RNF216 Inhibits the Replication of H5N1 Avian Influenza Virus and Regulates the RIG-I Signaling Pathway in Ducks.

The RING finger (RNF) family, a group of E3 ubiquitin ligases, plays multiple essential roles in the regulation of innate immunity and resistance to viral infection in mammals. However, it is still unclear whether RNF proteins affect the production of IFN-I and the replication of avian influenza virus (AIV) in ducks. In this article, we found that duck RNF216 (duRNF216) inhibited the duRIG-I signaling pathway. Conversely, duRNF216 deficiency enhanced innate immune responses in duck embryonic fibroblasts. duRNF216 did not interacted with duRIG-I, duMDA5, duMAVS, duSTING, duTBK1, or duIRF7 in the duck RIG-I pathway. However, duRNF216 targeted duTRAF3 and inhibited duMAVS in the recruitment of duTRAF3 in a dose-dependent manner. duRNF216 catalyzed K48-linked polyubiquitination of duck TRAF3, which was degraded by the proteasome pathway. Additionally, AIV PB1 protein competed with duTRAF3 for binding to duRNF216 to reduce degradation of TRAF3 by proteasomes in the cytoplasm, thereby slightly weakening duRNF216-mediated downregulation of IFN-I. Moreover, although duRNF216 downregulated the IFN-ß expression during virus infection, the expression level of IFN-ß in AIV-infected duck embryonic fibroblasts overexpressing duRNF216 was still higher than that in uninfected cells, which would hinder the viral replication. During AIV infection, duRNF216 protein targeted the core protein PB1 of viral polymerase to hinder viral polymerase activity and viral RNA synthesis in the nucleus, ultimately strongly restricting viral replication. Thus, our study reveals a new mechanism by which duRNF216 downregulates innate immunity and inhibits AIV replication in ducks. These findings broaden our understanding of the mechanisms by which the duRNF216 protein affects AIV replication in ducks.

HGCTNet: Handcrafted Feature-Guided CNN and Transformer Network for Wearable Cuffless Blood Pressure Measurement.

Biosignals collected by wearable devices, such as electrocardiogram and photoplethysmogram, exhibit redundancy and global temporal dependencies, posing a challenge in extracting discriminative features for blood pressure (BP) estimation. To address this challenge, we propose HGCTNet, a handcrafted feature-guided CNN and transformer network for cuffless BP measurement based on wearable devices. By leveraging convolutional operations and self-attention mechanisms, we design a CNN-Transformer hybrid architecture to learn features from biosignals that capture both local information and global temporal dependencies. Then, we introduce a handcrafted feature-guided attention module that utilizes handcrafted features extracted from biosignals as query vectors to eliminate redundant information within the learned features. Finally, we design a feature fusion module that integrates the learned features, handcrafted features, and demographics to enhance model performance. We validate our approach using two large wearable BP datasets: the CAS-BP dataset and the Aurora-BP dataset. Experimental results demonstrate that HGCTNet achieves an estimation error of 0.9 ± 6.5 mmHg for diastolic BP (DBP) and 0.7 ± 8.3 mmHg for systolic BP (SBP) on the CAS-BP dataset. On the Aurora-BP dataset, the corresponding errors are -0.4 ± 7.0 mmHg for DBP and -0.4 ± 8.6 mmHg for SBP. Compared to the current state-of-the-art approaches, HGCTNet reduces the mean absolute error of SBP estimation by 10.68% on the CAS-BP dataset and 9.84% on the Aurora-BP dataset. These results highlight the potential of HGCTNet in improving the performance of wearable cuffless BP measurements. The dataset and source code are available at https://github.com/zdzdliu/HGCTNet.

Chicken UFL1 Restricts Avian Influenza Virus Replication by Disrupting the Viral Polymerase Complex and Facilitating Type I IFN Production.

During avian influenza virus (AIV) infection, host defensive proteins promote antiviral innate immunity or antagonize viral components to limit viral replication. UFM1-specific ligase 1 (UFL1) is involved in regulating innate immunity and DNA virus replication in mammals, but the molecular mechanism by which chicken (ch)UFL1 regulates AIV replication is unclear. In this study, we first identified chUFL1 as a negative regulator of AIV replication by enhancing innate immunity and disrupting the assembly of the viral polymerase complex. Mechanistically, chUFL1 interacted with chicken stimulator of IFN genes (chSTING) and contributed to chSTING dimerization and the formation of the STING-TBK1-IRF7 complex. We further demonstrated that chUFL1 promoted K63-linked polyubiquitination of chSTING at K308 to facilitate chSTING-mediated type I IFN production independent of UFMylation. Additionally, chUFL1 expression was upregulated in response to AIV infection. Importantly, chUFL1 also interacted with the AIV PA protein to inhibit viral polymerase activity. Furthermore, chUFL1 impeded the nuclear import of the AIV PA protein and the assembly of the viral polymerase complex to suppress AIV replication. Collectively, these findings demonstrate that chUFL1 restricts AIV replication by disrupting the viral polymerase complex and facilitating type I IFN production, which provides new insights into the regulation of AIV replication in chickens.

Cuffless Blood Pressure Measurement Using Smartwatches: A Large-Scale Validation Study.

This study aimed to evaluate the performance of cuffless blood pressure (BP) measurement techniques in a large and diverse cohort of participants. We enrolled 3077 participants (aged 18-75, 65.16% women, 35.91% hypertensive participants) and conducted followed-up for approximately 1 month. Electrocardiogram, pulse pressure wave, and multiwavelength photoplethysmogram signals were simultaneously recorded using smartwatches; dual-observer auscultation systolic BP (SBP) and diastolic BP (DBP) reference measurements were also obtained. Pulse transit time, traditional machine learning (TML), and deep learning (DL) models were evaluated with calibration and calibration-free strategy. TML models were developed using ridge regression, support vector machine, adaptive boosting, and random forest; while DL models using convolutional and recurrent neural networks. The best-performing calibration-based model yielded estimation errors of 1.33 ± 6.43 mmHg for DBP and 2.31 ± 9.57 mmHg for SBP in the overall population, with reduced SBP estimation errors in normotensive (1.97 ± 7.85 mmHg) and young (0.24 ± 6.61 mmHg) subpopulations. The best-performing calibration-free model had estimation errors of -0.29 ± 8.78 mmHg for DBP and -0.71 ± 13.04 mmHg for SBP. We conclude that smartwatches are effective for measuring DBP for all participants and SBP for normotensive and younger participants with calibration; performance degrades significantly for heterogeneous populations including older and hypertensive participants. The availability of cuffless BP measurement without calibration is limited in routine settings. Our study provides a large-scale benchmark for emerging investigations on cuffless BP measurement, highlighting the need to explore additional signals or principles to enhance the accuracy in large-scale heterogeneous populations.

Genetic characterization and pathogenicity of H7N9 highly pathogenic avian influenza viruses isolated from South China in 2017.

Since 2017, the new H7N9 highly pathogenic avian influenza viruses (HPAIVs) have been responsible for more than 200,000 cases of chicken infection and more than 120,000 chicken deaths in China. Our previous study found that the Q26 was chicken-origin H7N9 HPAIV. In this study, we analyzed the genetic characterization of Q24, Q65, Q66, Q85, and Q102 H7N9 avian influenza viruses isolated from Guangdong, China in 2017. Our results showed that these viruses were highly pathogenic and belonged to two different genotypes, which suggested they occurred genetic reassortant. To investigate the pathogenicity, transmission, and host immune responses of H7N9 virus in chickens, we selected Q24 and Q26 viruses to inoculate chickens. The Q24 and Q26 viruses killed all inoculated chickens within 3 days and replicated effectively in all tested tissues. They were efficiently transmitted to contact chickens and killed them within 4 days through direct contact. Furthermore, we found that the expressions of several immune-related genes (e.g., TLR3, TLR7, MDA5, MAVS, IFN-ß, IL-6, IL-8, OAS, Mx1, MHC I, and MHC II) were upregulated obviously in the lungs and spleen of chickens inoculated with the two H7N9 viruses at 24 h post-inoculation (HPI). Among these, IL-6 and IFN-ß in lungs were the most upregulated (by 341.02-381.48-fold and 472.50-500.56-fold, respectively). These results suggest that the new H7N9 viruses isolated in 2017, can replicate and transmit effectively and trigger strong immune responses in chickens, which helps us understand the genetic and pathogenic variations of H7N9 HPAIVs in China.

Duck MARCH8 Negatively Regulates the RLR Signaling Pathway through K29-Linked Polyubiquitination of MAVS.

Mitochondrial antiviral signaling protein (MAVS) is a key adaptor in cellular innate immunity. Ubiquitination plays an important role in regulating MAVS-mediated innate immune responses; however, the molecular mechanisms underlying ubiquitination of MAVS have not been fully elucidated. In this study, we first identified the mitochondria-resident E3 ligase duck membrane-associated RING-CH 8 (duMARCH8) in ducks as a negative regulator of duck MAVS (duMAVS). Overexpression of duMARCH8 impaired the duMAVS-mediated signaling pathway, whereas knockdown of duMARCH8 resulted in the opposite effects. The suppression was due to duMARCH8 interacting with duMAVS and degrading it in a proteasome-dependent manner. We further found that duMARCH8 interacted with the 176-619 regions of duMAVS. Moreover, duMARCH8 catalyzed the K29-linked polyubiquitination of duMAVS at Lys 398 to inhibit the MAVS-mediated signaling pathway. Collectively, our findings reveal a new strategy involving MARCH8 that targets the retinoic acid-inducible gene-I-like receptor signaling pathway to regulate innate immune responses in ducks.

Duck TRIM29 negatively regulates type I IFN production by targeting MAVS.

The innate immune response is a host defense mechanism that induces type I interferon and proinflammatory cytokines. Tripartite motif (TRIM) family proteins have recently emerged as pivotal regulators of type I interferon production in mammals. Here, we first identified duck TRIM29, which encodes 571 amino acids and shows high sequence homology with other bird TRIM29 proteins. DuTRIM29 inhibited IFN-ß and IRF7 promoter activation in a dose-dependent manner and downregulated the mRNA expression of IFN-ß, IRF7, Mx and IL-6 mediated by duRIG-I. Moreover, duTRIM29 interacted and colocalized with duMAVS in the cytoplasm. DuTRIM29 interacted with duMAVS via its C-terminal domains. In addition, duTRIM29 inhibited IFN-ß and IRF7 promoter activation and significantly downregulated IFN-ß and immune-related gene expression mediated by duMAVS in ducks. Furthermore, duTRIM29 induced K29-linked polyubiquitination and degradation of duMAVS to suppress the expression of IFN-ß. Overall, our results demonstrate that duTRIM29 negatively regulates type I IFN production by targeting duMAVS in ducks. This study will contribute to a better understanding of the molecular mechanism regulating the innate immune response by TRIM proteins in ducks.

Layered Double Hydroxide Modified with Deoxycholic and Hyaluronic Acids for Efficient Oral Insulin Absorption.

INTRODUCTION: This study aimed to construct a layered double hydroxide (LDH) nanoparticle delivery system that was modified by deoxycholic acid (DCA) and hyaluronic acid (HA) to increase the bioavailability of oral insulin. METHODS: LDH-DCA-HA was synthesized by the hybridization of DCA and HA with LDH. Subsequently, insulin was loaded onto LDH-DCA-HA, resulting in the formation of INS@LDH-DCA-HA. The in vivo and in vitro mechanisms of insulin release, as well as the efficiency of insulin absorption, were analyzed before and after DCA-HA modification. RESULTS: MTT assay showed that there was satisfactory biocompatibility between LDH-DCA-HA and Caco-2 cells at a concentration below 1000 µg/mL. Flow cytometry analysis revealed that Caco-2 cells absorbed INS@LDH-DCA-HA more readily than insulin. Measurement of transepithelial electrical resistance indicated that INS@LDH-DCA-HA induced the reversible opening of tight cell junctions, thereby facilitating its absorption. This was confirmed via laser confocal microscopy analysis, revealing that a large amount of zonula occludens-1 tight junction (TJ) protein was utilized for the paracellular pathway of nanoparticles. We also measured the blood glucose levels of type I diabetic mice and found that oral INS@LDH-DCA-HA exerted a steady hypoglycemic effect lasting 12 h, with a small range of postprandial blood glucose fluctuation. Immunofluorescence analysis showed that the strong penetration ability of INS@LDH-DCA-HA allowed insulin to enter epithelial cells more readily than free insulin. Finally, immunohistochemical analysis of anti-SLC10A1 protein confirmed that the cholic acid transporter receptor protein played a key role in the functioning of INS@LDH-DCA-HA. CONCLUSION: LDH nanoparticles modified by DCA and HA improved the absorption efficiency of insulin by opening the TJs of cells and interacting with the cholic acid transporter receptor protein.

Overcoming Multiple Absorption Barrier for Insulin Oral Delivery Using Multifunctional Nanoparticles Based on Chitosan Derivatives and Hyaluronic Acid.

BACKGROUND: Although dynamics and uses of modified nanoparticles (NPs) as orally administered macromolecular drugs have been researched for many years, measures of molecule stability and aspects related to important transport-related mechanisms which have been assessed in vivo remain as relatively under characterized. Thus, our aim was to develop a novel type of oral-based delivery system for insulin and to overcome barriers to studying the stability, transport mechanisms, and efficacy in vivo of the delivery system. METHODS: NPs we developed and tested were composed of insulin (INS), dicyandiamide-modified chitosan (DCDA-CS), cell-penetrating octaarginine (r8), and hydrophilic hyaluronic acid (HA) and were physically constructed by electrostatic self-assembly techniques. RESULTS: Compared to free-insulin, levels of HA-DCDA-CS-r8-INS NPs were retained at more desirable measures of biological activity in our study. Further, our assessments of the mechanisms for NPs suggested that there were high measures of cellular uptake that mainly achieved through active transport via lipid rafts and the macropinocytosis pathway. Furthermore, investigations of NPs indicated their involvement in caveolae-mediated transport and in the DCDA-CS-mediated paracellular pathway, which contributed to increasing the efficiency of sequential transportation from the apical to basolateral areas. Accordingly, high efficiency of absorption of NPs in situ for intestinal loop models was realized. Consequently, there was a strong induction of a hypoglycemic effect in diabetic rats of NPs via orally based administrations when compared with measures related to free insulin. CONCLUSION: Overall, the dynamics underlying and influenced by HA-DCDA-CS-r8-INS may hold great promise for stability of insulin and could help overcome interference by the epithelial barrier, and thus showing a great potential to improve the efficacy of orally related treatments.

Pathogenicity of different H5N6 highly pathogenic avian influenza virus strains and host immune responses in chickens.

The H5N6 highly pathogenic avian influenza virus (HPAIV) has been circulating in China since 2013. In this report, we describe our recent chicken experimental studies investigating the pathogenicity and transmission of four H5N6 HPAIV field strains of different origins (GS39, CK44, DK47 and CK74) and the host immune responses. Four-week-old specific-pathogen-free chickens were inoculated intranasally with one of the four H5N6 HPAIV strains (one strain per group). Among the contact chickens, the GS39 and CK74 strains caused 100 % mortality, the CK44 strain caused 80 % mortality, and the DK47 strain caused 40 % mortality. The viruses were effectively replicated in multiple tissues of the inoculated chickens, in which high viral titers were detected in virus-infected tissues, and significantly upregulated expression of immune-related genes was found in the infected chickens at 24 hpi. The chicken serum antibody levels increased from 5log2 at 7 dpe to 7.67-8log2 at 14 dpe. The major histocompatibility complex molecules were upregulated 21.22- to 32.98-fold in lungs and 5.10- to 18.47-fold in spleens. In summary, H5N6 viruses can replicate within chickens and be effectively transmitted between chickens. Our study contributes to further understanding the pathogenesis of clade 2.3.4.4 H5N6 avian influenza viruses in chickens.

Duck TRIM32 Functions in IFN-ß Signaling Against the Infection of H5N6 Highly Pathogenic Avian Influenza Virus.

In mammals, tripartite motif 32 (TRIM32) is essential for regulating host innate immune responses to viral infections. However, the antiviral effect of TRIM32 in birds has not been reported. Here, we cloned the full-length duck TRIM32 (duTRIM32) cDNA from total spleen RNA of Peking duck. DuTRIM32 consists of 682 amino acids and has 95.5% similarity in amino acid sequences with chicken TRIM32 and 84.9% similarity with human TRIM32, respectively. DuTRIM32 mRNA was found to be ubiquitously expressed in all tested tissues from healthy ducks. Overexpression of duTRIM32 significantly activated the IFN-ß promoter and upregulated the mRNA levels of IFN-ß, IRF7, and Mx, which indicates that duTRIM32 is involved in the type I IFN pathway. Furthermore, duTRIM32 was found to directly interact with duck STING (duSTING) and to contribute to the expression of IFN-ß mediated by duSTING. The mRNA level of duTRIM32 was significantly upregulated in the lungs and spleens of H5N6 highly pathogenic avian influenza virus (HPAIV) infected ducks 3 days post-infection (DPI). Furthermore, overexpression of duTRIM32 could inhibit the replication of H5N6 HPAIV in duck embryo fibroblasts (DEFs). Therefore, these results indicate that duTRIM32 is involved in the type I IFN pathway and exhibit an antiviral effect against H5N6 HPAIV infection.

Phylogeny, Pathogenicity, Transmission, and Host Immune Responses of Four H5N6 Avian Influenza Viruses in Chickens and Mice.

H5Nx viruses have continuously emerged in the world, causing poultry industry losses and posing a potential public health risk. Here, we studied the phylogeny, pathogenicity, transmission, and immune response of four H5N6 avian influenza viruses in chickens and mice, which were isolated from waterfowl between 2013 and 2014. Their HA genes belong to Clade 2.3.4.4, circulated in China since 2008. Their NA genes fall into N6-like/Eurasian sublineage. Their internal genes originated from different H5N1 viruses. The results suggested that the four H5N6 viruses were reassortants of the H5N1 and H6N6 viruses. They cause lethal infection with high transmission capability in chickens. They also cause mild to severe pathogenicity in mice and can spread to the brain through the blood-brain barrier. During the infection, the viruses result in the up-regulation of PRRs and cytokine in brains and lungs of chickens and mice. Our results suggested that the high viral loads of several organs may result in disease severity in chickens and mice; there were varying levels of cytokines induced by the H5N6 viruses with different pathogenicity in chickens and mice.

Immune-Related Gene Expression in Ducks Infected With Waterfowl-Origin H5N6 Highly Pathogenic Avian Influenza Viruses.

Clade 2.3.4.4 H5 avian influenza viruses (AIVs) are widely prevalent and of significant concern to the poultry industry and public health in China. Nowadays, the clade 2.3.4.4 H5N6 virus has become a dominant AIV subtype among domestic ducks in southern China. We found that waterfowl-origin clade 2.3.4.4 H5N6 viruses (A/goose/Guangdong/16568/2016, GS16568 and A/duck/Guangdong/16873/2016, DK16873) isolated from southern China in 2016 could replicate in multiple organs of inoculated ducks. DK16873 virus caused mild infections and killed 2/5 of inoculated ducks, and GS16568 virus did not kill inoculated ducks. In addition, the two viruses could be transmitted via direct contact between ducks. DK16873 and GS16568 viruses killed 2/5 and 1/5 of contact ducks, respectively. Furthermore, ducks inoculated with the two H5N6 viruses exhibited different expressions of immune-related genes in their lungs. The expression of RIG-I, TLR3 and IL6 was significantly upregulated at 12 h post-inoculation (HPI) and most of the tested immune-related genes were significantly upregulated at 3 days post-inoculation (DPI). Notably, the expression of RIG-I and IL-6 in response to DK16873 virus was significantly higher than for GS16568 virus at 12 HPI and 3 DPI. Our research have provided helpful information about the pathogenicity, transmission and immune-related genes expression in ducks infected with new H5N6 AIVs.

Genetic characteristics, pathogenicity and transmission of H5N6 highly pathogenic avian influenza viruses in Southern China.

Since 2014, H5 highly pathogenic avian influenza viruses (HPAIVs) from clade 2.3.4.4 have been persistently circulating in Southern China. This has caused huge losses in the poultry industry. In this study, we analysed the genetic characteristics of seven H5N6 HPAIVs of clade 2.3.4.4 that infected birds in Southern China in 2016. Phylogenetic analysis grouped the HA, PB2, PA, M and NS genes as MIX-like, and the NA genes grouped into the Eurasian lineage. The PB1 genes of the GS24, GS25, CK46 and GS74 strains belonged to the VN 2014-like group and the others were grouped as MIX-like. The NP genes of GS24 and GS25 strains belonged to the ZJ-like group, but the others were MIX-like. Thus, these viruses came from different genotypes, and the GS24, GS25, CK46 and GS74 strains displayed genotype recombination. Additionally, our results showed that the mean death time of all chickens inoculated with 105 EID50 of CK46 or GS74 viruses was 3 and 3.38 days, respectively. The viruses replicated at high titers in all tested tissues of the inoculated chickens. They also replicated in all tested tissues of naive contact chickens, but their replication titers in some tissues were significantly different (p < 0.05). Thus, the viruses displayed high pathogenicity and variable transmission in chickens. Therefore, it is necessary to focus on the pathogenic variation and molecular evolution of H5N6 HPAIVs in order to prevent and control avian influenza in China.

Different Pathogenicity and Transmissibility of Goose-Origin H5N6 Avian Influenza Viruses in Chickens.

Highly pathogenic avian influenza H5N6 viruses have been circulating in poultry in Asia since 2013 and producing serious diseases in chickens. Here, we analyzed the genetic properties of 10 H5N6 subtypes AIVs from geese in 2015-2016 in Guangdong province. Phylogenic analysis showed that all HA genes of the 10 viruses belonged to clade 2.3.4.4, and their genes including HA, PA, PB1, M, NP, and NS all derived from Mix-like 1 (CH, VN, LS). Their PB2 genes come from Mix-like 2 (CH, VN, JP). The NA genes were classified into a Eurasian lineage. Therefore, the 10 viruses likely originate from the same ancestor and were all recombinant viruses between different genotypes. We selected A/Goose/Guangdong/GS144/2015(H5N6) (GS144) and A/Goose/Guangdong/GS148/2016(H5N6) (GS148) viruses to inoculate 5-week-old chickens intranasally with 104 EID50/0.1 mL dose intranasally to assess their pathogenicity and transmissibility. Inoculated chickens showed that the GS144 virus caused systematic infection with a lethality of 100%, but the lethality of GS148 virus was 0%. The two viruses were efficiently transmitted to contact chickens. The lethality of GS144 and GS148 virus in contact with chickens was 87.5% and 0%, respectively, which suggests that the transmissibility of GS144 virus was stronger than GS148 virus in chickens. Thus, different H5N6 viruses from the same waterfowl can show different pathogenicity and transmissibility in chickens. Continued surveillance and characteristic analysis of the H5N6 viruses will help us to keep abreast of evolution and variation in avian influenza viruses in the future.

Recent progress in synergistic chemotherapy and phototherapy by targeted drug delivery systems for cancer treatment.

Although it's pharmacological effect for cancer therapy, conventional chemotherapy has been compromised by a series of shortcomings such as limited stability, nonspecific tumour targeting ability and severe toxic side effects. To overcome these limitations, multifunctional targeted drug delivery systems for combinatorial therapeutics have been widely explored as novel cancer therapy strategies, showing encouraging results in many pre-clinical animal experiments. Among them, synergistic phototherapy and chemotherapy have demonstrated their abilities to enhance therapeutic efficacies and reduce unwanted side effects via a variety of mechanisms. In this review, we will summarize the latest progress in the development of targeted drug delivery systems with combinations of phototherapy and chemotherapy and discuss the important roles of phototherapy agents involved in those non-conventional therapeutic strategies.

Avian Influenza (H7N9) Viruses Co-circulating among Chickens, Southern China.

In April 2017, three avian influenza (H7N9) viruses were isolated from chickens in southern China. Each virus had different insertion points in the cleavage site of the hemagglutinin protein compared to the first identified H7N9 virus. We determined that these viruses were double or triple reassortant viruses.

[Clinical study of stress shielding after mini-plate internal fixation for mandibular fractures].

OBJECTIVE: To investigate the stress shielding after mini-plate internal fixation for mandibular fractures. METHODS: Eighteen patients with mandibular fractures were selected.X-rays were taken before operation and 3, 4, 6 months after operation when the plates were removed. The bone density of the mini plate area was examined and compared among different time interval groups. RESULTS: The bone density before operation (125.41 ± 2.47) and 3 months after operation (120.19 ± 3.52) was not significantly different, but became lower 4 and 6 months after operation than before operation. CONCLUSIONS: There appeared stress shielding after internal fixation for 4 and 6 months in mandibular fracture, and the mini plate should be removed at those times.