Comprehensive analysis and identification of subtypes and hub genes of high immune response in lung adenocarcinoma.

BACKGROUND: The advent of immunotherapy targeting immune checkpoints has conferred significant clinical advantages to patients with lung adenocarcinoma (LUAD); However, only a limited subset of patients exhibit responsiveness to this treatment. Consequently, there is an imperative need to stratify LUAD patients based on their response to immunotherapy and enhance the therapeutic efficacy of these treatments. METHODS: The differentially co-expressed genes associated with CD8 + T cells were identified through weighted gene co-expression network analysis (WGCNA) and the Search Tool for the Retrieval of Interacting Genes (STRING) database. These gene signatures facilitated consensus clustering for TCGA-LUAD and GEO cohorts, categorizing them into distinct immune subtypes (C1, C2, C3, and C4). The Tumor Immune Dysfunction and Exclusion (TIDE) model and Immunophenoscore (IPS) analysis were employed to assess the immunotherapy response of these subtypes. Additionally, the impact of inhibitors targeting five hub genes on the interaction between CD8 + T cells and LUAD cells was evaluated using CCK8 and EDU assays. To ascertain the effects of these inhibitors on immune checkpoint genes and the cytotoxicity mediated by CD8 + T cells, flow cytometry, qPCR, and ELISA methods were utilized. RESULTS: Among the identified immune subtypes, subtypes C1 and C3 were characterized by an abundance of immune components and enhanced immunogenicity. Notably, both C1 and C3 exhibited higher T cell dysfunction scores and elevated expression of immune checkpoint genes. Multi-cohort analysis of Lung Adenocarcinoma (LUAD) suggested that these subtypes might elicit superior responses to immunotherapy and chemotherapy. In vitro experiments involved co-culturing LUAD cells with CD8 + T cells and implementing the inhibition of five pivotal genes to assess their function. The inhibition of these genes mitigated the immunosuppression on CD8 + T cells, reduced the levels of PD1 and PD-L1, and promoted the secretion of IFN-γ and IL-2. CONCLUSIONS: Collectively, this study delineated LUAD into four distinct subtypes and identified five hub genes correlated with CD8 + T cell activity. It lays the groundwork for refining personalized therapy and immunotherapy strategies for patients with LUAD.

Myeloid-specific Hdac10 deletion protects against LPS-induced acute lung injury via P62 acetylation at lysine 165.

BACKGROUND: Aberrant activation of macrophages is associated with pathogenesis of acute lung injury (ALI). However, the potential pathogenesis has not been explored. OBJECTIVES: We aimed to identify whether histone deacetylase (HDAC) 10 is involved in lipopolysaccharide (LPS)-exposed ALI and reveal the underlying pathogenesis by which it promotes lung inflammation in LPS-exposed ALI via modifying P62 with deacetylation. METHODS: We constructed an ALI mice model stimulated with LPS to determine the positive effect of Hdac10 deficiency. Moreover, we cultured murine alveolar macrophage cell line (MH-S cells) and primary bone marrow-derived macrophages (BMDMs) to explore the pro-inflammatory activity and mechanism of HDAC10 after LPS challenge. RESULTS: HDAC10 expression was increased both in mice lung tissues and macrophage cell lines and promoted inflammatory cytokines production exposed to LPS. Hdac10 deficiency inhibited autophagy and inflammatory response after LPS stimulation. In vivo, Hdac10fl/fl-LysMCre mice considerably attenuated lung inflammation and inflammatory cytokines release exposed to LPS. Mechanistically, HDAC10 interacts with P62 and mediates P62 deacetylation at lysine 165 (K165), by which it promotes P62 expression and increases inflammatory cytokines production. Importantly, we identified that Salvianolic acid B (SAB), an HDAC10 inhibitor, reduces lung inflammatory response in LPS-stimulated ALI. CONCLUSION: These results uncover a previously unknown role for HDAC10 in regulating P62 deacetylation and aggravating lung inflammation in LPS-induced ALI, implicating that targeting HDAC10 is an effective therapy for LPS-exposed ALI.

[Population status and protection evaluation of Syzygium album, a species with extremely small population.] / 极小种群植物白果蒲桃种群现状与保护评价.

Syzygium album is a plant species with extremely small population and endemic to Yun-xiao County, Fujian Province. We examined its population status and conservation evaluation. The results showed that there were 25 individuals of S. album, with only one mother tree. Except for the mother tree and one small tree, the others were all young seedlings, which was rare in abundance and lack of age stage of middle and strong trees. The spatial distribution of young seedlings was not uniform, with most of individuals within the range of 10-25 m from the mother tree. The age class structure of the population was not continuous and the population development was unsustainable. The competition mainly came from other species. Cryptocarya chinensis accounted for 66.6% of the total competition index, which was much higher than other species. We established a comprehensive evaluation index system for the protection of rare and endangered plants with extremely small populations and evaluated the protection of S. album from three aspects, i.e., endangered degree, protection value, and protection priority. The endangered degree of S. album was level Ⅰ (the endangered degree index was 4.510), belonging to extremely endangered species, the protection value was level Ⅰ (the protection value index was 4.052), which was of great value, and the protection priority was level Ⅰ (the protection priority index was 4.016), which should be listed as the highest priority protection level.

Down-regulation of MicroRNA-592 in obesity contributes to hyperglycemia and insulin resistance.

BACKGROUND: Many studies have demonstrated that microRNAs, a class of small and non-coding RNA molecules, play an important role in the regulation of glucose and lipid homeostasis. In the present study, we sought to investigate the function of miR-592 in the development of obesity-associated metabolic disorders, including hyperglycemia andinsulin resistance. METHODS: The expression levels of miR-592 were measured in the liver of obese mice and humans by quantitative reverse transcription PCR. Loss- and gain-of function experiments were employed to explore the metabolic function of miR-592 using locked nucleic acids and adenovirus in lean and obese mice, respectively. The molecular target of miR-592 was determined by western blotting and luciferase reporter assays. FINDINGS: We found a significant decreased expression of miR-592 in the liver of obese mice and humans. Inhibition of miR-592 led to elevated blood glucose levels, enhanced gluconeogenesis and reduced insulin sensitivity in lean mice. In contrast, adenovirus-mediated overexpression of hepatic miR-592 improved metabolic disorders in obese mice. Mechanistically, we found that the transcription factor forkhead box O1 (FOXO1) is a direct target gene of miR-592 to mediate its metabolic functions. miR-592 was able to inhibit the mRNA and protein expression of FOXO1 by binding to its 3'-untranslated region. INTERPRETATIONS: Our findings demonstrate that obesity-associated down-regulation of miR-592 plays an important role in the progression of metabolic diseases. Restoration of hepatic miR-592 could improve glucose and lipid metabolism in obese mice. FUND: This work is supported by the National Key Research and Development Program of China (No. 2016YFC1304805 to Dr. Chen), Natural Science Foundation of China (No. 81771574 to Dr. Wu), Shanghai Science Foundation (No. 18ZR1437800 to Dr. Li), Science and Technology Commission of Shanghai Municipality (Nos.18dz2304400 and 15,411,970,700 to Dr. Yang).

Production of Wilson Disease Model Rabbits with Homology-Directed Precision Point Mutations in the ATP7B Gene Using the CRISPR/Cas9 System.

CRISPR/Cas9 has recently been developed as an efficient genome engineering tool. The rabbit is a suitable animal model for studies of metabolic diseases. In this study, we generated ATP7B site-directed point mutation rabbits to simulate a major mutation type in Asians (p. Arg778Leu) with Wilson disease (WD) by using the CRISPR/Cas9 system combined with single-strand DNA oligonucleotides (ssODNs). The efficiency of the precision point mutation was 52.94% when zygotes were injected 14 hours after HCG treatment and was significantly higher than that of zygotes injected 19 hours after HCG treatment (14.29%). The rabbits carrying the allele with mutant ATP7B died at approximately three months of age. Additionally, the copper content in the livers of rabbits at the onset of WD increased nine-fold, a level similar to the five-fold increase observed in humans with WD. Thus, the efficiency of precision point mutations increases when RNAs are injected into zygotes at earlier stages, and the ATP7B mutant rabbits are a potential model for human WD disease with applications in pathological analysis, clinical treatment and gene therapy research.

Effects of the histone deacetylase inhibitor 'Scriptaid' on the developmental competence of mouse embryos generated through round spermatid injection.

STUDY QUESTION: Can the histone deacetylase inhibitor Scriptaid improve the efficiency of the development of round spermatid injection (ROSI)-fertilized embryos in a mouse model? SUMMARY ANSWER: Treatment of ROSI mouse zygotes with Scriptaid increased the expression levels of several development-related genes at the blastocyst stage, resulting in more efficient in vitro development of the blastocyst and an increased birth rate of ROSI-derived embryos. WHAT IS KNOWN ALREADY: The full-term development of embryos derived through ROSI is significantly lower than that following ICSI in humans and other species. STUDY DESIGN, SIZE, DURATION: Oocytes, spermatozoa and round spermatids were collected from BDF1 (C57BL/6 × DBA/2) mice. For in vitro development experiments, mouse ROSI-derived zygotes were treated with Scriptaid at different concentrations (0, 125, 250, 500 and 1000 nM) and for different exposure times (0, 6, 10, 16 or 24 h). Next, blastocysts of the optimal Scriptaid-treated group and the non-treated ROSI group were separately transferred into surrogate ICR mice to compare in vivo development with the ICSI group (control). Each experiment was repeated at least three times. PARTICIPANTS/MATERIALS, SETTING, METHODS: Metaphase II (MII) oocytes, spermatozoa and round spermatids were obtained from sexually mature BDF1 female or male mice. The developmental potential of embryos among the three groups (the ICSI, ROSI and optimal Scriptaid-treated ROSI groups) was assessed based on the rates of obtaining zygotes, two-cell stage embryos, four-cell stage embryos, blastocysts and full-term offspring. In addition, the expression levels of development-related genes (Oct4, Nanog, Klf4 and Sox2) were analysed using real-time PCR, and the methylation states of imprinted genes (H19 and Snrpn) in these three groups were detected using methylation-specific PCR (MS-PCR) sequencing following bisulfite treatment. MAIN RESULTS AND THE ROLE OF CHANCE: The in vitro experiments revealed that treating ROSI-derived zygotes with 250 nM Scriptaid for 10 h significantly improved the blastocyst formation rate (59%) compared with the non-treated group (38%) and further increased the birth rates of ROSI-derived embryos from 21% to 40% in vivo. Moreover, in ROSI-derived embryos, the expression of the Oct4, Nanog and Sox2 genes at the blastocyst stage was decreased, but the optimal Scriptaid treatment restored expression to a level similar to their ICSI counterparts. In addition, Scriptaid treatment moderately repaired the abnormal DNA methylation pattern in the imprinting control regions (ICRs) of H19 and Snrpn. LARGE SCALE DATA: N/A LIMITATIONS, REASONS FOR CAUTION: Because of the ethics regarding the use of human gametes for ROSI studies, the mouse model was used as an approach to explore the effects of Scriptaid on the developmental potential of ROSI-derived embryos. However, to determine whether these findings can be applied to humans, further investigation will be required. WIDER IMPLICATIONS OF THE FINDINGS: Scriptaid treatment provides a new means of improving the efficiency and safety of clinical human ROSI. STUDY FUNDING/COMPETING INTERESTS: The study was financially supported through grants from the National Key Research Program of China (No. 2016YFC1304800); the National Natural Science Foundation of China (Nos: 81170756, 81571486); the Natural Science Foundation of Shanghai (Nos: 15140901700, 15ZR1424900) and the Programme for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. There are no conflicts of interest to declare.

Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous recombination and gene trapping through somatic cell nuclear transfer.

The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT(+/-) cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT(+/-) rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species.

The Oct4 promoter-EGFP transgenic rabbit: a new model for monitoring the pluripotency of rabbit stem cells.

The rabbit has long been used as a laboratory animal model for developing reproductive and stem cell-related technologies, as well as for studying human disease. The Oct4 transcription factor plays a crucial role in the maintenance and regulation of pluripotency in embryos and stem cells. We constructed a reporter plasmid containing the gene encoding the enhanced green fluorescent protein (EGFP) under the control of the rabbit Oct4 promoter (prOG) and transfected it into E14 mouse stem cells and rabbit ESCs. In addition, prOG transgenic fibroblasts were derived and prOG transgenic rabbits were produced by somatic cell nuclear transfer (SCNT). The pattern of expression of ectopic EGFP was similar in E14 mouse stem cells whether under the control of the rabbit (prOG) or mouse Oct4 promoter (pmOG). EGFP expression was observed in rabbit ESCs following prOG transfection. Both prOG transgenic SCNT embryos and F1 prOG transgenic embryos derived from adult transgenic rabbits expressed green fluorescence at the morula and blastocyst stages. EGFP was clearly detected in gonads isolated from fetuses at 27 dpc. The prOG transgenic rabbit represents a new model for studying the derivation and maintenance of rabbit pluripotent cells, and for investigating rabbit embryo development.

Noggin versus basic fibroblast growth factor on the differentiation of human embryonic stem cells.

The difference between Noggin and basic fibroblast growth factor for the neural precursor differentiation from human embryonic stem cells has not been studied. In this study, 100 µg/L Noggin or 20 µg/L basic fibroblast growth factor in serum-free neural induction medium was used to differentiate human embryonic stem cells H14 into neural precursors using monolayer differentiation. Two weeks after induction, significantly higher numbers of neural rosettes formed in the Noggin-induced group than the basic fibroblast growth factor-induced group, as detected by phase contrast microscope. Immunofluorescence staining revealed expression levels of Nestin, ß-III Tubulin and Sox-1 were higher in the induced cells and reverse-transcription PCR showed induced cells expressed Nestin, Sox-1 and Neurofilament mRNA. Protein and mRNA expression in the Noggin-induced group was increased compared with the basic fibroblast growth factor-induced group. Noggin has a greater effect than basic fibroblast growth factor on the induction of human embryonic stem cell differentiation into neural precursors by monolayer differentiation, as Noggin accelerates and increases the differentiation of neural precursors.

Parthenogenetic embryonic stem cells derived from cryopreserved newborn mouse ovaries: a new approach to autologous stem cell therapy.

OBJECTIVE: To evaluate whether cryopreserved newborn mouse ovaries can generate sufficient numbers of parthenogenetic mouse embryonic stem (pmES) cells for autologous stem cell therapy. DESIGN: Prospective study. SETTING: Reproductive clinic of Xinhua Hospital in Shanghai. ANIMAL(S): Kunming, C57BL/6J, BALB/c, and NOD-SCID mice. INTERVENTION(S): Cryopreserved newborn mouse ovaries were thawed, grafted into immunodeficient mice, treated with pregnant mare serum gonadotropin to promote follicular maturation, and collected oocytes activated in vitro to generate parthenogenetic embryonic stem cells. MAIN OUTCOME MEASURE(S): Preimplantation development and stem cell characterization. RESULT(S): This new protocol yielded a large number of oocytes from cryopreserved ovaries over a long period. These oocytes were used to derive pmES cell lines, which expressed embryonic stem cell-specific markers and differentiated into embryoid bodies in vitro and teratomas in vivo. The pmES cell line was propagated in an undifferentiated state for more than 30 passages and maintained a diploid karyotype. CONCLUSION(S): The pmES cells lines established by our protocol exhibited the same degree of pluripotency as standard embryonic stem cell lines. This approach may be used for exploring autologous stem cell therapies.

Quality of embryonic bodies and seeding density effects on neural differentiation of mouse embryonic stem cells.

Mouse embryonic stem (ES) cells can be differentiated into neural lineage cells, but the differentiation efficiency remains low. This study revealed two important factors that influence the neural differentiation efficiency of mouse ES cells: the first is the quality of embryonic bodies (EBs); good quality of EBs consistently originated from a suspension culture of 1x10(5) ES cells/ml serum-free chemically defined neural inducing medium and they exhibited a smooth round shape, with a dark central region surrounded by a light band. Such EBs are capable of attaining high neural differentiation efficiency. However, poor quality EBs originated from a suspension culture of 1x10(6) ES cells/ml serum-free chemically defined neural inducing medium and exhibited an irregular shape or adhered to the bottom of the dish; they displayed low neural differentiation efficiency. The second factor is the seeding density of EBs: a low seeding density (5 EBs/cm2) induced cells to differentiate into a more caudalized subtypes compared to the cells obtained from high seeding density (20 EBs/cm2). These findings provided fresh insight into the neural induction of mouse ES cells.

Rabbits generated from fibroblasts through nuclear transfer.

Somatic cell nuclear transfer offers new opportunities for genetic engineering and genome preservation in mammalian animal species. We show that, in addition to cumulus cells, cultured adult rabbit fibroblasts are also capable of supporting full-term development after nuclear transfer. Nuclear transfer embryos constructed using serum-starved fibroblasts showed a significantly higher developmental rate than non-starved fibroblasts through preimplantation stages. A total of 467 nuclear transfer embryos were transferred into the oviducts of pseudo pregnant mothers. Eight of the 20 surrogate rabbits carried the pregnancy to term and five of them gave birth naturally to a total of nine rabbits. However, all of the offspring died before postnatal day 10. A Caesarean section was performed on three surrogates, giving birth to a total of five rabbits, three of them survived and grew into healthy adults. DNA analyses confirmed that these rabbits were genetically identical to the donor male rabbit. The present study demonstrates that rabbits can be cloned from adult fibroblasts after culture.