Identification and effective regulation of scarb1 gene involved in pigmentation change in autotetraploid Carassius auratus

The autotetraploid Carassius auratus(4nRR,4n=200,RRRR)is derived from whole-genome duplication of Carassius auratus red var.(RCC,2n=100,RR).In the current study,we demonstrated that chromatophores and pigment changes directly caused the coloration and variation of 4nRR skin(red in RCC,brownish-yellow in4nRR).To further explore the molecular mechanisms underlying coloration formation and variation in 4nRR,we performed transcriptome profiling and molecular functional verification in RCC and 4nRR.Results revealed that scarb1,associated with carotenoid metabolism,underwent significant down-regulation in 4nRR.Efficient editing of this candidate pigment gene provided clear evidence of its significant role in RCC coloration.Subsequently,we identified four divergent scarb1 homeologs in 4nRR:two original scarb1 homeologs from RCC and two duplicated ones.Notably,three of these homeologs possessed two highly conserved alleles,exhibiting biased and allelespecific expression in the skin.Remarkably,after precise editing of both the original and duplicated scarb1homeologs and/or alleles,4nRR individuals,whether singly or multiply mutated,displayed a transition from brownishyellow skin to a cyan-gray phenotype.Concurrently,the proportional areas of the cyan-gray regions displayed a gene-dose correlation.These findings illustrate the subfunctionalization of duplicated scarb1,with all scarb1genes synergistically and equally contributing to the pigmentation of 4nRR.This is the first report concerning the functional differentiation of duplicated homeologs in an autopolyploidfish,substantiallyenrichingour understanding of coloration formation and change within this group of organisms.

Role of HCN channels in the nervous system:membrane excitability and various modulations

Hyperpolarization-activated and cyclic nucleotide-gated(HCN) channels,distributing in a variety of tissues,especially in excitable cells such as heart cells and many kinds of neurons,have an important role in the modulation of heart rate and neuronal excitability.Different from typical voltage-gated sodium channels and potassium channels,HCN channels were evoked inward currents when the cell was hyperpolarized.More and more recent studies have disclosed that HCN channels play important roles in the nervous system,which were linked with its special electrophysiological features as well as its regulatory effect on the cellular membrane excitability.HCN channels could be modulated by many factors including both extracellular molecules and intracellular signaling cascades,which made its functions complicated in the different condition.Based on its role,HCN channels are presumed to be a promising target for chronic pain and brain disorders.In this paper,we will focus on the advancement of roles of HCN channels in the neural system as well as its complex modulator factors.

Microinjection of a growth factor cocktail affects activated microglia in the neocortex of adult rats

Microglia, as the resident immune cells in the central nervous system, play important roles in regulating neuronal processes, such as neural excitability, synaptic activity, and apoptotic cell clearance. Growth factors can activate multiple signaling pathways in central nervous system microglia and can regulate their immune effects, but whether growth factors can affect the morphological characteristics and ultrastructure of microglia has not been reported. After microinjecting 300 nL of a growth factor cocktail, including 10 μg/mL epidermal growth factor, 10 μg/mL basic fibroblast growth factor, 10 μg/mL hepatocyte growth factor and 10 μg/mL insulin-like growth factor into adult rat cortex, we found that the number of IBA1-positive microglia around the injection area increased significantly, indicating local activation of microglia. All CD68-positive labeling co-localized with IBA1 in microglia. Cell bodies and protrusions of CD68-positive cells were strongly attached to or were engulfing neurons. Characteristic huge phagosomes were observed in activated phagocytes by electron microscopy. The phagosomes generally included non-degraded neuronal protrusions and mitochondria, yet they contained no myelin membrane or remnants, which might indicate selective phagocytosis by the phagocytes. The remnant myelin sheath after phagocytosis still had regenerative ability and formed "myelin-like" structures around phagocytes. These results show that microinjection of a growth factor cocktail into the cerebral cortex of rodents can locally activate microglia and induce selective phagocytosis of neural structures by phagocytes. The study was approved by the Institute of Laboratory Animal Science, Beijing Institute of Basic Medical Sciences(approval No. IACUC-AMMS-2014-501) on June 30, 2014.

No DCX-positive neurogenesis in the cerebral cortex of the adult primate

Whether endogenous neurogenesis occurs in the adult cortex remains controversial.An increasing number of reports suggest that doublecortin(DCX)-positive neurogenesis persists in the adult primate cortex,attracting enormous attention worldwide.In this study,different DCX antibodies were used together with NeuN antibodies in immunohistochemistry and western blot assays using adjacent cortical sections from adult monkeys.Antibody adsorption,antigen binding,primary antibody omission and antibody-free experiments were used to assess specificity of the signals.We found either strong fluorescent signals,medium-weak intensity signals in some cells,weak signals in a few perikarya or near complete lack of labeling in adjacent cortical sections incubated with the various DCX antibodies.The putative DCX-positive cells in the cortex were also positive for NeuN,a specific marker of mature neurons.However,further experiments showed that most of these signals were either the result of antibody cross reactivity,the non-specificity of secondary antibodies or tissue autofluorescence.No confirmed DCX-positive cells were detected in the adult macaque cortex by immunofluorescence.Our findings show that DCX-positive neurogenesis does not occur in the cerebral cortex of adult primates,and that false-positive signals(artefacts)are caused by antibody cross reactivity and autofluorescence.The experimental protocols were approved by the Institutional Animal Care and Use Committee of the Institute of Neuroscience,Beijing,China(approval No.IACUC-AMMS-2014-501).

Central projections and connections of lumbar primary afferent fibers in adult rats:effectively revealed using Texas red-dextran amine tracing

Signals from lumbar primary afferent fibers are important for modulating locomotion of the hind-limbs.However,silver impregnation techniques,autoradiography,wheat germ agglutinin-horseradish peroxidase and cholera toxin B subunit-horseradish peroxidase cannot image the central projections and connections of the dorsal root in detail.Thus,we injected 3-k Da Texas red-dextran amine into the proximal trunks of L4 dorsal roots in adult rats.Confocal microscopy results revealed that numerous labeled arborizations and varicosities extended to the dorsal horn from T12–S4,to Clarke＇s column from T10–L2,and to the ventral horn from L1–5.The labeled varicosities at the L4 cord level were very dense,particularly in laminae I–Ⅲ,and the density decreased gradually in more rostral and caudal segments.In addition,they were predominately distributed in laminae I–IV,moderately in laminae V–VⅡ and sparsely in laminae VⅢ–X.Furthermore,direct contacts of lumbar afferent fibers with propriospinal neurons were widespread in gray matter.In conclusion,the projection and connection patterns of L4 afferents were illustrated in detail by Texas red-dextran amine-dorsal root tracing.

A Gain-of-Function Mutation in OslAA11 Affects Lateral Root Development in Rice

Lateral roots are important to plants for the uptake of nutrients and water. Several members of the Aux/IAA family have been shown to play crucial roles in lateral root development. Here, a member of the rice Aux/IAA family genes, OslAA 11 （LOC_Os03g43400）, was isolated from a rice mutant defective in lateral root development. The gain-of-function mutation in OslAAll strictly blocks the initiation of lateral root primordia, but it does not affect crown root development. The expression of OslAAll is defined in root tips, lateral root caps, steles, and lateral root primordia. The auxin reporter DR5-GUS （^-glucuronidase） was expressed at lower levels in the mutant than in wild-type, indicating that OslAAll is involved in auxin signaling in root caps. The transcript abundance of both OsPINlb and OsPIN10a was diminished in root tips of the Osiaa 11 mutant. Taken together, the results indicate that the gain-of-function mutation in OslAA 11 caused the inhibition of lateral root development in rice.

Renal Phospholipase A2 Receptor and the Clinical Features of Idiopathic Membranous Nephropathy

Background： According to the renal phospholipase A2 receptor （PLA2R） immunohistochemistry, idiopathic membranous nephropathy （iMN） could be categorized into PLA2R-associated and non-PLA2R-associated iMN. This study aimed to examine whether the non-PLA2R-associated iMN had any difference in clinical features compared with PLA2R-associated iMN. Methods： A total of 231 adult patients diagnosed as iMN were recruited to this retrospective study. Renal PLA2R expression was examined by immunofluorescence. Among these patients, 186 （80.5%） with complete baseline clinical data were used for further study. Urinary protein excretion, serum albumin, and creatinine were analyzed. For those patients with follow-up longer than 1 year, the relationship between PLA2R and response to immunosuppressants were analyzed. The t-test was used for parametric analysis and the Mann-Whitney U-test was used for nonparametric analysis. Categorical variables were described as frequencies or percentages, and the data were analyzed with Pearson＇s Chi-squaxe test or Fisher＇s exact test. Results： Of the 231 iMN patients, 189 showed renal detectable PLA2R expression （81.8%）. The baseline serum creatinine, serum albumin, and urine protein excretion were not significantly different between PLA2R-associated （n = 145） and non-PLA2R-associated iMN patients （n = 41）. However, about 1/3 of the non-PLA2R-associated iMN had abnormal serological tests, significantly more common than PLA2R-associated iMN （31.7% vs. 8.3%, P = 0.000）. The non-PLA2R-associated iMN had lower C4 levels compared with PLA2R-associated iMN （P = 0.004）. The non-PLA2R-associated iMN patients also showed a better response to immunosuppressants （complete remission [CR] 42.9%; partial remission [PR] 14.3%） compared with PLA2R-associated iMN （CR 3.2%; PR 48.4%, P = 0.004） at the 3rd month. Conclusions： There were no significant differences in serum creatinine, albumin, and urine protein excretion between PLA2R-associated and non-PLA2R-associated iMN, while the non-PLA2R-associated iMN patients showed more abnormal serological tests. The non-PLA2R-associated iMN seemed to respond more quickly to the immunosuppressive therapy compared with P LA2R-associated iMN.

A rare case of spontaneous renal cholesterol crystallization embolism

To the Editor: Cholesterol crystal embolism (CCE) is a systemic disease caused by cholesterol crystal embolization leading to the occlusion of small arteries in a variety of organs. It may occur spontaneously, or more often, after intravascular procedures such as coronary angiography and carotid artery stenting, cardiovascular surgery such as coronary artery bypass grafting or aortic aneurysm surgery.[1] The kidneys were previously reported to be the most frequent target organ for CCE, and renal CCE was found during the course of clinical illness in approximately 50% of patients with CCE.

Identification of endothelial protein C receptor as a novel druggable agonistic target for reendothelialization promotion and thrombosis prevention of eluting stent

The commercially available drug-eluting stent with limus (rapamycin, everolimus, etc.) or paclitaxel inhibits smooth muscle cell (SMC), reducing the in-stent restenosis, whereas damages endothelial cell (EC) and delays stent reendothelialization, increasing the risk of stent thrombosis (ST) and sudden cardiac death. Here we present a new strategy for promoting stent reendothelialization and preventing ST by exploring the application of precise molecular targets with EC specificity. Proteomics was used to investigate the molecular mechanism of EC injury caused by rapamycin. Endothelial protein C receptor (EPCR) was screened out as a crucial EC-specific effector. Limus and paclitaxel repressed the EPCR expression, while overexpression of EPCR protected EC from coating (eluting) drug-induced injury. Furthermore, the ligand activated protein C (APC), polypeptide TR47, and compound parmodulin 2, which activated the target EPCR, promoted EC functions and inhibited platelet or neutrophil adhesion, and enhanced rapamycin stent reendothelialization in the simulated stent environment and in vitro. In vivo, the APC/rapamycin-coating promoted reendothelialization rapidly and prevented ST more effectively than rapamycin-coating alone, in both traditional metal stents and biodegradable stents. Additionally, overexpression or activation of the target EPCR did not affect the cellular behavior of SMC or the inhibitory effect of rapamycin on SMC. In conclusion, EPCR is a promising therapeutical agonistic target for pro-reendothelialization and anti-thrombosis of eluting stent. Activation of EPCR protects against coating drugs-induced EC injury, inflammatory cell, or platelet adhesion onto the stent. The novel application formula for APC/rapamycin-combined eluting promotes stent reendothelialization and prevents ST.