Functional significance of O-linked N-acetylglucosamine protein modification in regulating autophagy.

Autophagy is a core molecular pathway that preserves cellular and organismal homeostasis. Being susceptible to nutrient availability and stress, eukaryotic cells recycle or degrade internal components via membrane transport pathways to provide sustainable biological molecules and energy sources. The dysregulation of this highly conserved physiological process has been strongly linked to human disease. Post-translational modification, a mechanism that regulates protein function, plays a crucial role in autophagy regulation. O-linked N-acetylglucosamine protein modification (O-GlcNAcylation), a monosaccharide post-translational modification of intracellular proteins, is essential in nutritional and stress regulatory mechanisms. O-GlcNAcylation has emerged as an essential regulatory mechanism of autophagy. It regulates autophagy throughout its lifetime by targeting the core components of the autophagy regulatory network. This review provides an overview of the O-GlcNAcylation of autophagy-associated proteins and their regulation and function in the autophagy pathway. Therefore, this article may contribute to further understanding of the role of O-GlcNAc-regulated autophagy and provide new perspectives for the treatment of human diseases.

Nucleotide at position 66 of NS2A in Japanese encephalitis virus is associated with the virulence and proliferation of virus.

Most wild strains of Japanese encephalitis virus (JEV) produce NS1' protein, which plays an important role in viral infection and immune escape. The G66A nucleotide mutation in NS2A gene of the wild strain SA14 prevented the ribosomal frameshift that prevented the production of NS1' protein, thus reduced the virulence. In this study, the 66th nucleotide of the NS2A gene of SA14 was mutated into A, U or C, respectively. Both the G66U and G66C mutations cause the E22D mutation of the NS2A protein. Subsequently, the expression of NS1' protein, plaque size, replication ability, and virulence to mice of the three mutant strains were examined. The results showed that the three mutant viruses could not express NS1' protein, and their proliferation ability in nerve cells and virulence to mice were significantly reduced. In addition, the SA14(G66C) was less virulent than the other two mutated viruses. Our results indicate that only when G is the 66th nucleotide of NS2A, the JEV can produce NS1' protein, which affects the virulence.

Primary cutaneous anaplastic large-cell lymphoma resembling infratemporal space infection: a case report.

BACKGROUND: Primary cutaneous anaplastic large-cell lymphoma (PC-ALCL) is a rare T-cell lymphoma belonging to the CD30 + T-cell lymphoproliferative disorders. The case of PC-ALCL in the temporal region is exceedingly rare. Herein, we report a case of PC-ALCL involving the temporal region mimicking infratemporal space infection. CASE PRESENTATION: A 78-year-old woman presented to maxillofacial surgery service with a 6-month history of swelling and pain in the left side of her face. Laboratory investigations found an elevated C-reactive protein (CRP). Imaging findings showed enlarged lymph nodes and extensive thickening of subcutaneous tissue of the left temples. Based on these findings, the infratemporal space infection was suspected initially. The patient underwent incision and drainage, and we unexpectedly found no pus in the lesion area. Incisional biopsy showed necrosis and extensive involvement of the left temples by a diffuse infiltrate containing large, atypical cells. The tumor cells were positive for CD30, CD3, Ki67. They were negative for ALK (SP8), CD5, CD8, CD20 and PAX5. After considering these findings, a diagnosis of PC-ALCL was rendered. The patient was admitted to the lymphoma department for systemic chemotherapy and no relapse occurred during a follow-up period of six months. CONCLUSIONS: This report suggests that if there are suspicious intraoperative manifestations, carrying out a biopsy simultaneously, using Hematoxylin and eosin (HE) staining, and a comprehensive Immunohistochemistry (IHC) panel are essential to diagnosing PC-ALCL to prevent misdiagnosis.

A meta-analysis: laparoscopic versus open liver resection for large hepatocellular carcinoma.

BACKGROUND: The indication of laparoscopic liver resection (LLR) for treating large hepatocellular carcinoma (HCC) is controversial. In this study, we compared the short-term and long-term outcomes of LLR and open liver resection (OLR) for large HCC. MATERIAL AND METHODS: We searched eligible articles about LLR versus OLR for large HCC in PubMed, Cochrane Library, and EMBASE and performed a meta-analysis. RESULTS: Eight publications involving 1,338 patients were included. Among them, 495 underwent LLR and 843 underwent OLR. The operation time was longer in the LLR group (MD: 22.23, 95% CI: 4.14-40.33, p = 0.02). but the postoperative hospital stay time was significantly shorter (MD : -4.88, CI: -5.55 to -4.23, p < 0.00001), and the incidence of total postoperative complications and major complications were significantly fewer (OR: 0.49, 95% CI:0.37-0.66, p < 0.00001; OR: 0.54, 95% CI:0.36 - 0.82, p = 0.003, respectively). Patients in the laparoscopic group had no significant difference in intraoperative blood loss, intraoperative transfusion rate, resection margin size, R0 resection rate, three-year overall survival (OS) and three-year disease-free survival (DFS). CONCLUSION: LLR for large HCC is safe and feasible. This surgical strategy will not affect the long-term outcomes of patients.

Elevated NT-proBNP predicts unfavorable outcomes in patients with acute ischemic stroke after thrombolytic therapy.

OBJECTIVE: Few studies correlated n-terminal pro-brain natriuretic peptide (NT-proBNP) with early neurological deterioration (END) and prognosis of acute ischaemic stroke (AIS) patients with rt-PA intravenous thrombolysis. Therefore this study aimed to investigate the relationship between NT-proBNP and END, and prognosis after intravenous thrombolysis in patients with AIS. METHODS: A total of 325 patients with AIS were enrolled. We performed the natural logarithm transformation on the NT-proBNP [ln(NT-proBNP)]. Univariate and multivariate logistic regression analyses were performed to assess the relationship between ln(NT-proBNP) and END, and prognosis and receiver operating characteristic (ROC) curves were used to show the sensitivity and specificity of NT-proBNP. RESULTS: After thrombolysis, among 325 patients with AIS, 43 patients (13.2%) developed END. In addition, three months follow-up showed a poor prognosis in 98 cases (30.2%) and a good prognosis in 227 cases (69.8%). Multivariate logistic regression analysis showed that ln(NT-proBNP) was an independent risk factor for END (OR = 1.450,95%CI:1.072 ~ 1.963, P = 0.016) and poor prognosis at three months follow-up (OR = 1.767, 95%CI: 1.347 ~ 2.317, P < 0.001) respectively. According to ROC curve analysis, ln(NT-proBNP) (AUC 0.735, 95%CI: 0.674 ~0.796, P < 0.001) had a good predictive value for poor prognosis, with a predictive value of 5.12 and sensitivity and specificity of 79.59% and 60.35% respectively. When combined with NIHSS to predict END(AUC 0.718, 95%CI: 0.631 ~ 0.805, P < 0.001) and poor prognosis(AUC 0.780, 95%CI: 0.724 ~ 0.836, P < 0.001), the predictive value of the model is further improved. CONCLUSION: NT-proBNP is independently associated with END and poor prognosis in patients with AIS following intravenous thrombolysis and has a particular predictive value for END and poor prognosis.

High magnesium mitigates the vasoconstriction mediated by different types of calcium influx from monocrotaline-induced pulmonary hypertensive rats.

NEW FINDINGS: What is the central question of this study? What is the involvement of Mg2+ in mitigating the vasoconstriction in pulmonary arteries and smaller pulmonary arteries in the monocrotaline-induced pulmonary arterial hypertension (MCT-PAH) rat model? What are the main finding and its importance? Both store-operated Ca2+ entry- and receptor-operated Ca2+ entry-mediated vasoconstriction were enhanced in the MCT-PAH model. High magnesium inhibited vasoconstriction by directly antagonizing Ca2+ and increasing NO release, and this was more notable in smaller pulmonary arteries. ABSTRACT: Increased extracellular magnesium concentration has been shown to attenuate the endothelin-1-induced contractile response via the release of nitric oxide (NO) from the endothelium in proximal pulmonary arteries (PAs) of chronic hypoxic mice. Here, we further examined the involvement of Mg2+ in the inhibition of vasoconstriction in PAs and distal smaller pulmonary arteries (sPAs) in a monocrotaline-induced pulmonary arterial hypertension (MCT-PAH) rat model. The data showed that in control rats vasoconstriction in sPAs is more intense than that in PAs. In MCT-PAH rats, store-operated Ca2+ entry (SOCE)- and receptor-operated Ca2+ entry (ROCE)-mediated contraction were significantly strengthened. However, there was no upregulation of the vasoconstriction mediated by voltage-dependent calcium entry (VDCE). Furthermore, high magnesium greatly inhibited VDCE-mediated contraction in PAs rather than sPAs, which was the opposite of the ROCE-mediated contraction. Moreover, monocrotaline pretreatment partly eliminated the endothelium-dependent vasodilatation in PAs, which in sPAs, however, was still promoted by magnesium due to the increased NO release in pulmonary microvascular endothelial cells (PMVECs). In conclusion, the findings suggest that both SOCE- and ROCE-mediated vasoconstriction in the MCT-PAH model are enhanced, especially in sPAs. The inhibitory effect of high magnesium on vasoconstriction can be achieved partly by its direct role as a Ca2+ antagonist and partly by increasing NO release in PMVECs.

Synchronous detection of vascular tension and nitric oxide release in pulmonary artery: A combined application of confocal wire myograph with confocal laser scanning microscopy.

OBJECTIVES: To detect the vascular tension and nitric oxide (NO) release synchronously in mice pulmonary artery, we perform two experiments and present a novel application of confocal wire myograph coupled with the confocal laser scanning microscopy. METHODS: In the first experiment, viable endothelium-intact mouse pulmonary artery (outer diameter 100-300 µM) rings underwent a one-hour preincubation with a NO-specific fluorescent dye, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate Calbiochem (2.5 µM), and then precontracted with phenylephrine (Phen, 10-6 M), and subsequently dilated in acetylcholine (ACh, 10-6 M - 10-4 M). The endothelium-dependent vasorelaxation and NO generation in pulmonary artery rings were simultaneously recorded. In the second experiment, after 30-min incubation with the former NO fluorescent dye, the qualified pulmonary artery rings were co-incubated for another 30 min with a nitric oxide synthase inhibitor, 10-4 M Nω-nitro-L-arginine-methyl-ester (L-NAME), and then pretreated with Phen (10-6 M) followed by ACh (10-5 M). The Ach-induced vasodilation and NO release were recorded simultaneously. RESULTS: ACh (10-6 M - 10-4 M) promoted pulmonary artery relaxation and intracellular NO release in a dose-dependent manner. Additionally, L-NAME (10-4 M) significantly attenuated the vasodilatation and the intracellular NO release. CONCLUSIONS: This combined application visually confirms that the synchronous changes in Ach induced vasodilation and NO release, which provides a new method for cardiovascular research.

Increased caveolin-1 expression enhances the receptor-operated Ca2+ entry in the aorta of two-kidney, one-clip hypertensive rats.

NEW FINDINGS: What is the central question of this study? The aim was to examine and compare the contributions of caveolin-1 to the contractile responses mediated by L-type voltage-dependent calcium channels, store-operated Ca2+ channels and receptor-operated Ca2+ channels in two different types of arteries from two-kidney, one-clip hypertensive rats. What is the main finding and its importance? We demonstrated that the density of caveolae and caveolin-1 expression were significantly upregulated in the aorta of two-kidney, one-clip hypertensive rats, but not in the third-order branches of mesenteric arteries. We highlight that caveolin-1 plays an important role in aortic constriction by enhancing receptor-operated Ca2+ entry in the hypertensive rat model. ABSTRACT: Calcium and its multiple regulatory mechanisms are crucial for the development of hypertension. Among these regulatory mechanisms, store-operated Ca2+ entry (SOCE) and receptor-operated Ca2+ entry (ROCE) mediate agonist-induced calcium influx, contributing to vascular contraction. The SOCE and ROCE are regulated by a variety of mechanisms involving caveolin-1 (Cav1), which has been found to be strongly associated with hypertension in gene polymorphism. In the present study, we investigated the role of Cav1 during the enhanced activity of calcium channels in hypertensive arteries. We demonstrated that the expression level of Cav1 was significantly increased in the aorta of two-kidney, one-clip (2K1C) hypertensive rats. The disruption of caveolae by methyl-ß-cyclodextrin did not cause a marked difference in agonist-induced vasoconstriction in the third-order branches of the mesenteric arteries but strongly suppressed the aortic contractile response to endothelin-1 in the 2K1C group, which was not found in the control group. The increase in Cav1 by introduction of Cav1 scaffolding domain enhancing peptide promoted the 1-oleoyl-2-acetyl-glycerol-induced ROCE in hypertensive aortic smooth muscle cells but did not enhance the cyclopiazonic acid-induced SOCE. In the resistance arteries, similar changes were not observed, and no statistical changes of Cav1 expression were evident in the third-order branches of the mesenteric arteries. Our results indicate that increased Cav1 expression might promote the altered [Ca2+ ]i -induced aortic vasoreactivity by enhancing ROCE and be involved in the pathogenesis of hypertension.

Multiplex analysis of serum hormone and cytokine in patients with cervical cOPLL: towards understanding the potential pathogenic mechanisms.

Cervical ossification of the posterior longitudinal ligament (cOPLL) is one of the major causes of myelopathy. However, the mechanism underlying remains elusive. In the present study, using MILLIPLEX magnetic bead panel, we investigated four serum hormones and six serum cytokines in cOPLL patients and healthy subjects. The results showed that tumor necrosis factore-α (TNF-α) were significantly increased, and DDK-1 was significantly decreased in the serum from male and female cOPLL patients compared with those from healthy controls, respectively. Osteopontin (OPN) and fibroblast growth factor-23 (FGF-23) were significantly increased in male cOPLL patients compared with that in healthy male controls. Further analysis showed that FGF-23 and OPN significantly increased, dickkopf-1 (DKK-1) decreased in the extensive cOPLL group. In addition, a significant positive correlation between the OPN and FGF-23 was observed in male cOPLL patients. The results are useful for understanding the mechanism underlying cOPLL.

Polyamine-binding protein PotD2 is required for stress tolerance and virulence in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the cause of porcine contagious pleuropneumonia, which is one of the most important respiratory diseases in swine and causes huge economic losses in the swine industry. PotD, a polyamine-binding protein, has been well characterised in many pathogens of humans and animals. In this study, a ΔpotD2 mutant of A. pleuropneumoniae strain MS71 (serovar 1) was constructed successfully by homologous recombination. Growth curves of different strains showed that the growth of the ΔpotD2 mutant was affected greatly in the logarithmic phase compared with that of parental strain. In vitro stress assays revealed that the viability of ΔpotD2 mutant strain was significantly impaired under multiple environmental stresses, including high temperature, oxidation and hyperosmosis. Additionally, the ΔpotD2 mutant caused significantly decreased mortality in a mouse model. Taken together, the findings in this study suggest an important role of PotD2 in the growth, stress tolerance and virulence of A. pleuropneumoniae.

Review: Protein O-GlcNAcylation regulates DNA damage response: A novel target for cancer therapy.

The DNA damage response (DDR) safeguards the stable genetic information inheritance by orchestrating a complex protein network in response to DNA damage. However, this mechanism can often hamper the effectiveness of radiotherapy and DNA-damaging chemotherapy in destroying tumor cells, causing cancer resistance. Inhibiting DDR can significantly improve tumor cell sensitivity to radiotherapy and DNA-damaging chemotherapy. Thus, DDR can be a potential target for cancer treatment. Post-translational modifications (PTMs) of DDR-associated proteins profoundly affect their activity and function by covalently attaching new functional groups. O-GlcNAcylation (O-linked-N-acetylglucosaminylation) is an emerging PTM associated with adding and removing O-linked N-acetylglucosamine to serine and threonine residues of proteins. It acts as a dual sensor for nutrients and stress in the cell and is sensitive to DNA damage. However, the explanation behind the specific role of O-GlcNAcylation in the DDR remains remains to be elucidated. To illustrate the complex relationship between O-GlcNAcylation and DDR, this review systematically describes the role of O-GlcNAcylation in DNA repair, cell cycle, and chromatin. We also discuss the defects of current strategies for targeting O-GlcNAcylation-regulated DDR in cancer therapy and suggest potential directions to address them.

IL-1ra loaded chondroitin sulfate-functionalized microspheres for minimally invasive treatment of intervertebral disc degeneration.

Presently, the clinical treatment of intervertebral disc degeneration (IVDD) remains challenging, but the strategy of simultaneously overcoming the overactive inflammation and restoring the anabolic/catabolic balance of the extracellular matrix (ECM) in the nucleus pulposus (NP) has become an effective way to alleviate IVDD. IL-1ra, a natural antagonist against IL-1ß, can mitigate inflammation and promote regeneration in IVDD. Chondroitin sulfate (CS), an important component of the NP, can promote ECM synthesis and delay IVDD. Thus, these were chosen and integrated into functionalized microspheres to achieve their synergistic effects. First, CS-functionalized microspheres (GelMA-CS) with porous microstructure, good monodispersion, and about 200 µm diameter were efficiently and productively fabricated using microfluidic technology. After lyophilization, the microspheres with good local injection and tissue retention served as the loading platform for IL-1ra and achieved sustained release. In in vitro experiments, the IL-1ra-loaded microspheres exhibited good cytocompatibility and efficacy in inhibiting the inflammatory response of NP cells induced by lipopolysaccharide (LPS) and promoting the secretion of ECM. In in vivo experiments, the microspheres showed good histocompatibility, and local, minimally invasive injection of the IL-1ra-loaded microspheres could reduce inflammation, maintain the height of the intervertebral disc (IVD) and the water content of NP close to about 70% in the sham group, and retain the integrated IVD structure. In summary, the GelMA-CS microspheres served as an effective loading platform for IL-1ra, eliminated inflammation through the controlled release of IL-1ra, and promoted ECM synthesis via CS to delay IVDD, thereby providing a promising intervention strategy for IVDD. STATEMENT OF SIGNIFICANCE: The strategy of simultaneously overcoming the overactive inflammation and restoring the anabolic/catabolic balance of the extracellular matrix (ECM) in nucleus pulposus (NP) has shown great potential prospects for alleviating intervertebral disc degeneration (IVDD). From the perspective of clinical translation, this study developed chondroitin sulfate functionalized microspheres to act as the effective delivery platform of IL-1ra, a natural antagonist of interleukin-1ß. The IL-1ra loading microspheres (GelMA-CS-IL-1ra) showed good biocompatibility, good injection with tissue retention, and synergistic effects of inhibiting the inflammatory response induced by lipopolysaccharide and promoting the secretion of ECM in NPCs. In vivo, they also showed the beneficial effect of reducing the inflammatory response, maintaining the height of the intervertebral disc and the water content of the NP, and preserving the integrity of the intervertebral disc structure after only one injection. All demonstrated that the GelMA-CS-IL-1ra microspheres would have great promise for the minimally invasive treatment of IVDD.

Sustained release of basic fibroblast growth factor in micro/nanofibrous scaffolds promotes annulus fibrosus regeneration.

Tissue engineering has promising applications in the treatment of intervertebral disc degeneration (IDD). The annulus fibrosus (AF) is critical for maintaining the physiological function of the intervertebral disc (IVD), but the lack of vessels and nutrition in AF makes it difficult to repair. In this study, we used hyaluronan (HA) micro-sol electrospinning and collagen type I (Col-I) self-assembly techniques to fabricate layered biomimetic micro/nanofibrous scaffolds, which released basic fibroblast growth factor (bFGF) to promote AF repair and regeneration after discectomy and endoscopic transforaminal discectomy. The bFGF enveloped in the core of the poly-L-lactic-acid (PLLA) core-shell structure was released in a sustained manner and promoted the adhesion and proliferation of AF cells (AFCs). Col-I could self-assemble on the shell of the PLLA core-shell scaffold to mimic the extracellular matrix (ECM) microenvironment, providing structural and biochemical cues for the regeneration of AF tissue. The in vivo studies showed that the micro/nanofibrous scaffolds promoted the repair of AF defects by simulating the microstructure of native AF tissue and inducing endogenous regeneration mechanism. Taken together, the biomimetic micro/nanofibrous scaffolds have clinical potential for the treatment of AF defects caused by IDD. STATEMENT OF SIGNIFICANCE: The annulus fibrosus (AF) is essential for the intervertebral disc (IVD) physiological function, yet it lacks vascularity and nutrition, making repair difficult. Micro-sol electrospinning technology and collagen type I (Col-I) self-assembly technique were combined in this study to create a layered biomimetic micro/nanofibrous scaffold that releases basic fibroblast growth factor (bFGF) to promote AF repair and regeneration. Col-I could mimic the extracellular matrix (ECM) microenvironment, in vivo, offering structural and biochemical cues for AF tissue regeneration. This research indicates that micro/nanofibrous scaffolds have clinical potential for treating AF deficits induced by IDD.

Vanillin-based functionalization strategy to construct multifunctional microspheres for treating inflammation and regenerating intervertebral disc.

Intervertebral disc degeneration (IVDD) is one of the main causes of low back pain. Although local delivery strategies using biomaterial carriers have shown potential for IVDD treatment, it remains challenging for intervention against multiple adverse contributors by a single delivery platform. In the present work, we propose a new functionalization strategy using vanillin, a natural molecule with anti-inflammatory and antioxidant properties, to develop multifunctional gelatin methacrylate (GelMA) microspheres for local delivery of transforming growth factor ß3 (TGFß3) toward IVDD treatment. In vitro, functionalized microspheres not only improved the release kinetics of TGFß3 but also effectively inhibited inflammatory responses and promoted the secretion of extracellular matrix (ECM) in lipopolysaccharide-induced nucleus pulposus (NP) cells. In vivo, functionalized platform plays roles in alleviating inflammation and oxidative stress, preserving the water content of NP and disc height, and maintaining intact structure and biomechanical functions, thereby promoting the regeneration of IVD. High-throughput sequencing suggests that inhibition of the phosphatidylinositol 3-kinase (PI3K)-Akt signaling might be associated with their therapeutic effects. In summary, the vanillin-based functionalization strategy provides a novel and simple way for packaging multiple functions into a single delivery platform and holds promise for tissue regeneration beyond the IVD.

Targeting Endogenous Reactive Oxygen Species Removal and Regulating Regenerative Microenvironment at Annulus Fibrosus Defects Promote Tissue Repair.

The excessive reactive oxygen species (ROS) level, inflammation, and weak tissue regeneration ability after annulus fibrosus (AF) injury constitute an unfavorable microenvironment for AF repair. AF integrity is crucial for preventing disc herniation after discectomy; however, there is no effective way to repair the AF. Herein, a composite hydrogel integrating properties of antioxidant, anti-inflammation, and recruitment of AF cells is developed through adding mesoporous silica nanoparticles modified by ceria and transforming growth factor ß3 (TGF-ß3) to the hydrogels. The nanoparticle loaded gelatin methacrylate/hyaluronic acid methacrylate composite hydrogels eliminate ROS and induce anti-inflammatory M2 type macrophage polarization. The released TGF-ß3 not only plays a role in recruiting AF cells but is also responsible for promoting extracellular matrix secretion. The composite hydrogels can be solidified in situ in the defect area to effectively repair AF in rats. The strategies targeting endogenous ROS removal and improving the regenerative microenvironment by the nanoparticle-loaded composite hydrogels have potential applications in AF repair and intervertebral disc herniation prevention.

Engineering the viscoelasticity of gelatin methacryloyl (GelMA) hydrogels via small "dynamic bridges" to regulate BMSC behaviors for osteochondral regeneration.

The dynamic extracellular matrix (ECM) constantly affects the behaviors of cells. To mimic the dynamics of ECM with controllable stiffness and energy dissipation, this study proposes a strategy in which a small molecule, 3,4-dihydroxybenzaldehyde (DB), was used as fast "dynamic bridges'' to construct viscoelastic gelatin methacryloyl (GelMA)-based hydrogels. The storage modulus and loss modulus of hydrogels were independently adjusted by the covalent crosslinking density and by the number of dynamic bonds. The hydrogels exhibited self-healing property, injectability, excellent adhesion and mechanical properties. Moreover, the in vitro results revealed that the viscous dissipation of hydrogels favored the spreading, proliferation, osteogenesis and chondrogenesis of bone marrow mesenchymal stem cells (BMSCs), but suppressed their adipogenesis. RNA-sequencing and immunofluorescence suggested that the viscous dissipation of hydrogels activated Yes-associated protein (YAP) by stabilizing integrin ß1, and further promoted nuclear translocation of smad2/3 and ß-catenin to enhance chondrogenesis and osteogenesis. As a result, the viscoelastic GelMA hydrogels with highest loss modulus showed best effect in cartilage and subchondral bone repair. Taken together, findings from this study reveal an effective strategy to fabricate viscoelastic hydrogels for modulating the interactions between cells and dynamic ECM to promote tissue regeneration.

Outer Membrane Vesicles of Actinobacillus pleuropneumoniae Exert Immunomodulatory Effects on Porcine Alveolar Macrophages.

Outer membrane vesicles (OMVs) are spontaneously released by Gram-negative bacteria, including Actinobacillus pleuropneumoniae, which causes contagious pleuropneumonia in pigs and leads to considerable economic losses in the swine industry worldwide. A. pleuropneumoniae OMVs have previously been demonstrated to contain Apx toxins and proteases, as well as antigenic proteins. Nevertheless, comprehensive characterizations of their contents and interactions with host immune cells have not been made. Understanding the protein compositions and immunomodulating ability of A. pleuropneumoniae OMVs could help illuminate their biological functions and facilitate the development of OMV-based applications. In the current investigation, we comprehensively characterized the proteome of native A. pleuropneumoniae OMVs. Moreover, we qualitatively and quantitatively compared the OMV proteomes of a wild-type strain and three mutant strains, in which relevant genes were disrupted to increase OMV production and/or produce OMVs devoid of superantigen PalA. Furthermore, the interaction between A. pleuropneumoniae OMVs and porcine alveolar macrophages was also characterized. Our results indicate that native OMVs spontaneously released by A. pleuropneumoniae MIDG2331 appeared to dampen the innate immune responses by porcine alveolar macrophages stimulated by either inactivated or live parent cells. The findings suggest that OMVs may play a role in manipulating the porcine defense during the initial phases of the A. pleuropneumoniae infection. IMPORTANCE Owing to their built-in adjuvanticity and antigenicity, bacterial outer membrane vesicles (OMVs) are gaining increasing attention as potential vaccines for both human and animal use. OMVs released by Actinobacillus pleuropneumoniae, an important respiratory pathogen in pigs, have also been investigated for vaccine development. Our previous studies have shown that A. pleuropneumoniae secretes OMVs containing multiple immunogenic proteins. However, immunization of pigs with these vesicles was not able to relieve the pig lung lesions induced by the challenge with A. pleuropneumoniae, implying the elusive roles that A. pleuropneumoniae OMVs play in host-pathogen interaction. Here, we showed that A. pleuropneumoniae secretes OMVs whose yield and protein content can be altered by the deletion of the nlpI and palA genes. Furthermore, we demonstrate that A. pleuropneumoniae OMVs dampen the immune responses in porcine alveolar macrophages stimulated by A. pleuropneumoniae cells, suggesting a novel mechanism that A. pleuropneumoniae might use to evade host defense.

Fucoidan-loaded nanofibrous scaffolds promote annulus fibrosus repair by ameliorating the inflammatory and oxidative microenvironments in degenerative intervertebral discs.

Tissue engineering holds potential in the treatment of intervertebral disc degeneration (IDD). However, implantation of tissue engineered constructs may cause foreign body reaction and aggravate the inflammatory and oxidative microenvironment of the degenerative intervertebral disc (IVD). In order to ameliorate the adverse microenvironment of IDD, in this study, we prepared a biocompatible poly (ether carbonate urethane) urea (PECUU) nanofibrous scaffold loaded with fucoidan, a natural marine bioactive polysaccharide which has great anti-inflammatory and antioxidative functions. Compared with pure PECUU scaffold, the fucoidan-loaded PECUU nanofibrous scaffold (F-PECUU) decreased the gene and protein expression related to inflammation and the oxidative stress in the lipopolysaccharide (LPS) induced annulus fibrosus cells (AFCs) significantly (p<0.05). Especially, gene expression of Il 6 and Ptgs2 was decreased more than 50% in F-PECUU with 3.0 wt% fucoidan (HF-PECUU). Moreover, the gene and protein expression related to the degradation of extracellular matrix (ECM) were reduced in a fucoidan concentration-dependent manner significantly, with increased almost 3 times gene expression of Col1a1 and Acan in HF-PECUU. Further, in a 'box' defect model, HF-PECUU decreased the expression of COX-2 and deposited more ECM between scaffold layers when compared with pure PECUU. The disc height and nucleus pulposus hydration of repaired IVD reached up to 75% and 85% of those in the sham group. In addition, F-PECUU helped to maintain an integrate tissue structure with a similar compression modulus to that in sham group. Taken together, the F-PECUU nanofibrous scaffolds showed promising potential to promote AF repair in IDD treatment by ameliorating the harsh degenerative microenvironment. STATEMENT OF SIGNIFICANCE: Annulus fibrosus (AF) tissue engineering holds potential in the treatment of intervertebral disc degeneration (IDD), but is restricted by the inflammatory and oxidative microenvironment of degenerative disc. This study developed a biocompatible polyurethane scaffold (F-PECUU) loaded with fucoidan, a marine bioactive polysaccharide, for ameliorating IDD microenvironment and promoting disc regeneration. F-PECUU alleviated the inflammation and oxidative stress caused by lipopolysaccharide and prevented extracellular matrix (ECM) degradation in AF cells. In vivo, it promoted ECM deposition to maintain the height, water content and mechanical property of disc. This work has shown the potential of marine polysaccharides-containing functional scaffolds in IDD treatment by ameliorating the harsh microenvironment accompanied with disc degeneration.

Multifunctional Nanofibrous Scaffolds with Angle-Ply Microstructure and Co-Delivery Capacity Promote Partial Repair and Total Replacement of Intervertebral Disc.

There is an urgent clinical need for the treatment of annulus fibrosus (AF) impairment caused by intervertebral disc (IVD) degeneration or surgical injury. Although repairing injured AF through tissue engineering is promising, the approach is limited by the complicated angle-ply microstructure, inflammatory microenvironment, poor self-repairing ability of AF cells and deficient matrix production. In this study, electrospinning technology is used to construct aligned core-shell nanofibrous scaffolds loaded with transforming growth factor-ß3 (TGFß3) and ibuprofen (IBU), respectively. The results confirm that the rapid IBU release improves the inflammatory microenvironment, while sustained TGFß3 release enhances nascent extracellular matrix (ECM) formation. Biomaterials for clinical applications must repair local AF defects during herniectomy and enable AF regeneration during disc replacement, so a box defect model and total IVD replacement model in rat tail are constructed. The dual-drug delivering electrospun scaffolds are assembled into angle-ply structure to form a highly biomimetic AF that is implanted into the box defect or used to replace the disc. In two animal models, it is found that biomimetic scaffolds with good anti-inflammatory ability enhance ECM formation and maintain the mechanical properties of IVD. Findings from this study demonstrate that the multifunctional nanofibrous scaffolds provide inspirations for IVD repair.

Bacterial Outer Membrane Vesicles as a Versatile Tool in Vaccine Research and the Fight against Antimicrobial Resistance.

Gram-negative bacteria include a number of pathogens that cause disease in humans and animals. Although antibiotics are still effective in treating a considerable range of infections caused by Gram-negative bacteria, the alarming increase of antimicrobial resistance (AMR) induced by excessive use of antibiotics has raised global concerns. Therefore, alternative strategies must be developed to prevent and treat bacterial infections and prevent the advent of a postantibiotic era. Vaccines, one of the greatest achievements in the history of medical science, hold extraordinary potential to prevent bacterial infections and thereby reduce the need for antibiotics. Novel bacterial vaccines are urgently needed, however, and outer membrane vesicles (OMVs), naturally produced by Gram-negative bacteria, represent a promising and versatile tool that can be employed as adjuvants, antigens, and delivery platforms in the development of vaccines against Gram-negative bacteria. Here, we provide an overview of the many roles OMVs can play in vaccine development and the mechanisms behind these applications. Methods to improve OMV yields and a comparison of different strategies for OMV isolation aiming at cost-effective production of OMV-based vaccines are also reviewed.