Histone derived antimicrobial peptides identified from Mytilus coruscus serum by peptidomics.

Histones and their N-terminal or C-terminal derived peptides have been studied in vertebrates and presented as potential antimicrobial agents playing important roles in the innate immune defenses. Although histones and their derived peptides had been reported as components of innate immunity in invertebrates, the knowledge about the histone derived antimicrobial peptides (HDAPs) in invertebrates are still limited. Using a peptidomic technique, a set of peptide fragments derived from the histones was identified in this study from the serum of microbes challenged Mytilus coruscus. Among the 85 identified histone-derived-peptides with high confidence, 5 HDAPs were chemically synthesized and the antimicrobial activities were verified, showing strong growth inhibition against Gram-positive bacteria, Gram-negative bacteria, and fungus. The gene expression level of the precursor histones matched by representative HDAPs were further tested using q-PCR, and the results showed a significant upregulation of the histone gene expression levels in hemocytes, gill, and mantle of the mussel after immune stress. In addition, three identified HDAPs were selected for preparation of specific antibodies, and the corresponding histones and their derived C-terminal fragments were detected by Western blotting in the blood cell and serum of immune challenged mussel, respectively, indicating the existence of HDAPs in M. coruscus. Our findings revealed the immune function of histones in Mytilus, and confirmed the existence of HDAPs in the mussel. The identified Mytilus HDAPs represent a new source of immune effector with antimicrobial function in the innate immune system, and thus provide promising candidates for the treatment of microbial infections in aquaculture and medicine.

Effects of vitamin C on the pharmacokinetics and pharmacodynamics of nimodipine in rats.

In recent years, researchers have shown a growing interest in the interactions between different pharmaceutical agents. An intriguing instance lies in the possible interaction between nimodipine and vitamin C. To investigate the pharmacokinetic and pharmacodynamic effects of vitamin C on nimodipine in rats, rats were randomly divided into a nimodipine only group and a combination group (nimodipine + vitamin C). The two groups were given intragastric administration and nimodipine blood concentrations were determined using high-performance liquid chromatography-tandem mass spectrum at different time points. Blood pressure and heart rate were measured via carotid artery cannulation. Pharmacokinetic differences were observed between the nimodipine only group and the combination group at the same dose. Compared with the nimodipine only group, the combination group's main pharmacokinetic parameters of peak concentration and area under the curve increased significantly, and the difference was statistically significant (p < 0.05); furthermore, the combination group exhibited a significant reduction in average blood pressure, while no significant effects on heart rate were observed. Vitamin C did not affect the activity of CYP450 in rat liver. The pharmacokinetic characteristics and pharmacodynamics of nimodipine were changed by vitamin C administration in rats.

Myticofensin, a novel antimicrobial peptide family identified from Mytilus coruscus.

In this study, seven transcripts representing a novel antimicrobial peptide (AMP) family with structural features similar to those of arthropod defensins were identified from Mytilus coruscus. These novel defensins from the Mytilus AMP family were named myticofensins. To explore the possible immune-related functions of these myticofensins, we examined their expression profiles in different tissues and larval stages, as well as in three immune-related tissues under the threat of different microbes. Our data revealed that the seven myticofensins had relatively high expression levels in immune-related tissues. Most myticofensins were undetectable, or had low expression levels, in different larval mussel stages. Additionally, in vivo microbial challenges significantly increased the expression levels of myticofensins in M. coruscus hemocytes, gills, and digestive glands, showing different immune response patterns under challenges from different microbes. Our data indicates that different myticofensins may have different immune functions in different tissues. Furthermore, peptide sequences corresponding to the beta-hairpin, alpha-helix, and N-terminal loop of myticofensin were synthesized and the antimicrobial activities of these peptide fragments were tested. Our data confirms the diversity of defensins in Mytilus and reports the complex regulation of these defensins in the mussel immune response to different microbes in immune-related tissues. The immune system of Mytilus has been studied for years as they are a species with strong environmental adaptations. Our data can be regarded as a step forward in the study of the adaptation of Mytilus spp. to an evolving microbial world.

Molecular characterization of peptidoglycan recognition proteins from Mytilus coruscus.

Mytilus shows great immune resistance to various bacteria from the living waters, indicating a complex immune recognition mechanism against various microbes. Peptidoglycan recognition proteins (PGRPs) play an important role in the defense against invading microbes via the recognition of the immunogenic substance peptidoglycan (PGN). Therefore, eight PGRPs were identified from the gill transcriptome of Mytilus coruscus. The sequence features, expression pattern in various organs and larval development stages, and microbes induced expression profiles of these Mytilus PGRPs were determined. Our data revealed the constitutive expression of PGRPs in various organs with relative higher expression level in immune-related organs. The expression of PGRPs is developmentally regulated, and most PGRPs are undetectable in larvae stages. The expression level of most PGRPs was significantly increased with in vivo microbial challenges, showing strong response to Gram-positive strain in gill and digestive gland, strong response to Gram-negative strain in hemocytes, and relative weaker response to fungus in the three tested organs. In addition, the function analysis of the representative recombinant expressed PGRP (rMcPGRP-2) confirmed the antimicrobial and agglutination activities, showing the immune-related importance of PGRP in Mytilus. Our work suggests that Mytilus PGRPs can act as pattern recognition receptors to recognize the invading microorganisms and the antimicrobial effectors during the innate immune response of Mytilus.

Protective effect of SIRT6 on cholesterol crystal-induced endothelial dysfunction via regulating ACE2 expression.

Sirtuins are a family of highly conserved nicotinamide adenine dinucleotide (NAD+)-dependent enzymes. Among the sirtuins, SIRT1 and SIRT6 participate in the regulation of endothelial functions and play significant roles in the physiological and pathological processes of cardiovascular diseases (CVD). Recently, our study found that minute cholesterol crystals (CC) can be endocytosed by endothelial cells and further impair endothelial functions. Since previous studies have reported that angiotensin-converting enzyme (ACE2) involves Angiotensin (Ang) II-induced inflammation in endothelial cells, this study was designed to investigate the role of SIRT1 and SIRT6 in CC-induced variation of ACE2 expression and the related mechanism between SIRT6 and ACE2. We found that ACE2 is involved in CC-induced endothelial dysfunction, which inhibits decreases in nitric oxide (NO) level and endothelial nitric oxide synthase (eNOS) activity and increases in inflammatory factors and adhesion molecules. Besides, SIRT1 and SIRT6 regulated the protein expression of ACE2 in CC-stimulated human umbilical vein endothelial cells (HUVECs). Moreover, bioinformatics analysis from the Enrichr database indicated that activating transcription factor 2 (ATF2), is highly correlated with genes that significantly upregulated after infection with the SIRT6 adenovirus vector. In CC-induced HUVECs, ACE2 expression was up-regulated in cells transfected with ATF2 siRNA. However, further mechanism studies revealed that overexpression of SIRT6 decreases the accumulation of p-ATF2 in the nucleus, but did not affect p-ATF2 expression in the cytoplasm. Taken together, these data indicated that SIRT6 regulates ACE2 might via inhibiting the accumulation of nucleus p-ATF2 in CC-induced endothelial dysfunction.

Development and evaluation of poly adenosine 5'-diphosphate-ribose polymerase 1 immobilization-based receptor chromatography.

Yanghe decoction is a traditional Chinese medicine prescription and has been used for breast cancer treatment for many years. However, the effective ingredients in the decoction have not been identified. The expression of poly(ADP-ribose) polymerase-1 is highly related to breast cancer. Using poly(ADP-ribose) polymerase-1 as a probe, we expressed the haloalkane dehalogenase-tagged protein in BL21(DE3) E. coli, immobilized it on hexachlorocaproic acid-modified macroporous silica gel, and established a poly(ADP-ribose) polymerase-1 chromatographic model. The feasibility of the model was verified by testing the retention behaviors of five drugs on the protein column. We applied the model in screening the bioactive components in yanghe decoction. Rutin, liquiritin, and a compound ([M-H]- 681.7) were identified to be the potential bioactive ingredients. We studied the binding property between rutin and poly(ADP-ribose) polymerase-1 by injection amount dependent method, competitive studies, and molecular docking. We found that rutin can bind to the protein through the typical inhibitor binding site of the protein. Therefore, the chromatographic model is a useful tool to screen bioactive compounds from traditional Chinese medicine. The method is fast, reliable, and applicable to other functional proteins that can screen the potential lead compounds for the treatment of the related diseases.

Effects of isocorynoxeine, from Uncaria, on lower urinary tract dysfunction caused by benign prostatic hyperplasia via antagonism of α1A-adrenoceptors.

Medical therapy of lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH) targets smooth muscle contraction in the prostate, for which α1A-adrenoceptor (α1A-AR) antagonists have been considered to be the primary therapeutic method. We investigated the effects and underlying mechanisms of isocorynoxeine (ICN), one of indole alkaloids from Uncaria, on the treatment of LUTS secondary to BPH via α1A-ARs in mice. The effect of ICN on prostatic contractility was studied via myographic measurements in the prostates of rabbits. The effects of ICN on bladder function, serum-hormone levels, bladder histology, and prostate histology were determined in testosterone propionate-induced prostatic hyperplasic wild-type (WT) and α1A-AR knockout (α1A-KO) mice. The cytotoxicity of ICN in cultured human prostatic stromal cells (WPMY-1) was assessed by the following: a cell-counting kit, measuring the relaxant effect on WPMY-1 by a collagen gel contraction assay, intracellular Ca2+ mobilization indicated by Fluo-4, cytoskeletal organization by phalloidin staining, and expressions of α1A-AR-mediated key messengers by western blot analyses. ICN non-competitively antagonized the contractions of prostates induced by α1A-AR agonists. ICN treatment improved bladder functions in prostatic hyperplasic WT mice, whereas it failed to ameliorate bladder functions in prostatic hyperplasic α1A-KO mice. In WPMY-1, ICN relaxed cell contractions on collagen gels, disrupted F-actin organization, inhibited α1A-AR agonist-stimulated Ca2+ mobilization, and antagonized α1A-ARs via the RhoA/ROCK2/MLC signaling pathway. Our results suggest that ICN may be a promising therapeutic drug for targeting α1A-ARs in the treatment of BPH/LUTS.

Abundant members of Scavenger receptors family and their identification, characterization and expression against Vibrio alginolyticus infection in juvenile Larimichthys crocea.

Scavenger receptors (SRs) are crucial pattern recognition receptors (PRRs) to defense pathogen infection in fish innate immunity. In this paper, some members in SRs family of Larimichthys crocea were identified, including eight genes in the class A, B, D and F families. (G + C) % of all SRs members held 51% ∼ 59%, and these genes were no obvious codon bias by analyzing the distribution of A-, T-, G- and C-ended codons. The order of Enc for all SRs members by sequencing was LycCD68 > LycSCARA5 > LycSCARB1 > LycCD163 > LycMARCO > LycSREC1 > LycSCARA3 > LycSREC2. Moreover, different lengths and numbers of exons and introns led to the diverse mRNAs and respective functional domains or motifs, for example, an optional cysteine-rich (SRCR) domain in LycMARCO and LycSCARA5, an epidermal growth factor (EGF) and EGF-like domain in LycSREC1 and LycSREC2. The sub-cellular localization demonstrated SRs members mainly located in plasma membrane or extracellular matrix. Further, all of the SRs members in L. crocea were almost low expressed in heart, gill and intestine, whereas high in spleen and liver. After stimulation by Vibrio alginolyticus, the class A and F families were induced significantly, but the class B and D families expressed less even none after pathogenic infection. All the findings would pave the way to understand not only the evolution of the SR-mediated immune response, but also the complexity of fish immunity.

The cooperative expression of Heat Shock Protein 70 KD and 90 KD gene in juvenile Larimichthys crocea under Vibrio alginolyticus stress.

Heat shock proteins (HSPs) play significant roles in the immune response of fish in defending against diverse environmental threats or stresses. In this study, two complete HSP70 and HSP90 genes of Larimichthys crocea (designated as LycHSP70 and LycHSP90) were identified and characterized (GenBank accession no. KT456551 and KT456552). The complete open reading frame (ORF) fragments of LycHSP70 and LycHSP90 were 1917 bp and 2151 bp, encoding 638 and 716 amino acids residues respectively. Many significant functional domains and motifs were found, such as Hsp70 family signatures, Hsp90 family signatures, ATP-GTP binding site and EEVD motif regions, and they were associated with relative functions. Phylogenetic relationship and BLASTp analysis interpreted that they were unambiguously assigned to HSP70 and HSP90 family. The total length DNA of LycHSP70 was 7889bp, LycHSP90 was 5618 bp, and the gene location mapping were analyzed based on the whole-genomic DNA sequence of L. crocea. LycHSP70 and LycHSP90 were constantly expressed in eight tested tissues, with their expression peaks appearing in liver. Spleen, brain and head kidney also witnessed higher expression level. LycHSP70 and LycHSP90 were significantly induced by pathogenic bacteria V. alginolyticus, and they were both up-regulated in liver and spleen from 0 to 72 h post-injection. All the findings would contribute to better understanding the biologic function of HSPs in defending against pathogenic bacteria challenge and further exploring the innate immune response in fish.

Anti-infective mannose receptor immune mechanism in large yellow croaker (Larimichthys crocea).

Mannose receptor (MR) is a pattern recognition receptor (PRR) that plays a significant role in immunity responses. Its role has been described extensively in mammals, but very rarely in fish. Recently, with the rapid development of an aquaculture industry cultivating large yellow croaker (Larimichthys crocea), infectious diseases caused by viruses, bacteria and parasites are becoming more frequent and more severe, in particular bacterial infections caused by Vibrio anguillarum, resulting in great economical losses. Extensive use of antibiotics as conventional treatment has led to microenvironment imbalances, development of drug-resistant bacteria and deposition of drug residues, which cause environmental pollution and ultimately affect human health. The purpose of this pilot study was to detect the transcriptional levels of C-type mannose receptor genes MRC1 (4710-bp ORF; encoding 1437 aa; a signal peptide, a SMART RICIN domain, a SMART FN2 domain, eight SMART CLECT domain, and a transmembrane helix region) and MRC2 (3996-bp ORF; encoding 1484 aa; a SMART FN2 domain, eight SMART CLECT domains, and a transmembrane region) in the liver, kidney and spleen tissues of L. crocea challenged by V. anguillarum, to explore the effective domain and the molecular response mechanisms of MRC1 and MRC2, and, ultimately, to explore the possibility of developing a vaccine targeting V. anguillarum infections.

Combining Sprague-Dawley rat uterus cell membrane chromatography with HPLC/MS to screen active components from Leonurus artemisia.

CONTEXT: Leonurus artemisia (Lour.) S.Y.Hu (Lamiaceae) (YiMuCao in Chinese) is a traditional Chinese medicine. Leonurus artemisia has been shown to have many pharmacological effects such as increasing uterine contraction amplitude, and tension, but the active components are still unknown. OBJECTIVE: The objective of this study is to determine active components of L. Artemisia that are responsible for the biological activity using HPLC and cell membrane-based system. MATERIALS AND METHODS: The whole L. artemisia ethanol extract and its eight fractions were screened using Sprague-Dawley rat uterus cell membrane chromatography (CMC) combined with the HPLC/MS system. Oxytocin was used to investigate the activity of CMC column. The effect of active components screened from L. artemisia was studied by tension measurement of isolated rat uterine strips in vitro at a dose of 10(-7)-10(-4 )mol/L with oxytocin as a control. RESULTS: The acetone extract showed obvious activity when compared with the eight extracts of L. artemisia. From the acetone extract, in the negative ionization mode, the active compound was identified as genkwanin, with a molecular weight of 283. In vitro pharmacological experiments proved that genkwanin promoted uterine contractions at a dose from 10(-7) to 10(-4 )mol/L. The EC50 value was 4.86 ± 4.21 µmol/L for genkwanin and 4.30 ± 3.65 µmol/L for oxytocin on the contractile amplitude of uterine strips isolated from rats. DISCUSSION AND CONCLUSION: Genkwanin was identified as the active compound in L. artemisia by this method. In vitro pharmacological experiments proved that genkwanin promoted uterine contractions. Genkwanin may be used to uterine inertia and may have an effect on postpartum hemorrhage.

The imperatorin derivative OW1, a new vasoactive compound, inhibits VSMC proliferation and extracellular matrix hyperplasia.

Chronic hypertension induces vascular remodeling. The most important factor for hypertension treatment is reducing the risk of cardiovascular disease. OW1 is a novel imperatorin derivative that exhibits vasodilative activity and antihypertensive effects in two-kidney one-clip (2K1C) renovascular hypertensive rats. It also inhibited vascular remodeling of the thoracic aorta in a previous study. Here, the inhibitory effects and mechanisms of OW1 on arterial vascular remodeling were investigated in vitro and in 2K1C hypertensive rats in vivo. OW1 (20µM, 10µM, 5µM) inhibited Ang II-induced vascular smooth muscle cells (VSMCs) proliferation and ROS generation in vitro. OW1 also reversed the Ang II-mediated inhibition of α-SMA levels and stimulation of OPN levels. Histology results showed that treatment of 2K1C hypertensive rats with OW1 (20, 40, and 80mg/kg per day, respectively for 5weeks) in vivo significantly decreased the number of VSMCs, the aortic cross-sectional area (CSA), the media to lumen (M/L) ratio, and the content of collagen I and III in the mesenteric artery. Western blot results also revealed that OW1 stimulated the expression of α-SMA and inhibited the expression of collagen I and III on the thoracic aorta of 2K1C hypertensive rats. In mechanistic studies, OW1 acted as an ACE inhibitor and affected calcium channels. The suppression of MMP expression and the MAPK pathway may account for the effects of OW1 on vascular remodeling. OW1 attenuated vascular remodeling in vitro and in vivo. It could be a novel candidate for hypertension intervention.

Identification and analysis of icCu/Zn-SOD, Mn-SOD and ecCu/Zn-SOD in superoxide dismutase multigene family of Pseudosciaena crocea.

Superoxide dismutases (SODs) belong to a significant and ubiquitous family of metalloenzymes for eliminating excess reactive oxygen species (ROS). In this paper, the complete open reading frames (ORFs) of intracellular Cu/Zn-SOD (icCu/Zn-SOD), Mn-SOD and extracellular Cu/Zn-SOD (ecCu/Zn-SOD) were identified from the large yellow croaker (Pseudosciaena crocea, designated as LycSOD1, LycSOD2 and LycSOD3). The sequences were 465 bp, 678 bp and 645 bp (GenBank accession no. KJ908287, KJ908285 and KJ908286), encoding 154, 225 and 215 amino acid (aa) residues respectively. The deduced aa sequences of LycSOD1, LycSOD2 and LycSOD3 shared high identity to the known icCu/Zn-SODs, Mn-SODs and ecCu/Zn-SODs with BLASTp and Phylogenetic analysis. Two conserved Cu-/Zn-binding sites (H-44, H-47, H-64, H-121 for Cu binding and H-64, H-72, H-81, D-84 for Zn binding in LycSOD1, H-98, H-100, H-115, H-164 for Cu binding and H-115, H-163, H-166, D-169 for Zn binding in LycSOD3) and one conserved manganese coordinating sites (H-57, H-101, D-186, H-190 in LycSOD2) were identified. The total length of DNA sequences of LycSOD1, LycSOD2 and LycSOD3 were 3447 bp, 3387 bp and 3886 bp respectively, and there were 4 introns and 5 exons in Cu/Zn-SODs (LycSOD1 and LycSOD3), but only 3 exons and 2 introns in LycSOD3. Spatial expression analysis indicated the highest mRNA expression of three SODs all appeared in liver among eight detected tissues, the highest expression level was LycSOD1, then LycSOD2 and the lowest was LycSOD3 for almost each tissue. The expression of LycSOD1, LycSOD2 and LycSOD3 mRNA were all up-regulated in liver after Vibrio alginolyticus stimulation. The temporal expression peak of LycSOD1 and LycSOD2 were around 9-fold and 8-fold compared to control respectively, whereas, LycSOD3 got the highest level at 48 h post-injection (about 4.2-fold). All the results gave several new and useful evidences for further understanding the regulatory mechanism of superoxide dismutases in the innate immune system of sciaenidae fish.

The SREC-I and SREC-II associated with epidermal growth factor in scavenger receptor family are the potential regulative transmembrane receptors in Larimichthys crocea.

In innate immunity, the regulation of the immunologic gene expression plays a vital role in defense against pathogenic threat. The class F scavenger receptors (SCARFs), a kind of crucial immunologic type I transmembrane receptors, mainly involve in the signal transmission and eliminating pathogens in host immune system. In this study, the SREC-I and SREC-II of SCARFs in Larimichthys crocea (designated as LycSREC1 and LycSREC2 respectively) were first identified, the potential genetic locus relationships with other species were depicted and the features of gene expression after Vibrio alginolyticus stimulation were tested. The results demonstrated that the complete ORF sequences of two candidates were 3024 bp and 2832 bp (KM884873 and KM884874) respectively including some important domains and motifs, such as EGF/EGF-like domains, TRAF2-binding consensus motif, generic motif and atipical motif. The gene location maps and genetic locus interpreted that the DNA sequences of LycSREC1 and LycSREC2 were 7603 bp and 4883 bp, and some locus had changed compared with human being, but three more crucial genetic locus were conservative among ten species. Furthermore, quantitative real-time PCR (qRT-PCR) analysis indicated that the highest mRNA expression of LycSREC1 and LycSREC2 were both in liver among eight detected tissues, and their expression were up-regulated by V. alginolyticus stimulation. All these findings would contribute to better understanding the biologic function of SCARFs in defending against pathogenic bacteria challenge and further exploring the innate immune of sciaenidae fish.

Molecular characterization and expression analysis of a complement component C3 in large yellow croaker (Larimichthys crocea).

The complement system has been discovered in invertebrates and vertebrates, and plays a crucial role in the innate defense against common pathogens. Complement component 3 is a key molecule in the complement system, whose activation is essential for all the important functions performed by this system. In this study, the complete C3 cDNA sequence was isolated from the large yellow croaker (Larimichthys crocea), which was high similarity to other complement C3. We reported the primary sequence, tissue expression profile, polypeptide domain architecture and phylogenetic analysis of L. crocea complement component C3 (L.c-C3) gene. Its open reading frame (ORF) is 4962 bp and encodes for 1653 amino acids with a putative signal peptide of 23 amino acid residues. The deduced amino acid sequence showed that L.c-C3 has conserved residues and domains known to be crucial for C3 function. Phylogenetic analysis showed that L. crocea was closely related to Miichthys miiuy. The mRNA expressions of L.c-C3 was detectable at different tissues. L.c-C3 was expressed in a wide range of adult tissues, it showed highest expression in the liver. But the different developmental stages from fertilized egg to newborn larvae of the large yellow croaker the highest expression levels of L.c-C3 gene were not found. Bacterial challenge experiments showed that the levels of L.c-C3 mRNA expression were up-regulated in the liver, spleen and brain of adult large yellow croaker respectively. The results showed that L.c-C3 mRNA expression in the large yellow croaker is influenced by bacterial stress and L.c-C3 might play an important role in immunity mechanisms. This study will further increase our understanding of the function of L.c-C3 and molecular mechanism of innate immunity in teleosts.

A novel biomarker for marine environmental pollution of pi-class glutathione S-transferase from Mytilus coruscus.

Glutathione S-transferases (GSTs) are the superfamily of phase II detoxification enzymes that play crucial roles in innate immunity. In this study, a pi-class GST homolog was identified from Mytilus coruscus (named as McGST1, KC525103). The full-length cDNA sequence of McGST1 was 621bp with a 5' untranslated region (UTR) of 70bp and a 3'-UTR of 201bp. The deduced amino acid sequence was 206 residues in length with theoretical pI/MW of 5.60/23.72kDa, containing the conserved G-site and diversiform H-site. BLASTn analysis and phylogenetic relationship strongly suggested that this cDNA sequence was a member of pi class GST family. The prediction of secondary structure displayed a preserved N-terminal and a C-terminal comprised with α-helixes. Quantitative real time RT-PCR showed that constitutive expression of McGST1 was occurred, with increasing order in mantle, muscle, gill, hemocyte, gonad and hepatopancreas. The stimulation of bacterial infection, heavy metals and 180CST could up-regulate McGST1 mRNA expression in hepatopancreas with time-dependent manners. The maximum expression appeared at 6h after pathogenic bacteria injected, with 10-fold in Vibrio alginolyticus and 16-fold in Vibrio harveyi higher than that of the control. The highest point of McGST1 mRNA appeared at different time for exposure to copper (10-fold at day 15), cadmium (9-fold at day10) and 180 CST (10-fold at day 15). These results suggested that McGST1 played a significant role in antioxidation and might potentially be used as indicators and biomarkers for detection of marine environmental pollution.

Identification of SCARA3, SCARA5 and MARCO of class A scavenger receptor-like family in Pseudosciaena crocea.

The class A scavenger receptors are important pattern recognition receptors of the innate immune system in living organisms. According to the whole-genome data of large yellow croaker (Pseudosciaena crocea), three kinds of scavenger receptors, SCARA3, SCARA5 and MARCO were cloned from the spleen, designated severally as TycSA3, TycSA5 and TycMAC. The complete cDNAs open reading frames (ORF) of TycSA3, TycSA5 and TycMAC were 1938 bp, 1677 bp and 1218 bp (GenBank accession no. KJ467772, KJ467773 and KJ467771), encoding 645, 558 and 405 amino acid (aa) residues respectively. The BLASTp analysis strongly suggested that the sequences shared high similarity with known SCARA3, SCARA5 and MARCO. The phylogenetic relationship analysis illustrated that different subtype of SRs formed their own separate branches, TycSA3 and TycSA5 were placed in SCARA3 and SCARA5 branch of Osteichthyes fish respectively with strong bootstrap support. Curiously, the TycMAC was clustered with Alligator sinensis. ClustalW analysis with amino acid sequences revealed that the proportion of identity with other species was 59-71% for TycSA3 and 55-72% for TycSA5, but the scale of TycMAC was considerable lower than those of other two genes (only approximately 38%). The SR family motifs, such as transmembrane helix region, colied coli region and collagens region in the TycSA3, TycSA5 and TycMAC were conserved. There was an optional cysteine-rich (SRCR) domain (from 457 to 557 residues) containing 6 conserved cysteines (C-482, C-495, C-526, C-536, C-546 and C-556) in TycSA5. Likewise, the SRCR domains of TycMAC (from 310 to 405 residues) also contained C-333, C-346, C-374, C-384, C-394 and C-404 cysteines residues. Particularly, there were the major TRAF2-binding consensus motif and two main motifs on internalization of receptor in TycSA3 and TycSA5. The gene structures of different species were analyzed with GeneMaper v2.5, and the number of introns and exons of TycSA3, TycSA5 and TycMAC in DNA sequences were different, for example some corresponding exon regions were divided into several smaller exon portions. Furthermore, quantitative real-time PCR (qRT-PCR) analysis indicated the highest mRNA expression of TycSA3, TycSA5 and TycMAC all appeared in spleen among eight detected tissues, and the expression of them were up-regulated in spleen after Vibrio alginolyticus injection. All these results demonstrated that class A SRs played a significant role in the defense against pathogenic bacteria infection in innate immune of sciaenidae fish.

Antihypertensive and vascular remodelling effects of the imperatorin derivative OW1 in renovascular hypertension rats.

OW1 is a novel imperatorin derivative that exhibits vasodilator activity. In the present study, the antihypertensive effect of and inhibition of vascular remodelling by OW1 were investigated in two-kidney, one-clip (2K1C) renovascular hypertensive rats. Rats were subjected to the 2K1C procedure and treated with OW1 (40 or 80 mg/kg per day) for 8 weeks. Blood pressure was measured in conscious rats. Microalbumin (mALB) and total protein (U-TP) concentrations were determined in the urine, as were plasma concentrations of angiotensin (Ang) II, calcitonin gene-related peptide (CGRP) and angiotensin-converting enzyme 1 (ACE). The unclipped kidney was stained with haematoxylin and eosin and Masson trichrome, whereas aortic sections were stained with Masson trichrome. In addition, OW1-induced vasodilatation was evaluated in vitro in rat mesenteric and renal arteries. Immunohistochemical analysis was used to quantify collagen I and III expression. OW1 relaxed rat mesenteric and renal arterial rings in vitro. Treatment of 2K1C hypertensive rats with OW1 (40 and 80 mg/kg per day) for 8 weeks significantly decreased blood pressure. In addition, OW1 reduced plasma AngII and ACE concentrations and increased plasma CGRP concentrations. At 80 mg/kg per day, OW1 decreased blood urea nitrogen, mALB and U-TP levels. Histological analysis revealed that OW1 reduced renal arteriolar thickness and relieved the structural hypertrophic arteries. Moreover, OW1 had an inhibitory effect on vascular remodelling and renal lesions in hypertensive rats. In conclusion, the results suggest that OW1 could potentially be a novel candidate for hypertension intervention.

[Effect of Shenkang Injection on Kidney Function in Hypertensive Renal Damage Rats].

OBJECTIVE: To observe the effect of Shenkang Injection on the blood pressure, metabolism, blood biochemistry and renal pathology in hypertension renal damge rats, then to provide theoretical basis for clinical trials. METHODS: 75 spontaneously hypertensive nephropathy rats were randomly divided into five groups with 15 rats in each group: model group (SHR group) rats were intragastrically treated with the vehicle (4 mL/kg normal saline per day) of Shenkang Injection per day; Benazepril group( positive control group, 8 mg/ kg Benazepril per day) ;Shenkang Injection low-dose group (6.7 mL/kg Shenkang Injection per day); middle-dose group (13.3 mL/kg Shenkang Injection per day); high-dose group (26.6 mL/kg Shenkang Injection per day); and WKY rats were normal control group (n = 15) (4 mL/kg normal saline per day). RESULTS: After 3 months intraperitoneal injection treatment, SHR rats blood pressure were in- hibited; the levels of microalbumin (m-ALB), total protein (U-TP), serum creatinine (Ser) and urea nitrogen (BUN) were decreased significantly in Shenkang Injection treated groups rats. Shenkang Injection significantly improved the levels of creatinine clearance rate (Ccr), serum albumin (ALB) and superoxide dismutase (SOD), decreased the content of methane dicarboxylic aldehyde (MDA), aldosterone (Ald), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), transforming growth factor-ß1 (TGF-ß1) and C-reactive protein (CRP), and had histologic improvement compared with model group. CONCLUSION: Shenkang Injection can improve the kidney function, decrease the levels of serum inflammatory factors,improve the oxidative status and reduce the degree of hypertensive renal damage.

Harnessing 3D in vitro systems to model immune responses to solid tumours: a step towards improving and creating personalized immunotherapies.

In vitro 3D models are advanced biological tools that have been established to overcome the shortcomings of oversimplified 2D cultures and mouse models. Various in vitro 3D immuno-oncology models have been developed to mimic and recapitulate the cancer-immunity cycle, evaluate immunotherapy regimens, and explore options for optimizing current immunotherapies, including for individual patient tumours. Here, we review recent developments in this field. We focus, first, on the limitations of existing immunotherapies for solid tumours, secondly, on how in vitro 3D immuno-oncology models are established using various technologies - including scaffolds, organoids, microfluidics and 3D bioprinting - and thirdly, on the applications of these 3D models for comprehending the cancer-immunity cycle as well as for assessing and improving immunotherapies for solid tumours.