Expression vectors encoding wild-type PEBP1 and PEBP1 with Ser153Ala mutation (S153A), PEBP1 specific siRNAs, and human recombinant PEBP1 (rhPEBP1) were administered intracerebroventricularly. Endogenous PEBP1 level and its phosphorylation at Ser153 were increased within penumbra tissue and cultured neurons after I/R, accompanied by decreased interaction between PEBP1 and Raf-1. There was a trend toward increased Raf-1/MEK/ERK/NF-κB signaling pathway and phosphatidylcholine-phospholipase C (PC-PLC) activity after I/R, which was enhanced by wild-type PEBP1overexpression and rhPEBP1 treatment and inhibited by PEBP1 (S153A) overexpression. And PEBP1 (S153A) overexpression increased its interaction with Raf-1, reduced infarct size, neuronal death and inflammation, and improved neurological function after I/R, while wild-type PEBP1overexpression exerted opposite effects, suggesting that phosphorylation at Ser153 may exert as a functional switch of PEBP1 by switching PEBP1 from Raf-1 inhibition to PC-PLC activation following I/R. Compared with PEBP1 knockdown, PEBP1 (S153A) overexpression exerted a better rescue effect on I/R injury, which further proved that PEBP1 may be a good protein gone bad with phosphorylation at S153 as a functional switch following I/R. Collectively, our findings suggest that PEBP1 contributed to neuronal death and inflammation after I/R. Selective inhibition of PEBP1 phosphorylation may be a novel approach to ameliorate I/R injury.This study aimed to estimate the role of phosphatidylethanolamine binding protein 1 (PEBP1) in cerebral ischemia-reperfusion (I/R) injury and the underlying mechanisms. Middle cerebral artery occlusion/reperfusion (MCAO/R) model in adult male Sprague Dawley rats (250–280 g) were established and cultured neurons were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) to mimic I/R injury  Stroke is a devastating disease that can cause cognitive and motor dysfunction, and even acute death, and is becoming the leading cause of mortality and morbidity worldwide . In thisIt is generally accepted that inflammatory responses and apoptosis play crucial roles in the cascade reaction of cerebral I/R injury, and the Raf-1/MEK/ERK/nuclear factor-κB (NF-κB) signaling pathway is an important mechanism of both inflammatory responses and apoptosis . The intIn conclusion, accumulating researches suggested a therapeutic potential of PEBP1 in cerebral I/R injury. However, until now, no study has investigated the contribution of PEBP1 to I/R injury, and no study has been performed on the effects of PEBP1 phosphorylation, especially at ser153, on Raf-1/MEK/ERK/NF-κB signaling pathway, PC-PLC activation, and autophagy pathway during I/R. Therefore, the aim of this study was to investigate the role of PEBP1 and to assess the therapeutic potential of PEBP1 in cerebral I/R injury.All experiments were approved by the Ethics Committee of the First Affiliated Hospital of Soochow University and were in accordance with the guidelines of the National Institutes of Health on the care and use of animals. Adult male Sprague–Dawley (SD) rats weighing 250–280 g were purchased from Animal Center of Chinese Academy of Sciences, Shanghai, China. The rats were housed in temperature- and humidity-controlled animal quarters with a 12-h light/dark cycle.Figure 1A). In this model, around ischemic core, there is a penumbra (peri-infract) region, which could be rescued . Neurons were maintained in a 5% CO2 atmospheric incubator at 37°C. We renewed half of the medium every 2 days for 2 weeks. Finally, the neurons were exposed to the indicated treatments and harvested.As described previously , primary2 and 95% N2 atmospheric incubator for 2 h at 37°C. After that, neurons were cultured in neurobasal medium again and maintained in 5% CO2 atmospheric incubator for indicated time periods. Control groups were cultured in neurobasal medium in 5% CO2 atmospheric incubator for the same period. The pH of culture medium was maintained at 7.2.Briefly, neurobasal medium was replaced with DMEM  and cells were transferred to a 5% COFigure 1B, in vivo, 36 SD rats  were randomly assigned to six groups of six SD rats each: a sham group and five experimental groups arranged by reperfusion time: 2, 6, 12, 24, and 48 h. The brain penumbra of six SD rats in each group was achieved at the indicated time point and used for western blot analysis and double immunofluorescence analysis. In vitro, experiment was performed to evaluate the effect of OGD/R treatment on the level of p-PEBP1 and PEBP1 in cultured neurons.As shown in Figure 1C, in vivo, 270 SD rats  were randomly assigned to nine groups (n = 30 per group): sham group, MCAO/R group, MCAO/R + GFP-vector group, MCAO/R + GFP-PEBP1 group, MCAO/R + GFP-PEBP1 (S153A) group, MCAO/R + control-siRNA group, MCAO/R + PEBP1-siRNA group, MCAO/R + vehicle group, and MCAO/R + human recombinant PEBP1  group. The transfection of plasmids and siRNAs was given at 48 h before surgery and the injection of rhPEBP1 was given immediately following reperfusion intracerebroventricularly. Based on the previous time course study, 6 SD rats per group were extracted for western blot analysis of the level of p-PEBP1 and PEBP1 at 6 h after MCAO/R surgery. 10 SD rats were sacrificed at 12 h after MCAO/R surgery and used for western blot analysis of intranuclear accumulation of NF-κB p65 subunit and PC-PLC activity assay. Another 14 SD rats per group were examined for behavioral impairment and sacrificed at 72 h after MCAO/R surgery. Among them, 8 SD rats per group were used for TTC staining, 6 SD rats per group were used for fluoro-jade B (FJB) staining and western blot analysis of the level of active-caspase 3. The dose of rhPEBP1 was chosen based on the preliminary experiment results, which showed that 15 μg/kg body weight is the most cost-efficient dose of rhPEBP1 to induce a significant increase in the protein level of PEBP1 in brain tissues at 24 h after MCAO/R surgery  group, OGD/R + control-siRNA group and OGD/R + PEBP1-siRNA group. The transfection of plasmids and siRNAs were given at 48 h before OGD/R. Based on the previous time course study, at 6 h after reperfusion, we performed western blot analysis and co-immunoprecipitation analysis to test the level of p-PEBP1 and PEBP1 and the interaction between PEBP1 and Raf-1. At 12 h after reoxygenation, western blot analysis was performed to test the level of Raf-1/MEK/ERK/NF-κB signaling pathway and autophagy. At 24 h after reoxygenation, sulforhodamine B (SRB) assay and hoechst 33258 staining were performed to test cell viability and neuronal apoptosis, and all the culture supernatants were collected for lactate dehydrogenase (LDH) activity assay and enzyme-linked immunosorbent assay (ELISA) to evaluate neuronal necrosis and inflammatory response.As shown in Rabbit anti-p-PEBP1 (phospho S153) antibody (ab75971), rabbit anti-PEBP1 antibody (ab76582), rabbit anti-GFP antibody (ab6556), rabbit anti-Histone H3 antibody (ab8580), rabbit anti-NeuN antibody (ab177487) and mouse anti-NeuN (1B7) antibody (ab104224) were from Abcam . Mouse anti-PEBP1(D-5) antibody (sc-365973), rabbit anti-NF-κB p65 (C20) antibody (sc-372), rabbit anti-p-ERK 1/2 antibody (sc-23759-R), mouse anti-ERK 1/2 (MK1) antibody (sc-135900), mouse anti-β-actin (C4) antibody (sc-47778) and mouse anti-GAPDH (G-9) antibody (sc-365062) were from Santa Cruz Biotechnology . Mouse monoclonal anti-Raf-1/c-Raf antibody (R2404) was from Sigma–Aldrich Corporation . Rabbit anti-LC3B antibody (2775) and rabbit anti-cleaved-Caspase3 antibody (9661) were from Cell Signaling Technology . Normal mouse IgG (sc-2025) and normal rabbit IgG (sc-2027) were from Santa Cruz Biotechnology. Secondary antibodies for western blot analysis, including goat anti-rabbit IgG-HRP (sc-2004) and goat anti-mouse IgG-HRP (sc-2005), were from Santa Cruz Biotechnology. Secondary antibodies for immunofluorescence microscopy, including Alexa Fluor-555 donkey anti-rabbit IgG antibody (A31572), Alexa Fluor-488 donkey anti-rabbit IgG antibody (A21206), Alexa Fluor-555 donkey anti mouse IgG antibody (A31570) and Alexa Fluor-488 donkey anti-goat IgG antibody (A11055) were from Invitrogen. rhPEBP1 (PRO-722) was from ProSpec-Tany (Israel). PKC inhibitor (sc-3007) was from Santa Cruz Biotechnology .g for 15 min at 4°C, collected the supernatants, and determined the protein concentration of the supernatants by standard BCA  method. Then, the samples (100 μg/lane) were loaded onto a 15% SDS polyacrylamide gel, separated, and electrophoretically transferred to a polyvinylidene difluoride membrane . The membrane was blocked in 5% bovine serum albumin  for 2 h at room temperature. Subsequently, the membrane was further incubated with the primary antibody overnight at 4°C and then incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. The membrane was revealed with the enhanced chemiluminescence kit (Beyotime Institute of Biotechnology) and the relative quantities of proteins were analyzed with Image J software.The brain homogenate collected from SD rats and the cultured neurons were lysed in RIPA lysis buffer . We centrifuged the samples at 12,500 × To test the level of p-PEBP1 by western blot easily, we enriched PEBP1 from total proteins by immunoprecipitation using PEBP1 antibody before electrophoresis.The nuclear content of NF-κB was tested by western blot assay of nuclear protein. Nuclear protein extraction was performed using a Nuclear and Cytoplasmic Protein Extraction Kit from Beyotime (P0027).The brain samples were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 4 μm sections. The cultured neurons were fixed in 4% paraformaldehyde. Then, the sections and neurons were incubated with primary antibodies and secondary antibodies. Normal mouse IgG and normal rabbit IgG were used as negative controls for the immunofluorescence assay. The sections and neurons were observed in a fluorescence microscope  or a laser scanning confocal microscope . The relative fluorescence intensity was analyzed with the Image J program. The quantitative analysis was performed by an observer who was blind to the experimental group.The coding region of rat PEBP1 cDNA was sub-cloned into pGFP-N2 expression vector to produce the pGFP-N2-PEBP1 construct. In addition, a rat PEBP1 cDNA construct with a mutation in a possible key phosphorylation site (Ser153) was prepared. S153A mutant  was also sub-cloned into the pGFP-N2 expression vector as the wild-type PEBP1 cDNA, which allowed us to measure their location by fluorescence assay. All of the constructs were confirmed by DNA sequencing.in vitro transfection and western blot analysis. The most efficient one was used in the following study.Specific siRNAs against PEBP1 were obtained from Ribobio. The knockdown efficiency of all the three siRNAs was separately tested by PEBP1 siRNA sequences:(1) Sense: 5′ CCCACCCAGGTCATGAATA dTdT 3′Antisense: 3′ dTdT GGGTGGGTCCAGTACTTAT 5′(2) Sense: 5′ GCACTGTCCTCTCCGAATA dTdT 3′Antisense: 3′ dTdT CGTGACAGGAGAGGCTTAT 5′(3) Sense: 5′ GCAACAAGTCTGGAGACAA dTdT 3′Antisense: 3′ dTdT CGTTGTTCAGACCTCTGTT 5′®3000 Transfection Reagent  or siRNAs using Lipofectamine RNAi MAX  according to the manufacturer’s instructions. At 48 h after transfection, neurons were performed OGD/R treatment. Then, the cultured neurons were harvested and analyzed.Cultured neurons were transfected with indicated expression vectors using LipofectamineImmunoprecipitation analysis was performed as described previously . First, Neurons were fixed in 4% paraformaldehyde for 10 min, stained by Hoechst 33258 at room temperature for 15 min, and observed by a fluorescence microscope . Normal neuron nuclei were homogeneously stained blue, while the nuclei of apoptotic neurons displayed nuclear fragmentation or chromatin condensation. Apoptosis was assessed in ≥300 cells. The quantitative analysis was performed by an observer who was blind to the experimental group.Cell viability was examined by SRB assay. After indicated treatments, neurons were fixed with 50% trichloroacetic acid and stained with a SRB solution. Finally, SRB was measured with a Bio-Rad Micro plate reader. The cell viability was performed in triplicate and repeated three independent times at least.The concentration of LDH in medium was determined with LDH assay kit . These assays were performed according to manufacturer’s instructions, and these data were expressed relative to standard curves prepared for them.The concentrations of IL-1β, IL-6, and TNF-α in medium were determined by corresponding ELISA kits . These assays were performed according to manufacturer’s instructions, and these data were expressed relative to standard curves prepared for them.After indicated treatments, brains were removed and frozen in -20°C for 10 min, and sectioned coronally into 2 mm thick slices starting from the frontal pole. The olfactory bulb and cerebellum were discarded. Then, the brain slices were immersed in TTC solution  for 15–30 min at 37°C. After staining, the brain slices were washed three times and captured with a digital camera. To calculate the infarct volume, first, we calculated the total mean infarct area of each section as the average of the infarct area on the rostral and the caudal side. Then, we calculate the total area by adding the average area from each section and calculate the infarct volume as the multiplication of the total area by 2 mm (thickness of the sections). At last, the infarct volume was expressed as a percentage of the ipsilateral hemispheric volume.The Brain sections were deparaffinized, rehydrated, and incubated in 0.06% K permanganate for 15 min. Then, the brain sections were rinsed in deionized water, immersed in 0.001% FJB working solution (0.1% acetic acid) for 30 min, and dried in incubator (50–60°C) for 10 min. The brain sections were cleared in xylene, cover slipped with 1 drop of mountant and observed by a fluorescence microscope .As described previously , at the According to the manufacturer’s protocol as described previously , the PC-post hoc test, the former for comparisons to a single control group, the latter to compare across multiple groups. p < 0.05 was considered to be significant difference.Graph pad prism 7.00 was used for all statistical analysis. Neurobehavioral scores were shown as median with interquartile range. All the other data are presented as mean ± SD. Frequency distribution for the neurobehavioral score assay. One-way analysis of variance followed by either a Dunnett’s or a Tukey’s No significant changes in blood pressure, body temperature and heart beat were detected in any of the experimental groups . Consistent with the in vivo data, western blot assay showed that the protein levels of PEBP1 and p-PEBP1 (ser153) in cultured neurons were significantly increased by OGD/R at 2–12 h and peaked at 6 h . Double immunofluorescence with the neuronal marker protein NeuN further verified the MCAO/R-induced increase in the protein level of PEBP1 in neurons . Immunofluorescence staining of PEBP1 on cultured neurons treated with OGD/R stimulus in vitro also showed an OGD/R-induced increase in PEBP1 when compared with control group . In addition, previous studies have indicated a critical role of PEBP1 phosphorylation at ser153 in PEBP1 binding properties .To detect the expression and phosphorylation of PEBP1 after MCAO/R, through western blot analysis of protein samples from penumbra tissue, we first tested the protein level of PEBP1 and p-PEBP1 (ser153) in the penumbra tissue. The results demonstrated that, compared with the sham group, both the protein level of PEBP1 and p-PEBP1 (ser153) in the penumbra tissue reached the highest point at 6 h, and then rebounded gradually . Notably, compared with that in endogenous PEBP1 , OGD/R exerted a similar induction of the phosphorylation of GFP-PEBP1 at Ser153 and inhibition of the interaction between GFP-PEBP1 and Raf-1, which was almost abolished by the mutation of Ser153 to Ala , therefore supporting Ser153 as a key site for OGD/R-induced PEBP1 phosphorylation.To further elucidate the role of phosphorylation at Ser153 in PEBP1 functions, expression vectors encoding rat wild-type PEBP1 and PEBP1 with Ser153Ala mutation were prepared. First, both wild type GFP-PEBP1 and the mutant protein were strongly expressed in transfected neurons (51 KD), and exogenous expression of PEBP1 did not affect the level of endogenous PEBP1 (23 kD) .Finally, the interference efficiency of three different siRNAs specific for PEBP1 was tested, and the most efficient one (siRNA-3) was used in the following study . In addition, compared with the sham group, a significant increase in the levels of PEBP1 and p-PEBP1 (ser153) was observed in MCAO/R group, which was attenuated by PEBP1 knockdown and exacerbated by exogenous rhPEBP1 treatment .Then, we tested the transfection efficiency of exogenous expression vectors in vitro. First, TUNEL staining showed that only a few TUNEL-positive apoptotic cells were observed in the control group, while the apoptotic index was found to be significantly higher in the OGD/R group . As compared with the OGD/R group, the apoptotic index was significant exacerbated by wild type GFP-PEBP1 overexpression and attenuated by GFP-PEBP1 (S153A) overexpression, suggesting phosphorylation at Ser153 may be a functional switch of PEBP1 in OGD/R-induced neuronal injury. Cell viability assay also identified the rescue effects of the Ser153Ala mutation in PEBP1 in neurons under OGD/R stimulus . Consistently, the necrosis index showed the same trend with that in the apoptotic index . Finally, inflammatory cytokines, including IL-1β, IL-6, and TNF-α, were found to be significantly higher in the culture supernatant of the OGD/R group than that in the control group. Compared with OGD/R group, the mean inflammatory cytokine contents were higher in wild type GFP-PEBP1 overexpression group and lower in GFP-PEBP1 (S153A) overexpression group . In addition, in vitro rescue effects of PEBP1 knockdown on OGD/R-induced neuronal injury were also detected .We then investigated the function of PEBP1 in OGD/R-induced neuronal injury Figure 6A). To further examine the role of PEBP1 in MCAO/R-induced brain injury, FJB staining, caspase 3 activation and clinical behavior scores were tested. Compared to the sham group, FJB-positive cells were increased in the penumbra tissue in the MCAO/R group, which was significantly attenuated by PEBP1 (S153A) overexpression and PEBP1 knockdown and enhanced by wild type GFP-PEBP1 overexpression and exogenous rhPEBP1 treatment . Consistently, western blot assay of caspase 3 activation showed the same trend . Finally, clinical behavior scores assay showed the rescue effects of PEBP1 (S153A) overexpression and PEBP1 knockdown on the neurobehavioral function of rats subjected to MCAO/R, while wild type GFP-PEBP1 overexpression and exogenous rhPEBP1 treatment exerted opposite effects .First, TTC staining displayed the benefit of PEBP1 (S153A) overexpression and PEBP1 knockdown on the infarct volume, while wild type GFP-PEBP1 overexpression and exogenous rhPEBP1 treatment exerted opposite effects . The results showed the phosphorylation level of ERK1/2 in neurons subjected to OGD/R to be significantly higher than that in the control group. Wild type GFP-PEBP1 overexpression and exogenous rhPEBP1 treatment significantly increased the level of ERK1/2 phosphorylation, while PEBP1 (S153A) overexpression exerted opposite effects . Consistently, the nuclear content of NF-κB in MCAO/R group was significantly higher than that in the sham group, which was significantly attenuated by PEBP1 (S153A) overexpression and enhanced by wild type GFP-PEBP1 overexpression and exogenous rhPEBP1 treatment . Notably, compared with OGD/R + control-siRNA group, PEBP1 knockdown did not induce significant changes in the phosphorylation of ERK1/2 and the nuclear transfer of NF-κB .As shown in Figure 7C, PC-PLC activity was significantly higher in MCAO/R group than that in the sham group. Compared with MCAO/R group, the PC-PLC activity was significantly enhanced in MCAO/R + PEBP1 group and MCAO/R + rhPEBP1 group, and weakened significantly in MCAO/R + PEBP1 (S153A) group and MCAO/R + PEBP1-siRNA group . In addition, the results also revealed increases in the LC3-II/LC3-I conversion in OGD/R group, which was inhibited by wild type GFP-PEBP1 overexpression and exogenous rhPEBP1 treatment, while PEBP1 (S153A) overexpression and PEBP1 knockdown exerted opposite effects .As showed in Ischemic stroke is becoming the leading cause of mortality and morbidity worldwide with early recanalization as the preferred treatment strategy. However, I/R often involves unavoidable damages and leads to unsatisfactory clinical outcome of the therapy. At present, penumbra rescue by attenuating I/R injury is fully acknowledged and there is an urgent demand of efficient remedies for I/R injury. Using a clinically relevant rat MCAO/R model, we are exploring a new neuroprotective strategy targeting PEBP1. PEBP1 may be a good protein gone bad with phosphorylation at Ser153 as a functional switch following I/R. By attenuating neuronal death and inflammation, selective PEBP1 phosphorylation inhibition would limit I/R complications and benefit neurological outcomes.Figure 2). In addition, there was a decrease in the interaction between PEBP1 and Raf-1 accompanied by the increase in the phosphorylation at ser153 of PEBP1 . Then, using site-directed mutagenesis of Ser153 to alanine in PEBP1, we found the adverse effect of wild type PEBP1 overexpression and the rescue effect of PEBP1 (S153A) overexpression on I/R-induced brain injury , suggesting that the phosphorylation at Ser153 may exert as a functional switch of PEBP1. And, as previously reported . Finally, there was a trend toward increased Raf-1/MEK/ERK/NF-κB signaling pathway and PC-PLC activity after I/R with PEBP1 phosphorylation at Ser153 switching PEBP1 from Raf-1 inhibition to PC-PLC activation . The results also showed that, under I/R condition, PEBP1 knockdown did not further increase the Raf-1/MEK/ERK/NF-κB pathway, suggesting PEBP1 barely interacts with Raf-1 following I/R. By this binding partner switching, PEBP1 abolished its anti-inflammatory and anti-apoptotic effects, which is mediated by its binding to Raf-1, and obtained a pro-inflammatory and pro-apoptotic effect by binding to and activating PC-PLC. Notably, compared with PEBP1 knockdown, PEBP1 (S153A) overexpression seems to exert a better rescue effect on I/R-induced neuronal injury , all which further proved that PEBP1 might be a good protein gone bad with phosphorylation at Ser153 as a functional switch following I/R .Our present study showed the role of PEBP1 in the pathogenesis of cerebral I/R injury in a rat MCAO/R model. First, results showed elevated levels of PEBP1 and p-PEBP1 (p-ser53) in penumbra tissue after MCAO/R and cultured neuron suffered from OGD/R , suggesting that I/R condition increased the protein level of PEBP1 at a transcriptional level during 0–6 h after reperfusion. At the same time, PEBP1 phosphorylation reached a peak 6 h after reperfusion . After 6 h of reperfusion, there was a gradual decrease in both the level of PEBP1 and p-PEBP1 (ser153) . In order to explain the possible mechanisms underlying the decrease, we tested the changes of the level of GFP-PEBP1 and p-GFP-PEBP1 (ser153) after 6 h of reperfusion . Most secretory proteins contain amino internal or terminal signal peptides that direct their sorting to endoplasmic reticulum (ER) and are transported to the plasma membrane or extracellular space via a process known as conventional secretion pathway or ER-Golgi secretory pathway  group or OGD/R (48 h) group, suggesting there may be changes in the protein level of PEBP1 in other brain cell following I/R. In this study, we only focused on the role of PEBP1 in cerebral I/R injury via binding to Raf-1 and PC-PLC in neurons. Fourth, a critical gap in our knowledge of PC-PLC is the unavailability of its gene sequence and protein structure. As shown in this study, performing the activity assay has been an important strategy for the investigation of PC-PLC functions in mammalian cells  in penumbra tissue after MCAO/R, suggest that therapies targeting PEBP1 hold significant promise for the treatment and prevention of pathologic processes characterized by I/R injury.GC and HL conceived and designed the study, including quality assurance and control. ZW and JB performed the experiments and wrote the paper. XY and CL designed the study’s analytic strategy. HS and XL helped conduct the literature review and prepare the Section “Materials and Methods” of the text. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This paper focuses on the fabrication via simple melt blending of thermotropic liquid crystal polyester (TLCP) nanocomposites reinforced with a very small quantity of modified carbon nanotube (CNT) and the unique effects of the modified CNT on the physical properties of the nanocomposites. The thermal, mechanical, and rheological properties of modified CNT-reinforced TLCP nanocomposites are highly dependent on the uniform dispersion of CNT and the interactions between the CNT and TLCP, which can be enhanced by chemical modification of the CNT, providing a design guide of CNT-reinforced TLCP nanocomposites with great potential for industrial uses. Liquid-crystalline polymers (LCPs) have a phase of matter intermediate between the isotropic liquid and crystalline solid states termed as a mesophase or mesomorphic phase, and this LC state offers broad perspectives for various application materials that exhibit anisotropic but liquid properties . In geneThermotropic LCPs, that are a class of the materials used to produce anisotropic melts of relatively low viscosity, have been extensively investigated because of their excellent mechanical properties, superior chemical resistance, low gas and liquid permeability, low controllable coefficient of thermal expansion, excellent dimensional stability under temperature or in severe environment, and easy processability with high precision by extrusion and injection molding ,3,4,5. TCarbon nanotubes (CNTs) have attracted a great deal of scientific interest as advanced materials since they were first reported by Iijima . In partin situ polymerization, and melt blending. Among these processing techniques, melt blending process has been accepted as the simplest and the most effective method from an industrial perspective, because this process makes it possible to fabricate high performance polymer nanocomposites at low process costs, and facilitates commercial scale-up  versus θ as shown in Ea values of pure TLCP and TLCP/CNT nanocomposites were estimated to be 290.8, 293.7, 303.8, and 322.6 kJ/mol, respectively. As compared to pure TLCP, higher Ea value of TLCP nanocomposites suggested that they were more thermally stable than pure TLCP. In addition, TLCP nanocomposites containing modified CNT showed higher Ea values as compared to TLCP nanocomposites containing pristine CNT, suggesting that TLCP nanocomposites with more uniform dispersion of modified CNT were more thermally stable than those containing pristine CNT. This feature was also attributed to the interactions between modified CNT and TLCP matrix, which increase the activation of the thermal decomposition of TLCP matrix relative to pristine CNT. Similar observation has been reported that the enhanced thermal stability of CNT-reinforced poly (PEN) nanocomposites was associated with the increased interfacial interactions between modified CNT and PEN , where Gʹ and Gʺ are storage and loss moduli, respectively. The frequency ranges were varied between 0.05 and 450 rad/s, and the strain amplitude was applied to be within the linear viscoelastic ranges. The mechanical properties of TLCP nanocomposites were measured using an Instron 4465 testing machine, according to the procedures in the ASTM D 638 standard. Thermogravimertic analysis of TLCP nanocomposite were performed with a TA instrument SDF 2960 TGA over the temperature rage of 30~800 °C at a heating rate of 10 °C/min under N2.The chemical structure of CNT was characterized by means of room temperature FT-IR measurements in the range of 400~4,000 cmThis paper focused on the fabrication and characterization of thermotropic liquid crystal polymer (TLCP) nanocomposites reinforced with a very small quantity of modified carbon nanotube (CNT). The TLCP nanocomposites exhibited higher complex viscosity and more distinct shear thinning behavior than pure TLCP, resulting from strong interactions between CNT and TLCP matrix, which significantly influenced the relaxation behavior of polymer chains in the nanocomposites. The extent of the increase in the complex viscosity, storage modulus, and loss modulus of TLCP nanocomposites by the presence of modified CNT was more pronounced at low frequency as compared to those at high frequency. The decrease in the slopes of storage and loss moduli for TLCP/CNT nanocomposites with the introduction of modified CNT was attributed to the formation of the interconnected or network-like structures via the nanotube-nanotube and polymer-nanotube interactions, resulting in the pseudo solid-like behavior of TLCP nanocomposites. The incorporated CNT has a significant effect on the enhancement of thermal stability by acting as effective physical barriers against the thermal decomposition in TLCP nanocomposites. The improvement in the mechanical properties of TLCP nanocomposites resulted from the enhanced interfacial adhesion between modified CNT and TLCP as well as uniform dispersion of modified CNT in TLCP matrix, and their synergistic effect combining good interfacial interaction and uniform dispersion was more effective at lower CNT content relative to higher CNT content. This study demonstrates that the optimization of straight morphology and preferential alignment of CNT during melt processing as well as the enhanced interfacial adhesion between CNT and TLCP and uniform dispersion of CNT in TLCP matrix plays a key role in further improving the overall mechanical properties of TLCP nanocomposites.


BK virus is ubiquitously present in the latent state in humans, and awareness of the importance of BK polyomavirus is emerging among the kidney transplant community. First discovered in 1971 in the urine of a renal transplant recipient, BK virus nephropathy (BKVN) has come to be recognized as a significant cause of genitourinary disease and potential graft loss in the kidney transplant patient. In this review, we discuss the risk factors, available methods of diagnosis and therapeutic monitoring, and current approaches to therapy of BKVN.


Both the analytical and the simulated results indicate that the stiffness k increases with thickness h and width b, while decreasing with an increase of length l, and it is related to the shape. The inertial micro-switches with different kinds of stationary electrodes were simulated using ANSYS software and fabricated using surface micromachining technology. The dynamic simulation indicates that the contact time will decrease with the increase of thickness h and width b, but increase with the length l, and it is related to the shape. As a result, the contact time decreases with the stiffness k of the stationary electrode. Furthermore, the simulated results reveal that the stiffness k changes more rapidly with h and l compared to b. However, overlarge dimension of the whole microswitch is contradicted with small footprint area expectation in the structure design. Therefore, it is unreasonable to extend the contact duration by increasing the length l excessively. Thus, the best and most convenient way to prolong the contact time is to reduce the thickness h of the stationary electrode while keeping the plane geometric structure of the inertial micro-switch unchanged. Finally, the fabricated micro-switches with different shapes of stationary electrodes have been evaluated by a standard dropping hammer system. The test maximum contact time under 288 g acceleration can reach 125 µs. It is shown that the test results are in accordance with the simulated results. The conclusions obtained in this work can provide guidance for the future design and fabrication of inertial microswitches.A novel micro-electro-mechanical systems (MEMS) inertial microswitch with a flexible contact-enhanced structure to extend the contact duration has been proposed in the present work. In order to investigate the stiffness  Recently, MEMS inertial microswitches based on surface micromachining technology have been attracting much attention due to many advantages, such as lower cost, less power consumptions, larger volume production, smaller size, better performance, and more functionality than conventional mechanical inertial switches fabricated with precision machining processes . Therefoh), width (b), length (l) of the stationary electrode on the contact duration will be fully analyzed by analytical and finite-element simulation methods. Finally, the fabricated inertial micro-switch devices will be also evaluated by a standard drop hammer system.In traditional designs, both the movable electrode and the stationary electrode in the inertial microswitch are usually rigid, which will result in an extremely short contact duration. However, this extremely transient contact time will become a significant obstacle for subsequent signal processing if the microswitch is applied in an integrated circuit ,11,12,13k of the stationary electrode in sample 1 is equal to the total sum stiffness of two linear cantilever beams  of the cantilever beam is constrained to be zero, and the center point (O) is applied by a concentrated force F in z direction as shown in x is in x direction along the length of cantilever beam. The total length of the cantilever beam (PN) is approximately 2l. According to the data [F can be obtained by Equation (1):Sample 1 is the originally-designed stationary electrode as shown in the data , the maxz direction can be expressed by Equation (2) according to Hooke’s Law:E and I are the Young’s modulus and the bending moment of inertia of the linear cantilever beam, respectively. I can be defined as:The stiffness k of the stationary electrode in sample 1 can be written as:Substituting Equation (3) into Equation (2), the stiffness k of the stationary electrode in sample 1 is proportional to b and k of the stationary electrode in sample 1 varies with h, b, and l. It reveals that h and l more easily affects the stiffness k compared to b. However, the dimension of the fabricated microswitch cannot be too large because of the expected small footprint in the structure design. Therefore, the length l cannot be too long. Thus, the best way to control the stiffness k is to change the thickness h instead of b and l. The analytical models neglect the deformation from beam elongation, beam shortening, and shear. This assumptions is reasonable because these deformations are smaller than the deformation from bending moment and the deflection from torsion. The limitations of the analytical models neglect a series of holes in the cantilever beam.The analytical stiffness The mouth-shape crossbeam in sample 2 see d is usedh), width (b), and length (l) of the stationary electrode are chosen as variables. Two vertically-driven inertial microswitches with differently-shaped crossbeams as the stationary electrode for extending contact duration are designed. It can be seen from z direction), the sensing movable system can be described as follows [m stands for the proof mass, c is the damping coefficient, k is the whole system, stiffness and a(t) represents the applied acceleration.When the mass-spring system is shocked by an acceleration in its sensing direction ( follows :(5)mx¨+−3 [One-quarter of the inertial microswitch will be simulated by the commercial ANSYS software in order to save computing time, as shown in −3 .h, b, l) of the stationary electrode. In order to study the effect of the stationary electrode’s shape on the contact time, the same acceleration For a microswitch device, the shape of the stationary electrode is a crucial factor which can affect the contact time. In the designed inertial microswitch, samples 1 and 2 have the same geometric parameters (h) of the stationary electrode on the contact time, the width (b) and length (l) of the stationary electrode in samples 1 and 2 are invariant and set as 51.5 µm and 550 µm, respectively. The stiffness of the two designs of elastic cross cantilever beams was simulated by ANSYS software [k = h) is shown in h). That is to say, the contact time of the inertial micro-switch device decreases with the increase of the stiffness of the stationary electrode. It can be seen from the plot in In addition, the thickness of the stationary electrode has also a great impact on the contact time. For the inertial microswitch, decreasing the thickness of the stationary electrode is a key method to extend contact duration. In order to study the impact of the thickness (software . The simb) of the stationary electrode also has a great impact on the contact time. For the inertial micro-switch, minimizing the stiffness of the stationary electrode is a crucial way to prolong the contact time. One of the best ways to lessen the stiffness of the stationary electrode is to lessen its width (b). It can be seen from b). b) curves of samples 1 and 2 under the applied acceleration 288 g. The contact time in samples 1 and 2 decreases with the increase of the width (b) of the stationary electrode. That is to say, the contact time decreases with the increase of the stiffness of the stationary electrode. It is evident that the contact time can be controlled by changing the width (b) of the stationary electrode. It can be seen from It is well known that the width (l) of the stationary electrode is a key factor affecting the contact time of the inertial micro-switch. In the designed inertial microswitch, samples 1 and 2 have the same geometric parameters  of the stationary electrode, but length (l) is variable. For a microswitch device, length  of the stationary electrode. Therefore, the stiffness relates to the shape of the electrode. In conclusion, the contact time decreases as the stiffness of the stationary electrode increases. It has been proven that the simulated stiffness in sample 1 is in agreement with the above analytical calculation in sample 1. The slight deviation may be attributed to the following reasons: Firstly, the center cross part of the simulated model shown in The thickness, width, and length of the stationary electrode in samples 1 and 2 are key factors affecting the stiffness of the stationary electrode. Firstly, it reveals that the stiffness of the stationary electrode increases with the thickness and width of the stationary electrode. Secondly, the stiffness decreases with the increase of the length. Thirdly, the stiffness in sample 1 is larger than the one in sample 2 for the same geometric parameters The Cr/Cu was sputtered on the insulated quartz substrate as the first seed layer. We can electroplate the anchors of the spring and an array of raised strips after 10 µm photoresist  spining-on and patterning.(b)x1) can be formed.The anchors which suspended the spring and the stationary electrode were electroplated in Ni metal. Therefore, the first suspending layer gap ((c)The four serpentine springs were electroplated after the second chromium/copper seed layer was sputtered.(d)The proof mass was electroplated up to its required thickness (70 µm) in terms of multiple electroplated technology.(e)x2 (15 µm) between the proof mass and the stationary electrode was formed after the anchors which suspend the stationary electrode were electroplated higher (15 µm) than the proof mass.A gap (f)The stationary electrode was electroplated up to the required thickness.(g)The photoresist was dissolved in 7% sodium hydroxide solution. Then the completed released device which was rinsed in isopropyl alcohol was dried in the oven to avoid stiction.The prototype of the vertically-driven inertial microswitch was fabricated by low-cost nickel-electroplating technology. In this work, we selected PR AZP4620 as the mold and sacrificial layer. We used electroplating nickel as the main structure material due to its excellent mechanical property. The electroplating-nickel residue can be reduced by optimizing the electroplating conditions. In the present work, the electroplating nickel’s condition can be listed as follows: temperature 45 °C, current density 125 mA/cmThe inertia microswitch was sealed using an all-metal cap that was fabricated by ultraviolet Lithographie Galvanoformung Abformung technology. The dimension of the packaged single device by epoxy adhesive bonding is about 2.4 mm × 1.9 mm × 1.1 mm, which is smaller than existing devices . The staH as shown in H. The drop hammer can drop freely from a different pre-set height H, and an array of heights correspond to a series of acceleration amplitudes. The amplitude of the acceleration increases with H. The acceleration signal and the trigger signal can be detected by the oscilloscope when the test microswitch switches on.It can be seen from g which was applied to the test device could be generated. The oscilloscope records the test results of the fabricated micro-switches with different stationary electrode’s geometric parameters h, b, and l.When the drop hammer falls from the required height of 52 cm, a half sine wave acceleration with the amplitude of 288 h = 12 µm, b = 51.5 µm, and l = 550 µm) was shocked by the applied acceleration of 288 g. As is shown in h = 8 µm, b = 51.5 µm, and l = 550 µm) can be triggered with 125 µs contact time under the applied acceleration of 288 g as shown in h) test result is the average value of three samples’ repeated experiments. According to the former analytical and the simulation results, it reveals the thickness h easily affects the stiffness k and the plane geometric structure cannot be changed. The simulated results indicate that the contact time decreases with the increase of the thickness (h). That is to say, the contact time of the inertial microswitch device decreases with the increase of the stiffness of the stationary electrode. It can be seen from the plot in The original design inertial micro-switch in sample 1 (b) and thickness (h), but decreases with the increase of length (l). Meanwhile, the contact time decreases with the increase of the stiffness of the stationary electrode. Both the analytical results and the simulated results indicate that among all of the factors, h and l more easily affect the stiffness k compared to b. However, the dimension of the whole microswitch cannot be too large considering the miniature structure design. Therefore, it is not a workable way to prolong the contact time by increasing the length l too much. It is evident that to reduce the thickness h will not affect the plane geometric dimension of the inertial microswitch. Thus, the best and most convenient way to extend the contact duration is to reduce the thickness h. Finally, the fabricated microswitch has been tested by a drop hammer system. The redesigned inertial micro-switch of sample 2  can realize the contact time of 125 µs when a half-sine wave acceleration of 288 g is applied, which would facilitate the subsequent signal processing in its application system. The test results indicate that the contact time increases when decreasing the stiffness of the stationary electrode, which is in accordance with the simulated ones. These conclusions obtained in this work will provide some guidance for the future design and fabrication of the inertial microswitch.A MEMS inertial microswitch with an elastic crossbeam stationary electrode for extending the contact duration has been designed, simulated, and fabricated on a quartz substrate using surface micromachining technology. The simulation reveals that the stiffness of the stationary electrode relates to the shape of the stationary electrode and increases with the width (


Cystinosis is a rare autosomal recessive lysosomal disorder characterized by the accumulation of cystine in lysosomes. Cystinosis is much rarer in Asian than Caucasian populations. There are only 14 patients have with cystinosis alive in Japan. Most cystinosis is the nephropathic infantile form, as indicated by its apparent and severe clinical manifestations, including renal and ocular symptoms. Patients with the nephropathic juvenile form account for 5% of those with cystinosis. Their diagnosis is frequently delayed and difficult because of slower progression to end-stage renal disease and fewer cystine crystals in the cornea. Molecular analysis and a cysteine-binding protein assay should be performed when patients with proximal tubulopathy of an unknown origin are encountered.CTNS gene showed two novel heterozygous single nucleotide substitutions of c.329G > C and c.329 + 2 T > C. Both were splicing site variants causing exon 6 skipping proven by transcript analysis, although the functional prediction site showed c.329G > C, p.(Gly110Ala) as a benign missense substitution. The patient’s estimated glomerular filtration rate was 66.8 mL/min/1.73 m2. He was immediately treated with cysteamine after diagnosis.A 12-year-old boy had been suffering from Fanconi syndrome since he was 3 years old. He was only recently diagnosed despite repeated ophthalmological examinations. Corneal cystine crystals were found when he was 12 years old, and he was diagnosed with cystinosis by high free cystine content in granulocytes . Analysis of the Even if no ophthalmological abnormalities are present, nephropathic juvenile cystinosis should be suspected in children with Fanconi syndrome. Transcript analysis was useful to detect pathogenic splicing variants in this patient. CTNS gene, which encodes the lysosomal cystine carrier protein cystinosin. Cystinosis is classified into three forms according to the age at onset and severity. Nephropathic infantile cystinosis is the most frequent form and patients progress to ESRD in the first decade of life. The nephropathic juvenile form is rare and accounts for 5% of all patients. These patients show much slower progression to ESRD than those with the nephropathic infantile form. Early diagnosis is often difficult and most patients are only diagnosed after they reach 10 years of age [CTNS gene in Middle Eastern and East Asian people [Cystinosis is an autosomal recessive lysosomal disorder characterized by the accumulation of cystine in lysosomes throughout the body. This results in various clinical manifestations, such as Fanconi syndrome, end-stage renal disease (ESRD), hypothyroidism, hypogonadism, insulin-dependent diabetes mellitus, muscle weakness, swallowing dysfunction, central nervous system complications, keratopathy, and pigmentary retinopathy –3. Cystis of age . Addition people , 5. To d2. Levels of serum copper ceruloplasmin, lactic acid, and pyruvic acid indicated that Wilson disease and mitochondrial disease were not the cause of Fanconi syndrome.The patient was a 12-year-old boy who was the first child of non-consanguineous healthy parents. His growth and development had been normal. He had been followed at another hospital since the age of 3 years because of glycosuria and proteinuria of unknown cause. At the first visit to our hospital, there were no specific physical findings. Laboratory findings showed a low serum uric acid level (2 mg/dL), a high urinary β-2 microglobulin level , glycosuria (4+), proteinuria , and aminoaciduria consistent with Fanconi syndrome. Serum potassium and phosphate levels were within the lower normal limits. Blood gas analysis showed a normal bicarbonate ion value. His estimated glomerular filtration rate (eGFR) was 66.8 mL/min/1.73 mA recent slit-lamp examination showed cystine crystals in the cornea Fig. . He had CTNS gene showed two novel heterozygous single nucleotide substitutions of c.329G > C and c.329 + 2 T > C. The former is the last nucleotide of exon 6 and the latter is at the consensus sequence of the splicing donor site at intron 6  might be a single nucleotide polymorphism of p.(Gly110Ala) because the two functional prediction sites of the amino acid variant (PolyPhen2 and SIFT) showed this as benign. However, c.329 is the last nucleotide of exon6 and an effect of the splicing process from the exonic last nucleotide variant is possible. We subsequently conducted transcript analysis using mRNA extracted from leukocytes. cDNA reverse transcribed from mRNA was amplified by nested PCR using the following CTNS primer pairs: first PCR forward TTTCCCCTGAAGCTCGTAGA-3′ and reverse 5′-AAGACCCCGAGTCCAAACTT, and second PCR forward TCAGCCTCACTGTTCCTCCT and reverse CGAAGCTCAGACCAATGACA. PCR-amplified products were purified and subjected to direct sequencing. We found that both heterozygous mutations resulted in exon 6 skipping  in granulocytes at 6 h after taking cysteamine. eGFR improved to 77.8 mL/min/1.73 m2 from 66.8 mL/min/1.73 m2.The patient was administered cysteamine 1.3 g/mAmong the three forms of cystinosis, diagnosis of the nephropathic juvenile form is especially difficult because of the low frequency and mild clinical manifestations compared with the nephropathic infantile form. Delayed diagnosis often results in irreversible renal damage.Patients with nephropathic infantile cystinosis are born without any complications. At the age of 6–12 months, Fanconi syndrome causes polyuria, thirst, failure to thrive, growth retardation, vomiting, dehydration, constipation, developmental delays, and rickets. Untreated infantile patients rapidly progress to ESRD in the first decade of life . Renal tThere have been few reports of nephropathic juvenile cystinosis. Servais et al. reported 14 patients with the late-onset nephropathic form . The firA slit-lamp examination is useful for diagnosing nephropathic infantile cystinosis once a patient is 1–2 years old , but theA renal biopsy may help diagnosis the nephropathic juvenile form. Our patient showed multinucleated podocytes, cystine crystals, atrophy of tubules, interstitial fibrosis, and glomerular solidification. Among them, multinucleated podocytes suggested the possibility of nephropathic cystinosis, even in the absence of a history of Fanconi syndrome or chronic tubulointerstitial nephropathy .CTNS gene is a powerful tool for early diagnosis and can be used for prenatal diagnosis of cystinosis. In our patient, we observed two novel heterozygous mutations. One of these was an apparent pathogenic mutation at a splicing consensus site (c.329 + 2 T > C). The other mutation ) was suggested as a benign variant by two of the most popular prediction sites, PolyPhen2 and SIFT, although it was considered a missense mutation. It is unclear whether c.329G > C single base substitution may lead to a splicing abnormality. This was finally proven as pathogenic by transcript analysis. Therefore, transcript analysis was a good tool in the diagnosis of cystinosis in our patient. Servais et al. reported that four of 14 patients with late-onset nephropathic cystinosis showed only one mutation [Molecular analysis of the mutation . TranscrC, p.Gly1Ala) was Among Asians, cystinosis is extremely rare compared with people from the United States and Northern Europe, because the 57-kb deletion is rare in Asians [Cysteamine is a specific treatment for cystinosis. Cysteamine depletes lysosomal cystine content by reacting with cystine, and forming the mixed disulfide cysteine-cysteamine. Cysteamine treatment does not cure Fanconi syndrome, but prevents or delays progression to ESRD . This trPhysicians need to recognize the difficulty of early diagnosis and limitations of a slit-lamp examination to identify the nephropathic juvenile form. Patients with the nephropathic juvenile form show slow progression of renal involvement and mild urinary abnormalities, such as proteinuria or glycosuria. Molecular analysis and a cystine-binding protein assay should be performed when patients with proximal tubulopathy of an unknown origin are encountered.Proteinuria and glycosuria could indicate nephropathic juvenile cystinosis. A slit-lamp examination may help in the diagnosis of this condition, but cystine crystals may not be observed before adolescence in some cases. To the best of our knowledge, this is the first report of two novel mutations involved in cystinosis, one of which was proven by transcript analysis as pathogenic.


Peaks at 1545 cm−1 and 1562 cm−1 were found to refer to zirconia binding with 10-MDP. The presence of 10-MDP in both experimental ceramic primer and self-adhesive resin cement improved microshear bond strength to zirconia ceramic. It can be concluded that the nondestructive method of micro-Raman spectroscopy was able to characterize chemical bonds of 10-MDP with zirconia, which improves the bond strengths of resin cement.So far, there is no report regarding the micro-Raman vibrational fingerprint of the bonds between 10-methacryloyloxy-decyl dihydrogen phosphate (10-MDP) and zirconia ceramics. Thus, the aim of this study was to identify the Raman vibrational peaks related to the bonds of 10-MDP with zirconia, as well as the influence on microshear bond strength. Micro-Raman spectroscopy was employed to assess the vibrational peak of 10-MDP binding to zirconia. Microshear bond strength of the dual-cure resin cement to zirconia with the presence of 10-MDP in composition of experimental ceramic primer and self-adhesive resin cement was also surveyed. Statistical analysis was performed by one-way ANOVA and Tukey's test ( Overall, these features make zirconia the ideal material for core and frameworks of prosthesis in anterior and posterior region propane (Bis-GMA) and triethylene-glycoldimethacrylate (TEGDMA) donated by Esstech Inc. , used as received. Acidic monomer 10-MDP (10-methacryloyloxy-decyl-dihydrogen-phosphate) was donated by FGM . Photoinitiator system was composed of camphorquinone , chemical initiator benzoyl peroxide , and coinitiator ethyl 4-(dimethylamino)benzoate . Silanated barium borosilicate glass particles  ceramic blocks  with 13.2 × 13.2 × 3.2 mm dimensions were obtained and sintered according to manufacturers' instructions. They were embedded and fixed in acrylic resin . The exposed flat zirconia surfaces were polished for 30 s with 600-, 800- and 1200-grit SiC papers under water irrigation and then ultrasonicated for 10 minutes.n = 6 for group). Either experimental primer (EP) or the commercial primer (CP) was actively applied for 20 s followed by a gentle air-blast for 5 s. In negative control group, no primer was used before resin cement bonding.Following, eighteen Y-TZP blocks were divided into three groups. 10-MDP containing ceramic primers were applied onto Y-TZP surfaces (μ-SBS) specimens were bonded to the zirconia surfaces using cylindrical translucent moulds  as previously reported [n = 6) using the dual-cure resin cement RelyX ARC .Microshear bond strength , bonded with the commercial cement (CC) RelyX U200 (3M-ESPE), and the control resin cement (ECN) group. Resin cements were manipulated according to the manufacturer's instructions using the same volume of each paste and mixing for 30 s until obtaining homogeneous mixture. The experimental cements also followed this protocol, and then cements were carefully inserted in the Tygon tubes to avoid blisters.2; Dabi Atlante, Ribeirão Preto, Brazil) for 40 s. After 30 minutes, each Tygon tube was removed with scalpel blades. Cylinders were analysed by stereomicroscopy and those with defects were discarded and replaced. Before bond strength test, all specimens were immersed in distilled water at 37°C for 48 h to wait for the chemical cure of the resin cement.All specimens for both groups were light-cured with LED light-curing unit DB-685  adapted in a universal testing machine . An orthodontic wire (0.4 mm diameter) was positioned surrounding and in contact with half of the cylinder and connected to the load cell (50N) of the machine to exert shear force in upward direction. Each cylinder was tested individually with 1 mm/min crosshead speed up to fracture. Maximum μ-SBS was recorded in N and transformed to MPa by dividing the results in N by the cross-sectional area of each specimen.Bonded specimens were mounted in a device for α = 0.05), one-way ANOVA, and Tukey's test (α = 0.05).After debonding, all zirconia surfaces were examined with a stereomicroscope  to determine the mode of failure that was classified into three types: A, adhesive fracture between ceramic and cement without signs of residual cement on zirconia surface; C, cohesive failure of the cement with full area presenting cement remnants; M, mixed fracture with areas depicting adhesive debonding and some residual cement indicating partial cohesive failure. Microshear bond strength outcomes for each study were statistically analysed by Kolmogorov-Smirnov normality test , and 60 × 70 μm field area.To obtain the vibrational analysis of the bonds between 10-MDP and zirconia, the micro-Raman spectrophotometer  was calibrated internally in zero using the silicon standard sample provided by the manufacturer. The configurations of the equipment were HeNe laser with 3.2 mW power, 633 nm laser wavelength, 10 s acquisition time, 3 accumulations, 1.5 n = 3) were conducted in experimental primer-treated samples (Y-TZP + EP) and those treated with commercial primer (Y-TZP + CP). Initially, the spectral range was set from 100 to 4000 cm−1. Following initial observations, the range was narrowed to 1500–2000 cm−1, region that depicted differences between spectra. The chemical shift and intensity of each peak were processed for baseline correction and normalized.For observation of Raman peak referring to the bond of zirconia with phosphate functionality of 10-MDP, three Y-TZP blocks were used for group. From each block, three initial readings (Y-TZP) were performed. Afterwards, the 10-MDP primers were applied as aforementioned for 20 s. After 10 minutes, the primed blocks were thoroughly rinsed with absolute ethanol and dried with air-blast to remove unbound monomer. Further three readings per group (p < 0.001). The results showed that the presence of 10-MDP in ceramic primer applied previously to the dual-cure resin cement increased the μ-SBS to Y-TZP for both experimental primer and commercial one when compared to negative control . The presence of 10-MDP as a component of self-adhesive resin cement showed statistically significant higher mean μSBS when compared with other cements  and presence of 1630 cm−1 and 1710 cm−1 peaks from 10-MDP. However, a wide peak was observed between 1545 cm−1 and 1562 cm−1, which was not detected in pure 10-MDP and Y-TZP alone. Therefore, it is suggested that these wide peaks are referring to coordinates bond between 10-MDP phosphate functionality and zirconia ceramic.Micro-Raman spectra MDP-zirconia bond are presented in In the present investigation, 10-MDP was employed as chemical method to improve the bonding of resin cement to Y-TZP, which actually occurred when included either in ceramic primers and in self-adhesive resin cement, increasing statistically the bond strength to zirconia ceramic. Therefore, the study hypothesis needs to be accepted. In this study, the micro-Raman spectroscopy was also used to find the peak of this bond interaction.2O3), the so-called Y-TZP. This augments fracture toughness, once there is phase transformation (t → m) when a crack or microcrack appears. Such transformation is followed by volume increase (3–5%) sufficient to reduce crack propagation [Currently, zirconia frameworks for dental prosthesis are well-established , 16 due pagation , 19.In metal-free indirect restorations, feldspathic ceramic is applied in high temperatures as covering to zirconia frameworks . Residua2O3), alumina air-abrasion, and diamond burs in high-speed handpieces [However, all these procedures change zirconia in a difficulty substrate to be bonding using resin-based cements and the luting on dental substrate , once trndpieces . Thermalndpieces .10-MDP incorporation in ceramic primers or self-adhesive resin cements improves effectively bonding to zirconia , 28 by tFunctional monomer 10-MDP is nowadays one of the most efficient functional monomers in terms of chemical interaction to calcium  providinμ-SBS, in presence of 10-MDP either in ceramic primer or in self-adhesive resin cement. The present study also showed that the presence of 10-MDP in the primer is essential for attaining superior bond strength compared to using 10-MDP-containing self-adhesive resin cement  decreased and a wide shoulder appeared with peaks at 1545 cm−1 and 1562 cm−1. These peaks were not found in commercial primer, neat 10-MDP, and experimental primer. It is suggested that these new peaks are related to –P–O–Zr bonds, likely representing simple and double coordinate bonds showed by Xie et al. (2015) [Micro-Raman has some advantages like nondestructive sample preparation, analysis of liquids, powders, and no need for previous preparations. It utilizes the inelastic dispersion of light from a visible laser near to infrared or ultraviolet range in order to yield vibrational information of chemical bonds . In this. (2015) , also sh−1 and 1562 cm−1. The presence of 10-MDP in primer and self-adhesive resin cement increases the microshear bond strength to Y-TZP ceramic.It is worth concluding that micro-Raman identification of bonds formed between 10-MDP and Y-TZP attained the formation of peaks 1545 cm


Motivation: Influenza A viral heterogeneity remains a significant threat due to unpredictable antigenic drift in seasonal influenza and antigenic shifts caused by the emergence of novel subtypes. Annual review of multivalent influenza vaccines targets strains of influenza A and B likely to be predominant in future influenza seasons. This does not induce broad, cross protective immunity against emergent subtypes. Better strategies are needed to prevent future pandemics. Cross-protection can be achieved by activating CD8+ and CD4+ T cells against highly conserved regions of the influenza genome. We combine available experimental data with informatics-based immunological predictions to help design vaccines potentially able to induce cross-protective T-cells against multiple influenza subtypes.Results: To exemplify our approach we designed two epitope ensemble vaccines comprising highly conserved and experimentally verified immunogenic influenza A epitopes as putative non-seasonal influenza vaccines; one specifically targets the US population and the other is a universal vaccine. The USA-specific vaccine comprised 6 CD8+ T cell epitopes  and 3 CD4+ epitopes . The universal vaccine comprised 8 CD8+ epitopes:  and the same 3 CD4+ epitopes. Our USA-specific vaccine has a population protection coverage  of over 96 and 95% coverage of observed influenza subtypes. The universal vaccine has a PPC value of over 97 and 88% coverage of observed subtypes.Availability and Implementation:http://imed.med.ucm.es/Tools/episopt.html.Contact:d.r.flower@aston.ac.uk Vaccines are the most efficacious and efficient medical intervention known. While vaccines based on inactivated or attenuated whole microbes or single protein subunits have proved widely useful, vaccines based on ensembles of epitopes remain underexploited. We have recently developed a powerful new approach to the design of epitope ensemble vaccines combining available experimental data with informatics-based immunological predictions . Treatment when necessary includes antivirals to relieve symptoms and reduce complications. However, prevention through vaccination is currently the most effective strategy to combat influenza viruses  and neuraminidase (NA) surface glycoprotein antigens , as wellhttp://www.who.int/csr/don/2009_09_18/en/). Formulating a ‘universal vaccine’ where multiple strains are targeted to induce so-called heterosubtypic immunity (HSI) may prove more effective in combating novel strains before they can go on to cause pandemics . There are two main types of T cells: cytotoxic CD8+ T cell lymphocytes (CTLs) and CD4+. CTLs recognize antigens presented by HLA class I (HLA I) molecules while CD4 T cell recognize antigens presented by HLA class II (HLA II) molecules. The role of CTL in cellular immunity includes the direct clearance of virally infected cells and the indirect recruitment of other immune cells via chemokine and cytokine secretion. CD4+ T cells' primary roles include B cell stimulation leading to specific antigen antibody production as well as stimulating CD8+ proliferation and memory responses. CD4+ T cells also mediate direct and indirect viral clearance, and symptom severity reduction in secondary infection . Initially this seemed highly conserved and induced broadly acting antibodies. However, over 20 variants of M2e were later identified in newly emerging influenza A strains, raising questions over its usefulness as a ‘universal’ vaccine. Nevertheless, regions of M2e may still be useful for inducing broadly acting protection .Conserved targets would be useful in formulating a ‘universal’ vaccine, as they would cover multiple viral subtypes . Such ‘uinter alia filoviruses  (http://weizhong-lab.ucsd.edu/cdhit_suite/cgi-bin/index.cgi?cmd=cd-hit) , full length influenza A polyproteins were collected from the Influenza Viral Resource at the National Centre for Biotechnology Information [NCBI] (=cd-hit) .For each influenza protein, MSA data were subjected to sequence variability analysis using the Protein Variability Server [PVS] (http://imed.med.ucm.es/EPISOPT.html) and the IEDB PPC prediction tool (http://tools.immuneepitope.org/tools/population/iedb_input). EPISOPT computes PPC in one step after predicting epitope-HLA I binding profiles using HLA I frequencies for the five main ethnic groups present in the USA population  (http://tools.immuneepitope.org/mhci/). A class I HLA reference set was used as these alleles were frequently found in the population . Many other prediction methods are available , obtaining two different cluster sets, and two different sets of conserved regions, one from BLASTclust and the other from CD-HIT. We used these conserved regions to identify matching conserved T cell epitopes.First, we examined CD8+ T cell epitopes, identifying 11 conserved epitopes common to both sets of conserved regions. Additionally, two extra epitopes were identified in the BLASTclust set  and another two from CD-HIT . EPISOPT was used to select CD8+ T cell epitopes for our USA-specific vaccine. To reach a PPC > 80% a minimum of two epitopes were needed. To achieve a PPC > 95% at least six epitopes are required. EPISOPT identified four different combinations of which two combinations reached a PPC of 95.7% and the other two 95.4%. We selected set one to define the CD8+ arm of our USA-specific ensemble vaccine.We collected 816 influenza A CD4+ T cell epitopes from the IEDB database and selected 7 conserved CD4+ T cell epitopes by comparing with the conserved regions identified by BLASTClust and CD-HIT clustering routes were used. These conserved epitopes of varying amino acid length were submitted to IEDB the class II HLA binding prediction tool . Many epAll epitope-HLA II binding profiles were then entered into the IEDB PPC tool for analysis. NPLIRHENRMVLAST, TGTGYTMDTVNRTHQ, TYQRTRALVRTGMDP and MAFLEESHPGIFENS have class II HLA binding profiles that are completely covered by the other three remaining epitopes and therefore they were not considered when calculating PPC with multiple CD4+ T cell epitopes. The remaining three CD4+ T cell epitopes gave a combined PPC of 76.4%. The resulting combination of selected CD8+ and CD4+ epitopes form a population-weighted potential candidate USA-specific multiple epitope ensemble vaccine capable of inducing region-specific cross protection against influenza A .Table 3To derive the equivalent universal vaccine, the class I HLA binding profiles of each epitope were predicted using the IEDB class I HLA binding prediction tool. We then used the IEDB PPC tool to estimate PPC values against the world population, rather than the North American-only coverage provided by EPISOPT. GMDPRMCSL, YINTALLNA and IRHENRMVL were excluded as they did not bind to any class I HLA allele within the top 1% percentile rank. Excluding those epitopes that gave a PPC of 0%, a maximum PPC of 91.04% was possible with the remaining 12 CD8+ epitopes. This was different result to EPISOPT v.1, where just 6 epitopes gave a PPC > 95%. This reflects the lesser diversity within the North America population compared to the world population as a whole. Epitopes which gave an individual PPC > 10% using the IEDB PPC tool were considered; and together reached a PPC value of 90.2%. These constituted the CD8+ component of our universal influenza vaccine . The selWe have intentionally not specifically targeted subtypes. However, due to their wide sequence conservation, the identified epitopes are well represented across most major subtypes. Apart from subtypes of influenza A unique to bats, there are 16 haemagglutinin and 9 neuraminidase subtypes transmissible to humans making 144 possible H*N* combinations, of which 128 have been observed in the wild, as indicated by the presence of at least one designated sequence in the Influenza Virus Resource , nucleoprotein (NP), matrix 1 protein (M1) or polymerase basic protein 1 (PB1). The large PPC value found for GILGFVFTL located on M1 protein is not surprising as previous research concluded it was broadly recognized by the population  has also been tested in clinical trials involving healthy adults  or neuraminidase (N). While it is often assumed wrongly that H or N epitopes are all important, this is not the case with T-cell responses. While N and H are the key proteins for antibody binding, cellular immunity can respond to viral infection before viral shedding, opening the whole influenza genome to immune surveillance. The absence of N and H epitopes is explained by the low sequence conservation between different strains , due to Recent studies suggest immunodominant influenza epitopes are less conserved generally than their subdominant counterparts, which is thought to result from immune pressure  or in geographically restricted populations, in this case the USA (PPC = 96.3%). Our putative vaccines contain both CD8+ and CD4+ epitopes, as both types of T cells play important roles in viral clearance. Although all identified epitopes are known to be immunoreactive, further studies are needed to assess their efficacy. The extent of cross protection will require further analysis to verify the efficacy against particular influenza A subtypes. Despite these limitations the results are a promising vindication of our approach, and the extensions we detail, should prove of interest in future influenza vaccination strategies.


The CCT decreased from 720.3 ± 58.1 μm preoperatively to 511.5 ± 50.6 μm at 6 months postoperatively (p = 0.0001). The ECD decreased from 2,754 ± 296 cells/mm2 to 1,708 ± 426 cells/mm2 at 6 months postoperatively (p < 0.001). The ECD loss was 37.9 ± 16.3%. The outcomes using BBG were comparable to those of earlier reports that employed TB; thus, BBG may be a viable alternative to TB. Vital staining of the endothelial graft is essential during Descemet's membrane endothelial keratoplasty (DMEK) to ensure surgical success. DMEK surgeons worldwide commonly use trypan blue (TB) to this end. However, TB may exert toxic effects on both the cornea and retina. Recently, Brilliant Blue G (BBG) has become recognized as an alternative stain for use during vitreoretinal surgery; BBG is associated with lower levels of toxicity. We retrospectively analyzed the utility of BBG staining during DMEK. We used 0.1% (w/v) BBG to stain the DMEK grafts of 12 patients. We evaluated the best spectacle-corrected visual acuity (BSCVA), central corneal thickness (CCT), and endothelial cell density (ECD) before and 3 and 6 months after surgery. BBG was effective in terms of graft visualization during DMEK. The BSCVA (log  MAR) improved from 0.99 ± 0.57 to 0.01 ± 0.07 ( Descemet's membrane endothelial keratoplasty (DMEK) is a valuable method of corneal transplantation that corrects corneal endothelial dysfunction , 2. DMEKGood visualization of the DMEK graft is important; adequate graft staining facilitates successful surgery. Misdiagnosis of graft orientation causes both inverted graft attachment and primary graft failure . Vital dVital stains do not kill living cells. Vital dyes have been used both diagnostically and surgically by various specialists, including oncologists, when performing cataract and vitreoretinal surgery. TB is employed in the course of cataract surgery to visualize the anterior capsule during capsulorhexis , 18. DurIn the present study, we analyzed data from DMEK patients for whom BBG  was used to identify the grafts.BBG 250®  was dissolved in balanced saline solution  to 0.1% (w/v). All dye osmolarities were ca. 298 mOsm, and the pH values were 7.4. We used donor tissue from SightLife  for DMEK. All grafts were peeled as described previously. BBG  was used to stain the graft edges during peeling . A punchOur study protocol was approved by the Institutional Review Board of Yokohama Minami Kyosai Hospital . Written informed consent was obtained from all patients prior to enrolment in the study. We carefully followed all ethical principles of the Declaration of Helsinki. Twelve eyes of 12 patients with bullous keratopathy who underwent DMEK at Yokohama Minami Kyosai Hospital from February 2016 to August 2016 and who were followed up for more than 6 months were retrospectively analyzed. We treated 2 males and 10 females of mean age 75.9 ± 4.5 years. All eyes underwent DMEK performed by a single surgeon (TH). Eight eyes exhibited iatrogenic bullous keratopathy; five had undergone prior argon laser iridotomy (ALI) and three prior cataract surgery and intraocular lens implantation. Two eyes exhibited Fuchs' corneal endothelial dystrophy and two corneal endotheliopathy attributable to pseudoexfoliation syndrome (PEX).All surgeries were performed under local anesthesia. After establishing retrobulbar anesthesia and a Nadbath facial nerve block, 2 paracenteses and a 2.8 mm upper corneal or corneoscleral incision were made for the recipient cornea. Peripheral iridotomy was performed at the 6-o'clock position using a 25-gauge vitreous cutter to prevent the development of a postoperative pupillary block. The donor membrane graft stained with 0.1% (w/v) BBG (1.0 mg/ml) was placed into an intraocular lens injector  and inserted into the anterior chamber. A small amount of air was also injected between the host cornea and donor graft, and the rolled-up donor graft was then unfolded. Correct graft orientation was confirmed with reference to the preoperative marks. The anterior chamber was filled with air to allow the graft to adhere to the host cornea. Fifteen minutes later, the air was partially replaced with BSS. Finally, 0.4 mg of betamethasone  was subconjunctivally administered in 1.5% (w/v) levofloxacin eyedrops .Postoperative medications included 1.5% (w/v) levofloxacin (Cravit®), 0.1% (w/v) betamethasone sodium phosphate , and 2% (w/v) rebamipide ophthalmic solution , commencing at four times daily for 3 months and tapering thereafter.In addition to the standard ophthalmic examination, all of the best spectacle-corrected visual acuity (BSCVA), corneal ECD, central corneal thickness (CCT), and graft adaptation were evaluated both preoperatively and for up to 6 months postoperatively. Graft adaptation was assessed with the aid of both slit-lamp biomicroscopy and anterior segment OCT . Corneal thickness was measured via corneal tomography . Preoperative ECDs were retrieved from donor eye bank records. Intraoperative and postoperative complications were recorded and postoperative ECDs were measured with the aid of a specular microscope .The BBG staining capacity was evaluated by two experienced ophthalmologists (TH and KY) as follows: W, worse than TB; S, similar to TB; and B, better than TB.p value < 0.05 was considered to reflect statistical significance.The Wilcoxon test was used, when appropriate, to compare means among groups. All analyses were performed with the aid of StatView statistical software . A Detailed patient profiles are shown in p = 0.001). The CCT decreased from 720.3 ± 58.1 μm preoperatively to 511.5 ± 50.6 μm at 6 months postoperatively (p = 0.0001). The corneal ECD was 1,708 ± 426 cells/mm2 at 6 months postoperatively . No eye showed any sign of pupillary blockage, microbial infection, or endothelial rejection. Partial graft detachment requiring rebubbling of the anterior chamber was observed in only one eye 6 days after surgery; the graft attained complete reattachment after rebubbling. No significant intraocular pressure (IOP) elevation was detected during the follow-up period. No upside-down insertion was noted. Notably, we encountered no instance of primary graft failure.The BSCVA (log MAR) improved significantly from 0.99 ± 0.57 preoperatively to 0.01 ± 0.07 at 6 months postoperatively .Clinically, TB used during either cataract surgery or DMEK exhibits low toxicity. Indeed, we found, in a preliminary study, that neither TB nor BBG was toxic in terms of corneal ECD reduction after cataract surgery. In our preliminary work, the corneal endothelial cell losses 6 months after surgery were −2.2 ± 6.5% and −3.5 ± 9.8% in the TB and BBG groups, respectively , we earlier found that a severe ECD reduction was associated with long-term TB use.We make two useful points in the present study. First, 0.1% (w/v) BBG was not inferior to TB in terms of graft staining during DMEK. We noted no upside-down insertion of the DMEK graft. A recent report found that a lutein-based Brilliant Blue dye (lutein/zeaxanthin combined with Brilliant Blue [LZ/BB]), which is pale green in color, was valuable during both transplantation and graft preparation . In the Our work has three principal limitations. First, this was an in vivo study. We found that ECD decreased significantly after DMEK, as have earlier reports that employed TB staining. An in vitro study is required to determine if ECD loss is indeed influenced by the chosen surgical procedure, BBG staining, or both. Additional in vitro experiments are required to understand why the ECD falls after DMEK. Also, we did not prospectively compare DMEK outcomes after the use of BBG or TB. Ideally, the two vital dyes should be employed (during DMEK) in a prospective randomized fashion. Our work was retrospective and lacks a formal comparison of the two dyes; we switched from TB to BBG during the time course of our series. Finally, we applied 0.1% (w/v) BBG for an arbitrary 1 min; the dye concentration upon dilution with BSS remains unknown. Further work is essential to validate the safety of DMEK facilitated by BBG staining. Higher TB concentrations afford excellent staining without adverse effects, but only if staining persists for 1–3 min; a longer staining time (5 min) reduced the ECD .In conclusion, BBG is both efficacious and safe when used during DMEK. Additional studies are required in ocular surface surgeries, as with vitreoretinal surgeries.


Low-amylose and high-amylose potato starch (LAPS and HAPS) and low-amylose and high-amylose maize starch (LAMS and HAMS) were subjected to organocatalytic butyrylation. This resulted in products with an increasing degree of substitution (DS) measured by heterogenous saponification and back titration with the HCl  depending on reaction time. NMR analysis, however, showed that the major part of the acylation was induced by tartarate (75–89%) and only a minor part (11–25%) by butyrate. Generally, the chemical method overestimated the DS by 38% to 91% compared with the DS determination by NMR. Increasing the DS appeared to lower the in vitro fermentation capability of starches independent of the starch source and, therefore, do not seem to present a feasible method to deliver more butyrate to the colon than lower DS products.Intake of butyrylated starches may increase colonic butyrate supply, which can be of public health and clinical benefit by maintaining colonic health. The objective was to investigate if an organocatalytic method with tartaric acid as a catalyst could be applied to produce butyrylated products from different starch sources and to characterize their chemical structure and fermentation capability by using solid-state  The short chain fatty acids (SCFA) acetate, propionate, and butyrate produced by microbial fermentation of dietary fiber (DF) in the large intestine are believed to play a beneficial role in gut health . In partd-glucopyranosyl unit in starch is esterified with butyric acid.Strategies to increase the production of specific SCFA’s in the colon may be of public health and clinical benefit. One way to achieve this is to increase the consumption of foods high in resistant starch (RS) because RS fermentation generally favors butyrate production in the large intestine ,9. HowevMost commercial starch esters are produced by a reaction of starch in aqueous medium with anhydrides and NaOH as catalyst. However, Tupa et al.  describe13C MAS NMR spectroscopy and an in vitro fermentation model system, respectively.Therefore, the aim of this study was to examine if the methodology described by Tupa et al.  could be® 260, Ingredion, Bridgewater, NJ, USA), pre-gelatinized low-amylose potato starch , and high-amylose potato starch . The HAMS and HAPS are products of traditional plant breeding (hybrids), i.e., selection over generations and not derived from genetically modified plants. Dry matter (DM) content was determined by drying at 103 °C to constant weight and ash was analyzed according to the AOAC  method (923.03) [The four starches subjected to butyrylation were pre-gelatinized low-amylose maize starch , high-amylose maize starch   and RS bl-(+)-tartaric acid  and oven-dried starch (800 mg) were mixed in a glass vial and heated to 120 °C in a heat block with constant magnetic stirring. When the mixture achieved the target temperature, all tartaric acid dissolved and this was considered the beginning of the reaction. Esterification was run for different reaction times  as well as 0.5–2.5 h of reaction times in triplicate  and no replicates at 3 and 5 h of esterification. After the chosen reaction time, the mixture was allowed to cool to room temperature and the product was washed several times with distilled water by vacuum filtration in a Buchner funnel to guarantee the removal of the catalyst and unreacted acid. The solid was oven dried overnight at 40 °C to 50 °C.Esterification of LAMS, HAMS, LAPS, and HAPS with butyric acid was performed on a small scale in oven-dried 20 mL glass vials sealed with screw caps, according to a simple organocatalytic method described by Tupa et al. . ButyricBV = volume (mL) HCl required for titration of the blank (= non-treated starch), SV = volume (mL) HCl required for titration the sample, NHCl = normality of the HCl solution (= 0.1 M), Macyl = molecular weight of the acyl group (71 g/mol for butyryl), and W = mass of sample used (g).Determination of the acyl content and degree of substitution (DS) was performed by heterogeneous saponification and back titration with HCl, which was described by Tupa et al. . The acyd-glucopyranosyl unit in starch is esterified with butyric acid.The degree of substitution (DS) of an acylated starch is defined as the number of hydroxyl (OH) groups substituted by an acyl group per anhydroglucose unit of the starch polymer. Since the anhydroglucose unit possesses three hydroxyl groups at carbon 2, 3, and 6, the maximum DS value is 3. When DS = 0.25 for a butyrylated starch, ~8.3% of the hydroxyl groups on each The DS of acylated starches was calculated by using Equation (2)  assuming13C MAS NMR spectroscopy analysis. All solid-state NMR spectroscopic experiments were carried out using a Bruker Avance 400 (9.4 T) NMR spectrometer  operating at Larmor frequencies of 400.13 MHz and 100.63 MHz for 1H and 13C, respectively. 13C single-pulse (SP) MAS and cross-polarization (CP) MAS [1H and 13C, 600 scans, a spectral width of 50.13 kHz, and an acquisition time of 40.9 ms during which TPPM (two pulse phase modulation) [1H decoupling was applied. For the 13C CP/MAS experiments a contact time of 1.0 ms and a recycle delay of 16 s was utilized, a recycle delay of 128 s was used for the 13C SP/MAS experiments. Prior to Fourier Transformation, all FID (free induction decay)’s were apodized by a Lorentzian line-broadening of 10 Hz. All spectra were referenced to the carbonyl resonance of α-glycine at 176.5 ppm .Non-treated LAMS, HAMS, LAPS, and HAPS  and butyrylated LAMS, HAMS, LAPS, and HAPS at a low and high DS determined by the chemical method were subjected to solid-state (CP) MAS  NMR expeulation)  1H decou13C SP/MAS spectra. The ratio between the aliphatic carbons in butyrate and C1 was determined from the 13C CP/MAS spectra due to a higher S/N-ratio. Before calculating the ratios, the integrals were normalized according to the number of carbon sites contributing to the integrals.Determination of the relative ratios of glucose (starch), butyrate, and tartarate were obtained by integrating the region of the two aliphatic carbons not attached to the carbonyl in butyrate (5–25 ppm), the region of the anomeric carbon (C1) in starch (90–120 ppm), and the carbonyl carbon in the acid/ester (155–190 ppm). Due to the different longitudinal relaxation times for CH (hydrogen bearing carbon)’s and carbonyls, the ratio between C1 and the carbonyls was determined from the 2. Following 6 min of stomaching, the supernatant was filtered into a sterile bottle while flushing with N2. Triplicates of 100 mg starch samples were weighed and placed into Hungate tubes. The samples were dissolved in 7 mL sterile 0.1 M sodium phosphate buffer (pH 6.5). Three mL of the fecal inoculum slurry was added to the tubes, which resulted in 10% (weight/weight) fecal inoculum and 1% (weight/weight) substrate and flushed with N2. A positive control  and a negative control (7 mL sodium phosphate (pH 6.5) + 3 mL fecal slurry) were also included in the experiment. Tubes were placed horizontally in an incubator shaker and incubated at 37 °C for 24 h. Samples for SCFA determination were obtained at time 0 h and 24 h and stored at –18 °C until further analyzed.In vitro 24-hour batch fermentations using human fecal inoculum were conducted on the four non-treated starches and on four selected butyrylated starches with increasing DS from each starch source to determine if the DS measured by heterogenous saponification and back titration affected the fermentation pattern in vitro. The method described by Edwards et al. [Organic acid concentration was analyzed by gas chromatography  using an SGE-BP1 column  with 5% phenylpolysiloxane and 95% dimethylpolysiloxane and a flame ionization detector after submitting the samples to an acid-base treatment followed by ether extraction and derivatization, which was described by Canibe et al. [post hoc test was applied to adjust for multiple comparisons.One-way ANOVA was conducted to estimate the impact of butyrylation on butyric acid and total organic acid concentration following the in vitro fermentation using SAS . The Tukey-Kramer As expected, the content of amylose was 2–2.6 times higher in the HAMS and HAPS compared to the low-amylose starches with HAMS having the largest proportion of amylose  and only a minor part (11–25%) by butyrate. However, tartaric acid is a dicarboxylic acid and both of the acid groups may in principle react with a hydroxyl group on a glucose residue in starch. This makes DS with tartarate a more difficult parameter to handle and it is, therefore, more correct to provide the DS as the molar ratio between the acid and the anhydroglucose units, which is also the original output of the NMR-analysis (see As seen in ysis see . For botysis see .13C CP/MAS NMR spectra of the different starches. The spectrum of high-DS HAMS contains assignments of the six carbons (C1–C6) of the α-1,4 linked glucose units as well as the carbons originating from tartaric and buryric acid. Comparison of the four non-treated starch samples shows marked differences in the distribution of C1 resonances as well as the line width of the C6 resonance for the high and low amylose samples due to the pre-gelatinization of the latter. Furthermore, observation of lipid (~32 ppm) in the HAMS sample indicates the presence of lipid-amylose complexes in this sample. Apart from the observations on the non-treated starch samples, a significant decrease in the intensity of the C6 resonance as a function of the DS was observed for all samples. This demonstrates that the acylation takes place on C6, which was expected. Because of the spectral overlap of resonances from C2, C3, and C5 in starch and CH in tartaric acid, it was not possible to verify if acylation also takes place on C2 and C3.The aim of this study was first to examine if the organocatalytic methodology for butyrylation of starches  could be13C MAS NMR. Maize and potato starch differ in crystallinity (type A for maize and type B for potato) but there are many other morphological, structural, and molecular differences between the two types of starch [When attempting to develop starch products acylated with SCFA ,19,27 thf starch  includinf starch  that pot13C MAS NMR analysis revealed that the organocatalytic method involving tartaric acid as a catalyst resulted in much more tartarate (75–89% of the acylation) than butyrate (11–25% of the acylation) added to the starch polymer. This suggests a reaction mechanism involving both acids and it also suggests that results on DS measured by heterogeneous saponification and back titration with HCl  should be confirmed by other methods such as solid-state 13C MAS NMR. Another issue is that tartaric acid is a dicarboxylic acid and both of the acid groups may in principle react with a hydroxyl group on a glucose residue in the starch. Therefore, it is possible that the results on DS by tartarate  than the integrals of CH2 and CH3. The excess carbonyl must originate from tartaric acid, which demonstrates that tartaric acid also reacted with starch in their sample preparation. Lopez-Rubio et al. [13C MAS NMR analysis of acetylated, propionylated, and butyrylated starches produced by the organocatalytic method with tartaric acid should be performed. The characterization of the crystallinity and nanostructure should be used to investigate potential discrepancies in DS depending on the acyl group chain length.The solid-state rate see  are overrate see  are the nic acid  revealedo et al.  studied o et al. . OverallIn the current study, the overall rationale behind coupling butyrate to the starch polymer was to make the starch itself more resistant to enzymatic digestion in the small intestine and available for microbial fermentation and colonic SCFA production at the same time as butyrate is delivered directly . An addiOur esterified starch samples were not pre-digested with enzymes, according to the AOAC method (AOAC 991.43) prior to the in vitro fermentation since it is typically practiced in these types of study designs . We only13C MAS NMR. The butyrylated products, independent of a starch source, were only poorly fermented in an in vitro fermentation model and butyrylation to high DS with the organocatalytic method does not seem to potentially deliver more butyrate to the large intestine than lower DS products.The organocatalytic method can be used for butyrylation of both high amylose and low amylose maize and potato starch to yield products with increasing DS depending on reaction time. The method, however, resulted in much more tartarate than butyrate added to the starch polymer, which suggests a reaction mechanism involving both acids. The chemical method for DS determination highly overestimated the DS compared with solid-state