Temporal microRNA expression profile of pig peripheral blood during postnatal development.

Gene expression profiles of blood can reflect the physiopathologic status of the immune system. The dynamic microRNA (miRNA) expression profiles of peripheral blood from pigs at different developmental stages, and how differential expression of miRNAs might relate to immune system development, are unknown. In this study, peripheral blood samples taken at five developmental stages were used to construct 15 miRNA libraries (three biological replicates/stage): 0 days (newborn), 30 days (weaning), 60 days (weaned), and 180 and 360 days (puberty). We identified 295 known mature miRNAs. Hierarchical clustering of the miRNA expression profile showed significant differences between individuals at the neonatal and postnatal stages. Functional enrichment analysis revealed that miRNAs differentially expressed between pairwise comparisons of the developmental stages were over-represented in immune-related pathways such as toll-like receptor signaling. The time-course of expression of the over-representated miRNAs exhibited a pattern of steady decline over time, for both the complete miRNA compendium and immune-related miRNAs. We identified six marker miRNAs that were highly negatively correlated with chronologic age and enriched for genes involved in immune-related pathways. This study of a peripheral blood miRNA transcriptome offers insight into immune system development in swine and provides a resource for pig genome annotation.

Reinforcing the Induction of Immunogenic Cell Death Via Artificial Engineered Cascade Bioreactor-Enhanced Chemo-Immunotherapy for Optimizing Cancer Immunotherapy.

Traditional chemo-immunotherapy can elicit T cell immune response by inducing immunogenic cell death (ICD), however, insufficient ICD limits the lasting antitumor immunotherapeutic efficacy. Herein, tadpole-ovoid manganese-doped hollow mesoporous silica coated gold nanoparticles (Au@HMnMSNs) as biodegradable catalytic cascade nanoreactors are constructed to generate intratumoral high-toxic hydroxyl radicals combined with DOX and Aspirin (ASA) for enhancing the induction of ICD and maturation of dendritic cells (DCs). The released Mn2+ can catalyze endogenous H2 O2 to hydroxyl radicals, while internal gold nanoparticles mimetic glucose oxidase (GOx) converted glucose into H2 O2 to accelerate the generation of hydroxyl radicals. On the other hand, tadpole oval-structured Au@HMnMSNs can avoid the inactivation of gold nanoparticles due to strong protein adsorption. The introduction of ASA is to recruit DCs and cytotoxic T lymphocytes (CTLs) to tumor sites and restrain the intratumoral infiltration of immunosuppressive cells by decreasing the expression of prostaglandin E2 (PGE2 ). Accordingly, this work presents a novel insight to introduce GOx-like catalytic cascade ICD nano-inducer into antitumor immunotherapy for synergistic tumor therapy.

Functional characterization of protein kinase CK2 regulatory subunits regulating Penicillium oxalicum asexual development and hydrolytic enzyme production.

Casein kinase CK2 is a ubiquitous and conserved phosphate transferase that is critical for the growth and development of eukaryotic cells. In Penicillium oxalicum, one catalytic subunit (CK2A) and two regulatory subunits (CK2B1 and CK2B2) of CK2 were annotated. In this study, CK2 regulatory subunit-defective mutants Δck2B1 and Δck2B2 were constructed to investigate the biological function of CK2 in P. oxalicum. The Δck2B1 strain exhibited minimal changes in morphogenesis and conidiation, whereas the Δck2B2 strain showed delayed conidial germination and drastically reduced conidiation compared with the parent strain. The defect in conidiation in Δck2B2 could be attributed to the reduced expression of transcription factor BrlA. Both Δck2B1 and Δck2B2 showed delayed autolysis in carbon-starvation medium compared with the parent strain. Cellulase and amylase production were decreased considerably in both mutants. The transcript abundances of the main extracellular glycoside hydrolase genes cel7A-2, bgl1, and amy15A, as well as those of three related transcriptional activators (i.e., ClrB, XlnR, and AmyR), were reduced or delayed in the mutants. Epistasis analysis suggested that CK2B1 and CK2B2 might function upstream of transcription factor CreA by inhibiting its repressing activity. In summary, CK2 plays important roles in development and extracellular enzyme production in P. oxalicum, with both unique and overlapping functions performed by the two regulatory subunits.

Penicillium decumbens BrlA extensively regulates secondary metabolism and functionally associates with the expression of cellulase genes.

Penicillium decumbens has been used in the industrial production of lignocellulolytic enzymes in China for more than 15 years. Conidiation is essential for most industrial fungi because conidia are used as starters in the first step of fermentation. To investigate the mechanism of conidiation in P. decumbens, we generated mutants defective in two central regulators of conidiation, FluG and BrlA. Deletion of fluG resulted in neither "fluffy" phenotype nor alteration in conidiation, indicating possible different upstream mechanisms activating brlA between P. decumbens and Aspergillus nidulans. Deletion of brlA completely blocked conidiation. Further investigation of brlA expression in different media (nutrient-rich or nutrient-poor) and different culture states (liquid or solid) showed that brlA expression is required but not sufficient for conidiation. The brlA deletion strain exhibited altered hyphal morphology with more branches. Genome-wide expression profiling identified BrlA-dependent genes in P. decumbens, including genes previously reported to be involved in conidiation as well as previously reported chitin synthase genes and acid protease gene (pepB). The expression levels of seven secondary metabolism gene clusters (from a total of 28 clusters) were drastically regulated in the brlA deletion strain, including a downregulated cluster putatively involved in the biosynthesis of the mycotoxins roquefortine C and meleagrin. In addition, the expression levels of most cellulase genes were upregulated in the brlA deletion strain detected by real-time quantitative PCR. The brlA deletion strain also exhibited an 89.1 % increase in cellulase activity compared with the wild-type strain. The results showed that BrlA in P. decumbens not only has a key role in regulating conidiation, but it also regulates secondary metabolism extensively as well as the expression of cellulase genes.

Biodegradable copper-metformin nanoscale coordination polymers for enhanced chemo/chemodynamic synergistic therapy by reducing oxygen consumption to promote H2O2 accumulation.

Chemo/chemodynamic synergistic therapy is a promising strategy to improve the antitumor effect. However, hypoxia and a limited amount of hydrogen peroxide (H2O2) in the tumor microenvironment (TME) severely restrict the therapeutic efficacy of this combined treatment. Herein, we report biodegradable doxorubicin (Dox)-loaded copper-metformin (Met) nanoscale coordination polymers (Dox@Cu-Met NPs), which exert a chemo/chemodynamic synergistic therapeutic effect by reducing oxygen (O2) consumption to promote H2O2 accumulation in the tumor. Inside tumor cells, Met can inhibit the consumption of O2 to relieve tumor hypoxia by suppressing mitochondrial respiration. The alleviated-tumor hypoxia can not only elevate H2O2 content via the Dox-activated cascade reaction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) and superoxide dismutase (SOD), but also improve the efficacy of Dox. More importantly, the depletion of glutathione (GSH) accompanies the whole treatment process, which can realize the conversion of Cu2+ to Cu+ and boost reactive oxygen species (ROS) accumulation to improve chemodynamic therapy (CDT) efficacy. Meanwhile, Met is expected to cut off the energy supply by inhibiting respiration, leading to starvation therapy. In vivo investigations demonstrate that tumor growth is significantly inhibited through the enhanced chemo/chemodynamic synergistic treatment. This work provides a new paradigm for cancer therapy using an economical and straightforward method to construct a synergistic nanomedicine platform.

A metformin-based nanoreactor alleviates hypoxia and reduces ATP for cancer synergistic therapy.

Severe hypoxia in solid tumors limits the efficacy of oxygen (O2)-dependent photodynamic therapy (PDT). The overexpressed heat shock proteins (HSPs) in tumor cells hamper the effect of photothermal therapy (PTT). Herein, a tumor oxygenation-enhanced and ATP-reduced gelatin nanoreactor (MCGPD ∼ RGD NPs) for PDT/PTT-augmented combination cancer therapy is reported. In this nanosystem, the Arg-Gly-Asp (RGD) peptides of MCGPD ∼ RGD NPs can ensure accurate recognition and sufficient accumulation in the tumor site. After accumulation, doxorubicin (DOX) can be released from MCGPD ∼ RGD NPs in a mild acidic tumor microenvironment (TME) for highly efficient chemotherapy. Upon 808 nm laser irradiation, the overexpressed matrix metalloproteinase-2 (MMP-2) in the TME and the heat produced from the PDA coating trigger Gel NP degradation to expose chlorin e6 (Ce6) and Met from the cavity of MCGPD ∼ RGD NPs. The exposed Met elevates the O2 content and reduces ATP production in tumor sites to spur the successful O2-dependent PDT and HSP-mediated PTT. The heat generated by the PDA coating directly kills the tumor cells to ensure PTT and amplifies the chemotherapeutic effect. In vitro and in vivo assays indicate that MCGPD ∼ RGD NPs have excellent ability to promote cell apoptosis and to inhibit tumor growth. Overall, this smart responsive hydrogel nanosystem with hypoxia-relieving capacity and ATP-decreasing performance provides a promising strategy against cancer.

Electrochemical immunosensor for ochratoxin A detection based on Au octahedron plasmonic colloidosomes.

The accurate and rapid fungaltoxin detection at early stage can prevent people from getting cancer to a great extent. Here, an ultrasensitive electrochemical immunosensor for the detection of ochratoxin A was reported. Octahedral gold nanoparticles (Oct Au NPs) with edge and sharp corners as substrate materials can connect more primary antibodies and Au octahedron plasmonic colloidosomes (Au Oct PCs) were formed by the natural settlement of the Oct Au NPs in the inverse emulsion system. Au Oct PCs with high specific surface area and good electron transfer ability can hybridize with electron mediator toluidine blue to act as labels to amplify the output signal and ameliorate the stability of the immunosensor. The immunoassay was performed via the square wave voltammetry and showed a strong current response. The proposed immunosensor obtained a wide linear range from 0.1 pg mL-1 to 10 ng mL-1 and a low detection limit of 39 fg mL-1. This new strategy achieved favorable stability, reproducibility and selectivity, which had potential application in real sample analysis.

Knockdown expression of IL-10Rα gene inhibits PRRSV replication and elevates immune responses in PBMCs of Tibetan pig in vitro.

Increase of interleukin-10 (IL-10) induced by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection has been intensely studied to inhibit the anti-viral responses of host pigs. Blockade of expression of IL-10 receptor (IL-10R) by RNA interference (RNAi) may relieve the immunosuppression caused by excessive IL-10 in PRRSV infection. The recombinant short hairpin expressing plasmid targeted to pig IL-10Rα was transfected into peripheral blood mononuclear cells of Tibetan pig (Tp-PBMCs) prior to PRRSV inoculation, then the replication of PRRSV and immune responses in Tp-PBMCs were evaluated. The recombinant interfering plasmid greatly decreased PRRSV yield. The transcriptional level of IL-10Rαwas obviously inhibited by recombinant interfering plasmid; the expression of IL-10 was also down-regulated, while that of TGF-ß1 was not affected. Furthermore, the recombinant plasmid notably up-regulated the mRNA levels of TLR3, TLR7, IFN-α, IFN-γ, IL-2, IL-4, IL-12p40 and MyD88, while that of IL-8 was apparently decreased; In addition, cell viability of Tp-PBMCs was clearly enhanced by the interfering recombinant plasmid. Our results suggest that knockdown the expression of pig IL-10Rα can evidently inhibit the PRRSV infection and enhance the anti-viral immune responses of pig immune cells, which may be a promising way for preventing virus infection and developing new effective immune-regulator to strengthen the host immunity against PRRS.

Transcriptomic profiling in muscle and adipose tissue identifies genes related to growth and lipid deposition.

Growth performance and meat quality are important traits for the pig industry and consumers. Adipose tissue is the main site at which fat storage and fatty acid synthesis occur. Therefore, we combined high-throughput transcriptomic sequencing in adipose and muscle tissues with the quantification of corresponding phenotypic features using seven Chinese indigenous pig breeds and one Western commercial breed (Yorkshire). We obtained data on 101 phenotypic traits, from which principal component analysis distinguished two groups: one associated with the Chinese breeds and one with Yorkshire. The numbers of differentially expressed genes between all Chinese breeds and Yorkshire were shown to be 673 and 1056 in adipose and muscle tissues, respectively. Functional enrichment analysis revealed that these genes are associated with biological functions and canonical pathways related to oxidoreductase activity, immune response, and metabolic process. Weighted gene coexpression network analysis found more coexpression modules significantly correlated with the measured phenotypic traits in adipose than in muscle, indicating that adipose regulates meat and carcass quality. Using the combination of differential expression, QTL information, gene significance, and module hub genes, we identified a large number of candidate genes potentially related to economically important traits in pig, which should help us improve meat production and quality.

A novel bZIP transcription factor ClrC positively regulates multiple stress responses, conidiation and cellulase expression in Penicillium oxalicum.

Cellulase production in filamentous fungi is largely regulated at the transcriptional level, and several transcription factors have been reported to be involved in this process. In this study, we identified ClrC, a novel transcription factor in cellulase production in Penicillium oxalicum. ClrC and its orthologs have a highly conserved basic leucine zipper (bZIP) DNA binding domain, and their biological functions have not been explored. Deletion of clrC resulted in pleiotropic effects, including altered growth, reduced conidiation and increased sensitivity to oxidative and cell wall stresses. In particular, the clrC deletion mutant ΔclrC showed 46.1% ± 8.1% and 58.0% ± 8.7% decreases in production of filter paper enzyme and xylanase activities in cellulose medium, respectively. In contrast, 57.4% ± 10.0% and 70.9% ± 19.4% increased production of filter paper enzyme, and xylanase was observed in the clrC overexpressing strain, respectively. The transcription levels of major cellulase genes, as well as two cellulase transcriptional activator genes, clrB and xlnR, were significantly downregulated in ΔclrC, but substantially upregulated in clrC overexpressing strains. Furthermore, we observed that the absence of ClrC reduced full induction of cellulase expression even in the clrB overexpressing strain. These results indicated that ClrC is a novel and efficient engineering target for improving cellulolytic enzyme production in filamentous fungi.

Transcription analysis of lignocellulolytic enzymes of Penicillium decumbens 114-2 and its catabolite-repression-resistant mutant.

Penicillium decumbens 114-2 is a fast-growing filamentous fungus which secretes a variety of lignocellulolytic enzymes. Its catabolite-repression-resistant mutant JU-A10 with high secretion capacity of cellulolytic enzymes has been used industrially for biomass hydrolysis. Transcription levels of 6 important lignocellulolytic enzymes genes (cel5A, cel6A, cel7A, cel7B, xyn10A, and xyn11A) from both strains were determined on different carbon sources (glucose, sorbose, lactose, cellobiose, cellulose, and cellulose-wheat bran), by means of a real-time quantitative polymerase chain reaction. For both strains, the 6 genes are coordinately regulated at transcriptional level. Glucose and cellobiose repressed whereas cellulose and cellulose-wheat bran induced expression of 6 genes in both strains. Expression levels of all genes tested in the mutant strain JU-A10 were substantially higher than those in wild-type strain 114-2 on all carbon sources. On glucose repression condition, the mutant JU-A10 appeared obviously derepressed. Lactose was first proved to have an inductive effect on lignocellulolytic enzyme genes expression at lower concentration in Penicillium spp.