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In support of previous findings, we find that protracted learning is important for both cultural phenomena [21, 24]. On the one hand, protracted learning tends to limit the optimization of repertoires allowing for arbitrary variation between groups and hence traditional differences. On the other hand, the within-lifetime limitation of optimization of repertoires make cumulative cultural processes possible. In previous work we also found that protracted learning is important for the costs and benefits associated with particular SLMs . Thus multi-scale models with protracted learning allow us to study the adaptive and cultural impact of particular SLMs rather than assume them. In this way multi-scale models can be used to evaluate the assumptions we make about social learning in various top-down verbal and formal models.
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Previously we found that LE did not increase energy intake relative to solitary foraging , and concluded that grouping would probably evolve for other reasons, for example as an anti-predation strategy . If so, then our model here, like a previous model , predicts that traditional differences would evolve as a side-effect of grouping without any special cognitive adaptation besides those needed for living in groups.
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Relative to this baseline of traditional differences as an evolutionary byproduct, we showed in previous work that SE and OL can readily evolve because they enhance the level of foraging efficiency . Here we show that the evolution of such increased optimization need not generate greater traditional differences between groups, but could instead reduce them (Fig. 3 a). It is intuitive to assume that more accurate SLMs will increase within-group similarity (or conformity) and hence increase differences between groups . However, this overlooks the effect of SLMs on enhancing repertoire optimization . If all groups are able to correctly identify and eat the highest quality resources then behavioral repertoires will become similar , because the highest quality resources are always a limiting subset (Fig. 1 d). Despite this possible convergence between groups, we find that even when learning parameters evolve, optimization can still be sufficiently limited to allow for traditional differences.
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While our findings support the idea that traditions should be widespread in foragers in cohesive groups living in patchy environments, for cumulative cultural change we expect a large context dependency. Previous theoretical work on diet learning showed that cumulative culture could arise as a side-effect of grouping and therefore might commonly occur in animal societies . Our results here suggest that in the context of skill learning, cumulative cultural increases in energy intake may only arise for OL and only in environments with high task difficulty. The latter supports the idea that cumulative cultural processes may occur predominantly in species with cognitively demanding forms of social learning . In particular, since SE and LE are inviable in environments with high task difficulty, our results suggest that OL would need to evolve before niches with high task difficulty could be invaded, and only thereafter would cumulative cultural increases in energy intake evolve. Previously we have argued that through this process, the evolution of cognitively demanding forms of social learning could open up novel niches . Further modelling work is needed to confirm these expectations.
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Our measure of cumulative cultural change is very general and does not necessarily imply (i) the generation of behavioral complexity via the invention of novel behavioral combinations, or levels technological of complexity, nor (ii) open-ended change [4, 13, 33, 34]. In our model, the latter cannot arise because novel behavioural opportunities cannot be generated, and the cumulative cultural process is restricted to the opportunities that are available in the environment, and is ‘bounded’. Thus, the complexity of behavior remains limited in the sense that any single behavior could be invented within the lifetime of an individual . However, this behavior-level view contrasts with our repertoire-level perspective, where we consider culture cumulative if foragers exhibit a repertoire quality and overall skill level that they could not achieve within a lifetime of asocial learning. Thus while each single behavior could in principle be discovered by any forager, the level of repertoire optimization, or total ‘ecological knowledge’, cannot. In future, this ‘ecological knowledge’ perspective could be extended to spatial knowledge, in order to establish a more complete perspective on the scope of cumulative culture next to diet learning and skill learning (present study) in group foragers.
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Bounded contexts appear reasonable for considering cultural phenomena in many primate species and the kind of bounded cumulative culture observed here provides a putative evolutionary precursor to more open-ended forms of cumulative culture. However, our results suggest that precisely because the cumulative process is expressed at a repertoire level and bounded, detecting existing cumulative cultural processes empirically may be very difficult. We would expect a bounded cumulative cultural process to operate for some time, but then level off. Thus when observing primates in the wild, researchers may well be measuring the outcome of a cumulative cultural process, where the phenotypes observed cannot be achieved within a single lifetime, even though changes in time may not be detectable. Moreover, quantifying the difficulty of acquiring a particular repertoire and detecting social influences is extremely difficult , which could help to explain the lack of empirical evidence for such cumulative processes. Studying the reintroduction of animal species to the wild may be a promising setting in which to study the possibility of cumulative cultural change in animals across generations. The difficulty of successful re-introductions to the wild, especially those in great apes , could be an indication of a dependence on cumulative culture.
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If ecologically-bounded contexts are an evolutionary precursor to more open-ended forms of cumulative culture, then how can we use this to understand the transition between the two? At present many key variables have been proposed to explain this transition including, cognitive abilities for high fidelity copying [4, 13, 34], large population sizes [37, 38] and high rates of socialization and division of labour . What is lacking at present is a framework that explains how these factors originate and co-evolve. Extensions to the multi-scale simulation model presented here could help to address this question. In this sense our model represents a tangible ecologically-bounded baseline in which researchers could study how ecological bounds could be relaxed. In particular, we expect that niche construction processes [39, 40] will be critical in relaxing the bounds found in our model, because these appear to be needed for generating feedbacks between cultural inheritance and opportunities for cultural innovation. In this way, cultural processes can start to define their own possibilities for change.
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Chinese mitten crab (Eriocheir sinensis) is one of the most important aquaculture species, which is widely distributed in freshwater and low-salinity estuarine regions in China [1, 2]. Its culture under facility conditions started in the early 1980s and the annual output has increased during the past decade . In 2014, the domestic total production was more than 750,000 tons for aquaculture only. The male mitten crabs grow faster and are more aggressive than females in the current traditional culture system, which may result in high mortality of the females and thus influence the production of the crab. It is obvious that this culture of all-male or all-female populations would be more economically advantageous to crab aquaculture industry [4, 5].
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It was reported that monosex culture has been achieved through sex reversal mediated by steroid manipulations and has been practiced in fish aquaculture . Unlike fish, sexual differentiation in crustaceans is uniquely regulated by the male-specific androgenic gland (AG), which has been detected in male crustaceans. It plays a pivotal role in the regulation of male differentiation and in maintaining the male sexual characteristics [4, 7–13]. So far, the insulin-like androgenic gland hormone (IAG) was identified in many commercially important crustacean species [14–18]. IAG silencing in Macrobrachium rosenbergii proved to be useful in obtaining a full and functional sex reversal species , leading to the production of monosex populations. So far, AG cDNA libraries and transcriptome datasets serve as a basis for further studies [20, 21], but the molecular mechanism pertaining to the related regulatory mechanisms of AG in E. sinensis is still unclear.
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Transcriptome is a total set of transcripts, mRNA, and noncoding RNA, in one or a population of cells during a specific developmental stage or in response to a particular physiological condition using high-throughput technology . It is the connection between genome genetic information and proteomics biological function . In nonstandard model organisms where genomic resources are lacking, such as a fully sequenced genome, obtaining a transcriptome is an effective way to evaluate gene expression and perform comparative studies at the whole genome level . Comparative transcriptome analysis provides the foundation for gene structure and function research and determines when genes are expressed and how they are regulated . Now, it is being widely applied to elucidate genetic factors conferring economically significant traits in cultured crustacean species [26–28].
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In the current study, we generated AG transcriptomes using cDNA prepared from mRNAs isolated at the proliferation phase (PP) and secretion phase (SP) in E. sinensis and performed a comparative analysis. The objectives of the present study were to elaborate the genetic expression change between PP and SP during AG development and to reveal the molecular regulation mechanisms of stimulating spermatogenesis and maintaining male characteristics.
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All of the specimens were obtained from a farm in Ganyu, Jiangsu province, China, in October 2015. The PP and SP AG were identified and characterized under the microscope according to those described by Qiu et al. . Three replicated samples were prepared for PP and SP AG, respectively. Fifty individuals were included in each sample.
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Total RNA was isolated using TRIzol reagent according to the manufacturer's instructions. The isolated RNA was treated with RNase-free DNase I (Sangon, Shanghai, China) to eliminate possible genomic DNA contamination. Equal quantities of total RNA from three replicate samples were mixed to prepare the pooled RNA sample for RNA-Seq. RNA purity was checked using the Nano Photometer® spectrophotometer (IMPLEN, CA, USA) and RNA concentration was measured using Qubit® RNA Assay Kit in Qubit® 2.0 Fluorometer (Life Technologies, CA, USA). RNA integrity was determined using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
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The transcriptome libraries for sequencing were generated using the VAHTS™ mRNA-Seq v2 Library Prep Kit for Illumina® (Vazyme Biotech Co., Ltd., Nanjing, China) following the manufacturer's recommendations. The clustering of the indexed samples was carried out using the VAHTS RNA Adapters of Illumina (Vazyme Biotech Co., Ltd., Nanjing, China) according to the manufacturer's instructions. After clustering, the libraries were sequenced on Illumina HiSeq X Ten platform, with 150 bp pair-end reads produced.
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Raw images were transformed into raw reads by base calling using CASAVA (Version 1.8.2). Clean reads were obtained by removing reads with adapters, reads in which unknown bases were more than 5%, and low-quality reads (the percentage of low-quality bases was over 50% in a read, and we defined the low-quality base to be the base whose sequencing quality was no more than 10). At the same time, Q20, Q30, and GC content of the clean data were calculated.
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Gene functional annotation was performed by sequence comparison with public databases. The unigenes of the de novo assembly were searched against the NCBI nonredundant (NR), Swiss-Prot, KEGG, and COG protein databases by using BLASTX with a cut-off E value of 10−5.
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FPKM (fragments per kilobase per transcript per million mapped reads) method was performed to quantify the expression of two expression profiles . The False Discovery Rate (FDR) method was applied in hypothesis testing and multiple hypothesis testing to calibrate significant levels and eliminate influence of random fluctuations and errors . At the same time, according to the gene expression level (FPKM value), differentially expressed multiples in different samples were calculated. The differentially expressed genes (DEGs) were defined as FDR ≤ 0.001 and no less than 2-fold.
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First, we mapped differentially expressed genes to each term of the GO database (http://www.geneontology.org/) and calculated the gene number of each term, and thus we got a GO function gene list and gene number statistics. Then, we used hypergeometric inspection, compared with the entire genome background, to find out significantly enriched GO entries in the differentially expressed genes.
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KEGG pathway was the unit for pathway significant enrichment analysis. The P value calculation formula of the hypothesis test was similar to that of GO function significant enrichment analysis. After multiple testing corrections, we chose the pathway with Q-value ≤ 0.05 as a significant enrichment pathway in the differentially expressed genes.
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Six candidate DEGs were randomly selected for validation by QPCR. Total RNA from independent samples of PP and SP was extracted separately and reverse-transcribed using PrimeScript® RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). The SYBR Green RT PCR assay was carried out in an ABI PRISM 7300 Sequence Detection System (Applied Biosystems). Six pairs of gene-specific primers (IAG, IR, OA receptor, 5-HT receptor 1, Tra-2, and FTZ-F1; Table 1) designed using Premier Primer 5 were used to amplify the partial cDNA gene sequences, respectively. Three biological replicates for each sample and three technical replicates were performed. The β-actin from E. sinensis was chosen as a reference gene for internal standardization . PCR was carried out in a total volume of 10 μl, containing 5 μl of 2x SYBR Premix Ex Taq (TaKaRa, China), 0.2 μl of 50x ROX reference dye, 2 μl of the diluted cDNA mix, 0.2 μl of each primer (10 μM), and 2.4 μl of sterile distilled H2O. The PCR program was 95°C for 5 min, followed by 40 cycles of 95°C for 5 s and 60°C for 31 s. To confirm that only one PCR product was amplified and detected, dissociation curve analysis of amplification products was performed at the end of each PCR reaction. After the PCR program, data were analyzed with ABI7300 SDS software (Applied Biosystems). The relative expression level was calculated using the 2−ΔΔCt method. The results were subjected to one-way analysis of variance (one-way ANOVA) using SPSS 16.0, and P values less than 0.05 were considered statistically significant.
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21.392 and 55.29 μg RNA were obtained, respectively, whose qualities meet the requirements of the sequencing and building libraries. A total of 49,925,104 and 49,975,260 raw reads were obtained from the PP and SP transcriptomes. All of the raw reads were deposited into the Sequence Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/Traces/sra/) under accession numbers SAMN06204640 and SAMN06204641. After removing adaptors and low-quality reads, a total of 47,335,722 and 46,323,850 clean reads were obtained in each of the profiles, respectively. After de novo assembly, 72,000 unigenes were obtained by paired-end method of Trinity and TGICL clustering. Among them, a total of 30,576 unigenes were significantly matched with NR database.
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Based on GO analysis, 15,318 unigenes were annotated and divided into three categories: “biological process,” “cellular component,” and “molecular function” (Figure 1). The number of matched GO terms per unigene varied from 1 to 143. More than 89.1% of unigenes could be assigned to more than one GO term, with the majority of unigenes mapped to 2 to 7 GO terms. Based on GO analysis, three major functional groups were divided into 60 subcategories, including the dominant subcategories, cellular process (10955), cell (9486), cell part (9471), single-organism process (8860), metabolic process (8220), and binding (7958).
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For the eggNOG analysis, a total of 14,125 unigenes were assigned to 25 function categories (Figure S1 in Supplementary Material, available online at https://doi.org/10.1155/2017/4956216). Among them, 6,996 unigenes were assigned to general function prediction only, representing the largest group, followed by translation, ribosomal structure, and biogenesis (5065) and cell cycle control, cell division, and chromosome partitioning (3995). KEGG analysis revealed that 24,691 unigenes were assigned to six categories comprised of 258 metabolic or signaling pathways. The most prominent KEGG pathways were metabolic pathways (2683, 10.87%), regulation of actin cytoskeleton (1768, 7.16%), amoebiasis (1635, 6.62%), Vibrio cholerae infection (1409, 5.71%), focal adhesion (1225, 4.96%), RNA transport (1060, 4.29%), Salmonella infection (1048, 4.24%), adherens junction (958, 3.88%), bacterial invasion of epithelial cells (938, 3.8%), and chemokine signaling pathway (889, 3.6%).
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After comparison with fold change threshold value and FDR test, there were 4,027 genes from the total 70,915 genes (5.68% of the genes) with a significant difference (P < 0.05) between PP and SP stages, including 2,220 upregulated genes and 1,807 downregulated genes. The distribution of the significant changes of 70,915 unigenes was illustrated in a scatter diagram (Figure 2), where the statistical significance of each unigene was plotted against fold change. Functional distribution of these DEGs was further analyzed by GO and KEGG enrichment, respectively.
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To gain further insights into the molecular mechanisms of attractive quality formation during AG development between PP and SP, gene ontology enrichment analysis was performed to determine which kind of GO term DEGs were mainly enriched in. Finally, 2,345 DEGs were annotated to 3,588 GO terms. The functions of 854 DEGs with higher expression in SP than in PP AG were assigned to biological process, cellular component, and molecular function. In biological process, SRP-dependent cotranslational protein targeting to membrane (41 DEGs, GO: 0006614), nuclear-transcribed mRNA catabolic process, nonsense-mediated decay (42 DEGs, GO: 0000184), cotranslational protein targeting to membrane (41 DEGs, GO: 0006613), protein targeting to ER (41 DEGs, GO: 0045047), and establishment of protein localization to endoplasmic reticulum (41 DEGs, GO: 0072599) were the most prominent terms. Cytosolic ribosome (44 DEGs, GO: 0022626) was the most prominent term within the cellular component followed by ribosomal subunit (46 DEGs, GO: 0044391), cytosolic large ribosomal subunit (27 DEGs, GO: 0022625), ribosome (49 DEGs, GO: 0005840), and extracellular exosome (96 DEGs, GO: 0070062). In molecular function, most of the annotated unique sequences were assigned to phosphatidylserine binding (13 DEGs, GO: 0001786), structural constituent of ribosome (48 DEGs, GO: 0003735), and modified amino acid binding (14 DEGs, GO: 0072341).
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KEGG annotation is the process of mapping genes of interest to the metabolic pathways. In our study, 1,863 DEGs were mapped to 243 KEGG pathways. The significantly changed pathways included ribosome (46 DEGs, ko03010), cytosolic DNA-sensing pathway (21 DEGs, ko04623), neuroactive ligand interaction (52 DEGs, ko04080), RNA polymerase (36 DEGs, ko03020), pancreatic secretion (34 DEGs, ko04972), ECM-receptor interaction (53 DEGs, ko04512), and cell adhesion molecules (25 DEGs, ko04514), all of which should be the key metabolic networks leading to cell proliferation, secretion, and signal transduction in AG.
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The six DEGs were selected to verify the results of the RNA-Seq analysis by qRT-PCR using different RNAs from those used for RNA-Seq (Table 2). The results showed that the expression patterns of IAG, IR, OA receptor, Tra-2, 5-HT receptor 1, and FTZ-F1 all agreed well between RNA-Seq and qRT-PCR, which confirmed the data obtained from high-throughput sequencing (Figure 3).
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Although the mechanism of sex differentiation in crustaceans has yet to be defined, the androgenic gland (AG) is thought to be the exclusive organ that produces the androgenic hormone, which induces male sexual development. The AG system serves as a unique biological system for studying endocrine regulation of sex differentiation in decapods. GO and pathway analyses of DEGs will be helpful in the discovery of novel genes that play key roles in maintaining male characters and related behaviors during AG development.
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IAG was found to be expressed in the AG of male crustaceans, which belongs to the insulin superfamily of proteins and was considered as the key regulator of male sex differentiation [5, 12]. IAG is responsible for the development and continuation of male primary and secondary sexual characteristics, inhibiting the synthesis of Vg and stimulation of spermatogenesis. We found that a unigene from the AG transcriptome sequencing result has 44.2% and 42.4% identity with IAG of Callinectes sapidus and Scylla paramamosain, respectively. Further analysis found that the length of the gene sequence is 1392 bp and ORF length is 456 bp. The amino acid sequence alignment indicated that the novel gene had common conservative amino acids with Callinectes sapidus, Scylla paramamosain, and so forth (Figure 5), which proved the novel gene is IAG of E. sinensis.
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Transformer-2 (Tra-2) gene plays a key role in the regulatory hierarchy of sexual differentiation in somatic tissues and in the germline of Drosophila melanogaster. Sex-lethal (Sxl) plays a complicated and important role in embryogenesis, metamorphosis, somatic sexual development, and sex differentiation in insects and its highest expression levels were observed in testis and hepatopancreas [32, 33]. In our transcriptome database, we identified four Tra-2 isoforms designated as Estra-2a, Estra-2b, Estra-2c, and Estra-2d. Together with sex-lethal (Sxl) and double sex (dsx), most of ortholog genes in “Sxl-Tra/Tra-2-Dsx/Fru” pathway are detected. In the process of male differentiation, the expression of IAG and Tra-2 was significantly upregulated (Table 2), whereas the expression change of Sxl and Dsx was not significant, which illustrated that IAG and Tra-2 may play a more important role in promoting the development of testis and stimulating male related behaviors.
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DEAD box RNA helicase 20 (DDX20), forkhead transcription factor 2 (FOXL2), and fushi tarazu factor 1 (FTZ-F1) were investigated in the regulation of vitellogenesis (VTG). FOXL2 negatively regulates the VTG synthesis at developmental stage. FOXL2 downregulates VTG's expression by binding DDX20 in the regulation of follicular cell apoptosis and repress the synthesis of VTG when bound with FTZ-F1 at the mature stage [18, 34, 35]. According to our research, during the process of testis development, FOXL2 had no obvious change, and FTZ-F1 gene was downregulated. Therefore, FTZ-F1 may negatively regulate the testis development. Furthermore, cytochrome P450 upregulated significantly in the SP indicates that the mechanism of cytochrome P450 regulating gonad development in fish and crustaceans may be completely different.
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In C. elegans, Fem-1, encoding an ankyrin repeat protein, is a component of the signal transduction pathway controlling sex determination . There were three members in the Fem-1 gene family (designated as Fem-1a, Fem-1b, and Fem-1c) in our transcriptome database, which were homologs of the nematode Fem-1 protein . It is reported that Fem-1 might function in early sex determination and late gonad development in the crab . However, in the present study, the four homologs of Fem-1 are not significantly upregulated in the secretory phase of AG.
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Research about spermatogenesis and the structure of the androgenic gland in Eriocheir sinensis showed that there was a close relationship between the androgenic gland and spermatogenesis. There was no spermatid when the androgenic gland was in the primary development phase . With the AG development, a great number of primary and secondary spermatocytes appeared in the seminiferous tubules. Later, the sperms matured and were released when the androgenic gland was in the secretion phase.
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| 99.94 |
The ubiquitin-proteasome pathway influences many biological processes, including cell degradation and protein homeostasis maintenance. Many studies have shown that ubiquitin may be relevant to heat shock proteins [38, 39]. For instance, heat shock cognate 70 kDa proteins are synthesized in haploid cells during spermatogenesis and are mainly activated at the spermatid stage , which may help ubiquitin and target nonrepairable proteins to the proteasome . In the present study, a ubiquitin gene was found to be significantly upregulated in SP (Table 3). The serpin (serine proteinase inhibitor) family is reported to be exclusively expressed in the rat cauda epididymis and is upregulated, induced by androgens, and is secreted into the lumen to cover the sperm head. It can transform immotile spermatids into motile and fertilization-competent spermatozoa . Here, we identified two upregulated unigenes, CL4939.Contig2 and CL5743.Contig1, that were annotated as serine proteinase inhibitors.
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In crustaceans, cathepsin A functioned in the innate immune system . The purified cathepsin A proteins of E. sinensis can effectively digest the spermatophore wall . Cathepsin B was reported to control the developmental processes in insects and other arthropods. Cathepsin D is necessary for the formation of the yolk. Cathepsin L regulates the development of the ovary in many species including L. vannamei. Cathepsins A, B, C, D, F, and L were found in AG transcriptome. Cathepsin A was significantly upregulated while cathepsin D was significantly downregulated in SP AG, implying that these two types of cathepsins may play essential roles in the spermatogenesis process.
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These results indicated that sex-related genes, such as IAG, SXL, TRA-2, SRY, FTZ-F1, FOXL2, and cytochrome p450, might play important roles in maintaining the male physiology and morphology of Eriocheir sinensis. Ubiquitin, serpin, cathepsin A, and cathepsin D genes may have a direct influence on spermatogenesis.
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| 99.94 |
Ribosomes, the workplace for protein biosynthesis, are directly associated with translation, cell growth, cell cycle, localization, and cell proliferation . In our present study, the ribosome is the most prominent pathway (P value: 1.130046e − 12). In 2011, the first complete atomic structure of eukaryotic 80S ribosome from the yeast Saccharomyces cerevisiae was obtained by crystallography. The assembly model of 80S ribosomes requires the joining of 40S and 60S subunits , which reveals the architecture of eukaryote-specific elements and their interaction with the universally conserved core. During translation initiation, the 40S subunit is interactive with eukaryotic translation initiation factor 1 (eIF1) and 60S subunit is in complex with eIF6. The core of the 60S subunit is formed by the 28S ribosomal RNA, which contributes the active site of the ribosome [47, 48].
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Comparative transcriptomic analysis showed that a total of 44 DEGs, including 40s and 60s ribosomal protein genes, are significantly upregulated; only ribosomal protein L3 and 40S ribosomal protein S26-like are significantly downregulated in the secretion phase of AG (Figure 4). Histological observation has shown that the secretion phase of AG has developed more rough endoplasmic reticulum and abundant free ribosome, while the synthesis of material is more active to maintain the secretion function compared with the proliferation phase . The developmental cytology of the androgenic gland cells can be distinctly divided into proliferation phase and secretion phase . Cells of the newly formed parts of the androgenic gland are generally located on the surface of the subterminal portion of the ejaculatory duct near the base of the penis. And the structure of the gland changed greatly during testis development cycle. Later, cells of androgenic gland increase in size, becoming multinucleated and vacuolated .
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Sexual differentiation and maintenance of masculinity in crustaceans have been suggested to be regulated by IAG. However, downstream elements involved in the signaling cascade remained unknown. O. Sharabi and his colleges performed long-term RNA interference (RNAi) silencing in young male prawns to investigate the function of M. rosenbergii insulin receptor (Mr-IR). They found that the silencing of Mr-IR advanced the appearance of a male-specific secondary trait but had no effect on growth. The most noted effects of Mr-IR's silencing were hypertrophy of the AG and the associated increased production of Mr-IAG. These results suggested a role of Mr-IR in the regulation of the AG and implied sexual differentiation in crustaceans involving more than a single Mr-IAG receptor, which emphasized the complexity of sexual differentiation and maintenance . The pivotal protein in any insulin family-based signaling pathway is the insulin receptor that is responsible for mediating the signal carried by insulin-like peptides (ILPs) from the intercellular to the intracellular environment. In the present study, a total of three Es-IRs were identified for the first time in the AG comprehensive transcriptomic library.
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The insulin-like superfamily includes two major subgroups, ILPs and the insulin-like growth factors (IGFs) (Chandler et al., 2015). The role of the insulin pathway is very diverse and includes not only glucose homeostasis but also regulation of growth, longevity, and reproduction . IAG is the sole ILP widely found in decapod crustaceans thus far, with the exception of a Drosophila ILP7 ortholog of unknown function, identified in a spiny lobster species . IR and IGF1R are transmembrane receptors activated by insulin and insulin-like growth factor-1 (IGF-1). During the process of the AG development, 25 differentially expressed genes in the insulin signaling pathway were identified, among which were insulin receptor, translation initiation factor, factor facilitated glucose transporter, phosphorylase kinase gamma subunit, fatty acid synthase, and hormone-sensitive lipase genes. Unigene10231 and CL1931, mapped to the reference of insulin receptor cDNAs, are key genes in the insulin signaling pathway in E. sinensis.
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So far, IAG has been shown to be a key regulator of reproductive processes such as sexual differentiation, spermatogenesis, and sexual shifts in crustaceans. Insulin receptors are pivotal players in insulin signaling pathway (Figure S2), which mediate insulin and ILP signaling . However, apart from characterization of the hormone itself, little information regarding other elements of the signal transduction pathway is available. Unigene10231 is also identified as an insulin-like growth factor-1 receptor in progesterone-mediated oocyte maturation and oocyte meiosis pathways (Figure S3). Oocyte development can be inhibited by the androgenic glands when cultured in vitro. Unigene10231 was significantly downregulated in SP AG and participated in progesterone-mediated oocyte maturation and oocyte meiosis pathways. We can infer that Unigene10231 may indirectly regulate spermatogenesis and male secondary sexual characteristic development. In addition, whether CL1931 participates in sexual differentiation is unclear, which still needs further study.
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It is reported that a large amount of melatonin can lead to testicular atrophy in hamsters . While melatonin is produced in invertebrates and has influences on the physiology and behavior, little is known about its mechanisms of action . The eyestalks of crustaceans contain the optic lobes and the x-organ/sinus gland, a neuropeptide-secreting neurohemal structure that is analogous to the vertebrate hypothalamus-pituitary system. The concentration of melatonin is high in the eyestalk of crustaceans . The removal of eyestalk induces the hypertrophy of androgenic gland and precocious spermatogenesis in nonbreeding adult males .
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Melatonin acts mainly via high-affinity receptors coupled to heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins). Three high-affinity melatonin receptor subtypes have been cloned so far and classified as MT1a (also called MT1), MT1b (or MT2), and MT1c . The MT1a receptor subtype is believed to mediate major neurobiological functions. In our study, melatonin receptor gene (Unigene23893_All) was significantly downregulated. This means that melatonin has a negative effect on the development of AG and thus influences the development of testis in E. sinensis.
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Dopamine, octopamine, and serotonin are important signal molecules in the nervous systems of all multicellular animals. They are thought to exert hormonal control in a variety of behavioral contexts, including feeding behavior and aggression [60, 61]. Beltz et al. found that the biogenic amines 5-hydroxytryptamine (5-HT) and octopamine (OA) play a significant role in determining mating behavior in the lobster H. americanus [62, 63].
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Serotonin receptors, also known as 5-hydroxytryptamine receptor or 5-HT receptor, are a group of G protein-coupled receptors and ligand-gated ion channels found in the central and peripheral nervous systems. They mediate both excitatory and inhibitory neurotransmission. The serotonin receptors are activated by the neurotransmitter serotonin, which acts as their natural ligand . The serotonin receptors modulate the release of many neurotransmitters and influence various biological and neurological processes such as aggression, anxiety, appetite, cognition, learning, memory, mood, nausea, sleep, and thermoregulation. And great differences exist in 5-HT receptor subtypes; some of them play inhibitory roles, while others play exciting roles. In our study, the 5-HT receptor 1 was significantly downregulated in SP AG, implying that we found that the 5-HT receptor 1 gene negatively regulates the development of AG.
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In crickets, injection of the OA agonist chlordimeform causes normally submissive losers of fights to reengage in fighting faster than sham injected animals . Likewise, in honeybees, injection of two OA agonists, XAMI and DCDM, biases the likelihood of aggressive display toward nonnestmates over nestmates . Hoyer et al. found that aggression is almost abolished in mutant males lacking octopamine in Drosophila by using software to eliminate confounding effects . The expression of octopamine receptor was significantly upregulated in PP AG, indicating that the OA receptor positively regulated the aggression and mating behavior of E. sinensis.
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Barki and colleagues removed AG from juvenile intersex crayfish and observed that in the adult stage they did not fight with other males and did not initiate mating behaviors with females . We can see that AG has played a decisive role in male mating and aggressive behavior in E. sinensis. We found that the expression of octopamine receptor was significantly upregulated while 5-hydroxytryptamine receptor 1 was significantly downregulated in neuroactive ligand-receptor interaction pathway in SP AG (Figure S4). The function implementation of 5-HT and OA was regulated indirectly by AG. Neuroactive ligand-receptor interaction pathway analysis revealed the key regulation mechanism of melatonin, 5-HT, and OA, which have laid the foundation for further research.
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study
| 100.0 |
This is the first comprehensive transcriptome dataset report for the AG of proliferation phase and secretion phase in E. sinensis. A total of 72,000 unigenes and 4,027 DEGs were obtained. Comparative transcriptomic analysis showed that IAG, SXL, TRA-2, SRY, FTZ-F1, FOXL2, and cytochrome p450 might play important roles in maintaining the male physiology and morphology. Ubiquitin, serpin, cathepsin A, and cathepsin D genes may have a direct influence on spermatogenesis. Ribosomes are the most prominent pathway and their related genes might be the key elements in relation to cell growth and secretion. In insulin-like receptor signaling pathway, Unigene10231 may indirectly regulate spermatogenesis and male secondary sexual characteristic development, and whether CL1931 participates in sexual differentiation still needs further study. In neuroactive ligand-receptor interaction pathway, octopamine receptor was significantly upregulated and 5-HT receptor 1 and melatonin receptor were significantly downregulated. This revealed the key regulation mechanism in male mating and aggressive behavior. Our comparative transcriptomic analysis provides new insights into the genome-wide molecular mechanisms of AG development and the regulatory mechanisms of male important traits of E. sinensis.
|
study
| 99.94 |
Figure S1: COG function classification of Eriocheir sinensis androgenic gland transcriptome. Figure S2: Differentially regulated genes involved in insulin signaling pathway. The up-regulated and down-regulated genes are labeled in red and green, respectively. Figure S3: Differentially regulated genes involved in progesterone-mediated oocyte maturation. The up-regulated and down-regulated genes are labeled in red and green, respectively.
|
study
| 99.94 |
The increase in cytokine production caused by Helicobacter pylori has been studied before, including the anti-inflammatory cytokine. This cytokine is responsible for the decrease in immune response. IL-10 is an anti-inflammatory cytokine that is capable of decreasing the inflammatory responses caused by H. pylori, which will cause the rise in bacterial num-bers and the effects mediated by the epithelial cells of the gaster . Patients with chronic gastritis caused by H. pylori infection often develops peptic ulcers, as well as gastric carcinoma and lymphoma .
|
study
| 99.5 |
Matrix metalloproteinases (MMPs) are believed to have an essential role in the inflammation process and carcinogenesis, by causing degradation and remodelling of the extracellular matrix and basal membrane. MMP are secreted through transmembrane endoproteinases. MMP has a catalytic zinc domain, which is required for proteolytic activity. MMP are able to degrade at least one extracellular matrix component. MMP-7 and MMP-9 are members of the MMP family, which increase in H. pylori gastritis and in early gastric carcinoma .
|
study
| 92.56 |
A cross-sectional study was done on seventy consecutive gastritis patients that were admitted to endoscopy units at Adam Malik General Hospital and Permata Bunda Hospital, Medan, Indonesia from May-October 2014. All patients gave informed consent and the study was approved by the local ethical committee. None of the patients had received antibiotics, bismuth compounds, H2 antagonists, proton pump inhibitors or immune modulating drugs within the last four weeks before endoscopy. Patients with evidence of malignancy, immunosuppression, metabolic disorders, or gastrointestinal haemorrhage and patients who had a history of gastric surgery were excluded [2, 5-7].
|
study
| 99.94 |
Gastritis degree was evaluated from a biopsy of the mucosa of gastric antrum and body. The biopsy specimens were fixed in 10% formalin and embedded in paraffin. The samples were stained using Hematoxylin-Eosin and were evaluated by the pathologist of anatomic pathology referring to the visual analogue scale of the updated Sydney System. The higher degree was used if differences of degree were found between the body and antrum. The degree of chronic inflammation, neutrophil infiltration, atrophy, and intestinal metaplasia were scored 0 to 3, i.e., normal (0), mild (1), moderate (2), and severe (3) .
|
study
| 99.94 |
H. pylori were considered positive based on the positive results of the rapid urease test. In this study, we used Pronto Dry®. We used gastric antral biopsy specimen that was taken within 2 cm from the pylorus for Pronto Dry®. The results of the rapid urease test were read within 24 hours. The Pronto Dry® was considered to be positive if the colour changed from amber to pink-red .
|
study
| 100.0 |
Venous blood was drawn using a serum separator tube and allowed to clot for 30-45 minutes at room temperature before centrifugation for 15 minutes at approximately 1,000g. Serum was immediately stored frozen in aliquots at -20°C until assays for IL-10, MMP-7, and MMP-9 were performed. IL-10 was assayed by the high sensitive EBioscience technique, Bender MedSystems GmbH 1030 Vienna, Austria. Circulating MMP-7 and MMP-9 levels were examined in serum using the Quantikine Human MMP-7-ELISA and Quantikine Human MMP-9-ELISA (Quantikine, R&D System, Inc., Minneapolis). Serum levels were expressed as pg/ml. Levels above the mean were categorised as the high level, and levels below the mean were categorised as the low level.
|
study
| 100.0 |
SPSS version 22 (SPSS Inc., Chicago) was used for analysis. The data were analysed using univariate and bivariate analysis with 95% confidence intervals. The results were expressed as the mean ± standard deviation. Bivariate analysis was carried out using the independent t-test, Mann-Whitney test, and Chi-square test with a p-value < 0.05 was considered statistically significant.
|
other
| 52.7 |
There were 70 subjects, consisting of 35 males (50%) and 35 females (50%) subjects. The average age of the subjects was 49.9 ± 13.04 (SD) years. Most subjects were from the age group of 46-60 years old. 40% patients were infected with H. pylori. The occupation of most of the respondents was housewives (38.57%). The majority of subjects had an overweight or underweight nutritional status (58.57%) (Table 1).
|
study
| 100.0 |
There was no correlation found between IL-10 serum levels with the degree of chronic inflammation or neutrophil infiltration. There was no correlation found between MMP-7 serum levels with the degree of chronic inflammation or neutrophil infiltration. The same results were found for MMP-9 serum levels. There was no correlation found between MMP-9 serum levels with the degree of chronic inflammation or neutrophil infiltration (Table 3).
|
study
| 100.0 |
The average age of the subjects was 49.9 ± 13.04 (SD) years, which concludes that most of the gastritis patients are at their productive ages. The results of this study are similar to a study conducted by Garg B, et al, which reported that the average age of gastritis patients was 47 years old and the study reported by Mustapha SK, et al with an average age of 47 years old [7, 8].
|
study
| 99.94 |
In this study, a group of gastritis patients without major gastric disease (example: peptic ulcer and gastric carcinoma) were studied with the objective to examine the presence of H. pylori infection in gastritis patients. It is known that H. pylori are correlated with increased expressions of IL-10, MMP-7, and MMP-9 in the mucosa .
|
study
| 100.0 |
However, studies on the levels of IL-10, MMP-7 and MMP-9 in the serum are still limited and controversial. Owing to that, this study was conducted with the objective to find the differences between the serum levels of IL-10, MMP-7 and MMP-9 in patients with positive and negative H. pylori, and to analyse the correlations between the serum levels and the degree of gastritis based on histopathology appearance.
|
study
| 99.94 |
IL-10 as an anti-inflammatory cytokine has a role in the initial immune response which is mediated by B cell. Cytokine IL-10 is involved in the decrease of the inflammatory response . IL-10 inhibits the synthesis of IFN-c, IL-1, IL-6, IL-8 and TNF-α, and also acts as a feedback mechanism in reducing these cytokines [13, 14]. IL-10 might contribute to the failure of the immune response to clear H. pylori infection, and in a previous study we found the increased secretion of IL-10 in biopsy specimens in H. pylori infection, with the secretion of the cytokine correlating with the degree of chronic inflammation. Yamaoka et al have reported increased expression of IL-10 mRNA in biopsies from infected patients .
|
study
| 100.0 |
IL-10 helps to maintain bacterial colonisation and exerts its effects directly on gastric epithelial cells [2, 10, 11]. Goll et al reported that samples from H. pylori-positive patients showed increased production of IL-10 as much as 6.7 times the production of patients with negative H. pylori.
|
study
| 99.94 |
In this study, the serum levels of IL-10 increased significantly in positive H. pylori compared to negative H. pylori patients. This result was similar to that reported by Dlugovittzky et al, where the IL-10 serum levels of the positive H. pylori subjects were higher compared to negative H. pylori subjects (p<0.01) . However, other studies reported different conclusions, In the studies performed by Russo et al and Bayraktaroglu et al, they reported that there were no significant correlations between the positive H. pylori subjects and negative subjects, which might be caused by inhibition of further inflammation [17, 18].
|
study
| 99.94 |
H. pylori is able to trigger the expression of MMP, which is a proteolytic enzyme that has a role in maintaining and remodelling the interaction between epithelial cells and the basal membrane [19, 20]. A study, conducted by Bebb JR et al, found that gastric biopsy specimens from a positive H. pylori subject will show in a higher level of MMP-7 protein in the antrum and corpus . A similar result is found from the study conducted by Wroblewski et al, which concluded that the MMP-7 expression in the antrum and corpus increased with the presence of H. pylori .
|
study
| 99.7 |
In this study, there were no significant differences in MMP-7 serum levels between positive and negative H. pylori subjects. The same results were found in the study by Rautelin et al, which concluded that there were no significant differences in MMP-7 serum levels between positive and negative H. pylori gastritis subjects .
|
study
| 99.94 |
Studies by Rautelin HI et al and Li SL et al found that MMP-9 level increased significantly in the gastric mucosa of gastritis patients with positive H. pylori compared to the negative [22, 23]. In a study conducted by Oliviera, it was found that H. pylori through a T4SS (Type IV Secretion System) pathway increases the activity of MMP-9 as a response to the invasion of H. pylori on the gastric epithelium .
|
study
| 99.8 |
In this study, there was no difference in the serum levels of MMP-9 between the positive and negative H. pylori patients. Similar results were also found in the study reported by Rautelin et al and Ettehad et al, where it was concluded that there were no significant differences in the MMP-9 levels of the gastritis patients between the positive and negative H. pylori subjects [25, 26].
|
study
| 99.94 |
In conclusion, the IL-10 serum levels increased significantly in positive H. pylori subjects. There was no significant difference in the average serum levels of MMP-7 and MMP-9 between positive and negative infected H. pylori patients. There were no correlations found between serum levels of IL-10, MMP-7, MMP-9 with the degree of gastritis based on histopathology.
|
study
| 100.0 |
For more than 20 years, the incidence of thyroid cancer has steadily increased globally , exhibiting the most rapid rise of all major human cancer types. There is a strong suggestion that it is the increased detection of papillary carcinomas (PTCs), especially small PTCs, that is primarily responsible for this rising incidence . Cancer of the thyroid gland usually presents as a nodule, and thyroid nodules may either represent the presenting complaint or be an incidental finding. Fortunately, the majority (95%) of all thyroid nodules are benign. Nonetheless, thyroid nodule detection raises concerns regarding the possibility of an underlying malignancy and usually requires further investigation. The gold standard for thyroid nodule investigation is fine needle aspiration biopsy (FNAB). It is a critically important diagnostic method for distinguishing benign from malignant nodules and guiding their further treatment . In 2007, in order to reduce variation between pathologists in the reporting of cytopathological findings, and also to standardize diagnostic terminology, the National Cancer Institute held a consensus conference and developed recommendations for standardized reporting of thyroid cytopathological diagnoses. Having been named after the location of this conference, these thyroid cytopathology reporting guidelines have become known as the Bethesda System for Reporting Thyroid Cytopathology (BSRTC) . As stated in the 2015 American Thyroid Association (ATA) Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer, it is now a strong recommendation that all centers utilize the BSRTC for the reporting of thyroid nodule cytopathology . The BSRTC is a six-tiered classification system, with each diagnostic category having a specific associated risk of malignancy (ROM), as well as recommended management. These six BSRTC diagnostic categories are: unsatisfactory, benign, atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS), suspicious for follicular neoplasm/follicular neoplasm (sFN), suspicious for malignancy (sM), and malignant. The quoted ROM for each of these six BSRTC diagnostic categories are: 1%–4%, 0%–3%, 5%–15%, 15%–30%, 60%–75% and 97%–99%, respectively .
|
review
| 99.9 |
The most challenging BSRTC diagnostic categories for the clinician to manage are the three indeterminate categories (AUS/FLUS, sFN and sM). Due to an associated low cancer risk, it is the AUS/FLUS diagnostic group that is especially difficult to manage. Despite the recommendations that AUS/FLUS be used as a ‘last resort’ diagnostic category, and that it should represent no greater than 7% of all BSRTC diagnoses at a particular institution , there is considerable variability in its utilization at different centers, with reporting rates that range from 0.8% to 27.12% . Several studies have also reported a higher ROM for the AUS/FLUS diagnostic category than has been previously suggested [3, 7]. In the current ATA guidelines, for thyroid nodules diagnosed as AUS/FLUS, in addition to a repeat FNAB, a multidisciplinary approach that combines clinical, sonographic and molecular characteristics, is now recommended . While many of the variables that are taken into account to assist with making clinical decisions are subjected to confounding influences, patient variables such as age and gender remain consistent.
|
review
| 99.9 |
Thyroid cancer is more commonly diagnosed in younger people and in women . Unlike most other human cancer types, age represents an important disease prognosticator for individuals diagnosed with differentiated thyroid cancer (DTC). In the latest American Joint Commission on Cancer (AJCC) thyroid cancer staging system , cancer in patients 55 years and older is generally associated with a more aggressive clinical course and worse prognosis. Very recently, following large-scale studies, the 45-year age cut-off for cancer staging, and thus disease prognostication for differentiated thyroid cancers has been raised to 55 years . Studies have shown that this older age cut-off improves the accuracy of the prognostication system, and therefore should help prevent overtreatment of patients. The objective of this study was to review the distribution of the BSRTC diagnostic categories at our institution and to determine if patient age or gender significantly modifies the risk of malignancy in the indeterminate (AUS/FLUS, sFN, and sM) BSRTC diagnostic groups.
|
study
| 99.8 |
A retrospective chart and pathology review of consecutive patients at our center that underwent thyroid nodule FNAB and a subsequent thyroid operation was carried out. The final histopathological diagnoses of the thyroidectomy specimens were considered to be the gold standard for nodule diagnosis. To ensure consistency within the study with respect to cytopathological and histopathological evaluations, all cases that underwent thyroid FNAB and/or operation at other institutions were excluded from the study population. All cases of papillary thyroid microcarcinoma (defined as papillary carcinoma smaller than 1 cm in maximum diameter) that were not an index lesion of interest, and thus were incidentally diagnosed by histopathology, were grouped with the benign cases for the analysis. The study population was then grouped according to age (younger than 55 years or 55 years and older) and gender. The cytology slides were evaluated by one or more board-certified pathologists at our center. The FNABs were classified based upon their BSRTC diagnoses, and they were reported by the pathologists as being either unsatisfactory, benign, AUS/FLUS, sFN, sM, or malignant. For cases that underwent multiple FNABs in the current study, the diagnosis of AUS/FLUS was based upon the FNAB that most immediately preceded the thyroid operation. Though influenced by clinical factors, it is common practice at our institution to repeat the FNAB if the initial diagnosis is either unsatisfactory or AUS/FLUS. All cases not reported according to the BSRTC were excluded from the study population. The characteristics of the study population are summarized in Table 1. A ROM for each BSRTC diagnostic category was calculated after grouping cases by either their age or gender. Statistical analyses that compared the ROM between categories were calculated using the Fisher exact test. A p-value less than 0.05 was considered to be statistically significant.
|
study
| 100.0 |
A total of 3307 thyroid FNABs were performed and interpreted at our institution between May 2010 and December 2014. Most of the FNABs were interpreted as being either benign (62.1%) or unsatisfactory (23.0%). There were 283 (8.6%) AUS/FLUS cases diagnosed, 95 (2.8%) sFN cases diagnosed, 54 sM cases diagnosed (1.6%), and 60 cancers (2.2%) diagnosed. The overall proportion of the study population with an indeterminate BSRTC diagnosis was 11.1%. Of the total number of individuals undergoing FNAB at our center, 291/3307 (8.8%) also underwent a thyroid operation at our center. Of these 291 cases, 15 (5.2%) FNABs were unsatisfactory, 76 (26.1%) were benign, 65 (22.3%) were AUS/FLUS, 54 (18.6 %) were sFN, 22 (7.5%) were sM and 59 (20.3%) were malignant (Table 1). Of the 65 AUS/FLUS cases, 11 underwent repeat FNAB. Nine of the repeat FNABs were again diagnosed as AUS/FLUS, and two were benign. Overall, of the 291 operated cases, 113 were diagnosed as cancer by histopathology, making the overall rate of malignancy in the study population 38.8%. The majority of the thyroid cancer cases were diagnosed by final histopathology as being PTC (101/113, 89.4%) and the remaining 12 (10.6%) cases were follicular carcinomas (FTC). Of the PTC cases 11 (10.9%) were subcategorized as the follicular variant of PTC. During the study period, there were two anaplastic carcinoma cases and that were diagnosed by FNAB. These cases were excluded from the study population in order to eliminate a potential source of bias, as they were all readily diagnosed by preoperative cytological assessment.
|
study
| 99.94 |
In our study population there were 227 females and 64 males, and the mean study patient age was 50 years (range 20–83 years). There were 79 of 225 females (35%) who had a cancer, as diagnosed by histopathology. The mean age for females was 50 years (range 20–83 years), and for males was also 50 years (range 23–80 years). The rate of malignancy for each BSRTC category in females was; 14.2%, 3.2 %, 26.4 %, 19.5 %, 81.3% and 100% for non-diagnostic, benign, AUS/FLUS, sFN, sM, and malignant diagnoses, respectively (Table 2). There were 34 of 64 males (53.1%) who had a cancer diagnosed by postoperative histopathology. The rate of malignancy for each BSRTC diagnostic category in males was 0%, 7.7%, 25%, 46.2%, 66.7% and 95% for non-diagnostic, benign, AUS/FLUS, sFN, sM and malignant diagnoses, respectively (Table 2). Even though thyroid cancer was significantly more common in males (p=0.021), gender did not significantly influence specific BRSTC diagnostic category cancer risk estimation (Table 2).
|
study
| 100.0 |
Of the 291 patients that made up our study population 178 were younger than 55 years (45.5%) and 113 were 55 years or older (26.5%) (Table 3). The risk of malignancy in the non-diagnostic category was 20% for patients younger than 55 years and 0% for older patients. In the benign category, the risk of malignancy for the younger category was 5.6% vs 2.5% in the older category. In the AUS/FLUS category, the risk of malignancy was 36.8% in the younger category vs 7.4% in the older category. In the suspicious for follicular neoplasm category, the risk of malignancy in the younger group was 14.3% vs 42.1% in the older group. In the suspicious for malignancy category, the risk of malignancy was 92.3% in the younger group and 77.8% in the older group. In the malignant category, the risk of malignancy in the younger group was 100% vs. 92.3% in the older group. The overall risk of malignancy for patients younger than 55 years was 45.5%, and for patients 55 years or older was 26.5%. The difference in risk of malignancy between these two age groupings was statistically significant (p=0.0013). The malignancy risk was higher in the group of patients younger than 55 years. For the AUS/FLUS BSRTC diagnostic group, the cancer risk was significantly higher for patients younger than 55 years (p=0.0082) (Table 3).
|
study
| 99.94 |
At our Canadian center, over 700 thyroid FNABs are carried out and evaluated annually. Fortunately, most of the diagnoses are benign, which is consistent with reports from other institutions [4, 9]. Since the implementation and routine utilization of the BSRTC at our center in 2010, a subset of the total FNABs (13.6%) has been diagnosed as being indeterminate (AUS/FLUS or sFN or sM). Of these cases, our overall AUS/FLUS utilization rate was 8.3%, which is marginally higher than the recommended 7% utilization for this BSRTC diagnostic category . An especially challenging diagnosis, AUS/FLUS has a wide range of variability in utilization across institutions, and it has been suggested that the ROM associated with this diagnostic category is actually higher than the quoted 5-15%, being more in the 20-25% range [3-4, 7]. A meta-analysis reported by our group found that for 291 patients with thyroid lesions that underwent operation, the overall ROM for an AUS/FLUS diagnosis was 26.8% .
|
study
| 99.94 |
The clinical management of the AUS/FLUS diagnostic group has represented an ongoing challenge. Even though many cases with an AUS/FLUS BSRTC diagnosis undergo a repeat FNAB, there has also been much research that has focused on the application of adjunctive molecular testing for facilitating appropriate clinical management. Such strategies add significant complexity and cost to patient management. However, age is an easily accessible patient characteristic that has received little attention in the literature with regards to its influence on patient ROM within the BSRTC diagnostic categories. In the current study, all patients were stratified into two groups, those younger than 55 years, and those 55 years or older, a cut-off point we selected because it also represents an important thyroid cancer clinical prognosticator that has been recently incorporated into the eighth edition of the American Joint Committee on Cancer (AJCC) Differentiated Thyroid Cancer Staging System . We observed a statistically significant difference in the ROM in patients with an AUS/FLUS diagnosis who were younger than 55 years, with the ROM being 36.8% in the younger group, compared to 7.4% in the older group. This suggests that thyroid nodules with an AUS/FLUS diagnosis, although being more common and generally having a worse prognosis when diagnosed as malignancy in older people, tend to have a higher risk of actually being diagnosed as cancer when detected in younger people. Other groups have also made similar observations . Thus, when utilizing the BSRTC, patient age should be taken into account in order to facilitate a more accurate estimate of their actual ROM. Recently, when used in conjunction with other clinical parameters such as nodule size, and ultrasound characteristics, patient age has been found to be a predictive factor for malignancy in patients younger than 65 years .
|
study
| 99.94 |
With regards to gender, although we observed a significantly higher overall cancer risk in males, similar to observations made by other groups [7, 11], our study population is relatively small (291 cases) limiting our ability to study its interaction with age, though further investigation seems warranted.
|
study
| 100.0 |
The limitations of our study include a selection bias that exists because only the cases that went on to undergo an operation at our center were included in the study population. This leads to a higher than expected ROMs in the unsatisfactory, benign and AUS/FLUS groups, when compared to their expected BSRTC ROMs . Surgically resected AUS/FLUS nodules have actually been reported to have a ROM that ranges between 6% and 48% as highlighted in the current ATA guidelines. Similarly, a meta-analysis that was reported by our group compared 13 different centers following the implementation of the BSRTC and reported a ROM for AUS/FLUS nodules to be ranging between 19–38% .
|
study
| 100.0 |
Despite FNAB being the critical diagnostic test for thyroid nodules, decisions regarding the extent of thyroid surgery are also influenced by many other clinical factors. With regards to individual BSRTC diagnostic categories, the false negative rate in the benign category was low, and only marginally higher than the expected BSRTC ROM. The false negatives in this group were due to a follicular carcinoma case and a single follicular variant of PTC case. With respect to the AUS/FLUS diagnostic group, five of 17 (29.4%) cancer cases were diagnosed as follicular variant of PTC. In the malignant diagnostic group, 58/59 cases (98%) were malignant by final histopathological assessment. The single case within this group that had a benign pathological diagnosis was described as a ‘follicular adenoma with cytological atypia’ and this diagnosis was confirmed by an external pathology review. Despite the overlap in observations made across several studies, there has been the suggestion that the risk of malignancy in the AUS/FLUS category may be institution-specific due to variability in its utilization and application by cytopathologists . As the majority of thyroid lesions do not undergo an operation, there is really no consistent way to confirm whether or not many of the FNAB diagnoses were actually accurate. This is also true for the recently described benign pathological diagnosis: non-invasive follicular tumor with PTC-like nuclear features (NIFTP). Though reclassifying some of our malignant cases into the NIFTP category would decrease the ROM across all BSRTC groups, NIFTP is a histopathological diagnosis, and would not assist with cytology-based preoperative decision making. However, given efforts to clearly define NIFTP diagnostic criteria, along with its utilization in clinical practice, it is probable that the overall risks of malignancy across all BSRTC categories will decrease. Furthermore, a study limitation arises because some of the cases, especially those with an unsatisfactory, benign or AUS/FLUS BSRTC diagnosis may only be followed clinically, and because they did not undergo an operation, they are not included in our study population. In real-world practice, patient characteristics such as a history of prior neck radiation exposure, or a familial thyroid cancer history, influence the decision to operate and the extent of surgery, and this information is usually not available to reporting cytopathologists. Certainly, study of these variables, in combination with the BSRTC diagnosis, could potentially further augment cancer ROM estimation, and influence treatment.
|
study
| 99.94 |
In the current study, we found the ROM to be significantly higher in the AUS/FLUS category for patients younger than 55 years of age. However, the ROM presented across many studies, including our own, depends heavily upon the prevalence of thyroid cancer in the specific patient population being studied . Thus, it is of paramount importance for each institution that employs the BSRTC to guide clinical decision making to monitor its usage and to be aware of their ROM for each individual diagnostic category .
|
study
| 99.94 |
As our observations have demonstrated, the ROM for the AUS/FLUS BSRTC category is closer to the ROM of the sFN category in patients younger than 55 years. From a clinical perspective, these observations suggest that it may be appropriate for patients younger than 55 years of age with an AUS/FLUS diagnosis to be offered a diagnostic thyroid lobectomy, as opposed to observation or to a repeat FNAB, as recommended by the BSRTC. Irrespective of potential developments in this field, we believe that further study of the relationship between patient age, gender, and ROM within the BSRTC indeterminate diagnostic groups is warranted, and ultimately may lead to improved cancer risk estimation, and thus, better tailoring of treatment for individuals diagnosed with thyroid nodules.
|
study
| 99.94 |
Gene-environment (G×E) interactions may contribute to complex diseases, but their detection has proven challenging; hence, a variety of approaches have been developed to enhance power. Most G×E analyses focus on loci that are strong biological candidates or those with highly significant marginal effects . The latter approach is attractive because these loci are available in many large cohorts, and can be conveniently followed-up with interaction analyses if environmental data are accessible. Moreover, selecting SNPs with strong and reproducible marginal effect signals is a pragmatic data-reduction step that may improve power , although this approach risks omitting other promising candidates .
|
review
| 99.7 |
In a linear regression setting, the presence of interaction effects drives phenotypic variance heterogeneity by genotype . Exploiting variance heterogeneity as a signature of interactions is appealing because, unlike standard approaches for assessing G×E interactions, no explicit information about environmental exposures is needed and multiple exposures can be simultaneously considered.
|
study
| 99.94 |
Here we explored whether loci identified in large-scale genome-wide association studies (GWAS) of blood lipids and body mass index (BMI) are strong candidates for G×E interactions by comparing genome-wide variance heterogeneity P-value distributions generated using Levene’s test against P-value distributions for marginal effects and explicit G×E interaction effects (for smoking and physical activity).
|
study
| 100.0 |
We assessed between-genotype variance heterogeneity for up to 1,927,671 directly genotyped or imputed SNPs (HapMap II CEU reference panel ) that passed quality control (QC). Meta-analyses of Levene’s test summary statistics were performed for BMI (n≤44,211 participants), and blood concentrations of high-density lipoprotein cholesterol (HDL-C) (n≤34,315), low-density lipoprotein cholesterol (LDL-C) (n≤34,180), total cholesterol (TC) (n≤34,318) and triglycerides (TG) (n≤34,110). We then obtained marginal effects results for the same index traits and SNPs from publicly available GWAS summary data from the GIANT (Genetic Investigation of ANthropometric Traits) Consortium and GLGC (Global Lipids Genetics Consortium) .
|
study
| 100.0 |
We compared the genome-wide marginal effects with between-genotype variance heterogeneity results for each of the five cardiometabolic traits by calculating the association between marginal effects (Pm) and variance heterogeneity (Pv) P-values using the rank-based Spearman correlation (ρ). This was done using a set of 42,710 pruned SNPs produced using the--indep-pairwise command in PLINK (see Materials and Methods) to account for linkage disequilibrium (LD) among variants.
|
study
| 100.0 |
As shown in Table 1 (see also Fig 1A and S1 Table), the Spearman’s ρ for the association between Pm and Pv for all pruned SNPs was of very small magnitude and only statistically significant for BMI. The exclusion of SNPs based on progressively more conservative Pm thresholds (Pm<0.05; Pm<10−4; previously established loci with Pm<5×10−8 in external datasets), saw corresponding improvements in the magnitude of these correlations, which were statistically significant for all traits except TC when focusing on previously established loci. The BMI correlation at the Pm<0.05 threshold, as well as the test of equality with ρ for all SNPs, was statistically significant, suggesting concordance between marginal and variance signals at a nominal level of significance. The odds ratio (OR) for a SNP to have both Pm<0.05 and Pv<0.05 as compared to Pv≥0.05 was 1.33 (95% CI: 1.12, 1.57) for BMI while the 95% CIs of ORs for other traits included 1. On the other hand, the P-value for a non-zero ρ for TG was statistically significant when focusing on the established loci and at Pm<10−4, suggesting concordance between marginal and variance signals at more conservative Pm thresholds.
|
study
| 100.0 |
A. Percentile-scaled ranks of GWAS-derived SNPs for lipid traits on the genome-wide distribution of P-values from Levene’s meta-analysis. For each lipid trait (HDL-C, LDL-C, TG and TC on the vertical axis) we ranked Pv from Levene’s test for all SNPs from lowest to highest so that the lowest Pv for a given trait was assigned a rank equal to 1. We scaled ranks into percentiles such that the lowest Pv corresponded to the 100th percentile. We then plotted percentile-scaled ranks of GWAS-derived loci (black sticks on the blue axis) on the distribution of percentile-scaled ranks of genome-wide Pv (blue axis) for each trait and marked in red loci with Pv<0.05. Loci names are presented above the axis for Pv distribution of a given trait and are positioned in the same order as percentile-scaled ranks of GWAS-derived loci, but are equally spaced to facilitate cross-trait comparison (loci names with Levene’s test Pv<0.05 are highlighted in red). To the left of each axis we present counts of GWAS-derived loci with Pv<0.05 and total number of GWAS-derived loci in the analysis separated by a dash, as well as the P-value for the binomial test (Pbinomial). B. Percentile-scaled ranks of GWAS-derived SNPs for BMI on the genome-wide distribution of P-values obtained from Levene’s test (Pv) and between-strata difference test P-values (Pint) from the ‘SNP × Physical Activity’ and ‘SNP × Smoking’ interaction tests for BMI. For each analysis, we ranked P-values for all SNPs from lowest to highest so that the lowest P-value for a given trait was assigned a rank equal to 1. We scaled ranks into percentiles such that the lowest P-value corresponded to the 100th percentile. We then plotted percentile-scaled ranks of GWAS-derived loci (black sticks on the blue axis) on the distribution of percentile-scaled ranks of genome-wide P-values (blue axis) from all four approaches and marked in red loci with Pv<0.05 or Pint<0.05 (or 95th percentile for average rank between SNP × PA and SNP × Smoking). Loci names are presented above the axis for the P-value distribution of a given trait and are positioned in the same order as the percentile-scaled ranks of GWAS-derived loci, but are equally spaced to facilitate cross-trait comparisons (loci names with Pv<0.05 or Pint<0.05 are highlighted in red). To the left of each axis conveying each respective P-value distribution, we present counts of GWAS-derived BMI loci with Pv<0.05 or Pint<0.05 (or 95th percentile for the average rank of the SNP × PA and SNP × Smoking interaction tests) and the total number of GWAS-derived loci in the analysis separated by a dash, as well as the P-value for the binomial test (Pbinomial).
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We further compared Pm with interaction P-values from exposure-specific (smoking and physical activity) genome-wide interaction tests for BMI (Pint); this was only done for BMI owing to the requirement for an adequately powered external dataset (such a dataset was accessible through the GIANT consortium) (Table 2). Marginal effects GWAS were performed by strata of smokers vs. non-smokers and physically active vs. inactive participants (n = 210,316 European-ancestry adults ) respectively, and a heterogeneity test was used to generate exposure specific Pint distributions. Spearman ρ for the pruned set of SNPs in the SNP × physical activity and the SNP × smoking analyses were low and not statistically significant (Table 2). We also compared Pint values and Pv values for BMI. Spearman’s ρ for the pruned set of SNPs were low and not statistically significant.
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We next tested if the number of previously established marginal effect SNPs (Pm<5×10−8) that were also nominally significant (Pv<0.05) for variance heterogeneity was greater than expected by chance (Tables 3 and 4, Fig 1). For 4 out of the 5 index traits, we observed enrichment at the lower end of the Pv distribution (Pv<0.05) for the established GWAS-derived lead SNPs. Thus, the nominally significant regions of the Pv distributions were generally enriched for GWAS-derived loci.
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We also performed enrichment analyses to test if previously established marginal effects SNPs (Pm<5×10−8) are enriched for nominally significant (Pint<0.05) interactions in the SNP × physical activity or SNP × Smoking analyses, but no enrichment was observed (Table 3; Fig 1B). By contrast, for the physical activity and smoking interaction tests (using all pruned SNPs), the lower end of the Pint distribution (Pint<0.05) was enriched with SNPs that were nominally significant in the Levene’s test analysis (Pv<0.05) (Table 4). This enrichment translated into an OR of 1.08 (95% CI: 1.01, 1.14) for a SNP to have Pint<0.05 given Pv<0.05 vs. Pv≥0.05 for SNP × physical activity interaction. The corresponding OR for the SNP × smoking interaction test was not significant (OR = 1.02; 95% CI: 0.96, 1.08).
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Finally, in the pruned SNP-set we used the Mann–Whitney U test to probe for systematic differences in Pv and Pm ranks. P-values were ordered from least significant to most significant, and the lowest 100th centile (i.e. the most significantly associated SNPs) was compared to the remaining 99th percentile for each of the five traits. For BMI, SNPs in the lowest 100th centile of the Pm distribution had markedly higher Pv ranks (i.e. more significant Pv) than the remaining SNPs (PMann–Whitney = 1.46×10−5; Table 5). Even when excluding previously established lead SNPs (Pm<5×10−8) for BMI (or SNPs +/-500kb proximal), SNPs from the lowest 100th centile of the Pm rank-ordered distribution had higher Pv ranks than the remaining SNPs (PMann–Whitney = 4.30×10−4; Table 5). Conversely, no difference in Pv ranks was observed for SNPs from the lowest 100th centile of the Pm rank-ordered distribution for the four blood lipid traits; this may reflect trait-specific G×E effects or differences in statistical power by trait. No differences in Pv ranks between SNPs from the lowest 99th centile of the Pm rank-ordered distribution compared to SNPs from the 98th to 1st centiles of the distribution were observed for any trait (PMann–Whitney>0.05; Table 5). Similarly, no difference in Pm ranks was observed for SNPs from the lowest 100th centile of the Pv rank-ordered distribution for any traits (PMann–Whitney>0.05; Table 6).
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BMI: body mass index; HDL-C: low-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; SNP: single nucleotide polymorphism; TC: total cholesterol; TG: triglycerides; Pv: Variance (Levene’s) test P-value; Pm: marginal (linear regression) test P-value
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other
| 99.94 |
BMI: body mass index; HDL-C: low-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; SNP: single nucleotide polymorphism; TC: total cholesterol; TG: triglycerides; Pv: Variance (Levene’s) test P-value; Pm: marginal (linear regression) test P-value
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other
| 99.94 |
To assess whether a trait with a non-normal distribution (e.g. BMI) or strong marginal associations could cause spurious association between the marginal and variance signals, we recapitulated the analysis pipeline (correlation analysis, enrichment analysis, comparisons of rank Pm and Pv values) in simulations described in the Materials and Methods. Careful assessment of results emanating from these simulations did not reveal evidence of type I error inflation caused by the non-normal distribution of an outcome trait nor strong marginal effects. For instance, we extracted correlation P-values of Pm, Pv and Pint generated from 5,000 simulations. QQ-plots of the 5,000 correlation P-values, 2,500 binomial P-values, and 2,500 Mann-Whitney U test P-values revealed no inflation (S1A–S1C Fig, S2A and S2B Fig and S3A and S3B Fig, respectively). Repeating these analyses on subsets of SNPs with low Pm values did not materially change the results.
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Collectively, our analyses highlight a few variants with genome-wide significant marginal effects that may be strong candidates for G×E interactions owing to their strong concurrent variance heterogeneity P-values. For BMI, such SNPs are also overrepresented in the nominally significant part of the Pv distribution. FTO is an excellent example, as it conveys strong marginal effects , exhibits high between-genotype heterogeneity here (Tables 2 and 3 and Fig 1B) and elsewhere , and reportedly interacts with physical activity, diet and other lifestyle exposures and is associated with macronutrient intake .
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Although variance heterogeneity tests are potentially powerful screening tools for G×E interactions, like most interaction tests, they may be bias prone. For example, apparent differences in phenotypic variances across genotypes may be caused by scaling, particularly when the phenotypic means also differ substantially , such that the per-genotype means and variances for index traits are correlated. However, where necessary we transformed variables, and the correlations between Pm and Pv were generally weak, excluding this as a likely source of bias. Using simulated data, we investigated whether the non-normal distribution of a trait can cause a spurious association between marginal and variance signals, which we show is highly improbable. Through further simulations, we assessed whether SNPs with large marginal effects inflate Pv, but observed no inflation, indicating that large genetic marginal effects do not artificially inflate variance heterogeneity to a meaningful extent, and SNPs with low Pm and low Pv-values are thus likely to be strong candidates for G×E interactions, at least in the case of BMI. It might also be that combining populations from ancestral (e.g., hunter-gatherers) and contemporary environments increases variance heterogeneity owing to diversity in population substructure rather than G×E interactions per se . However, this seems unlikely here, as the cohorts examined are from Westernized European-ancestry populations.
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There are several additional explanations for between-genotype variance heterogeneity, such as variance misclassification that can occur when the index variant is located within a haplotype containing rare functional variants that convey strong marginal effects . Hence, although variance heterogeneity tests represent a useful data-reduction step, before conclusions are drawn about the presence or absence of G×E interactions, index variants should be validated by testing their interactions with explicit environmental exposures, as we did here with smoking and physical activity. However, genome-wide G×E interactions datasets are not comprised of functionally validated G×E interactions, as no such resource is currently available for human complex traits. This limitation inhibits the extent to which causal effects can be attributed to the top-ranking loci and their interactions with smoking or physical activity.
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| 100.0 |
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