Effects of bisphenols on lipid metabolism and neuro-cardiovascular toxicity in marine medaka larvae.

Bisphenols are environmental endocrine disruptors that have detrimental effects on aquatic organisms. Using marine medaka larvae, this study explored the effects of bisphenol compounds [bisphenol A (BPA), bisphenol S (BPS), bisphenol F (BPF), and bisphenol AF (BPAF)] on the early growth and development of aquatic organisms. Marine medaka larvae were exposed to bisphenol compounds at concentrations of 0.05, 0.5, and 5 µM for 72 h, and changes in heartbeat rate, behavior, hormone levels, and gene expression were determined. Bisphenols were shown to have a toxic effect on the cardiovascular system of larvae and can cause neurotoxicity and endocrine disruption, such as changes to thyroid-related hormones. Functional enrichment showed that bisphenols mainly affect lipid metabolism and cardiac muscle contraction of larvae, which implied that the main toxic effects of bisphenols on marine medaka larvae targeted the liver and heart. This study provides a theoretical foundation for evaluating the toxicological effects of bisphenols on the early development of aquatic organisms.

Protective effects of methylprednisolone-cyclophosphamide treatment on bleomycin-induced pulmonary fibrosis.

BACKGROUND: Methylprednisolone (MP) and cyclophosphamide (CTX) combination treatment has shown great benefits in improving pulmonary fibrosis (PF) and high safety. Currently, the mechanism underlying the effects of MP-CTX on improving PF remains unclear. This study determined the effects of MP-CTX combination treatment on the modulation of inflammation, oxidative stress, and T-cell immunity in PF. METHODS: PF rat models were induced by bleomycin stimulation. MP (3 mg/kg) and MP-CTX (MP: 3 mg/kg; CTX: 8 mg/kg) combination were administered in the PF + MP and PF + MP + CTX groups, respectively. Transmission electron microscopy, hematoxylin and eosin staining, Ashcroft score, and Masson trichrome staining were performed to measure lung morphology in PF. Enzyme-linked immunosorbent assay and quantitative polymerase chain reaction assay were performed to quantify inflammatory factors. Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) levels were determined using commercial kits. α-Smooth muscle actin (SMA) and collagen I levels were determined using western blotting and immunohistochemistry. The T-cell count was evaluated using flow cytometry. RESULTS: MP-CTX reduced lung injury, collagen deposition, and α-SMA and collagen I levels in a bleomycin-induced PF rat model. Additionally, MP-CTX decreased the levels of MDA and inflammatory factors (tumor necrosis factor-α, interleukin-1ß, and interleukin-6) but increased the activities of SOD and GSH-PX. Furthermore, MP-CTX changed T-cell types in lung tissues, such as increasing CD4+CD25+Foxp3+ cell count. CONCLUSIONS: MP-CTX combination treatment improved the degree of PF by reducing inflammation and oxidative stress and improving T-cell immunity. These findings provide novel insights into the mechanisms underlying the effects of MP-CTX on PF.

Appropriate empirical antifungal therapy is associated with a reduced mortality rate in intensive care unit patients with invasive fungal infection: A real-world retrospective study based on the MIMIC-IV database.

Objective: The study aimed to determine the prevalence and pathogens of invasive fungal infection (IFI) among intensive care unit (ICU) patients. The next goal was to investigate the association between empirical antifungal treatment and mortality in ICU patients. Methods: Using microbiological events, we identified all ICU patients with IFI and then retrieved electronic clinical data from the Medical Information Mart for Intensive Care IV (MIMIC-IV) database. The data were statistically analyzed using t-tests, chi-square tests, log-rank tests, and Cox regression. Results: The most commonly reported fungi were Candida (72.64%) and Aspergillus (19.08%). The most frequently prescribed antifungal medication was fluconazole (37.57%), followed by micafungin (26.47%). In the survival study of ICU patients and patients with sepsis, survivors were more likely to receive empirical antifungal treatment. In contrast, non-empirical antifungal therapy was significantly associated with poor survival in patients with positive blood cultures. We found that the current predictive score makes an accurate prediction of patients with fungal infections challenging. Conclusions: Our study demonstrated that empirical antifungal treatment is associated with decreased mortality in ICU patients. To avoid treatment delays, novel diagnostic techniques should be implemented in the clinic. Until such tests are available, appropriate empirical antifungal therapy could be administered based on a model that predicts the optimal time to initiate antifungal therapy. Additional studies should be conducted to establish more accurate predictive models in the future.

Comparative Mucous miRomics in Cynoglossus semilaevis Related to Vibrio harveyi Caused Infection.

Epidermal mucus is an important barrier and regulating mediator in fish. MicroRNAs (miRNAs) are proved to be involved in various biological processes, also as promising biomarkers for disease diagnosis. Vibrio harveyi has long been a noticeable bacterial pathogen in Cynoglossus semilaevis aquaculture. To find the evidence whether there are indicating miRNAs in mucus and whether the miRNAs are related to infections caused by V. harveyi, miRNA profiles of mucus from V. harveyi infected fish and healthy controls were screened by small RNA sequencing and verified by quantitative real-time PCR. This is the first report about miRNA profiling of flatfish mucus, aiming at illustrating the pathogenesis of V. harveyi caused infection and developing disease-related biomarkers. The results revealed significant differences in expression levels of some miRNAs between infected fish and healthy ones. Three hundred differentially expressed miRNAs were obtained after filtering through FC > 2 or FC < 0.5 and most of the differential miRNAs were downregulated. After verification through qRT-PCR, four unique miRNAs, dre-miR-451, dre-miR-184, dre-miR-205-5p > ssa-miR-205b-5p, and dre-miR-181a-5p > ssa-miR-181a-5p, were identified as V. harveyi infection-related signatures, consistent with sequencing trend. The expression levels of these four miRNAs in the infected fish were all significantly lower than controls. These miRNAs in mucus could be used to differentiate diseased and healthy fish in a non-invasive way with practical value for large-scale disease screening. They also provided new insights into the mechanism underlying the bacterial infections in fish.

Sex bias miRNAs in Cynoglossus semilaevis could play a role in transgenerational inheritance.

Alterations of non-coding RNA profiling in spermatozoa are candidate mechanisms related to changes in paternal environment and progeny. Transgenerational inheritance of sex in pseudomales of Cynoglossus semilaevis, a fish with significant sex dimorphism, is a typical example of non-Mendelian inheritance. In the present study, miRNA profiles of spermatozoa were compared between male and pseudomale of C. semilaevis. Differential miRNAs in sperm from F0 and F1 generation also provides clues for revealing the possible role of non-coding RNA mediated transgenerational inheritance. Four sexual bias miRNAs, dre-miR-26a-5p, dre-miR-27b-3p, dre-miR-125b-5p,pol-199a-5p, were identified and verified in F0 and F1 generation of C. semilaevis. All of them were highly expressed in male sperm compared with pseudomale sperm. Function of target genes indicates that target genes of these differential RNAs are highly correlated with sex differentiation, gametogenesis and maintenance of secondary sexual characteristics. In a word, identification of epigenetic markers in gametes has great prospects in predicting susceptibility and properties in offsprings, and providing an indicator of parentalgenetic property.

Extracellular vesicles piwi-interacting RNAs from skin mucus for identification of infected Cynoglossus semilaevis with Vibrio harveyi.

Extracellular vesicles play a regulatory role in intracellular and intercellular transmission through a variety of biological information molecules, including mRNA, small RNAs and proteins. piRNAs are one kind of regulatory small RNAs in the vesicles at the post transcriptional level. Hereby, we isolated the extracellular vesicles from skin mucus and screened the piRNA profiles of these vesicles, aiming at developing biomarkers related to bacterial infections in Cynoglossus semilaevis. The different profilings of piRNAs in mucous extracellular vesicles of C. semilaevis were compared through small RNA sequencing, between fish infected with Vibrio harveyi and healthy ones. The number of clean reads on the alignment of exosome sick (ES) group was 105, 345 and that of exosome control (EC) group was 455, 144. GO and KEGG pathway enrichment analysis showed that most of the target genes were involved in cellular process, response to stimulus, biological regulation, immune system process and signal transduction, signal molecular and interaction, transport and catabolism. The 45 final candidate piRNAs related to immunity or infectious diseases included 20 piRNAs with high expression in the ES group and 25 piRNAs with a low expression in the ES group. After verification by qRT-PCR, there was significant difference of five piRNAs expression level between infected fish and healthy fish, in line with the sequencing. The expression level of piR-mmu-16401212, piR-mmu-26829319 and piR-gga-244092 in infected fish were significantly lower than that of control group, while piR-gga-71717 and piR-gga-99034 were higher, which implying that these piRNAs in mucous extracellular vesicles can be used to identify diseased fish from normal ones. This work supplied a novel class of biomarker for infection diagnosis in fish, and it will be benefit for screening disease resistant breeding of C. semilaevis.

Proteomics of mucosal exosomes of Cynoglossus semilaevis altered when infected by Vibrio harveyi.

The cargo of exosomes contains proteins with various functions, which might be promising biomarkers for disease diagnosis and prognosis. To explore the impact of the Vibrio harveyi pathogen on Cynoglossus semilaevis from a different perspective and develop promising biomarkers for infection, the exosomes from epidermal mucus of healthy controls（EC）and sick fish（ES）were extracted and identified, coupled with proteomic screening through iTRAQ followed with LC-MS/MS. 1531 credible proteins were obtained relating to structural, metabolic and immunological functions. 359 different expressed proteins (DEPs) (FC > 2 or FC < 0.5) were found, with 161 up-regulated and 198 down-regulated in ES. Based on the database of C. semilaevis on Uniprot, 71 proteins were characterized as concrete names, including 19 up-regulated proteins and 52 down-regulated proteins, and were selected as subjects for further studies. Ferritin, Toll-like receptor 5S protein and Calcium-transporting ATPase were upregulated, while Histone H2B and Eukaryotic translation initiation factor 5A were downregulated, consistent with the expression levels of related mRNAs in skin tissue verified by qRT-PCR. The integrated analysis between miRomics and proteomics also provided possible regulatory relationships mediated by mucous exosomes during infection. The signature proteins in mucosal exosomes could make sense in the explanation of the infection defending mechanism and the development of biomarkers which can differentiate diseased and healthy C. semilaevis individuals.

Novel molecular marker for RAA-LFD visual detection of Cynoglossus semilaevis sex.

Due to the significant sex dimorphism, Cynoglossus semilaevis has long been a species of research interest in the field of artificial sex manipulation. The existence of pseudo-males both in the natural habitat and aquaculture enterprises also is indicative of the importance for identification of the genetic sex in this species. In the present study, there was elucidation of a novel molecular marker for utilizing the recombinase aided amplification-lateral flow dipstick (RAA-LFD) visual system to identify the genetic sex of C. semilaevis. This 533 bp novel marker is a differential single copy fragment between the Z and W chromosome of C. semilaevis and exists only in the W chromosome. After primer designing and probe labeling, this marker has been utilized in a RAA isothermal amplification system. There were 49 C. semilaevis specimens evaluated for genetic sex identification using both PCR-agarose gel electrophoresis based InDel marker detection and the novel RAA-LFD system. The results from conducting evaluations with the two methods were consistent in all samples. Also, results from sensitivity analysis with use of the RAA-LFD system indicated the detection system was effective and reliable from 108 copy number to 101. With use of the RAA reaction, there was only need to utilize a constant temperature of 37 ℃ for specific DNA amplification within 30 min. The combination use of RAA with LFD resulted in more efficient and convenient sex determination with there being a lesser technical threshold.

Single-nucleotide polymorphisms responsible for pseudo-albinism and hypermelanosis in Japanese flounder (Paralichthys olivaceus) and reveal two genes related to malpigmentation.

Paralichthys olivaceus is the kind of cold-water benthic marine fish. In the early stages of development, the symmetrical juveniles transform into an asymmetrical body shape through metamorphosis for adapting benthic life. After that, one side of the fish body is attached to the ground, and the eyes turn to the opposite side which is called ocular side. The body color also appears asymmetry. The skin on the ocular side is dark brown, and the skin on the blind side is white without pigmentation. Pseudo-albinism and hypermelanosis have been considered distinct body color disorders in flatfish. Pseudo-albinism and hypermelanosis in Paralichthys olivaceus are due to abnormal or uneven pigment distribution, due to the interaction of hereditary and environmental factors, rather than a single-nucleotide mutation of a specific gene. Here, we report three single-nucleotide polymorphisms (SNPs) responsible for both pseudo-albinism and hypermelanosis, which are located on two body color-related genes involved in melanogenesis-related pathways. c.2440C>A (P. V605I) and c.2271-96T>C are located on the Inositol 1,4,5-trisphosphate receptor type 2-like (ITPR2) (Gene ID: 109624047), they are located in exon 16 and the non-coding region, respectively, and c.2406C>A (P.H798N) is located in exon 13 of the adenylate cyclase type 6-like (AC6) gene(Gene ID: 109630770). ITPR2 and AC6 expression, which both participate in the thyroid hormone synthesis pathway associated with pseudo-albinism and hypermelanosis in P. olivaceus, were also investigated using qRT-PCR. In hypermelanotic fish, there were relatively higher levels of expression in ITPR2 and AC6 mRNA of hyper-pigmented skin of blind side than that of non-pigmented skin on the blind side and pigmented skin on the ocular side, while in pseudo-albino fish, expression level of ITPR2 and AC6 mRNA in pigmented skin of ocular side was significantly higher than that in non-pigmented skin both ocular and blind side. The results indicated that the expression of the two genes in abnormal parts of body color is positively correlated with pigmentation, suggesting that the influence of abnormal expression of two genes on the pigmentation in abnormal parts of body color deserves further study.

Detecting Cynoglossus semilaevis infected with Vibrio harveyi using micro RNAs from mucous exosomes.

Exosomes are important mediators of vesicle transportation and contain microRNAs (miRNAs) that mediate transcriptional gene knockout and silencing in biological processes. Moreover, exosomic miRNAs are promising biomarkers for disease diagnosis and physiological status indication in many species, including fish. The impact of the Vibrio harveyi pathogen on Cynoglossus semilaevis aquaculture is becoming more and more serious as the industry expands. To overcome this challenge, miRNAs in mucous exosomes were screened by small RNA sequencing and verified by quantitative real-time PCR to develop biomarkers. This is the first capture of exosomes from flatfish mucus coupled with miRNA profiling. The results revealed significant differences in expression levels of some miRNAs between infected and healthy fish. Three unique miRNAs were identified for V. harveyi infection diagnosis; expression levels of dre-miR-205-5p and dre-miR-205-5p in infected fish were significantly lower than controls, while dre-miR-100-5p expression was higher. These miRNAs in mucous exosomes could be used to differentiate diseased and healthy fish in an early screening method with practical value for breeding disease-resistant C. semilaevis.

Thrombocytopenia in 737 adult intensive care unit patients: A real-world study of associated factors, drugs, platelet transfusion, and clinical outcome.

OBJECTIVE: We aimed to identify and represent factors associated with thrombocytopenia in intensive care unit, especially the pathogens and drugs related to severe and extremely thrombocytopenia. Then, we aim to compare the mortality of platelet transfusion and non-transfusion in patients with different degrees of thrombocytopenia. METHODS: We identified all thrombocytopenic patients in intensive care unit by using platelet-specific values and then extracted electronic health records from our Hospital Information System. Data were statistically analyzed with t test, chi-square test, and logistic regression. RESULTS: We found that infections (32.7%) were the most frequent cause associated with thrombocytopenia, followed by sepsis shock (3.93%) and blood loss (2.99%). Meanwhile, antifungals (p = 0.002) and bacterial infection (p = 0.037) were associated with severe and extremely severe thrombocytopenia. Finally, we found that the mortality of platelet transfusion and non-transfusion in patients was statistically significant for patients with platelet counts between 30 and 49/nL (χ2 = 9.719, p = 0.002). CONCLUSION: Infection and sepsis emerged as two primary factors associated with thrombocytopenia in intensive care unit. Meanwhile, antifungals and bacterial infection were associated with platelet counts less than 49/nL. Finally, platelet transfusion may be associated with reduced mortality in patients with platelet counts between 30 and 49/nL.

A combination of genome-wide association study screening and SNaPshot for detecting sex-related SNPs and genes in Cynoglossus semilaevis.

Chinese tongue sole (Cynoglossus semilaevis) males and females exhibit great differences in growth rate and appearance. The species is heterogametic (ZW/ZZ) and has sex-reversed "pseudomales" that are genetically female and physiologically male. In this study, we identified eight sex-specific single nucleotide polymorphism (SNP) markers for the sex identification of C. semilaevis by using a combination of genome-wide association study (GWAS) screening and SnaPshot validation. Candidate SNPs were screened using genotyping by sequencing to perform GWAS of the differential SNPs between the sexes of C. semilaevis. The SNP loci were amplified using a multiplex PCR system and detected via SNaPshot, which enables multiplexing of up to 30-40 SNPs in a single assay and ensures high accuracy of the results. The molecular markers detected in our study were used to successfully identify normal males and pseudomales from 45 caught and 40 cultured C. semilaevis specimens. Linkage disequilibrium analysis showed that the eight SNP loci were related to each other, with a strong linkage. Moreover, we investigated the expression of prdm6 mRNA containing a missense SNP and confirmed that the gene is differentially expressed in the gonads of the different sexes of C. semilaevis; the expression of prdm6 mRNA was significantly higher in the males than in the females and pseudomales. This means prdm6 may be related to sex differentiation in C. semilaevis.

Large scale SNP unearthing and genetic architecture analysis in sea-captured and cultured populations of Cynoglossus semilaevis.

Knowledge on population structure and genetic diversity is a focal point for association mapping studies and genomic selection. Genotyping by sequencing (GBS) represents an innovative method for large scale SNP detection and genotyping of genetic resources. Here we used the GBS approach for the genome-wide identification of SNPs in a collection of Cynoglossus semilaevis and for the assessment of the level of genetic diversity in C. semilaevis genotypes. GBS analysis generated a total of 55.12 Gb high-quality sequence data, with an average of 0.63 Gb per sample. The total number of SNP markers was 563, 109. In order to explore the genetic diversity of C. semilaevis and to select a minimal core set representing most of the total genetic variation with minimum redundancy, C. semilaevis sequences were analyzed using high quality SNPs. Based on hierarchical clustering, it was possible to divide the collection into 2 clusters. The marine fishing populations were clustered and clearly separated from the cultured populations, and the cultured populations from Hebei was also distinct from the other two local populations. These analyses showed that genotypes were clustered based on species-related features. Differential significant SNPs were also captured and validated by GBS and SNaPshot, with linkage disequilibrium and haplotype analysis, seven SNPs have been confirmed to have obvious differentiation in two populations, which may be used as the characteristic evaluation sites of sea-captured and cultured Cynoglossus semilaevis populations. And SNP markers and information on population structure developed in this study will undoubtedly support genome-wide association mapping studies and marker-assisted selection programs. These differential SNPs could be also employed as the characteristic evaluation sites of sea-captured and cultured Cynoglossus semilaevis populations in future.

Identification and application of piwi-interacting RNAs from seminal plasma exosomes in Cynoglossus semilaevis.

BACKGROUND: Piwi-interacting RNAs (piRNAs) have been linked to epigenetic and post-transcriptional gene silencing of retrotransposons in germ line cells, particularly in spermatogenesis. Exosomes are important mediators of vesicle transport, and the piRNAs in exosomes might play an important role in cell communication and signal pathway regulation. Moreover, exosomic piRNAs are promising biomarkers for disease diagnosis and physiological status indication. We used Cynoglossus semilaevis because of its commercial value and its sexual dimorphism, particularly the sex reversed "pseudomales" who have a female karyotype, produce sperm, and copulate with normal females to produce viable offspring. RESULTS: To determine whether piRNAs from fish germ line cells have similar features, seminal plasma exosomes from half-smooth tongue sole, C. semilaevis, were identified, and their small RNAs were sequenced and analysed. We identified six signature piRNAs as biomarkers in exosomes of seminal plasma from males and pseudomale C. semilaevis. Bioinformatic analysis showed that all six signatures were sex-related, and four were DNA methylation-related and transposition-related piRNAs. Their expression profiles were verified using real-time quantitative PCR. The expression of the signature piRNAs was markedly higher in males than in pseudomales. The signature piRNAs could be exploited as male-specific biomarkers in this fish. CONCLUSIONS: These signatures provide an effective tool to explore the regulatory mechanism of sex development in C. semilaevis and may provide guidance for future research on the function of piRNAs in the generative mechanism of sex reversed "pseudomales" in C. semilaevis.

Identification and characterization of the melanocortin 1 receptor gene (MC1R) in hypermelanistic Chinese tongue sole (Cynoglossus semilaevis).

The Chinese tongue sole (Cynoglossus semilaevis) is a flatfish with distinctive asymmetry in its body coloration. The melanism (hyperpigmentation) in both the blind side and ocular side of C. semilaevis gives it an extremely low commercial value. However, the fundamental molecular mechanism of this melanism remains unclear. Melanocortin 1 receptor (MC1R), a GTP-binding protein-coupled receptor, is considered to play a vital role in the physiology of the vertebrate pigment system. In order to confirm the contribution of MC1R to the body coloration of C. semilaevis, the expression levels of Mc1r mRNA were measured in seven tissue types at different developmental stages of normal and melanistic C. semilaevis. The expression levels of Mc1r mRNA in the heart, brain, liver, kidney, ocular-side skin, and blind-side skin of melanistic C. semilaevis were significantly higher than that of normal C. semilaevis in all developmental stages. Moreover, the knocking down of Mc1r in the C. semilaevis liver cell line (HTLC) increased the expression of the downstream genes microphthalmia transcription factor (Mitf) and tyrosinase-related protein 1 (Tyrp1) in the pigmentation pathway. Thus, the present data suggest that MC1R might play important roles in Tyrp1- and Mitf-mediated pigment synthesis in C. semilaevis.

Comparison of the intestinal microbiota composition and function in healthy and diseased Yunlong Grouper.

Maintaining stabilization of the intestinal microbiota is important in preventing bacterial diseases in cultured fish. At present, there have been no reports on the composition and functional analysis of intestinal microbiota in Yunlong Grouper (Epinephelus moara♀ × Epinephelus lanceolatus♂). In this study we analyzed and compared the intestinal microbiota composition of healthy and diseased pond-reared fish to discern the functional profile of a healthy status. The richness and diversity of the intestinal microbiota did not differ significantly between diseased and healthy fish, yet the abundance of predominant phyla like the Proteobacteria were upregulated in the diseased Yunlong Grouper. At the genus level, a significant reduction of Cetobacterium was observed in the intestinal tracts of diseased fish, as Pseudomonas became the most dominant bacterium. To compare the intestinal microorganism abundances between the two health groups of fish, we first screened the gut bacteria and discerned 4 phyla and 12 genera to designate a healthy status in Yunlong Grouper. The environmental bacterial community influenced composition of the intestinal microbiota in Yunlong Grouper, and the intestinal microbiota of diseased fish was more susceptible to the influence of the culture water. In addition, the prediction of functional genes by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) indicated that the intestinal microbiota of Yunlong Grouper is related mainly to the terms "metabolism, environmental information processing, genetic information processing, human diseases, and cellular processing; moreover, the functions of the intestinal microbiota differed between the different health states of this fish. The overall results indicate that the occurrence of disease can affect the composition and function of the intestinal microbiota in a cultured fish.

Seminal Plasma Exosomes: Promising Biomarkers for Identification of Male and Pseudo-Males in Cynoglossus semilaevis.

In mammals, small RNAs enclosed in exosomes have been identified as appropriate signatures for disease diagnosis. However, there is limited information on exosomes derived from seminal plasma, and few studies have reported analyzed the composition of exosomes and enclosed small RNAs in fish. The half-smooth tongue sole (Cynoglossus semilaevis) is an economically important fish for aquaculture, and it exhibits sexual dimorphism: the female gender show higher growth rates and larger body sizes than males. Standard karyotype analysis and artificial gynogenesis tests have revealed that this species uses heterogametic sex determination (ZW/ZZ), and so-called sex-reversed pseudo-males exist. In this study, we successfully identified exosomes in the seminal plasma of C. semilaevis; to the best of our knowledge, this is the first report of exosomes in fish seminal plasma. Analysis of the nucleotide composition showed that miRNAs were dominant in the exosomes, and the miRNAs were sequenced and compared to identify signature miRNAs as sexual biomarkers. Moreover, target genes of the signature miRNAs were predicted by sequence matching and annotation. Finally, four miRNAs (dre-miR-141-3P, dre-miR-10d-5p, ssa-miR-27b-3p, and ssa-miR-23a-3p) with significant differential expression in the males and pseudo-males were selected from the signature candidate miRNAs as markers for sex identification, and their expression profiles were verified using real-time quantitative PCR. Our findings could provide an effective detection method for sex differentiation in fish.

Unsupervised boundary delineation of spinal neural foramina using a multi-feature and adaptive spectral segmentation.

As a common disease in the elderly, neural foramina stenosis (NFS) brings a significantly negative impact on the quality of life due to its symptoms including pain, disability, fall risk and depression. Accurate boundary delineation is essential to the clinical diagnosis and treatment of NFS. However, existing clinical routine is extremely tedious and inefficient due to the requirement of physicians' intensively manual delineation. Automated delineation is highly needed but faces big challenges from the complexity and variability in neural foramina images. In this paper, we propose a pure image-driven unsupervised boundary delineation framework for the automated neural foramina boundary delineation. This framework is based on a novel multi-feature and adaptive spectral segmentation (MFASS) algorithm. MFASS firstly utilizes the combination of region and edge features to generate reliable spectral features with a good separation between neural foramina and its surroundings, then estimates an optimal separation threshold for each individual image to separate neural foramina from its surroundings. This self-adjusted optimal separation threshold, estimated from spectral features, successfully overcome the diverse appearance and shape variations. With the robustness from the multi-feature fusion and the flexibility from the adaptively optimal separation threshold estimation, the proposed framework, based on MFASS, provides an automated and accurate boundary delineation. Validation was performed in 280 neural foramina MR images from 56 clinical subjects. Our method was benchmarked with manual boundary obtained by experienced physicians. Results demonstrate that the proposed method enjoys a high and stable consistency with experienced physicians (Dice: 90.58% ± 2.79%; SMAD: 0.5657 ± 0.1544 mm). Therefore, the proposed framework enables an efficient and accurate clinical tool in the diagnosis of neural foramina stenosis.