Transcriptomic Insights and the Development of Microsatellite Markers to Assess Genetic Diversity in the Broodstock Management of Litopenaeus stylirostris.

The Pacific blue shrimp (Litopenaeus stylirostris) is a premium product in the international seafood market. However, intensified farming has increased disease incidence and reduced genetic diversity. In this study, we developed a transcriptome database for L. stylirostris and mined microsatellite markers to analyze their genetic diversity. Using the Illumina HiSeq 4000 platform, we identified 53,263 unigenes from muscle, hepatopancreas, the intestine, and lymphoid tissues. Microsatellite analysis identified 36,415 markers from 18,657 unigenes, predominantly dinucleotide repeats. Functional annotation highlighted key disease resistance pathways and enriched categories. The screening and PCR testing of 42 transcriptome-based and 58 literature-based markers identified 40 with successful amplification. The genotyping of 200 broodstock samples revealed that Na, Ho, He, PIC, and FIS values were 3, 0.54 ± 0.05, 0.43 ± 0.09, 0.41 ± 0.22, and 0.17 ± 0.27, respectively, indicating moderate genetic variability and significant inbreeding. Four universal microsatellite markers (CL1472.Contig13, CL517.Contig2, Unigene5692, and Unigene7147) were identified for precise diversity analysis in Pacific blue, Pacific white (Litopenaeus vannamei), and black tiger shrimps (Penaeus monodon). The transcriptome database supports the development of markers and functional gene analysis for selective breeding programs. Our findings underscore the need for an appropriate genetic management system to mitigate inbreeding depression, reduce disease susceptibility, and preserve genetic diversity in farmed shrimp populations.

Washout CYFRA 21-1: A tool to improve diagnostic accuracy of fine needle aspiration in the diagnosis of metastatic lymph nodes in papillary thyroid cancer.

Thyroid carcinoma has an increasing incidence of endocrine system cancers. Fine needle aspiration cytology (FNAC) and thyroglobulin (Tg) are the primary diagnostic modalities employed for assessing metastatic lymph nodes (LNs) in thyroid cancer. Due to the limited accuracy, rare patients benefited from these procedures. In this research, we aimed to discover a dependable biomarker that could increase the accuracy of FNAC's ability to diagnose metastatic LNs among patients suffering from papillary thyroid cancer (PTC). From March 2021 to July 2023, 99 LNs from PTC patients who had thyroid ultrasonography suspicions of metastases were examined. All patients underwent FNAC, washout Tg and CYFRA 21-1 measurements. Surgical histology and a subsequent FNAC were utilized to validate the outcomes of LNs. In our study, the optimal cut-off value for CYFRA 21-1 washout fluid was 1.145 ng/mL, with a specificity of 94.00 % (slightly lower than Tg and FNAC at 98 %). However, CYFRA 21-1 demonstrated significantly higher diagnostic sensitivity (85.71 %) and accuracy (86.41 %) compared to Tg (71.43 %, 81.55 %) and FNAC (69.39 %, 80.58 %). Furthermore, FNAC plus washout CYFRA 21-1 performed better in diagnosing the metastatic LNs in PTC than FNAC plus Tg, which may indicate a novel solution for metastatic LNs diagnosis in PTC.

Establishing an astaxanthin-rich live feed strain of Pseudodiaptomus annandalei.

This study aimed to establish an astaxanthin-rich strain of the calanoid copepod Pseudodiaptomus annandalei, through selective breeding based on RGB (red, green and blue) value, a parameter indicating color intensity. We evaluated the RGB value frequency distributions of the copepod populations, and selected individuals with the highest 10% and the lowest 10% RGB value over six generations. The RGB value, nauplii production, clutch interval and clutch number were assessed, and the genetic gain was calculated across generations (G0-G5). Two strains of copepods were selected and defined as dark body copepod strain (DBS) and light body copepod strain (LBS) at the end of experiment. Results revealed significantly lower RGB values (male: 121.5 ± 14.1; female: 108.8 ± 15) in the G5 DBS population compared to the G0 (male: 163.9 ± 13.1; female: 162.2 ± 14.6), with higher genetic gains of RGB values during G0 to G2. While DBS females exhibited longer clutch intervals in the G3 and G4, there was no significant difference in nauplii production between the two strains across all generations. Significantly higher astaxanthin content was found in the DBS copepods (0.04 µg/ ind.) compared to the LBS copepods (0.01 µg/ ind.) and the non-selective copepods (0.02 µg/ ind.) 20 months post selective breeding, validating the stability of the desired trait in the DBS strain. This study successfully established an astaxanthin-rich strain of P. annandalei, which provides implications for enhancing marine and brackish larviculture production.

Renal-clearable nanoprobes for optical imaging and early diagnosis of diseases.

Optical imaging has played an indispensable role in clinical diagnostics and fundamental biomedical research due to its high sensitivity, high spatiotemporal resolution, cost-effectiveness, and easy accessibility. However, the issues of light scattering and low tissue penetration make them effective only for superficial imaging. To overcome these issues, renal-clearable optical nanoprobes have recently emerged, which are activated by abnormal disease-associated biomarkers and initiate a pharmacokinetic switch by undergoing degradation and eventually releasing signal reporters into urine, for simple imaging and sensitive optical in vitro urinalysis. In this review, we focus on the advancements of renal-clearable organic nanoprobes for optical imaging and remote urinalysis. The versatile design strategies of these nanoprobes are discussed along with their sensing mechanisms toward biomolecules of interest as well as their unique biological applications. Finally, challenges and perspectives are discussed to further advance the next-generation renal-clearable nanoprobes for in vivo imaging and in vitro urinalysis.

Anomalous behavior recognition of underwater creatures using lite 3D full-convolution network.

Global warming and pollution could lead to the destruction of marine habitats and loss of species. The anomalous behavior of underwater creatures can be used as a biometer for assessing the health status of our ocean. Advances in behavior recognition have been driven by the active application of deep learning methods, yet many of them render superior accuracy at the cost of high computational complexity and slow inference. This paper presents a real-time anomalous behavior recognition approach that incorporates a lightweight deep learning model (Lite3D), object detection, and multitarget tracking. Lite3D is characterized in threefold: (1) image frames contain only regions of interest (ROI) generated by an object detector; (2) no fully connected layers are needed, the prediction head itself is a flatten layer of 1 × [Formula: see text] @ 1× 1, [Formula: see text]= number of categories; (3) all the convolution kernels are 3D, except the first layer degenerated to 2D. Through the tracking, a sequence of ROI-only frames is subjected to 3D convolutions for stacked feature extraction. Compared to other 3D models, Lite3D is 50 times smaller in size and 57 times lighter in terms of trainable parameters and can achieve 99% of F1-score. Lite3D is ideal for mounting on ROV or AUV to perform real-time edge computing.

Application of split pancreatic duct stent in laparoscopic pancreaticoduodenectomy.

Laparoscopic pancreaticoduodenectomy (LPD) is a classic surgical method for diseases, such as tumors at the lower end of the common bile duct, pancreatic head, and benign and malignant tumors of the duodenum. Postoperative pancreatic fistula (POPF) is one of the most serious complications of LPD. To reduce the incidence of grade B or C POPF and other complications after LPD, we applied a split pancreatic duct stent combined with the characteristics of internal and external stent drainage. Between September 2020 and September 2022,12 patients underwent placement of the Split pancreatic duct stent during LPD. Data on basic characteristics of patients, surgical related indicators and postoperative POPF incidence were collected and analyzed. The results showed that the average operation time was 294.2 ± 36 minutes, average time for pancreaticojejunostomy was 35.9 ± 4.1 minutes, and average estimated blood loss was 204.2 ± 58.2 mL. Biochemical leakage occurred in 2 patients (16.7%), whereas no grade B or C POPF, 1 case (8.3%) had postoperative bleeding, and no death occurred within 30 days after the operation. Preliminary experience shows that the split pancreatic duct stent can effectively reduce the incidence of complications after LPD, especially grade B or C POPF.

Maintenance of Genetic Diversity of Black Sea Bream despite Unmonitored and Large-Scale Hatchery Releases.

Stock enhancement, used for replenishing depleted wild finfish populations, is an aggressive approach. Stock enhancement projects in Taiwan involve black sea bream (Acanthopagrus schlegelii), a major commercial species. During 2004-2015, even management agencies conducted stock enhancement projects, leading to numerous private releases that have not been recorded. Stock enhancement by a private hatchery without accurate genetic records may lead to a genetic structure change in wild populations. Using allele frequencies at nine microsatellite loci, we studied the genetic effects of stock enhancement in 19 samples collected from populations in the hatcheries and the wild. In 458 individuals from nine hatchery samples, most populations showed weak but significant genetic differences and complex clusters in structure analysis, indicating dramatic stock change within and among hatcheries. The 10 wild populations (n = 773) also had a complex genetic composition and were genetically different among sampling sites and times. However, a simple and clear cluster in structure analysis was found for only one sampling site, which had no release history. Thus, stock enhancement with complex genetic sources helps maintain genetic diversity but dramatically changes the genetic structure within and among wild populations, especially when stock enhancement is successful.

Identification of Genes Related to Cold Tolerance and Novel Genetic Markers for Molecular Breeding in Taiwan Tilapia (Oreochromis spp.) via Transcriptome Analysis.

Taiwan tilapia is one of the primary species used in aquaculture practices in Taiwan. However, as a tropical fish, it is sensitive to cold temperatures that can lead to high mortality rates during winter months. Genetic and broodstock management strategies using marker-assisted selection and breeding are the best tools currently available to improve seed varieties for tilapia species. The purpose of this study was to develop molecular markers for cold stress-related genes using digital gene expression analysis of next-generation transcriptome sequencing in Taiwan tilapia (Oreochromis spp.). We constructed and sequenced cDNA libraries from the brain, gill, liver, and muscle tissues of cold-tolerance (CT) and cold-sensitivity (CS) strains. Approximately 35,214,833,100 nucleotides of raw sequencing reads were generated, and these were assembled into 128,147 unigenes possessing a total length of 185,382,926 bp and an average length of 1446 bp. A total of 25,844 unigenes were annotated using five protein databases and Venny analysis, and 38,377 simple sequence repeats (SSRs) and 65,527 single nucleotide polymorphisms (SNPs) were identified. Furthermore, from the 38-cold tolerance-related genes that were identified using differential gene expression analysis in the four tissues, 13 microsatellites and 37 single nucleotide polymorphism markers were identified. The results of the genotype analysis revealed that the selected markers could be used for population genetics. In addition to the diversity assessment, one of the SNP markers was determined to be significantly related to cold-tolerance traits and could be used as a molecular marker to assist in the selection and verification of cold-tolerant populations. The specific genetic markers explored in this study can be used for the identification of genetic polymorphisms and cold tolerance traits in Taiwan tilapia, and they can also be used to further explore the physiological and biochemical molecular regulation pathways of fish that are involved in their tolerance to environmental temperature stress.

Characterization and phylogenetic analysis of the complete mitochondrial genome of Mytella strigata (Hanley 1843) (Bivalvia: Mytiloida: Mytilidae).

We sequenced and assembled the complete mitochondrial genome (mitogenome) sequence of the American brackish water mussel Mytella strigata. The mitogenome, reaching 16,302 bp in length, includes 13 protein-coding genes, 2 ribosomal RNA genes, and 23 transfer RNA genes. The overall nucleotide composition of mitogenome was 25.17% A, 41.86% T, 11.83% C, and 21.13% G. The most common start and stop codons were GTG and TAA, respectively. The phylogenetic analysis based on mitogenomes showed that the families Mytilidae, Ostreidae, and Veneridae are a monophyletic group. The phylogenetic position of M. strigata is sister to P. canaliculus and P. viridis. In this study, mitogenomic sequence data will provide a better understanding for future studies of population genetics, biogeography, and pest surveillance of M. strigata.

The complete mitochondrial genome of Metasepia tullbergi (Cephalopoda: Sepiidae).

The first complete mitochondrial genome of Metasepia tullbergi has been characterized in this study. The circular mitogenome is 16182 bp in length and comprises 13 protein-coding genes (PCGs), 22 transfer RNA genes, and two ribosomal RNA genes. The organization of these genes is highly consistent with that of other Sepiidae. The overall base composition of mitogenome is 39.20% A, 36.07% T, 8.98% G, and 15.75% C, with 75.27% AT. Phylogenetic analysis further suggests that M. tullbergi is placed within the Sepiidae and is closely related to Sepia latimanus and S. apama.

The complete mitochondrial genome of Laevistrombus canarium (Gastropoda: Stromboidae).

Laevistrombus canarium is a marine gastropod species with high economical value. The complete mitochondrial genome of L. canarium has been characterized in this study. The circular mitogenome is 15626 bp in length and comprises 13 protein-coding genes (PCGs), 22 transfer RNA genes, and two ribosomal RNA (rRNA) genes. The organization of these genes is consistent with that of other stromboidae species. The overall base composition of mitochondrial genome is 30.87% A, 38.99% T, 15.54% G, and 14.60% C, with 69.86% AT. Phylogenetic analysis further implies that L. canarium is placed within the Stromboidae.

Development of EST-Molecular Markers from RNA Sequencing for Genetic Management and Identification of Growth Traits in Potato Grouper (Epinephelus tukula).

The accuracy and efficiency of marker-assisted selection (MAS) has been proven for economically critical aquaculture species. The potato grouper (Epinephelus tukula), a novel cultured grouper species in Taiwan, shows large potential in aquaculture because of its fast growth rate among other groupers. Because of the lack of genetic information for the potato grouper, the first transcriptome and expressed sequence tag (EST)-derived simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were developed. Initially, the transcriptome was obtained from seven cDNA libraries by using the Illumina platform. De novo transcriptome of the potato grouper yielded 51.34 Gb and 111,490 unigenes. The EST-derived SSR and SNP markers were applied in genetic management, in parentage analysis, and to discover the functional markers of economic traits. The F1 juveniles were identified as siblings from one pair of parents (80 broodstocks). Fast- and slow-growth individuals were analyzed using functional molecular markers and through their association with growth performance. The results revealed that two SNPs were correlated with growth traits. The transcriptome database obtained in this study and its derived SSR and SNP markers may be applied not only for MAS but also to maintain functional gene diversity in the novel cultured grouper.

Development of Disease-Resistance-Associated Microsatellite DNA Markers for Selective Breeding of Tilapia (Oreochromis spp.) Farmed in Taiwan.

There are numerous means to improve the tilapia aquaculture industry, and one is to develop disease resistance through selective breeding using molecular markers. In this study, 11 disease-resistance-associated microsatellite markers including 3 markers linked to hamp2, 4 linked to hamp1, 1 linked to pgrn2, 2 linked to pgrn1, and 1 linked to piscidin 4 (TP4) genes were established for tilapia strains farmed in Taiwan after challenge with Streptococcus inae. The correlation analysis of genotypes and survival revealed a total of 55 genotypes related to survival by the chi-square and Z-test. Although fewer markers were found in B and N2 strains compared with A strain, they performed well in terms of disease resistance. It suggested that this may be due to the low potency of some genotypes and the combinatorial arrangement between them. Therefore, a predictive model was built by the genotypes of the parental generation and the mortality rate of different combinations was calculated. The results show the same trend of predicted mortality in the offspring of three new disease-resistant strains as in the challenge experiment. The present findings is a nonkilling method without requiring the selection by challenge with bacteria or viruses and might increase the possibility of utilization of selective breeding using SSR markers in farms.

Identification of Functional SSR Markers in Freshwater Ornamental Shrimps Neocaridina denticulata Using Transcriptome Sequencing.

The amazing colors and patterns are fascinating characteristics in all of the aquarium species. However, genetic and breeding molecular investigations of ornamental shrimps are rather limited. Here, we present the first transcriptomic analysis and application of microsatellites based on the chromatophore-encoded genes of Neocaridina denticulata to assist freshwater ornamental shrimp germplasm enhancement and its extensive applications. A total of 65,402 unigenes were annotated, and 4706 differentially expressed genes were screened and identified between super red shrimp and chocolate shrimp strains. Several gene ratios were examined to put in perspective possible genetic markers for the different strains of normal pigmentation development, including flotillin-2-like, keratin, the G protein-coupled receptor Mth2-like, annexin A7, and unconventional myosin-IXb-like. Five simple sequence repeat markers were effective for colored shrimps and were used to develop a marker-assisted selection platform for systematic breeding management program to maintain genetic diversity of the species. These markers could also be used to assist the identification of pure strains and increase the genetic stability of ornamental shrimp color phenotypes. Consequently, our results of microsatellite marker development are valuable for assisting shrimp genetic and selection breeding studies on freshwater ornamental shrimp and related crystal shrimp species.

The complete mitochondrial genome of Meretrix lusoria (Bivalvia: Veneroida: Veneridae) from Kumamoto, Japan.

We sequenced and assembled the complete mitochondrial genome sequence of the Meretrix lusoria, from Kumamoto, Japan. The length of mitogenome is 20,180 bp, including 13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes. The nucleotide composition of the mitogenome was 25.73% for A, 42.41% for T, 9.35% for C, and 22.49% for G. The AT and GC skewness of mitogenome sequence are -0.245 and 0.412, showing the T-skew and G-skew. The reconstructed phylogenetic relationships of 25 Bivalvia species based on 12 protein-coding genes were highly supported and the clade of all Meretrix clams included had a support value of 99%. Our results shall provide a better understanding in the evolutionary histories of the Veneroida and relative species.

[Corrigendum] CHD1L is associated with poor survival and promotes the proliferation and metastasis of intrahepatic cholangiocarcinoma.

Subsequent to the publication of the above paper, the authors have realized that the second affiliation for the second named author, Yi Chai, was not included with the affiliations. His second affiliation should have been listed as: "Department of Neurosurgery, Yuquan Hospital, School of Clinical Medicine, Tsinghua University, Beijing 100040, China." Therefore, the author affiliations for this paper should have appeared as follows: SHIMIAO LI1*, YI CHAI2,3*, YANBAO DING4, TINGHAO YUAN4, CHANGWEN WU5 and CHANGWEN HUANG1. 1Department of Hepatobiliary Surgery, Jiangxi Provincial People's Hospital, Nanchang, Jiangxi 330006; 2Department of Neurosurgery, Yuquan Hospital, School of Clinical Medicine, Tsinghua University, Beijing 100040; 3Department of Neurosurgery, Shangrao People's Hospital, Shangrao, Jiangxi 334000; 4Department of Hepatobiliary Surgery; 5Department of Urology Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China. The authors regret that this was not corrected prior to the publication of the paper, and apologize to the readers for any inconvenience caused. [the original article was published in Oncology Reports 42: 657­669, 2019; DOI: 10.3892/or.2019.7174].

CHD1L is associated with poor survival and promotes the proliferation and metastasis of intrahepatic cholangiocarcinoma.

Chromodomain helicase/ATPase DNA­binding protein 1­like gene (CHD1L) is a new oncogene which has been confirmed to be crucial to the progression of many solid tumors. In the present study, the expression of CHD1L was found to be upregulated in intrahepatic cholangiocarcinoma (ICC), which was significantly associated with histological differentiation (P=0.011), vascular invasion (P=0.002), lymph node metastasis (P=0.008) and TNM stage (P=0.001). Kaplan­Meier survival analysis revealed that ICC patients with positive CHD1L expression had shorter overall and disease­free survival than those with negative CHD1L expression. Functional study found that CHD1L exhibited strong oncogenic roles, including increased cell growth by CCK­8 assay, colony formation by plate colony formation assay, G1/S transition by flow cytometry and tumor formation in nude mice. In addition, RNAi­mediated silencing of CHD1L inhibited ICC invasion and metastasis by wound healing, Transwell migration and Matrigel invasion assays in vitro and in vivo. Collectively, our results show that CHD1L is upregulated and promotes the proliferation and metastasis of ICC cells. CHD1L acts as an oncogene and may be a prognostic factor or therapeutic target for patients with ICC.

Clinical significance of chromodomain helicase/ATPase DNA binding protein 1-like and human mutL homolog 1 gene expression in cholangiocarcinoma.

Cholangiocarcinoma is a highly malignant form of gastrointestinal cancer with an unfavorable prognosis. The novel oncogene chromodomain helicase/ATPase DNA binding protein 1-like (CHD1L) has been confirmed to serve a vital role in numerous types of cancer, including liver cancer. Mismatch repair (MMR) is a common DNA repair process that contributes to the preservation of the integrity and stability of genetic substances. Human mutL homolog 1 gene (hMLH1) is an important MMR protein family member. The present study aimed to evaluate the pathological and clinical features of cholangiocarcinoma, and to investigate the clinical significance of CHD1L and hMLH1 expression in cholangiocarcinoma. A total of 108 samples from cholangiocarcinoma tumor tissues and 60 samples from normal bile duct tissue were obtained from patients admitted to The Second Affiliated Hospital of Nanchang University between May 2005 and May 2014. All cholangiocarcinoma cases were pathologically confirmed. The expression of CHD1L and hMLH1 was examined by immunohistochemistry analysis. The expression of CHD1L in cholangiocarcinoma (94.44%) was significantly higher than in normal bile duct tissues (40.00%). CHD1L expression was associated with gallstone history, serum carbohydrate antigen 19-9 (CA19-9) level and Tumor-Node-Metastasis (TNM) stage (P<0.05). hMLH1 expression in cholangiocarcinoma (77.78%) was significantly lower than in normal bile duct tissues (96.67%), and was associated with gender, age, serum CA19-9 level, the presence of hepatitis B virus surface antigen, TNM stage and tumor diameter (P<0.05). Kaplan-Meier survival curve analysis indicated that the 3-year accumulative survival rates for CHD1L-positive and -negative patients differed significantly (P<0.05; 17.90 and 83.33%, respectively). There was no statistically significant difference (P>0.05) between the 3-year accumulate survival rates for hMLH1-positive and -negative patients (38.90 and 33.30%, respectively). High CHD1L expression and low hMLH1 expression levels were observed in patients with cholangiocarcinoma, and their abnormal expression patterns were associated with the progression of malignancy and an unfavorable disease prognosis. Therefore, CHD1L and hMLH1 may be potential prognostic biomarkers for cholangiocarcinoma.

Complete Genome Sequence of Streptococcus iniae 89353, a Virulent Strain Isolated from Diseased Tilapia in Taiwan.

Streptococcus iniae 89353 is a virulent strain isolated from diseased tilapia in Taiwan. The full-genome sequence of S. iniae 89353 is 2,098,647 bp. The revealed genome information will be beneficial for identification and understanding of potential virulence genes of Streptococcus iniae and possible immunogens for vaccine development against streptococcosis.

Double primary hepatic cancer (hepatocellular carcinoma and intrahepatic cholangiocarcinoma) originating from hepatic progenitor cell: a case report and review of the literature.

BACKGROUND: Synchronous development of primary hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) in different sites of the liver have rarely been reported before. The purpose of this study is to investigate the clinicopathological characteristics of synchronous double cancer of HCC and ICC. CASE PRESENTATION: A 56-year-old Chinese man without obvious liver cirrhosis was preoperation diagnosed with multiple HCC in segments VI (SVI) and VII (SVII) by the abdominal computed tomography (CT) and contrast-enhanced ultrasonography (CEUS). We performed hepatic resection of both segments. The tumors in SVI and SVII were pathologically diagnosed as ICC and HCC, respectively. Immunohistochemically, the HCC in SVII was positive for HepPar-1 and negative for CK19, while the ICC in SVI tumor was positive for CK19 and negative for HepPar-1. Interestingly, the immunohistochemical results also showed that the classic hepatic progenitor cell (HPCs) markers CD34 and CD117 were both positive of the two tumors. The patient still survived and at a 1-year follow-up did not show evidence of metastasis or new recurrent lesions. We speculate that the two masses may have originated from HPCs based on the findings of this patient. CONCLUSIONS: Synchronous development of HCC and ICC is very rare with unique clinical and pathological features. The correct preoperative diagnosis of double hepatic cancer of HCC and ICC is difficult. Hepatitis B virus (HBV) and hepatitis C virus (HCV) infection were both the independent risk factor to the development of double liver cancer. Hepatic resection is the preferred and most effective treatment choice. The prognosis of synchronous occurrence of double hepatic cancer was poorer than for either HCC or ICC, and the origin of it needs further study.