Host genotype-specific therapies can optimize the inflammatory response to mycobacterial infections.

Susceptibility to tuberculosis is historically ascribed to an inadequate immune response that fails to control infecting mycobacteria. In zebrafish, we find that susceptibility to Mycobacterium marinum can result from either inadequate or excessive acute inflammation. Modulation of the leukotriene A(4) hydrolase (LTA4H) locus, which controls the balance of pro- and anti-inflammatory eicosanoids, reveals two distinct molecular routes to mycobacterial susceptibility converging on dysregulated TNF levels: inadequate inflammation caused by excess lipoxins and hyperinflammation driven by excess leukotriene B(4). We identify therapies that specifically target each of these extremes. In humans, we identify a single nucleotide polymorphism in the LTA4H promoter that regulates its transcriptional activity. In tuberculous meningitis, the polymorphism is associated with inflammatory cell recruitment, patient survival and response to adjunctive anti-inflammatory therapy. Together, our findings suggest that host-directed therapies tailored to patient LTA4H genotypes may counter detrimental effects of either extreme of inflammation.

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.

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.

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.

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.

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.

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.

Roles of forkhead box protein L2 (foxl2) during gonad differentiation and maintenance in a fish, the olive flounder (Paralichthys olivaceus).

As an important maricultured fish, the olive flounder Paralichthys olivaceus shows sex-dimorphic growth. Thus, the molecular mechanisms involved in sex control in P. olivaceus have attracted researchers' attention. Among the sex-related genes, forkhead box protein L2 (foxl2) exhibits significant sex-dimorphic expression patterns and plays an important role in fish gonad differentiation and development. The present study first investigated the expression levels and promoter methylation dynamics of foxl2 during flounder gonad differentiation under treatments of high temperature and exogenous 17ß-oestradiol (E2). During high temperature treatment, the expression of flounder foxl2 may be repressed via maintenance of DNA methylation. Then, flounder with differentiated testis at Stages I-II were treated with exogenous 5ppm E2 or 5ppm E2+150ppm trilostane (TR) to investigate whether exogenous sex hormones could induce flounder sex reversal. The differentiated testis exhibited phenotypic variations of gonadal dysgenesis with upregulation of female-related genes (foxl2 and cytochrome P450 family 19 subfamily A (cyp19a)) and downregulation of male-related genes (cytochrome P450 family 11 subfamily B member 2 (cyp11b2), doublesex- and mab-3 related transcription factor 1 (dmrt1), anti-Mullerian hormone (amh) and SRY-box transcription factor 9 (sox9)). Furthermore, a cotransfection assay of the cells of the flounder Sertoli cell line indicated that Foxl2 was able alone or with nuclear receptor subfamily 5 group A member 2 (Nr5a2) jointly to upregulate expression of cyp19a. Moreover, Foxl2 and Nr5a2 repressed the expression of dmrt1. In summary, Foxl2 may play an important role in ovarian differentiation by maintaining cyp19a expression and antagonising the expression of dmrt1. However, upregulation of foxl2 is not sufficient to induce the sex reversal of differentiated testis.

Characteristics of Cyp11a during Gonad Differentiation of the Olive Flounder Paralichthys olivaceus.

The P450 side-chain cleavage enzyme, P450scc (Cyp11a) catalyzes the first enzymatic step for the synthesis of all steroid hormones in fish. To study its roles in gonads of the olive flounder Paralichthys olivaceus, an important maricultured fish species, we isolated the cyp11a genomic DNA sequence of 1396 bp, which consists of 5 exons and 4 introns. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) results indicated that the flounder cyp11a was exclusively expressed in gonad and head kidney tissues. Its expression level in the testis was higher than that in the ovary. According to the in situ hybridization patterns, cyp11a was mainly expressed in the Leydig cells of the testis, and the thecal cells of the ovary. Immunofluorescence analysis showed that Cyp11a was located in the cytoplasm of the cultured flounder testis cells. Further quantitative real-time PCR results presented the cyp11a differential expression patterns during gonad differentiation. Among different sampling points of the 17ß-estradiol (E2, 5 ppm) treatment group, cyp11a expression levels were relatively high in the differentiating ovary (30 and 40 mm total length, TL), and then significantly decreased in the differentiated ovary (80, 100 and 120 mm TL, p < 0.05). The pregnenolone level also dropped in the differentiated ovary. In the high temperature treatment group (HT group, 28 ± 0.5 °C), the cyp11a expression level fluctuated remarkably in the differentiating testis (60 mm TL), and then decreased in the differentiated testis (80, 100 mm TL, p < 0.05). In the testosterone (T, 5 ppm) treatment group, the cyp11a was expressed highly in undifferentiated gonads and the differentiating testis, and then dropped in the differentiated testis. Moreover, the levels of cholesterol and pregnenolone of the differentiating testis in the HT and T groups increased. The expression level of cyp11a was significantly down-regulated after the cultured flounder testis cells were treated with 75 and 150 µM cyclic adenosine monophosphate (cAMP), respectively (p < 0.05), and significantly up-regulated after treatment with 300 µM cAMP (p < 0.05). Both nuclear receptors NR5a2 and NR0b1 could significantly up-regulate the cyp11a gene expression in a dosage dependent way in the testis cells detected by cell transfection analysis (p < 0.05). The above data provides evidence that cyp11a would be involved in the flounder gonad differentiation and development.

Significant association of cyp19a promoter methylation with environmental factors and gonadal differentiation in olive flounder Paralichthys olivaceus.

The sex ratio of olive flounder Paralichthys olivaceus is sensitive to temperature or exogenous hormone exposures in the period of gonadal differentiation. Among sex-related genes, cyp19a, encoding cytochrome P450 aromatase, exhibits significant sex-dimorphic expression pattern and plays an important role in fish gonadal differentiation and development. The present study investigated the expression levels and promoter methylation dynamics of cyp19a and its regulators (nr5a2 and nr0b1), and sex-steroid hormone levels during flounder gonadal differentiation under the treatments of high temperature and estradiol-17ß (E2). The results showed that levels of flounder cyp19a expression and estradiol-17ß were repressed by high temperature treatment during this period. The up-regulation of nr5a2 by E2 treatment may be related to the all-female formation, and up-regulation of nr0b1 by high temperature treatment may be associated with masculinization. Co-transfection assay indicated that nr5a2 and nr0b1 were antagonist regulators of cyp19a. Furthermore, cyp19a promoter exhibited significant demethylation phenomenon at early stage of ovarian differentiation. While, high temperature could repress the demethylation process, resulting in hypermethylation maintenance in cyp19a promoter. The hypermethylation promoter was able to suppress cyp19a expression by blocking the nr5a2-mediated transactivation activity in vitro. The DNA methylation of epigenetic modification in cyp19a promoter might be the vital way linking environmental factors and gonadal differentiation in flounder.

Molecular cloning and sexually dimorphic expression of wnt4 in olive flounder (Paralichthys olivaceus).

WNT4 (wingless-type MMTV integration site family, member 4) is regarded as a key regulator of gonad differentiation in mammalians. However, the potential role of wnt4 in teleosts during gonad differentiation and development is still unclear. The full-length cDNA sequence of wnt4 in olive flounder (Paralichthys olivaceus) was obtained using RACE (rapid amplification of cDNA ends) technique. The wnt4 ORF contains 1059 nucleotides, encoding a protein with a signal peptide domain and a wnt family domain. Expression in tissues of adult flounders was analyzed by real-time RT-PCR. The results showed that wnt4 was widely expressed in multiple tissues of flounders, and the expression level was significantly higher in ovary than in testis. Then wnt4 expression pattern was investigated during gonadal differentiation period and at gonadal development stages (I-V). The results showed the expression levels were significantly higher in testis than in ovary during gonadal differentiation. Notably, wnt4 expression had a very significant increase before testis differentiation. At gonad different developmental stages, there was no expression signal at stage I or stage II, and the expression of wnt4 was much stronger in ovary than in testis at stage III and stage IV, followed by a faint expression in stage V in both sexes. Our results imply that cloned wnt4 could be wnt4a. It is a sex-related gene and its expression pattern in gonadal differentiation period of flounder is different from that in mammalians or other teleosts. Flounder wnt4 might play more important role in testis than in ovary during gonadal differentiation.

A new pattern of primordial germ cell migration in olive flounder (Paralichthys olivaceus) identified using nanos3.

The olive flounder (Paralichthys olivaceus) is an important cultured marine fish. However, little information is available on primordial germ cell (PGC) development and migration in this species; such information is vital for applications in artificial reproduction and for the preservation of genetic resources. Here, we sought to remedy this information deficit by isolating the germline-specific gene nanos3 and analyzing its expression in olive flounder. Sequencing analysis showed that olive flounder nanos3 contained a typical RNA-binding zinc finger domain. A phylogenetic analysis demonstrated that nanos3 of the olive flounder grouped with that of the barfin flounder (Verasper moseri). In the olive flounder, nanos3 was consistently expressed during embryogenesis. Whole-mount in situ hybridization showed that a new pattern of PGC migration was present in olive flounder, which combined elements of the PGC migration patterns of medaka, herring, and goby. In olive flounder, PGCs aligned along the lateral plate mesoderm in two loose, elongated lines at early embryogenesis, aggregated into a single loose cluster at mid-embryogenesis, then re-aligned into two tight clusters at late somitogenesis, and finally migrated to the genital ridge as two clusters. Furthermore, whole-mount in situ hybridization revealed that expression of stromal derived factor 1 (Sdf1) was important for guiding of PGC migration during somitogenesis. In particular, Sdf1 directed aggregation of PGCs into a single loose cluster from the two elongated lines during mid-embryogenesis. Additionally, PGCs in zebrafish were successfully visualized by injection of chimeric RNA containing the green fluorescent protein (GFP) and 3' untranslated region of olive flounder nanos3. These findings provide new insights into PGC migration and development in olive flounder and will also facilitate germ cell manipulation in this species.

Molecular cloning and sexually dimorphic expression patterns of nr0b1 and nr5a2 in olive flounder, Paralichthys olivaceus.

The nr0b1 and nr5a2 genes, members of the nuclear receptor superfamily, are strong candidate genes involved in gonadal differentiation in several vertebrate species. In this study, an nr0b1 complementary DNA (cDNA) of 1446 bp, which encodes a predicted 298 amino acid protein, and an nr5a2 cDNA of 2425 bp, which encodes a deduced 523 amino acid protein, were obtained from olive flounder Paralichthys olivaceus. Both genes were expressed in multiple organ tissues of adult flounder, with a higher expression in ovary than in testis. Quantitative real-time RT-PCR was performed to investigate their temporal expression profiles in gonads during differentiation and at five development stages. Results indicated that nr0b1 and nr5a2 were expressed in primitive gonad and in the ensuing gonadal differentiation periods. In general, both genes were more highly expressed in ovary than in testis at all observed development stages. The expression level of cyp19a correlated with the nr5a2/nr0b1 ratio over the course of flounder gonadal differentiation; hence, nr0b1 and nr5a2 genes may be involved in flounder ovarian differentiation by regulating the expression of cyp19a.

Construction and characterization of the angR mutant of Vibrio anguillarum via insertional inactivation.

Vibrio anguillarum strain M3 was isolated from cultured diseased flounder Paralichthys olivaceus. In order to determine whether chromosomal angR gene plays an important role in infecting flounder host, part fragment of a virulence regulatory gene angR from V. anguillarum M3 genome was cloned and ligated with suicide plasmids pNQ705 in the present study. The conserved fragment of angR gene was amplified from M3 genome and inserted into suicide plasmid pNQ705, a kind of conditional replicon. The recombinant plasmid was transferred into Vibrio anguillarum M3 strain genomic DNA through bacterial conjugation and homologous recombination. A mutant V. anguillarum strain with angR gene mutation was constructed and screened successfully using TCBS medium containing chloramphenicol. PCR identification and sequencing showed that the recombinant plasmid inserted into Vibrio anguillarum genome DNA, as expected design, resulted in insertional inactivation of angR gene. Results of artificial infection experiment showed that virulence of mutant Vibrio anguillarum strain reduced dramatically as compared with the wild strain M3.

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.

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.

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.

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.

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.