nQuant Enables Precise Quantitative N-Glycomics.

N-glycosylation is a highly heterogeneous post-translational modification that modulates protein function. Defects in N-glycosylation are directly linked to various human diseases. Despite the importance of quantifying N-glycans with high precision, existing glycoinformatics tools are limited. Here, we developed nQuant, a glycoinformatics tool that enables label-free and isotopic labeling quantification of N-glycomics data obtained via LC-MS/MS, ensuring a low false quantitation rate. Using the label-free quantification module, we profiled the N-glycans released from purified glycoproteins and HEK293 cells as well as the dynamic changes of N-glycosylation during mouse corpus callosum development. Through the isotopic labeling quantification module, we revealed the dynamic changes of N-glycans in acute promyelocytic leukemia cells after all-trans retinoic acid treatment. Taken together, we demonstrate that nQuant enables fast and precise quantitative N-glycomics.

Functional biomaterials for modulating the dysfunctional pathological microenvironment of spinal cord injury.

Spinal cord injury (SCI) often results in irreversible loss of sensory and motor functions, and most SCIs are incurable with current medical practice. One of the hardest challenges in treating SCI is the development of a dysfunctional pathological microenvironment, which mainly comprises excessive inflammation, deposition of inhibitory molecules, neurotrophic factor deprivation, glial scar formation, and imbalance of vascular function. To overcome this challenge, implantation of functional biomaterials at the injury site has been regarded as a potential treatment for modulating the dysfunctional microenvironment to support axon regeneration, remyelination at injury site, and functional recovery after SCI. This review summarizes characteristics of dysfunctional pathological microenvironment and recent advances in biomaterials as well as the technologies used to modulate inflammatory microenvironment, regulate inhibitory microenvironment, and reshape revascularization microenvironment. Moreover, technological limitations, challenges, and future prospects of functional biomaterials to promote efficient repair of SCI are also discussed. This review will aid further understanding and development of functional biomaterials to regulate pathological SCI microenvironment.

Potential mechanism of tea for treating osteoporosis, osteoarthritis, and rheumatoid arthritis.

Osteoporosis (OP), osteoarthritis (OA), and rheumatoid arthritis (RA) are common bone and joint diseases with a high incidence and long duration. Thus, these conditions can affect the lives of middle-aged and elderly people. Tea drinking is a traditional lifestyle in China, and the long-term intake of tea and its active ingredients is beneficial to human health. However, the mechanisms of action of tea and its active ingredients against OP, OA, and RA are not completely elucidated. This study aimed to assess the therapeutic role and related mechanisms of tea and its active ingredients in OP, OA, and RA. Moreover, it expanded the potential mechanisms of tea efficacy based on network pharmacology and molecular docking. Results showed that tea has potential anti-COX properties and hormone-like effects. Compared with a single component, different tea components synergize or antagonize each other, thereby resulting in a more evident dual effect. In conclusion, tea has great potential in the medical and healthcare fields. Nevertheless, further research on the composition, proportion, and synergistic mechanism of several tea components should be performed.

Confirmation of pain-related neuromodulation mechanism of Bushen Zhuangjin Decoction on knee osteoarthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Bushen Zhuangjin Decoction (BZD) are an herbal compound commonly used to treat osteoarthritis (OA) in China. AIM OF THE STUDY: This study aimed to verify the mechanism of Bushen Zhuangjin Decoction in relieving the pain of knee osteoarthritis. MATERIALS AND METHODS: Network pharmacology evaluation was used to discover the potential targets of BZD to relieve pain in KOA. The therapeutic effects of BZD treatment on KOA pain using histomorphology, behavioral assessments, suspension chip analysis, and ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) assays. The functional magnetic resonance imaging was used to explore the effects of BZD treatment on brain function associated to KOA. RESULTS: Network pharmacological analysis revealed the association between the analgesic effect of BZD on KOA and the pain signaling neurotransmitter 5-HT. Subsequently, we conducted experiments to verify the therapeutic effect of BZD on pain in KOA animal models. Behavioral tests demonstrated that the pain threshold of knee osteoarthritis rats decreased in PWT and PWL, but BZD was able to increase the pain threshold. Histopathological staining indicated thinning of the cartilage layer and sparse trabeculae in the subchondral bone. Suspension chip analysis revealed a significant increase in pro-inflammatory factors of IL-1α, IL-5, IL-12, IL-17A, RANTES, TNF-α and M-CSF in KOA, along with a significant decrease in anti-inflammatory factor of IL-13. However, BZD treatment decreased the expression of pro-inflammatory factors and increased the content of anti-inflammatory factor. UHPLC-MS/MS analysis showed a significant decrease in the serum levels of GABA, E, GSH, Kyn, Met, and VMA in KOA, which were significantly increased by BZD. Conversely, the serum levels of TrpA, TyrA, Spd, and BALa were significantly increased in KOA and significantly decreased by BZD. ELISA and Western blot analysis showed increased expression of subchondral bone pain-related neuropeptides SP, CGRP, TH, NPY, VEGFA, 5-HT3 in KOA, which were decreased in BZD. Functional magnetic resonance imaging demonstrated that BZD exerts its therapeutic effect on KOA by modulating the activity and functional connections of the cortex, hypothalamus, and hippocampus. CONCLUSIONS: This study confirmed the significant role of pain-related neuromodulation mechanisms in the analgesic therapy of BZD and provides a theoretical foundation for using BZD as a traditional Chinese medical treatment for KOA pain.

Rehmannia alcohol extract inhibits neuropeptide secretion and alleviates osteoarthritis pain through cartilage protection.

Osteoarthritis (OA) is a common joint disease characterized by chronic pain, and the perception of pain is closely associated with brain function and neuropeptide regulation. Rehmannia is common plant herb with anti-inflammatory and analgesic properties that is used to treat OA. However, it is unclear whether Rehmannia alleviates OA-related pain via regulation of neuropeptides and brain function. We examined the pain relief regulatory pathway in OA after treatment with Rehmannia by verifying the therapeutic effect of Rehmannia alcohol extract in vivo and vitro and exploring of the potential mechanism underlying the analgesic effect of Rahmanian using functional magnetic resonance imaging and measuring neuropeptide secretion. Our results showed that Rehmannia alcohol extract and the related active ingredient, Rehmannioside D, can delay cartilage degradation and alleviate inflammation in OA rats. The Rehmannia alcohol extract can also relieve OA pain, reduce the secretion of calcitonin gene-related peptide (CGRP) and substance P (SP), and reverse the pathological changes in the cerebral cortex and hippocampus. Our research results demonstrate that Rehmannia alleviates OA pain by protecting cartilage, preventing the stimulation of inflammatory factors on neuropeptide secretion, and influencing the relevant functional areas of the brain.

Investigations of brain-wide functional and structural networks of dopaminergic and CamKIIα-positive neurons in VTA with DREADD-fMRI and neurotropic virus tracing technologies.

BACKGROUND: The ventral tegmental area (VTA) contains heterogeneous cell populations. The dopaminergic neurons in VTA play a central role in reward and cognition, while CamKIIα-positive neurons, composed mainly of glutamatergic and some dopaminergic neurons, participate in the reward learning and locomotor activity behaviors. The differences in brain-wide functional and structural networks between these two neuronal subtypes were comparatively elucidated. METHODS: In this study, we applied a method combining Designer Receptors Exclusively Activated by Designer Drugs (DREADD) and fMRI to assess the cell type-specific modulation of whole-brain neural networks. rAAV encoding the cre-dependent hM3D was injected into the right VTA of DAT-cre or CamKIIα-cre transgenic rats. The global brain activities elicited by DREADD stimulation were then detected using BOLD-fMRI. Furthermore, the cre-dependent antegrade transsynaptic viral tracer H129ΔTK-TT was applied to label the outputs of VTA neurons. RESULTS: We found that DREADD stimulation of dopaminergic neurons induced significant BOLD signal changes in the VTA and several VTA-related regions including mPFC, Cg and Septum. More regions responded to selective activation of VTA CamKIIα-positive neurons, resulting in increased BOLD signals in VTA, Insula, mPFC, MC_R (Right), Cg, Septum, Hipp, TH_R, PtA_R, and ViC_R. Along with DREADD-BOLD analysis, further neuronal tracing identified multiple cortical (MC, mPFC) and subcortical (Hipp, TH) brain regions that are structurally and functionally connected by VTA dopaminergic and CamKIIα-positive neurons. CONCLUSIONS: Our study dissects brain-wide structural and functional networks of two neuronal subtypes in VTA and advances our understanding of VTA functions.

An auxiliary diagnostic technology and clinical efficacy evaluation in knee osteoarthritis based on serum surface-enhanced Raman spectroscopy.

Knee osteoarthritis (KOA), a progressive joint disease, is a leading source of chronic pain and disability, and its diagnosis mainly depends on medical imaging findings and clinical symptoms. This study aimed to explore an auxiliary diagnostic technology and clinical efficacy evaluation in KOA based on surface-enhanced Raman scattering (SERS). Three sequential experiments were performed: 1) preliminary observation of the therapeutic effects of icariin (ICA); 2) using serum SERS spectra obtained from rat models belonging to sham group, KOA group and icariin treatment group, respectively, to analyze the KOA-related expression profiles; 3) employing partial least squares (PLS) and support vector machines (SVM) algorithms to establish KOA diagnosis model. Pathological changes verified the efficacy of icariin in KOA. Raman peak assignment combined with spectral difference analysis reflected the biochemical changes associated with KOA, including amino acid, carbohydrates and collagen. ICA intervention significantly reversed these changes, although full recovery could not be achieved. Based on PLS-SVM approach, the sensitivity, specificity and accuracy of 100%, 98.33% and 98.89%, respectively, were obtained for screening KOA. This work proves that SERS has great potential to be used as an auxiliary diagnostic technology for KOA, and is also helpful for the exploration of novel KOA treatment agent.

The ERAD Pathway Participates in Fungal Growth and Cellulase Secretion in Trichoderma reesei.

Trichoderma reesei is a powerful fungal cell factory for the production of cellulolytic enzymes due to its outstanding protein secretion capacity. Endoplasmic reticulum-associated degradation (ERAD) plays an integral role in protein secretion that responds to secretion pressure and removes misfolded proteins. However, the role of ERAD in fungal growth and endogenous protein secretion, particularly cellulase secretion, remains poorly understood in T. reesei. Here, we investigated the ability of T. reesei to grow under different stresses and to secrete cellulases by disrupting three major genes (hrd1, hrd3 and der1) involved in the critical parts of the ERAD pathway. Under the ER stress induced by high concentrations of DTT, knockout of hrd1, hrd3 and der1 resulted in severely impaired growth, and the mutants Δhrd1 and Δhrd3 exhibited high sensitivity to the cell wall-disturbing agents, CFW and CR. In addition, the absence of either hrd3 or der1 led to the decreased heat tolerance of this fungus. These mutants showed significant differences in the secretion of cellulases compared to the parental strain QM9414. During fermentation, the secretion of endoglucanase in the mutants was essentially consistent with that of the parental strain, while cellobiohydrolase and ß-glucosidase were declined. It was further discovered that the transcription levels of the endoglucanase-encoding genes (eg1 and eg2) and the cellobiohydrolase-encoding gene (cbh1) were not remarkedly changed. However, the ß-glucosidase-encoding gene (bgl1) was significantly downregulated in the ERAD-deficient mutants, which was presumably due to the activation of a proposed feedback mechanism, repression under secretion stress (RESS). Taken together, our results indicate that a defective ERAD pathway negatively affects fungal growth and cellulase secretion, which provides a novel insight into the cellulase secretion mechanism in T. reesei.

Clinical and arthroscopic outcomes of single-bundle anterior cruciate ligament reconstruction using autologous hamstrings augmented with ligament augmentation and reconstruction systems compared with four-strand hamstring tendon grafts alone.

PURPOSE: To compare the clinical, radiological, and second-look arthroscopic outcomes in patients who underwent anterior cruciate ligament (ACL) reconstruction using a four-strand hamstring tendon graft (hamstring group) either without augmentation or with ligament augmentation and reconstruction system (LARS) augmentation (LARS augmentation group). METHODS: From January 2018 to December 2019, patients who underwent ACL reconstruction were included. Patient-reported outcome measures (PROMs) were undertaken pre-operatively and at three, six, 12, and 24 months post-operatively. Arthroscopic evaluation was performed focusing on the morphology of the graft based on graft tension, graft tear, and synovial coverage. RESULTS: A total of 178 consecutive patients received single-bundle ACL reconstruction, 89 patients in each group, and 20 patients were lost to follow-up in the first two years. At the three month follow-up, the LARS augmentation group had significantly higher Lysholm scores, IKDC scores, and KOS-ADLS scores than the hamstring group (P < 0.001). At the three, six and 12-month follow-ups, there were significantly higher Tegner scores and ACL-RSI scores in the LARS augmentation group than in the hamstring group (P < 0.05). At the three and six month follow-ups, the LARS augmentation group had significantly higher rates of return to sports and return to sports at their preinjury level (P < 0.05). There were no between-group differences in other outcomes, including arthroscopic outcomes, graft signal intensity, post-operative complications or rerupture rates. CONCLUSIONS: Autologous hamstring augmented with the LARS augmentation technique provides good and realistic clinical and functional results during the early post-operative period with high levels of satisfaction of patients, including participation in sports and physical activity, and high rates of return to sports at the preinjury level, without any apparent complications compared with hamstring ACL reconstruction alone. No increases in complication, reinjury rates, or increased lateral laxity were observed at the 12-month or 24-month follow-up.

Minimalistic fully convolution networks (MFCN): pixel-level classification for hyperspectral image with few labeled samples.

Most of the existing deep learning methods for hyperspectral image (HSI) classification use pixel-wise or patch-wise classification. In this paper, we propose an image-wise classification method, where the network input is the original hyperspectral cube rather than the spectral curve of each pixel (i.e., pixel-wise) or neighbor region of each pixel (i.e., patch-wise). Specifically, we propose a minimalistic fully convolution network (MFCN) and a semi-supervised loss function, which can perform pixel-level classification for HSI with few labeled samples. The comparison experiments demonstrated the progress of our methods, using three new benchmark HSI datasets (WHU-Hi-LongKou, WHU-Hi-HanChuan and WHU-Hi-HongHu) with wavelength range from 400 to 1000nm. In the comparison experiments, we randomly selected 25 labeled pixels from each class for training, equivalent to only 0.11%, 0.16%, and 0.14% of all labeled pixels for the three datasets, respectively. In addition, through ablation studies and theoretical analysis, we verified and analyzed the effectiveness and superiority of our design choices.