These 3-DOM IrO2 honeycomb arrays showed a large surface area and ordered macropores (155 nm in diameter) cross-linked by secondary mesopores. Internal structures of 3-DOM IrO2 material were observed microscopically through these secondary pores. According to the X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) spectra, 3-DOM IrO2 has a rutile crystal structure and is mainly composed of iridium dioxide. In acidic electrolytes, the overpotential of 3-DOM IrO2 material at 0.5 mV cm−2 was only 0.22 V. Accelerated durability tests demonstrated excellent durability of 3-DOM IrO2 as an oxygen evolution reaction (OER) catalyst.Three-dimensional ordered macroporous (3-DOM) IrO Hydrogen, as an environmentally friendly energy carrier with high thermal value, has many advantages, which solves the energy problems and environmental issues associated with other energy resources ,2,3. TheDespite numerous research studies in this area, high anodic overpotential remains a great challenge for oxygen evolution reaction (OER). Additionally, the stability of electrocatalysts is another factor restricting widespread SPE applications. The majority of metals and their oxides cannot be stabilized under high anode potential and strong acidic conditions; thus, choices for anode catalysts are very limited. Platinum metals have good catalytic performance because of their excellent adsorption affinity to hydrogen and excellent catalytic properties. Thus, Pt has been widely used in SPE water electrolysis ,7, fuel 2 and RuO2 were selected, most of which were oxides of transition metals. Although these materials have excellent chemical stability, their conductivity is poor, which makes OER electron transfer dependent on IrO2 and RuO2. The result does not reduce the amount of precious metals, and only when the content of IrO2 in the IrO2-ZrO2 solid solution reaches 80% does the catalyst demonstrate good performance [2 and/or RuO2 are TiO2 [2 [2O5 [2O5 [To increase the stability of the catalyst at the anode during SPE water electrolysis, a lot of work has been performed especially involving dimensionally stable anodes (DSA). Durable material to load on IrOformance . Other sare TiO2 ,17,18,19are TiO2 , SnO2 [2 TiO2 [2 ,22,23,24 [2 [2O5 ,26, and 2O5 [2O5 , etc. Re2 catalyst based on a template-free ammonia complex. Unlike the Adams fusion method, ammonia was used as a chelating agent to replace Cl in H2IrCl4. This IrO2 catalyst demonstrated the highest specific surface area ever reported (363.3 m2 g−1) as well as outstanding performance. Ortel et al. [2 film templated by PEO–PB–PEO block copolymers. The films possess nanocrystalline walls and ordered pores, which enhance the durability and activity of OER.To increase the catalytic activity during SPE water electrolysis, the activity of precious metal catalysts needs to be improved, for example by studying and analyzing its surface area and micropore structure ,29,30,312 using a SiO2 microsphere template [2 exhibited an ordered honeycomb array of macropores. The overpotential of this OER catalyst was only 0.22 V at 0.5 mA cm−2. However, organic templates have not been reported for the preparation of 3-DOM IrO2. Herein, we synthesized 3-DOM IrO2 using a PMMA template. Compared to the SiO2 template, PMMA templates can be removed by annealing, which makes the preparation process very simple, thereby reducing its cost. However, the decomposition temperature of the PMMA template is under 300 °C, which is too low for iridium chloride to form iridium oxide. Because the formation of 3-DOM structures is difficult under such conditions, we used ammonia as a chelator, which reacts with iridium chloride to form ammonium chloride and iridium hydroxide. Ammonium chloride is volatile and easily decomposed. Iridium hydroxide is easily dehydrated to produce iridium oxide under 300 °C, which can protect the 3-DOM structure against collapse. 3-DOM IrO2 was characterized by SEM, Brunauer-Emmett-Teller (BET), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). Ordered macropores were clearly seen with a significant decrease in overpotential.Three-dimensional ordered macroporous (3-DOM) materials are novel molecular sieves with uniform and ordered pores and well-controlled pore structures. The ordered porosity of the 3-DOM material is very conducive to an even distribution of the electrolyte between the electrode, which improves the mass transfer of ions to the electrode surface . This catemplate ,36, in w Chemical Reagent Co., Ltd., Shanghai, China) and methyl methacrylate  were added in a ratio of 1:118. The whole mixture was stirred for 1 h under nitrogen. After that, the PMMA emulsion was centrifuged and washed three times with deionized water, following which PMMA microspheres were dispersed ultrasonically in deionized water. A certain amount of PMMA emulsion was placed into a 25 mL beaker, which was then covered by tin foil with holes. PMMA microspheres were settled down and dried at room temperature. Bright fluorescence on the surface indicated that the PMMA microspheres were hexagonally close-packed.Conventional emulsifier-free emulsion polymerization was used to prepare polymethyl methacrylate (PMMA) templates . First, 4·2H2O  was added to H2O and isopropyl alcohol  (H2O: IPA = 3:1). Then, 4 mL of IrCl4·2H2O 0.03 mol L−1 was added to PMMA templates until it was immersed. After 30 min, the excess solution was removed by a dropper. PMMA templates were put into the oven at 70 °C. After drying, the above steps were repeated three times. Finally, the templates were heated to 450 °C in air at a rate of 5 °C/min in a tube furnace for 4 h. The final product was a black powder (marked as non-ammonia IrO2). Ammonia  (IrCl4·nH2O: NH4·H2O = 1:4) was then added to the above solution that contains isopropyl alcohol, H2O, and IrCl4·2H2O. 3-DOM IrO2 was prepared by repeated preparation of non-ammonia IrO2. Colloidal IrO2 was prepared by the colloidal method [IrCll method .2 gas absorption/desorption data collected by Micromeritics ASAP 2020 (BELSORP-max).Scanning electron microscopy (SEM) was performed using a Hitachi S-4800 microscope operated at 8 kV. X-ray photoelectron spectroscopy (XPS) spectra were recorded by an ESCALAB250Xi spectrometer using Mg Kα as an X-ray source. X-ray diffraction (XRD) spectra were recorded by Bruker D8 Advance using Cu–Kα radiation. Particle sizes were measured by the dynamic light scattering (DLS) technique. Specific surface areas and pore distribution were obtained by implementing Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) methods, respectively, using N−1 H2SO4 solution. The counter electrode was platinum foil, and a saturated calomel electrode  was a reference electrode. The working electrode was a glassy carbon (GC) 5 mm in diameter. The catalyst ink was prepared using 5 mg of testing material, 980 µL of isopropanol, and 20 µL of 5% Nafion. Both catalysts had a load of 106 µg cm−2. Cyclic voltammograms (CV) were measured between 0 V and 1.4 V at a 50 mV s−1 scan rate. Steady-state polarization curves for OER were obtained at a 5 mV s−1 scan rate. Before all tests, the electrode was activated by performing 20 cycles of the CV curve at the scanning rate (500 mV s−1). All electrochemical tests were performed using the CHI660 workstation.Electrochemical properties were tested using a three-electrode system at room temperature. The electrolyte was 0.5 mol LSince PMMA microspheres serve as a synthesis template, their particle size distribution is an important physical property. Analysis of the particle size distribution of PMMA by DLS demonstrates that the average diameter of PMMA microspheres is ~220 nm a. Polydi2 exhibits a three-dimensional ordered honeycomb macroporous structure  classification [2 belongs to the type IV with H3-type hysteresis, which is usually associated with highly consistent mesopore structures. Non-ammonia IrO2 also shows the type IV with H3-type hysteresis. However, the hysteresis loop is smaller than that in 3-DOM IrO2, showing that 3-DOM IrO2 has more mesopores. According to the BJH pore-size distribution analysis , (101), (200), (211), and (220) planes, can be easily seen.2 pattern does not show rutile IrO2 peaks. However, characteristic peaks of the elemental Ir at 2θ of ~40°, 47°, and 69° (which correspond to (111), (200), and (220) planes) could be clearly seen. This indicates that 3-DOM IrO2 contained metallic Ir formed because of template carbonization; after PMMA carbonization, internal Ir4+ had no access to air. Thus, a small amount of IrO2 was reduced to Ir. However, Ir4+ in the precursor that has no ammonia is IrCl4, and since Cl is less electronegative than O, Ir4+ was reduced almost entirely.Non-ammonia IrO2 is present in the catalyst , accompanied by protonation/deprotonation. The CV curve of 3-DOM IrO2 demonstrated a distinct redox peak at a low potential, which was related to the elemental Ir in 3-DOM IrO2. Ir, similar to Pt and Ru, can absorb hydrogen in the hydrogen underpotential deposition region [2 showed significantly larger current density in comparison to colloidal IrO2. Current responses consist of two effects, the redox pseudo-capacitance and the double-layer capacitance, both of which are related to the electroactive surface areas (ESAs).The electrocatalytic performances of two materials are shown in 2 a clearlyn region ,40. 3-DO2 demonstrates superior OER activity compared to colloidal IrO2. Compared to the thermodynamic minimum potential of OER, the overpotential of 3-DOM IrO2 is 0.22 V at 0.5 mA cm−2, which outperforms colloidal IrO2 (0.25 V) and is the same as the 3-DOM IrO2 material synthesized by the silica colloidal crystal template [2 templated by PEO–PB–PEO copolymers [2 prepared by the PMMA template did not require acid solution treatment, thereby reducing the cost of preparation. 2 (106 µg cm−2) has the same catalytic activity as colloidal IrO2 (265 µg cm−2), while the OER activity of 3-DOM IrO2 is outstanding compared to that of colloidal IrO2, because of their different ESAs . The Tafel slope is similar to that of nanoporous IrO2 and within the normal range of conventional IrO2 [2, indicating a faster rate for the OER.The linear sweep voltammetry (LSV) of two catalysts demonstrating their OER activities are shown in template  and the polymers . Howeverent ESAs a. The rumetal Ir ; therefomV dec−1 d, which nal IrO2 . This al2 still showed higher current density than that of colloidal IrO2. Slopes of the fitting curves in the range of 15,000–20,000 s (insert in  × 10−4 and −2.37 × 10−4, respectively. Thus, the current change of colloidal IrO2 deceases faster and 3-DOM IrO2 exhibits superior stability than that of colloidal IrO2.2 using a PMMA template and ammonia sol has been performed. The issue of the organic template having difficulty in maintaining the 3-DOM IrO2 structure has been solved by the ammonia sol. The sol consists of iridium chloride, isopropanol, and ammonia. IrCl4 and ammonia form a stable complex solution, in which NH4+ and Cl− form ammonium chloride, and ammonium chloride can easily be removed. The prepared 3-DOM IrO2 material has a honeycomb array macroporous structure with an average pore diameter of 155 nm and a large surface area. Many mesoscale pores are observed on the wall of the macropores. 3-DOM IrO2 demonstrates superior OER activity compared to colloidal IrO2, and the overpotential of 3-DOM IrO2 is lower than that of colloidal IrO2 by about 30 mV at 0.5 mA cm−2. In the same catalytic activity, the loading of 3-DOM IrO2 is much lower than that of colloidal IrO2. Our novel method to synthesize the 3-DOM IrO2 catalyst can reduce the anode overpotential of water electrolysis. The results suggest that the 3-DOM IrO2 catalyst is a promising material for OER.Synthesis of 3-DOM IrO


The striatum is critically involved in execution of appropriate behaviors, but its internal structures remain unmapped due to its unique structural organization, leading to ambiguity when interpreting heterogeneous properties of striatal neurons that differ by location. We focused on site-specific diversity of striosomes/matrix compartmentalization to draw the striatum map. Five types of striosomes were discriminated according to diverse immunoreactivities for the µ-opioid receptor, substance P (SP) and enkephalin, and each type occupied a particular domain inside the striatum. Furthermore, there was an additional domain lacking striosomes. This striosome-free space was located at the dorsolateral region and received afferents preferentially from the primary motor and sensory cortices, whereas the striosome-rich part received afferents from associational/limbic cortices, with topography inside both innervations. The proportion of dopamine D1 receptor-expressing, presumptive striatonigral neurons was approximately 70% in SP-positive striosomes, 40% in SP-deficient striosomes, 30% in the striosome-free space, and 50% in the matrix. In contrast, the proportion of D2 receptor-expressing, presumptive striatopallidal neurons was complementary to that of D1 receptor-expressing cells, indicating a close relationship between the map and the direct and indirect parallel circuitry. Finally, the most caudal part of the striatum lacked compartmentalization and consisted of three lamina characterized by intense and mutually exclusive immunoreactivities for SP and enkephalin. This tri-laminar part also received specific afferents from the cortex. The newly obtained map will facilitate broad fields of research in the basal ganglia with higher resolution of the three-dimensional anatomy of the striatum.The online version of this article (10.1007/s00429-018-1749-3) contains supplementary material, which is available to authorized users. The striatum is the major input structure of the basal ganglia and plays pivotal roles in optimizing behaviors through learning. It occupies a large portion of the subcortical gray matter in the mammalian telencephalon and receives driving inputs from the entire neocortex. These anatomical characteristics indicate versatile functions of the striatum such as control of voluntary movements, goal-directed behaviors, decision-making, cognition, and habituation Graybiel . HoweverAlternatively, functional division of the striatum into sensorimotor, associational, and limbic parts has been adopted in primates Parent . AnalogoRecently, these particular circumstances have driven researchers to draw an extensive map of the striatum based on the specific connectivity between diverse neocortical areas and striatal subdomains , substance P (SP), and enkephalin (Enk). Importantly, these substances are key molecules in basal ganglia circuitry; specifically, axon terminals of striatonigral, direct pathway neurons and striatopallidal, indirect pathway neurons contain SP and Enk, respectively , and all of the protocols were approved by the Institutional Animal Care and Use Committees at Kumamoto University and Kurume University. All efforts were made to minimize the number of animals used and their suffering.1R-DARPP-32-Flag/D2R-DARPP-32-Myc  and four male transgenic mice tagged for DFor fixation of brains, animals were deeply anesthetized with sodium pentobarbital  and perfused via the ascending aorta with 0.01 M phosphate-buffered saline  followed by 50 ml of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer  at room temperature. Brains were removed from the skull and stored overnight in the same fixative at 4 °C. The next day, the fixative was replaced by PBS containing 0.1% sodium azide.The anterograde tracers used were biotinylated 10 kDa dextran amine  or phaseolus vulgaris-leucoagglutinin . BDA and Phal were injected stereotaxically into ten different cortical areas as shown in Table 2. The sections were then rapidly thawed in 25% sucrose in PBS, and then processed for triple-fluorescent immunohistochemistry, as previously described  from the brain block that contained the entire striatum. After cryo-protection in 25% sucrose in PBS, the sections were placed on aluminum foil and rapidly frozen in the vapor of liquid NThe second set of immunostaining was prepared from the animals injected with the tracers. Sections from Phal-injected animals were incubated in a mixture of goat anti-Phal  and mouse anti-calbindin  antibodies and then in a mixture of Rhodamine red-conjugated donkey anti-goat IgG  and Alexa 488-conjugated donkey anti-mouse IgG . Sections from BDA-injected animals were incubated in mouse anti-CB antibody and then in a mixture of Alexa 488-conjugated donkey anti-mouse IgG and streptavidin-Alexa 647 . Note that BDA labeling in all figures are displayed with a pseudocolor of red as in color for Phal.The third set of immunostaining was prepared for sections from the transgenic mice. To investigate the distributions of D1R- and D2R-positive cells within the diverse compartments of the striatum, three sets of triple-immunostaining were performed in 4 adjacent sections using a mixture of mouse anti-Flag  and goat anti-c-Myc  antibodies combined with one of the following antibodies: guinea pig anti-MOR1, rat anti-SP, and rabbit anti-Met-Enk antibodies. The sections were further incubated with the secondary antibodies listed in Table Immunostained sections were mounted in Vectashield (Vector Laboratories) and examined using a confocal laser-scanning light microscope , which was equipped with three single laser beams, 488, 543, and 633 nm in wavelength, and a filter set of BA 515/30, BA 590/50, and 650 LP. Control sections were prepared by omission of primary antibodies and by mismatching secondary antibodies; both provided only weak nonspecific staining.Images for confocal laser scanning light microscopy (CLSM) were obtained using the × 4  and × 40  objective. The × 4 objective was used to visualize the whole striatum in a single frame in CLSM, whereas the × 40 objective was used to identify and analyze striatal neurons and tracer-labeled axon terminals of cortical afferents. The size of each frame was 1024 × 1024 pixels, and the intensity of the signal in each pixel was recorded at 8-bits for each channel. All of the image data from the same animal were obtained by maintaining the CLSM settings constant, such as the laser power and the gains of the photomultipliers. Images of the optical slices were acquired from the section surface to the bottom at the preset optimal step size and were stored as a stacked file for each frame using the three single laser beams alternately at each z-position of the stage to collect images for the different fluorescence signals.To determine the rostrocaudal extent of the striatum, the CLSM images were acquired with a × 4 objective from the serial coronal sections containing the whole striatum in animals, and the sections were triple-labeled for MOR, SP, and Met-Enk. The distance of each section from the reference section that contained the center of the anterior commissure crossing the midline was estimated by multiplying the section thickness (50 µm) by the number of intervening sections.Striosomes were defined in MOR-, SP-, and Enk-immunohistochemistry as patchy domains that showed higher immunoreactivity than the surrounding tissue (matrix), with a clear boundary and with morphological features common to the previous observations: they had round, oval or elongated shapes with an occasional bifurcation or intervening thin bridges and had a relatively constant transverse diameter . Then, we reconstructed the five types from complete serial sections covering the entire striatum Fig. , leadingThere has been accumulating evidence that striosomes that appear as patchy profiles in individual sections actually form a continuous labyrinth in many species, including the cat  the striosome-rich part, (2) striosome-free space, and (3) the most caudal tri-laminar part. The striosome-rich part contained five types of striosomes, each occupying a particular domain. According to these classifications, we drew an internal structure-based map of the striatum along the entire rostrocaudal axis of the striatum Fig. .As a next step, we determined that this map was in good correspondence to the distribution of afferent axons from diverse sources in the cerebral cortex. First, and most characteristically, axons labeled by anterograde tracers injected into the primary motor area were distributed predominantly in the striosome-free space Figs. , 10b–e. This remarkable projection pattern was in sharp contrast to the distribution of afferents from the associational/limbic cortical areas. Axons from the prelimbic area targeted the medial region of the striosome-rich part in the rostral and middle striatum Figs. , 10g–k, Axons from the secondary motor (M2) area were distributed in the subregion of the striosome-rich part that has been so far regarded as the “dorsolateral sensorimotor part” Fig. g–k, but Projections from the anterior cingulate cortex targeted the dorsomedial region of the striosome-rich part Fig. g–l. ThesAxons form the medial orbital area distributed in the most ventromedial region of the striosome-rich part Fig. g–i, wherThe present results show good topographical correspondence between the corticostriatal projections and the striatal division: the primary motor and sensory areas innervated the striosome-free space, whereas the associational/limbic areas targeted the striosome-rich part of the striatum. Further, the topography could be subdivided within both the striosome-free space and striosome-rich part as summarized in Fig. n = 22 striosomes, 1667 cells), 69.0 ± 6.9% , 71.6 ± 8.6% , respectively; whereas that in MOR-only and MOR/Enk striosomes was 42.3 ± 5.3%  and 42.3 ± 5.1% , respectively. On the other hand, the proportion of D1R-expressing cells in the striosome-free space, Enk-positive islands in the matrix, and CB-intense matrix was 33.2 ± 11.8% , 52.2 ± 3.2% , and 48.9 ± 6.2% , respectively. Thus, the proportion of D1R-expressing neurons was approximately 70% in all SP-containing striosomes irrespective of other striosomal markers, approximately 40% in SP-negative striosomes, as low as nearly 30% in striosome-free space, and approximately 50% in the matrix. The proportion of D1R-expressing cells in each of the three types of SP-containing striosomes was significantly higher than that in other compartments composed of SP-negative striosomes, the striosome-free space, Enk-positive islands in matrix, and CB-intense matrix . On the other hand, the proportion of D2R-expressing cells in the striosome-free space (65.0 ± 10.5%) was significantly higher than that in any other compartments. These findings strongly support the fact that the compartment-based map of the striatal internal structure is closely related to the structural organization of the striatum in terms of the direct and indirect parallel streams.Finally, we further assessed the validity of the internal structure-based map of the striatum shown here in terms of its relationship to the composition of dopamine D1R- and D2R-expressing neurons, which is another key to understand striatal organization, using D1-DARPP-32-Flag/D2-DARPP-32-Myc transgenic mice  of D1R-expressing neurons were located in the dorsomedial to ventromedial domains in the rostral precommissural striatum and in the medial laminar domain in the postcommissural striatum. On the other hand, two types of SP-weak striosomes, with a proportion of 40% for D1R-expressing neurons, were located in the dorsolateral to ventrolateral domains in the rostral striatum. The lateral decrease in the D1R proportion was accompanied by a lateral increase in the D2R proportion that reached the largest value in the striosome-free space


This study is aimed at investigating the lymphocyte subsets of cerebrospinal fluid (CSF) to provide possible differential diagnostic values and better understand the pathophysiological mechanism underlying autoimmune encephalitis (AE) and infectious lymphocytic encephalitis. A series of CD markers, including CD3/4/8/20 representing different types and developmental stages of lymphocytes, were used to count the corresponding subpopulations of CSF from clinical and laboratory confirmed cases of anti-N-methyl-D-aspartate receptor AE (NMDAR-AE), herpes simplex virus encephalitis (HSVE), and tuberculous meningitis (TBM). The percentages of lymphocytes observed and the CD4 : CD8 ratios were compared between the three groups. There were no significant differences of the percentage of total lymphocytes, CD3 cells, and CD4 cells of CSF among each group. However, there were strongly statistical differences of the CD4 : CD8 ratio in CSF of each group with 0.6 : 1 in NMDAR-AE, 0.9 : 1 in HSVE, and 3.2 : 1 in TBM. The percentage of CD20 B lymphocytes in NMDAR-AE was statistically higher than that of other groups. The distinct percentages of lymphocyte subpopulations of CSF appeared to be characteristic and could potentially serve as diagnostic indicators. Further verification and research will be necessary to clarify the significance and nature of CD4 : CD8 ratios and B lymphocytes in CSF between AE and the infectious lymphocytic encephalitis. Autoimmune encephalitis (AE) is a group of newly recognized encephalitis syndromes associated with the autoantibodies to the antigen of neurons . Among tCSF examination is still an exclusive golden diagnostic method in patients with the CSN infectious diseases or AE . But theIn the current study, the immunocytochemistry (ICC) method of CSF was extended to investigate the different percentages of lymphocyte subpopulations and CD4 : CD8 ratios in the CSF of patients with NMDAR-AE, HSVE, and TBM for new early diagnostic insights. Second, this study would also provide the explanations for the potential mechanisms of NMDAR-AE and the association with other infectious encephalitis from this regard.A total of 66 patients including 21 NMDAR-AE cases, 23 HSEV cases, and 22 TBM cases, were enrolled in this study. The basic clinical information is shown in 10.3760/cma.j.issn.1006-7876.2017.02.004). The initial criteria to be met for the consideration of possible AE include are as follows: (1) a compatible clinical syndrome of subacute/rapidly progressive memory loss, psychiatric symptoms, or consciousness disorders; (2) one or more of (A) focal CNS findings, (B) new seizures, (C) speech disturbance, (D) severe dyskinesia, and (E) autonomic dysfunction; (3) electroencephalogram abnormalities; and (4) pathologic neuroimaging findings of MRI. Apart from the clinical information, the final definite diagnosis was made based on antibody testing results. Anti-NMDAR was tested in both the CSF and serum using an indirect immunofluorescence testing (IIFT) method. The postfixed sagittal mouse brain sections and IIFT detection kit in the form of biochips were commercially available from the company of Euroimmun . But at least, the CSF results of anti-NMDAR positive were the reference indicator.The criteria for AE were based on a Lancet paper of “A Clinical Approach to Diagnosis of Autoimmune Encephalitis”  and “ChiThe criteria for HSV was as follows : (1) theThe excluded criteria were as follows: (1) the high proportions of nonviable cells or insufficient cell numbers are to be evaluated; (2) anti-NMDAR antibody was solely positive in CSF or blood, but without related clinical encephalitis evidence; and (3) when the first CSF was collected of the patient admitted, it was over than two weeks after disease onset.CSF samples were collected on the first day of admission without any special or nonspecial treatment as a natural history of the disease, including anti-virus, anti-TB, or any types of steroids. CSF evaluating team was blinded to the diagnosis. The slides of CSF were prepared as follows: (i) inhaling 200 microliters of CSF into a cell centrifuge (Statspin Cytofuge 12 or Cytospin 3) at a low speed of 1000 rpm/3 min ; (ii) drying the slides at room temperature (RT); (iii) fixing them with cold acetone for 3-5 min at 4°C in the refrigerator; and (iv) setting aside to prepare for use. The total CSF leukocyte counts and the percentage of lymphocytes in the CSF were observed in each patient by using the Wright–Giemsa stain method with centrifuged sediment of the CSF as described previously.The detail protocols were as follows: staining was performed using the SP kit according to the manufacturer's instructions. After 10 min blocked with 5% normal goat serum at RT, the prepared slide above was incubated with primary antibody. Mouse anti-CD3 monoclonal antibody  represents the mature T cell type. Mouse anti-human CD4 monoclonal antibody  represents T helper cells or inducer T cells, as the functional cells for executing and regulating immune systems. Mouse anti-CD8 monoclonal antibody  is the marker of T cells responsible for directly killing infected and abnormal cells. Mouse anti-CD20 monoclonal antibody  is the special marker of B cell. All the four kinds of primary antibodies were purchased from Maixin Bio Co., Ltd., Fuzhou, China.The primary antibodies were at a dilution ratio of 1 : 100 overnight at 4°C. After washing, the slices were incubated with biotin-labelled second antibody, reagent-labelled goat anti-mouse, and anti-rabbit IgG polymer (as an enhanced polymer)  at 37°C for 30 min. And then, the immunoreactivity was tested with the avidin-biotin-peroxidase technique, using 3,30-diaminobenzidine as the chromogen.The percentages of CD4, CD3, and CD8 T cells and CD20 B-positive cells were counted, respectively. The method has been described in our published work [one-way ANOVA, and further multiple comparisons for post hoc tests used the Least Significant Difference (LSD) method. A chi-square test was used for analysing categorical variables. The P value reported was two-sided, and P < 0.05 was considered statistically significant. All analyses were performed using the SPSS software .Measurement data were expressed as the mean ± SD. Qualitative data were described as percentages or rations. Differences among groups with continuous data were analysed by P = 0.784 and P = 0.535, respectively); however, the total WBC count in the NMDAR-AE group is lower than that of the HSVE and TBM groups (both P = 0.000). This suggests that it may be difficult by using the traditional method of CSF cytology to different NMDAR-AE from the infectious lymphocytic encephalitis including HSVE and TBM if they are presented with the similar WBC count in CSF.The distributions of age and gender were not statistically different among the three groups . There wP = 0.024), but with no significance within the TBM group (P = 0.220). The percentage of CSF CD8 T cell count was significantly higher in patients with NMDAR-AE (P = 0.0002) and HSVE (P = 0.000) than in patients with TBM. There were strongly statistical differences of the CD4 : CD8 ratio in CSF of each group with 0.6 : 1 in NMDAR-AE, 0.9 : 1 in HSVE, and 3.2 : 1 in TBM . The percentage of CD20 B lymphocytes in NMDAR-AE was higher than that of other groups. The proportion of CD20 B cells was not obviously different between the groups of TBM and HSVE (P = 0.650). These results suggested that the CD4:CD8 ratios and B lymphocytes in CSF may be useful for the differential diagnosis for the AE with infectious CNS disease  of CD3 cells of CSF in patients with the neurological disease  compared with that in bacterial infection patients . We specThe other useful information given by our present study is that the proliferation and differentiation of various lymphocytes may mediate the development of the CNS immune or infectious inflammatory disorders. T cells have different abilities to recognize antigen and are varied in terms of their function. CD4 cells represent a mature type of T cells . CD8 celIn summary, the current study showed that there were different percentages of lymphocyte subpopulations and CD4 : CD8 ratios in the CSF of patients with NMDAR-AE, HSVE, and TBM, which may play an important potential role in diagnostic aspect. It may be helpful to use the immunocytochemistry method of CSF but it still need to be extensively verified in clinical practice with larger populations. This study also provides the clue of different types of immune response undergoing the pathophysiological mechanisms of NMDAR-AE and the association with other infectious encephalitis.


The analysis performed clearly showed that both AQPs have a significant participation in the CSF production, and additionally revealed that the double AQP1-AQP4 mutation alters the CSF drainage and the ventricular compliance. The data reported here indicate a significant extra-choroidal CSF formation mediated by AQP4, supporting the idea of an important and constant CSF production/absorption process, sustained by efflux/influx of water between brain capillaries and interstitial fluid. Moreover, our results suggest the participation of AQPs in structural functions also related with CSF homeostasis such as the distensibility capacity of the ventricular system.Aquaporin 1 (AQP1), expressed in epithelial cells of the choroid plexus, and aquaporin 4 (AQP4) present in ependymal cells and glia limitants have been proposed to play a significant role in cerebrospinal fluid (CSF) production and homeostasis. However, the specific contribution of each water channel to these functions remains unknown, being a subject of debate during the last years. Here, we analyzed in detail how AQP1 and AQP4 participate in different aspects of the CSF homeostasis such as the load and drainage of ventricles, and further explored if these proteins play a role in the ventricular compliance. To do that, we carried out records of intraventricular pressure and CSF outflow, and evaluated ventricular volume by magnetic resonance imaging in AQP1 Cerebrospinal fluid (CSF) and interstitial fluid (ISF) are crucial components of the cerebral extracellular fluid, being in dynamic equilibrium and bathing neurons and glial cells. The continuous and strictly regulated interchange between CSF and ISF provides a stable brain volume and fluid content of ions and other solutes for neuronal and glial function ,2,3. CSF−/− mice showed decreased CSF production and intraventricular pressure, suggesting a prominent role of the AQP1 on CSF formation , was placed in the parietal cortex above the lateral ventricle . When the pipette reached the parietal cortex, the pressure gradually rose. Once the pressure was >50 cm H2O, the infusion was stopped and the pipette was slowly advanced. The pressure fell promptly when the micropipette reached the lateral ventricle. Henceforth, IVP was recorded. A detailed example of an in vivo record of IVP, and the procedure used to infer the CSF outflow and ventricular compliance  filled with artificial CSF (aCSF), which was inserted into the lateral ventricle through the cerebral cortex  as previously shown ,19. The ance see  is shownDynamic of the CSF outflow was studied by the constant-rate infusion method reported before ,19. Conc1 − t0) of aCSF infusion after increase the flow rate from 7 to 14 μL/min  produced after 1.75 μL of volume increase. This increment in the ventricular volume was induced by 15 s  are displayed in each figure legend. Data are presented as mean ± standard error of the mean (SEM), and the statistical test performed is specified in each figure legend. For all the analysis carried out, data were tested for normality (Kolmogorov–Smirnov test) and equal variance. When these properties were confirmed, analysis of variance was carried out with Tukey HSD post hoc analysis for multiple group comparisons. All statistical analyses were conducted by IBM SPSS Statistics for Windows (Version 20) or GraphPad Prism 6 packages.−/−, AQP4−/− and double-AQP−/− KO mice; −/− mice . Moreover, double-AQP−/− KO mice presented an additive decrease in the ventricle size (~30% of reduction with respect to wt controls). Interestingly, the double-AQP−/− KO mice presented diminished ventricle size whether compared with AQP1−/− or AQP4−/− mice, indicating a similar contribution of AQP1 and AQP4 to the ventricular volume.To study the specific contribution of AQP1 and AQP4 to the ventricular volume, the volume occupied for the cerebral ventricular system was assessed in wt, AQP1l groups A,B. As i−/− mice showed a significant lower IVP value than wt controls , confirming the relevant participation of this protein in the CSF formation and the ventricular volume. Nevertheless, double-AQP−/− KO mice did not present further lower IVP values than AQP1−/− or AQP4−/−, which can be attributed to the extremely reduced ventricular system existing in these double mutant mice. Taken together, the MRI and IVP analysis performed in the different AQPs mutants clearly showed that both AQP1 and AQP4 contribute similarly to the volume of the brain ventricular system.We also analyzed the IVP of the previously described experimental groups . As repocontrols ,19). Sim−/−, AQP4−/− and double-AQP−/− KO mice. In accordance with previously reported data by our group and other authors [−/− and AQP4−/− mice  indicated a decreased CSF outflow in these mice with respect to wt controls. To perform a deeper analysis of the CSF drainage, we compared the IVP obtained under low  produced in the ventricle after 1.75 μL of aCSF volume increase . Similar−/− and AQP4−/− mice showed a comparable reduction in the ventricular volume and IVP suggesting that, in terms of CSF production, choroidal-AQP1 and parenchymal-AQP4 have a similar contribution to the CSF formation. In accordance with the participation of both cerebral AQPs in the ventricular volume, double-AQP−/− KO mice displayed lower ventricular volume than single AQP1 or AQP4 mutants. Despite the reduction in the ventricular volume observed in double-AQP−/− KO mice, with respect to AQP1−/− or AQP4−/− KO mice, these animals present similar IVP values to those observed in the single AQPs KO mutants. These observations suggest a functional coupling between the CSF production, facilitated either by choroidal AQP1 or astroglial and ependymal AQP4, and the ventricular volume to maintain the intracranial pressure under homeostatic values.AQP1 and AQP4 are the principal water-transporter channels in the central nervous system showing a distinct and complementary expression pattern within the brain. Both proteins have been proposed to be principal regulators of the cerebral fluid homeostasis in health as well as pathophysiological conditions. AQP1, exclusively expressed in the apical side of epithelial choroid plexus cells, and AQP4, mainly expressed in astrocytes and ependymal cells ,32, have−/− mice [−/− mice [The study of the CSF drainage performed by pressure-dependent CSF outflow revealed that single AQP1 or AQP4 mutants do not present significant alterations in the CSF drainage. These results are in accordance with previously reported analysis performed by Verkman’s group for AQP1−/− mice  and our −/− mice . However−/− mice ,34,35,36−/− mice ,37 have −/− KO mice suggest, unexpectedly, that cerebral AQPs could regulate the ventricular compliance. The mechanism by which these AQPs could alter the ventricular system distensibility is unidentified at the present time. Additional experimental analysis should be performed to understand if the reduced ventricular volume could be related with this alteration, or if the absence of cerebral AQPs alters the biophysical properties of the cerebral parenchyma; or even if this modification could be associated with systemic alterations that potentially modify the cerebral blood flow, and secondarily alter the cerebral compliance.Regarding the contribution of AQP1 and AQP4 to the ventricular compliance, the data obtained in the double-AQP−/− KO could be related with compensation of other AQPs and/or alterations in the brain water content.In addition to AQP1 and AQP4, the aquaglyceroporin AQP9 is also expressed in the brain, being localized in astrocytes in the glial limitants, endothelial cells of the pial vessels and specific groups of neurons ,39. AgaiIn conclusion, our results clearly indicate that glial- and ependymal-expressed AQP4 facilitate the CSF formation, having a quantitatively similar contribution to this process that choroidal-expressed AQP1. The fact that AQP4 facilitates the CSF production is aligned with the recently formulated “Bulat–Klarica–Oreskovic hypothesis” that predicts a significant CSF production by water filtration from the cerebral capillaries. However, the results obtained from the AQP1 mutant mice, by us and others , also im


How do different proposed measurements of tau brain pathologic abnormalities  and neurodegeneration  change over the course of Alzheimer disease?This cohort study of 465 participants with dominantly inherited Alzheimer disease found that the rates of change for CSF levels of total tau and phosphorylated tau 181 had a different pattern across the course of Alzheimer disease. The association between the rates of change of CSF levels of total tau and phosphoryated tau 181 and brain atrophy varied by disease stage.These results may provide a better understanding of the dynamics of Alzheimer disease and have important implications as trials targeting tau brain pathologic abnormalities move forward. This cohort study of participants with dominantly inherited Alzheimer disease examines the rates of change in cerebrospinal fluid levels of total tau (tTau) and phosphorylated tau 181 (pTau181) across the disease course and how the rates of change are associated with brain atrophy. The amyloid/tau/neurodegeneration (A/T/N) framework uses cerebrospinal fluid (CSF) levels of total tau (tTau) as a marker of neurodegeneration and CSF levels of phosphorylated tau 181 (pTau181) as a marker of tau tangles. However, it is unclear whether CSF levels of tTau and pTau181 have similar or different trajectories over the course of Alzheimer disease.To examine the rates of change in CSF levels of tTau and pTau181 across the Alzheimer disease course and how the rates of change are associated with brain atrophy as measured by magnetic resonance imaging.This cohort study was set in tertiary research clinics. Each participant was a member of a pedigree with a known mutation for dominantly inherited Alzheimer disease. Participants were divided into 3 groups on the basis of the presence of a mutation and their Clinical Dementia Rating score. Data analysis was performed in June 2019.Rates of change of CSF tTau and pTau181 levels and their association with the rate of change of brain volume.P = .05] and 0.7 [0.3] for pTau 181 [P = .04]) and 15 years before onset , respectively. The rate of change of pTau181 was positive and increased at the early stages of the disease, showing a positive rate of change starting at 15 estimated years before onset until 5 estimated years before onset , followed by a positive but decreasing rate of change at year 0  and then negative rates of change at 5 years  and 10 years  after symptom onset. In individuals without symptoms , the rates of change of CSF tTau and pTau181 were negatively associated with brain atrophy (high rates of change in CSF measures were associated with low rates of change in brain volume in asymptomatic stages). After symptom onset , an increased rate of brain atrophy was not associated with rates of change of levels of both CSF tTau and pTau181.Data from 465 participants (283 mutation carriers and 182 noncarriers) were analyzed. The mean (SD) age of the cohort was 37.8 (11.3) years, and 262 (56.3%) were women. The mean (SD) follow-up duration was 2.7 (1.5) years. Two or more longitudinal CSF and magnetic resonance imaging assessments were available for 160 and 247 participants, respectively. Sixty-five percent of mutation carriers (183) did not have symptoms at baseline . For mutation carriers, the annual rates of change for CSF tTau and pTau181 became significantly different from 0 approximately 10 years before the estimated year of onset (mean [SE] rates of change, 5.5 [2.8] for tTau [These findings suggest that CSF tTau and pTau181 may have different associations with brain atrophy across the disease time course. These results have implications for understanding the dynamics of disease pathobiology and interpreting neuronal injury biomarker concentrations in response to Alzheimer disease progression and disease-modifying therapies. Dominantly inherited AD (DIAD) is considered clinically similar to sporadic AD except for a younger age at onset (AAO).6 Mutation carriers (MCs) have a somewhat predictable age at AD symptom onset.7The neuropathologic hallmarks of Alzheimer disease (AD) are the presence of neuritic amyloid plaques, primarily of the amyloid-β peptide, and the intraneuronal accumulation of neurofibrillary tangles (NFTs) and neuropil threads composed of hyperphosphorylated, aggregated tau protein.8 have contributed to hypothesized trajectories of fluid and imaging biomarker changes that occur over the course of the disease, from the preclinical phase to the end stages characterized by advanced dementia. However, models of biomarker change in AD have been based mostly on cross-sectional data.10 More recently, many models have classified biomarkers according to their proposed association with the biological underpinnings of the disease; the recent amyloid/tau/neurodegeneration (A/T/N) framework11 was developed to provide a more biological rationale to the classification of the disease. Phosphorylated tau 181 (pTau181) in the cerebrospinal fluid (CSF) has been suggested to represent NFT pathologic abnormalities, whereas total tau (tTau) in the CSF is thought to be a marker of neurodegeneration that is passively released with cell death or injury.12 Given these putative mechanisms, it might be expected that levels of both tTau and pTau181 would continue to become more abnormal with disease progression, as NFT pathologic abnormalities increase and neurodegeneration accelerates. However, recent longitudinal studies17 from the Dominantly Inherited Alzheimer Network (DIAN) and Alzheimer’s Disease Neuroimaging Initiative (ADNI) cohorts have challenged the linear model from previous cross-sectional studies,18 which have consistently found higher CSF levels of tTau and pTau181 as the disease progresses.Biomarker studies19These findings highlight a need for accurate determination of the evolution of longitudinal changes in CSF levels of tTau and pTau181 and their association with disease progression. In the present study, we assessed the longitudinal pattern of changes in CSF levels of tTau and pTau181 and their association with brain atrophy as measured by magnetic resonance imaging (MRI). We hypothesized that if CSF tTau and pTau181 were passively released with neurodegeneration, they should be associated with MRI measures of neurodegeneration . To evaluate this hypothesis, we used a well-characterized cohort with DIAD from the DIAN study.20 Participants provided written informed consent or assent with proxy consent. The institutional review boards for each of the participating DIAN sites approved all aspects of the study. This study follows the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.All participants were recruited as part of the DIAN study.21For our analysis, 465 participants with at least 1 CSF measurement in the DIAN cohort were available from the data freeze 12 . Each participant was a member of a pedigree with a known mutation for DIAD. Genotyping was performed to determine the genetic status for each participant at risk; the presence or absence of a DIAD mutation was determined using polymerase chain reaction–based amplification of the appropriate exon followed by Sanger sequencing methods on a genetic analyzer .22 score at baseline, with a CDR score of 0 indicating no symptoms and a CDR score of 0.5 or higher indicating the presence of symptoms. The participant’s estimated years before expected symptom onset (EYO) was defined as the participant’s age at baseline minus their expected AAO.23 The AAO was calculated according to either the family mutation–specific expected age at dementia onset or parental age at first progressive cognitive decline if the expected AAO for the mutation was unknown. Negative values indicate that an individual is younger than his or her expected AAO.Participants were divided into 3 groups according to the presence of a mutation and their Clinical Dementia Rating (CDR)24 Clinicians performing the assessments were blinded to mutation status of participants. Full details of participating sites, enrollment, and assessments in DIAN have been published elsewhere.20Participants underwent extensive clinical evaluation, which included family history of AD, personal medical history, and physical and neurological examination. Clinical dementia status was determined with the CDR in accordance with standard protocols and criteria.25 Sites used a 3-T scanner and were required to pass regular quality control assessments. Volumetric segmentation and cortical surface reconstruction were performed using FreeSurfer image analyzing software version 5.3 ,26 and subcortical volumes were corrected for intracranial volume using a regression approach.27 Cortical thickness and volume measures were averaged across hemispheres because there were no a priori laterality predictions. Because the focus of our study was to assess the association between longitudinal CSF tTau and pTau181 levels with atrophy rate, a limited number of regions of interest (ROIs) reflecting brain atrophy patterns across AD stages were included. We followed an ROIs approach to include posterior areas with earlier tau deposition  and atrophy vs anterior areas with later tau deposition  and atrophy. The ROIs were defined according to areas of early vs late neurodegeneration and tau deposition in the literature and our previous work.31Participants underwent volumetric T1-weighted MRI, using the magnetization-prepared rapid acquisition with gradient-echo sequence defined in the ADNI second phase.14 To also test whether those findings were specific to the method of measurement, here we used a fully automated, high-performance electroluminescence immunoassay  of tTau and pTau181 in the laboratory of Leslie Shaw, PhD, in the ADNI Biomarker Core at the University of Pennsylvania according to the kit manufacturer’s instructions and as described in previous studies.34 Analyses were performed in a series of runs, with each sample run 1 time for each of the tau analytes, over the period of October 18, 2017, through November 9, 2017; acceptance criteria as documented according to the Roche Protocol in the University of Pennsylvania ADNI Biomarker Laboratory were followed. In each of the 14 days of performing analyses, the manufacturer’s quality control results were within stated limits to meet acceptance criteria for precision and accuracy. There were 2 runs each day of approximately 40 samples per run. Precision performance was documented according to analysis of pristine aliquots of a CSF pool in each of the 28 runs for each of the biomarkers. The percentage coefficients of variation for this CSF quality control pool were less than 2%.The CSF was collected in the morning under fasting conditions by lumbar puncture and immediately placed on ice. Samples were shipped on dry ice to the DIAN biomarker core laboratory. We have previously reported a longitudinal decrease in CSF tTau and pTau181 levels using commercially available immunoassays.P values for comparing the difference between MCs and noncarriers (NCs) were obtained using generalized linear mixed-effects models with random intercepts for family clusters to take into account the associations between participants within the same family; the tests were 2-sided. The annualized rate of change over the longitudinal follow-up period was estimated for each participant separately using linear regression and then plotted against baseline EYO to evaluate the trajectories of the biomarker changes over EYO. To visualize the differences in the rates of change of different biomarkers as a function of EYO, locally estimated scatterplot smoothing curves of the standardized difference of individual annual rates of change for MCs for different biomarkers were plotted against baseline EYO. A linear or linear spline mixed-effects model for each biomarker (depending on the curve fit) was then used to estimate and test the rates of change at each integer EYO point. The fixed effects in the models included mutation group (MC or NC), baseline EYO, time since baseline, and all possible 2-way or 3-way interactions. Sex, years of education, and apolipoprotein ε4 allele status were considered as covariates, but only significant effects were retained in the models. Random effects included in the models are random intercepts for family clusters, individual random intercept, and random slope. An unstructured covariance matrix was used to account for the within-participant association due to repeated measures. Further details for the linear and linear spline mixed-effects models can be found in the article by McDade et al.14 Data analysis was performed in June 2019. Baseline characteristics of the participants are summarized as mean (SD) for continuous variables and number (column percentage) for categorical variables. 36 Statistical analyses were conducted with the PROC MIXED and PROC NLMIX procedures in SAS statistical software version 9.4 (SAS Institute Inc). A P < .05 was considered to be statistically significant. For CSF biomarkers (145 NCs and 235 MCs), the effect size that could be detected with 80% power was 0.3. For MRI biomarkers (177 NCs and 270 MCs), the effect size that could be detected with 80% power was 0.27.Associations between the individual annual rates of change of tTau and pTau181 concentrations and the annualized rates of brain atrophy were evaluated for MCs with symptoms  and MCs without symptoms  separately, using bivariate linear mixed-effects models with random intercepts for family clusters.We analyzed data from 465 participants, including 283 MCs (183 [65%] without symptoms and 100 with symptoms) and 182 NCs . The mea17 of this cohort have found that CSF tTau and pTau181 levels were increased in MCs 15 years before expected symptom onset (EYO = −15). We also examined the longitudinal change in CSF tTau and pTau181 levels in terms of annual rate of change across the EYO (P = .05] and 0.7 [0.3] for pTau 181 [P = .04]) and EYO −15 , respectively , followed by rate of change in hippocampal volume , entorhinal thickness , and superior-frontal thickness  around EYO −15. The longitudinal rate of change for all measured brain ROI increased 5 years before symptoms appeared (EYO −5) and followed a similar trend through the disease . For allTo visually compare the differences in the trajectories of the rates of change of CSF tTau and pTau181 levels and structural MRI measures, locally estimated scatterplot smoothing curves were constructed for the standardized rate of change as a function of baseline EYO . NotablyWe found differences in nearly all posterior and limbic or paralimibic regions for associations in rates of change with tTau and pTau181 according to stage of disease when comparing MCs without symptoms vs those with symptoms .38The use of biomarkers has become an essential component of AD research20 we evaluated the trajectories of CSF tTau and pTau181 over the course of AD. Notably, we included a bigger sample and used newer CSF tTau and pTau181 values generated with a fully automated, high-performance electroluminescence immunoassay. We compared the trajectories of CSF tTau and pTau181 with the atrophy of brain structures as measured by MRI. First, consistent with previous reports,39 our study found mean concentrations of CSF tTau and pTau181 to be higher in MCs from the early stage of AD, supporting the use of CSF tau as a marker of AD risk and progression. Second, we found that the positive rate of change of CSF tTau remained constant after EYO −10, whereas CSF pTau181 had a positive rate of change early in the disease course, which then reversed and became negative later in the disease. This indicates that our previous results14 were not likely to be an artifact of the measurement used because we used a different method for this study. Third, the associations identified between the rates of change of CSF tTau and pTau181 with brain atrophy do not support the assumption that CSF tTau changes follow a pattern similar to that of structural brain changes. Our findings indicate that neither CSF tTau nor pTau181 has the same pattern of change as brain measures and should be considered as associated but distinct biomarkers in AD. In addition, these findings indicate that, within the current biomarker classification, tTau is an important marker of AD but may not be the ideal marker of neurodegeneration.In this study, on the basis of longitudinal data from the DIAN study,41 have suggested that in the presence of amyloid pathologic abnormalities, more CSF tTau and pTau181 is released. Moreover, increased tau in CSF seems to be dependent of amyloid deposition and occurs in the absence of tau brain pathologic abnormalities.42Recent studies44 and inflammation, with higher levels of tTau and pTau181 in CSF representing a response.45 However, if CSF tTau directly reflected neurodegeneration, it would be expected that the rate of change of CSF tTau would increase in concert with brain atrophy during the period of maximal rate of atrophy (EYO >0). It is possible that during disease progression and neurodegeneration, the loss of neuronal cells results in less neuronal substrate to produce tau. Although this might account for some of the slowing in longitudinal changes in CSF tTau that we observed, it is unlikely that the degree of neurodegeneration is sufficient to fully explain our findings. Early elevations may also be associated with acute neuronal membrane damage, whereas apparent later reductions reflect the death of a smaller number of neurons that remain. Acute neuronal injury may be associated with a stronger inflammatory response at early stages of the disease.46Early disease stages may also be characterized by higher cellular stress47 we found evidence of a decrease in the rate of change, arguing against the use of these measures as a reflection of a passive release from neuronal death and NFT. These data support the relevance of CSF tTau and pTau181 as markers of amyloid deposition and accompanying changes 11; however, the complex rates of change identified here and in our previous work suggest that using them as measures of therapeutic response requires further investigation, because levels vary as a function of where an individual is in the neuropathological cascade. The apparent disconnect between CSF tTau and MRI measures may reflect the fact that they are measuring different stages of the neurodegenerative process, with CSF tTau accounting for the active phase of neuron injury and damage, and MRI measuring the subsequent structural sequelae of the active death process.The present findings challenge some previous assumptions about AD progression and its association with both pTau181 and tTau. Contrary to the idea that tTau and pTau181 levels continue to increase with greater neurodegeneration and the spread of NFT,50 of sporadic late-onset AD. Seppälä et al,48 in a longitudinal study in Finland, described higher levels of pTau181 level in mild cognitive impairment stages when compared with AD dementia, whereas levels of pTau181 decreased over a period of about 3 years. Similarly, Toledo et al38 reported a decrease in tTau in those at the dementia stage from the ADNI cohort. More recently, in a similar analysis of ADNI participants with longitudinal follow-up and using similar methods to determine CSF tTau and pTau181 levels , Sutphen et al15 reported that tTau and pTau181 showed consistent increases in the amyloid-positive participants with normal cognition and those with mild cognitive impairment, whereas pTau181 decreased substantially in those with AD dementia.Our findings are consistent with previous studies52 suggests that neurofilament light chains may be an advantageous marker of neurodegeneration in therapeutic trials of AD.53This study has potential implications for AD trials using tau-based therapies and other putative disease-modifying therapies. First, during trial design, the active group and the placebo group will have to be randomized by disease stage (according to disease severity measured using biomarkers and the severity of cognitive impairment), because minor differences in the neurodegeneration cascade stage might translate into major differences in biomarker trajectories, and as a result, might be misinterpreted as a treatment effect. Second, one must consider how to interpret changes in biomarkers during clinical trials readout; in other words, one must consider how a successful treatment would be expected to affect CSF tau levels or rate of change. This study, along with recent work assessing neurofilament light chains,57 These differences may be associated with biomarker profiles or natural progression, so the total generalizability of the present findings to late-onset AD will have to be confirmed in future cohorts. Nevertheless, the results highlight the importance of gathering longitudinal data to refine current biomarker models.The present study is not without limitations. First, our results are dependent on the accuracy of the DIAN EYO and, like any measure, it is subject to error . Second, we recognize that DIAD and late-onset AD are similar but not identical. Sporadic AD disease occurs later in life, and individuals often exhibit a wide range of comorbidities, including vascular disease and the presence of transactive response DNA-binding protein of 43 kDa.The results of this study support the A/T/N framework, whereby increases in CSF levels of tTau and pTau181 are robust diagnostic markers of pathologic abnormalities and neuronal injury in early Alzheimer disease. However, our data suggest that levels of CSF tTau and pTau181 diverge from atrophy-based measures of neurodegeneration later in the disease. This is an important result to consider in clinical trials targeting tau brain pathologic abnormalities. Future studies should explore the longitudinal rate of change of CSF pTau181 levels and positron emission tomography of tau as markers of tau brain pathologic abnormalities.


All participants had to perform three TT on different days, receiving either 6 mmol nitrate as BR or NIT or water as a placebo. Time-to-complete the TT, power output (PO), as well as oxygen uptake (VO2) were measured. No significant differences in time-to-complete the TT were found between the three interventions in able-bodied individuals (p = 0.80) or in paracyclists (p = 0.61). Furthermore, VO2 was not significantly changed after the ingestion of BR or NIT in either group (p < 0.05). The PO to VO2 ratio was significantly higher in some kilometers of the TT in able-bodied individuals (p < 0.05). The ingestion of BR or NIT did not increase handcycling performance in able-bodied individuals or in paracyclists.This study aimed to investigate the influence of a single dose of either beetroot juice (BR) or sodium nitrate (NIT) on performance in a 10 km handcycling time trial (TT) in able-bodied individuals and paracyclists. In total, 14 able-bodied individuals [mean ± SD; age: 28 ± 7 years, height: 183 ± 5 cm, body mass (BM): 82 ± 9 kg, peak oxygen consumption (VO During −1 (1549 g) and 4 °C for 10 min within 1 min of collection. Blood plasma was harvested and stored at −80 °C in 1.5 mL tubes . Plasma nitrate ([NO3−]) and nitrite ([NO2−]) concentrations were assessed by chemiluminescence NO analyzer . [NO2−] was reduced to NO in Brown’s solution , and subsequent photons produced as NO interacted with O3 were detected. Detection of [NO3−] included one more step in which [NO3−] was quantitatively reduced to [NO2−] using World Precisions Instrument’s Nitralyzer kit . Reduction of [NO3−] to [NO2−] was performed on copper-coated cadmium beads. Thereafter, total NO metabolites level (NOx) = ([NO2−] + [NO3−]) was measured, and [NO3−] concentrations were calculated as a difference between [NO2−] levels before and after the reduction step. Calibration solutions of sodium nitrite (NaNO2−)  and sodium nitrate (NaNO3−)  were prepared in bi-distilled water to produce calibration plots.Venous blood samples were drawn into lithium-heparin tubes . All samples were immediately centrifuged at 3000 r·min2, mean PO/VO2 ratio, and other cardiorespiratory parameters were assessed using a one-way repeated-measures ANOVA. Pairwise t-tests were used for post hoc analysis, and Bonferroni corrections were applied for multiple testing. The PO and PO/VO2 ratio data were graphed for the 10 km distance and analyzed using a two-way (supplement x distance) repeated-measures ANOVA with Bonferroni correction. In case of violation of sphericity, Greenhouse–Geisser correction was applied. The effect of order on TT performance was analyzed using a one-way repeated-measures ANOVA and pairwise t-tests with Bonferroni correction.The statistical analysis was performed using the IBM SPSS Statistics software for Windows . Statistical significance was set at an α-level of 0.05. The analysis with the Q-Q plot and the Shapiro-Wilk test showed that data for the able-bodied individuals were normally distributed, and data from paracycling athletes were not normally distributed. The data were presented as mean ± standard deviation (SD). The data of the able-bodied participants were analyzed using parametric testing. To analyze changes in blood pressure and heart rate from pre- to post-ingestion between NIT, BR, and PLA, a one-way repeated-measures ANOVA was performed. Differences in completion time, mean PO, mean VOThe data from paracyclists were analyzed using non-parametric testing. The Friedman test was applied to detect differences between the three groups for all parameters. If significant differences occurred, the Wilcoxon post-hoc test was applied with Bonferroni correction. For comparison between able-bodied individuals and paracyclists, the Mann-Whitney-U test was used to detect any differences.2peak: 33.9 ± 4.2 mL/min/kg, POmax: 152 ± 20 W] who were used to upper body exercises volunteered to participate in this study. They trained 4 ± 2 times per week with a total weekly duration of 6 ± 3 h. Additionally, eight paracyclists from the national team : 82 ± 9 kg, VOp > 0.05) in systolic and diastolic blood pressure pre and post ingestion of the supplements between the interventions were found in able-bodied participants or paracyclists. Neither in able-bodied individuals nor in paracyclists did the change in blood pressure from pre to post ingestion significantly differ (p > 0.05) in all three interventions. No significant difference in systolic and diastolic blood pressure was found between able-bodied individuals and paracyclists (p > 0.05). No significant differences between the three interventions in able-bodied individuals and in paracyclists were observed for the change of heart rate from pre to post ingestion of the supplement (p < 0.05). No significant difference between able-bodied individuals and paracyclists was found (p < 0.05). Data are presented in No significant differences  or in paracyclists (p = 0.19). A significantly higher [NO2−] and [NO3−] concentration was found in able-bodied individuals  and in paracyclists  in the BR intervention compared to PLA. No significant difference was found post ingestion between plasma [NO3−] and [NO2−] concentration in BR and NIT in either group (p > 0.05). Able-bodied individuals showed significantly higher [NO2−] concentrations after BR consumption compared to paracyclists (p < 0.05). Paracyclists showed significantly higher [NO3−] concentrations after BR and NIT consumption compared to able-bodied individuals (p < 0.05).Plasma [NOp < 0.05). [Lac] in the warm-up was significantly lower in paracyclists compared to able-bodied individuals (p = 0.029). Maximal and average heart rate (HR) as well as PO during the warm-up were all significantly different between the two groups of participants (p < 0.05). Data are presented in The warm-up parameters were not significantly different in the three intervention groups in paracyclists or in able-bodied individuals (p = 0.61). Furthermore, no significantly different time to complete the TT in paracyclists was found (p = 0.80). Paracyclists completed the 10 km TT significantly faster in all three trials compared to able-bodied individuals (p < 0.05) and therefore showed a significantly higher POaverage compared to able-bodied individuals (p < 0.05).No significant difference in time to complete the 10 km handcycling TT  was founp = 0.31) after the time trial nor rated perceived exertion (RPE) (p = 0.41) for the trials were significantly different between the three interventions in able-bodied individuals. The same was true in paracyclists between the three interventions . No significant differences were found in RPE (p = 0.08) and [Lac] (p = 0.86) concentration between able-bodied individuals and paracyclists. No significant differences between the three interventions were found in HFaverage in able-bodied individuals (p = 0.25) or in paracyclists (p = 0.69) during the TT, and there was no difference between the two groups of participants (p = 0.11). Data are presented in Neither [Lac] (p = 0.07) and in paracyclists (p = 0.14). POaverage was significantly greater in the third TT compared to the second in able-bodied individuals  but not in paracycling athletes (p = 0.11).Out of all 22 participants, only one suffered from gastrointestinal side effects. The participant reported nausea. No significant order effect was found comparing time to complete the first, the second, and the third TT regardless of the supplement in able-bodied individuals  or in paracyclists (p = 0.07). VO2average was found to be significantly higher in paracyclists  compared to able-bodied  individuals (p = 0.042). Furthermore, no significant difference was found in PO to VO2 ratio between the three interventions in able-bodied individuals (p = 0.62) or in paracyclists (p = 0.88). PO to VO2 ratio was significantly different in some kilometers over the course of the TT in able-bodied individuals (p < 0.05). No significant differences in the same parameter were found in the paracyclists (p > 0.05) during the TT  did not improve 10 km handcycling TT performance in able-bodied, upper body trained individuals or in trained paracyclists with a spinal cord injury . To date3−] to NO is enhanced in hypoxic and acidic conditions  concentration in participants with a spinal cord injury. Therefore, we might only speculate about the reason for such a difference. Possible reasons might be the lower energy or the nitrate intake in general due to lower active muscle mass resulting in lower energy expenditure. Another explanation might involve the slower gastrointestinal transition time in those participants due to the impaired nervous system [2−] and [NO3−] concentrations after NIT and BR were found in both groups of athletes. Interestingly, able-bodied individuals showed significantly higher [NO2−] concentrations after BR consumption compared to paracyclists, whereas paracyclists showed significantly higher [NO3−] concentrations after BR and NIT ingestion compared to able-bodied individuals. It seems possible that reduction from [NO3−] to [NO2−] was reduced in the paracyclists and therefore a higher [NO3−] was present 3 h after the ingestion. Such a hypothesis needs to be confirmed in future studies. No significant differences were found in [NO2−] and [NO3−] concentrations between NIT and BR ingestion in the same group. These findings seemed to be very similar to the previous study with a 6 mmol NIT and BR intervention in able-bodied individuals [2−] and [NO3−] concentrations in this study seem to be consistent with the literature.Resting levels of [NOs system . This miividuals . Thus, tOne major limitation of this study was that we did not use a nitrate-depleted BR as a placebo supplement. The participants were possibly able to distinguish between BR and NIT or between BR and PLA. Therefore, we cannot exclude the possibility that our findings were influenced by a potential placebo effect in the BR group. However, the participants were not able to distinguish between PLA and NIT because of similar taste, smell, and appearance.Additionally, there was a significant difference between the two groups. Able-bodied individuals showed a significantly higher body mass compared to paracyclists. This fact evolved mainly from the loss of muscle mass in the legs of athletes with a spinal cord injury due to immobilization. Furthermore, the age was significantly different between the two groups, as paracyclists were older. This is a known phenomenon in Paralympic sports, as most athletes start their career after the incident of the injury. Last but not least, both groups showed a different fitness level, as paracyclists appeared to be more highly trained than able-bodied individuals. All these issues might have influenced the outcome of the study and therefore needed to be acknowledged.2 in the present study were masked or blunted by the observed order effect.Finally, although our able-bodied participants were used to upper body exercise, we still reported a significant order effect on 10 km handcycling TT performance in this group of participants. Therefore, we assume that only one familiarization trial was insufficient in the present study. We cannot exclude the possibility that the effects of dietary nitrate on exercise performance and VO2 ratio in some km of the TT compared to PLA in able-bodied individuals but not in paracyclists. Overall, the present results do not support previous findings where acute nitrate supplementation enhanced short-term, high-intensity TT performance [Our study demonstrated that acute nitrate supplementation either by BR or NIT (6 mmol) did not improve 10 km handcycling TT performance in recreationally active men or in paracyclists. BR ingestion seemed to increase PO to VOformance ,37,38 in


Staphylococcus aureus (S. aureus) is a major cause of infection in hospitals and communities. Widespread dissemination of multi-drug resistant S. aureus is a serious threat to the health of humans and animals. An anti-virulence strategy has been widely considered as an alternative therapeutic approach. Inhibitors of virulence factors are able to treat S. aureus infections without influencing the growth or viability of bacteria and rarely lead to bacterial resistance. Sortase A (SrtA) is a membrane-associated cysteine transpeptidase that catalyzes up to 25 surface proteins that covalently bind to cell wall peptidoglycans. In S. aureus, most of these surface proteins have been identified as important virulence factors that are vital in bacterial pathogenesis. In the present study, we show that acacetin, a natural flavonoid compound, inhibits the activity of SrtA in S. aureus  which affects the assembly of protein A (SpA) to cell walls and reduces the binding of S. aureus to fibrinogen (Fg). The mechanism of the interaction between acacetin and SrtA were preliminarily discussed using molecular dynamics simulations. The results suggested that acacetin adopted a compact conformation binding at the pocket of the SrtA via residues Arg-139 and Lys-140. By performing an animal infection model, we demonstrated that acacetin was able to protect mice from renal abscess formation induced by S. aureus and significantly increased survival rates. Taken together, these findings suggest that acacetin may be a promising candidate for the development of anti-S. aureus drugs. Anti-virulence strategies have been proposed recently to combat the increasing drug resistance caused by conventional antibiotics combined with a salient lack of research and development of new antibiotics to counteract bacterial pathogens . Many suS. aureus (Sa-SrtA) has been extensively studied since its discovery in 1999 [S. aureus infection with promising pharmaceutical prospects.SrtA from  in 1999 ,8. Sever in 1999 . Additio in 1999 ,12. ThessrtA mutants that failed to anchor proteins to the cell wall peptidoglycans [Earlier work identified oglycans , displayoglycans ,14,15,16oglycans ,18,19. 50 = 72 μM and 96 μM, respectively). Accordingly, this paper aimed to enlarge the screening range, select a new natural compound with anti-virulence capacity in vivo and in vitro, clarify the mechanisms, and provide a new lead compound or drug candidate for the treatment of S. aureus infection.Several strategies have been developed for the identification and characterization of new SrtA inhibitors. The most widely used strategies include screening of natural product or small compound libraries based on enzyme activity, screening based on molecular modelling, pharmacophore hypotheses, 3D-QSAR models and virtual screening, which led to the discovery of natural and chemically synthesized products (reviewed in ). In a pRobinia pseudoacacia, Chrysanthemi indici Flos, Tunera diffusa and Betula pendula [By performing the FRET assay, acacetin emerged as the best candidate among 50 types of natural compounds with diverse structures that we screened. Acacetin  were both greater than 1024 μg/mL. Further, a growth curve was studied to reveal whether the growth states varied after the addition of acacetin. According to the results, the growth rate of S. aureus WT and ΔSrtA strain was analogous to WT + acacetin (256 μg/mL), even when the concentration was seven times its IC50 , which are vital for the attachment of Staphylococci to host Fg, which is the initiation of infection. A virulence reduction of SrtA-deficient S. aureus was found in a previous study [Previous conclusions illustrated that acacetin was able to significantly inhibit the SrtA-catalyzed transpeptidation at sub-MIC concentrations. us study . One impO” at the 4-position and the hydroxyl group at the 7-position of acacetin formed two hydrogen bond interactions with the residues Arg-139 (bond length: 2.6 Å) and Lys-140 (bond length: 1.8 Å), respectively, which were the main interactions between acacetin and SrtA. In summary, the above molecular dynamics simulation gave us a rational explanation of the interactions between acacetin and SrtA, which provided valuable information for the further development of SrtA inhibitors.SrtA-acacetin complex was equilibrated after 20 ns MD simulation, and the plot of root-mean-square deviation (RMSD) (in Ångstroms) of the complex is shown to determine that the system is stabilized during the simulation in S. aureus-infected mice in vivo, we conducted both fatal and non-fatal infections in the present study. Six days after injection with 2 × 109 CFU of S. aureus, which is a lethal concentration for animals, 90% of the mice infected with the WT strain had died. In contrast to the WT strain, the mortality rate of ΔSrtA was still 0% by the observation deadline  after initial infection and then died of S. aureus bacteremia five days after infection. The detailed results suggested that the acacetin-treated mice experienced a significant survival advantage compared to the WT group  was first hypodermically injected 30 min after the initial S. aureus infection. Six days after the injection, the mice were euthanized and dissected to excise the kidneys. The left kidneys of the infected mice were homogenized, distributed in PBS, and then plated for the enumeration of CFUs. The CFUs results show that the WT group exhibited a mean of 6.2 × 105 CFU/g in the kidneys, which is remarkably larger than in the ΔSrtA group (11.6 CFU/g) and acacetin-treated group (103 CFU/g) (S. aureus is surrounded by polymorphonuclear leukocytes (PMNs), amorphous material and eosinophils in kidneys from WT challenged mice  P. The ri3 CFU/g) A, which 3 CFU/g) C. The ΔS3 CFU/g) B. The reged mice D,E,J,K [ylococci H,I,N,O. S. aureus is notorious as a major source of hospital- and community-acquired infections [S. aureus infection is becoming increasingly difficult [fections . Bacteriifficult . Anti-viO-methylated flavone, was able to significantly inhibit SrtA activity both in vitro and ex-vivo without showing a noticeable antimicrobial effect. To our knowledge, only a few articles have reported in vivo therapeutic effects of SrtA inhibitors and validated their inhibitory activity in vitro [Combretum vendae [We have screened potent SrtA inhibitors from various types of natural compounds utilizing the FRET-based cleavage of LPETG as a measure of sortase activity. Acacetin, an in vitro ,35. Althin vitro , the flam vendae . HoweverS. aureus-infected mice in vivo. The results suggested that acacetin was able to prolong the survival of mice and alleviate the symptoms of renal abscesses  was used in this work. The ΔSrtA strain was constructed using ATCC25904 in our previous work [ΔN59 was obtained by cloning the SrtAΔN59 sequence into a pGEX-6P-1 vector. S. aureus ATCC25904 and ΔSrtA strains were cultured in brain-heart infusion (BHI) solution  at 37 °C with shaking at 220 rpm. The SrtAΔN59 protein was expressed from pGSrtAΔN59 vector transformed E. coli strain BL21 (DE3) and purified by GST affinity chromatography column. Fluorescent-quenched peptide substrate Dabcyl-QALPETGEE-Edans was purchased from Shanghai GL Biochem . The acacetin compound was obtained from Chengdu Pufeide Biotech Company .The reference strains ous work . The Srt50 value. This assay was based on a fluorescence resonance energy transfer (FRET) method that has been described previously [ΔN59 protein and the assay buffer  in a final volume of 300 μL. Increasing concentrations of acacetin and SrtAΔN59 protein were added to the plate and incubated at 37 °C for 1 h; the peptide substrate was then added, and the reaction proceeded at 37 °C for 1 h. The increase in fluorescence intensity was measured using a microplate reader , applying 495 nm as the emission wavelength and 350 nm as the excitation wavelength. Each reaction was repeated three times in this assay to ensure reproducibility.Sortase A activity inhibition assay was performed in 96-well black plates. Fluorescent peptide substrate Dabcyl-QALPETGEE-Edans was used to determine the ICeviously ,30. BrieS. aureus was determined by broth microdilution on the basis of NCCLS guideline M31-A2. To plot the growth curves of S. aureus, overnight-cultured S. aureus was diluted by 1:100 into sterile BHI broth that contained or did not contain appropriate concentrations of acacetin. Absorbance at 600 nm was measured at different intervals of time.The MIC of acacetin against S. aureus ATCC25904 strain was diluted 1:100 into sterile BHI broth and incubated along with variable concentrations of acacetin at 37 °C until A600 reached 0.5. Positive control was the ΔSrtA strain cultured using the same conditions. Then, all of the cell suspensions were removed, deposited by centrifugation (5000× g for 5 min), and suspended in Phosphate buffered saline (PBS) to an A600 of 1.0. The resuspended cells were added to the wells of a fibrinogen-coated  polystyrene Costar 96-well plate and incubated at 37 °C for 2 h. The liquid was removed. Bacteria were washed with PBS, and 25% (v/v) formaldehyde was added to fix samples. Adherent bacterial cells were stained with crystal violet solution (12.5 g/L) for 10 min, the wells were washed again with PBS and dried, and the absorbance at 570 nm of disparate samples was subsequently measured. The results were shown using the percentage of the tested group compared to the wild type group.Overnight cultured S. aureus was diluted by 1:100 into sterile BHI broth and incubated with or without appropriate concentrations of acacetin at 37 °C until A600 reached 0.5. The bacteria were washed twice with PBS and then blocked with 2% BSA for 1 h. The bacteria were centrifuged and suspended in PBS containing a 1:100 dilution of FITC-labelled goat anti-rabbit IgG (eBioscience). After 1 h of incubation at room temperature, the S. aureus were washed twice with PBS and added to poly-l-lysine-coated glass slides. The fluorescence was observed using a confocal laser-scanning microscope .Overnight cultured t-test was used to assess the significance of Fg-binding data and bacterial burden data; p < 0.05 was considered statistically significant. The molecular dynamics and animal model experimental methods that were used are described in the The log-rank test (Mantel-Cox) was used to analyze the survival rate data, and the two-tailed Student’s S. aureus-induced fatal infection and renal abscesses made acacetin worthy for further development into a novel therapeutic agent. We will improve its physical and chemical properties and biological activities through structural modification and other optimization measures to develop it for use as an alternative or complementary treatment of S. aureus infections.Overall, our results suggest that acacetin is a potent and promising small molecule anti-SrtA agent which prevents the surface protein Spa from anchoring in the cell wall and prevents the clumping factors (ClfA and ClfB) from playing an adhesion function. The potential therapeutic effect of acacetin on


Compared with group NC, the levels of RBP4, TG, LDL-c, FPG, FINS, CRP, AI1, AI2, SBP, and HOMA-IR increased, while the levels of HDL-c, 25(OH)D, and HOMA-β decreased in groups DM1 and DM2. After 8 weeks of vitamin D supplementation in group DM3, the levels of 25(OH)D and HOMA-β increased and the levels of LDL-c, TC, HOMA-IR, FINS, CRP, RBP4, AI1, AI2, and SBP decreased significantly when compared with group DM2 (P < 0.05); Pearson analysis showed that serum RBP4 was positively correlated with TG, FINS, HOMA-IR, SBP, CRP, and AI and negatively correlated with 25(OH)D. In addition, multivariable logistic regression analysis showed that serum RBP4, SBP, and HDL-c were predictors for the presence of diabetic atherosclerosis. These findings suggested that RBP4 could involve in the improvement of diabetic atherosclerosis; vitamin D had the ability to decrease the level of RBP4 and eventually played an important role in preventing atherosclerosis in diabetes.The purposes of this study were to evaluate the expression of retinol-binding protein 4 (RBP4) in diabetic rats with atherosclerosis and to investigate the role of vitamin D intervention. Male Wistar rats were randomly divided into 4 groups, including the control group (NC), the diabetic rats (DM1), the untreated diabetic atherosclerosis rats (DM2), and the vitamin D-treated diabetic atherosclerosis rats (DM3). The levels of serum and adipose RBP4, fasting insulin (FINS), fasting plasma glucose (FPG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), 25-hydroxyvitamin D [25(OH)D], C-reactive protein (CRP), and systolic blood pressure (SBP) were measured. Homeostasis model assessment of insulin resistance (HOMA-IR), homeostasis model assessment  It is reported that the worldwide prevalence of diabetes keeps increasing and may lead to a high incidence of macrovascular complications, such as heart failure, ischemic stroke, and nonfatal myocardial infarction. Diabetic macrovascular disease is a heterogeneous disorder characterized by multifactorial etiology and various processes, it has become the main leading cause of death and disability in diabetes, and its underlying potential pathological basis is atherosclerosis . Thus, iAdipose tissue is mainly responsible for energy storage and has been found to secrete a variety of biologically active substances, such as resistin, adiponectin, and leptin. Retinol-binding protein 4 (RBP4) is a newly discovered adipokine secreted by adipocytes and hepatocytes, and it is a specific transport protein for serum retinol . MultiplVitamin D or also known as the “sunshine vitamin” is a kind of secosteroid, and serum 25-hydroxyvitamin D [25(OH)D] is a biomarker of vitamin D status in human body. Recent studies have found that besides the role of regulating calcium-phosphorus metabolism and bone calcification, vitamin D also has multiple biological effects. Meanwhile, it has been found related to nonbone metabolic diseases like cancer, obesity, and diabetes –16. As vThis research was approved by the Ethics Committee of Anhui Medical University, Medical Institution Animal Care and Research Advisory Committee . According to the guidelines of Animal Institute of Nutrition (AIN) of USA, the animals were kept in the area with a temperature of 23 ± 2°C, a relative humidity of 55 ± 5%, and a light cycle of 14/10 h.n = 8), the diabetes group , and the diabetic atherosclerosis group . The rats in groups NC, DM1, and DAM were fed up with a normal diet, a high-fat diet , and a high-fat diet with different components , respectively. In the 4th week, a dose of streptozotocin  was added for rats in groups DM1 and DAM through intraperitoneal injection, whereas an equivalent volume of citric acid buffer was added for the rats in group NC. After 72 h, blood samples of rat tail veins in the 3 groups were collected to evaluate the level of plasma glucose and fasting plasma glucose (FPG) > 7.8 mmol/L was chosen as the T2DM model. Totally, 10 and 23 rats were successfully modeled in groups DM1 and DAM, respectively. Then, rats in group DAM were subjected to thoracic artery balloon injury. After the 8-week feeding, we subdivided the rats in DAM into 2 groups: the untreated diabetic atherosclerosis group  and the vitamin D-treated diabetic atherosclerosis group . A dose of 0.025 μg·kg−1·d−1 vitamin D [Two-week-old male Wistar rats with the weight of 160–200 g were purchased from the experimental animal center of Anhui Medical University and were divided into 3 groups randomly after adaptive feeding, including the normal control  kit  to evaluate the level of C-reactive protein (CRP), serum RBP4, and 25(OH)D. HOMA-IR (homeostasis model assessment insulin resistance index) and HOMA-β (homeostasis model assessment β-cell function index) were used for estimating IR and the function of β-cells, respectively, and they were calculated by HOMA-IR = FINS × FPG/22.5 and HOMA-β = 20 × FINS/FPG − 3.5. As predictors of atherosclerosis, atherogenic indexes (AI) including AI1 and AI2 were estimated by the formulas AI1 = TC − HDL-c/HDL-c and AI2 = LDL-c/HDL-c. Subsequently, we isolated the thoracic aorta and performed hematoxylin and eosin (HE) staining. Finally, the mRNA and the quantitative protein expressions of RBP4 in visceral adipose tissues were detected by reverse transcription polymerase chain reaction (RT-PCR) analysis  and Western blot analysis, respectively.In the 16th week, all the experimental rats were fasted for 12 hours and then anesthetized with somnopentyl  and finally received euthanasia by collecting total blood from the left ventricle. After that, the blood samples were collected, then centrifuged to separate the serum, and stored at −80°C for detecting the levels of relevant serum components. Meanwhile, the visceral adipose tissue and other organs were collected, weighed, washed in saline, immersed in liquid nitrogen, and immediately stored at −80°C until use. Automatic biochemical analyzer  was applied to detect the levels of low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), triglycerides (TG), and total cholesterol (TC). Additionally, the insulin radioimmunoassay kit  was performed for the level of fasting insulin (FINS). We then centrifuged the remaining samples at 3000To detect the expression of serum RBP4, we performed ELISA assay according to the manufacturer's process. The first process was incubation in which the RBP in the plasma samples was bound to a polyclonal rabbit anti-RBP antibody fixed on a microtitration plate. We then washed the plates and added a peroxidase-conjugated anti-RBP antibody, which was incubated for a certain time and then being washed. We applied tetramethylbenzidine as a peroxidase substrate for detection and quantification and calculated a dose-response curve of absorbance unit  with spline fitting from the standard values, which was then used to measure the RBP4 concentration of the samples. The intra- and intervariation of the assay were 5% and 9.75%, respectively.μg RNA were used for the synthesis of complementary deoxyribonucleic acid (cDNA). Syber Green II was used to perform the PCR reaction in PerkinElmer PCR System 9700  with the following steps: initial denaturation at 94°C for 30 seconds, followed by 30 PCR cycles of 94°C for 30 seconds, 60°C for 45 seconds, and 72°C for 45 seconds. The oligonucleotide primers were listed as follows: 5′-GACAAGGCTCGTTTCTCTGG-3′ (sense) and 5′-AAAGGAGGCTACACCCCAGT-3′ (antisense) for RBP4 and 5′-CACGATGGAGGGGCCGGACTCATC-3′ (sense) and 5′-TAAAGACCTCTATGCCAACACAGT-3′ (antisense) for β-actin. The amplified products were used for electrophoresis with 1.5% agarose gel , and the corresponding bands were obtained for optical density analysis .Total RNA of the visceral adipose tissues was extracted by TRIzol  according to the manufacturer's instructions, and the concentration and purity of the extracted RNA were detected by the ratio of OD 260 and 280. The MMLV reverse transcriptase and 2 The tissue samples were firstly added to the tissue lysate solution  at a ratio of 1 : 20, and then, the lysates were centrifuged at 14000 ×g for 30 min at 4°C. BCA Protein Assay Reagent Kit  was used to measure the protein concentrations of the samples, and sodium dodecyl sulfate-polyacrylamide gel was applied for protein electrophoresis. Then, we transfer the protein to a polyvinylidene fluoride  membrane, incubate the membrane in TPBS  with 5% non-fat milk at room temperature for 2 h to block nonspecific binding, and use the anti-RBP4 antibody as the primary antibody at a dilution of 1 : 3000. After washing three times with TPBS, the PVDF membrane was then incubated with a horseradish peroxidase-conjugated secondary antibody at a dilution of 1 : 4000 at room temperature for 2 h. Finally, the Gel-Pro Analyzer software was used to analyze the bands captured on X-ray films.t-test or one-way analysis of variance (ANOVA) was performed for differences between different groups. Pearson correlation analysis was used for correlation between different variables. Multivariable logistic regression analysis was performed to measure the ORs and 95% confidence intervals of diabetic atherosclerosis. All P values were two-tailed, and P < 0.05 was considered statistically significant.SPSS 19.0  was used for all the statistical analysis. All the experimental data were represented as mean ± standard deviation. Student's Results showed that in group NC , the thiβ decreased in groups DM1 and DM2 compared to group NC (P < 0.05). Compared with group DM2, we found that the levels of 25(OH)D and HOMA-β increased significantly while the levels of LDL-c, TC, HOMA-IR, CRP, AI1, AI2, FINS, and SBP decreased in group DM3 after 8 weeks of treatment with vitamin D (P < 0.05).The weights of rats in groups DM1 and DM2 were greater than those in group NC at the 4th, 8th, and 16th weeks, and the increase was more significant in group DM2. After vitamin D supplementation, the weights of rats in group DM3 decreased compared to the ones in group DM2. The weight curves of all the rats are presented in P < 0.05).As shown in P < 0.05).Results shown in  revealedP < 0.05) D. However, the level of 25(OH)D was positively associated with HDL-c and HOMA-shown in .Multivariate logistic regression analysis is shown in Diabetes and atherosclerosis may exhibit common pathology, although the underlying mechanisms are still being explored. Systemic factors accompanied with diabetes, such as dyslipidaemia and hypertension, are thought to affect the development of diabetic vascular diseases. In addition, IR and hyperglycemia are the most two important factors . In the α [κB pathways occurs independently of STRA6 and retinol [RBP4 is a 21 kDa secreted protein elevated in insulin resistant states such as obesity and T2DM. In the present study, we found that the levels of adipose tissue and serum RBP4 in the DM groups especially in the diabetic atherosclerosis group were higher than those in the NC group. This phenomenon was in accordance with the previous results that RBP4 increased in diabetes patients with coronary heart disease and cerebral infarction and was negatively associated with carotid intima-media thickness . The corα –35, whic retinol .The function of 25(OH)D on adipokines is a research hotpot currently. Several researches have shown that vitamin D is correlated with adiponectin, leptin, and other adipokines , but fewβ and improved histological aspect of thoracic aorta compared with those without the treatment, which demonstrates that it is helpful to prevent atherosclerosis by controlling blood glucose, blood pressure, IR, and inflammation after vitamin D supplements. The following aspects might be important for the antiatherosclerotic effects of vitamin D. (1) As an endocrine suppressor of the renin angiotensin aldosterone system (RAAS), vitamin D can reduce the blood pressure by downregulating the RAAS [β-cells [β, and RBP4, and after vitamin D therapy, the three indexes were all improved. Vitamin D can also reduce peripheral IR by inhibiting the expression of peroxisome proliferator-activated receptor γ (PPAR-γ), which might partly contribute to the regulation of RBP4 [Multiple researches have confirmed the relationship between vitamin D deficiency and an increased risk for cardiovascular diseases in diabetes patients , 43, 44,the RAAS . (2) Vitthe RAAS . Our resthe RAAS . (4) Vit[β-cells . In the  of RBP4 . (5) LDL of RBP4 , 51. VitThe results revealed that RBP4 may contribute to the development of diabetes complicated with cardiovascular diseases through some potential mechanisms, such as insulin secretion and resistance, inflammatory reaction, dyslipidaemia, and lowering RBP4 levels, which may be an effective strategy for the prevention and treatment of T2DM macrovascular complications. Besides the bone metabolism role, vitamin D also has the ability to reduce the level of RBP4 and to provide vascular protection, which is partly associated with inhibiting cholesterol biosynthesis, relieving inflammation reaction, improving insulin resistance, and increasing insulin sensitivity. Detailed vitro experiments and longitudinal studies with larger sample size are still needed to confirm and extend these mechanisms in the future.