Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis.

Histone acetylation plays critical roles in chromatin remodeling, DNA repair, and epigenetic regulation of gene expression, but the underlying mechanisms are unclear. Proteasomes usually catalyze ATP- and polyubiquitin-dependent proteolysis. Here, we show that the proteasomes containing the activator PA200 catalyze the polyubiquitin-independent degradation of histones. Most proteasomes in mammalian testes ("spermatoproteasomes") contain a spermatid/sperm-specific α subunit α4 s/PSMA8 and/or the catalytic ß subunits of immunoproteasomes in addition to PA200. Deletion of PA200 in mice abolishes acetylation-dependent degradation of somatic core histones during DNA double-strand breaks and delays core histone disappearance in elongated spermatids. Purified PA200 greatly promotes ATP-independent proteasomal degradation of the acetylated core histones, but not polyubiquitinated proteins. Furthermore, acetylation on histones is required for their binding to the bromodomain-like regions in PA200 and its yeast ortholog, Blm10. Thus, PA200/Blm10 specifically targets the core histones for acetylation-mediated degradation by proteasomes, providing mechanisms by which acetylation regulates histone degradation, DNA repair, and spermatogenesis.

LTA and PGN from Bacillus siamensis can alleviate soybean meal-induced enteritis and microbiota dysbiosis in Lateolabrax maculatus.

An eight-week feeding trial was designed to assess which component of commensal Bacillus siamensis LF4 can mitigate SBM-induced enteritis and microbiota dysbiosis in spotted seabass (Lateolabrax maculatus) based on TLRs-MAPKs/NF-кB signaling pathways. Fish continuously fed low SBM (containing 16 % SBM) and high SBM (containing 40 % SBM) diets were used as positive (FM group) and negative (SBM group) control, respectively. After feeding high SBM diet for 28 days, fish were supplemented with B. siamensis LF4-derived whole cell wall (CW), cell wall protein (CWP), lipoteichoic acid (LTA) or peptidoglycan (PGN) until 56 days. The results showed that a high inclusion of SBM in the diet caused enteritis, characterized with significantly (P < 0.05) decreased muscular thickness, villus height, villus width, atrophied and loosely arranged microvillus. Moreover, high SBM inclusion induced an up-regulation of pro-inflammatory cytokines and a down-regulation of occludin, E-cadherin, anti-inflammatory cytokines, apoptosis related genes and antimicrobial peptides. However, dietary supplementation with CW, LTA, and PGN of B. siamensis LF4 could effectively alleviate enteritis caused by a high level of dietary SBM. Additionally, CWP and PGN administration increased beneficial Cetobacterium and decreased pathogenic Plesiomonas and Brevinema, while dietary LTA decreased Plesiomonas and Brevinema, suggesting that CWP, LTA and PGN positively modulated intestinal microbiota in spotted seabass. Furthermore, CW, LTA, and PGN application significantly stimulated TLR2, TLR5 and MyD88 expressions, and inhibited the downstream p38 and NF-κB signaling. Taken together, these results suggest that LTA and PGN from B. siamensis LF4 could alleviate soybean meal-induced enteritis and microbiota dysbiosis in L. maculatus, and p38 MAPK/NF-κB pathways might be involved in those processes.

Paraprobiotic and postbiotic forms of Bacillus siamensis improved growth, immunity, liver and intestinal health in Lateolabrax maculatus fed soybean meal diet.

Live commensal Bacillus siamensis LF4 showed reparative potentials against high SM-induced negative effects, but whether its paraprobiotic (heat-killed B. siamensis, HKBS) and postbiotic (cell-free supernatant, CFS) forms had reparative functions and potential mechanisms are not yet known. In this study, the reparative functions of HKBS and CFS were investigated by establishing an injured model of spotted seabass (Lateolabrax maculatus) treated with dietary high soybean meal (SM). The results showed that HKBS and CFS effectively mitigated growth suppression, immune deficiency, and liver injury induced by dietary high SM. Simultaneously, HKBS and CFS application positively shaped intestinal microbiota by increased the abundance of beneficial bacteria (Fusobacteria, Firmicutes, Bacteroidota, and Cetobacterium) and decreased harmful bacteria (Proteobacteria and Plesiomonasare). Additionally, HKBS and CFS improved SM-induced intestinal injury by restoring intestinal morphology, upregulating the expression of tight junction proteins, anti-inflammatory cytokines, antimicrobial peptides, downregulating the expression of pro-inflammatory cytokines and apoptotic proteins. Furthermore, HKBS and CFS intervention significantly activated TLR2, TLR5 and MyD88 signaling, and eventually inhibited p38 and NF-κB pathways. In conclusion, paraprobiotic (HKBS) and postbiotic (CFS) from B. siamensis LF4 can improve growth, immunity, repair liver and intestinal injury, and shape intestinal microbiota in L. maculatus fed high soybean meal diet, and TLRs/p38 MAPK/NF-κB signal pathways might be involved in those processes. These results will serve as a base for future application of paraprobiotics and postbiotics to prevent and repair SM-induced adverse effects in fish aquaculture.

Commensal Bacillus siamensis LF4 induces antimicrobial peptides expression via TLRs and NLRs signaling pathways in intestinal epithelial cells of Lateolabrax maculatus.

Antimicrobial peptides (AMPs) play an important role in modulating intestinal microbiota, and our previous study showed that autochthonous Baccilus siamensis LF4 could shape the intestinal microbiota of spotted seabass (Lateolabrax maculatus). In the present study, a spotted seabass intestinal epithelial cells (IECs) model was used to investigate whether autochthonous B. siamensis LF4 could modulate the expression of AMPs in IECs. And then, the IECs were treated with active, heat-inactivated LF4 and its supernatant to illustrate their AMPs inducing effects and the possible signal transduction mechanisms. The results showed that after 3 h of incubation with 108 CFU/mL B. siamensis LF4, lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT), glutamic propylic transaminase (GPT) activities in supernatant decreased significantly and obtained minimum values, while supernatant alkaline phosphatase (AKP) activity, ß-defensin protein level and IECs Na+/K+-ATPase activity, AMPs (ß-defensin, hepcidin-1, NK-lysin, piscidin-5) genes expression increased significantly and obtained maximum values (P < 0.05). Further study demonstrated that the active, heat-inactivated LF4 and its supernatant treatments could effectively decrease the LDH, GOT, and GPT activities in IECs supernatant, increase AKP activity and ß-defensin (except LF4 supernatant treatment) protein level in IECs supernatant and Na+/K+-ATPase and AMPs genes expression in IECs. Treatment with active and heat-inactivated B. siamensis LF4 resulted in significantly up-regulated the expressions of TLR1, TLR2, TLR3, TLR5, NOD1, NOD2, TIRAP, MyD88, IRAK1, IRAK4, TRAF6, TAB1, TAB2, ERK, JNK, p38, AP-1, IKKα, IKKß and NF-κB genes. Treatment with B. siamensis LF4 supernatant also resulted in up-regulated these genes, but not the genes (ERK, JNK, p38, and AP-1) in MAPKs pathway. In summary, active, heat-inactivated and supernatant of B. siamensis LF4 can efficiently induce AMPs expression through activating the TLRs/NLRs-MyD88-dependent signaling, active and heat-inactivated LF4 activated both the downstream MAPKs and NF-κB pathways, while LF4 supernatant only activated NF-κB pathway.

Probiotic components of Bacillus siamensis LF4 mitigated ß-conglycinin caused cell injury via modulating TLR2/MAPKs/NF-κB signaling in Lateolabrax maculatus.

ß-conglycinin is a recognized factor in leading to intestinal inflammation and limiting application of soybean meal in aquaculture. Our previous study reported that heat-killed B. siamensis LF4 could effectively mitigate inflammatory response and apoptosis caused by ß-conglycinin in spotted seabass (Lateolabrax maculatus) enterocytes, but the mechanisms involved are not fully understood. In the present study, therefore, whole cell wall (CW), peptidoglycan (PG) and lipoteichoic acid (LTA) and cell-free supernatant (CFS) have been collected from B. siamensis LF4 and their mitigative function on ß-conglycinin-induced adverse impacts and mechanisms underlying were evaluated. The results showed that ß-conglycinin-induced cell injury, characterized with significantly decreased cell viability and increased activities of lactate dehydrogenase, glutamic oxaloacetic transaminase, glutamic propylic transaminase (P < 0.05), were reversed by subsequent heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS treatment. Enterocytes co-cultured with heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS (especially PG) significantly increased expressions of anti-inflammatory genes (IL-2, IL-4, IL-10 and TGF-ß1), tight junction proteins (ZO-1, occludin and claudin-b) and antimicrobial peptides (ß-defensin, hepcidin-1, NK-lysin and piscidin-5), and decreased expressions of pro-inflammatory genes (IL-1ß, IL-8 and TNF-α) and apoptosis-related genes (caspase 3, caspase 8 and caspase 9) (P < 0.05), indicating their excellent mitigation effects on ß-conglycinin-induced cell damages. In addition, heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS significantly increased TLR2 mRNA level (especially in PG treatment), and decreased MAPKs (JNK, ERK, p38 and AP-1) and NF-κB related genes expressions. In conclusion, heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS could modulating TLR2/MAPKs/NF-κB signaling and alleviating ß-conglycinin-induced enterocytes injury in spotted seabass (L. maculatus), and PG presented the best potential.

Commensal Bacillus siamensis LF4 ameliorates ß-conglycinin induced inflammation in intestinal epithelial cells of Lateolabrax maculatus.

ß-conglycinin and glycinin, two major heat-stable anti-nutritional factors in soybean meal (SM), have been suggested as the key inducers of intestinal inflammation in aquatic animals. In the present study, a spotted seabass intestinal epithelial cells (IECs) were used to compare the inflammation-inducing effects of ß-conglycinin and glycinin. The results showed that IECs co-cultured with 1.0 mg/mL ß-conglycinin for 12 h or 1.5 mg/mL glycinin for 24 h significantly decreased the cell viability (P < 0.05), and overstimulated inflammation and apoptosis response by significantly down-regulating anti-inflammatory genes (IL-2, IL-4, IL-10 and TGF-ß1) expressions and significantly up-regulated pro-inflammatory genes (IL-1ß, IL-8 and TNF-α) and apoptosis genes (caspase 3, caspase 8 and caspase 9) expressions (P < 0.05). Subsequently, a ß-conglycinin based inflammation IECs model was established and used for demonstrating whether commensal probiotic B. siamensis LF4 can ameliorate the adverse effects of ß-conglycinin. The results showed ß-conglycinin-induced cell viability damage was completely repaired by treated with 109 cells/mL heat-killed B. siamensis LF4 for ≥12 h. At the same time, IECs co-cultured with 109 cells/mL heat-killed B. siamensis LF4 for 24 h significantly ameliorated ß-conglycinin-induced inflammation and apoptosis by up-regulating anti-inflammatory genes (IL-2, IL-4, IL-10 and TGF-ß1) expressions and down-regulated pro-inflammatory genes (IL-1ß, IL-8 and TNF-α) and apoptosis genes (caspase 3, caspase 8 and caspase 9) expressions (P < 0.05). In summary, both ß-conglycinin and glycinin can lead to inflammation and apoptosis in spotted seabass IECs, and ß-conglycinin is more effective; commensal B. siamensis LF4 can efficiently ameliorate ß-conglycinin induced inflammation and apoptosis in IECs.

GSK872 and necrostatin-1 protect retinal ganglion cells against necroptosis through inhibition of RIP1/RIP3/MLKL pathway in glutamate-induced retinal excitotoxic model of glaucoma.

BACKGROUND: Glaucoma, the major cause of irreversible blindness worldwide, is characterized by progressive degeneration of retinal ganglion cells (RGCs). Current treatments for glaucoma only slow or partially prevent the disease progression, failing to prevent RGCs death and visual field defects completely. Glutamate excitotoxicity via N-methyl-D-aspartic acid (NMDA) receptors plays a vital role in RGCs death in glaucoma, which is often accompanied by oxidative stress and NLRP3 inflammasome activation. However, the exact mechanisms remain unclear. METHODS: The glutamate-induced R28 cell excitotoxicity model and NMDA-induced mouse glaucoma model were established in this study. Cell counting kit-8, Hoechst 33342/PI dual staining and lactate dehydrogenase release assay were performed to evaluate cell viability. Annexin V-FITC/PI double staining was used to detect apoptosis and necrosis rate. Reactive oxygen species (ROS) and glutathione (GSH) were used to detect oxidative stress in R28 cells. Levels of proinflammatory cytokines were measured by qRT-PCR. Transmission electron microscopy (TEM) was used to detect necroptotic morphological changes in RGCs. Retinal RGCs numbers were detected by immunofluorescence. Hematoxylin and eosin staining was used to detect retinal morphological changes. The expression levels of RIP1, RIP3, MLKL and NLRP3 inflammasome-related proteins were measured by immunofluorescence and western blotting. RESULTS: We found that glutamate excitotoxicity induced necroptosis in RGCs through activation of the RIP1/RIP3/MLKL pathway in vivo and in vitro. Administration of the RIP3 inhibitor GSK872 and RIP1 inhibitor necrostatin-1 (Nec-1) prevented glutamate-induced RGCs loss, retinal damage, neuroinflammation, overproduction of ROS and a decrease in GSH. Furthermore, after suppression of the RIP1/RIP3/MLKL pathway by GSK872 and Nec-1, glutamate-induced upregulation of key proteins involved in NLRP3 inflammasome activation, including NLRP3, pro-caspase-1, cleaved-caspase-1, and interleukin-1ß (IL-1ß), was markedly inhibited. CONCLUSIONS: Our findings suggest that the RIP1/RIP3/MLKL pathway mediates necroptosis of RGCs and regulates NLRP3 inflammasome activation induced by glutamate excitotoxicity. Moreover, GSK872 and Nec-1 can protect RGCs from necroptosis and suppress NLRP3 inflammasome activation through inhibition of RIP1/RIP3/MLKL pathway, conferring a novel neuroprotective treatment for glaucoma.

Preventive and reparative functions of host-associated probiotics against soybean meal induced growth, immune suppression and gut injury in Japanese seabass (Lateolabraxjaponicus).

A 56-day feeding trial was conducted to examine the preventive and reparative functions of host-associated probiotics against high soybean meal (SM)-induced negative effects in Japanese seabass (Lateolabrax japonicus). Fish continuously fed low SM (containing 16% SM) and high SM (containing 40% SM) diets were named as positive (PC) and negative (C) control, respectively. Preventive functions of probiotics were evaluated by continuously feeding diets LF3 (Lactococcus petauri LF3 supplemented in high SM diet, group PLF3) and LF4 (Bacillus siamensis LF4 supplemented in high SM diet, group PLF4), while reparative functions were estimated by feeding the high SM diet during 0-28 days, then feeding diets LF3 (group RLF3) and LF4 (group RLF4) until day 56. Compared with the group PC, suppressed growth and immunity, and damaged intestinal health were observed in the group C on days 28 and 56. Fish in groups PLF3 and PLF4, rather than in groups RLF3 and RLF4, showed higher growth compared with the group C and displayed similar immune status to the group PC, indicating that the initial and continued application of probiotic LF3 and LF4 can efficiently improve high SM induced growth and immune deficiency in Japanese seabass, but probiotics had limited reparative benefits when they were administrated at the middle of the feeding trial (28 d). Furthermore, probiotics showed good preventive functions and limited reparative functions on gut health via improving intestinal morphology and inflammation markers, for example, decreasing diamine oxidase activity and d-lactate content, while up-regulating anti-inflammatory TGF-ß1 expression and down-regulating pro-inflammatory TNF-α, IL-1ß, and IL-8 expressions. Moreover, dietary supplementation of probiotics (especially on day 56) could effectively shape the gut microbiota, such as significantly decreasing abundances of opportunistic pathogens (phylum Actinobacteria, genera Pseudomonas and Moheibacter on day 28, phylum Proteobacteria, genus Plesiomonas on day 56), significantly increasing gut microbial diversity and abundances of possible beneficial bacteria (phylum Bacteroidetes and genus Lactobacillus on day 28, phyla Firmicutes, Bacteroidetes and Cyanobacteria, genera Bacillus, Lactobacillus and Bacteroides on day 56). In conclusion, we evidenced for the first time that host-associated L. petauri LF3 and B. siamensis LF4 can provide effectively preventive and certain reparative functions against high SM-induced adverse effects in L. japonicus.

Pathogenesis and prospects for therapeutic clinical application of noncoding RNAs in glaucoma: Systematic perspectives.

Noncoding ribonucleic acids (ncRNAs) are an increasingly studied class of RNA molecules with extensive biological activities, including important roles in human development, health, and disease. Glaucoma is a neurodegenerative disease of the retina, and one of the leading causes of blindness worldwide. However, the specific roles of ncRNAs in the development and progression of glaucoma are unclear, and related reports are fragmented. An in-depth understanding of ncRNAs participating in the pathogenesis and progression of glaucoma would be helpful for opening up new avenues to facilitate the early diagnosis and clinical treatment. Therefore, in this review, we aimed to discuss the current research progress, the potentialfuture clinical applications and the research limitations of three critical classes of ncRNAs in glaucoma, namely microRNAs, long noncoding RNAs, and circular RNAs.

CaMK II -induced Drp1 phosphorylation contributes to blue light-induced AIF-mediated necroptosis in retinal R28 cells.

Retinal damage caused by blue light has become an important public health concern. Mitochondria have been found to play a key role in light-induced retinal cell death. In this study, we aimed to clarify the molecular mechanism involved in mitochondrion-related retinal cell damage caused by blue light, the major component of light-emitting diodes (LEDs). Our results show that blue light (450 nm, 300lux)-induced R28 cell death is caspase independent and can be attenuated by necrostatin-1. Apoptosis-inducing factor (AIF) cleavage and translocation to the nucleus are involved in the cell death progress. Blue light exposure causes mitochondrial fragmentation, which is mediated by phosphorylation at dynamin-related protein 1 (Drp1) Ser616 site, but it does not alter the protein levels of fission or fusion machinery. Knocking down Drp1 or treatment with Drp1 inhibitor Mdivi-1 protects R28 cells from blue light. Overproduction of reactive oxygen species (ROS) is induced by blue light. The ROS scavenger Trolox decreases Drp1 Ser616 phosphorylation level and mitochondrial fragmentation upon blue light exposure. Moreover, Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 blocks Drp1 phosphorylation and rescues mitochondrial fragmentation and AIF-mediated cell death caused by blue light. In conclusion, our data suggest that the CaMKII-Drp1 pathway plays a major role in blue light-induced AIF-mediated retinal cell damage.

Normal vitreous promotes angiogenesi via the epidermal growth factor receptor.

Vitreous, a transparent tissue in our body, contains anti-angiogenesis factors. Our previous work reported that vitreous activates the signaling pathway of epidermal growth factor receptor (EGFR), which plays a critical role in angiogenesis. The aim of this study was to determine the role of EGFR in vitreous-induced angiogenesis-related cellular responses in vitro. Using a pharmacologic and molecular approach, we found that vitreous increased proliferation and migration via EGFR in human umbilical vein endothelial cells (HUVECs). Furthermore, we demonstrated that vitreous promoted tube formation via EGFR in HUVECs. Subsequently, depletion of EGFR using CRISPR/Cas9 and blockage with EGFR inhibitor AG1478 suppressed vitreous-induced Akt activation and cell proliferation, migration, and tube formation in HUVECs. The significance of the angiogenic effect derived from vitreous demonstrates the importance of vitreous in the ocular physiology and the pathobiology of angiogenesis-related ophthalmic diseases, such as proliferative diabetic retinopathy.

RSK3 mediates necroptosis by regulating phosphorylation of RIP3 in rat retinal ganglion cells.

Receptor-interacting protein 3 (RIP3) plays an important role in the necroptosis signaling pathway. Our previous studies have shown that the RIP3/mixed lineage kinase domain-like protein (MLKL)-mediated necroptosis occurs in retinal ganglion cell line 5 (RGC-5) following oxygen-glucose deprivation (OGD). However, upstream regulatory pathways of RIP3 are yet to be uncovered. The purpose of the present study was to investigate the role of p90 ribosomal protein S6 kinase 3 (RSK3) in the phosphorylation of RIP3 in RGC-5 cell necroptosis following OGD. Our results showed that expression of RSK3, RIP3, and MLKL was upregulated in necroptosis of RGC-5 after OGD. A computer simulation based on our preliminary results indicated that RSK3 might interact with RIP3, which was subsequently confirmed by co-immunoprecipitation. Further, we found that the application of a specific RSK inhibitor, LJH685, or rsk3 small interfering RNA (siRNA), downregulated the phosphorylation of RIP3. However, the overexpression of rip3 did not affect the expression of RSK3, thereby indicating that RSK3 could be a possible upstream regulator of RIP3 phosphorylation in OGD-induced necroptosis of RGC-5 cells. Moreover, our in vivo results showed that pretreatment with LJH685 before acute high intraocular pressure episodes could reduce the necroptosis of retinal neurons and improve recovery of impaired visual function. Taken together, our findings suggested that RSK3 might work as an upstream regulator of RIP3 phosphorylation during RGC-5 necroptosis.

Inhibition of connexin 43 attenuates oxidative stress and apoptosis in human umbilical vein endothelial cells.

BACKGROUND: Previous studies demonstrated an important role for connexin 43 (Cx43) in the regulation of apoptosis by influencing mitochondrial functions. This study aimed to investigate the relationship between Cx43 and lipopolysaccharide (LPS)-induced oxidative stress and apoptosis in human umbilical vein endothelial cells (HUVECs). METHODS: Western blot was performed to determine mitochondrial Cx43 (MtCx43) protein level and phosphorylation (p-MtCx43). Gap19, a selective Cx43 inhibitor, was used to examine the effects of Cx43 on LPS-induced oxidative stress and apoptosis in HUVECs. Expression of regulatory genes associated with oxidative stress was examined by quantitative polymerase chain reaction (qPCR) and Western blot. Apoptosis was assessed by flow cytometry. RESULTS: LPS stimulation resulted in increased levels of MtCx43 and p-MtCx43. Interestingly, Gap19 antagonized the upregulation of glutathione S-transferase Zeta 1 (GSTZ1) and cytochrome b alpha beta (CYBB), and the downregulation of antioxidant 1 (ATOX1), glutathione synthetase (GSS) and heme oxygenase 1 (HMOX1) induced by LPS or Cx43 overexpression. Moreover, the increased production of reactive oxygen species (ROS) and apoptosis elicited by LPS or Cx43 overexpression were reduced following treatment with Gap19. CONCLUSIONS: Selective inhibition of Cx43 hemichannels protects HUVECs from LPS-induced apoptosis and this may be via a reduction in oxidative stress production.

Characterization and genomic function analysis of phenanthrene-degrading bacterium Pseudomonas sp. Lphe-2.

A stain of Pseudomonas sp. Lphe-2, which could degrade phenanthrene as the main carbon and energy source, was isolated from the aerobic sludge of a coking plant. Then its biodegradation characteristics, whole genome sequence and biodegradation pathway were examined. The Lphe-2 strain exhibited broad-spectrum degradation activities for various polycyclic aromatic hydrocarbons (PAHs), including naphthalene (NAP), phenanthrene (PHE), and pyrene (PYR). Under the optimal conditions, the degradation efficiency of phenanthrene (100 mg/L) is 92.76% on the 7th day, and 2-carboxybenzaldehyde and 1-hydroxy-2-naphthoic acid are the major metabolites found in phenanthrene metabolism. Genomic analysis of Pseudomonas sp. Lphe-2 showed that a total of 3879 genes from the Lphe-2 strain were annotated based on the COG classification, and the genomic information was annotated to 185 metabolic pathways. Based on the intermediate metabolites detected by Gas Chromatography-Mass Spectrometer (GC-MS) and all potential phenanthrene-degrading genes identified by BLAST search, a phenanthrene biodegradation pathway of Lphe-2 strain was proposed. These results suggested that Lphe-2 strain has a good prospect in the bioremediation of PAHs pollution.

Bacillus pumilus SE5 originated PG and LTA tuned the intestinal TLRs/MyD88 signaling and microbiota in grouper (Epinephelus coioides).

The normal microbiota plays a key role in the health of host, but little is known of how the fish immune system recognizes and responds to indigenous bacteria/probiotics. Our previous studies have showed that heat-inactivated indigenous Bacillus pumilus SE5 activate the TLR2 signaling pathways and modulate the intestinal microbiota in grouper (Epinephelus coioides), suggesting microbial-associated molecular patterns (MAMPs) involved. In this study, whole cell wall (CW) and two possible MAMPs, peptidoglycan (PG) and lipoteichoic acid (LTA) have been extracted from B. pumilus SE5 and their effects on intestinal immune related genes expression and microbiota were evaluated in a 60 days feeding trial. Significantly elevated expression of TLR1, TLR2, TLR5 and MyD88 was observed in fish fed the CW, PG and LTA containing diets, and the highest expression was observed in groups PG and LTA. At the same time, significantly upregulated expression of antimicrobial effectors, such as antimicrobial peptides (epinecidin-1, hepcidin-1 and ß-defensin), C-type Lectin and IgM was observed in fish fed PG and LTA containing diets. This induced activation of intestinal immunity was consistent with the microbiota data showing that CW, PG and LTA originated from SE5 modulated the overall structure of intestinal microbiota, and the relative abundance of potentially pathogenic Vibrio decreased significantly while beneficial Lactobacillus increased significantly in fish fed PG and LTA. In conclusion, both the PG and LTA originated from B. pumilus SE5 could activate TLRs/MyD88 signaling and expression of wide-ranging antibacterial effectors, and therefore shape the intestinal microbiota in grouper.

Synthesis of N-hydroxycinnamoyl amide derivatives and evaluation of their anti-oxidative and anti-tyrosinase activities.

Twelve N-hydroxycinnamoyl amino acid amide ethyl esters (CAES) were synthesized by using l-amino acid ethyl ester hydrochloride and corresponding cinnamic acid (ferulic acid, acetylferulic acid and caffeic acid) as raw materials in the presence of a catalytic amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-hydrochloride (EDC) and 1-hydroxybenzotriene (HOBt). The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activities of CAES were evaluated. The anti-tyrosinase activities of N-feruloyl amino acid ethyl esters and the hydroxyl (OH) free radical scavenging activities of N-caffeoyl amino acid ethyl esters were also examined. DPPH free radical scavenging activity was shown in all CAES, of which N-caffeoyl amino acid ethyl esters demonstrated higher radical scavenging activity than N-feruloyl amide derivatives, and (E) -N-(caffeic acid)-l-glycinate ethyl ester (c5) had the strongest ability to scavenge free radicals with an IC50 value of 18.6 µM. The acetylferuloyl amino acid esters exhibited the highest tyrosinase inhibition activity among the tested amides.

[Quality assessment of commercial rosemary based on liquid/gas chromatography and mass spectrography with multivariate statistical analysis].

A GC-MS fingerprint of rosemary oil and an UPLC fingerprint of non-volatile compounds of rosemary were established. Sixty-three kinds of volatile compounds and thirty-eight kinds of non-volatile compounds were identified tentatively using GC-MS and UPLC-Q-TOF-MS, respectively, and most of them are flavonoids, oxygenated monoterpenes, and diterpenoids, etc. According to multivariate data analysis, chemical differences existed among commercial rosemary samples, for example, the essential oil of imported rosemary sample belongs to Morocco/Tunisian type and the one of domestic sample belongs to Spanish type. The results of principal components analysis and partial least squares discriminant analysis showed that the imported and domestic rosemary samples were classified into two groups and fourteen components including eucalyptol, (+)-α-pinene, and carnosic acid, were found as the discrimination markers. In comparison with imported rosemary, it can be found that the contents of ten markers such as (+)-α-pinene, o-cymene, and carnosic acid were higher, nonetheless, lower contents for the other markers, in those domestic rosemary samples. Moreover, rosmarinic acid, an important bioactive component in rosemary extract, extensively varied among different samples, indicating that the quality evaluation of rosemary should be concerned.

Long Non-Coding RNA-MALAT1 Mediates Retinal Ganglion Cell Apoptosis Through the PI3K/Akt Signaling Pathway in Rats with Glaucoma.

BACKGROUND/AIMS: The aim of the present study is to investigate the effect of long non-coding RNA-MALAT1 (LncRNA-MALAT1) on retinal ganglion cell (RGC) apoptosis mediated by the PI3K/Akt signaling pathway in rats with glaucoma. METHODS: RGCs were isolated and cultured, and monoclonal antibodies (anti-rat Thy-1, Brn3a and RBPMS) were examined by immunocytochemistry. An overexpression vector MALAT1-RNA activation (RNAa), gene knockout vector MALAT1-RNA interference (RNAi), and control vector MALAT1-negative control (NC) were constructed. A chronic high intraocular pressure (IOP) rat model of glaucoma was established by episcleral vein cauterization. The RGCs were divided into the RGC control, RGC pressure, RGC pressure + MALAT1-NC, RGC pressure + MALAT1-RNAi and RGC pressure + MALAT1-RNAa groups. Sixty Sprague-Dawley (SD) rats were randomly divided into the normal, high IOP, high IOP + MALAT1-NC, high IOP + MALAT1-RNAa and high IOP + MALAT1-RNAi groups. qRT-PCR and western blotting were used to detect the expression levels of LncRNA-MALAT1 and PI3K/Akt. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and flow cytometry were used to detect RGC apoptosis. RESULTS: Immunocytochemistry revealed that the cultured RGCs reached 90% purity. Compared with the RGC pressure + MALAT1-NC group, the RGC pressure + MALAT1-RNAa group exhibited elevated expression levels of MALAT1, lower total protein levels of PI3K and Akt and decreased RGC apoptosis, while these expression levels were reversed in the RGC pressure + MALAT1-RNAi group. RGC numbers and PI3K/Akt expression levels in the high IOP model groups were lower than those in the normal group. In the high IOP + MALAT1-RNAa group, the mRNA and protein expression levels of PI3K/Akt were reduced but higher than those in the other three high IOP model groups. Additionally, RGC numbers in the high IOP + MALAT1-RNAa group were lower than those in the normal group but higher than those in the other three high IOP model groups. CONCLUSION: Our study provides evidence that LncRNA-MALAT1 could inhibit RGC apoptosis in glaucoma through activation of the PI3K/Akt signaling pathway.

Safety assessment of widely used fermented virgin coconut oil (Cocos nucifera) in Malaysia: Chronic toxicity studies and SAR analysis of the active components.

Fermented Virgin Coconut Oil (FVCO) is widely used in the Southeast Asia as food and traditional medicine. The objective of the present study is the evaluation of chronic safety of the commercialized FVCO of Malaysia and other Southeast Asian countries. A single dose of 5000 mg/kg of FVCO was administered orally in rats (each group, n = 5) for the acute toxicity study and 175, 550 and 2000 mg/kg for sub-chronic and chronic studies (each group, n = 10), respectively. The behavior, mortality, and body weight of the rats were assessed to determine the toxic effects of FVCO. The haematology, biochemistry and histopathology of the treated rats were evaluated. The treated rats were safe with the dose of 5000 mg/kg in acute, sub-chronic and chronic indication. Abnormal clinical signs and morphology (gross necroscopy), changes of organ weight, anomalous haematology and biochemistry indexes were not found in comparison with the control (p > 0.05). In general, food and water intake were higher in the treated rats related to control. It was concluded that the presence of the antioxidant active compounds of FVCO might be the reason of safety. The structure activity relationship (SAR) provides a comprehensive mechanism to determine the safety that is the presence of the electron donating phenolic groups, carbonyl groups, and carboxylic acid in the ortho and meta position of the aromatic rings. The SAR showed the antioxidant properties of myristic acid and lauric acid determined by GC-MS analysis. This result suggests the safety of FVCO for chronic use, nutritional activity that FVCO formulation complies the requirements of regulatory agencies.

[Optimization of Aqueous Two-Phase Extraction of Polysaccharide from Schisandra chinensis Using Response Surface Methodology with Box-Behnken Design].

Objective: To optimize the extraction condition of polysaccharide from Schisandra chinensis. Aqueous two-phase extraction（ ATPE） method was used, based on Box-Behnken design with Response surface methodology（ BBD-RSM）. Methods: solvent volume,K2HPO4 and PEG6000 were selected as the investigation factors by the single-factor experiment, and the overall desirability（ OD）of phase volume ratio, partition coefficient and extraction rates were the reponse value. BBD-RSM was used to optimize the extraction process. . Results: The optimal parameters were as follows,the solvent volume was 5 m L,the addition amount of K2HPO4 was 1. 0 g, the addition amount of PEG6000 was 1. 8 g and centrifugation time was 9 min, which indicated that the model had a good predictability. The predicted value was 0. 950,and the deviation between observed and predicted values was 3. 94% Conclusion: The ATPE technology is easy to operate and cost-effective for the extraction of polysaccharides from Schisandra chinensis.