Detection and differentiation of seven porcine respiratory pathogens using a multiplex ligation-dependent probe amplification assay.

Respiratory diseases due to viral or bacterial agents, either alone or in combination, cause substantial economic burdens to the swine industry worldwide. Rapid and reliable detection of causal pathogens is crucial for effective epidemiological surveillance and disease management. This research aimed to employ the multiplex ligation-dependent probe amplification (MLPA) assay for simultaneous detection of seven distinct pathogens causing respiratory problems in swine, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV2), Pasteurella multocida, Actinobacillus pleuropneumoniae, and Glässerella parasuis. The results indicated no probe cross-reactivity among the seven target agents with other swine pathogens. The detection limits ranged from 5 to 34 copies per assay for the target organisms. The MLPA assay was evaluated with 88 samples and compared to real-time or multiplex PCR for the target pathogens. The MLPA assay demonstrated high relative test sensitivities (100%) and reasonable to good relative specificities at 62.5%, 95.1%, 86.8%, and 97.6% for PRRSV, P. multocida, G. parasuis, and PCV2, respectively, relative to comparator PCR assays. In 72 samples where MLPA and comparator PCR assays matched exactly, infections were detected in 65 samples (90%), with PRRSV being the most commonly found virus and 50% of the samples showing co-infection with two to five of the pathogens. This approach serves as a valuable tool for conducting differential diagnoses and epidemiological investigations of pathogen prevalence within swine populations.

The nucleocapsid protein facilitates p53 ubiquitination-dependent proteasomal degradation via recruiting host ubiquitin ligase COP1 in PEDV infection.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of the coronavirus family and caused severe economic losses to the global swine industry. Previous studies have established that p53 is a host restriction factor for PEDV infection, and p53 degradation occurs in PEDV-infected cells. However, the underlying molecular mechanisms through which PEDV viral proteins regulate p53 degradation remain unclear. In this study, we found that PEDV infection or expression of the nucleocapsid protein downregulates p53 through a post-translational mechanism: increasing the ubiquitination of p53 and preventing its nuclear translocation. We also show that the PEDV N protein functions by recruiting the E3 ubiquitin ligase COP1 and suppressing COP1 self-ubiquitination and protein degradation, thereby augmenting COP1-mediated degradation of p53. Additionally, COP1 knockdown compromises N-mediated p53 degradation. Functional mapping using truncation analysis showed that the N-terminal domains of N protein were responsible for interacting with COP1 and critical for COP1 stability and p53 degradation. The results presented here suggest the COP1-dependent mechanism for PEDV N protein to abolish p53 activity. This study significantly increases our understanding of PEDV in antagonizing the host antiviral factor p53 and will help initiate novel antiviral strategies against PEDV.

In-silico simulation of nanoindentation on bone using a 2D cohesive finite element model.

This study proposed and validated a 2D finite element (FE) model for conducting in-silico simulations of in-situ nanoindentation tests on mineralized collagen fibrils (MCF) and the extrafibrillar matrix (EFM) within human cortical bone. Initially, a multiscale cohesive FE model was developed by adapting a previous model of bone lamellae, encompassing both MCF and EFM. Subsequently, nanoindentation tests were simulated in-silico using this model, and the resulting predictions were compared to AFM nanoindentation test data to verify the model's accuracy. The FE model accurately predicted nanoindentation results under wet conditions, closely aligning with outcomes obtained from AFM nanoindentation tests. Specifically, it successfully mirrored the traction/separation curve, nanoindentation modulus, plastic energy dissipation, and plastic energy ratio obtained from AFM nanoindentation tests. Additionally, this in-silico model demonstrated its ability to capture alterations in nanoindentation properties caused by the removal of bound water, by considering corresponding changes in mechanical properties of the collagen phase and the interfaces among bone constituents. Notably, significant changes in the elastic modulus and plastic energy dissipation were observed in both MCF and EFM compartments of bone, consistent with observations in AFM nanoindentation tests. These findings indicate that the proposed in-silico model effectively captures the influence of ultrastructural changes on bone's mechanical properties at sub-lamellar levels. Presently, no experimental methods exist to conduct parametric studies elucidating the ultrastructural origins of bone tissue fragility. The introduction of this in-silico model presents an invaluable tool to bridge this knowledge gap in the future.

Characterizing Conformational Change of a Thermoresponsive Polymeric Nanoparticle with Raman Spectroscopy.

Molecular conformational changes in the collapsing and reswelling processes occurring during the phase transition at the lower critical solution temperature (LCST) of the polymer are not well understood. In this study, we characterized the conformational change of Poly(oligo(Ethylene Glycol) Methyl Ether Methacrylate)-144 (POEGMA-144) synthesized on silica nanoparticles using Raman spectroscopy and zeta potential measurements. Changes in distinct Raman peaks associated with the oligo(Ethylene Glycol) (OEG) side chains (1023, 1320, and 1499 cm-1) with respect to the methyl methacrylate (MMA) backbone (1608 cm-1) were observed and investigated under increasing and decreasing temperature profiles (34 °C to 50 °C) to evaluate the polymer collapse and reswelling around its LCST (42 °C). In contrast to the zeta potential measurements that monitor the change in surface charges as a whole during the phase transition, Raman spectroscopy provided more detailed information on vibrational modes of individual molecular moieties of the polymer in responding to the conformational change.

Assessment of glycosaminoglycan content in bone using Raman spectroscopy.

Glycosaminoglycans (GAGs) are responsible for preserving bone tissue toughness as well as regulating collagen formation and mineralization in the extracellular matrix. However, current methods for characterization of GAGs in bone are destructive, thus unable to capture in situ changes or differences in GAGs between experimental groups. As an alternative, Raman spectroscopy is a non-destructive method and can detect concurrent changes in GAGs and other bone constituents. In this study, we hypothesized that the two most prominent Raman peaks of sulfated GAGs (at ~1066 cm-1 and at ~1378 cm-1) could be used to detect differences in GAGs content of bone. To test this hypothesis, three experimental models were utilized: an in vitro model (enzymatic removal of GAGs from human cadaver bone), an ex vivo mouse model (biglycan KO vs. WT), and an ex vivo aging model (comparing cadaveric bone samples from young and old donors). All Raman measurements were compared to Alcian blue measurements to confirm the validity of Raman spectroscopy in detecting GAGs changes in bone. Irrespective of different models, it was found that the ~1378 cm-1 peak in Raman spectra of bone was uniquely sensitive to changes of GAGs content in bone when normalized with respect to the phosphate phase (~960 cm-1); i.e., 1378 cm-1/960 cm-1 (peak intensity ratio) or 1370-1385 cm-1/930-980 cm-1 (integrated peak area ratio). In contrast, the 1070 cm-1 peak, which includes another major peak of GAGs (1066 cm-1), seemed to be compromised to detect changes of GAGs in bone due to concurrent changes of carbonate (CO3) in the similar peak range. This study validates the ability of Raman spectroscopy to detect in situ treatment-, genotype-, and age-related changes in GAG levels of bone matrix.

Porcine epidemic diarrhea virus activates PERK-ROS axis to benefit its replication in Vero E6 cells.

Of the three branches of unfolded protein response (UPR) that were reportedly activated by porcine epidemic diarrhea virus (PEDV), PERK is recently shown to act as an upstream regulator of oxidative response of the cells. However, it remains unknown if and how PERK activation during PEDV infection would result in oxidative stress, and whether activation of PERK and its downstream molecules affect PEDV replication. Here, we demonstrate that infection with the PEDV strain YJH/2015 triggered UPR in Vero E6 cells by activating the PERK/eIF2α pathway and led to significant increase in the expression of proapoptotic protein C/EBP homologous protein (CHOP) and ER oxidoreductase 1 alpha (ERO1α). Inhibition of PERK by short hairpin RNA (shRNA) or GSK2606414 and knockdown of CHOP by small interfering RNA reduced expression of ERO1α and generation of ROS in PEDV-infected cells. Inhibition of ERO1α by shRNA or EN460 decreased PEDV-induced ROS generation. Genetic or pharmacological inhibition of each component of PERK, CHOP, ERO1α, and ROS led to significant suppression of PEDV replication. Collectively, our study provides the first evidence that PEDV manipulates endoplasmic reticulum to perturb its redox homeostasis via the PERK-CHOP-ERO1α-ROS axis in favor of its replication.

Genetic characterization of porcine reproductive and respiratory syndrome virus from Eastern China during 2017-2022.

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease caused by PRSS virus (PRRSV). PRRSV mainly causes reproductive disorders in pregnant sows and respiratory diseases in piglets. Recently, it has emerged as one of the most important diseases of the pig industry across the globe. In this study, we have collected 231 samples from differently sized pig farms in Eastern China from 2017 to 2022 to investigate the epidemic characteristics of the disease. All samples were screened by RT-PCR and analyzed further using Nsp2 and ORF5 genes. The result showed that the positive rate of PRRSV was 24% (54/231). Phylogenetic analysis (13 positive samples) revealed that all isolates belonged to genotype 2, and they were mainly distributed in four lineages (i.e., lineage 1, 3, 5, and 8). Nsp2 is the most variable protein among all PRRSV NSPs, several isolates from this study had amino acid deletions within Nsp2 compared to that of strain VR-2332. The major structural protein glycoprotein (GP5) protein is encoded by ORF5. Epitope analysis of the 13 isolated strains and additional reference strains revealed that all 13 strains had some mutations on the decoy epitope, the primary neutralizing epitope, T cell epitopes, and B cell epitopes. This study showed that the prevalent PRRSV strain in Eastern China was still HP-PRRSV, while the proportion of NADC30-like and NADC34-like strains have increased. This study further enriches the epidemiological data of PRRS in Eastern China and provides a theoretical basis for vaccine development and prevention and control of the disease across the region.

Machine learning based personalized promotion strategy of piglets weaned per sow per year in large-scale pig farms.

BACKGROUND: The purpose of this study was to analyze the relationship between different productive factors and piglets weaned per sow per year (PSY) in 291 large-scale pig farms and analyze the impact of the changes in different factors on PSY. We chose nine different algorithm models based on machine learning to calculate the influence of each variable on every farm according to its current situation, leading to personalize the improvement of the impact in the specific circumstances of each farm, proposing a production guidance plan of PSY improvement for every farm. According to the comparison of mean absolute error (MAE), 95% confidence interval (CI) and R2, the optimal solution was conducted to calculate the influence of 17 production factors of each pig farm on PSY improvement, finding out the bottleneck corresponding to each pig farm. The level of PSY was further analyzed when the bottleneck factor of each pig farm changed by 0.5 standard deviation (SD). RESULTS: 17 production factors were non-linearly related to PSY. The top five production factors with the highest correlation with PSY were the number of weaned piglets per litter (WPL) (0.6694), mating rate within 7 days after weaning (MR7DW) (0.6606), number of piglets born alive per litter (PBAL) (0.6517), the total number of piglets per litter (TPL) (0.5706) and non-productive days (NPD) (- 0.5308). Among nine algorithm models, the gradient boosting regressor model had the highest R2, smallest MAE and 95% CI, applied for personalized analysis. When one of 17 production factors of 291 large-scale pig farms changed by 0.5 SD, 101 pig farms (34.7%) can increase 1.41 PSY (compared to its original value) on average by adding the production days, and 60 pig farms (20.6%) can increase 1.14 PSY on average by improving WPL, 45 pig farms (15.5%) can increase 1.63 PSY by lifting MR7DW. CONCLUSIONS: The main productive factors related to PSY included WPL, MR7DW, PBAL, TPL and NPD. The gradient boosting regressor model was the optimal method to individually analyze productive factors that are non-linearly related to PSY.

Porcine circovirus type 2 induces CHOP-ERO1α-ROS-mediated apoptosis in PK-15 cells.

Porcine circovirus type 2 (PCV2) infection induces endoplasmic reticulum (ER) stress and oxidative stress. These cellular responses could be connected with apoptosis. However, the mechanisms that link ER stress and oxidative stress in PCV2-induced apoptosis are poorly characterized. Here, we demonstrate that PCV2 infection increased expression of proapoptotic protein C/EBP homologous protein (CHOP) and ER oxidoreductase 1 alpha (ERO1α). Inhibition of CHOP by RNA silencing or inhibition of ERO1α by short hairpin RNA or EN460 repressed PCV2-induced reactive oxygen species (ROS) generation, cytosolic calcium level, and apoptotic rate in PK-15 cells. Overexpression of ERO1α enhanced PCV2-induced oxidative stress, caspase-3 cleavage, and apoptosis rate. Treatment of PCV2-infected cells with ROS scavenger N-acetyl-L-cysteine downregulated PCV2-induced ROS production, cytosolic calcium level, and apoptosis rate, but intriguingly decreased expression of CHOP and ERO1α. Thus, we propose that PCV2 induces apoptosis through ER Stress via CHOP-ERO1α-ROS signaling in host cells.

Research advances on interferon (IFN) response during BVDV infection.

Bovine viral diarrhea virus (BVDV) is an important pathogen responsible for significant economic loss to cattle. BVDV infection in pregnant cattle leads to fetal infection and reproductive losses, including early embryonic death, abortion, and stillbirth. Importantly, vaccinated heifers could not provide fetal protection against BVDV. It can be divided into two genotypes (BVDV-1 and BVDV-2) and two biotypes (cytopathic (CP) and non-cytopathic (NCP)). Infection with NCP-BVDV during gestation, the fetus becomes persistently infected (PI) and sheds BVDV throughout life, serving as the main source of infection for other cattle. BVDV potentially induces immunosuppression and aggravates bovine respiratory disease (BRD). Accordingly, BVDV infection results in a heterogeneous range of clinical signs and immune responses. Interferon (IFN) plays a vital role by mediating the innate immune response against antiviral infection through the Janus Kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. BVDV infection can reportedly exert variable degrees of influence on IFN response. Interestingly, reports have suggested that IFN can exert a significant inhibitory effect on various viruses. Human IFN-α was used to restrain BVDV in vitro. In this article, we summarized the latest researches on IFN response during BVDV infection.

Chitosan-Coated Selenium Nanoparticles Attenuate PRRSV Replication and ROS/JNK-Mediated Apoptosis in vitro.

Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly prevalent and endemic swine pathogen that causes significant economic losses to the global swine industry. Selenium nanoparticles (SeNPs) have attracted increasing attention in the biomedical field, given their antiviral effects. This study aimed to investigate the inhibitory effect of chitosan-coated SeNPs (CS-SeNPs) on PRRSV replication. Methods: In this study, CS-SeNPs were synthesized by chemical reduction and characterized by assessing the morphology, size distribution, zeta potential, and element composition. Marc-145 cells were infected with r-PRRSV-EGFP (0.1 MOI) and inoculated with CS-SeNPs (10 µM). Subsequently, the concentrations of hydrogen peroxide (H2O2) and glutathione (GSH), and glutathione peroxidase (GSH-Px) activity were measured using specific commercial assay kits. ORF5 RNA expression, viral titer, and nucleocapsid (N) protein expression were assessed using qRT-PCR, TCID50, and Western blot. ROS generation, apoptosis rates, and JNK /caspase-3/PARP protein expression were evaluated using dihydroethidium staining, flow cytometry, and Western blot. Results: The results showed that CS-SeNPs treatment significantly suppressed oxidative stress induced by r-PRRSV-EGFP infection by increasing GSH-Px activity, promoting GSH production, and inhibiting H2O2 synthesis. CS-SeNPs treatment significantly inhibited ORF5 gene expression, viral titers, and N protein of r-PRRSV-EGFP at 24 and 48 hours post-infection (hpi) in Marc-145 cells. The increase in apoptosis rates induced by r-PRRSV-EGFP infection was significantly decreased by CS-SeNPs inoculation through inhibiting ROS generation, JNK phosphorylation levels, and cleavage of caspase-3 and PARP mainly at 48 hpi. Conclusion: These results demonstrated that CS-SeNPs suppress PRRSV-induced apoptosis in Marc-145 cells via the ROS/JNK signaling pathway, thereby inhibiting PRRSV replication, which suggested the potential antiviral activity of CS-SeNPs that deserves further investigation for clinical applications.

Probability-based approach for characterization of microarchitecture and its effect on elastic properties of trabecular bone.

Trabecular bone is a random cellular solid with an interconnected network of plate-like and rod-like components. However, the structural randomness and complexity have hindered rigorous mathematical modeling of trabecular bone microarchitecture. Recent advancements in imaging processing techniques have enabled us to define the size, orientation, and spatial location of individual trabecular plates and rods in trabecular bone. Based on the essential information, this study proposed a probability-based approach to define the size, orientation, and spatial distributions of trabecular plates and rods for trabecular bone cubes (N = 547) acquired from six human cadaver proximal femurs. Using two groups of probability-based parameters, it was attempted to capture microarchitectural details, which could not be captured by the existing histomorphometric parameters, but crucial to the elastic properties of trabecular bone. The elastic properties of the trabecular bone cubes in three principal axes were estimated using microCT based finite element (FE) simulations. Based on the results of multivariate multiple regression modeling, the efficacy of the two groups of probability-based parameters in prediction of the elastic properties was verified in comparison with that of the existing histomorphometric parameters (BV/TV, Tb.Th, Tb.Sp, DA, EF.Med, and Conn.D). The results indicated that the regression models trained using the probability-based parameters had a comparable and even better accuracy (rMSE = 0.621 and 0.548) than that of the histomorphometric parameters (rMSE = 0.647). More importantly, the probability-based parameters could provide more insights into some unexplored microarchitectural features, such as individual trabecular size, orientation, and spatial distributions, which are also critical to the elastic properties of trabecular bone.

Protective Effect of Isoorientin on Oleic Acid-Induced Oxidative Damage and Steatosis in Rat Liver Cells.

The harm of nonalcoholic fatty liver disease to human health is increasing, which calls for urgent prevention and treatment of the disease. Isoorientin is an effective ingredient of Chinese herbal medicine with anti-inflammatory and antioxidant effects. However, the effect of isoorientin in nonalcoholic fatty liver disease is still unclear. In this study, combined in vivo and in vitro experiments, through pathological observation, flow cytometry, immunofluorescence and western blot analysis to explore the role of isoorientin in steatosis and reveal its molecular mechanism. The results demonstrated that oleic acid treatment significantly increased the content of ROS and lipid droplets in rat hepatocytes, and promoted the expression of γH2AX, HO-1, PPARγ, SREBP-1c, FAS. The ROS content in the cells of co-treated with isoorientin and oleic acid was significantly reduced compared to the oleic acid group, and the expression of γH2AX, HO-1, PPARγ, SREBP-1c, FAS, and the nuclear translocation of NF-κB p65 were also significantly inhibited. Our data showed that oleic acid induce oxidative damage and steatosis in hepatocytes both in vitro and in vivo, and activate the PPARγ/NF-κB p65 signal pathway. Moreover, isoorientin can significantly reduce oleic acid -induced oxidative damage and steatosis by regulating the PPARγ/NF-kB p65 signal pathway.

Supplemental Dietary Selenohomolanthionine Improve Antioxidant Activity and Immune Function in Weaned Beagle Puppies.

The purpose of this study was to investigate the effects of dietary Selenohomolanthionine (SeHLan) on antioxidant status and immune response in canine parvovirus (CPV) vaccinated puppies. In this study, 30 weaned puppies were randomly divided into six groups: control group (-Se/-Vacc), immunization group (-Se/+Vacc), supplementation of sodium selenite group (SS/+Vacc, 0.35 mg/kg DM), low-dose SeHLan group (SeHLan-L/+Vacc, 0.35 mg/kg DM), mid-dose SeHLan group (SeHLan-M/+Vacc, 1.0 mg/kg DM), and high-dose SeHLan group (SeHLan-H/+Vacc, 2.0 mg/kg DM). The puppies were fed for 42 days and vaccinated with Vanguard Plus 5 on day 0 and day 21. Blood samples were collected on 7, 14, 21, 28, 35, 42 days post-immunization (PI) for determination of antioxidant indicators, lymphocyte proliferation index, serum cytokine concentration (IL-2, IL-4), canine polymorphonuclear neutrophils (PMN) phagocytic function, and the level of CPV antibody titers. The results showed that SeHLan supplementation raised the serum Se concentration and glutathione peroxidase (GSH-Px) activity in a dose-dependent manner (P < 0.05). It also increased the activity of serum superoxide dismutase (SOD) and decreased serum malondialdehyde (MDA) content, especially in SeHLan-M/+Vacc group (1.0 mg/kg DM) (P < 0.01). SeHLan supplementation significantly increased lymphocyte proliferation, IL-2, and IL-4 levels in canine serum, and enhanced phagocytosis of PMN in vaccinated puppies (P < 0.05). Moreover, SeHLan supplementation shortened the CPV antibody production time and increased the CPV antibody titers (P < 0.05). Of note, the beneficial effects of SeHLan were superior to those of SS. In conclusion, dietary SeHLan supplementation improved antioxidant activity, increased CPV antibody titers, and enhanced immune function in puppies after weaning. An appropriate dosage of SeHLan (1~2 mg/kg DM) may confer nutritional benefits in puppies.

Can DXA image-based deep learning model predict the anisotropic elastic behavior of trabecular bone?

3D image-based finite element (FE) and bone volume fraction (BV/TV)/fabric tensor modeling techniques are currently used to determine the apparent stiffness tensor of trabecular bone for assessing its anisotropic elastic behavior. Inspired by the recent success of deep learning (DL) techniques, we hypothesized that DL modeling techniques could be used to predict the apparent stiffness tensor of trabecular bone directly using dual-energy X-ray absorptiometry (DXA) images. To test the hypothesis, a convolutional neural network (CNN) model was trained and validated to predict the apparent stiffness tensor of trabecular bone cubes using their DXA images. Trabecular bone cubes obtained from human cadaver proximal femurs were used to obtain simulated DXA images as input, and the apparent stiffness tensor of the trabecular cubes determined by using micro-CT based FE simulations was used as output (ground truth) to train the DL model. The prediction accuracy of the DL model was evaluated by comparing it with the micro-CT based FE models, histomorphometric parameter based multiple linear regression models, and BV/TV/fabric tensor based multiple linear regression models. The results showed that DXA image-based DL model achieved high fidelity in predicting the apparent stiffness tensor of trabecular bone cubes (R2 = 0.905-0.973), comparable to or better than the histomorphometric parameter based multiple linear regression and BV/TV/fabric tensor based multiple linear regression models, thus supporting the hypothesis of this study. The outcome of this study could be used to help develop DXA image-based DL techniques for clinical assessment of bone fracture risk.

Removal of glycosaminoglycans affects the in situ mechanical behavior of extrafibrillar matrix in bone.

Previous studies have shown that glycosaminoglycans (GAGs) in bone matrix, coupling with water in bone matrix, may play a significant role in toughening bone tissues. Since GAGs are most likely present only in the extrafibrillar matrix (EFM) of bone, we hypothesized that GAGs in EFM would have a major impact on bone tissue toughness. To confirm this conjecture, we removed GAGs ex vivo from human cadaveric bone samples using a protein deglycosylation mix kit and then examined the in situ mechanical behavior of mineralized collagen fibrils (MCFs) and the surrounding EFM of the samples, using a high-resolution atomic force microscopy (AFM). By testing the bone samples before and after removal of GAGs, we found that under the wet condition removal of GAGs resulted in an increase in the elastic modulus of both EFM and MCFs, whereas a significant decrease in plastic energy dissipation was observed mainly in EFM. In contrast, under the dry condition the removal of GAGs had little effects on the mechanical properties of either MCFs or EFM. These results suggest that both MCFs and EFM contribute to the plastic energy dissipation of bone, whereas in the presence of matrix water removal of GAGs significantly reduces the capacity of EFM in plastic energy dissipation, but not MCFs. In addition, GAGs may affect the elastic modulus of both EFM and MCFs. These findings give rise to new understanding to the underlying mechanism of GAGs in toughening of bone tissues.

Re-emergence of H5N8 highly pathogenic avian influenza virus in wild birds, China.

In mid-November 2020, deaths of whooper swan were reported in the Yellow River Reservoir Area, China. In the present study, we describe the genetic characterizations and phylogenetic relationships of four clade 2.3.4.4b H5N8 highly avian influenza viruses (HPAIVs) identified from a sick whooper swan and environmental samples collected in the Yellow River Reservoir Area in late November 2020. They were closely related to recent H5Nx HPAIVs causing outbreaks in Eurasia in the 2020-2021 influenza season, suggesting these isolates might be imported into China via migratory birds. The newly identified H5N8 HPAIVs possessed Q226 and G228 (H3 numbering), indicating that they prefer to avian-like receptors. However, they had three mutations falling within known antigenic regions, including T144A in antigenic region A, T192I in antigenic region B, and N240D in antigenic region D. Our study highlights the risk of the rapid global spread of H5N8 HPAIVs and the necessity for continuous monitoring of avian influenza viruses in wild birds.

Intrafibrillar mineralization deficiency and osteogenesis imperfecta mouse bone fragility.

Osteogenesis imperfecta (OI), a brittle bone disease, is known to result in severe bone fragility. However, its ultrastructural origins are still poorly understood. In this study, we hypothesized that deficient intrafibrillar mineralization is a key contributor to the OI induced bone brittleness. To test this hypothesis, we explored the mechanical and ultrastructural changes in OI bone using the osteogenesis imperfecta murine (oim) model. Synchrotron X-ray scattering experiments indicated that oim bone had much less intrafibrillar mineralization than wild type bone, thus verifying that the loss of mineral crystals indeed primarily occurred in the intrafibrillar space of oim bone. It was also found that the mineral crystals were organized from preferentially in longitudinal axis in wild type bone to more randomly in oim bone. Moreover, it revealed that the deformation of mineral crystals was more coordinated with collagen fibrils in wild type than in oim bone, suggesting that the load transfer deteriorated between the two phases in oim bone. The micropillar test revealed that the compression work to fracture of oim bone (8.2 ± 0.9 MJ/m3) was significantly smaller (p < 0.05) than that of wild type bone (13.9 ± 2.7 MJ/m3), while the bone strength was not statistically different (p > 0.05) between the two genotype groups. In contrast, the uniaxial tensile test showed that the ultimate strength of wild type bone (50 ± 4.5 MPa) was significantly greater (p < 0.05) than that of oim bone (38 ± 5.3 MPa). Furthermore, the nanoscratch test showed that the toughness of oim bone was much less than that of wild type bone (6.6 ± 2.2 GJ/m3 vs. 12.6 ± 1.4 GJ/m3). Finally, in silico simulations using a finite element model of sub-lamellar bone confirmed the links between the reduced intrafibrillar mineralization and the observed changes in the mechanical behavior of OI bone. Taken together, these results provide important mechanistic insights into the underlying cause of poor mechanical quality of OI bone, thus pave the way toward future treatments of this brittle bone disease.

Characterization of Microstructural Changes on Biglycan Induced Mice Bone by Low-Field Nuclear Magnetic Resonance.

A NMR spin-spin (T2) relaxation technique has been described for determining the porosity, and the bound water distribution in biglycan induced mouse bone and correlate to their mechanical properties. The technique of low-field proton NMR involves spin-spin relaxation and free induction decay (FID) measurements, and the computational inversion methods for decay data analysis. The CPMG T2 relaxation data can be inverted to T2 relaxation distribution and this distribution then can be transformed to a pore size distribution with the longer relaxation times corresponding to larger pores. The FID T2 relaxation data of dried bone (mobile water removed) can be inverted to T2 relaxation distribution and this distribution then can be transformed to bound and solid-like water distribution with the longest relaxation time corresponding to bound water component. These techniques are applied to quantify apparent changes in porosity, and bound water in controlled and biglycan knockout mouse bone. Overall bone porosity from CPMG T2 relaxation is determined using the calibrated NMR fluid volume from the proton relaxation data divided by overall bone volume. Ignore the physical sample differences, from the inversion FID T2 relaxation spectrum, the ratio of the bound to solid-like water components is used to calibrate the bound water inside bone, and the results can be used to correlated bone mechanical properties. Hydration status significantly affects the toughness of bone, and bound water has been considered as a biomarker for prediction of bone fragility fractures. In addition to the collagen phase, recent evidence shows that glycosaminoglycans (GAGs) of proteoglycans (PGs) in the extracellular matrix also play a pivotal role in regulating the tissue-level hydration status of bone, there by affecting the tissue-level toughness of bone. Furthermore, biglycan and decorin are two major types of PGs in bone reports. Biglycan knockout induced changes in GAGs, bound water, as well as bone tissue toughness. Among all subtypes of PGs, biglycan is identified as a major subtype in the bone mineral matrix. In this study, we used a biglycan mouse model and the obtained bone samples were measured by low-field NMR to determine the bone porosity and bound water changes, and used to predict if knockout of biglycan may affect the amount of bound water and subsequently lead to reduce toughness of bone.