Stem cell factor and erythropoietin-independent production of cultured reticulocytes.

Cultured reticulocytes can supplement transfusion needs and offer promise for drug delivery and immune tolerization. They can be produced from induced pluripotent stem cells (iPSCs), but the 45-day culture time and cytokine costs make large-scale production prohibitive. To overcome these limitations, we have generated IPSCs that express constitutive SCF receptor and jak2 adaptor alleles. We show that iPSC lines carrying these alleles can differentiate into self-renewing erythroblast (SRE) that can proliferate for up to 70 cell-doubling in a cost-effective, chemically-defined, albumin- and cytokine-free medium. These kitjak2 SREs retain the ability to enucleate at a high rate up to senescence. Kitjak2 derived cultured reticulocytes should be safe for transfusion because they can be irradiated to eliminate residual nucleated cells. The kitjak2 cells express blood group 0 and test negative for RhD and other clinically significant RBCs antigens and have sufficient proliferation capacity to meet global RBC needs.

NOX2 oxidase inhibitor GSK2795039 possess antiviral activity against H1N1 influenza A virus in vitro and vivo.

The continuous zoonotic circulation and reassortment potential of influenza A viruses (IAV) in nature represents an enormous public health threat to humans. Beside vaccination antivirals are needed to efficiently control spreading of the disease. The previous research has shown that NOX2 involved in IAV replication, but the detailed mechanism has not been reported. In the present study we investigated the roles of NOX2 in host inflammatory response and IAV replication using a novel inhibitor GSK2795039. The drug significantly reduced H1N1 virus induced NOX2 activity and ROS release in human lung epithelial cells. The results of time course experiments suggested that GSK2795039 inhibited an early post-entry step of viral infection. Concomitantly, there was a decreased expression of pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interferon (IFN)-ß and interleukin (IL)-6) in NOX2 suppressed cells. In vivo, compared with control groups, suppression of NOX2 improved the survival rate of mice infected with H1N1 virus (42.9% in GSK2795039 treated mice versus >0% of control mice) and viral burden also decreased in the GSK2795039 treated group. Thus, our data demonstrated a critical role for NOX2 in the establishment of H1N1 infection and subsequent inflammatory reactions, which suggest that GSK2795039 may be a potential therapeutic drug for IAV infection.

CD44 deficiency represses neuroinflammation and rescues dopaminergic neurons in a mouse model of Parkinson's disease.

CD44 is a transmembrane protein that transduces extracellular stimuli to immune response. Neuroinflammation is a causative factor in neurodegenerative diseases, such as Parkinson's disease (PD). Owing to its role in inflammation, this study investigated whether CD44 is involved in the pathological progression of PD. Our data showed that CD44 deficiency largely abolished proinflammatory cytokine expression in primary microglia and astrocytes. In PD model mice, CD44 knockout improved behavioral defects, prevented TH loss in the SNpc and striatum, and blocked activation of microglia and astrocytes. Moreover, CD44 neutralization by anti-CD44 antibody recapitulated the phenotypes observed in CD44 knockout mice. Mechanistically, CD44 neutralization blocked TLR4 expression and NF-κB p65 nuclear translocation induced by lipopolysaccharide in BV2 cells. Overall, our results indicate that CD44 deficiency has a beneficial role against PD, which is likely due to repression of the TLR4/NF-κB axis, leading to reduced neuroinflammation. Therefore, CD44 might be a therapeutic target for the development of anti-PD agents.

Baicalin suppress growth and virulence-related factors of methicillin-resistant Staphylococcus aureus in vitro and vivo.

A Staphylococcus aureus (S.aureus) was isolated from pigs suffered in pneumonia that can't be cured by antibiotic such as methicillin and vancomycin. It was demonstrated that baicalin, an active natural compound extracted from the traditional Chinese medicinal, possess antimicrobial activity. In the present study, we evaluate it efficacy in vitro and vivo against this isolated methicillin-resistant S.aureus (MRSA). Our findings demonstrated that baicalin can inhibit S. aureus growth in a dose-dependent manner and attenuate the biofilm formation. Scanning electron microscopies showed that cell membrane was damaged and accompany with contents leaks after treated with high concentration of baicalin. In addition, baicalin exerted inhibitory effects on the expression of S.aureus virulence-related factors. Moreover, baicalin treated mice had enhanced survival after a lethal dose of S.aureus infection compared with untreated mice. Simultaneously, the pathological tissue damage and bacterium burden were decrease in baicalin treated mice. These data demonstrated that baicalin displayed a high effectiveness in vitro and vivo against MRSA infection, suggesting that baicalin may potentially be used to treat MRSA infection.

miR-31 shuttled by halofuginone-induced exosomes suppresses MFC-7 cell proliferation by modulating the HDAC2/cell cycle signaling axis.

Traditional Chinese medicine (TCM) are both historically important therapeutic agents and important source of new drugs. Halofuginone (HF), a small molecule alkaloid derived from febrifugine, has been shown to exert strong antiproliferative effects that differ markedly among various cell lines. However, whether HF inhibits MCF-7 cell growth in vitro and underlying mechanisms of this process are not yet clear. Here, we offer the strong evidence of the connection between HF treatment, exosome production and proliferation of MCF-7 cells. Our results showed that HF inhibits MCF-7 cell growth in both time- and dose-dependent manner. Further microRNA (miRNA) profiles analysis in HF treated and nontreated MCF-7 cell and exosomes observed that six miRNAs are particularly abundant and sorted in exosomes. miRNAs knockdown experiment in exosomes and the MCF-7 growth inhibition assay showed that exosomal microRNA-31 (miR-31) modulates MCF-7 cells growth by specially targeting the histone deacetylase 2 (HDAC2), which increases the levels of cyclin-dependent kinases 2 (CDK2) and cyclin D1 and suppresses the expression of p21. In conclusion, these data indicate that inhibition of exosome production reduces exosomal miR-31, which targets the HDAC2 and further regulates the level of cell cycle regulatory proteins, contributing to the anticancer functions of HF. Our data suggest a new role for HF and the exosome production in tumorigenesis and may provide novel insights into prevention and treatment of breast cancer.

Influenza A virus infection induces liver injury in mice.

Respiratory infections such as SARS-CoV in humans are often accompanied by mild and self-limiting hepatitis. As a respiratory disease, influenza A virus (IAV) infection can lead to hepatitis, but the mechanism remains unclear. This study aimed to investigate the occurrence of hepatitis by establishing a model for infected mice for three different subtypes of respiratory IAVs (H1N1, H5N1, and H7N2). Histological analysis was performed, and results showed increase serum aminotransferase (ALT and AST) levels and evident liver injury on days 3 and 7, especially on day 5 post infection. Immunohistochemistry (IHC) results indicated a wide distribution of IAV's positive signals in the liver of infected mice. Real-time PCR results further revealed a similar viral titer to IHC that presented a remarkedly positive correlation with histology injury. All these data showed that the mouse model suitably contributed valuable information about the mechanism underlying the occurrence of hepatitis induced by respiratory influenza virus.

Highly Sensitive Detection of PCV2 Based on Tyramide Signals and GNPL Amplification.

The frequent emergence of secondary infection and immunosuppression after porcine circovirus type 2 (PCV2) infection highlights the need to develop sensitive detection methods. A dual-signal amplification enzyme-linked immunosorbent assay (ELISA) based on a microplate coated with gold nanoparticle layers (GNPL) and tyramide signal amplification (TSA) was established. Results confirmed that the microplates coated with GNPL have a strong binding ability to the antibody without affecting the biological activity of the antibody. The microplates coated with GNPL have strong binding ability to the antibody, and the amplification of the tyramide signal is combined to further improve the sensitivity of PCV2. The PCV2 antibody does not crossreact with other viruses, demonstrating that the method has good specificity. A dual-signal amplification strategy is developed using microplates modified with GNPL and TSA to sensitively detect PCV2.

The role of natural antimicrobial peptides during infection and chronic inflammation.

Natural antimicrobial peptides (AMPs), a family of small polypeptides that are produced by constitutive or inducible expression in organisms, are integral components of the host innate immune system. In addition to their broad-spectrum antibacterial activity, natural AMPs also have many biological activities against fungi, viruses and parasites. Natural AMPs exert multiple immunomodulatory roles that may predominate under physiological conditions where they lose their microbicidal properties in serum and tissue environments. Increased drug resistance among microorganisms is occurring far more quickly than the discovery of new antibiotics. Natural AMPs have shown promise as 'next generation antibiotics' due to their broad-spectrum curative effects, low toxicity, the fact that they are not residual in animals, and the low rates of resistance exhibited by many pathogens. Many types of synthetic AMPs are currently being tested in clinical trials for the prevention and treatment of various diseases such as chemotherapy-associated infections, diabetic foot ulcers, catheter-related infections, and other conditions. Here, we provide an overview of the types and functions of natural AMPs and their role in combating microorganisms and different infectious and inflammatory diseases.

DNAzymes Dz13 target the c-jun possess antiviral activity against influenza A viruses.

The emergence of anti-influenza A virus drugs resistant strain highlights the need for more effective therapy. Our earlier study demonstrated that c-jun, a downstream molecule of JNK, might be important in viral infections and inflammatory responses. In the present study, we explored the function of DNAzymes Dz13 that target c-jun in influenza A virus infected mice. Dz13 displayed non-toxic side effects on A549 cells and BALB/c mice. Moreover, Dz13-treated mice had enhanced survival after influenza compared with untreated mice. Simultaneously, the pulmonary inflammatory responses and viral burden were decreased in Dz13 treated mice. Furthermore, proliferation levels of infection-induced CD4+ and CD8+ T cells were impaired. These data demonstrated that Dz13 could reduce viral replication and inflammatory response in vivo, suggesting that Dz13 may potentially be used to treat influenza A viral infection.

Mice with type 1 diabetes exhibit increased susceptibility to influenza A virus.

Type 1 diabetes (T1D) is a metabolic disease induced by abnormal insulin secretions from damaged islet B cells. Clinical observations have shown that T1D patients are more easily infected by influenza A virus (IAV) and suffer more serious symptoms than non-T1D patients. To investigate the susceptibility of T1D mice to IAV, a T1D mouse model was built by intraperitoneal injection of diluted streptozotocin (STZ) over 5 consecutive days, followed by infection with three subtypes of IAV (H1N1/H5N1/H7N2). The T1D-infected mice showed more serious clinical symptoms and lower survival rates than the non-T1D infected mice. The hematoxylin and eosin (H&E) staining results revealed an increase in serious pathological damage to the lung and pancreas in T1D-infected mice. Immunohistochemistry results indicated higher IAV loads and a more extensive distribution of positive signals in the lungs and pancreas of T1D-infected mice than in those of non-T1D infected mice. Furthermore, according to real-time quantitative polymerase chain reaction (PCR) results, viral replication appeared to occur more easily in the lungs of T1D-infected mice. Thus, T1D-infected mice exhibited higher susceptibility to IAV than did normal mice. This study contributes a mouse model suitable for T1D research as well as valuable information about the mechanism underlying T1D patients' increased susceptibility to IAV.

Transcription profiles of the responses of chicken bursae of Fabricius to IBDV in different timing phases.

BACKGROUND: Infectious bursal disease virus (IBDV) infection causes immunosuppression in chickens and increases their susceptibility to secondary infections. To explore the interaction between host and IBDV, RNA-Seq was applied to analyse the transcriptional profiles of the responses of chickens' bursas of Fabricius in the early stage of IBDV infection. RESULTS: The results displayed that a total of 15546 genes were identified in the chicken bursa libraries. Among the annotated genes, there were 2006 and 4668 differentially expressed genes in the infection group compared with the mock group on day 1 and day 3 post inoculation (1 and 3 dpi), respectively. Moreover, there were 676 common up-regulated and 83 common down-regulated genes in the bursae taken from the chickens infected with IBDV on both 1 and 3 dpi. Meanwhile, there were also some characteristic differentially expressed genes on 1 and 3 dpi. On day 1 after inoculation with IBDV, host responses mainly displayed immune response processes, while metabolic pathways played an important role on day three post infection. Six genes were confirmed by quantitative reverse transcription-PCR. CONCLUSIONS: In conclusion, the differential gene expression profile demonstrated with RNA-Seq might offer a better understanding of the molecular interactions between host and IBDV during the early stage of infection.

The c-Jun N-terminal kinase (JNK) is involved in H5N1 influenza A virus RNA and protein synthesis.

The activation of c-jun N-terminal kinases (JNK) was previously shown to be required for efficient influenza A virus replication, although a detailed mechanism has not been reported. In this study, we found that replication of H5N1 influenza virus was influenced by the JNK inhibitor SP600125. The results of time course experiments suggested that SP600125 inhibited an early post-entry step of viral infection but did not affect nucleocytoplasmic trafficking of the viral ribonucleoprotein complex. The levels of influenza virus genomic RNA (vRNA), but not the corresponding cRNA or mRNA, were specifically reduced by SP600125 in virus-infected cells, indicating that the JNK protein is intimately involved in vRNA synthesis. Additionally, SP600125 affected H5N1 virus protein synthesis, because NS1, PB1, PB2, HA and M1 protein production was impaired. Thus, our data demonstrated a critical role of the JNK protein in the regulation of vRNA and protein synthesis during virus infection. This enhances our understanding of the complicated signal transduction network involved in influenza A virus replication.

Regulatory roles of c-jun in H5N1 influenza virus replication and host inflammation.

The cytokine storm which is a great burden on humanity in highly pathogenic influenza virus infections requires activation of multiple signaling pathways. These pathways, such as MAPK and JNK, are important for viral replication and host inflammatory response. Here we examined the roles of JNK downstream molecule c-jun in host inflammatory responses and H5N1 virus replication using a c-jun targeted DNAzyme (Dz13). Transfection of Dz13 significantly reduced H5N1 influenza virus replication in human lung epithelial cells. Concomitantly, there was a decreased expression of pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interferon (IFN)-ß and interleukin (IL)-6) in c-jun suppressed cells, while the expression of anti-inflammatory cytokines, such as IL-10, was increased. In vivo, compared with control groups, suppression of c-jun improved the survival rate of mice infected with H5N1 virus (55.5% in Dz13 treated mice versus ≤11% of control mice) and decreased the CD8(+) T cell proliferation. Simultaneously, the pulmonary inflammatory response and viral burden also decreased in the Dz13 treated group. Thus, our data demonstrated a critical role for c-jun in the establishment of H5N1 infection and subsequent inflammatory reactions, which suggest that c-jun may be a potential therapeutic target for viral pneumonia.

Small intestinal injury in mice infected with respiratory influenza A virus: evidence for virus induced gastroenteritis.

OBJECTIVES: Influenza in humans is often accompanied by gastroenteritis-like symptoms such as diarrhea and abdominal pain nausea, but the underlying mechanism remains unclear. RESULTS: Mice infected with three subtypes of respiratory influenza A virus (IAV), particularly H5N1 and H7N2, developed intestinal injury. The avian H5N1 and H7N2 IAV were detected in the small intestine, whereas the human H1N1 was not detected. Section staining with the sialic acid (SA) receptor demonstrated that the small intestine mainly expressed SA α2, 3 Gal instead of SA α2, 6 Gal which preferentially binds to avian IAV. The number of goblet and sIgA cells in the small intestine increased, whereas CD4(+) and CD8(+) T cells decreased in all infected mice except for CD8(+) T cells increased in H7N2 infected mice. CONCLUSIONS: Respiratory IAV infection, particularly infected by avian IAV, can cause small intestine structural damage and modify the local immune response, thereby resulting in gastroenteritis-like symptoms.

Rhein kills Actinobacillus pleuropneumoniae, reduces biofilm formation, and effectively treats bacterial lung infections in mice.

Background. Actinobacillus pleuropneumoniae, a member of the Pasteurellaceae family, is known for its highly infectious nature and is the primary causative agent of infectious pleuropneumonia in pigs. This disease poses a considerable threat to the global pig industry and leads to substantial economic losses due to reduced productivity, increased mortality rates, and the need for extensive veterinary care and treatment. Due to the emergence of multi-drug-resistant strains, Chinese herbal medicine is considered one of the best alternatives to antibiotics due to its unique mechanism of action and other properties. As a type of Chinese herbal medicine, Rhein has the advantages of a wide antibacterial spectrum and is less likely to develop drug resistance, which can perfectly solve the limitations of current antibacterial treatments.Methods. The killing effect of Rhein on A. pleuropneumoniae was detected by fluorescence quantification of differential expression changes of key genes, and scanning electron microscopy was used to observe the changes in A. pleuropneumoniae status after Rhein treatment. Establishing a mouse model to observe the treatment of Rhein after A. pleuropneumoniae infection.Results. Here, in this study, we found that Rhein had a good killing effect on A. pleuropneumoniae and that the MIC was 25 µg ml-1. After 3 h of action, Rhein (4×MIC) completely kills A. pleuropneumoniae and Rhein has good stability. In addition, the treatment with Rhein (1×MIC) significantly reduced the formation of bacterial biofilms. Therapeutic evaluation in a murine model showed that Rhein protects mice from A. pleuropneumoniae and relieves lung inflammation. Quantitative RT-PCR (Quantitative reverse transcription polymerase chain reaction is a molecular biology technique that combines both reverse transcription and polymerase chain reaction methods to quantitatively detect the amount of a specific RNA molecule) results showed that Rhein treatment significantly downregulated the expression of the IL-18 (Interleukin refers to a class of cytokines produced by white blood cells), TNF-α, p65 and p38 genes. Along with the downregulation of genes such as IL-18, it means that Rhein has an inhibitory effect on the expression of these genes, thereby reducing the activation of inflammatory cells and the production of inflammatory mediators. This helps reduce inflammation and protects tissue from further damage.Conclusions. This study reports the activity of Rhein against A. pleuropneumoniae and its mechanism, and reveals the ability of Rhein to treat A. pleuropneumoniae infection in mice, laying the foundation for the development of new drugs for bacterial infections.

Complete genome sequence of a subgenotype VIId Newcastle disease virus circulating predominantly in chickens in China.

At least 11 genotypes of class II viruses have been identified since the discovery of Newcastle disease virus (NDV) in 1926. Here, we reported the complete genome sequence of a prevalent NDV variant from China, belonging to subgenotype VIId in class II. The similar viruses have been the predominant strains circulating in China for the past decade, which occupied over 80% of Chinese prevalent strains and were phylogenetically different from currently available vaccines.

Analysis of energy loss in the helical hedge flow channel of fruit tree root emitter.

This paper proposes the design of a helical hedge flow channel with a high energy loss, which shows promising potential for application in fruit tree root emitters. The aim is to investigate the relationship between the energy loss form in the channel and its influencing factors. The hydraulic performance testing method is employed to analyze the factors that affect energy loss. The main influencing factors are determined using the response surface methodology (RSM) for experimental design. Based on the obtained experimental results, the energy loss form and influencing factors are analyzed, and a prediction model for the energy loss coefficient (ξ) is established. The results indicate that the ξ exhibits a decreasing trend with an increase in the diversion angle (α), a trend of first increasing and then decreasing with an increase in the channel width (b), and an increasing trend with an increase in the number of channel units (n). The effects of the straight section length (l1), convergence section length (l2), and bend radius (r) on the ξ can be neglected. The ranking of the geometric parameters' influence on the ξ is as follows: n > b > α > l1 > r > l2. The experimental results reveal that the ξ ranges from 19.2 to 234.3. Furthermore, the head loss along the flow channel constitutes merely 0.06-0.47% of the local head loss, The main form of energy loss in the spiral counter flow channel is local head loss. There is a significant linear relationship between α, b, n and the ξ, The established prediction model (R2 = 0.9691) can accurately predict the ξ of the channel.

Antimicrobial Peptide MPX with Broad-Spectrum Bactericidal Activity Promotes Proper Abscess Formation and Relieves Skin Inflammation.

Bacteria have developed antibiotic resistance during the large-scale use of antibiotics, and multidrug-resistant strains are common. The development of new antibiotics or antibiotic substitutes has become an important challenge for humankind. MPX is a 14 amino acid peptide belonging to the MP antimicrobial peptide family. In this study, the antibacterial spectrum of the antimicrobial peptide MPX was first tested. The antimicrobial peptide MPX was tested for antimicrobial activity against the gram-positive bacterium S. aureus ATCC 25923, the gram-negative bacteria E. coli ATCC 25922 and Salmonella enterica serovar Typhimurium CVCC541, and the fungus Candida albicans ATCC 90029. The results showed that MPX had good antibacterial activity against the above four strains, especially against E. coli, for which the MIC was as low as 15.625 µg/mL. The study on the bactericidal mechanism of the antimicrobial peptide revealed that MPX can destroy the integrity of the cell membrane, increase membrane permeability, and change the electromotive force of the membrane, thereby allowing the contents to leak out and mediating bacterial death. A mouse acute infection model was used to evaluate the therapeutic effect of MPX after acute infection of subcutaneous tissue by S. aureus. The study showed that MPX could promote tissue repair in S. aureus infection and alleviate lung damage caused by S. aureus. In addition, skin H&E staining showed that MPX treatment facilitated the formation of appropriate abscesses at the subcutaneous infection site and facilitated the clearance of bacteria by the skin immune system. The above results show that MPX has good antibacterial activity and broad-spectrum antibacterial potential and can effectively prevent the invasion of subcutaneous tissue by S. aureus, providing new ideas and directions for the immunotherapy of bacterial infections.

Effect of Gan Cao (Glycyrrhiza uralensis Fisch) polysaccharide on growth performance, immune function, and gut microflora of broiler chickens.

Glycyrrhiza uralensis Fisch, also called Gan Cao, is a commonly prescribed herb in traditional Chinese medicine. Gan Cao is associated with immune-modulation and antitumor potential though its mechanism of action is not well-known. To explore the effects of different dietary levels of Gan Cao polysaccharide (GCP) on broilers, a total of 400 Avian broiler chickens were randomly divided into 4 groups with 10 replicates of 10 broilers each. The broilers in the control group were fed a basal diet, while those in the experimental groups were fed the basal diet supplemented with 0.5%, 1.0%, and 1.5% GCP, respectively, for 42 d. The results showed a significant increase in the growth performance in the GCP groups. The antibody titer of NDV and the phagocytosis index was higher in the birds with GCP treatment than in the control group, with the 1% GCP addition displaying the highest titer. The Lactobacillus and Bifidobacteria count in the cecum content of the birds in the 1% GCP group was higher compared to the other groups. In conclusion, dietary supplementation with GCP had a substantial impact on the growth performance, immune response, and microflora population in the cecum of the birds, especially at a level of 1% addition.

Combined Antibody Tagged HRP Gold Nanoparticle Probe for Effective PCV2 Screening in Pig Farms.

Introduction: Porcine circovirus type 2 (PCV2) causes immune repression and intercurrent infections in pigs, resulting in a huge economic loss to the pig breeding industry. Additionally, the spread of PCV2 in pig farms can pollute the living environment of the residents in the farm's vicinity, which increases the rate of infections. Therefore, rapid and sensitive detection methods are needed for disease prevention and timely environmental cleaning. Methods: This research describes a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) that utilizes gold nanoparticles (AuNPs) in a functional, specific antibody labeled probe for the detection of PCV2. Due to their high specific surface area and histocompatibility, AuNPs were used as carriers of HRP labeled anti-PCV2 antibodies to amplify the detection signal. Results: Compared to conventional sandwich ELISA procedures, this method resulted in higher sensitivity (51-fold) and a shorter assay time with a limit of detection of 195 TCID50/mL. The cross-reactivity assay demonstrated that this assay was PCV2 specific. Conclusion: The amplified Ab (HRP) labeled AuNPs probe provides a sensitive analytical approach for the determination of the traces of the PCV2 antigen in early diagnosis.