PHB2 inhibits WSSV replication by promoting the nuclear translocation of STAT.

Prohibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses and cell proliferation. However, the function of the PHBs in immune regulation has largely not been determined. In the present study, we identified PHB2 in the red swamp crayfish Procambarus clarkii. PHB2 was found to be widely distributed in several tissues, and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge. PHB2 significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. Here, we observed that PHB2 promotes the nuclear translocation of STAT by binding to STAT. After blocking PHB2 or STAT with antibodies or interfering with PHB2 or STAT, the expression levels of the antiviral genes ß-thymosin (PcThy-4) and crustin2 (Cru2) decreased. The gene sequence of PHB2 was analyzed and found to contain a nuclear introgression sequence (NIS). After in vivo injection of PHB2 with deletion of NIS (rΔNIS-PHB2), the nuclear translocation of STAT did not change significantly compared to that in the control group. These results suggest that PHB2 promoted the nuclear translocation of STAT through NIS and mediated the expression of antiviral proteins to inhibit WSSV infection.

PcTrim prevents early infection with white spot syndrome virus by inhibiting AP1-induced endocytosis.

Viruses have evolved various strategies to achieve early infection by initiating transcription of their own early genes via host transcription factors, such as NF-κb, STAT, and AP1. How the host copes with this immune escape has been a topic of interest. Tripartite motif (TRIM) family proteins with RING-type domains have E3 ubiquitin ligase activity and are known as host restriction factors. Trim has been reported to be associated with phagocytosis and is also believed to be involved in the activation of autophagy. Preventing the virus from entering the host cell may be the most economical way for the host to resist virus infection. The role of TRIM in the early stage of virus infection in host cells remains to be further interpreted. In the current study, a crayfish TRIM with a RING-type domain, designated as PcTrim, was significantly upregulated under white spot syndrome virus (WSSV) infection in the red swamp crayfish (Procambarus clarkii). Recombinant PcTrim significantly inhibited WSSV replication in crayfish. RNAi targeting PcTrim or blocking PcTrim with an antibody promoted WSSV replication in crayfish. Pulldown and co-IP assays showed that PcTrim can interact with the virus protein VP26. PcTrim restricts the expression level of dynamin, which is involved in the regulation of phagocytosis, by inhibiting AP1 entry into the nucleus. AP1-RNAi effectively reduced the expression levels of dynamin and inhibited host cell endocytosis of WSSV in vivo. Our study demonstrated that PcTrim might reduce early WSSV infection by binding to VP26 and then inhibiting AP1 activation, resulting in reduced endocytosis of WSSV in crayfish hemocytes. Video Abstract.

KCTD7 mutations impair the trafficking of lysosomal enzymes through CLN5 accumulation to cause neuronal ceroid lipofuscinoses.

Lysosomes are central organelles for cellular degradation and energy metabolism. Neuronal ceroid lipofuscinoses (NCLs) are a group of the most common neurodegenerative lysosomal storage disorders characterized by intracellular accumulation of ceroid in neurons. Mutations in KCTD7, a gene encoding an adaptor of the CUL3-RING E3 ubiquitin ligase (CRL3) complex, are categorized as a unique NCL subtype. However, the underlying mechanisms remain elusive. Here, we report various lysosomal and autophagic defects in KCTD7-deficient cells. Mechanistically, the CRL3-KCTD7 complex degrades CLN5, whereas patient-derived KCTD7 mutations disrupt the interaction between KCTD7-CUL3 or KCTD7-CLN5 and ultimately lead to excessive accumulation of CLN5. The accumulated CLN5 disrupts the interaction between CLN6/8 and lysosomal enzymes at the endoplasmic reticulum (ER), subsequently impairing ER-to-Golgi trafficking of lysosomal enzymes. Our findings reveal previously unrecognized roles of KCTD7-mediated CLN5 proteolysis in lysosomal homeostasis and demonstrate that KCTD7 and CLN5 are biochemically linked and function in a common neurodegenerative pathway.

Flavonoids derived from Anemarrhenae Rhizoma ameliorate inflammation of benign prostatic hyperplasia via modulating COX/LOX pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Flavonoids are the main components of the traditional Chinese medicine Anemarrhenae Rhizoma (dried rhizome of Anemarrhena asphodeloides Bge.), which has been reported to possess activity against inflammation and tumor. AIM OF STUDY: Regulation of the arachidonic acid (AA) cascade through cyclooxygenase (COX) and lipoxygenase (LOX) represent the two major pathways to treat inflammatory of benign prostatic hyperplasia (BPH). In this study, Anemarrhenae Rhizoma flavonoids and its main compounds (mangiferin, neomangiferin and isomangiferin) were investigated for effects on AA metabolism. METHODS: Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used to monitor AA metabolites in BPH rats and in PC-3 cells. COX-2 and 5-LOX protein and mRNA levels were measured by Western blot and qPCR, respectively, along with histopathological assessment of prostate tissues. RESULTS: Treatment with flavonoids significantly ameliorated BPH-associated prostate inflammation and inhibited the expression of COX-2 and 5-LOX at the protein and mRNA levels. Quantitative metabolomic analysis of blood plasma showed flavonoids treatment decreased AA levels and its metabolites associated with the COX and LOX pathways. Further exploration of the flavonoid compounds mangiferin, neomangiferin and isomangiferin showed they inhibited AA metabolism to varying degrees in PC-3 cell cultures. CONCLUSION: Anemarrhenae Rhizoma flavonoids act to inhibit BPH-related inflammation in vivo and in vitro by targeting AA metabolism and interfering with COX and LOX pathways. The identification of mangiferin, neomangiferin and isomangiferin as anti-inflammatory components suggests flavonoids interventions represent a promising therapeutic approach for BPH.

The bioactive alkaloids identified from Cortex Phellodendri ameliorate benign prostatic hyperplasia via LOX-5/COX-2 pathways.

BACKGROUND: The bioactive alkaloids identified from Cortex Phellodendri (CP) were highly effective in treating rats with benign prostatic hyperplasia (BPH). Specifically, lipoxygenase-5 (LOX-5) and cyclooxygenase-2 (COX-2) were identified as two primary targets for alleviating inflammation in BPH rats. However, it remains unknown whether the alkaloid components in CP can interact with the two target proteins. PURPOSE: To further identify bioactive alkaloids targeting LOX/COX pathways. METHODS: An affinity-ultrafiltration mass spectrometry approach was employed to screen dual-target LOX-5/COX-2 ligands from alkaloid extract. The structures of bioactive alkaloids were characterized by high-resolution Fourier transform ion cyclotron resonance mass spectrometry. To understand the molecular mechanisms underlying the effects of bioactive alkaloids, the expression levels of LOX-5 and COX-2 in BPH model rats were investigated at both protein and mRNA levels. The LOX-5/COX-2 enzymes activity experiments and molecular docking analysis were performed to fully evaluate the interactions between bioactive alkaloids and LOX-5/COX-2. RESULTS: After comprehensive analysis, the results showed that bioactive alkaloids could suppress the expression of LOX-5 and COX-2 simultaneously to exert an anti-inflammatory effect on the progression of BPH. In addition, the screened protoberberine, demethyleneberberine was found to exhibit prominent inhibitory activities against both LOX-5 and COX-2 enzymes, palmatine and berberine with moderate inhibitory activities. Molecular docking analysis confirmed that demethyleneberberine could interact well with LOX-5/COX-2. CONCLUSION: This study is the first to explore the inhibitory effects of bioactive alkaloids from CP on LOX-5 and COX-2 activities in BPH rats. Our findings demonstrate that the bioactive alkaloids from CP can ameliorate BPH via dual LOX-5/COX-2 pathways, which serves as an efficient approach for the discovery of novel drug leads from natural products with reduced side effects.

Effects of Airflow Ultrafine-Grinding on the Physicochemical Characteristics of Tartary Buckwheat Powder.

Five different ultrafine milled flours (UMFs) were prepared from Tartary buckwheat via airflow ultrafine-grinding at different grinding pressures. The airflow ultrafine-grinding resulted in marked differences in particle size (from 100 to 10 µm). The UMFs were all brighter in appearance (higher L*) than Tartary buckwheat common flour (TBCF). Illustrated by the example of 70 °C, the UMFs were also found to have a greater water holding capacity (from 4.42 g/g to 5.24 g/g), water solubility (from 12.57% to 14.10%), and water solubility index (from 5.11% to 6.10%). Moreover, as the particle sizes reduced, the moisture content decreased (from 10.05 g/100 g DW to 7.66 g/100 g DW), as did the total starch content (from 68.88 g/100 g DW to 58.24 g/100 g DW) and the protein content (from 13.16% to 12.04%). However, the grinding process was also found to have negative effects on the mineral content of the Tartary buckwheat. Additionally, several substantial variations were found in their hydration properties along with grinding pressure changes in the differently ground UMFs. Consequently, fine Tartary buckwheat powders of a bright yellow color, with superior food processing properties, were prepared in this study by airflow ultrafine-grinding.

Hepatopancreas-Specific Lectin Participates in the Antibacterial Immune Response by Regulating the Expression of Antibacterial Proteins.

The hepatopancreas is an important digestive and immune organ in crustacean. There were low but stable numbers of microbes living in the hemolymph of crustacean, whereas the organs (including hepatopancreas) of crustacean were immersed in the hemolymph. It is very important to study the immune mechanism of the hepatopancreas against bacteria. In this study, a novel CTL (HepCL) with two CRDs, which was mainly expressed in the hepatopancreas, was identified in red swamp crayfish (Procambarus clarkii). HepCL binds to bacteria in vitro and could enhance bacterial clearance in vivo. Compared with the C-terminal CRD of HepCL (HepCL-C), the N-terminal CRD (HepCL-N) showed weaker bacterial binding ability in vitro and stronger bacterial clearance activity in vivo. The expression of some antimicrobial proteins, such as FLP, ALF1 and ALF5, was downregulated under knockdown of HepCL or blocked with Anti-HepCL after challenge with Vibrio in crayfish. These results demonstrated that HepCL might be involved in the antibacterial immune response by regulating the expression of antimicrobial proteins.

A crayfish ALF inhibits the proliferation of microbiota by binding to RPS4 and MscL of E. coli.

Antimicrobial peptides (AMPs), most of which are small proteins, are necessary for innate immunity against pathogens. Anti-lipopolysaccharide factor (ALF) with a conserved lipopolysaccharide binding domain (LBD) can bind to lipopolysaccharide (LPS) and neutralize LPS activity. The antibacterial mechanism of ALF, especially its role in bacteria, needs to be further investigated. In this study, the antibacterial role of an anti-lipopolysaccharide factor (PcALF5) derived from Procambarus clarkii was analyzed. PcALF5 could inhibit the replication of the microbiota in vitro and enhance the bacterial clearance ability in crayfish in vivo. Far-western blot assay results indicated that PcALF5 bound to two proteins of E. coli (approximately 25 kDa and 15 kDa). Mass spectrometry (MS), far-western blot assay, and pull-down results showed that 30S ribosomal protein S4 (RPS4, 25 kD) interacted with PcALF5. Further studies revealed that another E. coli protein binding to PcALF5 could be the large mechanosensitive channel (MscL), which is reported to participate in the transport of peptides and antibiotics. Additional assays showed that PcALF5 inhibited protein synthesis and promoted the transcription of ribosomal component genes in E. coli. Overall, these results indicate that PcALF5 could transfer into E. coli by binding to MscL and inhibit protein synthesis by interacting with RPS4. This study reveals the mechanism underlying ALF involvement in the antibacterial immune response and provides a new reference for the research on antibacterial drugs.

Variable beam entrance Faraday cup system for pulsed electron beam current profile characterization.

A high accuracy variable beam entrance Faraday cup (VBEFC) system is designed in this work. The presented VBEFC system is designed for the beam current profile measurement of the transient hollow cathode discharge (THCD) generated pulsed electron beam, which is a new source of high energy flux for metallic material processing. By proper designs of the beam entrance array, fast response electron collector, grounding, and shielding, this VBEFC system is capable of determining the radial profile and its temporal evolution of the THCD generated electron beam. The results of the electron beam current and current density distributions collected at varying radial locations and times under multiple voltages are presented in this paper. The experimental results show that both the amplitude and the current density of the THCD electron beam at a given radius always increased with the increase in the accelerating voltage, which is coincident with the self-focused propagation of the electron beam promoted by the voltage.

Genetic environments and related transposable elements of novel cfr(C) variants in Campylobacter coli isolates of swine origin.

The cfr(C) is a cfr-like gene that confers cross-resistance to antibiotics targeting the 23S rRNA through methylation of nucleotide A2503. Here, we identified 7 C. coli isolates containing 4 novel cfr(C) variants from swine farm and slaughterhouses samples. Of the 7 cfr(C)-carrying isolates, one had a frame-shift mutation, while the other 6 had intact genes. However, one of the 6 intact genes did not show a PhLOPSA phenotype in the original isolate, but was fully functional when cloned into C. jejuni NCTC 11168. Cloning of cfr(C) variants into C. jejuni NCTC 11168 and conjugative transfer of the two cfr(C)-containing plasmids further confirmed their role in conferring resistance to PhLOPSA antimicrobials, and resulted in an 8-128-fold increase in their MICs. In all cfr(C)-carrying isolates, cfr(C) genes were located in the downstream of the kanamycin resistant gene aphA3. IS607* and IS1595-like were located immediately upstream of aphA3 gene and seemed to play a role in its recombination. A novel transposable element named ISCco7, which located immediately downstream of cfr(C) in two isolates, was probably associated with the integration of cfr(C). However, neither insertion sequence nor other transposable elements were identified near cfr(C) in the remaining five cfr(C)-positive isolates, indicating the mechanism underlying the integration of cfr(C) into plasmids or chromosomal DNA requires further investigation. These results reveal novel cfr(C) variants and their associated genetic environments in C. coli isolates and indicate the flexibility of C. coli in acquiring new antibiotic resistance genes.

Characterization of spirostanol glycosides and furostanol glycosides from anemarrhenae rhizoma as dual targeted inhibitors of 5-lipoxygenase and Cyclooxygenase-2 by employing a combination of affinity ultrafiltration and HPLC/MS.

BACKGROUND: Modulation of the arachidonic acid (AA) cascade via 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) represent the two major pathways for treatments of inflammation and pain. The design and development of inhibitors targeting both 5-LOX and COX-2 has gained increasing popularity. As evidenced, 5-LOX and COX-2 dual targeted inhibitors have recently emerged as the front runners of anti-inflammatory drugs with improved efficacy and reduced side effects. Natural products represent a rich resource for the discovery of dual targeted 5-LOX and COX-2 inhibitors. By combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS), an efficient method was developed to identify spirostanol glycosides and furostanol glycosides as the 5-LOX/COX-2 dual inhibitors from saponins extract of Anemarrhenae Rhizoma (SEAR). METHODS: A highly efficient method by combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS) was first developed to screen and characterize the 5-LOX/COX-2 dual targeted inhibitors from SEAR. The structures of compounds in the ultrafiltrate were characterized by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). In addition, in vitro 5-LOX/COX-2 inhibition assays and their dual expression in vivo were performed to confirm the inhibitory activities of the compounds screened by AUF-LC-MS. Molecular docking studies with the corresponding binding energy were obtained which fit nicely to both 5-LOX and COX-2 protein cavities and in agreement with our affinity studies. RESULTS: A total of 5 compounds, timosaponin A-II, timosaponin A-III, timosaponin B-II, timosaponin B-III and anemarrhenasaponin I, were identified as potential 5-LOX/COX-2 dual targeted inhibitors with specific binding values > 1.5 and IC50 ≤ 6.07 µM. CONCLUSION: The present work demonstrated that spirostanol glycoside and furostanol glycoside were identified as two novel classes of dual inhibitors of 5-LOX/COX-2 enzymes by employing a highly efficient screening method of AUF-LC-MS. These natural products represent a novel class of anti-inflammatory agents with the potential of improved efficacy and reduced side effects.

Screening for natural inhibitors of 5-lipoxygenase from Zi-shen pill extract by affinity ultrafiltration coupled with ultra performance liquid chromatography-mass spectrometry.

ETHNOPHARMACOLOGICAL RELEVANCE: Zi-shen pill (ZSP), a traditional Chinese medicine, is widely used for the treatment of benign prostatic hyperplasia (BPH) and has remarkable curative effect. AIM OF THE STUDY: To screen the potential 5-Lipoxygenase(5-LOX) inhibitors from ZSP extract. MATERIALS AND METHODS: A new approach based on affinity ultrafiltration-ultra performance liquid chromatography-mass spectrometry(UPLC-MS) was established and validated. Zileuton and glipizide were chosen as positive and negative control drug, respectively. For better screening result, the concentration of 5-LOX enzyme, incubation temperature and time, pH and ion strength were optimized. In addition, 5-LOX inhibitory assay in vitro and molecular docking technique were used for further verification. RESULTS: 20 compounds were characterized in the ultrafiltrate by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and 16 ligands showed binding ability to 5-LOX. Among them, six ligands were deduced as high-potential 5-LOX inhibitors with their high specific binding values (>2.0). The inhibitory activities of anemarrhenasaponin I, timosaponin AI, nyasol and demethyleneberberine were confirmed by the 5-LOX inhibitory assay for validating the reliability of affinity ultrafiltration approach and the computer-simulated molecular docking technique further clarified the possible mechanism of action between the active compounds and the 5-LOX active sites.

PcLys-i3, an invertebrate lysozyme, is involved in the antibacterial immunity of the red swamp crayfish, Procambarus clarkii.

Antimicrobial peptides (AMPs) play important roles in innate immunity against pathogens and lysozymes are a particularly type of AMP. Lysozymes are hydrolytic enzymes that are characterized by their ability to cleave the beta-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, which is the major bacterial cell wall polymer. In this work, a lysozyme was identified from Procambarus clarkii and designated PcLys-i3. Quantitative RT-PCR was used to analyze the tissue distribution and expression profiles of PcLys-i3. PcLys-i3 was present in all tested tissues and had high expression levels in gills, stomach and intestine. The expression levels of PcLys-i3 were up-regulated in gills and intestine after challenge with Vibrio parahaemolyticus, Staphylococcus aureus and Aeromonas hydrophila. PcLys-i3 and PcFer proteins can enhance the bacterial elimination in crayfish, whereas the bacterial elimination was weakened when the expression level of PcLys-i3 or PcFer RNAs was suppressed by RNAi. Recombinant PcLys-i3 and PcFer significantly reduced the mortality of crayfish with bacterial infections. Further study found that PcLys-i3 could interact with PcFer in vitro. Finally, the PcLys-i3 and PcFer proteins could bind to bacteria and inhibit bacterial replication. These results suggest that both PcLys-i3 and PcFer play important roles in the antibacterial immunity of red swamp crayfish.

Bioinspired Multifunctional Membrane for Aquatic Micropollutants Removal.

Micropollutants present in water have many detrimental effects on the ecosystem. Membrane technology plays an important role in the removal of micropollutants, but there remain significant challenges such as concentration polarization, membrane fouling, and variable permeate quality. The work reported here uses a multifunctional membrane with rejection, adsorption, and catalysis functions to solve these problems. On the basis of mussel-inspired chemistry and biological membrane properties, a multifunctional membrane was prepared by applying "reverse filtration" of a laccase solution and subsequent "dopamine coating" on a nanofiltration (NF) membrane support, which was tested on bisphenol A (BPA) removal. Three NF membranes were chosen for the preparation of the multifunctional membranes on the basis of the membrane properties and enzyme immobilization efficiency. Compared with the pristine membrane, the multifunctional membrane exhibited significant improvement of BPA removal (78.21 ± 1.95%, 84.27 ± 7.30%, and 97.04 ± 0.33% for NT103, NF270, and NF90, respectively), all of which are clearly superior to the conventional Fenton treatment (55.0%) under similar conditions and comparable to soluble laccase coupled with NF270 membrane filtration (89.0%). The improvement would appear to be due to a combination of separation (reducing the enzymatic burden), adsorption (enriching the substrate concentration as well as prolonging the residence time), and lastly, catalysis (oxidizing the pollutants and breaking the "adsorption saturation limits"). Furthermore, the synergistic effect of the polydopamine (PDA) layer on the enzymatic oxidation of BPA was confirmed, which was due to its enhanced adsorption and electron transfer performance. The multifunctional membrane could be reused for at least seven cycles with an acceptable activity loss, demonstrating good potential for removal of micropollutants.

A facile microwave-assisted fabrication of fluorescent carbon nitride quantum dots and their application in the detection of mercury ions.

A facile microwave-assisted solvothermal method was used to prepare fluorescent carbon nitride quantum dots (CNQDs) using oleic acid as the reaction media at moderate reaction temperature in a short time (5 min). Citric acid monohydrate and urea were used as the precursors. The as-prepared CNQDs were characterized by multiple analytical techniques. The CNQDs exhibited an uncommon excitation wavelength-dependent fluorescence with two maximum emission peaks at 450 and 540 nm. The CNQDs with a quantum yield of 27.1% could serve as an effective fluorescent sensing platform for label-free sensitive detection of Hg(2+) ions with a detection limit of 0.14 µM. This method was also applied to the detection of Hg(2+) ions in tap water samples.

One-pot fabrication of hollow cross-linked fluorescent carbon nitride nanoparticles and their application in the detection of mercuric ions.

Hollow cross-linked fluorescent carbon nitride nanoparticles (CNNPs) were fabricated via a facile one-pot solvothermal process. The obtained CNNPs were characterized by multiple analytical techniques including transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), solid-state nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR). The excitation-dependent fluorescence emission spectra showed significant differences for the CNNPs derived from various proportions of citric acid monohydrate and urea. The fluorescence quantum yield of the obtained CNNPs could reach 31%. The CNNPs exhibited good fluorescence quenching selectivity to mercuric ions. Concentration experiments showed that there existed two parts of linear relationship between fluorescence intensity and concentration of Hg(2+) ions in the range of 0.1-8 and 8-32 µM. The limit of detection (LOD) was estimated to be 0.094 µM. This method can be applied to the detection of Hg(2+) ions in tap water samples.