Chicken miR-148a-3p regulates immune responses against AIV by targeting the MAPK signalling pathway and IFN-γ.

MicroRNAs are involved in the immune systems of host animals and play essential roles in several immune-related pathways. In the current study, we investigated the systemic biological function of the chicken miRNA gga-miR-148a-3p on immune responses in chicken lines resistant and susceptible to HPAIV-H5N1. We found that gga-miR-148a expression in the lung tissue of H5N1-resistant chickens was significantly downregulated during HPAIV-H5N1 infection. Overexpression of gga-miR-148a and a reporter construct with wild type or mutant IFN-γ, MAPK11, and TGF-ß2 3' untranslated region (3' UTR)-luciferase in chicken fibroblasts showed that gga-miR-148a acted as a direct translational repressor of IFN-γ, MAPK11, and TGF-ß2 by targeting their 3' UTRs. Furthermore, miR-148a directly and negatively influenced the expression of signalling molecules related to the MAPK signalling pathway, including MAPK11, TGF-ß2, and Jun, and regulated antiviral responses through interferon-stimulated genes and MHC class I and class II genes by targeting IFN-γ. Downstream of the MAPK signalling pathway, several proinflammatory cytokines such as IL-1ß, IFN-γ, IL-6, TNF-α, IFN-ß, and interferon-stimulated genes were downregulated by the overexpression of gga-miR-148a. Our data suggest that gga-miR-148a-3p is an important regulator of the MAPK signalling pathway and antiviral response. These findings improve our understanding of the biological functions of gga-miR-148a-3p, the mechanisms underlying the MAPK signalling pathway, and the antiviral response to HPAIV-H5N1 infection in chickens as well as the role of gga-miR-148a-3p in improving the overall performance of chicken immune responses for breeding disease-resistant chickens.

Factors Associated With Voriconazole Concentration in Pediatric Patients.

BACKGROUND: Serum concentrations of voriconazole are difficult to predict, especially in pediatric patients, because of its complex pharmacokinetic characteristics. This study aimed to identify the factors associated with the concentration of voriconazole in pediatric patients. METHODS: This cohort study was based on retrospective data collection and involved the administration of voriconazole to pediatric patients younger than 18 years, between January 2010 and August 2017. Electronic medical records of the patients were reviewed to collect demographic characteristics, voriconazole treatment regimen, and factors that could potentially influence voriconazole trough concentrations. A voriconazole trough serum concentration of less than 1.0 mcg/mL or greater than 5.5 mcg/mL was defined as outside the therapeutic range and was set as the outcome of this study. RESULTS: Among the 114 patients enrolled, 61 patients were included in the analysis. Oral administration of a maintenance dose of voriconazole and C-reactive protein (CRP) level were significantly and independently associated with a low initial trough concentration of voriconazole (<1.0 mcg/mL). Alanine aminotransferase levels were a significant factor associated with a high initial trough concentration of voriconazole (>5.5 mcg/mL) after adjusting for sex, age, weight, and serum creatinine (odds ratio 5.42; 95% confidence interval 1.34-21.97). CONCLUSIONS: Considering the variability of voriconazole concentrations in pediatric patients, monitoring certain parameters and considering the route of administration could help determine the therapeutic range of voriconazole and subsequently avoid unwanted effects.

Eyelid squinting improves near vision in against-the-rule and distance vision in with-the-rule astigmatism in pseudophakic eyes: an eye model experimental study.

BACKGROUND: To elucidate whether eyelid squinting improves near and distance vision in against-the-rule (ATR) and with-the-rule (WTR) simple myopic astigmatism in pseudophakic eyes. METHODS: A refraction-model eye was mounted on a wavefront analyzer. The eyelid fissure was simulated using a slit placed horizontally in front of the model eye. Four different refractive statuses [- 1.50 diopters (D) and - 3.00 D of both WTR and ATR simple myopic astigmatism] were set using cylindrical lenses. For each refractive status (emmetropia, - 1.50 D WTR, - 1.50 D ATR, - 3.00 D WTR, and - 3.00 D ATR astigmatism), wavefront aberrations were measured, both with and without the slit, 40 times each. RESULTS: The 2 mm horizontal slit caused a hyperopic focus shift (+ 6.69 µm) in - 1.50 D WTR astigmatism, whereas, in - 1.50 D ATR astigmatism, it caused a myopic focus shift (- 2.01 µm). The astigmatism was decreased in the ATR astigmatism groups and increased in the emmetropia and WTR astigmatism groups, respectively. Total aberrations were decreased in the emmetropia and WTR astigmatism groups and increased in the ATR astigmatism groups. When the reference plane was set to the near plane, total aberrations were decreased in the ATR astigmatism groups. CONCLUSION: As the horizontal slit was placed in front of the model eye, the focus moves nearer in ATR astigmatism and farther in WTR astigmatism. These effects of eyelid cause improvement of near vision of pseudophakic eyes with ATR astigmatism.

Robust gene selection methods using weighting schemes for microarray data analysis.

BACKGROUND: A common task in microarray data analysis is to identify informative genes that are differentially expressed between two different states. Owing to the high-dimensional nature of microarray data, identification of significant genes has been essential in analyzing the data. However, the performances of many gene selection techniques are highly dependent on the experimental conditions, such as the presence of measurement error or a limited number of sample replicates. RESULTS: We have proposed new filter-based gene selection techniques, by applying a simple modification to significance analysis of microarrays (SAM). To prove the effectiveness of the proposed method, we considered a series of synthetic datasets with different noise levels and sample sizes along with two real datasets. The following findings were made. First, our proposed methods outperform conventional methods for all simulation set-ups. In particular, our methods are much better when the given data are noisy and sample size is small. They showed relatively robust performance regardless of noise level and sample size, whereas the performance of SAM became significantly worse as the noise level became high or sample size decreased. When sufficient sample replicates were available, SAM and our methods showed similar performance. Finally, our proposed methods are competitive with traditional methods in classification tasks for microarrays. CONCLUSIONS: The results of simulation study and real data analysis have demonstrated that our proposed methods are effective for detecting significant genes and classification tasks, especially when the given data are noisy or have few sample replicates. By employing weighting schemes, we can obtain robust and reliable results for microarray data analysis.

The effects of proinflammatory cytokines on the apoptosis of corneal endothelial cells following argon laser iridotomy.

The aim of this study was to evaluate the relationship between the expression of proinflammatory cytokines and the apoptosis of corneal endothelial cells after argon laser iridotomy (ALI). ALI was performed on each quadrant of the iris in the right eye of mice (ALI1 group). Left eyes were used as control group. The levels of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß, and interferon (IFN)-γ in mice eyes were measured, and TUNEL staining was performed 12 h after ALI. Mice in the ALI-Dexa group were pretreated daily with an intraperitoneal injection of dexamethasone for 4 days before undergoing ALI and compared with mice without dexamethasone pretreatment (ALI2 group). Twelve corneas from six rabbits were incubated ex vivo with (n = 6) or without (n = 6) IL-1ß. TUNEL staining was performed 24 h after ex vivo incubation. In the mice experiment, the levels of IL-1ß, TNF-α, TGF-ß, and IFN-γ were increased in the ALI1 group compared to the control group. Although many TUNEL-positive cells were observed in the ALI1 group, those were not detected in the control group. Dexamethasone pretreatment inhibited the increase in the levels of all four proinflammatory cytokines and reduced TUNEL-positive cells. In the rabbit experiment, TUNEL-positive cells were increased in the incubated corneas with IL-1ß compared to those without IL-1ß. Expression of proinflammatory cytokines following ALI seems to play a role in the apoptosis of corneal endothelial cells after ALI. Dexamethasone pretreatment inhibited increases in proinflammatory cytokines and reduced the apoptosis of corneal endothelial cells.

The effect of posterior corneal flat meridian and astigmatism amount on the total corneal astigmatism estimated from anterior corneal measurements.

BACKGROUND: To evaluate the effects of posterior corneal astigmatism and the absolute flat meridian difference between anterior and posterior corneal surfaces (AMDAnt-Post) on the estimation of total corneal astigmatism using anterior corneal measurements (simulated keratometry [K]). METHODS: Ninety-nine eyes of 99 healthy participants were enrolled. Anterior, posterior, and total mean corneal power, cylinder power, flat meridian, and vector components J0, and J45 measured by a dual Scheimpflug camera were analyzed. The correlation between the posterior corneal cylinder power, AMDAnt-Post, and the difference in the cylinder power between simulated K and total cornea (cylinder power differenceSimK-Tot) were evaluated. RESULTS: The cylinder power differenceSimK-Tot was positively correlated with the posterior corneal cylinder power (rho = 0.704 and P < 0.001) and negatively correlated with AMDAnt-Post (rho = -0.717 and P < 0.001). In the multivariate linear regression analysis, anterior corneal J0 was strongly associated with the posterior corneal cylinder power and the AMDAnt-Post. When corneal J0 had a positive value, the cylinder power of simulated K tended to be larger than the total corneal cylinder power. In comparison, the opposite trend was presented in eyes with negative anterior corneal J0. When anterior corneal J0 was larger than 1.0 or smaller than -0.9, the errors from estimating the total corneal cylinder power using anterior corneal measurements tended to be larger than 0.25 D. CONCLUSION: Posterior corneal astigmatism should be considered for more accurate corneal astigmatism predictions, especially in eyes with anterior corneal astigmatism greater than 2.0 D of with-the-rule astigmatism or greater than 1.8 D of against-the-rule astigmatism.

Use of corneal power-specific constants to improve the accuracy of the SRK/T formula.

PURPOSE: To evaluate the effect of average corneal power (K) and axial length (AL) in a data-adjusted A-constant for improving the refractive outcome in the Sanders-Retzlaff-Kraff (SRK)/T formula. DESIGN: Retrospective, consecutive, case series. PARTICIPANTS: A total of 637 patients (637 eyes) who underwent uncomplicated phacoemulsification with implantation of the Acrysof IQ (IQ; Alcon, Fort Worth, TX; 314 eyes) or Akreos AO (AO; Bausch & Lomb, Rochester, NY; 323 eyes) intraocular lens (IOL) by a single surgeon. METHODS: The correlation among the K, AL, and predicted refractive error in the SRK/T formula was analyzed. Patients were divided into 2 subgroups, the first to calculate the different data-adjusted A-constants based on the K and the second to compare the median absolute error (MedAE) based on different A-constants with the traditional A-constant in the SRK/T formula. MAIN OUTCOME MEASURES: The data-adjusted A-constant and the MedAE (diopters [D]). RESULTS: The data-adjusted A-constant showed a decreasing trend as K increased. The data-adjusted A-constant was 119.04 in the IQ group and 118.27 in the AO group. The calculated A-constant was 119.33 in the IQ group and 118.57 in the AO group when the cornea was flatter than 43.0 D and 43.2 D, respectively. The A-constant was 118.71 in the IQ group and 117.96 in the AO group when the cornea was steeper than or equal to 44.7 D and 45.0 D, respectively. The MedAE decreased from 0.29 D to 0.23 D in the IQ group (P = 0.001) and from 0.44 D to 0.38 D in the AO group (P < 0.001) when different A-constants were used. The MedAE further decreased from 0.36 D to 0.24 D in the IQ group (P = 0.005) and from 0.58 D to 0.37 D in the AO group (P < 0.001) when subjects with K 1.00 D or more above or 1.00 D below the most accurate K in each group were compared. CONCLUSIONS: For a steep cornea, the calculated A-constant was smaller than that of the entire K, but for a flat cornea, a larger A-constant was calculated. Using different A-constants based on the K improved the refraction outcomes relying on the SRK/T formula.

Inactivation of foodborne pathogens in ground pork tenderloin using 915 MHz microwave heating depending on power level.

The main purpose of this research was to investigate the effect of power level of 915 MHz microwave heating on the inactivation of foodborne pathogens in ground pork and its bactericidal mechanism. It was demonstrated that the heating rate was proportional to the power level. For instance, the heating rates observed at microwave heating powers of 2, 3, 4, and 5 kW were 1.70, 2.77, 3.35, and 4.03℃/s, respectively. The bactericidal effect of microwave heating also significantly (P < 0.05) increased with increasing power level. In particular, when ground pork was subjected to microwave heating at 5 kW, the reduction level of pathogens was>2 log units higher than at 2 kW. To determine the bactericidal mechanism of microwave heating depending on power level, changes in transmembrane potential and DNA damage were determined using fluorescence. The extent of depolarization in membrane potential of pathogens significantly (P < 0.05) increased as power level increased. There was no significant difference in degree of DNA damage at different power levels. However, the percentage of DNA damage was>86% at all power levels. The transmembrane potential assay indicates that the bacteria exhibited dramatic pore formation on the membrane at 5 kW. Through transmission electron microscopy, the electroporation effect was enhanced as power level increased. Moreover, the quality of ground pork subjected to microwave heating at 2-5 kW was determined by measuring the moisture content, cooking loss, and texture profile.

Exosome-mediated delivery of gga-miR-20a-5p regulates immune response of chicken macrophages by targeting IFNGR2, MAPK1, MAP3K5, and MAP3K14.

OBJECTIVE: This study aims to evaluate the target genes of gga-miR-20a-5p and the regulated immune responses in the chicken macrophage cell line, HD11, by the exosome-mediated delivery of miR-20a-5p. METHODS: Exosomes were purified from the chicken macrophage cell line HD11. Then, mimic gga-miR-20p or negative control miRNA were internalized into HD11 exosomes. HD11 cells were transfected with gga-miR-20a-5p or negative control miRNA containing exosomes. After 44 h of transfection, cells were incubated with or without 5 µg/mL poly(I:C) for 4 h. Then, expression of target genes and cytokines was evaluated by quantitative real-time polymerase chain reaction. RESULTS: Using a luciferase reporter assay, we identified that gga-miR-20a-5p directly targeted interferon gamma receptor 2 (IFNGR2), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase kinase kinase 5 (MAP3K5), and mitogen-activated protein kinase kinase kinase 14 (MAP3K14). Moreover, the exosome-mediated delivery of gga-miR-20a-5p successfully repressed the expression of IFNGR2, MAPK1, MAP3K5, and MAP3K14 in HD11 cells. The expressions of interferon-stimulated genes (MX dynamin like GTPase 1 [MX1], eukaryotic translation initiation factor 2A [EIF2A], and oligoadenylate synthase-like [OASL]) and proinflammatory cytokines (interferon-gamma [IFNG], interleukin-1 beta [IL1B], and tumor necrosis factor-alpha [TNFA]) were also downregulated by exosomal miR-20a-5p. In addition, the proliferation of HD11 cells was increased by exosomal miR-20a-5p. CONCLUSION: The exosome-mediated delivery of gga-miR-20a-5p regulated immune responses by controlling the MAPK and apoptotic signaling pathways. Furthermore, we expected that exosomal miR-20a-5p could maintain immune homeostasis against highly pathogenic avian influenza virus H5N1 infection by regulating the expression of proinflammatory cytokines and cell death.

Analysis of miRNA expression in the trachea of Ri chicken infected with the highly pathogenic avian influenza H5N1 virus.

BACKGROUND: Highly pathogenic avian influenza virus (HPAIV) is considered a global threat to both human health and the poultry industry. MicroRNAs (miRNA) can modulate the immune system by affecting gene expression patterns in HPAIV-infected chickens. OBJECTIVES: To gain further insights into the role of miRNAs in immune responses against H5N1 infection, as well as the development of strategies for breeding disease-resistant chickens, we characterized miRNA expression patterns in tracheal tissues from H5N1-infected Ri chickens. METHODS: miRNAs expression was analyzed from two H5N1-infected Ri chicken lines using small RNA sequencing. The target genes of differentially expressed (DE) miRNAs were predicted using miRDB. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis were then conducted. Furthermore, using quantitative real-time polymerase chain reaction, we validated the expression levels of DE miRNAs (miR-22-3p, miR-146b-3p, miR-27b-3p, miR-128-3p, miR-2188-5p, miR-451, miR-205a, miR-203a, miR-21-3p, and miR-200a-3p) from all comparisons and their immune-related target genes. RESULTS: A total of 53 miRNAs were significantly expressed in the infection samples of the resistant compared to the susceptible line. Network analyses between the DE miRNAs and target genes revealed that DE miRNAs may regulate the expression of target genes involved in the transforming growth factor-beta, mitogen-activated protein kinase, and Toll-like receptor signaling pathways, all of which are related to influenza A virus progression. CONCLUSIONS: Collectively, our results provided novel insights into the miRNA expression patterns of tracheal tissues from H5N1-infected Ri chickens. More importantly, our findings offer insights into the relationship between miRNA and immune-related target genes and the role of miRNA in HPAIV infections in chickens.

Chicken miR-26a-5p modulates MDA5 during highly pathogenic avian influenza virus infection.

MicroRNAs play crucial roles in immune-related pathways in host animals. In this study, we aimed to investigate the systemic biological function of gga-miR-26a-5p, a chicken miRNA, in the immune responses to HPAIV H5N1 infection in the Vietnamese Ri chicken line. Our results showed a significant downregulation in gga-miR-26a expression in the lung tissue of Ri chickens during HPAIV H5N1 infection. Overexpression of gga-miR-26a and the reporter construct, either containing the wildtype or mutant melanoma differentiation-associated protein 5 (MDA5) 3' untranslated region (3' UTR)-luciferase, into a chicken fibroblast cell line, revealed that gga-miR-26a can act as a direct translational repressor of MDA5 by targeting the 3' UTRs. Additionally, miR-26a negatively regulated the expression of the signaling molecules related to the MDA5 signaling pathway, including MDA5, mitochondrial antiviral-signaling (MAVS), interferon regulatory factor 7 (IRF7), p38 mitogen-activated protein kinases, and nuclear factor-kappa B (NF-κB). Moreover, downstream of the IRF7 and NF-κB signaling pathway, the proinflammatory cytokines such as IL-1ß, IFN-γ, IFN-α, IFN-ß, and the interferon-stimulated gene (Mx1) were, likewise, downregulated by the overexpression of gga-miR-26a. These findings suggest that gga-miR-26a-5p serves as an important regulator in the MDA5 signaling pathway and antiviral response. Overall, our results contribute to an improved understanding of the biological functions of gga-miR-26a-5p, alongside the mechanisms underlying the MDA5 signaling pathway, and the antiviral response to HPAIV-H5N1 infection in chickens.

HPAI-resistant Ri chickens exhibit elevated antiviral immune-related gene expression.

BACKGROUND: Highly pathogenic avian influenza viruses (HPAIVs) is an extremely contagious and high mortality rates in chickens resulting in substantial economic impact on the poultry sector. Therefore, it is necessary to elucidate the pathogenic mechanism of HPAIV for infection control. OBJECTIVE: Gene set enrichment analysis (GSEA) can effectively avoid the limitations of subjective screening for differential gene expression. Therefore, we performed GSEA to compare HPAI-infected resistant and susceptible Ri chicken lines. METHODS: The Ri chickens Mx(A)/BF2(B21) were chosen as resistant, and the chickens Mx(G)/BF2(B13) were selected as susceptible by genotyping the Mx and BF2 genes. The tracheal tissues of HPAIV H5N1 infected chickens were collected for RNA sequencing followed by GSEA analysis to define gene subsets to elucidate the sequencing results. RESULTS: We identified four differentially expressed pathways, which were immune-related pathways with a total of 78 genes. The expression levels of cytokines (IL-1ß, IL-6, IL-12), chemokines (CCL4 and CCL5), type interferons and their receptors (IFN-ß, IFNAR1, IFNAR2, and IFNGR1), Jak-STAT signaling pathway genes (STAT1, STAT2, and JAK1), MHC class I and II and their co-stimulatory molecules (CD80, CD86, CD40, DMB2, BLB2, and B2M), and interferon stimulated genes (EIF2AK2 and EIF2AK1) in resistant chickens were higher than those in susceptible chickens. CONCLUSIONS: Resistant Ri chickens exhibit a stronger antiviral response to HPAIV H5N1 compared with susceptible chickens. Our findings provide insights into the immune responses of genetically disparate chickens against HPAIV.

MicroRNA expression profiling in the lungs of genetically different Ri chicken lines against the highly pathogenic avian influenza H5N1 virus.

The highly pathogenic avian influenza (HPAI) virus triggers infectious diseases, resulting in pulmonary damage and high mortality in domestic poultry worldwide. This study aimed to analyze miRNA expression profiles after infection with the HPAI H5N1 virus in resistant and susceptible lines of Ri chickens.For this purpose, resistant and susceptible lines of Vietnamese Ri chicken were used based on the A/G allele of Mx and BF2 genes. These genes are responsible for innate antiviral activity and were selected to determine differentially expressed (DE) miRNAs in HPAI-infected chicken lines using small RNA sequencing. A total of 44 miRNAs were DE after 3 days of infection with the H5N1 virus. Computational program analysis indicated the candidate target genes for DE miRNAs to possess significant functions related to cytokines, chemokines, MAPK signaling pathway, ErBb signaling pathway, and Wnt signaling pathway. Several DE miRNA-mRNA matches were suggested to play crucial roles in mediating immune functions against viral evasion. These results revealed the potential regulatory roles of miRNAs in the immune response of the two Ri chicken lines against HPAI H5N1 virus infection in the lungs.

Small RNA sequencing and profiling of serum-derived exosomes from African swine fever virus-infected pigs.

African swine fever (ASF) virus (ASFV) is responsible for one of the most severe swine diseases worldwide, with a morbidity rate of up to 100%; no vaccines or antiviral medicines are available against the virus. Exosomal miRNAs from individual cells can regulate the immune response to infectious diseases. In this study, pigs were infected with an ASFV Pig/HN/07 strain that was classified as acute form, and exosomal miRNA expression in the serum of infected pigs was analyzed using small RNA sequencing (small RNA-seq). Twenty-seven differentially expressed (DE) miRNAs were identified in the ASFV-infected pigs compared to that in the uninfected controls. Of these, 10 were upregulated and 17 were downregulated in the infected pigs. All DE miRNAs were analyzed using gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and the DE miRNAs were found to be highly involved in T-cell receptor signaling, cGMP-PKG signaling, Toll-like receptor, MAPK signaling, and mTOR signaling pathways. Furthermore, the Cytoscape network analysis identified the network of interactions between DE miRNAs and target genes. Finally, the transcription levels of four miRNA genes (ssc-miR-24-3p, ssc-miR-130b-3p, ssc-let-7a, and ssc-let-7c) were examined using quantitative real-time PCR (qRT-PCR) and were found to be consistent with the small RNA-seq data. These DE miRNAs were associated with cellular genes involved in the pathways related to immune response, virus-host interactions, and several viral genes. Overall, our findings provide an important reference and improve our understanding of ASF pathogenesis and the immune or protective responses during an acute infection in the host.

African swine fever is a viral disease caused by African swine fever virus (ASFV) which induces a big threat to the pig industry in the world. To date, there are no vaccines or antiviral medicines against the ASFV. Therefore, it is important to improve the understanding of the pathogenesis of ASFV and host­pathogen interaction using miRNA that may regulate genes related to the immune system. This study aimed to investigate the differentially expressed (DE) miRNA in serum-derived exosomes from African swine fever virus infected pigs. We successfully infected pigs with an ASFV Pig/HN/07 strain and identified the DE miRNAs in serum-derived exosomes using small RNA sequencing. Our results showed that total of 27 miRNAs were differentially expressed in serum-derived exosomes from ASFV-infected pigs. We analyzed the small RNA sequencing results using gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and found that most DE miRNA may regulate the expression of genes related with the immune response pathway (T-cell receptor signaling pathway, cGMP-PKG signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, etc.).

Comparative analysis of exosomal miRNAs derived from lipopolysaccharide and polyinosinic-polycytidylic acid-stimulated chicken macrophage cell line.

Exosomes play important roles in cellular communication by delivering exosomal proteins and nucleic acid molecules to cells. In particular, exosomal miRNAs can modulate various biological processes in recipient cells by repressing target gene expression. In this study, to identify the composition of exosomal miRNAs and their regulatory mechanisms against bacterial and viral infections, profiles of exosomal miRNAs from lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly(I:C))-stimulated chicken macrophage cell line (HD11) were analyzed by small RNA sequencing. Exosomes were purified after stimulation with LPS (1 µg/mL) and poly(I:C) (50 µg/mL) for 24 h. Then, exosomal RNA were analyzed for small RNA sequencing using the HiSeq 2500 System. Thirty six differentially expressed miRNAs (DE miRNAs) were obtained by comparing LPS-stimulated exosomes (LPS-EXO) and unstimulated exosomes (CTRL-EXO), 42 DE miRNAs in poly(I:C)-stimulated exosomes (POLY-EXO) and CTRL-EXO, and 45 DE miRNAs in LPS-EXO and POLY-EXO. Target genes of DE miRNAs were predicted using miRDB and TargetScan. KEGG pathway analysis showed that most of the target genes were related to mitogen-activated protein kinase and Wnt signaling pathways. Moreover, results of qRT-PCR for miRNAs (gga-miR-142-3p, gga-miR-19a-3p, gga-miR-21-3p, gga-miR-301a-3p, gga-miR-338-3p, and gga-miR-3523) were consistent with the sequencing results. This study will provide knowledge about immuno-regulatory mechanisms of exosomal miRNAs derived from macrophages against pathological insults such as bacterial and viral infections.

Profiling and analysis of exosomal miRNAs derived from highly pathogenic avian influenza virus H5N1-infected White Leghorn chickens.

Exosomes are small cell membrane-derived vesicles; they play important roles as mediators of cell-to-cell communication via delivery of their contents, such as proteins and microRNAs (miRNAs). In particular, exosomal miRNAs regulate the gene expression of recipient cells by inhibiting the expression of target mRNAs. In this study, we investigated the miRNA expression profiles of highly pathogenic avian influenza virus (HPAIV) H5N1-infected White Leghorn chickens and analyzed the functions of their target genes. After 3 d of infection with A/chicken/Vietnam/NA-01/2019 (H5N1), exosomes were isolated from the blood serum of White Leghorn chickens for small RNA sequencing. We accordingly identified 10 differentially expressed miRNAs (DE miRNAs; 5 upregulated and 5 downregulated) by comparing the exosomes derived from infected and noninfected chickens. The target genes of DE miRNAs were predicted using miRDB and TargetScan for Gene Ontology and KEGG pathway enrichment analyses. A majority of the target genes was found to be associated with the MAPK signaling pathway; several immune-related genes were identified as being regulated by these DE miRNAs. Moreover, we predicted DE miRNA binding sites in HPAIV RNA segments using the RNAhybrid algorithm. The findings of this study provide a theoretical basis for gaining insights into the regulatory mechanisms of exosomal miRNAs in response to HPAIV H5N1 infection and the identification of novel vaccine candidates.

Exosomes from H5N1 avian influenza virus-infected chickens regulate antiviral immune responses of chicken immune cells.

Exosomes (membrane-derived vesicles) enable intracellular communication by delivering lipids, proteins, DNA, and RNA from one cell to another. Highly pathogenic avian influenza virus (HPAIV) H5N1 causes considerable economic loss in the poultry industry and poses a public health concern. The host innate immune system defends against H5N1 infection by activating antiviral immune responses. This study aimed to demonstrated that immunomodulatory effects of exosomes from HPAIV H5N1-infected White Leghorn chickens on chicken macrophages, fibroblasts, T cell, and B cell lines. The expression of type I interferons (IFN-α and -ß) were highly upregulated in immune-related cell lines after treatment with exosomes derived from H5N1-infected chickens. Levels of pro-inflammatory cytokines, such as IFN-γ, IL-1ß, and CXCL8, were also elevated by the exosomes. The mitogen-activated protein kinase (MAPK) signaling pathway was stimulated in immune-related cells by such exosomes via phosphorylation of extracellular regulated kinases 1/2 and p38 signaling molecules. Furthermore, the H5N1 viral proteins, nucleoprotein (NP) and non-structural protein (NS1), were packaged in exosomes and successfully transferred to non-infected immune-related cells. Therefore, exosomes from H5N1-infected chickens induced pro-inflammatory cytokine expression and stimulated the MAPK signaling pathway by delivering key viral proteins. These findings would aid better understanding of the mechanism underlying the modulation of antiviral immune responses of host immune-related cells by viral-protein-carrying exosomes and support their further application as a novel exosome-based H5N1 AIV vaccine platform.

A Serum Marker for Early Pancreatic Cancer With a Possible Link to Diabetes.

BACKGROUND: Pancreatic cancer (PC) has a grim prognosis, and an early diagnostic biomarker has been highly desired. The molecular link between diabetes and PC has not been well established. METHODS: Bioinformatics screening was performed for a serum PC marker. Experiments in cell lines (5 PC and 1 normal cell lines), mouse models, and human tissue staining (37 PC and 10 normal cases) were performed to test asprosin production from PC. Asprosin's diagnostic performance was tested with serums from multi-center cohorts (347 PC, 209 normal, and 55 additional diabetic patients) and evaluated according to PC status, stages, and diabetic status, which was compared with that of CA19-9. RESULTS: Asprosin, a diabetes-related hormone, was found from the bioinformatics screening, and its production from PC was confirmed. Serum asprosin levels from multi-center cohorts yielded an age-adjusted diagnostic area under the curve (AUC) of 0.987 (95% confidence interval [CI] = 0.961 to 0.997), superior to that of CA19-9 (AUC = 0.876, 95% CI = 0.847 to 0.905), and a cut-off of 7.18 ng/mL, at which the validation set exhibited a sensitivity of 0.957 and a specificity of 0.924. Importantly, the performance was maintained in early-stage and non-metastatic PC, consistent with the tissue staining. A slightly lower performance against additional diabetic patients (n = 55) was restored by combining asprosin and CA19-9 (AUC = 0.985, 95% CI = 0.975 to 0.995). CONCLUSIONS: Asprosin is presented as an early-stage PC serum marker that may provide clues for PC-induced diabetes. Larger prospective clinical studies are warranted to solidify its utility.

Undergraduate nursing students' experience of learning respiratory system assessment using flipped classroom: A mixed methods study.

BACKGROUND: Knowledge and skill acquisition to perform an accurate respiratory system assessment is a key competency expected in undergraduate nursing students. Learning physical assessment requires the integration of multiple knowledge bases and skills; hence, applying an innovative teaching approach, such as the flipped-classroom (FC) approach, fosters an active and student-centered learning environment for physical assessment class. OBJECTIVES: This study evaluated FC's feasibility in delivering respiratory system assessment content in a health assessment course and explored the changes in nursing students' perceptions regarding student-centeredness and active learning environments before and after applying FC. DESIGN: A single group pre- and post-test concurrent mixed-methods design was used. SETTINGS: This study was conducted in a private nursing college in South Korea. PARTICIPANTS: A convenience sample of 91 second year undergraduate nursing students enrolled in a health assessment course. METHODS: FC was offered at one didactic session of a physical assessment course. In the FC, students completed a self-directed pre-class activities using online lecture videos and reading materials prior to the class and participated in interactive team-based learning activities inside the classroom. Skills lab practicum took place after the FC. Students' perceptions regarding student-centeredness and active learning environments, in terms of teaching, social, and cognitive presences were measured before (T1) and after (T2) conducting the FC. Qualitative data were obtained at T2 using free-response questions, which required students to comment on their FC experience. RESULTS: Participants' perceptions of student-centeredness significantly increased from T1 to T2. Although student-perceived teaching and social presence in their learning environment showed upward trends from T1 to T2, these changes were not statistically significant. Students considered FC an acceptable approach to foster active learning in a supportive learning environment. CONCLUSIONS: This study revealed that incorporating FC to deliver respiratory system assessment content was feasible and considered acceptable by undergraduate nursing students.

Telomere shortening reflecting physical aging is associated with cognitive decline and dementia conversion in mild cognitive impairment due to Alzheimer's disease.

We investigated whether telomere length (TL) reflecting physical rather than chronological aging is associated with disease progression in the different cognitive stages of Alzheimer's disease (AD). Study participants included 89 subjects with amyloid pathology (A+), determined through amyloid PET or cerebrospinal fluid analysis, including 26 cognitively unimpaired (CU A+) individuals, 28 subjects with mild cognitive impairment (MCI A+), and 35 subjects with AD dementia (ADD A+). As controls, 104 CU A- individuals were selected. The participants were evaluated annually over two years from baseline. Compared to the highest TL quartile group of MCI A+ participants, the lowest TL quartile group yielded 2-year differences of -9.438 (95% confidence interval [CI] = -14.567 ~ -4.309), -26.708 (-41.576 ~ -11.839), 3.198 (1.323 ~ 5.056), and 2.549 (0.527 ~ 4.571) on the Mini-Mental State Examination, Consortium to Establish a Registry for AD, Clinical Dementia Rating-Sum of Boxes, and Blessed Dementia Scale-Activities of Daily Living, respectively. With this group, the lowest TL quartile group had a significantly greater probability of progressing to ADD than the highest TL quartile group (hazard ratio = 13.16, 95% CI = 1.11 ~ 156.61). Telomere shortening may be associated with rapid cognitive decline and conversion to dementia in MCI A+.