rats moved freely in the cage. The body mass of rats was recorded within 20 d after modeling, and behavior was assessed by the foot-fault test. Some of the rats were euthanized 18 d after modeling, and high performance liquid chromatography (HPLC) was used to detect monoamine neurotransmitters (5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIVV), homovanillic acid (HVA) ), and amino acid neurotransmitters (glutamic acid (Glu), asparaginic acid (ASP), glutamine (Gln), glycine (Gly), taurine (Tau), gamma aminobutyric acid (GABA) ) in the motor cortex and striatum, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of total P70 ribosomal protein S6 kinase (p70s6k) and p70s6k phosphorylated protein (p-p70s6k) in motor cortex and striatum, respectively. RESULTS: Compared with the control group, the body mass of rats in the mCIMT group was comparable (P >0.05) within 21 d after modeling, foot-fault rate of the mCIMT group was significantly lower at 17 d after modeling (P<0.05). At 18 d after modeling, compared with the control group, the level of 5-HIVV in the motor cortex increased significantly (P<0.05), and the relative content of amino acid neurotransmitters (the ratio of Glu) in the motor cortex including Gln, Gly, Tau and GABA to Glu increased significantly (P<0.05 or P<0.01) except for decreased ASP/Glu (P<0.05). Moreover, compared with the control group, the expression of p-p70s6k in the motor cortex of the mCIMT was significantly decreased (P<0.05). There were no significant differences in monoamine neurotransmitters and amino acid neurotransmitters in the striatum between two groups (P>0.05). CONCLUSION: mCIMT improved the motor function of MCAO rats, and the mechanism might be related to the increase of amino acid neurotransmitters and 5-HIVV and decrease of p-p70s6k expression in the motor cortex.

Paired associated magnetic stimulation promotes neural repair in the rat middle cerebral artery occlusion model of stroke.

Paired associative stimulation has been used in stroke patients as an innovative recovery treatment. However, the mechanisms underlying the therapeutic effectiveness of paired associative stimulation on neurological function remain unclear. In this study, rats were randomly divided into middle cerebral occlusion model (MCAO) and paired associated magnetic stimulation (PAMS) groups. The MCAO rat model was produced by middle cerebral artery embolization. The PAMS group received PAMS on days 3 to 20 post MCAO. The MCAO group received sham stimulation, three times every week. Within 18 days after ischemia, rats were subjected to behavioral experiments-the foot-fault test, the balance beam walking test, and the ladder walking test. Balance ability was improved on days 15 and 17, and the foot-fault rate was less in their affected limb on day 15 in the PAMS group compared with the MCAO group. Western blot assay showed that the expression levels of brain derived neurotrophic factor, glutamate receptor 2/3, postsynaptic density protein 95 and synapsin-1 were significantly increased in the PAMS group compared with the MCAO group in the ipsilateral sensorimotor cortex on day 21. Resting-state functional magnetic resonance imaging revealed that regional brain activities in the sensorimotor cortex were increased in the ipsilateral hemisphere, but decreased in the contralateral hemisphere on day 20. By finite element simulation, the electric field distribution showed a higher intensity, of approximately 0.4 A/m2, in the ischemic cortex compared with the contralateral cortex in the template. Together, our findings show that PAMS upregulates neuroplasticity-related proteins, increases regional brain activity, and promotes functional recovery in the affected sensorimotor cortex in the rat MCAO model. The experiments were approved by the Institutional Animal Care and Use Committee of Fudan University, China (approval No. 201802173S) on March 3, 2018.

Modified constraint-induced movement therapy alters synaptic plasticity of rat contralateral hippocampus following middle cerebral artery occlusion.

Modified constraint-induced movement therapy is an effective treatment for neurological and motor impairments in patients with stroke by increasing the use of their affected limb and limiting the contralateral limb. However, the molecular mechanism underlying its efficacy remains unclear. In this study, a middle cerebral artery occlusion (MCAO) rat model was produced by the suture method. Rats received modified constraint-induced movement therapy 1 hour a day for 14 consecutive days, starting from the 7th day after middle cerebral artery occlusion. Day 1 of treatment lasted for 10 minutes at 2 r/min, day 2 for 20 minutes at 2 r/min, and from day 3 onward for 20 minutes at 4 r/min. CatWalk gait analysis, adhesive removal test, and Y-maze test were used to investigate motor function, sensory function as well as cognitive function in rodent animals from the 1st day before MCAO to the 21st day after MCAO. On the 21st day after MCAO, the neurotransmitter receptor-related genes from both contralateral and ipsilateral hippocampi were tested by micro-array and then verified by western blot assay. The glutamate related receptor was shown by transmission electron microscopy and the glutamate content was determined by high-performance liquid chromatography. The results of behavior tests showed that modified constraint-induced movement therapy promoted motor and sensory functional recovery in the middle cerebral artery-occluded rats, but had no effect on cognitive function. The modified constraint-induced movement therapy upregulated the expression of glutamate ionotropic receptor AMPA type subunit 3 (Gria3) in the hippocampus and downregulated the expression of the beta3-adrenergic receptor gene Adrb3 and arginine vasopressin receptor 1A, Avpr1a in the middle cerebral artery-occluded rats. In the ipsilateral hippocampus, only Adra2a was downregulated, and there was no significant change in Gria3. Transmission electron microscopy revealed a denser distribution the more distribution of postsynaptic glutamate receptor 2/3, which is an α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor, within 240 nm of the postsynaptic density in the contralateral cornu ammonis 3 region. The size and distribution of the synaptic vesicles within 100 nm of the presynaptic active zone were unchanged. Western blot analysis showed that modified constraint-induced movement therapy also increased the expression of glutamate receptor 2/3 and brain-derived neurotrophic factor in the hippocampus of rats with middle cerebral artery occlusion, but had no effect on Synapsin I levels. Besides, we also found modified constraint-induced movement therapy effectively reduced glutamate content in the contralateral hippocampus. This study demonstrated that modified constraint-induced movement therapy is an effective rehabilitation therapy in middle cerebral artery-occluded rats, and suggests that these positive effects occur via the upregulation of the postsynaptic membrane α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor expression. This study was approved by the Institutional Animal Care and Use Committee of Fudan University, China (approval No. 201802173S) on March 3, 2018.

Association of UGTs, ABCC2, SLCO1B1 and IMPDH Gene Polymorphisms with the Metabolism and Adverse Reactions of Mycophenolate Ester in Renal Transplant Patients / 中国药学杂志

OBJECTIVE: To investigate the association of UGTs, ABCC2, SLCO1B1 and IMPDH gene polymorphisms with metabolism and adverse reaction of mycophenolate ester in early renal transplant recipients. METHODS: A total of 233 patients who received tacrolimus+glucocorticoid+mycophenolate mofetil treatment after living kidney transplantation were enrolled. Single nucleotide polymorphisms of 13 gene were detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Kruskal-Wallis H test was used to analyze the effect of 13 single nucleotide polymorphisms on the exposure/dose (AUC/D) and adverse reactions of mycophenolic acid in early recipients after renal transplantation. RESULTS: The AUC/D of mycophenolic acid in UGT2B7 802C>T CC and CT genotype recipients was (36.03±16.19) and (38.06±15.41) mg•h•L-1/g•d-1, which was significantly lower than that in TT genotype recipients (43.63±15.10) mg•h•L-1/g•d-1 (P=0.021). The AUC/D of mycophenolic acid in SLCO1B1 521T > C TT, TC and CC genotype recipients were (36.78±15.70), (41.27±12.92) and (45.10±21.32) mg•h•L-1/g•d-1, respectively. There was significant difference between groups (P=0.036). There was no significant difference between 13 single nucleotide polymorphisms and adverse reactions (P>0.05). CONCLUSION: The AUC/D of mycophenolic acid in early recipients after kidney transplantation are associated with the polymorphism of UGT2B7 802C > T and SLCO1B1 521T > C.

Fluoxetine adjunct to therapeutic exercise promotes motor recovery in rats with cerebral ischemia: Roles of nucleus accumbens.

The study aimed to explore the molecular mechanism of fluoxetine as an adjunct to therapeutic exercise to improve motor recovery using a rat cerebral ischemic model with middle cerebral artery occlusion (MCAO). We hypothesized that the nucleus accumbens (NAc) may be one of the responding areas to fluoxetine where relevant elevations in 5-hydroxytryptamine (5-HT) and ΔFosB were associated with motor behavioral recovery. Male Sprague-Dawley rats were randomly divided into five groups: rats without intervention; rats that underwent MCAO without exercise or fluoxetine; rats that underwent MCAO treated only with fluoxetine; rats that underwent MCAO treated only with exercise; and rats that underwent MCAO treated with both exercise and fluoxetine. Motor function and motivation were assessed by the fault footsteps test and the forced swimming test. 5-HT level in the bilateral NAc and the expression of 5-HT2C receptor (5-HT2CR) and ΔFosB in the ipsilesional (left) NAc were measured. Correlation was explored by Pearson correlation analysis. Our results indicated that either treatment helped improve the grasp dexterity of the affected limb, motor motivation, and resilience to adverse environment in MCAO rats. The dual treatment with fluoxetine and exercise may hasten the recovery process. The dual treatment helped restore the balance of 5-HT level between the bilateral NAc by significantly increasing its level in the ipsilesional side. Either treatment could resume the expression of 5-HT2CR in the ipsilesional side of the NAc close to the normal level, which was correlated with motor recovery. The dual treatment significantly increased the expression of ΔFosB in the ipsilesional side of the NAc, which was correlated with the balance of 5-HT in the bilateral NAc, but not directly with motor recovery. In conclusion, the NAc may play an important role in driving physical motivation, which was possibly related to motor recovery after stroke. Fluoxetine may hasten the effectiveness of therapeutic exercise, possibly via regulating 5-HT and its receptors in the NAc.