Identification and Validation of an Immunological Expression-Based Prognostic Signature in Breast Cancer.

Background: Emerging evidence suggests that the immune system plays a crucial role in the regulation of the response to therapy and long-term outcomes of patients with breast cancer (BRCA). In this study, we aimed to identify a significant signature based on immune-related genes to predict the prognosis of BRCA patients. Methods: The expression data were downloaded from The Cancer Genome Atlas (TCGA). The immune-related gene list, the transcription factor (TF) gene list, and the immune infiltrate scores of samples in the TCGA database were acquired from the ImmPort database, the Cistrome Cancer database, and the TIMER database, respectively. Univariate Cox regression analysis was utilized to identify prognostic immune-related differentially expressed genes (DEGs) (PIRDEGs) in BRCA. A prognostic immune signature containing 15 PIRDEGs in BRCA was established using the least absolute shrinkage and selection operator (LASSO) model with 1,000 iterations followed by a stepwise Cox proportional hazards model with a training set of 508 samples in TCGA. An independent assessment of the prognostic prediction ability of the signature was conducted using Kaplan-Meier survival analysis with a testing set of 505 samples in TCGA. Results: We identified 466 PIRDEGs and 80 TFs among the DEGs. A gene signature containing 15 PIRDEGs was constructed. Risk scores of BRCA patients were calculated using this model, which showed a high accuracy of prognosis prediction in both the training set and testing set and could be an independent prognostic factor of BRCA patients. Conclusions: Our study revealed that a PIRDEG signature could be a candidate prognostic biomarker for predicting the overall survival (OS) of patients with BRCA.

Mycoplasma gallisepticum Infection Impaired the Structural Integrity and Immune Function of Bursa of Fabricius in Chicken: Implication of Oxidative Stress and Apoptosis.

Mycoplasma gallisepticum (MG) induces a dysregulated immune response in the lungs and air ways of poultry. However, the mechanism of MG-induced immune dysregulation is still not completely understood. In the present study, the effect of MG-infection on chicken bursa of fabricius (BOF) is investigated. Histopathology, electron microscopy, TUNEL assay, qRT-PCR and western blot were employed to examine the hallmarks of oxidative stress and apoptosis. The data revealed that MG-infection induced oxidative stress and decreased antioxidant responses in BOF tissues compared to control group. Histopathological study showed pathological changes including reduction in lymphocytes and increased inflammatory cell infiltration in MG-infection group. Ultrastructural assessment represents obvious signs of apoptosis such as mitochondrial swelling, shrinkage of nuclear membrane and fragmentation of nucleus. Increased cytokine activities were observed in MG-infection group compared to control group. Meanwhile, the mRNA and protein expression level of apoptosis-related genes were significantly (p < 0.05) upregulated in MG-infection group. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay further confirmed that MG induced apoptosis in BOF tissues as TUNEL-stained positive nuclei were remarkably increased in MG-infection group. In addition, MG-infection significantly reduced the number of CD8+ lymphocytes in chicken BOF at day 7. Moreover, bacterial load significantly increased at day 3 and day 7 in MG-infection group compared to control group. These results suggested that MG-infection impaired the structural integrity, induced oxidative stress and apoptosis in chicken BOF tissues, which could be the possible causes of damage to immune function in chicken BOF.

Arachidonic acid metabolism is elevated in Mycoplasma gallisepticum and Escherichia coli co-infection and induces LTC4 in serum as the biomarker for detecting poultry respiratory disease.

Outbreaks of multiple respiratory diseases with high morbidity and mortality have been frequently reported in poultry industry. Metabolic profiling has showed widespread usage in metabolic and infectious disease for identifying biomarkers and understanding of complex mechanisms. In this study, the non-targeted metabolomics were used on Mycoplasma gallisepticum (MG) and Escherichia coli (E.coli) co-infection model in serum, which showed that Leukotriene C4 (LTC4), Leukotriene D4 (LTD4), Chenodeoxycholate, Linoleate and numerous energy metabolites were varied significantly. KEGG enrichment analysis revealed that the metabolic pathways of linoleic acid, taurine and arachidonic acid (AA) were upregulated. To further characterize the consequences of co-infection, we performed an AA metabolic network pathway with metabolic products and enzyme genes. The results showed that the expression of LTC4 increased extremely significant and accompanied with different degree of infection. Meanwhile, the AA network performed the changes and differences of various metabolites in the pathway when multiple respiratory diseases occurred. Taken together, co-infection induces distinct alterations in the serum metabolome owing to the activation of AA metabolism. Furthermore, LTC4 in serum could be used as the biomarker for detecting poultry respiratory disease. ABBREVIATIONS: MG: Mycoplasma gallisepticum; E.coli: Escherichia coli; AA: Arachidonic acid; LTC4: Leukotriene C4; CRD: chronic respiratory diseases; KEGG: Kyoto Encyclopedia of Genes and Genomes; LTs: leukotrienes; PGs: prostaglandins; NO: nitric oxide; HIS: histamine; PCA: Principal Component Analysis; PLS-DA: Partial Least Squares Discriminant Analysis; CCU: color change unit; UPLC: ultra-performance liquid chromatography; MS: mass spectrometry; DEMs: differentially expressed metabolites; ELISA: enzyme-linked immunosorbent assay; SD: standard deviation; VIP: Variable importance in the projection.

Co-infection of Mycoplasma gallisepticum and Escherichia coli Triggers Inflammatory Injury Involving the IL-17 Signaling Pathway.

Mycoplasma gallisepticum and Escherichia coli are well known respiratory disease-inducing pathogens. Previous studies have reported that co-infection by MG and E.coli causes significant economic loss in the poultry industry. In order to assess the respiratory toxicity of co-infection in chicken lung, we established a co-infection model to investigate changes in the inflammatory cytokines, lung tissue structure, and transcriptome profiles of chicken lung. The results showed that co-infection caused a wider range of immune damage and more severe tissue lesions than single-pathogen infection. Differentially expressed gene (DEG) analysis indicated that 3,115/1,498/1,075 genes were significantly expressed among the three infection groups, respectively. Gene ontology and KEGG analysis showed genes enriched in response to immune response, cytokine-cytokine receptor interaction, and inflammation-related signaling pathways. Among these pathways, IL-17 signaling was found to be significantly enriched only in co-infection. The expression of IL-17C, CIKS, TRAF6, NFκB, C/EBPß, and inflammatory chemokines were significantly up-regulated in response to co-infection. Taken together, we concluded that co-infection increased the expression of inflammatory chemokines in lungs through IL-17 signaling, leading to cilia loss and excessive mucus secretion. These results provide new insights into co-infection and reveal target proteins for drug therapy.

Population pharmacokinetics for danofloxacin in the intestinal contents of healthy and infected chickens.

Avian pathogenic Escherichia coli could cause localized and systemic infection in the poultry, and danofloxacin is usually used to treat avian colibacillosis through oral administration. To promote prudent use of danofloxacin and reduce the emergence of drug-resistant E. coli strains, it is necessary to understand the population pharmacokinetics (PopPK) of danofloxacin in chicken intestines. In this study, reversed-phase high performance liquid chromatography (HPLC) with fluorescence detection was used to detect the concentrations of danofloxacin in the contents of duodenum, jejunum, and ileum of the healthy and infected chickens after single oral administration (5 mg/kg body weight). Then, the PopPK of danofloxacin in intestines were analyzed using NONMEM software. As a result, a two-compartment PK model best described the time-concentration profile of duodenal, jejunal, and ileal contents. Interestingly, absorption rate (Ka ), distribution volume (V), and clearance (CL) for danofloxacin from duodenal, jejunal to ileal contents were sequentially decreased in the healthy chickens. However, the trend of Ka , V, and CL of danofloxacin was changed dramatically in the intestine of infected chickens. Ka and V of danofloxacin in the jejunum were higher than in the ileum and duodenum. Compared with healthy chickens, Ka and V of danofloxacin in the duodenum decreased significantly, while increased in jejunum, respectively. It has been noted that Ka decreased and V increased in the ileum of infected chickens. Besides, CL in the duodenum, jejunum, and ileum of infected chickens was, respectively, lower than those of healthy chickens. Interestingly, the relative bioavailability (F) of danofloxacin in the ileum was relatively higher in both healthy and infected chickens. In addition, F in the duodenal, jejunal, and ileal contents of infected chickens was respectively higher than healthy chickens. In summary, the PopPK for danofloxacin in infected chicken intestines was quite different from healthy chickens. The absorption, distribution, and clearance of danofloxacin in healthy chickens decreased from duodenum to jejunum and to ileum. Moreover, the pharmacokinetic characteristics in the intestine of infected chickens changed significantly, and the pharmacokinetic characteristics in the ileum can be used as a representative of all intestinal segments.

A tomato B-box protein SlBBX20 modulates carotenoid biosynthesis by directly activating PHYTOENE SYNTHASE 1, and is targeted for 26S proteasome-mediated degradation.

Carotenoids play important roles in many biological processes, such as light harvesting, photoprotection and visual attraction in plants. However, the regulation of carotenoid biosynthesis is still not fully understood. Here, we demonstrate that SlBBX20, a B-box (BBX) zinc-finger transcription factor, is a positive regulator of carotenoid accumulation in tomato (Solanum lycopersicum). Overexpression of SlBBX20 leads to dark green fruits and leaves and higher levels of carotenoids relative to the wild-type. Interactions between SlBBX20 and DE-ETIOLATED 1 (SlDET1) lead to the ubiquitination and 26S proteasome-mediated degradation of SlBBX20. Moreover, deficiencies in the components of the CUL4-DDB1-DET1 complex enhanced the stability of the SlBBX20 protein. Thus, we conclude that SlBBX20 is a substrate of the CUL4-DDB1-DET1 E3 ligase. SlBBX20 can activate the expression of PHYTOENE SYNTHASE 1, encoding a key enzyme in carotenoid biosynthesis, by directly binding to a G-box motif in its promoter, which results in the elevated levels of carotenoids in SlBBX20 overexpression lines. We identified a key regulator of carotenoid biosynthesis and demonstrated that the stability of SlBBX20 is regulated by ubiquitination. These findings provide us a new target for the genetic improvement of the nutritional quality of tomato fruit.

Baicalin Attenuates Mycoplasma gallisepticum-Induced Inflammation via Inhibition of the TLR2-NF-κB Pathway in Chicken and DF-1 Cells.

BACKGROUND: Previous reports demonstrated that baicalin possesses potential anti-inflammatory properties. The present study was conducted to determine the effects of baicalin against inflammatory responses in chicken and DF-1 cells infected with Mycoplasma gallisepticum (MG). METHODS: An MG infection model was developed in chickens to study the anti-inflammatory mechanism of baicalin. Baicalin was mixed in water at a dose of 450 mg/kg per day, and the treatment is continued for 7 consecutive days. Samples were taken at 1, 4, and 7 days post treatment. RESULTS: By using transmission electron microscopy, ultrastructure of lung and tracheal cells has been examined. It can be seen that the cilia cells in the MG-infected group have pyknosis, degeneration, and necrosis. In the lung tissues, alveolar type-I epithelial cells were severely damaged. In the baicalin-treated group, cilia were swollen, mushroom-shaped edema bubbles formed on the apex, and fused together. Alveolar type I epithelial cells injury was significantly reduced. Compared to MG-infection group, the levels of proinflammatory cytokines IL-1ß and TNF-α were significantly decreased (P < 0.01). The corresponding proteins TLR2 and P-p65 decreased in the baicalin-treated group after 1 (p > 0.05), 4 (p < 0.05), and 7 days (p < 0.05), respectively. CONCLUSION: The results showed that baicalin can interfere with inflammatory injury by suppressing the release of inflammatory cytokines IL-1ß and TNF-α during MG infection both in vivo and in vitro. Meanwhile, baicalin suppressed TLR2-NFκB signaling pathway by inhibiting the phosphorylation of p65 and IκB, thereby affecting the expression of inflammatory factors. The results suggested that baicalin acts as a potential anti-inflammatory agent against MG infection in chicken and DF-1 cells.

Preventive effects of nerve growth factor against colistin-induced autophagy and apoptosis in PC12 cells.

In this study, the preventive effects of NGF against colistin-induced autophagy and apoptosis in PC12 cells have been investigated. Fluorescence microscopy, real-time PCR, transmission electron microscopy (TEM), flow cytometery, and western blotting technique were used. The results showed that large amounts of autophagosomes and apoptotic markers were triggered by colistin. Consistently, a significant increase has been noted at mRNA and protein levels in autophagy and apoptosis-related genes. Besides, TEM analysis showed that autophagic vacuoles were obvious at 12 h, while nuclear chromatin condensation and edge accumulation were clearly seen at 24 h in colistin alone group. Importantly, the visual autophagy and apoptosis were markedly reduced with NGF treatment in a dose-dependent manner. Moreover, colistin-induced reduction in mitochondrial membrane potential was partly attenuated by NGF in a dose dependent manner. In summary, NGF ameliorated colistin-induced apoptosis and autophagy, and partially recovered MMP in PC12 cells.

Species identification of polygonati rhizoma in China by both morphological and molecular marker methods.

Morphological markers as well as two types of molecular markers, inter-sample sequence repeat (ISSR) and start codon targeted (SCoT) are suitable for species identification of the polygonati rhizoma germplasms. In this paper, we adopted these methods for the identification of rhizomes collected from 47 areas in China. Based on their morphological characters, the collected germplasms were classified into two populations, one with alternate leaf arrangement and the other with verticillate leaf arrangement, and they were comprised of five species and fourteen subgroups. Of the five species identified: Polygonatum kingianum, P. cirrhifolium, P. alternicirrhosum, and P. sibiricum belonged to one cluster, and P. cyrtonema belonged to a different cluster. According to the analysis of both ISSR and SCoT markers, all germplasms with greater genetic similarity were classified into one group. Especially, P. sibiricum and P. cirrhifolium, which shared ∼80% similarity, were clustered together, whereas the germplasms identified as P. kingianum with ∼86% similarity formed a separate clade. P. kingianum showed a much greater genetic similarity with P. cyrtonema than with P. sibiricum. The multidimensional scaling analysis further verified the accuracy and reliability of the molecular marker-based results. Thus, both morphological and molecular methods should be combined for the differentiation of germplasms such as those of polygonati rhizoma.

Electroacupuncture decreases cognitive impairment and promotes neurogenesis in the APP/PS1 transgenic mice.

BACKGROUND: Alzheimer's disease (AD) is a severe neurodegenerative disease for which there is currently no effective treatment. The purpose of this study was to investigate whether repeated electroacupuncture (EA) stimulation would improve cognitive function and the pathological features of AD in amyloid precursor protein (APP)/presenilin 1 (PS1) double transgenic mice. METHODS: Cognitive function of APP/PS1 double transgenic mice was assessed using the Morris water maze test before and after EA treatment. Levels of amyloid ß-peptide (Aß) deposits in the hippocampus and cortex were evaluated by immunofluorescence, western blot and enzyme-linked immunosorbent assay. Expression of brain-derived neurotrophic factor (BDNF) was also examined by immunofluorescence and western blot. The neurogenesis was labeled by the DNA marker bromodeoxyuridine. RESULTS: EA stimulation significantly ameliorated the learning and memory deficits of AD mice by shortening escape latency and increasing the time spent in the target zone during the probe test. Additionally, decreased Aß deposits and increased BDNF expression and neurogenesis in the hippocampus and cortex of EA-treated AD mice were detected. The same change was detected in wild-type mice after EA treatment compared with wild-type mice without EA treatment. CONCLUSIONS: Repeated EA stimulation may improve cognitive function, attenuate Aß deposits, up-regulate the expression of BDNF and promote neurogenesis in the APP/PS1 double transgenic mice. This suggests that EA may be a promising treatment for AD.

Spatio-temporal expression of paired immunoglobulin-like receptor-B in the adult mouse brain after focal cerebral ischaemia.

PRIMARY OBJECTIVE: Paired immunoglobulin-like receptor-B (PirB) is another receptor, except for the Nogo receptor, that is involved in inhibition of axons regeneration after central nervous system injury. However, the expression of PirB in focal cerebral ischaemic brain remains unclear. Herein, this study investigated spatial-temporal expression of PirB in the mouse brain following transient focal cerebral ischaemia. METHODS AND PROCEDURE: Adult male C57BL/6 mice underwent a 60-minute transient occlusion of middle cerebral artery. Mice were killed and brain samples were harvested at 30 minutes, 2 hours, 24 hours, 3 days and 7 days after reperfusion. Expression of PirB in the brain was determined by reverse transcriptase-polymerase chain reaction (RT-PCR), western blot analysis and immunohistochemical staining. MAIN OUTCOMES AND RESULTS: The results showed that PirB was mainly expressed in ischaemic penumbra. PirB mRNA and protein expression began to increase at 2 hours, peaked at 24 hours and lasted for 7 days after reperfusion in the ischaemic penumbra. By using immunofluorescence, PirB signals were co-localized with NeuN-positive neurons. CONCLUSION: PirB expression is up-regulated in ischaemic penumbra following transient focal cerebral ischaemia. PirB expression in neurons may play important pathological roles in the inhibition of axonal regeneration after stroke, suggesting that the inhibition of PirB expression may enhance axonal regeneration and functional recovery after stroke.

NDRG2 is a novel p53-associated regulator of apoptosis in C6-originated astrocytes exposed to oxygen-glucose deprivation.

N-myc downstream-regulated gene 2 (NDRG2) has been documented to be a pro-differentiative and anti-proliferative gene in cancer research. Our previous study found a significant NDRG2 up-regulation in reactive astrocytes of penumbra after transient focal cerebral ischemia, which was parallel to the enhancement of TUNEL-positive signals. However, it is still uncertain whether NDRG2 participates in cellular apoptosis induced by ischemia-reperfusion injury in brain. In this study, we investigated the role of NDRG2 in cellular apoptosis induced by oxygen-glucose deprivation (OGD) in IL-6-differentiated C6 glioma cells. The results showed that NDRG2 was up-regulated and translocated from the cytoplasm to the nucleus after OGD exposure. NDRG2 over-expression exhibited an anti-proliferative effect and increased the Bax/Bcl-2 ratio after OGD exposure, while NDRG2 silencing promoted the cellular proliferation and attenuated the up-regulation of Bax/Bcl-2 ratio. The pro-apoptotic effect of p53 was verified by the results in which p53 silencing greatly reduced the percentage of OGD-induced apoptotic cells. p53 silencing also reduced the OGD-induced NDRG2 up-regulation. However, over-expression of p53 did not further improve the NDRG2 up-regulation. In conclusion, NDRG2 is a p53-associated regulator of apoptosis in C6-originated astrocytes after OGD exposure. These findings bring insight to the roles of NDRG2 in ischemic-hypoxic injury and provide potential targets for future clinical therapies on stroke.

Electroacupuncture pretreatment attenuates cerebral ischemic injury through α7 nicotinic acetylcholine receptor-mediated inhibition of high-mobility group box 1 release in rats.

BACKGROUND: We have previously reported that electroacupuncture (EA) pretreatment induced tolerance against cerebral ischemic injury, but the mechanisms underlying this effect of EA are unknown. In this study, we assessed the effect of EA pretreatment on the expression of α7 nicotinic acetylcholine receptors (α7nAChR), using the ischemia-reperfusion model of focal cerebral ischemia in rats. Further, we investigated the role of high mobility group box 1 (HMGB1) in neuroprotection mediated by the α7nAChR and EA. METHODS: Rats were treated with EA at the acupoint "Baihui (GV 20)" 24 h before focal cerebral ischemia which was induced for 120 min by middle cerebral artery occlusion. Neurobehavioral scores, infarction volumes, neuronal apoptosis, and HMGB1 levels were evaluated after reperfusion. The α7nAChR agonist PHA-543613 and the antagonist α-bungarotoxin (α-BGT) were used to investigate the role of the α7nAChR in mediating neuroprotective effects. The roles of the α7nAChR and HMGB1 release in neuroprotection were further tested in neuronal cultures exposed to oxygen and glucose deprivation (OGD). RESULTS: Our results showed that the expression of α7nAChR was significantly decreased after reperfusion. EA pretreatment prevented the reduction in neuronal expression of α7nAChR after reperfusion in the ischemic penumbra. Pretreatment with PHA-543613 afforded neuroprotective effects against ischemic damage. Moreover, EA pretreatment reduced infarct volume, improved neurological outcome, inhibited neuronal apoptosis and HMGB1 release following reperfusion, and the beneficial effects were attenuated by α-BGT. The HMGB1 levels in plasma and the penumbral brain tissue were correlated with the number of apoptotic neurons in the ischemic penumbra. Furthermore, OGD in cultured neurons triggered HMGB1 release into the culture medium, and this effect was efficiently suppressed by PHA-543,613. Pretreatment with α-BGT reversed the inhibitory effect of PHA-543,613 on HMGB1 release. CONCLUSION: These data demonstrate that EA pretreatment strongly protects the brain against transient cerebral ischemic injury, and inhibits HMGB1 release through α7nAChR activation in rats. These findings suggest the novel potential for stroke interventions harnessing the anti-inflammatory effects of α7nAChR activation, through acupuncture or pharmacological strategies.

[Involvement of cerebral neuroglobin in electroacupuncture preconditioning-induced protection effect in cerebral ischemia-reperfusion rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) preconditioning on cerebral ischemia and the role of cerebral neuroglobin (NgB) in EA-induced brain protection in focal cerebral ischemia and reperfusion injury (CI/RI) rats. METHODS: Male SD rats were randomly assigned to sham control, CI/RI 6 h, CI/RI 24 h and CI/RI 72 h groups (n = 6) for observing changes of NgB at different time-points. Additional SD rats were randomly assigned to sham, model, and EA preconditioning (EA-PC) groups (n = 16) for observing changes of cerebral NgB positive cell counts in the ischemic penumbra region 24 h after reperfusion. EA pre-conditioning was applied to "Baihui" (GV 20) for 30 min, once daily for 5 days before CI/RI. CI/RI model was established by occlusion of the right middle cerebral artery and reperfusion for 6 h, 24 h and 72 h respectively. The neurological behavior scores (NBS) of all the rats were evaluated according to Garcia's methods. The cerebral infarct volume was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The number of cerebral NgB positive cells was detected by immunofluorescent staining. RESULTS: No infarct loci were found in the sham group. The cerebral infarction volume percentage was significantly higher in the model group than in the EA-PC group (P < 0.01), while the NBS was significantly lower in the model group than in the EA-PC group (P < 0.01). The number of cerebral NgB positive cells in the ischemic penumbra was up-regulated 6 h after CI/RI injury, peaked at 24 h and continued at 72 h. Compared with the sham group, the number of cerebral NgB positive cells of the model group was increased significantly, whereas that of the EA-PC group up-regulated further obviously in comparison with the model group (P < 0.01). CONCLUSION: EA pretreatment has a significant neuroprotective effect on cerebral ischemia-reperfusion, which is closely related to its effect in up-regulating NgB protein expression.