Keratan sulfate (KS) is a sulfated linear polymer of N-acetyllactosamine. Proteoglycans carrying keratan sulfate epitopes were majorly observed in cornea, cartilage and brain; and mainly involved in embryonic development, cornea transparency, and wound healing process. Recently, expression of KS in cancer has been shown to be highly associated with advanced tumor grade and poor prognosis. Therefore, we aimed to identify the expression of KS epitope in human pancreatic cancer primary and metastatic tumor lesions. Immunohistochemical analysis of KS expression was performed on primary pancreatic tumors and metastatic tissues. We observed an increased expression of KS epitope on primary tumor tissues compared to uninvolved normal and tumor stroma; and is associated with worse overall survival. Moreover, lung metastatic tumors show a higher-level expression of KS compared to primary tumors. Interestingly, KS biosynthesis specific glycosyltransferases expression was differentially regulated in metastatic pancreatic tumors. Taken together, these results indicate that aberrant expression of KS is predictive of pancreatic cancer progression and metastasis and may serve as a novel prognostic biomarker for pancreatic cancer. KS is widely distributed in both extracellular matrix (ECM) of cornea, bone, cartilage, brain, and on surface of the epithelial tissues3. Structurally, KS consists of a linear polymer of N-acetyllactosamine backbone of Galβ1-4GlcNacβ1-3 that has a sulfate moiety at C-6 positions of galactose  and N-acetylglucosamine (GlcNAc)3. KS biosynthesis occurs in two stages: production of GlcNAc-sulfated poly-N-acetyllactosamine chain by the sequential action of β1,3-N-aceltylglucosaminyltransferase 7 (β3GnT7), N-acetylglucosaminyl-6-sulfotransferase (GlcNAc6ST) and β1,4-galactosyltranferase 4 ; and production of highly sulfated KS by galactosyl-6-sulfotransferase 5. In tissues, KS chains can be either N- or O-linked to the proteoglycan core protein. KS proteoglycans (KSPGs) with the family of small leucine-rich ECM PGs  are mainly composed of KS chains, whereas aggrecan with large chondroitin sulfate composition contains few KS chains3. In addition, epithelial mucin (MUC1)6 and an isoform of CD447 have KS chains in their structure. These KSPGs are widely distributed in tissues and have diverse range of functions2.Keratan Sulfate (KS) is a glycosaminoglycan (GAG) first detected in bovine cornea9. KS also plays a role in regulation of inflammation and has been reported to have potential in the treatment of inflammatory conditions such as rheumatoid arthritis and Chronic Obstructive Pulmonary Disease (COPD)10. Dysregulation of KS biosynthesis has been associated with numerous diseased conditions, including macular degeneration and keratoconus5, amyotrophic lateral sclerosis11, Alzheimer’s diseases12, and mucopolysaccharidosis IV13. In addition, several studies have reported that aberrant expression of KS is highly associated with malignant conditions such as Burkitt’s lymphoma14, astrocytic tumors15, glioblastoma16, and in human embryonic cancer cells17. Miyamoto et al. (2011) reported a consistent expression of KS in carcinomas of the female genital tract and the absence of KS expression in carcinoma of gastrointestinal tract, liver, and urinary tract suggesting that KS might be a potential cancer biomarker for differential diagnosis of these cancers18. Furthermore, the study also reported that carcinomas of lung, mesothelium, mammary gland, thyroid, and pancreas express KS18. Addition of sulfate group on the KS side chains induces p38 MAPK and PI3K mediated anti-apoptotic signaling pathways in Burkitt’s lymphoma cells14. KS specific sulfotransferases (CHST) 2/6/7, which catalyze the transfer of a sulfate group to the GlcNAc residues of KS side chains have been shown to play a vital role in tumor malignancy16. Differential expression of specific β1,3-N-aceltylglucosaminyltransferases (β3GnTs) involved in KS biosynthesis have also been reported in cancers21. Moreover, KSPGs like lumican are also upregulated in several cancer tissues and cell lines including pancreatic cancer27. However, the functional importance of KS and its biosynthetic glycosyltransferases expression in primary and metastatic pancreatic tumors remains elusive.KS plays an important role in corneal transparency, developmental biology, cell signaling, adhesion, and migration28. Of the multiple histologic subtypes described, pancreatic ductal carcinoma (PDAC) is the most common and deadliest form of pancreatic cancer. Therefore, understanding the underlying molecular changes and the potential molecules involved in disease progression is critical for early diagnosis and therapeutic intervention. In this study, we explored the expression levels of KS and its biosynthesis specific enzymes in human PDAC primary and metastatic tumors. We detected differential expression of KS associated with organ specific metastasis. We also observed a differential expression of KS specific glycosyltransferases between primary and metastatic tumors. In addition to this, we also observed the correlation of aberrant expression of KS with increase risk and reduced overall patients’ survival.Pancreatic cancer is the fourth most common cause of cancer related death in the United States mainly due to disseminated diseased conditions, and has a 8% five-years survival rateWe examined the cellular distribution of keratan sulfate (KS) expression in pancreatic tumors immunohistochemically by using KS epitope specific antibody, I-22. For this, 35 pancreatic cancer patient samples were obtained through rapid autopsy program (RAP) as described in materials and methods section  involved in KS synthesis were measured in TMA samples of 12 primary tumors and corresponding lymph nodes, liver and lung metastatic deposits  cell line  are found in numerous tissues8. Aberrant expression of KS results in numerous pathophysiological conditions including cancers9. Recent studies have shown that addition of sulfate group on the KS side chains induce p38 MAPK and PI3K mediated anti-apoptotic signaling pathways in cancer cells14. However, little is known about the correlation of KS expression with pancreatic cancer. The study presented here investigated the potentials of KS GAG as a non-invasive prognostic and metastatic predictive biomarker in patients with pancreatic cancer. We showed for the first time the correlation of aberrant KS expression with pancreatic cancer progression and metastasis and with worse overall patient survival status.KS is a major GAG shown to play an important role in developmental and physiological processes including tissue hydration, cell signaling and migrationExperiments performed in a cohort of 35 human pancreatic cancers patients demonstrate a significant increase in KS expression levels in tumor cells than in uninvolved normal cells or tumor stroma. Moreover, KS expression levels were correlated with pancreatic cancer progression with significantly higher levels in patients with stage III/IV than in stage I/II. Interestingly, positive KS levels were significantly predictive of overall survival; and the Cox proportional hazard regression analysis showed patients with KS positive group has 4.48 times the risk of experiencing events compare to KS negative group. In addition, KS expression levels were upregulated in lung metastasis as compared to matched sets of primary tumors and / or liver metastases. Therefore, the study reported here clearly demonstrate that KS expression level is highly correlated with pancreatic cancer progression and metastasis as well as with worst patients’ survival status. More than 91% (32 out of 35) of these pancreatic cancer patients  were given different regimen of chemotherapeutic drugs during the course of disease progression and did not show any statistical significance on KS expression 39. In this study, we demonstrated that a higher-level expression of B3GNT5 mRNA in KRAS and p53 mutated highly metastatic FG, T3M-4, and HPAF pancreatic cancer cell lines  were obtained with consent and Institutional Review Board approval from University of Nebraska Medical Center’s Tissue Bank through the Rapid Autopsy Pancreatic (RAP) program. Of these, 32 patients >91%) received different combinations of chemotherapy  were prepared from these tissue sections using 2- to 2.5- mm punch. The arrays contained cores of matched normal and tumor tissues. Once completed, TMA blocks are cut into 4-micron thick sections with a microtome and mounted on charged slides.All immunohistochemistry analyses were performed using Dako EnVision kits . Keratan sulfate (KS) specific mouse monoclonal antibody (I-22) was obtained from Developmental Studies Hybridoma Bank, Iowa University. Slides with serial sections of tissue microarrays were stained using KS mouse monoclonal antibody with standard IHC procedures. Briefly, tissues were deparaffinized in histoclear and rehydrated in descending grades of ethanol followed by antigen retrieval using an alkaline citrate buffer and microwave treatment. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide and slides were blocked with 5% BSA. Following incubation with primary (1:100 dilution) and secondary antibodies substrate chromogen 3,3′-diaminobenzidine (DAB) was added followed by counterstaining with Harris hematoxylin and dehydration with alcohol gradients ending with xylene. Primary antibody concentrations and incubation conditions were optimized using positive control tissues. Specimens were processed on the same day to eliminate any variability in conditions. Whole slides scanning was performed using Ventana’s Coreo Au Slide Scanner in UNMC Tissue Science Facility.29. The data are publicly available in Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc = GSE21501). The two color Agilent arrays were used to quantify the relative gene expressions between samples from the metastasis sites or primary cancer sites versus the reference human mRNA samples (Stratagene). The array data were preprocessed by filtering the data to eliminate poor quality spots or genes that did not have mean intensity greater than 10 for either channel in at least 70% of the experiments. Lowess normalization were used to normalize the filtered data, and log2 ratio between the metastasis sites or primary cancer vs the reference mRNA samples were calculated. The ratios with at least 30 for the normalized raw intensity from both channels were included for further heat map analysis, which provide a visual comparison in the gene expression between metastasis site and primary pancreatic cancer.We have utilized human whole genome 4 × 44 K DNA microarrays data from UNMC RAP tissue specimens -producing cell line was established from a primary PDAC transplanted into nude mice and then cultured51. HPAF cell line was originally derived from the ascetic fluid of patient with pancreatic cancer52. All cell lines were maintained in DMEM (Dulbecco’s Modified Eagle Medium)  supplemented with 10% heat deactivated fetal bovine serum , penicillin G (100 U/mL), and streptomycin (100 µg/mL)  at 37 °C with 5% CO2 in a humidified atmosphere as described previously35.HPNE cell line is an hTERT (active telomerase) immortalized human pancreatic Nestin expressing (HPNE) ductal progenitor cell line is undifferentiated and non-tumorigenic53. Briefly, total cellular RNAs were isolated from HPNE, FG, T3M-4 and HPAF pancreatic cancer cell lines and pancreatic tumor and uninvolved normal tissues using TRI reagent  as per the manufacturer’s protocol. RNA sample integrity was measured by Nanodrop spectrophotometry and Aligent Bioanalyzer and cDNA was synthesized from total cellular RNAs using VersoTM cDNA kit . QRT-PCR was performed on a Master cycler realplex2  using SYBER Green PCR kit . Forward and reverse primers, 5′-TATTCACATGGGAGAGCCGCATGA-3′ and 5′-TGCGGAAGAATGGAACACCCTGTA-3′ respectively, were used for selective amplification of B3GNT5. The data were analyzed using Eppendorf realplex software, version 1.5 (Eppendorf). Amplification of GAPDH  was used to normalize the samples for comparison.Quantitative Real-Time PCR (qRT-PCR) analysis was performed as described previouslyHistological sections were annotated by two independent pathologists. Relative KS antigen expression level was semi-quantified based on percentage of cells staining positive for KS antigen. A scale of 0–3 was used to indicate relative percentage of cells positive with 0 being no detectable expression and 3 indicating ≥67% of the total cell population expressed KS antigen. Discrepancies between two pathologists were rare and were resolved by averaging the results. 2–4 sections from each tumor were present on tissue microarrays and the final score for each sample was based on the mean score from all sections stained, to best represent the spectrum of tumor heterogeneity. Staining intensity scores were converted into heat maps for better visualization.Statistical analysis was performed using SAS software  and Graph Prism 7 software. Comparison between immunohistochemical scores of different groups in the matched autopsy samples were analyzed using paired t-tests between two groups or linear mixed models across more than two groups. Post-hoc tests with Turkey’s method were used for multiple comparisons when appropriate. Chi-square tests or Fisher’s exact tests were used to compare the categorical clinicopathological parameters between KS staining positive and KS staining negative groups. Kaplan-Meier plot was used to describe overall survival between groups and log-rank test was used for comparison. The Cox proportional hazard regression model was utilized to assess the association between KS staining with overall survival after accounting for the confounding effects of age, gender, stage, and grade.Supplemental Information


Soluble epoxide hydrolase (sEH) is a bifunctional enzyme with COOH-terminal hydrolase and NH2-terminal lipid phosphatase activities. It is expressed in various cell types in the brain and is involved in the pathogenesis of inflammatory and neurodegenerative diseases. Alzheimer’s disease (AD) is a progressive neuroinflammatory and neurodegenerative disease. However, the pathological significance of sEH and underlying molecular mechanism in AD remain unclear.sEH−/−) and two mouse models of AD, including amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic (APP/PS1 Tg) and APP/PS1 Tg/sEH−/− mice. Western blotting analysis and immunohistochemistry assay were performed to evaluate the protein expression. Locomotion, nesting building ability, Y-maze, and Morris water maze tests were conducted to study mouse behavior. The levels of interleukin (IL)-1β, IL-4, IL-6, and IL-10 and the activities of NF-κB and nuclear factor of activated T cells (NFAT) were measured by commercial assay kits. The quantitative protein level profiling in the brain lysate was analyzed using LC-MS/MS approaches.To examine the role of sEH in pathogenesis of AD, we used wild-type (WT) mice, soluble epoxide hydrolase deficient (APP/PS1 Tg mice. Genetic ablation of sEH in APP/PS1 Tg mice delayed the progression of AD as evidenced by the alleviation in behavior outcomes and Aβ plaque deposition. In addition, loss of the function of sEH in APP/PS1 Tg mice increased astrogliosis and the production of astrocyte-derived anti-inflammatory cytokines including IL-1β, IL-4, and IL-10, as well as the activity of NF-kB and NFAT. Moreover, analysis of gene ontology in the AD brain revealed that important signaling pathways and processes related to AD pathogenesis such as translational regulation, oxidative stress, cytoskeleton reorganization, and small GTPase signal transduction were altered in APP/PS1 Tg/sEH−/− mice compared with APP/PS1 Tg mice.We demonstrated that the level of sEH was increased in the brain and predominantly appeared in hippocampal astrocytes of Our results suggest that sEH is a crucial regulator in the progression of AD and might be a potential therapeutic target for the treatment of AD. Soluble epoxide hydrolase (sEH), an enzyme with COOH-terminal hydrolase (EH) and NH2-terminal lipid phosphatase (PT) activities, is expressed in several tissues, including the brain, heart, and kidney –4. In thAlzheimer’s disease (AD) is a progressive neurodegeneration disease with typical symptoms, including impairment of learning, memory, and cognition abilities , 18. RecAPP/PS1 Tg mice. We demonstrated that genetic deletion of sEH in the APP/PS1 Tg mice rescued the impairment of AD pathologies, including Aβ plaque deposition, cytokines production, and dysfunction of behavioral outcomes. The quantitative proteomic analysis of brain samples from APP/PS1 Tg/sEH−∕− and APP/PS1 Tg mice was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mapping of the deregulated proteins with bioinformatics tools revealed that differentially abundant proteins were significantly enriched for pathways and processes known to be important in AD pathology, including translational regulation, oxidative stress, cytoskeleton reorganization, and small GTPase-mediated signal transduction. Collectively, our results suggest that sEH is a key regulator of astrocytes-derived inflammation in AD progression.In this study, we aimed to investigate the potential role of sEH and possible molecular mechanisms during AD pathogenesis by using the mouse models. Here, we report that the protein level of sEH was increased in astrocytes of 6-month-old Rabbit anti-sEH (sc-25797), mouse anti-von Willebrand factor (vWF) (sc-365712), goat anti-rabbit IgG FITC-conjugated (sc-2012), Texas red-conjugated (sc-2780), and goat anti-mouse IgG Texas red-conjugated (sc-2781) antibodies were obtained from Santa Cruz Biotechnology . Rabbit anti-glial fibrillary acidic protein (GFAP) (AB5804), mouse anti-GFAP (MAB360), anti-NeuN (MAB377B), and anti-ionized calcium-binding adapter molecule 1 (IBA-1) (MABN92) antibodies were obtained from Millipore . Rabbit anti-LDLR-related protein 1 (LRP-1) (L2170) antibody, mouse anti-α-tubulin (T9026) antibody, bovine serum albumin (BSA), and phosphatase inhibitor cocktails 1 and 2 were obtained from Sigma-Aldrich . Mouse anti-Aβ (SIG-39320) antibody was from Covance . Rabbit anti-β-APP C-terminal fragment (βCTF) (802801) antibody was obtained from BioLegend . Mouse anti-ATP-binding cassette transporter A1 (ABCA1) (ab18180), rabbit anti-BACE1 (ab2077), anti-IL-1β (ab9722), anti-IL-4 (ab9622), anti-IL-6 (ab6672), and anti-IL-10 (ab9969) antibodies were obtained from Abcam . Rabbit anti-apolipoprotein E (apoE) (1930-1) antibody was obtained from Epitomics . Retrieval buffer was from Biocare Medical . The mounting medium with DAPI was from Vector Laboratories . The ELISA kit for NF-κB activity was from Cayman Chemical  and for NFAT activity from Active Motif . ELISA kits for IL-1β, IL-4, IL-6, and IL-10 were obtained from R&D systems .tm1/Gon2/J (sEH−∕−) mice, and B6.Cg-Tg85Dbo/J (APP/PS1 Tg) mice were purchased from Jackson Laboratory . For the generation of APP/PS1 Tg/sEH−∕− mice, sEH−∕− mice were crossed with APP/PS1 Tg background, and the genotypes were confirmed by PCR of genomic DNA isolated from these animals. Mice were housed in barrier facilities, maintained in a 12-h/12-h light and dark cycle. Temperature (22 °C) and humidity (40–60%) of the vivarium were tightly controlled. Mice were group-housed 3–4 per cage and fed a regular chow diet, which contained 4.5% fat by weight (0.02% cholesterol) . At the end of the experiments, mice were euthanized with CO2, and then brain tissues were collected for histological analysis or were stored at − 80 °C. The isolated brain tissues were homogenized, and lysates were subjected to western blot analysis.The investigation conformed to the Guide for the Care and Use of Laboratory Animals , and all animal experiments were performed in accordance with the approved guidelines by the Animal Care and Utilization Committee of the National Yang-Ming University. C57BL/6 J WT, Ephx2Brain tissues were lysed in immunoprecipitation lysis buffer . Aliquots of brain lysates were separated on SDS-PAGE, transferred to membranes, incubated with primary antibodies, and then horseradish peroxidase-conjugated secondary antibody. Bands were detected using an enzyme-linked chemiluminescence detection kit  and the band signal was quantified by Imagequant 5.2 .The brain tissues were fixed in 4% paraformaldehyde and 15-μm cross sections were prepared. Sections were incubated with retrieval buffer for 10 min, blocked with 2% BSA for 60 min, and incubated with primary antibody overnight at 4 °C, then FITC- or Texas Red-conjugated secondary antibody for 1 h at 37 °C. Antigenic sites were visualized under a Nikon TE2000-U microscope (Tokyo) with QCapture Pro 6.0 software .The concentrations of pro-inflammatory cytokines, including IL-1β, IL-4, IL-6, and IL-10 in brain lysates, were measured by using the ELISA kits according to the manufacturer’s instructions.The DNA-binding activities of NF-κB and NFAT in brain lysates were measured by using the ELISA kit according to the manufacturer’s instructions.The protocol of nest building has been described . Each moThe protocol of Y-maze was followed as previously described with modifications . The Y-mThe protocol of Morris water maze (MWM) has been described  and modiAPP/PS1 Tg mice and APP/PS1 Tg/sEH−∕− mice was performed by researchers of Visual Protein Company .The LC-MS/MS analysis of brain tissues from APP/PS1 Tg and APP/PS1 Tg/sEH−∕− mice was performed by researchers of Visual Protein Company .The gene ontological analysis of brain tissues from U test was used to compare two independent groups. Kruskal-Wallis followed by Bonferroni post hoc analyses were used to account for multiple testing. SPSS v20.0  was used for analysis. Differences were considered statistically significant at P < 0.05.Results are presented as mean ± SEM. Mann-Whitney APP/PS1 Tg mice. The results of western blot analysis showed that the protein level of sEH was increased in 6-month-old APP/PS1 Tg mice   Fig. . These dAPP/PS1 Tg mice, we generated the APP/PS1 Tg/sEH−∕− mice by an inbred of sEH−∕− mice with APP/PS Tg mice  –4. RecenAPP/PS1 Tg/sEH−∕− mice. However, the protein levels of ABCA1 and LRP-1 were not changed. These results suggest that the decreased levels of neurotoxic Aβ in the brain of APP/PS1 Tg/sEH−∕− mice could be attributed to a decrease in Aβ biosynthesis but not an increase in Aβ clearance. Nevertheless, the detailed mechanism underlying sEH-mediated regulation in regulating Aβ generation and its clearance needs further investigation.APP can be rapidly metabolized by posttranslational proteolysis via an amyloidogenic or non-amyloidogenic pathway in neurons . In the APP/PS1 Tg/sEH−∕− mice. We also found that deletion of sEH in APP/PS1 Tg mice increased DNA-binding activity of NF-κB and NFAT, two key transcriptional factors for in the expression of IL-4 gene [APP/PS1 Tg/sEH−∕− mice were mediated by the increased anti-inflammatory responses. Nevertheless, further investigations are required for evaluating the exact mechanism underlying sEH-mediated inflammation and AD pathology.Astrocyte-mediated inflammation has been shown to play a key role in the progression of AD pathology , 42, 43.L-4 gene –31. ThusAPP/PS1 Tg/sEH−∕− mice than that in APP/PS1 Tg mice. In parallel, the levels of the anti-inflammatory cytokines were increased in the brain of APP/PS1 Tg/sEH−∕− mice. We thus suggest that sEH plays a critical role in regulating astrogliosis, which protects the neuronal cells from Aβ stimulation and delays the progression of AD.Astrogliosis (referred to as reactive astrocytes) occurs prominently in the central nervous system in response to pathological insults , 45. AltAPP/PS1 Tg/sEH−∕− mice as compared with that of APP/PS1 Tg mice. Oxidative stress is an event that precedes the appearance of the pathologic hallmark of AD and forming a vicious cycle that promotes the initiation and progression of AD [sEH deregulates the ribosome-related functions in APP/PS1 Tg mice, which are agreement with Ding et al. who found that a posttranslational regulation-dependent impairment in ribosome function was detected in cortical areas of AD subjects [To examine the possible molecular mechanism of sEH in AD pathogenesis, we used LC-MS/MS and gene ontology analysis to evaluate the involvement of sEH in cellular pathways during AD progression. Our results showed that the pathways of oxidative stress, cytoskeleton reorganization, small GTPase-mediated signal transduction, and translational regulation are regulated in the brain of on of AD . Besideson of AD . Numerouon of AD , 49. Theon of AD –50. For on of AD . In addiAPP/PS1 Tg/sEH−∕− mice compared with APP/PS1 Tg mice. Our data also showed that APP/PS1 Tg/sEH−∕− mice exhibited improved AD-related behavior outcomes, Aβ production, and aggregation compared with APP/PS1 Tg mice. Collectively, our findings suggested that sEH is critical in regulating the biological response of oxidative stress, cytoskeleton-dependent synaptic efficacy, and translational regulation during AD progression. However, extensive investigations are required for clarifying the participation and the regulatory mechanisms of sEH in these signaling pathways.Furthermore, several lines of evidence support the close relationship between oxidative stress and AD pathology , 51. AccAPP/PS1 Tg mice and APP/PS1 Tg/sEH−∕− mice due to the technical limitation. In addition, from the point in search of drug targets, the roles of EH activity or PT activity of sEH in the progression of AD should be investigated by using pharmacological inhibitors targeting EH activity and PT activity of sEH in AD mice. To this end, further investigations describing the implications of sEH in AD are warranted.Nevertheless, our study contains several limitations because we only examined the effect of genetic deletion of sEH on the progression of AD pathology. We did not examine the levels of epoxide fatty acids in the brain of We provide several lines of novel evidence for the role of sEH in the pathogenesis of AD, which advances our knowledge for better understanding of the biological significance of sEH in AD. Our findings suggest a broader biological role of sEH and the underlying molecular mechanisms in the initiation and progression of neuroinflammatory and neurodegenerative diseases. Overall, sEH might be a potential pharmacological target for treating AD and related neurological diseases.


Silicone modified polyurethane (PUSX) has attracted interest as a useful material by various properties, which are combined with silicone and polyurethane. In this paper, we tried to optimize the electrospinning process of silicone modified polyurethane (PUSX) nanofibers on a lab scale device and a multinozzle pilot scale set-up to investigate the potential and limitations of preparing PUSX nanofibrous sheets using different equipment. The morphology and diameter of the obtained fibers were studied via scanning electron microscopy (SEM). Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) was also carried out to analyze the chemical structure of PUSX nanofibers. As a result, we successfully figured out the optimal parameters of PUSX electrospinning process and demonstrated the great potential of the process for mass production of PUSX nanofibrous sheets from solutions. Silicones are highly functional resins that combine the characteristics of both inorganic and organic substances and exhibit an array of useful properties, including heat resistance, cold resistance, weather ability, dielectric properties, release properties, and water repellency. By introducing silicones into other resins, such as polyamide, polyimide, polyester, polycarbonate, and polyurethane, new resin materials can be created with enhanced functionality that include not only heat resistance, weather ability, and flame resistance, but improved impact resistance, lubricity, and flexibility. With their enhanced functionality, these high-performance composite resins are lightweight and have excellent fabricating characteristics and are used in a myriad of fields, including electric and electronics applications, automotive applications, electrical wire, and construction . For insPolyurethane (PU) is formed by reaction of polyisocyanates with hydroxyl-containing compounds. Desired properties can be tailored by selecting the type of isocyanate and polyols, or combination of isocyanates and polyols . Strong Here, we tried to get the advantages of both PU and silicone by introducing silicone groups into polyurethane polymer chains. Silicone modified polyurethane (PUSX) has attracted as useful material by various properties, which combined with silicone and polyurethane, such as heat resistance, weather resistance, peeling of molding, oil resistance, and high mechanical strength . As one In this research, we tried to synthesize the silicone modified polyurethane (PUSX) copolymers instead of using blends of two polymers and we optimized the electrospinning process of PUSX nanofibers on a lab scale device and a multinozzle pilot scale set-up to investigate the potential and limitations of preparing PUSX nanofibrous sheets using different equipment. The morphology and diameter of the obtained fibers were studied via scanning electron microscopy (SEM). Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) was also carried out to analyze the chemical structure of PUSX nanofibers.5 to 2.33 × 105 were synthesized as described in All the 12 kinds of silicone modified polyurethane (PUSX) solutions were kindly synthesized and provided by Shin-Etsu Chemical Co., Ltd  and Dainichiseika Color & Chemicals Mfg. Co., Ltd . The weight average molecular weight (Mw) is a polymethyl methacrylate (PMMA) equivalent value measured by gel permeation chromatography (GPC). GPC measurements were carried out with an HLC-8320 GPC system , tetrahydrofuran (THF) as the solvent, and at a resin concentration of 0.1%. Defined PUSX with Mw ranging from 1.48×101 and R2 are methyl group (-CH3) while R3 is a -OC2H4- group.The carbinol-modified silicone (silicone polyol) used in the synthesis process of PUSX was hydrosilylated by organopolysiloxane (with Si-H group) and unsaturated alcohol compound, catalyzed by platinum catalyst. The synthesis of block type silicone polyol  is shown in Polytetramethylene glycol  in DMF/MEK mixed solvent and stirring at room temperature for 48 h to obtain homogeneous solutions. To investigate the optimized parameters, all electrospinning experiments were performed by a mononozzle lab scale device at room temperature (22 °C) and the deposited nanofibers were collected on a drum shape rotating metallic collector. A 10–20 kV voltage was applied while the needle tip to collector distance was 10 cm with the irradiation angle of 30° and air flow rate in the spinning environment was 0.1 mL/min. The detailed relevant electrospinning parameters applied for each sample are discussed in The apparent viscosity of the polymer solutions with different concentrations was measured using a digital rotational viscometer . Software was used for the complete external control of the viscometer. Before the measurements, the viscometer was calibrated with standard oil , and the computed maximum uncertainty in the viscosity measurement was lower than 5%.The measurement of the surface tension of the solution was performed by a simple surface and interfacial tensiometer . The Wilhelmy plate method was employed. It is similar to the du Noüy ring method, but it is simpler and does not require correction. In this method, the plate is oriented perpendicular to the interface, and the force exerted on it is measured. This instrument was computer controlled, and it was calibrated with a known weight of 400 mg. The results we collected were the average of more than three specimens of each sample, with the computed maximum uncertainty lower than 5%.For both viscosity and surface tension measurement, the number of replicates tested for each solution was three.The surface morphology of nanofibers was investigated by scanning electron microscope  at an accelerating voltage of 10 kV. The prepared sample before SEM analysis was coated using a platinum sputter coater  under 30 mA and 30 seconds to be observed by SEM. The diameters of nanofibers were measured by Image J . US). The average fiber diameters and their standard deviations were calculated from data of at least 50 measurements per sample. At least three specimens were observed for each sample.−1 with a resolution of 4 cm−1 and accumulation of 20 scans. The number of specimens observed for each polymer was three.Attenuated total reflectance-Fourier transform infrared spectroscopy  was carried out to analyze the chemical structure of PUSX nanofibers. All spectra were taken in an absorption mode between the wavenumber range of 4000–700 cmThe electrospinning process is influenced by many parameters that consecutively also affect the morphology and the diameter of the obtained fibers. These parameters can be subdivided into polymer related and processing parameters. The predominant polymer related parameters, including the molecular weight, polymer concentration, and the solution viscosity, were varied as shown in In the first part of this work, the influence of the polymer concentration and applied voltage, viscosity on the fiber diameter was studied. Then, the optimized electrospinning parameters were transferred to a multinozzle pilot scale set-up to investigate the reproducibility of the fiber production method, the transferability of the electrospinning parameters, and the feasibility of upscaling the procedure.Solution concentration is one of the most significant parameters for electrospinning and affects the viscosity. Electrospinning of polymer solutions with too low concentrations results in the formation of beads and heterogeneous fibers, and upon increasing the concentration, their shape evolves from spheres to spindles until uniform fibers are produced at the appropriate concentration. Conversely, high concentration leads to too viscous solutions for which continuous flow cannot be maintained, leading to electrospinning instability and the formation of thick and inhomogeneous fibers. Thus, an optimal concentration range for the electrospinning of polymers, resulting in stable and reproducible fiber production, must be determined. The optimal range for successful electrospinning of all the different PUSX fibers was around 10 wt %~20 wt % according to w/v) polymer concentration. PUSX Si01-20 samples were dissolved in the given solvents at room temperature (25 ± 2 °C) with 48 h stirring to obtain homogeneous solutions. –5 S/m) than that of DMF (2.5 × 10–4 S/m), leading to a trend of stable flow of the solution, stable whipping, stable taylor cone, and improvement of the fiber quality during electrospinning. While increasing the amount of DMF, the stretching of the solution jet was increased as a result of a higher level of charges carried by the solution. The same factor also encouraged the reduction of the fiber diameter [Another significant factor that influences the electrospinning process a lot is the selection of solvents. In this study, in order to dissolve PUSX to get an easier electrospinning process, instead of using single solvent DMF, different types of solvents for electrospinning were prepared by using a mixture of DMF and MEK in the weight ratios of 64:36, 70:30, 80:20, and 90:10 in 13%  by using ATR for different kinds of PUSX nanofibers was used to observe the chemical structural differences due to the incorporation of a silicone group to the PU polymer chain and electrospinning process. From In the present study, we successfully prepared 12 kinds of PUSX nanofibers under the optimized conditions and investigated the effects of solvents and solution concentrations on the electrospinnability of PUSX solutions along with the morphological appearance of the as-spun nanofibers and chemical structures were characterized. This is the first time this way of preparing PUSX nanofibers has been suggested during past decades instead of making silicone and PU into composite and then preparing it into nanofibers or films. We reported a new material, which has the advantages of both PU nanofiber and silicone. Silicone groups had greater effects on block type PUSX than graft type. The optimal polymer concentration for electrospinning and the average diameters of the obtained nanofibers decreased with the increase of both the silicone chain length and silicone concentration in block type PUSX nanofibers as well as the decrease of viscosity and surface tension. Graft type PUSX nanofibers did not show a clear influence of the silicone chain length on electrospinning parameters. In addition, a study of the reproducibility and feasibility of the electrospinning parameters was performed on a pilot scale electrospinning set-up. On both the lab scale device and pilot scale set-up, PUSX solutions could be successfully electrospun. Hence, the present work demonstrated the great potential of the electrospinning process for the production of PUSX nanofibrous sheets from solutions. The physical properties and biocompatibilities of PUSX nanofibers and films are under investigation and will be discussed in a later manuscript. We believe that PUSX nanofibers might become a new alternative to PU nanofibers or composites in many potential fields.


For each compound, we provide the General Amber Force Field parameters for the major species at physiological pH, together with an analysis of properties of interest as extracted from μs-long molecular dynamics simulations in explicit water solution. The properties include number and population of structural clusters, molecular flexibility, hydrophobic and hydrophilic molecular surfaces, the statistics of intra- and inter-molecular H-bonds, as well as structural and dynamical properties of solvent molecules within first and second solvation shells. In addition, the database contains several key molecular parameters, such as energy of the frontier molecular orbitals, vibrational properties, rotational constants, atomic partial charges and electric dipole moment, computed by Density Functional Theory. The present database  is part of a wider project aiming to build-up a database containing structural, physico-chemical and dynamical properties of medicinal compounds using different force-field parameters with increasing level of complexity and reliability. The database is freely accessible at  The study of dynamical interactions between drugs and their biological targets (such as nucleic acids and proteins) is of paramount importance in medicinal chemistry and related fields . AmonDynamical interactions can be described in MD simulations by means of different models : from fab-initio calculations has indeed allowed the fitting to any property, including those not or partly accessible through experiments. An example in this direction was the use of DFT to calculate all vibrational frequencies and related eigenvectors of a chromophore [etc.) remains often a non-trivial task [In particular, MD simulations based on all-atom empirical “force-fields”, nowadays routinely performed in the microsecond timescale, have been proven to yield structural, dynamical, thermodynamical, and kinetic information with good accuracy ,14,15. Iomophore , that onomophore . The foromophore , nucleicomophore , and memomophore , althougomophore . Howeverial task , despiteial task ,28 and tial task  databaseial task  to classial task  and/or qial task  at diffeRecently, as part of the activity of the TRANSLOCATION consortium within the Innovative Medicines Initiative antimicrobial resistance programme, New Drugs for Bad Bugs , we haveetc., as extracted from μs-long molecular dynamics simulations in explicit water solution. In addition, the database includes several key molecular parameters, such as energy of the frontier molecular orbitals, normal modes of vibration, rotational constants, atomic partial charges and electric dipole moment, computed by Density Functional Theory. This database is freely accessible on-line at the address http://www.dsf.unica.it/translocation/db/ and is suitable for further inclusion of new data.This work constitutes the first step of the aforementioned wider project: we report a homogenous database of the computed molecular properties for a sample of 40 different antimicrobial compounds ranging in size from ∼20 to ∼80 atoms see . For eacFor each compound we obtained the 3D structure data file (SDF format) from the Pubchem database . We then(Symmetry=None), adopted very restrictive convergence criteria for both self-consistent-field iterations (10SCF(Conver=8)) and geometry optimizations (Opt(VeryTight)), and used a pruned  grid (Int=UltraFine) for numerical integration. For each compound we optimized the ground-state structure employing the Polarizable Continuum Model [SCRF=) particularly to avoid formation of strong intra-molecular H-bonds. To confirm the geometry obtained to be a global minimum on the potential energy surface we performed full vibrational analyses obtaining real frequencies in all cases. We processed the output of Gaussian09 with GaussSum [The structure of the major species determined in the above step has been subsequently used as an input to quantum-chemical calculations at the Density Functional Theory level . For thiasis-set . The comnal cost ,43. In aum Model  as to miGaussSum  and GabeGaussSum  to extraPop=MK) and CHELPG [Pop=CHelpG) schemes to construct a grid of points around the molecule. Atomic partial charges were then generated through the two-step restrained electrostatic potential (RESP) method [On the optimized geometry we then performedB3LYP/6-31G-Kollman  (Pop=MK)d CHELPG  (Pop=CHe) method  implemen) method . Followi) method . The der) method ,28. Fina program , and pol program .tleap of AmberTools14 [We performed all-atom molecular dynamics simulations in the presence of explicit water solution (0.1 M KCl) using the Amber14 package . Model srTools14  adoptingrTools14  and the rTools14 . For therTools14 . For allbarostat  and the barostat , respectg_rmsf of GROMACS [i.e., asphericity, acylindricity, and kappa2, as implemented in the PLUMED plugin [From the all-atom MD simulations we obtained structural and dynamical features of the compounds investigated by means of the PTRAJ and CPPTRAJ programs . In detaectively ,60,61,62ectively . To eval GROMACS . During D plugin . AspheriD plugin . The dynD plugin  and ChemD plugin . MoleculD plugin  and VMD D plugin  packagesEscherichia coli, Pseudomonas aeruginosa, Salmonella aenterica and Klebsiella pneumoniae. The complete set of compounds included in the current version of the database, together with some of their general properties are listed in The total of 40 species presently included in the database, ranging in size from clavulanic acid  to tigecycline , covers eight different classes of antibiotics and related compounds, namely: carbapenems, cephalosporins, monocyclic beta-lactams, oxazolidinones, penicillines, quinolones, tetracyclines, and beta-lactamase inhibitors. Antimicrobial compounds of these classes are the most widely used against infections caused by Gram-negative bacteria, such as http://www.dsf.unica.it/translocation/db/ contains the full list of compounds ordered, within each class, by increasing molecular weight (see .prep and .frcmod formats [molecule_mk_hf.prep) and the B3LYP/6-31G charges fitting the molecular electrostatic potential using the CHELPG (molecule_chelpg.prep) and Merz-Kollman (molecule_mk.prep) schemes (see The main-page ight see A. As shoight see B. The la formats ) generat formats  can be demes see ..xyz and MDL .sdf formats. For the above molecular structure we report: energy of the highest-occupied molecular orbital (HOMO) and lowest-unoccupied molecular orbital (LUMO), HOMO-LUMO gap, rotational constants, and electric dipole moment computed in vacuum and implicit water. The whole set of orbital data and the corresponding plots  extracted with GaussSum [gabedit [A separate table reports general properties as well as molecular descriptors extracted from both quantum-mechanics and molecular-dynamics simulations. As shown in GaussSum  . However, to the best of our knowledge, the present database is the first extensive one reporting structural, physico-chemical, and especially dynamical properties obtained combining different computational tools (in particular μs-long long MD simulations). There are basically three different levels of active use of our data. First of all the obvious stand-alone use of the tabulated static and dynamical properties of selected compounds. Second, the availability of the (at the moment only) GAFF parameters for the major microspecies at physiological pH, makes possible to straightforwardly perform MD simulations with ready-to-use input files. Finally, the knowledge of the dynamical behavior of a large number of molecular descriptors extracted from μs-long MD runs allows performing statistical analyses that go beyond the availability of one static value. This piece of information can find a plethora of possible applications, for example in the field of molecular docking, just to mention one. Most of the available docking programs take into account ligand flexibility only in terms of rotatable bonds of one given input structure  in protein-ligand docking [Among the antimicrobial compounds included in this release of the database, those for which ., rDock  and Auto., rDock ) or in t., rDock . However., rDock ,85,86,87 docking ,89,90, cdynamical values extracted from the MD trajectories and compares them with the static ones publicly available from Chemicalize [The knowledge of the values distribution for specific molecular descriptors can be also useful in the study of structure-dynamics-activity relationships by means of specific molecular descriptors. For instance, the minimal projection area (MPA) has been recently used  in the cmicalize . As showIn the near future we plan to extend the amount of information present in the database. For instance, in order to guarantee reproducibility, we plan to make available the input and output files of our simulations. Furthermore, the database will be extended towards two main directions. From one side we will include more compounds, covering additional antimicrobial classes. At the same time, for each compound, we will setup parameters at different pHs including at least two more cases (low and high-pH) in addition to the physiological pH already considered. We plan to include in the individual page of each compound the relevant literature reporting on molecular properties from both experimental and computational studies. From the technical point of view we plan to improve the key parameters reported, in particular dihedral angles, by comparison with quantum-mechanical dynamics in presence of implicit and explicit solvent molecules, using biased molecular dynamics techniques ,101,102.


An electrospinning technique was employed to fabricate the multi-layer biodegradable drug-eluting nanofibers. After fabrication, the nanofibrous membranes were characterized. The drug release characteristics were also investigated. In addition, the in vivo efficacy of nanofibers for pain relief and hemostasis in palatal oral wounds of rabbits were evaluated. Histological examinations were also completed. The experimental results suggested that all nanofibers exhibited good biocompatibility and eluted effective levels of lidocaine and epinephrine at the initial stages of wound recovery.This study developed multi-layered lidocaine- and epinephrine-eluting biodegradable poly[( A free gingival graft (FGG) is a dental procedure that involves harvesting soft tissue from a distant site in the mouth and grafting it over a localized recession defect. FGG was first introduced in 1963 and found the clinical applications to treat mucogingival lesions one year later ,3. AfterFor decades, science has recognized that wound dressings are extremely important components of wound care and are necessary for optimal wound healing. Open palatal wound recovery consists of a dynamic and intense process, which involves blood clot deposition, inflammation, and angiogenesis to constitute a vascularized granulation tissue. After that, an approximation of epithelium and tissue remodeling is completed to create a mature recovery process in four weeks . Wound dLidocaine is a drug widely used to numb tissue in a specific area and may be applied directly to the skin for numbing . EpinephThis study developed biodegradable lidocaine, epinephrine, and collagen loaded nanofibrous membranes that offered a sustainable elution of hemostatic and analgesic drugs at the oral wound sites to decrease postoperative pain and accelerate palatal donor site wound healing. An electrospinning technique was employed to fabricate the multi-layer biodegradable drug-eluting nanofibrous membranes. The technique has been widely employed to fabricate nanofibers of various materials as well as nanofibrous membranes encapsulating living cells . After fd,l)-lactide-co-glyco lide] (PLGA) polymer (lactide/glycolide: 50/50) . Epinephrine and lidocaine hydrochloride were included, while type I collagen was from calf skin .The polymeric materials included poly(ε-caprolactone) (PCL)  and poly[, PLGA/epinephrine (300 mg:50 mg), and PLGA/collagen (300 mg:50 mg) were primarily dissolved in 1 mL of hexafluoroisopropanol (HFIP) (Sigma-Aldrich), respectively. The PLGA/lidocaine solution was spun and collected by the collection plate in a nonwoven form. The procedure was duplicated to manufacture subsequently the PLGA/epinephrine and PLGA/collagen nanofibers. All procedures were performed at room temperature. On the other hand, the preparation of nanofibrous membranes with 3:1 and 2:1 polymer-to-drug ratios followed the same electrospinning procedure, except that the polymer/drug used were 300 mg/100 mg and 300 mg/150 mg, respectively.A field emission scanning electron microscope (FE-SEM)  was employed to quantify the electrospun nanofibers. The diameter compositions of nanofibers were acquired by characterizing the microphotos of 50 arbitrarily picked fibers for each specimen.−1 and 32 scans. Nanofibrous membranes were pressed as KBr discs, and spectra were analyzed over the 400–4000 cm−1 range.The spectra of electrospun nanofibrous membranes were characterized using a Fourier Transform Infrared (FTIR) spectrometry. FTIR analysis was completed on a Nicolet iS5 spectrometer  at a resolution of 4 cmThe tensile characteristics of electrospun nanofibrous membranes were quantified on a Lloyd tensiometer .The water contact angles of the nanofibers were assessed by a contact angle measuring apparatus  (N = 5).The elution behavior of lidocaine and epinephrine from the drug-loaded nanofibers with various PLGA/drug combinations, namely 6:1, 3:1 and 2:1, respectively, was evaluated. Specimens, with an approximate size of 2.0 mm × 3.0 mm and weight of 1 g, were sliced from the electrospun membrane and stored in glass tubes. Each tube contained one specimen along with 1 mL of buffered solution . All test tubes were maintained at 37 °C for 24 h. Then the eluent was collected for analysis, and fresh buffered solution (1 mL) was added. The procedure was repeated every 24 h until the specimen was fully dissolved. The experiment was triplicated (N = 3).v/v)) were adopted as the mobile phase. The absorbency of monitoring was set at a wavelength of 210 nm, while the flow rate was maintained at 1.0 mL/min. To characterize epinephrine, a Discovery BIO Wide C18-5, 25 cm × 4.6 mm column was used. The mobile phase contained water, methanol, and acetic acid (Sigma-Aldrich) in a volume ratio of 85:10:5. The pH value of the mobile phase was adjusted by adding ammonium acetate (Sigma-Aldrich) to reach 3.1. The absorbency was maintained at 280 nm, while the flow rate was set at 1.0 mL/min. All specimens were assayed in triplicate (N = 3).The lidocaine levels in the eluents were quantified using a high performance liquid chromatography (HPLC) assay. The HPLC analyses were carried out on a Hitachi L-2200R system, using an ATLANTIS dC18, 4.6 cm × 150 mm column . Ammonium formate and methanol  in DMEM at 37 °C under 5% CO2/95% air conditions until cell confluence. Cell viability was observed on Days 1, 2, 3, and 7 by MTT assays and evaluated using an ELISA reader. The number of cells was also calculated under an optical microscope .Cytotoxicity of fabricate nanofibers was assessed by MTT assay . Electrospun nanofibers were cut out with punch and placed onto 24-well culture plates. Human fibroblasts were seeded  and Ketamine-HCl . The operative area was cleaned and sterilized. All surgical procedures were carried out using a #15 blade after the local injection of 0.5 mL 2% Xylestesin-A containing epinephrine at a concentration of 1:100,000. A rectangular shape incision of 6 mm × 8 mm (width × length) and 1–1.5 mm in thickness was created on the palate of rabbits. After eliminating the graft, wet gauze was applied onto the donor site for 60 s with moderate finger pressure.The 12 rabbits were stochastically separated into two groups: In the control group, the excision wound was covered by a PCL stent without membrane and sutured with 6-0 polypropylene suture at the corners of the stent . In the In the test group, the tissue fluid at the palatal excision wound was extracted using #30 standardized sterile paper point  on Days 1, 2, 4, 7, 10 and 14. Sampling was performed in situ for 30 s during each procedure. Immediately after collection, the paper point was eluted with 0.1 mL phosphate buffer solution and then stored at −20 °C until it was analyzed. The HPLC analysis was employed to characterize the drug concentrations at each time period, using the same procedure as the in vitro study.The daily changes of body weight, and food/water intakes were monitored post-surgery for 14 days. In addition, the hemostasis analysis was completed following the work of Saroff et al. .On Days 1, 3, 7 and 14 after operation, photos of the wound for each rabbit were taken. To evaluate and compare the tissue responses at a microscopically level, standard biopsy was done at the end of Days 1, 3, 7 and 14. The specimens were harvested and fixed in 10% formalin and embedded in paraffin, cut into 4 μm frontal section. The epithelial and connective tissue characteristics were examined by employing the Hematoxylin and Eosin (H&E) staining. The interpretation was completed at ×10 and ×20 magnification by an independent examiner.The differences between groups were assessed using a least significance difference test, by employing SPSS software .−1 can be attributed to the N-H bonds of lidocaine and epinephrine, while the vibration at 3250 cm−1 of the O–H bond was improved with the presence of the drugs. In addition, the absorbance at the region of 3200 cm−1 could be resulted from the benzene of lidocaine [idocaine  and epinidocaine . The resMeasured water contact angles of pure PLGA, and 6:1, 3:1 and 2:1 PLGA:drug ratio loaded nanofibrous membranes were 127.4°, 118.5°, 87.6° and 54.6°, respectively. The addition of lidocaine and epinephrine increased the hydrophilicity of the PLGA nanofibers. Furthermore, the water contact angle (hydrophilicity) increased with the content of drugs in electrospun nanofibers.The tensile test results demonstrated that the maximum strength (elongation at break) of the pure PLGA, and 6:1, 3:1 and 2:1 PLGA:drug ratio loaded nanofibers were 4.53 MPa (229.7%), 2.85 MPa (51.2%), 1.87 MPa (37.0%) and 1.40 MPa (26.1%), respectively. Obviously, the mechanical strengths decreased with the content of incorporated drugs.Cytotoxicity experiments from MTT assays of the composite nanofibers were examined. The measured result in The hemostasis efficacy of the drugs loaded nanofibrous membranes was evaluated . For theThe preoperative body weights in the control group and the test group were comparable: 2.97 ± 0.43 kg in the control group A and 3.04 ± 0.68 kg in the test group B. No significant difference was found in the mean body weights of the rabbits between the control group and the test group by the end of the study.The food intake and water consumption were recorded. On Day 1, food intake by rabbits in the control group decreased significantly when compared to those in the test group. The amount of food intake increased gradually for all rabbits in both groups. On Day 14, rabbits of both groups regained their normal food intake (approximately 135 g). Throughout the study period, the rabbits in the test group exhibited greater food intake than did the rabbits in the control group. However, the difference did not reach significant level.On the other hand, all rabbits reduced their water consumption by 50% at one day post-operation, after which the water consumption increased gradually. On Day 14, all rabbits regained their normal amount of water consumption (approximately 1100 mL). Again, the rabbits in the test group (composite membrane group) showed greater amount of water consumption than did those in the control group, although the difference is not statistically significant.Postoperative pain and bleeding are the most common complications following soft tissue grafting procedures. Pain control is important during the early phase in soft tissue surgery. In the early operative period, free gingival graft accounts for the greater incidence of donor site pain. The pain results in stress and impairs the neutrophil function. Furthermore, the stress affects the immune system and down regulates IL-1 gene expression and impairs wound healing . After tThis study developed multi-layered nanofibrous lidocaine/epinephrine-eluting PLGA membranes that provide good conformity to underlying gingiva, reduce postoperative pain and hemorrhage, and protect the clot from the forces applied during chewing. The drug release kinetics for the biodegradable drug-eluting nanofibrous membranes comprise of two different phases: an initial burst and a degradation-dominated phase. During electrospinning, most drugs are encapsulated and distributed in the nanofibers. Nevertheless, some drug compounds may be located on the nanofiber surface, thus resulting in the initial burst. After that, the drug-elution behavior is mainly decided by polymer degradation. The drugs are released as the polymers degrade with time . The nanMalmquist et al.  studied Recent studies used platelet-rich fibrin (PRF) for promoting palatal donor wound healing. PRF is a fibrin matrix incorporating and releasing platelet cytokines, growth factors, and cells. The fibrin matrix is the natural guide of initial angiogenesis modulated by the binding to various growth factors. Fibrin plays an important role in modulating neutrophil activity and inducing epithelial cell migration to enhance the process of wound healing . KulkarnCollagen is a natural substrate of extracellular matrix. The collagen dressing has the ability to realize hemostasis, chemotactic to fibroblasts and platelets, and induce the proliferation and differentiation of mesenchymal cells. Shanmugam et al.  demonstrIt has been proposed that human skin fibroblasts exhibited higher growth on nanofibrous membranes possessing fiber diameters in the range of 350–1100 nm . The meaThis study developed multi-layered lidocaine- and epinephrine-eluting PLGA/collagen nanofibers and evaluated their efficacy for pain relief and hemostasis in palatal oral wounds of rabbits. All nanofibers exhibited good biocompatibility and eluted effective levels of lidocaine and epinephrine at the early stages of wound healing. Rabbits in the test group showed faster hemostasis as well as recovery of food and water intake postoperatively, compared with those in the control group. The experimental results demonstrate that the multi-layered biodegradable nanofibrous membranes offered adequate efficacy of hemostasis and sustainable pain relief for the initial healing of palatal oral wounds.


The ability to understand speech relies on accurate auditory processing of complex sounds. Individuals with Parkinson’s disease suffer from speech perception deficits, suggesting that dopamine is involved in the encoding of complex sounds. Recent studies have demonstrated that dopamine has heterogeneous effects on the responses of many neurons in the inferior colliculus (IC) of mice, although the strongest effect is to suppress neural activity. However, it was previously unknown which dopamine receptors are involved in modulating neuronal responses, and whether the observed preponderance of depressive effects reflects the endogenous dopamine system in the IC. In this study, we tested whether dopamine acts via D1- and/or D2-like receptors to alter responses of IC neurons in female and male mice. We also tested the effect of optogenetically induced dopamine release on auditory responses in the IC. We found that the effects of dopamine in the IC occur via D2-like receptors. In iontophoretic and freely behaving experiments, the single-unit and multi-unit effects of dopamine and a D2-like agonist were heterogeneous as both either increased or decreased responses of IC neurons to tones, while a D2-like antagonist had opposite effects. We also found that optogenetic activation of the endogenous dopamine system in the IC alters responses of auditory neurons. Similar to the effects of exogenous dopamine application, optogenetic induction of endogenous dopamine release heterogeneously altered auditory responses in the majority of cells in mice expressing channelrhodopsin-2 (ChR2). Understanding how dopamine modulates auditory processing will ultimately inform therapies targeting mechanisms underlying auditory-related communication disorders. Understanding the neural mechanisms of normal and disrupted sound processing is a fundamental goal of auditory research. In this study, we examine the neuronal effects and underlying cellular and synaptic actions of dopamine on auditory processing in the auditory midbrain of normal-hearing mice. Our results increase our mechanistic understanding of auditory processing of sounds and how this encoding may change with different contexts, physiologic states, and disorders such as Parkinson’s disease. Understanding the mechanisms underlying normal dopaminergic modulation will help guide novel diagnostic and therapeutic approaches for disorders associated with abnormal dopamine signaling. Additionally, these studies using mice with normal hearing will facilitate future use of genetically modified mouse strains to target specific communication and neurologic disorders.Communication through speech and language is a hallmark of human society and being unable to communicate can rapidly lead to social isolation and a high risk of depression . UnderstDopaminergic inputs and receptors reside in multiple auditory structures, including the inner ear, auditory brainstem, auditory thalamus, and auditory cortex . The priIn this study, we tested whether (1) the heterogeneous effects of exogenously applied dopamine are also seen following activation of the endogenous dopamine system in the IC of mice, and (2) whether dopamine acts via D1-like and/or D2-like receptors to alter auditory responses of IC neurons. We compared the effects of exogenous dopamine application via iontophoresis with the effects of optogenetically induced endogenous dopamine release on response properties of single IC neurons. We also analyzed the effects of dopamine receptor subtype agonists and antagonists on single-unit and multi-unit responses in restrained and freely behaving mice. We found that both optogenetic stimulation of the endogenous dopamine system and exogenous dopamine application produce heterogeneous effects. We also found that D2-like receptors mediate the effects of dopamine on responses of IC neurons, and that the dopamine system actively modulates auditory processing in the IC. While the function of normal dopamine signaling in the IC is not well understood, our results provide a crucial first step toward understanding how dopamine exerts its effects on auditory processing, which may guide development of therapies for disordered dopamine signaling underlying communication-related and auditory-related neurologic disorders.ad libitum access to food and water. We performed all animal care and use procedures in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the Institutional Animal Care and Use Committee of Washington State University, an AAALAC-accredited research institution. Beginning time of experiments in Zeitgeber time (ZT), where ZT0  =  lights on and ZT12  =  lights off, was approximately ZT17. Experimental sessions lasted no >5 h, and each mouse was used for one to four recording sessions. We monitored mice frequently, and if signs of distress were observed, the experiments were terminated. All founder breeder mice were purchased from The Jackson Laboratory. For iontophoretic and freely behaving experiments, we used CBA/CaJ mice . For optogenetic experiments, we generated mice expressing the H134R variant of channelrhodopsin-2 (ChR2) selectively driven by the DA transporter (DAT) promoter of DA neurons by crossing B6.SJL-Slc6a3tm1.1(cre)Bkmn/J (DAT::Cre) heterozygous mice with B6;129S-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J (Ai32(ChR2-YFP)) homozygous mice to create DAT:ChR2 mice  and wild-type littermates  on a C57Bl6/J background  administered subcutaneously immediately before surgery and the opioid buprenorphine (0.1 mg/kg) administered subcutaneously 1 h before surgery. We anesthetized mice with inhaled isoflurane (5% for induction and 1.5–2% for maintenance) and placed them in a stereotaxic apparatus. We administered an analgesic injection  at the incision site, and attached a lightweight metal head post to the skull using dental cement . A sharphttps://github.com/portfors-lab/sparkle.We controlled stimulus generation and data acquisition via custom-written software (Sparkle) run on an IBM PC computer with a Windows 7 Professional operating system. The code/software described in the paper is freely available online at We presented tone bursts  output through a 16-bit digital-to-analog converter , sent through a power amplifier (Parasound HCA-1000A) to a programmable attenuator (Tucker Davis Technologies PA5), and routed to a free field speaker (LCY-K100). For iontophoretic and optogenetic experiments, we placed the speaker 10 cm from the ear contralateral to the IC under investigation. For freely behaving experiments, we mounted the speaker onto a plastic cage lined with sound attenuating foam. To confirm that stimuli did not contain artifacts, we calibrated the speaker output over a range of 5–110 kHz using a conditioning amplifier (Brüel & Kjær 2690) and calibrated microphone (Brüel & Kjær 4231) positioned at the location or height normally occupied by the mouse’s ear. Any stimulus distortion components were buried in the noise floor, at least 50 dB below the signal level, as determined by power spectral analysis of the microphone signals.We administered an intraperitoneal injection of acepromazine (2 mg/kg) to provide mild sedation, and then placed the mouse in a foam body mold and secured its head post to a stereotaxic device housed in a sound-attenuating chamber . We obtained extracellular recordings using glass pipettes  filled with 1 M NaCl recording solution. For the application of pharmacologic agents, we constructed “piggyback” electrodes  consistin = 6 neurons, and allowed neuronal responses to recover to baseline between each concentration. We observed dose-dependent effects on response rate both in the increase (n = 2) and decrease (n = 4) directions. To evaluate the magnitude of the effects of different dopamine concentrations independent of directional effect, we calculated absolute percentage change relative to baseline at each concentration divided by the maximum percentage change in each neuron. We observed robust effects of dopamine on response rate at 500 mM (p = 0.09a).To determine the optimal concentration of dopamine, we performed concentration curve experiments in which we constructed seven-barrel piggyback electrodes filled with five different concentrations of dopamine . One of the remaining barrels was filled with 1 M NaCl and used as a sum channel to balance the currents. We randomly applied current through each barrel to t 500 mM A. We didWe also used dopamine at this concentration (500 mM) as a reference point to evaluate the specificity of the dopamine receptor agonists and antagonists of interest. Since dopamine receptor agonists produce effects that are the same as or similar to dopamine, and since dopamine receptor antagonists oppose the effects of dopamine, alternative effects suggested that the pharmaceutical of interest produced non-specific effects. When this occurred we adjusted concentrations until such effects were no longer observed. Final concentrations used throughout the study were: dopamine (500 mM), SKF-38393 , quinpirole , SCH-23390 (5 mM), and eticlopride . For the remainder of iontophoretic experiments, we constructed three-barrel piggyback electrodes and filled two of the three barrels of with combinations of pharmacologic agents . One barrel always contained dopamine, while the other barrel contained either the D1 agonist SKF hydrobromide, the D2 agonist QP hydrochloride, the D1 antagonist SCH-23390 hydrochloride, or the D2 antagonist EP hydrochloride. The remaining barrel was filled with 1 M NaCl and used as a sum channel to balance the currents.To evoke the release of endogenous dopamine in the IC and record responses of single units before and after the dopamine release, we used either a custom-built optrode consisting of a microelectrode coupled to a glass fiber , or a glass fiber fed into the tip of a glass pipette  via the Optopatcher (A-M Systems). Other than different electrodes, and stimulating the release of dopamine via blue light instead of applying exogenous dopamine iontophoretically, the optogenetic experiments used the same calibrations, recording equipment, and recording protocols as in the iontophoretic experiments.2) for each neuron, and allowed neuronal responses to recover to baseline between each intensity. To verify that effects were dependent on not only light intensity but also opsin expression, we recorded single units in both DAT:ChR2 (n = 15) and wild-type littermate (n = 7) mice. Methods to compute effect magnitude as a function of different light intensities were the same as those in iontophoretic experiments, with the most robust effects occurring in DAT:ChR2 mice at 42 mW/mm2; no effects were observed in wild-type mice (p = 0.59b).To determine the optimal light stimulation parameters, we varied the pulse duration and wavelength of blue light (470 nm) through an optrode until we observed effects on response rate, with the most robust effects occurring with 75-ms pulses delivered at 6 Hz. We then performed intensity curve experiments in which we randomly stimulated at five different intensities of blue light , and collected sections serially in 0.1 M phosphate buffer. We rinsed sections three times for 5 min in 0.1 M PBS, incubated sections in 50% ethanol for 30 min to permeabilize cell membranes, and again rinsed sections three times for 5 min in 0.1 M PBS. We then incubated sections for 1 h in blocking buffer [3% normal donkey serum (Millipore) in PBS], and rinsed sections three times for 10 min in 0.1 M PBS. We then incubated sections for 2 h in a solution of rabbit anti-GFP  and 2% normal donkey serum in PBS. We mounted sections on Superfrost Plus microscope slides (Fisher Scientific), dehydrated and cleared sections, and coverslipped with DPX (Electron Microscopy Sciences). We observed labeling using a confocal microscope (Leica Microsystems TCS SP8).We briefly anesthetized the experimental mouse in the induction chamber (∼5 min) using isoflurane gas (5%) to allow for attachment of lightweight wires to the recording electrode  and ground pin implanted during surgical procedures. We then placed the mouse into a testing box (45 × 25 cm) lined with foam and fashioned with a flush-mount speaker, and placed the testing box inside a sound-attenuating booth . Recording and calibration equipment and parameters were the same as in iontophoretic and optogenetic experiments except that neuronal responses were acquired using a different preamplifier (Dagan Industries). The length and weight of the attached wires allowed the mouse to behave freely in the testing box. We monitored the behavior of the mouse with a computer camera (Logitech C615).Recording began 10 min after the mouse had recovered from anesthesia and acclimated to the testing box. After recording baseline responses to different sounds from populations of neurons, we removed mice from the testing box and briefly anesthetized them (∼5 min) using isoflurane gas (5%) to allow for pressure injection of pharmacological agents (1 μl administered over the course of ∼1.5 min). Since we verified via iontophoretic controls that observed effects were due to pharmacological manipulations and not due to non-specific effects of the drugs or drug delivery, we used the same concentrations of dopamine (500 mM), QP (10 mM), and EP (10 mM) in freely behaving experiments. We then returned mice to the testing box and allowed them to recover. We monitored multi-unit responses over the course of ∼1 h following drug application. To verify that effects were due to receptor manipulation and not damage from drug delivery, we recorded from mice over multiple sessions using pressure injection of dopamine, agonists/antagonists, or vehicle.To counterbalance between sexes, we non-randomly selected 38 female and 37 male mice for the study. We analyzed recorded waveforms and detected spiking events using a threshold feature that reliably separated single-unit or multi-unit activity with waveforms from the noise floor (Sparkle). We determined the characteristic frequency  and threshold  for each unit. We measured rate, probability , mean first spike latency, and jitter (defined as the SD of first spike latency) in response to CF tone bursts at 10 dB above MT before and after application of pharmacological agents or stimulation with blue light.U or the Wilcoxon matched-pairs signed rank test to assess statistical significance in all cases. Details for each statistical test are provided in p < 0.05, **p < 0.01, ***p < 0.001). For exemplar neurons and dose-response curves we computed within-subjects effects of dopamine manipulation, and values in the text represent the sample mean and SEM. For population analyses, we computed the fractional change caused by drug or light treatment in the response to sound. Values in the text represent the population mean and SD. Because we did not observe any sex differences during either iontophoretic or optogenetic experiments (Statistics were applied using either SSHF (Sparkle) or graphing software used to generate data plots . We performed the Shapiro–Wilk test to determine whether each data set fit a normal distribution, and evaluated whether all data sets in a group of analyses met this assumption. Because there were no instances in which data from all sets of analyses were normally distributed, for consistency we used either the Mann–Whitney eriments B,D, we pn = 118; 60 from females and 58 from males), blue light pulses , or dopamine plus selective D1 and D2 receptor agonists or antagonist . We targeted units where the tone-evoked response could be clearly distinguished from spontaneous activity, and observed spiking patterns resembling those previously reported in the central nucleus of the IC including onset, sustained, pauser, and chopper-like responses (We recorded tone-evoked activity from 245 single-units and three multi-unit sites (∼5–10 units at each site) to investigate how dopamine affects auditory response properties in the IC. Recordings were conducted before, during, and when possible, following the application of either dopamine (esponses .p < 0.05), whereas 30% showed an increase in firing rate . In the six neurons in which evoked firing changed by 10% or less, the changes were not significant . We observed CF representation similar to previous reports in the IC  of the 118 neurons that changed their response rate with the application of dopamine, we obtained measures of response probability, latency, and jitter. Iontophoretic application of dopamine had heterogeneous effects as some neurons showed a decrease in probability and increase in response latency (p = 4.66 × 10−10e), while neurons that increased in rate also increased in probability (p = 0.0078f). Additionally, neurons that displayed a decreased spiking rate during dopamine application (blue circles) showed a significant increase in first spike latency (p = 5.82 × 10−11 ×g) and jitter (p = 5.82 × 10−11h), while neurons exhibiting an increased effect of dopamine on spike rate (red circles) had decreased response latency (p = 2.44 × 10−4i) and jitter (p = 2.44 × 10−4j).In a subset  exhibited significant changes in evoked spike rate during blue light stimulation (p = 1.19 × 10−7°) and 31% increasing in response rate (p = 4.88 × 10−4p).Consistent with results from iontophoretic experiments, optogenetically induced release of endogenous dopamine had heterogeneous effects on spiking rate A,B. Impol bodies D. We obsl bodies E. In DATmulation F, with 6p = 9.54 × 10−7q), while neurons that increased firing (red circles) also increased in response probability (p = 0.0039r). Also similar to iontophoretically applied dopamine, endogenous dopamine inversely altered response rate relative to response latency (p = 1.19 × 10−7s) and jitter (p = 3.58 × 10−7t), while neurons exhibiting an increased effect of dopamine on spike rate had decreased response latency (p = 4.88 × 10−4u) and jitter (p = 4.88 × 10−4v). We found no significant effects of blue light stimulation in n = 30 neurons from wild-type littermate mice .In neurons where there was a significant effect of blue light on response rate to CF tones, we compared the effects of light stimulation on response probability, latency, and jitter. Consistent with results from exogenous application of dopamine, optogenetically induced dopamine release altered response probability in the same relative direction as response rate G. Neuron latency H and jit latency I. Neuronp = 6.10 × 10−5ee), and dopamine had effects in the same direction and to a similar degree (p = 6.10 × 10−5ff). The D1 agonist SKF had no effect on spiking rate (p = 0.18gg), but response rate was significantly altered by subsequent dopamine application (p = 0.0039hh). The heterogeneous effects of dopamine (p = 0.002ii) were reversed by the D2 antagonist EP, and this effect had a significant overshoot relative to baseline (p = 0.002jj). The effects of dopamine (p = 0.0078kk) were not affected by the D1 antagonist SCH (p = 0.11ll).To determine the type(s) of dopamine receptors involved in the observed effects of exogenous and endogenous dopamine, we applied dopamine and selective D2/D1 agonists or antagonists to 58 of the original 245 neurons. Because of the length of the experimental paradigm applying multiple drugs, we focused our data collection and analyses only on response rate. The D2 agonist QP produced heterogeneous effects on auditory spiking rate (p = 0.0039mm), and dopamine reversed these effects beyond baseline (p = 0.0039nn). Activity of endogenous dopamine might also result in maximum receptor occupancy of D1 receptors, which could account for the lack of effects given SKF application. However, SCH had no effect on response rate (p = 0.69oo), although spiking was altered by dopamine in these neurons (p = 0.016pp).The observed overshoot given EP application suggested that the endogenous dopamine system might be contributing to baseline measurements. Indeed, initial application of EP produced heterogeneous effects on rate (p < 1 × 10−15qq). Although the global effect was a decreased response to dopamine injection, application of vehicle in a subsequent recording session produced no significant effects over multiple time points .To confirm that results from iontophoretic and/or optogenetic experiments were not affected by restraint and/or sedative drugs, we injected dopamine into the IC of three freely behaving mice. Consistent with single-unit results from awake, yet sedated and restrained mice, dopamine altered multi-unit responses in the IC of normally behaving mice. The example in p < 1 × 10−15 for both dopaminett and QPuu). The D2 antagonist EP, however, had the opposite effect . Clearly, these results indicate that the low level of acepromazine used in restrained experiments had no major effect on D2 receptors in the IC.To confirm that results from experiments in single IC neurons reflect the activation/inhibition of D2-type dopamine receptors in the absence of acepromazine, a known allosteric inhibitor of D2 receptors , we compThe IC receives dopaminergic projections from the SPF , containPharmacologic and optogenetic manipulation of dopamine could increase or decrease sound-evoked firing rate, probability of firing, first spike latency, and first spike jitter of single neurons in the IC. Both endogenous and exogenous dopamine caused heterogeneous effects, where neuronal spiking rate and probability were relatively directionally similar and spiking latency and jitter were relatively opposite. Additionally, both types of dopamine manipulation revealed similar proportions of increased and decreased effects. Such similarities (or differences) were critical to establish, as iontophoretic application of pharmacological agents may lead to the spread of drugs to other neurons, potentially resulting in auditory and/or dopaminergic feedback loops . Our resi/o  cation current, which can increase or decrease the excitability of different cells, respectively. Additionally, D2 receptor activation can inhibit the calcium-activated potassium current (Potential explanations for heterogeneous effects of dopamine on IC neurons following activation of a single type of dopamine receptor include activation of D2-like receptors inducing a number of different intracellular cascades. For example, activation of the D2 receptor can inhibit  or stimu current  or can s current , which c current . Finally current , and als current . Thus, tOur data demonstrate a basal level of dopamine modulation occurring in IC neurons. In both restrained mice and freely behaving mice, we found that application of a D2-like antagonist following dopamine application resulted in an overshoot of spiking rate beyond that of baseline, and application of a D2-like antagonist produced heterogeneous effects that were opposite the effect of dopamine. These results are consistent with previous studies demonstrating that injection of a D2-like antagonist into the IC alters auditory-evoked potentials recorded in the IC . AdditioThese previous studies in conjunction with our results strongly suggest that basal activity of the endogenous dopamine system in the IC contributes to baseline measurements of IC neurons. However, given that optogenetic induction of endogenous dopamine release, as well as pharmacologic application of dopamine and/or D2-like agonists, can result in significant effects, it is evident that such basal dopamine activity does not produce maximal physiologic effects. Combined, these results indicate that the dopamine system can be further activated, and/or perhaps suppressed, likely given the activity of inputs to the SPF, the source of dopamine in the IC.While behavioral studies are required to understand the precise functional role of dopamine signaling in the IC, understanding how dopamine modulates auditory responses of IC neurons is likely to be an integral part of understanding and interpreting results from behavioral studies. The IC contains both tyrosine hydroxylase-positive terminals  originat


Klinefelter syndrome (KS) or 47, XXY is a chromosomal disorder in males. Persons with KS have an additional X chromosome creating karyotype 47, XXY and 46, XY/47, XXY mosaics. According to existing epidemiological studies KS is one of the most common genetic disorders, affecting ~1 in 500 men [see ]. WhilstLearning difficulties, low self-confidence and issues relating to social interaction are also reported in relation to those with KS –6. WhilsIn order to identify literature for this review we searched the following key databases: Academic Search Premier, CINAHL, MEDLINE, PsychINFO. The search terms “Klinefelter's syndrome+ PsychosocialMuch of the literature examined discusses the challenges of getting and managing a diagnosis for KS. Fewer than 10% of cases of KS are diagnosed before puberty , 25, witWhilst diagnosis can then be a relief for those with KS, literature relating to the experience of parents of boys with KS shows that diagnosis can be uncertain and complex which can be a source of frustration for parents , 30. EveQuality of life (QoL) outcomes are reported as being worse for men with KS than for the general population , 27, 28.Turriff et al.  found thPhysical health outcomes for those with KS can include lower physical activity levels and higher BMIs  as well There is seen to be widespread lack of knowledge about KS by HCPs , 14, 29,Knowledge amongst healthcare professionals around treatment options is also now seen to be outdated  and not This narrative review suggests that a lack of or late diagnosis remains a critical problem in relation to KS. Whilst prenatal screening techniques may improve future diagnosis , currentThe experience with healthcare for persons with KS is described as poor , 14, 29,Given the prevalence of KS within the population, greater research focus on the disorder in the future, particularly in relation to reproductive health and the psychosocial impact of KS, would have a significant impact for patients and their families. There are inevitably limitations to a short review of this nature, and not all papers which may be relevant to KS, particularly those which are more clinically focused [such as ] appeareEH led the analysis and drafting and all other authors equally contributed to the writing, editing, and refining of the work. All authors contributed to the preparation of 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.


Although endovascular therapy (EVT) is commonly used in treatment of peripheral artery disease (PAD), severely calcified lesions pose a challenge, in spite of the technical advancement. In this report, we discuss the case of a 74-year-old male with coronary artery disease and end-stage renal disease who presented at our institution with bilateral intermittent claudication. Angiography showed chronic total occlusion (CTO) of the right superficial femoral arteries (SFA). Because the bilateral external iliac arteries demonstrated moderate stenosis, we performed endovascular therapy on the right SFA-CTO using a contralateral approach. With the antegrade wire progressing into the subintimal space, direct distal-SFA puncture was performed and wire externalization was established. However, no devices  were able to pass the lesion in antegrade or retrograde manner, even though the child catheter support or needle cracking technique from outside/inside was applied. Therefore, we used a combination of an excimer laser and high-speed rotational atherectomy to overcome the severely calcified lesion. First, the excimer laser catheter (Turbo Elite 0.9 mm) ablated the entry to the CTO; however, it did not pass through completely. Thereafter, the thin microcatheter (Caravel) succeeded in crossing the CTO in an antegrade manner using the BAlloon Deployment using FORcible Manner (BADFORM) technique. After wire-exchange to the Rota-wire, rotational atherectomy (RotaLink Plus 1.5 mm) passed through the CTO. Subsequently, we could dilate the CTO lesion with a conventional balloon followed by bare metal stent deployment. The right ankle-brachial index of the patient improved from being unmeasurable to 0.79, and the intermittent claudication disappeared. This combination therapy, described as the “RASER” technique in coronary section, is accepted for reimbursement. However, these devices in EVT section are considered off-label use in Japan. Therefore, we have to refrain from frequent use of this strategy; however, this method provides an option for severely calcified lesions. Endovascular therapy (EVT) has developed over the past decade because of advancement in techniques and devices , 2. TherHere, we report the case in which combination therapy using ELA and RA successfully treated chronic total occlusion (CTO) of the superficial femoral artery (SFA) with severe calcification.A 74-year-old male with hypertension, dyslipidemia, diabetes mellitus, coronary artery disease, and end-stage renal disease was transferred to our institution due to the bilateral intermittent claudication 6 months before admission. He had been given 75 mg of clopidogrel and 200 mg of cilostazol per day. His right ankle-brachial index was unmeasurable, and echography was suggestive of right SFA-CTO. Although this lesion was classified as class D based on the Transatlantic Inter-Society Consensus Document (TASC) [Hence, we applied the ELA using the Turbo Elite 0.9 mm . This device also could not pass the lesion; however, it modified the CTO entry-morphology . After EThereafter, we dilated the entire SFA lesion with conventional balloons and simultaneously achieved hemostasis of the distal-SFA puncture point. A bare nitinol stent was deployed due to severe dissection of the partial SFA. The final angiography revealed acceptable result without delay in blood-flow or bleeding complications  and S4.Thereafter we treated the bilateral iliac arteries using the bare nitinol stents. The right ABI of the patient improved from being unmeasurable to 0.79, and his intermittent claudication disappeared. His ABI and symptom has remained satisfactory eight months after current EVT.The application of EVT is widespread with novel techniques and devices; however, severely calcified lesion is still a vulnerable point. To overcome this lesion, several atherectomy devices have been developed . Most ofThe application of ELA in EVT has been mainly limited to in-stent restenosis (ISR) lesions. The first prospective, multicenter study of 90 patients with ISR of nitinol stents in the femoropopliteal arteries treated by ELA and adjunctive percutaneous transluminal angioplasty (PTA) showed 97% procedural success, 7% residual stenosis, 2.2% 30-day major complication rate, and 64% 1-year freedom from target lesion revascularization . With reThe application of high-speed RA in EVT section has been limited to a few case reports. Fukuda et al. reported a case of calcified SFA lesion after femoropopliteal bypass grafting treated by RA . A guideOur case demonstrates a successful combination therapy of ELA and high-speed RA in severely calcified SFA-CTO. The modification of the CTO entry by ELA complemented the disadvantage of the need for the dedicated wire in RA and aided in crossing the microcatheter to the distal CTO. In fact, this combined strategy is known as the RASER  technique in coronary section , 16. AftThis is a treatment option for severely calcified lesions. To the best of our knowledge, the RASER technique in EVT section has not been reported in literature.Although the above technique is technically feasible and beneficial, it should be considered as a last-resort technique in case of complications, like uncontrollable bleeding or absolute contraindications of surgical revascularization, because both ELA and RA in EVT are considered off-label use in Japan.Combined therapy with ELA and high-speed RA in EVT section is effective in CTO of the SFA and could be a useful option in managing severely calcified lesions.