Whole genome identification of olive flounder (Paralichthys olivaceus) cathepsin genes: Provides insights into its regulation on biotic and abiotic stresses response.

Cathepsins are major lysosomal enzymes involved in essential physiological processes, including protein degradation, tissue differentiation, and innate or adaptive responses. Several kinds of cathepsins have been reported in teleost fishes, but no characterization have been performed for the inflammatory response of cathepsin family in olive flounder until now. In our current study, a total of 17 cathepsins in olive flounder were systematically identified and characterized. Phylogenetic analysis clearly indicated that the cathepsin genes was highly conserved. Analysis of structure and motifs exhibited high sequence similarity of cathepsin genes in olive flounder. Expression profiles of cathepsin genes in different tissues and developmental stages showed that cathepsins were temporally and spatially specific. RNA-seq analysis of bacteria and temperature stresses revealed that members of cathepsin were involved in inflammatory responses. Collectively, our findings would provide a further reference for understanding the molecular mechanisms of cathepsins in olive flounder.

Integration of transcriptome and proteome analyses reveals the regulation mechanisms of Larimichthys polyactis liver exposed to heat stress.

Small yellow croaker (Larimichthys polyactis) is one of the most economically important marine fishery species. L. polyactis aquaculture has experienced stress response and the frequent occurrence of diseases, bringing huge losses to the aquaculture industry. Little is known about the regulation mechanism of heat stress response in L. polyactis. In this study, to provide an overview of the heat-tolerance mechanism of L. polyactis, the transcriptome and proteome of the liver of L. polyactis on the 6 h after high temperature (32 °C) treatment were analyzed using Illumina HiSeq 4000 platform and isobaric tag for relative and absolute quantitation (iTRAQ). A total of 3700 upregulated and 1628 downregulated genes (differentially expressed genes, DEGs) were identified after heat stress in L. polyactis. Also, 198 differentially expressed proteins (DEPs), including 117 upregulated and 81 downregulated proteins, were identified. Integrative analysis revealed that 72 genes were significantly differentially expressed at transcriptome and protein levels. Functional analysis showed that arginine biosynthesis, tyrosine metabolism, pentose phosphate pathway, starch and sucrose metabolism, and protein processing in the endoplasmic reticulum were the main pathways responding to heat stress. Among the pathways, protein processing in the endoplasmic reticulum was enriched by most DEGs/DEPs, which suggests that this pathway may play a more important role in the heat stress response. Further insights into the pathway revealed that transcripts and proteins, especially HSPs and PDIs, were differentially expressed in response to heat stress. These findings contribute to existing data describing the fish response to heat stress and provide information about protein levels, which are of great significance to a deeper understanding of the heat stress responding regulation mechanism in L. polyactis and other fish species.

Whole-genome assembly and annotation for the little yellow croaker (Larimichthys polyactis) provide insights into the evolution of hermaphroditism and gonochorism.

The evolutionary direction of gonochorism and hermaphroditism is an intriguing mystery to be solved. The special transient hermaphroditic stage makes the little yellow croaker (Larimichthys polyactis) an appealing model for studying hermaphrodite formation. However, the origin and evolutionary relationship between of L. polyactis and Larimichthys crocea, the most famous commercial fish species in East Asia, remain unclear. Here, we report the sequence of the L. polyactis genome, which we found is ~706 Mb long (contig N50 = 1.21 Mb and scaffold N50 = 4.52 Mb) and contains 25,233 protein-coding genes. Phylogenomic analysis suggested that L. polyactis diverged from the common ancestor, L. crocea, approximately 25.4 million years ago. Our high-quality genome assembly enabled comparative genomic analysis, which revealed several within-chromosome rearrangements and translocations, without major chromosome fission or fusion events between the two species. The dmrt1 gene was identified as the male-specific gene in L. polyactis. Transcriptome analysis showed that the expression of dmrt1 and its upstream regulatory gene (rnf183) were both sexually dimorphic. Rnf183, unlike its two paralogues rnf223 and rnf225, is only present in Larimichthys and Lates but not in other teleost species, suggesting that it originated from lineage-specific duplication or was lost in other teleosts. Phylogenetic analysis shows that the hermaphrodite stage in male L. polyactis may be explained by the sequence evolution of dmrt1. Decoding the L. polyactis genome not only provides insight into the genetic underpinnings of hermaphrodite evolution, but also provides valuable information for enhancing fish aquaculture.

Preparation of animal model of chicken for lymphatic anastomosis technique training.

Background: To discuss the preparation of a simple and practical animal model for lymphatic anastomosis technology training. At present, there is no widely used animal model for training lymphatic anastomosis. Methods: The neck of chicken is long enough, and there are abundant lymphatic lymph nodes. The caliber of lymphatic vessels in the neck of chicken is similar to the caliber of lymphatic vessels in human limbs, so multi-stage anastomosis practice can be carried out. The lymphatic vessels of chicken are easier to stain and fully exposed, which meets the requirements of lymphatic vein anastomosis model. This model allows for training of end-to-end anastomosis (LVA), end-to-side anastomosis, and lymph node vein anastomosis under microscope to be carried out. In this study, the trainees were divided into six groups, with five animals in each group. After 14-28 days of training, the average training was 21 days. Data were statistically analyzed by SPSS15. Results: Obvious and clear lymphatic staining could be obtained after animal modeling, and the success rate of dissecting and staining lymphatic vessels under microscope could reach 100%. After training, the trainees could perform the anastomosis of lymphatic vessel and vein and lymph node vein under ultra-microscopic conditions, and the immediate patency rate of the anastomosis of the trainees can reach 80% during the examination. Conclusions: This animal model can be used for effective lymphatic vein anastomosis training, which is a simple, practical, and effective microscopic lymphatic anastomosis technology training model.

Loss of Nobox prevents ovarian differentiation from juvenile ovaries in zebrafish.

As a species without master sex-determining genes, zebrafish displays high plasticity in sex differentiation, making it an excellent model for studying the regulatory mechanisms underlying gonadal differentiation and gametogenesis. Despite being a gonochorist, zebrafish is a juvenile hermaphrodite that undergoes a special phase of juvenile ovary before further differentiation into functional testis and ovary. The mechanisms underlying juvenile ovary formation and subsequent gonadal differentiation remain largely unknown. In this study, we explored the role of Nobox/nobox (new born ovary homeobox protein), another oocyte-specific transcription factor in females, in early zebrafish gonadogenesis using CRISPR/Cas9 technology. As in mammals, nobox is specifically expressed in zebrafish gonads with a dimorphic pattern at juvenile stage. In contrast to the mutant of figla (factor in the germline alpha, another oocyte-specific transcription factor), the nobox mutants showed formation of typical perinucleolar (PN) follicles at primary growth (PG) stage in juvenile gonads, suggesting occurrence of follicle assembly from cystic oocytes (chromatin nucleolar stage, CN). These follicles, however, failed to develop further to form functional ovaries, resulting in all-male phenotype. Despite its expression in adult testis, the loss of nobox did not seem to affect testis development, spermatogenesis and male spawning. In summary, our results indicate an important role for Nobox in zebrafish ovarian differentiation and early folliculogenesis.

Growth and gonadal development retardations after long-term exposure to estradiol in little yellow croaker, Larimichthys polyactis.

Endocrine disrupting chemicals (EDCs) including 17ß-estradiol (E2) are widely distributed in the aquatic environment and are known to negatively affect the reproductive system of many animals, including fish. EDCs leading to feminization, altered sex ratio and reduced fecundity, it is possibly posing potential risks to the ecosystems. To investigate the potentially toxic effects of E2 exposure on little yellow croaker (Larimichthys polyactis, L. poliactis) who have a unique gonadal development pattern that males undergo a hermaphroditic stage. An experiment was set up where L. poliactis were maintained in tanks and exposed to E2 concentrations of 10 µg/L or no E2 exposure (the ethanol and control groups) from 30 to 90 days post-hatching (dph). After exposure, the E2 withdrawal and continual cultured to 150 and 365 dph. The morphological and histological analyses were used to compare the changes in the fish body and gonad under E2 exposure. The results showed that E2 exposure caused three major phenotypes at 30 and 60 days after treatment (dat), including ovary, ovotestis and gonadal development retardation compared with the control groups. The average ratio of these three phenotypes is 60.6%, 11.97% and 27.43%, respectively. The body length and weight of E2 exposure groups were repressed during the E2 exposure period, while it can recover after E2 withdrawal. However, the gonadal development (Gonadosomatic Index) of E2 exposure groups testis were retarded at 60 dat and doesn't recover until 365 dph. The sex determination/differentiation-related genes erα, erßI, erßII, fshß and cyp11b2 were significantly decreased in E2-exposure male fish. This research highlights the E2 leads to feminization, disrupts testis maturation and spermatogenesis, this effect persisted into the stage of sexual maturity. Collectively, our findings provide insights into the molecular mechanisms underlying E2 disturbance of a marine economic fish reproduction.

KIFC1 functions in nuclear reshaping and midpiece formation during the spermatogenesis of small yellow croaker Larimichthys polyactis.

The C-terminal kinesin motor protein (KIFC1) has essential functions in spermatogenesis. To evaluate molecular mechanisms of KIFC1 during teleost fish spermatogenesis, there was cloning and sequencing the kifc1 cDNA in the testis of Larimichthys polyactis. Quantitative PCR results indicated there were Lp-kifc1 mRNA transcripts in the testes. Results from conducting fluorescence in situ hybridization and immunofluorescence procedures indicated there were trends in relative abundance changes in Lp-kifc1 mRNA transcripts that were associated with abundance of Lp-KIFC1 protein during spermatogenesis. The Lp-KIFC1 protein was detected at all stages of spermatogenesis. There was minimal Lp-KIFC1 in the cytoplasm of spermatogonia, with content being greater and concentrated in the perinuclear region in spermatocytes and during early/mid-stages of development of spermatids. There were large abundances of Lp-KIFC1 in spermatids at the mid-developmental stage. In late-developing spermatids, Lp-KIFC1 content was less and concentrated in the bottom of the nucleus, where the midpiece formed. There was a small Lp-KIFC1 in the midpiece of mature sperm. These findings indicate Lp-KIFC1 may have functions in L. polyactis spermatogenesis. Results from conducting immunofluorescence procedures indicated Lp-KIFC1 was co-localized microtubules and mitochondria throughout spermatogenesis. There were large abundances of Lp-KIFC1 and tubulin in spermatids during the mid-developmental stage, when there is a decrease in size and reshaping of the nucleus. During midpiece formation, there was co-localization of the Lp-KIFC1 and mitochondria in the spermatid perinuclear region to the midpiece. These findings indicate Lp-KIFC1 is involved in nuclear reshaping and midpiece formation during spermatogenesis in L. polyactis.

A first genetic linage map construction and QTL mapping for growth traits in Larimichthys polyactis.

Larimichthys polyactis is a commercially important marine fish species in Eastern Asia, yet very few genetic resources exist. In particular, genetic linkage maps are critical tools for genetic breeding. In this study, we generated a high resolution linkage map from a family of 110 individuals and their parents by resequencing the individuals. 3,802 effective SNPs were mapped to 24 linkage groups (LGs). The map spanned 2,567.39 cm, with an average marker interval of 0.66 cm. We used the map to conduct QTL analysis for growth traits, and found 31 markers were significantly associated with growth-related traits. Specifically, three SNPs were identified for total length, nineteen SNPs for body length, and nine SNPs for body weight. The identified SNPs could explain 15.2-22.6% of the phenotypic variation. SNPs associated with growth traits were distributed on LG6 and LG11, and candidate genes included, kif26b, bat1, gna1, gbgt1, and amfr, which may regulate growth. The linkage map and mapped QTLs would be useful for improving the quality of L. polyactis via marker-assisted selection.

A Transient Hermaphroditic Stage in Early Male Gonadal Development in Little Yellow Croaker, Larimichthys polyactis.

Animal taxa show remarkable variability in sexual reproduction, where separate sexes, or gonochorism, is thought to have evolved from hermaphroditism for most cases. Hermaphroditism accounts for 5% in animals, and sequential hermaphroditism has been found in teleost. In this study, we characterized a novel form of the transient hermaphroditic stage in little yellow croaker (Larimichthys polyactis) during early gonadal development. The ovary and testis were indistinguishable from 7 to 40 days post-hatching (dph). Morphological and histological examinations revealed an intersex stage of male gonads between 43 and 80 dph, which consist of germ cells, somatic cells, efferent duct, and early primary oocytes (EPOs). These EPOs in testis degenerate completely by 90 dph through apoptosis yet can be rescued by exogenous 17-ß-estradiol. Male germ cells enter the mitotic flourishing stage before meiosis is initiated at 180 dph, and they undergo normal spermatogenesis to produce functional sperms. This transient hermaphroditic stage is male-specific, and the ovary development appears to be normal in females. This developmental pattern is not found in the sister species Larimichthys crocea or any other closely related species. Further examinations of serum hormone levels indicate that the absence of 11-ketotestosterone and elevated levels of 17-ß-estradiol delineate the male intersex gonad stage, providing mechanistic insights on this unique phenomenon. Our research is the first report on male-specific transient hermaphroditism and will advance the current understanding of fish reproductive biology. This unique gonadal development pattern can serve as a useful model for studying the evolutionary relationship between hermaphroditism and gonochorism, as well as teleost sex determination and differentiation strategies.

Transcriptome analyses provide the first insight into the molecular basis of cold tolerance in Larimichthys polyactis.

Larimichthys polyactis is one of the most economically important marine fish species that have become newly cultured in China in recent years. The gene expression changes that L. polyactis experiences in cold toleranceis still unknown, limiting the expansion of its cultivation, fast growth, and high yield. To investigate the molecular mechanism behind L. polyactis's cold tolerance and to provide a resource for conducting genetic research on L. polyactis, transcriptome sequencing using RNA-seq was performed on individuals that survived cold stress at 4 °C (cold tolerant, CT), and individuals that barely survived 4 °C (cold sensitive, CS), which was considered as the control. A number of 387,607,550 clean reads were obtained from the transcriptomes, and comparative transcriptomic analysis identified 141 differently expressed genes (DEGs), of which 67 were up-regulated and 74 were down-regulated in CT compared to CS under cold stress. Furthermore, ten differently expressed genes were selected from the RNA-Seq analysis to be further validated by real-time PCR. Functional network analysis indicated that L. polyactis adapted to cold stress by employing a series of mechanisms to minimize damages caused by exposure to cold temperatures. The molecular mechanisms identified through RNA-Seq included Extracellular matrix (ECM) receptor interaction, glycerolipid metabolism, regulation of autophagy and focal adhesion pathway as playing vital roles in cold tolerance in L. polyactis. This study may help elucidate how L. polyactis tolerates cold, which is of value for breeding cold-tolerant L. polyactis stocks for cultivation.

Expression and potential functions of KIF3A/3B to promote nuclear reshaping and tail formation during Larimichthys polyactis spermiogenesis.

KIF3A and KIF3B are homologous motor subunits of the Kinesin II protein family. KIF3A, KIF3B, and KAP3 form a heterotrimeric complex and play a significant role in spermatogenesis. Here, we first cloned full-length kif3a/3b cDNAs from Larimichthys polyactis. Lp-kif3a/3b are highly related to their homologs in other animals. The proteins are composed of three domains, an N-terminal head domain, a central stalk domain, and a C-terminus tail domain. Lp-kif3a/3b mRNAs were found to be ubiquitously expressed in the examined tissues, with high expression in the testis. Fluorescence in situ hybridization (FISH) was used to analyze the expression of Lp-kif3a/3b mRNAs during spermiogenesis. The results showed that Lp-kif3a/3b mRNAs had similar expression pattern and were continuously expressed during spermiogenesis. From middle spermatid to mature sperm, Lp-kif3a/3b mRNAs gradually localized to the side of the spermatid where the midpiece and tail form. In addition, we used immunofluorescence (IF) to observe that Lp-KIF3A protein co-localizes with tubulin during spermiogenesis. In early spermatid, Lp-KIF3A protein and microtubule signals were randomly distributed in the cytoplasm. In middle spermatid, however, the protein was detected primarily around the nucleus. In late spermatid, the protein migrated primarily to one side of the nucleus where the tail forms. In mature sperm, Lp-KIF3A and microtubules accumulated in the midpiece. Moreover, Lp-KIF3A co-localized with the mitochondria. In mature sperm, Lp-KIF3A and mitochondria were present in the midpiece. Therefore, Lp-KIF3A/KIF3B may be involved in spermiogenesis in L. polyactis, particularly during nuclear reshaping and tail formation.

Elastic fixation of mallet finger fractures using two K-wires: A case report of a new fixation technique.

RATIONALE: Mallet finger fracture is a common sports-related injury that may lead to the tearing of extensor tendon and protrusion of a bony fragment located at the base of the distal phalanx. We affirmed that the elastic fixation of with two K-wires technique is a good method to deal with Mallet Finger fractures that fractures could gain effective fixation than the conventional treatment method and avoid surgical incision complication PATIENT CONCERNS:: We reported a 33-year-old female patient came to our hospital complaining of mild pain, swelling and her right little finger was deformed because of sport's injury. DIAGNOSIS: Acute mallet finger fracture type IV B according to Doyle classification of mallet injuries. INTERVENTIONS: We performed an emergency operation for the elastic fixation of the mallet finger fractures with two K-wires. OUTCOMES: After the surgery, the patient showed functional recovery. No evidence of recurrence was noted 6 months after the operation, and the patient showed no symptoms of sports-related injuries. LESSONS: We discuss the clinical diagnosis, treatment, and follow-up of the patient and suggest that elastic fixation with two K-wires is a good method to treat mallet finger fractures.

Aucubin suppresses Titanium particles­mediated apoptosis of MC3T3­E1 cells and facilitates osteogenesis by affecting the BMP2/Smads/RunX2 signaling pathway.

Aucubin represents an iridoid glucoside separated from multiple Chinese herbs, which has been demonstrated to possess numerous pharmacological activities. In the present study, the aim was to investigate the roles and mechanisms of aucubin in the suppression of mouse MC3T3­E1 osteoblast apoptosis induced by Titanium particles and the promotion of bone formation. MTT assay and flow cytometry were performed to analyze cell viability and apoptosis, respectively. ELISA and para­nitrophenyl phosphate colorimetry were carried out to evaluate the oxidative stress markers and alkaline phosphatase (ALP). Western blotting and reverse transcription­quantitative polymerase chain reaction assays were used to evaluate the associated mRNA and protein expression. The results revealed that aucubin enhanced the cell activity of MC3T3­E1 cells treated with Ti particles. Aucubin suppressed the apoptosis of Ti particles­induced MC3T3­E1 cells and facilitated osteogenesis by affecting the B­cell lymphoma­2 (Bcl­2), Bcl­2 associated X protein, ALP and associated osteogenic factors expression. Aucubin reduced the oxidative stress in Ti particles­induced MC3T3­E1 cells. In addition, aucubin upregulated the bone morphogenetic protein 2 (BMP2)/Smads/runt related transcription factor 2 (RunX2) pathway in Ti particles­induced MC3T3­E1 cells. In conclusion, the present study confirmed that aucubin suppressed the Ti particles­mediated apoptosis of MC3T3­E1 cells and facilitated osteogenesis by affecting the BMP2/Smads/RunX2 signaling pathway.

Oncoplastic breast reconstruction with omental flap: A retrospective study and systematic review.

The increasing frequency of early breast cancer cases has driven an increasing number of patients to choose immediate reconstruction with an autologous flap. Omentum-flap-based breast reconstruction is a unique strategy that is highly suitable for repairing moderate tissue defects. However, all available evidence comes from individual reports with small numbers of cases, and the overall effectiveness and safety of the procedure have yet to be reported. Here, we reported 7 cases of laparoscopically harvested omental free flap breast reconstruction and performed a systematic review to assess the applicability and safety of this approach. The data were gathered from MEDLINE, Ovid, Google Scholar and the China Knowledge Resource Integrated Database. In total, we combined 15 articles (410 cases) for analysis. The data revealed that almost all patients (87.6%) were reported to have undergone laparoscopy instead of laparotomy; pedicle flaps were used in 90.9% of the cases, while only 5 (37 cases) used free flaps for reconstruction; and 96.6% (396/410) of all reconstruction procedures were immediate. Almost all of these cases had a small tumour burden (T0/Tis/T1 59.8%; T2 36.8%), and the distribution of tumour location was similar among the four quadrants. The cosmetic outcomes were desirable in most cases (83.9%). There were 41 complications identified in the dataset: partial graft necrosis accounted for the largest percentage (41.5%) of all events, followed by skin necrosis (19.5%), haematoma (12.2%) and wound infection (9.8%). During the follow-up period, which had a short median duration, 2 cases of tumour recurrence were reported. Overall, our systematic review found that omentum-flap-based breast reconstruction could achieve a satisfactory aesthetic outcome, especially for small breasts and tissue replacement after breast-conserving surgery, and the safety of the procedure was also acceptable.

A new surgical treatment for abdominal wall defects: A vascularized ribs-pleural transfer technique that can be used with or without a thoracic umbilical flap a case report.

RATIONALE: Abdominal wall defects are common after tumor resection. PATIENT CONCERNS: We report an 83-year-old male patient with recurrent tumors in his abdomen, and who had an incision wound that could not be directly closed. Mesh was not suitable because the wound was infected. DIAGNOSES: Abdominal wall defect result from the resection of recurrent tumor. INTERVENTIONS: We carried out a vascularized ribs-pleural transfer operation. OUTCOMES: After the surgery, the patient gained a functional recovery. No evidence of recurrence was noted 1 year after operation, and the patient showed no symptoms of abdominal compression syndrome. LESSONS: We discuss the clinical diagnosis, treatment, and follow up and argue that the vascularized ribs-pleural transfer technique is a good method to deal with abdominal wall defects.