Bovine parainfluenza virus type 3 infections induce ER stress-mediated autophagy to facilitate virus replication.

Bovine Parainfluenza Virus Type 3 (BPIV3) serves as a crucial pathogen in cattle, adept at triggering severe respiratory symptoms. This investigation explores the intricate interplay of endoplasmic reticulum stress (ER stress), unfolded protein response (UPR), and autophagy upon BPIV3 infection. In this study, we initially confirm a substantial increase in glucose regulatory protein 78 (GRP78) expression, accompanied by noticeable morphological changes and significant expansion of the ER lumen observed through transmission electron microscopy upon BPIV3 infection. Our findings indicate that ER Stress is induced during BPIV3 infection in vitro. Subsequently, we illustrate that BPIV3 triggers ER Stress to facilitate viral replication through heightened autophagy through treatment with the ER stress inhibitor 4-phenylbutyrate (4-PBA) and utilizing small interfering RNA (siRNA) technology to knock down GRP78. Additionally, we observe that the activation of ER stress initiates the UPR via PERK and ATF6 pathways, with the IRE1 pathway not contributing to the regulation of ER stress-mediated autophagy. Moreover, intervention with the PERK inhibitor GSK2606414, ATF6 inhibitor Ceapin-A7, and siRNA technology successfully reverses BPIV3-induced autophagy. In summary, these findings propose that BPIV3 induces ER stress to enhance viral replication through increased autophagy, with the PERK and ATF6 pathways playing a significant role in ER stress-mediated autophagy.

Towards interpretable machine learning for observational quantification of soil heavy metal concentrations under environmental constraints.

Monitoring heavy metal concentrations in soils is central to assessing agricultural production safety. Satellite observations permit inferring concentrations from spectrum, thereby contributing to the prevention and control of soil heavy metal pollution. However, heavy metals exhibit weak spectral responses, particularly at low and medium concentrations, and are predominantly influenced by other soil components. Machine learning (ML)-driven modelling can produce predictions but lacks interpretability. Here, we present an interpretable ML framework for concentration quantification modelling and investigated the contributions of spectral and environmental factors-pH and organic carbon-to the estimation of metals with multiple concentration gradients, as analysed through SHAP (SHapley Additive exPlanations) data derived from four learning-based scenarios. The results indicated that scenarios SHC (spectral, pH, and organic carbon) and SH (spectral and pH) were the most optimal for chromium (Cr) [RPD = 1.42, Adj R2 = 0.62], and cadmium (Cd) [RPD = 1.80, Adj R2 = 0.80]. Under environmental constraints, the spectral predictability for Cr and Cd was improved by 67 % and 87 %, respectively. We concluded that interpretable modelling, utilising both spectral and soil environmental factors, holds significant potential for estimating heavy metals across concentration gradients. It is recommended that samples with higher organic carbon content and lower pH be selected to enhance Cr and Cd predictions. An advanced grasp of interpretable predictions facilitates earlier warning of heavy metal contamination and guides the formulation of robust sampling strategies.

Transcriptomic analysis and oxidative stress induced by sodium dichloroisocyanurate in the intestine of Phascolosoma esculenta.

Sodium dichloroisocyanurate (NaDCC, C3Cl2N3NaO3) is a solid chlorine-containing product that is widely used as a disinfectant in living environments, which has potential toxic effects on human and rats. Phascolosoma esculenta is a species native to the southeast coast of China and can be used as an indicator organism. In the present study, 150 P. esculenta were used to determine the LC50 of NaDCC for P. esculenta, then 100 P. esculenta were used to analysis the change of histopathology, oxidative stress and transcriptome after NaDCC exposure. The results showed that the LC50 of NaDCC for 48 h was 50 mg/L. NaDCC stress induced pathological events in P. esculenta, including blisters, intestinal structural damage and epithelial cell ruptured or even loss. The highest and lowest intestinal activity of superoxide dismutase in individual survivors was detected at 12 h and 72 h, respectively. Malondialdehyde levels in the intestine declined gradually from 3 h and increased at 9 h, and peaked at 12 h. Total antioxidant capacity declined at 3 h and dropped below the levels of control group after 9 h. Transcriptome sequencing analysis yielded a total of 48.65 Gb of clean data. A total of 34,759 new genes were found including 957 differentially expressed genes (DEGs). The DEGs were significantly enriched in ferroptosis, response to chemicals, response to stress, immune system, ion transport, cell death, oxidation-reduction, cellular homeostasis, protein ubiquitination, and protein neddylation. Additionally, the levels of detoxification enzymes, such as glutathione-S-transferase, cytochrome P450, ABC, UDP-glycosyltransferase and SLC transporters of endogenous and exogenous solutes were significantly changed. Overall, the results provide reference for reasonable use of disinfectants during farming, and also provide insight into the mechanisms related to NaDCC toxicity in P. esculenta.

IL-4 activates ULK1/Atg9a/Rab9 in asthma, NLRP3 inflammasomes, and Golgi fragmentation by increasing autophagy flux and mitochondrial oxidative stress.

During asthma, there is an intensification of pulmonary epithelial inflammation, mitochondrial oxidative stress, and Golgi apparatus fragmentation. However, the underlying mechanism remains largely unknown. Therefore, this study investigated the roles of ULK1, Atg9a, and Rab9 in epithelial inflammation, mitochondrial oxidative stress, and Golgi apparatus fragmentation. We found that ULK1 gene knockout reduced the infiltration of inflammatory cells, restored the imbalance of the Th1/Th2 ratio, and inhibited the formation of inflammatory bodies in the lung tissue of house dust mite-induced asthma mice. Moreover, we demonstrated that Atg9a interacted with ULK1 at S467. ULK1 phosphorylated Atg9a at S14. Treatment with ULK1 activator (LYN-1604) and ULK1 inhibitor (ULK-101) respectively promoted and inhibited inflammasome activation, indicating that the activation of inflammasome induced by house dust mite in asthma mice is dependent on ULK1. For validation of the in vivo results, we then used a lentivirus containing ULK1 wild type and ULK1-S467A genes to infect Beas-2b-ULK1-knockout cells and establish a stable cell line. The results suggest that the ULK1 S467 site is crucial for IL-4-induced inflammation and oxidative stress. Experimental verification confirmed that Atg9a was the superior signaling pathway of Rab9. Interestingly, we found for the first time that Rab9 played a very important role in inflammation-induced fragmentation of the Golgi apparatus. Inhibiting the activation of the ULK1/Atg9a/Rab9 signaling pathways can inhibit Golgi apparatus fragmentation and mitochondrial oxidative stress in asthma while reducing the production of NLRP3-mediated pulmonary epithelial inflammation.

Transcriptome analysis of salivary glands of rabies-virus-infected mice.

Rabies is a fatal zoonotic disease that poses a threat to public health. Rabies virus (RABV) is excreted in the saliva of infected animals, and is primarily transmitted by bite. The role of the salivary glands in virus propagation is significant, but has been less studied in the pathogenic mechanisms of RABV. To identify functionally important genes in the salivary glands, we used RNA sequencing (RNA-seq) to establish and analyze mRNA expression profiles in parotid tissue infected with two RABV strains, CVS-11 and PB4. The biological functions of differentially expressed genes (DEGs) were determined by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, which revealed 3,764 DEGs (678 up-regulated and 3,086 down-regulated) in the CVS-11 infected group and 4,557 DEGs (874 up-regulated and 3,683 down-regulated) in the PB4 infected group. Various biological processes are involved, including the salivary secretion pathway and the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) signaling pathway. This study provides the first mapping of the transcriptome changes in response to RABV infection in parotid tissue, offering new insights into the study of RABV-affected salivary gland function and RABV pathogenic mechanisms in parotid tissue. The salivary gland-enriched transcripts may be potential targets of interest for rabies disease control.

Prolyl isomerase Pin1 sculpts the immune microenvironment of colorectal cancer.

Pin1, a peptide prolyl cis-trans isomerase, is overexpressed and/or overactivated in many human malignancies. However, whether Pin1 regulates the immunosuppressive TME has not been well defined. In this study, we detected the effect of Pin1 on immune cells and immune checkpoint PD-L1 in the TME of CRC and explored the anti-tumor efficacy of Pin1 inhibitor ATRA combined with PD-1 antibody. We found that Pin1 facilitated the immunosuppressive TME by raising the proportion of myeloid-derived suppressor cells (MDSCs) and declining the percentage of CD8+ T cells and CD4+ T cells. Pin1 restrained PD-L1 protein expression in CRC cells and the effect was tempered by endoplasmic reticulum (ER) stress inducers. Mechanically, Pin1 overexpression decreased the stability of PD-L1 and promoted its degradation by mitigating ER stress. Silencing or inhibiting Pin1 promoted PD-L1 protein expression by inducing ER stress. Hence, Pin1 inhibitor ATRA enhanced the anti-tumor efficacy of PD-1 antibody in the CRC allograft by upregulating PD-L1. Our results reveal the critical and pleiotropic effects of Pin1 on managing the immune cells and immune checkpoint PD-L1 in the TME of CRC, providing a new promising candidate for combination with immunotherapy.

Aging-Related Rotator Cuff Tears: Molecular Mechanisms and Implications for Clinical Management.

Shoulder pain and disabilities are prevalent issues among the elderly population, with rotator cuff tear (RCT) being one of the leading causes. Although surgical treatment has shown some success, high postoperative retear rates remain a great challenge, particularly in elderly patients. Aging-related degeneration of muscle, tendon, tendon-to-bone enthesis, and bone plays a critical role in the development and prognosis of RCT. Studies have demonstrated that aging worsens muscle atrophy and fatty infiltration, alters tendon structure and biomechanical properties, exacerbates enthesis degeneration, and reduces bone density. Although recent researches have contributed to understanding the pathophysiological mechanisms of aging-related RCT, a comprehensive systematic review of this topic is still lacking. Therefore, this article aims to present a review of the pathophysiological changes and their clinical significance, as well as the molecular mechanisms underlying aging-related RCT, with the goal of shedding light on new therapeutic approaches to reduce the occurrence of aging-related RCT and improve postoperative prognosis in elderly patients.

Mechanism of phenanthrene degradation by the halophilic Pelagerythrobacter sp. N7.

PAHs has shown worldwide accumulation and causes a significant environmental problem especially in saline and hypersaline environments. Moderately halophilic bacteria could be useful for the bioremediation of PAH pollution in hypersaline environments. Pelagerythrobacter sp. N7 was isolated from the PAH-degrading consortium 5H, which was enriched from mixed saline soil samples collected in Shanxi Province, China. 16S rRNA in the genomic DNA revealed that strain N7 belonged to Pelagerythrobacter. Strain N7 exhibited a high tolerance to a wide range of salinities (1-10%) and was highly efficient under neutral to weak alkaline conditions (pH 6-9). The whole genome of strain N7 was sequenced and analyzed, revealing an abundance of catabolic genes. Using the whole genome information, we conducted preliminary research on key enzymes and gene clusters involved in the upstream and downstream PAH degradation pathways of strain N7, thereby inferring its degradation pathway for phenanthrene and naphthalene. This study adds to our understanding of PAH degradation in hypersaline environments and, for the first time, identifies a Pelagerythrobacter with PAH-degrading capability. Strain N7, with its high efficiency in phenanthrene degradation, represents a promising resource for the bioremediation of PAHs in hypersaline environments.

Bovine parainfluenza type 3 virus induces incomplete autophagy to promote viral replication by activated beclin1 in vitro.

Bovine Parainfluenza virus Type 3 (BPIV3) is one of the most important pathogens in cattle, capable of causing severe respiratory symptoms. Numerous studies have shown that autophagy plays a diverse role in the infection process of various pathogens. The influence of autophagy machinery on BPIV3 infection has not yet been confirmed. In the present study, we initially demonstrated that the expression of LC3 was significantly increased and exhibited a notable increase in double or single-membrane vesicles under a transmission electron microscope during BPIV3 infection. These observations unequivocally establish the induction of steady-state autophagy in vitro consequent to BPIV3 infection. Furthermore, quantification of autophagic flux substantiates the induction of an incomplete autophagic process during BPIV3 infection. Additionally, through targeted interventions, we demonstrate the regulatory impact of pharmacological agents influencing autophagy and RNA interference targeting an autophagy-associated protein on viral replication. Intriguingly, our data revealed that BPIV3 infection enhanced the phosphorylation of rapamycin kinase (mTOR). This result demonstrated that mTOR does not operate as a counteractive regulator of BPIV3-induced autophagy. Instead, we discern an augmentation in the expression of Beclin1, a key autophagy initiator, which complexes with Vps34, constituting a Class III phosphatidylinositol 3-kinase. This phenomenon serves as a hallmark in the inaugural phase of autophagy initiation during BPIV3 infection. Collectively, these discernments underscore that BPIV3 infection actively stimulates autophagy, thereby enhancing viral replication through the activation of Beclin1, independently of the mTOR signaling pathway. This nuanced comprehension significantly contributes to unraveling the intricate molecular mechanisms governing BPIV3-induced autophagy.

Scaled consensus for second-order multi-agent systems subject to communication noise with stochastic approximation-type protocols.

This work is dedicated to the leaderless/leader-following stochastic scaled consensus issue of second-order stochastic multi-agent systems (SMASs) in a noisy environment. Scaled consensus represents that the ratios among agents asymptotically tend to designated constants rather than the common convergence value. To lessen the influence of communication noise, some stochastic approximation protocols with time-varying gain are designed for our underlying system, where the time-varying gain remove the restriction of nonnegative value. Compared with the existing consensus results with communication noise, the major challenge is that the introduction of time-varying gain results in the inapplicability of Lyapunov-based technique. To cope with it, a state decomposition method is utilized, and a series of sufficient necessary conditions are set up for interacting agents with constant velocity and zero velocity if the topology includes a spanning tree. Furthermore, it is conducted that the consensus and bipartite consensus can be seen as two special cases of our work. Finally, the validity of our results is demonstrated by a simulation example.

Erratum: MiR-375 Has Contrasting Effects on Newcastle Disease Virus Growth Depending on the Target Gene : Erratum.

[This corrects the article DOI: 10.7150/ijbs.25106.].

Decoding the temporal representation of facial expression in face-selective regions.

The ability of humans to discern facial expressions in a timely manner typically relies on distributed face-selective regions for rapid neural computations. To study the time course in regions of interest for this process, we used magnetoencephalography (MEG) to measure neural responses participants viewed facial expressions depicting seven types of emotions (happiness, sadness, anger, disgust, fear, surprise, and neutral). Analysis of the time-resolved decoding of neural responses in face-selective sources within the inferior parietal cortex (IP-faces), lateral occipital cortex (LO-faces), fusiform gyrus (FG-faces), and posterior superior temporal sulcus (pSTS-faces) revealed that facial expressions were successfully classified starting from ∼100 to 150 ms after stimulus onset. Interestingly, the LO-faces and IP-faces showed greater accuracy than FG-faces and pSTS-faces. To examine the nature of the information processed in these face-selective regions, we entered with facial expression stimuli into a convolutional neural network (CNN) to perform similarity analyses against human neural responses. The results showed that neural responses in the LO-faces and IP-faces, starting ∼100 ms after the stimuli, were more strongly correlated with deep representations of emotional categories than with image level information from the input images. Additionally, we observed a relationship between the behavioral performance and the neural responses in the LO-faces and IP-faces, but not in the FG-faces and lpSTS-faces. Together, these results provided a comprehensive picture of the time course and nature of information involved in facial expression discrimination across multiple face-selective regions, which advances our understanding of how the human brain processes facial expressions.

Effect of the telemedicine-supported multicomponent exercise therapy in patients with knee osteoarthritis: study protocol for a randomized controlled trial.

INTRODUCTION: The rising prevalence of knee osteoarthritis is placing a considerable strain on the global healthcare system. To address this issue, telemedicine-supported multicomponent exercise therapy has emerged as a promising approach. This therapy combines exercise, patient education, and health coaching to empower knee osteoarthritis patients to manage their condition from the comfort of their homes. Nevertheless, there are some existing limitations in the current research on this approach, including challenges related to patient compliance and the absence of objective evaluation methods. METHODS AND ANALYSIS: Patients diagnosed with knee osteoarthritis, who have not undergone knee surgery in the past year, will be recruited for a randomized controlled trial. The trial will include an intervention group and a control group. The intervention group will receive an mHealth app-based multicomponent exercise therapy, consisting of exercise therapy, patient education, and health coaching. Meanwhile, the control group will receive usual care, involving drug therapy and patient education. The primary outcome of the trial will be the measurement of pain intensity, assessed using a visual analog scale at baseline and at 4, 8, and 12 weeks of the post-intervention. To analyze the data, a two-factor, four-level repeated measures ANOVA will be used if the assumptions of homogeneity of variance and sphericity are met. If not, a mixed effects model will be employed. DISCUSSION: The aim of the study is to evaluate the effectiveness of multicomponent exercise therapy aimed at enhancing pain self-management for knee osteoarthritis patients in the comfort of their own homes. The intervention incorporate wearable devices equipped with advanced deep learning systems to monitor patients' adherence to the prescribed at-home exercise regimen, as well as to track changes in outcomes before and after the exercise sessions. The findings from this trial have the potential to enhance both the accessibility and quality of care provided to knee osteoarthritis patients, offering valuable insights for future improvements in their treatment and management. TRIAL REGISTRATION: Chinese Clinical Trials Registry, ChiCTR2300073688. Registered on 19 July 2023, https://www.chictr.org.cn/bin/project/edit?pid=199707 . World Health Organization International Clinical Trials Registry Platform, https://trialsearch.who.int/Trial2.aspx?TrialID=ChiCTR2300073688 .

Macroporous Granular Hydrogels Functionalized with Aligned Architecture and Small Extracellular Vesicles Stimulate Osteoporotic Tendon-To-Bone Healing.

Osteoporotic tendon-to-bone healing (TBH) after rotator cuff repair (RCR) is a significant orthopedic challenge. Considering the aligned architecture of the tendon, inflammatory microenvironment at the injury site, and the need for endogenous cell/tissue infiltration, there is an imminent need for an ideal scaffold to promote TBH that has aligned architecture, ability to modulate inflammation, and macroporous structure. Herein, a novel macroporous hydrogel comprising sodium alginate/hyaluronic acid/small extracellular vesicles from adipose-derived stem cells (sEVs) (MHA-sEVs) with aligned architecture and immunomodulatory ability is fabricated. When implanted subcutaneously, MHA-sEVs significantly improve cell infiltration and tissue integration through its macroporous structure. When applied to the osteoporotic RCR model, MHA-sEVs promote TBH by improving tendon repair through macroporous aligned architecture while enhancing bone regeneration by modulating inflammation. Notably, the biomechanical strength of MHA-sEVs is approximately two times higher than the control group, indicating great potential in reducing postoperative retear rates. Further cell-hydrogel interaction studies reveal that the alignment of microfiber gels in MHA-sEVs induces tenogenic differentiation of tendon-derived stem cells, while sEVs improve mitochondrial dysfunction in M1 macrophages (Mφ) and inhibit Mφ polarization toward M1 via nuclear factor-kappaB (NF-κb) signaling pathway. Taken together, MHA-sEVs provide a promising strategy for future clinical application in promoting osteoporotic TBH.

JTE-013 Alleviates Pulmonary Fibrosis by Affecting the RhoA/YAP Pathway and Mitochondrial Fusion/Fission.

Pulmonary fibrosis may be due to the proliferation of fibroblasts and the aggregation of extracellular matrix, resulting in the stimulation of inflammation damage, destroying lung tissue structure, seriously affecting the patient's respiratory function, and even leading to death. We investigated the role and mechanism of JTE-013 in attenuating bleomycin (BLM)-induced pulmonary fibrosis. BLM-induced pulmonary fibrosis was established in mice. Type 2 alveolar epithelial cells (MLE-12) were stimulated with sphingosine monophosphate (S1P) in vitro. JTE-013, an S1PR2 (sphingosine 1-phosphate receptor 2) antagonist, and Verteporfin were administered in vivo and in vitro. IL-4, IL-5, TNF-α, and IFN-γ were measured by ELISA. IL-4 and IFN-γ positive cells were detected by flow cytometry. Inhibition of S1PR2 with JTE-013 significantly ameliorated BLM-induced pathological changes and inflammatory cytokine levels. JTE-013 also significantly reduced the expression of RHOA/YAP pathway proteins and mitochondrial fission protein Drp1, apoptosis, and the colocalization of α-SMA with YAP, Drp1, and Tom20, as detected by immunohistochemistry, immunofluorescence staining, TUNEL, and Western blot. In vitro, S1PR2 and YAP knockdown downregulated RHOA/YAP pathway protein expression, Drp1 phosphorylation, and Drp1 translocation, promoted YAP phosphorylation and phenotypic transformation of MFN2, and inhibited the up-regulation of mitochondrial membrane potential, reactive oxygen species production, and cell apoptosis (7.13% vs. 18.14%), protecting the integrity of the mitochondrial dynamics. JTE-013 also inhibited the expression of fibrosis markers α-SMA, MMP-9, and COL1A1, and alleviated the symptoms of pulmonary fibrosis. Conclusively, JTE-013 has great anti-pulmonary fibrosis potential by regulating RHOA/YAP and mitochondrial fusion/fission.

3D-Printed Filters for Efficient Heavy Metal Removal from Water Using PLA@CS/HAP Composites.

Chitosan/Hydroxyapatite composites, enriched with relatively active -NH2 and -OH groups, have emerged as promising adsorbents for heavy metal removal. In this study, we harnessed the potential of CS/HAP composites by developing monolithic PLA@CS/HAP filters utilizing 3D printing and freeze-drying techniques. These filters possess both macroscopic and microscopic porous structures, endowing them with exceptional capabilities for removing heavy metals from water. The adsorption properties of CS/HAP composites were explored by varying the dosage, duration, and initial concentrations of copper ions. The maximum adsorption capacity for Cu2+ was determined to be approximately 119+/-1 mg/g at the natural pH and 298 K. Notably, the monolithic PLA@CS/HAP filters demonstrated remarkable efficiency in the removal of copper ions, with 90% of copper ions effectively removed within a mere 2-h period in a cyclic adsorption experiment. Furthermore, the PLA@CS/HAP filters exhibited a robust dynamic Cu2+ removal capacity (80.8% or even better in less than 35 min) in a dynamic adsorption experiment. Importantly, all materials employed in this study were environmentally friendly. In summary, the PLA@CS/HAP filter offers advantages such as ease of preparation, eco-friendliness, versatility, and broad applicability in diverse wastewater treatment scenarios, thereby presenting a significant potential for practical implementation.

Attachment, Entry, and Intracellular Trafficking of Classical Swine Fever Virus.

Classical swine fever virus (CSFV), which is a positive-sense, single-stranded RNA virus with an envelope, is a member of the Pestivirus genus in the Flaviviridae family. CSFV causes a severe and highly contagious disease in pigs and is prevalent worldwide, threatening the pig farming industry. The detailed mechanisms of the CSFV life cycle have been reported, but are still limited. Some receptors and attachment factors of CSFV, including heparan sulfate (HS), laminin receptor (LamR), complement regulatory protein (CD46), MER tyrosine kinase (MERTK), disintegrin, and metalloproteinase domain-containing protein 17 (ADAM17), were identified. After attachment, CSFV internalizes via clathrin-mediated endocytosis (CME) and/or caveolae/raft-dependent endocytosis (CavME). After internalization, CSFV moves to early and late endosomes before uncoating. During this period, intracellular trafficking of CSFV relies on components of the endosomal sorting complex required for transport (ESCRT) and Rab proteins in the endosome dynamics, with a dependence on the cytoskeleton network. This review summarizes the data on the mechanisms of CSFV attachment, internalization pathways, and intracellular trafficking, and provides a general view of the early events in the CSFV life cycle.

Optical coherence elastography and its applications for the biomechanical characterization of tissues.

The biomechanical characterization of the tissues provides significant evidence for determining the pathological status and assessing the disease treatment. Incorporating elastography with optical coherence tomography (OCT), optical coherence elastography (OCE) can map the spatial elasticity distribution of biological tissue with high resolution. After the excitation with the external or inherent force, the tissue response of the deformation or vibration is detected by OCT imaging. The elastogram is assessed by stress-strain analysis, vibration amplitude measurements, and quantification of elastic wave velocities. OCE has been used for elasticity measurements in ophthalmology, endoscopy, and oncology, improving the precision of diagnosis and treatment of disease. In this article, we review the OCE methods for biomechanical characterization and summarize current OCE applications in biomedicine. The limitations and future development of OCE are also discussed during its translation to the clinic.

Catalytic site flexibility facilitates the substrate and catalytic promiscuity of Vibrio dual lipase/transferase.

Although enzyme catalysis is typified by high specificity, enzymes can catalyze various substrates (substrate promiscuity) and/or different reaction types (catalytic promiscuity) using a single active site. This interesting phenomenon is widely distributed in enzyme catalysis, with both fundamental and applied importance. To date, the mechanistic understanding of enzyme promiscuity is very limited. Herein, we report the structural mechanism underlying the substrate and catalytic promiscuity of Vibrio dual lipase/transferase (VDLT). Crystal structures of the VDLT from Vibrio alginolyticus (ValDLT) and its fatty acid complexes were solved, revealing prominent structural flexibility. In particular, the "Ser-His-Asp" catalytic triad machinery of ValDLT contains an intrinsically flexible oxyanion hole. Analysis of ligand-bound structures and mutagenesis showed that the flexible oxyanion hole and other binding residues can undergo distinct conformational changes to facilitate substrate and catalytic promiscuity. Our study reveals a previously unknown flexible form of the famous catalytic triad machinery and proposes a "catalytic site tuning" mechanism to expand the mechanistic paradigm of enzyme promiscuity.

NAMPT-Improved Mitochondrial Function Alleviates Degenerative Rotator Cuff Tendinopathy in Aged Mice.

BACKGROUND: Age-related rotator cuff tendinopathy (RCT) is associated with increased rotator cuff tear and postoperative retear rates. This study aimed to determine whether nicotinamide phosphoribosyltransferase (NAMPT) can alleviate degenerative RCT and prevent postoperative retears by reversing mitochondrial dysfunction in aged mice. METHODS: We assigned 32 young (4 months) and 64 aged (19 to 20 months) male wild-type C57BL/6 mice to young, aged, and aged NAMPT-treated (ANAMPT) groups (n = 32 each). Mice in the ANAMPT group underwent subacromial injection with NAMPT-loaded fibrin gel, whereas the other 2 groups were injected with fibrin gel alone. Histological staining and each of the biomechanical and mitochondrial function tests were performed using 8 samples each. RESULTS: Histological staining in the aged group revealed decreased cellularity, disrupted fiber architecture, and reduced type-I collagen content inside tendon tissues proximal to the enthesis, demonstrating the spontaneous development of age-related degenerative RCT. Compared with the young group, the maximum tendon-to-bone failure load (4.22 ± 0.81 versus 5.52 ± 0.81 N, p = 0.0106) and maximum suture cut-through force (0.83 ± 0.08 versus 1.07 ± 0.10 N, p = 0.0006) of degenerated tendon tissues in the aged group were significantly lower. Significantly reduced nicotinamide adenine dinucleotide (NAD + ) levels, adenosine triphosphate (ATP) production, and citrate synthase activity indicated that mitochondrial dysfunction was closely related to the development of the degenerative RCT. Furthermore, NAMPT-improved mitochondrial function alleviated age-induced degenerative histological changes and increased the maximum failure load (5.32 ± 0.68 N, p = 0.0375) and maximum suture cut-through force (0.99 ± 0.13 N, p = 0.0285). CONCLUSIONS: Spontaneously developed degenerative RCT in aged mice mimicked the clinical situation in elderly patients. NAMPT-improved mitochondrial function could alleviate age-induced degenerative RCT and prevent postoperative suture cut-through of tendons with degenerative RCT. CLINICAL RELEVANCE: This study confirmed the spontaneous development of degenerative RCT in aged mice, which will facilitate future studies of this condition. The results also suggest that NAMPT offers a novel therapeutic approach for treating age-related degenerative RCT.