We used inductively coupled plasma mass spectroscopy (ICP-MS) to determine macro- and microelements in CSF and plasma, and enzyme-linked immunosorbent assays (ELISA) to determine protein biomarkers of AD in CSF. This study included 193 participants . Simple correlation, as well as machine learning algorithms ), demonstrated that levels of heavy metals , essential metals , and essential non-metals  are positively associated with CSF phosphorylated tau isoforms, VILIP-1, S100B, NFL, and YKL-40 in AD.Various metals have been associated with the pathogenesis of Alzheimer’s disease (AD), principally heavy metals that are environmental pollutants  and essential metals whose homeostasis is disturbed in AD . Although there is evidence of the involvement of these metals in AD, further research is needed on their mechanisms of toxicity. To further assess the involvement of heavy and essential metals in AD pathogenesis, we compared cerebrospinal fluid (CSF) AD biomarkers to macro- and microelements measured in CSF and plasma. We tested if macro- and microelements’ concentrations , essential metals , essential non-metals , and other non-essential metals ) are associated with CSF AD biomarkers that reflect pathological changes in the AD brain (amyloid β Macroelements are those elements that the body needs more than any other mineral and include sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), chlorine (Cl), phosphorus (P), and sulfur (S). Microelements are those elements that are required for good health in very small amounts, such as chromium (Cr), copper (Cu), fluorine (F), iodine (I), iron (Fe), manganese (Mn), molybdenum (Mo), selenium (Se), zinc (Zn), and cobalt (Co). Some of these microelements are also metals and are regarded as essential for human health in trace amounts, for example, Fe, Zn, Cu, Mn, Cr, Mo, Se, and Co . However, non-essential metals are considered harmful to human health and the environment. These include four heavy metals: arsenic (As), cadmium (Cd), lead (Pb), and mercury (Hg) that can cause neurodegenerative changes .There is evidence that essential metal homeostasis is disturbed in the brain of AD patients ,5,6. Herhttp://www.alzforum.org/news/research-news/pbt2-takes-dive-phase-2-alzheimers-trial (accessed on 20 November 2022)). Clioquinol and PBT2 remove excess copper and zinc by acting as ionophores, and clioquinol probably also removes iron by chelation [in vivo and in vitro, and deferoxamine has also been tested on patients , and healthy controls (HC). We assessed such associations with AD-related pathological changes reflected by the levels of eleven AD biomarkers in CSF, whose association with AD is established. p-values ≤ 0.001. These correlations are presented in Several macro- and microelements correlated with CSF AD biomarkers. However, after applying Bonferroni correction for multiple comparisons, we considered statistically significant only those correlations with Principal component analysis (PCA) showed differences in all macro- and microelements measured in CSF  and plasma. Factor analysis of CSF macro- and microelements included 23 parameters that were classified into 6 groups (factors) after analysis . Factors181), Thr 231 (p-tau231), visinin-like protein 1 (VILIP-1), pregnancy-associated plasma protein A, pappalysin-1 (PAPP-A) and albumin, and negatively with the Aβ1–42/p-tau181 ratio. The metals measured in plasma (As and Hg) showed a positive association with VILIP-1 and neurofilament light chain (NFL) levels. PCA also showed that Ni measured in plasma was positively associated with various CSF AD biomarkers. In CSF, B and Li grouped together (Factor 3) and showed a positive association with NFL, S100 calcium-binding protein B (S100B), and PAPP-A.The results of linear regression analysis that compared the factors obtained by PCA analysis of CSF macro- and microelements with CSF AD biomarkers are given in APOE (apolipoprotein E) genotype were included in the analysis. Redescription mining gave us in total of 2648 redescriptions. Redescriptions that depict more closely: (1) AD patients, (2) both AD and MCI patients, (3) MCI patients, (4) MCI patients and HC, and (5) HC were extracted  was associated with Se and Cu in 52 and 41 redescriptions, respectively. When analyzing the association of CSF AD biomarkers with macro- and microelements measured in plasma, PAPP-A occurred together with the following elements (the number of redescriptions is shown in parentheses): Li (281), Ca (279), Na (192), P (191), Ni (177), Hg (172), Se (164), Sr (130), and Mo (125) (only the most significant associations are presented). Total tau (t-tau) occurred together with Ca (181), Li (166), P (126), and Na (125); YKL-40 with Ca (173), Li (171), P (129), and Na (118); p-tau231 with Li (168), Ca (159), P (122), and Se (116); and p-tau181 with Li (122), and Ca (125).Redescription mining showed an association of CSF AD biomarkers with macro- and microelements measured in CSF , and plaxtracted , Table 7181, p-tau231, VILIP-1, PAPP-A, and albumin, and negatively with the Aβ1–42/p-tau181 ratio. The metals measured in plasma (As and Hg) showed a positive association with VILIP-1 and NFL levels. PCA also showed that Ni measured in plasma was positively associated with various CSF AD biomarkers. The redescription mining algorithm successfully clustered individuals by the combination of CSF AD biomarkers with macro- and microelements measured in CSF, CSF AD biomarkers with macro- and microelements measured in plasma, and CSF AD biomarkers with macro- and microelements measured both in CSF and in plasma. Using this algorithm, we extracted those redescriptions that depict more closely: AD patients, both AD and MCI patients, MCI patients, MCI patients and HC, and HC. Additionally, redescription mining showed the association of Ca, Li, P, and Na measured in plasma with various CSF AD biomarkers.In this study, we used three different statistical methods to test the association of macro- and microelements with CSF biomarkers of AD. All three methods  demonstrated some association of macro- and microelements with CSF AD biomarkers. Macro- and microelements that positively correlated with a high number of CSF AD biomarkers were As, Hg, Zn, Cu, Fe, S, K, Se, Co, Mn, Ni, Na, Mg, Tl, and Li . PCA further confirmed the association of these macro- and microelements with CSF AD biomarkers. Following PCA, heavy metals are distinctly grouped . The metals measured in CSF  associated positively with p-tau1–42 (Aβ1–42) levels [1–42, and a positive correlation with CSF t-tau levels [1–42, t-tau and p-tau levels in 68 AD patients [1–42 levels [1–42 levels  [1–42, t-tau, and p-tau [1–42 levels and no association with CSF t-tau and p-tau181  levels . A studyu levels , while opatients . Strozyk2 levels . A recen in CSF) . Kushnirnd p-tau , while M 413 HC; ). Blood u levels , while Cu levels . CSF Mn 2 levels . Blood M0 levels , while s0 levels . Mielke  in life . Shams ein ratio .Rhizoprionodon terraenovae) showed that brain Hg levels positively correlated with CSF S100B levels [Additionally, it was shown that VILIP-1 is a neuronal calcium sensor protein that contains an EF-hand structural domain. This domain can bind metal ions , such asB levels , while rB levels ). StudieB levels , while iB levels . Levels B levels . A studyB levels , while mB levels . AdditioB levels  while inB levels  and neonB levels , this trB levels . RegardiB levels , while AB levels .181 strongly correlated with Cu, p-tau199 with As, and p-tau231 with Co measured in CSF. Vinceti et al. also reported a positive association of CSF Se with CSF p-tau181 levels [2S) is beneficial in AD [2S levels in AD patients [2S can be produced by some bacteria [Porphyromonas gingivalis and Helicobacter pylori; reviewed in [All phosphorylated tau isoforms showed a strong positive correlation with Se, while p-tau1 levels . However1 levels ,61,62,631 levels . Previou1 levels ,66 and A1 levels ,67,68 in1 levels . The neu1 levels . Similar1 levels , selenop1 levels , and as 1 levels ,73,74,751 levels . However1 levels . To our 1 levels . Most st1 levels ). Thus, 1 levels . A recenpatients . Additiobacteria  that haviewed in ). Damageiewed in . Whether1–42, p-tau, and t-tau  [The strength of our study is in the analysis of 24 macro- and microelements measured in CSF and 21 measured in plasma, in addition to 11 CSF AD biomarkers determined in nearly 200 participants. Only two studies ,32 that cipants) . A limitIn conclusion, our study showed that essential metals , heavy metals , and essential non-metals  are positively associated with CSF AD biomarkers, mainly phosphorylated tau isoforms, VILIP-1, S100B, NFL, and YKL-40, suggesting new diagnostic opportunities and therapeutic targets in future studies on AD.g for 10 min, CSF samples were aliquoted in polypropylene tubes and stored at −80 °C. Venous blood samples were collected from 143 participants in the morning on an empty stomach. Samples were collected using plastic syringes (with 1 mL of acid citrate dextrose as an anticoagulant). Thrombocyte-free plasma was collected by centrifugation, first at 1100× g for 3 min and then at 5087 g for 15 min. Plasma samples were stored at −20 °C. We included 193 patients who were admitted to the University Hospital Center Zagreb and General Hospital Varaždin. Patients underwent thorough neurological testing, including MMSE, complete blood tests , and VDRL testing for syphilis, as described previously . NINCDS-v/v) Triton X-100, 0.7 mM ammonia, and 2 µg/L of internal standards  in ultrapure water. We used a MicroMist nebulizer combined with a Peltier standard quartz spray chamber  and a quartz torch with a 2.5-mm diameter injector with a Shield Plate system and Ni sampler and skimmer cones. Daily optimization of ICP-MS working conditions was achieved using a tuning solution of 1 µg/L 7Li, 59Co, 89Y, 140Ce, and 205Tl. HVAC systems  combined with HEPA filters were used for sample preparation and analysis. Quantification of elements concentration in samples was done by the standard addition method . Commercially available reference materials were used to confirm the accuracy of the measurements: ClinChek Serum Controls (Level I and II) and ClinChek Plasma Controls (Level I and II) from RECIPE ; Seronorm Trace Elements Serum (Level I and II) .Inductively coupled plasma mass spectroscopy (ICP-MS) was used for the measurement of CSF and plasma levels of As, B, Ca, Cd, Co, Cu, Fe, Hg, Li, Mg, Mn, Mo, Na, Ni, P, Pb, S, Se, Sr, Tl, and Zn, and CSF levels of Al, Ba, and K . Cr leve1–42, VILIP-1, t-tau, p-tau181, p-tau231, tau phosphorylated at Ser 199 (p-tau199), NFL, S100B, YKL-40, PAPP-A, and albumin. Following ELISA kits were used for the measurement of CSF AD biomarkers; Aβ1–42 , p-tau181 (Innotest Phospho-Tau (181P), Fujirebio), p-tau231 (Tau (pT231) Phospho-ELISA Kit, Human, Thermo Fisher Scientific, Waltham, MA, USA), p-tau199 , t-tau , VILIP-1 , NFL , S100B , YKL-40 (Chitinase 3-like 1 Quantikine ELISA Kit R&D Systems), PAPP-A , and albumin .Eleven AD biomarkers were measured in CSF by the enzyme-linked immunosorbent assays (ELISA); Aβ2 test of sphericity and the Kaiser–Meyer–Olkin index. The list of 23 macro- and microelements measured in CSF  and 21 macro- and microelements measured in plasma were reduced to key factors (groups). If a factor loading for an element was ≥0.4, it was considered that the element loads in the given group (factor). Factor loading measures the correlation between the element and a factor. Additionally, factor-specific scores were calculated for each individual. Multiple linear regression analyses were used for testing the association between PCA-obtained macro- and microelements’ groups (factors) and CSF AD biomarkers (with CSF AD biomarkers as dependent variables and macro- and microelements’ groups as independent variables). Statistical analyses were performed using SPSS 19.0.1  and R , with the α value set at 0.05 for statistical significance.Correlation (Spearman’s and Pearson’s correlation coefficient) and linear regression were used for testing the association between CSF AD biomarkers and macro- and microelements measured in CSF and plasma. Additionally, principal component analysis  was used to reduce the dimensionality of macro- and microelements measured in CSF and plasma. PCA analyses were performed for the macro- and microelements measured in CSF, and for the macro- and microelements measured in plasma. Orthogonal (varimax) rotation was used in PCA, while the adequacy of test items and sample size for factor analysis was assessed by Bartlett’s χThree redescription sets were created using the redescription mining algorithm CLUS-RM ,91. The In all experiments, we performed 10 runs with different random initialization and 30 iterations of the CLUS-RM algorithm for each run. In each run, the algorithm creates one starting initial clustering of patients that is used to create initial pair of Predictive Clustering trees (PCTs). Initial pairs of PCTs are used as a starting point for a sequence of iterations  that create two pairs of matching PCTs per iteration, used to construct redescriptions. A supplement random forest, consisting of twenty trees and a conjunctive refinement procedure was used to obtain more diverse and accurate redescriptions . Thep-value) measures how probable it would be to obtain a redescription at random (by a random choice of rules that form it), so that each rule in a randomly created redescription describes the same number of patients as the original, and that the resulting redescription has a support set size equal or larger to the support set size of the original redescription [p-value of 0.01. To maximize interpretability and to allow analyses of strong associations between different indicators (measurements), we construct redescriptions with rules containing only logical AND operator. As it can be seen from the example above, when only AND operator is used, each patient described by a rule must have all measurements for all indicators in the exactly specified interval. On the other hand, the rule NOT PAPP-A (122.39–511.26) describes all patients such that either PAPP-A < 122.39 OR PAPP-A >511.26, and understanding such rules would be much more difficult.In this work, we use a minimal support set size of 30 and a maximal support set size of 155 for the first set, a minimal ort set size of 30 and a maximal support set size of 115 for the second set, and a minimal support set size of 20 and a maximal support set size of 110 for the third set. Redescription accuracy measures what fraction of patients, described by either of the rules forming some redescription, is described by all these rules. The corresponding measure that captures this property is called the Jaccard index . In thiscription . In this


In addition, we choose a popular seaweed extract—fucoidan was extracted from the same brown algae—Sargassum aquifolium collected from Taiwan-Pingtung-Kenting-Chuanfan Rock. In this research, we used acid hydrolysis to prepared two different molecular weight fucoidan, the small molecular fucoidan (Fus) as drug, and the molecular weight approximately 1 kDa fucoidan (Ful) as the nanoparticle gatekeeper, and as targeting molecule for overexpressed P-selectin on the surface of the metastatic tumors. The results of the cell cytotoxicity experiment showed that HCT116 cancer cells have a survival rate of approximately 58.12% when treated with 200 μg/mL fucoidan. Dual-imaging rice husk mesoporous silica nanoparticles (rMSN-EuGd) were modified with 1 kDa fucoidan (Ful) as the gatekeeper and target, and the small molecule fucoidan (Fus) was loaded into nanoparticles (Ful-Fus@rMSN-EuGd) at a concentration of 200 μg/mL. The HCT116 cancer cells had a survival rate of approximately 55.56%. The cell cytotoxicity experiment results show that Ful-Fus@rMSN-EuGd can improve the anticancer effect of fucoidan, and the nanoparticle drug delivery system using fucoidan as a drug, target, and gatekeeper was successfully synthesized.In this study, we used the nanoparticle delivery system to reduce the side effect of conventional cancer treatment- radiation therapy and chemotherapy. We used rice husk silicon source mesoporous silica nanoparticle doped in Eu Cancer has always been the one of the leading causes of death globally, and cancer treatment methods include chemotherapy , surgicaSargassum aquifolium collected from Chuanfan Rock in Kenting-Taiwan to carry out the experiments. There are various extraction methods used to produce fucoidan, such as the acid extraction method [Taiwan is an island surrounded by the sea, which provides a lot of algae resources for algae research –13. Durin method , enzyme n method , and hotn method –21, and 3+ to make a metal–ligand complex. Fucoidan itself is negatively charged because it contains sulfate. Sulfate (SO42−) interacts with the metal–ligand complex formed by EDA to become the gatekeeper of the nanoparticle, and it will decompose due to the H+ ion in the acidic environment of the tumor tissue to release the drug from the carrier [In metastatic tumor cells, P-selectin is overexpressed , and poo carrier . In thisNext, mesoporous silica nanoparticles were used as the combined therapy nanocarriers. To improve the advantages of traditional mesoporous silica nanoparticles, we used recycled agricultural waste rice husk as the silica source to replace tetraethyl orthosilicate. Rice husk is an agricultural waste that cannot easily be decomposed. At present, it is disposed of using a very environmentally friendly incineration method. It contains organic matter such as cellulose and lignin and minerals such as silicon dioxide and trace elements. The ash content of the calcined rice husk obtained afterward is not only 10–20% higher than that of other biomass fuels, but more importantly, silicon dioxide accounts for 80% of the ash . Rice huThis study also used the lanthanide metals europium (Eu) and gadolinium (Gd) to confirm the treatment effect and in vivo location tracking. By doping europium and gadolinium ions into the rMSN structure to archeive red fluorescence  and magn3·6H2O) and 99.9% Gadolinium (III) chloride hexahydrate (GdCl3·6H2O) were purchased from Alfa Aesar; Sodium hydroxide (NaOH), Ethanol (C2H5OH), calcium chloride (CaCl2), barium chloride (BaCl2), gelatin, hydrochloric acid (HCl), sodium sulfate (Na2SO4), trifluoroacetic acid (TFA), 1-Phenyl-3-methyl-5-pyrazolone (PMP), methanol (CH3OH), chloroform (CHCl3), acetonitrile (ACN), sodium nitrate (NaNO3), sodium hydrogen phosphate (Na2HPO4), sodium chloride (NaCl), potassium dihydrogen phosphate (KH2PO4) and dimethyl sulfoxide (DMSO) were purchased from J.T. Baker; potassium chloride (KCl) were purchased from Mallinckrodt Chemical; minimum essential medium (MEM) were purchased from Biowes; fetal bovine serum (FBS), non-essential amino acids (NEAA), antibiotic–antimycotic (AA) and trypsin were purchased Gibco; mouse fibroblasts cell (L929) and human colorectal carcinoma cells (HCT116) were purchased ATCC; Sargassum aquifolium from Kenting-Chuanfan Rock.98% Tetraethyl orthosilicate (TEOS), (3-Glycidyloxypropyl) trimethoxysilane (GPTMS), ethylenediamine (EDA), 98% Hexadecyl trimethyl ammonium bromide (CTAB), mannose, fucose, glucose, galactose, xylose, McCoy’5A medium and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich; 99.9% Europium (III) chloride hexahydrate  × 100%. .This experiment uses the phenol–sulfuric acid colorimetric method to estimate the total sugar content of the molecule.The 10 mg sample (Fucose is the standard) was dissolved in 10 mL of deionized water, 0.4 mL samples were mixed with 20 μL 5% phenol and 1 mL of concentrated sulfuric acid, and then stirred at room temperature 20 min, later measure the 490 nm absorbance.This experiment uses the turbidimetric method to estimate the concentration of sulfate in the sample to calculate the content. The principle is the barium ions added to the solution contain sulfate, the barium sulfate will form and the solution becomes cloudy.2 into the solution at room temperature, and store at 4 °C. Then 0.1 g sample dissolved in 10 mL HCl and 100 °C refluxed for 1 h, mixed 0.2 mL sample solution with 1 mL BaCl2-Gelatin solution for 15 min, and measured the absorbance at 360 nm. For the standard curve, we used sodium sulfate to replace the sample.First, 0.5 g gelatin was dissolved with 100 mL 60 °C deionized water, added 0.5 g BaCl−1 and the resolution is 8 cm−1, scanning 16 times.The crude fucoidan was ground with grade potassium bromide  and pressed into a disc under vacuum using a pelletizer. In addition, 0.1 g KBr is used as a background value. The FTIR spectra were obtained using a FTIR spectrophotometer (BRUKER TENSOR Series FT-IR Spectrometer). The infrared spectrum was recorded the wavenumber from 400 and 4000 cmThe 20 mg sample was mixed with 5 mL 2 M TFA, and hydrolyzed for 4 h at 120 °C. After cooling to room temperature, neutralized the hydrolysate to pH 7 for later subsequent experiments.At 70 °C water bath, 1 mL of the hydrolysate with 0.5 mL 0.03 M NaOH and 0.05 M PMP-methanol mixed for 60 min. After cooling, the samples with 0.5 mL 0.03 M HCl and then evaporated in vacuo, and then used 1 mL deionized water dissolved. Add 1 mL of deionized water and chloroform to remove excess derivatizing agent. After centrifugation , the supernatant through 0.22 μm membrane filter, the filtrate was collected for HPLC analysis.The monosaccharides composition of fucoidan determined by high performance liquid chromatography (HPLC) was performed using a Hypersil BDS-C18 column (4.6 mm × 250 mm) at a wavelength of 245 nm. The flow rate was 1.0 mL/min. The monosaccharide mixture containing fucose, xylose, glucose, galactose, and mannose was used as the standard.3 (0.1%) solution, the flow rate was 1 mL/min. Then, 1 mg of the sample was dissolved in 1 mL of deionized water and filtered through a 0.22-μm filter. We used pullulan (0.18–1284 kDa) as standards.Molecular weight (MW) was determined by using a high-performance liquid chromatogram with a refractive index detector (HPLC-RI) using an OHpak SB-806M HQ column (8.0 mm × 300 mm). Mobile phase: NaNO3⋅6H2O and GdCl3⋅6H2O were added at 80 °C and stirred. After that, it was washed several times, and finally calcined at 650 °C for 6 h to obtain MSN-EuGd [To synthesize MSN-EuGd, 97 mL of deionized water was mixed with 1.4 mL of 1 M NaOH and 1 g of CTAB stirring at 80 °C for 1 h until clear, then add 1 mL of 98% TEOS, 3 mL of 0.05 M EuClMSN-EuGd .Adding 10% HCl solution to the rice powder and reflux for 48 h, then make the solution neutral, and then dry and calcine to obtain a white powder. 3.8 g of white powder and 1 g of NaOH were added to 38 mL of deionized water, and then refluxed and stirred at 110 °C for one day to obtain a sodium silicate solution (rice husk).3⋅6H2O and GdCl3⋅6H2O were added at 80 °C and stirred. After that, it was washed with water and ethanol several times, and finally calcined at 650 °C for 6 h to obtain rMSN-EuGd [97 mL of deionized water was mixed with 1.4 mL of 1 M NaOH and 1 g of CTAB stirring at 80 °C until clear, then add 3 mL sodium silicate solution (rice husk), 3 mL of 0.05 M EuClMSN-EuGd .The rMSN-EuGd was added to anhydrous toluene, GPTMS was added dropwise, and then inert gas was continuously introduced at heated 85 °C for 12 h. Then the solution was cooled at room temperature and washed with toluene and ethanol several times in sequence. After drying, GPTMS-rMSN-EuGd was obtained.The GPTMS-rMSN-EuGd is added to THF, ultrasonically shaken to disperse, add EDA dropwise, heated to reflux at 45 °C, then cool the solution at room temperature and wash it with ethanol several times, then dry the product to obtain EDA-rMSN-EuGd.3, stir at room temperature to form Fe3+-EDA complex, precipitate the supernatant to obtain the product, wash with deionized water, add Ful, the product was captured with deionized water several times, and then lyophilized to obtain the final product Ful-Fus@rMSN-EuGd.The EDA-rMSN-EuGd and Fus were added to deionized water, shaken to disperse, stirred at room temperature, locked several times with deionized water and ethanol, and freeze-dried to obtain the product Fus@rMSN-EuGd. The Fus@rMSN-EuGd to deionized water, then add 24 mM FeCl3 stir at room temperature to form Fe3+-EDA complex. Centrifuge to remove the supernatant to obtain the product, wash with deionized water and ethanol for several times, and freeze-dry the product to obtain the final product Ful-Rh@rMSN-EuGd.The EDA-rMSN-EuGd and Rhodamine to deionized water, ultrasonically disperse, and stir at room temperature. After 24 h, wash with deionized water and ethanol for several times, then freeze-dry the product to obtain Rh@rMSN-EuGd. The Rh@rMSN-EuGd add to deionized water mixed with 24 mM FeClIn the experiment, two groups of 20 mg Ful-Rh@rMSN-EuGd were pressed into ingot tablets using an inverting machine, and then put into pH = 7.4 and 5.5 PBS respectively, then shaken evenly, and aspirated 100 μl of the supernatant and transferred to a 96-well plate. Measured the absorbance at 652 nm, and substitutes it into the calibration curve to calculate the total drug release in a cumulative manner.3+ and Gd3+ ions doped into rMSN were measured by inductively coupled plasma mass spectrometry  and reported as mass percentages.Use Bruker D2 phase instrument to perform X-ray powder diffraction (XRD) to identify the property of the pore of MSN-EuGd and rMSN-EuGd. Particle size and Zeta potential analysis were performed using dynamic light scattering . Tecnai F30 instrument was used to take transmission electron microscope (TEM) and energy dispersive X-ray (EDX) spectra. Barrett-Joyner-Halenda (BJH) analysis  was used for nitrogen adsorption isotherm analysis. The surface area and pore size distribution curves of MSN-EuGd and rMSN-EuGd are determined by Brunauer–Emmett–Teller (BET) method. The BRUKER TENSOR II series spectrometer  was used to confirm the Fourier Transform Infrared (FTIR) spectrum of the characteristic peak of rMSN-EuGd and Ful-Fus@rMSN-EuGd. A Jasco FP-6300 photoluminescence spectrophotometer  was used to record the luminescence excitation spectrum at an excitation wavelength of 395 nm. Use Netzsch TG 209 F3 equipment to obtain thermogravimetric analysis (TGA) curve to determine the drug loading efficiency when the temperature rises to 700 °C. A 7 T scanner  was used to comfirm the rMSN-EuGd MRI imaging ability. The concentrations of Eu2 at 37 ℃. The colon cancer cell line HCT116 was maintained in F-12K medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% antibiotics (AA) in an incubator with 5% CO2 at 37 °C.Normal L929 cells were maintained in MEM medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% antibiotics (AA) in an incubator with 5% COA normal cell model (L929 cells), and cancer cell models (HCT116 cells) were selected as test cells. The experimental steps were as follows:4 per well of cells were seeded in 96-well culture plates, overnight incubated at 37 °C. After overnight incubation, exposed to serial concentrations of materials 4 h, and add 50 μl MTT to each well and incubated for 4 h. Then, add 100 μl DMSO to each well, shake for 15 min, and measured the absorbance values at 540 nm by used enzyme immunoassay analyzer (ELISA reader). The ability of cells to reduce MTT can be used as an indicator of cell viability [For both L929 and HCT116 cells, 2 × 10iability .The cell survival rate is calculated as follows:4 cells per well incubate for overnight, each well was added with a mixture of materials and culture medium for co-cultivation, after the completion of the culture, aspirate the culture medium and rinse with PBS twice, then add 3.7% Formaldehyde/PBS solution and react for 10 min. After the reaction is completed, absorb the Formaldehyde solution and rinse with PBS twice, then add 0.1% Triton X100/PBS. After the reaction is completed, absorb the Triton X100 solution and rinse with PBS twice. Stain the cell nucleus with DAPI/PBS and rinse twice with PBS again, place the removed cover slip on the glass slide pre-dropped with mounting glue and mount it on the slide with nail polish, and use Confocal Laser Scanning Microscopy, CLSM (Leica TCS SP5) Observe the phagocytosis of cells and materials.Put the sterilized glass coverslip into a 24-well culture dish. Then planted 2 × 10Sargassum aquifolium were collected Pingtung-Chuanfan Rock in March 2018, and then the fucoidan was obtained by acid extraction. As shown in Table S. aquifolium was 35.52 ± 0.25 wt %, since the sulfate content is known to be related to the collection site [S. aquifolium in Chuanfan rock is 33.20 ± 0.91 wt %.ion site . To analTo determine the monosaccharide composition of the fucoidans after acid extraction, they were analyzed by HPLC. Table −1 shows the O–H group of the polysaccharide, the peak at 2960 cm−1 indicates the C–H of the carbohydrate, the two peaks at 1641 cm−1 and 1420 cm−1 are C=O, and the peaks at 1260 and 1056 cm−1 are S=O, which are the characteristic peaks of fucoidan and sulfated polysaccharides. The peak at 802 cm−1 is C–O–S, confirming the sulfate group, and thus, the results of this study are consistent with the literature [The FTIR spectrum of fucoidan extracted by acid extraction is shown in Fig. terature .Fig. 1FTFigure 2/g and 670.18 m2/g, and the pore volume are 0.60 cm3/g and 0.55 cm3/g. The inner wall diameters of MSN-EuGd and rMSN-EuGd are 2.37 nm and 1.76 nm. From Table BET was used to analyze MSN-EuGd and rMSN-EuGd, Fig. Through the use of TEM to observe and confirm the structure and size of the nanoparticles , Fig. 2cThe particle size and surface charge of rMSN-EuGd were measured by DLS. As Table Energy dispersive spectroscopy (EDS) was used to analyze the elements contained in rMSNs and rMSN-EuGd. As Fig. 3+ receives 395 nm UV, the energy level will transition from 5D0 → 7F2 (611 nm) to the red emission wave [3+ is successfully doped into rMSNs. After confirming the luminescence characteristics of rMSN-EuGd, we used a noninvasive in vivo molecular imaging system  to observe the luminescence properties of rMSN-EuGd. Figure A photoluminescence spectrometer (PL), was used to observe the emission characteristics of rMSN-EuGd and the results are shown in Fig. ion wave . If the 3+ paramagnetism, and five different Eu3+ and Eu3+ were synthesized experimentally. Gd3+ doping ratio of rMSN-EuGd, Eu3+: Gd3+  = 1: 0, Eu3+: Gd3+  = 2: 1, Eu3+: Gd3+  = 1: 1, Eu3+: Gd3+  = 1: 2 and Eu3+: Gd3+  = 0: 1. Five groups, as shown in Fig. 3+ and Gd3+ ratio configurations. As the ratio of Gd3+ increases, it tends to become paramagnetic. These experimental results prove that rMSNs doped with Gd3+ has paramagnetism.Next, a superconducting quantum interference device (SQUID) was used to confirm whether rMSN-EuGd exhibits Gd3+ is paramagnetic, it is often used as an imaging agent for magnetic resonance imaging (MRI) in medicine. After confirming by SQUID that rMSN-EuGd has paramagnetism, MRI can be used to confirm whether rMSN-EuGd doped with lanthanide metal ion Gd3+ can also be used for in vitro MRI. Set the experimental parameters in a 7 T magnetic field, TR/TE = 300 ms, FOV = 7 cm, NEX = 1, slice thickness = 2.00 mm, matrix = 256 × 256, and the material concentration is from 0.25 to 4 mg/mL. Figure Because Gd−1, Si–O–Si signal at 1100 cm−1, and Si–OH signal at 960 cm−1 [−1 and 2877 cm−1 due to the stretching vibration signal of the C–H bond, which means that GPTMS [−1, and there is a strong C–H absorption peak of sugar molecules at 1420 cm−1, which proves that the sample contains fucoidan [Fourier transform infrared spectroscopy (FTIR) was used to analyze the characteristics, chemical compounds, and functional groups of rMSN-EuGd and Ful-Fus@rMSN-EuGd. Figure 960 cm−1 ; Ful-Fusat GPTMS  is succefucoidan .Fig. 5a For determined the surface modification rate and drug loading rate of rMSN-EuGd, GPTMS-rMSN-EuGd, EDA-rMSN-EuGd, Fus@rMSN-EuGd and Ful-Fus@rMSN-EuGd through the thermogravimetric analysis. The analysis results of materials are shown in Fig. To prove that Ful-Fus@rMSN-EuGd can control drug release in different pH value environments, this experiment loaded rhodamine  instead of Fus and prepared Ful-Rh@rMSN-EuGd. The drug release experiment was conducted in two PBS solutions at pH = 5.5 and pH = 7.4. From the results in Fig. To determine whether the nanoparticle material has good biocompatibility, this study was conducted by culturing the nanoparticles with mouse fibroblasts (L929) and human colon cancer cells (HCT116) for 72 h to confirm whether the materials were compatible with L929 and HCT116 cells. From Fig. Sargassum aquifolium is non-cytotoxic to normal cells, Fig. 50) of Fus on HCT116 cells was 6 × 102 µM. The concentration of fucoidan was 200 μg/mL, and the survival rate of the HCT116 cells was 58.12%.To verify that fucoidan acid extracted from Taiwan-Pingtung-Kenting-Chuanfan rock collected from Sargassum aquifolium has a relatively low molecular weight after two hours of acid hydrolysis. Most fucoidans with low molecular weights have better anticancer activity than those with high molecular weights [The anticancer activity is affected by many factors, and one of these factors is using a better degradation method to reduce the molecular weight of fucoidan to increase its drug activity . We spec weights . In thisTo confirm that the materials can be taken up by cells, this experiment compared the results of L929 and HCT116 cells cultured with rMSN-EuGd and Ful-rMSN-EuGd for 4 h. Figure As the experiment results, we successfully synthesized the rice husk silica source mesoporous silica nanoparticle with dual imaging functions, and compared to other research, the rice husk silica source appears more environmentally friendly. But the size of nanoparticles still needs to improve smaller particles size to increase the cellular uptake –50 and gPrevious experiments have focused on combining fucoidan in a therapeutic system to obtain gatekeeper , target Therefore, renewable resources and marine active extracts still have a lot of value worthy of research.3+ and Gd3+ to synthesize the rice husk mesoporous silica nanoparticles (rMSN-EuGd) for required in vivo fluorescence and magnetic resonance dual imaging functions. Then, fucoidan successfully was loaded into and modified on the surface of rMSN-EuGd as an anticancer drug, gatekeeper, and target molecule. Modified large molecular fucoidan  on the surface of the nanoparticles with EDA combined with Fe3 + to form a metal coordination bond complex. From the result of the drug release system, the Ful-Rh@rMSN-EuGd was easily released under the pH 5.5 PBS environment relative to pH 7.4 which proves that the material has a controlled release function.In this study, fucoidan was used as an anticancer drug, nanoparticle carrier gatekeeper, and target molecule. This study used rice husk as biological silica sources and doped EuThe cell viability results showed that the survival rate of HCT116 cells was 58.12% when cocultured with 200 mg/mL fucoidan, and 55.56% when cocultured with 200 μg/mL Ful-Fus@rMSN-EuGd. From the TGA results, the amount of fucoidan loaded in 200 μg Ful-Fus@rMSN-EuGd was 10.9 μg/mL, which proves that loading fucoidan into the nanoparticle carrier can achieve better results with a smaller amount of drug. Finally, confocal laser scanning microscopy imaging confirmed that Ful-rMSN-EuGd targets HCT116 cells. The signal of Ful-rMSN-EuGd overlapped with that of HCT116 cells, but there was no such phenomenon in L929 cells, and rMSN-EuGd did not overlap with the HCT116 cell signal, which proves that the surface-modified fucoidan material can target HCT116 cells via targeting the overexpressed P-selectin.Hopefully, this biosource nanodrug carrier can successfully become a potential delivery material for cancer treatment.


Levels of phospholipid (PL) and sphingomyelin (SM) species were decreased in CSF of RTT patients, whereas the lipidomics profile of plasma samples was unaltered in RTT patients compared to healthy controls. This study shows that the CSF lipidomics profile is altered in RTT, which is the basis for future  studies to validate selected lipid species as CSF biomarkers for RTT. Rett syndrome (RTT) is defined as a rare disease caused by mutations of the methyl-CpG binding protein 2 (MECP2). It is one of the most common causes of genetic mental retardation in girls, characterized by normal early psychomotor development, followed by severe neurologic regression. Hitherto, RTT lacks a specific biomarker, but altered lipid homeostasis has been found in RTT model mice as well as in RTT patients. We performed LC-MS/MS lipidomics analysis to investigate the cerebrospinal fluid (CSF) and plasma composition of patients with RTT for biochemical variations compared to healthy controls. In all seven RTT patients, we found decreased CSF cholesterol levels compared to age-matched controls ( MECP2) located on chromosome X [Rett syndrome OMIM 312750, RTT) is defined as an orphan disease with a prevalence of 1:10,000 to 1:15,000 live female births [2750, RTTmosome X ,5. In thmosome X . Symptoms in RTT progress over time and appear in four progressive stages. Following a period of seemingly normal infantile psychomotor development which lasts for 6–18 months after birth, the disease is characterized by a phase of stagnation with developmental progress delay . In the stage of rapid regression acquired motor and mental skills are lost and breathing abnormalities and seizures occur. These stages are followed by a plateau stage and a stage of motor deterioration. Epilepsy occurs in 60–80% of all cases with RTT whereas incidences are even higher in early onset RTT and in RTT with more severe developmental disabilities . Due to Mecp2-null mice display perturbed lipid homeostasis, including high serum triglyceride and cholesterol levels, and elevated cholesterol in whole brain homogenates. Further, Buchovecky et al. showed that nonsense mutations in Squalene monooxygenase, one of the rate-limiting enzymes in cholesterol biosynthesis, as well as statin treatment rescue a disrupted lipid metabolism and improve symptoms in Mecp2-null mice [Previous studies in RTT model mice reported an altered cholesterol metabolism similar to RTT patients ,14,15. Mull mice . Additioull mice . To dateMECP2 variants are classified as VUS [Multi-omics approaches, including Next Generation Sequencing (NGS) and mass spectrometrical (MS) analysis, offer enormous potential for in-depth analysis of rare diseases. The investigation of disease-associated pathomechanisms is especially important for the development of treatment options but can also be useful in case NGS reveals variants of unknown significance (VUS). To date, 215 d as VUS . Sophistd as VUS . Deep biRecent advances in MS, bioinformatics tools and software enable huge diagnostic progress through an unbiased, comprehensive and rapid analysis of the global metabolic content of a distinct biological sample. The metabolome represents the global spectrum of low molecular-weight (<1 kDa) metabolites and provides a snapshot of its biochemical composition in the respective body fluid (5). As such, alterations of the metabolome reflect genomic, transcriptomic and proteomic changes and can accelerate the diagnosis of neurometabolic disorders . LipidomThe aim of this study was to establish metabolomics/lipidomics profiles of CSF and plasma samples from RTT patients and healthy individuals in order to obtain detailed and deep insights into metabolic perturbation. In this study, CSF samples were taken from 13 female disease controls and seven female patients suffering from RTT with a median age of 12 years and 13 years, respectively A. AdditiR and the lipidr package [Data obtained by UHPLC-MS/MS underwent metabolite and lipid identification with the Lipid Data Analyzer (LDA) software , followed by statistical univariate and multivariate analysis with the program  package . In a pi package . OPLS-DA analysis revealed a clear separation of the patient group (blue) versus the control group (red) in both, the CSF A and pla2 values of total area counts and are additionally listed as Lipidomics data obtained from UHPLC-MS/MS analysis further underwent univariate analysis using Wilcoxon rank-sum test using FDR as multiple comparison correction and are depicted as boxplots. Results are presented as logLipids detected in the CSF and plasma of RTT patients and controls are presented as lipid classes and lipid species. Results are shown as lipid classes (=sum of lipid species detected in one lipid class) should provide an overview of lipids detected in CSF and plasma samples and changes in the lipid distribution. We identified 14 lipid classes in the CSF A and 16 Significantly changed lipid species identified in the CSF of RTT patients compared to healthy controls are depicted in UVA (pFDR) and MVA (Variable Projection of Importance-VIP) metrics were compared graphically . In totaPrevious studies on metabolic perturbations in RTT reported, that a subset of patients shows elevated levels of peripheral cholesterol, triglycerides and/or LDL upon routine diagnostic analysis ,25. To dMecp2-deficient mice, which showed an increase of cholesterol in whole brain homogenates, whereas a reduction of cholesterol precursors and decreased cholesterol synthesis rate to unaffected littermates were also reported [Mecp2-deficient rat oligodendrocytes showed downregulation of myelin gene expression and hence an impact on myelination [In this study, we describe for the first time a detailed lipidomics analysis of plasma and CSF in RTT patients. MVA, but not UVA lipidomics analysis confirmed increased DG and TG species in the plasma. As a novelty, MS revealed decreased cholesterol levels in the CSF of RTT patients. This is in contrast to studies in reported ,32,33. Ireported . Interesreported , and mayreported . The brareported . Cholestreported . Any choreported . Imagingreported . In contlination . Mecp2-deficient mice, small abnormalities in cholesterol metabolism were found to have major effects on neuronal function, but the regulatory link is yet to be elucidated [In addition to its function in myelin sheaths, cholesterol is an essential component of the membranes of astrocytes and neurons. Even though imaging studies of patients with RTT display no evidence of malformation or neurodegeneration, cell morphologic changes with smaller and more closely packed neurons with reduced dendritic complexity have been shown . In Mecpucidated ,12. MECP2 mutation [Our lipidomics study demonstrates that beyond cholesterol various other membranes- and myelin-forming lipid classes and species were reduced in CSF of RTT patients. The most significant finding in lipidomics analysis was the reduction of SM in the CSF compared to healthy controls. Inline, Cappuccio et al. detected altered sphingolipid metabolism using 14 plasma samples of RTT patients carrying the mutation . Furthermutation . SM is mmutation ,45. In amutation ,47,48. Imutation . These pmutation . It coulMecp2-knockdown mice reported a crucial role of the PC synthesis pathway by enabling improved neuronal morphology [Mecp2-deficient mice, studies on PL levels were contradictory with Seyfried et al. reporting no change in brain PL levels [In addition, MS analysis in the CSF of RTT patients compared to healthy controls revealed a decrease of various PL and ether-linked PL including plasmalogens. PL and their subclass, ether-linked PL, are one of the three major classes of membrane lipids in myelin sheaths and are responsible for membrane mobility, cholesterol trafficking, exo- and endocytosis due to their fusogenic ability . Previourphology . Our liprphology . In MecpL levels , whereasL levels . Mecp2-deficient mice, metabolic fingerprinting of the cortex showed affected amino acid, carbohydrate, as well as lipid metabolism and altered levels of several neurotransmitters [Previously reported metabolomics analysis on the plasma of 34 RTT patients identified metabolic pathway abnormalities in oxidative stress, mitochondrial dysfunction, and alterations in the gut microbiome compared to their 37 unaffected age- and gender-matched siblings . In Mecpsmitters . In thisMECP2 [MECP2 variants, including 215 variants of unknown significance (VUS) have been described [To date diagnosis for RTT is based on proof of pathogenic variants in the regulatory gene function of MECP2 . Accordiescribed . Lipidom®, Novartis, Basel, Switzerland) in children with RTT aged above six years along with CSF and MRI investigations. This study design opened the opportunity for a sub-study located at the Medical University of Graz by employing both, targeted and untargeted UHPLC-MS/MS of the metabolome and lipidome in plasma and CSF. In collaboration with the Department of Pediatric Neurology, Klinikum Kassel, Germany and the Department of Pediatrics, Medical University of Innsbruck, Austria we were able to increase the sample size of CSF and plasma samples obtained from RTT patients and healthy controls. Clinical parameters of patients enrolled in the study are listed in This study was conducted as part of a phase I clinical study based at the Department of Neurology, University Hospital Basel, Switzerland . The maiPatients and/or their legal guardians were asked for informed consent prior to blood collection and/or the spinal tap performed according to the study protocol of the main study. The remaining samples were taken after informed consent in the context of routine diagnostic procedures which included 0.5–1.0 mL of additional CSF and 1.0 mL of whole blood for plasma isolation (in lithium heparin tubes). The study population involved a control group of healthy children and one study group. CSF and plasma samples were stored at −80 °C prior to analysis.All chemicals used for sample preparation (metabolite and lipid extraction) were purchased from Merck .v/v) to the original and 0.5 sample volume, respectively, and subjected to MS analysis or immediately placed at −80 °C until further analysis.For metabolomics analysis, a 3:1 volume of ice-cold ACN/MeOH/acetone (1/1/1) was added to 50 µL of plasma and 100 µL of CSF and vortexed for 15 s as described previously ,60. The g and the upper phase was transferred to a new test tube. The lower aqueous phase was re-extracted with 2 mL of the upper phase of MTBE/methanol/deionized water 10:3:2.5 (v/v/v). The upper phases were combined, evaporated in a vacuum centrifuge and dissolved in 500 µL chloroform/methanol 1:1 (v/v) for storage at −80 °C. For MS analysis of the lipidome, plasma and CSF samples were evaporated under a stream of nitrogen and the resultant pellet was resuspended in the original or 0.5 sample volume, respectively, of isopropanol/methanol/water (95:5:5). For lipidomics analysis, the liquid extraction protocol with methyl-tert-butyl ether (MTBE) was applied . For thim/z 400. For MS/MS experiments, the 10 most abundant ions of the full scan spectrum were sequentially fragmented in the ion trap using He as collision gas  and centroided product spectra were collected. The exclusion time was set to 10 s. This protocol generated three complementary datasets for each sample collected and provided a complete and data-rich profile of the individuals’ metabolome and lipidome at the given time point. Respective pooled CSF and plasma samples were used as quality controls (QC). These QC were repeatedly measured at every 10th position in the acquisition line to examine and if necessary to correct for systematic errors.A full-scan mass-spectrometric analysis of each sample’s metabolite and lipid components was achieved by Dionex Ultimate XRS UHPLC (ultra-high performance liquid chromatography)-Orbitrap Velos Pro hybrid mass spectrometer  operated in Data Dependent Acquisition mode using a HESI II ion source ,62,63. TAll raw files obtained from high-resolution MS were either imported into the Compound discoverer 2.0 software  for metabolite identification, or into the Lipid Data Analyzer (LDA) software   for lipilipidr package [p < 0.05, ** p < 0.01, *** p < 0.001To evaluate the variance, the study and the healthy control group datasets obtained underwent statistical analysis using the programming language R (version 4.1.1) and the  package . VariablTaking into account that cholesterol, various PL species including plasmalogens and SM are decreased in the CSF of RTT patients we hypothesize, that the composition of membrane lipids is impaired and is a major cause of CNS dysfunction in RTT. Future functional studies are aiming to address this issue. In contrast to CSF data, MVA of plasma samples identified various PL and acylglycerols as major separators between the RTT patient and healthy control group, whereas UVA did not detect significant lipids in the patient group. In conclusion, this study provides insights into changes of the lipidome occurring in the CSF of RTT patients. In view of emerging therapies, this study may provide a basis for future biomarker discovery that may help in the delineation of VUS in MeCP2 and serve as surrogate parameters to study treatment effects.


In the event of an acute ischemic stroke, saving the penumbra is the most important aspect of early treatment. The rapid and accurate identification of ischemic penumbra plays a key role in its comprehensive treatment. At present, the identification method and evaluation standard of ischemic penumbra have not been unified. Numerous pieces of software identifying ischemic penumbra have been developed, such as rapid processing of perfusion and diffusion (RAPID), Sphere, Vitrea, and computed tomography perfusion+ (CTP+). The RAPID software, analyzing and integrating multi-mode image data (mainly based on perfusion weighted imaging (PWI) or computed tomography perfusion (CTP) images, shows good performance in identifying ischemic penumbra and has been utilized for the assessment of ischemic penumbra in many ischemic stroke clinical studies, achieving good outcomes and promoting the transition from “time window” to “tissue window” in the treatment of early stage AIS. To obtain a comprehensive understanding of the RAPID software and its accuracy in evaluating ischemic penumbra, this paper reviews the background and development of the RAPID software, summarizes the published acute cerebral infarction trials using the RAPID software, generalizes the threshold parameters in different time windows, and further discusses its application and limitations. Moreover, in recent years, the RAPID software has been proved and applied in many international clinical studies to provide the software with a good chance for optimizing an appropriate parameter to identify penumbra. The current paper reviews the background, development, application, and limitation of the RAPID software and summarizes the history of clinical trials using RAPID to identify penumbra.Acute strokes pose the greatest threat to human health. They are characterized by high morbidity, disability, death, and recurrence rates. Acute ischemic stroke (AIS) is the most common subtype of acute stroke characterized by the reversible or irreversible loss of cerebral tissue and neurological function caused by an inadequate supply of cerebral blood flow . In 2019In 2006, the diffusion and perfusion imaging evaluation for understanding stroke evolution (DEFUSE) study , based oTmax>6s lesion volume/DWI lesion volume > 1.2, DWI or PWITmax>8s lesion ≤ 100 mL, and |PWITmax>6s lesion volume − DWI lesion volume| ≥ 10 mL) achieved a better clinical prognosis following intravenous tissue plasminogen activator (tPA) reperfusion therapy, which could slow down the increase in infarction volume.In 2011, Lansberg MG et al.  continue−6 mm2/s and critically hypoperfused tissue as Tmax in PWI > 6 s. The criteria for identifying target mismatches were as follows: critically hypoperfused tissue/ischemic core ≥ 1.8; a |critically hypoperfused tissue − ischemic core| ≥ 15 mL; ischemic core volume < 70 mL; and lesion volume Tmax>10s ≤ 100 mL. Among a total of 138 enrolled patients, 104 patients had reliable MR image quality (MRI profile cohort) and the consistent rate between automatic analysis by the RAPID software and the manual identification of lesion range reached 97% (101/104), which proved that the RAPID software accurately identifies hypoperfused and ischemic lesions and their locations [In 2011, the DEFUSE 2 study defined the ischemic core as ADC < 600 × 10In 2013, the RAPID software obtained the FDA 510 K clinical use license and is, at present, a medical image processing software package approved by the FDA. In 2015, the extending the time for thrombolysis in emergency neurological deficits (EXTEND) study confirmed that the automatic analysis of CTP-based hypoperfusion/infarction mismatches by the RAPID software was also rapid and reliable, and the processing time of RAPID was quicker with CTP data than MR data [In 2017, several large trials of acute cerebral infarction successively used the RAPID software to select patients with target mismatches, and further explored the threshold parameter adjustment of the software in different time windows. The timeline of the main RAPID-related clinical trials is summarized in RAPID has been used as an image processing platform for hypoperfusion/infarction mismatches in large clinical studies, such as DEFUSE1, 2, 3; DAWN; SWIFT PRIME; EXTEND; and EXTEND-IA using MR or CT perfusion images.When the Tmax threshold is 6 s, it is the most accurate value used to determine the hypoperfused area based on the voxel ,9. Recen−2 mm2/s, which was most consistent with the literature data [−2 mm2/s has a low segmentation sensitivity and an ADC higher than 640 × 10−2 mm2/s has a low segmentation specificity [Based on CTP, when the rCBF threshold was 0.30–0.34, the predicted infarct range presented the greatest consistency with the infarct range 27 h later . The MR-ure data ,19. In oThe DEFUSE3 study  enrolled3, and NIHSS ≥ 10; age < 80 years, infarction area 0–31 cm3, and NIHSS ≥ 10; and age < 80 years, infarction area 31–51 cm3, and NIHSS ≥ 20. The results show that patients with anterior circulation AIS with a small infarct size and mismatched clinical function scores can still take advantage of endovascular therapy within 6–24 h of the last normal time.DAWN study  defined −6 mm/s [In SWIFT PRIME study ,20, pati−6 mm/s , which p−6 mm/s . Within −6 mm2/s for patients undergoing an MR scan [The EXTEND study  was desi MR scan . The resThe EXTEND-IA study  determinhttps://doi.org/10.3760/cma.j.issn.1005-1201.2019.11.002, accessed on 18 October 2022) and Chinese Stroke Association guidelines for clinical management of cerebrovascular disorders: executive summary and 2019 update of clinical management of ischaemic cerebrovascular diseases [The accurate estimation of the extent of the ischemic zone and infarct core area is the core problem and key purpose in the evaluation of ischemic penumbra. Compared with traditional image techniques, RAPID has apparent advantages. It only spends several minutes to evaluate an AIS patient, which could reduce the clinical decision time and improve the prognosis of AIS patients (demonstrated by the above-mentioned RCTs). RAPID also has good performance compared with other recently developed evaluation software. Rava et al. divided patients with AIS into endovascular and conservative treatment groups and compared the assessment of the ischemic zone and infarct core zone using RAPID, Sphere, Vitrea, and CTP+ software, respectively. The results show that, in the endovascular treatment group, RAPID has the least chance to overestimate the volume of the infarct core area, which presents patients with the greatest possibility for receiving endovascular treatment ,26. Nowadiseases  suggest diseases . Severaldiseases . (3) SevThis paper summarized the background, development, application, and limitations of the RAPID software in clinical research. Several studies have shown that RAPID software can rapidly and accurately judge the state of ischemic penumbra at the early stage of AIS, providing a histological basis for thrombolysis or thrombectomy. However, in the future, more clinical randomized controlled studies are needed to verify the evaluation power of RAPID software on ischemic penumbra and further optimize the “mismatch” parameter and its threshold, which may allow the software to be formally recommended by relevant guidelines and lay a foundation for extensive introduction into medical institutions and cost reduction. Moreover, it remains to be further explored whether RAPID can also accurately identify ischemic penumbra in most patients who have exceeded the time window for vascular recanalization. At present, our research team is exploring the use of RAPID software to evaluate the effects of butylphthalide  ,30,31 on


This study investigated the correlation between the levels of long noncodingribonucleic acids (lncRNAs) AF131217.1 and coronary slow flow (CSF).Conclusion: Thus, lncRNA AF131217.1promoted inflammation in the regulated CSF via KLF4 by miR-128-3p.A total of 22 patients in the high-sensitivity C-reactive protein (hsCRP)group diagnosed with CSF from January 2018 to December 2018 were enrolled inthis study. Coronary flow velocity was determined using the thrombolysis inmyocardial infarction frame count (TFC) method. Results: LncRNA AF131217.1expression in the CSF model was activated. Mean TFC was positivelycorrelated with lncRNA AF131217.1 levels and hsCRP levels. LncRNA AF131217.1induced inflammation factor levels in the in vitro model. Micro ribonucleicacid (miR)-128-3p is a target spot of lncRNA AF131217.1 on the inflammationin vitro model via Kruppel-like factor (KLF) 4. MiR-128-3p reducedinflammation factor levels .  The exactpathological mechanism of CSF is still unclear. At present, there is no standardizedtherapeutic measures for CSF in clinical practice, with inconsistent reportedefficacy of drug treatment.Studies have shown that CSF is strongly associated with cardiovascular adverseevents, such as arrhythmia, ACS, and sudden cardiac death. In addition, the levelsof high-sensitivity C-reactive protein (hsCRP), vascular cell adhesion molecules,and intercellular adhesion molecules are significantly increased in CSF patientsthan in healthy individuals, which is also positively correlated with thrombolysisin myocardial infarction frame count (TFC), indicating endothelial activation andinflammatory response in CSF patients[2,.The coronary slow flow (CSF) phenomenon is defined as the presence of delayedperfusion of peripheral coronary artery identified by coronary arteriography afterexcluding coronary artery disease, cardiomyopathy, valvular disease, congenitalheart disease, connective tissue disease, etc. Although coronary angiography inpatients with slow blood flow shows no stenosis of coronary artery, recurrentcardiovascular events still occur, generally clinically manifested by angina,arrhythmia, or acute coronary syndrome (ACS), which may be associated with CSF andusually requires emergency admission. KLF familymembers are widely involved in the regulation of multiple life activities, includingcell proliferation, apoptosis, differentiation, and embryonic development. The abnormal function of KLFfamily members is strongly associated with metabolic diseases, cardiovasculardiseases, and cancer. KLF4, originally isolated from the gastrointestinal tract, isone of the transcriptional regulators required for the early differentiation ofadipocytes. It activatesthe expression of the CCAAT/enhancer binding protein beta (C/EBPβ) gene bybinding itself to the promoter of C/EBPβ, thereby activating the downstreamcascade of fat cell differentiation to promote this differentiation[6,. In recent years, KLF4 has become a research hotspot inrelevant fields due to its regulatory roles on chronic inflammatory responses ofvarious cells.Kruppel-like factors (KLF) are a type of transcription factors with zinc-fingerstructure, which is characterized by the three C2H2 zinc-finger structures at thecarboxyl terminal. To date, hundredsof thousands of eukaryotic lncRNAs have been found, most of which are lowlyconservative and involved in the pathogenesis and progression of a series ofdiseases, including tumors, nervous system disorders, metabolic diseases,reproductive development, and cardiovascular diseases. Long intergenic noncoding RNA-p21, a p53-inducedlncRNA, is able to inhibit cell proliferation and promote apoptosis duringatherosclerosis[9,. Thisstudy investigated the correlation between the levels of lncRNA AF131217.1 andCSF.Long noncoding ribonucleic acids (lncRNAs) are a type of noncoding RNA with over 200bp in lengthA total of 22 patients in the hsCRP group diagnosed with CSF at the AffiliatedChinese Medicine Hospital of Xinjiang Medical University, from January 2018 toDecember 2018, were enrolled in this study. All patients had stenosis of lumendiameter < 40% (no significant vascular lesion). The control group was 24patients who did not meet the abovementioned criteria. All procedures performedin studies involving human participants were in accordance with the ethicalstandards of the institutional and/or National Research Committee and with the1964 Helsinki Declaration and its later amendments or comparable ethicalstandards. This study was approved by the Ethics Committee of the AffiliatedChinese Medicine Hospital of Xinjiang Medical University. Written informedconsent was obtained from all individual participants included in the study. TFCwas used to quantitatively measure coronary blood flow using a cineangiography.CSF was diagnosed if TFC > 27 in at least one coronary artery. Threecardiologists who were blinded to the clinical findings independently assessedthe TFC.http://lgsun.grc.nia.nih.gov/ANOVA).Total RNA was purified with the RNeasy Kit (Qiagen). Complementarydeoxyribonucleic acid (cDNA) and complementary RNA were generated and hybridizedto HumanHT-12 v4 BeadChips (Illumina). Cubic spline-normalized  data were analyzed by the NIA Array Analysis tool(Micro RNA (miR)-128-3p was inserted into the luciferase gene psiCHECK2 vector,named as KLF4-WT (the wild type) or KLF4-Mut (mutant) 3'-UTR. Next, miR-128-3pwas inserted into the luciferase gene psiCHECK2 vector, named as lncRNAAF131217.1-WT or lncRNA AF131217.1-Mut 3'-UTR. After transduction, at 48 hours,cell was transduced using Lipofectamine® 2000 Reagent . The luciferase assay was performed using Luciferase ReporterGene Detection Kit .-△△Ctmethod.Total RNA was isolated with the RNeasy Micro Kit (Qiagen) and cDNA wassynthesized with the Maxima First Strand cDNA Synthesis Kit. Quantitativereal-time polymerase chain reaction was performed using SYBR Green Master Mix.Relative fold changes were calculated using the 22. HUVECs (1×105/well) were transientlytransfected with 50 nM of lncRNA AF131217.1, silencing lncRNA AF131217.1,miR-128-3p, and negative control using Lipofectamine® 2000 reagent. After 48 hours of transfection,HUVECs were treated with 100 µg/ml oxidized low-density lipoprotein for24 hours.Human umbilical vein endothelial cells (HUVECs) were cultured in Roswell ParkMemorial Institute 1640 medium  supplementedwith 10% fetal bovine serum  at 37°C with 5%COCells were lysed with radioimmunoprecipitation buffer, and protease andphosphatase inhibitors (Sigma-Aldrich) were added. Protein concentration wasmeasured using a bicinchoninic acid protein assay kit . Equal amounts of proteins were separated by 10% sodium dodecylsulphate-polyacrylamide gel electrophoresis and transferred to polyvinylideneﬂuoride membranes. After blocking with no-fat skim milk, the membranes wereincubated with primary antibodies  diluted in TrisBuffered Saline with Tween 20 overnight at 4°C and then with the horseradishperoxidase-conjugated secondary antibody for two hours at room temperature.Protein bland was detected by electrochemiluminescence chromogenic substratekits and quantified by Image Lab 3.0 .Serum samples were collected at 1000 g for 10 min and used to measure tumornecrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1β, andIL-18 levels. Cell samples were also collected at 1000 g for 10 min and used tomeasure TNF-α, IL-6, IL-1β, and IL-18 levels. TNF-α, IL-6,IL-1β, and IL-18 ELISA kits were purchased from Nanjing JianchengBiological Engineering Research Institute Co. LTD .All data are expressed as the mean ± standard error of mean. P<0.05 wasconsidered statistically significant. Statistical significance between groups ofdata was calculated by Student’s t-test or one-way analysis of variance andTukey's post-test.Initially, we sought to validate lncRNA AF131217.1 expression in the CSF model.We found using gene chip that six genes were downregulated and 11 genes wereupregulated in the CSF model . LncRNA Moreover, in the lipopolysaccharide (LPS)-induced in vitro model, lncRNAAF131217.1 plasmid increased lncRNA AF131217.1 expression and inducedinflammation factor levels  . Then, iIn our search to define the possible target spot of lncRNA AF131217.1 oninflammation of CSF, miR-128-3p expression had a negative correlation withlncRNA AF131217.1 levels in the CSF model . The relIn the rat model of CSF, miR-128-3p expression was downregulated and KLF4expression was upregulated, compared with the control group . MiR-128The study defined the possible target spot of miR-128-3p on inflammation of CSF,the relative luciferase activity in KLF4-WT and miR-128-3p groups was lower thanthat of KLF4-Mut and miR-NC cotransfection groups . NonetheLncRNA AF131217.1 Regulated Inflammation in the In Vitro Model via KLF4 bymiR-128-3pIn an attempt to delineate whether miR-128-3p was required for lncRNA AF131217.1induction of KLF4 signaling in CSF, we found that miR-128-3p overexpressioninduced miR-128-3p expression and reduced inflammation factor levels in the in vitro model of lncRNAAF131217.1 . Meanwhi11-. We showed that lncRNA AF131217.1expression in the CSF model was activated. Mean TFC was positively correlated withlncRNA AF131217.1 levels and hsCRP levels. These characteristics suggest that lncRNAAF131217.1 participated in the pathophysiological process of CSF.CSF is defined as the presence of delayed perfusion of peripheral coronary artery butwithout obvious lesions in the coronary arteries diagnosed by coronary arteriographyafter excluding coronary spasm, coronary artery dilatation, coronary angioplasty,cardiomyopathy, heart valvular disease, autoimmune diseases, tumors, and otherimportant organs or systemic diseases. More and more attention are being paid to theresearch of CSF, however, the etiology and pathogenesis of CSF remainunclear showed thatshear-sensitive lncRNA AF131217.1 reduced inflammation in HUVECs via regulation ofKLF4.Lu et al.. Theannual number of people dying from cardiovascular disease ranks the firstglobally. LncRNAs area type of noncoding RNAs with over 200 nt in length, which are also the mostabundantly expressed RNA[16,.Accumulative studies have revealed that lncRNAs play an important regulatory role inthe pathogenesis and development of cardiovascular diseases. Interestingly, lncRNA AF131217.1induced inflammation factor levels and played an important role in the inflammationof CSF.Cardiovascular disease is a common disease that threatens the health of human beings,especially the elderly showed thatmiR-128-3p accelerates cardiovascular calcification in type 2 diabetes mellitusrats. Studies have shown that CSF is closely associated with cardiovascular adverseevents, including arrhythmia, ACS, and sudden cardiac death[19,. When coronary angiography is performed onpatients with suspected cardiovascular disease, the detection rate of CSF isapproximately 1%. Recentstudies have demonstrated that inflammatory response plays an important role in thepathogenesis of CSF[19,. Our study showed that miR-128-3pis a target spot of lncRNA AF131217.1 on the inflammation in vitro model.Wang et al.. In endothelial cells and immunecells, P300 can bind with the active subunit P65 of NF-κB to induceacetylation of P65, thereby promoting the transcriptional activation on downstreaminflammatory factors by P65.Overexpressed KLF4 can competitively bind to P300, inhibiting the binding of P300with P65 to attenuate its transcriptional activity, subsequently exerting ananti-inflammatory effect. Asmembers of the KLF family, KLF2 and KLF15 can also competitively bind withP300. It has beenreported that KLF4 can bind to the active subunit P65 of NF-κB in vascularendothelial cells, to promote the binding of P65 to its downstream inflammatoryfactor vascular cell adhesion molecule 1, thereby playing a pro-inflammatoryrole. Meanwhile, wefound that KLF4 is a target spot of miR-128-3p on the inflammation in vitromodel.KLF4 has been reported to physically bind to the active subunit P65 of transcriptionfactor NF-κB in vascular endothelial cells to prevent the nucleartranslocation of P65, thereby exerting its anti-inflammatory role by inhibiting thetranscriptional activation on downstream inflammatory factors of P65In summary, lncRNA AF131217.1 expression in the CSF model was activated and promotedinflammation by the suppression of miR-128-3p via KLF4/RhoF/NF-κB signalpathway, and this may play an important role in the pathogenesis of CSF. An elevatedplasma lncRNA AF131217.1 level may indicate the presence of CSF. Further studies areneeded to establish clinical significance of increased plasma lncRNA AF131217.1levels and to investigate the therapeutic efficacy of targetingmiR-128-3p/KLF4/RhoF/NF-κB signal pathway.


Streptococcus suis ) and Japanese encephalitis virus (JEV) ). Commonly reported animal contact included keeping, raising or handling ) and handling, cooking or consuming raw meat, blood or viscera in the 2 weeks prior to symptom onset ), with the latter most commonly from pigs . There was no association between an unknown aetiology and exposure to animals in a multivariate logistic regression. Further testing for unknown or undetected pathogens may increase diagnostic yield, however, given the high proportion of zoonotic pathogens and the presence of risk factors, increasing public awareness about zoonoses and preventive measures can be considered.An estimated 73% of emerging infections are zoonotic in origin, with animal contact and encroachment on their habitats increasing the risk of spill-over events. In Vietnam, close exposure to a wide range of animals and animal products can lead to acquisition of zoonotic pathogens, a number of which cause central nervous system (CNS) infections. However, studies show the aetiology of CNS infections remains unknown in around half of cases. We used samples and data from hospitalised patients with CNS infections, enrolled into the Vietnam Initiative on Zoonotic Infections multicentre study, to determine the association between aetiology and animal contact including those in whom the cause was unknown. Among 933 patients, a pathogen or an antibody response to it was identified in 291 . The most common pathogens were The online version contains supplementary material available at 10.1007/s10393-022-01611-w. Streptococcus suis  infections in Vietnam are caused by a number of pathogens, many of which are zoonotic  was developed with the aim of characterizing endemic infections, novel infections and infections of unknown origin through a prospective hospital-based surveillance programme. Patients with either a respiratory tract infection, enteric infection, central nervous system (CNS) infection or jaundice were enrolled as part of the hospital surveillance component , Ha Noi; Ba Vi District Hospital, Ha Noi (Ba Vi); Hue Central Hospital, Hue (Hue); Dak Lak General Hospital, Buon Me Thuot (Dak Lak); Khanh Hoa General Hospital, Nha Trang (Khanh Hoa); and Dong Thap General Hospital, Cao Lanh (Dong Thap) Fig. .Figure 1Informed written consent was obtained from patients  included in the study. Diagnostic results from this study were previously described in a paper by Robertson et al.  who lookhttps://clires.oucru.org). GPS coordinates were also provided for the location of the hospitals.A standard Case Report Form (CRF) detailing demographics, admission to an intensive care unit, self-reported HIV status, drinking water source and animal contact was completed on enrolment (supplementary data). The questions relating to animal exposure included raising, keeping or handling animals; or slaughtering an animal; or handling, cooking or consuming the raw meat, blood or viscera from a list of twenty-nine domestic, livestock and wild animals within the 2 weeks prior to the onset of symptoms. An additional CRF was completed at discharge with the diagnosis and outcome. Global positioning system (GPS) coordinates for the centre of the patients’ commune were obtained using a conversions system via the CliRes Data Management system  were collected as soon as possible. Initial analysis was undertaken at the hospital of admission including a full blood and CSF cell count, and CSF Gram/Ziehl–Neelsen stain. Blood and CSF culture was performed at the hospital sites including Dak Lak, Dong Thap, Khanh Hoa and Hue with a repeat culture performed at the Oxford University Clinical Research Unit (OUCRU) in Ho Chi Minh City (HCMC) although a blood culture was not obtained for all patients. Discrepancies in culture results between the hospital sites and the putative causal pathogen identified at OUCRU were reviewed to predict the causal pathogen for the purpose of the analysis. In Ba Vi and NHTD, blood and CSF were transported to OUCRU Ha Noi on daily basis where these were cultured.Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus suis, Haemophilus influenzae type b, herpes simplex virus (HSV), varicella zoster virus (VZV), and enteroviruses on CSF samples from all hospital sites at OUCRU HCMC  or Ha Noi. The Panbio Dengue IgM Capture Enzyme-linked immunosorbent assay (ELISA)  value of < 40, or ELISA with an optical density ratio defined as positive according to the manufacturers’ instructions.3 and those with a CSF white cell count of ≥ 5 cells/mm3 to account for any potential misdiagnoses of encephalopathy or a CNS infection without a CSF pleocytosis. Age was categorised into five categories: under 5 years; 5–17 years; 18–49 years; 50–69 years and 70 years and over. All the pathogens found in less than six patients were pooled into a category ‘other’. Statistical differences in the demographics of the patients including age category, gender and site of hospital admission by aetiology; and age category and gender by site of hospital admission were determined using Fisher’s exact test and the p value simulated with 2000 replicates. Univariate binomial regression was used to calculate the odds ratio (OR) of the presence of each of the aetiologies between age, gender, site of recruitment and type of animal exposure. Univariate binomial regression was also used to calculate the OR between each type of exposure to pigs including keeping, raising or handling; slaughtering or eating, cooking or handling the raw meat, blood or viscera and S. suis and JEV.All analyses were performed using R statistical software version 3.6.0 Core . In the 3 and a known aetiology; and patients with an unknown aetiology and a CSF white cell count of < 5 cells/mm3; age and gender were included as fixed effects. The hospital site was added as a random effect. An outcome of any type of exposure to animals was included as the sample size for the independent variables was too small to perform multivariable logistic regression modelling for exposures including slaughtering an animal or handling, cooking or consuming raw, meat, blood or viscera.A mixed-effects multivariate binomial regression model was used to determine the association between any type of animal of exposure and aetiology of the CNS infection. The four most common pathogens, patients with an unknown aetiology and a CSF white cell count of ≥ 5 cells/mmn = 296, 31.7%). An aetiology was established in 291/933 (31.2%). Among those in whom a pathogen was detected, 247/291 (87.9%) had CSF white cell count of ≥ 5 cells/mm3 and 44/291 (15.1%) had a CSF white cell count of < 5 cells/mm3. Among those in whom no pathogen was detected, 435/642 (67.8%) had a CSF white cell count of ≥ 5 cells/mm3 and 207/642 (32.2%) had a CSF white cell count of < 5 cells/mm3. A total of 12 different bacterial pathogens were found with the most common being S. suis (n = 91 (9.8%)) and S. pneumoniae (n = 49 (5.3%)). Of the five viruses tested, the most detected were JEV (n = 72 (7.7%)) and enterovirus (n = 25 (2.7%)) . Infections with S. suis were predominantly seen in adult males, with median age of 49 years [IQR = 39–58]. Infections with S. pneumoniae, JEV and enterovirus were predominantly seen in paediatric patients, with median age of 12 years [IQR = 2–33 years], 11 years [IQR = 6.8–15] and 13 years [IQR = 8–22], respectively. The median age of those in whom the aetiology was unknown was 23 years [IQR = 10–45] in those with a CSF white cell count of ≥ 5 cells/mm3 and 11 years [IQR = 2–36.5] in those with a CSF white cell count of CSF white cell count of < 5 cells/mm3. Half of the cases of JEV were recruited from Dak Lak General Hospital (n = 36 (50%)) and nearly half of the cases of S. suis were recruited from Hue Central Hospital (n = 43 (47.3%)). However, although there was statistical evidence of a difference in the aetiology of CNS infection by age of the patient and also, the site of recruitment (p = 0.001) there was not by gender of the patient (p = 0.062)  with a median age ranging from 16 years [IQR = 7–37] in Khanh Hoa to 29.5 years [IQR = 17–42] in the NHTD. No difference was seen between hospital site and gender (p = 0.102) ), and those with S. pneumoniae or an unknown aetiology with a CSF white cell count of < 5mm3 were less likely to keep, raise or handle an animal  and OR 0.67 , respectively). Those with an unknown aetiology with a CSF white cell count of < 5mm3 were less likely to have contact with an animal compared to those with a known pathogen or unknown aetiology with a CSF white cell count of > 5mm3 (OR 1.47 (1.08–2), p = 0.015)) , have slaughtered an animal (OR 5.34 (3.28–8.91), p < 0.001), and handled, cooked or consumed raw meat, blood or viscera compared to other sites (OR 2.73 (2.06–3.63), p < 0.001). Patients admitted to the hospital in Dong Thap were also more likely to have handled, cooked or consumed raw meat, blood or viscera (OR 1.51 (1.04–2.18), p = 0.028) whereas those admitted to the hospital in Hue or Khanh Hoa were more likely to have had no contact with an animal (OR 1.82 (1.34–2.47), p < 0.001 and OR 2.44 (1.72–3.48), p < 0.001, respectively) . Adults aged 50 years and over were more likely to keep, raise or handle animals compared to other age groups  and 70 years and over: OR 1.92 ) with those aged 18–69 years more likely to have slaughtered an animal  and 50–69 years: 2.5 ) and handled, cooked or consumed raw meat, blood or viscera  and 50–69 years: 1.7 . Children aged under 5 years were more likely to have had no contact with animals  . Females were more likely to have no contact with animals  with males more likely to keep, raise or handle animals  but with no difference between the genders in terms of slaughtering animals or handling, cooking or consuming raw meat, blood or viscera .Three hundred and sixty-four patients 39%) raised an animal, 78 (8.4%) had slaughtered an animal and 371 (39.8%) handled, cooked or consumed raw meat, blood or viscera. In univariate analyses, patients with 9% raisedn = 344/371 (92.7%)) again both in those with and without a known aetiology  and rat ). However, the raw meat, blood or viscera of wild/farmed wild animals were handled, cooked or consumed by those with a known aetiology  and pigeon ); those with an unknown aetiology and CSF white cell count of ≥ 5 cells/mm3 , other wild bird , pigeon  and squirrel ; and those unknown aetiology and CSF white cell count of < 5 cells/mm3  and pigeon . In the univariate binomial regression, those with S. suis were more likely to have slaughtered a pig . However, there was no difference between those with and without S. suis who kept or raised pigs; or ate, cooked or handled the raw meat, blood or viscera from pigs  and OR 1.02 , respectively). No difference was between those with and without JEV and keeping or raising pigs , slaughtering pigs  or eating, cooking or handling the raw meat blood or viscera from pigs .The most common animals kept, raised or handled were dogs (264/364 patients 72.5%)) and chickens (204/364 patients (56.0%)) both in those with and without a known aetiology. Chickens were the most common animals slaughtered (61/78 patients (78.2%)), and the raw meat, blood or viscera from pigs were the most common product handled, cooked or consumed (%) and chS. suis, S. pneumoniae, JEV or enterovirus. Similarly, there was no difference between animal exposure and an unknown aetiology, either with or without a CSF white cell count of ≥ 5 cells/mm3. Men were more likely than women to have exposure to animals ; however, there was no difference by age  of adults and 2% (n = 11) of children diagnosed with MTB  in the previous year , rats (n = 1) and squirrels  had a CNS infection with unknown aetiology and a CSF white cell count of ≥ 5 cells/mm3. Rodents are known hosts for a number of zoonotic pathogens with a study in the Mekong delta, Vietnam, detecting antibodies to Tick-borne encephalitis virus (TBEV) and hantavirus in both humans and rodents  and indirect immunofluorescence assays for O. tsutsugamushi and qPCR and microscopic agglutination tests for Leptospira spp. as described by Dittrich et al. . Figure S2. The effect of animal exposure by age of the patient as shown by odds ratios with 95% confidence intervals from univariate binomial regression with an outcome of animal contact . Figure S3. The effect of animal exposure by gender of the patient as shown by odds ratios with 95% confidence intervals from univariate binomial regression with an outcome of animal contact . (DOCX 44 kb)Below is the link to the electronic supplementary material.


Hydrocephalus has been observed in rats with spontaneous hypertension (SHRs). It has been demonstrated that activation of the oxidative stress related protein retinoic acid receptor alpha (RARα) has neuroprotective impacts. Our investigation aims to determine the potential role and mechanism of RARα in hydrocephalus. The RARα-specific agonist (Am80) and RARα inhibitor (AGN196996) were used to investigate the role of RARα in cerebrospinal fluid (CSF) secretion in the choroid plexus of SHRs. Evaluations of CSF secretion, ventricular volume, Western blotting, and immunofluorescent staining were performed. Hydrocephalus and CSF hypersecretion were identified in SHRs but not in Wistar–Kyoto rats, occurring at the age of 7 weeks. The RARα/MAFB/MSR1 pathway was also activated in SHRs. Therapy with Am80 beginning in week 5 decreased CSF hypersecretion, hydrocephalus development, and pathological changes in choroid plexus alterations by week 7. AGN196996 abolished the effect of Am80. In conclusion, activation of the RARα attenuated CSF hypersecretion to inhibit hydrocephalus development via regulating the MAFB/MSR1 pathway. RARα may act as a possible therapeutic target for hydrocephalus. Hydrocephalus frequently develops following intracranial hemorrhage, resulting in poor clinical outcomes ,2. InvasHydrocephalus is a common CSF physiology disorder that results in abnormal enlargement of the cerebral ventricles ,6,7. ExcRetinoic acid receptors (RARs) are members of the nuclear receptor superfamily, and RARs are important oxidative stress-related proteins, which are regulated by the oxidative stress state of the body. However, studies on other mechanisms of activation of RARs are relatively limited. Studies have shown that RARs regulate cell differentiation, embryonic development, and metabolism . In addiMacrophage scavenger receptor 1 (MSR1) is a heterogeneous molecular found on the surface of phagocytes . Recent p < 0.05, p < 0.05, p < 0.05, We assessed the time point of CSF secretion by quantifying CSF production rates in WKY and SHR groups at weeks five, six, and seven. At weeks five and six, CSF production rates were equal in WKY rats and SHRs. However, at week seven, SHRs exhibited obvious CSF hypersecretion . All procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. SHRs and age-matched Wistar–Kyoto (WKY) rats  were maintained in a 12/12 h light/dark cycle environment with controlled humidity and temperature. All rats had a starting body weight of ninety–one-hundred grams. Blood pressure (BP) was determined utilizing the tail cuff technique . At weeks 5, 6, and 7, systolic BP (SBP) and mean arterial pressure (MAP) were assessed and documented.All rats were assigned into two experiments. First, 4-week-old SHRs and WKY rats were housed, and their CSF production was measured at weeks 5, 6, and 7 to determine the rates of CSF production . At weekStarting from week 5, Am80 was administered intraperitoneally (IP) every day at a dose of five milligrams per kilogram in SHRs . The dur3) = π·r2·d, in which r is the capillary tube’s radius and d is the distance that the CSF traveled in the capillary. The CSF production rate (μL/min) might be estimated from the slope of the volume–time curve.Following the early reported Karimy’s technique , rates o2, summed over fourteen levels, and multiplied by the distance of every 200 μm to determine ventricular volume [The brains were removed and kept in formalin  for one day at 4 °C. The samples were immersed in 30% sucrose until sinking to the bottom. To eliminate distortion caused by cryo-sectioning and slide mounting, we captured high-resolution images of serial coronal slices of the brain  while it was placed in the cryostat, utilizing standard criteria for camera location, magnification, and external illumination. ImageJ was utilized to count the number of pixels in the area of the lateral ventricle in every thick slice. Pixels were turned into area in mmr volume .g, 4 °C). The protein concentration was measured using a BCA Protein Assay Kit . Equal amounts (40 g) of protein samples were denatured in loading buffer  and isolated utilizing SDS-PAGE. The protein was transported to a membrane of polyvinylidene fluoride (PVDF). The PVDF membranes were blocked in 5% skim milk  for 1 h at room temperature. The membrane was treated overnight at 4 °C with primary antibodies: rabbit anti-RARα antibody , rabbit anti-MAFB antibody , rabbit anti-MSR1 antibody , and rabbit anti-β-actin antibody . The membranes were treated with horseradish peroxidase-conjugated secondary antibodies  for two hours at room temperature. Membranes were identified with the Immobilon enhanced chemiluminescence (ECL) Ultra Western HRP Substrate reagent kit  and photographs were examined using ImageJ software .Western blotting was conducted as reported earlier . Rats weRats were anesthetized and perfused with 0.1 mol/L cold PBS , followed by 4% paraformaldehyde (PFA) . The brains were harvested and then fixed with 4% PFA overnight and 30% sucrose until sinking to the bottom at 4 °C. Brain slices (8 μm thickness) were blocked for half an hour at room temperature with immunol staining blocking buffer  and washed with PBS three times. Sections were treated overnight at 4 °C with the primary antibody: rabbit anti-IBA-1 antibody . At room temperature, sections were treated with a secondary antibody for two hours: Goat anti-Rabbit IgG (H + L) Cross-Adsorbed, Alexa Fluor™ 488 . Finally, the sections were covered with 4′,6-diamidino2-phenylindole (DAPI) . A fluorescence microscope  was employed to observe the photographs. ImageJ program was used to examine the outcomes. To quantify the Iba-1-positive cells, we selected at least three sections per mouse from similar areas of the choroid plexus and analyzed three fields per section at a magnification of ×200 per section.t-test, comparisons between two groups were made for data with a normal distribution. If there were more than two groups being compared, a one-way analysis of variance (ANOVA) was conducted. Variables were evaluated using the Mann–Whitney U test or Kruskal–Wallis test, accompanied by the Dunn post hoc test, for the result that was not regularly distributed. Statistical evaluation was conducted employing GraphPad Prism 8 and SPSS. p < 0.05 was indicated as substantially significant.All outcomes are displayed as average ± standard deviation (SD) or median with an interquartile range. Using the student’s Our outcomes revealed that Am80, the agonist of RARα, attenuated the macrophage/microglia infiltration, and inhibited the cerebrospinal fluid hypersecretion of SHRs, possibly by regulating the MAFB/MSR1 pathway. Hence, RARα might be a possible therapeutic target to alleviate hydrocephalus.