Aggressive intraoperative “sculpting” is employed both “in situ” at the donor site and during the flap transfer to simulate the normal facial contours and planes. Comparisons of the author’s approach are made to the whole spectrum of reconstructive modalities ranging from conventional grafting to expanded pre-fabricated flaps and even to CTA face transplants; advantages/disadvantages of each are discussed. The pre-patterned, sculpted microvascular (MV) free flap offers the benefit of a single-stage transfer of composite skin/soft tissue hiding the seams at the junction of facial planes. When harvested from distant donor sites, the donor deformities can easily be concealed. The MV free tissue transfer offers the substrate that can be sculpted into nuanced facial components as well as the “palette” upon which the face can be painted with creative camouflage makeup. The soft contour and texture of the autogenous patterned transfers translates into a “natural” facial appearance while preserving fluid motions of facial expression.The author reviews his pioneering work in aesthetic restoration of the severely disfigured burn face first introduced in 1995 and refined over the past two decades. The reader will be exposed to the step by step approach to achieving cosmetic enhancement and functional rehabilitation of advanced facial burns. The “keystone” of the autogenous reconstruction is the  The severely disfigured burned face offers a unique challenge to the reconstructive surgeon. The complexities of thick hypertrophic plaques of scar, ambiguities on facial planes, functional burn contractures, architectural distortions and aesthetic disfigurement compel excellence in planning and execution of the restorative process. Less extensive procedures such as Z-plasties, local flaps , distortion of facial planes, and restricted facial movement and expression. Extensive burns often leave little adjacent skin for local/region flap transpositions. Traditional skin grafting often yields a corrugated, “woody texture” to the grafted skin as well as a poor color match and indistinct facial planes. Movement is compromised given the face a “flat,” artificial look with limited animation.in adjacent areas  or at distant site . While I was lecturing at the 10th People’s Hospital in Shanghai in May 2014, Professor Yixin Zhang showed me elegant patient examples of expanded skin with sufficient surface area to cover the entire face. Many well-crafted papers, of course, have been written in the Asian and world literature describing “super-thin” flaps [pre-fabricated expanded flaps [pre-expansion at the donor site along with the requisite lengthy amount of time to achieve adequate surface area expansion for coverage. Patients, by nature, must be exceptionally compliant and willing to accept the presence of a “melon size” balloon often in conspicuous body areas, not to mention the risk of exposure, infection, extrusion of the long-term tissue expander. Moreover, extensive scarring at the donor sites in visible area of the upper chest, shoulder, or supraclavicular regions is often very conspicuous and deforming.In my recent travels to China, I was impressed by the use of “mega” expanded blocks of composite skin pre-fabricated n” flaps –9, perfoed flaps  in resurimmediacy of a free tissue transfer. In 1995, I described the application of large “pre-patterned” microvascular free flaps for resurfacing of large surface facial defects [look and feel of normal facial skin and provide the palette to sculpt the nuances of facial components and the substrate for camouflage makeup. Donor sites are routinely hidden in inconspicuous areas of the back, scalp, forehead, forearm, etc., that can be covered with hair styles or clothing.In my experience, most Western patients do not have the temperament for long-term placement of a “mega” tissue expander nor the fortitude to withstand the “embarrassment” of the visible expansion balloon; rather, when given the option, will often choose the “instant gratification” offered by the  defects . Flap de defects . Aggress defects . These sOne-stage transfersymmetry and balanceImmediate facial Release of contracturesSculpted facial planes and contoursSoft skin texture as a “palette” for corrective makeupHidden seamsDistant inconspicuous donor sitesNatural facial appearanceFluid facial expressionNormal appearance at conversation distanceRebuild deep facial architecture or deep facial foundation preferably with autogenous materials pre-patterned MV free tissue transfersSegmental replacement of “aesthetic” facial units with Aggressive sculpting in situ at donor site and during transfer at recipient site Fig. Fig. 1DesSeams hidden at junction of facial planesSecondary debulking, contouring of transferred units to achieve “natural” facial contourLaser resurfacing of scars at periphery of the flapApplication of camouflage makeup to “blend” flap into facial profileThe facial unit to be excised is marked at its peripheral margins with a felt tip pen. A template of the facial defect pattern is cut into a transparent film sheet (10/10 steridrape). The pattern is centered over the auscultated axial vessels or perforators at the donor site and positioned for optimal vascularity. Key landmarks are marked on the template to orient flap to the excised facial unit.scapula or DIEP flaps for the cheek, forehead, or hemiface; radial forearm or scapula for neck; temporoparietal for ear or scalp [axial forehead or radial forearm for nose. Supraclavicular perforator flaps or submental flaps can be used for smaller, more localized facial defects. Generally, the closer the donor flap is to the face, the closer the color match. However, after 1–2 years of flap positioning in the face, the flap often takes on the color hue of the face due to environmental exposure.Donor sites are chosen based on compatibility with the recipient facial unit—thickness, color match, hair density, texture, etc. My preferences are or scalp ; axial fThe pre-patterned flap is thinned in situ at the donor site (except directly overlying the vascular pedicle). If perforator flaps are used, flaps can be thinned to the subdermal plexus. The flap is raised from the periphery utilizing frequent Doppler auscultation to determine depth and position of donor vessels. Flap perfusion can be ascertained by observation of capillary bleeding at the cut edges, Doppler auscultation of the pedicle within the flap, and by SPY perfusion imaging if necessary. Division of vascular pedicle to the flap is not divided until flap circulation is assured and recipient artery and vein have been isolated.Only after the donor flap is raised and flap perfusion assured (see above paragraph) is the facial scar removed. The inferior third of the hypertrophic scar is elevated to allow dissection of the facial artery and vein. The pre-patterned flap is transposed to the recipient site and “tacked” at key sites to retain the orientation of the facial pattern. After the MV anastomoses have been successfully performed, the remainder of the facial scar is excised. This approach is failsafe and precludes premature creation of a huge facial defect before high probability of flap success is assured.The recipient vessels are exposed and the MV anastomoses carried out with minimal elevation of the keloid scar (see above paragraph). Only after patency of the vessels has been assured is the remaining keloid scar excised. If fascial suspension or architectural modifications are necessary, the slings, cartilage, or bone grafts are inset prior to flap closure. The edges of the flap are inset into the “pieces of the puzzle” and meticulously closed with deep and cutaneous stitches.In addition to the in situ fat removal, additional “sculpting” is carried out during final inset of the flap to simulate the replaced aesthetic unit. Donor sites are closed by direct advancement or temporary STSGs. Additional refinement of the restored facial unit at 4–6 months includes additional debulking, laser resurfacing of scars, scar revision, etc. After final facial contour is achieved, patients are taught application of camouflage makeup by a professional aesthetician.Donor sites are closed by direct advancement and/or temporary STSGs. Dimensions of the donor site defect can usually be diminished by 50–60 % by undermining and advancement of the adjacent tissue. Temporary split skin grafts complete the closure. Residual skin grafted sites may be selectively removed by insertion of skin expanders as secondary procedures, leaving only curvilinear scars at the donor site.A 12-year-old Irish boy suffered a near total facial burn as a toddler when his Halloween costume caught fire. Eleven prior surgeries for correction of lip and eyelid ectropion were marginally successful. On initial exam, dense keloid scars were present over the entire face, chin, jaw, and neck Fig. . Centralpre-patterned, sculpted MV scapular flaps to the RT and LT hemiface, respectively  is mitigated. Moreover, there are compelling financial reasons to consider multi-stage autogenous facial restoration over CTA face transplants. Preliminary data shows that while the initial operative costs incurred during facial transplants are comparable to conventional reconstructions of similar facial defects, post face transplantation costs are “significant and progressive and their final values indeterminable” [In recent years, composite tissue allotransplantation (CTA) face transplants have been popularized by Siemienow , Pribaz  defects . The oneminable” .sculpted and is the “keystone” of the restorative process. By replacing the aesthetic units of the facial “puzzle,” the composite flap completes the “palette” to shape the face and camouflage with creative makeup. In restoring the beauty and function of the disfigured face, the reconstructive and aesthetic disciplines are truly merged, yielding a truly natural facial appearance.The pre-patterned microvascular free flap is the “clay” from which the shape of the face is Written informed consents were obtained from the parents (or guardians) of the 2 patients for the publication of the case report, along with all corresponding figures. Copies of the consents are available for review by the editors of this journal.


The results indicated that, after exposure of oMWCNTs to normal mice, oMWCNTs could not induce the liver to produce metallothionein (MT). When exposing Cd-MT mouse to different doses of oMWCNTs oMWCNTs could cause Cd2+ release from the accumulated Cd-MT; subsequently, one part of the free Cd2+ was eliminated with blood circulation; the other part adsorbed by oMWCNTs would remain in the tissues together with oMWCNTs. The results of the activities changes of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), and blood urea nitrogen (BUN) in plasma showed that the hepatotoxicity of coexposure was lower than that of single exposure, and the hepatotoxicity and accumulation of oMWCNTs in livers depended strongly on the exposure dosage of oMWCNTs. The histology of liver and kidney tissue also confirmed the previous results. Therefore, the author inferred that MT could be connected with oMWCNTs to reduce their hepatotoxicity, but the detailed mechanism needs to be further studied.The effects of oxidized multiwalled carbon nanotubes (oMWCNTs) on the behavior and hepatotoxicity of Cd Cdl metals , includil metals . When Cdic doses –9. There2+ can be released from its bond with MT in some tissues such as liver, in which toxicity may appear again [ in vivo by using isotope tracer technique or fluorescence imaging and find that carbon nanotubes are mainly in the liver, spleen, and lung tissues with a long retention [2+ should be easy into the living things with nanomaterials in real environment, and that may aggravate toxicity of single carbon nanotubes or Cd2+; meanwhile nanomaterials may change the toxicity and metabolic behavior of heavy metal ions in vivo; thereby, it was important for human health to study the cotoxicity of carbon nanotubes and heavy metal ions in the organism.However, the physiological environment is a very complex system. Under certain conditions, Cdar again . As wellar again –14. Throetention –20; at tetention . Therefo2+ or oMWCNTs in livers or kidneys.In the present work, the effects of oxidation multiwalled carbon nanotubes (oMWCNTs) on the behavior and hepatotoxicity of cadmium (Cd) were investigated. Because the hormone levels  in blood would change when there was some damage in liver or kidney tissues –24, the μm in length, with a diameter of 10 to 30 nm. Purity was >96% (wt. %), containing <3% amorphous carbon and <0.2% ash. The as-grown MWCNTs (named as untreated MWCNTs) were added into the solution of 3 mol/L HNO3 to remove the metal hemispherical caps on the nanotubes  that was a byproduct of the synthetic procedure. The mixture of 3 g MWCNTs and 400-mL 3 mol/L HNO3 was ultrasonically stirred for 24 h. The suspension was filtrated and rinsed with deionized water until the pH of the suspension reached about 6 and then dried at 80°C. The treated MWCNTs (named oMWCNTs) were calcined at 450°C for 24 h to remove the amorphous carbon [3, alcohol, and chloroform (ultrapure grade), were purchased from Tianjin Fuyu Chemical Co., Ltd., China; TRIS (ultrapure grade), from Sigma; Sephadex G-75 medium, from Pharmacia 17-0050-02; AST/ALT/BUN/TB (ELISA), from Shanghai Hengdailao Trade Co., Ltd., China.Multiwalled carbon nanotubes (MWCNTs) were purchased from Shenzhen Nanotech Port Co. Ltd., Guangdong, China, and prepared using chemical vaporization deposition method. According to the product specification, as-received MWCNTs were determined with transmission electron microscopy (TEM) to be 1 to 2 s carbon . And the ad libitum. All animal protocols were in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC), and approved by Institutional Animal Care and Use Committees of Gansu Province Medical Animal Center and Lanzhou University Animal Committees Guideline. Mice were pretreated with CdCl2 (1 mg Cd/kg once daily subcutaneously for 6 days) to accumulate Cd-MT and those mice were referred to as Cd-MT accumulated mice.Kunming mice  initially weighing 15 to 18 g were provided by Laboratory Center for Medical Science, Lanzhou University, Gansu, China. All animals were housed in individual cages in a temperature (21°C to 22°C) and light (from 08:00 to 20:00 hours) controlled environment and were fed food and tap waterμg/mouse) to Cd-MT model (4 mice) and normal mice (4 mice), respectively. And the content changes curve of Cd-MT and Cd2+ in liver was measured, according to the following steps to extract Cd-MT [3CH2OH): Volume (CH3Cl) = 1 : 1.03 : 0.08), homogenate. After adding 20 mL of the mixed liquid into the liver homogenate and mixing them, the liver homogenate was centrifuged for 20 min at 4000 ×g in 4°C; the supernatant was heated at 80°C for 5 min, after cooling in cold water, and centrifuged for 20 min at 4000 ×g in 4°C; and the supernatant was diluted to 4 times volume with −20°C precooled ethanol, stayed overnight at −20°C, and then centrifuged for 30 min at 4000 ×g in 4°C; the precipitation was dissolved fully in 0.01 mol/L Tris-HCl buffer solution and then centrifuged for 20 min at 4000 ×g in 4°C. The supernatant was applied to a Sephadex G-75 column (2 cm × 80 cm) equilibrated with 0.01 mol/L Tris-HCl (pH8.2); the column was eluted with the same buffer solution, and 4 mL fractions were collected to measure the absorbance by Ultraviolet Absorption Spectrophotometry  and the Cd content of fractions was determined through Atomic Absorption Spectrophotometry .The livers were harvested at 4 h after intravenous exposure of oMWCNTs , liver, and kidney were harvested and the blood with anticoagulant was used to measure the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), and blood urea nitrogen (BUN) by ELISA testing. The liver, kidney, and blood were digested with the mixture of perchloric acid and 30% of hydrogen peroxide  [Three groups of Cd-MT model mice and normal mice (6 mice/group) were exposed to oMWCNTs about 100  volume) , and the t-test and a two-tailed analysis of variance (ANOVA). Differences were considered significant at P < 0.05.Data were analyzed for significance using Student'sFrom It was reported that the UV adsorption peak of Cd-MT was about 250 nm , the maxHowever, the maximum absorption in elution was 260 nm for normal and single exposure groups  and therP < 0.05), indicating that oMWCNTs could release Cd from Cd-MT in tissues; the Cd content of exposure group to 100 μg/mouse oMWCNTs was less than that of 500 and 750 μg/mouse for liver tissue, but higher than that for kidney and blood, which showed that the low-dosage of oMWCNTs had a stronger influence on Cd-MT in vivo. Because free Cd was absorbed onto oMWCNTs and agglomerated in organism together with oMWCNTs, the influence of exposure group to 750 μg/mouse oMWCNTs was obviously higher than that to 500 μg/mouse.The results of μg/mouse oMWCNTs, the ALT content showed no difference with the Cd-MT model group. Compared to the single oMWCNTs exposure group, the ALT content of the coexposure group was significantly lower  in presence of oMWCNTs in mice. It was proposed that the maximum absorption peak appeared around 250 nm for Cd-MT [2+ induced successfully Cd-MT synthesis in liver tissue in mice  exhibiting different structures and functions independently, and Cd preferred to bind with α domains compared to β domains. In addition, the absorption peak for β domains at 254 nm was less significant than that for α domains [The present work studied preliminarily the behavior and hepatotoxicity of heavy metal ions  exposure groups.It was reported that oMWCNTs would be retained for long time in liver, lung, spleen, and other tissues after entering into body and damaged the tissues through inducing inflammation, granuloma, cell apoptosis and DNA damage, and so on , 20. Cd2 Figures  and 6, b Figures  and 8. S reduced . Therefo2+ and then they were exposed to different dosages of oMWCNTs. The results showed that oMWCNTs could cause Cd2+ releasing from the accumulated Cd-MT. After the oMWCNTs and Cd2+ were injected together or respectively, the content changes of ALT, AST, TB, and BUN in plasma and the histology of liver and kidney both indicated that the hepatotoxicity of coexposure was lower than that of single exposure. It can be deduced that MT could be connected with oMWCNTs to reduce their hepatotoxicity, but the detailed mechanisms needed further study.The model mice with Cd-MT were made via injection with Cd+2 release from the accumulated Cd-MT; in addition, after the oMWCNTs and Cd injected together or respectively, the content changes of hormone in plasma and liver tissues indicated that the hepatotoxicity of co-exposure was lower than that of single exposure. The detailed flow chart was provided in Supplementary Material.In sum, oMWCNTs could cause Cd


NSE and S100B belong among the so-called structural proteins of the central nervous system (CNS). Lately, this group of structural proteins has been profusely used as specific biomarkers of CNS tissue damage. So far, the majority of the research papers have focused predominantly on the concentrations of these proteins in blood in relation to CNS damage of various origins. Considering the close anatomic and functional relationship between the brain or spinal cord and cerebrospinal fluid (CSF), in case of a CNS injury, a rapid and pronounced increase of the concentrations of structural proteins specifically in CSF takes place. This study inquires into the physiological concentrations of NSE and S100B proteins in CSF, carried out on a sufficiently large group of 601 patients. The detected values can be used for determination of a normal reference range in CSF in a clinical laboratory diagnostics. Approximately from the 80s, there has been a notable increase of interest in structural proteins of the central nervous system (CNS), including S100B and NSE as biomarkers of CNS tissue damage [The exact term is S100 calcium binding protein B or S100B. This protein is the first identified member of the S100 protein multigenic family and participates in an extracellular and intracellular regulation of a cellular calcium metabolism . ProteinS100B might have either a trophic or toxic effect depending on the local concentration. In a low nanomolar physiological concentration it seems to have a neurotrophic effect, it stimulates the growth of neurons, and it increases their survival during development and also during an injury –6. On thα-γ or γ-γ, which belongs to the group of hydrolytic enzymes. NSE is an isoenzyme of enolase (2-phospho-D-glycerate hydrolase), which catalyzes the transition of 2-phosphoglycerate into phosphoenolpyruvate [It is a dimer formed in neurons with subordinate units pyruvate . It is ppyruvate , 14.In case of a CNS injury, accompanied by a nervous tissue and cellular damage, these structural proteins are released from cells, and their concentrations increase extracellularly—including CSF and blood. In these consequences these proteins could be called “biomarkers of brain damage” , 15–18.Considerable number of research papers, engaged in monitoring the concentrations of NSE and S100B protein after a traumatic brain injury (TBI), was published , 19–23.The concrete value and dynamics of the concentrations of S100B protein and NSE also play an important role in the prediction of the outcome of patients who had brain ischemia , 23–28 oProteins S100B and NSE are also produced by some tumorous cells of neuroectodermal origin , 37.Furthermore, structural proteins of CNS, including NSE and S100B protein, were researched in relation to prion diseases of CNS .Major part of the research studies focuses on monitoring the concentrations of these biomarkers in blood. Sporadic studies that address the determination of their physiological concentrations in CSF are carried out on relatively small groups of patients .Considering not only the close anatomic relationship between the brain or spinal cord and CSF, but also the small volume of a CSF reservoir, when CNS tissue is damaged, a rapid and pronounced increase of the concentrations of structural proteins specifically in CSF takes place.In our opinion, a substantial study of the physiological concentrations of NSE and S100B protein in CSF, carried out on a sufficiently large group of patients, is missing.In the Laboratory for CSF and Neuroimmunology, Prague, there are approximately 5000 CSF samples analyzed per year. Biochemical analysis , CSF cytological analysis , immunological analysis , microbiological CSF analysis , and determination of structural proteins of CNS, namely, S100B, NSE are performed in this laboratory workplace.We were able to compile a large enough investigated group of approximately 600 patients, on which we determined sufficiently reliable range of normal values of NSE and S100B in CSF.From the group of altogether 28.394 patients whose CSF was obtained and analyzed for diagnostic purposes within the years 2008–2014 , a file of 601 patients was selected. There were no pathological findings in these patients as well as no clinical or CT/MRI signs of CNS tissue damage. Biochemical, cytological, and immunological values in CSF were normal. μL.Total cell count in CSF <= 4/ Normal cytological finding: 60–80% of lymphocytes, no plasma cells, monocytes without signs of activation, no phagocytosis, no granulocytes, no atypical or tumorous cells. Total CSF protein <= 0.4 g/L. Lactate in CSF <= 2 mmol/L. Glucose in CSF >= 2.2 mmol/L AND <= 4.2 mmol/L. Coefficient of energy balance (CEB) in normal range (>27) , 41. Negative finding of oligoclonal bands (OCB) in CSF on isoelectric focusing (IEF) of IgG . IL 6 in CSF < 18 pg/mL.Patients signed an informed consent with a further scientific use of their CSF samples.For determination of the concentrations of S100B and NSE in CSF, the sensitive electrochemiluminescence immunoassay  on Elecsys 2010 analyzer was used.The method is based on the use of a ruthenium-complex and tripropylamine (TPA). The chemiluminescence reaction for the detection of the reaction complex is initiated by applying a voltage to the sample solution resulting in a precisely controlled reaction.The standard kits for in vitro diagnostics, NSE catalogue number: 12133113 and S100B catalogue number: 03175243 by Roche Diagnostics GmbH, Sandhofer Strasse 116, D-68305 Mannheim, were used .Interlaboratory comparisons of measurements of the concentrations of S100B and NSE were done in the frame of EQA  organized by SEKK, Pardubice, Czech Republic.The investigated group was divided into two groups of men and women to determine sex dependency and then into two age groups (20–59 and over 60 years) to establish age consequences.The obtained data were statistically analyzed and scatter diagrams were created using program MS Excel. We estimated the reference limit as the 2.5th and the 97.5th percentiles for sex and age dependency.See In the investigated group it was possible to observe similar tendencies in relation to age, as it had already been observed in previously published studies , 43, 44,Explanation of different allocation of both proteins, based on sex, could be most likely found in the dissimilar physiological tasks of each structural protein, eventually in their different distribution within the frame of the CNS—the source of S100B protein is astroglial cells above all, whereas NSE comes from the cellular body of neurons for the most part.NSE and S100B are considered as quite established and specific markers of the CNS tissue damage. So far, clinical-laboratory interpretations mostly relied on studies based on monitoring of these structural proteins in blood.Nonetheless, besides taking into account the close anatomic relationship between the brain or spinal cord and CSF, and also the small volume of a physiological CSF reservoir, in case of a CNS injury, the most sensitive and specific compartment accessible for a routine laboratory analysis is CSF.With regard to the intercompartmental dynamics of a protein transportation from CNS to CSF , after aThis study evaluated the physiological concentrations of NSE and S100B protein in CSF, carried out on a sufficiently large group of 601 patients. Correlations of the concentrations of S100B and NSE depending upon age and sex were established as well.The values detected in this study can be used for stating the normal reference range in CSF in a clinical laboratory diagnostics.Reliable reference ranges of S100B and NSE are necessary for a clinical interpretation of laboratory findings in CSF in patients with suspected damage of CNS tissue. Preference of determination of concentrations of these proteins in CSF, to their determination in blood, is meaningful especially in a more discrete impairment of CNS . Into a large group of diseases, with a possible tissue damage of CNS, where CSF diagnostics is preferred, belong neuroinflammations, both neuroinfections and autoimmune processes .Laboratory analysis used in this study is based on an established and widely accessible ECLIA methodology used in a routine laboratory practice. Presently, there are lots of regular external quality control assessments (EQA) for S100B and NSE available, so that interlaboratory comparability of results is guaranteed. Introduction of these new parameters into the CSF diagnostics brings, in the authors' opinion, a new and potent diagnostic tool for a detection of a CNS tissue damage, which is both independent of and complementary to neuroimaging methods like CT or MRI.The precise determination of the presence and degree of a structural damage of CNS has a clinical significance for both prognosis of a patient's outcome and a choice of therapeutic approaches.


Granulocyte-colony-stimulating-factor (G-CSF) induces mobilization of progenitor cells but may also exert pro-inflammatory and pro-thrombotic effects. Treatment with recombinant G-CSF after acute myocardial infarction is currently under examination and has been associated with in-stent restenosis. However, it is not known whether plasma levels of endogenous G-CSF are also associated with an increased cardiovascular risk. Therefore we included 280 patients with angiographically proven stable coronary artery disease. G-CSF was measured by specific ELISA and patients were followed for a median of 30 months for the occurrence of major adverse cardiovascular events . Those with cardiac events during follow-up showed significant higher G-CSF levels  at baseline. Patients with G-CSF plasma levels above the median had a 2-fold increased risk for MACE (p<0.05). This was independent from established cardiovascular risk factors. In addition, G-CSF above the median was a predictor of clinical in-stent restenosis after implantation of bare-metal stents  but not of drug-eluting stents . This data suggests that endogenous plasma levels of G-CSF predict cardiovascular events independently from established cardiac risk factors and are associated with increased in-stent restenosis rates after implantation of bare metal stents. Granulocyte-colony-stimulating-factor (G-CSF) is produced by monocytes, macrophages, fibroblasts, endothelial cells and bone marrow stromal cells, respectively. It induces mobilization of progenitor cells and acts as proliferative agent for stem cells. On the other side G-CSF has well known pro-inflammatory properties, which might be explained due to its ability in controlling neutrophil numbers as well as their activity during inflammation. As a cliTreatment with recombinant G-CSF has been under examination after myocardial infarction and a large number of clinical trials have tested its effects on myocardial infarct size. It is suHowever, despite various studies using recombinant G-CSF, it is not known whether plasma levels of circulating endogenous G-CSF are associated with an increased cardiovascular (CV) risk. Therefore, it was our aim to evaluate whether endogenous G-CSF plasma levels are associated with CV events and the occurrence of in-stent restenosis in patients with stable CAD.We included consecutive 280 patients with angiographically proven stable CAD undergoing elective stent implantation for stable angina pectoris. Patients with unstable angina or myocardial infarction within the previous six months were not included. PCI and stent implantation (PCIs) was performed according to standard techniques and stent type was left to the decision of the experienced interventionalist. Study subjects received a loading dose of 600 mg clopidogrel pre-PCIs. Intravenous unfractionated heparin was administered as to standard practice. After the procedure, patients were maintained on aspirin 100 mg indefinitely, and clopidogrel 75 mg according to the guidelines of the European Society of Cardiology (ESC) (ref). Other medications such as beta-blockers and angiotensin-converting-enzyme inhibitors were given as appropriate. Statin therapy was routinely administered to all patients. The study was approved by the institutional ethics committee for human subjects (EK 10-046-VK_NZ).Blood from the cubital vein was taken under fasting conditions immediately before PCIs into serum seperator tubes. Samples were allowed to clot for 30 minutes (min), were then centrifugated at 1500g for 15min and stored at −80°C until use. G-CSF was measured by specific ELISA , according to the manufacturer’s instructions. Laboratory determinations were performed by investigators that were blinded to clinical characteristics and patients' outcome.Patients were followed for a median of 30 (20–38) months for the occurrence of a combined clinical endpoint  of 0.05. ContinuoBaseline characteristics of study population are given in Patients with CV events during follow-up showed significantly higher endogenous G-CSF levels before PCIs . Median G-CSF levels for patients with or without MACE during follow up are shown in In addition, G-CSF plasma levels above the median were associated with a higher rate of in-stent restenosis in patients with bare-metal stents  but not in patients with drug-eluting stents .We were able to show for the first time that pre-existing plasma levels of G-CSF predict MACE in patients with stable CAD, independently from established CV risk factors. Furthermore, endogenous G-CSF levels predict clinical in-stent restenosis after implantation of BMS, but not after implantation of DES.vasa vasorum neovascularization.[While other colony-stimulating factors like M-CSF and GM-CSF have been extensively studied in animal and clinical studies of atherosclerotic diseases, G-CSF has received hardly any attention and results are conflicting. Additionrization. In additrization. In the lrization. Recentlyrization. In additrization. In our sAnimal studies have shown that G-CSF might prevent restenosis by acceleration of re-endothelialization and a decrease of neo-intima formation after vascular injury ,14; HoweSome limitations of the present study have to be acknowledged. First, our study is of observational nature. Accordingly, our results may be explained by unmeasured confounding factors. Therefore, we tried to control for baseline imbalances by multivariate modeling. However, the possibility of residual or undetected confounding is small but cannot be ruled out completely. Although inflammation is a well-known contributor to atherosclerosis and G-CSF is known to increase numbers of neutrophils, differential blood count was not available in our patients, therefore it is not possible to analyse whether the observed effects could have been mediated through circulating neutrophils. On the other hand this investigation represents high-quality as patients were included consecutively and loss-to-follow-up was less than 1%.In conclusion, endogenous plasma levels of G-CSF predict CV events independently from established CV risk factors and are associated with in-stent restenosis in patients who underwent BMS implantation.


The study of hand and finger movement is an important topic with applications in prosthetics, rehabilitation, and ergonomics. Surface electromyography (sEMG) is the gold standard for the analysis of muscle activation. Previous studies investigated the optimal electrode number and positioning on the forearm to obtain information representative of muscle activation and robust to movements. However, the sEMG spatial distribution on the forearm during hand and finger movements and its changes due to different hand positions has never been quantified. The aim of this work is to quantify 1) the spatial localization of surface EMG activity of distinct forearm muscles during dynamic free movements of wrist and single fingers and 2) the effect of hand position on sEMG activity distribution. The subjects performed cyclic dynamic tasks involving the wrist and the fingers. The wrist tasks and the hand opening/closing task were performed with the hand in prone and neutral positions. A sensorized glove was used for kinematics recording. sEMG signals were acquired from the forearm muscles using a grid of 112 electrodes integrated into a stretchable textile sleeve. The areas of sEMG activity have been identified by a segmentation technique after a data dimensionality reduction step based on Non Negative Matrix Factorization applied to the EMG envelopes. The results show that 1) it is possible to identify distinct areas of sEMG activity on the forearm for different fingers; 2) hand position influences sEMG activity level and spatial distribution. This work gives new quantitative information about sEMG activity distribution on the forearm in healthy subjects and provides a basis for future works on the identification of optimal electrode configuration for sEMG based control of prostheses, exoskeletons, or orthoses. An example of use of this information for the optimization of the detection system for the estimation of joint kinematics from sEMG is reported. The human hand shows highly complex motor skills, which are essential for many daily activities. The study of hand and finger movement is an important topic with potential applications in prosthetics, rehabilitation, and ergonomics It has been proven that the classification accuracy of movements based on sEMG improves by increasing the number of electrodes, up to a limit beyond which a plateau is reached The aim of this work was to quantify, by means of a sEMG multi-channel detection system, 1) whether it is possible to spatially localize the sEMG activity related to the activation of distinct forearm muscles during dynamic free movements of the wrist and single fingers, and 2) the effect of hand position  on the sEMG activity in terms of changes in amplitude and spatial distribution. The study provides quantitative knowledge about sEMG activity distribution on the forearm as a basis for future works on the identification of optimal electrode number and positioning for sEMG based control of prostheses, exoskeletons, or orthoses. Preliminary tests about the use of the obtained knowledge in the optimization of the detection system for the estimation of joint kinematics from sEMG are shown.C. Hand kinematics recordings).Eight male healthy subjects, aged between 25 and 40 years, participated in the study. All subjects provided written informed consent and the protocol was within a program of research approved by the local Regional Ethics Committee . The subjects were seated in a height adjustable chair in a comfortable position with the shoulder abducted and flexed ∼30°, the elbow flexed ∼120° and with the elbow and the forearm resting on a horizontal surface. The subjects were instructed to perform cyclic dynamic single DOF tasks involving the wrist and the fingers . The wrisEMG signals have been acquired from the left forearm muscles using a wearable detection system consisting of a grid of 112 silver circular electrodes : 15 mm LISiN unpublished internal report) integrated into a stretchable textile sleeve  with the 14 columns of electrodes placed around the forearm circumference . Before The sensorized hand glove HumanGlove  was used to record the kinematics of the hand and of the fingers. HumanGlove is a wireless sensing glove designed to measure the hand’s posture using twenty-two Hall Effect sensors. A glove calibration procedure was performed for each subject using the Graphical Virtual Hand (GVH) calibration software provided by Humanware. Kinematic data acquisition has been performed through the GVH software  storing the angle values for each joint together with the sampling time. The joint angle signals from index finger MCP and wrist flexion/extension were acquired also through two auxiliary inputs of the EMG-USB system in order to perform the off-line synchronization of sEMG and kinematics signals.The sEMG and kinematics signals were processed offline with MATLAB R2010b .Kinematic signals were re-sampled to 2048 Hz and synchronized with sEMG through the common synchronization signals. For each task, the angular signal detected from the joint involved in the movement was used to identify the movement cycles. A movement cycle was defined between the beginnings of two subsequent flexion/adduction dynamic phases . The begth order Butterworth non-causal filter (20–450 Hz) and power line interference (50 Hz and its four higher harmonics) was removed using spectral interpolation th order Butterworth filter with 1 Hz cutoff frequency.The monopolar sEMG signals were digitally filtered with a 4The envelopes of sEMG epochs corresponding to each movement cycle were extracted and time-normalized in order to obtain 500 data points for each cycle. The repeatability of the sEMG envelopes among the movement cycles was evaluated using the Coefficient of Multiple Correlation (CMC) for all electrodes and all tasks. The mean sEMG envelope was calculated for each subject, task, and channel by averaging the sEMG envelopes on all cycles.m channels, n samples), m, that represent invariant patterns of activity across the channels), ij, specifies the contributions of each component j to the measured activation pattern iM), M and WC.a) Identification of sEMG clusters. Non Negative Matrix Factorization (NNMF) has been used as a dimensionality reduction pre-processing step for clustering. NNMF decomposes a non-negative matrix In the present study, the NNMF basis and the associated coefficients were extracted, for each subject, by applying the NNMF algorithm to the concatenation of the sEMG mean envelopes of all channels and tasks. To avoid local minima, the NNMF algorithm was repeated 100 times for each subject. The result showing the lowest squared error between original and reconstructed sEMG envelopes was kept.N of modules needed for the approximation of the sEMG envelope was assessed by repeating, for all subjects, the NNMF analysis with the number of modules N varying between 1 and 10. The quality of envelope reconstruction with N modules was quantified by means of the variation accounted for (VAF) defined as the variation that can be explained by the model: VAF = 1–SSE/SST, where SSE (sum of squared errors) is the unexplained variation and SST  is the pooled variation of the data The number C maps of coefficients to identify the electrode clusters where each module was mostly represented. The algorithm consists in two steps: the first step identifies the areas with different amplitude by applying watershed segmentation to the equalized amplitude C map. In the second step, the electrodes whose coefficients are higher than a threshold  are considered as belonging to a cluster of activity.The segmentation algorithm proposed by Vieira et al elx is the electrode position in ulnar–radial direction  Comparison of sEMG activity areas for different movements and conditions. In order to study the effect of different hand positions  on the sEMG activity distribution the centre of gravity (COG) of electrode clusters was estimated. The COG’s coordinates for each electrode cluster were defined as:The degree of overlapping of the sEMG areas identified for tasks involving different fingers or joints was calculated as the intersection of the clusters (that is the number of electrodes common to each pair of clusters). This number was normalized to 1) the number of channels in the smallest of the two clusters to obtain the degree of inclusion of the smallest area into the biggest one and 2) the number of channels in the largest of the two clusters to obtain a measure of the area not common to both clusters.In order to test the importance of identifying specific areas of sEMG activity during hand and finger movements, preliminary tests were performed comparing the performance in the reconstruction of joint angles from sEMG of two sets of electrodes: a) one ring of 14 electrodes placed around the forearm 2 cm from the elbow crease and b) a set of electrodes whose position has been selected corresponding to the barycenter of the identified areas of activity.2r.In order to reconstruct the joint angles from monopolar sEMG envelopes, a set of feed-forward multilayer perceptrons (MLPs), one for each joint, were used 2r).The Wilcoxon test was used to study the effect of the hand position on the COG coordinates with statistics significance level set to 0.05. Analysis of variance (ANOVA) followed by the post-hoc Student Newman-Kelus test was applied to investigate the effect of channel selection on the joint angle reconstruction quality  with broken connection. On an average, 2–3 additional electrodes per subject were discarded because of bad electrode-skin contact.Kinematic signals showed a good repeatability among movement cycles for all subjects and all movements. For three subjects a percentage of cycles reported in the following (median (IQR 25%–75%)) was excluded from the analysis: subject AlFa (2.5% (0.0–33.3)), subject PaMa (8.6% (0–20%)), and subject RoSt: (5.5% (0–30%)).N modules is essentially preserved in the set with N+1 modules. N and N+1 modules. Motor module 1 is primarily active during the second half of the movement cycle; it mainly represents extensors activity during extension/flexion tasks and abductor activity during abduction/adduction tasks. Motor module 2 is active during the first half of the movement cycle; it mainly represents the extensor activity during extension/flexion tasks and adductor activity during abduction/adduction tasks. The dimensionality analysis determined that three motor modules are sufficient to reconstruct the sEMG envelopes with VAF>0.9. From On the other hand, 2ringr: (69±12%); 2COGr: (80±6%); p<0.01) and the best reconstruction was obtained with the electrode set positioned in correspondence of the barycenters of the identified sEMG activity areas.Statistical analysis was performed on the quality of reconstruction of joint angles from sEMG detected using two different sets of electrodes: a) one ring of 14 electrodes equally spaced and positioned 2 cm from the elbow crease and b) one set of electrodes whose position has been optimally selected in correspondence of the barycenter of the identified areas of sEMG for each finger. Results showed the quality of joint angle reconstruction depends on the electrode set  whether it is possible to spatially identify distinct areas of sEMG activity in the forearm during dynamic free movements of the wrist and single fingers, 2) the effect of the hand position on the sEMG activity. The results show that specific distinguishable areas of activity can be identified during wrist and single finger movements suggesting the use of targeted positioning of electrodes could improve the performances of sEMG-based movement classification systems. Considering the main application could be in prosthetics, these results must be validated in amputees.Most studies on hand prosthesis myoelectric control make use of eight or less pairs of standard electrodes In literature, some studies focused on the effect of the number of electrodes on the movement classification accuracy for prosthesis control and on the identification of the minimum number of sEMG electrodes that allow maintaining good classification performances. The general approach is based on the evaluation of the performances of a movement classifier changing the number and set of electrodes in an iterative way. Liu and Zhou In this study, a different approach has been adopted; the NNMF technique has been applied to sEMG signals recorded using a multi-channel detection system to identify the clusters of sEMG activity during different wrist and finger movements. Multi-channel high density sEMG provides detailed topographical maps of sEMG activity and allows to verify if distinguishable sEMG spatio-temporal patterns are produced during different tasks and to identify the electrode positions detecting the higher sEMG activity. The use of NNMF for linear factorization of forearm sEMG signals even without targeting specific muscles for electrode positioning has been shown to be appropriate in a recent work N modules are essentially preserved in the set with N+1 modules. This result shows that the choice of the number of modules is not critical and the analysis performed with a particular number of modules will be consistent with the analysis based on larger sets.The dimensionality analysis on NNMF results showed that three modules account for 90% of VAF and the modules identified in the set with The first two modules extracted with NNMF show a high level of similarity among all subjects . Module The hand shows substantial mechanical coupling across adjacent fingers Some works in literature indicate that sEMG cannot be used to distinguish single finger muscle activity because of high levels of crosstalk The results of this work about single finger sEMG activity, showed that the use of multi-channel detection systems allows to identify distinct areas of sEMG activity in the forearm for different fingers, in accordance with recent findings The results of the comparison of sEMG areas detected during movement involving PIP and MCP joints show a high superposition of the activity areas and a distance between the barycenters of the activity areas lower than one IED. This is in accordance with Kamper et al. In most studies for sEMG-based multifunctional prosthesis control, the tests are performed with the subject in a reference position and the training and testing sEMG signals are recorded in a constant position of the arm. However, in daily living activities the user’s arm position changes and different forearm muscle activities could be observed during the same movement. The changes in sEMG activity distribution can be due to two main factors: 1) the electrode displacement due to skin and muscle shift and 2) the different effect of gravity on body segments.The works published in literature evaluate the effect of different arm positions on the classification accuracy or simulate the shift of the electrode. Fougler et al. In this work the use of multi-channel sEMG allowed to investigate the changes in sEMG activity distribution during the same movement when hand position changes . The results showed a shift of the COG of the sEMG activity areas of approximately 1 IED for all considered wrist movements. Moreover, we investigated if sEMG activity level was influenced by the hand position. The ratio between the NNMF weights associated with each module for all tasks in prone versus neutral position highlight the influence of the hand position on the muscle activation. For flexion/extension and adduction/abduction movements the ratios reflect the influence of gravity that facilitates flexion and opposes to extension in prone position and facilitates abduction and opposes adduction in neutral position. These results must be carefully considered in the development of sEMG-based control for orthoses or prostheses. Forearm pronation-supination is, in fact, one of the most common movements carried out in conjunction with grasps and hand movements.The main limitation of the present study, considering one of the primary applications could be the control of prosthesis, is the tested subjects are all healthy subjects while amputees may have significantly different muscle maps than able-bodied individuals. However, this work serves as a methodological foundation for future work. The feasibility and real advantages of the targeted selection of electrode positioning with respect to an electrode ring shown in this work must be verified in amputees. Moreover, this work could contribute in the development of orthoses and new human-computer interfaces.In this work the sEMG signals were recorded in monopolar modality and are probably e affected by crosstalk. However, due to the close interlacing of the narrow extensor muscles Only a male population has been studied because of the availability of only one size (medium) of the prototype of sleeve for sEMG detection. However, the uniform distribution of the electrodes around the forearm circumference, allows sampling the sEMG activity in the same relative positions regardless of the forearm circumference. For this reason the obtained results are expected to be generalizable to a population with smaller or bigger forearm.The textile sleeve used in this work provided a fast setup and a reliable detection of multi-channel sEMG signals from forearm muscles during dynamic tasks. The effect of hand position on the distribution and on the level of sEMG activity during wrist and hand movements has been quantified. The results give new information, not available in literature, that are independent from the characteristics and robustness of a specific classification system and provides a basis for a future work on the identification of optimal electrode number and positioning for sEMG based prosthesis control.


Although it is known that chemical mixture can evidently change the band gaps of alloyed Mo1−xWxS2 crystals, the successful growth of Mo1−xWxS2 monolayers with tunable Mo/W ratios has not been realized by conventional chemical vapor deposition. Herein, we developed a low-pressure chemical vapor deposition (LP-CVD) method to grow monolayer Mo1−xWxS2 (x = 0–1) 2D crystals with a wide range of Mo/W ratios. Raman spectroscopy and high-resolution transmission electron microscopy demonstrate the homogeneous mixture of Mo and W in the 2D alloys. Photoluminescence measurements show that the optical band gaps of the monolayer Mo1−xWxS2 crystals strongly depend on the Mo/W ratios and continuously tunable band gap can be achieved by controlling the W or Mo portion by the LP-CVD.Band gap engineering of monolayer transition metal dichalcogenides, such as MoS Thus, the CVD temperature for WS2 growth is not lower than 800 °C by using WO3 or WO3−x as the precursor26282 and WS2 growth makes it difficult to control the supply of the two oxide precursors for the synthesis of monolayer Mo1−xWxS2 alloys. As a result, MoS2 is usually deposited prior to WS2, resulting in the formation of two-phase MoS2/WS2 heterostructures, rather than Mo1−xWxS2 alloys with a uniform composition and a well-defined band gap1−xWxS2 alloys have been obtained by atomic layer deposition1−xWx thin films381−xWxS2 monolayers with tunable compositions and band gaps have not been realized by the conventional CVD method. In this study, we report a facile method for the synthesis of compositionally homogeneous monolayer Mo1−xWxS2 (x = 0–1) single crystals based on low-pressure (LP) CVD in a conventional CVD system. The band gaps of the synthesized monolayer alloys can be tailored by changing the Mo/W ratios, demonstrating the feasibility of realizing band gap engineering in 2D materials by the conventional CVD method.Band gap engineering is indispensable in nowadays electronics and optoelectronics since a variety of artificial band gaps build on the basics for the optimization and innovation in diverse device applications. Recently, the emergence of two dimensional 2D) materials with a direct band gap opens up a new avenue for improving the integrity in devices. Similar to the bulk semiconductors, the band gaps of 2D materials are changeable and can be modified by applying strains1D materia34689101112131415163 powders were used as the W source, in this study a highly volatile W-containing precursor WCl6 was chosen. WCl6 sublimates at much lower temperature below 100 °C27302 growth can be reduced to ~700 °C, which is very close to the CVD temperature of MoS2 using MoO3 as the precursor3 and WCl6 powders can be simultaneously used as the Mo and W sources for the CVD growth of monolayer Mo1−xWxS2 alloys and the Mo/W ratio can be tailored by tuning the MoO3 and WCl6 supplies. 3−x and WCl6 are transported to the glass substrate, adsorb to and diffuse on the surface before being completely sulfurized and rearranged to form a Mo1−xWxS2 alloy layer. Details about the CVD setup   images  . In contcroscope , which a) images . The as-1−xWxS2 alloys was visualized by HAADF-STEM  from the entire sample. The atomic images reveal that the as-grown Mo1−xWxS2 monolayer alloys contain two phases: metallic 1T and semiconductor 1H. Although the 1T phase has been observed in pristine MoS2 and WS2 before414243444546474849501−xWxS2 grown by CVD. The phase identification is based on the contrast of S atoms. Since S atoms are much lighter than Mo and W, they are almost invisible in the metallic 1T phase due to the misaligned two sulfur layers as shown in 2414522 monolayer470.33W0.67S2 2D crystals is statistically analyzed by using the HAADF-STEM images randomly acquired from different regions of 1T and 1H phases. Both phases have a similar and homogeneous composition with small variation of local W proportion in the range from ~60% to ~80%. Based on a typical HAADF-STEM image, spatial distribution of W proportion x is shown in The atomic structure of the monolayer MoADF-STEM  in which1−xWxS2 alloys. 0.33W0.67S2 alloy. For comparison, we also measured the XPS spectra of monolayer MoS2  and W 4f7/2 (at 33.1 eV) from the 1H phase in Mo0.33W0.67S2 are ~0.2 eV lower than those in the 1H WS2 while W 4f5/2 at 34.7 eV and W 4f7/2 at 32.5 eV from the 1T phase deviate for ~0.6 eV from the corresponding 1H signals in Mo0.33W0.67S2. According to the integral intensities of Mo and W peaks from each phase, the volume ratios of 1T to 1H phases and the compositions of each phase can be approximately estimated. By plotting the volume ratio of 1T phase against the W fraction, one can see that the fraction of 1T phase shows the maximum at the composition of about the half substitution of Mo atoms   , while t1−xWxS2 monolayers can be customized by changing supply rates of Mo and W, Mo1−xWxS2 monolayers with various Mo/W ratio (x ranging from 0–1) were grown by simply changing the amount of MoO3 and WCl6 precursors  and A’1 Raman modes show blue-shift and MoS2-like E’ shows red-shift. Meanwhile, the relative intensity of WS2-like E’ (+2LA) increases and MoS2-like E’ decreases with the increase of W. From the PL results shown in 1−xWxS2 can be calculated, which exhibits a bowing effect with the increase of Mo/W ratios. By fitting the curve with the following equation:Since the composition of the Moecursors . The depb was calculated to be ~0.26 eV, in good agreement with the reported value1−xWxS2 can be achieved by the LP-CVD method. Note that the full width at half maximum (FWHM) of the exciton peak in the PL spectra of the monolayer Mo1−xWxS2 alloys  is obviously larger than those of the pristine MoS2 (57 meV) and WS2 (60 meV) (the bowing factor 60 meV) . The enl meV . Th1−xWxS2 alloys with homogeneously mixed W and Mo in 2D crystals benefits from the LP-CVD process that allows the simultaneous deposition of W and Mo at a constant atomic ratio. Moreover, the preference of hetero-atomic bonding under kinetic equilibrium conditions results in the random mixture of Mo and W atoms, which is maintained in Mo1−xWxS2 crystals without MoS2 and WS2 phase separation1−xWxS2 monolayers with tunable compositions and band gaps can be obtained by the LP-CVD method. After lowering down the deposition rate by reducing the amount of both precursors, growth of multilayers can be largely suppressed  and mechanical/chemical exfoliation. Finally, the appearance of metastable 1T phase enables the incorporation of phase engineering into the alloying of TMDCs for device applications.The successful growth of the monolayer Moppressed . And at ea sheet . Our LP-1−xWxS2 crystals using a conventional CVD system. The monolayer Mo1−xWxS2 alloys with different compositions have intense A excitonic photoluminescence, which shows the strong dependence on Mo/W ratios of the monolayer alloys. As a result, the direct band gaps of the 2D Mo1−xWxS2 can be continuously tuned by altering W proportions in the 2D alloys. This work may pave a new way for fulfilling the applications of band gap engineering in 2D materials by LP-CVD growth of composition-controllable monolayer alloys. In addition, the co-existence of semiconducting 1H and metallic 1T phase is for the first time observed in CVD grown 2D dichalcogenides, which may be important in tuning electronic structures and exploring superconductivity in 2D alloy crystals6162In summary, we have successfully developed a facile LP-CVD route for growing monolayer Mo6 , sulfur , MoO3  and micro slide glass substrate  from upstream to downstream in a 2-inch quartz tube as shown in 6, 130 °C for sulfur, 520 °C for MoO3 and 700 °C for substrate) in 10 min under the Ar flow rate of 2000 sccm (corresponding to 5.1 mbar). In the growth stage, Ar flow rate was switched to 500 sccm (2.1 mbar) to initiate the supply of all the species to substrate for film growth. The temperatures of the precursor sources of sulfur and MoO3 and the substrate were kept constant for 15 min while the temperature of WCl6 gradually dropped from 35 °C to 25 °C. Finally, in the cooling down stage, WCl6 was rapidly cooled down to 15 °C and the others to room temperature in 5 min to end the CVD growth. As-grown samples were transferred by using the standard PMMA-assisted transfer method. The as-grown samples were first spin-coated with poly(methyl methacrylate) . 2M NaOH aq. was used to assist the detachment of samples from glass substrate. After fishing out the PMMA coated Mo1−xWxS2 samples by other substrates, PMMA was dissolved by acetone.Three precursor sources and a substrate were placed in the sequence of WCl5/2:3d3/2 = 3:2 and 4f7/2:4f5/2 = 4:3 respectively. Sensitive factors for Mo 3d and W 4f are 3.321 and 3.523 respectively.Raman and PL spectra were acquired by a confocal Raman spectroscopic system (Renishaw InVia RM 1000) with 514.5 nm excitation laser at the power of 10 mW and 2 mW respectively. The atomic structure and chemical analysis of the 2D alloys were characterized by HAADF-STEM and STEM-EDS by utilizing a high resolution TEM system (JEOL JEM-2100F) with double spherical aberration correctors for imaging and probing lenses. The TEM observations were performed at the acceleration voltage of 200 kV and the spatial resolution of the STEM is ~0.1 nm. XPS measurements were performed by AXIS ultra DLD (Shimazu) with Al Ka using X-ray monochromator. The peaks were fitted by Voigt functions with Lorentzian ratio of 30% and Gaussian ratio of 70% using the software CasaXPS from Casa software Ltd. The branching ratios for spin orbit split components of 3d and 4f orbitals are 3dHow to cite this article: Wang, Z. et al. Chemical Vapor Deposition of Monolayer Mo1-xWxS2 Crystals with Tunable Band Gaps. Sci. Rep.6, 21536; doi: 10.1038/srep21536 (2016).


Pestivirus, is one of the major causes of economic losses for pig farming. The aim of this work was to validate a RT-qPCR using Taqman for detection of CSF in swine tissues. The parameters for the validation followed the specifications of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (OIE) and the guide ABNT NBR ISO/IEC 17025:2005. The analysis of the 5′NTR region of CSF virus was performed in 145 samples from 29 infected pigs and in 240 samples from 80 pigs originated in the Brazilian CSF-free zone. The tissues tested were spleen, kidney, blood, tonsils, and lymph nodes. Sequencing of the positive samples for 5′NTR region was performed to evaluate the specificity of the RT-qPCR. Tests performed for the RT-qPCR validation demonstrated that the PCR assay was efficient in detecting RNA from CSF virus in all materials from different tissues of infected animals. Furthermore, RNA from CSF virus was not detected in samples of swine originated from the Brazilian CSF-free zone. Hence, it is concluded that RT-qPCR can be used as a complementary diagnostic for CSF.The viral disease classical swine fever (CSF), caused by a  Pestivirus genre [Classical swine fever is a highly contagious disease that affects domestic and wild swine and is caused by a virus from Flaviviridae family andus genre , 2. The us genre –6. The ius genre .The trade of live pigs and their byproducts in national and international scope represents a risk to areas or country free of CSF. Laboratory diagnosis of CSF is essential once many symptoms are not exclusively associated with the disease and may vary depending on the virus strain, age, and health status of the animals. Such diagnosis must be fast and accurate, especially in countries that has eradication programs already established, since the fast diagnosis helps reducing the potential for transmission for uninfected herds and therefore prevents the spread of the disease. Pestivirus genome has a highly conserved nontranslated region (NTR), which has been amplified by various studies that performed polymerase chain reaction from reverse transcription of viral RNA (RT-PCR) [ Pestivirus NTR by RT-PCR represents a valuable complimentary alternative for CSF diagnosis. Advantages of using this molecular technique include the possibility of working with deteriorated samples, which otherwise is not possible when performing virus isolation [The gold standard diagnosis of CSF performed by virus isolation can be problematic, particularly due to biosecurity risks involved with handling live infectious agents. In addition, serological assays performed to diagnose CSF may yield confounding results because antibodies for other diseases caused by Pestiviruses, such as bovine viral diarrhea (BVDV) and border disease, can cross-react with the CSF virus , 9. In o(RT-PCR) –12. Thersolation , and theThus, this study aimed to validate a RT-qPCR detection of the region 5′NTR of CSF virus from Brazilian swine. The validation of this assay was performed according to the recommendations of the Brazilian Society for Technical Standards  and inclIn order to perform the validation of RT-qPCR detection of the region 5′NTR of CSF virus, samples from CSF-infected pigs (positive control) and from pigs originated from a CSF-free zone (negative control) were used. The CSF-infected pig samples were kindly provided by the National Agricultural Laboratory of Pernambuco , a laboratory located outside the Brazilian CSF-free zone and that routinely inoculates swine for tests of CSF vaccine potency. Twenty-nine pigs were intramuscularly infected with the Brescia strain of CSF virus and euthanasied three days later. At necropsy, a total of 145 samples of tissues including spleen, kidney, blood, tonsils, and a pool of lymph nodes were obtained from the CSF-infected pigs. The CSF-free zone samples were in total 240, comprising blood, tonsils, and kidney, from 80 pigs reared on Brazilian CSF-free zone and originated from a slaughterhouse certified by the Federal Inspection Service (SIF) and located in Minas Gerais, Brazil.All samples were inactivated using TRIzol  before being sent to analysis. RNA was extracted from 100 mg of samples following the TRIzol protocol  according to the manufacturer's instructions. The concentration of the extracted material was estimated by spectrophotometer .Samples were subjected to a RT-qPCR to the normalizing gene beta-actin, following the protocol described by Bielanski et al.  in orderNTR region was chosen as the target for RT-qPCR, according to Hoffmann et al.  and as dTo evaluate the specificity of the test, nine samples of different genotypes were analyzed. The strains were CSFV RNA Alfort187 strain ; Brescia strain ; CSF0653 strain ; Paderborn strain ; CSF0849 strain ; Parma98 strain ; Spain 2001 strain (genotype 2.3); CSF1045 strain ; CSF0864 strain . The samples were obtained from the reference laboratory for CSF diagnosis, Instituto Nacional de Investigación y Tecnologia Agrária y Alimentaria, located in Spain. RT-qPCR products were sequenced in an automated sequencer ABI Prism 3130 Genetic Analyzer  using Big Dye Terminator Kit from the same manufacturers. Specific primers for the sequencing are CSF 52F: CCCTGGGTGGTCTAAGTCCT and CSF 281R: CTCCATGTGCAATGTACARC. Sequences were edited in Bioedit program  and compSpecificity was also evaluated after the sequencing of 12 clinical samples from an outbreak of CSF that occurred in 2009, Rio Grande do Norte, Brazil. The analytical specificity of the RT-qPCR was checked by the absence of amplification of RNA of the BVDV-I NADL cytopathic strain, the BVDV-I W2 noncytopathic strain, viral DNA from the Paracambi strain of the African swine fever virus (ASF), and the viral DNA from Shope strain of the pseudorabies virus. All these viruses were growth in MDBK and PK15 cell lines tested for contamination as described by Pinheiro de Oliveira et al. .5.8 TCID50/mL titer, and it was serially diluted until 10−6. Each dilution was tested in triplicate and the concentration was estimated by spectrophotometer UV light . The assay was repeated in three different days. Standard curves were made for the determination of the optimal concentration of primers, defining 0.99 as minimum R2 and 0.90 and 1.10 as minimum and maximum efficiency, respectively.Determination of the LD  was performed using a blood sample collected from a CSFV infected swine. This sample had 10Assays were carried out with samples of seven animals. The RNA from the blood samples was extracted as previously described and subjected to the RT-qPCR. Repeatability was accessed by testing samples in triplicates and in different days as suggested previously . Tests ok = 2, which for a normal distribution corresponds to a coverage probability of about 95.45%.Calculation of uncertainty was carried out using the Cts obtained from repeatability and reproducibility assays. Uncertainty value was calculated from the maximum standard deviation values found for each sample in repeatability and reproducibility assays. The uncertainty is stated as the standard uncertainty of measurement multiplied by the coverage factor The extracted RNAs showed good quality, given that all tested samples were positive on RT-qPCR for beta-actin. Different from that observed by Deng et al. , it was E value 2e − 23 when the sequence was submitted to Blast). Moreover, no cross-reactivity of the CSFV RT-qPCR with the BVDV I and II RNA, ASFV or pseudorabies virus DNA on RT-qPCR was observed.Analytical specificity was confirmed through sequencing, where it was observed that the sequence of the amplicons obtained showed high similarity with the CSF virus reference genome available in GenBank : U = 3.12 when comparing the results of reproducibility and U = 1.37 when comparing the results of robustness. Thus it was found that variance is larger when comparing results between technicians than when comparing results obtained from the same equipment and with analyst variation.Results were similarly found for all the three replicates of the dilution and also in the replicates evaluated in three different days. The repeatability, reproducibility, and robustness tests yielded the following values of expanded uncertainty (U = 3.12.Given the results, the following criteria were established in order to validate the analysis: samples were considered positive for CSF when their amplification curves were similar to the positive control curve and exceeded the threshold with Ct values lower that 41, considering the measurement uncertainty The quality of a laboratorial result is linked to the use of procedures such as validated methods, quality internal controls, participation in interlaboratorial comparison programs, the proper use of certified reference materials, and the compliance with requirements of standards. The understanding of the variables involved is extremely important to produce reliable results. Validation is the confirmation by examination and supply of objective evidence that the particular requirements for a specific use are met. Some of the parameters used in the validation are the uncertainty of the results, the detection limit, and specificity of the method, linearity, repeatability, reproducibility, and robustness . FurtherThe validation of RT-qPCR performed in this study followed the parameters described in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the OIE  and the


Borrelia miyamotoi is a newly recognized human pathogen in the relapsing fever group of spirochetes. We investigated a case of B. miyamotoi infection of the central nervous system resembling B. burgdorferi–induced Lyme neuroborreliosis and determined that this emergent agent of central nervous system infection can be diagnosed with existing methods. Borrelia miyamotoi was described in 1994 but only recently was recognized as a human pathogen  that could be attributed to B. miyamotoi only by molecular–biological methods.The tickborne relapsing fever spirochete 2 every 8 weeks) until November 2014. The monoclonal antibody rituximab that targets the CD20 antigen expressed by B cells leads to anti-CD20–mediated B cell depletion. This treatment, together with the underlying hematologic disease, resulted in an immunocompromised condition. The patient reported 2 tick bites while in Elsteraue municipality  during June and July 2015 but had no recent travel history. On examination, she had slight neck stiffness but no other notable findings.On July 31, 2015, a 74-year-old woman with a history of non-Hodgkin lymphoma  came to the Burgenlandkreis Hospital . She reported dizziness, vomiting, and a headache of 5 days’ duration. She had been treated for her lymphoma with cyclophosphamide, doxorubicin, vincristine, and prednisone with rituximab during May–August 2012. Thereafter, she received rituximab maintenance therapy . However, because the CSF constellation was typical for LNB, including the slight intrathecal IgM synthesis  showed a pleocytosis of 70 leukocytes/μL (reference 0–5 leukocytes/μL) and elevated protein at 1,718 mg/L (reference 150–400 mg/L); albumin quotient  34.8% (reference <9%), lactate 5.58 mmol/L (1.2–2.1 mmo/L), and glucose quotient 0.45 (>0.5); and an intrathecal IgM synthesis of 18%. The greatly elevated lactate and highly increased Qalb and protein made a viral cause unlikely. Pappenheim staining of a cytospin preparation of CSF revealed a mixed cell population  with heterogeneous morphology of lymphocytes that did not suggest lymphomatous meningitis. Serum and CSF were negative for B. burgdorferi–specific antibodies in serum or CSF, and B. burgdorferi–specific PCR of the first CSF specimen showed no amplification product.To exclude early tuberculous meningitis, we examined the CSF for mycobacteria by PCR, culture, and staining. To further substantiate the LNB diagnosis, we determined the B-cell attractant chemokine CXCL13 from CSF. The measured value of 1,155 pg/mL was clearly higher than the minimum concentration of 250 pg/mL, indicating neuroborreliosis and thus supporting our working diagnosis of LNB . Howeverhttp://blast.ncbi.nlm.nih.gov/Blast.cgi) in GenBank, we found, to our surprise, that the sequence was identical to that for B. miyamotoi. Moreover, sequences of 16S rRNA, flaB, and 6 housekeeping genes revealed the European type of B. miyamotoi  (Germany) .Borrelia is divided into 2 groups: B. burgdorferi sensu lato, which causes Lyme disease, and a group of species that cause relapsing fever. B. miyamotoi belongs to the second group and was first described in 2011 as a human pathogen in several patients from Russia who had an unspecific febrile illness where few patients experienced febrile relapses, which is the typical sign for relapsing fever  of short duration, the other published cases had decline in mental status , and disturbed gait developing gradually over several months. All 3 B. miyamotoi cases had CSF pleocytosis with elevated CSF protein concordant with CSF changes found in LNB (Detection of specific antibodies is a useful complementary diagnostic method. Only recently it was shown that assays based on the GlpQ protein, which is present in relapsing fever d in LNB . We evenB. burgdorferi and Treponema pallidum (B. burgdorferi in CSF, B. miyamotoi, as a member of the relapsing fever spirochete group, was detectable by microscopy and by PCR in the CSF in this patient, as well as in the 2 previously reported (,B. miyamotoi infection in a patient from Germany indicates that this emergent agent of central nervous system infection can be diagnosed by using existing methods if clinicians are aware of it.In recent years, CXCL13 has been identified as a potentially sensitive and specific biomarker for diagnosing acute LNB. It was a useful indicator in our investigation and might be a suitable indicator for diseases caused by spirochetes in general, as was already shown for pallidum . In contBorrelia miyamotoi, Elsteraue, Germany, 2015.GenBank accession numbers for nucleotide sequence of


Experiments were carried out in an In Vitro model of CSF flow through VP shunts (Medtronic Inc) to investigate the influence of three factors on shunt function: 1) CSF protein concentration; 2) CSF contamination and; 3) Time of shunt incubation in the artificial CSF.Two components of a shunt were tested separately: 1) Ventricular catheter and; 2) Medium pressure, non-programmable valve connected to a peritoneal catheter. In the “sterile” artificial CSF groups, each shunt was incubated in one of six different sterile solutions of varying protein conc. , prepared by adding egg protein  to normal saline, and incubated in sterile artificial CSF for periods of 0, 7, 14 and 46 days.A “Contaminated” group of shunts were similarly prepared but exposed to skin microbes by handling them with ungloved hands and incubating them in contaminated artificial CSF. The same contaminated CSF solutions were used over the total 46 days of incubation but only 3 different protein conc.  were prepared.Both the sterile and contaminated shunts were tested after four different incubation periods . Each of the two shunt components  were tested separately by connecting them with I.V. tubing to a manometer, all filled with the test CSF. The manometer was prefilled to a height of 30 cm H20 and CSF was allowed to flow through each shunt component. The elapsed time (s) was recorded when the CSF passed each 5cm increment from 30 to 0 cm H20 or until flow stopped above 0 cm. On a few occasions, CSF did not drop from the 30 cm level. This “shunt blockage” occurred only when testing the valve / peritoneal catheter complex. Height of CSF column (cm) was plotted against time and the best linear fit determined for each data set. The slope of each curve approximated the average rate of CSF flow (cm/s).In this In Vitro model, CSF protein of 5g/L or higher was the threshold level to adversely affect CSF flow through the valve/peritoneal catheter component: Incubation for 46 days in a protein conc. of 10 g/L or higher, resulted in a 75% decrease in CSF flow rate (.07 cm/s) and an elevated closing pressure of 11 cm H20, compared to testing at day 0 in 0.5 g/L . Microbial contamination did further slow flow but only after 46 days incubation in CSF with a protein conc. of greater then 5 g/L. Absence of cellular and humoral mediators of inflammation in this artificial CSF could be addressed in future studies by using CSF from patients with proven shunt infection. Flow through the ventricular catheter was not affected in any situation in this model.


The co-occurrence of multiple pathologies in the pelvic viscera in the same patient, such as, irritable bowel syndrome and interstitial cystitis, indicates the complexity of viscero-visceral interactions and the necessity to study these interactions under multiple pathological conditions. In the present study, the effect of distal colon irritation (DCI) on the urinary bladder interaction with distal esophagus distention (DED), distal colon distention (DCD), and electrical stimulation of the abdominal branches of vagus nerve (abd-vagus) were investigated using cystometry parameters. The DCI significantly decreased the intercontraction time (ICT) by decreasing the storage time (ST); nonetheless, DED and Abd-vagus were still able to significantly decrease the ICT and ST following DCI. However, DCD had no effect on ICT following the DCI. The DCI, also, significantly decreased the Intravesical pressure amplitude (P-amplitude) by increasing the resting pressure (RP). Although DED has no effect on the P-amplitude, both in the intact and the irritated animals, the abd-vagus significantly increased the P-amplitude following DCI by increasing the maximum pressure (MP). In the contrary, 3mL DCD significantly increased the P-amplitude by increasing the MP and lost that effect following the DCI. Concerning the pressure threshold (PT), none of the stimuli had any significant changes in the intact animals. However, DCI significantly decreased the PT, also, the abd-vagus and 3mL DCD significantly decreased the PT. The results of this study indicate that chemical irritation of colon complicates the effects of mechanical irritation of esophagus and colon on urinary bladder function. Due to viscero-visceral interactions, many inflammatory conditions have manifestations affecting not only the pathological organ but extends onto other viscera within the same vicinity or even further away. The spread of the symptoms onto other viscera complicates the pathological condition and make it more difficult to diagnose and manage. For example, chronic pelvic pain could be attributed to many conditions affecting urinary bladder/interstitial cystitis, gastrointestinal tract/irritable bowel syndrome, prostate gland/prostatitis or any other pelvic organ pathology .Urinary bladder function can be affected by stimuli from other viscera. For example, colon inflammation in rats was shown to increase urinary bladder frequency . In addiThe complexity of the viscero-visceral interactions requires the study of these interactions under different pathological conditions. Most studies were designed to show the simple viscero-visceral interaction between viscera or the direct effect of a pathological condition of one viscera on the function of another; however, it is rare to find a study on how a pathological condition of one viscera can affect the viscero-visceral interaction of other viscera. In the present study, the influence of distal colon chemical irritation on the interaction of urinary bladder with distal colon and distal esophagus distentions was investigated.Eighteen male Wistar rats (300-350g) were used in the present study. Ten of these animals had distal colon irritation and the other eight animals remained intact. Animals were purchased and housed under standard conditions in the animal house at The Hashemite University. All experimental methods were approved by the Hashemite University Institutional Board and Animal Ethical Committee, which meet the requirements of the National Institute of Health  guide for the use and care of laboratory animals.At the day of experiment, each rat was anesthetized with urethane (1.2g/kg of 50% urethane in water); the anesthetic solution was divided into two halves, of which, one half was administered intraperitoneally and the other was given subcutaneously . UrethanAn abdominal incision was made, in each animal, in order to expose the urinary bladder and ureters. A 20-gauge needle was introduced into the dome of the urinary bladder and connected to a programmable pump . Cystometry was done by bumping normal saline at a rate of 0.25mL/min to the urinary bladder. Intravesical pressure was monitored using a pressure monitor  that was connected to the needle through a pressure transducer. The temperature and hydration of the urinary bladder were preserved throughout the experiment by surrounding the urinary bladder with cotton pallets that were soaked in a warm normal saline. The ureters were tied proximal to the urinary bladder, then ureters were cut and drained distal to it.Distal colon distention was done by a 10mm long balloon, which was made from latex material and attached to a 25GX¾” catheter . The colDistal esophagus distention was done by balloon, similar to the distal colon balloon. The esophagus balloon was inserted about 7cm from the upper incisors, intra-orally, to the distal esophagus . The balThe abdominal branches of the vagus nerve were stimulated, at the level of the esophageal opening of the diaphragm, using digital stimulator  and isolated using isolated current source  with isolation. The stimulation was done indirectly through the esophageal wall using a bipolar electrode that was introduced orally down to the esophagus. The stimulation was conducted at frequency of 1 train/sec with 100msec train duration. Each train was composed of 14 pulses (at 70pulses/sec). Each pulse intensity was set at 8mA with 2msec duration . This stTen rats had distal colon chemical irritation. Chemical irritation of the distal colon was done in the anesthetized animals by infusing 1mL of 2% acetic acid into the distal colon through a catheter (comprised of PE 60 tubing attached to a syringe). A cotton pallet was attached to the distal end of the catheter by fixing part of it to the distal end of the catheter tub, while leaving the remaining free part in direct contact with colon mucosa. This procedure insures the localization of the irritant within the distal colon.At the beginning of each experiment, normal cystometry recordings were performed for 30 minutes. After that, cystometry recordings were done for another 30 minutes following the infusion of the irritant . Then, cystometry recordings were done for 10 minutes with esophagus distention, followed by 10 minutes recordings without esophagus distention. In the same way, cystometry recordings for another 10 minutes were done with vagal nerve stimulation, followed by normal recordings for 10 minutes. At the end, cystometry recordings were done for 30 minutes with distal colon distentions . Cystometry was recorded for 10 more minutes following colon balloon deflation.All data obtained from the pressure monitor were amplified and recorded on a computer using data acquisition system . Cystometrograms were recorded using a BioBench software program . The micturition cycle timing parameters, intercontraction time (the whole micturition cycle time) (ICT); the time of the voiding phase (VT); and the time of the storage phase (ST), and the intravesical pressure parameters, resting pressure (RP); pressure threshold (PT); maximum pressure (MP); and pressure amplitude  were measured, offline, for statistical analysis.All the parameters of the micturition cycles in ten minutes after each stimulus and the micturition cycles in the last ten minutes preceded the stimulus from each animal were considered for statistical analysis for each stimulus. The measurements of micturition cycles parameters preceded the stimulus were considered as control for the measurements following the stimulus. Statistical analysis was performed using two tailed, unpaired student t-test (t). Results were considered significant when P<0.05. All data are presented as mean±standard error.Distal colon irritation significantly increased the ICT. In addition, DCI significantly increased the ST, however, there was no significant changes to the VT following distal colon irritation. Compared to intact animals , in the In the intact animals, distal esophagus distention and electrical stimulation of the abdominal branches of vagus nerve didn’t have any significant effect on the intravesical pressure parameters , except for a significant increase in the RP to vagal stimulation. At the same time, only 3mL distal colon distention significantly increased the MP and the P-amplitude, without any significant effect on the RP or PT. Distal colon distention with 1mL and 2mL didn’t have any significant effects on the intravesical pressure.In the irritated animals, irritation of distal colon significantly increased the RP, while significantly decreased the PT and P-amplitude. However, Irritation of distal colon had no significant effect on the MP. Following distal colon irritation, distal esophagus distention still had no significant effect on the intravesical pressure parameters. Simultaneously, distal colon distention almost had no significant effect on the intravesical pressure parameters following distal colon irritation, except for a significant increase in the PT with 3mL distal colon distention. However, in the irritated animals, electrical stimulation of the vagus nerve significantly decreased the PT, though, significantly increased the MP and P-amplitude. Vagal stimulation had no significant effect on the RP. The effect of esophagus, vagal, and distal colon stimuli on the P-amplitude are presented in The results of this study shows how the changes of the urinary bladder function in response to mechanical stimuli from other viscera, either in the same region  or in another region (esophagus distention), can be affected by chemical irritation to the distal colon.The chemical irritation of the distal colon significantly increased the urinary bladder frequency. The increase in bladder frequency was attributed by the significant decrease in the storage time of the micturition cycle, where the voiding time did not have any significant changes. Since the storage phase is mainly a spinally mediated reflex , these rThe neuronal sensitization can also explain the effect of distal colon irritation on intravesical pressure, where colon irritation significantly decreased the pressure amplitude by significantly increasing the resting pressure. In addition, distal colon irritation significantly decreased the pressure threshold. The effect of colon irritation on pressure threshold could be related to sensitization of the urinary bladder afferents as well. Ustinova et al.  demonstrAll the same, distal colon irritation had an influence on the bladder responses to the distal colon distention and distal esophagus distention, as well as to electrical stimulation of the abdominal branches of vagus nerve. Following distal colon irritation, the distal esophagus distention and electrical stimulation of vagus nerve were still able to significantly increase the bladder frequency as it did in the intact animals. This increase in the bladder urinary frequency is still attributed to the decrease in the storage time as well . So, desOn the other hand, following distal colon irritation, distal colon distention didn’t have any significant effect on bladder frequency, which means that distal colon distention lost its inhibitory effect on the bladder function. On the opposite, 3mL distal colon distention significantly decreased the pressure threshold following colon irritation. The ability of colon irritation to sensitize the convergent neurons for urinary bladder and colon again can explain these results. Wang et al.  showed tFinally, the setup of the present study has some limitations, mainly related to the distal colon irritation and the conscious level of the animals. More studies must be done about chronic irritation of the colon to resemble more the real pathological conditions. In addition, these experiments could be done in conscious animals, in order to exclude the effect of anesthesia on the viscero-visceral interactions. Moreover, electrophysiological studies must be carried on, in order to elaborate the changes in the neural circuitries that control the viscero-visceral interactions.In conclusion, the results of the present study demonstrate that viscero-visceral interaction effects on the urinary bladder function depends on the variability of the mechanical and chemical stimuli from other viscera and the degree of neural overlapping that controls the different viscero-visceral interactions. The results of this study also indicate the necessity of taking in consideration the different nociceptive stimuli from other viscera in managing pathological conditions affecting certain visceral organ such as urinary bladder.


HLA-haploidentical hematopoietic stem cell transplantation (HSCT) is suitable for patients lacking related or unrelated HLA-matched donors. Herein, we investigated whether plerixafor (MZ), as an adjunct to G-CSF, facilitated the collection of mega-doses of hematopoietic stem cells (HSC) for TCR-αβ/CD19-depleted haploidentical HSCT, and how this agent affects the cellular graft composition.+ HSCs/μL after 4 daily G-CSF doses and/or with predicted apheresis yields ≤12.0x106 CD34+ cells/kg recipient’s body weight.Ninety healthy donors were evaluated. Single-dose MZ was given to 30 ‘poor mobilizers’ (PM) failing to attain ≥40 CD34+ counts in PM. Naïve/memory T and B cells, as well as natural killer (NK) cells, myeloid/plasmacytoid dendritic cells (DCs), were unchanged compared with baseline. MZ did not further promote the G-CSF-induced mobilization of CD16+ monocytes and the down-regulation of IFN-γ production by T cells. HSC grafts harvested after G-CSF + MZ were enriched in myeloid and plasmacytoid DCs, but contained low numbers of pro-inflammatory 6-sulfo-LacNAc+ (Slan)-DCs. Finally, children transplanted with G-CSF + MZ-mobilized grafts received greater numbers of monocytes, myeloid and plasmacytoid DCs, but lower numbers of NK cells, NK-like T cells and Slan-DCs.MZ significantly increased CD34+ HSCs for haploidentical HSCT, while affecting graft composition.MZ facilitates the collection of mega-doses of CD34The online version of this article (doi:10.1186/s12967-014-0240-z) contains supplementary material, which is available to authorized users. Interestingly, PMs given single-dose MZ had higher day-5 monocyte and lymphocyte counts compared with GMs receiving G-CSF alone  and 5.4% (1.0-9.1), respectively] and post-mobilization samples , irrespective of the mobilization protocol  and 30.9% (1.0-58.7), and for 32.2% (2.4-56.4) and 26.6% (2.0-53.4) of the CD4+ and CD8+ T cells before and after treatment with G-CSF  undergoing a standard large-volume 15-20 L) leukapheresis, the graft was extensively characterized in terms of immune effector cells with particular relevance to the setting of haploidentical HSCT, such as NK cells, CD3and DCs. -39 For t-20 L leu+ monocytes were contained at higher frequency in TCR-αβ/CD19-depleted grafts, compared with normal BM samples  DCs constitute 0.5-2% of all PBMCs, are CD14lowCD16+ and are a highly phagocytic monocyte subset inducing potent pro-inflammatory Th1 and Th17 responses [Both conventional and CD16esponses . As illuFinally, Figure +CD16− monocytes  compared with children without chronic GVHD  nor the number of monocytes and DCs infused correlated with either the occurrence of acute GVHD or the reactivation of viral infections (data not shown). By contrast, patients who developed chronic GVHD had received lower numbers of conventional CD14Herein, we show that the addition of ‘immediate salvage’ MZ to a standard, G-CSF-based mobilization regimen augments HSC yield in HLA-haploidentical donors showing less-than-optimal CD34-cell mobilization, and that MZ administration affects the graft cell composition. Thirty-two percent of our donors were operationally defined as PMs and were given MZ to increase HSC mobilization. More than 95% of the donors collected the required mega-dose of HSCs with a single apheresis session.+ HSCs by 8.2-fold (range 1.4-29.2), compared with that measured after 4 days of G-CSF treatment. This is remarkably similar to the 8-fold increase of CD34+ HSCs reported in donors given MZ only [Currently, anecdotal reports describe the ‘just-in-time’ application of MZ to healthy donors, either as single agent or after mobilization failure with G-CSF -24. In o MZ only .+ T-cell release of IFN-γ and TNF-α may be higher in autologous grafts collected after G-CSF and MZ, compared with G-CSF alone [+ T-cell production of IFN-γ was not potentiated by single-dose MZ. Furthermore, IL-17 and IL-4 release by CD4+ T cells were not affected by G-CSF and/or MZ administration. These observations are in line with pre-clinical data showing that MZ alone, in contrast to G-CSF, is unable to alter the phenotype and cytokine polarization of T cells, as well as T-cell’s ability to induce acute GVHD [+ T cells. Notably, studies in mice suggest that G-CSF may separate GVHD and graft-versus-leukemia (GVL) responses by exerting suppressive effects on CD4+ T cells, that are implicated in GVHD, while preserving the cytolytic pathways of CD8+ T cells that are critical for effective GVL [The few available data on immunological effects of MZ are mostly limited to cancer patients and show that CD8SF alone . We prevSF alone ,19,41. TSF alone . In our ute GVHD . It musttive GVL . At varitive GVL , we weretive GVL  nor our + T cells, a phenomenon that was mainly accounted for by a reduction of fully mature CD56+CD16+ and tissue-resident CD56+CD16− NK cells, with preserved frequencies of immature CD56−CD16+ NK cells. Interestingly, the frequency of both NK cells and NK-like CD56+ T cells was reduced in the PM group receiving single-dose MZ. Although the number of NK cells collected was not significantly different when comparing donors given G-CSF alone with those receiving G-CSF in combination with MZ, higher numbers of both NK cells and NK-like CD56+ T cells were infused in children transplanted with G-CSF-mobilized HSC products. A potential clinical implication of this finding pertains to the field of graft engineering, insofar donor mobilization with G-CSF alone might offer an advantage over the use of G-CSF + MZ for patients with NK-susceptible hematological malignancies [In our cohort of 90 donors, HSC mobilization with G-CSF translated into lowered frequencies of both NK cells and NK-like CD56gnancies .+ subset of intermediate/non-conventional monocytes. Although the frequency of both conventional and CD16+ monocytes was higher in TCR-αβ/CD19-depleted HSC grafts compared with normal BM samples, the overall number of conventional and CD16+ monocytes infused in our haploidentical HSCT recipients was not correlated with the mobilization regimen used in the donor. It has been demonstrated that macrophages generated from CD16+ monocytes manifest higher phagocytic activity compared with macrophages derived from classical monocytes [dimCD16+ monocytes are endowed with a unique patrolling function, as they detect virally infected and damaged cells and produce pro-inflammatory cytokines [+ monocytes infused with the TCR-αβ/CD19-depleted haploidentical grafts may protect the recipient from infectious episodes, while contributing to prevention of GVHD [As previously published, G-CSF-mobilized monocytes are functionally defective . In our onocytes . In addiytokines . In ligh of GVHD .in vivo cleavage of the N-terminus of CXCR4 on BM-resident HSCs [+ HSCs and monocytes [in vivo administration of G-CSF, when compared to G-CSF plus MZ, this likely favoring Slan-DC retention into tissues. The CD184-12G5 mAbs recognize an epitope involving the first and second extracellular domains of CXCR4, and inhibit MZ binding to CXCR4. A different anti-CXCR4 mAb, termed 1D9, binds to the N terminus of CXCR4 and is not affected by MZ [in vivo [There is also evidence that G-CSF mobilizes IL-12/TNF-α-producing, pro-inflammatory Slan-DCs . Thus, Sent HSCs , it is tonocytes ,50, may ed by MZ . In a ph[in vivo . By cont+ MDC1, CD141+ MDC2 and plasmacytoid DCs [+ DCs in donors given G-CSF compared with baseline [Finally, the frequency of CD1ctoid DCs  was unchbaseline . The TCRbaseline . Conversbaseline . Importabaseline .Thus far, we have transplanted 23 children with non-malignant disorders using TCR-αβ/CD19-depleted HSC grafts . Primary+ HSCs to be transplanted into haploidentical recipients. Furthermore, our data shed some light into the optimal clinical use of MZ, insofar differences in graft cellular composition after mobilization with G-CSF and MZ are expected to quantitatively and/or qualitatively influence the immune processes that occur after allogeneic HSCT.Collectively, our study shows that MZ is highly effective at mobilizing mega-doses of CD34