Spatial Proteomics Analysis of Soft and Stiff Regions in Human Acute Arterial Thrombus.

BACKGROUND: The soft regions of a thrombus tend to be more susceptible to r-tPA (recombinant tissue-type plasminogen activator)-mediated thrombolysis and are more easily removed by mechanical thrombectomy than the stiff counterpart. This study aimed to understand the molecular pathological differences between the soft and stiff regions of human arterial thrombus. METHODS: We developed a spatial proteomic workflow combining proteomics with laser-captured microdissection to analyze human arterial thrombi with Masson trichrome staining to identify stiff and soft regions from 2 independent cohorts of patients with acute myocardial or cerebral infarction. Dysregulated proteins in a C57BL6/J male mouse model of arterial thrombosis were identified by pathway enrichment and pairwise analyses from the common gene ontology enrichment and dysregulated proteins between carotid and coronary arterial thrombi, and validated by immunohistochemistry. RESULTS: Spatial proteomics of the coronary arterial thrombi collected from 7 patients with myocardial infarct revealed 7 common dysregulated proteins in 2 cohorts of patients, and upregulation of TGF-ß1 (transforming growth factor ß1) was the most prominent fibrosis-related protein. Inhibition of TGF-ß1 resulted in delayed arterial thrombosis and accelerated blood flow restoration in mouse model. We further expanded the spatial proteomic workflow to the carotid artery thrombi collected from 11 patients with cerebral infarction. Pairwise proteomic analysis of stiff and soft regions between carotid and coronary arterial thrombi further revealed 5 common gene ontology clusters including features of platelet activation, and a common dysregulated protein COL1A1 (collagen type 1 alpha 1) that was reported to be influenced by TGF-ß1. We also verified the expression in human and mice carotid arterial thrombi. CONCLUSIONS: This study demonstrates the spatially distinct composition of proteins in the stiff and soft regions of human arterial thrombi, and suggests that TGF-ß1 is a key therapeutic target for promoting arterial thrombolysis.

Characterization of Pathogenic Pseudomonas alcaligenes Isolated from Koi Carp in China.

Pseudomonas alcaligenes infection is rare in aquaculture. In this study, we provide the first report on the characterization of P. alcaligenes from koi (a variant of Common Carp Cyprinus carpio) in China. A gram-negative bacterium was isolated from the diseased koi and was named KCP-516. Morphological and biochemical tests as well as phylogenetic tree analyses derived from 16S ribosomal RNA, gyrase subunit A, and gyrase subunit B gene sequencing all strongly indicated that the isolate KCP-516 was P. alcaligenes. In liquid medium, the optimal growth conditions were 25°C, 2.5% NaCl, and pH 8. The pathogenicity of the isolate was demonstrated in koi, with 7.0 × 104 CFU/g fish weight identified as the dose lethal to 50% of test fish. The results will provide a scientific reference for the diagnosis and treatment of P. alcaligenes infection.

Molecular characterization of carp edema virus disease: An emerging threat to koi Cyprinus carpio in China.

Carp edema virus disease (CEVD) has resulted in great economic losses in koi (Cyprinus carpio koi) and common carp (Cyprinus carpio carpio) populations in the world. In this study, the diseased koi were diagnosed as CEV infection based on 5' untranslated region (5'UTR) and 4a protein genes by the conventional PCR, nested PCR and quantitative PCR (qPCR) analyses. Phylogenetic tree analysis showed that the TJ201708 strain was classified into the genogroup IIa. Furthermore, qPCR of 5'UTR gene revealed that the lowest detection limit was 4.0 fg/µL. The pathogenicity of CEV for koi was demonstrated in the infection experiments. Histopathological examination revealed the petechial hemorrhages of liver and spleen, vacuolization of lamina propria of intestine and swelling and necrosis of respiratory epithelial cells of gills. To our knowledge, this is the first report the qPCR of 5'UTR gene in the detection of carp edema virus.

Skin proteome profiling of tongue sole (Cynoglossus semilaevis) challenged with Vibrio vulnificus.

Vibrio vulnificus is a major pathogen of cultured Cynoglossus semilaevis and results in skin ulceration and haemorrhage, but the proteomic mechanism of skin immunity against V. vulnificus remains unclear. In this study, we investigated the histopathology and skin immune response in C. semilaevis with V. vulnificus infection at the protein levels, the differential proteomic profiling of its skin was examined by using iTRAQ and LC-MS/MS analyses. A total of 951 proteins were identified in skin, in which 134 and 102 DEPs were screened at 12 and 36 hpi, respectively. Selected eleven immune-related DEPs (pvß, Hsp71, MLC1, F2, α2ML, HCII, C3, C5, C8ß, C9 and CD59) were verified for their immune roles in the V. vulnificus infection via using qRT-PCR assay. KEGG enrichment analysis revealed that most of the identified immune proteins were significantly associated with complement and coagulation cascades, antigen processing and presentation, salivary secretion and phagosome pathways. To our knowledge, this study is the first to describe the proteome response of C. semilaevis skin against V. vulnificus infection. The outcome of this study contributed to provide a new perspective for understanding the molecular mechanism of local skin mucosal immunity, and facilitating the development of novel mucosal vaccination strategies in fish.

MicroRNA expression profile analysis of skin immune response in crucian carp (Carassius auratus) infected by Aeromonas hydrophila.

MicroRNAs (miRNAs) are non-coding RNA molecules that regulate gene expression in fish, but its regulatory mechanism of the skin mucosal immune response remains poorly understood. In order to investigate the immunological role of miRNAs, three sRNA libraries (mSC, mST1, mST2) from skin samples of crucian carp (Carassiusauratus) infected with Aeromonas hydrophila at three time points (0, 6 and 12 hpi) were constructed and examined using Illumina Hiseq 2000 platform. All of the identified miRNA, rRNA and tRNA were 69444 (13.39%), 29550 (5.70%) and 10704 (2.06%) in skin, respectively. At 6 and 12 hpi, 829 and 856 miRNAs were differentially expressed, respectively. Among these DEMs, 53 known and 10 novel miRNAs were all significantly differentially expressed during early infection (p < 0.01). GO and KEGG enrichment analyses revealed that 118111 target-genes were primarily involved in cellular process, metabolic process, biological regulation and stress response, such as antigen processing and presentation, complement and coagulation cascades, phagosome, MAPK, TLR, NF-κB and JAK-STAT signaling pathways. These results will help to elucidate the mechanism of miRNAs involved in the skin mucosal immune response of crucian carp against Aeromonas hydrophila infection.

Isolation and genomic characterization of a pathogenic Providencia rettgeri strain G0519 in turtle Trachemys scripta.

Providencia rettgeri infection has occurred occasionally in aquaculture, but is rare in turtles. Here, a pathogenic P. rettgeri strain G0519 was isolated from a diseased slider turtle (Trachemys scripta) in China, and qPCR assay was established for the RTX toxin (rtxD) gene. Histopathological examination showed that many inflammatory cells were infiltrated into heart, liver and intestine, as well as the necrosis of liver, kidney and spleen. The genome consisted of one circular chromosome (4.493 Mb) and one plasmid (18.8 kb), and predicted to contain 4170 and 19 protein-coding genes, respectively. Multiple pathogenic and virulence factors (e.g., fimbria, adhesion, invasion, toxin, hemolysin, chemotaxis, secretion system), multidrug-resistant genes (e.g., ampC, per-1, oxa-1, sul1, tetR) and a novel genomic resistance island PRI519 were identified. Comparative genome analysis revealed the closest relationship was with P. rettgeri, and with P. heimbachae closer than with other Providencia spp. To our knowledge, this was first report on genomic characterization of multidrug-resistant pathogenic P. rettgeri in cultured turtles.

First report on the characterization of pathogenic Rahnella aquatilis KCL-5 from crucian carp: Revealed by genomic and proteomic analyses.

Rahnella aquatilis is an important pathogen of several aquatic organisms and is found widely distributed in the freshwater, soil, fish and human clinical samples. Our previously published study reported a novel pathogenic R. aquatilis strain KCL-5 to crucian carp (Carassius auratus). To further investigate the characteristics and pathogenesis caused by R. aquatilis, we here report on the pathological changes, bacterial genomic and proteomic analyses of strain KCL-5. Significantly pathological changes in liver, intestine, spleen and gills were observed in infected fish. The genome consists of one circular chromosome 5,062,299 bp with 52.02% GC content and two plasmids (506,827 bp, 52.16%; 173,433 bp, 50.00%) and predicted 5,653 genes, 77 tRNAs and 22 rRNAs. Some virulence factors were characterized, including outer membrane protein, haemolysin, RTX toxin, chemotaxis and T3SS secretion system. Antimicrobial resistance genes such as EmrAB-TolC, MexABC-OpmB and RosAB efflux pump were found in strain KCL-5. KEGG analysis showed that mainly functional modules were ABC transporters, biosynthesis of amino acids, two-component system, quorum sensing, flagellum assembly and chemotaxis, in which most of them were identified by using 2-DE/MS analyses. To our knowledge, this was first report on the molecular characteristics of R. aquatilis by multi-omics approaches, which will provide insights into the pathogenic mechanism of R. aquatilis infection in fish.

Skin transcriptome, tissue distribution of mucin genes and discovery of simple sequence repeats in crucian carp (Carassius auratus).

Crucian carp (Carassius auratus) is one of the major freshwater species and important food fish in China. Fish skin acts as the first line of defence against pathogens, yet its molecular and immune mechanism remains unclear. In this study, a de novo transcriptome assembly of C. auratus skin was performed with the Illumina Hiseq 2000 platform. A total of 49,154,776 clean reads were assembled, among which 60,824 (46.86%), 37,103 (28.59%), 43,269 (33.33%) unigenes were annotated against National Center for Biotechnology Information, Gene Onotology and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively. KEGG Orthology categories were significantly involved in immune system (20.50%), signal transduction (18.04%) and mucosal mucin genes (e.g., muc2, muc5AC, muc5B, muc17, muc18). The high expression of muc18 gene was observed in brain; that of muc2 in intestine; and that of muc5AC in skin, liver, spleen, intestine and muscle. Moreover, the potential 28,928 simple sequence repeats with the three most abundant dinucleotide repeat motifs (AC/GT, AG/CT, AT/AT) were detected in C. auratus. To authors' knowledge, this is the first report to describe the transcriptome analysis of C. auratus skin, and the outcome of this study contributed to the understanding of mucosal immune response of the skin and molecular markers in cyprinid species.

Transcriptome analysis in the skin of Carassius auratus challenged with Aeromonas hydrophila.

Skin plays an important role in the innate immune responses of fish, particularly towards bacterial infection. To understand the molecular mechanism of mucosal immunity of fish during bacterial challenge, a de novo transcriptome assembly of crucian carp Carassius auratus skin upon Aeromonas hydrophila infection was performed, the latter with Illumina Hiseq 2000 platform. A total of 118111 unigenes were generated and of these, 9693 and 8580 genes were differentially expressed at 6 and 12 h post-infection, respectively. The validity of the transcriptome results of eleven representative genes was verified by quantitative real-time PCR (qRT-PCR) analysis. A comparison with the transcriptome profiling of zebrafish skin to A. hydrophila with regards to the mucosal immune responses revealed similarities in the complement system, chemokines, heat shock proteins and the acute-phase response. GO and KEGG enrichment pathway analyses displayed the significant immune responses included TLR, MAPK, JAK-STAT, phagosome and three infection-related pathways (ie., Salmonella, Vibrio cholerae and pathogenic Escherichia coli) in skin. To our knowledge, this study is the first to describe the transcriptome analysis of C. auratus skin during A. hydrophila infection. The outcome of this study contributed to the understanding of the mucosal defense mechanisms in cyprinid species.

First report of Rahnella aquatilis infection in crucian carp Carassius auratus in China.

Rahnella aquatilis infection is rare in aquaculture. Here, a Gram-negative rod-shaped bacterium was isolated from diseased crucian carp Carassius auratus in Xuzhou City, Jiangsu Province, eastern China. The isolate was tentatively named strain KCL-5, and subsequently identified as R. aquatilis by biochemical properties and molecular techniques. The results showed that the isolate KCL-5 was most closely related to the type strain ATCC33071 (= DSM4594) of R. aquatilis, which shared 99.67, 96.26 and 99.58% nucleotide sequence identities for 16S rDNA, gyrB and toxin yhaV genes, respectively. Experimental challenges were conducted which demonstrated pathogenicity of the isolate in crucian carp. Antimicrobial susceptibility testing showed that the isolated strain was susceptible to piperacillin, gentamicin, kanamycin, nalidixic acid, norfloxacin, ofloxacin, azithromycin and erythromycin. To our knowledge, this is the first report on R. aquatilis infection in crucian carp, and the first evidence of pathogenicity in fish.

Tissue distribution of olive flounder VDAC2 and its expression in fish cell lines.

Voltage-dependent anion channel (VDAC) is located in the mitochondrial outer membrane, which plays a crucial role in regulating cell life and death. In this study, the tissue distribution of olive flounder Paralichthys olivaceus VDAC2 (PoVDAC2) was detected by quantitative real-time PCR and Western blot analysis. The qRT-PCR results showed that the expression level of PoVDAC2 was abundant in heart, muscle and gill tissues. Western blot analysis revealed a protein of 32 kDa detected in all six tissues. Furthermore, a recombinant eukaryotic expression plasmid pEGFP-N3-PoVDAC2 was successfully constructed and transiently expressed the fusion protein in fish cell lines. Subcellular localization indicated that PoVDAC2-GFP was distributed in a punctate mitochondria-like pattern throughout the cytoplasm in flounder embryonic cells (FEC). The distribution of native VDAC2 in untransfected fish cells was also investigated by immunofluorescence microscopy. The punctate VDAC2 fluorescence signals of both FEC and EPC cells were identically observed in the cytoplasm but not in the nucleus. These results laid a foundation for investigating the functional relevance of VDAC response to pathogens in flounder.

[Two-Dimensional Hetero-Spectral Near-Infrared and Mid-Infrared Correlation Spectroscopy for Discrimination Adulterated Milk].

New approach for discriminant analysis of adulterated milk is proposed based on combining hetero-spectral two-dimensional (2D) near-infrared (NIR) and mid-infrared (IR) correlation spectroscopy along with multi-way partial least squares discriminant analysis (NPLS-DA). Firstly, 36 pure milk samples were collected and 36 adulterated milk with starch samples (0.01 to 1 g · L(-1)) were prepared by adding appropriate mass of starch into pure milk. Then, one-dimensional NIR transmittance spectra and IR attenuated total reflection spectra of pure milk and adulterated milk with starch were measured at room temperature. And the synchronous 2D NIR-IR (4200~4800 vs. 900~1700 cm(-1)) correlation spectra of all samples were calculated. Due to the trace of adulterants, the synchronous 2D IR-NIR correlation spectral differences between adulterated milk with starch and pure milk are very subtle. Consequently, it was impossible to directly distinguish whether the sample was pure milk or adulterated milk. Finally, 2D IR-NIR correlation spectra were to build a discriminant model to classify adulterated milk and pure milk. The classification accuracy rates of samples in calibration set and in prediction set were 95.8% and 100% respectively. Also, the NPLS-DA models were built based on 2D NIR and 2D IR correlation spectra, respectively. The classification accuracy rates of samples in prediction set were 95.8%. Comparison results showed that the NPLS-DA model could provide better results using 2D NIR-IR correlation spectra than using 2D NIR, and 2D IR correlation spectra. The proposed method can not only effectively extract the feature information of adulterants in milk, but also explores a new perspective method for detection of adulterated food.

iTRAQ analysis of gill proteins from the zebrafish (Danio rerio) infected with Aeromonas hydrophila.

The gills are large mucosal surfaces and very important portals for pathogen entry in fish. The aim of this study was to determine the gill immune response at the protein levels, the differential proteomes of the zebrafish gill response to Aeromonas hydrophila infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1338 proteins were identified and classified into the categories primarily related to cellular process (15.36%), metabolic process (11.95%) and biological regulation (8.29%). Of these, 82 differentially expressed proteins were reliably quantified by iTRAQ analysis, 57 proteins were upregulated and 25 proteins were downregulated upon bacterial infection. Gene ontology (GO) enrichment analysis showed that approximately 33 (8.8%) of the differential proteins in gills were involved in the stress and immune responses. Several upregulated proteins were observed such as complement component 5, serpin peptidase inhibitor clade A member 7, annexin A3a, histone H4, glyceraldehyde 3-phosphate dehydrogenase, creatine kinase, and peroxiredoxin. These protein expression changes were further validated at the transcript level using microarray analysis. Moreover, complement and coagulation cascades, pathogenic Escherichia coli infection and phagosome were the significant pathways identified by KEGG enrichment analysis. This is first report on proteome of fish gills against A. hydrophila infection, which contribute to understanding the defense mechanisms of the gills in fish.

Gene expression profiling in the skin of zebrafish infected with Citrobacter freundii.

Skin is considered the largest immunologically active organ, but its molecular mechanism remains unclear in fish. Here, Affymetrix Zebrafish GeneChip was used to assess gene expression in the skin of zebrafish (Danio rerio) infected with the bacterium Citrobacter freundii. The results showed that 229 genes were differentially expressed, of which 196 genes were upregulated and 33 genes were downregulated. Gene Ontology and KEGG pathway analyses indicated 88 genes significantly associated with skin immunity involved in complement activation and acute phase response, defense and immune response, response to stress and stimulus, antigen processing and presentation, cell adhesion and migration, platelet activation and coagulation factors, regulation of autophagy and apoptosis. When compared with transcriptional profiles of previously reported carp (Cyprinus carpio) skin, a similar innate immunity (e.g., interferon, lectin, heat shock proteins, complements), and several different acute phase proteins (transferrin, ceruloplasmin, vitellogenin and alpha-1-microglobulin, etc.) were detected in zebrafish skin. The validity of the microarray results was verified by quantitative real-time PCR analysis of nine representative genes. This is first report that skin play important roles in innate immune responses to bacterial infection, which contribute to understanding the defense mechanisms of the skin in fish.

Multi-substituted N-phenyl-2, 2-dichloroacetamide analogues as anti-cancer drugs: design, synthesis and biological evaluation.

Our earlier research has shown that mono-substituted N-phenyl-2, 2-dichloroacetamide exhibited much higher anti-cancer activity than the lead compound sodium dichloroacetate (DCA). In this paper, a variety of multi-substituted N-phenyl-2, 2-dichloroacetamides were synthesized and biologically evaluated. The results showed that 3, 5-disubstituted N-phenyl-2, 2-dichloroacetamide analogues had satisfactory potency. Among them, N-(3, 5-diiodophenyl)-2, 2-dichloroacetamide had an IC50 of 2.84 micromol x L(-1) against non-small cell lung cancer cell line A549 and could induce cancer cell apoptosis.

Case Report: Stress-Induced Childhood-Onset Neurodegeneration With Ataxia-Seizures Syndrome Caused by a Novel Compound Heterozygous Mutation in ADPRHL2.

ADPRHL2 gene mutations have been demonstrated as the cause of stress-induced childhood-onset neurodegeneration with variable ataxia and seizures (CONDSIAS), an autosomal recessive genetic disorder characterized by an abnormal gait, intellectual disability, seizures, ataxia, other nervous system degenerative diseases, and axonal sensorimotor neuropathy. Since first reported in 2018, ADP-ribosylhydrolase like 2 (ADPRHL2) gene mutations in previous cases were all diallelic homozygous. Here, we report a case of CONDSIAS with a novel compound heterozygous mutation in the ADPRHL2 gene. This patient is presented with autonomic nervous dysfunction manifested as polyuria, gastrointestinal disturbance, and sinus arrhythmia, which may be considered as new clinical manifestations in addition to the above classical manifestations. Muscle biopsy revealed myogenic lesions, which is a previously unreported feature.

Herpes simplex virus type 2 encephalitis in a child with chronic progressive white matter lesions: A case report.

RATIONALE: This case is a rare manifestation of central nervous system infection of Herpes simplex virus (HSV)-2. Due to few studies in China, it provides a pathological basis for further diagnosis and treatment of HSV-2. PATIENT CONCERNS: We describe a patient with HSV-2 virus infection who was diagnosed with HSV-2 encephalitis in a Chinese patient. DIAGNOSIS: Based on brain biopsy and pathological findings, the patient was diagnosed with HSV-2 encephalitis. INTERVENTIONS: Hormone and antiviral therapy were given. OUTCOME: The patient eventually died. LESSONS: The diagnosis and differential diagnosis of the disease is very difficult. Its differential diagnosis include cerebrovascular disease, bacteria or fungi and other viral infection of the brain.

Design, synthesis and docking studies on benzamide derivatives as histone deacetylase inhibitors.

A series of benzamide derivatives including two scaffolds were designed and synthesized as potential histone deacetylase inhibitors. Most of synthesized compounds showed moderate enzymatic potency at the same order of magnitude, and compound 12b possessed better potency to the positive control (3.8 µM vs 13.0 µM). It also showed a 50-fold increase in vitro anticancer activity against DU-145 cell-lines. Molecular docking studies were carried out and used to explain the structure-activity relationships observed in vitro. Then we found that the cavity surrounded by ASP104, HIS33, PRO34 and PHE155 may be crucial for the inhibitors' activity. The docking results provide some useful information for future design of more potent inhibitors.

Single nucleotide polymorphisms of the prolactin receptor (PRLR) gene and its association with growth traits in Chinese cattle.

Genetic polymorphism of the prolactin receptor (PRLR) gene was detected by PCR-SSCP and DNA sequencing methods in 665 individuals from five Chinese cattle breeds. The results showed that at the P1 locus, three observed genotypes (AA, AB and BB), two linked SNPs (G1267A and T1268C), and one missense mutation (S18N) within a putative signal peptide were determined. The frequencies of haplotypes A and B in the five breeds were 0.596-0.802 and 0.198-0.404, respectively. Polymorphism of the PRLR gene was shown to be significantly associated with growth traits in the Nanyang breed. Individuals with genotype BB had greater hucklebone width, body weight and average daily gain than those with genotype AA at 6 months old (P<0.01), as well as better body height, body length and heart girth when 6 months (P<0.05). This study revealed for the first time that the PRLR gene is a promising candidate gene that affects growth traits in cattle.

Novel SNPs of the bovine PRLR gene associated with milk production traits.

The single nucleotide polymorphisms (SNPs) within exon 10 of the prolactin receptor gene (PRLR) were detected in Chinese Holstein cows using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods, and their genetic effects on milk production traits were evaluated in this study. Two newly detected SNPs (g.9206G→A and g.9681C→T) caused amino acid variations E378K and A536V, respectively, which were then preliminarily predicted at the topological level. Statistical results indicated that the two SNPs were significantly associated with milk yields, and cows with the combined genotype GGCC showed superior milk performance. A putative phosphorylation site was identified at residue 378K ([ST]-×-[RK]), which offers a partial explanation for the associations. These results suggest that the two novel SNPs within exon 10 of the PRLR gene associated with milk production traits are useful genetic markers in a selection program for Holstein dairy cattle.