Distinct fibroblast subpopulations associated with bone, brain or intrapulmonary metastasis in advanced non-small-cell lung cancer.

BACKGROUND: Bone or brain metastases may develop in 20-40% of individuals with late-stage non-small-cell lung cancer (NSCLC), resulting in a median overall survival of only 4-6 months. However, the primary lung cancer tissue's distinctions between bone, brain and intrapulmonary metastases of NSCLC at the single-cell level have not been underexplored. METHODS: We conducted a comprehensive analysis of 14 tissue biopsy samples obtained from treatment-naïve advanced NSCLC patients with bone (n = 4), brain (n = 6) or intrapulmonary (n = 4) metastasis using single-cell sequencing originating from the lungs. Following quality control and the removal of doublets, a total of 80 084 cells were successfully captured. RESULTS: The most significant inter-group differences were observed in the fraction and function of fibroblasts. We identified three distinct cancer-associated fibroblast (CAF) subpopulations: myofibroblastic CAF (myCAF), inflammatory CAF (iCAF) and antigen-presenting CAF (apCAF). Notably, apCAF was prevalent in NSCLC with bone metastasis, while iCAF dominated in NSCLC with brain metastasis. Intercellular signalling network analysis revealed that apCAF may play a role in bone metastasis by activating signalling pathways associated with cancer stemness, such as SPP1-CD44 and SPP1-PTGER4. Conversely, iCAF was found to promote brain metastasis by activating invasion and metastasis-related molecules, such as MET hepatocyte growth factor. Furthermore, the interaction between CAFs and tumour cells influenced T-cell exhaustion and signalling pathways within the tumour microenvironment. CONCLUSIONS: This study unveils the direct interplay between tumour cells and CAFs in NSCLC with bone or brain metastasis and identifies potential therapeutic targets for inhibiting metastasis by disrupting these critical cell-cell interactions.

Relevance of pyroptosis-associated genes in nasopharyngeal carcinoma diagnosis and subtype classification.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a highly aggressive and metastatic malignancy originating in the nasopharyngeal tissue. Pyroptosis is a relatively newly discovered, regulated form of necrotic cell death induced by inflammatory caspases that is associated with a variety of diseases. However, the role and mechanism of pyroptosis in NPC are not fully understood. METHODS: We analyzed the differential expression of pyroptosis-related genes (PRGs) between patients with and without NPC from the GSE53819 and GSE64634 datasets of the Gene Expression Omnibus (GEO) database. We mapped receptor operating characteristic profiles for these key PRGs to assess the accuracy of the genes for disease diagnosis and prediction of patient prognosis. In addition, we constructed a nomogram based on these key PRGs and carried out a decision curve analysis. The NPC patients were classified into different pyroptosis gene clusters by the consensus clustering method based on key PRGs, whereas the expression profiles of the key PRGs were analyzed by applying principal component analysis. We also analyzed the differences in key PRGs, immune cell infiltration and NPC-related genes between the clusters. Finally, we performed differential expression analysis for pyroptosis clusters and obtained differentially expressed genes (DEGs) and performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. RESULTS: We obtained 14 differentially expressed PRGs from GEO database. Based on these 14 differentially expressed PRGs, we applied least absolute shrinkage and selection operator analysis and the random forest algorithm to obtain four key PRGs (CHMP7, IL1A, TP63 and GSDMB). We completely distinguished the NPC patients into two pyroptosis gene clusters (pyroptosis clusters A and B) based on four key PRGs. Furthermore, we determined the immune cell abundance of each NPC sample, estimated the association between the four PRGs and immune cells, and determined the difference in immune cell infiltration between the two pyroptosis gene clusters. Finally, we obtained and functional enrichment analyses 259 DEGs by differential expression analysis for both pyroptosis clusters. CONCLUSIONS: PRGs are critical in the development of NPC, and our research on the pyroptosis gene cluster may help direct future NPC therapeutic approaches.

Efficacy and safety of durvalumab + chemotherapy vs. atezolizumab + chemotherapy in the treatment of small­cell lung cancer: a retrospective comparative cohort study.

Background: Durvalumab and atezolizumab have recently been approved in extensive small cell lung cancer (SCLC) with moderate median overall survival (OS) improvements. However, only limited data exist regarding the impact of immunotherapy in real-world SCLC patients. This study sought to assess the efficacy and safety of atezolizumab plus chemotherapy and durvalumab plus chemotherapy in the treatment of SCLC in a real-world setting. Methods: A retrospective cohort study of all patients treated for SCLC with chemotherapy with PD-L1 inhibitor, at 3 centers in China between February 1, 2020 and April 30, 2022. Patient characteristics, adverse-events and survival analyses were conducted. Results: A total of 143 patients were enrolled in this study, 100 were treated with durvalumab and the remainder with atezolizumab. The baseline characteristics of the two groups were fundamentally balanced before using PD-L1 inhibitors (P>0.05). The median OS (mOS) of the patients who received durvalumab or atezolizumab as the first-line treatment were 22.0 and 10.0 months, respectively (P=0.03). Survival analysis of patients with brain metastasis (BM) revealed that the median progression-free survival (mPFS) of patients without BM treated with durvalumab plus chemotherapy (5.5 months) was longer than that of those with BM (4.0 months) (P=0.03). In contrast, in the atezolizumab plus chemotherapy regimen, BM did not affect survival. In addition, the addition of radiotherapy to treatment with PD-L1 inhibitors in combination with chemotherapy has a tendency to improve long-term survival. As for safety analysis, there was no significant difference in the incidence of immune-related adverse events (IRAEs) during PD-L1 inhibitor therapy between the 2 groups (P>0.05). And during treatment with immunochemotherapy, radiotherapy was not associated with the development of IRAE (P=0.42) but increased the risk of immune-related pneumonitis (P=0.026). Conclusions: The implication of this study for clinical practice is a preference for durvalumab in first-line immunotherapy for SCLC. In addition, appropriate radiotherapy during treatment with PD-L1 inhibitors in combination with chemotherapy may prolong long-term survival, but the occurrence of immune-related pneumonitis should be vigilant. Data from this study are limited and the baseline characteristics of the two populations still need to be more finely classified.

Expression characteristics of Hsd3b7 in the gonads of Paralichthys olivaceus.

Steroidogenesis is an important biological process for gonadal differentiation and development. In mammals, 3ß-hydroxysteroid dehydrogenase 7 (HSD3B7) could convert 3ß-hydroxy of 7α-hydroxycholesterol into a ketone and form 7α-hydroxy-4-cholesten-3-one, which may affect steroidogenesis. However, in fish, the study of Hsd3b7 is still lacking. In this study, Hsd3b7 was identified in the olive flounder Paralichthys olivaceus, an important mariculture fish. According to bioinformatics analysis, Hsd3b7 belongs to a Rossmann-fold NAD(P)(+)-binding protein and can interact in a predictable manner with Hsd17b2, -3, and - 4, which play a role in steroidogenesis. In the adult flounder, Hsd3b7 was expressed in various tissues, at particularly high level in male muscle. The expression levels of Hsd3b7 at gonadal development stages I-V initially increased and then decreased, with an inflection point in the ovary at stage III and in the testis at stage IV. At stage III, the expression level of Hsd3b7 was significantly higher in the ovary than in the testis (P < 0.01). The results of in situ hybridization (ISH) revealed that it was mainly expressed in oocytes of phases I-IV or around oocytes of phases IV-V in the ovaries and around spermatid lobules at stages IV-V in the testes. Three regulatory sites of SRY-box transcription factor 9 (Sox9), a transcription factor involved in steroidogenesis and gonadal differentiation, were predicted in the promoter of Hsd3b7. After intraperitoneal injection with the recombination flounder Sox9a, the expression of Hsd3b7 was significantly up-regulated (P < 0.01). During the flounder gonadal differentiation, 17ß-estradiol (E2, 5 µg/g feed) and 17α-methyltestosterone (T, 5 µg/g feed) were used to obtain the phenotypic female or male flounder, and the results showed that in the E2 group, Hsd3b7 expression was highest at 2 cm TL, the primordial gonad stage, which was significantly higher than that at 12 cm TL (P < 0.05). In the T group, Hsd3b7 expression level was also highest at 2 cm TL and significantly higher than at 10 and 12 cm TL (P < 0.05). Moreover, Hsd3b7 was detected to be localized mainly around oogonia and spermatogonia during the differentiated gonads with ISH. These findings first introduce the expression characteristics of Hsd3b7 and the effect of Sox9a on its expression, which contribute to our understanding of the function of Hsd3b7 in fish gonads.

Real-world data on efficacy and safety of osimertinib in non-small cell lung cancer patients with EGFR T790M mutation detected by first and repeat rebiopsy.

BACKGROUND: Osimertinib could effectively target epidermal growth factor receptor (EGFR) T790M resistance mutations in non-small cell lung cancer (NSCLC), indicating that rebiopsy may be particularly important. However, the clinical benefit of repeat rebiopsy in T790M-negative patients with NSCLC detected by the first rebiopsy is still unclear, and data on the efficacy and safety of osimertinib in patients with NSCLC who are T790M-positive patients on a repeat rebiopsy remain rare. METHODS: We retrospectively collected the clinical data of advanced NSCLC patients with common EGFR mutation who were treated with 1/2-generation (1/2G) EGFR-tyrosine kinase inhibitors (TKIs) in first-line therapy in our center from January 2018 to December 2020. The detection rate of T790M by first and repeat rebiopsy was recorded, and we also analyzed the efficacy and safety of osimertinib for T790M-positive patients. RESULTS: Among 190 common EGFR-mutant patients who received 1/2G EGFR-TKIs with advanced NSCLC in the first-line treatment, 141 patients developed progressive disease. In total, 110 of 141 accepted the first rebiopsy, with a T790M prevalence of 50.9% (56/110). In total, 43 T790M-positive patients who received osimertinib were included in first rebiopsy group. Of 54 T790M-negative patients detected by the first rebiopsy, 28 underwent repeated rebiopsy in subsequent clinical treatment, and 10 (35.7%) T790M-positive cases were confirmed. In total, eight T790M-positive patients treated with osimertinib were included in repeat rebiopsy group. Overall, 66 (60%) of 110 patients acquired a T790M mutation. In patients with the T790M mutation discovered by the first and repeat rebiopsy, osimertinib resulted in median progression-free survival of 7 (95% confidence interval [CI]: 5.3-8.7) and 6 (95% CI: 4.7-7.3) months, respectively (p = .656). The median overall survival since osimertinib initiation for T790M-positive patients at first rebiopsy was 20 (95% CI: 15.1-24.9) months and 19 (95% CI: 16.9-21.1) months, for those at repeated rebiopsy (p = .888). Adverse events of grade 3 or higher were similar in the two groups (25.6% vs. 12.5%, p = .616). There was no treatment-related death in the two groups. CONCLUSIONS: Repeat rebiopsy can increase the T790M mutation positivity rate. Osimertinib showed similar efficacy and safety in T790M-positive patients whether detected by the first or repeat rebiopsy.

Expression of scp3 and dazl reveals the meiotic characteristics of the olive flounder Paralichthys olivaceus†.

Olive flounder Paralichthys olivaceus is an important cultured marine fish. We found that the meiosis marker scp3 and its intrinsic regulator dazl were mainly expressed in the gonads. During the ovarian differentiation, scp3 signal was detected first in pre-meiotic oogonia at 60-mm total length (TL) and then in primary oocytes at 80- and 100-mm TL, with a sharp increase in scp3 expression level observed at 80- and 100-mm TL. Dazl signal was detected in primordial germ cells at 30-mm TL and oogonia at 60-mm TL, but no significant change of expression was observed. During the testicular differentiation period, scp3 and dazl expression remained at low levels, and scp3 signal was weakly detected in spermatogonia at 80-mm TL, whereas dazl signal was not found. During the ovarian developmental stages, the highest expression levels of scp3 and dazl were detected at stages I and II, respectively, and strong signals of scp3 and dazl were detected in primary oocytes and oocytes at phases I and II. In the testis, the high expression of scp3 and dazl was detected at stages II-IV and II-III, respectively. Scp3 signal was weakly observed in pre-meiotic spermatogonia at stages I and II and strongly detected in primary spermatocytes at stages III-V. Dazl was detected in the nuclei of spermatogonia and spermatids at stages II-IV. Furthermore, scp3 expression in the ovary could be promoted by 17α-ethynylestradiol and tamoxifen, whereas dazl expression could be downregulated by tamoxifen.

Rxrs and their partner receptor genes inducing masculinization plausibly mediated by endocrine disruption in Paralichthys olivaceus.

Retinoid X receptors (RXRs) can form homo- or heterodimers with orphan receptors involved in multiple intertwined signaling pathways. However, there is limited study on the formation of sex phenotypes and the regulation of steroidogenesis by RXRs in fish. Here, in Paralichthys olivaceus, we first indicated that PPARγ::RXRα was predictably a transcription factor for steroidogenesis genes, and Foxl2 and Dmrt1 were also transcription factors for rxrs and their partner receptor genes. When the flounder fry were exposed to LG100268 (LG, RXRs agonist, 50 mg/kg diet), the percentage of males increased from 50% to 71.4%. Before histological differentiation of the flounder ovary (3 cm TL) and testis (6 cm TL), the transcripts of rar ß and rar γ (P < 0.05) were activated, and the steroidogenesis gene Hsd3b1 was down-regulated (P < 0.05). The ratios of testosterone (T)/17ß-estradiol (E2) were all greatly increased (P < 0.05), and the ratio of 11-ketotestosterone (11-KT)/E2 was elevated at 3 cm TL. Moreover, LG was used to treat the cultured gonads in vitro (10 µM) and the fish with intraperitoneal injection in vivo (12 mg/kg body weight), respectively. LG was able to up-regulate rxr γ, rar γ, and ppar δ, and Hsd3b1 was significantly up-regulated (P < 0.05). The ratios of 11-KT/E2 in the culture medium and in the ovaries of the fish were decreased. Furthermore, the recombinant flounder Foxl2 protein was able to significantly down-regulate ppar γ (P < 0.05) and tr ß (P < 0.01) in the ovaries in vitro, and the result of the Dmrt1 in the testes was opposite to that of the Foxl2, probably indicating a feedback loop between RXRs' partner receptors and Foxl2/Dmrt1. These findings introduce for the first time the mode of action of RXRs on the flounder steroidogenesis and provide important data to learn the potential function of RXRs in fish sex differentiation and the potential role of RXRs in aquatic animals in the presence of water pollutants.

Evaluation of nitroreductase activity in nasopharyngeal carcinoma progression by an activatable two-photon fluorescent probe.

Nasopharyngeal carcinoma (NPC) originating from the epithelium cells is the most common malignant tumor of the head and neck. Small-molecule fluorescent probes for early diagnosis of NPC can effectively improve the 5-year survival rate of patients, which makes it become a research hotspot in recent years. Previous studies have suggested the expression levels of NTR in hypoxic tissues or cells and tumors increased relative to the normal state and were positively correlated with the degree of hypoxia. Regarding the mentioned above, we designed a two-photon fluorescent probe NaT-NTR for the detection of NTR in nasopharyngeal cell lines and tissues at different hypoxia levels. NaT-NTR showed high selectivity and sensitivity toward NTR in a complex physiological environment. Furthermore, imaging NTR in different cell lines revealed that the level of intracellular NTR might be positively correlated with the malignancy of nasopharyngeal carcinoma. More importantly, NaT-NTR was successfully applied to detect and image NTR in human nasopharyngeal carcinoma with a penetration depth of 100 µm. On this basis, NaT-NTR might be a powerful chemical tool for the early diagnosis of nasopharyngeal carcinoma.

Function of Foxl2 and Dmrt1 proteins during gonadal differentiation in the olive flounder Paralichthys olivaceus.

Study on fish sex differentiation is important both from academic and practical aspects. Foxl2 and Dmrt1 are important transcription factors that should be involved in fish gonadal differentiation, but there is still no direct evidence to clarify their protein functions. Olive flounder Paralichthys olivaceus, an important mariculture fish in China, Japan, and Korea, shows sex-dimorphic growth. In this study, the Foxl2 and Dmrt1 proteins were detected in granulosa cells of the ovary and Sertoli cells of the testis, respectively, showing significant sex-dimorphic expression patterns. Then, bioactive high-purity Foxl2 and Dmrt1 recombinant proteins were obtained in vitro. Furthermore, effects of the recombinant Foxl2 and Dmrt1 during gonadal differentiation period were evaluated by intraperitoneal injection in juvenile fish. Compared with the control group, the male rate in the Dmrt1 group increased from 0 % to 82 %, showing for the first time in fish that the recombinant Dmrt1 could alter the sex phenotype. In addition, transcription levels of cyp19a and its transcription factors also changed after the recombinant Foxl2 and Dmrt1 injection. These findings reveal that Foxl2 and Dmrt1 are vital regulators for fish gonadal differentiation by regulating cyp19a expression, and also provide a new approach for sex control in fish aquaculture.

Construction and validation of immune-related LncRNAs classifier to predict prognosis and immunotherapy response in laryngeal squamous cell carcinoma.

BACKGROUND: Rapid advances in transcriptomic profiles have resulted in recognizing IRLs (immune-related long noncoding RNAs), as modulators of the expression of genes related to immune cells that mediate immune inhibition as well as immune stimulatory, indicating LncRNAs play fundamental roles in immune modulation. Hence, we establish an IRL classifier to precisely predict prognosis and immunotherapeutic efficiency in laryngeal squamous cell carcinoma (LSCC). METHODS: LSCC RNA-seq (RNA sequencing) datasets, somatic mutation data, and corresponding clinicopathologic information were acquired from TCGA (the Cancer Genome Atlas) and Gene Expression Omnibus (GEO) databases. Spearman correlation analysis identified LncRNAs associated with immune-related genes (IRG). Based on Lasso penalized regression and random forest (RF), we constructed an IRL classifier associated with prognosis. GEO database was utilized to validate the IRL classifier. The predictive precision and clinical application of the IRL classifier were assessed and compared to clinicopathologic features. The immune cell infiltration of LSCC was calculated via CIBERSORTx tools and ssGSEA (single-sample gene set enrichment analysis). Then, we systematically correlated the IRL classifier with immunological characteristics from multiple perspectives, such as immune-related cells infiltrating, tumor microenvironment (TME) scoring, microsatellite instability (MSI), tumor mutation burden (TMB), and chemokines. Finally, the TIDE (tumor immune dysfunction and exclusion) algorithm was used to predict response to immunotherapy. RESULTS: Based on machine learning approach, three prognosis-related IRLs (BARX1-DT, KLHL7-DT, and LINC02154) were selected to build an IRL classifier. The IRL classifier could availably classify patients into the low-risk and high-risk groups based on the different endpoints, including recurrence-free survival (RFS) and overall survival (OS). In terms of predictive ability and clinical utility, the IRL classifier was superior to other clinical characteristics. Encouragingly, similar results were observed in the GEO databases. Immune infiltration analysis displayed immune cells that are significantly richer in low-risk group, CD8 T cells and activated NK cells via CIBERSORTx algorithm as well as activated CD8 T cell via ssGSEA. Additionally, compared with the high-risk group, immune score, CD8 T effector was higher in the low-risk group, yet stromal score, score of p53 signaling pathway and TGFher in the Tx algorithm, was lower in the low-risk group. Corresponding results were confirmed in GEO dataset. Finally, TIDE analysis uncovered that the IRL classifier may be effectually predict the clinical response of immunotherapy in LSCC. CONCLUSION: Based on BARX1-DT, KLHL7-DT, and LINC02154, the IRL classifier was established, which can be used to predict the prognosis, immune infiltration status, and immunotherapy response in LSCC patients and might facilitate personalized counseling for immunotherapy.

Sexual dimorphism of DNA and histone methylation profiles in the gonads of the olive flounder Paralichthys olivaceus.

DNA methylation and histone methylation are two types of the most important epigenetic modifications. However, research on their differential expression in gonads of male and female fish is limited. In this study, we examined the characteristics of DNA methylation and tri-methylation of lysine 4 of histone H3 (H3K4me3) modification profiles in the gonads of the wild-type and meio-gynogenetic olive flounders Paralichthys olivaceus. Enzyme-linked immunosorbent assay (ELISA) analysis revealed that the global DNA methylation level was higher in the testis than in the ovary. Real-time quantitative PCR (qPCR) results indicated that maintenance DNA methyltransferase gene dnmt1 and de novo DNA methyltransferase gene dnmt3a are highly expressed in the ovary, while DNA demethyltransferase genes tets are highly expressed in the testis. The inconsistency of DNA methylation and methyltransferase genes in the gonads might associate with the differential distribution in the testis. 5-mC mainly located in the spermatids of the testis was shown with immunohistochemistry (IHC). Furtherly, dnmt3a and tets are mainly located in spermatocytes and oocytes with in situ hybridization (ISH) analysis. As for H3K4me3, total level is higher in the ovary detected with western blot assay. IHC results showed that the signals of H3K4me3 in Sertoli cells of the testis were stronger than those in spermatocytes and spermatids. Methyltransferase gene kmt2b and demethylase genes kdm5a and kdm5c also exhibit much higher expression in the testis with qPCR, and ISH stronger signals of kmt2b and kdm5s were detected in spermatocytes. These results implied that DNA methylation and H3K4me3 are involved in the flounder sex differences and gametogenesis.

Sexually dimorphic expression and regulatory sequence of dnali1 in the olive flounder Paralichthys olivaceus.

Dynein axonemal light intermediate chain 1 (dnali1) is an important part of axonemal dyneins and plays an important role in the growth and development of animals. However, there is little information about dnali1 in fish. Herein, we cloned dnali1 gene from the genome of olive flounder (Paralichthys olivaceus), a commercially important maricultured fish in China, Japan, and Korea, and analyzed its expression patterns in different gender fish. The flounder dnali1 DNA sequence contained a 771 bp open reading frame (ORF), two different sizes of 5' untranslated region (5'UTR), and a 1499 bp 3' untranslated region (3'UTR). Two duplicated 922 nt fragments were found in dnali1 mRNA. The first fragment contained the downstream coding region and the front portion of 3'UTR, and the second fragment was entirely located in 3'UTR. Multiple alignments indicated that the flounder Dnali1 protein contained the putative conserved coiled-coil domain. Its expression showed sexually dimorphic with predominant expression in the flounder testis, and lower expression in other tissues. The gene with the longer 5'UTR was specifically expressed in the testis. The highest expression level in the testis was detected at stages IV and V. Transient expression analysis showed that the 922 bp repeated sequence 3'UTR of dnali1 down-regulated the expression of GFP at the early stage in zebrafish. The flounder dnali1 might play an important role in the testis, especially in the period of spermatogenesis, and the 5'UTR and the repetitive sequences in 3'UTR might contain some regulatory elements for the cilia.

Effects of busulfan on somatic cells after inhibiting germ cells in the gonads of the young olive flounder Paralichthys olivaceus.

Busulfan is widely used in some species to inhibit germ cell proliferation. This study was conducted to evaluate effects of busulfan on germ and somatic cells in gonads of olive flounder, Paralichthys olivaceus, one of the most economically important mariculture fish species. After intraperitoneal injection with 80 (80B) or 120 (120B) mg/kg busulfan, both gonads were atrophied, and ovaries were discolored with adhesion to the visceral mass. Histological results indicated that germ cells in the gonads were detached, and there was a larger nucleus size and smaller cytoplasmic volume in spermatogonia. Numbers of oocytes and somatic cells in the ovary were both less (P < 0.05), while in the testis, numbers of spermatogonia and somatic cells were markedly lesser and greater, respectively (P < 0.05). In ovaries of the flounder treated with 80B and 120B, relative abundance of vasa and cyp19a1a mRNA transcripts was very small in the cytoplasm of oocytes, while the cyp19a1a transcript was still present in theca cells. In the testis of flounder treated with 80B and 120B, abundance of vasa was markedly less (P < 0.05) with there being very little vasa in spermatogonia and disruption of the spermatogonium structure. In the 80B treatment group, amh was in lesser abundance with there being very little amh in spermatogonia, however, with the 120B treatment there was a large amh abundance in spermatogonium with there being disruption of structure of these germ cells and Sertoli cells. Busulfan, therefore, might inhibit the development of spermatogonia in the flounder testis.

A dual responsive fluorescent probe for selective detection of cysteine and bisulfite and its application in bioimaging.

A coumarin-based dual responsive fluorescent probe with a simple structure was developed for the detection of Cys and HSO3 -. Under simulated physiological conditions, Cou-F displayed an on-off fluorescence response to Cys at 521 nm and an off-on fluorescence response to HSO3 - at 500 nm. Furthermore, Cou-F had the advantages of high sensitivity, strong specificity and rapid response. The detection limits of Cou-F toward Cys and HSO3 - were 0.54 µM and 0.65 µM, respectively. Cou-F enabled high selective responses to Cys and HSO3 - over other biologically related species. The response times of Cou-F toward Cys and HSO3 - were 80 s and 100 s. The fluorescence imaging of Cys and HSO3 - was achieved in living RAW246.7 cells.

Sex-Dependent RNA Editing and N6-adenosine RNA Methylation Profiling in the Gonads of a Fish, the Olive Flounder (Paralichthys olivaceus).

Adenosine-to-inosine (A-to-I) editing and N6-methyladenosine (m6A) are two of the most abundant RNA modifications. Here, we examined the characteristics of the RNA editing and transcriptome-wide m6A modification profile in the gonads of the olive flounder, Paralichthys olivaceus, an important maricultured fish in Asia. The gonadal differentiation and development of the flounder are controlled by genetic as well as environmental factors, and the epigenetic mechanism may play an important role. In total, 742 RNA editing events were identified, 459 of which caused A to I conversion. Most A-to-I sites were located in 3'UTRs, while 61 were detected in coding regions (CDs). The number of editing sites in the testis was higher than that in the ovary. Transcriptome-wide analyses showed that more than one-half of the transcribed genes presented an m6A modification in the flounder gonads, and approximately 60% of the differentially expressed genes (DEGs) between the testis and ovary appeared to be negatively correlated with m6A methylation enrichment. Further analyses revealed that the mRNA expression of some sex-related genes (e.g., dmrt1 and amh) in the gonads may be regulated by changes in mRNA m6A enrichment. Functional enrichment analysis indicated that the RNA editing and m6A modifications were enriched in several canonical pathways (e.g., Wnt and MAPK signaling pathways) in fish gonads and in some pathways whose roles have not been investigated in relation to fish sex differentiation and gonadal development (e.g., PPAR and RNA degradation pathways). There were 125 genes that were modified by both A-to-I editing and m6A, but the two types of modifications mostly occurred at different sites. Our results suggested that the presence of sex-specific RNA modifications may be involved in the regulation of gonadal development and gametogenesis.

Amh dominant expression in Sertoli cells during the testicular differentiation and development stages in the olive flounder Paralichthys olivaceus.

The olive flounder Paralichthys olivaceus, an important marine fish, shows gender differences in growth. The mechanism on its gonadal differentiation direction affected with exogenous factors still needs to be clarified. The anti-Müllerian hormone (amh) gene is involved in fish testicular differentiation and maintenance. The aim of this study was to investigate the expression of the flounder amh in tissues and the gonads. The quantitative expression analysis results showed that it was highly expressed in the testis, especially in the testis at stages I - IV (P < 0.05). Also, amh was detected in Sertoli cells surrounding spermatogonia and peripheral seminiferous lobule of the testis with in situ hybridization (ISH) and immunohistochemistry (IHC). During the differentiation period, the amh expression in the testis of the tamoxifen treatment group (100 ppm) was higher than that in the ovary of the 17ß-estradiol (E2, 5 ppm) group, and the expression levels of amh during process of the male differentiation in the tamoxifen group were higher than those in the 17ɑ-methyltestosterone (MT, 5 ppm) group (P < 0.05). ISH results also exhibited that amh was expressed in the somatic cells that surrounded the germ cells of juvenile flounder similar to adult ones. Furthermore, the flounder gonads in the tamoxifen group maintained more germ cells and somatic cells than those in the MT group from 20 to 80 mm total length (TL). Especially, at 60 and 80 mm TL, the numbers of germ and somatic cells in the tamoxifen group were significantly higher than those in the MT group (P < 0.05). In summary, amh might initiate the process of testicular differentiation, and is involved in the early development and maintenance of testis.

Molecular Fluorescent Probes for Imaging and Evaluation of Hypochlorite Fluctuations during Diagnosis and Therapy of Osteoarthritis in Cells and in a Mouse Model.

The early diagnosis of osteoarthritis (OA) can halt or delay the progression of the disease, and it is essentially beneficial to its treatment. However, biomarkers with sufficient sensitivity for dynamically identifying early OA are still yet to be determined. The overproduced hypochlorous acid (HOCl) has been proposed as an obvious symptom in early OA. Herein, based on the oxidation reaction of the sulfur atom in phenothiazine into sulfoxide, we design and synthesize a phenothiazine-derived coumarin fluorescent probe PDC for the detection of ClO- in cells and in an OA mouse model. The probe PDC exhibits excellent selectivity and sensitivity for ClO- detection with a limit of detection as low as 16.1 nM. Taking advantage of the probe PDC, we visualize and evaluate the level changes of ClO- in macrophage cells, which is stimulated by various inflammatory factors. The anti-inflammatory and therapeutic effects of selenocysteine and methotrexate in inflamed cells are also confirmed. Finally, with in vivo imaging of ClO- concentration changes in OA BALB/c mouse models, we successfully inspected the relationship between OA phenotypes and the burst of ClO-. We suggest that abnormal changes in HOCl concentration may be considered as a new biomarker for the early OA diagnosis.

Characteristics and sex dimorphism of 17ß-hydroxysteroid dehydrogenase family genes in the olive flounder Paralichthys olivaceus.

Sex steroid hormones play important roles in fish sex differentiation, gonadal development and secondary sexual characteristics. Olive flounder Paralichthys olivaceus is a valuable commercial marine fish species and has marked sexual dimorphism. However, the mechanisms of action of sex hormones in flounder sex are still unclear. In this study, a total of ten Hsd17b family genes, including Hsd17b3, -4, -7, -8, -9, -10, -12a, -12b, -14 and -15, were identified in the flounder, which encoded critical enzymes acting on sex steroid synthesis and metabolism. Hsd17b genes were distributed on eight chromosomes. Hsd17b12a and -12b were located on chromosomes 19 and 7, respectively. It was speculated that these two genes were just highly similar rather than different transcripts derived from the same gene. According to the results of domain and motif analyses, they all belonged to the SDR superfamily and contained conserved Hsd17b motifs TGxxxGxG, PGxxxT, NNAG and YxxxK. Analysis of amino acid sequences predicted that Hsd17b1, -4, -7, -12a and -14 were hydrophilic proteins. The stability of Hsd17b1, -3 and -12b proteins was predicted to be low. The various Hsd17b family genes differed in tissue expression pattern, and Hsd17b10, -12a and -12b were highly expressed in the flounder ovary. Moreover, throughout gonadal development, Hsd17b3 was highly expressed in the testis, and Hsd17b1, -12a and -12b were highly expressed in the ovary, suggesting that they might play an important role in testosterone synthesis in the testis or estrogen synthesis in the ovary. Activities of Hsd17b3 at stages I-V were all significantly higher in the testis than in the ovary (P < 0.05, P < 0.01). Transfection analysis in HEK293T cells showed that Hsd17b1 and -3 were located in both the cytoplasm and nucleus. Additionally, after challenging fish with tamoxifen, Hsd17b3 expression level in the testis decreased significantly (P < 0.01), and in the ovary no significant change was observed. Moreover, the expression of Hsd17b1 in the ovary was significantly upregulated after injection with flutamide (P < 0.05). These findings introduce the characteristics of the flounder Hsd17b in subfamily, which contribute to our understanding of the regulation of sex steroid hormone synthesis in fish gonadal development.

Bioimaging of Glutathione with a Two-Photon Fluorescent Probe and Its Potential Application for Surgery Guide in Laryngeal Cancer.

As the most abundant nonprotein biothiol in living cells, glutathione (GSH) prevents cellular components from oxidative damage and maintains the intracellular redox homeostasis. For further exploring whether GSH can be employed as a bioindicator to discriminate tumor lesion at a cellular level, the highly selective detection and accurate quantification of GSH under pathological conditions are critical. Herein, we design a coumarin derivative-based two-photon fluorescent probe Cou-Br for the detection of GSH in living cells, mice models, and clinical specimens. The prepared probe is capable of sensitively and selectively detecting GSH in complex biological systems. Cou-Br displays a good linear relationship in response to GSH and a low limit of detection. With the fluorescence signal positively associated with intracellular GSH levels, the probe enables real-time imaging of GSH in various cell lines. Under the condition of CS2 stimulation, Cou-Br can rapidly respond to the fluctuation of intracellular GSH induced by oxidative damage. Furthermore, the in situ and in vivo bioimaging performances of Cou-Br are demonstrated. Typically, relying on the different cellular concentrations of GSH, the probe is successfully employed to identify the human laryngeal cancer lesion with outstanding capabilities of deep tissue imaging and tumor margin recognition. We assume that the abnormal expression level of GSH may be utilized as a potential bioindicator to discriminate tumor tissues from the surrounding disease-free tissues. To conclude, the proposed probe Cou-Br may potentially serve as a powerful chemical tool for the surgical navigation of cancer in clinic.

Analyses of mRNA-seq and miRNA-seq of the brain reveal the sex differences of gene expression and regulation before and during gonadal differentiation in 17ß-estradiol or 17α-methyltestosterone-induced olive flounder (Paralichthys olivaceus).

Olive flounder (Paralichthys olivaceus) is a commercially important flatfish species cultured in East Asia. Female flounders generally grow more rapidly than males, therefore control of the sex ratio seems to be a proposed way to increase production. However, the sex determination gene and sex determination mechanism have yet been elucidated. The brain is an important organ that is involved in gonadal development. To explore the sex differences of gene expression in the brain before and during the flounder gonadal differentiation, we used messenger RNA (mRNA)-seq technology to investigate transcriptomes of male and female brains. Between female and male brains, 103 genes were differentially expressed before ovarian differentiation, 16 genes were differentially expressed before testicular differentiation, and 64 genes were differentially expressed during gonadal differentiation. According to annotation and Kyoto Encyclopedia of Genes and Genomes information, the differentially expressed genes (DEGs) were involved in circadian rhythm, circadian rhythm-fly, circadian entrainment, dopaminergic synapse, calcium signaling, glutamatergic synapse, taste transduction, herpes simplex infection, long-term depression, retrograde endocannabinoid signaling, and the synaptic vesicle cycle pathways. MicroRNA (miRNA)-seq was performed during the gonadal differentiation and the target genes of miRNAs were predicted. Integrated analysis of mRNA-seq and miRNA-seq showed that 29 of the 64 DEGs were regulated by the differentially expressed miRNAs during the gonadal differentiation. Our study provides a basis for further studies of brain sex differentiation and the molecular mechanism of sex determination in olive flounder.