Multi-level cell operation is possible, but the stability of the high resistance state is also an important reliability concern. These devices show a good data retention of >105 s at >85°C. However, more study is needed to achieve a 10-year guarantee of data retention for non-volatile memory application. The crossbar memory is benefited for high density with low power operation. Some CBRAM devices as a chip have been reported for proto-typical production. This review shows that operation current should be optimized for few microamperes with a maintaining speed of few nanoseconds, which will have challenges and also opportunities for three-dimensional (3D) architecture.The performances of conductive-bridging random access memory (CBRAM) have been reviewed for different switching materials such as chalcogenides, oxides, and bilayers in different structures. The structure consists of an inert electrode and one oxidized electrode of copper (Cu) or silver (Ag). The switching mechanism is the formation/dissolution of a metallic filament in the switching materials under external bias. However, the growth dynamics of the metallic filament in different switching materials are still debated. All CBRAM devices are switching under an operation current of 0.1 μA to 1 mA, and an operation voltage of ±2 V is also needed. The device can reach a low current of 5 pA; however, current compliance-dependent reliability is a challenging issue. Although a chalcogenide-based material has opportunity to have better endurance as compared to an oxide-based material, data retention and integration with the complementary metal-oxide-semiconductor (CMOS) process are also issues. Devices with bilayer switching materials show better resistive switching characteristics as compared to those with a single switching layer, especially a program/erase endurance of >10 In 1999, Kozicki et al. [z+, z = 1, 2, or Ag+). The cations drift through the solid electrolyte switching layer under the electric field, and the metal ions are reduced on the inert electrodes. As this process continues, a metallic filament is established in between the two electrodes and the device switches from the high resistance state (HRS) to the low resistance state (LRS). By changing the polarity of the voltage, an electrochemical dissolution of the conductive bridges takes place, resetting the device from the LRS to the HRS. The resistance state changes by applying external bias, and the switching is reversible. It is true that device reliability including stable switching characteristics, program/erase endurance, and data retention at 85°C for 10 years at a low current of few microamperes is a very challenging issue for production. In addition, the switching mechanism in different structures including materials and electrodes is not understood clearly. Especially, the growth kinetics of the metallic filament in oxide- and non-oxide-based materials are still debated. Although the memory performances of a designed structure with low operation current have huge opportunities to fulfill the requirements of ITRS, they are not achieved yet. Even though many papers have been reported by several groups, a complete review on CBRAM performances including switching characteristics, reliability, mechanism, and so on for real production is needed, which is not reported yet.In recent days, resistive random access memory (RRAM) technology is one of the most promising and reliable alternative solutions to overcome the scaling bottleneck of FLASH . The advi et al.  reportedi et al. , or CBRAHere, the challenges and opportunities of CBRAM devices using different switching materials such as chalcogenides, oxides, and bilayers in different structures have been reviewed. Memory performances such as switching characteristics, endurance, multi-level cell operation, and data retention have been discussed. Memory devices can be operated with an operation current of 0.1 μA to 1 mA under an operation voltage of ±2 V. Filament growth dynamics in different switching materials have been discussed. Device reliabilities such as switching uniformity, endurance, and data retention under a few microamperes are very challenging issues. The device can be scaled down below the 11 nm technology node and has a very good opportunity for non-volatile crossbar memory for 3D architecture in the near future. Finally, CBRAM as a chip has been discussed for future production.x [2 [2S [2S [2O5 [2 [2 [x [3N4 [x [x [x/GdOx [x/TaSiOy [x/TaOx [x [2O3 [2O3 [2O3 [CBRAM performance depends on switching materials including different non-oxides and oxides. The non-oxides, i.e., chalcogenides, are GeSex -12, GeS2x [2 ,14, GeTex [2 , Cu2S [1x [2 [2S , Ag2S [8[2S [2O5 ,17, SiO2 [2O5 [2 , ZrO2 [1O5 [2 [2 , GeOx [2[2 [2 [x ,21, and [2 [2 [x  and Si3N [x [3N4  have bee [3N4 [x , Cu-Te/SN4 [x [x , MoOx/Gd [x/GdOx , TiOx/Tax/TaSiOy , GeSex/T [x/TaOx , Ti/TaOx/TaOx [x , Cu-Te/A [x [2O3 , TiW/Al22O3 [2O3 , and CuT2O3 [2O3  have bee33Ge20Se47/Ni PMC device. A 100-nm-thick Ni as a bottom electrode (BE) was fabricated on SiO2/Si substrates. Then, 50-nm-thick Ge70Se30 was evaporated with the help of a Knudsen-type cell under 10−6 Torr vacuum. This approach will help in maintaining almost the same level of elementary content of the switching material. A very low deposition rate of 0.03 nm/s was maintained to enhance the step coverage and filled into the narrow via-holes. Immediately after that, a 30-nm-thick Ag film was deposited to form a Ag:GeSe solid electrolyte. In this case, Ag diffusion by exposure to 405-nm ultraviolet (UV) radiation was time consuming and cost effective. To avoid this issue, Schindler et al. [2/Si substrates. A Si3N4 layer was deposited on Pt by plasma-enhanced chemical vapor deposition (PECVD) method with a deposition rate of 0.2 nm/s. Via-holes with varying diameters from 2.5 to 50 μm were patterned by optical lithography. The solid electrolyte layer was 50-nm-thick Ge0.3Se0.7 and was deposited by radio-frequency sputtering or physical vapor deposition (PVD) at a rate of 0.2 nm/s. The applied power ranged from 5 to 25 W. The top electrodes were patterned by optical lithography. Then, the Ag layer was deposited by thermal evaporation at a deposition rate of 0.5 nm/s. After that, a lift-off process was carried out to obtain the final device. Due to the low melting point of Se (221°C), it is difficult to control the composition of GeSe after deposition by sputtering. To keep the same composition, the GeSe material was deposited by using an electron beam evaporator, which we have also reported previously [2 layer with a thickness of approximately 200 nm. The SiO2 layer was grown by thermal oxidation. Then, W or TiN metal was used as a BE on the SiO2/Si substrates. The thickness of the BE was approximately 200 nm. To design the memory devices, a SiO2 layer with a thickness of approximately 150 nm was deposited. Lithography was used to pattern via-holes with different nominal areas from 200 × 200 nm2 to 8 × 8 μm2 of the active switching region. After defining the via-hole regions, reactive-ion etching (RIE) process was used to etch the uncovered SiO2 films and to open the W BE contact. Following this, a GexSex1 −  solid electrolyte with a thickness of approximately 40 nm was deposited using an electron beam evaporator. Pure GexSex1 −  (x = 0.2 to 0.5) granules  with different compositions such as Ge0.2Se0.8, Ge0.3Se0.7, Ge0.4Se0.6, and Ge0.5Se0.5 were used for deposition. The chamber pressure was 5 × 10−6 Torr prior to deposition. The deposition rate was 1 to 2 Å/s. Then, a Cu top electrode (TE) with a thickness of approximately 40 nm was deposited using a thermal evaporator. A 160-nm-thick layer of Al was then deposited in situ using the same thermal evaporator. Otherwise, a 100- to 200-nm-thick Cu electrode was used. Finally, a lift-off process was performed to obtain the resistive switching memory device. To investigate the memory device and microstructure of the solid electrolyte films, transmission electron microscopy (TEM) was carried out using a FEI Tecnai G2F-20 field emission system with an energy of 200 kV. The memory devices for TEM observation were prepared using an FEI Helios-400s system with an operating voltage of 5 kV and using a Ga+ ion source. Figure 0.4Se0.6/W structure [0.2Se0.8 solid electrolyte films is approximately 38 nm. A small via-hole size is about 140 nm in width, which is lower (slightly) than our design size (200 nm). The Ge0.4Se0.6 film is amorphous, as shown in the outside  owing to increased Se content [3d (29 eV) electrons are 1.1, 1.6, 2.0, and 2.1 eV for Ge0.5Se0.5, Ge0.4Se0.6, Ge0.3Se0.7, and Ge0.2Se0.8 films, respectively. A similar chemical shift of Ge 3d electrons was reported by Ueno et al. [3d electrons are 2.2 ± 0.1 eV for GeSe2 and 1.7 ± 0.1 eV for GeSe, while those of Se 3d electrons are negative, which is similar to our results. The corresponding binding energies of Se 3d core-level electrons for Ge0.5Se0.5, Ge0.4Se0.6, Ge0.3Se0.7, and Ge0.2Se0.8 films are found to be 54.2, 54.5, 54.7, and 54.8 eV, respectively  electrons are found to be −1.0, −0.7, −0.5, and −0.4 eV for Ge0.5Se0.5, Ge0.4Se0.6, Ge0.3Se0.7, and Ge0.2Se0.8 films, respectively. However, the chemical shifts of Se 3d (55.5 eV) electrons are −1 eV for GeSe2, as reported by Ueno et al. [1s core-level electron signal is not observed in the GexSex1 −  film. To search better thermal stability, other switching materials are discussed below.Memory devices using chalcogenide-based switching materials are reported below. Chalcogenide glasses are based on the chalcogen elements, and they have a wide range of properties such as optical and electrical ones, as reported in the literature. Several types of chalcogenide-based solid electrolytes have been reported for future resistive switching memory applications. Kozicki et al.  reportedr et al.  reportedeviously . The res content ,35. The o et al. . On the 2/Si substrates by chemical vapor deposition (CVD) method and then covered it with a 100-nm-thick SiO2 layer. Via-holes with different diameters ranging from 180 nm to 5 μm were opened on the SiO2 layer by optical projection lithography and RIE. Then, 40- and 50-nm-thick GexSx1 −  (0.30 < x < 0.35) films were deposited by PVD. Next, a 25-nm-thick Ag (or Cu) layer was deposited by PVD. The chamber pressure was maintained at 10−6 Torr. It is important to note that the deposition of the switching layer is a difficult process as the vapor pressure of the S component is very high. Vianello et al. [2/W-plug structure, and 1T1R configuration in 8 × 8 arrays integrated in the complementary metal-oxide-semiconductor (CMOS) process was done. The plug size was 0. 2 μm. The thickness of the Ag-doped GeS2 layer, which was deposited by co-sputtering, was 30 nm. The Sb material was doped 2% to 20% in GeS2. A thin Ag layer was dissolved into GeS2 using a photo-diffusion process [2 layer with a thickness of 100 nm was grown on a Si substrate. Corresponding materials such as TaN, GeTe, and SiO2 were deposited (in the given order) onto the prepared substrate using a radio-frequency (RF) sputtering system. The thicknesses of TaN, GeTe, and SiO2 layers were 30, 50, and 100 nm, respectively. Via-hole devices with different sizes of 200 nm, 400 nm, 10 μm, and 50 μm were patterned by e-beam lithography. Banno et al. [− α2 S/Pt structure. Terabe et al. [2S-coated Ag wire was crossed by a Pt wire. They mentioned that the ‘crossbar’ structure was convenient for integrating switches to be used in actual devices. Some oxide-based materials for CBRAM devices are explained below.Kozicki et al.  also repo et al.  reported process . Then, t process  reportedo et al.  reported2O5/Pt structure for their CBRAM devices. A 15-nm-thick Ta2O5 film with an O/Ta ratio of 2.5 was deposited on a Pt electrode by RF sputtering. Next, a 50-nm-thick Cu electrode was deposited and covered by a 50-nm-thick Pt film to avoid oxidization of Cu. In our previous work [2O5/W structure. The via-holes were patterned as mentioned above in our work. Then, 15- to 20-nm-thick Ta2O5 films were deposited using an e-beam evaporator. The Ta2O5 granules were used. Next, a Cu TE with a thickness of 100 nm was deposited using a thermal evaporator. Finally, a lift-off process was done to obtain the memory devices. A post metal annealing treatment was performed at a temperature of 350°C for 1 min in N2 ambient. Schindler et al. [2/W structure. A 100-nm-thick W BE was deposited by CVD on SiO2/Si substrates. Then, a SiO2 switching material with thicknesses varying from 12 to 50 nm was grown by electron beam evaporation. A Cu TE with a thickness of 35 to 45 nm was deposited. Then, Cu was diffused into SiO2 by thermal annealing at 610°C to have a Cu/Cu:SiO2/W structure. Li et al. [2/Au structure. First, a 50-nm-thick Ag layer was deposited using an electron beam evaporator. A ZrO2 film was also deposited. The vacuum pressure and the deposition rate were 2 × 10−6 Torr and 1 Å/s, respectively. After that, a 50-nm-thick Au TE with different areas such as 100 × 100 μm2 to 800 × 800 μm2 was deposited. A GeOx switching material as CMOS compatible was reported by us [2 to 8 × 8 μm2 were reported. A GeOx film with a thickness of approximately 10 nm was then deposited by RF sputtering. The GeOx film showed to be polycrystalline. The chamber pressure was 2 × 10−5 Torr. The deposition power and pressure were 50 W and 2 × 10−2 Torr, respectively. Argon (Ar) gas with a flow rate of 25 sccm was used. A Ge target was used to deposit the GeOx solid electrolyte. Then, a Cu TE with a thickness of approximately 200 nm was deposited using a thermal evaporator. A lift-off process was next performed to obtain the final via-hole devices in a Cu/GeOx/W structure. Cross-point memory devices using an Al/Cu/GeOx/W structure were also reported [2 layer with a thickness of approximately 200 nm was grown by a wet oxidation process. After the masking process, a GeOx film with a thickness of 10 nm was deposited by the same RF sputtering. A Cu layer with a thickness of 40 nm was deposited using a thermal evaporator, and then, an Al layer with a thickness of approximately 160 nm was deposited in situ using a thermal evaporator. Finally, a lift-off process was performed to get the cross-point devices. A schematic illustration of the fabricated GeOx-based cross-point memory device is shown in Figure x solid electrolyte was sandwiched in between Cu and W electrodes. An optical micrograph (OM) image of the 5 × 5 cross-points is shown in Figure xGex1 −  film of 20 nm was deposited by thermal CVD at 425°C. An amorphous Si (a-Si) as a resistive switching material was deposited by PECVD method. The thickness of the a-Si film was approximately 20 nm. Finally, a Ag TE was deposited. This complete stack was patterned by electron beam lithography and plasma etching process. The thickness of all the layers was 50 nm half-pitch arrays. Sun et al. [3N4/Pt memory device. The device size was 100 × 100 μm2. Even though many groups reported a single-layer switching material, bilayer switching materials showed to be promising.Banno et al.  reportedous work , we haver et al.  reportedi et al.  fabricatreported . First, n et al.  also repx/W structure. In this case, both TE and switching materials were deposited on the BE by a sputtering process. Device sizes of 20 and 50 nm were designed. The Cu-Te layer behaved as a buffer layer, and the GdOx layer was used as a switching layer. The Cu filament formation/dissolution in the GdOx layer was controlled by a buffer layer through external bias. Similarly, Cu-Te/SiOx bilayers were also reported [x/GdOx bilayers in a Cu/MoOx/GdOx/Pt structure. A 10-nm-thick GdOx layer was deposited on a Pt/Ti/SiO2/Si substrate using an electron beam evaporator. A 100-nm-thick Pt was also deposited. After a lift-off process, a device size of 50 × 50 μm2 was fabricated. A NanoBridge CBRAM device was reported by using a Cu/Ta/TaSiOy/Ru structure [y solid electrolyte was deposited by RF sputtering in Ar/O2 gas. The CBRAM devices were connected through contact holes and controlled the switching through a CMOS transistor. Therefore, the 1T1R configuration was developed. In our previous work [x/TaOx bilayer materials in an Al/Cu/GeSex/TaOx/W structure. Although this CBRAM device using GeSex/TaOx bilayers shows promising resistive switching characteristics, the thermal stability of the Ge20Se80 material is an issue. To have a CMOS-compatible structure, we reported a Al/Cu/TiO2/TaOx/W CBRAM device [2O5 film with a thickness of 18 nm was deposited from pure Ta2O5 granules using an electron beam evaporator. The resulting Ta2O5 film was mixed with Ta metal , as characterized by XPS. The Cu as mobile ions plays a major role in the Al/Cu/TaOx/W structure resistive switching memory device. The Cu can be oxidized at the Cu/TaOx interface during deposition and hinders the resistive switching memory performance. Therefore, Cu oxidation is expected to be avoided by inserting a Ti nanolayer at the Cu/TaOx interface. A thin Ti layer with a thickness of approximately 3 nm was deposited in situ using an electron beam evaporator with Ti granules. Finally, a device with an Al/Cu/TiO2/TaOx/W structure was obtained. These memory devices were annealed by rapid thermal annealing (RTA) at 350°C in N2 ambient for 1 min. Oxygen accumulated in the Ti nanolayer and formed TiO2. Figure x interface [2. All layers were covered well at the active and outer regions . Using a shadow mask, CuxTex1 −  dots were deposited by using a co-sputtering system. Cu and Cu0.1Te0.9 targets were used. The size of the dots was 3 mm wide and 50 nm thick. Belmonte et al. [2O3/W structure in a 90 nm technology node. Firstly, a 3-nm-thick Al2O3 layer was deposited on a W BE by ALD. After that, a 10-nm-thick TiW alloy with 25 at % of Ti was deposited by using a sputtering system. Then, a Cu seed layer was deposited by a sputtering process. At last, a TiN TE was deposited to get the final device. Devulder et al. [0.6Te0.4-C/Al2O3/Si structure to enhance the thermal stability and switching behavior of memory devices. Table Aratani et al.  investigreported . Yoon etreported  reportedtructure . The memous work , we repoM device . The viaM device ,28. A Tanterface . The devs Figure b,c. Oxygx et al.  reportede et al.  fabricatr et al.  reportedz+ or Ag+ ions exhibit high mobility. In our previous study [I-V) characteristics of a Cu/Ge0.4Se0.6/W structure, as shown in Figure 2. It is noted that the formation voltage is not required for GexSex1 −  solid electrolyte-based CBRAM devices. The I-V hysteresis loop can be explained as follows by using arrows 1 to 9. Initially, a voltage applied to the TE is swept from 0 to +0.56 V (arrow 1); this is the HRS. Beyond the voltage of +0.56 V, there is an instantaneous switching from the HRS to the LRS (arrow 2). The resistive switching is observed beyond a voltage of 0.56 V, which is called SET voltage (VSET). To form a Cu metallic filament into the Ge0.4Se0.6 solid electrolyte, the applied bias should be larger than the SET voltage (arrow 3). The memory device keeps the LRS up to a negative voltage of −0.18 V (arrows 3 to 6). The resistive memory device reaches a HRS if the voltage applied is more than the negative voltage or RESET voltage (VRESET) < −0.16 V (arrow 7). A large RESET current (IRESET) of −85 μA confirms that the strong Cu metallic filament is formed into the Ge0.4Se0.6 solid electrolyte. To reach a HRS, a negative voltage of −1.2 V should be large enough (arrow 8). The metallic filament is gradually dissolved by applying a higher negative voltage. To get a second switching, the sweeping voltage can be followed using arrows 9 and 1. The corresponding resistance vs sweeping voltage (R-V) hysteresis characteristics are plotted in Figure 6 is observed, which is useful for MLC operation. Choi et al. [3 to 105. Kund et al. [VSET was +0.2 V. Kozicki et al. [VSET of +0.4 V and a CC of approximately 10 μA. One hundred consecutive I-V switching cycles at a CC of 100 μA are shown in Figure VSET varied from 0.3 to 0.5 V. Variations of the resistance states and RESET current are also observed, due to the higher Se content (80%). Figure 5 P/E cycles. All memory devices using GexSex1 −  solid electrolytes are performed more than >105 P/E cycles, which is beneficial by using GexSex1 −  solid electrolytes. Kozicki et al. [10 read endurance using a Ag/Ag33Ge20Se47/Ag structure. Kund et al. [6 P/E endurance using a GeSex-based CBRAM device. Vianello et al. [2-based chalcogenide-based CBRAM cell, as shown in Figure 5 P/E cycles at 30-ns pulse width with a resistance ratio of >10. However, there is voltage-time dilemma. For a high-speed operation, the program/erase voltage needs to be higher. Hasegawa et al. [2S- and Cu2S-based gap-type atomic switch as a function of bias voltage measured at room temperature, as shown in Figure ts) is expressed as function of switching bias (Vs > VSET), as shown in Equation Germanium (Ge)-based chalcogenide glasses have been widely studied because of their well-known mechanism and also they offer several advantages, whereas Cuus study , we havei et al.  and Liawi et al.  also repi et al.  have repo et al.  have als V arrow . A largea et al.  have stuβ1 and β2 are 67.7 and 32.9 for low-voltage and high-voltage regions, respectively. Similar phenomena are also reported in a Cu2S switching material, as shown in Figure β1 and β2 are 54 and 25 for low-voltage and high-voltage regions, respectively. These two exponential decay factors exhibit different rate limiting processes for the low-voltage and high-voltage regions. It is well known that there are only few mechanisms such as metallic filamentary switching that require few nanoseconds (<10 ns) to switch a device from SET to RESET and vice versa. Thus, for the 10-year data retention criterion, the device needs to maintain the ratio of the retention time to the SET time of around 1016 and VSET/Vread of around 10. Again, the read voltage needs to be high enough to maintain the ratio of Vprogram/Vread at 10 for circuit constraint. It is also required that the cell withstand the read voltage for 10 years. Schindler et al. [2-based CBRAM cells. Therefore, CBRAM devices can be operated at low current and stable data retention is also an important part. We have reported data retention characteristics of >11 h at a CC of 200 μA for a Cu/Ge0.4Se0.6/W CBRAM device [5 s at 130°C using a Sb-doped GeS2 solid electrolyte. Jameson et al. [6 s at 200°C in a Ag/GeS2/W memory cell. In addition, MLC capability is also important for high-density memory application. Russo et al. [5 s with CC (or Icompl) varying from 5 to 500 μA, as shown in Figure Rc is not so stable at 5 μA as compared to 50 and 500 μA. This suggests that the filament size is smaller for lower current compliance as compared to higher current compliance. However, HRS maintains the same level at different CCs for more than 104 s. Kund et al. [0.2Se0.8 solid electrolytes in an Al/Cu/Ge0.2Se0.8/W structure which can be operated at a very low CC of 1 nA. The sweeping voltage direction is shown by arrows 1 to 2. Figure 0.2Se0.8 and Ge0.5Se0.5 devices. For both devices, LRS decreases with increasing CCs from 1 nA to 1 mA owing to filament diameter increase. At a CC of <10 μA, the LRS of the Ge0.2Se0.8 device is lower than that of the Ge0.5Se0.5 device. This suggests that Cu ions can be easily migrated through a higher Se content, which results in a large variation of HRS. However, the average HRS of Ge0.2Se0.8 devices decreases faster than that of Ge0.5Se0.5 devices. This indicates that the filament dissolution length of Ge0.5Se0.5 devices is larger than that of Ge0.2Se0.8 devices because of the thinner filament diameter or less overshoot effect. So resistive switching properties strongly depend on the compositional variations of GexSex1 −  solid electrolytes. However, the SET voltage, endurance, data retention, and so on may be controlled using different compositions of GeSex solid electrolytes as per the requirement of the applications.The exponential nature of the time vs voltage characteristics suggests that a higher SET voltage can drastically reduce the time to SET the memory cell. Even a 0.1-V increment can result in around one to two orders of reduction in SET time. The switching time can be distinguished into two decay components when the bias voltage is increased Figure a. The var et al.  have alsM device . The memM device  have shon et al.  have repo et al.  have repseconds < ns to swseconds < ns to sw2O5 [2 [2 [x [2O5/Pt structure at a CC of 100 μA. However, a large IRESET of >1 mA was reported. In our previous study [x/W structure at a CC of 100 μA and a small operating voltage of ±1 V, as shown in Figure IRESET is approximately 43 μA, and it is good for application. The values of VSET and VRESET are 0.75 and −0.2 V, respectively. This device is operated at a very low CC of 5 pA  growth and reduce the randomness of the CF formation and rupture processes, leading to improved stability and uniformity of the memory devices. Li et al. [2/Ag devices. Even though I-V switching has shown to be good with low operation current, P/E endurance and data retention are also important, which are discussed below.It is observed that the chalcogenide-based CBRAM device shows great potential for next-generation non-volatile memory, but one of the discrepancies is compatibility with the CMOS process. To mitigate this issue, binary metal oxides like Ta2O5 ,17, SiO22O5 [2 , ZrO2 [1O5 [2 [2 , GeOx [2[2 [2 [x ,21, and [2 [2 [x  have repA Figure b. The IRr et al. ,44 have a et al.  have repus study  have repi et al.  have rep4 P/E cycles with 100-μs pulse width, as shown in Figure 7 P/E cycles in a Cu/SiO2/W CBRAM cell. To analyze the data retention of an oxide-based CBRAM device, Schindler et al. [5 s data retention at a CC of 1 μA using a Cu/Cu:SiO2/W structure. Both HRS and LRS exhibit stable data retention with a large resistance ratio of >104. Tsunoda et al. [4 s data retention at a CC of 100 μA in a Ag/TiO2/Pt cell. We have reported good data retention at a CC of 50 μA of more than 200 h at 85°C with a high resistance ratio of >108 using a Cu/GeOx/W structure [x films [x films. This suggests that our resistive switching memory device can be applicable for MLC. The RESET current increases with increasing CC from 5 pA to 700 μA, which indicates that a metallic filament can be formed by applying larger current. Figure Banno et al.  and Sakar et al.  have repa et al.  have sho[x films . The valx [x/GdOx [x [x/TaOx [2O3 [2O3 [x bilayer materials in 1T1R configuration for 20-nm  shows a leakage current of approximately 1.5 pA at a Vread of +0.1 V for a pristine device . An improved LRS dispersion is also observed for Al/Cu/TiO2/TaOx/W devices owing to Cu migration controlled through a TiOx nanolayer under external bias. A low operation current of 10 μA using a Cu/TiW/Al2O3/W structure is also reported by limiting the possible Cu diffusion and has shown resistive switching with limited variability [IRESET. This is attributed to the higher value of HRS and broadened memory window. An Al/GeSex/TaOx/W device shows a low operation current of 100 nA [x in an Al/Cu/GeSex/W structure and TaOx in an Al/Cu/TaOx/W structure, improved uniformity of LRS/HRS with switching cycles is observed for GeSex/TaOx bilayer materials, as shown in Figure x, TaOx/GeSex, and TaOx devices, respectively. A stable LRS/HRS of the GeSex/TaOx device is observed, as compared to that of pure GeSex and TaOx devices. This is due to the Cu nanofilament formation in the TaOx switching layer at the GeSex/W interface. The LRS values are different because of defect control in switching materials. The GeSex/TaOx bilayer device can be operated with different CCs varying from 1 nA to 500 μA. The average (or standard deviation) values of the LRS for device-to-device are found to be 0.14 GΩ (4.7 × 107), 5.2 kΩ (1.7 × 103), and 601 Ω (117), and those of the HRS are 11.9 GΩ (9 × 109), 0.34 GΩ (5 × 108), and 9.6 MΩ (9.5 × 106) at CCs of 1 nA, 50 μA, and 500 μA, respectively. The average resistance ratios are 85, 6.5 × 104, and 1.6 × 104 at CCs of 1 nA, 50 μA, and 500 μA, respectively. Due to such a high resistance ratio, this resistive switching memory device can be useful for MLC applications in the future with a wide range of CCs from 1 nA to 500 μA.Several researchers have studied CBRAM phenomena using bilayer materials to control Cu or Ag diffusion and improve switching uniformity and other electrical performances as well. Different bilayers such as Cu-Te/GdOx , MoOx/Gd [x/GdOx , Ti/TaOx/GdOx [x , GeSex/T [x/TaOx , Cu-Te/AaOx [2O3 , TiW/Al22O3 [2O3 , and so m Figure a and 50-m Figure b cell siOx layer . Improveed by us . Figure e Figure a, lower e Figure b. Becausiability . The staf 100 nA . Compare7 P/E cycles are reported, as shown in Figure 5 s at 130°C in a Cu:Ge-Te/Al2O3/TiN CBRAM device with a small cell size of 20 nm is also reported [5 s at 85°C under a small CC of 50 μA is reported [x bilayer materials [VSET and VRESET voltages are also important to control the switching characteristics as well as power. The device is operated with a high speed of 10 ns for the SET and RESET. Table VSET and VRESET of CBRAM devices with different switching materials. This indicates that bilayer materials show more reasonable VSET and VRESET voltages for application and the operation voltage of CBRAM devices is very good (±2 V).Introducing bilayer materials, long >10reported . Many dereported . Figure aterials . Levels 0.33Ge0.20Se0.47 solid electrolyte material, as shown in Figure V > VSET), Ag atoms become electrically oxidized and drift through the Ag0.33Ge0.20Se0.47 solid electrolyte and finally reduce at the Au BE due to incoming electrons from the BE. As a result, the metallic filament forms due to the gradual process and the memory device switches from HRS to LRS, which is known as the SET process , Ag atoms in the filament become oxidized and migrate towards the TE, which results in the device to get back from LRS to HRS , the Cu ions are generated at the Cu/Ge0.4Se0.6 interface from the Cu TE following the oxidation method as mentioned in Equation xSex1 −  solid electrolyte through this redox reaction. It is reported that the base of the filament is at the cathode side and the thinner part of the filament at the anode side [V < VRESET), the electric field will be higher at the Cu/filament interface and the Cu filament starts to dissolve at the Cu/GexSex1 −  interface by Joule heating first and then the electrochemical oxidation at the top of the filament or the GexSex1 − /filament interface. Basically, Joule heating at the weak part  of a metallic filament will play a role to dissolve the filament first. However, further study is necessary to understand the RESET mechanism. Then, the Cu ions are collected in the Cu electrode through the reduction method as mentioned in Equation 0.4Se0.6/W devices by TEM [0.4Se0.6 film in the filament region shows to be crystalline owing to Cu ion migration and Cu filament formation after the SET condition. The diameter of the Cu nanofilament is approximately 11 nm. Some plausible explanations of Cu diffusion have also been discussed from other reported results in the literature. The diffusion occurs when copper or silver is in contact with chalcogenides, as reported by McHardy et al. [2 film and polycrystalline materials such as Cu2Se (FCC) formed as small crystallites surrounding the amorphous film. In contrast, we observed that the Ge0.4Se0.6 film shows to be amorphous, which suggests that there is no diffusion of Cu initially unless external bias is applied. By applying external bias, structural changes  occur in an amorphous Ge0.4Se0.6 solid electrolyte. A huge amount of Cu signal is obtained into the crystalline region, due to the formation of a Cu cluster or Cu nanofilament into the Ge0.4Se0.6 solid electrolyte. There are mainly two diffusion mechanisms involved: one is a fast interstitial mechanism and the other is a slow substitutional mechanism. However, Cu or Ag exhibits high ionic mobility in chalcogenides; therefore, Ag or Cu ions diffuse rapidly as interstitial species through these chalcogenides until a vacant site is encountered where substitution can occur [0.3Se0.7 film after deposition of a 160-nm Ge0.3Se0.7 film on a 50-nm-thick Cu layer. They also observed a Ag filament with a diameter of 20 nm in the Ge0.3Se0.7 solid electrolyte by conductive atomic force microscopy (CAFM) [2. The crystalline Cu nanofilament and GeOx film (without Cu nanofilament) are shown. The clear crystallization in the GeOx film shows a truncated conically shaped Cu nanofilament, which was also confirmed by EDX analysis  in the Cu nanofilament than it would be without the Cu nanofilament in the GeOx film. This nanofilament has Cu clusters  embedded in the GeOx film or it is a Cu:GeOx mixture. The Cu clusters are also confirmed by fast Fourier transform (FFT) analysis  and 2.4506 Å (Cu2O) are observed inside the nanofilament  of 2.0880 Å [d-spacing value of 2.1953 Å was observed at the Cu filament/W interface, which is in between the d-spacing value of Cu (111) and W (110) and was approximately 2.2378 Å  under the electric field and finally reach the inert electrode. The filament formation/removal mechanism is reported first using a Agmentclasspt{minimay et al. . The Cu an occur ,52. Theran occur  have alsy (CAFM) . The Cu s Figure f. The Cus Figure b,c,i, wht Figure b,i, whic2.0880 Å  and simiÅ Figure c. A trund Figure j. Furthem Figure k,l, and m Figure m are als2/Pt CBRAM device  and reduction (ER) remain the same, as shown in Figure + cations move towards the surface and hence the concentration of the Ag+ ions increases at the Ag2S surface. According to the formula of electrochemical potential, μ = μ0 + Fφ + RT lnγC, where μ0 is the potential for γC = 1, F is the Faraday constant, φ is the electrical potential, R is the molar gas constant, T is the absolute temperature, γ is the activity co-efficient, and C is the concentration of the metal cation, the potential of Ag+ ions increases due to the increase of the concentration resulting in ER < EO, and the Ag nanowire starts to grow inside the gap  layers [2 chips are the supply voltages of 1.2 and 5 V with an on-chip charge pump providing 6.6 V required for programming. It has two BEs which are driven by the two buried word line MOS transistors to write or erase the memory cells. The memory cell size is designed to be 6 F2. This storage device can be combined high performance and high bit density whereas its other counterparts are failing to achieve the same goal. Those CBRAM chips can be used as an economical alternative to the conventional Electrically Erasable Programmable Read-Only Memory (EEPROM) technology and may be applicable to electronic toys, games, consumer electronics, wireless LAN, data storage, etc. The concept of this dual cell is similar to that of complementary resistive switching [A CBRAM with a 32-kb chip has been reported by using I2C (Inter-Integrated Circuit) and SPI  bus architecture . The ope) layers . The opewitching , where t5 cycles, and a good data retention of >105 s at >85°C. The memory device also shows multi-level operation with varying current compliance from CCs of 5 to 500 μA. It is observed that the oxide-based memory device performs at a low CC of 5 pA, good device-to-device uniformity, good data retention at low current of 1 μA, and good P/E cycles as well. It is found that bilayer switching materials show excellent device-to-device uniformity, robust data retention, and better P/E cycles of >107 with a high speed of few nanoseconds. The performances of CBRAM devices using different switching materials and structures are listed in Table In this article, we have reviewed CBRAM switching characteristics and switching mechanism, introducing chalcogenide, oxide, and bilayer switching layers. In the first section, we review the switching performance of chalcogenide-based CBRAM. It is observed that a chalcogenide-based memory device shows good bipolar resistive switching, long P/E endurance of >10


PRF1, UNC13D, STX11, STXBP2, SH2D1A, XIAP, and ITK were reported to be primary HLH. Due to the different treatment options, the differentiation between primary and secondary HLH is critical. Our previous studies have showed that a Th1/Th2 cytokine profile is diagnostic for HLH, yet the cytokine profiles between primary and secondary HLH have not been compared. The aim of the study was to test whether the Th1/Th2 cytokine profile could be used as a tool to differentiate between primary and secondary HLH.Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder of immune regulation, and HLH patients with mutations in genes including A total of 45 hospitalized Chinese children with HLH during the period of February 2010 through September 2012 were enrolled in the study. Fifty healthy children were enrolled as controls. Primary HLH related genes were sequenced using genomic DNA samples. The Th1/Th2 cytokine levels including interferon-γ (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-10, IL-6, IL-4 and IL-2 were quantitatively determined by cytometric bead assay techniques.n = 4) included one patient with biallelic heterozygous mutations in PRF1 gene, and three patients with hemizygous mutation in SH2D1A gene. Based on the available genetic data, the other 41 patients were classified into the secondary HLH group. When compared the cytokine levels between the two groups, IL-4 level in primary-HLH was significantly lower than that in secondary HLH (P = 0.025), while IFN-γ level in primary HLH had a tendency of statistically lower than that in secondary HLH (P = 0.051). Area under receiver operating characteristic (ROC) curves of IL-4 and IFN-γ, IL-10, TNF-α, IL-2, and IL-6 levels were 0.841, 0.799, 0.506, 0.494, 0.457, and 0.250, respectively. ROC curves showed that 1.7 pg/ml of IL-4 had sensitivity and specificity for differentiation between primary and secondary HLH as 70.7 and 100.0 %, while 433.9 pg/ml of IFN-γ had sensitivity and specificity as 51.2 and 100.0 %, respectively.Primary HLH group  contains supplementary material, which is available to authorized users. Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder of immune regulation, characterized by a highly stimulated, but ineffective immune response to antigens, which results in cytokine storm and inflammatory reaction . HLH is PRF1 gene (FHL2) [UNC13D gene [STX11 gene and STXBP2 gene are found to be the causes of FHL4 [SH2D1A, XIAP, and ITK genes, respectively.Primary HLH can be further divided into familial hemophagocytic lymphohistiocytosis (FHL) and immune deficiencies associated HLH . Five gee (FHL2) , a gene  of FHL4  and FHL5 of FHL4 , 16, res of FHL4 , XLP typ of FHL4 , and IL- of FHL4 , which aPRF1, UNC13D, STX11, STXBP2, SH2D1A, XIAP, and ITK genes, together with cytokine determination for HLH patients to test whether cytokine profile could be used as a tool to distinguish between primary HLH and secondary HLH patients.Hypercytokinemia is a hallmark of HLH. Henter JI et al. suggested that hypercytokinemia may be caused by a genetic defect in cytokine regulation as FHL patients showed high cytokine levels . Using tThe diagnoses of all our patients were made based on the HLH-2004 criteria [PRF1, UNC13D, STX11, STXBP2, SH2D1A, XIAP, and ITK genes, and the deep intronic sequences of intron 1 in UNC13D gene were amplified by a polymerase chain reaction (PCR) machine (BIO-RAD) in our laboratory, and the PCR products were sequenced by a DNA sequencer in Invitrogen Company . We analyzed PRF1, UNC13D, STX11, STXBP2, and ITK genes in all 45 patients and 50 controls, and all the male patients (n = 27) and male controls (n = 30) were sequenced in their SH2D1A and XIAP genes. The mutations were validated by re-sequencing an independent PCR-generated amplicon from the subjects. The variants were named according to the Human Genome Variation Society and journal requirements.Genomic DNA was isolated from peripheral blood using QIAamp DNA Blood Mini Kit . The coding regions and flanking intronic sequences of http://exac.broadinstitute.org, March, 2016 accessed), previous reports of the same variants in the literature checked from Human Gene Mutation Database  and Google Scholar , in silico prediction results by Polymorphism Phenotyping-2  and Sorting Intolerant From Tolerant . For PolyPhen-2, higher scores mean more deleterious. For SIFT, amino acids with probabilities < 0.05 were predicted to be deleterious.Database investigation and in silico prediction of mutations were checked, including the frequency of each variant in the general population investigated by Exome Aggregation Consortium . Concentrations of the six cytokines aforementioned were quantitatively determined using the CBA Human Th1/Th2 Cytokine Kit II  as described previously . The min2 for 2 h, and washed in 1 × PBS . K562 cells were obtained from the American Type Culture Collection  and cultured in RPMI1640 medium  supplemented with 10 % FBS. All the antibodies including CD3-FITC, CD8-PerCP, CD56-APC, CD107a-PE , and isotype controls were purchased from Becton Dickinson .Degranulation assay were tested in 36 HLH patients and 43 controls. The assay was performed as previously described with modifications . Periphe5 PBMCs were mixed with 5 × 104 sensitive target cells (K562) as stimulants in 12 flat bottom well plates and incubated for 2 h at 37°C. An equal volume of RPMI-1640 culture medium to replace the volume of K562 cells were used as negative control. Then the cells were stained with 10μl of CD3-FITC, 10μl of CD8-PerCP, 2.5μl of CD56-APC, and 10μl of CD107a-PE (isotype IgG1-PE as negative control) for 30 min at 4 °C in the dark. After two washes with 1 × PBS of the above samples, flow cytometric analyses were performed by a FACSCalibur cytometer . CD3-CD56+ and CD3+CD8+ T cells were gated as NK cells and Cytotoxic T cells (CTLs), respectively. Data were acquired with CellQuest software (BD Bioscience).5 × 10Information at diagnosis of age, sex, with fever or not, hemoglobin levels, platelets numbers, white blood cell counts, percentage of neutrophils, total neutrophil counts, percentage of lymphocytes, total lymphocyte counts, triglyceride levels, fibrinogen levels, LDH levels, ferritin levels, sIL2R levels, and sIL2R/ferritin ratio were collected. Quantitative real-time PCR was used to detect EBV-DNA and CMV-DNA copies in sera.P-value <0.05 was considered to be statistically significant.Serum concentrations of individual cytokines, clinical data were compared between groups using the Mann-Whitney U test. A chi-square test was used to assess ratio differences between groups. Receiver operating characteristic (ROC) curves were derived from the cytokine levels for all HLH patients. In a ROC curve, the sensitivity and specificity of all six cytokines were calculated for the differentiation between primary and secondary HLH. All statistical analyses were performed using SPSS 17.0 software . A two-sided PRF1 gene (P2), and three patients with hemizygous mutation in SH2D1A gene  were categorized into primary HLH group (n = 4). Based on the available genetic data, the other 41 patients were classified into the secondary HLH group, including nine HLH patients with single heterozygous mutation , three HLH patients with only SNPs , and the remaining 29 HLH patients without any SNPs.The genetic findings of the 45 HLH patients were shown in Table PRF1, UNC13D, STX11, STXBP2, SH2D1A, XIAP, and ITK genes of all the 45 HLH cases accounted for 2/45 (4.4 %), 6/45 (13.3 %), 0/45 (0.0 %), 2/45 (4.4 %), 3/45 (6.7 %), 0/45 (0 %), and 0/45 (0.0 %), respectively. For the four primary HLH cases, mutations in PRF1 and SH2D1A genes accounted for 1/4 (25 %) and 3/4 (75 %), respectively. Three missense heterozygous variants in PRF1 gene were found in one male and one female  and c.1061A>T  in PRF1 gene were found in P2, a 6-year-3-month female, with c.757G>A inherited from her father and c.1061A>T from her mother. Five missense mutations of UNC13D gene  in P4, c.760C>T (p.Arg254Cys) in P5, c.3259C>T (p.Arg1087Trp) in P7, and c.118-307G>A in P45, were found in four males and one female, respectively. Another heterozygous mutation c.2296C>T (p.Glu766Ter) in exon23 of UNC13D gene of an 8-day male patient (P6), with the cDNA study revealed that this mutation had a deleterious effect on splicing, c.2295_2298delGCAG (p.Glu765Aspfs*27) , c.163C>T (p.Arg55Ter), and c.191G>A (p.Trp64Ter), were identified in three male patients  with c.497C>T  of STXBP2 gene, and two male patients (P7 and P38) with c.1268A>C  of XIAP gene, were identified in this cohort .Database investigation and in silico prediction of mutations in HLH patients were shown in Table P < 0.05) while those of IL-6, IL-10 and IFN-γ were significantly higher (P < 0.05) .The mean  of serum IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ concentrations for healthy controls were 2.4 (2.2–2.7), 3.2 (2.9–3.5), 6.0 (4.0–7.9), 3.4 (3.0–3.9), 2.9 (2.5–3.4), 9.2 (8.3–10.0) pg/ml, respectively. As compared to control group, the levels of IL-4 in both primary and secondary HLH groups were significantly lower (P = 0.025), with mean (95 % CI) in primary and secondary HLH groups showed as 1.1 (0.6–1.6) and 2.3 (1.9–2.7), respectively. Additionally, IFN-γ level in primary HLH had a tendency of statistically lower than that in secondary HLH (P = 0.051), with mean (95 % CI) in primary and secondary HLH groups showed as 106.6 (minus-393.9) pg/ml and 905.7 (530.7–1280.6) pg/ml, respectively. The 95 % CI gap of IL-4 between primary and secondary HLH groups was 1.6–1.9 pg/ml, while the gap of IFN-γ between the two groups was 393.9–530.7 pg/ml. The levels of the remaining four cytokines including IL-2, IL-6, IL-10, and TNF-α were not significantly different between primary and secondary HLH groups .When we compared the levels of the cytokines between the primary and secondary HLH groups, the IL-4 level in primary-HLH was significantly lower than that in secondary HLH  and the percentage of lymphocytes (P = 0.012), there was no significant difference between primary HLH and secondary HLH groups in any other factors.Information of surface CD107a level in resting NK cells, age at diagnosis, sex, with fever or not, hemoglobin levels, platelet counts, white blood cell counts, percentage of neutrophils, absolute neutrophil counts, percentage of lymphocytes, absolute lymphocyte counts, triglyceride levels, fibrinogen levels, LDH levels, ferritin levels, sIL2R levels, sIL2R/ferritin ratio, EBV-DNA, and CMV-DNA copies were shown in Table The degranulation assay results in healthy controls, primary HLH cases, and secondary HLH cases were shown in Fig. n = 16), and HLH with CD107a>5 % (n = 20). Comparisons of serum cytokine concentrations (pg/ml) among Control, HLH with CD107a<5 %, and HLH with CD107a>5 % were shown in Additional file P < 0.05). Between HLH with CD107a<5 % and CD107a>5 % groups, all six cytokines showed no statistical significance.Meanwhile, we re-grouped HLH patients based on their CD107a levels in resting NK cells: HLH with CD107a<5 %  than those of controls, and no patient was overlapped with our previous study, which further confirmed the usefulness of this cytokine profile for the diagnosis of HLH.In our previous study, we found that HLH patients presented a specific cytokine profile of highly increased levels of IFN-γ and IL-10, and a moderately increased level of IL-6 . In thisP = 0.025), with the gap of IL-4 between the two groups was 1.6–1.9 pg/ml, and IFN-γ level in primary HLH had a tendency of statistically lower than that in secondary HLH (P = 0.051), with the gap of IFN-γ between the two groups was 393.9–530.7 pg/ml. AUCs of six cytokines indicated that levels of IL-4 and IFN-γ may be used as additional tools for the quick differential diagnosis between primary and secondary HLH. Taken the results of IL-4 and IFN-γ together, we propose that HLH patients with IL-4 below 1.7 pg/ml and IFN-γ below 433.9 pg/ml have a higher possibility to be primary HLH.When we compared the levels of the cytokines between the primary and secondary HLH groups, the IL-4 level in primary-HLH was significantly lower than that in secondary HLH (P = 0.012), and lower percentage of neutrophils , which is consistent with Yasumi et al’s findings.There have been studies trying to find other discriminators to differentiate primary from secondary cases of HLH. According to the report by Bryceson et al. , degranuOur study has some limitations. First, the risk of having missed pathogenic variants is high, and possible additional variants, such as deep intronic variants, regulatory variants, and complex structural variants, might have been missed for some cases, which would have great impact on the grouping strategy and then influence the interpretation of the results. Second, as this is a single-center study, and the results are based on data from small number of samples and incomplete genetic sequencing results, a multi-center study was required to be performed to validate the results.PRF1, UNC13D, STX11, STXBP2, SH2D1A, XIAP, and ITK genes account for 2/45 (4.4 %), 6/45 (13.3 %), 0/45 (0.0 %), 2/45 (4.4 %), 3/45 (6.7 %), 0/45 (0 %), and 0/45 (0.0 %), respectively. HLH patients with lower IL-4 level, lower IFN-γ level, higher percentage of lymphocytes, and lower percentage of neutrophils have higher possibility to be primary HLH. The cytokine profiles can be used as an additional tool for the quick differential diagnosis between primary and secondary HLH.Among 45 HLH patients, four HLH patients are identified as primary HLH with hemizygous or compound heterozygous mutations, and 41 HLH patients belong to secondary HLH. Variants in


Corynebacterium glutamicum shows great potential for the production of the glutamate-derived diamine putrescine, a monomeric compound of polyamides. A genome-scale stoichiometric model of a C. glutamicum strain with reduced ornithine transcarbamoylase activity, derepressed arginine biosynthesis, and an anabolic plasmid-addiction system for heterologous expression of E. coli ornithine decarboxylase gene speC was investigated by flux balance analysis with respect to its putrescine production potential. Based on these simulations, enhancing glycolysis and anaplerosis by plasmid-borne overexpression of the genes for glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase as well as reducing 2-oxoglutarate dehydrogenase activity were chosen as targets for metabolic engineering. Changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and changing threonine 15 of OdhI to alanine reduced 2-oxoglutarate dehydrogenase activity about five fold and improved putrescine titers by 28%. Additional engineering steps improved further putrescine production with the largest contributions from preventing the formation of the by-product N-acetylputrescine by deletion of spermi(di)ne N-acetyltransferase gene snaA and from overexpression of the gene for a feedback-resistant N-acetylglutamate kinase variant. The resulting C. glutamicum strain NA6 obtained by systems metabolic engineering accumulated two fold more putrescine than the base strain, i.e., 58.1 ± 0.2 mM, and showed a specific productivity of 0.045 g·g−1·h−1 and a yield on glucose of 0.26 g·g−1. In contrast to isocyanate and vinyl groups, which are rarely compatible with biological systems, the hydroxyl, carboxyl, and amino functional groups of polyamides and polyesters occur throughout the biological world, and their total synthesis by either biocatalysis or fermentation appears feasible  (pH 7.2), 10 mM MgCl2, 3 mM cysteine, 10 vol-% glycerol. The assay mixture (1 mL) was 50 mM TES (pH 7.7), 10 mM MgCl2, 3 mM cysteine, 2 mM NAD, 0.9 mM thiamine diphosphate, 0.05 mM chlorpromazine, and 2.5 mM 2-oxoglutarate. The reaction was performed at 30 oC, started with 0.2 mM CoA, and extinction followed at 340 nm.2-oxoglutarate dehydrogenase activity was assayed as described previously . Only fr+, 50 mM Na2HPO4, 0.2 mM EDTA, and 0.5 mM glyceraldehyde 3-phosphate in 50 mM triethanolamine hydrochloride (TEA) buffer pH 8.5, at 30 oC. One unit of enzyme activity corresponds to 1 μmol NADH formed per minute.Glyceraldehyde 3-phosphate dehydrogenase activity was measured as previously reported . The ass3OH-HCl, 400 mM Tris (base), 40 mM N-acetyl-l-glutamate, 20 mM MgCl2, 10 mM ATP and up to 0.03 unit of enzyme in a final volume of 1.0 mL. The pH of the incubation mixture containing all components except N-acetyl-l-glutamate and enzyme was adjusted to 7.2 with NaOH at 25 °C. The reaction was started by the addition of N-acetyl-l-glutamate. After incubation at 30 °C for 10–20 min the reaction was terminated by the addition of 1.0 mL of a solution containing 5% (w/v) FeCl3 hexahydrate, 8% (w/v) trichloroacetic acid, and 0.3 M HCl. When necessary the precipitate was removed by centrifugation. The volume of all reagents was halved for the assay of purified enzyme. The absorbance of the resulting hydroxamate-Fe3+ complex was measured at 540 nm. One unit of N-acetylglutamate 5-phosphotransferase is defined as the amount of enzyme which catalyzes the formation of of 1 µmol of product per min under assay conditions.N-Acetylglutamate 5-phosphotransferase activity was performed based on previous report . The inc600) was measured and cells were removed by centrifugation . The supernatant was analyzed using a high-pressure liquid chromatography system .For quantification of extracellular putrescine, aliquots of the culture were withdrawn, the optical density . The respective fluorescent isoindole derivatives were detected by excitation at 330 nm (emission at 450 nm). For elution gradients of 2.5 g·Lstandard .C. glutamicum PUT21 was improved by combinatorial engineering guided by flux balance analysis. In shake flask cultivations 58.1 ± 0.2 mM or about 5.1 g·L−1 putrescine accumulated and C. glutamicum NA6 produced putrescine with 56% of the maximal product yield . An overall volumetric productivity of 0.21 g·L−1·h−1, an overall specific productivity of 0.045 g·g−1·h−1, and a biomass yield of 0.23 g·g−1 were observed (Taken together, the previously available putrescine producing strain observed . Three o


As lysosomal protein transmembrane 4 beta (LAPTM4B) is an important biomarker for many solid tumours, development of small-molecule fluorescence light-up probes for detection and imaging of LAPTM4B proteins is particularly valuable. In this work, we reported the design and synthesis of a far-red/near-infrared (FR/NIR) fluorescence light-up probe DBT-2EEGIHGHHIISVG, which could specifically visualize LAPTM4B proteins in cancer cells and tumour-bearing live mice. DBT-2EEGIHGHHIISVG was synthesized by the conjugation of two LAPTM4B-binding peptide ligands (EEGIHGHHIISVG) with one environment-sensitive fluorogen, 4,7-di(thiophen-2-yl)-2,1,3-benzothiadiazole (DBT). Owing to the intramolecular charge transfer character of DBT, DBT-2EEGIHGHHIISVG is weakly emissive in aqueous solution, but switches to fluoresce upon LAPTM4B proteins specifically bind to the peptide ligand of the probe, which provide the DBT with hydrophobic microenvironment, greatly reducing its charge transfer effect with water. It is found that DBT-2EEGIHGHHIISVG can achieve targeted imaging of LAPTM4B proteins in HepG2 cancer cells and visualize LAPTM4B protein-expressed tumour tissues of live mice in a selective and high-contrast manner. However, the fluorogens could alter to show strong fluorescence when located in hydrophobic environment, which significantly reduces ICT between the fluorogen and polar media205in vivo application of environment-sensitive fluorogen-based light-up probes is that most of the currently available environment-sensitive fluorogens are low-wavelength emitters (<650 nm), which often suffer from relatively high biological background fluorescence and low tissue permeability222325in vivo bioimaging.In recent years, great interest has been focused on the design of small-molecule fluorescence light-up probes by employment of environment-sensitive molecules161718via incorporation of DBT with a specific LAPTM4B-binding peptide IHGHHIISVGin vivo bioimaging study using a tumour-bearing mouse model reveals that DBT-2EEGIHGHHIISVG can also serve as an FR/NIR fluorescence light-up probe to selectively visualize the LAPTM4B protein-expressed tumour. To the best of our knowledge, this study reports the first example of fluorescence light-up probe for in vivo LAPTM4B imaging, which may inspire more exciting in vivo use probes in the future research.Lysosomal protein transmembrane 4 beta (LAPTM4B) is a vital biomarker for many solid tumours272815303-EEGIHGHHIISVG) was firstly synthesized via standard solid-phase Fmoc peptide chemistry. DBT-2EEGIHGHHIISVG was then synthesized through “click” chemistry between peptides of N3-EEGIHGHHIISVG and bisethynyl-bearing DBT. N3-EEGIHGHHIISVG and DBT-2EEGIHGHHIISVG were characterized by 1H NMR, HPLC, LC-MS and HRMS  of DBT-2EEGIHGHHIISVG after treatment with LAPTM4B proteins as a function of LAPTM4B concentration gives a good linear line. The limit of detection (LOD) for DBT-2EEGIHGHHIISVG is then determined to be 2.4 μg mL−1 based on the slope in via a 3-sigma methodThe photoluminescence spectra of DBT-2EEGIHGHHIISVG in phosphate buffered saline (PBS) buffer and in cell culture medium (DMEM) are displayed in −1  that are overexpressed LAPTM4B proteins were employed15via pre-treatment with IHGHHIISVG peptides verifies the specific binding interaction of the peptide ligand in the probe and LAPTM4B protein  was purchased from Nankai University Resin Co. Ltd. Human LAPTM4B full length protein was bought from Abcam. All the other starting materials were bought from Sigma-Aldrich. Chemical reagents and solvents were utilized as received from commercial sources. Bisethynyl-functionalized DBT was synthesized according to previous literatureFmoc-OSu and other Fmoc-amino acids were purchased from GL Biochem . 2-Cl-trityl chloride resin (1.0–1.2 mmol g1H NMR spectra were recorded on a Bruker ARX 400. HPLC was carried out at a LUMTECH HPLC (Germany) system using a C18 RP column with MeOH (0.1% of TFA) and water (0.1% of TFA) as the eluents. LC-MS was conducted at the LCMS-20AD (Shimadzu) system. High resolution mass spectra (HR-MS) were received from VG ZAB-HS system (England). UV-vis spectra were measured on a Shimadzu UV-1700 spectrometer. Photoluminescence spectra were measured on a Perkin-Elmer LS 55 spectrofluorometer. Cellular imaging was performed by confocal laser scanning microscopy . Non-invasive in vivo fluorescence imaging was conducted on a Maestro EX system .via HPLC (79% yield). 1H NMR  δ: 8.94–9.00 , 8.41–8.46 , 8.27–8.36 , 7.97–8.18 , 7.87–7.93 , 7.71–7.77 , 7.30–7.37 , 4.56–4.71 , 4.41–4.45 , 4.17–4.30 , 4.10–4.14 , 3.70–3.80 , 3.04–3.12 , 2.93–3.02 , 2.54 , 2.22–2.26 , 2.10–2.17 , 1.97–2.03 , 1.68–1.79 , 1.47–1.56 , 1.24–1.33 , 1.00–1.11 , 0.71–0.89 . MS: calcd M+ = 1523.65, obsvd 1/2(M + H)+ = 762.50.The azide-functionalized EEGIHGHHIISVG was synthesized by solid-phase 9-fluorenylmethoxycarbonyl (Fmoc) peptide synthesis (SPPS) using 2-chlorotrityl chloride resin and the corresponding N-Fmoc protected amino acids with side chains properly protected by a tert-butyl group. Azide caproic acid was synthesized according to a previous report4 and 50 μL of 1 M sodium ascorbate aqueous solution were added to the mixture to initiate the click chemistry. The reaction was proceeded at room temperature under stirring for 16 h to yield the final product (80% yield), which was purified by HPLC. 1H NMR  δ: 8.85–8.92 , 8.63 , 8.26–8.36 , 8.17–8.21 , 8.00–8.07 , 7.72–7.76 , 7.55–7.58 , 7.24–7.33 , 7.13 , 7.00 , 4.60–4.66 , 4.39–4.44 , 4.19–4.31 , 4.10–4.16 , 3.67–3.84 , 2.65–2.69 , 2.21–2.31 , 2.12–2.19 , 1.98–2.03 , 1.86–1.92 , 1.69–1.76 , 1.52–1.58 , 1.22–1.32 , 1.15–1.20 , 1.00–1.11 , 0.71–0.88 . MS: calcd M+ = 3395.77, obsvd 1/3(M + 3H)+ = 1133.00.Azide-bearing EEGIHGHHIISVG  and bisethynyl-functionalized DBT  were dissolved in 2 mL of DMSO. After that, 50 μL of 0.1 M CuSO2, respectively. Before experiments, the cells were pre-cultured until confluence was reached.Human hepatocelluar carcinoma cells (HepG2), hepatic L02 cells and human smooth muscle cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in a humidified environment containing 5% COHepG2 cancer cells were seeded and cultured in confocal imaging chambers and then the cells were divided into two groups. One was placed at 37 °C and the other was placed at 0 °C for 30 min. Subsequently, the cells were washed three times with 1× PBS buffer and then incubated with DBT-2EEGIHGHHIISVG (10 μM) in serum-free cell culture medium at 0 °C or 37 °C. After incubation for 90 min, the cells were washed three times with 1× PBS and fixed using 4% paraformaldehyde for 20 min at 0 °C. The cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature, which was followed by imaging with confocal laser scanning microscopy upon excitation at 488 nm and a collection of fluorescence signals above 650 nm. The cell imaging studies of hepatic L02 cells and smooth muscle cells was carried out following the same experimental procedure as that for HepG2 cancer cells.3 cells of each well. After adherence, the cells were exposed to the probe at a pre-determined range of concentrations at 37 °C. After incubation for 48 h, the cell culture medium was removed and the cells were washed twice with sterile 1× PBS. 100 μL of MTT dissolved in serum-free culture medium (0.5 mg/mL) was added into each well. After 4 h incubation, the MTT solution was removed cautiously and then 100 μL of DMSO was added into the wells, followed by gently shaking for 10 min. The absorbance of MTT at 570 nm was determined by the microplate reader (Genios Tecan). To dispel the absorption interference of the probe at 570 nm, the cells were also incubated with the same concentrations of DBT-2EEGIHGHHIISVG as the control. After 48 h incubation, the control cells were not treated with MTT and then the absorbance at 570 nm were monitored by microplate reader. The absorbance of MTT in sample well was determined by the differentiation between the absorbance of the sample well and that of the corresponding control well. Cell viability was expressed by the ratio of the absorbance of MTT in the sample wells to that of the cells incubated with culture medium only.The cytotoxicities of DBT-2EEGIHGHHIISVG against HepG2 cancer cells and hepatic L02 cells were evaluated by 3--2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, HepG2 cancer cells or L02 cells were seeded in 96-well plates at an appropriate density of 5 × 107 HepG2 cancer cells. When the tumours reached to 200–400 mm3, the mice were used for in vivo imaging study.All animal studies were performed in compliance with the guidelines set by Tianjin Committee of Use and Care of Laboratory Animals and the overall project protocols were approved by the Animal Ethics Committee of Nankai University. 8 weeks old male BALB/c nude mice ) were utilized to establish the tumour-bearing mouse model. In brief, the mice were subcutaneously inoculated in the right axillae with 1 × 10via the tail vein. Subsequently, scans were conducted with a Maestro EX in vivo fluorescence imaging system at designated time intervals after administration. When taking images, the mice were temporarily anesthetized with isoflurane and placed appropriately. The 523 nm excitation filter (ranging from 503 to 548 nm) was selected and spectral imaging from 560 nm to 900 nm (10 nm step) was performed with an exposure time of 200 ms for each image frame. Additionally, the HepG2 tumour-bearing mice intravenously injected with DBT-2EEGIHGHHIISVG or pure DBT were sacrificed at 1 h post-administration. Subsequently, the main organs and tumour tissue were isolated and imaged with Maestro system.The HepG2 tumour-bearing mice were injected with 200 μL of DBT-2EEGIHGHHIISVG or pure DBT at 120 μM How to cite this article: Chen, C. et al. Far-red/near-infrared fluorescence light-up probes for specific in vitro and in vivo imaging of a tumour-related protein. Sci. Rep. 6, 23190; doi: 10.1038/srep23190 (2016).


Lactobacillus sp. strain TCF032-E4 (= CCTCC AB2015090 = DSM 100358), isolated from a Chinese fermented radish. The total length of the 57 contigs is about 2.9 Mb, with a G+C content of 43.5 mol% and 2,797 predicted coding sequences (CDSs).Here, we report the draft genome sequence of  Lactobacillus sp. strain TCF032-E4 was isolated in 2013 from a homemade fermented white radish (brine pH 3.7) originating from Guilin, Guangxi Province, China. The 16S rRNA gene sequence of this strain shared high similarity with that of Lactobacillus plantarum subsp. plantarum ATCC 14917T (99.2%), Lactobacillus pentosus NCDO 363T (99.2%), and Lactobacillus paraplantarum CNRZ 1885T (99.1%). However, an abnormal amplification pattern (no product) was obtained with the recA multiplex PCR, a method described by Torriani et al.  and sugar fermentation pattern . These distinguishing phenotypic characters motivated a deeper analysis of TCF032-E4 through whole-genome sequencing.Lactobacillus sp. TCF032-E4 was sequenced by Majorbio Biopharm Technology Co., Ltd.  using an Illumina HiSeq 2500 platform. A paired-end library with a DNA insert length of 300 bp was generated, and 11,896,210 reads with a mean length of 101 nucleotides (nt) were obtained, representing ~413-fold genome coverage. Velvet 1.2.10 . The resulting draft genome sequence comprised 57 contigs >500 bp in length, with a maximum contig length of 279,565 bp. The total length of all contigs is 2,904,660 bp, with a G+C content of 43.5 mol%. The contigs were ordered with Mauve 2.3.1 (L. plantarum WCFS1 (The genome of LFEE00000000. The version described in this paper is version LFEE01000000.The strain is publicly available from two collections under references CCTCC AB2015090 and DSM 100358. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Fusarium fungi. Consumption of food and feed contaminated with T-2 toxin causes diseases in humans and animals.T-2 toxin is a mycotoxin that is produced by the In this study T-2 toxin was analyzed in poultry and cattle feedstuff in cities of Mazandaran province , Northern Iran.In this study, 90 samples were analyzed for T-2 toxin contamination by the ELISA method.Out of 60 concentrate and bagasse samples collected from various cities of Mazandaran province, 11.7% and 3.3% were contaminated with T-2 toxin at concentrations > 25 and 50 µg/kg, respectively. For mixed poultry diets, while 10% of the 30 analyzed samples were contaminated with > 25 µg/kg, none of the tested samples contained T-2 toxin at levels > 50 µg/kg.The results obtained from this study show that poultry and cattle feedstuff can be contaminated with different amounts of T-2 toxin in different conditions and locations. Feedstuff that are contaminated by this toxin cause different diseases in animals; thus, potential transfer of mycotoxins to edible by-products from animals fed mycotoxin-contaminated feeds drives the need to routinely monitor mycotoxins in animal feeds and their components. This is the basis on which effective management of mycotoxins and their effects can be implemented. F. acuminatum, F. poae and F. sporotrichioides. The basic structure of T-2 toxin is tetracyclic, with a sesquiterpenoid 12, 13 epoxytrichothec-9-ene ring system. This toxin is mainly found in wheat, maize, barley, oats and rye and processed grains  and complete poultry feed samples were randomly collected during winter of 2013, from Sari, Babol and Chalus cities of Mazandaran province, Nothern Iran. For each foodstuff, 10 samples were collected from each city; 60 cattle feedstuff samples were gathered in this order: 20 samples from Babol , 20 samples from Sari  and 20 samples from Chalus . Also 30 mixed poultry feedstuff samples consisting of 10 samples from Babol, 10 samples from Sari and 10 samples from Chalus were gathered, making a total of 90 samples. Samples were then put into sterile plastic bags to be protected from light. Samples were put into sealed sterile plastic bags and transported to a Mycology laboratory where they were ground to fine powder.T-2 toxin was extracted from samples and analyzed according to the manufacturer's specifications. Accordingly, 20 g of the toxin was weighed and added to a blender containing 100 mL of 70% methanol and blended for three minutes. The extract was allowed to stand for a few minutes and the content was filtered through a Whatman No. 1 filter paper. T-2 toxin contamination in extracts was measured by competitive enzyme-linked immunosorbent assay (ELISA) using the Agraquant T-2 toxin assay kit .T-2 toxin contamination rate of the samples was measured by competitive enzyme-linked immunosorbent assay (ELISA) using the Agraquant T-2 toxin assay kit (purchased from the Romer Singapore Company). Firstly, we matched the number of samples to the number of wells in the kit. Next, 200 μL of the conjugated solution was added to uncoated-antibody microplate wells. 100 μL of each standard solution  was diluted in distilled water with a ratio of 1:10, in the test tubes. Then, 100 μL of the above solution was transferred to coated-antibody microplate wells. Toxins in samples and control standards competed with the enzyme conjugate for binding to the solid phase antibody. They were incubated at room temperature (37˚C) and washed after 15 minutes by distilled water with a sprinkler. After the washing step, 100 μL of the enzyme substrate was added to wells and incubated at room temperature for another five minutes. Tetra methylbenzidine/hydrogen peroxide was used as a substrate for color development. After the washing step, a blue color was observed in the wells. Finally, 100 μL of stopping solution was added to stop the reaction and then the blue color turned yellow. The color intensity was inversely proportional to the mycotoxin concentration and measured with the ELISA reader (450-630 nm). Toxin concentration in the samples was compared with standard concentrations and absorption by using a standard curve. Optical density (OD) readings were recorded for each microwell  standard, a dose-response curve with the five standards was constructed). Since the amount of T-2 in each standard was known, the unknowns could be measured by interpolation from this standard curve. Concentration of T-2 toxin was calculated by analysis of variance (ANOVA) using the SPSS software package (P < 0.05) (according to manufacturer's instruction).Of the 20 samples collected from Babol, only 5% showed positive contamination with > 25 µg/kg of T-2 toxin and none of the samples were contaminated with > 50 µg/kg of the T-2 toxin (mean ± SE: 10.40 ± 1.82). Out of 20 samples gathered from Sari, 15% were contaminated with > 25 µg/kg and 5% with > 50 µg/kg (mean ± SE: 12.12 ± 3.05). Also in Chalus 15% of samples were contaminated with > 25 µg/kg while 5% of samples were contaminated with > 50 µg/kg (mean ± SE: 11.48 ± 2.92). Based on the experiments done on the cattle feedstuff samples, gathered from the 3 mentioned cities of the Mazandaran province, we concluded that out of 60 samples, 11.7% and 3.3% were infected with > 25 µg/kg and > 50 µg/kg of the toxin, respectively. The highest contamination level of the T-2 toxin was observed for Sari and Chalus samples. Samples collected from Babol had a lower concentration of toxin compared to samples from Sari and Chalus .Tests for T-2 toxin contamination in the cattle feedstuffs revealed that 13.3% of 30 concentrate samples had > 25 µg/kg of contamination and 3.3% were contaminated with > 50 µg/kg (mean ± SE: 10.24 ± 2.21). Tests on the beetroot bagasse samples showed that, 10% had > 25 µg/kg and 3.3% had > 50 µg/kg of T-2 toxin infection (mean ± SE: 12.76 ± 2.07). In total, from the 60 samples, 11.7% had > 25 µg/kg and 3.3% had > 50 µg/kg of contamination (mean ± SE: 11.45 ± 1.51) .Also, of the ten mixed poultry feedstuff samples that were collected from Babol, two (10%) samples showed > 25 µg/kg of T-2 toxin contamination, with this value being higher than the Iran Standard Institute limit (mean ± SE: 13.05 ± 3.43). In Sari, none of the mixed poultry feedstuff samples were contaminated with > 25 µg/kg and > 50 µg/kg of this toxin. Despite the contamination of samples, there was no contamination higher than the standard limit of European Union (EU) and Iran Standard Institute (mean ± SE: 11.03 ± 2.96). Of the ten collected mixed poultry feedstuff samples from Chalus, only one (10%) sample was contaminated with > 25 µg/kg (mean ± SE: 11.20 ± 3.53). Of the 30 poultry samples, three (10%) were contaminated with > 25 µg/kg of T-2 toxin (mean ± SE: 11.76 ± 1.86). > 25 µg/kg of T-2 toxin contamination was observed in Babol and Chalus, respectively. None of the mixed poultry feedstuff samples were contaminated with > 50 µg/kg .Fusarium spp. growth  for T-2 toxin production and can produce this mycotoxin during overwintering, under a snow cover in the field and/or during storage (T-2 toxin is toxic to humans, mammals, birds, invertebrates, plants and eukaryotic cells. Symptoms of T-2 toxin poisoning depend on the dose and way of exposure . Results. growth , 12. Aks. growth . In our f plants . Also cof plants . Howeverf plants . Cortinof plants . In our  storage . Since s storage . Our stu storage . Greco e storage . In our  storage , 18. Sel storage . In Nort storage . Thus, d


Lactobacillus species for disease treatment and prevention as well as health restoration and maintenance is not new. Probiotics have been used therapeutically to modulate immunity, lower cholesterol, treat rheumatoid arthritis, prevent cancer, improve lactose intolerance, and prevent or reduce the effects of atopic dermatitis, Crohn’s disease, diarrhea, and constipation as well as candidiasis and urinary tract infections.The concept of using L. acidophilus IMV B-7279 and L. delbrueckii subsp. bulgaricus IMV B-7281 probiotic strains to influence on Th1-type cytokines production.was study the ability of 9 cells/ml) into diluted blood. Cytokines level were investigated in cells supernatants via IFA-method.Experimental studies were performed on blood of clinically healthy donors . Venous blood was diluted in a ratio 1:4 of cell medium RPMI-1640. Cytokines were induced in diluted blood 500 µl via PHA 500 µl (40 µg/ml). Ability of peripheral blood cells to produce IFN-γ and IL-2 was studied via added probiotic strains (5 × 10L. delbrueckii subsp. bulgaricus IMV B-7281. The concentration of IFN-γ in the culture medium increased to 322.4 ± 7.5 pg/ml against 123.2 ± 13.8 pg/ml (p < 0.05) in control; IL-2 – 39.3 ± 1.1 pg/ml . L. acidophilus IMV B-7279 had no activating effect on the production of these cytokines by means of peripheral blood cells. The concentration of IFN-γ in the culture medium was 147.3 ± 15.9 pg/ml (p> 0.05), and IL-2 was at the control level .Thus, the ability of peripheral blood cells to produce IFN-γ and IL-2 was increased under the influence of L. acidophilus IMV B-7279 and L. delbrueckii subsp. bulgaricus IMV B-7281 probiotic strains have no activating effect on production of TNF-α. The concentration of this cytokine in the culture medium of peripheral blood cells, which were treated with L. acidophilus IMV B-7279 or L. delbrueckii subsp. bulgaricus IMV B-7281, amounted to 8.7 ± 0.1 and 8.9 ± 0.3 pg/ml against 8.5 ± 0.5 pg/ml in control (p > 0.05). L. delbrueckii subsp. bulgaricus IMV B-7281 probiotic strain induced in vitro production of Th1-type cytokines in the culture of peripheral blood cells.The results of our study suggested that Therefore, this strain is prospective for correction of immune response in infectious and other diseases with clinically unfavorable course accompanied by inhibition of Th1-type T-helpers/inductors.


Objective. High glucose- (HG-) induced neuronal cell death is responsible for the development of diabetic neuropathy. However, the effect of HG on metabolism in neuronal cells is still unclear. Materials and Methods. The neural-crest derived PC12 cells were cultured for 72 h in the HG (75 mM) or control (25 mM) groups. We used NMR-based metabolomics to examine both intracellular and extracellular metabolic changes in HG-treated PC12 cells. Results. We found that the reduction in intracellular lactate may be due to excreting more lactate into the extracellular medium under HG condition. HG also induced the changes of other energy-related metabolites, such as an increased succinate and creatine phosphate. Our results also reveal that the synthesis of glutamate from the branched-chain amino acids  may be enhanced under HG. Increased levels of intracellular alanine, phenylalanine, myoinositol, and choline were observed in HG-treated PC12 cells. In addition, HG-induced decreases in intracellular dimethylamine, dimethylglycine, and 3-methylhistidine may indicate a downregulation of methyl group metabolism. Conclusions. Our metabolomic results suggest that HG-induced neuronal cell death may be attributed to a series of metabolic changes, involving energy metabolism, amino acids metabolism, osmoregulation and membrane metabolism, and methyl group metabolism. Diabetes mellitus is a group of metabolic disorder diseases affecting an increasing number of the population in the world. According to survey results from Shaw et al. , approxiHyperglycemia, as a common diabetes symptom, plays a great important role in the development of diabetic neuropathy . Hypergl1H NMR-based metabolomics method was applied to examine both intracellular and extracellular metabolic changes in PC12 cells under high glucose in order to explore underlying metabolic mechanisms of high glucose-induced neuronal cell death.The PC12 cell, as a mature cell line from a rat pheochromocytoma, has been commonly used as a cellular model for studying the mechanisms underlying neuronal cell death and neurotransmitter secretion  as well PC12 cells were obtained from the Chemical Biology Research Center, Wenzhou Medical University. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and 0.25% trypsin-EDTA (1x) were purchased from GIBCO BRL . Penicillin-streptomycin solution containing 10.000 units/mL penicillin and streptomycin and phosphate buffer saline (PBS) were purchased from HyClone. Methanol and chloroform were purchased from the Sinopharm Chemical Reagent Co., Ltd. . In addition, ultrapure water was prepared using the Millipore MilliQ purification system.μL MTT (0.5 mg/mL) and placed at 37°C for 4 h. Then, the medium was removed carefully and 150 μL DMSO was added in each well to dissolve the formazan product. Finally, formazan absorbance was assessed at a wavelength of 570 nm using a microplate reader . Each experiment was repeated three times. The cell viability was assessed with 3-2,5-diphenyl-tetrazolium bromide (MTT) assay for the selection of high glucose culture condition. In brief, PC12 cells were seeded at a density of 5,000 per well in 96-well microplates. After overnight incubation, the culture media were replaced with fresh DMEM media containing different concentrations of glucose  and PC12 cells were cultured for 24, 48, and 72 h, respectively. Subsequently, each culture well was added with 20 2 at 37°C. When confluent, these cells were trypsinized and seeded in 12 T-175 culture flasks (5 × 105 cells per flask). After 24 h seeding, PC12 cells were washed three times by PBS, and the culture media were replaced by fresh DMEM media containing 25 mM glucose as the control group (n = 6) and 75 mM as the HG group (n = 6). To reduce the hyperosmolar effect of glucose, 50 mmol/L mannitol was supplemented in the control group. After 72 h of culture, these cells were trypsinized, centrifuged at 300 ×g for 5 min at 4°C, and harvested for metabolomic analysis. Meanwhile, the corresponding culture media were also collected for NMR analysis.PC12 cells were routinely thawed and cultured in T-75 culture flasks with DMEM media containing 25 mM glucose, 10% FBS, 1% penicillin, and 1% streptomycin in a humidified atmosphere containing 5% COμL of ice-cold 2 : 1 (v/v) methanol/chloroform solution and then transferred into a 1.5 mL Eppendorf tube. After vortexing, the tubes were incubated on a mixer for 10 min at 4°C. Then, 250 μL of ice-cold 1 : 1 (v/v) chloroform/H2O was added and mixed using a vortex mixer. The mixture was ultrasonicated on ice for 10 min and centrifuged at 13,000 ×g for 20 min at 4°C. The supernatant was collected, lyophilized, and stored at −80°C until analysis.The harvested cells were washed three times with cold PBS and then methanol-chloroform-water extraction was used as previously described . BrieflyMetabolites were extracted both from the blank medium (DMEM + 10% FBS) and from the conditioned culture medium using a modified Bligh-Dyer extraction method . BrieflyμL D2O and centrifuged at 12,000 ×g for 10 min at 4°C. Then, 550 μL aliquots of the supernatant were pipetted into 5 mm NMR tubes for 1H NMR experiments. All 1H NMR spectra were recorded at 25°C using a Bruker AVANCE III 600 spectrometer operating at 600.13 MHz equipped with a triple resonance probe. A one-dimensional ZGPR pulse sequence was applied to achieve satisfactory water suppression in aqueous extracts. Moreover, the other main acquisition parameters included data points, 32 K; relaxation delay, 8 sec; spectral width, 12,000 Hz; number of scans, 512; acquisition time, 2.66 sec per scan; exponential line-broadening function, 0.3 Hz. http://dx.doi.org/10.1155/2016/4125731, illustrate the typical 1H NMR spectra obtained from intracellular and extracellular extracts, respectively.The lyophilized intracellular and extracellular extracts were reconstituted with 600 3) at 1.33 ppm. Then, the spectra were integrated to binning data with a size of 0.01 ppm from 0.4 to 10.0 ppm excluding the residual water region from 4.66 to 5.02 ppm by using the Bruker Topspin 2.1 software . Finally, prior to multivariate data analysis, the binned data of each NMR spectrum were normalized to the total sum of the spectral intensity.All spectra were phase- and baseline-corrected and referenced to the methyl peak of lactate (CHR2) and the predictive ability (Q2) of the model were calculated and these two parameters close to 1.0 mean an excellent model. In addition, the absolute value of the correlation coefficient, |r|, in PLS-DA was used to identify variables mainly contributing to the separation between two groups.Partial least squares-discriminate analysis (PLS-DA) was performed to identify the separation between the control and HG groups using the SIMCA 13.0 software . Leave-one-out cross validation (LOOCV) and permutation tests (200 cycles) were conducted to assess the performance of PLS-DA. The explained variance  was applied to evaluate the difference between the control and HG groups using SPSS software (version 16.0). The difference was considered statistically significant when The change of PC12 cell viability with increases in glucose concentration and culture time was shown in 1H NMR spectra detected from intracellular extracts in the control and HG groups, and a total of 26 metabolites were identified, involving energy metabolism, amino acid metabolism, osmoregulation, and membrane metabolism as well as methyl group metabolism. Then, multivariate data analysis (PLS-DA) was employed to analyze the metabolic difference between the control and HG groups. Results based on PLS-DA show that the HG group was clearly separated from the control group , methionine (−2.31 ± 0.05 versus −2.01 ± 0.04), lysine (−9.34 ± 0.28 versus −7.30 ± 0.21), myoinositol (−2.26 ± 0.16 versus −1.66 ± 0.10), threonine (−2.03 ± 0.05 versus −1.24 ± 0.08), tyrosine (−2.77 ± 0.03 versus −2.65 ± 0.03), phenylalanine (−1.90 ± 0.03 versus −1.44 ± 0.03), and tryptophan (−0.41 ± 0.05 versus −0.27 ± 0.03) more than the control in the extracellular media, while opposite results were obtained in valine (−6.96 ± 0.19 versus −9.07 ± 0.60), ethanol (−20.92 ± 3.39 versus −32.02 ± 1.96), creatine phosphate (−0.35 ± 0.07 versus −0.63 ± 0.02), creatine (−0.13 ± 0.04 versus −0.42 ± 0.02), and choline (−1.18 ± 0.07 versus −1.81 ± 0.05). In addition, relative to the blank media, acetate level (−0.12 ± 0.19 versus 0.44 ± 0.05) in the extracellular media was increased in the control group but decreased in the HG group. Table S1 shows that both of the control and HG groups had increased levels of alanine (2.57 ± 0.13 versus 7.35 ± 0.15) and lactate (31.54 ± 0.36 versus 25.84 ± 0.51) compared with the blank media. However, the increased amount of lactate in the HG group (31.54 ± 0.36 versus 25.84 ± 0.51) was significantly higher than that in the control group, while the HG group had a lower amount of increased alanine level (2.57 ± 0.13 versus 7.35 ± 0.15), as shown in Table S1.Figure S1 shows typical Hyperglycemia plays a critical role in the development of diabetic neuropathy, which may be attributed to HG-induced neuronal cell death . In this db/db mice compared with wild-type mice -lactate infusion [ in vivo mice study, where a significant increase in aspartate was observed in the brain of db/db mice as compared with wild-type mice [The branched-chain amino acids (BCAAs) such as isoleucine and valine are important nitrogen sources for glutamate synthesis via transamination . In the infusion . Glutamainfusion . The gluinfusion . Thus, Hype mice .In addition, intracellular phenylalanine level was increased and the corresponding extracellular concentration was reduced under HG condition. A high phenylalanine level has been found to cause cell death in cultured neurons , which iOsmoregulation plays a vital role in the maintenance of cell structure and function . Myoinos in vitro study, we found that HG resulted in reduced intracellular dimethylamine, dimethylglycine, and 3-methylhistidine levels as compared with the control group, suggesting that a downregulation of methyl group metabolism may be induced by HG stress. Formate can be produced from many ways. In mitochondria, it is mainly derived from serine and glycine as well as two intermediates of choline catabolism, sarcosine and dimethylglycine [Disturbance of methyl group metabolism has been implicated in various pathological conditions . For inslglycine . In the  in vitro study, so it needs to be further verified by in vivo studies; (3) metabolites were limited to be detected by a single analytical technique, so an integrated analytical platform such as LC-MS and GC-MS is recommended to achieve a more detailed metabolic analysis; (4) the present results were only based on metabolomic analysis but combining with genomic or proteomic analysis will advance deep understanding of the mechanisms underlying HG-induced neuronal cell death.Several limitations need to be considered as follows: (1) PC12 is a mature cell line from a rat pheochromocytoma, but it could be of great interest to use a primary neuronal cell line to study the mechanisms underlying HG-induced cell death; (2) these findings were based on an in vitro results proposed in the present study may advance understanding of the pathogenesis of diabetic neuropathy. However, these findings should be further verified in in vivo studies.In the present study, we expectedly found that HG induced PC12 cells death. Thus, NMR-based metabolomics was applied to explore the metabolic mechanisms underlying this phenomenon. A disturbance of energy metabolism and a reduction in methyl group metabolism were found in the HG-treated PC12 cells. HG treatment may enhance glutamate synthesis from BCAAs, resulting in the increase in glutamate level in PC12 cells. Moreover, our results suggest that phenylalanine-induced PC12 cells death may also occur under HG stress. However, increases in myoinositol and choline may indicate a self-protection behavior of PC12 cells under HG stress. The1H NMR spectra in extracellular extracts of the control and high glucose (HG) groups were illustrated in Figure S1. A clear separation was observed between the control and HG groups by multivariate analysis (PLS-DA) based on the extracellular metabolite profiles and the contributed metabolites were identified from its loading plot . In addition, Table S1 lists the changes in extracellular metabolite levels between the control and HG groups.In the Supplementary Material, typical