The sudden stiffness reduction in a structure may cause the signal discontinuity in the acceleration responses close to the damage location at the damage time instant. To this end, the damage detection on sudden stiffness reduction of building structures has been actively investigated in this study. The signal discontinuity of the structural acceleration responses of an example building is extracted based on the discrete wavelet transform. It is proved that the variation of the first level detail coefficients of the wavelet transform at damage instant is linearly proportional to the magnitude of the stiffness reduction. A new damage index is proposed and implemented to detect the damage time instant, location, and severity of a structure due to a sudden change of structural stiffness. Numerical simulation using a five-story shear building under different types of excitation is carried out to assess the effectiveness and reliability of the proposed damage index for the building at different damage levels. The sensitivity of the damage index to the intensity and frequency range of measurement noise is also investigated. The made observations demonstrate that the proposed damage index can accurately identify the sudden damage events if the noise intensity is limited. The widely used vibration-based damage assessment methods require modal properties that are obtained from signals via the traditional Fourier transform (FT) , 2. TherThe earliest work on applying wavelet analysis in SHM dated back to the work of Yamamato and his group in 1995. The cumulative damage of a building with bilinear restoring force subjected to a real earthquake ground motion was estimated in terms of the accumulated ductility ratio, which is related to the number of spikes in the wavelet results , 9. The The sudden stiffness reduction in a structure may cause the signal discontinuity in the acceleration responses close to the damage location at the damage time instant. In reality, the signal discontinuity around damage instant due to sudden stiffness loss can be taken as a kind of signal singularity and can be detected by the WT. However, the severity of damage events cannot be depicted by the current developed WT based detection approaches. To this end, the damage detection on sudden stiffness reduction of building structures has been actively investigated in this study. The signal discontinuity of the structural acceleration responses of an example building is extracted based on the discrete wavelet transform (DWT). It is proved that the variation of the first level detail coefficients of the WT at damage instant is linearly proportional to the magnitude of the stiffness reduction. A new damage index is developed and implemented in this paper to detect the damage time instant, location, and severity of a structure due to a sudden change of structural stiffness. Numerical simulation using a five-story shear building under different types of excitation is carried out to assess the effectiveness and reliability of the proposed damage index for the building at different damage levels. The sensitivity of the damage index to the intensity and frequency range of measurement noise is also investigated. The made observations demonstrate that the proposed damage index can accurately identify the damage time instant and location in the building due to a sudden loss of stiffness. The relation between the damage severity and the proposed damage index is linear. The proposed damage index can identify the damage events from the contaminated acceleration responses if the noise intensity is limited.ψ(t) is a waveform that has limited duration and an average value of zero and the wavelet kernel can be expressed bya and b are dilation and translation parameters, respectively. Both are real numbers and a must be positive. Similar to the short time Fourier transform, one can analyze square-integrable function f(t) with wavelet transform, which decomposes a signal in the time domain into a two-dimensional function in the time-scale plane  as follows:Morlet and Grossmann initially proposed wavelet theory and Meyer developed the mathematical foundations of wavelets. The two America-based researchers, Daubechies , 20 and ψ(t) should satisfy the following admissibility condition to ensure existence of the inverse wavelet transform such asψ(t). The existence of the integral in  can be reconstructed by an inverse wavelet transform of C as defined byThe signal a and the translation parameter b in real signal processing. The procedure becomes much more efficient if dyadic values of a and b are used. That is,Z is a set of integers. This sampling of the coordinates  is referred to as dyadic sampling because consecutive values of the discrete scales differ by a factor of 2. Using the discrete scales of WT, one can define the discrete wavelet transform (DWT) [The calculating wavelet coefficients at every possible scale will generate a lot of redundant data. A discrete version of the wavelet is often utilized by discretizing the dilation parameter rm (DWT)  as folloa = 2j−, and the integer j is referred to as the level. The signal can be reconstructed from the wavelet coefficients Cj,k and the reconstruction algorithm is called the inverse discrete wavelet transform as follows:The signal resolution is defined as the inverse of the scale 1/ϕ(t), referred to as the scaling function, is important for the numerical implementation of the fast wavelet transform [a and b, and consider a reference level J. Applying (Another function ransform . SupposeApplying  for thiscDj(k) is known as the level-J detail coefficients. Using the dyadic scale level J yields the level-J approximation coefficients as follows:The coefficient j is defined asj is defined asIn the DWT, a signal can be represented by its approximations and details. The detail at level m = 1.3 × 106 kg and k = 4.0 × 109 N/m, respectively. The Rayleigh damping assumption is adopted to construct the structural damping matrix, and the damping ratios in the first two modes of vibration of the building are set as 0.05. The original building is supposed to suffer a sudden 20% stiffness reduction in the first story with the horizontal stiffness reducing from 4.0 × 109 N/m to 3.2 × 109 N/m, while the horizontal stiffness in other stories remains unchanged. The frequency reduction due to 20% stiffness reduction in the first story is small with a maximum reduction of no more than 5% in the first natural frequency.The dynamic responses of a five-story shear building subjected to a sudden stiffness reduction at its first story under three different external excitations are computed. The mass and horizontal stiffness of the undamaged building are uniform for all stories as shown in 2. A sinusoidal excitation expressed by the following equation with 10-second duration is assumed to act on each floor of the building:The sinusoidal excitation, seismic excitation, and impulse excitation are, respectively, utilized to calculate the acceleration responses of the example building to examine the signal features due to sudden stiffness reduction. The seismic excitation used is the first 10-second portion of the El-Centro 1940 earthquake ground acceleration (S-N component) with a peak amplitude of 1.0 m/sβ method with a time interval of 0.002 s. The two factors in the Newmark-β method are selected as α = 1/2 and β = 1/4 [An impulse excitation represented by 0.1 m/s initial velocity is supposed to occur at the first floor of the building. The damage time instant of the building is set as 6.0 s for seismic excitation and sinusoidal excitation and as 0.2 s for impulse excitation. The equation of motion of the example building with a 20% sudden stiffness reduction at its first story at the given time instant is established. The dynamic responses under each type of external excitation are computed by using the Newmark- β = 1/4 , 23.The computed acceleration time histories of the first floor under seismic excitation are displayed in Since the signal discontinuity is of very high frequency, the wavelet transform is applied to decompose the original acceleration responses. m, the damping ratio ξ of the system is supposed to remain unchanged before and after sudden damage, and the stiffness is denoted as k which will have a sudden reduction at time instant ti as follows:Let us consider a SDOF system subjected to a sudden stiffness reduction under impulse excitation. The mass of the system is denoted as ku and kd are the stiffness of undamaged and damaged system, respectively. The initial velocity and displacement due to the impulse excitation are assumed to be 0 and v0, respectively. The circular frequency of the system before and after sudden damage can be expressed asin which α that varies from 0 to 1 as follows:Define a frequency reduction coefficient The stiffness reduction can be expressed asThe equation of motion of the SDOF system before sudden damage isThe above equation can be solved in terms of the given initial conditions, and the structural dynamic responses arein whichti as the starting point of the SDOF system after sudden damage and use a new time axis t1 = t − ti. Then, the equation of motion of the system after damage becomesLet us take the time instant The initial conditions for  can be et1 isThe damping ratio of a civil engineering structure is often very small; that is, Furthermore, the time interval should be very small to describe the sudden stiffness reduction properly; thusti+1 isTherefore, the acceleration response at the time instant The first level detail coefficients of wavelet transform of the acceleration responses before sudden damage The first level detail coefficients of wavelet transform of the acceleration responses after sudden damage The variation of first level detail coefficients of the WT before and after the sudden damage event can be given asConsidering that the damping ratio of a civil engineering structure is often very small, the above expression can be simplified ast for sudden damage is further regarded as a fixed value,  and y represents damage index. It is observed that there exists a linear relationship between damage index and damage severity for the building under either seismic or sinusoidal or impulse excitation. The magnitudes of the damage index increase with the increasing extents of the stiffness reduction for a given external excitation and the slope of the linear fit is different for the building under different excitations. The proposed damage index and detection approach can be used to find the damage time instant and damage location from the measured structural responses. Then, one can measure the external excitation and input to the structural model with a sudden stiffness reduction at the identified damage location and at the identified damage time instant to determine the slope of the linear relationship between the damage severity and damage index. The linear relationship can be used finally to determine the damage severity in the actual structure in terms of the damage index identified from the actual structure.The magnitudes of damage index of the example building subjected to different damage severities in the first story under seismic, sinusoidal, and impulse excitations are computed and listed in The effect of measurement noise on the quality of the damage detection is a key issue needed to be addressed in real application. Hou et al.  reportedDisplayed in Figures The effects of measurement noise on the damage detection are assessed and the results indicate that both the noise intensity and the frequency range have a remarkable influence on the results from DWT based detection approach. If the noise frequency range is too narrow to overlap the sudden damage signal with high frequency components, the influence of noise intensity on detection accuracy is small. Otherwise, the detection accuracy will decrease with the increase of noise intensities.The damage detection on sudden stiffness reduction of building structures has been actively investigated in this study. The signal jump of the structural acceleration responses of an example building due to sudden damage is examined and the signal discontinuity is extracted based on the discrete wavelet transform. It is proved that the variation of the first level detail coefficients of the wavelet transform at damage instant is proportional to the magnitude of the stiffness reduction. In this regard, a new damage index based on the DWT is proposed in terms of the slope of the decomposed detail coefficients of wavelet transform to detect the damage time instant, damage location, and damage severity.Extensive numerical simulations have been executed on a five-story shear building to assess the performance of DWT based detection approach. The made observations indicate that the DWT based detection index proposed in this study can accurately identify the damage time instant and damage location due to a sudden stiffness reduction in terms of the occurrence time and spatial distribution of damage index spikes. The proposed damage index is linearly proportional to damage severity, but the slope of the linear function depends on external excitation and damage time instant. A possible way of determining damage severity has been suggested using the calibrated structural model and the measured excitation. The proposed damage index can identify the damage events from the contaminated acceleration responses if the noise frequency range is limited. If the noise frequency range is wide enough, the reliability of damage detection using the proposed approach remarkably deteriorates with the increase of noise intensity.


In this study, a new reliable, economic, and environmentally-friendly one-step synthesis is established to obtain carbon nanodots (CNDs) with well-defined and reproducible photoluminescence (PL) properties via the microwave-assisted hydrothermal treatment of starch and Tris-acetate-EDTA (TAE) buffer as carbon sources. Three kinds of CNDs are prepared using different sets of above mentioned starting materials. The as-synthesized CNDs: C-CND (starch only), N-CND 1 (starch in TAE) and N-CND 2 (TAE only) exhibit highly homogenous PL and are ready to use without need for further purification. The CNDs are stable over a long period of time (>1 year) either in solution or as freeze-dried powder. Depending on starting material, CNDs with PL quantum yield (PLQY) ranging from less than 1% up to 28% are obtained. The influence of the precursor concentration, reaction time and type of additives on the optical properties  is carefully investigated, providing insight into the chemical processes that occur during CND formation. Remarkably, upon freeze-drying the initially brown CND-solution turns into a non-fluorescent white/slightly brown powder which recovers PL in aqueous solution and can potentially be applied as fluorescent marker in bio-imaging, as a reduction agent or as a photocatalyst. Since their first description in 2006 carbon dots (CNDs) have attracted considerable attention of both fundamental research and industry56681011121356711101418792068Among various kinds of CNDs, nitrogen-doped carbon nanodots (N-CNDs) have emerged to be attractive materials due to their excellent photoluminescence (PL) properties1026282967915679682223323334561521α-D-glucose and organic acids (e.g. acetic acid)36λmax = 312 nm). After freeze-drying for three days the former dark yellow solutions turned into a white powder. The powder did not exhibit any PL under UV-light  can presumably be attributed to n-π* transition within the C=O bonds, located either on the C-CND surface or within the C-CND coreπ-systems, which also absorb in the 300 nm to 400 nm region, can be excluded as XRD and Raman measurements  have emerged to be complementary to C-CNDs due to their high PLQYs3334414225The highest PLQYs were observed for the combination of Tris and EDTA as TAE-buffer  without starch addition. The 10X TAE buffer contains acetic acid (200 mM), EDTA (10 mM) and Tris (400 mM), and the TAE based CNDs are here referred to as N-CND 1 (starch in TAE) and N-CND 2 (TAE only). The overall yield of N-CND 2 was calculated to be ~80% (w/w), matching the results from previous CNDs2332π* transitions in C=O bonds, while the peak at around 225 nm is assigned to C-N bonds located either on the N-CND surface or in the N-CND core. Noticeably, the typical peak at around 275 nm , 1357 cm−1 , 1150 cm−1 (C-H wag) and 1026 cm−1  indicate C-H bonds. The peak at 1597 cm−1 can be correlated with C=C double bonds in an aromatic ring, which is backed up by the peak at 860 cm−1 for C-H “loop” in aromatic systems. Further carboxyl groups are confirmed by the small peak at 1076 cm−1, which can be ascribed to C=O double bonds and the peak at 936 cm−1, which is ascribed to O-H bending in carboxylic acids. As shown in −1  is due to N-H stretching vibrations appearing in addition to C-H stretching vibrations. The broad bands at 2500–3000 cm−1  are much stronger for N-CNDs than for C-CNDs, due to less aromatic structures in N-CNDs. The strong peaks at 1568 cm−1 (N-H bend) and 1398 cm−1 (C-N = stretching vibrations) indicate successful nitrogen-doping. The bands at 1337 cm−1 (C-N stretch), 1286 cm−1 (aromatic amines) and 1020 cm−1  further show the diverse character of N-CND structures. Additionally, the strong peaks at 900–625 cm−1 can be assigned to N-H wag vibrations. As nitrogen content increases the PLQY of N-CNDs, the logical progression was to increase the content of EDTA and Tris in our synthesis. Both N-additives, EDTA (C10H16O8) and Tris (C4H11NO3), can serve also as carbon sources; especially if used at high concentrations. Therefore, we synthesized N-CNDs without using starch as a carbon source in the precursor solutions.The influence of reaction temperature on the UV-Vis absorption spectra and PLQY for N-CND 2 is shown in 5 nm see  derivingctra see  show thactra see  suggestsctra see , confirmThe elemental composition of C-CNDs and N-CND 2 was analyzed by energy-dispersive X-ray spectroscopy (EDX) as shown in π* transition of the C=O bonds  and the peak for π-π* transition of C=C bonds in aromatic sp2 domains (260 nm). This suggests that N-CND 2 have a different molecular structure than N-CND 1, despite their similar PL emission properties. This indicates again, that the nature of the surface states determines the emission wavelength of CNDs. This is confirmed by the excitation vs emission wavelength measurements , have been prepared using three different sets of starting materials, referred to as C-CND (starch), N-CND 1 (starch in TAE) and N-CND 2 (TAE only). The as-prepared CNDs are highly homogenous in their photoluminescence properties and ready to use without need for further purification, modification or surface passivation agents (e.g. PEG). The CNDs are comparatively small (C-CND: (2.0 ± 0.24) nm; N-CNDs (2.4 ± 0.25) nm) with narrow size distributions ; are staPotato starch (20 wt% amylose); wheat starch (28 wt% amylose); corn starch wild type (approx. 27% amylose); waxy corn starch (76 wt% amylose) and low amylose corn starch (0–1 wt% amylose) and TAE buffer were purchased from Sigma-Aldrich Chemie GmbH . Modified Starches: Alimentamyl 2002 (distarch phosphate); Amylex 20/20 (depolymerized potato starch); Fibraffin K105 (cationic starch ether); Licocat P (quaternary cationic starch solution); Sobocat (quaternary starch ether); Sobotex 5305 NN (starch acetate) and Sobotex CM (starch ether) were provided by Südstärke GmbH . The undefined starch was purchased in a supermarket in Germany. Double deionized water from a Milli-Q water system  was used throughout all experiments.μL of Lugol’s Iodine solution. Finally, the CND-solutions were freeze dried and stored at room temperature for further measurements.In a typical CND synthesis 0.014 mg/mL to 10 mg/mL of the respective starch were dissolved in 4 mL of deionized water (C-CND) or 4 mL of TAE buffer (N-CND 1) at room temperature (RT) for several minutes. To obtain type 2 photoluminescent nitrogen-doped CNDs (N-CND 2) the precursor solutions consisted solely of pure TAE buffer at various concentrations (1X to 50X), all originating from a 50X stock solution containing 242 g Tris base (2 M), 57.1 mL glacial acetic acid (1 M) and 14.612 g of EDTA (50 mM) dissolved in 1 L of water. The pH values of the starting solutions were set to 7.0 and 8.0 for C-CND and N-CND-synthesis, respectively. The as-prepared precursor solutions were then transferred into a pressure resistant glass vial and heated up to 200–230 °C for 5–120 min with a Monowave 300 synthesis microwave oven . After cooling down to RT, the resulting clear light yellow to brown aqueous solutions needed no further purification and were ready for further characterization. In order to verify total starch conversion, 0.5 mL of the obtained solutions  were added to 50 −1.The UV-Vis absorption spectra were collected on a Perkin-Elmer Lambda 750 and a Nanodrop200c spectrophotometer . Emission spectra were collected using a FluoroMax-P fluorescence spectrometer . PL quantum yield (PLQY) measurements were carried out on a modular C9920-02/-03  including an integrating sphere, a PMA-12 Photonic Multi-Channel-Analyser C10027-01  as detector and a 150 W/CW Xe lamp L10092  as excitation source. Samples were placed in a 1.0 × 1.0 cm quartz cuvette and measurement data was collected, processed, visualized and analysed automatically by built-in software. Time-correlated single photon counting measurements were done on a FLS920 Fluorescence spectrophotometer . The samples were measured in a 90° setup using a white light source  as excitation source and a Multi-Channel-Plate  as detector. The Fluorescence decays were fitted using the FAST software . To avoid filter effects and other effects that might distort the signal readout, the PL emission and PLQY measurements were carried out at an optical density (OD) equal to 0.1 at the corresponding excitation wavelength. Fourier transform infrared (FT-IR) spectra were collected using a Bruker Tensor 27 FT-IR spectrometer  using diamond in attenuated total reflection (ATR) in the range of 400–4000 cm2 coated copper grids . Evaluation of obtained TEM images  was performed using Pebbles and PebbleJugglerα (λ = 0.15405 nm) radiation at scanning speed of 0.47 °/min in the range from 4° to 70°. X-ray microanalysis was performed on a Zeiss ULTRA55 PLUS scanning electron microscope , operating at 5 to 10 kV and a working distance of 10 mm. EDX microanalyses with an acquisition time of 20–30 min and a Quantax 400  as detector have been obtained for both the C-CND and the N-CND 2. The elemental quantitative analysis used an automatic background subtraction and a correction has been used to calculate the elemental composition in weight percents and atomic percents.Elemental analysis was performed on Elementar Vario EL III . Laser diffraction particles size analysis (SLS) was performed on LS 13 320 Laser Diffraction Particle Size Analyser . TEM images were recorded on a JEOL JEM 1011 microscope  operated at 200 kV with samples prepared on 400 mesh, SiOHow to cite this article: Meiling, T. T. et al. White carbon: Fluorescent carbon nanoparticles with tunable quantum yield in a reproducible green synthesis. Sci. Rep.6, 28557; doi: 10.1038/srep28557 (2016).


In the course of the millennia, fire, a natural as well as a man-made entity, has been promoting human cultural and technological development. However, just like other powerful forces, fire has two sides: one that is positive or helpful and another that is negative or harmful. Obviously, as human beings survive and prosper better with fire than without it, it follows that possible burn injuries will stay with human beings.th Meeting of the International Society for Burn Injuries (ISBI) held in Edinburgh in 2012. That proposal outlines the ideal state of affairs, and indeed it is also our dream. On the contrary, all over the globe, patients with burn wounds are at present exposed to widely dissimilar conditions stemming from broadly different ethnic cultures, personal economic conditions, national health policies, and local medical traditions, which together end up determining profound dissimilarities in burn care. This inconsistent care is the reason for us launching an open-access journal, Burns & Trauma, as we wish to share on this platform several experiences, different data, various cases, and diverse ideas in a fast and easy way. Doctors, scientists, nurses, therapists, and social workers could improve the care of burn patients, promote the prevention of fire disasters, decrease postburn disabilities, and pursue a common dream: “No Burn Injury” or at least “One World, One Standard”.For a number of well-known reasons, burn injury remains a quite severe social and medical problem. “One World, One Standard” was the proposal as the aim of the 16Burns & Trauma is an open access journal that aims to cover the latest and best achievements in all aspects of burn and trauma research. This new journal will provide a unique platform for the rapid publication of guidelines, original articles, review articles, case reports, letters, commentaries, and thematic issues on scientific research related to burn and trauma research. Themes include but are not limited to emergency medicine, wound healing, tissue engineering, intensive care, nutrition, bioengineering, shock, rehabilitation, immunology, infection, psychology, stem cells, organ damage, injury epidemiology, regeneration, and so on.Burns & Trauma: Antonino Gullo, Zhiyong Sheng, Zhengguo Wang, Xiaobing Fu, David Mackie, Basil A. Pruitt, Ronald G. Tompkins, Yongmin Yu, Ubaldo Armato, Xianchang Li, Xuetao Pei and the whole editorial team. Their suggestions, encouragement, and hard work will promote the growth of this forthcoming journal absolutely.I wish to acknowledge the people who have enthusiastically contributed to the launch of that Burns & Trauma will act as a successful vehicle for sharing our knowledge and thereby significantly contributing to the prevention and the management of burn injuries.I sincerely hope


Cytotoxic T lymphocyte (CTL) epitopes within the HCV non-structural proteins were identified, and proteasomal cleavage sites and helper T cell (Th) epitopes at close proximity to these CTL epitopes were analyzed using multiple prediction algorithms. This combined in silico analysis enhances the precision of identification of functional HCV-specific CTL epitopes. This approach will be applicable to the design of human vaccines not only for HCV, but also for other antigens in which T-cell responses play a crucial role.Tumor vaccine design requires prediction and validation of immunogenic MHC class I epitopes expressed by target cells as well as MHC class II epitopes expressed by antigen-presenting cells essential for the induction of optimal immune responses. Epitope prediction methods are based on different algorithms and are instrumental for a first screening of possible epitopes. However, their results do not reflect a one-to-one correlation with experimental data. We combined several  Hepatitis C virus (HCV) primarily infects and persists in human hepatocytes. Clearance of HCV involves killing virus-infected cells through cellular immunity, particularly cytotoxic T lymphocytes (CTLs). However, 80% of HCV-infected patients are not able to clear the virus, meaning HCV infection persists. Development of chronic HCV results in liver cirrhosis, and 10% to 15% of chronically infected patients develop hepatocellular carcinoma in 10 to 15 years after the primary infection. During disease progression, viral mutants arise due to the high mutation rate of the HCV genome and immune pressure .In 2011 two antiviral HCV protease inhibitors, telaprevir and boceprevir, were approved for treatment for chronic HCV infection next to/combined with the standard treatment with pegylated interferon alpha and ribavirin. Although the sustained antiviral response was improved when using these new drugs, two third of chronically HCV-infected patients are still non-responders to all available treatments ,4. AntivSpontaneous resolution of HCV infection is positively correlated to the activation of a wide T-cell repertoire recognizing multiple HCV epitopes ,7. CTL ein vitro. Furthermore, we determined the immunogenicity of the identified epitopes in vivo.In this study, using a murine model, we aimed to set up a procedure to define and subsequently verify protective HCV CTL epitopes. We selected immunogenic epitopes within conserved HCV proteins based on the results from several mathematical algorithms available. Next, we measured the binding affinity of the predicted CTL peptides to MHC class I molecules b and H-2Kb) were predicted with SYFPEITHI, NetMHCpan 2.8 and Immune epitope database and analysis resource (IEDB). HCV peptides associated with MHC class II molecules (H-2-IAb) were predicted with IEDB. Proteasomal cleavage sites of the selected HCV peptides were predicted with MAPPP, PAProC I and Netchop. Protein analyzed: nsPs of HCV genotype 1 (H77) (Genomic sequence: NC_004102.1), ovalbumin (Genomic sequence: NC_006089.3) and E7 protein of Human papillomavirus type 16 (Genomic sequence: NC_001526.2).HCV peptides associated with MHC class I molecules (H-2DLong synthetic peptides (13 to 18-mers) were kindly received from BEI Resources, NIAID, NIH: Peptide Array, Hepatitis C Virus, H77, NS2 protein, NR-3751; NS3 protein, NR-3752; NS4A protein, NR-3753; NS4B protein, NR-3754; NS5A protein, NR-3755; NS5B protein, NR-3756. Short synthetic peptides (8 to 9-mers) were manufactured by the Leiden University Medical Center, the Netherlands. The purities of the synthetic peptide were analyzed with HPLC. All peptides had a purity of >90%.b Ab (clone: AF6-88.5.5.3) and FITC-anti-H-2Db Ab (clone: 28-14-8) . The surface expression of MHC class I molecules were analyzed by FACSCalibur cytometer  and data analyzed with FlowJo software . Fluorescence index was calculated by dividing the median fluorescence intensity (MFI) value of cells incubated with SLPs by the MFI value of cells incubated with equivalent concentration of DMSO.RMA-S cells were maintained in IMDM  supplemented with 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin (Life Technologies). RMA-S cells were cultured at 26 °C for 48 h to induce expression of MHC class I molecules. Cells were then incubated with various concentrations of synthetic peptides at 26 °C for 4 h, followed by a 1-h culture at 37 °C. Cells were stained with APC-anti-H-2Kb) mice expressing both MHC class I Kb and Db molecules were obtained from Harlan CPB  and kept under the institute guidelines. Mice were 8 to 10 weeks of age at the start of all experiments. Animal experiments were approved by the Institutional Animal Care and Use Committee of the University Medical Center of Groningen (DEC 5946).Specific pathogen-free female inbred C57BL/6JOlaHsd (H-26 rSFV in 50 μL (25 μL/thigh muscle) under anesthesia (isoflurane/O2). For negative controls, PBS was injected intramuscularly.SFV plasmids  containing HCV non-structure proteins were constructed by sub-cloning the HCV fragments from the full-length cDNA of HCV H77 genotype 1a consensus sequence (H/FL) into pSFVe  . H/FL wa® 660-anti-CD107a Ab (clone: eBio1D4B) and eFluor® 660-anti-CD107b Ab (clone: eBioABL-93) in a 96-well plate at 37 °C with 5% CO2. As a negative control, splenocytes were cultured without peptides but in the presence of anti-CD28 Ab. One hour after culture, brefeldin A (10 μg/mL) was added and the cultures were incubated for another 4 h. Cells were harvested, washed and stained with LIVE/DEAD® fixable violet dead cell stain kit (Life Technologies), followed by surface staining with PE-Cy7-anti-CD8a Ab (clone: 53-6.7) and intracellular staining with PerCP-Cyanine5.5-anti-IFN-γ Ab (clone: XMG1.2). Unless otherwise indicated, antibodies were purchased from eBioscience. FACS analysis was conducted with LSR-II flow cytometer (BD Biosciences), and data was analyzed with FlowJo software .Splenocytes isolated from immunized mice were cultured with 10 μg/mL of synthetic peptides in the presence of anti-CD28 Ab , eFluorb/Db. The first round of HCV CTL epitope selection was based on predictions from SYFPEITHI (1–16 (H-2d) [46–63  [1–16 and NS5B46–63, were predicted as strong binders by both NetMHCpan 2.8 and IEDB.The MHC class I epitope prediction algorithms SYFPEITHI , NetMHCpYFPEITHI . Eighteealleles) , which ab and H-2Kb binders. HCV SLPs were classified as weak, intermediate and strong binders according to the ability to stabilize MHC class I molecules on the surface of RMA-S cells. Known CTL epitope peptides of ovalbumin (OVA257–264) and human papillomavirus (HPV E749–57) were included as positive controls for H-2Kb and H-2Db binding, respectively. Among 13 predicted HCV H-2Db binders, predicted by at least one algorithm , there was one strong binder (NS3507–524), two intermediate binders (NS3547–563 and NS5B329–346) and three weak binders . Three predicted H-2Kb binders (open circles) stabilized H-2Db molecules and were considered intermediate binders  , one was classified as an intermediate binder (NS5B152–169) and two were weak binders . Two predicted H-2Db binders (solid circle) stabilized H-2Kb molecules and were considered weak binders (NS3165–180 and NS5B329–346)  containing the selected CTL epitopes based on the prediction of the three algorithms  were det525–542) a. In ten329–346) b.323–340, NS3507–524, NS3601–618, NS5B1–16, NS5B46–63, NS5B423–439); (ii) CTL epitope has to be predicted by at least 1 algorithm and classified as intermediate or strong binder from the result of MHC I stabilization assay (NS5B152–169). Since the accuracy of prediction greatly increased when peptides were analyzed with two or more algorithms, four extra CTL epitopes, which were predicted by at least two algorithms as strong binders, were selected  (257–264) and human papillomavirus (HPV E749–57), which are strong H-2Kb and H-2Db binders, respectively, were selected as positive controls.Six out of nine SLPs that contain a CTL epitope predicted by two or more algorithms were able to bind and stabilize MHC class I molecules . However280–287) . And alsb and H-2Kb molecules increased with increasing concentration of peptide. The kinetic of the stabilization of H-2Db molecules was similar for all selected short peptides. The plateau of stabilization was reached at approximately 30 μM of peptides , there was a dose dependent increase of FI and the plateau of stabilization was not detectable even at high peptide concentration (100 μM). For intermediate binders , stabilization of H-2Kb molecules was modest at low peptide concentration (0.3–10 μM). However, the stabilization effect increased exponentially once the concentration was above 30 μM. This observation may explain why we did not see strong H-2Kb binders when SLPs at 10 μM were used in the MHC I stabilization assay  (Binding affinity of selected short synthetic peptides on MHC class I molecules was determined by MHC I stabilization assay with various concentrations of peptide . As expepeptides a. Two kion assay . Resultsmatched) .Under physiological conditions, proteins have to be cleaved by proteasomes into short peptides in order to be loaded onto MHC class I molecules. Proteasomal cleavage sites were predicted with MAPPP, PAProC I and Netchop. MAPPP and Netchop predict the cleavage site of constitutive proteasomes and PAProC I predicts cleavage site of both constitutive proteasomes and immunoproteasomes. From the result of at least two prediction algorithms, proteasomal cleavage sites were present at all strong MHC class I binders (+++) .in vivo [b and H-2Kb), overlap with a predicted MHC class II epitope (IEDB rank < 10) (NS3514–522 and NS5B2–10). Flanking of MHC class I epitopes with predicted MHC class II epitopes was also observed in the positive control, OVA257–264  against NS2139–147, NS3603–611, NS5B2–10 and NS5B157–165. When no peptides were added to the splenocytes, we already observed the presence of endogenous CD107a/b+IFN-γ+CD8+ cells from mice immunized with any rSFV particles but not from mice immunized with PBS. This indicates that rSFV immunizations in general induced functional CD8+ T-cell responses. To check for a specific response against peptide, background (without peptide re-stimulation) subtraction was applied. Frequencies above zero indicate specific response against the re-stimulating peptide. CD8+ T-cell response against NS3603–611 [2–10 and NS5B157–165 were low and induced in mice immunized with rSFVeNS5A/B' or rSFVeNS2'-5B'. A very low response against NS2139–147 was observed only in mice immunized with rSFVeNS2'-5B', not in mice with other immunizations. Of note, all responding peptides were predicted by at least one algorithm for MHC class I prediction. Proteasomal cleavages sites were presented in the carboxyterminals of all four peptides. Three peptides  contained predicted MHC class II epitopes in close proximity to their CTL epitopes with relatively high rankings . Ideally one should identify HCV epitopes expressed by the patients’ infected cells and induce T-cell responses against the identified epitopes with vaccine immunizations. Identification of HCV epitopes from cell lines expressing HLA-A2 and HCV proteins has been performed with electro-spray ionization quadrupole time-of-flight mass spectrometry/tandem mass spectrometry (ESI Q-TOF-MS/MS) and the data were consistent with the results predicted from mathematical algorithms . This meb molecules on RMA-S cells. We next showed that immunization with rSFV expressing all nsPs induced a strong epitope-specific T-cell response against one out of ten H-2b binders and a weak response against three epitopes.Here we show that the prediction accuracy for the identification of potential CTL epitopes is improved by combining several algorithms for MHC class I epitope and proteasomal cleavage site prediction, and possibly even MHC class II epitope prediction. We narrowed down all HCV nsPs to 22 CTL epitopes using prediction algorithms, of which 10 were proven to bind to H-2i.e., the programs were trained with large databases for reliable results. Therefore, most algorithms favor the detection of immunodominant epitopes. Predictions were made with allele-specific motif methods based on amino acid sequences [Computational analyses used in this study are machine-learning algorithms, equences ,31. For equences . With alb and H-2Kb binders contained proteasomal cleavage sites predicted by at least two algorithms.Antigen processing involves a complex regulation of antigen degradation, interaction with chaperones, binding to the peptide-loading complex, loading onto MHC class I molecules and transport to the cell surface . For bot+ T cells relies on both recognition of MHC I-peptide complexes on APCs and co-stimulatory activation signals provided by adjacent cells such as APCs and/or CD4+ helper T cells. Activated CD4+ helper T cells orchestrate the activation of CD8+ T cells through secretion of IL-2 and/or activation of dendritic cells for up-regulation of MHC I molecules and antigen presentation [+ T cell epitopes such as OVA257–264 contain predicted high-affinity Th epitopes that overlap or are in close proximity to one another [+ and CD4+ T cells simultaneously [+ T-cells responses [+ T cells [+ T cells as helper T cells provide both antigen stimulation and co-stimulation [139–147 and NS5B2–10) overlapped with strong predicted Th epitopes (603–611) contains intermediate predicted Th epitopes at the 5' ends of the CTL epitope. Remarkably, MHC class II epitope prediction by IEDB may not identify all possible Th epitopes, since, for example, it has been shown that E744–62, binds to MHC class II molecules in vitro but was not predicted as a strong binder [in vitro experiments such as an MHC class II binding assay. For the design of vaccines expressing specific CTL epitopes, this could be considered to include Th epitopes that overlap or are in close proximity with CTL epitopes to enhance immune responses. In a separate study, we demonstrated that inclusion of strong universal helper-epitopes in an SFV replicon vaccine expressing human papillomavirus (HPV) antigens strongly enhanced the frequency of HPV specific CTLs [Activation of CD8entation . Interes another ,38. Thisaneously . Alternaesponses ,41,42. I T cells ,43,44. Tmulation . Peptideepitopes . The immg binder . As MHC + T-cell responses (NS3603–611) and 3 out of 22 inducing minor CD8+ T-cell responses . Subdominant CTL epitopes could not be identified with rSFVeNS2'-5B', rSFVeNS3/4A or rSFVeNS5A/B' immunizations. Inclusion of more epitopes may increase competition between T cells of different specificity, resulting in a reduced response to subdominant epitopes [+ T cells and APC and thus inability to fully activate T cells [in silico predictions is to immunize with synthetic long peptides expressing only one particular CTL epitope and its flanking sequences. In this way, the immune response will be directed to that specific epitope.In this study, we identified 1 out of 22 predicted CTL epitopes inducing strong CD8epitopes . Further T cells . Althougin silico approaches to epitope prediction in vivo. We showed that responses against predicted dominant CTL epitopes were induced in mice immunized with HCV-nsPs expressing rSFV. In vivo relevance of subdominant epitopes, which were predicted by mathematical algorithms, may have to be confirmed with peptide immunizations. Combining epitope prediction algorithms with proteasomal cleavage site algorithms increases the accuracy of CTL epitope identification. The predictive value of strong Th epitopes overlapping with, or in close proximity to, CTL epitopes remains to be established. Thus, potential CTL with or without Th epitopes as candidates for therapeutic human vaccine development can be identified and narrowed down. For clinical practice, the establishment of an ex vivo stimulation assay with patients’ PBMCs is warranted to select the rationally designed candidate vaccine that is most immunogenic for that specific patient.To conclude, we validated


X-ray diffraction and electron diffraction spectroscopy results indicate that doping with 2 mol% Bi2O3 and adding 10 mol% yttria result in a stable zirconia cubic phase. Adding Bi2O3 as a dopant increases the density of zirconia to above 96%, while reducing its normal sintering temperature by approximately 250 °C. Moreover, electrical impedance analyses show that adding Bi2O3 enhances the conductivity of zirconia, improving its capability as a solid electrolyte for intermediate or even lower temperatures.Bismuth oxide (Bi Because of its good high-temperature electrical conductivity, thermal stability, and chemical stability, 8 mol% yttria-stabilized zirconia (8YSZ) ceramic material has been used as an oxygen ion conductor in oxygen sensors ,2,3,4 an2O3 as a sintering aid during sintering can effectively reduce the sintering temperature of zirconia, as well as increase its density. Hirano et al. [2O3 decreases the sintering temperature of scandia-stabilized zirconia ceramics by 300 °C, while allowing its electrical conductivity at 1000 °C and 800 °C to reach 0.33 and 0.12 S/cm, respectively. Yeh et al. [2O3 were able to effectively reduce sintering temperature and promote ceramic densification. Besides serving as a sintering aid, Bi2O3 is also a very good oxygen ion conductor, and adding Bi3+ to zirconia can also produce oxygen vacancies. Bai et al. [2O3 on the physical and electrochemical properties of scandia-stabilized zirconia, and they found that the conductivity of samples doped with 2 mol% Bi2O3 was also enhanced. Similar results were also discovered by Raghvendra and Prabhakar Singh in Bi2O3 doped calcia-stabilized zirconia [et al. [et al. [2O3 on the ionic conductivity of YSZ, but in those studies it was found that the enhancing effect of Bi2O3 on the ionic conductivity of YSZ was very limited. Verkerk et al. [2O3 has a negative influence on the electrical conductivity of YSZ, but they did not study the properties of Bi2O3 doped YSZ in a completely stable cubic phase [2O3 can exert on the electrical conductivity of YSZ are not well understood.Using Bio et al.  reportedi et al.  studied zirconia  and Sara [et al. . Winnubs [et al.  studied k et al.  discover2O3 might also have a influence like that and be beneficial to the improment of the conductivity of YSZ.In addition, some researchers ,21 have 2O3 on the polymorphic phasing and ionic conductivity of YSZ via X-ray diffraction (XRD), electron diffraction, electron microscopy, dielectric spectroscopy, etc. Then we report the effects of bismuth oxide doping on reducing zirconia sintering temperature, increasing the density and conductivity.This study investigates the influence of Bi2O3 doped YSZ. 8 mol% YSZ (8YSZ) and 10 mol% YSZ (10YSZ) were each mixed with 2 mol% [2O3 powder , adding ethanol as an abrasive, and ground in a planetary ball mill for 10 h. The ground powder was then pressed into disks with a diameter of 10 mm and a thickness of 2 mm via cold isostatic pressing. The disks were then sintered in air atmosphere for 2 h at 1100, 1200, 1300, and 1400 °C, respectively. An 8YSZ ceramic disk was sintered at 1550 °C for 4 h as a control.In this experiment, zirconia powder  was used as a reactant in the solid state reaction to prepare Bih 2 mol% ,22,23 BiThe density of the ceramic disks was determined via the Archimedes method. Crystal structure was studied via X-ray diffraction . Microscopic morphology was characterized with a scanning electron microscope . Samples for transmission electron microscopy (TEM) and spectroscopy were prepared via a dual beam-focused ion beam  instrument. A G2 F20 transmission electron microscope  was used to perform TEM and high angle annular dark field scanning transmission electron microscopy (HADDF-STEM) analysis on Bi10YSZ samples sintered at 1300 °C. X-ray Energy Dispersive Spectroscopy (EDS) data was acquired at STEM model. For electrodes, the sample disks were first painted with a thin and homogeneous silver slurry lamella as big as the disk surface, and then sintered at 700 °C for 30 min to get bright and smooth silver electrodes. The electrical properties of the samples were measured using a VersaSTAT electrochemical workstation  at 50 °C intervals from 350 °C to 650 °C, with the frequency range of 0.1 Hz–1 MHz and a voltage of 10 mV.2O3-doped 8YSZ (2Bi-8YSZ) samples sintered at varying temperatures and 8YSZ sintered at 1550 °C. Diffraction shows that the primary structure of the sample is cubic phase (c), with small amounts of the monoclinic phase (m). The monoclinic phase exhibits low electrical conductivity and causes material instability due to the polymorphic transitions, thereby making it an undesirable phase for zirconia as a solid electrolyte material. The purpose of the stabilization with yttria is to reduce and eliminate the monoclinic phase in zirconia. 2O3 failed to produce a zirconia sample with a complete cubic phase.2; these results were also verified via selected area electron diffraction analysis.2O3 can significantly improve material density and promote grain growth because of introducing the liquid phase sintering effect [2O3 improved the sample density, and the grain size increased as the sintering temperature increased. Analysis of the XRD spectra and the SEM picture show that 2Bi-10YSZ can form stabilized zirconia with a uniform, dense, and complete cubic phase structure. This type of homogeneous, stable full cubic zirconia will likely exhibit better electrical properties and stability.g effect ,12,18. T2O3 presented at the grain boundaries can act as fillers in the triangular boundary and increase the material density with the liquid sintering. The high resolution pictures in 2O3 grain. The high resolution pictures show that there are only 2–3 atomic layers at the transition interphase between the zirconia grain boundaries, while the transition interphase is slightly widened between the zirconia and Bi2O3 grains. There was no amorphous layer in both grain boundaries. The impurities present in an amorphous layer are high resistance materials, which will block the conduction and migration of oxygen vacancies. Thus, the lack of an amorphous layer at the grain boundary can also enhance conductivity. As 2O3 grains contact and converge nicely at the grain boundary, which makes the Bi2O3 located in the boundary looks like a bridge linking the zirconia grains together.As 2O3 is dissolved in the zirconia lattice, that is, Bi2O3 and yttria have a co-doping effect in the zirconia lattice. EDS spectrum analysis (2O3 is present in grains and grain boundaries, and enhances the density and conductivity of the material.analysis a,c show i.e., of about 10−10 F and 10−7 F for the grains and the grain boundaries, respectively.2O3 doped calcium oxide stabilized zirconia materials; and it has also been discovered by Bai et al. [2O3-doped scandia stabilized zirconia.The figure shows that compared with the 8YSZ sample, which has almost the highest electrical conductivity among all YSZ materials ,5,26, thi et al.  in Bi2O32O3 improves the conductivity to about four times the original conductivity.Comparison between 2Bi-10YSZ samples of different sintering temperatures shows that the sample sintered at 1300 °C exhibits the lowest total resistance. 2O3, affording a high density material. Another important reason for the boundary improvement is attributed to the aggregation and segregation of some Bi2O3. Bismuth oxide-based electrolyte is a widely used solid electrolyte material, which has a higher ionic conductivity than zirconia-based materials. Because these materials contain more oxygen vacancies on one side. On the other side, Bi3+ has a high polarization capability due to the influence of 6s2 lone pair electrons and a strong ability to adapt and integrate to a relatively disordered surroundings [2O3 segregated at the grain boundaries can remove impurities, such as aluminum oxide, silicon oxide, etc. [2O3 segregated at grain boundaries bridges the grains for oxygen transportation and increases the sample conductivity. And for the part of grain conductivity, the Bi2O3 infiltrated into the zirconia grains can also generate oxygen vacancies due to the charge compensation mechanism, of which the defect reaction can be represented by the following equation: 2O3BiO.In oundings ,28,29. Tde, etc. ,31, so t2O3 is added in YSZ, the activation energies increase. The increace may be ascribed to the added oxygen vacancies with more correlation effects and the lattice distortion in both the grain and grain boundary caused by the solution of Bi2O3 in the grain and the segregation of Bi2O3 in the grain boundary, respectively. Thus, there are more vacancies and defect positions in the Bi10YSZ, which is benificial to the ions transport and then the conductivity of the sample becomes more sensitive to the temperature.However, at low temperatures, these oxygen vacancies cannot migrate freely, and most of which will bind and interact with cations. Thus, at lower temperatures, the 2Bi-10YSZ grain conductivity has no big difference with that of 8YSZ. When the temperature becomes higher, more vacancies get free from association to migrate. The conductivity of the 2Bi-10YSZ grain increases notably in the latter range and the grain conductivity of 2Bi-10YSZ also become higher than that of 8YSZ. 2O3 enhances the grain growth of 10YSZ, reduces its sintering temperature and improves its ionic conductivity. 10 mol% YSZ doped with 2 mol% Bi2O3 exists in a completely stable cubic phase after sintering at 1300 °C. Due to the filling effect of Bi2O3 at grain boundaries, 2Bi-10YSZ has a higher conductivity of 0.013 S/cm at 650 °C, which is about four times higher than that of 8YSZ.Doping with Bi


There are errors in the author affiliations. The affiliations should appear as shown here:1,2,4,5, Ying Li1,2,4,5, Yingke Zhang1,2,4,5, Lin Zhang1,2,4,5, Zilian Wang3, Xuzhi Zhang1,2,4,5, Lian Gui1,2,4,5, Junqi Huang1,2,4,5*.Yiming Qi1 Guangdong Provincial Key Laboratory of Organ Donation and Transplant Immunology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, PR China, 2 Organ Transplant Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, PR China, 3 Department of Obstetrics and Gynecology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, PR China, 4 Key Laboratory of Tropical Diseases Control, Ministry of Education, Guangzhou, PR China, 5 Institute of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, PR Chinahttp://isis.nsfc.gov.cn; Natural Science Foundation of Guangdong Province  http://gdsf.gdstc.gov.cn; Guangdong Province Scientific Technology Project  http://www.gdstc.gov.cn; Guangzhou City Scientific Technology Project  http://apply.gysi.gov.cn; The Fundamental Research Funds for the Central Universities (no. 10YKPY31) http://mso.sysu.edu.cn; Guangdong Provicial Key Laboratory Construction Projection on Organ Donation and Transplant Immunology (no.2013A061401007) http://www.gdstc.gov.cn. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.2. There are errors in the Funding section. The correct funding information is as follows: This work was supported by the National Natural Science Foundation of China (no.30872350 and no.31370870)


Journal of Medical Radiation Sciences (JMRS). The JMRS is a joint journal publication of the Australian Institute of Radiography (AIR) and New Zealand Institute of Medical Radiation Technology (NZIMRT). The JMRS constitutes the amalgamation of The Radiographer in Australia and Shadows in New Zealand  of There were many possible journal names suggested by members of the AIR and NZIMRT. The name that received the most votes from members of the ERB was JMRS. This was approved by both boards of the AIR and NZIMRT and officially became the JMRS on 3 September 2012.Journal of Medical Radiation Sciences, as the name implies, encompasses the three strands of diagnostic radiography, radiation therapy, and nuclear medicine. Diagnostic radiography includes medical imaging modalities that utilize x-rays plus sonography and magnetic resonance imaging.Radiation therapy encompasses all the techniques and technologies in external beam radiation therapy and brachytherapy. Nuclear medicine and molecular imaging involve the use of radiopharmaceuticals, gamma, and hybrid cameras for the diagnosis and treatment of disease.The topics covered range from technical, education, and patient care–related issues. The journal aim and scope of JMRS was created to reflect clinical practice where collaboration between members of the multi-disciplinary team occurs Box . TherefoJournal of Medical Radiation Sciences (JMRS) is an international and multi-disciplinary, peer-reviewed journal that accepts manuscripts related to medical imaging/diagnostic radiography, radiation therapy, nuclear medicine, medical ultrasound/sonography, and the complementary disciplines of medical physics, radiology, radiation oncology, nursing, psychology, and sociology. 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The practice of psychosomatic medicine is closely related to that of behavioral medicine, particularly in terms of the bio-psycho-social aspect of health, as has been demonstrated in the previous articles of BioPsychoSocial Medicine –3. BehavFor example, among the major psychosomatic medicine journals, the percentage of articles focusing on behavioral medicine [MeSH] was estimated by a PubMed search in September 2015, and the results were as follows; 4.5 % (=212/4706) for “Psychosomatic Medicine”, 3.7 % (=164/4388) for “Psychosomatics”, 2.9 % (=129/4488) for “Journal of Psychosomatic Research”, and 2.2 % (=60/2768) for =2.2 %. It was 8.8 % (=16/181) for this journal, “BioPsychoSocial Medicine”, which was launched in 2007.In the same way, the percentage of articles focusing on psychosomatic OR mind/body [text word] was estimated for the major behavioral medicine journals, and the results were as follows; 2.3 % =12/531) for “Behavioral Medicine”, 1.3 % (=11/856) for “International Journal of Behavioral Medicine”, and 0.7 % (=12/1626) for Journal of Behavioral Medicine. The percentages were smaller than those of the psychosomatic medicine journals. These figures mean that it is necessary for specialists engaging in psychosomatic medicine not only to sharpen their own research into behavioral medicine but also to broaden their findings toward specialists in behavioral medicine. Psychosomatic medicine encompasses all aspects of the interrelationships between the biological, psychological, social, and behavioral factors of health and illness, and is not limited to mind and body connections in humans. Thus, it is meaningful that behavioral and social aspects of health and illness are discussed in the current issue of this journal [2/531 forIn Japan, a total of 80 medical schools exist, and curriculums for medical students have been changed in accordance with international standards. In the revised curriculums, aspects of behavioral and social sciences must be included and it is a great chance to facilitate behavioral medicine. In these circumstances, the Japanese Society of Behavioral Medicine established a working group to develop a core curriculum for behavioral medicine and has discussed the clinical, psychological, and public health aspects of behavioral medicine. Specifically, two purposes are intended in this special series. The first is to summarize the behavioral and social aspects of health and illness in the psychosomatic filed in order to achieve full cooperation among different specialists in terms of prevention and treatment of psychosomatic illnesses as well as promotion of mind/body health. The second is to emphasize the importance of education on behavioral medicine, exemplified by the establishment of a core curriculum for behavioral medicine in Japanese medical schools.


Acinetobacter baumannii as a problematic microorganism in inpatient facilities, particularly intensive care units, has increased over time. This review aims to draw attention to (i) the historical emergence of carbapenem-resistant Acinetobacter baumannii, (ii) the current status of surveillance needs in Latin America, and (iii) recent data suggesting that A. baumannii continues to spread and evolve in hospital settings. First, we present synopsis of the series of events leading to the discovery and precise identification of this microorganism in hospital settings. Then key events in the acquisition of antibiotic-resistant genes by this microorganism are summarized, highlighting the race between new antibiotic generation and emergence of A. baumannii resistant strains. Here we review the historical development of this species as an infectious threat, the current state of its distribution, and antibiotic resistance characteristics, and we discuss future prospects for its control.Antibiotic-resistant infectious bacteria currently imply a high risk and therefore constitute a strong challenge when treating patients in hospital settings. Characterization of these species and of particular strains is a priority for the establishment of diagnostic tests and preventive procedures. The relevance of Acinetobacter infections in a hospital setting, within an intensive care unit (ICU), dates back to the 1960s [ Acinetobacter baumannii as a nosocomial pathogen was underestimated. The persistent presence of A. baumannii in the hospital sector allowed it to come into contact with antibiotics. These environmental conditions exerted such a selective pressure that successful clones with particular antibiotic resistance characteristics emerged. Previous reviews have focused on A. baumannii as a successful pathogen, and its biological aspects, epidemiology, pathogenicity factors, and global spread have also been addressed. Here we review the increase of antibiotic-resistant Acinetobacter in recent reports and summarize the multilateral work being carried out in order to control this pathogen. As multinational networks have emerged based on the information provided by these reports, we update the available information and give an example of the importance of local reports. This review is organized as a timeline divided in three periods. The first period includes the decades of the 60s and 70s when the Acinetobacter gender started to be reported in hospitals. The second period includes the decades of the 80s and 90s during which baumannii species was determined and the reports of the organism's carbapenem resistance started. The last period includes years 2000 to 2015, during which World Health Organization (WHO) has issued calls for containment of antimicrobial resistance.The first report of Acinetobacter was first described in 1911. The bacterium isolated from soil was called Micrococcus calcoaceticus, Achromobacter, Alcaligenes, Bacterium anitratum, Moraxella glucidolytica, Neisseria winogradsky, Alcaligenes haemolysans, Mima polymorpha, and Moraxella lwoffii [ Acinetobacter  was proposed in 1954 by Brisou and Prevot and it was accepted in 1968 [ Bergey's Manual of Systematic Bacteriology, where it was described as having only one species: Acinetobacter calcoaceticus [ Acinetobacter. These inconsistencies arose due to the fact that the members of this genus have different catabolic pathways that allow them to adapt to most substrates [ A. calcoaceticus-baumannii includes four genospecies: genospecies 1, A. calcoaceticus; genospecies 2, A. baumannii; genospecies 3, A. pittii; and genospecies 13TU, A. nosocomialis. Among these, A. baumannii is the most important in the clinical context [The genus lwoffii . The cur in 1968 . Finally context , since i A. baumannii in nosocomial infections in reports before the year 1974. At that time, there was a specific designation for a single species: A. calcoaceticus. Moreover, the routine laboratory diagnostic identification was carried out by phenotypic methods that are not useful for species designation. Particularly, these methods do not allow a distinction within the calcoaceticus-baumannii complex. Here we use identification and antibiotic resistance data arising from hospital settings where the pathogen has become successful.It is difficult to determine the presence of A. baumannii in hospital settings is the following: How did it become a pathogen? Olson has pointed out at least three contributing elements. First, water is an important reservoir for clinically significant Gram-Negative Nonfermenting (GN-NF) bacteria. Second, this species is a water-related organism, and solutions as well as surgical, medical, and clinical instruments can harbor high levels of bacteria. With bacteria already present, debilitated patients became susceptible to infection by this opportunistic microorganism, giving rise to nosocomial outbreaks [ Acinetobacter outbreaks were easily controlled with common antibiotics including β-lactams and sulfonamides. However, these treatments became ineffective very soon, coincident with early reports of emerging microbial resistance to these antibiotics. Unfortunately, since the 1970s, microbial resistance has only increased; at the same time, the creation of effective new antibiotics has declined  strains and the creation of international surveillance networks [ Acinetobacter. The worldwide presence of antibiotic-resistant microbes has made surveillance shift from a local scale to a global one  out of 12,000 annual Acinetobacter infections were MDR, which in turn has led to about 500 annual deaths attributed to these infections [http://www.cdc.gov/drugresistance/tatfar/about.html) [ A. baumannii was noticed for the first time in 2001 by Gales and collaborators [In the United States, the national public health surveillance system is a collaborative project between CDC, FDA, USDA, and state and local health departments. The CDC together with the Interagency Task Force on Antimicrobial Resistance (ITFAR) coordinates different agencies for the containment of resistance. The 2013 CDC report shows thatfections . Acknowlut.html) . The higut.html) , 43. Addborators . Acinetobacter isolates, specific reports have rarely arisen from Chilean agencies. In Brazil, apparently decreasing rates were reported between 1997 and 2011 (13.6% to 2.2%) [ Acinetobacter surveillance in hospital settings [Despite a general increase of carbapenem-resistantto 2.2%) . Howeverto 2.2%) . In addisettings . This ne A. baumannii is one of the most common pathogens [ A. baumannii [Currently, some countries in Asia are carrying out surveillance at a national level. These include Malaysia, Thailand, Pakistan, India, and Taiwan, whereathogens . In 2011athogens . The exiaumannii . Internaaumannii , 52. Howaumannii .The data collected by international networks have allowed researchers to generate a global picture of the circulating antibiotic-resistant strains. Recent studies have shown the presence of three successful clones called ribogroups or World Wide Types distributed in some countries: WWI, WWII, and WWIII . Interna A. baumannii took place [ A. baumannii outbreaks after the arrival of military patients from Iraq [The beginning of the 21st century was marked by a sharp increase in international mobility. The intense travelling across countries and continents has had a strong effect on the dissemination of infectious bacteria throughout the world . This isok place . Anotherrom Iraq . Howeverrom Iraq .A positive outcome of the establishment of international surveillance networks is that member countries have to provide information. This baseline information is useful to understand the development and dissemination of antibiotic-resistant strains. Furthermore, the main positive impact perceived is the standardization of methods across countries and an improved laboratory capacity, both of which enhance the quality of testing. This is important because surveillance information allows the establishment of specific strategies targeting each country and/or region. These strategies can be designed according to impact, and they can be followed by particular indicators that can be measured at the surveillance centers belonging to the network . Despite A. baumannii in this country is presented in  A. baumannii incidence was 74% for 2010 [Secretaría de Salud (SESA)) [Instituto Nacional de Referencia Epidemiológica (INDRE)) program, as part of the Latin American network for monitoring resistance to antibiotics (ReLAVRE). However, no recent information on A. baumannii was available from these sources [ Acinetobacter in hospital settings in Mexico. Furthermore, a multicenter study carried out recently provides valuable information to be integrated into the global picture [ A. baumannii in various hospitals from different regions in this country. It found a predominance of the Iberian-American clonal complex CC636 and the international clonal complex CC92. It also reported a regional distribution of oxacilinases OXA-72 and OXA-239 in the North and South of Mexico, respectively. An important observation derived from this study is the presence of different STs constituting two clonal complexes among the hospitals included. It is important to note that this kind of distribution had been previously reported in the USA, in Brazil [ A. baumannii [Different countries face different challenges; here we illustrate that although continuous surveillance programs are ideal, contributions of individual hospitals and research groups can provide valuable information. Here available data on Mexico is described as an example to illustrate the value of these efforts. An early limitation in the case of Mexico (1990s) was the use of phenotypic methods; as a consequence, such information should at least be used with caution when integrated with more recent data , 65. A sfor 2010  and it bfor 2010 . Also, tfor 2010 . Two addfor 2010 , 46. In  (SESA)) . There a (SESA))  and the  sources . Neverth picture . This stn Brazil , and morn Brazil . In addiaumannii .Increased mobilization of people worldwide is an intrinsic component of globalization. The increase in medical tourism increases the probability of MDR bacteria exchange. A clear example is the case of Mexico, which is considered one of the top medical tourism destinations , 73. As Bacteria causing specific diseases should be closely monitored through the establishment of precise programs. Such has been the case with tuberculosis: multidisciplinary action groups, like the Directly Observed Treatment Short-Course (DOTS) program, have established appropriate programs to prevent the spread of resistance. This strategy includes five important components: government, case detection, standardized treatment observed by healthcare workers, drug supply, and a standardized recording and reporting system. The implementation of a public health policy at an international level increases the potential success of the intervention . The res A. baumannii and make them available.Multicenter collaboration among hospitals remains a relevant component of surveillance and its continuity should be supported. However, it is critical to start working towards the establishment of a national surveillance program as part of the Global Strategy for Containment of Antimicrobial Resistance in medical centers . This gl


Brucella melitensis. BSR0602, one sRNA that was highly induced in stationary phase, was further examined and found to modulate the intracellular survival of B. melitensis. BSR0602 was present at very high levels in vitro under stresses similar to those encountered during infection in host macrophages. Furthermore, BSR0602 was found to be highly expressed in the spleens of infected mice, suggesting its potential role in the control of pathogenesis. BSR0602 targets the mRNAs coding for gntR, a global transcriptional regulator, which is required for B. melitensis virulence. Overexpression of BSR0602 results in distinct reduction in the gntR mRNA level. B. melitensis with high level of BSR0602 is defective in bacteria intracellular survival in macrophages and defective in growth in the spleens of infected mice. Therefore, BSR0602 may directly inhibit the expression of gntR, which then impairs Brucellae intracellular survival and contributes to Brucella infection. Our findings suggest that BSR0602 is responsible for bacterial adaptation to stress conditions and thus modulate B. melitensis intracellular survival.Bacterial small non-coding RNAs (sRNAs) are gene expression modulators respond to environmental changes, stressful conditions, and pathogenesis. In this study, by using a combined bioinformatic and experimental approach, eight novel sRNA genes were identified in intracellular pathogen  The survival of pathogens within a host is highly dependent upon their ability to sense and adapt to changes in the host environments. This entails a coordinated regulation of virulence genes in response to various environmental stresses. Recently, small non-coding RNAs (sRNAs) have attracted a great interest as important regulators in both eukaryotes and prokaryotes Storz, . In diffBrucella spp. is facultative intracellular pathogenic α-proteobacteria that causes undulant fever, endocarditis, arthritis, and osteomyelitis in humans and abortion in domestic animals  at 37°C. Escherichia coli strain DH5α was grown on Luria–Bertani (LB) medium. Plasmid pBBR1MCS-4, a broad host range plasmid capable of replicating in Brucella, was kindly provided by Professor Kenneth M. Peterson   as recommended by the manufacturer. Then, RNA samples were treated with DNAse I (Promega) to eliminate contaminating genomic DNA. RNA quantity and quality were assessed using ND-1000 Spectrophotometer Nanodrop (Technologies) and agarose gel electrophoresis.Total RNA was extracted from + membranes (GE) via electroblotting. The membranes were UV-cross-linked and prehybridized for 45 min, and 3′-end DIG-labeled RNA probes were added. The membranes were then hybridized overnight at 68°C in a DIG Easy Hyb according to the manufacturer’s protocols.Northern blot analyses were carried out using a DIG northern starter kit (Roche) according to the manufacturer’s protocol as described previously  as recommended by manufacturer’s instructions. Prior to initiating the 3′ rapid amplification of cDNA ends (RACE) protocol, total RNA was polyadenylated by treatment with poly(A) polymerase (Ambion) at 37°C for 1 h. The PCR products were cloned into pMD19-T Vector , and then the clones were sequenced and analyzed. For each RACE analysis, 6–10 clones were sequenced, and the farthest 5′ (3′) end was considered as the 5′ (3′) end of the sRNA.in vitro environmental stress were compared by quantitative RT-PCR (qRT-PCR). Samples were amplified in a 25 μL volumes containing 12.5 μL of 2× SYBR Green I Master Mix , 100 nM each primer, and 1 μL of cDNA sample. Thermocycling conditions were as follows: 10 min at 95°C for pre-incubation, and then 45 cycles of amplification . The primers used for qRT-PCR are listed in Table S1 in Supplementary Material. All primer sets showed standard curves with R2 values of >0.980, 90–110% reaction efficiencies, and only one peak in dissociation curves. Relative transcriptional level was determined by the methods of 2−ΔΔCt as described previously  extraction method. Further qRT-PCR analysis was carried out as described above.For transcription analysis during mouse infection, 6- to 8-week-old female BALB/c mice (five per time point) were infected intraperitoneally with 2 × 10B. melitensis 16M , and the primer sets BSR0602-express-For/Rev or nonsense sRNA-express-For/Rev, respectively. The amplified DNA fragments were digested with XbaI and treated with polynucleotide kinase, and the digested/treated fragments were then cloned into a derivative of pZE12 as described previously , and specific primers . The PCR-amplified DNA fragments were digested with NsiI and NheI, and ligated into pXG10-SF  were transformed either with a target gfp fusion plasmid alone or with a combination of a target gfp fusion plasmid and a sRNA expression plasmid. The levels of GFP in the E. coli strains were assessed by immunoblot analysis using the methods described previously  at 37°C in TSB medium. The effects of various stresses were tested as follows: to determine the effect of high-salinity or high-osmolarity stress, the cells were incubated at 37°C for 20 min in the presence of NaCl (1.5 M); for acidification stress, the cells were incubated at 37°C for 15 min in TSB medium at pH 3.0; for oxidative stress, the cells were incubated at 37°C for 40 min in the presence of 440 mM H2O2; for heat shock, the cells were transferred to pre-warmed 50°C tubes and incubated at 50°C for 60 min. After treatment, cells were diluted and plated on TSA plates to determine viability. Results are expressed as a mean percentage ±SD from three independent experiments.The susceptibility of 5 cells per well. Wild type and mutant strains or wild type and overexpression cells obtained at mid-log phase were mixed in a 1:1 ratio to generate the inoculum (2.5 × 107 CFU) for competition assays. Macrophages were infected with bacterial suspension at a MOI of 50. At 45 min post-infection, the cells were washed three times with PBS and then incubated with 50 μg/mL of gentamycin for 60 min to kill extra-cellular bacteria. The cultures were then replaced with DMEM with 20 μg/mL of gentamycin. At 4 and 24 h post the infection, the supernatant was discarded and cells were lysed, and the live bacteria were enumerated by plating in duplicate on TSA plates with or without kanamycin or ampicillin. Colony counts on plates containing antibiotics represent kanamycin-resistant ΔBSR0602 mutant or ampicillin-resistant BSR0602 overexpression strains, and these values were subtracted from the colony counts determined on plates representing both the mutant and the wild-type strains. Data are presented as a log10 value of CFU averaged over five wells.Murine macrophage-like RAW264.7 were used to assess survival capability of 16MΔBSR0602 mutant, 16M-BSR0602 and their wild type strain 16M. In brief, monolayers of macrophages were seeded in 24 well plates 1 day prior to infection at 5 × 106 CFU/mL) representing a 1:1 ratio of wild-type B. melitensis 16M to mutant or overexpression strains. At 24 h post the inoculation, the infected mice were sacrificed by cervical dislocation and spleens were removed aseptically and homogenized with PBS containing 0.1% Triton X-100. Serial dilutions of spleen homogenates were prepared and plated in duplicate on TSA plates with or without kanamycin or ampicillin, and the CFU were counted after 4 days of infection at 37°C. The competitive index (CI) values were calculated from the ratios of total input and recovered wild-type and kanamycin-resistant ΔBSR0602 mutant (or ampicillin-resistant BSR0602 overexpression strains) CFU as previously described  based on genome annotation. The intergenic regions (IGRs) with a minimal size of 80 bp were then subjected to BLAST against all Brucella spp. genomic sequences available at the NCBI web site. Hits with an E value <0.001 were thought to be B. melitensis specific and thus exclude from further analyses. The replication origin, putative pseudogenes, and intergenic sequences encoding tRNAs or rRNAs were also excluded. Promoters were identified in IGRs with pftools 2.3Intergenic sequences were extracted from the Brucella small non-coding RNA,” followed by the gene number of the adjacent downstream protein-coding gene. The sequences of these sRNA candidates were conserved in all Brucella species. Furthermore, it seems that these sequences could not encode small peptides as they lacked appropriately positioned start and stop codons. The genomic coordinates in B. melitensis 16M, the transcription orientation, flanking genes, and predicted lengths of these sRNAs candidates are shown in Table Based upon these analysis criteria, a total of 21 candidate sRNA genes were predicted. These sRNA candidates were named BSR for “B. melitensis 16M grown in TSB7.0 at 37°C in exponential and stationary phases. For each putative sRNA region, a 3′-end DIG-labeled RNA probe was prepared for the most highly conserved portion of the sequence. Among 21 IGRs thus analyzed, 15 were reproducibly found to express transcripts . The remaining six sRNAs candidates could not be detected by northern blot analysis, possibly resulted from that these candidate sRNAs were probably expressed at very low levels or not expressed under the present conditions.Northern blot hybridization was employed to verify the presence of these 21 putative sRNA candidates. Total RNAs were isolated from B. melitensis.Of the 15 sRNA candidate genes, the direction of BSR0653, BSR1073, BSR0626, BSR0602, and BSR1141 were opposite to the 2 flanking ORFs, suggesting that these 5 sRNA candidates could not be co-transcribed with the flanking ORFs. As expected, RT-PCR showed that the five sRNA candidates were transcribed independently. Then, the remaining 10 sRNA candidates were examined whether to be co-transcribed with their upstream and/or downstream genes. Using primer combinations that located in the sRNA and the adjacent gene, no products were observed for BSR0709, BSR1350, and BSR0739, indicating that they were transcribed independent of flanking genes  were affected in the Δhfq mutant, particularly, transcriptional levels of BSR1350, BSR0602, and BSR1141 was significantly decreased when Hfq was inactivated, indicating that the expression or stability of these sRNAs was dependent on Hfq , acid stress (TSB 4.0), heat shock (induced by high temperature), oxidative stress (induced by H2O2), and standard in vitro growth condition (TSB7.0). Total RNA was isolated, and then the expression of BSR0602 under environmental stress was examined using qRT-PCR. Acidic pH condition is an environmental stress that Brucella encounters in host macrophages, and it could trigger an expression response required for successful adaptation to the intracellular environment. Exposure of B. melitensis to pH 4.0 (TSB4.0) for 30 min resulted in increased transcription of BSR0602  of mRNAs of annotated ORFs within a given genome  was cloned into pZE12-luc. E. coli Top10 strains were transformed either with the gfp fusion construct alone or with a combination of both the gfp fusion construct and sRNA-encoding construct. Then, western blot was used to assess the amount of GFP. Prior testing showed that neither BSR0602 nor the nonsense sRNA impact expression of gfp in the pXG10-SF (Data not shown), suggesting that the detected expression is specific to the cloned targets. For gntR-gfp fusion, the amount of GFP was strongly reduced in cells co-expressing BSR0602, while the amounts of GFP had no change between the strain carrying the gfp fusion and the nonsense sRNA and the “fusion-only” strain  and gntR (C−5A−4U−3 > G−5U−4A−3) to generate mutant m-BSR0602 and m-gntR  has any influence on B. melitensis virulence, a gntR (BMEI0106) mutant was constructed and its virulence was evaluated. BALB/c mice were inoculated intraperitoneally with B. melitensis and spleen colonization was assessed at different time point postinfection. Compared to the wild-type strain 16M and the complementary strain 16MΔgntR-C, splenic CFU in 16MΔgntR infected mice were significantly reduced  . Under acid stress, the survival percentage of 16M-BSR0602 even decreased about 50% compared to the wild-type strain. These data demonstrated that overexpression of BSR0602 could lead to the reduced survival of B. melitensis in vitro. Then, competitive assays were performed by intraperitoneally inoculating BALB/c mice with 2 × 106 CFU of an equally mixture of two different strains, (i) wild type (16M) plus ΔBSR0602 mutant (16MΔBSR0602) and (ii) wild type (16M) plus BSR0602 overexpression strain (16M-BSR0602). As shown in Figure in vitro CI value of 0.76 in GEM medium (Data not shown). This data demonstrated that the BSR0602 overexpression strain was significantly attenuated when compared to the WT B. melitensis. In contrast, the deletion of BSR0602 leads to increased colonization of spleen, suggesting that BSR0602 is a negative regulator of virulence. As the ability to replicate within host phagocytes is essential to the pathogenicity of Brucella, we also tested the survival ability of 16MΔBSR0602 and 16M-BSR0602 in macrophages. RAW264.7 macrophages were infected with an equal mixture of (i) 16M plus 16MΔBSR0602 and (ii) 16M plus 16M-BSR0602. The ratio of wild type to BSR0602 overexpression strain reveals a 3.3 and 2.0-log difference in survival at 4 and 8 h of mixed infection, respectively  was identified to be the target for BSR0602. The majority of the regulation by the known trans-encoded sRNAs is negative (Gottesman, gntR was also negatively regulated by BSR0602, since overexpression of BSR0602 results in nearly full repression of BMEI0106. Results from GFP reporter system demonstrated that BSR0602 directly inhibit the expression of gntR mRNA and this direct sRNA–mRNA interactions were further confirmed by compensatory mutant experiments. The location of the hypothesized interaction between BSR0602 and gntR indicated that BSR0602 may assert its negative regulatory effect on gntR expression by hindering ribosome binding. Many bacterial trans-encoded sRNAs required the RNA chaperone Hfq to stabilize the sRNA–mRNA interaction. BSR0602 was determined to be an Hfq-dependent sRNA both in vitro and in vivo. Examine the role of Hfq in the BSR0602–gntR interaction will help uncover its full role in post-transcriptional regulation.A growing number of mRNAs encoding transcription regulators appear to be targets of multiple sRNAs (Battesti et al., gntR family in Brucella have proved to be associated with the virulence of the pathogen (Haine et al., gntR contribute to intracellular survival in spleens of infected mice as 16MΔgntR mutant was rapidly cleared from the spleen in BALB/c mice, which was inconsistent with the results of previously reported (Haine et al., gntR mutant. Haine et al. (In vivo transcriptional analysis indicated that gntR accumulated to high levels during infection, again confirming its role in pathogenesis. So, BSR0602 may modulate B. melitensis intracellular survival through repressing gntR. Considering GntR function as a transcriptional regulatory protein in B. melitensis, regulation by BSR0602 may be a two-step process in which BSR0602 regulates GntR and then affects transcription of downstream genes.GntR family of regulators has been shown to be involved in the regulation of many different biological processes (An et al., e et al.  used thee et al. . In vivoin vitro and in vivo. Data presented here would be consistent with the suggestion that decreased intracellular survival was due to the significant declined expression levels of gntR in BSR0602 overexpression strains. Although many sRNAs result in impaired virulence upon deletion, sRNAs could be negative regulators for bacterial virulence. For example, the deletion of Listeria monocytogenes RliB (Toledo-Arana et al., Vibrio cholerae VrrA (Song et al., M. tuberculosis could be lethal (Arnvig and Young, BSR0602 overexpression strains showed significant decreased survival under all stress conditions when compared with the wild type strain 16M and the BSR0602 deletion mutants. Moreover, high level of BSR0602 impairs bacterial survival both gntR. Eleven putative target mRNAs were identified using TargetRNA; however, these predictions need to be further validated. In summary, we have identified a novel sRNA BSR0602 modulating B. melitensis intracellular survival and this provide significant insights for unraveling the sRNA-mediated regulatory networks in B. melitensis.The widespread utilization of RNA-based regulation of diverse processes has a number of potential advantages for bacterial (Beisel and Storz, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fmicb.2015.00164/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.Click here for additional data file.