Dual RNA-seq provides novel insight into the roles of dksA from Pseudomonas plecoglossicida in pathogen-host interactions with large yellow croakers ( Larimichthys crocea).

Pseudomonas plecoglossicida is a rod-shaped, gram-negative bacterium with flagella. It causes visceral white spot disease and high mortality in Larimichthys crocea during culture, resulting in serious economic loss. Analysis of transcriptome and quantitative real-time polymerase chain reaction (PCR) data showed that dksA gene expression was significantly up-regulated after 48 h of infection with Epinephelus coioides (log 2FC=3.12, P<0.001). RNAi of five shRNAs significantly reduced the expression of dksA in P. plecoglossicida, and the optimal silencing efficiency was 96.23%. Compared with wild-type strains, the symptoms of visceral white spot disease in L. crocea infected with RNAi strains were reduced, with time of death delayed by 48 h and mortality reduced by 25%. The dksA silencing led to a substantial down-regulation in cellular component-, flagellum-, and ribosome assembly-related genes in P. plecoglossicida, and the significant up-regulation of fliC may be a way in which virulence is maintained in P. plecoglossicida. The GO and KEGG results showed that RNAi strain infection in L. crocea led to the down-regulation of inflammatory factor genes in immune-related pathways, which were associated with multiple immune response processes. Results also showed that dksA was a virulence gene in P. plecoglossicida. Compared with the wild-type strains, RNAi strain infection induced a weaker immune response in L. crocea.

Novel insights into host-pathogen interactions of large yellow croakers ( Larimichthys crocea) and pathogenic bacterium Pseudomonas plecoglossicida using time-resolved dual RNA-seq of infected spleens.

Host-pathogen interactions are highly complex, involving large dynamic changes in gene expression during infection. These interactions are fundamental to understanding anti-infection immunity of hosts, as well as the pathogenesis of pathogens. For bacterial pathogens interacting with animal hosts, time-resolved dual RNA-seq of infected tissue is difficult to perform due to low pathogen load in infected tissue. In this study, an acute infection model of Larimichthys crocea infected by Pseudomonas plecoglossicida was established. The spleens of infected fish exhibited typical symptoms, with a maximum bacterial load at two days post-injection (dpi). Time-resolved dual RNA-seq of infected spleens was successfully applied to study host-pathogen interactions between L. crocea and P. plecoglossicida. The spleens of infected L. crocea were subjected to dual RNA-seq, and transcriptome data were compared with those of noninfected spleens or in vitro cultured bacteria. Results showed that pathogen-host interactions were highly dynamically regulated, with corresponding fluctuations in host and pathogen transcriptomes during infection. The expression levels of many immunogenes involved in cytokine-cytokine receptor, Toll-like receptor signaling, and other immune-related pathways were significantly up-regulated during the infection period. Furthermore, metabolic processes and the use of oxygen in L. crocea were strongly affected by P. plecoglossicida infection. The WGCNA results showed that the metabolic process was strongly related to the entire immune process. For P. plecoglossicida, the expression levels of motility-related genes and flagellum assembly-related genes were significantly up-regulated. The results of this study may help to elucidate the interactions between L. crocea and P. plecoglossicida.

Histopathological changes and piscidin 5-like location in infected Larimichthys crocea with parasite Cryptocaryon irritans.

Cryptocaryon irritans infection could cause huge economic losses to the marine fish industry. Larimichthys crocea, a special economic species in China, suffered from the threat of serious infection, and L. crocea could enhance the level of piscidin 5-like to defense against the infection. This study set out to observe the main histopathological changes of some key tissues caused by infection, and determineed how an ectoparasite affected the expression of piscidin-5 like in its hosts. Pathological changes and immune response were assessed using histological and in situ hybridization (ISH) technologies. The infection induced inflammation occurring, especially in the gill where epithelium cells swell, hyperplasia, necrosis shedding adjacent to the parasites attachment sites. Infected hepatic cells grown big vacuoles in the cytoplasm. The boundary between red pulp and white pulp turned indistinct, splenic corpuscle lost the normal structure, the number and size of melano-macrophage centers increased apparently in the infected spleen. The whole structure of head kidney became loose. Immunostaining with RNA probes against piscidin 5-like showed subpopulations of mast cells (MCs) were positive. Piscidin 5-like-positive MCs existed mainly in the head kidney where they distributed around melano-macrophage center, followed in the gill located at different positions they also distributed in the margin of spleen, and randomly and sparsely existed in the liver. After being infected by C. irritans, the gill arch arose positive MCs groups, and they also migrated to spleen, while the positive staining deepen in other detected tissues. Therefore, organism enhanced the expression level through improving expression ability of positive MCs, or increasing the number of positive MCs.

A novel stimulator of interferon gene (STING) from Larimichthys crocea and their involvement in immune response to ectoparasite Cryptocaryon irritans infection.

The marine white spot disease caused by protozoan ectoparasite Cryptocaryon irritans is a severe problem to the large yellow croaker farming industry. To understand the molecular immune mechanisms and improve its immune capacity are particular important. STING, one of the important second messengers in innate immune response process, plays pivotal roles in defensing against different pathogenic microorganisms. Many reports have pointed that STING could not only combine the uncovered dsDNA, ssDNA directly in the cytoplasm from the pathogens or biology itself, but it also could recognize cyclic diguanylate monophosphate (c-di-GMP), cyclic diadenylate monophosphate (c-di-AMP). Based on the STING sequence, a variant of the L. crocea STING (termed LcSTING2) was found by accident. RACE was used to clone the full-length cDNA of LcSTING2 which contained a 5'- UTR of 154 bp, a 3'-UTR of 592 bp and an ORF of 1227 bp encoding 408 amino acids. The predicted protein molecular weight (Mw) was 45.83 KDa and the estimated theoretical isoelectric point (pI) was 6.24. The deduced protein of LcSTING2 contains no signal peptide. One transmembrane motif (TM) in the N-terminal region, a TMEM173 domain and five putative motifs (RXR) found in resident endoplasmic reticulum proteins were also conserved. According to the partial genomic sequence, the unknown sequences were amplified, it contained 6 exons and 5 introns, and all the exon-intron boundaries were in accordance with classical GT-AG rule (GT/intron/AG). The similarity shared with fishes was higher than other high vertebrates. qRT-PCR results showed that LcSTING (two variants) distributed in all examined tissues, and it was the most abundant in gill. After challenged by C. irritans, LcSTING mRNA only appeared instantaneous up-regulation during the infective stage of theronts in the head kidney and was also up-regulated during the whole infectious cycle of C. irritans in the liver, which implied LcSTING gene was likely to be involved in the defensing against C. irritans infection, it is the first time to explore the STING taking part in the immune response to ectoparasite rather than bacterium or viruses.

Analysis of liver and gill miRNAs of Larimichthys crocea against Cryptocryon irritans challenge.

The white-spot disease caused by marine ciliate Cryptocryon irritans hindered the sustainable development of large yellow croaker Larimichthys crocea industry. Better understandings about the parasite-host interactions in the molecular level will facilitate the prevention of mass mortality of the L. crocea caused by white-spot disease. MicroRNAs (miRNAs) are a class of small RNA molecules about 20-22 nucleotides which post-transcriptionally regulated many protein-coding genes and involved in many biological processes, especially in host-pathogen responses. In this study, we identified known and novel miRNAs in the gill and liver of L. crocea challenged by C. irritans by high throughput sequencing using Solexa technology. The data were further studied to screen differentially expressed miRNAs, and predict their target genes. The differential expression (p < 0.05) between libraries was observed in 103 miRNAs in liver tissue, among which 65 and 38 were conserved and novel miRNAs, 67 and 36 were up- and down-regulated miRNAs. While in gill tissue, 122 significant differentially expressed miRNAs were identified, among which 83 and 39 were conserved and novel miRNAs, 79 and 43 were up- and down-regulated miRNAs. In addition, these differentially expressed miRNAs target a series of genes which involved in many important biological processes including immune response. Here via deep sequencing, we for the first time characterize L. crocea miRNAs in response to C. irritans challenge, the results should help for better understandings about the immune response of the L. crocea under C. irritans challenge.

Identification, expression and antibacterial activities of an antimicrobial peptide NK-lysin from a marine fish Larimichthys crocea.

As fundamental immunologic mechanism, the innate immunity system is more important than the specific immunity system in teleost fishes during pathogens infection. Antimicrobial peptides are integral parts of the innate immune system, and play significant roles against pathogens infection. NK-lysin, the compounds of the natural killer cells and cytotoxic T cells, are potent and effective antimicrobial peptides widely distributed in animals. In this study, we reported the sequence characteristics, expression profiles and antibacterial activities of a NK-lysin gene (Lc-NK-lysin) from a commercially important marine fish, the large yellow croaker (Larimichthys crocea). The open reading frame of Lc-NK-lysin cDNA sequence was 447 bp in length, coding 148 amino acids. The genomic DNA of Lc-NK-lysin has the common features of NK-lysin family, consisting of five exons and four introns, and in its deduced mature peptide, there are six well-conserved cysteine residues and a Saposin B domain. Lc-NK-lysin was expressed in all tested tissues (skin, muscle, gill, brain, head kidney, heart, liver, spleen, stomach and intestine) with different expression patterns. In pathogens infection the expression profiles of Lc-NK-lysin varied significantly in gill, head kidney, spleen and liver, indicating its role in immune response. Two peptides (Lc-NK-lysin-1 and Lc-NK-lysin-2) divided from the core region of the Lc-NK-lysin mature polypeptide were chemically synthesized and their antibacterial activities were examined; the potential function on the inhibition of bacteria propagation was revealed. Our results suggested that Lc-NK-lysin is a typical member of the NK-lysin family and as an immune-related gene it involves in the immune response when pathogens invasion.

Transcriptome analysis of the Larimichthys crocea liver in response to Cryptocaryon irritans.

The large yellow croaker (Larimichthys crocea) is an economically important marine fish cultured in China and East Asian countries and is facing a serious threat from Cryptocaryon irritans, which is a protozoan ectoparasite that infects most reared marine fish species. To understand the molecular immune mechanisms underlying the response to C. irritans, we first performed a comparative gene transcription analysis using livers from C. irritans-immunized L. croceas and from a control group through RNA-Seq technology. After the removal of low-quality sequences and assembly, 51360 contigs were obtained, with an average length of 1066.93 bp. Further, a blast analysis indicates that 30747 contigs can be annotated based on homology with matches in the NT, NR, gene, and string databases. A gene ontology analysis was used to classify 21598 genes according to three major functional categories: molecular function, cellular component, and biological process. Moreover, 14470 genes were found in 303 KEGG pathways. We used RSEM and EdgeR to determine that 3841 genes were significantly differentially expressed (FDR < 0.001), including 2129 up-regulated genes and 1712 down-regulated genes. A significant enrichment analysis of these differentially expressed genes and isogenes revealed major immune-related pathways, including the toll-like receptor, complement and coagulation cascades, and chemokine signaling pathways. In addition, 28748 potential simple sequence repeats (SSRs) were detected from 12776 transcripts, and 62992 candidate single nucleotide polymorphisms (SNPs) were identified in the L. croceas liver transcriptome. This study characterized a gene expression pattern for normal and C. irritans-immunized L. croceas for the first time and not only sheds new light on the molecular mechanisms underlying the host-C. irritans interaction but also facilitates future studies on L. croceas gene expression and functional genomics.

Molecular characterization and expression analysis of interferon-gamma in the large yellow croaker Larimichthys crocea.

The large yellow croaker Larimichthys crocea is an important mariculture fish species in China, and the bacterium Vibrio harveyi (V. harveyi) and the ciliate protozoan Cryptocaryon irritans (C. irritans) are the two major pathogens in its aquaculture sector. Interferon-gamma (IFN-γ) plays important roles in regulating both innate and cell mediated immune responses as an inflammatory cytokine. In this study, we obtained the nucleotide sequence of IFN-γ from the large yellow croaker (LcIFN-γ). The phylogenetic relationship tree of 18 available IFN-γ genes was constructed based on their sequences. Expression analyses in 10 various tissues were conducted after the croaker challenged with V. harveyi and C. irritans, respectively. Real time PCR analysis showed that the expression of LcIFN-γ was observed broadly in health individuals. After injected with V. harveyi, the 10 tissues had a higher expression of IFN-γ at the first day (1 d); only spleen, muscle, intestine, heart and skin had higher expressions after infected with C. irritans at 1 d. Major immune tissues (skin, gill, head kidney and spleen) and detoxification tissue (liver) were sampled at 0 h, 6 h, 1 d, 2 d, 3 d, 4 d, 5 d and 7 d to understand the expression trends of LcIFN-γ after challenged with C. irritans. The expressions of LcIFN-γ in skin and gill (the primary immune organs) showed a clear correlative relationship with the life cycle of C. irritans.

Exploring novel targets of basal-like breast carcinoma by comparative gene profiling and mechanism analysis.

The heterogeneity of breast cancer makes its diagnosis and treatment far from being optimal. Analysis of traditional pathological and prognostic markers based on immunohistochemistry (IHC) is inadequate in elucidating the inherent heterogeneity of breast cancer, especially basal-like breast carcinoma (BLBC) which displays complex and unique epidemiological, phenotypic, and molecular features with distinctive relapse patterns and poor clinical outcomes. Gene expression profiling opened an avenue in research as independent predictors by classifying breast cancers into discrete groups with prognostic references, but it is not cost-effective in clinical application. It is necessary to develop an effective predictive gene list from gene profiling to optimize the treatment with traditional markers. In this report, we analyzed the correlation between IHC and gene profiling of breast cancer with an emphasis on the BLBC, highlighting the potential discovery of diagnostic markers and cellular mechanisms that may guide the development of BLBC-targeted therapy. Random forest-based classification and PAM50 gene-sets were used in the comparison analysis of traditional prognostic markers including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and microarray profiles. An intrinsic 40-gene set was developed to classify breast cancer subtypes, and genes expression differentiations were used to explore the different mechanisms between the BLBC and non-BLBC subtypes based on the comparison of clinicopathological markers and microarray profiling. Pathways and DNA repairs were analyzed to evaluate the biological mechanisms in BLBC and other breast cancer subtypes. It is reasonable to define BLBC as those tumors that are negative for ER, PR, and HER2 by IHC for their accordance with gene expression profiles. Focal adhesion kinase, ERBB, and their signaling pathways may play crucial role in BLBC. The intrinsic 40-gene set can be used to classify breast cancer and help to optimize therapeutic management of BLBC.

[Clinical analysis of 217 patients with gastrointestinal stromal tumor].

OBJECTIVE: To study the clinical characteristics, diagnosis, treatment and prognostic factors of gastrointestinal stromal tumor(GIST). METHODS: Clinicopathological data of 217 GIST patients from January 2005 to September 2010 in Wuhan Union Hospital were analyzed retrospectively and the prognostic factors were evaluated. RESULTS: There were 103 males and 114 females with a median age of 55 years old. Two hundred and thirteen patients underwent R0 resection and 4 R1 resection due to extensive invasion. Thirty-five patients underwent laparoscopic resection. Forty-eight patients received imatinib mesylate therapy after surgery. A total of 178 patients(82.0%) were followed up for 3 to 74 months. Sixteen patients(9.0%) developed recurrence or metastasis. Logistic regression analysis showed that tumor location (OR=2.547, 95% CI:1.466-4.424) and mitotic count(OR=6.556, 95% CI:2.974-14.449) were independent factors for post-operative recurrence or metastasis. Five patients survived with tumor, and 11 patients(6.2%) died of GIST including intestinal GIST(n=7) and extraintestinal GIST(n=4). Cox regression analysis showed that the mitotic count (RR=2.654, 95% CI:1.094-6.438) and post-operative recurrence or metastasis (RR=32.988, 95% CI:3.879-280.529) were independent prognostic factors. CONCLUSIONS: Tumor location and mitotic count are independent risk factors for post-operative recurrence or metastasis in GIST. Mitotic count and post-operative recurrence or metastasis are independent indicators of poor prognosis. Surgical radical resection combined with targeted therapy can achieve satisfactory outcomes in patients with GIST.

Pathological and molecular analysis of sporadic hepatic angiomyolipoma.

Hepatic angiomyolipoma (HAML) is an unusual mesenchymal lesion that can be misdiagnosed as hepatocellular carcinoma or sarcoma. We sought to better define the morphological variations and immunohistochemical and molecular features of this unusual tumor. Forty-nine sporadic HAMLs were investigated for immunopathologic characteristics with EnVision Plus. The clonal analysis was based on the methylation pattern of the polymorphic X chromosome-linked human androgen receptor gene, loss of heterozygosity (LOH), and microsatellite instability (MSI) detected with polymerase chain reaction using the MegaBACE 500 automatic system on microdissected tissues. Histologically, HAML is composed of a heterogeneous mixture of blood vessels, smooth muscle, and adipose cells. The myomatous or myoid cells were the most variable. Most of the tumor cells were positive for HMB-45 (100%) and SMA (100%). There was a typical monoclonal pattern in 35 of the 40 tumors. No LOH or MSI was found. Hepatic AML is a benign neoplasm with varied morphology and monoclonal growth. HMB-45 is the best marker available for diagnosis. Neither LOH nor MSI appears to play an important role in the pathogenesis of this tumor.

Elastin distribution in the normal uterus, uterine leiomyomas, adenomyosis and adenomyomas: a comparison.

OBJECTIVE: To describe the histologic distribution of elastin in the nonpregnant human uterus, uterine leiomyomas, adenomyosis and adenomyomas. STUDY DESIGN: Uteri were obtained from women undergoing hysterectomy for benign conditions, including 26 cases of uterine leiomyomas, 24 cases of adenomyosis, 18 adenomyomas and 6 cases of autopsy specimens. Specific histochemical staining techniques were employed in order to demonstrate the distribution of elastin. RESULTS: The distribution of elastin components in the uterus was markedly uneven and showed a decreasing gradient from outer to inner myometrium. No elastin was present within leiomyomas, adenomyomas or adenomyosis. CONCLUSION: The distribution of elastin may help explain the normal function of the myometrium in labor. It implies that the uneven distribution of elastin components and absence of elastin within leiomyomas, adenomyomas and adenomyosis could be of some clinical significance. The altered elastin distribution in disease states may help explain such symptoms as dysmenorrhea in uterine endometriosis.

Age-related effects of estrogen on the expression of estrogen receptor alpha and beta mRNA in the ovariectomized monkey hypothalamus.

In the present study, we reported distribution of ER alpha and ER beta mRNAs in the hypothalamus of young and old ovariectomized (OVX) rhesus macaques. The ER alpha were detected in all six major vestiblular nuclei which included arcuate nucleus (ARC) , paraventricularis nucleus (PVN) , periventricular nucleus (PeriV) , supraoptic nucleus (SON) , medial prioptic nucleus (MPN) and lateral hypothalamus area (LHA). However, the ER beta mRNA can also detected in those nuclei excerpt SON, but the signals of ER beta mRNA were weaker than those of ER alpha mRNA. We observed that the degree of expression of ERs mRNA were different in most nucleus of old and young monkeys. The ER alpha mRNAs were highly expressed in ARC and SON in young monkeys compared with old monkeys. Moderate amount of ERalpha mRNAs hybridization signals and weak signals were observed in LHA, and MPN both in young and old monkeys. In contrast, only lower level of ER alpha hybridization signal were observed in PVN and PeriV in young monkeys, and the signals of ER alpha were very low in those nucleus of old monkeys. In general, the expression of ER beta mRNA were weaker than that of ER alpha mRNA in above nucleus excerpt LHA. The relatively higher density of ER beta hybridization signals have been observed in the LHA in young monkey compared with old monkeys. Low amount of ER beta mRNA hybridization signals were observed in the ARC, PVN and MPN, and no age differences were seen in PVN and MPN of those monkeys. In PeriV, we observed some signals in young monkey and a few signals in old monkeys. It was different from the rodent in which we did not found ER beta hybridization signal in SON. This study showed that both of the two estrogen receptors not only had the same pattern of expression but also had many different patterns of expression. The different expression of ER alpha and ER beta mRNAs in the young and old monkey brain may imply diverse functions in different regions of the monkey brain.

Relationship between levels of Skp2 and P27 in breast carcinomas and possible role of Skp2 as targeted therapy.

Estrogen receptor-negative breast carcinomas are more aggressive and are unresponsive to anti-estrogens. Thus, they clearly require new therapies targeted against specific genes and proteins actively engaged in the pathophysiology of cancer. The S-phase kinase-associated protein Skp2 is required for the ubiquitin-mediated degradation of the cdk-inhibitor p27 and is a bona fide proto-oncoprotein. We attempted to explore whether Skp2 may be a potential specific therapeutic target in the subset of aggressive breast carcinomas by investigating the possible relationship between expression of Skp2 and p27 proteins and estrogen receptor (ER). Immunohistochemical analysis of tumor tissues was employed to determine the expression of Skp2, p27, and ER proteins in 82 cases of primary breast carcinoma. Higher levels of Skp2 were detected more frequently in ER-negative tumors and tumors metastatic to the axillary lymph nodes. The expression of p27 was inverse with the histologic grade. Statistical analysis showed that the percentage of high Skp2 expressors was significantly greater in the group with low p27 expression than in the group with high p27 expression. The current study, together with the results from a previous study, demonstrated the existence of a subtype of high-grade, negative ER breast carcinomas with high Skp2 and low p27 levels. This implies that Skp2 may be a potential specific therapeutic target in a subset of aggressive breast carcinomas. Thus far, there is no specific therapy for the ER-negative and HER-2/neu resistant groups, which are among the subset of aggressive tumors.

[The expression and significance of structural proteins, VEGF and Ang-1 in cavernous venous malformations of the body surface].

OBJECTIVE: To study the expression and significance of structural proteins, VEGF and Ang-1 in cavernous venous malformations of the body surface. METHODS: Tissue samples came from 25 cases of cavernous venous malformations, 12 cases of normal moderate veins and 12 cases of normal small veins. Envision immunohistochemical stain was used to investigate the expression of IV collagen, fibronectin, laminin, VEGF and Ang-1. The results were analyzed semi-quantitatively. RESULTS: The distribution of structural proteins in cavernous venous malformations is similar to moderate and small veins, but the expression in venous malformations is less obviously. VEGF expression in cavernous venous malformations and small veins is stronger obviously than moderate veins. Ang-1 expression in small veins is stronger remarkably than cavernous venous malformations and moderate veins. CONCLUSION: The abnormal expression of structural proteins may be an important factor in etiopathology and progress of cavernous venous malformations. There is disturbance of blood vessel remodelling in the sinusoid of cavernous venous malformations, with which the less expression of Ang-1 may be related.

[A clinical pathological study on cavernous venous malformation of the body surface].

OBJECTIVE: To investigate the clinical pathology of cavernous venous malformations of the body surface. METHODS: Tissue samples of cavernous venous malformations from 42 cases were stained with hematoxylin and eosin to observe the pathologic structure. The clinical manifestations and case history were summarized accordingly. RESULTS: There was no distribution difference of the malformation in sex and body sides, but with obvious difference in anatomic sites. The malformation occurred most frequently at the head and neck, more frequently at extremities and least frequently at the trunk. According to pathologic structure, cavernous venous malformations of the body surface can be divided into three types: the cellular, the canaliform and the mixed. CONCLUSION: The cause of distribution difference in anatomic sites remains unclear. Internal hemorrhage and infection may account for the increased growth and ache of the lesion. The different pathologic structure of the malformation may cause different clinical manifestations.

Correlation between laminin and cathepsin D expressions in breast carcinoma.

AIMS AND BACKGROUND: Laminin is a major glycoprotein component of basement membrane which is an important barrier to tumor cells which must be breached before metastatic spread can occur. Proteolytic enzymes play an important role in mediating the passage of cancer cells through the basement membrane (BM) and extracellular matrix. We have compared the patterns of laminin and cathepsin D (CD) expressions in a range of benign and malignant breast lesions to better understand the process of tumor progression. METHODS: One hundred and sixty-two cases of breast samples comprising 18 fibroadenomas, 22 cases of fibrocystic disease, 96 cases of invasive ductal carcinoma and 26 carcinomas with intraductal components were evaluated for laminin and cathepsin D expressions by immunohistochemical staining. RESULTS: The prevalence of CD positivity in both neoplastic and stromal cell components were significantly higher in higher histological grade tumors compared to lower grades (P <0.01). Various severity of BM disruption correlated with histological grade of the carcinomas (P <0.001). There was a negative correlation between the laminin expression and CD presence. CONCLUSIONS: It was confirmed that in a process of cancer cell invasion and metastasis, it could be necessary with the basement membrane disruption by proteinase secreted by cancer cells and especially by stroma cells of cancer.