Immunosensing of neuron-specific enolase based on signal amplification strategies via catalysis of ascorbic acid by heteropolysate COF.

In view of the importance of quantification of neuron-specific enolase (NSE), an electrochemical NSE immunosensor was developed. The sandwich voltammetric immunosensor utilized vinyl-functionalized crystalline covalent organic framework (COFTAPT-Dva) modified electrode to load lots of Ab1 via thiol-ene "click" reaction as matrix. A crystalline cationic EB-COF:Br was used to load Au nanoparticles (AuNPs) and H3[PMo12O40] (PMo12) as immunoprobe. The AuNPs with the size of about 30 nm were firstly grown on EB-COF:Br and then a large number of electroactive PMo12 were uniformly assembled on AuNPs/EB-COF:Br via ion exchanging reaction. The AuNPs not only facilitated the bonding of Ab2 based on Au-S bond, but also improved performance of Ab2/AuNPs/EB-COF:PMo12 immunoprobe. The sensitivity of sandwich electrochemical immunosensor could be primarily amplified based on loaded abundant PMo12. Secondary sensitivity amplification of immunosensor could be achieved by using PMo12 to catalyze ascorbic acid. The linear range of sandwich voltammetric immunosensor based on current change of differential pulse voltammetry is 500 ± 36 fg mL-1 - 100 ± 8 ng mL-1. Thanks to the dual sensitivity amplification strategy, the sensitivity is as high as 54.06 ± 3.2 µA cm-2/lg(cNSE/ng mL-1), and the detection limit is as low as 166 ± 10.8 fg mL-1. It proves that it is completely feasible to amplify sensitivity of sandwich voltammetric immunosensors using polyoxometalate-COF and its catalytic substrate.

A Single Amino Acid Replacement Boosts the Analgesic Activity of α-Conotoxin AuIB through the Inhibition of the GABABR-Coupled N-Type Calcium Channel.

α-conotoxin AuIB is the only one of the 4/6 type α-conotoxins (α-CTxs) that inhibits the γ-aminobutyric acid receptor B (GABABR)-coupled N-type calcium channel (CaV2.2). To improve its inhibitory activity, a series of variants were synthesized and evaluated according to the structure-activity relationships of 4/7 type α-CTxs targeting GABABR-coupled CaV2.2. Surprisingly, only the substitution of Pro7 with Arg results in a 2-3-fold increase in the inhibition of GABABR-coupled CaV2.2 (IC50 is 0.74 nM); substitutions of position 9-12 with basic or hydrophobic amino acid and the addition of hydrophobic amino acid Leu or Ile at the second loop to mimic 4/7 type α-CTxs all failed to improve the inhibitory activity of AuIB against GABABR-coupled CaV2.2. Interestingly, the most potent form of AuIB[P7R] has disulfide bridges of "1-4, 2-3" (ribbon), which differs from the "1-3, 2-4" (globular) in the isoforms of wildtype AuIB. In addition, AuIB[P7R](globular) displays potent analgesic activity in the acetic acid writhing model and the partial sciatic nerve injury (PNL) model. Our study demonstrated that 4/6 type α-CTxs, with the disulfide bridge connectivity "1-4, 2-3," are also potent inhibitors for GABABR-coupled CaV2.2, exhibiting potent analgesic activity.

A Paper-Based Electrochemical Sensor Based on PtNP/COFTFPB-DHzDS@rGO for Sensitive Detection of Furazolidone.

Herein, a paper-based electrochemical sensor based on PtNP/COFTFPB-DHzDS@rGO was developed for the sensitive detection of furazolidone. A cluster-like covalent organic framework (COFTFPB-DHzDS) was successfully grown on the surface of amino-functional reduced graphene oxide (rGO-NH2) to avoid serious self-aggregation, which was further loaded with platinum nanoparticles (PtNPs) with high catalytic activity as nanozyme to obtain PtNP/COFTFPB-DHzDS@rGO nanocomposites. The morphology of PtNP/COFTFPB-DHzDS@rGO nanocomposites was characterized, and the results showed that the smooth rGO surface became extremely rough after the modification of COFTFPB-DHzDS. Meanwhile, ultra-small PtNPs with sizes of around 1 nm were precisely anchored on COFTFPB-DHzDS to maintain their excellent catalytic activity. The conventional electrodes were used to detect furazolidone and showed a detection limit as low as 5 nM and a linear range from 15 nM to 110 µM. In contrast, the detection limit for the paper-based electrode was 0.23 µM, and the linear range was 0.69-110 µM. The results showed that the paper-based electrode can be used to detect furazolidone. This sensor is a potential candidate for the detection of furazolidone residue in human serum and fish samples.

A novel ω-conotoxin Bu8 inhibiting N-type voltage-gated calcium channels displays potent analgesic activity.

ω-Conotoxins inhibit N-type voltage-gated calcium (CaV2.2) channels and exhibit efficacy in attenuating neuropathic pain but have a low therapeutic index. Here, we synthesized and characterized a novel ω-conotoxin, Bu8 from Conus bullatus, which consists of 25 amino acid residues and three disulfide bridges. Bu8 selectively and potently inhibits depolarization-activated Ba2+ currents mediated by rat CaV2.2 expressed in HEK293T cells (IC50 = 89 nmol/L). Bu8 is two-fold more potent than ω-conotoxin MVIIA, a ω-conotoxin currently used for the treatment of severe chronic pain. It also displays potent analgesic activity in animal pain models of hot plate and acetic acid writhing but has fewer side effects on mouse motor function and lower toxicity in goldfish. Its lower side effects may be attributed to its faster binding rate and higher recovery ratios. The NMR structure demonstrates that Bu8 contains a small irregular triple ß-strand. The structure-activity relationships of Bu8 Ala mutants and Bu8/MVIIA hybrid mutants demonstrate that the binding mode of CaV2.2 with the amino acid residues in loop 1 and loop 2 of Bu8 is different from that of MVIIA. This study characterizes a novel, more potent ω-conotoxin and provides new insights for designing CaV2.2 antagonists.

Competitive coordination of the dual roles of the Hedgehog co-receptor in homophilic adhesion and signal reception.

Hedgehog (Hh) signaling patterns embryonic tissues and contributes to homeostasis in adults. In Drosophila, Hh transport and signaling are thought to occur along a specialized class of actin-rich filopodia, termed cytonemes. Here, we report that Interference hedgehog (Ihog) not only forms a Hh receptor complex with Patched to mediate intracellular signaling, but Ihog also engages in trans-homophilic binding leading to cytoneme stabilization in a manner independent of its role as the Hh receptor. Both functions of Ihog (trans-homophilic binding for cytoneme stabilization and Hh binding for ligand sensing) involve a heparin-binding site on the first fibronectin repeat of the extracellular domain. Thus, the Ihog-Ihog interaction and the Hh-Ihog interaction cannot occur simultaneously for a single Ihog molecule. By combining experimental data and mathematical modeling, we determined that Hh-Ihog heterophilic interaction dominates and Hh can disrupt and displace Ihog molecules involved in trans-homophilic binding. Consequently, we proposed that the weaker Ihog-Ihog trans interaction promotes and stabilizes direct membrane contacts along cytonemes and that, as the cytoneme encounters secreted Hh ligands, the ligands trigger release of Ihog from trans Ihog-Ihog complex enabling transport or internalization of the Hh ligand-Ihog-Patched -receptor complex. Thus, the seemingly incompatible functions of Ihog in homophilic adhesion and ligand binding cooperate to assist Hh transport and reception along the cytonemes.

A role of color vision in emmetropization in C57BL/6J mice.

Spectral composition affects emmetropization in both humans and animal models. Because color vision interacts the effects of chromatic defocus, we developed a method to bypass the effects of longitudinal chromatic aberration by placing a spectral filter behind the optics of the eye, using genetic tools. Newborn C57BL/6J (B6) mice were reared in quasi-monochromatic red (410-510 nm) or blue (585-660 nm) light beginning before eye-opening. Refractive states and ocular dimensions were compared at 4, 6, 8, and 10 weeks with mice reared in normal white light. Cre recombinase-dependent Ai9 reporter mice were crossed with Chx10-Cre to obtain Chx10-Cre;Ai9 mice, expressing red fluorescent protein in retinal Cre-positive cells. Ai9 offsprings, with and without Cre, were reared under a normal visual environment. Refraction and axial components were measured as described above. Expression levels of M and S opsin were quantified by western blotting at 10 weeks. Compared with those reared in white light, B6 mice reared in red light developed relative hyperopia, principally characterized by flattening of corneal curvature. Emmetropization was not affected by blue light, possibly because the reduction in vitreous chamber depth compensated for the increase in corneal curvature. Compared with Cre-negative littermates, the refraction and axial dimensions of Chx10-Cre;Ai9 mice were not significantly different at the follow-up timepoints. M opsin levels were higher in Chx10-Cre;Ai9 mice at 10 weeks while S opsin levels were not different. Red light induced a hyperopic shift in mouse refractive development. Emmetropization was not impacted in mice with perturbed color vision caused by intrinsic red-fluorescent protein, suggesting that color vision may not be necessary in mouse emmetropization when other mechanisms are present.

Effects of the Tyrosinase-Dependent Dopaminergic System on Refractive Error Development in Guinea Pigs.

Purpose: To determine if myopia in albino guinea pigs is linked to altered ocular dopamine (DA) levels in both the retinal and uveal dopaminergic systems. Methods: Retina and retinal pigment epithelium (RPE)/choroid were dissected from eyes of 2-week-old albino myopic (AM) and pigmented hyperopic (PH) guinea pigs. The levels of DA, dihydroxy-phenyl acetic acid (DOPAC), and homovanillic acid (HVA) were determined. Tyrosine hydroxylase (TH) and tyrosinase activities were also measured. PH animals received daily unilateral peribulbar injections of either kojic acid (tyrosinase inhibitor) or vehicle for 2 to 4 weeks. Refractive errors and ocular axial dimensions were measured by eccentric infrared photoretinoscopy and A-scan ultrasonography. Results: Retinal DA levels were similar between the two strains, but AM eyes had higher levels of DOPAC. RPE/choroid DA and tyrosinase activity in AM eyes were lower than in PH eyes (P < 0.01); however, the DA turnover was higher (P < 0.05). After 2 weeks of kojic acid treatment, PH eyes developed significant myopia, accompanied by elongated vitreous chambers and axial lengths. Inhibition of tyrosinase activity was linearly correlated with a myopic refraction shift (R = 0.79, P < 0.01). PH animals that received 62.5 ng/mL kojic acid treatment daily for 2 weeks followed by 625 ng/mL for 2 more weeks became more myopic and had deeper anterior chambers compared to those that received the 62.5 ng/mL dose over this period (P = 0.04). Conclusions: The uveal tyrosinase-dependent dopaminergic system is involved in the development of guinea pig refraction. Enhancing uveal tyrosinase activity might slow down the development of myopia.

Prostaglandin F2α Receptor Modulation Affects Eye Development in Guinea Pigs.

Retinal arachidonic acid (ARA) levels in form-deprived eyes decline in guinea pigs. As prostaglandin F2α (PGF2α) is an ARA metabolite and endogenous agonist of prostaglandin F receptor (FP), we have been suggested that down-regulation of PGF2α-FP receptor signalling pathway contributes to myopia onset. To test this hypothesis, this study determines whether: (i) retinal PGF2α levels decline during the development of form deprivation myopia (FDM) in guinea pigs; (ii) FP receptor agonism and antagonism alter emmetropization and myopia development. Pigmented guinea pigs were randomly assigned to normal vision and form-deprived groups. Ultraperformance liquid chromatography coupled with a mass spectrometer (UPLC-MS) measured retinal PGF2α levels 2 weeks after form deprivation (FD). The selective FP agonist, latanoprost acid (LAT) and its corresponding antagonist, AL8810, were peribulbarly injected into each group. An eccentric infrared photorefractor (EIR) monitored refraction. A-scan ultrasonography measured axial elongation (AL) and vitreous chamber depth (VCD). Tonometry measured the intraocular pressure (IOP). Retinal PGF2α levels declined in form-deprived eyes compared to those in normal eyes. Neither LAT nor AL8810 affected IOP with or without FD. On the other hand, after 4 weeks of daily 0.5 µg AL8810 treatment, a myopia of -1.99 ± 0.34 dioptre (D) developed, but LAT had no effect on emmetropization in a normal visual environment. Nevertheless, daily 30 µg LAT treatment for 4 weeks inhibited FDM development by 41% (vehicle control: -8.39 ± 0.45 D; LAT: -4.95 ± 0.39 D; two-way anova with repeated measures, p < 0.05). Down-regulation of PGF2α-FP receptor signalling pathway may contribute to myopia onset as retinal PGF2α declined in myopic eyes and antagonism of FP receptor by AL8810 induced a myopic shift in normal vision environment. Meanwhile, up-regulation of this pathway by LAT inhibited FDM development. However, the mechanism underlying LAT-induced FDM inhibition needs further clarification. This uncertainty exists because its inhibition of FDM suggests that LAT strengthens the scleral framework which reduces axial elongation. On the other hand, its IOP-lowering effect is attributed to thinning and weakening the scleral framework in glaucoma treatment.

Are gastric mucosal macrophages responsible for gastric injury in acute pancreatitis?

AIM: To investigate the protective effect of clodronate-containing liposomes against severe acute pancreatitis (SAP)-triggered acute gastric mucosal injury (AGMI) in rats. METHODS: Clodronate- and phosphate-buffered saline (PBS)-containing liposomes were prepared by reverse-phase evaporation. The SAP rat model was established by injecting sodium taurocholate into the pancreatic subcapsular space. Sprague-Dawley rats were randomly divided into three groups: control (C), SAP plus PBS-containing liposome (P) and SAP plus clodronate-containing liposome (T). Serum tumor necrosis factor (TNF)-α levels were estimated by ELISA. Pathological changes in the gastric mucosa and pancreas were observed by hematoxylin and eosin (HE) staining. Apoptotic cells were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The numbers of macrophages in the gastric mucosa were analyzed by CD68 immunohistochemical staining. RESULTS: The liposomes had a mean diameter of 150 ± 30 nm. The TNF-α levels were significantly higher in the P group than that in the C group (2 h, 145.13 ± 11.50 vs 23.2 ± 2.03; 6 h, 245.06 ± 12.11 vs 30.28 ± 6.07, P < 0.05), and they were significantly lower in the T group than that in the P group (2 h, 93.24 ± 23.11 vs 145.13 ± 11.50; 6 h, 135.18 ± 13.10 vs 245.06 ± 12.11, P < 0.05). The pathological scores of the pancreas were lower in the T group than in the P group (2 h, 1.88 ± 0.83 vs 4.13 ± 0.83; 6 h, 2.87 ± 0.64 vs 6.25 ± 0.88, P < 0.01). The pathological scores of the gastric mucosa were also lower in the T group than in the P group (2 h, 1.12 ± 0.64 vs 2 ± 0.75; 6 h, 1.58 ± 0.53 vs 3 ± 1.31, P < 0.05). In addition, increased CD68 levels were observed in the gastric mucosa of the P group compared with the C group. Clodronate-containing liposomes decreased the CD68 levels in the mucosa of the T group. The apoptotic indexes of the gastric mucosa were higher in the T group than in the P group (2 h, 15.7 ± 0.92 vs 11.5 ± 1.64; 6 h, 21.12 ± 1.06 vs 12.6 ± 2.44, P < 0.01). CONCLUSION: Gastric macrophages contribute to the pathogenesis of gastric injury in SAP. Clodronate-containing liposomes have protective effects against AGMI in rats with SAP.

Clodronate-superparamagnetic iron oxide-containing liposomes attenuate renal injury in rats with severe acute pancreatitis.

BACKGROUND AND OBJECTIVE: It has been shown that macrophages play an important role in the development of severe acute pancreatitis (SAP), and eventually lead to multiple organ failure (MOF). Clodronate-liposome selectively depleted macrophages. This study was to investigate the role of renal macrophage infiltration in acute renal injury in rats with SAP and to evaluate the potential of superparamagnetic iron oxide (SPIO)-enhanced magnetic resonance imaging (MRI) for diagnosis. METHODS: Superparamagnetic Fe3O4 nanoparticles were prepared by chemical coprecipitation. SPIO-liposomes and SPIO-clodronate-liposomes were prepared by the thin film method. SAP models were prepared by injection of sodium taurocholate into the subcapsular space of rat pancreas. Sprague-Dawley rats were randomly divided into a control group, SAP plus SPIO-liposome (P) group, and SAP plus SPIO-clodronate-containing liposome (T) group. Kidney injury was evaluated by T2-weighted MRI scan. The levels of serum amylase (SAM), blood urea nitrogen (BUN), and serum creatinine (SCr) were measured by an automated enzymatic method. Serum tumor necrosis factor-α (TNF-α) was measured by enzyme-linked immunosorbent assay (ELISA). Pathological changes in the pancreas and kidney were observed using hematoxylin and eosin (H&E) staining, while cell apoptosis was detected with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. In addition, the macrophage markers (CD68) of the renal tissue were detected with immunohistochemistry. RESULTS: The pathological changes in the pancreas and kidneys of rats in the T group were milder than those in the P group. The MRI signal intensity of the kidneys in the P and T groups was significantly lower than that in the control group. There were significant changes in the two experimental groups (P<0.01). The levels of SAM, Bun, SCr, and TNF-α in rats in the P group were higher than those in the control group (P<0.01) and in the T group (P<0.01). The apoptosis of the kidney in the T group was higher than that in the P group at 2 and 6 h (P<0.01). CONCLUSIONS: Clodronate-containing liposomes protected against renal injury in SAP rats, and SPIO can be used as a tracer for MRI examination to detect renal injury in SAP rats. SPIO-aided MRI provided an efficient non-invasive way to monitor the migration of macrophages after renal injury in rats with SAP.

Detection of serum autoantibodies against AT1A-receptor during the development of the four types of hypertensive rat models.

Using two-kidney one-clip renal hypertensive (2K1C group), stress-induced hypertensive (neural group), DOCA-salt treated hypertensive (DOCA group) and spontaneously hypertensive rats (SHR group), to investigate the change in AT(1A)-receptor autoantibodies (AT(1A)-AAs) during the development of the four types of hypertension. The biological activities of AT(1A)-AAs were examined. It was shown that the frequency of occurrence and titres of AT(1A)-AAs increased significantly during the development of hypertension. In the four hypertensive groups studied, the occurrence of AT(1A)-AAs was most prominent in SHR, 2K1C and neural groups. The biological effects of AT(1A)-AAs were shown to increase the beating frequency of cultured neonatal myocardial and vascular contractile tension. It is suggested that autoimmune mechanisms are involved the pathogenesis of different types of hypertension and the AT(1A)-AAs may be one of the mechanisms leading to cardiac hypertrophy.

Changes in autoantibodies against beta1-adrenoceptor and M2-muscarinic receptor during development of renovascular hypertension in rats.

In an experimental rat's renovascular hypertension model, we studied the genesis of anti-cardiac beta1-adrenoceptor and M2-muscarinic receptor autoantibodies in relation to the changes in immunological function during the development of renal hypertension. The biological activities of these autoantibodies were also examined. It was shown that after two weeks of operation both the frequency of occurrence and the titre of autoantibodies to cardiac beta1-adrenoceptor and M2-muscarinic receptor were significantly increased as compared with the control of pre-treatment. The increased autoantibodies lasted for several weeks and then automatically decreased gradually to the pre-clipping level at 10 weeks. Meanwhile the ratio of CD4+/CD8+ was also undergone an initial increase followed by gradual recovery and correlated well with the changes in antibody titre. The biological effects of these autoantibodies displayed an "gonistic-like" activities on the beating frequency of cultured neonatal cardiomyocyte. It is suggested that autoimmune mechanisms are involved in the pathogenesis of renal hypertension and the cardiac receptor autoantibodies might be one of the mechanisms leading to cardiac dysfunction.

Comparative studies of vasodilating effects of angiotensin-(1-7) on the different vessels.

The purpose of this study was to compare the vasodilating effects of angiotensin-(1-7) [Ang-(1-7)] on the different vessels and to clarify its mechanisms by using relaxing responses of preconstricted vascular rings. The results showed: (1) Ang-(1-7) dose-dependently induced vasorelaxation in all the vessels studied. However, there is apparent heterogeneity in the responsiveness of vessels from different origin. (2) The Ang-(1-7)-induced vasorelaxation was endothelium dependent and largely mediated by NO system. (3) The vasodilator action of Ang-(1-7) was not mediated by AT1 or AT2 receptor subtypes. It is suggested that the Ang-(1-7)-induced vasorelaxation is endothelium dependent by some other unclarified angiotensin receptor subtypes and is largely mediated by NO system.

[Molecular mechanism of cleft palate induced by retinoic acid].

OBJECTIVE: To explore the molecular mechanism of cleft palate induced by chemicals. METHODS: Retinoic acid was used as a known teratogen to induce cleft palate in ICR mice and a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes that related to cleft palate of ICR mice. RESULTS: 14 reverse differently and 9 forward differentially expressed clones were obtained. Some clones were selected to be sequenced and aligned to GenBank. CONCLUSION: In this study, suppressed Gpc3 and Insulin-Induced protein 1 could affect growth of palate shelves and resulted in cleft palate by reducing the size of the palate shelves. Down-regulation of Ptprs interfered with a cell signal pathway and down-regulation of Tn C inhibited the cell de-adhesion and expression of Egfr, then suppressed Egfr prevented the normal expression of MMPs that influenced the medial edge epithelium disruption and caused cleft palate. Tn C could bind to Ptprs and Gpcs, and HSPGs were ligands for Ptrps. Up-regulate of Rps25 might play a role in cleft palate by excessively apoptosis.