Large T antigen mediated target gene replication improves site-specific recombination efficiency.

With advantages of high-fidelity, monoclonality and large cargo capacity, site-specific recombination (SSR) holds great promises for precise genomic modifications. However, broad applications of SSR have been hurdled by low integration efficiency, and the amount of donor DNA available in nucleus for SSR presents as a limiting factor. Inspired by the DNA replication mechanisms observed in double-stranded DNA virus SV40, we hypothesized that expression of SV40 large T antigen (TAg) can increase the copy number of the donor plasmid bearing an SV40 origin, and in consequence promote recombination events. This hypothesis was tested with dual recombinase-mediated cassette exchange (RMCE) in suspension 293F cells. Results showed that TAg co-transfection significantly enhanced SSR in polyclonal cells. In the monoclonal cell line carrying a single landing pad at an identified genomic locus, 12% RMCE efficiency was achieved, and such improvement was indeed correlated with donor plasmid amplification. The developed TAg facilitated RMCE (T-RMCE) was exploited for the construction of large libraries of >107 diversity, from which GFP variants with enhanced fluorescence were isolated. We expect the underlying principle of target gene amplification can be applicable to other SSR processes and gene editing approaches in general for directed evolution and large-scale genomic screening in mammalian cells.

Identification of a Fully Human Antibody VH Domain Targeting Anaplastic Lymphoma Kinase (ALK) with Applications in ALK-Positive Solid Tumor Immunotherapy.

The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC50 of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer.

Two new megastigmane glycosides from the leaves of Nicotiana tabacum and their anti-inflammatory activities.

Two new megastigmane glycosides, (6 R,7E,9R)-3-oxo-α-ionyl-9-O-α-L-rhamnopyranosyl-(1''→4')-ß-D-glucopyranoside (1) and (6 R,7E,9R)-3-oxo-α-ionyl-9-O-ß-D-glucopyranosyl-(1''→6')-ß-D-glucopyranoside (2), together with six known analogues (3-8) were isolated from the leaves of Nicotiana tabacum. The structures of all metabolites were determined by comprehensive analysis of NMR and MS spectroscopic data as well as by comparison with those of previously reported. The in vitro anti-inflammatory activity of all isolates was evaluated using a lipopolysaccharide (LPS)-induced RAW264.7 cell inflammatory model, and the compounds 1, 3, 7, and 8 exhibited inhibition of LPS-induced NO production in RAW264.7 macrophage cells with IC50 values of 42.3-61.7 µM (positive control, dexamethasone, IC50 = 21.3 ± 1.2 µM).

One-step production of fully biotinylated and glycosylated human Fc gamma receptors.

Initiating and regulating humoral immunity, Fc gamma receptors (FcγRs) have been identified both as therapeutics and as drug targets, and thus production of biologically active FcγRs is highly demanded for biopharmaceutical development. Focusing on low-affinity FcγRs IIA (131H/R allotypes), IIB, and IIIA (176F/V), this study used human 293-F cells to achieve correct post-translational modifications (PTMs) including biotinylation, N-glycosylation, and disulfides. Approaches involving co-expression of FcγR-AviTag and Escherichia coli biotin ligase BirA, endoplasmic reticulum retention, stable and transient transfections, and optimization of transgene ratio were investigated. Protein electrophoresis under reducing and non-reducing conditions, enzymatic deglycosylation, streptavidin pull-down assays, and binding kinetic analysis collectively indicated that the produced FcγR ectodomains were fully biotinylated, N-glycosylated, had formed disulfide bond, and exhibited expected binding affinities toward IgG1 trastuzumab and its Fc mutants. A clear trade-off between production yield and PTM quality was also observed. Achieving multiple types of PTMs completely by one-step cell culture should have applications for the production of a variety of complex proteins of biomedical importance.

High-fidelity large-diversity monoclonal mammalian cell libraries by cell cycle arrested recombinase-mediated cassette exchange.

Mammalian cells carrying defined genetic variations have shown great potentials in both fundamental research and therapeutic development. However, their full use was limited by lack of a robust method to construct large monoclonal high-quality combinatorial libraries. This study developed cell cycle arrested recombinase-mediated cassette exchange (aRMCE), able to provide monoclonality, precise genomic integration and uniform transgene expression. Via optimized nocodazole-mediated mitotic arrest, 20% target gene replacement efficiency was achieved without antibiotic selection, and the improved aRMCE efficiency was applicable to a variety of tested cell clones, transgene targets and transfection methods. As a demonstration of this versatile method, we performed directed evolution of fragment crystallizable (Fc), for which error-prone libraries of over 107 variants were constructed and displayed as IgG on surface of CHO cells. Diversities of constructed libraries were validated by deep sequencing, and panels of novel Fc mutants were identified showing improved binding towards specific Fc gamma receptors and enhanced effector functions. Due to its large cargo capacity and compatibility with different mutagenesis approaches, we expect this mammalian cell platform technology has broad applications for directed evolution, multiplex genetic assays, cell line development and stem cell engineering.

oppHeatmap: Rendering Various Types of Heatmaps for Omics Data.

Heatmaps are preferred visualization modes for biologists to display high-dimensional information from high-throughput omics data. Many software including website services and R packages are available to generate various types of customized heatmaps. Here, we describe oppHeatmap (omics pilot platform of heatmap), a new tool constructed to render different kinds of heatmaps through MATLAB. oppHeatmap is available for plotting ordinary heatmaps, hierarchical clustering, TreeMaps, microplates graph, sample correlation (full heatmap and upper and lower triangle parts), gene correlation (between columns or tables), and polar heatmaps. oppHeatmap can support the modification of borders, fonts, and colors to customize the final plots. oppHeatmap can not only read data from Microsoft Excel to generate specific heatmaps but also make Excel heatmaps by coloring each cell in Excel. The graphs can be stored in SVG (supported vector graph) format and modified by other SVG recognition software. oppHeatmap is designed by MATLAB AppDesigner with GUI (graphical user interface) operation. The program for oppHeatmap was available at https://github.com/HangZhouSheep/oppHeatmap .are diagramed by All kinds of heatmaps oppHeatmap. The main function of oppHeatmap is to enable visualization of two types of heatmaps and a total of 11 graphs. The first type is a heatmap with a polar or rectangular coordinate system, including ordinary heatmap, microplates graph, polar heatmap, and Excel heatmap. The last one involves writing the heatmap in Excel and others in MATLAB. The second type is with rearranged rows and columns, which includes hierarchical clustering, TreeMaps, and sample/gene correlation between columns or rows. All plots support row standardization for better color contrast. Eleven base functions implement heatmap plotting in the command line environment of MATLAB. GUI designed by AppDesigner supports the interactive construction of heatmaps.

Development of an efficient method for selection of stable cell pools for protein expression and surface display with Expi293F cells.

Compare with transient expression, stable cell lines generally have higher productivity and better quality for protein expression. However, selection of stable cell line is time-consuming and laborious. Here we describe an optimized selection method to achieve high-efficient stable cell pools with Expi293F suspension cells. This method only takes 2-3 weeks to generate stable cell pools with 2- to 10-fold higher productivity than transient gene expression (TGE). In fed-batch culture with Yeastolate, >1 g/L yield was achieved with our KTN0239-IgG stable cell pool in shaker flasks. This method can be also applied to efficiently display proteins on the cell surface.

An MMP-9 exclusive neutralizing antibody attenuates blood-brain barrier breakdown in mice with stroke and reduces stroke patient-derived MMP-9 activity.

Rapid upregulation of matrix metalloproteinase 9 (MMP-9) leads to blood-brain barrier (BBB) breakdown following stroke, but no MMP-9 inhibitors have been approved in clinic largely due to their low specificities and side effects. Here, we explored the therapeutic potential of a human IgG monoclonal antibody (mAb), L13, which was recently developed with exclusive neutralizing specificity to MMP-9, nanomolar potency, and biological function, using mouse stroke models and stroke patient samples. We found that L13 treatment at the onset of reperfusion following cerebral ischemia or after intracranial hemorrhage (ICH) significantly reduced brain tissue injury and improved the neurological outcomes of mice. Compared to control IgG, L13 substantially attenuated BBB breakdown in both types of stroke model by inhibiting MMP-9 activity-mediated degradations of basement membrane and endothelial tight junction proteins. Importantly, these BBB-protective and neuroprotective effects of L13 in wild-type mice were comparable to Mmp9 genetic deletion and fully abolished in Mmp9 knockout mice, highlighting the in vivo target specificity of L13. Meanwhile, ex vivo co-incubation with L13 significantly neutralized the enzymatic activities of human MMP-9 in the sera of ischemic and hemorrhagic stroke patients, or in the peri-hematoma brain tissues from hemorrhagic stroke patients. Overall, we demonstrated that MMP-9 exclusive neutralizing mAbs constitute a potential feasible therapeutic approach for both ischemic and hemorrhagic stroke.

Survey of Acetylation for Thermoanaerobacter tengcongensis.

Non-histone protein acetylation is involved in key cellular processes both in eukaryotes and prokaryotes. Acetylation in bacteria is used to modify proteins involved in metabolism and allow the bacteria to adapt to their environment. TTE (Thermoanaerobacter tengcongensis) is an anaerobic, thermophilic saccharolytic bacterium that grows at extreme temperature range between 50 and 80 â„ƒ. The annotated TTE proteome contains less than 3000 proteins. We analyzed the proteome and acetylome of TTE using 2DLC-MS/MS (2-dimensional liquid chromatography mass spectrum). We evaluated the ability of mass spectrometry technology to cover a relatively small proteome as much as possible. And we also observed wide spread of acetylation in TTE, which changed under different temperatures. A total of 2082 proteins were identified, which accounts for about 82% of the database. A total of 2050 (~ 98%) proteins were quantified in at least one culture condition and 1818 proteins were quantified in all 4 conditions. The result also consisted 3457 acetylation sites corresponding to 827 distinct proteins, which covered 40% of the proteins identified. Bioinformatics analysis reported that proteins related to replication, recombination, repair, and extracellular structure cell wall biogenesis had more than half members acetylated, while energy production, carbohydrate transport, and metabolism related proteins were least acetylated. Our result suggested that acetylation affects the ATP-related energy metabolism and energy-dependent biosynthesis process. Comparing the enzymes related with lysine acetylation and acetyl-CoA (acetyl-coenzyme A) metabolism, we suggested that the acetylation of TTE took a non-enzymatic mechanism and affected by abundance of acetyl-CoA.

oppOntology: a MATLAB Toolbox for Enrichment Analysis.

Biologists often use systems of ontologies to classify gene lists obtained by high-throughput gene or protein-sequencing instruments, and then enrichment scores were used to rank the ontology system. Therefore, the important molecular functional categories related to the phenotype can be conveniently viewed in the ontology system. Since the birth of GO (Gene Ontology) organization, various types of ontology software have been developed to calculate enrichment scores for the target gene list in the GO system. Herein, we provide an enrichment calculation application oppOntology (Omics Pilot Platform for Ontology) developed by MATLAB. oppOntology supports simultaneous calculation of multiple samples with manifold enrichment scores (GeneCount, GeneRatio, EnrichFactor, HypergeometricTest, and FisherExactTest). oppOntology can not only calculate enrichment scores for generic functional databases, such as GO, KEGG, HPO, and MsigDB, but also for self-defined functional category databases and customized GO Slim. Moreover, oppOntology supports online mapping of KEGG pathway diagrams in a batch way. The GUI (Graphical User Interface) of oppOntology is developed on the architecture of AppDesigner in MATLAB, and all input and output files are Microsoft Excel. oppOntology is an independent, easy-to-use enrichment calculation software, that can be available at https://github.com/HangZhouSheep/oppOntology .

Control of SARS-CoV-2 infection by MT1-MMP-mediated shedding of ACE2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Angiotensin-converting enzyme 2 (ACE2) is an entry receptor for SARS-CoV-2. The full-length membrane form of ACE2 (memACE2) undergoes ectodomain shedding to generate a shed soluble form (solACE2) that mediates SARS-CoV-2 entry via receptor-mediated endocytosis. Currently, it is not known how the physiological regulation of ACE2 shedding contributes to the etiology of COVID-19 in vivo. The present study identifies Membrane-type 1 Matrix Metalloproteinase (MT1-MMP) as a critical host protease for solACE2-mediated SARS-CoV-2 infection. SARS-CoV-2 infection leads to increased activation of MT1-MMP that is colocalized with ACE2 in human lung epithelium. Mechanistically, MT1-MMP directly cleaves memACE2 at M706-S to release solACE218-706 that binds to the SARS-CoV-2 spike proteins (S), thus facilitating cell entry of SARS-CoV-2. Human solACE218-706 enables SARS-CoV-2 infection in both non-permissive cells and naturally insusceptible C57BL/6 mice. Inhibition of MT1-MMP activities suppresses solACE2-directed entry of SARS-CoV-2 in human organoids and aged mice. Both solACE2 and circulating MT1-MMP are positively correlated in plasma of aged mice and humans. Our findings provide in vivo evidence demonstrating the contribution of ACE2 shedding to the etiology of COVID-19.

opplncRNA: A MATLAB Package for Comprehensive Pathway Analysis of lncRNA-miRNA-mRNA in Humans.

The discovery of new lncRNAs (long noncoding RNAs) and their regulatory pathways has always been a hotspot in the field of ceRNA (competing endogenous RNA). Herein, we report opplncRNA (Omics Pilot Platform of lncRNA), a novel and rapid tool for investigating lncRNA-miRNA-mRNA interactions based on the architecture of MATLAB AppDesigner. opplncRNA is useful to analyze the regulatory interaction networks of lncRNA with a friendly GUI (graphical user interface). There are three lncRNA databases (ENCORI, LncBase, and miRcode) about lncRNA-miRNA interactions that have been integrated into opplncRNA, as well as seven miRNA databases (miRcode, ENCORI, TarBase, miRTarBase, miRDB, miRanda, and miRecords) about miRNA-mRNA interactions as also. opplncRNA can read expression data from any profile techniques, such as microarray or RNA-seq. Then, the relationships between lncRNA-miRNA and miRNA-mRNA can be directly calculated through the profile data of lncRNA, miRNA, and mRNA by the threshold of correlation coefficients. Integrated databases can be used to filter calculation outcomes to obtain more reliable pathways. Moreover, opplncRNA has the functionality of directly demonstrating 3 layers network from lncRNA to mRNA in command line form.

Regulation of age-associated insulin resistance by MT1-MMP-mediated cleavage of insulin receptor.

Insulin sensitivity progressively declines with age. Currently, the mechanism underlying age-associated insulin resistance remains unknown. Here, we identify membrane-bound matrix metalloproteinase 14 (MT1-MMP/MMP14) as a central regulator of insulin sensitivity during ageing. Ageing promotes MMP14 activation in insulin-sensitive tissues, which cleaves Insulin Receptor to suppress insulin signaling. MT1-MMP inhibition restores Insulin Receptor expression, improving insulin sensitivity in aged mice. The cleavage of Insulin Receptor by MT1-MMP also contributes to obesity-induced insulin resistance and inhibition of MT1-MMP activities normalizes metabolic dysfunctions in diabetic mouse models. Conversely, overexpression of MT1-MMP in the liver reduces the level of Insulin Receptor, impairing hepatic insulin sensitivity in young mice. The soluble Insulin Receptor and circulating MT1-MMP are positively correlated in plasma from aged human subjects and non-human primates. Our findings provide mechanistic insights into regulation of insulin sensitivity during physiological ageing and highlight MT1-MMP as a promising target for therapeutic avenue against diabetes.

Comparative Proteomic Analysis of Drug Trichosanthin Addition to BeWo Cell Line.

Trichosanthin (TCS) is a traditional Chinese herbal medicine used to treat some gynecological diseases. Its effective component has diverse biological functions, including antineoplastic activity. The human trophoblast cell line BeWo was chosen as an experimental model for in vitro testing of a drug screen for anticancer properties of TCS. The MTT method was used in this study to get a primary screen result. The result showed that 100 mM had the best IC50 value. Proteomics analysis was then performed for further investigation of the drug effect of TCS on the BeWo cell line. In this differential proteomic expression analysis, the total proteins extracted from the BeWo cell line and their protein expression level after the drug treatment were compared by 2DE. Then, 24 unique three-fold differentially expressed proteins (DEPs) were successfully identified by MALDI-TOF/TOF MS. Label-free proteomics was run as a complemental method for the same experimental procedure. There are two proteins that were identified in both the 2DE and label-free methods. Among those identified proteins, bioinformatics analysis showed the importance of pathway and signal transduction and gives us the potential possibility for the disease treatment hypothesis.

Body weight regulation via MT1-MMP-mediated cleavage of GFRAL.

GDNF-family receptor a-like (GFRAL) has been identified as the cognate receptor of growth/differentiation factor 15 (GDF15/MIC-1), considered a key signaling axis in energy homeostasis and body weight regulation. Currently, little is known about the physiological regulation of the GDF15-GFRAL signaling pathway. Here we show that membrane-bound matrix metalloproteinase 14 (MT1-MMP/MMP14) is an endogenous negative regulator of GFRAL in the context of obesity. Overnutrition-induced obesity increased MT1-MMP activation, which proteolytically inactivated GFRAL to suppress GDF15-GFRAL signaling, thus modulating the anorectic effects of the GDF15-GFRAL axis in vivo. Genetic ablation of MT1-MMP specifically in GFRAL+ neurons restored GFRAL expression, resulting in reduced weight gain, along with decreased food intake in obese mice. Conversely, depletion of GFRAL abolished the anti-obesity effects of MT1-MMP inhibition. MT1-MMP inhibition also potentiated GDF15 activity specifically in obese phenotypes. Our findings identify a negative regulator of GFRAL for the control of non-homeostatic body weight regulation, provide mechanistic insights into the regulation of GDF15 sensitivity, highlight negative regulators of the GDF15-GFRAL pathway as a therapeutic avenue against obesity and identify MT1-MMP as a promising target.

Ki20227 aggravates apoptosis, inflammatory response, and oxidative stress after focal cerebral ischemia injury.

The survival of microglia depends on the colony-stimulating factor-1 receptor (CSF1R) signaling pathway under physiological conditions. Ki20227 is a highly selective CSF1R inhibitor that has been shown to change the morphology of microglia. However, the effects of Ki20227 on the progression of ischemic stroke are unclear. In this study, male C57BL/6 mouse models of focal cerebral ischemic injury were established through the occlusion of the middle cerebral artery and then administered 3 mg/g Ki20227 for 3 successive days. The results revealed that the number of ionized calcium-binding adaptor molecule 1/bromodeoxyuridine double positive cells in the infarct tissue was reduced, the degree of edema was increased, neurological deficits were aggravated, infarct volume was increased, and the number of peri-infarct Nissl bodies was reduced. The number of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells in the peri-infarct tissue was increased. The expression levels of Bax and Cleaved caspase-3 were up-regulated. Bcl-2 expression was downregulated. The expression levels of inflammatory factors and oxidative stress-associated factors were increased. These findings suggested that Ki20227 blocked microglial proliferation and aggravated the pathological progression of ischemia/reperfusion injury in a transient middle cerebral artery occlusion model. This study was approved by the Animal Ethics Committee of Lanzhou University Second Hospital (approval No. D2020-68) on March 6, 2020.

High-Throughput Screening and Characterization of Phenolic Compounds in Stone Fruits Waste by LC-ESI-QTOF-MS/MS and Their Potential Antioxidant Activities.

Stone fruits, including peach (Prunus persica L.), nectarine (Prunus nucipersica L.), plum (Prunus domestica L.) and apricot (Prunus armeniaca L.) are common commercial fruits in the market. However, a huge amount of stone fruits waste is produced throughout the food supply chain during picking, handling, processing, packaging, storage, transportation, retailing and final consumption. These stone fruits waste contain high phenolic content which are the main contributors to the antioxidant potential and associated health benefits. The antioxidant results showed that plum waste contained higher concentrations of total phenolic content (TPC) (0.94 ± 0.07 mg gallic acid equivalents (GAE)/g) and total flavonoid content (TFC) (0.34 ± 0.01 mg quercetin equivalents (QE)/g), while apricot waste contained a higher concentration of total tannin content (TTC) (0.19 ± 0.03 mg catechin equivalents (CE)/g) and DPPH activity (1.47 ± 0.12 mg ascorbic acid equivalents (AAE)/g). However, nectarine waste had higher antioxidant capacity in ferric reducing-antioxidant power (FRAP) (0.98 ± 0.02 mg AAE/g) and total antioxidant capacity (TAC) (0.91 ± 0.09 mg AAE/g) assays, while peach waste showed higher antioxidant capacity in 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay (0.43 ± 0.09 mg AAE/g) as compared to other stone fruits waste. Qualitative and quantitative phenolic analysis of Australian grown stone fruits waste were conducted by liquid chromatography coupled with electrospray-ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS) and HPLC-photodiode array detection (PDA). The LC-ESI-QTOF-MS/MS result indicates that 59 phenolic compounds were tentatively characterized in peach (33 compounds), nectarine (28), plum (38) and apricot (23). The HPLC-PDA indicated that p-hydroxybenzoic acid (18.64 ± 1.30 mg/g) was detected to be the most dominant phenolic acid and quercetin (19.68 ± 1.38 mg/g) was the most significant flavonoid in stone fruits waste. Hence, it could be concluded that stone fruit waste contains various phenolic compounds and have antioxidant potential. The results could support the applications of these stone fruit wastes in other food, feed, nutraceutical and pharmaceutical industries.

A Comparative Investigation on Phenolic Composition, Characterization and Antioxidant Potentials of Five Different Australian Grown Pear Varieties.

Pear (Pyrus communis L.) is widely spread throughout the temperate regions of the world, such as China, America and Australia. This fruit is popular among consumers due to its excellent taste and perceived health benefits. Various bioactive compounds, which contribute to these health benefits, have been detected in the pear fruits, including a range of phenolic compounds. Five Australian grown pear varieties, which include Packham's Triumph, Josephine de Malines, Beurre Bosc, Winter Nelis and Rico were selected for this study to examine the phenolic compounds in pears. Beurre Bosc exhibited the highest total polyphenol content (TPC) (3.14 ± 0.02 mg GAE/g), total tannin content (TTC) (1.43 ± 0.04 mg CE/g) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH) (5.72 ± 0.11 mg AAE/g), while the Josephine de Malines variety was high in total flavonoid content (TFC) (1.53 ± 0.09 mg QE/g), ferric reducing antioxidant power (FRAP) (4.37 ± 0.04 mg AAE/g), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (4.44 ± 0.01 mg AAE/g) and total antioxidant capacity (TAC) (5.29 ± 0.09 mg AAE/g). The liquid chromatography coupled with electrospray-ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS) data indicate that a total of 73 phenolic compounds were detected in Beurre Bosc (37 compounds), Josephine de Malines (34), Rico (22), Packham's Triumph (15) and Winter Nelis (9), respectively. From HPLC-PDA quantification, the Beurre Bosc pear variety showed significantly higher in phenolic acids (chlorogenic acid; 17.58 ± 0.88 mg/g) and while flavonoids were significantly higher in Josephine de Malines (catechin; 17.45 ± 1.39 mg/g), as compared to other pear varieties. The analyses suggest that the Australian grown pears might contain an ideal source of phenolic compounds which benefit human health. The information provided by the present work can serve as practical supporting data for the use of pears in the nutraceutical, pharmaceutical and food industries.

Pharmacological Targeting of CSF1R Inhibits Microglial Proliferation and Aggravates the Progression of Cerebral Ischemic Pathology.

Ischemic stroke can induce rapid activation of the microglia. It has been reported that the microglia's survival is dependent on colony-stimulating factor 1 receptor (CSF1R) signaling and that pharmacological inhibition of CSF1R leads to morphological changes in the microglia in the healthy brain. However, the impact of CSF1R inhibition on neuronal structures and motor ability after ischemia-reperfusion remains unclear. In this study, we investigated microglial de-ramification, proliferation, and activation after inhibition of CSF1R by a tyrosine kinase inhibitor (ki20227) in a mouse model of global cerebral ischemia induced by bilateral common carotid artery ligation (BCAL). In addition to microglial morphology, we evaluated the mRNA expression of cytokines, chemokines, and inflammatory receptors. Our results show that pharmacological inhibition of CSF1R in ischemic mice resulted in the blockade of microglial proliferation and a shift in microglial morphology reflected by excessive de-ramification and a more activated phenotype accompanied by an enhanced innate immune response. Furthermore, we show that pharmacological inhibition of CSF1R in ischemic mice resulted in the aggravation of neuronal degeneration and behavioral impairment. Intravital two-photon imaging revealed that although pharmacological inhibition of CSF1R did not affect the recovery of dendritic structures, it caused a significant increase in spine elimination during reperfusion in ischemic mice. These findings suggest that pharmacological inhibition of CSF1R induces a blockade of microglial proliferation and causes acute activation of the microglia accompanied by a severe inflammatory response. It aggravates neuronal degeneration, loss of dendritic spines, and behavioral deficits after transient global cerebral ischemia.

Lactic acid, neuron-specific enolase, and blood-brain barrier index after a severe traumatic brain injury: a prospective study.

OBJECTIVE: To explore the clinical significance of dynamic monitoring of cerebrospinal fluid (CSF) and serum Lactic acid（Lac), neuron-specific enolase (NSE), and the blood-brain barrier (BBB) index in evaluating the condition and prognosis after a severe traumatic brain injury (TBI). METHODS: A total of 52 severe TBI patients admitted to the Department of Neurosurgery within 24 hours after injury were dynamically monitored. CSF and serum samples were collected on the 1st, 3rd, and 7th day after a severe TBI to monitor the changes in Lac, NSE, and the BBB index. Intracranial pressure (ICP), Glasgow coma scale (GCS), and 6-month Glasgow outcome scale-extended (GOS-E) were tested. According to the results of GOS-E, the patients were divided into two groups (i.e. the poor prognosis group and good prognosis group). Statistical analysis was conducted to investigate the clinical significance of dynamic monitoring of CSF and serum Lac, NSE, and BBB index after a severe TBI. RESULTS: After a severe TBI, the levels of Lac, NSE, and BBB in CSF and serum were significantly higher than those in the normal range. Lac, NSE, and the BBB index did not correlate with ICP (except serum Lac) but had correlations with GCS and post-injury 6 months post-injury (except serum Lac). Moreover, the correlations between Lac, NSE, and BBB index were statistically significant (p < 0.05): CSF Lac and CSF NSE; CSF Lac and serum NSE; Lac and BBB index of CSF; Lac and BBB index of CSF; NSE and CSE of serum; CSF NSE and BBB index; and serum NSE and BBB index. Additionally, serum NSE is correlated with NSE in CSF (p < 0.05). CONCLUSION: After a severe TBI, dynamic monitoring of CSF and serum Lac, NSE, and BBB index has the potential to assess the condition, predict the prognosis, and have clinical significance.