Ex vivo drug sensitivity screening predicts response to temozolomide in glioblastoma patients and identifies candidate biomarkers.

BACKGROUND: Patient-derived glioma stem-like cells (GSCs) have become the gold-standard in neuro-oncological research; however, it remains to be established whether loss of in situ microenvironment affects the clinically-predictive value of this model. We implemented a GSC monolayer system to investigate in situ-in vitro molecular correspondence and the relationship between in vitro and patient response to temozolomide (TMZ). METHODS: DNA/RNA-sequencing was performed on 56 glioblastoma tissues and 19 derived GSC cultures. Sensitivity to TMZ was screened across 66 GSC cultures. Viability readouts were related to clinical parameters of corresponding patients and whole-transcriptome data. RESULTS: Tumour DNA and RNA sequences revealed strong similarity to corresponding GSCs despite loss of neuronal and immune interactions. In vitro TMZ screening yielded three response categories which significantly correlated with patient survival, therewith providing more specific prediction than the binary MGMT marker. Transcriptome analysis identified 121 genes related to TMZ sensitivity of which 21were validated in external datasets. CONCLUSION: GSCs retain patient-unique hallmark gene expressions despite loss of their natural environment. Drug screening using GSCs predicted patient response to TMZ more specifically than MGMT status, while transcriptome analysis identified potential biomarkers for this response. GSC drug screening therefore provides a tool to improve drug development and precision medicine for glioblastoma.

Overexpression of wild-type IL-7Rα promotes T-cell acute lymphoblastic leukemia/lymphoma.

Tight regulation of IL-7Rα expression is essential for normal T-cell development. IL-7Rα gain-of-function mutations are known drivers of T-cell acute lymphoblastic leukemia (T-ALL). Although a subset of patients with T-ALL display high IL7R messenger RNA levels and cases with IL7R gains have been reported, the impact of IL-7Rα overexpression, rather than mutational activation, during leukemogenesis remains unclear. In this study, overexpressed IL-7Rα in tetracycline-inducible Il7r transgenic and Rosa26 IL7R knockin mice drove potential thymocyte self-renewal, and thymus hyperplasia related to increased proliferation of T-cell precursors, which subsequently infiltrated lymph nodes, spleen, and bone marrow, ultimately leading to fatal leukemia. The tumors mimicked key features of human T-ALL, including heterogeneity in immunophenotype and genetic subtype between cases, frequent hyperactivation of the PI3K/Akt pathway paralleled by downregulation of p27Kip1 and upregulation of Bcl-2, and gene expression signatures evidencing activation of JAK/STAT, PI3K/Akt/mTOR and Notch signaling. Notably, we also found that established tumors may no longer require high levels of IL-7R expression upon secondary transplantation and progressed in the absence of IL-7, but remain sensitive to inhibitors of IL-7R-mediated signaling ruxolitinib (Jak1), AZD1208 (Pim), dactolisib (PI3K/mTOR), palbociclib (Cdk4/6), and venetoclax (Bcl-2). The relevance of these findings for human disease are highlighted by the fact that samples from patients with T-ALL with high wild-type IL7R expression display a transcriptional signature resembling that of IL-7-stimulated pro-T cells and, critically, of IL7R-mutant cases of T-ALL. Overall, our study demonstrates that high expression of IL-7Rα can promote T-cell tumorigenesis, even in the absence of IL-7Rα mutational activation.

The mRNA-lncRNA landscape of multiple tissues uncovers key regulators and molecular pathways that underlie heterosis for feed intake and efficiency in laying chickens.

BACKGROUND: Heterosis is routinely exploited to improve animal performance. However, heterosis and its underlying molecular mechanism for feed intake and efficiency have been rarely explored in chickens. Feed efficiency continues to be an important breeding goal trait since feed accounts for 60 to 70% of the total production costs in poultry. Here, we profiled the mRNA-lncRNA landscape of 96 samples of the hypothalamus, liver and duodenum mucosa from White Leghorn (WL), Beijing-You chicken (YY), and their reciprocal crosses (WY and YW) to elucidate the regulatory mechanisms of heterosis. RESULTS: We observed negative heterosis for both feed intake and residual feed intake (RFI) in YW during the laying period from 43 to 46 weeks of age. Analysis of the global expression pattern showed that non-additivity was a major component of the inheritance of gene expression in the three tissues for YW but not for WY. The YW-specific non-additively expressed genes (YWG) and lncRNA (YWL) dominated the total number of non-additively expressed genes and lncRNA in the hypothalamus and duodenum mucosa. Enrichment analysis of YWG showed that mitochondria components and oxidation phosphorylation (OXPHOS) pathways were shared among the three tissues. The OXPHOS pathway was enriched by target genes for YWL with non-additive inheritance of expression in the liver and duodenum mucosa. Weighted gene co-expression network analysis revealed divergent co-expression modules associated with feed intake and RFI in the three tissues from WL, YW, and YY. Among the negatively related modules, the OXPHOS pathway was enriched by hub genes in the three tissues, which supports the critical role of oxidative phosphorylation. Furthermore, protein quantification of ATP5I was highly consistent with ATP5I expression in the liver, which suggests that, in crossbred YW, non-additive gene expression is down-regulated and decreases ATP production through oxidative phosphorylation, resulting in negative heterosis for feed intake and efficiency. CONCLUSIONS: Our results demonstrate that non-additively expressed genes and lncRNA involved in oxidative phosphorylation in the hypothalamus, liver, and duodenum mucosa are key regulators of the negative heterosis for feed intake and RFI in layer chickens. These findings should facilitate the rational choice of suitable parents for producing crossbred chickens.

Identification of miRNA Associated with Trichomonas gallinae Resistance in Pigeon (Columba livia).

Trichomonas gallinae (T. gallinae) has a great influence on the pigeon industry. Pigeons display different resistance abilities to T. gallinae, so the study of the molecular mechanism of resistance is necessary in breeding disease resistant lines. MiRNA plays important roles in the immune response, but there are still no reports of miRNA regulating trichomonosis resistance. We used small RNA sequencing technology to characterize miRNA profiles in different groups. T. gallinae was nasally inoculated in one day old squabs, and according to the infection status, the groups were divided into control (C), susceptible (S) and tolerant (T) groups. We identified 2429 miRNAs in total, including 1162 known miRNAs and 1267 new miRNAs. In a comparison among the C, S and T groups, the target genes of differentially expressed miRNAs were analyzed via GO and KEGG annotation. The results showed that the target genes were enriched in immune-response-related pathways. This indicated that the differentially expressed miRNAs had a critical influence on T. gallinae infection. Novel_miR_741, which could inhibit the expression of PRKCQ, was down-regulated in the T group compared to the C group. It was proven that a decreased novel_miR_741 expression would increase the expression of PRKCQ and increase the immune response. This study brings new insights into understanding the mechanism of trichomonosis resistance.

Seminal Plasma Proteome as an Indicator of Sperm Dysfunction and Low Sperm Motility in Chickens.

Molecular mechanisms underlying sperm motility have not been fully explained, particularly in chickens. The objective was to identify seminal plasma proteins associated with chicken sperm motility by comparing the seminal plasma proteomic profile of roosters with low sperm motility (LSM, n = 4) and high sperm motility (HSM, n = 4). Using a label-free MS-based method, a total of 522 seminal plasma proteins were identified, including 386 (∼74%) previously reported and 136 novel ones. A total of 70 differentially abundant proteins were defined, including 48 more-abundant, 15 less-abundant, and seven proteins unique to the LSM group (specific proteins). Key secretory proteins like less-abundant adhesion G-protein coupled receptor G2 (ADGRG2) and more-abundant serine peptidase inhibitor Kazal-type 2 (SPINK2) in the LSM suggested that the corresponding secretory tissues played a crucial role in maintaining sperm motility. Majority (80%) of the more-abundant and five specific proteins were annotated to the cytoplasmic domain which might be a result of higher plasma membrane damage and acrosome dysfunction in LSM. Additionally, more-abundant mitochondrial proteins were detected in LSM seminal plasma associated with lower spermatozoa mitochondrial membrane potential (ΔΨm) and ATP concentrations. Further studies showed that the spermatozoa might be suffering from oxidative stress, as the amount of spermatozoa reactive oxygen species (ROS) were largely enhanced, seminal malondialdehyde (MDA) concentrations were increased, and the seminal plasma total antioxidant capacity (T-AOC) were decreased. Our study provides an additional catalogue of chicken seminal plasma proteome and supports the idea that seminal plasma could be as an indicator of spermatozoa physiology. More-abundant of acrosome, mitochondria and sperm cytoskeleton proteins in the seminal plasma could be a marker of sperm dysfunction and loss of motility. The degeneration of spermatozoa caused by the reduced seminal T-AOC and enhanced oxidative stress might be potential determinants of low sperm motility. These results could extend our understanding of sperm motility and sperm physiology regulation.

Metagenomic insights into effects of thiamine supplementation on ruminal non-methanogen archaea in high-concentrate diets feeding dairy cows.

BACKGROUND: Overfeeding of high-concentrate diet (HC) frequently leads to subacute ruminal acidosis (SARA) in modern dairy cows' production. Thiamine supplementation has been confirmed to attenuate HC induced SARA by increasing ruminal pH and ratio of acetate to propionate, and decreasing rumen lactate, biogenic amines and lipopolysaccharide (LPS). The effects of thiamine supplementation in HC on rumen bacteria and fungi profile had been detected in our previous studies, however, effects of thiamine supplementation in HC on rumen non-methanogen archaea is still unclear. The objective of the present study was therefore to investigate the effects of thiamine supplementation on ruminal archaea, especially non-methanogens in HC induced SARA cows. RESULTS: HC feeding significantly decreased dry matter intake, milk production, milk fat content, ruminal pH and the concentrations of thiamine and acetate in rumen fluid compared with control diet (CON) (P < 0.05), while the concentrations of propionate and ammonia-nitrogen (NH3-N) were significantly increased compared with CON (P < 0.05). These changes caused by HC were inversed by thiamine supplementation (P < 0.05). The taxonomy results showed that ruminal archaea ranged from 0.37 to 0.47% of the whole microbiota. Four characterized phyla, a number of Candidatus archaea and almost 660 species were identified in the present study. In which Euryarchaeota occupied the largest proportion of the whole archaea. Furthermore, thiamine supplementation treatment significantly increased the relative abundance of non-methanogens compared with CON and HC treatments. Thaumarchaeota was increased in HC compared with CON. Thiamine supplementation significantly increased Crenarchaeota, Nanoarchaeota and the Candidatus phyla, however decreased Thaumarchaeota compared with HC treatment. CONCLUSIONS: HC feeding significantly decreased ruminal pH and increased the content of NH3-N which led to N loss and the increase of the relative abundance of Thaumarchaeota. Thiamine supplementation increased ruminal pH, improved the activity of ammonia utilizing bacteria, and decreased Thaumarchaeota abundance to reduce the ruminal NH3 content and finally reduced N loss. Overall, these findings contributed to the understanding of thiamine's function in dairy cows and provided new strategies to improve dairy cows' health under high-concentrate feeding regime.

Single SNP- and pathway-based genome-wide association studies for beak deformity in chickens using high-density 600K SNP arrays.

BACKGROUND: Beak deformity, typically expressed as the crossing of upper and lower mandibles, is found in several indigenous chicken breeds, including the Beijing-You chickens studied here. Beak deformity severely impairs the birds' growth and welfare. Although previous studies shed some light on the genetic regulation of this complex trait, the genetic basis of this malformation remains incompletely understood. RESULTS: In this study, single SNP- and pathway-based genome-wide association studies (GWASs) were performed using ROADTRIPS and SNP ratio test (SRT), respectively. A total of 48 birds with deformed beaks (case) and 48 normal birds (control) were genotyped using Affymetrix 600 K HD genotyping arrays. As a result, 95 individuals and 429,539 SNPs were obtained after quality control. The P-value was corrected by a Bonferroni adjustment based on linkage disequilibrium pruning. The single SNP-based association study identified one associated SNP with 5% genome-wide significance and seven suggestively associated SNPs. Four high-confidence genes, LOC421892, TDRD3, RET, and STMN1, were identified as the most promising candidate genes underlying this complex trait in view of their positions, functions, and overlaps with previous studies. The pathway-based association study highlighted the association of six pathways with beak deformity, including the calcium signaling pathway. CONCLUSIONS: Potentially useful candidate genes and pathways for beak deformity were identified, which should be the subject of further functional characterization.

IL-7 Receptor Mutations and Steroid Resistance in Pediatric T cell Acute Lymphoblastic Leukemia: A Genome Sequencing Study.

BACKGROUND: Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood cancer and the leading cause of cancer-related mortality in children. T cell ALL (T-ALL) represents about 15% of pediatric ALL cases and is considered a high-risk disease. T-ALL is often associated with resistance to treatment, including steroids, which are currently the cornerstone for treating ALL; moreover, initial steroid response strongly predicts survival and cure. However, the cellular mechanisms underlying steroid resistance in T-ALL patients are poorly understood. In this study, we combined various genomic datasets in order to identify candidate genetic mechanisms underlying steroid resistance in children undergoing T-ALL treatment. METHODS AND FINDINGS: We performed whole genome sequencing on paired pre-treatment (diagnostic) and post-treatment (remission) samples from 13 patients, and targeted exome sequencing of pre-treatment samples from 69 additional T-ALL patients. We then integrated mutation data with copy number data for 151 mutated genes, and this integrated dataset was tested for associations of mutations with clinical outcomes and in vitro drug response. Our analysis revealed that mutations in JAK1 and KRAS, two genes encoding components of the interleukin 7 receptor (IL7R) signaling pathway, were associated with steroid resistance and poor outcome. We then sequenced JAK1, KRAS, and other genes in this pathway, including IL7R, JAK3, NF1, NRAS, and AKT, in these 69 T-ALL patients and a further 77 T-ALL patients. We identified mutations in 32% (47/146) of patients, the majority of whom had a specific T-ALL subtype (early thymic progenitor ALL or TLX). Based on the outcomes of these patients and their prednisolone responsiveness measured in vitro, we then confirmed that these mutations were associated with both steroid resistance and poor outcome. To explore how these mutations in IL7R signaling pathway genes cause steroid resistance and subsequent poor outcome, we expressed wild-type and mutant IL7R signaling molecules in two steroid-sensitive T-ALL cell lines (SUPT1 and P12 Ichikawa cells) using inducible lentiviral expression constructs. We found that expressing mutant IL7R, JAK1, or NRAS, or wild-type NRAS or AKT, specifically induced steroid resistance without affecting sensitivity to vincristine or L-asparaginase. In contrast, wild-type IL7R, JAK1, and JAK3, as well as mutant JAK3 and mutant AKT, had no effect. We then performed a functional study to examine the mechanisms underlying steroid resistance and found that, rather than changing the steroid receptor's ability to activate downstream targets, steroid resistance was associated with strong activation of MEK-ERK and AKT, downstream components of the IL7R signaling pathway, thereby inducing a robust antiapoptotic response by upregulating MCL1 and BCLXL expression. Both the MEK-ERK and AKT pathways also inactivate BIM, an essential molecule for steroid-induced cell death, and inhibit GSK3B, an important regulator of proapoptotic BIM. Importantly, treating our cell lines with IL7R signaling inhibitors restored steroid sensitivity. To address clinical relevance, we treated primary T-ALL cells obtained from 11 patients with steroids either alone or in combination with IL7R signaling inhibitors; we found that including a MEK, AKT, mTOR, or dual PI3K/mTOR inhibitor strongly increased steroid-induced cell death. Therefore, combining these inhibitors with steroid treatment may enhance steroid sensitivity in patients with ALL. The main limitation of our study was the modest cohort size, owing to the very low incidence of T-ALL. CONCLUSIONS: Using an unbiased sequencing approach, we found that specific mutations in IL7R signaling molecules underlie steroid resistance in T-ALL. Future prospective clinical studies should test the ability of inhibitors of MEK, AKT, mTOR, or PI3K/mTOR to restore or enhance steroid sensitivity and improve clinical outcome.

Study on LOC426217 as a candidate gene for beak deformity in chicken.

BACKGROUND: The beak deformity (crossed beaks) was found in some indigenous chickens of China, such as Beijing-You (BJY), Qingyuan Partridge, and Huxu Chickens. Birds with deformed beaks have reduced feed intake and drinking, impeded growth rate, and poor production performance. Beak deformity reduces the economy of poultry industry and affects animal welfare as well. The genetic basis of this malformation remains incompletely understood. LOC426217, also named claw keratin-like, was the most up-regulated gene in the deformed beaks from a previous digital gene expression (DGE) analysis and was selected as an important candidate gene for further analysis. RESULTS: In the present study, quantitative real-time PCR (qRT-PCR) was firstly performed to determine the expression pattern of LOC426217 gene in deformed and normal beaks to verify the DGE results. Tissue-specific expression profile of this gene in 14 tissues was also determined using qRT-PCR. The LOC426217 was amplified from the genomic DNA of 171 deformed and 164 normal beaks, and sequenced to detect the single nucleotide polymorphisms (SNPs). The results showed that LOC426217 was significantly high-expressed in the deformed beaks, which was in good agreement with the DGE results. This gene was specifically high-expressed in beaks than other tissues. Eight SNPs were detected in LOC426217: -62G > T, 24 T > C, 36G > C, 192A > T, 204C > T, 222 T > C, 285G > T, and 363 T > C. Genotype frequency of G-62 T, T24C, G36C, T222C, and T363C loci was significant different between deformed and normal beaks. Haplotype analysis revealed one block with SNPs T24C and G36C, and one block with SNPs A192T, C204T, T222C, and G285T in normal birds, while the block with SNPs G36C and A192T in deformed ones. CONCLUSIONS: It was concluded from these results that the over-expression of LOC426217 in the beak maybe related to the malformation. The polymorphisms of LOC426217 gene were associated with the beak deformity trait where the SNPs of G-62 T, T24C, G36C, T222C, and T363C loci maybe used as markers. The specific haplotype block in deformed birds may be a potential linkage marker for this trait.

Potential therapeutic targets for hypoxia-induced pulmonary artery hypertension.

BACKGROUND: Hypoxic pulmonary artery hypertension (PAH) as a severe pulmonary disease is characterized by changes of pulmonary vascular reconstruction. Mitochondrial ATP-sensitive potassium channel (mitoKATP) was considered as one of factors responsible for the proliferation of hypoxic pulmonary arterial smooth muscle cells (PASMCs), although the exact mechanisms remain unclear. METHODS: Pulmonary artery hypertension was induced in rats with or without 5-hydroxydecanoate (5-HD). The mean pulmonary artery pressure, morphologic changes, mRNA and protein expressions of voltage-gated potassium channels (Kv1.5 channel), were measured. The concentrations of monocyte chemo-attractant protein-1 (MCP-1) and transforming growth factor-beta1 (TGF-ß1) were detected. Furthermore, pulmonary arterial smooth muscle cells (PASMCs) were isolated and cultured with or without hypoxia pretreated with or without 5-HD or/and Kv1.5 inhibitor 4-aminopyridine (4-AP). Mitochondrial membrane potential (Δψm) and the proliferation of PASMCs were detected. RESULTS: 5-HD significantly prevented the development of PAH by blocking the mitochondrial membrane depolarization, increased the expression of voltage-gated potassium channels, and reduced pulmonary hypertension mediated by TGF-ß1 or MCP-1 signaling pathway. CONCLUSION: The MitoKATP plays an important role in the development of PAH and may be therapeutic target for the treatment of disease.

Immature MEF2C-dysregulated T-cell leukemia patients have an early T-cell precursor acute lymphoblastic leukemia gene signature and typically have non-rearranged T-cell receptors.

Three distinct immature T-cell acute lymphoblastic leukemia entities have been described including cases that express an early T-cell precursor immunophenotype or expression profile, immature MEF2C-dysregulated T-cell acute lymphoblastic leukemia cluster cases based on gene expression analysis (immature cluster) and cases that retain non-rearranged TRG@ loci. Early T-cell precursor acute lymphoblastic leukemia cases exclusively overlap with immature cluster samples based on the expression of early T-cell precursor acute lymphoblastic leukemia signature genes, indicating that both are featuring a single disease entity. Patients lacking TRG@ rearrangements represent only 40% of immature cluster cases, but no further evidence was found to suggest that cases with absence of bi-allelic TRG@ deletions reflect a distinct and even more immature disease entity. Immature cluster/early T-cell precursor acute lymphoblastic leukemia cases are strongly enriched for genes expressed in hematopoietic stem cells as well as genes expressed in normal early thymocyte progenitor or double negative-2A T-cell subsets. Identification of early T-cell precursor acute lymphoblastic leukemia cases solely by defined immunophenotypic criteria strongly underestimates the number of cases that have a corresponding gene signature. However, early T-cell precursor acute lymphoblastic leukemia samples correlate best with a CD1 negative, CD4 and CD8 double negative immunophenotype with expression of CD34 and/or myeloid markers CD13 or CD33. Unlike various other studies, immature cluster/early T-cell precursor acute lymphoblastic leukemia patients treated on the COALL-97 protocol did not have an overall inferior outcome, and demonstrated equal sensitivity levels to most conventional therapeutic drugs compared to other pediatric T-cell acute lymphoblastic leukemia patients.

Effects of Supplementing Drinking Water of Parental Pigeons with Enterococcus faecium and Bacillus subtilis on Antibody Levels and Microbiomes in Squabs.

Enterococcus faecium (E. faecium) and Bacillus subtilis (B. subtilis) are widely used as probiotics to improve performance in animal production, but there have been few reports of their impacts on pigeon milk. In this study, twenty-four pairs of parental pigeons were randomly divided into four groups, with six replicates, and each pair feeding three squabs. The control group drank normal water. The E. faecium group, B. subtilis group, and mixed group drank water supplemented with 3 × 106 CFU/mL E. faecium, 2 × 107 CFU/mL B. subtilis, and a mixture of these two probiotics, respectively. The experiment lasted 19 days. The results demonstrated that the IgA and IgG levels were significantly higher in the milk of Group D pigeons than in the other groups. At the phylum level, Fimicutes, Actinobacteria, and Bacteroidetes were the three main phyla identified. At the genus level, Lactobacillus, Bifidobacterium, Veillonella, and Enterococcus were the four main genera identified. In conclusion, drinking water supplemented with E. faecium and B. subtilis could improve immunoglobulin levels in pigeon milk, and this could increase the ability of squabs to resist disease. E. faecium and B. subtilis could be used as probiotics in the pigeon industry.

Bone Marrow Mesenchymal Stem Cell Extracellular Vesicle-derived miR-27b- 3p activates the Wnt/Β-catenin Pathway by Targeting SMAD4 and Aggravates Hepatic Ischemia-reperfusion Injury.

BACKGROUND: To investigate the roles of extracellular vesicles (EVs) secreted from bone marrow mesenchymal stem cells (BMSCs) and miR-27 (highly expressed in BMSC EVs) in hepatic ischemia‒ reperfusion injury (HIRI). APPROACHES AND RESULTS: We constructed a HIRI mouse model and pretreated it with an injection of agomir-miR-27-3p, agomir-NC, BMSC-EVs or control normal PBS into the abdominal cavity. Compared with the HIRI group, HIRI mice preinjected with BMSC-EVs had significantly decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and alleviated liver necrosis (P<0.05). However, compared with HIRI+NC mice, HIRI+miR-27b mice had significantly increased ALT and AST levels, aggravated liver necrosis, and increased apoptosis-related protein expression (P<0.05). The proliferation and apoptosis of AML-12 cells transfected with miR-27 were significantly higher than the proliferation and apoptosis of AML-12 cells in the mimic NC group (P<0.01) after hypoxia induction. SMAD4 was proven to be a miR-27 target gene. Furthermore, compared to HIRI+NC mice, HIRI+miR-27 mice displayed extremely reduced SMAD4 expression and increased levels of wnt1, ß-catenin, c-Myc, and Cyclin D1. CONCLUSION: Our findings reveal the role and mechanism of miR-27 in HIRI and provide novel insights for the prevention and treatment of HIRI; for example, EVs derived from BMSCs transfected with antimiR- 27 might demonstrate better protection against HIRI.

Multi-tissue transcriptome profiling linked the association between tissue-specific circRNAs and the heterosis for feed intake and efficiency in chicken.

Heterosis has been widely utilized in chickens. The nonadditive inheritance of genes contributes to this biological phenomenon. However, the role of circRNAs played in the heterosis is poorly determined. In this study, we observed divergent heterosis for residual feed intake (RFI) between 2 crossbreds derived from a reciprocal cross between White Leghorns and Beijing You chickens. Then, circRNA landscape for 120 samples covering the hypothalamus, liver, duodenum mucosa and ovary were profiled to elucidate the regulatory mechanisms of heterosis. We detected that a small proportion of circRNAs (7.83-20.35%) were additively and non-additively expressed, in which non-additivity was a major inheritance of circRNAs in the crossbreds. Tissue-specific expression of circRNAs was prevalent across 4 tissues. Weighted gene co-expression network analysis revealed circRNA-mRNA co-expression modules associated with feed intake and RFI in the hypothalamus and liver, and the co-expressed genes were enriched in oxidative phosphorylation pathway. We further identified 8 nonadditive circRNAs highly correlated with 16 nonadditive genes regulating negative heterosis for RFI in the 2 tissues. Circ-ITSN2 was validated in the liver tissue for its significantly positive correlation with PGPEP1L. Moreover, the bioinformatic analysis indicated that candidate circRNAs might be functioned by binding the microRNAs and interacting with the RNA binding proteins. The integration of multi-tissue transcriptome firstly linked the association between tissue-specific circRNAs and the heterosis for feed intake and efficiency in chicken, which provide novel insights into the molecular mechanism underlying heterosis for feed efficiency. The validated circRNAs can act as potential biomarkers for predicting RFI and its heterosis.

Integrated transcriptomic analysis on chicken ovary reveals CYP21A1 affects follicle granulosa cell development and steroid hormone synthesis.

Egg production is an economically important trait in poultry breeding and production. Follicular development was regulated by several hormones released and genes expressed in the granulosa cells, impacting the egg production and fecundity of hens. However, the molecular functions of these candidate genes that modulate these processes remain largely unknown. In the present study, bioinformatics analyses were performed to identify the candidate genes related to egg production in the ovarian tissue of White Leghorns with high egg production and Beijing You chicken with low egg production during sexual maturity and peak laying periods. The ovarian granulosa cells were used to assess the function of CYP21A1 by transfecting with CYP21A1-specific small interfering RNAs (siRNAs) and overexpression plasmids. We identified 514 differentially expressed genes (|Log2(fold change) | >1, P <0.05) between the 2 chicken breeds in both laying periods. Among these genes, CYP21A1, which is involved in the steroid hormone biosynthesis pathway was consistently upregulated in White Leghorns. Weighted gene co-expression network analysis (WGCNA) further suggested that CYP21A1 was a hub gene, which could positively respond to treatment with follicle stimulation hormone (FSH), affecting egg production. The interference of CYP21A1 significantly inhibited cell proliferation and promoted cell apoptosis. Overexpression of CYP21A1 promotes cell proliferation and inhibits cell apoptosis. Furthermore, the interference with CYP21A1 significantly downregulated the expression of STAR, CYP11A1, HSD3B1, and FSHR and also decreased the synthesis of progesterone (P4) and estradiol (E2) in granulosa cells. Overexpression of CYP21A1 increased the synthesis of P4 and estradiol E2 and the expression of steroid hormone synthesis-related genes in granulosa cells. Our findings provide new evidence for the biological role of CYP21A1 on granulosa cell proliferation, apoptosis, and steroid hormone synthesis, which lays the theoretical basis for improving egg production.

Pigeon novel-miR-741 targets OTUD1 to inhibit proliferation and promote apoptosis of crop fibroblasts.

Trichomonas gallinae (T. gallinae) is a globally distributed protozoan parasite and could cause serious damage to the pigeon industry. MiRNAs have important roles in regulating parasite infection, but its impacts on T. gallinae resistance have rarely been reported. In the present study, we identified a new miRNA (novel-miR-741) and its predicted target OTU deubiquitinase 1 (OTUD1) that might be associated with immunity to T. gallinae in pigeon. Novel-miR-741 and OTUD1 over-expression vectors and interference vectors were constructed. Results from dual luciferase activity assay demonstrated that OTUD1 was a downstream target of novel-miR-741. The Cell Counting Kit-8 and apoptosis assays showed that novel-miR-741 inhibited the proliferation and promoted apoptosis of pigeon crop fibroblasts. Meanwhile, mRNA levels of OTUD1 were significantly reduced in novel-miR-741 mimic-transfected fibroblasts, while mRNA levels of OTUD1 were significantly increased in the novel-miR-741 inhibitor-transfected fibroblasts. The regulatory roles of si-OTUD1 on fibroblasts proliferation, apoptosis, and migration were similar to novel-miR-741 mimic. Our findings demonstrated that novel-miR-741 inhibited the proliferation, and migration of crop fibroblasts, while OTUD1 promoted the proliferation and migration of crop fibroblasts. Therefore, the regulation of OTUD1 by novel-miR-741 was proposed as a potential therapeutic strategy for T. gallinae.

Apatinib plus chemotherapy is associated with an improved tumor response, survival and tolerance compared with chemotherapy alone for advanced lung adenocarcinoma treatment.

Apatinib plus chemotherapy demonstrates good efficacy in multiple advanced carcinomas; however, its use in patients with advanced lung adenocarcinoma (LUAD) has not yet been assessed. The present study evaluated the potential benefits of apatinib plus chemotherapy in patients with advanced LUAD. A total of 145 patients with advanced LUAD and negative driver genes who received apatinib plus chemotherapy (n=65) or chemotherapy alone (n=80) were analyzed. The overall response rate was significantly improved by apatinib plus chemotherapy vs. chemotherapy alone (53.8 vs. 36.3%; P=0.034). Moreover, progression-free survival (PFS) was significantly longer in patients who received apatinib plus chemotherapy, compared with those who received chemotherapy alone [median (95% CI), 13.4 months (11.5-15.3) vs. 8.2 months (6.9-9.5); P<0.001], as was overall survival (OS) [median (95% CI), 23.1 months (not reached) vs. 17.0 months (14.6-19.4; P=0.001). Following adjustment by multivariate Cox regression analysis, apatinib plus chemotherapy was associated with a significantly longer PFS [hazard ratio (HR), 0.444; P<0.001] and OS (HR, 0.347; P<0.001), compared with chemotherapy alone. Subgroup analyses revealed that PFS and OS were significantly improved following apatinib plus chemotherapy vs. chemotherapy alone (all P<0.05) in patients receiving first- or second-line treatment. Notably, the incidence of hypertension was significantly increased following apatinib plus chemotherapy vs. chemotherapy alone (43.1 vs. 25.0%; P=0.021), whereas the incidence of other adverse events was not significantly different between the two treatment groups (all P>0.05). In conclusion, apatinib plus chemotherapy is associated with an improved treatment response and survival compared with chemotherapy alone, with a tolerable safety profile in patients with advanced LUAD.

Transcriptome analysis of ovarian tissues highlights genes controlling energy homeostasis and oxidative stress as potential drivers of heterosis for egg number and clutch size in crossbred laying hens.

Heterosis is the major benefit of crossbreeding and has been exploited in laying hens breeding for a long time. This genetic phenomenon has been linked to various modes of nonadditive gene action. However, the molecular mechanism of heterosis for egg production in laying hens has not been fully elucidated. To fill this research gap, we sequenced mRNAs and lncRNAs of the ovary stroma containing prehierarchical follicles in White Leghorn, Rhode Island Red chickens as well as their reciprocal crossbreds that demonstrated heterosis for egg number and clutch size. We further delineated the modes of mRNAs and lncRNAs expression to identify their potential functions in the observed heterosis. Results showed that dominance was the principal mode of nonadditive expression exhibited by mRNAs and lncRNAs in the prehierarchical follicles of crossbred hens. Specifically, low-parent dominance was the main mode of mRNA expression, while high-parent dominance was the predominant mode of lncRNA expression. Important pathways enriched by genes that showed higher expression in crossbreds compared to either one or both parental lines were cell adhesion molecules, tyrosine and purine metabolism. In contrast, ECM-receptor interaction, focal adhesion, PPAR signaling, and ferroptosis were enriched in genes with lower expression in the crossbred. Protein network interaction identified nonadditively expressed genes including apolipoprotein B (APOB), transferrin, acyl-CoA synthetase medium-chain family member (APOBEC) 3, APOBEC1 complementation factor, and cathepsin S as hub genes. Among these potential hub genes, APOB was the only gene with underdominance expression common to the 2 reciprocal crossbred lines, and has been linked to oxidative stress. LncRNAs with nonadditive expression in the crossbred hens targeted natriuretic peptide receptor 1, epidermal differentiation protein beta, spermatogenesis-associated gene 22, sperm-associated antigen 16, melanocortin 2 receptor, dolichol kinase, glycine amiinotransferase, and prolactin releasing hormone receptor. In conclusion, genes with nonadditive expression in the crossbred may play crucial roles in follicle growth and atresia by improving follicle competence and increasing oxidative stress, respectively. These 2 phenomena could underpin heterosis for egg production in crossbred laying hens.

Increased vascular smooth muscle cell senescence in aneurysmal Fibulin-4 mutant mice.

Aortic aneurysms are dilatations of the aorta that can rupture when left untreated. We used the aneurysmal Fibulin-4R/R mouse model to further unravel the underlying mechanisms of aneurysm formation. RNA sequencing of 3-month-old Fibulin-4R/R aortas revealed significant upregulation of senescence-associated secretory phenotype (SASP) factors and key senescence factors, indicating the involvement of senescence. Analysis of aorta histology and of vascular smooth muscle cells (VSMCs) in vitro confirmed the senescent phenotype of Fibulin-4R/R VSMCs by revealing increased SA-ß-gal, p21, and p16 staining, increased IL-6 secretion, increased presence of DNA damage foci and increased nuclei size. Additionally, we found that p21 luminescence was increased in the dilated aorta of Fibulin-4R/R|p21-luciferase mice. Our studies identify a cellular aging cascade in Fibulin-4 aneurysmal disease, by revealing that Fibulin-4R/R aortic VSMCs have a pronounced SASP and a senescent phenotype that may underlie aortic wall degeneration. Additionally, we demonstrated the therapeutic effect of JAK/STAT and TGF-ß pathway inhibition, as well as senolytic treatment on Fibulin-4R/R VSMCs in vitro. These findings can contribute to improved therapeutic options for aneurysmal disease aimed at reducing senescent cells.

Effects of 28 h ahemeral light cycle on production performance, egg quality, blood parameters, and uterine characteristics of hens during the late laying period.

This study aimed to systematically determined the effect of 28 h ahemeral light cycle on production performance, egg quality, blood parameters, uterine morphological characteristics, and gene expression of hens during the late laying period. At 74 wk, 260 Hy-Line Brown layers were randomly divided into 2 groups of 130 birds each and in duplicates. Both a regular (16L:8D) and an ahemeral light cycle (16L:12D) were provided to the hens. The oviposition pattern in an ahemeral cycle shifted into darkness, with oviposition mostly occurring 3 to 5 h after light out. Production performance was unaffected by light cycle (P > 0.05). Nonetheless, compared to the normal group, the ahemeral group exhibited increased egg weight, eggshell weight, eggshell percentage, yolk percentage, eggshell thickness, and eggshell strength (P < 0.05). There were rhythmic changes in the uterine morphological structure in both cycles, however, the ahemeral group maintained a longer duration and had more uterine folds than the normal group. In the ahemeral cycle, the phases of the CLOCK and PER2 genes were phase-advanced for 3.96 h and 4.54 h compared to the normal cycle. The PHLPP1 gene, which controls clock resetting, exhibited a substantial oscillated rhythm in the ahemeral group (P < 0.05), while the expression of genes presenting biological rhythm, such as CRY2 and FBXL3, was rhythmically oscillated in normal cycle (P < 0.05). The ITPR2 gene, which regulates intracellular Ca2+ transport, displayed a significant oscillated rhythm in ahemeral alone (P < 0.05), while the CA2 gene, which presents biomineralization, rhythmically oscillated in both cycles (P < 0.05). The ahemeral cycle caused 2.5 h phase delays in the CA2 gene compared to the normal cycle. In conclusion, the 28 h ahemeral light cycle preserved the high condition of the uterine folds and changed the uterine rhythms of CLOCK, PER2, ITPR2, and CA2 gene expression to improve ion transport and uterine biomineralization.