CPD-002, a novel VEGFR2 inhibitor, relieves rheumatoid arthritis by reducing angiogenesis through the suppression of the VEGFR2/PI3K/AKT signaling pathway.

Synovial angiogenesis is a key player in the development of rheumatoid arthritis (RA), and anti-angiogenic therapy is considered a promising approach for treating RA. CPD-002 has demonstrated efficacy in suppressing tumor angiogenesis as a VEGFR2 inhibitor, but its specific impacts on RA synovial angiogenesis and possible anti-RA effects need further study. We examined the influences of CPD-002 on the migration and invasion of human umbilical vein endothelial cells (HUVECs) and its impacts on HUVECs' tube formation and vessel sprouting ex vivo. The therapeutic potential of CPD-002 in adjuvant-induced arthritis (AIA) rats and its suppression of synovial angiogenesis were examined. The involvement of the VEGFR2/PI3K/AKT pathway was assessed both in HUVECs and AIA rat synovium. Here, CPD-002 inhibited the migration and invasion of VEGF-stimulated HUVECs, decreased their chemotactic response to RA fibroblast-like synoviocyte-released chemoattractants, and exhibited anti-angiogenic effects in vitro and ex vivo. CPD-002's targeting of VEGFR2 was confirmed with molecular docking and cellular thermal shift assays, supported by the abolishment of CPD-002's effects upon using VEGFR2 siRNA. CPD-002 relieved paw swelling, arthritis index, joint damage, and synovial angiogenesis, indicating its anti-arthritic and anti-angiogenic effects in AIA rats. Moreover, the anti-inflammatory effects in vivo and in vitro of CPD-002 contributed to its anti-angiogenic effects. Mechanistically, CPD-002 hindered the activation of VEGFR2/PI3K/AKT pathway in VEGF-induced HUVECs and AIA rat synovium, as evidenced by reduced p-VEGFR2, p-PI3K, and p-AKT protein levels alongside elevated PTEN protein levels. Totally, CPD-002 showed anti-rheumatoid effects via attenuating angiogenesis through the inhibition of the VEGFR2/PI3K/AKT pathway.

Shikonin suppresses rheumatoid arthritis by inducing apoptosis and autophagy via modulation of the AMPK/mTOR/ULK-1 signaling pathway.

BACKGROUND: The overproliferation of fibroblast-like synoviocytes (FLS) contributes to synovial hyperplasia, a pivotal pathological feature of rheumatoid arthritis (RA). Shikonin (SKN), the active compound from Lithospermum erythrorhizon, exerts anti-RA effects by diverse means. However, further research is needed to confirm SKN's in vitro and in vivo anti-proliferative functions and reveal the underlying specific molecular mechanisms. PURPOSE: This study revealed SKN's anti-proliferative effects by inducing both apoptosis and autophagic cell death in RA FLS and adjuvant-induced arthritis (AIA) rat synovium, with involvement of regulating the AMPK/mTOR/ULK-1 pathway. METHODS: SKN's influences on RA FLS were assessed for proliferation, apoptosis, and autophagy with immunofluorescence staining (Ki67, LC3B, P62), EdU incorporation assay, staining assays of Hoechst, Annexin V-FITC/PI, and JC-1, transmission electron microscopy, mCherry-GFP-LC3B puncta assay, and western blot. In AIA rats, SKN's anti-arthritic effects were assessed, and its impacts on synovial proliferation, apoptosis, and autophagy were studied using Ki67 immunohistochemistry, TUNEL, and western blot. The involvement of AMPK/mTOR/ULK-1 pathway was examined via western blot. RESULTS: SKN suppressed RA FLS proliferation with reduced cell viability and decreased Ki67-positive and EdU-positive cells. SKN promoted RA FLS apoptosis, as evidenced by apoptotic nuclear fragmentation, increased Annexin V-FITC/PI-stained cells, reduced mitochondrial potential, elevated Bax/Bcl-2 ratio, and increased cleaved-caspase 3 and cleaved-PARP protein levels. SKN also enhanced RA FLS autophagy, featuring increased LC3B, reduced P62, autophagosome formation, and activated autophagic flux. Autophagy inhibition by 3-MA attenuated SKN's anti-proliferative roles, implying that SKN-induced autophagy contributes to cell death. In vivo, SKN mitigated the severity of rat AIA while also reducing Ki67 expression, inducing apoptosis, and enhancing autophagy within AIA rat synovium. Mechanistically, SKN modulated the AMPK/mTOR/ULK-1 pathway in RA FLS and AIA rat synovium, as shown by elevated P-AMPK and P-ULK-1 expression and decreased P-mTOR expression. This regulation was supported by the reversal of SKN's in vitro and in vivo effects upon co-administration with the AMPK inhibitor compound C. CONCLUSION: SKN exerted in vitro and in vivo anti-proliferative properties by inducing apoptosis and autophagic cell death via modulating the AMPK/mTOR/ULK-1 pathway. Our study revealed novel molecular mechanisms underlying SKN's anti-RA effects.

One-pot derivatization/extraction coupled with liquid chromatography-tandem mass spectrometry for furfurals determination.

Furfurals (5-hydroxymethylfurfural, furfural and 5-methyl furfural) have potential toxic effects to humans. This study developed a simple and rapid one-pot derivatization/extraction procedure for effective sample preparation of furfurals in complex samples prior to instrument analysis. The sample solution was incubated with 1-pyrenebutyric hydrazide (PBH) and hydroxyl-functionalized multi-walled carbon nanotubes (MWCNTs-OH) in a vial for 3 min. During this process, the furfurals were effectively derivatized by PBH and the furfural-PBH derivatives were selectively captured by MWCNTs-OH simultaneously. The detection selectivity and accuracy were greatly improved for the following liquid chromatography-tandem mass spectrometry analysis. Quantifying furfurals was validated over the 0.5-500 ng/mL concentration range with satisfactory linearities (R2 >0.99), accuracies (84.7%-119.0%) and precisions (<9.0%). The limits of quantification of 0.30, 0.36 and 0.20 ng/mL for 5-hydroxymethylfurfural, furfural and 5-methyl furfural, respectively, were achieved. Finally, the validated method was successfully applied to determine furfurals concentrations in various samples.

A fully green sample preparation method for synthetic antioxidants determination in edible oils based on natural feather fiber-supported liquid extraction.

The current study proposed a novel feather fiber-supported liquid extraction (FF-SLE) method for extracting analytes from oil samples. The natural feather fibers were used as the oil support material and directly loaded in the plastic tube of a disposable syringe to construct the low-cost extraction device (∼0.5 CNY). The edible oil without any pretreatment including dilution was added directly to the extraction device, followed by the addition of the green extraction solvent of ethanol. As an example, the proposed method was applied to extract nine synthetic antioxidants from edible oils. The optimized extraction conditions for processing 0.5 g of oil were obtained when the syringe dimension was 5 mL, the extraction solvent was 0.5 mL of ethanol, the amount of feather fibers was 200 mg of duck feather fibers and the static extraction time was 10 min. The applications to seven kinds of feathers and seven kinds of edible oils all indicated the excellent oil removal efficiencies (>98.0%). Combined with high-performance liquid chromatography-ultraviolet, a quantification method was validated with satisfied linearity (R2≥0.994), accuracy (95.8-114.6%) and precision (≤8.3%) with the limits of detection ranging from 50 to 100 ng/g. The proposed FF-SLE method was simple, effective, convenient, low-cost, green and environmental-friendly for the extraction of analytes from oil samples prior to instrument analysis.

Kapok fiber-supported liquid extraction for convenient oil samples preparations: A feasibility and proof-of-concept study.

In this study, a novel kapok fiber-supported liquid extraction (KF-SLE) method was developed for conveniently extracting analytes from oil samples. Natural kapok fiber without any pretreatment was directly used as an oil support medium. The extraction device was conveniently constructed by directly packing some kapok fibers into a syringe tube. Due to the fibrous property of the kapok fiber, no filter plate was needed. The cost of a KF-SLE device was as low as 0.5 CNY. The KF-SLE process was conveniently conducted using a simple three-step protocol: (1) the oil sample without any pretreatment including dilution was added directedly; (2) then, the oil-immiscible extractant was added; (3) after waiting a certain time for static extraction, the extractant was eluted out by pressing the kapok fibers with the syringe plunger. The extractant could be directly transferred for subsequent instrumental detection. For the feasibility and proof-of-concept study, the method was applied to quantify four synthetic flavor chemicals in edible oils. Satisfied quantification results were obtained with the correlation coefficient (R2) being greater than 0.996, the relative recoveries ranging from 92.90% to 107.53% and intra- and inter-day RSDs being less than 7.56%. All in all, for the first time, the SLE technique was expanded to process oil samples and the method has the characteristics of low cost, environmental friendliness, high sample processing throughput and ease of automation, offering a promising approach for edible oil sample preparations.

On-tissue pyrene-1-boronic acid labeling assisted MALDI imaging of catecholamines in porcine adrenal gland.

In this study, an on-tissue chemical labeling - matrix assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) method was developed for visualization of the distribution of three catecholamine (CA) compounds (dopamine, epinephrine and norepinephrine) in porcine adrenal gland. Commercially available pyrene-1-boronic acid (PBA) was employed as an effective in situ derivatizing reagent dissolved in acetonitrile containing 0.1% pyridine for the chemical labeling and the matrix coating. Without extra matrix coating, the tissue section was directly analyzed by MALDI-MS. The detection specificity and sensitivity were greatly improved with the on-tissue PBA labeling and successful imaging of the three CAs in porcine adrenal gland was achieved. Compared with previously reported methods for MALDI-MSI of the CAs, the analytical strategy proposed in the study provided a robust, easy-to-use and low-cost on-tissue chemical derivatization method that facilitated simultaneous molecular imaging of the three compounds.

Melatonin improves meiosis maturation against diazinon exposure in mouse oocytes.

AIMS: Organophosphorus pesticide diazinon (DZN) has adverse effects on animals and humans by direct contact or the spread of food chain. The antioxidant melatonin has protective effects on female reproduction. This study aimed to explore the effects of DZN on meiosis maturation in mouse cumulus oocyte complexes (COCs) and the effects of melatonin. MAIN METHODS: Different concentrations of DZN and melatonin were added during the in vitro maturation of COCs. Then we detected the extrusion rate of the first polar body, the number of sperms binding to oocyte, mitochondrial membrane potential, reactive oxygen species (ROS), early apoptosis. Subsequently, the expression of Juno, CX37, CX43 and ERK1/2 were detected by immunofluorescence staining and Western blotting. KEY FINDINGS: DZN exposure results in the failure of nuclear and cytoplasmic maturation of oocyte meiosis. Destruction of repositioning and function of mitochondria increases the levels of ROS and early apoptosis. The DZN-exposed oocytes express less Juno resulting to bind less sperms than normal. The loss of gap junctions and failure to activate ERK1/2 also contribute to the failure of cytoplasmic maturation. All these ultimately lead to the poor oocyte quality and low fertility. Appropriate melatonin can effectively restore all these defects. SIGNIFICANCE: Under DZN exposure, melatonin can significantly improve the quality of oocytes, and melatonin promotes oocyte maturation by protecting gap junction and restoring ERK1/2 pathway, which is a new breakthrough for improving female fertility.

In-syringe cotton fiber-supported liquid extraction coupled with gas chromatography-tandem mass spectrometry for the determination of free 3-mono-chloropropane-1,2-diol in edible oils.

In the current study, natural cotton fiber was served as the supporter of water, and the water acted as an extractant for liquid-phase microextraction of polar components in low-polar edible oils. An in-syringe extraction device was constructed to facilitate the extraction process by simply loading a certain amount of cotton fibers between the syringe needle and the plastic syringe tube. Then, the extraction process can be conveniently conducted by pull-push the syringe plunger. It can be regarded as a new type of dynamic liquid-phase microextraction method while operated more convent. For the feasibility study, the novel in-syringe cotton fiber-supported liquid extraction (CF-SLECF-SLE) pretreatment method was applied to extract free 3-mono-chloropropane-1,2-diol (3-MCPD) in edible oils. Specifically, the cotton fibers supported a certain amount of water by successfully pulling-pushing 1 mL of water and 1 mL of HEX in/out twice, respectively. Then, 2.0 mL of diluted oil sample (containing 0.4 g oil) was loaded in and out four times for extraction, during which process 3-MCPD was extracted into the supported water. The extracted 3-MCPD was desorbed with 1 mL of ethyl acetate (EA), derivatized with trimethyl silane imidazole (TMSI), and analyzed by gas chromatography-tandem mass spectrometry (GC-MS/MS). For three different spiked edible oils, the internal standard normalized matrix effect (IS-normalized ME) values were in ranges of 96.3-104.8% with RSD being 4.3%, benefiting the accurate quantitative analysis. The limit of quantification (LOQ) was calculated to be 2 ng/g, which met the regular determination requirement of 3-MCPD in edible oils. Satisfied linearity was obtained in 2-500 ng/g, with correlation coefficients (R2) being 0.998. The relative recoveries were in the ranges of 96.9-110.5%. The intra-/inter-day RSDs were less than 8.2% and 10.2%, respectively. The proposed method provides an efficient, simple, low-cost, and easy to automate strategy for determining free 3-MCPD in edible oils.

Tea polyphenols alleviate the adverse effects of diabetes on oocyte quality.

Maternal diabetes mellitus reduces oocyte quality, such as abnormalities of spindle assembly and chromosome segregation, mitochondrial dysfunction, decrease of fertilization rate, increase of ROS, and so on. So, it is important to research how to restore the decreased oocyte quality induced by maternal diabetes mellitus. Polyphenols are the most abundant bioactive components of green tea. It is reported that tea polyphenols have many health functions, for instance anti-oxidation, anti-inflammation, anti-obesity, and anti-diabetes. Thus, we hypothesize that tea polyphenols may play a crucial role in alleviating adverse effects of diabetes on oocyte quality. In the present study, we researched the effects of tea polyphenols on diabetic oocyte maturation in vitro. Compared with the control, oocytes from diabetic mice displayed a lower maturation rate and a higher frequency of spindle defects and chromosome misalignment. However, tea polyphenols significantly increased the oocyte maturation rate, and reduced the incidence of abnormal spindle assembly and chromosome segregation. Tea polyphenols also obviously decreased the reactive oxygen species (ROS) levels in diabetic oocytes, and increased the expression of antioxidant genes (Sod1 and Sod2). Abnormal mitochondrial membrane potential was also alleviated in diabetic oocytes, and the expression of genes regulating mitochondrial fusion (Opa1, Mfn1 and Mfn2) and fission (Drp1) was significantly increased while tea polyphenols were added. Meanwhile, tea polyphenols reduced DNA damage in diabetic oocytes which may be mediated by the increased expression of Rad51, related to DNA damage repair. Our results suggest that tea polyphenols would, at least partially, restore the adverse effects of diabetes mellitus on oocyte quality.

Diagnostic Value of Circulating Antigens in the Serum of Piglets with Experimental Acute Toxoplasmosis.

Toxoplasmosis, caused by Toxoplasma gondii, an apicomplexan parasite, infects all warm-blooded animals, including a third of the human population. Laboratory diagnosis of acute toxoplasmosis is based on the detection of anti-T. gondii IgM and IgG and T. gondii nucleic acid; however, these assays have certain limitations. Circulating Ags (CAgs) are reliable diagnostic indicators of acute infection. In this study, we established a model of acute T. gondii infection in Large White pigs. CAg levels peaked between 3 and 5 d after inoculation, and 28 CAgs were identified using an immunoprecipitation-shotgun approach, among which dolichol-phosphate-mannose synthase family protein (TgDPM), C3HC zinc finger-like protein (TgZFLP3), and ribosomal protein RPL7 (TgRPL7) were selected to further investigate their value in the diagnosis of acute toxoplasmosis. Immunofluorescence assays revealed that TgDPM and TgRPL7 were localized in the membrane surface, while TgZFLP3 was localized in the apical end. Western blotting revealed the presence of the three proteins in the serum during acute infection. Indirect ELISA results indicate that TgZFLP3 is likely to be a novel candidate for the diagnosis of acute toxoplasmosis. However, these three proteins may not be useful as candidate vaccines against toxoplasmosis owing to their low protective ability. In addition, deletion of the zflp3 gene partially attenuated virulence in Kunming mice. Collectively, we identified 28 CAgs in the serum of piglets with experimental acute toxoplasmosis and confirmed that TgZFLP3 is a potential biomarker for acute T. gondii infection. The results of this study provide data to improve the detection efficiency of acute toxoplasmosis.

Genome-Wide CRISPR/Cas9 Screen Identifies New Genes Critical for Defense Against Oxidant Stress in Toxoplasma gondii.

Toxoplasma gondii is one of the most widespread apicomplexans and can cause serious infections in humans and animals. Its antioxidant system plays an important role in defending against oxidant stress imposed by the host. Some genes encoding the antioxidant enzymes of T. gondii have been identified; however, critical genes that function in response to reactive oxygen species (ROS) stress are still poorly understood. Here, we performed genome-wide CRISPR/Cas9 loss-of-function screening in the T. gondii RH strain to identify potential genes contributing to the ROS stress response. Under hydrogen peroxide treatment, 30 single guide RNAs targeting high-confidence genes were identified, including some known important antioxidant genes such as catalase and peroxiredoxin PRX3. In addition, several previously uncharacterized genes were identified, among which five hypothetical protein-coding genes, namely, HP1-HP5, were selected for further functional characterization. Targeted deletion of HP1 in T. gondii RH led to significant sensitivity to H2O2, suggesting that HP1 is critical for oxidative stress management. Furthermore, loss of HP1 led to decreased antioxidant capacity, invasion efficiency, and proliferation in vitro. In vivo results also revealed that the survival time of mice infected with the HP1-KO strain was significantly prolonged relative to that of mice infected with the wild-type strain. Altogether, these findings demonstrate that the CRISPR/Cas9 system can be used to identify potential genes critical for oxidative stress management. Furthermore, HP1 may confer protection against oxidative damage and contributes to T. gondii virulence in mice.

Feasibility of Ultrasound-Guided Peritoneal Perfusion with Ozone in the Treatment of Chronic Pelvic Pain: A Bicenter Retrospective Analysis.

BACKGROUND: Numerous therapies have been developed for the treatment of chronic pelvic pain (CPP). Oxygen-ozone therapy is a new method for the treatment of CPP. OBJECTIVES: This article evaluated the feasibility of ultrasound-guided peritoneal perfusion with ozone in patients with CPP. STUDY DESIGN: This is a bicenter retrospective study. SETTING: The study was conducted at 2 pain centers of a university hospital. METHODS: The medical records of patients with CPP (n = 60) from March 2016 until October 2018 were collected and reviewed. Group A contained 19 patients who were treated with a 1500 mcg dose of ozonated water (10 mcg/mL concentration and 150 mL volume), group B contained 23 patients using the same dose of ozonated water but a 15 mcg/mL concentration and 100 mL volume. Group C included 18 patients using a similar ozone dose but delivered in an oxygen-ozone mixture (15 mcg/mL concentration and 100 mL volume oxygen-ozone mixture). Visual Analog Scale (VAS) scores for pain of the 3 groups were compared at pretreatment, posttreatment, 1, 3, and 6 months posttreatment. The injection pain was evaluated using a 4-point verbal rating scale. Quality of life (QoL), anxiety, and depression were assessed at pretreatment and at 6 months posttreatment. RESULTS: The VAS scores of the 3 groups decreased over time following treatment. Group A showed much higher pain scores compared with groups B and C at 1, 3, and 6 months posttreatment. However, the injection pain for groups B and C was higher than group A, but there was no difference seen between group B and C. At 6 months posttreatment, the QoL for all patients improved compared with pretreatment, whereas the anxiety and depression did not demonstrate differences. LIMITATIONS: The main limitations of this study are the retrospective study design, limited case number, and short follow-up period. CONCLUSIONS: Ultrasound-guided peritoneal perfusion with ozone is a feasible therapy for patients with CPP.

Complete mitochondrial genome of the Ruff, Calidris pugnax (Aves, Scolopacidae).

This paper reports the mitochondrial genome of Calidris pugnax, which is a closed circular molecule of 16,902 bp. The overall base composition of this species is 25.2% T, 30.6% C, 32.0% A, and 12.2% G, with an A + T content of 55.2%. The structure of the mitogenome is a typical vertebrate mitochondrial gene arrangement. Phylogenetic analysis of complete mitochondrial genome concatenated sequences from 13 species from 6 genera was conducted using the maximum-likelihood (ML) model. The topology demonstrated that the mitogenome of this species was genetically closest to that of Calidris tenuirostris. Calidris pugnax mitogenome can contribute to our understanding of the phylogeny and evolution of this species.

Cones structure and seed traits of four species of large-seeded pines: Adaptation to animal-mediated dispersal.

Seed dispersal selection pressures may cause morphological differences in cone structure and seed traits of large-seeded pine trees. We investigated the cone, seed, and scale traits of four species of animal-dispersed pine trees to explore the adaptations of morphological structures to different dispersers. The four focal pines analyzed in this study were Chinese white pine (Pinus armandi), Korean pine (P. koraiensis), Siberian dwarf pine (P. pumila), and Dabieshan white pine (P. dabeshanensis). There are significant differences in the traits of the cones and seeds of these four animal-dispersed pines. The scales of Korean pine and Siberian dwarf pine are somewhat opened after cone maturity, the seeds are closely combined with scales, and the seed coat and scales are thick. The cones of Chinese white pine and Dabieshan white pine are open after ripening, the seeds fall easily from the cones, and the seed coat and seed scales are relatively thin. The results showed that the cone structure of Chinese white pine is similar to that of Dabieshan white pine, whereas Korean pine and Siberian dwarf pine are significantly different from the other two pines and vary significantly from each other. This suggests that species with similar seed dispersal strategies exhibit similar morphological adaptions. Accordingly, we predicted three possible seed dispersal paradigms for animal-dispersed pines: the first, as represented by Chinese white pine and Dabieshan white pine, relies upon small forest rodents for seed dispersal; the second, represented by Korean pine, relies primarily on birds and squirrels to disperse the seeds; and the third, represented by Siberian dwarf pine, relies primarily on birds for seed dispersal. Our study highlights the significance of animal seed dispersal in shaping cone morphology, and our predictions provide a theoretical framework for research investigating the coevolution of large-seeded pines and their seed dispersers.

Polymer monolith microextraction online coupled to hydrophilic interaction chromatography/mass spectrometry for analysis of beta2-agonist in human urine.

An analytical method based on online combination of polymer monolith microextraction (PMME) technique with hydrophilic interaction LC (HILIC)/MS is presented. The extraction was performed with a poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolithic column while the subsequent separation was carried out on a Luna silica column by HILIC. After 1:1 v/v dilution with 20 mM phosphate solution at pH 7.0 and centrifugation, urine sample was directly used for extraction. After optimization, 85% ACN (containing 0.3% formic acid v/v) was used for rapid online elution, which was also the mobile phase in HILIC to avoid band broadening during separation or carry-over that was usually observed in PMME-RP LC system. Online automation of extraction and separation procedures was realized under the control of a program in this study. The developed method was applied to rapid and sensitive monitoring of three beta2-agonist traces in human urine. The LODs (S/N = 3) of the method were found to be 0.05-0.09 ng/mL of beta2-agonists in urine. The recoveries of three beta2-agonists spiked in five different urine samples ranged from 79.8 to 119.8%, with RSDs less than 18.0%.

Monitoring of sulfonamide antibacterial residues in milk and egg by polymer monolith microextraction coupled to hydrophilic interaction chromatography/mass spectrometry.

A simple, rapid, and sensitive method for the determination of traces of thirteen sulfonamide antibacterials in milk and eggs is presented. This method is based on the combination of polymer monolith microextraction (PMME) technique with hydrophilic interaction chromatography/mass spectrometry (HILIC/MS). The extraction was performed with a poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column while the subsequent separation was carried out on a Luna NH2 column by HILIC. To obtain optimum results, several parameters relating to HILIC and PMME were investigated. After optimization, acetonitrile (contain 0.05% formic acid, v/v) was used as the elution solution, which was well compatible with the mobile phase in HILIC. Good linearities were obtained for thirteen SAs with the correlation coefficients (R2) above 0.997. The limits of detection (S/N=3) of the method were found to be 0.4-5.7 ng mL(-1) of SAs in whole milk and 0.9-9.8 ng g(-1) of SAs in eggs. The recoveries of thirteen SAs in two matrices ranged from 80.4 to 119.8%, with relative standard deviations less than 11.8%.