Expression pattern analysis of anti-Mullerian hormone in testis development of pearlscale angelfish (Centropyge vrolikii).

In vertebrates, anti-Mullerian hormone (Amh) secreted by Sertoli cells (SC) performs a pivotal function in male sex differentiation. Compared with that of higher vertebrates, the expression pattern of Amh is more diversified in fish. In this study, the full-length complementary DNA (cDNA) of Amh in Centropyge vrolikii (Cv-Amh) was cloned and analysed, which was 2,470 bp, including a 238 bp 5'UTR, a 1,602 bp ORF and a 633 bp 3'UTR; the similarity of Amh between Cv-Amh and other fish is relatively high. The quantitative real-time PCR (qRT-PCR) results of healthy tissues and gonads at sex reversal stages in C. vrolikii showed that the expression level of Amh in the testis was significantly higher than that in other tissues (P < 0.05). Amh was weakly expressed in the vitellogenic stage ovary and perinucleolus stage ovary, but its expression significantly increased in the gonads at the hermaphroditic stage, and finally reached the highest in the pure testis after sexual reversal. The results of in situ hybridization indicated that the positive signal of Amh was strongly concentrated in SCs of testis. After Amh knockdown in the gonads, the effect on sex-related genes was tested using qRT-PCR. Among these, the expression of Dmrt1, Cyp11a, Hsd11b2, Sox8 and Sox9 significantly decreased, whereas that of Cyp19a, Sox4, Foxl2 and Sox3 increased. These results suggested that Amh could be the pivotal gene in reproductive regulation in C. vrolikii, and the data will contribute to sex-related research of C. vrolikii in the future.

Establishment and characterization of the ovary cell line derived from two-spot puffer Takifugu bimaculatus and its application for gene editing and marine toxicology.

Takifugu bimaculatus is a marine fish with high nutritional value. Its ovary contains tetrodotoxin (TTX) which is a severe neurotoxin that limits its edible value of it. To understand the mechanism of oogenesis and production of TTX in T. bimaculatus, an ovarian cell line named TBO from an adolescent ovary was established. TBO was composed of fibroblast-like cells that expressed the ovarian follicle cells marker gene Foxl2 and highly expressed TTX binding protein 2 (PSTBP2) but did not express the germ cells marker gene Vasa. Therefore, TBO seems to be mainly composed of follicle cells and possibly a small percentage of oocytes. Electroporation was used to successfully transfect the pEGFP-N1 and pNanog-N1 vectors into the TBO cell line with a high transfection efficiency. The morphological changes and survival rates of the exposed cells proved that this cell line was effective for exposure to conotoxins (CTXs), another group of toxins related to food safety. Furthermore, PSTBP2 was knocked out in TBO using CRISPR/Cas9 technology, showing that sgRNA2 could mutate PSTBP2. The results suggested that TBO will be more convenient, efficient, and rapid for reproduction and toxicology investigation, and gene editing. This study laid the groundwork for future research into the fish gonadal cell culture and food-related marine toxins. In conclusion, a cell line has been generated from T. bimaculatus, which might represent a valuable model for fish studies in the fields of toxicology and gene editing.

dmrtb1 is involved in the testicular development in Larimichthys crocea.

In brief: dmrtb1 performs critical functions in sex determination/differentiation and gonadal development in many organisms, but its role in teleost is rarely studied. Through gene cloning, in situ hybridization, and RNA interference technology, the function of dmrtb1 in testicular development of large yellow croaker (Larimichthys crocea) was studied; our study will be helpful in understanding further the molecular regulation mechanism of Lcdmrtb1/Lcdmrt6 in testicular development in L. crocea, and our results enrich the theory of fish dmrts involved in reproductive regulation and provide a new idea for sex control breeding of L. crocea by manipulating reproductive pathway. Abstract: Doublesex- and mab-3-related transcription factor B1 (dmrtb1/dmrt6) belongs to one of the members of DMRT family, which performs critical functions in sex determination and differentiation, gonadal development, and functional maintenance. However, knowledge of its exact mechanism remains unclear in teleost. Very little is known about the role of dmrtb1 in the gonad development of Larimichthys crocea. In this study, a dmrtb1 homolog in L. crocea named as Lcdmrtb1 with the full-length cDNA was isolated and characterized. Except for the conserved DM domain, the other regions had low homology. Of the tissues sampled, Lcdmrtb1 was only found to be highly expressed in the testis. In situ hybridization of testis revealed Lcdmrtb1 in both spermatogonia and spermatocytes. After Lcdmrtb1 interference in the testis cells (LYCT) of L. crocea, the expression levels of Lcdmrtb1 and Lcdmrt1 were significantly decreased; subsequently, testicular cell morphology changed from fibrous to round and their growth rate slowed. Similarly, the expression levels of Lcdmrtb1, Lcdmrt1, sox9a/b, and amh were significantly decreased after RNAi in the testis. Furthermore, it was discovered that the spermatogonia had disappeared, and the Sertoli cells had been reduced. The results of immunohistochemistry showed that the expression of Sox9 protein in the testis was not detected after dmrtb1 was knocked down. These results indicated that the absence of Lcdmrtb1 not only greatly inhibited cell growth and destroyed the morphology of testis cells but also down-regulated Lcdmrt1 expression in the testis. This study will be helpful in understanding further the molecular regulation mechanism of Lcdmrtb1/Lcdmrt6 in testicular development in L. crocea.

The molecular regulation mechanism of dmrt1-based on the establishment of the testis cell line derived from two-spot puffer Takifugu bimaculatus.

The establishment of fish cell lines can provide an important in vitro model for developmental biology, pathology, and genetics and also an effective tool to investigate the interactions and related functions of genes. Two-spot puffer Takifugu bimaculatus is a high economic and nutritional value marine fish in Fujian in recent years. Nevertheless, dmrt1 plays a key role in the male differentiation from invertebrates to vertebrates. To understand the molecular regulatory mechanisms of dmrt1 in T. bimaculatus, a testis cell line called TBTc from a juvenile testis of this organism was established with modified Leibovitz's L-15 medium supplemented with 20% FBS, fish serum, embryo extract, and other growth factors. The TBTc with a stable karyotype can be passaged continuously, which was composed of fibroblast-like cells and expressed the marker genes of male-special cells, dmrt1, and amh, and the absence of vasa expression may rule out the possibility of the presence of germ cells. Therefore, TBTc appeared to consist of the mixture of the Sertoli cell and germ cell of the testis. The dmrt1 was significantly expressed in the testes and slightly expressed in the late embryonic development, illustrating that the dmrt1 may participate in the molecular regulation of gonads development and sex differentiation. With the high transfection efficiency of TBTc by electroporation, the cell lines could be used effectively in the study for the expression of exogenous and endogenous genes. Meanwhile, after the knockdown of dmrt1, the morphological changes and survival rates of cells proved that dmrt1 could affect the growth of testicular cells. Furthermore, with the loss of dmrt1, the expression of male-bias genes amh, sox9, and cyp11a was significantly decreased, and the expression of female-bias genes foxl2, sox3, and cyp19a was increased, which suggested that dmrt1 upregulates amh, sox9, and cyp11a and downregulates foxl2, sox3, and cyp19a to participate in the testis development. As a first fish gonadal cell lines of T. bimaculatus, which can be a more convenient, efficient, and rapid model for the investigation of the expression and function of genes, the results will lay a foundation for the next study of the molecular regulation mechanism in gonadal development and sex determination of fish in the future.

Characterization of the transcription factor Sox3 regulating the gonadal development of pearlscale angelfish (Centropyge vrolikii).

As a member of the Sox gene family, Sox3 plays a vital role in gonadal development and gametogenesis. Nevertheless, the exact expression pattern of this gene in fish is still unknown. Here, we identified the Sox3 gene of Centropyge vrolikii, namely, Cv-Sox3. The Cv-Sox3 mRNA expression in the ovary and testis was detected by reverse transcription-polymerase chain reaction (RT-PCR) analysis, and the mRNA expression level of Cv-Sox3 in the ovary in the resting stage was significantly higher than that in other tissues. The phylogenetic tree and alignment of multiple sequences were constructed to analyze the evolutionary relationships of Cv-Sox3. Cv-Sox3 was relatively conserved in the evolution of teleost fish, indicating the importance and similarity of its function. The in situ hybridization results demonstrate that Cv-Sox3 was present in the follicle cells and cytoplasm of oocytes in the ovary of different stages, and the positive signals occurred in germ cells of the testis. After interfering with Cv-Sox3, the growth rate of ovarian cells in culture became slow, and the expression of ovary-bias-related genes Cyp19a and Foxl2 significantly increased. Meanwhile, the expression of testis-bias-related genes Dmrt1, Sox9, Cyp11a, Amh, and Sox8 significantly decreased. These results suggest that Cv-Sox3 gene might be expressed in the germ cells of male and female gonads during gonadal development. This study provides a precise expression pattern of Cv-Sox3 and demonstrates that Cv-Sox3 might play a significant role in the reproductive regulation of C. vrolikii. In this study, Sox3 of C. vrolikii (Cv-Sox3) was cloned to understand the expression pattern in the gonadal development, which is expressed in germ cells, involved in the process of gonadal development. The results demonstrated that Cv-Sox3 may play a significant role in the reproductive regulation of C. vrolikii.

Characterization of the Nanog gene involved in the gonadal development in pearlscale angelfish (Centropyge vrolikii).

The homeodomain transcription factor Nanog plays a crucial role in the embryonic and gonadal development and the maintenance of embryonic stem cells (ESCs), interacting with transcription factors such as Oct4 and Sox2 in mammals. Nevertheless, its pathways to molecular mechanisms remain unclear as to teleosts. This study investigates the role of the Nanog gene in gonadal development and sex reversal of pearlscale angelfish (Centropyge vrolikii). To understand the expression pattern of gonadal development, we identified the Nanog gene of C. vrolikii, which we named Cv-Nanog. The full-length cDNA sequence of Cv-Nanog was 2,136 bp in length and encoded a homeodomain protein of 436 amino acid residues. The gene structure and western blot prove results that Cv-Nanog was homologous to the Nanog gene of mammalians. The protein sequence comparison demonstrates that the Cv-Nanog shared a high degree of similarity with orthologs from other vertebrates in the conserved homeodomain. The Cv-Nanog gene was substantially expressed in gonads, and the expression was significantly higher in the ovaries than in the testis, according to quantitative real-time PCR (qRT-PCR) and western blot analyses. In situ hybridization reveals that the transcripts were located in the cytoplasm and membrane of the oocytes in the ovaries and testes. The expression of Cv-Nanog mRNA was weak in Sertoli cells but strong in germ cells. After overexpression of Cv-Nanog, the expression levels of pluripotent factors Sox2 and Oct4 increased significantly with 21.5-fold and 12.2-fold, respectively. Simultaneously, the TGF-beta signaling pathway was activated, and the gonadal cell growth was promoted. The expression of ovary-bias genes Cyp19a and Foxl2 was upregulated, and the expression of testis-bias genes Sox9 and Dmrt1 was downregulated to promote ovarian development. These results imply that the Nanog gene might play a crucial role in the process of gonadal development and sexual reversion in C. vrolikii. This study provides new insight to understand the molecular regulatory mechanism of the Nanog gene further and important clues for the future studies in gonadal development.

Neuropeptides as the Shared Genetic Crosstalks Linking Periodontitis and Major Depression Disorder.

BACKGROUND: The aim of this study was at investigating the association between major depressive disorder (MDD) and periodontitis based on crosstalk genes and neuropeptides. METHODS: Datasets for periodontitis (GSE10334, GSE16134, and GSE23586) and MDD (GSE38206 and GSE39653) were downloaded from GEO. Following batch correction, a differential expression analysis was applied (MDD: ∣log2FC | >0 and periodontitis ∣log2FC | ≥0.5, p < 0.05). The neuropeptide data were downloaded from NeuroPep and NeuroPedia. Intersected genes were potential crosstalk genes. The correlation between neuropeptides and crosstalk genes in MDD and periodontitis was analyzed with Pearson correlation coefficient. Subsequently, regression analysis was performed to calculate the differentially regulated link. Cytoscape was used to map the pathways of crosstalk genes and neuropeptides and to construct the protein-protein interaction network. Lasso regression was applied to screen neuropeptides, whereby boxplots were created, and receiver operating curve (ROC) analysis was conducted. RESULTS: The MDD dataset contained 30 case and 33 control samples, and the periodontitis dataset contained 430 case and 139 control samples. 35 crosstalk genes were obtained. A total of 102 neuropeptides were extracted from the database, which were not differentially expressed in MDD and periodontitis and had no intersection with crosstalk genes. Through lasso regression, 9 neuropeptides in MDD and 43 neuropeptides in periodontitis were obtained. Four intersected neuropeptide genes were obtained, i.e., ADM, IGF2, PDYN, and RETN. The results of ROC analysis showed that IGF2 was highly predictive in MDD and periodontitis. ADM was better than the other three genes in predicting MDD disease. A total of 13 crosstalk genes were differentially coexpressed with four neuropeptides, whereby FOSB was highly expressed in MDD and periodontitis. CONCLUSION: The neuropeptide genes ADM, IGF2, PDYN, and RETN were intersected between periodontitis and MDD, and FOSB was a crosstalk gene related to these neuropeptides on the transcriptomic level. These results are a basis for future research in the field, needing further validation.

Comparison of differential expression genes in ovaries and testes of Pearlscale angelfish Centropyge vrolikii based on RNA-Seq analysis.

Pearlscale angelfish Centropyge vrolikii is a kind of protogynous hermaphrodite fish with a natural sexual reversion. Under appropriate social conditions, a female fish can transform into a male fish spontaneously. It is an important prerequisite for artificial breeding to understand the process of its gonadal development and sexual reversion. Gonadal development is regulated by many sex-related genes. In this study, we used unreferenced RNA-Seq technology to sequence the ovary at the perinucleolus stage (OII), ovary at the yolk vesicle stage (OIV),IV and testis (T), respectively; screened the gonadal differential expression genes (DEGs); and analyzed the expression of these genes in different developmental stages of ovary and different sex gonads. The results showed that a total of 142,589 all-unigene samples were assembled, and gene annotation was performed by COG, GO, KEGG, KOG, Pfam, Swissprot, eggNOG, and NR functional database. Comparative analysis revealed that there were 1919 genes that were up-regulated and 1289 genes were down-regulated in comparison to OIV vs OII, while there were 3653 genes that were up-regulated and 2874 genes were down-regulated in comparison of OIV vs T, there were 3345 genes that were up-regulated and 2995 genes were down-regulated in comparison of the OII vs the T. At the same time, the results verified by RT-qPCR were consistent with the variation trend of transcriptome data. Among the results, amh, sox9b, dmrt1, dmrt2, cyp11a, cyp17a, and cyp19a were significantly expressed in the testes, while sox3, sox4, sox11, sox17, and hsd3b7 were significantly expressed in the ovaries. And, the expression of the amh, sox9b, dmrt2, and dmrt1 were low in the OII and OIV, while significantly increased during the ovotestis in the hermaphroditic period (OT), and finally reached the highest level in pure testis after sex reversal. The expression of sox3, sox4, hsd3b7, sox11, and sox17 was significantly reduced during the hermaphroditic period (OT). These results suggested that these genes may play an important role in the process of sex reversal. This study is helpful to further understand the molecular regulation mechanism of gonadal development and sexual reversion in Pearlscale angelfish and also provide important clues for future studies.

[Treatment of severe diabetic foot ulcer using tibia transverse transport combined with nose ring drain].

OBJECTIVE: To investigate the effectiveness of tibial transverse transport (TTT) combined with nose ring drain (NRD) in the treatment of severe diabetic foot ulcer. METHODS: The clinical data of 60 patients with severe diabetic foot (Wagner grade 3 or 4) ulcer who were admitted between April 2017 and August 2020 and met the selection criteria were retrospectively analyzed. Among them, 30 cases were treated with TTT combined with NRD (TTT+NRD group), and 30 cases were treated with TTT (TTT group). There was no significant difference in gender, age, diabetes duration, preoperative glycated hemoglobin, comorbidities, wound area, and duration, side, and grade of diabetic foot ( P>0.05). The wound healing time, wound healing rate, amputation rate, recurrence rate, duration of antibiotic therapy, hospital stay, number of hospitalizations, and number of operations were recoreded and compared between the two groups. RESULTS: No obvious surgical complications occurred in the two groups. Patients in both groups were followed up 3-13 months, with an average of 5.7 months. The duration of antibiotic therapy and hospital stay in the TTT+NRD group were significantly shorter than those in the TTT group ( P<0.05). There was no significant difference in wound healing time, wound healing rate, number of hospitalizations, and number of operations between the two groups ( P>0.05). During follow-up, there was no recurrence of ulcer in the TTT+NRD group while 2 recurrent cases (6.7%) in the TTT group. The difference in recurrence rate was not significant ( P=0.492). One case (3.3%) in the TTT+NRD group underwent amputation due to acute lower extremity vascular embolism, and 1 case (3.3%) in the TTT group underwent amputation due to secondary necrosis. The difference in amputation rate was not significant between the two groups ( P=1.000). CONCLUSION: TTT combined with NRD is an effective method for the treatment of severe diabetic foot ulcers with deep infections or relatively closed cavities or sinuses. It can shorten the time of antibiotic use and the length of hospitalization; and the NRD has a good drainage effect without obvious comorbidities, procedure and the postoperative care are simple and easy to obtain materials.

Depth-independent internal fingerprint based on optical coherence tomography.

Optical coherence tomography (OCT) was used for imaging three-dimensional fingerprint to overcome the effects of different skin states and fake fingerprint. However, the OCT-based fingerprint features depend on the depth of fingertip skin which is still challenging for biometric recognition and encryption. In this work, we presented a new approach of maximum intensity projection (MIP) image of the epidermal-dermal junction (DEJ) to extract the internal fingerprint that is independent of the depth of fingertip skin. To begin with, the surface and DEJ were segmented based on the deep learning algorithm. Then the internal fingerprint was extracted by the MIP image of DEJ which has a more accurate structural similarity by quantitative analysis. The experimental results showed that internal fingerprint acquired by MIP of DEJ can be applied for scar-simulation fingertip and encryption since it is not sensitive to the states of surface skin and independent of the depth.

Tibial cortex transverse transport facilitating healing in patients with recalcitrant non-diabetic leg ulcers.

OBJECTIVE: The treatment of recalcitrant not-diabetic leg ulcers remains challenging. Distraction osteogenesis is accompanying by angiogenesis and neovascularization in the surrounding tissues. We previously applied tibial cortex transverse transport (TTT) to patients with recalcitrant diabetic foot ulcers and found neovascularization and increased perfusion in the foot and consequently enhanced healing and limb salvage and reduced recurrence. However, the effects of TTT on recalcitrant non-diabetic leg ulcer remains largely unknown. METHODS: Consecutive patients (n â€‹= â€‹85) with recalcitrant non-diabetic leg ulcers (University of Texas Grade 2-B to 3-D, ie, wound penetrating to the tendon, capsule, bone, or joint with infection and/or ischemia) were recruited and divided into TTT (n â€‹= â€‹42) and control (n â€‹= â€‹43) groups based on the treatment they received. There were 36 (85.7%) arterial ulcers, 4 (9.5%) venous ulcers and 2 (4.8%) mixed ulcers in the TTT group and 32 (74.4%) arterial ulcers, 7 (16.7%) venous ulcers and 4 (9.3%) mixed ulcers in the control group (p â€‹> â€‹0.05). The two groups were matched on demographic and clinical characteristics. Patients in the TTT group underwent tibial corticotomy followed by 4 weeks of distraction medially then laterally, while those in the control group received conventional surgeries (debridements, revascularization, reconstruction with flaps, or skin grafts or equivalents). Ulcer healing and healing time, limb salvage, recurrence, and patient death were evaluated at a 1-year follow-up. Changes in leg small vessels were assessed in the TTT group using computed tomography angiography (CTA). RESULTS: TTT group had higher healing rates at 1-year follow-up than the control group (78.6% [33/42] vs. 58.1% [25/43], OR 2.64 [95% CI 1.10 to 6.85], p â€‹= â€‹0.04). The healing time of the TTT group was shorter than the control group (4.5 vs. 6.1 months, mean difference -1.60 [95% CI -2.93 to -0.26], p â€‹= â€‹0.02). There were no significant differences in rates of major amputation, reulceration, or mortality between the groups (p â€‹> â€‹0.05). TTT group displayed more small vessels 4 weeks postoperatively at the wound area, the foot, and the calf of the ipsilateral side in CTA. All patients in the TTT group achieved good union at the osteotomy site and had no skin or soft tissue necrosis or infection around the incision area. CONCLUSION: The findings showed that TTT facilitated the healing of recalcitrant non-diabetic leg ulcers and reduced the healing time compared with conventional surgeries. They suggest that TTT is an effective procedure to treat recalcitrant non-diabetic foot ulcers compared with standard surgical therapy. The procedure of TTT is relatively simple. Randomized controlled trials are required to confirm these findings. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: TTT can be used as an effective treatment for recalcitrant non-diabetic leg ulcers in patients. The mechanism may be associated with the neovascularization in the ulcerated foot induced by TTT and consequently increased perfusion. Together with previous findings from recalcitrant diabetic leg ulcers, the findings suggest TTT as an effective procedure to treat recalcitrant chronic leg ulcers.

Ferroptosis-Related Long Non-Coding RNA Signature Contributes to the Prediction of Prognosis Outcomes in Head and Neck Squamous Cell Carcinomas.

Background: Head and neck squamous cell carcinoma (HNSCC) is a malignant tumor, which makes the prognosis prediction challenging. Ferroptosis is an iron-dependent form of non-apoptotic regulated cell death, which could affect cancer development. However, the prognostic value of ferroptosis-related long non-coding RNA (lncRNA) in HNSCC is still limited. Methods: In the current study, we employed the DESeq2 method to characterize the differentially expressed ferroptosis-related genes (FEGs) between cancer and normal samples. Next, the FEG-related lncRNAs (FElncRNAs) were identified using Spearman's correlation analysis and multiple permutation hypotheses. Subsequently, LASSO and stepwise multivariate Cox regression analyses were undertaken to recognize the prognosis-related FElncRNA signature (PFLS) and risk scores. Results: Herein, we first identified 60 dysregulated FEGs and their co-expressed FElncRNAs in HNSCC. Then, we recognized a set of six FElncRNAs PFLS (SLCO4A1-AS1, C1RL-AS1, PCED1B-AS1, HOXB-AS3, MIR9-3HG, and SFTA1P) for predicting patients' prognostic risks and survival outcomes. We also assessed the efficiency of PFLS in the test set and an external validation cohort. Further parsing of the tumor immune microenvironment showed the PFLS was closely associated with immune cell infiltration abundances. Notably, the low-risk group of the PFLS showed a higher MHC score and cytolytic activity (CYT) score than the high-risk group, implying the low-risk group may have greater tumor surveillance and killing ability. In addition, we observed that the expression levels of two immune checkpoints (ICPs), i.e., programmed cell death protein 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1), showed significant associations with patients' risk score, prompting the role of the PFLS in ICP blockade therapy. Finally, we also constructed a drug-PFLS network to reinforce the clinical utilities of the PFLS. Conclusion: In summary, our study indicated that FElncRNAs played an important role in HNSCC survival prediction. Identification of PFLS will contribute to the development of novel anticancer therapeutic strategies.

Genome-wide investigation of Dmrt gene family in large yellow croaker (Larimichthys crocea).

The Dmrt (Doublesex and Mab-3 related transcription factor) gene family is a class of crucial transcription factors characterized by a conserved DM (Doublesex/Mab-3) domain. Previous researches indicate this gene family is involved in various physiological processes, especially in sex determination/differentiation and gonad development. Despite the vital roles of the Dmrt gene family in physiological processes, the comprehensive characterization and analysis of the dmrt genes in large yellow croaker (Larimichthys crocea), one of the most commercially important marine fish in China, have not been described. In this study, we performed the first genome-wide systematic analysis of L. crocea dmrt genes through the bioinformatics method. A total of seven members of the Dmrt gene family including Lcdmrt1, Lcdmrt2a, Lcdmrt2b, Lcdmrt3, Lcdmrt4, Lcdmrt5, and Lcdmrt6 were excavated based on the genome data of L. crocea. Further analysis revealed that the dmrt genes of L. crocea were distributed unevenly across four chromosomes. There were three dmrt genes (Lcdmrt1, Lcdmrt2a, and Lcdmrt3) on 3rd chromosome, one (Lcdmrt6) on 13th chromosome, one (Lcdmrt4) on 14th chromosome, two on (Lcdmrt5 and Lcdmrt2b) 17th chromosome. The gene structure analysis indicated that the number of introns of different dmrt genes of L. crocea had some differences: Lcdmrt1 had four introns, Lcdmrt2a, Lcdmrt2b, and Lcdmrt6 had two introns, Lcdmrt3, Lcdmrt4, and Lcdmrt5 had only one intron. The expression pattern analysis with published gonad transcriptome datasets and further confirmed by qRT-PCR revealed that these members of the Dmrt gene family except for Lcdmrt4 were all sexually dimorphic and preferred expressing in testis. Furthermore, the expression pattern analysis also revealed that the expression level of Lcdmrt1 and Lcdmrt6 was significantly higher than that of other members, suggesting that these two genes may play a more important role in testis. Overall, our studies provide a comprehensive insight into the Dmrt gene family members and a basis for the further study of their biological functions in L. crocea.

Semi-quantitative evaluation of seafood spoilage using filter-paper strips loaded with an aggregation-induced emission luminogen.

The method for seafood spoilage detection is far from satisfactory for ensuring food safety and security. Here, we develop a simple and cost-effective method using the filter papers loaded with a dihydroquinoxaline derivative (H + DQ2) to monitor salmon spoilage. The correlation between the content of solid biogenic amines and the photoluminescence intensity (PL) of H + DQ2 induced by amine vapours showed that the PL intensities of H + DQ2 increased with the increase of spoilage, which indicates that it is feasible to evaluate the spoilage degree of salmon based on the PL intensity of H + DQ2-loaded filter papers by semi-quantitation. The optimum detection condition is 75, 50 and 50 g of salmon, 75, 25 and 10 µM H + DQ2 at 0, 4 and 25 °C, respectively. This study provides a quick and simple way for testing amine vapour from fish and provides baseline information for developing an easy-to-use on-site method to evaluate seafood quality for customers.

Evidence of virus-responsive pathways in response to poly I: C challenge in a muscle cell line derived from large yellow croaker Larimichthys crocea.

In this study, a new continuous muscle cell line, LYCMS (large yellow croaker muscle cell line), derived from the muscle tissue of larva of large yellow croaker (Larimichthys crocea) was developed with modified DMEM/F12 medium at 27 °C. The muscle cell line could be passaged at different ratios for different growth rates. Karyotype analysis showed that a large proportion of LYCMS cells had 48 chromosomes. The proliferation of LYCMS cell line could be affected by mammalian growth factors such as human basic fibroblast growth factor (b-FGF), epidermal growth factor (EGF), and hepatocyte growth factor (HGF). GFP expression experiments indicated that the LYCMS cell line could be used for exogenous genes' expression. Different virus response-related genes tested in this study showed diverse change types in expression before and after (0-24 h) polycytidylic acid (poly I: C) challenge of LYCMS cells. This is the first study of virus response signaling pathways of large yellow croaker based on the muscle cell line. The results showed that compared with the in vivo experiments, the use of the LYCMS cell line for immune research is more convenient, efficient, and rapid. By using this model, we demonstrated that MDA5-IPS1-TRAF6-NFκB-cytokines, MDA5-IPS1-TRAF3-IRF3-interferon or TLR22-TRIF-IRF3-interferon, TLR8-MyD88-NFκB-cytokines, and TLR3-TRIF-IRF3-interferon pathways were able to response to poly I: C challenge in the muscle cell line of large yellow croaker.

[The effectiveness of Ilizarov technique-based transverse tibial bone transport on treatment of severe diabetic foots complicated with systemic inflammation response syndrome].

Objective: To evaluate the effectiveness of Ilizarov technique-based transverse tibial bone transport on the treatment of severe diabetic foot ulcer (Wagner grades 3 to 5) complicated with systemic inflammatory response syndrome (SIRS). Methods: Between August 2014 and December 2017, 33 patients with severe diabetic foot and SIRS were treated with Ilizarov technique-based transverse tibial bone transport. There were 27 males and 6 females, with a mean age of 60.6 years (range, 34-79 years). All of them suffered from type 2 diabetes mellitus. The duration of diabetes was 1-28 years (mean, 10 years) and the duration of diabetic foot was 1-12 months (mean, 2.7 months). According to Wagner classification, there were 8 cases in grade 3, 23 cases in grade 4, and 2 cases in grade 5. The wound healing condition was observed after operation, and the limb salvage rate was calculated. The changes in body temperature, heart rate, respiratory rate, white blood cell count, erythrocyte sedimentation rate, and C-reactive protein concentration were assessed. The skin temperature of the dorsum of the foot was measured, and the visual analogue scale (VAS) score was used to evaluate the improvement of foot pain. Results: All 33 patients were followed up 3-30 months (mean, 14.1 months). All ulcers healed and the healing time was 3-12 months (mean, 5.3 months); the limb salvage rate was 100%. Postoperative body temperature, heart rate, respiratory rate, white blood cell count, erythrocyte sedimentation rate, and C-reactive protein concentration were significantly lower than those before operation ( P<0.05). The skin temperature of the dorsum of the foot was (32.64±2.17)℃ at 1 month after operation, which was significantly improved when compared with preoperative value [(31.28±1.99)℃] ( t=0.05, P=0.00); but there was no significant difference in skin temperature compared with healthy side [(32.46±2.10)℃] ( t=2.04, P=0.41). The VAS score was 2.4±0.7 at 1 month after operation, which was significantly improved when compared with preoperative score (4.3±0.8) ( t=3.10, P=0.00). Conclusion: Ilizarov technique-based transverse tibial bone transport is an effective way to treat severe diabetic foot complicated with SIRS. It can promote foot ulcer healing and avoid amputations.

Quantitative analysis on the characteristics of targets with FDA approved drugs.

Accumulated knowledge of genomic information, systems biology, and disease mechanisms provide an unprecedented opportunity to elucidate the genetic basis of diseases, and to discover new and novel therapeutic targets from the wealth of genomic data. With hundreds to a few thousand potential targets available in the human genome alone, target selection and validation has become a critical component of drug discovery process. The explorations on quantitative characteristics of the currently explored targets (those without any marketed drug) and successful targets (targeted by at least one marketed drug) could help discern simple rules for selecting a putative successful target. Here we use integrative in silico (computational) approaches to quantitatively analyze the characteristics of 133 targets with FDA approved drugs and 3120 human disease genes (therapeutic targets) not targeted by FDA approved drugs. This is the first attempt to comparatively analyze targets with FDA approved drugs and targets with no FDA approved drug or no drugs available for them. Our results show that proteins with 5 or fewer number of homologs outside their own family, proteins with single-exon gene architecture and proteins interacting with more than 3 partners are more likely to be targetable. These quantitative characteristics could serve as criteria to search for promising targetable disease genes.

Huge proteins in the human proteome and their participation in hereditary diseases.

Protein lengths vary considerably from a few to thousands of amino acids and length variations are documented to have multiple effects. A computational approach to investigate the functional impact of protein length variation in genetic disorders is presented. The genes for huge proteins are found to have more introns. Our analysis also shows greater involvement of huge proteins in hereditary diseases.

[A clinical trial of anti-CD4 CD8 monoclonal antibodies combined with cyclosporine A in treatment of severe aplastic anemia].

OBJECTIVE: To explore the safety and efficacy of a therapeutic regimen for treating severe aplastic anemia (SAA) in its early stage. METHODS: Two groups of SAA patients were enrolled in the present study. One was a treatment group including 21 patients being treated with anti-CD(4), CD(8) monoclonal antibodies as well as cyclosporine A. Another was a control group including 20 patients being treated with anti-lymphocyte globulin and cyclosporine A. RESULTS: The response rates in the two groups were more or less some, being 76.2% for the treatment group and 65.0% for the control group (P > 0.05), but the blood routine examination results showed quicker recovery in the treatment group than in the control group (P < 0.05) and the incidences of side effects related to therapy such as fever were less in the treatment group than in the control group (P < 0.01). CONCLUSIONS: The combination of anti-CD(4), CD(8) monoclonal antibodies and cyclosporine A is safe and efficient in treating SAA.