Construction of Xylose-Utilizing Cyanobacterial Chassis for Bioproduction Under Photomixotrophic Conditions.

Xylose is a major component of lignocellulose and the second most abundant sugar present in nature after glucose; it, therefore, has been considered to be a promising renewable resource for the production of biofuels and chemicals. However, no natural cyanobacterial strain is known capable of utilizing xylose. Here, we take the fast-growing cyanobacteria Synechococcus elongatus UTEX 2973 as an example to develop the synthetic biology-based methodology of constructing a new xylose-utilizing cyanobacterial chassis with increased acetyl-CoA for bioproduction.

Experimental and theoretical insight into carbamazepine degradation by chlorine-based advanced oxidation processes: Efficiency, energy consumption, mechanism and DBPs formation.

Chlorine has been widely used in different advanced oxidation processes (AOPs) for micropollutants removal. In this study, different chlorine-based AOPs, namely medium pressure (MP) UV/chlorine, low pressure (LP) UV/chlorine, and in-situ chlorination, were compared for carbamazepine (CBZ) removal efficiency, energy consumption, and disinfection by-products (DBPs) formation. All three processes could achieve nearly 100% CBZ removal, while the reaction time needed by in-situ chlorination was double the time required by UV/chlorine processes. The energy consumed per magnitude of CBZ removed (EE/O) of MP UV/chlorine was 13 times higher than that of LP UV/chlorine, and relative to that of in-situ chlorination process. Accordingly, MP and LP UV/chlorine processes generated one to two orders of magnitude more hydroxyl radicals (•OH) and reactive chlorine species (RCS) than in-situ chlorination. Besides, RCS were the dominant reactive species, contributing to 78.3%, 75.6%, and 71.6% of CBZ removal in MP, LP UV/chlorine, and in-situ chlorination, respectively. According to the Gibbs free energy barriers between CBZ and RCS/•OH calculated based on density functional theory (DFT), RCS had more reaction routes with CBZ and showed lower energy barrier in the main CBZ degradation pathways like epoxidation and formation of iminostilbene. When applied to secondary wastewater effluent, UV/chlorine and in-situ chlorination produced overall DBPs ranging from 104.77 to 135.41 µg/L. However, the production of chlorate during UV/chlorine processes was 15 times higher than that during in-situ chlorination.

Enhanced infection efficiency and cytotoxicity mediated by vpx-containing lentivirus in chimeric antigen receptor macrophage (CAR-M).

Genetically modified macrophage infusion has been proven to be a novel treatment for cancer. One of the most important processes in macrophage-based therapy is the efficient transfer of genes. HIV-1-derived lentiviruses were widely used as delivery vectors in chimeric antigen receptor T and NK cell construction. While macrophages are relatively refractory to this lentiviral vector transduction as a result of the myeloid-specific restriction factor SAMHD1, which inhibited the virion cycle through exhausting the dNTPs pool and degradating RNAs. An efficient macrophage transduction strategy has been developed via packaging the HIV-2 accessory protein Vpx into the virion. Vpx counteracts SAMHD1 through CRL4 (DCAF1) E3 ubiquitin ligase mediated SAMHD1 degradation, yet the influence by the introduction of Vpx on macrophage has not been fully evaluated. Here, we constructed the chimeric lentiviral vector HIV-1-Vpx and systematically analyzed the infection efficiency of this vector in time-dependent manner. Our results showed that the simplified chimeric virus exhibited dramatically enhanced infection in human macrophages compared to normal lentivirus. Moreover, transcriptome sequencing was performed to evaluate the cellular status after chimeric virus infection. The sequencing results indicated that Vpx introduction promoted macrophage remodeling towards a proinflammatory phenotype, without affecting classic M1/M2 cell surface markers. Our results suggest that the Vpx-containing lentivirus could be used as an ideal tool for the generation of genetically engineered macrophages with high gene transfer efficiency and poised proinflammatory gene sets, especially for solid tumor treatment.

Andrographolide exerts a neuroprotective effect by regulating the LRP1-mediated PPARγ/NF-κB pathway.

Low-density lipoprotein receptor-associated protein 1 (LRP1) is widely expressed in neurons, microglia and astrocytes. Studies have revealed that the suppression of LRP1 expression in the brain significantly exacerbates Alzheimer's disease (AD)-related neuropathology. Andrographolide (Andro) has been demonstrated to possess neuroprotective properties, although its underlying mechanisms remain largely unknown. This study aims to investigate whether Andro can inhibit neuroinflammation in AD by modulating the LRP1-mediated PPARγ/NF-κB pathway. In Aß-induced BV-2 cells, Andro was found to increase cell viability and enhance the expression of LRP1, while decreasing the expression of p-NF-κB (p65) and NF-κB(p65), as well as IL-1ß, IL-6 and TNF-α levels. In addition, when Aß was cotreatment with Andro to BV2 cells with either LRP1 or PPARγ knockdown, increased mRNA and protein expression of p-NF-κB(p65) and NF-κB(p65), NF-κB DNA binding activity as well as IL-1ß, IL-6 and TNF-α levels were observed. These findings suggested that Andro could attenuate Aß induced cytotoxicity by reducing neuroinflammation which may be partly attributed to its effects on this LRP1 mediated PPARγ/NF-κB pathway.

Colletotrichum Species Associated with Anthracnose in Salix babylonica in China.

Salix babylonica L. is a popular ornamental tree species in China and widely cultivated in Asia, Europe, and North America. Anthracnose in S. babylonica poses a serious threat to its growth and reduces its medicinal properties. In 2021, a total of 55 Colletotrichum isolates were isolated from symptomatic leaves in three provinces in China. Phylogenetic analyses using six loci (ITS, ACT, CHS-1, TUB2, CAL, and GAPDH) and a morphological characterization of the 55 isolates showed that they belonged to four species of Colletotrichum, including C. aenigma, C. fructicola, C. gloeosporioides s.s., and C. siamense. Among them, C. siamense was the dominant species, and C. gloeosporioides s.s. was occasionally discovered from the host tissues. Pathogenicity tests revealed that all the isolates of the aforementioned species were pathogenic to the host, and there were significant differences in pathogenicity or virulence among these isolates. The information on the diversity of Colletotrichum spp. that causes S. babylonica anthracnose in China is new.

Research progress of PPARγ regulation of cholesterol and inflammation in Alzheimer's disease.

Peroxidase proliferator receptors (PPARs) are defined as key sensors and regulators of cell metabolism, transcription factors activated by ligands, involved in lipid, glucose and amino acid metabolism, participating in the processes of cell differentiation, apoptosis, inflammation regulation, and acute and chronic nerve damage. Among them, PPARγ is expressed in different brain regions and can regulate lipid metabolism, mitochondrial disorders, oxidative stress, and cell apoptosis. It has anti-inflammatory activity and shows neuroprotection. The regulation of Aß levels in Alzheimer's disease involves cholesterol metabolism and inflammation, so this article first analyzes the biological functions of PPARγ, then mainly focuses on the relationship between cholesterol and inflammation and Aß, and elaborates on the regulation of PPARγ on key proteins and the corresponding molecules, which provides new ideas for the treatment of AD.

Extracting Useful Emergency Information from Social Media: A Method Integrating Machine Learning and Rule-Based Classification.

User-generated contents (UGCs) on social media are a valuable source of emergency information (EI) that can facilitate emergency responses. However, the tremendous amount and heterogeneous quality of social media UGCs make it difficult to extract truly useful EI, especially using pure machine learning methods. Hence, this study proposes a machine learning and rule-based integration method (MRIM) and evaluates its EI classification performance and determinants. Through comparative experiments on microblog data about the "July 20 heavy rainstorm in Zhengzhou" posted on China's largest social media platform, we find that the MRIM performs better than pure machine learning methods and pure rule-based methods, and that its performance is influenced by microblog characteristics such as the number of words, exact address and contact information, and users' attention. This study demonstrates the feasibility of integrating machine learning and rule-based methods to mine the text of social media UGCs and provides actionable suggestions for emergency information management practitioners.

Medicinal fungus Phellinus igniarius alleviates gout in vitro by modulating TLR4/NF-kB/NLRP3 signaling.

Background: Phellinus igniarius (P. igniarius) is a valuable medicinal and edible fungus with various biological activities such as anti-inflammation, antioxidation, and immune regulation. In this study, we explored the effects of P. igniarius on a gout model in vitro. Methods: The DPPH, ABTS, and FRAP methods were combined to determine and compare the antioxidant activities of wild P. igniarius total polyphenols (WPP) and cultivated P. igniarius total polyphenols (CPP) in vitro. Spectrophotometry was used to compare the inhibitory effect of WPP and CPP on xanthine oxidase (XO) activity to evaluate anti-hyperuricemia activity in vitro. HUVECs were stimulated with monosodium urate (MSU) crystals for 24 h to establish an acute gouty inflammation model in vitro. The protective effects were compared by measuring cell viability; the contents of ICAM-1, IL-1ß, IL-6 and VCAM-1; the protein expressions of TLR4 and NLRP3; reactive oxygen species production; and the nuclear translocation of NF-κB p65. UHPLC-QE-MS technology was used to explore the potential metabolic mechanism of P. igniarius against gout. Results: WPP and CPP had strong antioxidant capacity, and the antioxidant capacity of CPP was similar to that of WPP. In a comparative experiment of xanthine oxidase activity inhibition by WPP and CPP, the IC50 values were 88.19 µg/ml and 108.0 µg/ml, respectively. At a dose of 40 µg/ml, WPP and CPP significantly improved the decrease in cell viability induced by monosodium urate (150 µg/ml) and inhibited the increase in inflammatory factors such as ICAM-1, IL-1ß, IL-6, and VCAM-1. The increase in TLR4 and NLRP3 protein expression induced by MSU crystals in HUVECs was also significantly inhibited by total polyphenols from wild and cultivated P. igniarius. In addition, both significantly improved MSU-induced ROS overproduction and NF-κB p65 nuclear translocation. WPP and CPP may primarily be involved in phenylalanine metabolism and lysophosphatidylcholine metabolism in their role in the treatment of gout. Conclusion: CPP and WPP both showed good antioxidant activity and xanthine oxidase inhibitory activity and had good therapeutic effects on the gout model in vitro. Furthermore, this study indicated that cultivated P. igniarius had a protective effect similar to that of wild P. igniarius, which would be expected to improve the shortage of wild P. igniarius and promote the development of the cultivated P. igniarius industry and product development.

Comparison of biological characteristics of human adipose- and umbilical cord- derived mesenchymal stem cells and their effects on delaying the progression of osteoarthritis in a rat model.

BACKGROUND: The prevalence of osteoarthritis (OA) is constantly increasing with age. Adipose-derived (AD-) and umbilical cord-derived (UC-) mesenchymal stem cells (MSCs) are attractive alternatives in OA therapy and regenerative medicine. However, whether there are differences in the efficacy of MSCs derived from different tissues in the cartilage regeneration, and the frequency of administration of MSCs needs to be further studied. EXPERIMENT: UC-MSCs and AD-MSC were isolated from the umbilical cord and subcutaneous fatty tissue of humans respectively and identified by flow cytometry. In vitro, the proliferation ability and chondrogenic potential of AD-MSCs and UC-MSCs were analyzed. In vivo, forty-three Sprague-Dawley rats were used for the OA model induced by ACLT surgery. OA rats were divided into a sham group, an ACLT model group, and two therapy groups (treated with AD-MSCs or UC-MSCs). Therapy groups were treated using a single or repeated twice injection of AD-MSCs and UC-MSCs at a concentration of 1.0 × 106 cells and were followed up for 12 weeks. Serial sections of knees were examined for histological, immunohistochemical and TUNEL analysis. RESULTS: We demonstrated that the proliferation of UC-MSCs was higher than that of AD-MSCs, consistent with the bigger pellets from UC-MSCs in a chondrogenic induction medium. Degeneration of articular cartilage was observed in histological appearance of Safranine O and Toluidine blue staining, and quantitative results of modified Mankin's Score. Importantly, both AD-MSCs and UC-MSCs transplantation significantly attenuated ACLT surgical-induced OA development. In addition, ACLT-induced reduction in cartilage extracellular matrix synthesis (aggrecan) was significantly suppressed by AD-MSCs or UC-MSCs transplantation. TUNEL assay showed that AD-MSCs and UC-MSCs treatments significantly protected chondrocytes against apoptosis compared with the ACLT group. No significant differences were observed between single injections and repeated twice injections. CONCLUSIONS: The current study suggested that, in vitro, AD-MSCs and UC-MSCs showed a comparable chondrogenic potential, although UC-MSCs displayed a superior proliferation capacity. Furthermore, our results confirmed that the injection of AD-MSCs and UC-MSCs, either single or repeated twice, could significantly inhibit the progression of ACLT-induced osteoarthritis with a similar effect, and MSCs transplantation can decrease the apoptosis of articular chondrocytes caused by ACLT.

Engineering a Xylose-Utilizing Synechococcus elongatus UTEX 2973 Chassis for 3-Hydroxypropionic Acid Biosynthesis under Photomixotrophic Conditions.

Photomixotrophic cultivation of cyanobacteria is considered a promising strategy to achieve both high cell density and product accumulation, since cyanobacteria can obtain carbon and energy sources from organic matter in addition to those obtained from CO2 and sunlight. Acetyl coenzyme A (acetyl-CoA) is a key precursor used for the biosynthesis of a wide variety of important value-added chemicals. However, the acetyl-CoA content in cyanobacteria is typically low under photomixotrophic conditions, which limits the productivity of the derived chemicals. In this study, a xylose utilization pathway from Escherichia coli was first engineered into fast-growing Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 2973), enabling the xylose based photomixotrophy. Metabolomics analysis of the engineered strain showed that the utilization of xylose enhanced the carbon flow to the oxidative pentose phosphate (OPP) pathway, along with an increase in the intracellular abundance of metabolites such as fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (FBP), ribose-5-phosphate (R5P), erythrose-4-phosphate (E4P), and glyceraldehyde-3-phosphate (G3P). Then, the native glycolytic pathway was rewired via heterologous phosphoketolase (Pkt) gene expression, combined with phosphofructokinase (Pfk) gene knockout and fructose-1,6-bisphosphatase (Fbp) gene overexpression, to drive more carbon flux from xylose to acetyl-CoA. Finally, a heterologous 3-hydroxypropionic acid (3-HP) biosynthetic pathway was introduced. The results showed that 3-HP biosynthesis was improved by up to approximately 4.1-fold (from 22.5 mg/L to 91.3 mg/L) compared with the engineered strain without a rewired metabolism under photomixotrophic conditions and up to approximately 14-fold compared with the strain under photoautotrophic conditions. Using 3-HP as a "proof-of-molecule", our results demonstrated that this strategy could be applied to improve the intracellular pool of acetyl-CoA for the photomixotrophic production of value-added chemicals that require acetyl-CoA as a precursor in a cyanobacterial chassis.

First report of leaf spot caused by Colletotrichum siamense on Salix matsudana in China.

Salix matsudana Koidz. (Chinese willow) is an important landscaping tree species widely grown in China (Zhang et al. 2017). In October 2019, a characteristic leaf spot disease of S. matsudana was found on the campus of Nanjing Forestry University. Most 25-year-old S. matsudana trees (13 out of 21, approximately 62%) on campus showed the leaf spot disease. On average, 70% of the leaves per individual tree were affected by this disease. Foliar symptoms began as dark brown, irregular spots and the centers were gray-white, gradually enlarging with time. Leaf spot symptomatic leaves were collected from three infected S. matsudana trees (10 leaves/tree), and small infected tissues (3-4 mm2) were surface-sterilized in 75% ethanol for 30 s, 1% NaClO for 90 s, rinsed in ddH2O, dried on sterilized filter paper, and plated on potato dextrose agar (PDA), and then incubated at 25°C. Three isolates (NHY1-1, NHY1-2, and NHY1-3) of the same fungus were obtained in 85% of the samples and deposited in China's Forestry Culture Collection Center (NHY1-1: cfcc55354, NHY1-2: cfcc55355, NHY1-3: cfcc55359). The colonies of three isolates were white, but the reverse side was grayish-white. The conidia of NHY1-1 were one-celled, straight, subcylindrical, hyaline, 14.4 ± 0.9 × 5.4 ± 0.4 µm (n = 50), with a rounded end. Conidiophores were hyaline to pale brown, septate, and branched. Appressoria were one-celled, ellipsoidal, brown or dark brown, thick-walled, 8.0 ± 0.9 × 5.9 ± 0.5 µm (n = 50). The conidia and appressoria of the other two isolates weralmost identical to NHY1-1. The morphological characters of the three isolates were matched with those of the Colletotrichum gloeosporioides complex (Weir et al. 2012). For accurate identification, the DNA of the three isolates was extracted. The internal transcribed spacer region (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), superoxide dismutase (SOD2), and ß-tubulin 2 (TUB2) genes were amplified using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-345R, GDF1/GDR1, SODglo2-F/SODglo2-R, and Bt2a/Bt2b, respectively (Weir et al. 2012). The sequences were deposited in GenBank [Accession Nos. MW784679 and MW808959 to MW808964 for NHY1-1; MW784726 and MW808965 to MW808970 for NHY1-2; MW784729 and MW808971 to MW808976 for NHY1-3]. A BLAST search of GenBank showed that ITS, ACT, CAL, GAPDH, SOD2, and TUB2 sequences of the three isolates were identical to Colletotrichum siamense at a high level (>99%), and CHS-1 sequences of three isolates were consistent with Colletotrichum fructicola at a high level (>99%). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, ACT, CAL, CHS-1, GAPDH, SOD2, and TUB2) placed NHY1-1, NHY1-2, and NHY1-3 in the clade of C. siamense with high bootstrap support values (ML/BI = 93/1). The pathogenicity of three isolates were tested on potted 2-yr-old seedlings (50-cm tall) of S. matsudana, which were grown in a greenhouse. Healthy leaves were wounded with a sterile needle and then inoculated with 10 µL of conidial suspension (106 conidia/mL). Controls were treated with ddH2O (Zhu et al. 2019). In total, 12 seedlings were inoculated including controls. Three seedlings/isolate and 10 leaves/seedling were used for each treatment. The plants were covered with plastic bags after inoculation and sterilized H2O was sprayed into the bags twice/day to maintain humidity and kept in a greenhouse at the day/night temperatures at 25 ± 2 / 16 ± 2°C. Within 7 days, all the inoculated points showed lesions similar to those observed in field, whereas controls were asymptomatic. The infection rate of each of the three isolates is 100%. C. siamense was re-isolated from the lesions, whereas no fungus was isolated from control leaves. The diseases caused by C. siamense often occur in tropical and subtropical regions of China, with a wide range of hosts, such as Hevea brasiliensis and Coffea arabica, etc. (Cao et al. 2019; Liu et al. 2018). This is the first report of C. siamense causing leaf spot of S. matsudana in China and the world. These data will help to develop effective strategies for managing this newly emerging disease.

Conserved Role of the Large Conductance Calcium-Activated Potassium Channel, KCa1.1, in Sinus Node Function and Arrhythmia Risk.

BACKGROUND: KCNMA1 encodes the α-subunit of the large-conductance Ca2+-activated K+ channel, KCa1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of KCa1.1 are limited, and KCNMA1 has not been investigated as an AF candidate gene. METHODS: The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of KCa1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized. RESULTS: A complex KCNMA1 variant was identified in 1 kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of KCa1.1 in normal hearts using immunostaining and immunogold electron microscopy. KCa1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the KCa1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the KCa1.1 ortholog, kcnma1b, in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila KCa1.1 ortholog, slo, systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of slo-deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the KCa1.1 loss-of-function models. CONCLUSIONS: Our data point to a highly conserved role of KCa1.1 in sinus node function in humans, mice, zebrafish, and fly and suggest that KCa1.1 loss of function may predispose to AF.

Intraperitoneal injection of Desferal® alleviated the age-related bone loss and senescence of bone marrow stromal cells in rats.

BACKGROUND: Age-related bone loss plays a vital role in the development of osteoporosis and osteoporotic fracture. Bone marrow stromal cell (BMSC) senescence is highly associated with osteoporosis and limits the application of BMSCs in regenerative medicine. Hypoxia is an essential component for maintaining the normal physiology of BMSCs. We have reported that activation of hypoxia-induced factor by deletion of von Hippel-Lindau gene in osteochondral progenitor cells protected mice from aging-induced bone loss. However, whether pharmacologically manipulation of hypoxic niche would attenuate age-related bone loss and dysfunction of BMSCs is not well understood. METHODS: Twelve-month-old Sprague-Dawley rats were used as an aged model and were intraperitoneally injected with Desferal® (20, 60 mg/kg weight or vehicle), three times a week for a continuous 8-week period. Two-month-old young rats were set as a reference. After 8 weeks, micro-CT and HE staining were performed to determine the effect of Desferal® on bone loss. In order to investigate the effects of Desferal® on BMSC senescence, 12-month-old rats were treated with high-dose Desferal® (60 mg/kg weight) daily for 10 days. BMSCs were isolated and evaluated using CCK-8 assay, colony-forming cell assay, cell differentiation assay, laser confocal for reactive oxygen species (ROS) level, senescence-associated ß-galactosidase (SA-ß-gal) staining, and molecular expression test for stemness/senescence-associated genes. RESULTS: Micro-CT and HE staining showed that high-dose Desferal® significantly prevented bone loss in aged rats. Compared with vehicle group, the ex vivo experiments showed that short-term Desferal® administration could promote the potential of BMSC growth (proliferation and colony formation ability) and improve the rebalance of osteogenic and adipogenic differentiation, as well as rejuvenate senescent BMSCs (ROS level and SA-ß-gal staining) and revise the expression of stemness/senescence-associated genes. The potential of BMSCs from 12M-H-Desferal® group at least partly revised to the level close to 2-month-old group. CONCLUSIONS: The current study suggested that Desferal®, an iron-chelating agent, could alleviate age-related bone loss in middle-aged rats. Meanwhile, we found that short-term intraperitoneal injection of Desferal® partly rejuvenate BMSCs from aged rats. Overall, we demonstrated a novel role of Desferal® in rejuvenating aged BMSCs and preventing age-related bone loss.

Blockade of Fgfr1 with PD166866 Protects Cartilage from the Catabolic Effects Induced by Interleukin-1ß: A Genome-Wide Expression Profiles Analysis.

OBJECTIVE: Previously we showed that genetic deletion of Fgfr1 in chondrocytes protected mice from progression of osteoarthritis (OA). The aim of this study is to evaluate the effect of PD166866, a potent selective inhibitor of Fgfr1, on cartilage degeneration induced by interleukin-1ß (IL-1ß) and to clarify underlying global gene expression pattern. DESIGN: Cartilage explants and primary rat chondrocytes were stimulated with IL-1ß to establish an inflammatory OA in vitro model. The effects of PD166866 were determined by measuring the release of glycosaminoglycans (GAG) in cartilage explants and primary rat chondrocytes, and the underlying molecular mechanism was analyzed by microarray and RT-PCR analysis in primary chondrocytes. RESULTS: In cartilage explants, PD166866 significantly counteracts IL-ß stimulated GAG release. In addition, PD166866 impede IL-1ß-stimulated nuclear translocation of p65 in rat chondrocytes. Based on microarray analysis, a total of 67 and 132 genes with more than 1.5-fold changes were identified in IL-1ß-treated versus control and PD166866 cotreatment versus IL-1ß treatment alone, respectively. Only 19 thereof were coregulated by IL-1ß and PD166866 simultaneously. GO and KEGG pathway analysis showed that some pathways, including "cytokine-cytokine receptor interaction," "chemokine signaling pathway," and "complement and coagulation cascades," as well as some key genes like chemokines, complement, and matrix metalloproteinases may relevant for therapeutic application of Fgfr1 blockade in IL-1ß-stimulated chondrocytes. CONCLUSION: Our results clearly demonstrated that blockade of Fgfr1 with PD166866 could effectively suppress the catabolic effects induced by IL-1ß, and elucidated whole genomic targets of Fgfr1 inhibition responsible for the therapeutic effects of Fgfr1 blockade against inflammatory OA.

Anti-Gout Effects of the Medicinal Fungus Phellinus igniarius in Hyperuricaemia and Acute Gouty Arthritis Rat Models.

Background: Phellinus igniarius (P. igniarius) is an important medicinal and edible fungus in China and other Southeast Asian countries and has diverse biological activities. This study was performed to comparatively investigate the therapeutic effects of wild and cultivated P. igniarius on hyperuricaemia and gouty arthritis in rat models. Methods: UPLC-ESI-qTOF-MS was used to identify the chemical constituents of polyphenols from wild P. igniarius (WPP) and cultivated P. igniarius (CPP). Furthermore, WPP and CPP were evaluated in an improved hyperuricaemia rat model induced by yeast extract, adenine and potassium oxonate, which was used to examine xanthine oxidase (XO) activity inhibition and anti-hyperuricemia activity. WPP and CPP therapies for acute gouty arthritis were also investigated in a monosodium urate (MSU)-induced ankle swelling model. UHPLC-QE-MS was used to explore the underlying metabolic mechanisms of P. igniarius in the treatment of gout. Results: The main active components of WPP and CPP included protocatechuic aldehyde, hispidin, davallialactone, phelligridimer A, hypholomine B and inoscavin A as identified by UPLC-ESI-qTOF-MS. Wild P. igniarius and cultivated P. igniarius showed similar activities in reducing uric acid levels through inhibiting XO activity and down-regulating the levels of UA, Cr and UN, and they had anti-inflammatory activities through down-regulating the secretions of ICAM-1, IL-1ß and IL-6 in the hyperuricaemia rat model. The pathological progression of kidney damage was also reversed. The polyphenols from wild and cultivated P. igniarius also showed significant anti-inflammatory activity by suppressing the expression of ICAM-1, IL-1ß and IL-6 and by reducing the ankle joint swelling degree in an MSU-induced acute gouty arthritis rat model. The results of metabolic pathway enrichment indicated that the anti-hyperuricemia effect of WPP was mainly related to the metabolic pathways of valine, leucine and isoleucine biosynthesis and histidine metabolism. Additionally, the anti-hyperuricemia effect of CPP was mainly related to nicotinate and nicotinamide metabolism and beta-alanine metabolism. Conclusions: Wild P. igniarius and cultivated P. igniarius both significantly affected the treatment of hyperuricaemia and acute gouty arthritis models in vivo and therefore may be used as potential active agents for the treatment of hyperuricaemia and acute gouty arthritis.

Delivery of dimethyloxalylglycine in calcined bone calcium scaffold to improve osteogenic differentiation and bone repair.

As hypoxia plays a vital role in the angiogenic-osteogenic coupling, using proline hydroxylase inhibitors to manipulate hypoxia-inducible factors has become a strategy to improve the osteogenic properties of biomaterials. Dimethyloxallyl glycine (DMOG) is a 2-ketoglutarate analog, a small molecular compound that competes for 2-ketoglutaric acid to inhibit proline hydroxylase. In order to improve the osteogenic ability of calcined bone calcium (CBC), a new hypoxia-mimicking scaffold (DMOG/Collagen/CBC) was prepared by immersing it in the DMOG-Collagen solution, followed by freeze-drying. All coated CBC scaffolds retained the inherent natural porous architecture and showed excellent biocompatibility. A slow release of DMOG by the DMOG-loaded CBC scaffolds for up to one week was observed inin vitroexperiments. Moreover, the DMOG/Collagen/CBC composite scaffold was found to significantly stimulate bone marrow stromal cells to express osteogenic and angiogenic genesin vitro. In addition, the osteogenic properties of three kinds of scaffolds, raw CBC, Collagen/CBC, and DMOG/Collagen/CBC, were evaluated by histology using the rabbit femoral condyle defect model. Histomorphometric analyses showed that the newly formed bone (BV/TV) in the DMOG/Collagen/CBC group was significantly higher than that of the Collagen/CBC group. However, immunostaining of CD31 and Runx2 expression between these two groups showed no significant difference at this time point. Our results indicate that DMOG-coated CBC can promote osteogenic differentiation and bone healing, and show potential for clinical application in bone tissue engineering.

First Report of Leaf Blotch of Salix babylonica Caused by Botryosphaeria dothidea in China.

Salix babylonica L. (weeping willow) is an important ornamental tree commonly planted in China. Since 2018, a new disease with a high incidence has been observed on S. babylonica at the campus of Nanjing Forestry University (NFU), Nanjing, Jiangsu, China. The symptoms began as small dark brown lesions formed along the leaf margins and tips; and later became gray to brown in the center with dark brown borders. Small samples (3 to 4 mm2) from the lesion margins were surface-sterilized with 75% ethanol for 30 s and 1% NaClO for 90 s. Subsequently samples were, rinsed with sterile H2O, plated on potato dextrose agar (PDA) and incubated at 25°C. The same fungus was isolated in 95% of the samples. Pure cultures were obtained by monosporic isolation. A representative isolate, NFS1 was used for morphological and molecular characterization and deposited in China's Forestry Culture Collection Center (cfcc 54212). On PDA, colonies were initially white and gradually became grayish-green to dark gray from the center to the edge. After 1 week, colonies turned dark, and after 3 weeks black pycnidia developed on the surface of media. Conidia were one-celled, hyaline, smooth, and fusoid to ellipsoid. Conidia measurements were 23.0 ± 1.9 × 5.8 ± 0.7 µm (n = 50). The morphology matched the description of Botryosphaeria dothidea (Slippers et al. 2004). For an accurate identification, genomic DNA of NSF1 was extracted to amplify the internal transcribed spacer (ITS) region, the transcription eongation factor (tefa-1), beta-tubulin (ß-tub), the large subunit (LSU), and small subunit (SSU) genes with the specific primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn, 1999), ßt2a/ßt2b (Glass and Donaldson, 1995), LR0R/LR05 (Schoch et al. 2009), and NS1/NS4 (White et al. 1990), respectively. The sequences were deposited in GenBank (Accession Nos. MN826233 for ITS, MN855215 for tefa-1, MN855216 for ß-tub, MN886965 for LSU, and MN886966 for SSU). A BLAST search of GenBank showed that the ITS, tefa-1, ß-tub, LSU and SSU sequences of NSF1 were similar to those of B. dothidea KY788304 (Identity = 527/532; 99%), MG459974 (Identity = 247/247; 100%), MH724212 (Identity = 404/404; 100%), DQ377850 (Identity = 865/867; 99%), and KX091154 (Identity = 1,043/1,045; 99%), respectively. A maximum likelihood and Bayesian posterior probability-based phylogenetic analyses using IQ-tree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, tefa-1, ß-tub, LSU, and SSU) placed NFS1 in the clade of B. dothidea. Based on the multi-gene phylogeny and morphology, NFS1 isolate was identified as B. dothidea. To fulfill Koch's postulates, 20 detached and 20 attached healthy 10-week-old leaves from three 30-year-old S. babylonica plants at the campus of NFU were inoculated with 5-mm mycelial plugs of isolate NFS1 of 3-day-old cultures grown on PDA. Control leaves were treated with agar plugs. The detached inoculated leaves were placed in Petri dishes on a piece of wet filter paper and incubated at 25°C. The attached leaves were enclosed in a plastic bag along with the branches with a wet cotton ball inside. Sterile H2O was sprayed into the plastic bags twice daily to keep moisture conditions and incubated for 5 days. The experiment was repeated two times. Within 5 days, all the inoculated points showed lesions similar to those obsrved in the field, whereas controls were asymptomatic. The same fungus was re-isolated from these lesions with a frequency of 100%. B. dothidea has been reported to infect a broad range of hosts, including S. babylonica in the USA (Grand 1985). This is the first report of B. dothidea on S. babylonica in China. This finding provides crucial information on this high risk disease to willow and basis for identifying management strategies.

Applications of Matrix-Assisted Laser Desorption Ionization In-Source Decay Mass Spectrometry.

Matrix-Assisted Laser Desorption Ionization In-Source Decay (MALDI-ISD) Mass Spectrometry is a very powerful tool for providing terminal sequence information of biomolecules with minimal sample preparations. Fragmentation is induced at the position where hydrogen radical transfers from matrix to analyte in the MALDI-ISD process by proposed mechanism. Uniform fragmentation in MALDI-ISD generates relative simple ion spectra of readable sequence ladders with labile modifications retained, which is advantageous over other fragmentation methods such as collision-induced dissociation (CID) for characterizing modifications. MALDI-ISD has been applied to de novo sequencing of a 13.6 kDa protein and fully validate sequences of therapeutic antibodies, showing its promising potential in examining reference sequences of biotherapeutics unambiguously. It has also been successfully applied to the analysis of modifications such as post-translational modifications (PTMs) and PEGylation. Here we discuss the applications of MALDI-ISD in protein sequencing and modification analysis by featuring representative studies in details.

Relative interfacial cleavage energetics of protein complexes revealed by surface collisions.

To fulfill their biological functions, proteins must interact with their specific binding partners and often function as large assemblies composed of multiple proteins or proteins plus other biomolecules. Structural characterization of these complexes, including identification of all binding partners, their relative binding affinities, and complex topology, is integral for understanding function. Understanding how proteins assemble and how subunits in a complex interact is a cornerstone of structural biology. Here we report a native mass spectrometry (MS)-based method to characterize subunit interactions in globular protein complexes. We demonstrate that dissociation of protein complexes by surface collisions, at the lower end of the typical surface-induced dissociation (SID) collision energy range, consistently cleaves the weakest protein:protein interfaces, producing products that are reflective of the known structure. We present here combined results for multiple complexes as a training set, two validation cases, and four computational models. We show that SID appearance energies can be predicted from structures via a computationally derived expression containing three terms (number of residues in a given interface, unsatisfied hydrogen bonds, and a rigidity factor).

Localization of Protein Complex Bound Ligands by Surface-Induced Dissociation High-Resolution Mass Spectrometry.

Surface-induced dissociation (SID) is a powerful means of deciphering protein complex quaternary structures due to its capability of yielding dissociation products that reflect the native structures of protein complexes in solution. Here we explore the suitability of SID to locate the ligand binding sites in protein complexes. We studied C-reactive protein (CRP) pentamer, which contains a ligand binding site within each subunit, and cholera toxin B (CTB) pentamer, which contains a ligand binding site between each adjacent subunit. SID dissects ligand-bound CRP into subcomplexes with each subunit carrying predominantly one ligand. In contrast, SID of ligand-bound CTB results in the generation of subcomplexes with a ligand distribution reflective of two subunits contributing to each ligand binding site. SID thus has potential application in localizing sites of small ligand binding for multisubunit protein-ligand complexes.