Material purity is confirmed by X-ray powder diffraction and thermogravimetric analyses. Images of transmission electron microscopy show samples with a spider-net shape consisting of very fine interconnected nanoneedles. The nanostructure is characterized by crystallite of 4.4 nm in diameter and large surface area of 160.7 m2 g−1. The material delivers an initial capacity of 211 mAh g−1 with high Coulombic efficiency of 99% and 82% capacity retention after 100 cycles. Thus, α-KyMnO2 synthesized via a green process exhibits very promising electrochemical performance in terms of initial capacity, cycling stability and rate capability.With the aim to reduce the entire cost of lithium-ion batteries and to diminish the environmental impact, the extract of broccoli is used as a strong benign reducing agent for potassium permanganate to synthesize α-K Lithium-ion batteries (LiBs) have been widely used to power many systems  ,2,3. How−1 based on the transfer of one electron per formula in the potential range of 1.5–4.0 V (vs. Li+/Li). In addition to applications in batteries, MnO2 has also been used as a component in composite electrodes for electrochemical supercapacitors [2, etc., which differ from each other by the different assembly of octahedral MnO6 units. Thus, the electrochemical performance of MnO2 strongly depend on the number of sites available for Li-ion insertion in its tunnel framework. According to different conditions of reaction or synthesis methods, MnO2 can be obtained with various morphologies , nanoneedles (NNs), nanofibers (NFs), nanoflakes (NKs), nanosheets (NSs), nanorods (NRs), etc.) that promote suitable electrode material for LiBs [2 [Manganese dioxide (MDO) is an abundant, rather cheap and green material, which is not only used in primary lithium batteries , but alspacitors . It crysfor LiBs . Accordi LiBs [2 . The nan LiBs [2 .2 polymorphs, the tetragonal α-phase has attracted special attention because its large 2 × 2 tunnels are favorable for the transportation and accommodation of foreign ions [+ in the cryptomelane-type KxMn8O16 compounds [0.25Mn2O4 nanofibers exhibit excellent lithium insertion properties with high charge capacities and good rate capability [Among different MnOign ions ,18, suchompounds . Zhang epability .2 nanostructured particles has been realized by different techniques, including co-precipitation [2 via biological reducing agents such as extract of green and black tea [The preparation of α-MnOpitation , comproppitation , hydrothpitation , sonochepitation , micro-epitation , simple pitation  and reflpitation . Green spitation ,28,29. Ppitation . Previoulack tea , citrus lack tea , and extlack tea . Broccollack tea . Such velack tea . Figure lack tea ,34. ThesyMnO2 nanoneedles with a low concentration of potassium ions using a two-step green synthesis approach with the extract of broccoli plant as reducing agent, and to investigate the electrochemical performance of the as-prepared sample. The structure and morphology of the prepared sample are investigated by X-ray diffraction (XRD), thermal gravimetric analysis (TGA), Brunauer–Emmett–Teller (BET) specific surface area method, and transmission electron microscopy (TEM). Cyclic voltammetry (CV) and galvanostatic charge-discharge (GCD) experiments are carried out in lithium-metal cells with α-KyMnO2 as the positive electrode. Finally, the rate capability and cyclability of α-KyMnO2 NNs are further investigated.The aim of this work is to prepare α-K4 (oxidizing agent) and the extract of broccoli (reducing agent) was directly employed to prepare KyMnO2 nanowires without templates or surfactants, thus the synthesis can be considered as a green approach. A concentrated extract of broccoli sprouts was obtained through fresh seasonal vegetables cleaned in distilled water followed by 10 min boiling at 100 °C [4 (19 mmol) dissolved in 100 mL distilled water (DI) and acidified with 2 mL of 2.5 mol L−1 H2SO4. The entire mixture was stirred vigorously for 1 h at room temperature. Then, a change in color from purple to black was observed as KMnO4 was completely reduced by the broccoli extract and black precipitate was formed. The precipitate was isolated by filtration and washed several times by distilled water, then was dried overnight at 90 °C and further calcined at 300 °C for 5 h in air.All chemicals were analytical grade and used without further purifying. The redox reaction between KMnOt 100 °C . After fα X-ray source (λ = 1.54056 Å) in the 2θ range of 10–80°. TGA measurements were carried out using a thermal gravimetric analyzer  in the temperature range of 50–1000 °C at a heating rate of 10 °C min−1 in air. The morphology of the prepared sample was investigated by transmission electron microscopy . The BET specific surface area was measured by nitrogen adsorption/desorption at 77 K in a relative pressure P/P0 = 0.0–1.0, with P and P0 being the equilibrium and saturation pressure, respectively, using a Quantachrome NOVA Automated Gas Sorption analyzer . The pore size distribution and pore volume were estimated using the Barrett–Joyner–Halenda (BJH) method [The crystal structure of the final product was determined by XRD using Philips X’Pert apparatus  equipped with a CuK) method .yMnO2 active material, 10 wt.% conductive carbon C65 (TIMCAL), and 5 wt.% polyvinylidene fluoride (PVdF) onto an Al foil. The dried sheet was punched into ∅ 12 mm discs, and the mass loading of active material was evaluated to be 1.2–1.3 mg cm−2. These positive electrodes were assembled inside 2016 coin cells with Li metal foil as the counter electrode and 1 mol L−1 LiPF6 in ethylene carbonate:dimethyl carbonate  as electrolyte. Cyclic voltammetry measurements were carried out at scan rate of 0.2 mV s−1 using a coin-cell with a 2-electrode configuration, that implies cumulative electrochemical response of both electrodes. The cycling and rate performance were obtained by galvanostatically discharging and charging cells on a Maccor series 4000 battery tester  between 4.0 and 1.5 V at 25 °C under different current densities from 0.1 to 10 C rate (1 C = 320 mAh g−1). Electrodes for electrochemical testing were prepared by casting a slurry (N-methyl-2-pyrrolidone (NMP) used as the processing solvent) composed with 85 wt.% α-KyMnO2 were investigated by XRD, TGA, BET and TEM measurements. Particular attention was paid to characterize the crystal-chemistry of KyMnO2 synthesized via the reduction of potassium permanganate, which always induces a residual fraction of K+ ions (VIIIK ionic radius of 1.51 Å) located in the 2 × 2 tunnels of the α-MnO2 lattice, whose main functions are templating and stabilizing the tunneled network [2 phases [2 (Mn8O16) lattice . Thus, the degree of insertion of K+ ions located in the crystal tunnels depends on the synthesis process as it will be discussed in infra.The structure, composition and morphology of the as-prepared K network . Althoug2 phases ,38,39,40yMnO2 are shown in yMnO2 architecture is an assembly of nanoneedles interwoven into a mesoporous network as it will be characterized in infra. The HRTEM bright-field image of a selected well-crystallized nanoneedle  has been investigated in previous works [2 is synthesized in a basic solution. Portehault at al. obtained cryptomelane nanowires (15–40 nm in diameter) when the pH value of the solution has been controlled in the range of 0.9–1.25 [4 by H2O2 under acidic condition produced NNs with width of a few tens of nanometers [2 NNs (20–30 nm in size) without K+ inclusion using a sonochemical hydrolysis of Mn(3)acetate with a solution close to neutral pH followed by mild drying [SEM and TEM images of Knoneedle d displayof KMnO4  and the us works ,44,45,460.9–1.25 . It appeyMnO2 NNs. The main characteristic peak appearing at 2θ value of 37° is indexed to the (211) plane, while other diffraction peaks at 42.0°, 55.5° and 66.4° with low intensity in pattern are assigned to the (301), (600) and (002) planes, respectively, of the tetragonal α-MnO2 phase  with no apparent impurities. No diffraction pattern attributable to other crystallographic MnO2 is observed. The low intensity is due to the poor crystallinity and the peak broadening indicates the nanosized character of α-KyMnO2 samples. The refined lattice parameters are a = 9.95(2) Å, c = 2.76(7) Å and V = 274.1 Å3. A comparison with values of the α-MnO2 crystal  confirms the lattice expansion along the a-axis direction, which may have originated by the presence of bigger six-coordinated Mn3+ ions  than Mn4+ (rMn4+ = 0.53 Å). Meanwhile, the elementary unit volume matches well with values of the α-KyMnO2 structure, in which a low concentration of K+ ions is inserted in the 2 × 2 tunnels  [3 was reported by Vicat et al. for K1.33Mn8O16 [θ ≈ 37.6° is found to be of 4.4 nm, showing the polycrystalline character of α-KyMnO2 NNs. The combination of XRD data and HRTEM image reveals that NNs are composed of a few aggregated crystallites. This result is comparable with those obtained from α-KyMnO2 nanoneedles prepared by the mild synthetic method using the extract of tea as reducing agent, in which XRD patterns are dominated by the (211) Bragg line and the crystallite size is found to be ~4 nm [yMn8O16) . For exa33Mn8O16 . Scherre2 adsorption desorption isotherm and Barrett–Joyner–Halenda (BJH) pore-size distribution for as-prepared α-KyMnO2. It can be noticed that the isotherm curve displays an increase in the amount of adsorbed N2 with increase of the P/P0 value; the appearance of a hysteresis loop indicates the hierarchical mesoporous structure of α-KyMnO2 NNs. At P/P0 = 0.97, the volume of N2 adsorbed is found to be 450 cm3 g−1. The mesoporous nature of the as-prepared α-KyMnO2 is also illustrated by the graph in inset of 2 nanoparticles (nanorods or nanoneedles) along the lateral faces. The mesopore-size distribution in the range 1–20 nm displays a single peak centered at 10.4 nm, with total pore volume of 0.950 cm3 g−1. The α-KyMnO2 samples (powders) are composed of secondary particles (agglomerates), which are interconnected nanoneedles (primary particles) with diameters in the range of 5–20 nm and lengths of 100–300 nm. The mesopores (10 nm in size) correspond to the intraconnecting voids existing between randomly-packed nanoneedles [oneedles .2 materials were studied extensively  [2 g−1) [2 g−1) [The effects of the particle size and the surface area of the nanostructured MnOexample, ,53,54,55perature . Li et a6 m2 g−1  The BET  m2 g−1) , hydroth [2 g−1) , silica  [2 g−1) , mild re [2 g−1) ).yMnO2 NNs with relatively small particles. The K-birnessite-type MnO2 hydrothermally synthesized at 160 °C for 30 min exhibits a narrow pore size distribution around 5.4 nm [2 nanorods (NRs) synthesized by reduction of KMnO4 with a triblock polymer assisted by sonication  [2 nanorods prepared by the solvent-free synthesis method show a unique pore-size distribution peak positioned at 16 nm [Thus, the synthesis technique used in this work appears to be efficient in providing mesoporous pure α-phase Kd 5.4 nm . Regardi13.5 nm) . The mesat 16 nm . yMnO2 NNs analyzed by inductively-coupled plasma (ICP) measurements shows 52.62 wt.% Mn and 1.45 wt.% K. This result confirms that the cryptomelane-type sample contains a small concentration of potassium. The formula can be calculated as α-K0.03MnO2, taking into account the error of measurements. The presence of K+ ions inside 2 × 2 tunnels is considered to be positive to stabilize the tetragonal structure and enhance the Li-diffusion kinetics [2 to the non-conductive Mn2O3 (dense bixbyite structure) occurring with a release of oxygen (2MnO2→Mn2O3 + ½O2) via an exothermic reaction [yMn8O16 structure [2→Mn2O3 transformation occurs in the temperature range of 480–670 °C, 480 °C being the decomposition temperature for a material without any large stabilizing cations and 670 °C being the transformation temperature of K2Mn8O16 to bixbyite [+ ions in α-KxMn8O16. According to x = 0.035), which is close to the value obtained from ICP measurement. Thus, the amount of electrochemically inactive K+ ions (1.45 wt.%) is confirmed to be much smaller than that of Mn (52.82 wt.%), and does not obviously affect the specific capacity of the electrode.The chemical composition of Kkinetics . This coreaction . Severaltructure ,58. Genebixbyite . The decyMnO2 nanoneedles as a cathode material for lithium batteries was tested electrochemically by cyclic voltammetry (CV) and galvanostatic charge discharge (GCD) experiments using 2016-type coin cells. All measurements were carried out at room temperature. Note that without reference electrode, the choice of cell voltage range should exclude that of the lithium electrode. yMnO2 NNs recorded at scan rate of 0.1 mV s−1 within the voltage range of 1.0–4.0 V. The redox peak is attributed to the Li insertion associated with the Mn4+/Mn3+ couple. The initial open circuit voltage (OCV) of the cell with α-KyMnO2 NNs electrode was 3.08 V. The broadening of CV peaks is attributed to the poor crystallinity of α-KyMnO2 electrode [2 tunnels. The second cycle and subsequent ones differ notably from the first one with cathodic peaks shifted to higher voltage at 2.85 and 1.73 V. The voltage shift is very small for the anodic peak at the second cycle indicating a stabilization of the insertion/extraction mechanism in K0.03MnO2 host lattice. Poyraz et al. [+ content in the tunnels. Sample with high K+ content have smaller initial discharge capacities compared to low K+ containing sample. The initial electrochemical modification (1st cycle) is assigned to the “Li-cell formation” with the structural change that results from the so-called “loss of lithium inventory” [+ ions inserted during the first discharge remains trapped in the tunnel structure. Both structural K+ and trapped Li+ ions stabilize the MnO2 framework for the second and further discharge-charge cycles. The inserted Li+ ions occupying the 8h and 8h’ Wyckoff sites of the tetragonal lattice requires different energies of formation [The suitability of the α-Klectrode  rather tlectrode . Two redz et al.  demonstrventory” . A fractormation .0.25MnO2 at about 2.22 V vs. Li+/Li toward higher potential in subsequent cycles was reported by Zhang et al. [yMnO2 lattice, more Mn3+ ions remain at the end of the redox process. Mn3+ is a Jahn–Teller ion, which induce a lattice distortion and, consequently, a change in the Mn4+/3+ redox potential. (ii) The redox potential shift could be due to the reduction of Mn3+/Mn2+. Let us consider the standard potentials Mn4+/Mn3+ and Mn3+/Mn2+ redox reactions in solids. For aqueous systems, the MnIVO2 → Mn3+ → Mn2+ redox reactions occur at the standard potential of 0.96 and 1.51 V vs. NHE, respectively. Mn3+/Mn4+ displays an oxidation potential at 0.8 V vs. RHE in perovskites. Spinel materials containing Mn3+ and Mn2+ also exhibit high activities at 0.8 V vs. RHE. The reduction of β-MnO2 and the formation of Mn3O4  occurs at 0.70 V vs. RHE. For aprotic systems, the lithium pyrophosphate Li2MnP2O7 and LiMnPO4 olivine display a Mn3+/Mn2+ redox potential centered at 1.17 and 0.82 V vs. SCE, respectively. Wang et al. [2: the anode and cathodic peaks at 0.45 and 0.22 V vs. SCE are assigned to the Mn4+/Mn3+ reaction, while 0.0 and −0.25 V are the anodic and cathodic peaks of the Mn3+/Mn2+ reaction. Thus, the difference in the cathodic peak positions of 0.47 V is higher than the potential shift (0.27 V) observed in our voltammograms.However, during the first cycle, the cathodic peak shift has been observed by several workers, but the reason for such trend is not exactly clear ,63,64. Tg et al. . Some log et al.  reported0.25MnO2 [Eox (2.85 V) and Ered (2.58 V) is ΔE = 0.27 V in the first cycle, but diminishes to as low as 0.05 V in forthcoming cycles, which is the other good evidence for high reversibility of as prepared α-KyMnO2. Furthermore, the good reversibility is due to the presence of K+ ions within the 2 × 2 tunnels of the α-MnO2 structure maintained stable towards Li insertion and extraction during cycling. The excellent redox reversibility in the electrode is also demonstrated by the equal charge calculated by integration of the area under the reductive (discharge) and oxidative (charge) CV peaks. It is worthy to note that the polarization ΔE of 50 mV at a sweep rate of 0.1 mV s−1 for K0.03MnO2 NNs is smaller than ΔE = 170 mV of α-MnO2 synthesized by acid digestion of Mn2O3 powders, due to the higher electronic conductivity of K-doped MnO2 [+ ions [E is irrespective of the degree of structural order [Ip vs. scan rate ν−1/2 plot shows a linear behavior, which corresponds to a diffusion-controlled process.The oxidation process occurs with the appearance of corresponding anodic peaks at 2.90 and 2.38 V. The overlapping of the charge–discharge profile of the second cycle with subsequent ones implies a good reversibility. Similar electrochemical features have been observed previously for K0.25MnO2 . In addiped MnO2  and the  [+ ions . It was 0.03MnO2//Li cells cycled in the voltage range of 1.5–4.0 V at constant current density of 30 mA g−1 at 25 °C. We can observe a slow decrease of the cell voltage with the presence of two pseudo-plateaus, which are evidenced as an S-shaped profile indicating a topotactic reaction for the Li insertion in the MnO2 framework, in which two inequivalent sites are available to coordinate Li+. Furthermore, the electrochemical behavior observed in CV measurement is confirmed by GCD experiment. There is a notable difference between the first discharge profile and forthcoming ones with a shift of plateaus to higher potential starting from the second cycle. The material also exhibits a capacity loss in initial four cycles from 211 to 198 mAh g−1. The Faraday yield of 0.68 F mol−1 for MnO2 is consistent with the mean oxidation state +3.885 for Mn ions determined by elemental analysis assuming an equal number of K+ and Mn3+ cations.h and 8h’ Wyckoff position , (ii) an ion-exchange reaction inducing a partial extraction of potassium from the centered 4e site, and (iii) a fraction of inserted lithium ions become trapped within the crystal structure of MnO2 during the first charge that enhances the lattice stabilization. Johnson [2 prepared by the acid digestion of Mn2O3 by H2SO4 but did not justify this claim by complementary experiments. Considering the small amount of K+ ions, the pronounced change in electrochemical behaviors in the second cycle (vs. the first cycle) could be signed to the difference in the site occupation by Li+ ions rather than the ion exchange in our sample. Recently, the CV peak shift during first scans has also been observed in the Li-KyMnO2 system due to the irreversible local activation in the intercalated lattice and the formation of the solid interphase (SEI) layer [The pronounced changes in the electrochemical profile of the second cycle have been reported several times ,68,69,70 Johnson  assignedI) layer ,72.Q/dV), of the first and second lithiation processes). This analysis can be considered as an efficient tool to determine the electrochemical spectroscopy of an electrode [yMnO2 framework.lectrode . For inslectrode ,75. The 0.03MnO2 electrode were tested in the voltage range 1.5–4.0 V at various current densities in the range 0.1–10 C. Results are presented in 0.03MnO2 electrode delivers a specific capacity of ~32 mAh g−1 at 10 C. 0.03MnO2 electrode carried out at 30 mA g−1 up to 100 cycles. Except for the first few cycles, the capacities decrease steadily. The fade rate of 0.3 mAh g−1 per cycle for α-K0.03MnO2 NNs electrode is much smaller than that for the unstabilized α-MnO2 (2.3 mAh g−1 per cycle) [8O16 nanofibers [8O16 nanorods [0.03MnO2 NNs is evidenced by the relevant coulombic efficiency that remains around 98.8% after several cycles. Considering the satisfying electrochemical stability, it seems that the cationic exchange  is negligible during the Li insertion in the α-K0.03MnO2 framework.The rate capability and cycling stability of the α-Kr cycle) . This benofibers  and for nanorods . The gooyMnO2 nanoparticles have been widely investigated in supercapacitor, to the best our knowledge, there is a limited number of studies devoted as cathode materials in LiBs [yMnO2 has been successfully prepared by the reduction of MnO4− ions in acidic medium at room temperature. Nanoneedles are well formed even within a short reaction duration of 1 h. A lot of literature has been reported on the synthesis of nanoparticle α-MnO2 based materials, a brief summary is given here. Gao and Norby reported that the growth of α-MnO2 nanofibers started after 30 min as a mixture of birnessite and cryptomelane phase via hydrothermal route at 140 °C. Relatively pure cryptomelane nanofibers would only be obtained after a reaction time of ~4 h [2 nanowires (average diameter of 30 nm) were synthesized by only using potassium permanganate and sodium nitrite (molar KMNO4:NaNO2 ratio of 2:1) in acidic solution, while nanorods (50–100 nm diameter) were formed for decreasing molar ratio to 2:5 [2 nanoneedles with diameter of 10–50 nm and length of 200–500 nm were prepared by a two-step synthesis at temperature of 83 °C with a more complex procedure using MnCl2·4H2O, isopropanol and KMnO4 raw materials [Although the application of cryptomelane K in LiBs ,75,76,77 of ~4 h . Single-o to 2:5 . Similaraterials .yMnO2 have been obtained using a much simple synthesis process, in which the biologic reducing agent allows shorter reaction duration in ambient conditions. Let compare our material with others. Cryptomelane-type K0.106Mn2O4 nanoneedles were prepared via a template-free, one-step hydrothermal method (HTM) using a complex chemistry composed of manganese(II) acetate tetrahydrate (MnAc24H2O) 99%, oxone monopersulfate compound , and potassium nitrate (KNO3). Needle-like samples were obtained by HTM at 200 °C for 20 h [0.25Mn2O4 nanofibers with high potassium content with diameters in the range of 10–20 nm [0.3MnO2 nanocrystals were obtained using PVA [α-MnO2 NWs using a hydrothermal method, in which potassium permanganate is reduced by acetic acid. The NWs with a length about 6–10 mm and an average diameter of 20 nm were obtained using 3 mmol KMnO4 dissolved in 0.4 mol L−1 CH3COOH solution (30 mL) [α-MnO2 nanowires (width of ~30 nm and length of ~10 µm) with exposed (110) crystal plane using KMnO4 and NH4F in hydrothermal conditions but did not report the presence of tunneled K+ ions [Excellent properties of α-Kfor 20 h . Similarsing PVA . Cheng e (30 mL) . Li et a K+ ions .α-KyMnO2 appeared to be favorable of enhanced electrochemical performance partly due to the modified lattice parameters . Similar results have been reported by Feng et al. for the urchin-like MnO2 formed by the assembly of nanoneedles prepared in acidic conditions [2 g−1 is greater than values reported for MnO2 prepared by different methods [2 g−1 was reported for mesoporous α-MnO2 hollow urchins prepared by mild reaction route [α-MnO2 nanorods synthesized by reduction of KMnO4 with a triblock polymer assisted by sonication  [2 g−1 for α-MnO2 NWs were shown to be dependent on the growth conditions [α-MnO2 nanotubes with large surface area (226 m2 g−1) fabricated by exfoliation of α-MnO2 nanoflowers exhibited enhanced lithium storage properties [2 with fiber-like morphology growing along the c-axis [+ ions concentration in the tetragonal crystal [α-MnO2 nanoparticles is time- and temperature-dependent, it is noteworthy that the use of extract of broccoli favors the formation of nanoneedle-shape. During the synthesis, the purple color of KMnO4 disappeared quickly after less than 1 h at room temperature and nanoneedles with diameter of about 5 nm and length of about 100 nm were formed. On the contrary, for the synthesis of K0.08MnO2 from the reduction of KMnO4 in sulfuric acid without chelating agent, the final product was obtained after a reaction at high temperature of 60 °C for longer duration of 8 h [The nanoneedles morphology of nditions . A secon methods ,51,52,5313.5 nm) . Specifinditions . α-MnO2 operties  The morpe c-axis . Dai et  crystal . As the + ions in the cryptomelane structure maintains the integrity of the lattice and is suitable for improving electrochemical properties of α-MnO2 when applying as the cathode material for LIBs [+ ions. Tompsett and Islam performed a comprehensive ab initio study of Li insertion in hollandite α-MnO2 but did not consider a lattice with K+ ions in the tunnels [2. Due to the lattice expansion and the electron donation, higher concentration of K+ ions not only improves the Li+ diffusion coefficient (DLi+) and enhances the electronic conductivity by boosting electrons hopping via Mn3+/Mn4+ couples, but also reduces the specific discharge capacity because the addition of inactive K+ ions. An enhanced DLi+ has been estimated from 2.8 × 10−15 to 1.9 × 10−12 cm2 s−1 when pure α-MnO2 was doped with 0.25 K+ ion per formula [DLi+ ≈ 10−10 cm2 s−1 for the hydrated α-phase K0.062MnO2 using cyclic voltammetry. Recently, using electrochemical impedance spectroscopy, Abuzeid et al. reported DLi+ values of 5.2 × 10−11 and 2.1 × 10−11 cm2 s−1 for K0.11MnO2 and K0.06MnO2, respectively, showing the diffusivity enhancement with the higher content of K+ ions [2 with different particle shapes, particle sizes and particle size distributions [Several studies have demonstrated that introduction of Kfor LIBs ,39,72,83 tunnels . However formula . Bach et formula  calculat K+ ions . Meanwhiibutions ,86. Addiibutions ,88.α-KyMnO2 nanoneedle-like material reported herein synthesized with the assistance of extract of broccoli plant as reducing agent with a small concentration of potassium has shown excellent electrochemical performances. However, the use of nanoneedles as electrode requested careful fabrication. It is worth noting that the composite hardly adheres to the Al foil during the electrode coating. This may attribute to the rather soft aluminum foil and the weak interaction between the very fine MnO2 nanoneedle and aluminum . Thus, the preparation of α-KyMnO2 electrode with ultra-high mass loading would be difficult and calls for the further study.The yMnO2 comes from the insertion of lithium ions on available empty sites inside the (2 × 2) tunnels, i.e., off-center 8h Wyckoff positions [yMnO2 + xLi+ + xe− → LixKyMnO2. However, the real situation seems to be more complex due to the presence of K+ ions in the MnO2 tunnels. We observed a drastic change of the discharge voltage profile after the first cycle. Such a behavior has been revealed by several reports on α-MnO2 materials with different tunneled cations [0.00MnO2 reported by Poyraz et al. [+ concentration . However, data of the literature are quite controversial showing the major influence of the central cation in the tunnel and the main role of the material morphology. In the early work, Rossouw et al. [2 0.106MnO2·0.3H2O) upon Li insertion that implies similar free energies for the different hosting Li sites after several cycles [α-MnO2 0.143MnO2), while the discharge profile change is less pronounced for ammonia-stabilized sample 0.2MnO2). Kijima et al. confirmed the loss of the plateau with cycling of α-(Li2O)0.12MnO2) [α-K1.0Mn8O16 synthesized by hydrothermal route at 120 °C that showed a voltage plateau at ca. 2.3 V and an initial discharge capacity of 360 mAh g−1 at current density of 0.1 A g−1. A non-negligible amount of K+ ions were extracted from the K1.0Mn8O16 nanofibers cathode in the first charge reaction but showed a Coulombic efficiency of ~50% at C/3 rate [8O16 nanorods [2 nanowires (30 nm in diameter) prepared by oxidation of Mn2+ by MnO4− and an initial capacity of 251 mAh g−1 at C/2 rate but the K+ content was not mentioned. Kumakai et al. demonstrated a two S-shaped discharge behavior with plateaus at 2.6 and 2.3 V for K0.72Mn8O16 prepared by hydrothermal method at 80 °C [α-KyMnO2 and its Co-doped specimen (KyMn0.88Co0.12O2), the Li is inserted into two types of empty sites of the small (1 × 1) and large (2 × 2) tunnels with an initial discharge capacity of 250 mAh g−1 at 10 mA g−1.It is well known that the discharge capacity of α-Kositions  with the cations ,90. Aftez et al. , who show et al.  observedw et al.  revealedl cycles . Johnsonl cycles  indicatenanorods . In contat 80 °C . They styMnO2 prepared in this work to previous reported cryptomelane-type α-KyMnO2 cathode materials. In the early work by Ohzuku et al. [yMn8O16 cathode materials prepared by two different methods showed S-shaped discharge curves with the relatively high voltage plateaus at 3.0 and 2.45 V. Sol–gel K1.2Mn8O16 and acid digestion K1.3Mn8O16 exhibit modest initial discharge capacities of 160 and 188 mAh g−1, when cycled at 50 mA g−1, respectively, due to the micrometer-sized particles [α-KyMn8O16 nanorods  synthesized using a reflux method with capacity of 159 mAh g−1 at 50 mA g−1 [0.25MnO2 prepared by hydrothermal method at 160 °C and MnO2 obtained by acid digestion (HNO3 treatment) of the K-doped material shows specific discharge capacities of 143 and 170 mAh g−1 at current density of 0.1 A g−1, while in terms of cyclability, the capacity retention of 62% at 5 C rate is better for K0.25MnO2 than for the undoped cathode [0.14Mn0.9Co0.1O2 from MnSO4 and K2S2O8 solution showed higher initial discharge capacity 180–200 mAh g−1 at 50 mA g−1 [0.84Mn8O16·0.25H2O nanowire-type electrode material fabricated by thermal regeneration process of MnO2 waste delivered a specific capacity of 120 mAh g−1 at 50 mA g−1 [yMnO2 nanorods (100 nm in size) prepared by template-based sol-gel method delivered an initial discharge capacity of 183 mAh g−1, which stabilized on subsequent cycles to 134 mAh g−1 [0.04MnO2 formed by nanoneedles synthesized by sol-gel assisted by a redox reaction between ascorbic acid and KMnO4 showed an outstanding initial specific capacity of 230 mAh g−1 and 45% capacity retention at cycle one hundred [α-KyMnO2 cathode prepared via biological reducing agent (broccoli extract) assisted synthesis shows good electrochemical performance.u et al. , KyMn8O1articles . Similar0 mA g−1 . A compa cathode . The Co-0 mA g−1 . After 20 mA g−1 . KyMnO2  mAh g−1 . Urchin- hundred . CompareyMnO2 nanoneedles in pure cryptomelane phase were successfully prepared by a facile, one pot, scalable and environmentally-friendly method. An alternative biological reducing agent, i.e., extract of broccoli plant, was used instead of a traditional chemical chelate. This green-synthesis route is a straightforward and inexpensive facilitating mass production of nanostructured α-MnO2 particles stabilized by potassium ions without templates. The nanostructure characterized by crystallite of 4.4 nm, large surface area 160.7 m2 g−1, high porosity and low potassium content of 0.03 per formula in the 2 × 2 tunnels favors good electrochemical performance. Electrochemical tests show an initial capacity ca. 211 mAh g−1 with high Coulombic efficiency of 99% and good reversibility with a capacity retention 82% after 100 cycles. Thus, α-K0.03MnO2 nanoneedle-shaped material synthesized via the broccoli-extract-assisted green process could be considered as a promising cathode for LiBs.In this work, one-dimensional α-K


Compared to the control, CYP isoforms and their transcriptional regulators mRNA expressions significantly increased when the Hep G2 cells were treated with DPTM for a certain period of time. In the Ames test, Salmonella strains TA97, TA98, TA100, TA102 and TA1535 were treated with or without the metabolic activation (S9). Analysis showed the average number of revertant colonies per plate was less in double in the groups treated with DPTM than that in the negative control plate and showed no dose-related increase.14-O-[thioacetyl] mutilin (DPTM) is a promising drug candidate with excellent antibacterial activity against Gram-positive bacteria. The present study was designed to characterize its Cytochrome P450 (CYP) enzymes inhibition activities and the genotoxicity with the standard Ames test. We determined the inhibitory effects of DPTM on CYP1A2, CYP2D1/6, CYP2E1, CYP2C11/9 and CYP3A/4 in rat liver microsomes (RLMs) and in human liver microsomes (HLMs). The mRNA expressions of the above CYP isoforms and their transcriptional regulators were also evaluated using the Hep G2 cell model. The results showed DPTM exhibited a moderate inhibitory potential against CYP3A/4 (IC Pleuromutilin class of antibiotics selectively inhibits bacterial protein synthesis by interacting with the peptidyl transferase centre (PTC) of prokaryotic ribosomes at the A- and P-site through hydrophobic interactions and hydrogen bonds4. The modifications of pleuromutilin side chain led to tiamulin  inhibition and limited bioavailability13. Tiamulin is also a potent inhibitor and inducer of CYP, via the formation of a metabolic intermediate complex14. Lefamulin showed relative high inhibition for CYP3A4 with IC50 < 5 μM15. Therefore, the investigation of inhibition potency of pleuromutilin analogues against CYP was subsequently emphasized to avoid the risk for development a new drug.Azamulin Fig. , an azolO-[thioacetyl] mutilin , Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), as well as in vivo activity using systemic infection mode in mice16. As previous studies reported that pleuromutilin analogues showed high CYP inhibition effect18, we evaluated the inhibitory effects of DPTM on the major CYP enzymes, including CYP1A2, CYP2D1, CYP2E1, CYP2C11 and CYP3A in rat liver microsomes (RLMs) and CYP1A2, CYP2D6, CYP2E1, CYP2C9 and CYP3A4 in human liver microsomes (HLMs). Furthermore, mRNA expressions of CYP enzymes and their transcriptional regulators, including the aryl hydrocarbon receptor (AhR), the pregnane X receptor (PXR) and the peroxisome proliferator-activated receptor (PPAR) were investigated after treatment by DPTM using the human HepG2 cell-line. Moreover, the genotoxic properties of DPTM were also assessed by the standard Ames test with and without metabolic activation (S9).A novel pleuromutilin derivative, 14-50) value. On the whole, along with the increase of concentrations of DPTM, the cell viability became lower with the IC50 value of 10 ± 2 mM.Using the MTT -2,5-diphenyl-2H-tetrazolium bromide) assay, the cytotoxic potential of DPTM was evaluated on Hep G2 cells. The viability percentages of cells treated with different dilutions of DPTM were shown in Fig. 50 values with 95% confidence intervals from eight-point concentration–response curves  within the reference ranges19. In RLMs and HLMs, DPTM exhibited moderate inhibitory potentials against CYP3A and CYP 3A4 with IC50 values of 10 ± 2 and 8 ± 2 μM, respectively. In the cases of CYP1A2, CYP2D1/6, CYP2E1 and CYP2C11/9, DPTM showed weak inhibitory effects with IC50 ≥ 61 ± 8 μM.We used cocktail method to assess the direct inhibitory potential of DPTM on CYPs in RLMs and HLMs. The determined ICves Fig.  were lisTo further investigate the inhibitions of DPTM against CYP isoforms, the expressions of the regulated genes of CYP1A2, CYP2D6, CYP2E1, CYP2C9 and CYP3A4 and their transcriptional regulators  were evaluated using the Hep G2 cell model. Compared to the control, treatment for 2 and 4 h with 4.98, 0.16 and 0.08 mM DPTM resulted in significantly increase in CYP1A2, CYP2C9 and CYP3A4 mRNA expressions -mutilin, the main process impurities, could be neglected toxicity properties because they were usually used as pharmaceutical raw materials. Therefore, in spite of the low purity of DPTM, the biased results of its CYP enzymes inhibition activity and the genotoxicity could be negligible.In the present studies, the purity of DPTM (98.72%) was below 99.00%, which was not enough pure to examine its inhibitory effects and genotoxic property. According to the synthetic scheme16 and HPLC chromatography of DPTM  thioacetyl) mutilin 27. However, the selective inhibition for CYP3A4 may be a primary function of the pleuromutilin portion of the molecule28. Furthermore, the side chain structure of pleuromutilin derivative has some influence on CYP3A4 inhibition profiles29.CYP enzymes are very important for the metabolism of a wide range of endogenous compounds, as well as xenobiotics including drugs, environmental pollutants and dietary products31. The present study showed DPTM could increase the mRNA expressions of CYP isoforms. However, the significantly decrease of the mRNA expressions of CYP1A2, CYP 2D6 and CYP 2E1 when treated with DPTM for 12 h  of 1.7–1.9 h in rats after intravenous administration32. We speculated that HepG2 cells metabolize DPTM rapidly within a short time. After treatment for 12 h, the majority of prototype drug was changed to one or more new compounds that probably inhibited the metabolic function of the cells. However, this mechanism was obtained on the basis of speculation and should be further confirmed.For further evaluating the inhibition activities of DPTM on the CYP isoforms, we investigated CYP enzymes and their main transcription factors  mRNA expressions using human HepG2 cell-line. The main transcription factors AhR, PXR and PPAR are responsible for the induction of one to four CYP families The mRNA expressions of transcriptional regulators were also increased when HepG2 cells were treated by DPTM Fig. . Interesin vitro34. The present study was carried out using five S. typhimurium strains TA97, TA98, TA100, TA102 and TA1535 with and without S9 metabolic activation. We did not observe any treatment-related mutagenic activity in the histidine auxotrophy of the S. typhimurium strains up to 3.98 μmol/plate, indicating that under this study conditions, they do not produce reverse mutations in five S. typhimurium strains either in the presence or absence of metabolic activation system. Furthermore, no linear relationship of number of colonies induced by five doses of DPTM was observed with or without S9.Ames test is a rapid, effective and short-term bacterial reverse mutation assay which is commonly employed as an initial screening for genotoxic activity of chemical substances In conclusion, DPTM displayed moderate inhibitory potentials on CYP3A/4 and weak against CYP1A2, CYP2D1/6, CYP2E1 and CYP2C11/9 in RLMs and HLMs using cocktail method. Furthermore, this compound significantly increased the mRNA expressions of CYP isoforms and their transcriptional regulators when HepG2 cells were treated for a certain period of time. The Ames test showed that DPTM did not exhibit mutagenic response under the present experimental conditions. These results provide useful data and information for further developing DPTM to a potential anti-bacterial clinical candidate.16. The purity of this compound was checked by HPLC and quantitative NMR analyses with 98.72%  strains TA97, TA98, TA100, TA102 and TA1535 were purchased from Molecular Toxicology, Inc. (MolTox), USA 35.Hep G2 cells were purchased from Beina Biotechnology Co., Ltd. . RPMI-1640 culture media and SYBR Green Real-time PCR Master Mix were purchased from Thermo Fisher Scientific . Foetal bovine serum was purchased from Bovogen Biologicals Pty Ltd . Male Pooled SD Rat Liver S9, male SD rat liver microsomes and pooled human liver microsomes were all procured from Research Institute for Liver Diseases (Shanghai) Co., Ltd . PrimeScript RT reagent Kit was purchased from Takara Biotech Co., Ltd. . 38 with some modifications. Briefly, 100 μL/mL of RPMI-1640 medium containing 10% foetal bovine serum and Hep G2 cells was seeded in each well of a flat-bottom 96-well plate. After proliferating to logarithmic growth phase at 37 °C under 5% CO2, cells were seeded to a new 96-well plate with a density of 4 × 106 cells/hole and cultured for 24 h at the same condition. The media were replaced with 100 μL of serial dilutions of DPTM . The blank group only used 1% DMSO to replace the media. The obtained mixture was incubated for 24 h. After that, all media in cells were removed and 20 μL of MTT reagent was added for an additional 4 h. Then, supernatant fraction was removed and 100 μL dimethyl sulfoxide (DMSO) was added, followed by incubation at 37 °C for 15 min. The absorbance of each well was determined by a spectrophotometer at dual wavelengths of 570 and 279 nm for the background. The viability percentage was calculated by the following formula: cell viability % = (ODsample − ODblank/ODcontrol − ODblank) × 100. A sigmoid-shaped curve was fitted to the data, and the IC50 was calculated by fitting the Hill equation to the data with nonlinear regression (least-squares best fit modelling) using GraphPad Prism 4 . The measurements were repeated at least three times.Intrinsic toxicity of DPTM was conducted as described in the literature39. DPTM (250 μmol) was dissolved in 1.25 mL methanol to a solution and then diluted with methanol and water to 2, 10, 20, 100, 200, 400, 1,000 and 2000 μM, respectively. The obtained serial dilutions (10 μL) were added to the incubation mixtures , containing 100 μL RLMs or HLMs , 10 μL glucose-6-phosphate , 10 μL glucose-6-phosphate dehydrogenase , 20 μL potassium phosphate buffer  and 20 μL MgCl2 , to give the final concentrations were 0.1, 0.5, 1, 5 10, 20, 50, and 100 μM, respectively. For the groups that in the absence of DPTM, 10 μL methanol (1%) replaced the DPTM and was added to the incubation mixtures. The substrates  were dissolved methanol and added at final concentrations of 10 μM (55 μM for dapsone). The final concentration of methanol in all the incubation mixtures, including the blank group, was less than 0.2% (v/v). After being pre-incubated at 37 °C for 5 min, the reaction was initiated by adding 20 μL NADPH with the final concentration of 1 mM. The reaction was quenched by adding 400 μL of acetonitrile after being incubated for 15 min. The mixtures were centrifuged at 10,000 rpm for 10 min, and an aliquot of supernatant was transferred to an auto-injector vial for analyzing the five substrates using a previously validated HPLC method40. The determined metabolic substrates were used for calculating the relative enzyme activity (Erel) by the following formula: Erel (%) = Ci(n)/Ci(0) × 100%, in which Ci(n) was the determined metabolic substrates in the presence of varying concentrations of DPTM and Ci(0) was the determined metabolic substrates in the absence of DPTM. According to the obtained relative enzyme activities and inhibitor concentrations, the IC50 values were obtained as the same method as the cytotoxicity of DPTM. Various concentrations of fluvoxamine  and ketoconazole  which replaced DPTM for CYP1A2 and CYP3A4 were performed as the same procedure. The same procedure was repeated three times.We used the cocktail method to study the inhibition activities of DPTM against CYP enzymes in RLMs and HLMs with slight modification7 cells/well. After incubation at 37 °C under 5% CO2 for 24 h, the media were then replaced with 1 mL of serial dilutions of the DPTM  in fresh media. When cells were incubated for 2, 4 and 12 h at 37 °C under 5% CO2, the RNA was isolated using the Trizol reagent according to the manufacturer’s protocol. Total RNA (5 μL) was transcribed into cDNA by using the PrimeScript RT reagent Kit . The obtained cDNA (5 μL) was added for 45 cycles of PCR amplification to detect the mRNA expression  and presence of the plasmid pKM101 (ampicillin resistance) or pAQ1 (tetracycline resistance). Different strains were used to identify different types of mutations.The test was carried out at five different concentrations of DPTM. DPTM (39.84 μmol) was weighed accurately and dissolved in 2 mL DMSO. Then 1 mL solution was diluted with distilled water by two fold. The obtained serial dilutions (1 mL) were then incorporated into 9 mL melted top agar to dilute to the desired concentrations . The negative control used in this experiment was 1 mL DMSO which was directly incorporated into 9 mL melted top agar. 2-aminoanthracene was used as positive control for all bacterial strains in the presence of S9 with the concentration of 13.3% (v/v). Four different positive control chemicals were used for each tested strain without S9, including 4-nitro-o-phenylenediamine (10 μg/plate) for TA97, daunorubicin (2 μg/plate) for TA98, sodium azide (5 μg/plate) for TA 100 and TA 1535, and mitomycin (0.5 μg/plate) for TA102.43 with some modifications. Briefly, 1 mL of strain was inoculated into 5 mL of nutrient broth, followed by incubating overnight at 37 °C. The mixture comprised respective strains, drug, negative and positive controls with the S9 for testing in the presence of metabolic activation system. For testing without S9, 1 × phosphate buffer saline  replaced the S9 mix. The obtained mixtures were added into the tube, mixed well and incubated at 37 °C for 20 min. Then, the top agar was melted and 2 mL of this melted top agar was added to each tube and gently mixed. The mixture was poured onto the surface of glucose minimal (GM) agar plate . The plate was then swirled to distribute the overlay agar to all surfaces of GM agar, followed by invertion and incubation at 37 °C for 48 h when the top agar solidified. The resultant colonies were counted manually considering the high density of bacterial colony. The results were expressed as the number of revertant colonies per plate. The experiments were performed in triplicate.The Ames test of DPTM was conducted as described in the literaturesp < 0.05.The data were presented as mean ± standard deviation (SD). Statistical analyses were performed by One-way ANOVA followed by the Line Segment Detector (LSD) test using SPSS 17.0 software. Differences were considered significant at Supplementary information.


A superaerophobic heterostructured nanowrinkles of bimetallic selenides is developed.The 3D heterostructure exhibits excellent activity and stability towards oxygen evolution reaction (OER) in base.The online version of this article (10.1007/s40820-020-00442-0) contains supplementary material, which is available to authorized users. 2 and NiFe2Se4 grown on NiFe alloy (NiSe2/NiFe2Se4@NiFe) prepared by a thermal selenization procedure. In this unique 3D heterostructure, numerous nanowrinkles of NiSe2/NiFe2Se4 hybrid with a thickness of ~ 100 nm are grown on NiFe alloy in a uniform manner. Profiting by the large active surface area and high electronic conductivity, the superaerophobic NiSe2/NiFe2Se4@NiFe heterostructure exhibits excellent electrocatalytic activity and durability towards OER in alkaline media, outputting the low potentials of 1.53 and 1.54 V to achieve ultra-high current densities of 500 and 1000 mA cm−2, respectively, which is among the most active Ni/Fe-based selenides, and even superior to the benchmark Ir/C catalyst. The in-situ derived FeOOH and NiOOH species from NiSe2/NiFe2Se4@NiFe are deemed to be efficient active sites for OER.Cost-effective and stable electrocatalysts with ultra-high current densities for electrochemical oxygen evolution reaction (OER) are critical to the energy crisis and environmental pollution. Herein, we report a superaerophobic three dimensional (3D) heterostructured nanowrinkles of bimetallic selenides consisting of crystalline NiSeThe online version of this article (10.1007/s40820-020-00442-0) contains supplementary material, which is available to authorized users. With the ever-worsening energy and environmental crises, electrocatalytic water-splitting is believed as a promising method to resolve the global tremendous energy needs of future societies , 2. Howe−2) . Furthermore, the NiSe2/NiFe2Se4@NiFe sustained durable stability with a low voltage of 1.96 V to achieve 500 mA cm−2 in 10.0 M KOH at 60 °C after chronoamperometry testing Below is the link to the electronic supplementary material.


Karst caves host most European Paleolithic sites. Near the Eurasian-Arabian Plate convergence in the Caucasus’ Lower Chegem Formation, Saradj-Chuko Grotto (SCG), a lava tube, contains 16 geoarchaeologically distinct horizons yielding modern to laminar obsidian-rich Middle Paleolithic (MP) assemblages. Since electron spin resonance (ESR) can date MP teeth with 2–5% uncertainty, 40 sediment samples were analyzed by neutron activation analysis to measure volumetrically averaged sedimentary dose rates. SCG’s rhyolitic ignimbrite walls produce very acidic clay-rich conglomeratic silts that retain 16–24 wt% water today. In Layers 6A-6B, the most prolific MP layers, strongly decalcified bones hinder species identification, but large ungulates inhabited deciduous interglacial forests. Unlike in karst caves, most SCG’s layers had sedimentary U concentrations >4 ppm and Th, >12 ppm, but Layer 6B2 exceeded 20.8 ppm U, and Layer 7, >5 ppm Th. Such high concentrations emit dose rates averaging ~1.9–3.7 mGy/y, but locally up to 4.1–5.0 mGy/y. Within Layer 6, dose rate variations reflect bone occurrence, necessitating that several samples must be geochemically analyzed around each tooth to ensure age accuracy. Coupled with dentinal dose rates up to 3.7–4.5 mGy/y, SCG’s maximum datable ages likely averages ~500–800 ka. ESR (electron spin resonance) can establish absolute (chronometric) dates for fossils, like teeth and molluscs, which range from <10 ka to >4 Ma, depending on the radiation dose rates that the fossils experience in their depositional history. Like the other trapped charge dating methods, like thermoluminescence (TL), optically stimulated luminescence (OSL), and radioluminescence (RL), ESR measures the intensity of a radiation-induced signal recorded in the datable mineral compared with the rate at which that radiation irradiates that mineral when deposited in sediment’s can date the hydroxyapatite in tooth enamel, carbonate minerals in molluscs and other invertebrates, and quartz in fault gouge and quartz-rich sediment. Used mainly in Quaternary geology and archaeology, ESR can date teeth and molluscs from some late Pliocene sites. Using teeth, ESR has been used to date many archaeological, human paleontological sites, and mammalian paleontological sites g = 2.0018 ± 0.0002, which has a mean signal lifetime, 19 y (, should be measured for each layer or horizon. Nonetheless, [Wsed(0)] measurements do not guarantee that [Wsed(t)] has been constant over time, because paleoclimatic and hydrological changes may have altered the time-averaged sedimentary water concentrations Since water absorbs radiation, it reduces Dγ,ised,(t) over the 30 cm sphere and over time  immediately after opening the bags. To calculate [Wsed(0)], the sediment mass was measured and gently heated at ~50 °C for several days until no mass changes were recorded to find the mass loss.Immediately after collection in the field , sedimenUsed], [Thsed], and [Ksed], all associated sediment were powdered to ≤500 mesh and analyzed by neutron activation analysis (NAA). In JT5, 1–2 dentines/subsample and enamels/subsample were analyzed for their U concentrations, [Uden] and [Uen] respectively. After a 60.0 s irradiation and a 10.0 s delay, U was counted for 60.0 s in a delayed neutron counting system. Th and K were counted for 20.0 min in a γ counter. Th was counted after a 1.0 h irradiated and a 7.0 day delay. K had a 24–30 h delay after a 60.0 s irradiation. To ensure accuracy, all results were calibrated against NIST Standard 1633B , [Thsed], [Ksed], and [Wsed(0)]  , assuminWsed(0)]  and DataWsed(0)] . Both thx models  using Rox models ,6,25, anDβsed,(t) and Dγsed,(t) were calculated ] (Wsed(0)] at 15.5 ± 0.9 and 15.6 ± 0.4 wt% respectively, while Layer 1 averaged 15.9 ± 0.2 wt%, and Layer 4, at 16.5 ± 0.3 wt%. Probably due to its higher sand concentrations and less clay, Layer 7 had a water concentration at 17.1 ± 0.3 wt%. Within Layer 6, water concentrations ranged from 18.9 ± 0.3 to 23.8 ± 2.4 wt%, with higher uncertainties than seen in other layers. Meanwhile, Layers 1B, 1C, and 6B3b all had >22 wt% [Wsed(0)]. Within Layer 6, [Wsed(0)] ranged from 18.9 ± 0.3 to 23.8 ± 2.4 wt%, with higher uncertainties than seen in other layers. In most karst cave sediment, [Wsed(0)] tends to average 5–15 wt% [Wsed(0)] >12–15 wt%, the sediment feels damp, which tends to discourage both long-term human inhabitation and cave bear hibernation. Undoubtedly, the high clay concentrations in most of SCG’s sedimentary layers ensures that they stayed wet, which also promoted higher degradation of bone and other the organic remains, as well as the igneous minerals found in the ignimbrite constituting the cave rocks. Whether under different climate regimes, the sediment may not have stayed as wet as it is now. Nonetheless, wet sediment might have discouraged hominin and cave bear inhabitation in the grotto during all but the driest seasons and the coldest glacial periods. With the high bone dissolution, the dental seasonal analyses needed to answer this question have yet to be completed.Throughout the layers at SCG, the sediment retained high modern water concentrations, [Wsed(0)] a. Layers5–15 wt% ,31,32,33Ksed]  (see below).The SCG sediment also contained high K concentrations, [Ksed] b. In botsediment ,31,32,33Used], >4 ppm ], and its igneous source rocks that weather into sediment with high acidity, the SCG sediment contained up to 40–50% clay in many horizons. That acidity contributed to high dissolution rates for bone and other organic tissues, as shown by the prevalence of the bone ghosts seen in many layers. Once exposed to U in the sediment, however, bone, dentine, and dental cementum begin to scavenge U rapidly [Used], coupled with somewhat lower [Thsed]. These horizons also yielded the higher artefact numbers. Likely, in these horizons, the hominins contributed more bone and dental tissues to the sediment than in other horizons. Without a good proxy for the initial bone concentrations, however, estimating how much  derived from U scavenging by bone and dental issues from the groundwater compared to U scavenged from , including , after igneous rock dissolution is difficult. Nor can we estimate how high [Used] might have been for each horizon before the bones began to dissolve and release  again to the sediment, from where it may have been subsequently leached.Interestingly, within Layer 6, large variations in both  rose with depth, Dβsed,(t) reached a local maximum in Layer 1A due to high [Ksed] and Dγsed,(t) in Layer 1B due to [Thsed] (Wsed(0)] coupled with high [Thsed] produced high Dβsed,(t) and Dγsed,(t). In Layer 7, low [Wsed(0)] mixed with both high [Used] and [Thsed] again yielded high Dβsed,(t) and Dγsed,(t).In Layer 1, although [ [Thsed] ; Table 2Dβsed,(t) and Dγsed,(t), while Layers 6B1a, 6B2, and 6B3b gave higher Dβsed,(t) and Dγsed,(t) (Wsed(0)] combined with low [Used] to make low Dβsed,(t) and Dγsed,(t). Despite higher [Wsed(0)], the very high [Used] in Layers 6B2 and 6B3b produced the highest and second highest Dβsed,(t) and Dγsed,(t) within Layer 6.Within Layer 6, Layers 6a, 6B1b, 6B3a, and 6B3c had lower sed,γ(t) i–o. In LDβsed,(t), Dγsed,(t), and their uncertainties varied greatly from horizon to horizon, due in part to  associated with the sedimentary bone concentrations  and Dγsed,(t), the effect of  was examined by plotting the means for both Dβsed,(t) and Dγsed,(t) using all samples analyzed from the layer, as well as the means in the samples having higher amounts of bone, and those without the highest  samples  and Dγsed,(t) than for all other horizons and comparable those seen in Layer 7, which lacks hominin artefacts.Within Layer 6, trations i–o. Sinc samples i–o. In eDβsed,(t) typically ranges from 100 to 500 µGy/y, while Dγsed,(t) varies from 300 to 1000 µGy/y, partly due to the limestone and éboulis that tend to range at 30–60 µGy/y for Déb,βsed,(t) and 100–250 µGy/y for Déb, γsed,(t)   ranging from 3.751 ± 0.270 to 4.475 ± 0.322 mGy/y assuming an early U uptake model in the dentine. Again, [Uden] tend to range 100–150 ppm higher than comparable [Uden] values seen in dentine from karst caves  . Not surst caves ,38,39. J50, is ≲4 Gy of radiation, although a dose as low as 0.25 Gy produces measurable effects in the body  higher than 16 wt% on average, but as high as ~24 wt%. Since clays adsorb Th, the SCG sediment contains 10–20 ppm more [Thsed] than those seen in most karst cave sediment. At SCG, [Ksed] concentrations also far exceeded those in karst caves. Dissolution of bones that scavenged [Used] from the local groundwater has produced [Used] up to ~30 ppm, SCG’s [Used] averaged 5–10 times higher than [Used] seen in most karst caves. In SCG, Dden(t) measures. With high [Uden] near 130–160 ppm, Dden(t) also contribute radiation at 2–4 times faster than typical seen in karst caves. Therefore, for some SCG teeth found in the horizons rich in bone where the highest Unlike typical karst limestone caves, Saradj-Chuko Grotto nestles in rhyolitic ignimbrite. Due to its calcium carbonate, limestone buffers the sediment and its groundwater, but the rhyolitic ignimbrite at SCG lacks an effective geochemical buffer. Thus, in situ degradation of the ignimbrite has made the sediment very acidic. Thus, the acidic sediment contains high clay concentrations from the degraded silicates and obsidian in the lava. Acting as an aquatard, the clay also retains water well, producing [


Cancer patients often experience loss in body weight and also a decrease in muscle mass, which results in the reduction of physical activity and mobilization of the patient. To decelerate the loss of muscle mass, as part of the cancer treatment patients frequently undergo physical therapy and considering the physical capabilities of the patients, with moderate loads. Moreover, frequent studies also observed for cancer patients, together with the decrease in muscle mass a shift into fast-twitch muscle fibers from slow-twitch fibers. The aim of our study therefore was to determine how motor fibers behave under moderate isometric load executed until total exhaustion. 11 university students (G1), and 14 elite athletes (G2) participated in the study. 65% of the maximal voluntary contraction (MVC) was determined for the biceps brachii muscle, and with this load holding a weight, participants had to sustain a 90 deg. isometric elbow flexion in a standing posture until complete fatigue occurred. EMG activity for the biceps brachii muscle was measured and frequency analysis was performed. 3 windows were determined in the fatiguing protocol: the first (W1), middle (W2), and last (W3) 5 s, and also frequency analysis for MVC was performed (MAX) between 0 and 260 Hz with 20 Hz wide frequency bands. The results indicate, that as the protocol progressed in time and the effect of fatigue increased (from W1 to W3) the activity of low frequency muscle fibers significantly increased (0-40 Hz) while activity of high frequency muscle fibers (60-260 Hz) significantly decreased for G1 and G2 groups identically. We can conclude, that training applied with constant moderate tension as fatigue increases will result in the increased activation of the lower frequency slow–twitch muscle fibers, but the increase of fatigue in the lower frequency fibers will not result in the increase in the activation level of the higher frequency fast-twitch fibers. Consequently, because as slow-twitch fibers are being used at moderate loads and even when fatigue occurs in these fibers the fast-twitch fibers will not work, higher muscle loads are needed if the aim is to activate fast-twitch fibers. Considering the shift into fast-twitch muscle fibers from slow-twitch fibers for cancer patients, in some cases if the patient’s age and physical status allows during the physical treatment, higher loads and consequently higher levels of activation might be beneficial for the retardment of loss concerning the fast-twitch fiber mass. In cancer cachexia patients often face weight loss and also muscle atrophy, that results in the reduction of their quality of life, accelerates the progression of the cancer, results in worse surgical outcome and lower probability of survival –4.Although loss of muscle mass can often be observed in cancer patients, our present understanding of the phenomenon is restricted. Some studies conclude, that muscle atrophy is a result of cancer, but also cancer treatment can be associated with the loss of muscle mass , 6. To cConsequently, as in many cases for cancer patients, when high intensity training is not applicable, the question may arise as to whether exercises applied with moderate load will activate the fast-twitch muscles, and as a result reduce muscle mass loss in these fibers. When applying resistance training with moderate loads the slow-twitch fibers will be active, but it can be speculated, that if these slow-twitch fibers fatigue but the training continues, the fast-twitch fibers might take over the role and will become active.In the human neuromuscular system, the central nervous system regulates muscle force output by sending neural signals through alpha motoneurons to the muscles in two ways: by increasing the activation level if the signal, consequently recruiting and therefore activating more muscle fibers, and by altering the frequency of the stimuli . Low freThe aim of this study was to determine how the activation level of the biceps brahii muscle changes while the muscle reaches a completely fatigued state using moderate isometric load. To determine the activation level of different frequency muscle fibers, we have calculated the power spectrum content of the EMG data for 20 Hz wide frequency bands. The EMG signal, that can be recorded with surface electrodes, is a complex signal which consists of lower and higher frequency parts. Using the EMG software and Fourier analysis the lower and higher frequency components can be separated, and the amplitude, and consequently the intensity of the lower and higher frequency signals can be calculated, and as a result the activation level of the lower and higher frequency, slow and fast-twitch muscle fibers can be determined. Figure . Using m15 male and 10 female subjects participated in the study. The subjects were the students of the University of Physical Education. The subjects were in good physical condition, prior to the study they did not report any upper limb injury. Age: 23,83 ± 5,02 yrs., body mass: 70,71 ± 7,99 kg, height: 176,91 ± 7,13 cm. The sample was divided into two groups: recreational university students without sports background (G1) and professional handball players (G2) As the fiber characteristics for the two groups is different it can also be hypothesized, that the activation pattern of the frequency distribution for the two groups during the protocol would be different. As G1 group presumably has a mixed fiber type distribution, and G2 has a fast-twitch dominant fiber distribution, with this construction of the study we can replicate the phenomenon, that in the case of cancer patients with the decrease in muscle mass simultaneously a shift into fast-twitch muscle fibers from slow-twitch fibers occur [G1 consisted of 11 subjects , age: 21,63 ± 1,6 yrs., body mass: 69,45 ± 6,78 kg, height: 174,81 ± 5,09 cm, and G2 consisted of 14 subjects , age 25,57 ± 6,25 yrs., body mass 71,71 ± 10,74 kg, height 178,57 ± 9,33 cm, training age 13,71 ± 5,58 yrs. The subjects were informed about the study orally and signed a written agreement before participating in the study. The Ethical Committee of the University of Physical Education approved the protocol , which was in union with the Declaration of Helsinki.Surface EMG (sEMG) signals of the right biceps brachii muscle were recorded during the protocol. Before beginning the trial, the skin needed to be prepared to obtain better sEMG-recordings, to reduce the risk of imbalance between the electrodes, and noise. In order to achieve this, we removed the hair from the skin surface with a razor and the mortified epithelium with sandpaper, and finally washed the skin with alcohol . Two uniOur aim was to study muscle activation under submaximal isometric load with the use of a controlled external load. To achieve this first the subjects’ maximal voluntary contraction (MVC) of the elbow flexor muscles were measured in 90 degrees elbow flexion. The subjects had their arms flexed α = 90 degrees in supinated position, while standing on a Kistler Force Platform System 92–81 B and holding a fixed rod , middle 5 s (W2), and last 5 s (W3), and immediately after W3 signals for the maximal effort contraction was recorded (MAX), and also signals of the whole second MVC was processed. Between 0 and 260 Hz frequency spectrum intensity distribution was divided into 20 Hz frequency bands. Using the Noraxon software we have calculated the power spectrum content for the frequency analysis using Fourier transformation in the frequency bands.p < 0.05.The basic data was characterized by means and standard deviations. Because of the limited sample size and for the purpose of selecting the adequate statistical procedure Shapiro Wilk’s W test was calculated for checking the normal distribution of the data. The data was normally distributed, and for comparing the differences in different times for the same samples dependent T-test was used, for comparing the differences between the groups independent T-test was used. StatSoft STATISTICA 13.2 was used for the statistical analysis, Significance level was set for When comparing the data between the recreational university students (G1) and the professional handball players (G2) group, the G1 group had significantly greater activity in the W1 of 0–20 Hz frequency band, and MAX of 60–80 Hz frequency band, but we have not found significant difference for any other comparisons. As minimal difference could be observed in the EMG characteristics between the G1 and G2 groups, and taking into consideration the nature of EMG signals with relatively high standard deviations we have united the datasets for the two groups to calculate statistical difference between W1-W2-W3-Max to increase sample size (nG1 + G2 = 25 subjects), and consequently to increase the power of the statistical procedures and consequently the conclusions of the study. Comparing the results for the data of all of the participants significant increase  was found in 0–20 Hz frequency band between W1-W2 (inc. 162%), W1-W3 (inc. 618%), W2-W3 (inc. 174%); 20–40 Hz frequency band between W1-W2 (inc. 120%), W1-W3 (inc. 347%), W1-MAX (inc. 55%), W2-W3 (inc. 103%); 40–60 Hz frequency band between W1-W2 (inc. 78%), W1-W3 (inc. 28%); 60–80 Hz frequency band between W3-MAX (inc. 120%); 80–100 Hz frequency band between W3-MAX (inc. 163%); 100–120 Hz frequency band between W3-MAX (inc. 90%); significant decrease was found in 0–20 Hz frequency band between W2-MAX (dec. 50%), W3-MAX (dec. 82%); 20–40 Hz frequency band between W2-MAX (dec. 30%), W3-MAX (dec. 66%); 40–60 Hz frequency band between W2-W3 (dec. 28%), W2-MAX (dec. 30%); 60–80 Hz frequency band between W1-W3 (dec. 48%), W2-W3 (dec. 55%); 80–100 Hz frequency band between W1-W3 (dec. 70%), W2-W3 (dec. 63%); 100–120 Hz frequency band between W1-W3 (dec. 41%), W2-W3 (dec. 35%) ; 140–160 Hz frequency band between W3-MAX (inc. 143%); 160–180 Hz frequency band between W3-MAX (inc. 183%); 180–200 Hz frequency band between W2-MAX (inc. 65%), W3-MAX (inc. 176%); 200–220 Hz frequency band between W3-MAX (inc. 237%); 220–240 Hz frequency band between W3-MAX (inc. 195%); 240–260 Hz frequency band between W2-MAX (inc. 81%), W3-MAX (inc. 304%); significant decrease was found in 120–140 Hz frequency band between W1-W3 (dec. 57%), W2-W3 (dec. 53%); 140–160 Hz frequency band between W1-W3 (dec. 59%), W2-W3 (dec. 58%); 160–180 Hz frequency band between W1-W2 (dec. 36%), W1-W3 (dec. 72%), W2-W3 (dec. 56%); 180–200 Hz frequency band between W1-W2 (dec. 34%), W1-W3 (dec. 61%), W2-W3 (dec. 41%); 200–220 Hz frequency band between W1-W3 (dec. 61%), W2-W3 (dec. 52%); 220–240 Hz frequency band between W1-W2 (dec. 35%), W1-W3 (dec. 70%), W2-W3 (dec. 54%); 240–260 Hz frequency band between W1-W2 (dec. 52%), W1-W3 (dec. 69%), W2-W3 (dec. 55%)  of the individual is not a prohibiting factor, higher training load is beneficial for the retardment of loss in the fast-twitch fiber mass.


Rhodococcus jostii RHA1 was carried out in order to optimise the production of pyridine-2,4-dicarboxylic acid and pyridine-2,5-dicarboxylic acid bioproducts from lignin or lignocellulose breakdown, via insertion of either the Sphingobium SYK-6 ligAB genes or Paenibacillus praA gene respectively. Insertion of inducible plasmid pTipQC2 expression vector containing either ligAB or praA genes into a ΔpcaHG R. jostii RHA1 gene deletion strain gave 2–threefold higher titres of PDCA production from lignocellulose (200–287 mg/L), compared to plasmid expression in wild-type R. jostii RHA1. The ligAB genes were inserted in place of the chromosomal pcaHG genes encoding protocatechuate 3,4-dioxygenase, under the control of inducible Picl or PnitA promoters, or a constitutive Ptpc5 promoter, producing 2,4-PDCA products using either wheat straw lignocellulose or commercial soda lignin as carbon source. Insertion of Amycolatopsis sp. 75iv2 dyp2 gene on a pTipQC2 expression plasmid led to enhanced titres of 2,4-PDCA products, due to enhanced rate of lignin degradation. Growth in minimal media containing wheat straw lignocellulose led to the production of 2,4-PDCA in 330 mg/L titre in 40 h, with > tenfold enhanced productivity, compared with plasmid-based expression of ligAB genes in wild-type R. jostii RHA1. Production of 2,4-PDCA was also observed using several different polymeric lignins as carbon sources, and a titre of 240 mg/L was observed using a commercially available soda lignin as feedstock.Genetic modification of  The aromatic heteropolymer lignin accounts for 15–25% of plant cell wall lignocellulose, and is the most abundant renewable source of aromatic carbon in the biosphere. The conversion of lignocellulose from plant biomass into fuels and chemicals via the biorefinery concept requires the efficient conversion of cellulose, hemi-cellulose and lignin into high value products, but at present the majority of lignin produced by pulp/paper manufacture and biofuel production is burnt for energy. Hence there is considerable interest in the biocatalytic or chemocatalytic valorisation of lignin into useful chemical products [Rhodococcus jostii RHA1 in which the vanillin dehydrogenase gene was deleted was shown in 2013 to generate vanillin as a bio-product when grown on minimal media containing wheat straw lignocellulose [Pseudomonas putida KT2440 to produce polyhydroxyalkanoic acids [cis,cis-muconic acid, which can be chemically converted to adipic acid [Corynebacterium glutamicum to produce cis,cis-muconic acid has also been reported [One strategy for biocatalytic valorisation of lignin that has shown promise is to engineer lignin-degrading micro-organisms to produce target chemicals . A gene ic acids  and cis,pic acid . Metabolreported .Sphingobium SYK-6 ligAB genes) or protocatechuate 2,3-dioxygenase (Paenibacillus praA gene) in R. jostii RHA1, followed by ammonia cyclisation of the extradiol ring fission product, generates pyridine-2,4-dicarboxylic acid  or pyridine-2,5-dicarboxylic acid  bioproducts respectively  to block the competing β-ketoadipate pathway which metabolises protocatechuic acid; (3) to enhance the metabolic flux of lignin depolymerisation via overexpression of lignin-oxidising genes.We have previously reported that re-routing of protocatechuic acid via either protocatechuate 4,5-dioxygenase , likely to be the major competing pathway. The method of van der Geize [pcaHG gene deletion mutant strain of R. jostii RHA1, using vector pK18mobsacB. The ΔpcaHG gene deletion strain showed only slightly reduced growth on lysogeny broth, compared to wild-type R. jostii RHA1, but when grown on M9 minimal media containing 0.1% PCA as carbon source, showed little or no growth over 48 h  encoding vanillin dehydrogenase has previously been targeted for gene deletion, in order to generate vanillin, but is located in a different gene cluster [ligAB or praA  inserted in place of pcaHG genes. New constructs containing promoters and ligAB genes were assembled using the knock-in vector as shown in Fig. icl promoter has been used previously in R. erythropolis [nitA promoter from Rhodococcus rhodochrous J1 [Streptomyces species [bad promoter is commonly used in Escherichia coli [Rhodococcus opacus [tipA promoter from pTipQC2 was modified with 5 mutations in the -10 and -35 regions, as described by Nakashima and Tamura [tpc5 , R. jostii pcaHG:ligAB(PnitA) and R. jostii pcaHG:ligAB(Ptpc5) each showed LigAB enzyme activity in cell-free extract when grown in LB media using the appropriate inducer (shown for R. jostii pcaHG:ligAB(Picl) in Additional file bad promoter, inducing with 0.1% arabinose. Production of the LigAB proteins was observed by SDS-PAGE of cell extracts  and pcaHG:ligAB(Ptpc5) whole cells grown previously in LB media. In a 30 min biotransformation, efficient conversion of PCA to 2,4-PDCA was observed  cells, and 98% consumption of PCA for R. jostii pcaHG:ligAB(Ptpc5) cells.In order to verify the catalytic activity of the expressed LigAB enzyme, biotransformation of 0.05% PCA was carried out using R. jostii pcaHG:ligAB(Ptpc5) in LB media containing 0.1% 4-hydroxybenzoic acid resulted in efficient conversion to 2,4-PDCA, but growth in M9 minimal media in the presence of 0.1% glucose gave very weak production of 2,4-PDCA, likely due to catabolite repression by glucose [Having established the conversion of protocatechuic acid to 2,4-PDCA, the production of 2,4-PDCA was first tested using an aromatic precursor 4-hydroxybenzoic acid as carbon source. Growth of  glucose . HoweverligAB genes were then tested for production of 2,4-PDCA on M9 media containing Green Value Protobind lignin (GVPL), a commercially available soda lignin prepared from wheat straw/sarkanda. This lignin shows partial aqueous solubility which makes it more convenient for microbial transformation, and it has been structurally characterised as a S/G/H lignin containing predominantly β-O-4 units, MW 3270 Mn 620 g/mol [R. jostii pcaHG:ligAB(Ptpc5) in M9/1% GVPL resulted in the formation of 2,4-PDCA and PCA after 168 h as observed by HPLC analysis  was carried out in 4 mL incubations. Addition of 0.15 g/L FeSO4 was found to improve the titre of 2,4-PDCA observed using GVPL as carbon source  formed 2,4-PDCA (164 mg/L) and PCA in titres of 164 mg/L and 200 mg/L respectively after 240 h at 30 °C from 1% GVPL as feedstock, and 290 mg/L 2,4-PDCA was formed using 1% wheat straw lignocellulose as feedstock, verifying that 2,4-PDCA could be formed from a polymeric lignin feedstock.In order to optimise the growth media, screening of media additives , with induction by 5% methanol, was found to generate 70 mg/L 2,4-PDCA using 1% GVPL , with induction by 0.1% ε-caprolactam, was found to generate 100 mg/L 2,4-PDCA using 1% GVPL, and 79 mg/L 2,4-PDCA using 1% wheat straw lignocellulose. These data establish that the Ptpc5, Picl, and PnitA promoters are functional for ligAB expression in R. jostii RHA1. Although promoter Picl gave the most efficient conversion of PCA to 2,4-PDCA, catalysed by LigAB, constitutive promoter Ptpc5 gave highest titres of the desired 2,4-PDCA product from lignin or lignocellulose feedstocks.Constructs containing other gene promoters were then compared, in cultures grown in M9 containing either 1% wheat straw lignocellulose or 1% GVPL, for 168 h at 30 °C. Growth of Amycolatopsis sp. 75iv2 Dyp2 peroxidase, reported to have high manganese peroxidase activity for lignin oxidation [Amycolatopsis dyp2 gene was overexpressed in R. jostii pcaHG:ligAB(Ptpc5) using the pTipQC2 expression plasmid, and production of the Dyp2 enzyme was observed in cell extracts by SDS-PAGE  with and without pTipQC2-dyp2 were grown on M9 minimal media containing 1% wheat straw lignocellulose for 120 h at 30 °C, and 1.6-fold enhancement of 2,4-PDCA production was observed  strain with and without pTipQC2-dyp2 was then tested at small scale in M9 minimal media containing a wider range of lignocellulose and polymeric lignin feedstocks at 1% (w/v) concentration. As shown in Fig. dyp2 gave increases in titre of 60–160% on a small scale.The 2+, Cu2+ and Mn2+ salts to cultures of R. jostii pcaHG:ligAB(Ptpc5) grown in M9 media containing 0.2% GVPL on a small scale (4 mL) revealed that 20–40% enhancement in 2,4-PDCA titre could be achieved by inclusion of metal ions in the media, as shown in Fig. 2, and overexpression of dyp2 gave increases of 35–75% in titre on a small scale. Small amounts of PCA product were also observed by HPLC analysis  and R. jostii pcaHG:ligAB(Ptpc5)/pTipQC2-dyp2 strains was then scaled up to a 2.5L bioreactor, and grown on minimal media containing 1% wheat straw or Green Value Protobind lignin feedstocks, monitoring the time-course of 2,4-PDCA production. It was found that overexpression of dyp2 resulted in faster release of PDCA product as well as improved PDCA titre /pTipQC2-dyp2 from M9/1% wheat straw lignocellulose was 330 mg/L, from a 40 h bioconversion. The higher titres obtained by this method we believe are due to the binding of some PDCA product to hydrophobic lignin or lignocellulose particles, hence the estimates obtained from the earlier small scale HPLC analyses may have under-estimated the amount of product formed. The titres obtained in the course of this work are summarised in Table Recovery of the 2,4-PDCA bioproduct was previously reported via anion exchange , howeverR. jostii RHA1 strains containing either ligAB or praA genes overexpressed on the pTipQC2 plasmid [R. jostii RHA1. Therefore, the aims of this study were to insert the ligAB or praA genes onto the R. jostii RHA1 chromosome, achieve efficient gene expression, and optimise PDCA production.In our previous study, 2,4-PDCA or 2,5-PDCA were generated as bio-products from lignin degradation by  plasmid . The recpcaHG genes initiating the competing β-ketoadipate pathway was found to result in significant enhancements in PDCA titre, 4–eightfold from aromatic carbon sources, and 2–threefold from lignocellulose as carbon source. Interestingly, the ΔpcaHG gene deletion strain was still able to grow weakly on M9/PCA, indicating the presence of another competing pathway from PCA in R. jostii RHA1. In a separate study, we have recently identified the genes responsible for a pathway in R. jostii RHA1 and Agrobacterium sp. proceeding via conversion of protocatechuic acid to hydroxyquinol [Gene deletion of the xyquinol .ligAB genes integrated onto the R. jostii RHA1 chromosome, of which Ptpc5 is constitutive, and Picl and PnitA can be activated by inexpensive inducers methanol and ε-caprolactam respectively. These constructs generate 2,4-PDCA product using either wheat straw lignocellulose or a commercially available soda lignin as carbon source. 2,4-PDCA production from lignin feedstocks was enhanced by overexpression of the Amycolatopsis sp. 75iv2 dyp2 gene, and inclusion of Fe2+, Mn2+ and Cu+ salts in the media further enhances 2,4-PDCA production, presumably by activating lignin-oxidising Dyp-type peroxidase [Amycolatopsis dyp2 gene in the optimised production strain also reduces the time taken to reach maximum titre of 2,4-PDCA, from 168 h to 40 h. The productivity of 2,4-PDCA production from lignocellulose in the optimised production strain has therefore been improved > tenfold from 0.5–0.7 mg/L/h for wild-type R. jostii pTipQC2-ligAB [−1 h−1 for R. jostii ΔpcaHG:ligAB(Ptpc5)/pTipQC2-dyp2. These two effects of dyp2 overexpression demonstrate that the rate of lignin depolymerisation is a limiting factor in bioproduct formation. Overexpression of lignin-oxidising multi-copper oxidase enzyme SLAC in Amycolatopsis sp. 75iv3 has been reported by Singh et al., leading to increased acid-precipitable lignin formation and enhanced production of monocyclic aromatic compounds [Three promoters have been shown to be functional for expression of roxidase , 26 and ompounds . The titR. jostii RHA1 strain from a commercially available lignin feedstock is a significant step towards the generation of bioproducts from lignin or lignocellulose feedstocks. Literature studies on bioproduct generation from lignin using Pseudomonas putida KT2440 or Corynebacterium glutamicum have used biomass pretreated by alkali [R. jostii RHA1 to utilise polymeric lignin as feedstock allows the use of industrial lignins to generate 2,4-pyridinedicarboxylic acid. We have shown that commercially available Green Value Protobind soda lignin is an effective feedstock for R. jostii RHA1 bioconversions. Moreover, the ability of engineered R. jostii RHA1 strains to utilise Kraft lignin as carbon source is remarkable, since Kraft lignin has a condensed structure that is generally found to be more difficult to valorise [Rhodococcus jostii RHA1 is able to break down condensed as well as uncondensed polymeric lignins.The production of 2,4-PDCA using an engineered y alkali  or supery alkali , containvalorise . The abivalorise , confirmRhodococcus jostii RHA1 [Pseudomonas putida KT2440 [Escherichia coli K12. Corynebacterium glutamicum [Rhodococcus opacus [P. putida KT2440 has also been reported to produce 2,5-PDCA bioproducts, using 4-hydroxybenzoic acid or glucose as feedstocks [Rhodococcus jostii RHA1 strains to generate 2,4-PDCA bio-products from a range of lignin feedstocks makes this host well-suited to convert industrial lignins or lignocellulose to bioplastic monomers.Metabolic engineering for lignin degradation is still an emerging field, due to the limited number of microbial hosts available for lignin degradation, and the genetic tools available for those organisms. tii RHA1 , 8 and Pa KT2440  have bota KT2440 , a propeutamicum  and Rhods opacus  have alsedstocks . The abiAll chemicals were purchased form Sigma Aldrich unless otherwise stated. Green Value Protobind 1000 soda lignin was purchased from Green Value SA ; Na lignosulfonate was a gift from Borregaard LignoTech ; wheat straw organosolv lignin was a gift from CIMV .Rhodococcus jostii RHA1 was used as the ancestral strain. For routine growth and maintenance, R. jostii RHA1 cells were cultured in liquid or solid lysogeny broth (LB) medium, with the appropriate selection medium at 30 °C and with shaking at 180 rpm if required.pcaHG gene deletion mutant was made using the pk18mobsacB plasmid, which uses sacB (confers sucrose sensitivity) as a counter-selectable marker [pcaH and pcaG genes. The PCR products included restriction sites for cloning into the pK18mobsacB plasmid. The downstream 1 kb region included the restriction sites XbaI and PstI, while the upstream 1 kb region included the restriction sites PstI and HindIII. All three products were ligated together and the resulting construct confirmed by sequencing and restriction digestion , and recombinant colonies selected by kanamycin resistance. Isolation of the double cross-over gene deletion was carried out using sucrose resistance counter-selection [pcaHG markerless deletion was confirmed by PCR  methanol after 24 h, and further additions of 5% methanol made after each 24 h. The PnitA promoter was induced by addition of 0.1% (w/v) ε-caprolactam after 24 h.Constructs containing the Rhodococcus jostii RHA1 was transformed with exogenous DNA using electroporation. A single colony of R. jostii RHA1 was used to inoculate 10 ml of sterile lysogeny broth (LB) and grown overnight at 30 °C with shaking at 180 rpm. This overnight culture was used to inoculate 50 ml of lysogeny broth  which was then grown overnight at 30 °C, with shaking at 180 rpm. The cells were harvested using centrifugation (5000g) at 4 °C and the pelleted cells were then washed three times with sterile ice-cold 10% (v/v) glycerol at 4 °C. Following the final glycerol wash, the pelleted cells were re-suspended in the residual 10% glycerol and stored at − 80 °C. For the electroporation, 80 μL of cells was used for each transformation, with 3 μL of plasmid DNA. The electroporation was performed on ice, using 2 mm electroporation cuvettes, with the following conditions; 2.5 kV, 25 μF and 400 Ω. After electroporation, 1 mL of sterile ice-cold LB was added to the electroporation cuvette and incubated without shaking overnight at 30 °C. Following overnight incubation, 200 μL of cell culture was plated out onto LB plates with the appropriate selection and incubated at 30 °C. Colonies were usually visible after 2–3 days.R. jostii RHA1 strain and the ΔpcaHG deletion strain were compared. For growth rates, cultures were grown in either LB or M9 minimal medium with 0.1% (w/v) PCA as the sole carbon source. Single colonies were used to inoculate 10 mL of LB, which were grown overnight at 30 °C at 180 rpm. For growth in the minimal medium, the cells were pelleted using centrifugation and washed twice in sterile M9. 5 μL of the overnight culture was then used to inoculate 200 μL of culture in a 96-well deep-well plate containing M9 media (4 mL per well) with media additives as described in the text. The plates were then incubated at 30 °C for 48 h, and A600 measured.The growth of wild-type 18 reverse phase HPLC column. The HPLC solvents were water/0.1% trifluoracetic acid (solvent A) and methanol/0.1% trifluoracetic acid (solvent B). The applied gradient was 15% B for 15 min; 15–50% B over 1 min; 50–15% B over 8 min, at a flow rate of 0.8 mL/min. UV detection was at 270 nm. Analyses of 2,4-PDCA and 2,5-PDCA were compared with authentic standards of 2,4-PDCA and 2,5-PDCA (from Sigma-Aldrich), with detection at 270 nm. Product titres were determined at 4 mL scale (deep well microtitre plate) using triplicate biological replicates, and at 50–100 mL scale (shake flask) using duplicate biological replicates.For metabolite analysis, 500 μL aliquots of culture were removed and combined with 500 μL of HPLC grade methanol/0.1% trifluoracetic acid. Samples were vortexed and then centrifuged (microcentrifuge) for 15 min. HPLC analysis of the supernatant was performed using a Zorbax Eclipse plus (Agilent) CR. jostii RHA1 maintaining pTipQC2-ligAB or pTipQC2-praA were grown at 30 °C in 2 L of M9 minimal media  with 1 ml of trace elements  in an Electrolab FerMac 3010 bioreactor. Wheat straw lignocellulose pellets were added ) and yeast extract (0.25%). Chloramphenicol was added to 50 μg/mL and cultures were induced by addition of 5 μg/mL thiostrepton after 24 h. The culture was then grown for 40–168 h at 30 °C, supplementing with 5 μg/mL thiostrepton every 48 h. Samples were removed ascetically for HPLC analysis and estimation of cell growth from colony forming units.Cultures of g, 10 min). To the culture supernatant was added 30% (w/v) NaCl and 1 volume isopropanol, and the resulting mixture was stirred, forming two layers. The upper organic phase was separated, and an aliquot removed for HPLC analysis. Isopropanol was removed via rotary evaporation at reduced pressure, and residual water was then removed by freeze-drying, to give a solid residue, which was analysed by HPLC and 1H NMR spectroscopy.The culture medium was centrifuged (5000R. jostii RHA1 cells were collected by centrifugation (5000g) and washed three times with 5 mL of ice cold NaCl (150 mM). After the washes, the pelleted cells were resuspended into 500 μL of ice cold NaCl (150 mM) to which 500 μL of 0.1 mm/100 μm glass beads (Sigma-Aldrich) were added. The cells were then incubated on ice for 30 min before cell disruption by vigorous vortexing for 1 min followed by 1 min on ice, which was repeated 10 times. The lysed cells were then centrifuged for 1 min at 13,000g to pellet the cell debris and glass beads. 50 μL of the supernatant was then added to 950 μL of 0.4 mM PCA in 20 mM Tris buffer pH 8.0. The appearance of a visible yellow colour indicated dioxygenase activity, which was measured at 350 nm for PraA and 410 nm for LigAB [Recombinant Additional file 1:Figure S1. Vector for construction of the ΔpcaHG markerless deletion R. jostii strain; Figure S2. PCR confirmation of the ΔpcaHG markerless deletion; Figure S3. Growth characteristics of ΔpcaHG markerless deletion and wild-type R. jostii strains; Figure S4. HPLC analysis of ΔpcaHG markerless deletion and wild-type R. jostii strains grown in M9 minimal media containing 0.1% 4-hydroxybenzoic acid; Figure S5. Production of 2,4-PDCA observed by HPLC analysis in R. jostii ΔpcaHG containing pTipQC2ligAB; Figures S6. HPLC traces for production of 2,4-PDCA by R. jostii ΔpcaHG containing pTipQC2ligAB, and for production of 2,5-PDCA by R. jostii ΔpcaHG containing pTipQC2praA; Figure S7. Agarose gels showing loss of plasmid DNA from R. jostii ΔpcaHG containing pTipQC2ligAB; Figures S8,S9. Chromosomal integration vector (S8) for insertion of ligAB or praA genes onto R. jostii chromosome, and relevant PCR primers (S9); Figure S10. Gene expression for praA and ligAB genes for chromosomal gene insertion constructs (without promoter) by RT-PCR; Figure S11. Nucleotide sequence of constitutive promoter Ptpc5; Figures S12,S13. LigAB activity (S12) and protein production (S13) from pcaHG:ligAB(Picl) construct, induced with 1-8% methanol; Figure S14. LigAB activity observed using R. jostii ppcaHG:ligAB constructs containing four different promoters, grown in LB media; Figure S15. Whole cell biotransformation of protocatechuic acid (PCA) to 2,4-PDCA using R. jostiipcaHG:ligAB(Picl) and R. jostiipcaHG:ligAB(Ptpc5); Figure S16. Production of 2,4-PDCA using R. jostiipcaHG:ligAB(Ptpc5) grown on M9 minimal media containing 0.1% 4-hydroxybenzoic acid and 0.1% yeast extract, analysed by HPLC; Figure S17. Production of 2,4-PDCA from minimal media containing Green Value Protobind lignin by constructs containing chromosomal expression of ligAB genes, analysed by HPLC; Figure S18. SDS-PAGE gel of cell extracts of R. jostiipcaHG:ligAB(Ptpc5) with or without pTipQC2-dyp2, showing expression of recombinant Dyp2 peroxidase; Figure S19. Production of 2,4-PDCA by R. jostiipcaHG:ligAB(Ptpc5) with or without pTipQC2-dyp2, grown on M9 minimal media containing 1% wheat straw lignocellulose; Figure S20. Small scale (4 mL) testing of the production of 2,4-PDCA and protocatchuic acid (PCA) by R. jostiipcaHG:ligAB(Ptpc5) with or without expression of Amycolatopsis dyp2 gene; Figure S21. Titre of 2,4-PDCA vs time from 2.5L bioreactor for R. jostiipcaHG:ligAB(Ptpc5) grown on M9 minimal media containing 1% Green Value Protobind lignin at 30 °C; Figures S22–S24. Isolation of 2,4-PDCA product from fermentation broth: S22, Extraction of 2,4-PDCA into isopropanol; S23, HPLC analysis of extracted product; S24, 1H NMR analysis of isolated product.


RP2 mutations cause a severe form of X-linked retinitis pigmentosa (XLRP). The mechanism of RP2-associated retinal degeneration in humans is unclear, and animal models of RP2 XLRP do not recapitulate this severe phenotype. Here, we developed gene-edited isogenic RP2 knockout (RP2 KO) induced pluripotent stem cells (iPSCs) and RP2 patient-derived iPSC to produce 3D retinal organoids as a human retinal disease model. Strikingly, the RP2 KO and RP2 patient-derived organoids showed a peak in rod photoreceptor cell death at day 150 (D150) with subsequent thinning of the organoid outer nuclear layer (ONL) by D180 of culture. Adeno-associated virus-mediated gene augmentation with human RP2 rescued the degeneration phenotype of the RP2 KO organoids, to prevent ONL thinning and restore rhodopsin expression. Notably, these data show that 3D retinal organoids can be used to model photoreceptor degeneration and test potential therapies to prevent photoreceptor cell death.  •Isogenic RP2 knockouts match the phenotype of RP2 patient-derived organoids•Retinal organoids from RP2 nulls undergo rod photoreceptor cell death•Cell death correlates with rod photoreceptor differentiation•AAV gene therapy improved photoreceptor viability and increased rhodopsin expression Cheetham and colleagues show that 3D retinal organoids lacking the RP2 protein develop rod photoreceptor degeneration that can be prevented with AAV gene augmentation for RP2. The reprogramming of patient-derived cells into induced pluripotent stem cells (iPSCs) has enabled the derivation and differentiation of a range of somatic cell types and has revolutionized our ability to study inherited disease . The difRP2 account for approximately 15% of all cases of X-linked retinitis pigmentosa (XLRP)  . RP2 is a (XLRP) , which ia (XLRP) . ARL13B a (XLRP) . ARL3, wa (XLRP) . Rp2 kno4 months , whereas4 months . In cont4 months , highligRP2  showed changes in Golgi cohesion, Gbeta trafficking in RPE, and ciliary trafficking of KIF7 in ROs . AAV delivery of human RP2 to a mouse knockout model of RP2-XLRP preserved cone function, but had no effect on rod cell function and toxicity was observed at a higher viral dose  was selected for further study as it had the shortest frameshift extension before a premature stop codon. Western blotting of iPSCs showed efficient knockout, as no RP2 protein was detectable  carrying the nonsense mutation c.358C > T; p.R120X were reprogrammed into iPSCs by nucleofection . CRISPR/protocol . The loctectable A and 1B.RP2 mRNA expression in ROs at D180. The RP2 KO clone had only 20% RP2 mRNA relative to its isogenic control, similar to the R120X patient ROs (n = 3)  E, sugges (n = 3) F. These in vivo, the outermost cell layer of the ROs consists of a uniform compacted ONL, which terminates with photoreceptor synaptic pedicles that are separated from the inner retinal cells by a layer immunoreactive for synaptic structural protein Bassoon in the outer plexiform layer  was performed on D150 controls, RP2 KO, and R120X-A ROs. Principal-component analyses and sample to sample distance shows that the gene expression profiles of the RP2 KO ROs were between the R120X-A patient line and the parental isogenic control E and 2F.RHO) and rod transducin (GNAT1) expression by qPCR. In the controls both RHO and GNAT1 showed an age-dependent increase, with a major increase between D150 and D180. In contrast, the RP2 KO ROs had reduced expression of RHO and GNAT1 mRNA at D180, but not D120 or D150, confirming that this difference manifested from D150 onward. The percentage of photoreceptors that were immunoreactive for rhodopsin was quantified as a percentage of DAPI-positive nuclei across the full length of the ONL in sections from all ROs (ARR3) was also increased in the RP2 KO ROs at D150 and D180 , although the level of RP2 overexpression in transduced mouse eyes was not defined  was used to predict off-targets for the selected gRNA and the top 10 off-targets were assessed with Sanger sequencing, which detected no changes  as described previously . RP2 KO RP2 see . iPSC clRP2 see . iPSC cl changes . In addi changes . These s2. Emerging transparent pouches of neuroepithelium were isolated using a needle and cultured in suspension in retinal maturation media +0.5 μm retinoic acid up to D140, after which retinoic acid was removed. Alternatively EBs were generated by single-cell dissociation and forced aggregation in 96-well V-bottomed plates and cultured in suspension thereafter  sequence, and a minimal rabbit β-globin poly(A), was synthesized by GeneArt (Life Technologies) and cloned into pAAV-MCS using flanking NotI sites. More detail of sequences is in the 12 vg/mL. At 140 days, organoids with brush borders visible by light microscopy, which show inner segment and OS development, were transferred to the well of 96-well plate and incubated with 1E11 viral genomes in 75 μL media for 8 h before topping the media up to 200 μL. Followed by 50/50 medium changes every 2 days until day 180.RP2 AAV was prepared to a final titer 4.73 × 10POLR2A and MAN1B1 were chosen due to their consistency across the sample groups, which was determined using the GeNorm algorithm  and cDNA synthesis was performed using a Tetro cDNA synthesis kit (Bioline). qPCR was carried out on an Applied Biosystems 7900HT Fast Real-Time PCR system using the SYBR Green method using 1 μL cDNA per triplicate. Data from the qPCR were normalized to the geometric mean of the expression of two internal reference genes in each sample. lgorithm . Primerslgorithm .Three ROs from BJ controls and R120X cell lines and two ROs from the RP2 KO cell line were harvested at D150. The RNA was extracted with the RNeasy micro kit (QIAGEN) following the manufacturer’s instructions, followed by paired-end sequencing at 100 million read depth for each sample . Raw.fastq sequences were cleaned from any residual sequencing adapter using cutadapt with parameters -m 20 and -e 0.1. Fragments were then aligned to the human genome  using STAR and genes counted using featurecounts.https://amp.pharm.mssm.edu/Enrichr/). According to the KEGG there was a significant upregulation of the “p53 signaling pathway” in the RP2 KO and RP2-R120X retina . The differential expression analysis showed that 1,141 genes were differentially expressed between the R120X ROs and the control, whereas 979 genes were differentially expressed between the RP2 KO ROs and the isogenic control and 328 genes were common to the 2 RP2-depleted backgrounds. Of these, 232 genes were consistently upregulated and 56 were downregulated. The list of DE genes is shown in X retina .ROs were either fixed whole or bisected under a dissecting microscope using micro scissors (Fine Science Tools) with the other half processed for RNA extraction. ROs or half ROs were fixed in 4% paraformaldehyde at 4°C for 20–30 min before cryoprotection by immersion overnight in 30% sucrose/PBS. Following orientation under a dissecting microscope in OCT they were frozen and cryosectioned into 6-μm sections. For ICC, slides were blocked in 10% donkey serum, 0.1% Triton X for 1 h before incubation with primary antibodies for 2 h  twice for 5 min, the ROs were post-fixed in 1% aqueous osmium tetroxide for 2 h, dehydrated by passage through ascending alcohols  and two changes of propylene oxide, and infiltrated overnight with a 1:1 mixture of propylene oxide:araldite on a rotator. Finally, ROs were infiltrated with araldite resin over 4–6 h and embedded in fresh resin, which was then cured by overnight incubation at 60°C. Semithin sections (0.75 μm) were stained with a 1% mixture of toluidine blue-borax in 50% ethanol, and ultrathin sections were contrasted with Reynolds lead citrate for imaging in a JEOL 1010 TEM operating at 80 kV. Images were captured using a Gatan Orius CCD camera using Digital Micrograph software.Statistical analysis was carried out on GraphPad Prism. Data were subjected to two-way ANOVA and/or by multiple t tests using the Holm-Sidak method, with α = 5.00%. Each time point was analyzed individually, without assuming a consistent standard deviation (SD). Significance was determined at a p value of < 0.05.GSE148300.The RNA-seq data are available at GEO: A.L., K.J., C.S., D.O., A.B.P., N.S., R.G., M.J.H., N.E.S., A.P., and N.C. performed the experiments and/or analyzed the data. A.L., K.J., C.S., D.O., N.E.S., G.J.F., A.J.H., and M.E.C. conceived the hypothesis and designed the experiments. A.L., K.J., C.S., G.J.F., A.J.H., and M.E.C. drafted the manuscript. All authors edited the draft manuscript.