Azonal steatosis correlates with disease severity and poor outcome of nonalcoholic fatty liver disease.

OBJECTIVES: In this study we aimed to assess the clinicopathological characteristics and long-term prognosis of patients with nonalcoholic fatty liver disease (NAFLD) having distinct steatosis distribution patterns. METHODS: Clinicopathological data of 238 individuals with biopsy-confirmed NAFLD were collected. Nonalcoholic steatohepatitis-clinical research network (NASH-CRN) and steatosis, activity and fibrosis (SAF)/fatty liver inhibition of progression (FLIP) algorithm were used. Cumulative incidence of liver-related events (LREs) was compared by Kaplan-Meier analysis. Univariate and multivariate logistic regression analyses were used to identify independent predictors for steatosis distribution. RESULTS: Eligible patients were categorized into three groups based on their steatosis distribution, including azonal steatosis (AS) (62 [26.1%]), perivenular steatosis (PVS) (147 [61.8%]), and the pan-acinar steatosis (PAS) groups (29 [12.1%]). There were significantly higher ballooning grade and disease activity (P < 0.05), more severe fibrosis (P < 0.001), and a higher cumulative incidence of LREs (hazard ratio [HR] 8.0, 95% confidence interval [CI] 2.34-27.35, P < 0.0001) in the AS group than in the PVS and PAS groups after a median of 3.6-year follow-up. Multivariate logistic regression analysis revealed age (odds ratio [OR] 1.11, 95% CI 1.06-1.16, P < 0.001) might be independently associated with AS distribution, and PNPLA3 rs738409 CG/GG genotype (OR 3.36, 95% CI 0.98-11.47, P = 0.053) might also play a role. CONCLUSIONS: AS is associated with more severe disease activity and fibrosis stage in NAFLD, and predisposes toward poor prognosis. Age might be an independent predictor for AS in NAFLD, while PNPLA3 rs738409 CG/GG genotype might also play a role.

A remarkably diverse and well-organized virus community in a filter-feeding oyster.

BACKGROUND: Viruses play critical roles in the marine environment because of their interactions with an extremely broad range of potential hosts. Many studies of viruses in seawater have been published, but viruses that inhabit marine animals have been largely neglected. Oysters are keystone species in coastal ecosystems, yet as filter-feeding bivalves with very large roosting numbers and species co-habitation, it is not clear what role they play in marine virus transmission and coastal microbiome regulation. RESULTS: Here, we report a Dataset of Oyster Virome (DOV) that contains 728,784 nonredundant viral operational taxonomic unit contigs (≥ 800 bp) and 3473 high-quality viral genomes, enabling the first comprehensive overview of both DNA and RNA viral communities in the oyster Crassostrea hongkongensis. We discovered tremendous diversity among novel viruses that inhabit this oyster using multiple approaches, including reads recruitment, viral operational taxonomic units, and high-quality virus genomes. Our results show that these viruses are very different from viruses in the oceans or other habitats. In particular, the high diversity of novel circoviruses that we found in the oysters indicates that oysters may be potential hotspots for circoviruses. Notably, the viruses that were enriched in oysters are not random but are well-organized communities that can respond to changes in the health state of the host and the external environment at both compositional and functional levels. CONCLUSIONS: In this study, we generated a first "knowledge landscape" of the oyster virome, which has increased the number of known oyster-related viruses by tens of thousands. Our results suggest that oysters provide a unique habitat that is different from that of seawater, and highlight the importance of filter-feeding bivalves for marine virus exploration as well as their essential but still invisible roles in regulating marine ecosystems. Video Abstract.

Virus classification for viral genomic fragments using PhaGCN2.

Viruses are the most ubiquitous and diverse entities in the biome. Due to the rapid growth of newly identified viruses, there is an urgent need for accurate and comprehensive virus classification, particularly for novel viruses. Here, we present PhaGCN2, which can rapidly classify the taxonomy of viral sequences at the family level and supports the visualization of the associations of all families. We evaluate the performance of PhaGCN2 and compare it with the state-of-the-art virus classification tools, such as vConTACT2, CAT and VPF-Class, using the widely accepted metrics. The results show that PhaGCN2 largely improves the precision and recall of virus classification, increases the number of classifiable virus sequences in the Global Ocean Virome dataset (v2.0) by four times and classifies more than 90% of the Gut Phage Database. PhaGCN2 makes it possible to conduct high-throughput and automatic expansion of the database of the International Committee on Taxonomy of Viruses. The source code is freely available at https://github.com/KennthShang/PhaGCN2.0.

Histological assessment based on liver biopsy: the value and challenges in NASH drug development.

Nonalcoholic steatohepatitis (NASH) is increasingly recognized as a serious disease that can lead to cirrhosis, hepatocellular carcinoma (HCC), and death. However, there is no effective drug to thwart the progression of the disease. Development of new drugs for NASH is an urgent clinical need. Liver biopsy plays a key role in the development of new NASH drugs. Histological findings based on liver biopsy are currently used as the main inclusion criteria and the primary therapeutic endpoint in NASH clinical trials. However, there are inherent challenges in the use of liver biopsy in clinical trials, such as evaluation reliability, sampling error, and invasive nature of the procedure. In this article, we review the advantages and value of liver histopathology based on liver biopsy in clinical trials of new NASH drugs. We also discuss the challenges and limitations of liver biopsy and identify future drug development directions.

Novel RNA viruses in oysters revealed by virome.

Eighteen novel RNA viruses were found in Crassostrea hongkongensis. Phylogenic analysis shows evidence of recombination between major genes of viruses. Picobirnaviruses are ubiquitous and abundant in oysters.

Identification and analysis of two lebocins in the oriental armyworm Mythimna separata.

The insect immune system can produce defensive molecules, such as antimicrobial peptides (AMPs), to eliminate invading pathogens. Here, we report the identification of two cDNAs (MseLeb1, MseLeb2) that encode lepidopteral lebocin preproproteins in the oriental armyworm, Mythimna separata. Their open reading frames are 483/492 bp that encode 161/164 aa peptides. MseLeb1 is mainly expressed in the fat body and epidermis, while MseLeb2 is mainly expressed in the fat body, Malpighian tube, and epidermis. They were significantly induced by Escherichia coli, Staphylococcus aureus, and Beauveria bassiana in hemocytes. The preproproteins can be processed after RXXR motifs into mature peptides. Multiple sequence alignment indicates that MseLeb1 (18-42, 121-161) are potentially active peptides. Five peptides were synthesized for analyses: 18-42, 121-161, 121-154, 121-151, 121-146. Synthetic peptides showed agglutinating activity, but no hemolytic activity. Bacterial growth assay, colony formation assay, and electron microscopy revealed that synthetic peptides can inhibit bacterial growth and disrupt bacterial cell wall. B. bassiana conidia and blastospores were lysed by synthetic peptides. These results indicate that MseLeb1 and MseLeb2 are immune responsive lebocins, and the mature peptides have antibacterial and antifungal activities.

Characterization of the active fragments of Spodoptera litura Lebocin-1.

Insects can produce various antimicrobial peptides (AMPs) upon immune stimulation. One class of AMPs are characterized by their high proline content in certain fragments. They are generally called proline-rich antimicrobial peptides (PrAMPs). We previously reported the characterization of Spodoptera litura lebocin-1 (SlLeb-1), a PrAMP proprotein. Preliminary studies with synthetic polypeptides showed that among the four deductive active fragments, the C-terminal fragment SlLeb-1 (124-158) showed strong antibacterial activities. Here, we further characterized the antibacterial and antifungal activities of 124-158 and its four subfragments: 124-155, 124-149, 127-158, and 135-158. Only 124-158 and 127-158 could agglutinate bacteria, while 124-158 and four subfragments all could agglutinate Beauveria bassiana spores. Confocal microscopy showed that fluorescent peptides were located on the microbial surface. Fragment 135-158 lost activity completely against Escherichia coli and Staphylococcus aureus, and partially against Bacillus subtilis. Only 124-149 showed low activity against Serratia marcescens. Negative staining, transmission, and scanning electron microscopy of 124-158 treated bacteria showed different morphologies. Flow cytometry analysis of S. aureus showed that 124-158 and four subfragments changed bacterial subpopulations and caused an increase of DNA content. These results indicate that active fragments of SlLeb-1 may have diverse antimicrobial effects against different microbes. This study may provide an insight into the development of novel antimicrobial agents.

MG132 protects against renal dysfunction by regulating Akt-mediated inflammation in diabetic nephropathy.

Diabetic nephropathy (DN), the leading cause of end-stage renal disease (ESRD). To date, mounting evidence has shown that inflammation may contribute to the pathogenesis of DN. Recent reports have shown that proteasome inhibitors display cytoprotection by reducing the phosphorylation of Akt, a serine/threonine kinase, plays a critical role in cellular survival and metabolism and can crosstalk with inflammation. Therefore, we hypothesized that MG132, specific proteasome inhibitor, could provide renoprotection by suppressing Akt-mediated inflammation in DN. In vivo, male Sprague-Dawley rats were divided into normal control group (NC), diabetic nephropathy group (DN), DN model plus MG132 treatment group (MG132), and DN model plus deguelin treatment group (Deguelin)(deguelin, a specific inhibitor of Akt). In vitro, a human glomerular mesangial cell lines (HMCs) was exposed to 5.5 mmol/L glucose (CON), 30 mmol/L glucose (HG), 30 mmol/L glucose with 0.5 umol/L MG132 (MG132) and 30 mmol/L glucose with 5 umol/L deguelin (Deguelin). Compared with NC, DN showed a significant increase in the urinary protein excretion rate and inflammatory cytokines, as well as p-Akt. Compared with CON, HMCs co-cultured with HG was notably proliferated, which is in accord with α-smooth muscle actin (α-SMA) expression. These alterations were inhibited by administration of MG132 or deguelin. In conclusion, MG132 significantly inhibits the development of DN by regulating Akt phosphorylation-mediated inflammatory activation.

Characterization of PGRP-S1 from the oriental armyworm, Mythimna separata.

Peptidoglycan is the key component forming the backbone of bacterial cell wall. It can be recognized by a group of pattern recognition receptors, known as peptidoglycan recognition proteins (PGRPs) in insects and higher animals. PGRPs may serve as immune receptors or N-acetylmuramoyl-L-alanine amidases (EC 3.5.1.28). Here, we report the characterization of a short PGRP, PGRP-S1, from the oriental armyworm, Mythimna separata. MsePGRP-S1 cDNA encodes a protein of 197 amino acids (aa) with a PGRP domain of about 150 aa. MsePGRP-S1 was expressed in several tissues of naïve larvae, including hemocytes, midgut, fat body and epidermis. Bacterial challenges caused variable changes in different tissues at the mRNA level. The recombinant protein bound strongly to Staphylococcus aureus and purified peptidoglycans from Staphylococcus aureus and Bacillus subtilis. It can inhibit the growth of gram-negative and gram-positive bacteria by disrupting bacterial surface. It can degrade peptidoglycans from Escherichia coli and Staphylococcus aureus. Taken together, these data demonstrate that M. separata PGRP-S1 is involved in defending against bacteria.

Antimicrobial activities of a proline-rich proprotein from Spodoptera litura.

Antimicrobial peptides (AMPs) are produced by the stimulated humoral immune system. Most mature AMPs contain less than 50 amino acid residues. Some of them are generated from proproteins upon microbial challenges. Here, we report the antimicrobial activities of a proline-rich proprotein, named SlLebocin1 (SlLeb1), from the tobacco cutworm Spodoptera litura. SlLebocin1 cDNA contains a 477-bp open reading frame (ORF). It is mainly expressed in hemocytes and the midgut in naïve larvae. The transcript level was significantly induced in hemocytes but repressed in the midgut and fat body by bacterial challenges. The proprotein contains 158 amino acids with 3 RXXR motifs that are characteristic of some Lepidopteral lebocin proproteins. Four peptides corresponding to the predicted processed fragments were synthesized chemically, and their antimicrobial activities against two Gram-negative and two Gram-positive bacterial strains were analyzed. The peptides showed differential antimicrobial activities. For Escherichia coli and Bacillus subtilis, only the C-terminal fragment (124-158) showed strong inhibitory effects. For Staphylococcus aureus, all peptides showed partial inhibitions. None of them inhibited Serratia marcescens. Bacterial morphologies were examined by the scanning electron microscopy and confocal laser scanning microscopy. The antimicrobial peptides either disrupted cellular membrane or inhibited cell division and caused elongated/enlarged morphologies. The results may provide ideas for designing novel antibiotics.

Immune functions of insect ßGRPs and their potential application.

Insects rely completely on the innate immune system to sense the foreign bodies and to mount the immune responses. Germ-line encoded pattern recognition receptors play crucial roles in recognizing pathogen-associated molecular patterns. Among them, ß-1,3-glucan recognition proteins (ßGRPs) and gram-negative bacteria-binding proteins (GNBPs) belong to the same pattern recognition receptor family, which can recognize ß-1,3-glucans. Typical insect ßGRPs are comprised of a tandem carbohydrate-binding module in the N-terminal and a glucanase-like domain in the C-terminal. The former can recognize triple-helical ß-1,3-glucans, whereas the latter, which normally lacks the enzymatic activity, can recruit adapter proteins to initiate the protease cascade. According to studies, insect ßGRPs possess at least three types of functions. Firstly, some ßGRPs cooperate with peptidoglycan recognition proteins to recognize the lysine-type peptidoglycans upstream of the Toll pathway. Secondly, some directly recognize fungal ß-1,3-glucans to activate the Toll pathway and melanization. Thirdly, some form the 'attack complexes' with other immune effectors to promote the antifungal defenses. The current review will focus on the discovery of insect ßGRPs, functions of some well-characterized members, structure-function studies and their potential application.

Spexin Enhances Bowel Movement through Activating L-type Voltage-dependent Calcium Channel via Galanin Receptor 2 in Mice.

A novel neuropeptide spexin was found to be broadly expressed in various endocrine and nervous tissues while little is known about its functions. This study investigated the role of spexin in bowel movement and the underlying mechanisms. In functional constipation (FC) patients, serum spexin levels were significantly decreased. Consistently, in starved mice, the mRNA of spexin was significantly decreased in intestine and colon. Spexin injection increased the velocity of carbon powder propulsion in small intestine and decreased the glass beads expulsion time in distal colon in mice. Further, spexin dose-dependently stimulated the intestinal/colonic smooth muscle contraction. Galanin receptor 2 (GALR2) antagonist M871, but not Galanin receptor 3 (GALR3) antagonist SNAP37899, effectively suppressed the stimulatory effects of spexin on intestinal/colonic smooth muscle contraction, which could be eliminated by extracellular [Ca(2+)] removal and L-type voltage-dependent Ca(2+) channel (VDCC) inhibitor nifedipine. Besides, spexin dramatically increased the [Ca(2+)]i in isolated colonic smooth muscle cells. These data indicate that spexin can act on GALR2 receptor to regulate bowel motility by activating L-type VDCC. Our findings provide evidence for important physiological roles of spexin in GI functions. Selective action on spexin pathway might have therapeutic effects on GI diseases with motility disorders.

Resectoscopic ablation of the gallbladder mucosa after cholecystostomy.

PURPOSE: To investigate the feasibility of endoscopic ablation of the gallbladder mucosa after cholecystostomy. MATERIALS AND METHODS: The mucosa of the gallbladder was endoscopically electrocoagulated through the cholecystostomy tract. After ablation, repeated luminal irrigation with chymotrypsin solution was given before removal of the tube. The patients were followed up by ultrasonography after the procedure. RESULTS: Twenty patients accepted this operation. The mean operating time was 38 minutes (range, 25 to 55 min). There were no procedure-related mortality and complications including perforation, bleeding, and cholangitis. Eighteen gallbladders have completely obliterated in 3 months. The other 2 gallbladders developed asymptomatic retention cysts. During 48 months of follow-up period, no stone occurred and no signs of malignancy were found at the site of the gallbladder. Seven patients died from the other medical illness. CONCLUSION: Resectoscopic ablation of the gallbladder mucosa is a safe and promising approach to sclerosis of the gallbladder after cholecystostomy.

4-Phenylbutyric acid suppresses inflammation through regulation of endoplasmic reticulum stress of endothelial cells stimulated by uremic serum.

AIMS: Endoplasmic reticulum (ER) stress is involved in the pathogenesis of atherosclerosis (AS). Endothelial cell (EC) dysfunction and monocyte migration to the subendothelium are considered to be essential manifestations of AS. We conducted this study to determine whether ER stress was involved in uremic serum-induced EC dysfunction and whether the regulation of ER stress using a chemical chaperone 4-phenylbutyric acid (4-PBA) had a preventative effect. MAIN METHODS: Human umbilical vein endothelial cells (HUVECs) were divided into 4 groups: a control serum group (C.S), a uremic serum group (U.S), a uremic serum plus 4-PBA (5mM) treatment group (4-PBA), and a uremic serum plus pyrrolidine dithiocarbamate (PDTC:50 µM) treatment group (PDTC). KEY FINDINGS: Lower concentrations of uremic serum (<10%) facilitated the proliferation of HUVECs. In contrast, the proliferative capability of HUVECs was gradually decreased when we continuously increased the concentration of uremic serum. Compared with C.S, HUVEC incubation with uremic serum had high expression levels of GRP78, p-PERK, NF-κB, MCP-1, and VEGF. THP-1 migration was markedly higher than C.S over the indicated time. These alterations were inhibited by the administration of 4-PBA. SIGNIFICANCE: These findings suggest that regulation of ER stress coupled with inflammatory activation by 4-PBA would be a promising therapy to reverse the process and development of uremic serum-induced EC dysfunction.

Comparison of the anti-amyloidogenic effect of O-mannosylation, O-galactosylation, and O-GalNAc glycosylation.

Our aim was to explore the effects of functional groups at carbon-2 (C2) of a sugar on the conformational properties of the peptide backbone. Three monosaccharides, mannose, galactose, and N-acetylgalactosamine (GalNAc), were added separately to the serine side-chain of a hamster prion peptide because it is a sensitive model for comparing the effect of protein modification on the conformational properties of the polypeptide chain. In buffer, this prion peptide goes through a gradual coil-to-ß structural conversion and forms amyloid fibrils slowly during incubation. Our results showed that a sugar with an N-acetyl amino group in the equatorial configuration (GalNAc) or with a hydroxyl group in the axial configuration (mannose) on C2 had a greater inhibitory effect on the amyloidogenesis of the prion peptide than a sugar with the hydroxyl group in the equatorial configuration (galactose). We suggest that galactosylation has less effect than mannosylation or GalNAc glycosylation on promoting turn formation at the glycosylation site and on inhibition of amyloidogenesis. The anti-amyloidogenic property of mannose implies that protein mannosylation has an anti-aggregation function.

Endotoxin molecule lipopolysaccharide-induced zebrafish inflammation model: a novel screening method for anti-inflammatory drugs.

Lipopolysaccharide (LPS), an endotoxin molecule, has been used to induce inflammatory responses. In this study, LPS was used to establish an in vivo inflammation model in zebrafish for drug screening. We present an experimental method that conveniently and rapidly assesses the anti-inflammatory properties of drugs. The yolks of 3-day post-fertilization (dpf) larvae were injected with 0.5 mg/mL LPS to induce fatal inflammation. After LPS stimulation, macrophages were tracked by NR and SB staining and neutrophil migration was observed using the MPO:GFP line. Larval mortality was used as the primary end-point. Expression levels of key cytokines involved in the inflammatory response including IL-1ß, IL-6, and TNF-α, were measured using quantitative reverse transcription polymerase chain reaction (RT-PCR). Macrophages and neutrophils were both recruited to the LPS-injected site during the inflammatory response. Mortality was increased by LPS in a dose-dependent manner within 48 h. Analyses of IL-1ß, IL-6, and TNF-α expression levels revealed the upregulation of the inflammatory response in the LPS-injected larvae. Further, the anti-inflammatory activity of chlorogenic acid (CA) was evaluated in this zebrafish model to screen for anti-inflammatory drugs. A preliminary result showed that CA revealed a similar effect as the corticosteroid dexamethasone (DEX), which was used as a positive control, by inhibiting macrophage and neutrophil recruitment to the LPS site and improving survival. Our results suggest that this zebrafish screening model could be applied to study inflammation-mediated diseases. Moreover, the Traditional Chinese Medicine CA displays potential anti-inflammatory activity.

[Spatial localized distribution of modes in two-dimension random medium].

Based on the time dependent theory, FDTD algorithm was used to numerically solve Maxwell's equations and rate equations. By dividing the two-dimension random medium, the emission spectra of different region under different pumping intensities were obtained. The calculated results show that the emission spectra of different region are different, the energy of emission is mainly distributed in some certain region, and the pumping efficiency is different. Also spatial extent overlap of modes is reproduced. With this method, the dependence of random distribution on lasing can be analyzed and it should be useful for the preparation of pseudo-random medium.

Observation of protein folding/unfolding dynamics of ubiquitin trapped in agarose gel by single-molecule FRET.

A ubiquitin mutant with two Cys mutations, m[C]q/S65C, was site-specifically labeled with two dye molecules, Alexa Fluor 488 (donor) and Alexa Fluor 594 (acceptor), due to the different reactivity of these two Cys residues. This doubly dye-labeled ubiquitin has lower structural stability than wild-type ubiquitin. Taking advantage of this decreased stability, conformational heterogeneity of this protein under nondenaturing condition was observed at the single-molecule level using single-paired Förster resonance energy transfer (FRET) by trapping the protein in agarose gel. Three conformational populations corresponding to folded (E (ET) ≈ 0.95), loosely packed (E (ET) ≈ 0.72), and unfolded (E (ET) ≈ 0.22) structures, and the structural transitions between them were observed. Our results suggest that agarose immobilization is good for observing structural dynamics of proteins under native condition.

Impairment of perceptual and motor abilities at the end of a night shift is greater in nurses working fast rotating shifts.

OBJECTIVE: A three-shift work schedule with fast rotation is common among healthcare workers in Taiwan. This study compared cognitive performance at the time of maximum fatigue (3-4am on the last night shift of the rotation) between nurses working two, three, and four consecutive night shifts. METHODS: Sixty-two nurses [mean age 26.4 (standard deviation 2.0) years] were recruited from the acute psychiatric ward and assigned at random to three groups: two, three, and four consecutive night shifts. The exclusion criteria were: current use of hypnotic drugs, regular consumption of coffee, psychiatric illness, major systemic disease, and sleep disorders. Cognitive performance was assessed using the State-Trait Anxiety Inventory, Stanford Sleepiness Scale, Wisconsin Card Sorting Test, Taiwan University Attention Test, Digit Symbol Substitution Test, and Symbol Searching Test. RESULTS: Greater impairment of perceptual and motor ability was seen among subjects who worked two consecutive night shifts compared with those who worked four consecutive night shifts. No differences in demographic data, executive function or attention were found between the three groups. CONCLUSION: The main duties of nurses working night shifts at the study hospital include checking medical orders and prescriptions, which require perceptual and motor abilities. The results of this study suggested that a fast shift rotation may increase the risk of medical errors.