Furthermore, we highlight the importance of the fabrication technology and experimental conditions, such as the effects of pressure and operating parameters, on the electrochemical performance of all-solid-state Li batteries. In particular, we emphasize promising electrolyte systems based on sulfides and argyrodites, such as LiPS5Cl and β-Li3PS4, oxide electrolytes, bare and doped Li7La3Zr2O12 garnet, NASICON-type structures, and perovskite electrolyte materials. Moreover, we discuss the present and future challenges that all-solid-state batteries face for large-scale industrial applications.Energy storage materials are finding increasing applications in our daily lives, for devices such as mobile phones and electric vehicles. Current commercial batteries use flammable liquid electrolytes, which are unsafe, toxic, and environmentally unfriendly with low chemical stability. Recently, solid electrolytes have been extensively studied as alternative electrolytes to address these shortcomings. Herein, we report the early history, synthesis and characterization, mechanical properties, and Li They reported the presence of a three-dimensional (3D) pathway network for the long-range ion conduction of all Li6PS5X  phases, which consisted of interconnected low-energy local pathway cages S4 is an entropically stabilized fast-ion conductor. The β-LPS phase presents orthorhombic structure with the space group Pnma, and its lattice parameters have been reported to be a = 13.066(3) Å, b = 8.015(2) Å, and c = 6.101(2) Å ((ii) Homma et al.  studied 101(2) Å a–d 222]3.25S4 (0.15 < x < 0.33) prepared by solid solution methods using a mixture of Li2S, P2S5, Si, and S; 5 wt.% excess S was added to the mixture to fully oxidize Si. First, the powder was pelletized, then it was placed in a glassy-carbon crucible in a sealed quartz tube under vacuum. The sample was heated to 750 °C, slowly cooled to 725 °C for 18 h, and then cooled to room temperature at the rate of 5 °C min−1. The material was further characterized using XRD, neutron diffraction, NMR, bond valence calculations, and conductivity measurements. Crystal structure studies revealed that Lix3+[SixPx1−]S4 was isostructural with β-LPS  Å, b = 8.029(0) Å, and c = 6.129(1) Å  revealed the presence of peaks at the chemical shifts, of ∼5 and ∼86.5 ppm, which corresponded to the SiS44– and PS43– moieties, respectively.In 2019, Zhou et al.  investigth β-LPS ; however129(1) Å a,b 222]3+xS4  solid solutions  than β-LPS and suggested that the Li+ ion diffusion occurred both along the b-axis and in the  plane. Owing to its flexible and ductile nature, the Lix3+[SixPx1−]S4 electrolyte could be more easily processed and densified than sulfide and oxide electrolytes. Moreover, owing to its synthesis temperature being similar to that of the cathode, this electrolyte could be useful for the preparation of ASSB oxide/sulfide composite electrolytes.Liolutions a,b; more2S–25B2S3–45LiI–xSiO2 (Li1.05B0.5SixOx2S1.05I0.45) (0 ≤ x ≤ 1) quaternary superionic Li oxythioborate glasses. The prepared compositions presented negligible H2S evolution on pellets upon exposure to ambient air and a stable capacity of 230 mAh g−1 up to 230 cycles, at a rate of 0.1C when paired with a TiS2 intercalation cathode Li2S–xP2S5 (x = 30) glass composite and evaluated the effects of the ball milling time and crystallization temperature on the conductivity (~0.2 mS cm−1) and electrolytic stability for ASSBs. Ujiie et al. ..13]. Thee) scans A–F. Thisd LiCoO2 . Authors−2 (140 mAh g−1) and 1.62 mAh cm−2 (113 mAh g−1), respectively, and the irreversible capacity loss and at end of the 100th cycle was approximately 27 mAh g−1, because the capacity of 1.62 mAh cm−2 (36 mAh g−1) was retained ..+ ion co2.9B0.9S0.1O3.1 and LATP (mole ratio of 1:9) at 800 °C presented a total conductivity of 1.5 × 10−5 S cm−1 at room-temperature [(v) Additives have been reported to improve the ionic conductivity of LATP. For example, the product obtained by sintering of a mixture of Liperature .+ ion conductivity, LATP is an important ASSB ceramic electrolyte; however, when Li metal is used as the anode, the LATP membrane has to be separated from it using an additional protective layer to avoid the Ti4+/Ti3+ reduction reaction, because the presence of this redox couple during electrochemical cycling leads to slow structural phase transitions and lowers the Li+ ion conducting properties of the LATP electrolyte during cycling. The cycling performance of ASSBs at high charge–discharge rates remains challenging owing to the low conductivity of the decomposition products and small contact areas or space-charge layers. de Klerk and Wagemaker [(vi) Owing to its high Liagemaker  proposed−1, respectively. In addition, the films were sintered at 920 °C and it was concluded that the conductivity of the films increased with the sintering temperature. The microstructure of LATP ceramics fabricated by milling after spark plasma sintering at 950 and 1000 °C is shown in Recently, Dashjav et al.  reported1.3Al0.3Ti1.7(PO4)3 (LiATP), LCO, and Li metal were the electrolyte, cathode, and anode, respectively. They reported that the capacity of the cell was 150 mA g−1 at the rate of 0.1C. Moreover, the cell presented good charge–discharge profiles and cycling performances, similarly to that of liquid electrolyte cell showing the main redox couples (4.0/3.85 V) and hexagonal phase transformations of LiCoO2 around ~4.06, ~4.18, ~4.5 V vs. Li [2.9B0.9S0.1O3.1 glass additive on the structure and electrical properties of the LATP-based ceramics. Using high-resolution synchrotron-based X-ray and neutron powder diffraction, Monchak et al. [Recently, Kou et al.  reportedV vs. Li . We noteV vs. Li  examinedk et al.  characte4 thin films using the PLD technique and reported various surface analysis methods. Time-of-flight secondary-ion mass spectrometry studies on the as-deposited (unheated) films revealed well defined interfaces; conversely, the interdiffusion of Co and Ti ions was observed between the heat-treated electrolyte and cathode films. Atomic force microscopy analysis revealed that LATP presented well-defined smooth surface and XPS studies indicated that no changes occurred in the oxidation states of the ions at the electrode/electrolyte interface. Recently, Bock et al. [−1 m−1.Hofmann et al.  fabricatk et al.  reported+ ion conductivity of 1 × 10−3 S cm−1, bulk density of 2.92 g cm−3, and relative density of 99.4%. In contrast, the Li+ ion conductivities of the samples sintered at 800 and 850 °C were 1.1 × 10−4 and 4.8 × 10−4 S cm−1, respectively, and their relative and (bulk) densities were 87.4 % (2.57 g cm−3) and 96.1 % (2.824 g cm−3), respectively. Although the excellent ionic conductivity of the LATP pellets sintered at 100 °C was ascribed to the samples being homogeneous and crack-free .Recently, Case et al.  performe4 cathode and reported a capacity of 138 mAh g−1 after 250 cycles with 98% capacity retention at 60 °C. In addition, Wang et al. [3SO3–LATP composite membrane solid electrolyte and paired it with a LiFePO4 cathode. Moreover, they studied the effect of various LATP solid electrolyte and polymer compositions and reported that the highest ionic conductivity of 1.6 × 10−4 S cm−1 at 60 °C was achieved when the amount of LATP electrolyte was 25 wt.%; in addition, when the membrane was paired with a Li metal anode, it was stable for up to 1800 h , respectively, at 60 °C  as precursor provides the best LAGP phase purity and the highest ionic conductivity of ~5 × 10−4 S cm−1 at 25 °C. In addition, few studies on the synthesis, conductivity (PO4)3 solid electrolyte with x = 0.3–0.7. Recently, Paolella et al. [1.5Al0.5Ge1.5(PO4)3 at a low temperature of 650°C using hot-press technique (56 MPa applied pressure); this solid electrolyte was used in all-solid-state battery with LiFePO4 cathode without addition of any further polymer or liquid electrolyte additives.Bock et al.  reportede et al.  reporteda et al.  reportedEa, of the LAGP–30 wt.% PPC–SCE electrolyte are 5.5 × 10−4 S cm−1 at 50 °C and 0.506 eV, respectively, and Tg of 8.11 °C. An SSB was fabricated using LAGP–30 wt.% PPC–SCE, LiFePO4, and Li metal as the composite electrolyte, cathode, and anode, respectively. The battery was formatted at 80 °C for 12 h to ensure optimal contact between the electrodes and electrolyte, and then it was subjected to galvanostatic cycling at 55 °C. The cell presented good reversible charge–discharge profiles at ~3.5/3.4 V vs. Li and delivered a capacity of 151 mAh g−1 at a current rate of 0.05 C with 92.3% capacity retention (4)3) with PVDF and LiTFSI polymer-salt matrix. Xie et al. [2P3O12 and reported room-temperature conductivities in the range of 10−4–10−6 S cm−1. In addition, Abdel-Hameed et al. [− and B3+ ions and heat treatment on the enhancement of electrochemical and electrical properties of nanosized LiTi2(PO4)3 glass-ceramic for ASSB and Kahlaoui et al. [1+4xTi2−x(PO4)3 NASICON-type oxide solid electrolytes.In 2019, Wang et al.  studied etention B. Electre et al. ,442, Prae et al. , and Case et al.  studied d et al.  investigi et al.  examinedxM1/3Nbx1−TixO3  perovskite (ABO3)-type oxides. In addition, they investigated the ion conduction mechanism of these materials using the NMR technique. Furthermore, in 1984, Kochergina et al. [0.5La0.5TiO3. Subsequently, the Lix3Lax(2/3)−□x(1/3)−2TiO3 phase (with 0 < x < 0.16) (LLTO), where □ denotes a structural vacancy, and its related compounds, have been thoroughly studied by numerous workers [x3Lax(2/3)−□x(1/3)−2TiO3 (0.04 ≤ x ≤ 0.17) and performed conductivity measurements. In 1993, Inaguma et al. [0.34La0.5TiO2.94 electrolyte. Among all Lix3Lax(2/3)−□x(1/3)−2TiO3 structures, x ≈ 0.1 presented a conductivity of 1 × 10−3 S cm−1 at 25 °C [Ea of 0.40 eV. In 2003, Stramare et al. [In 1984, Latie et al.  reporteda et al.  publishe workers ,466,467. workers  studied a et al.  studied at 25 °C  and an Ee et al.  reviewedHerein, we summarize the findings of the previous reports and discuss a few recent additional studies as follows.x3Lax(2/3)−□x(1/3)−2TiO3 by (a) analyzing the effect of the preparation method: Solid-state [+ and K+ ions, or alkaline-earth ions, such as Sr2+ and Ba2+ ions, or Ag+ ions at the La sites; (c) in 2000, Mizumoto investigated the conductivity relaxation in various lithium ion conductors with the perovskite-type structure [3+) [4+), penta-  [6+). It was determined that the conduction mechanism of the LLTO compounds varied with the composition, A-site deficiency, Li+ and La3+ ions concentration, and dopants [aE and increase in ionic conductivity was noted with increasing the rare-earth metal ion size as follows: Sm3+ < Nd3+ < Pr3+ < La3+; furthermore, the microstructure, density, domain size, and composition of the domain boundaries affected the ionic conductivity and Ea values of the LLTO compounds [+ ions hopped between cages through the bottleneck in the ab plane at low temperature, whereas at high temperature, the Li+ ions hopped in all three directions. The reported conductivity values of Li0.34La0.56TiO3 range from ~7 × 10−4 to ~1 × 10−3 S cm−1 ((i) Many efforts have been invested to elucidate the crystal structure and conduction mechanism of Liid-state , sol–gelid-state , precipiid-state , electroid-state , and thiid-state , and reaid-state , using vid-state , or substructure , and (d)ure [3+) , tetra- +, Nb5+) , and hex dopants . For exaompounds ,469,470.3 S cm−1 .x3Lax(2/3)−□x(1/3)−2(A)Ti(B)O3 perovskite electrolyte presents three different types of polymorphs [a = 3.872 Å, for x = 0.97–0.11, tetragonal: a = b= 3.87 Å and c = 7.74 Å, for x = 0.11–0.2, and orthorhombic: a = 3.864 Å, b = 3.875 Å, c = 7.786 Å, for x = 0.03–0.09. Among all polymorphs, the cubic structure presented the highest conductivity followed by the tetragonal and orthorhombic ones, for the same bulk composition. The low ionic conductivity of the well-ordered phases was correlated with the uneven ordering of Li, La, and vacancies along the c-axis. The Lix3Lax(2/3)−□x(1/3)−2(A)Ti(B)O3 LLTO presents perovskite (ABO3)-type structure, where the A-sites consist of La, alkaline , or rare earth ions, which are arranged in the corners of a cube and the B-sites consist of transition metal (Ti) ions, which are located at the center of the cube; the face-center positions are occupied by O atoms. Typically, the A- and B-sites present 12- and 6-fold coordination (BO6), respectively, that share corners with each other (x3Lax(2/3)−□x(1/3)−2(A)Ti(B)O3 can be written as La2/3TiO3, which is intrinsically A-cation deficient, with 1/3 of vacant A-sites. The La vacancies are partitioned into alternating La-rich and La-poor layers along one axis to form a partially ordered super lattice structure at room temperature. Depending on the Li content of the materials, they present different symmetries. The Li-poor (0.03 ≤ x < 0.1) compositions present orthorhombic symmetry, with high La-site occupancy (≥90%) in the La-rich layer and antiphase tilting of the TiO6 octahedra. Conversely, the Li-rich (0.1 ≤ x < 0.167) compositions present tetragonal symmetry, and the occupancies of the two types of La layers become less dissimilar as the Li content increases [(ii) The Lilymorphs , viz. sich other a,b [469,ncreases .c-axis and indicated that the Li+ ions could also diffuse along c-axis. Computational studies offered further insight into the size of the bottleneck and indicated a possible increased using large rare-earth or alkaline-earth metal ions as A-site ions; moreover, changing the bond strength between the B-site cations and O also affects the conductivity of these electrolytes. In addition, Binninger et al. [(iii) The experimental observations were further validated by the results of the computational study performed by Jay et al. . They rer et al.  performe4+ to lower oxidation state. These studies revealed that the temperature dependence of the ionic conductivity can be modelized by a Vogel–Tamman–Fulcher (VTF)-type relationship. Klingler et al. [xLax/3) and Pr-, Tb-, Cr-, and Fe-doped compounds with the cycling lower limit of up to 1.1 V vs. Li. Lithium intercalation was noted for all analyzed electrolytes, which led to the formation of the Ti4+/Ti3+ redox couple, which is a drawback when this electrolyte is used for ASSBs.(iv) The good room-temperature ionic conductivity values motivated researchers to further elucidate the reactivity of LLTO electrolytes with Li metal anodes and the processes that occur at the electrode/electrolyte interface. According to the early study conducted by Bohnke et al.  on the gr et al.  analyzed4+ to Ti3+,2+, 0 using XPS analysis. Owing to this drawback, only few reports on the application of the bare LLTO electrolyte for ASSBs have been published. However, for academic purposes, the study conducted by Araki et al. [x3Lax1/3−MO3  revealed that the thermal expansion coefficient was ~3 × 10−6 K−1 above 400 K regardless of x. More studies were conducted to analyze modified synthesis methods, understand the interface mechanisms, and improve the conductivity using modified strategies [−1 LiPF6 in mixture of ethylene carbonate+dimethyl carbonate+diethyl carbonate (EC:DMC:DEC) electrolytes with LLTO, and in some cases using polymer separators. These batteries are typically termed “hybrid composite SSBs”, and the reduction of Ti in the LLTO electrolyte cannot be suppressed in these cells. Lai et al. [0.33La0.56TiO3 nanofiber (NF) layer deposited on the top of thin lithiophilic Al2O3 NF layer. This electrolyte was used to form a cell using 1 mol L−1 LiPF6 (EC:DMC:DEC) and Celgard 2500, LiNi0.8Co0.15Al0.05O2, and Li metal as the separator, cathode, and anode, respectively, and the capacity of the cell was 133 mAh g−1 at a current rate of 5C in the voltage range of 2.7–4.3 V. Xu et al. [0.33La0.57TiO3/LiMn2O4 half-cell. Jiang et al. [−1 with 86.2% capacity retention after 50 cycles; cycling was performed at 65 °C at the current rate of 0.1C. Li et al. [4|PVDF, LiTFSI-CPE (15 wt.% LLTO)|Li cell, in which the 15 wt.% electrospun LLTO fibers , and good thermal stability . The reversible capacities of the fabricated battery at the current rates of 0.2, 0.5, 1, 2, and 5C were 147, 129, 120, 107, and 91 mAh g−1, respectively O4.In 1972, West  publishe4SiO4 ,498,499 6Ti0.4O4  were rep14Zn(GeO4)4 and doped Lil6.2xA(BO4)4, in which A2+ = Mg, Zn, B4+ = Si, Ge, and x = 1, 2, or 3. Among the analyzed specimens, Li14ZnGeO4 presented good conductivity (8 S cm−1 at 300 °C). Ivanov-Shitz and Kireev [3.34P0.66Ge0.34O4 was ~1.8 × 10−6 and 3.7 × 10−2 S cm−1 at 40 and 400 °C, respectively.In 1978, Hong  reportedd Kireev  reportedx4±Six1−xXO4 , Li4SiO4, Li3.75Si0.75P0.25O4, Li4.25Si0.75Al0.25O4, Li4Al0.33Si0.33P0.33O4, and Li4Al1/3Si1/6Ge1/6P1/3O4. They observed that the conductivities of the P-, Al-, and Ge- doped samples were higher than those of the other samples. In addition, the MD simulation studies revealed that the conductivity of Li4Al1/3Si1/6Ge1/6P1/3O4 was 0.9 mS cm−1; furthermore, its Ea of 0.28 eV was the lowest of all analyzed samples. Recently, Zhao et al. [y3.75±(Ge0.75P0.25)x1−xMO4  LISICON-type structures and reported that Li3.53(Ge0.75P0.25)0.7V0.3O4 presented the highest ionic conductivity of 5.1 × 10−5 S cm−1 at 25 °C of all samples, and also the low Ea of 0.43 eV  LiPON thin films, and noted that the ionic conductivity increased with the incorporation of N2 into the glassy structure. The correlations between composition, local structure (by XPS), and the electrical properties were reported for lithium borophosphate  thin films and for xLiBO2:(1−x)Li3PO4  glasses [−6 S cm−1 and lowest Ea of 0.52 eV of all analyzed samples.Lithium phosphorous-oxynitride (LiPON) is one of the most studied oxide-based electrolytes owing to its reasonably good ionic conductivity and stability when paired with Li metal anode Bates et al.  reportedu et al.  further ectively . Hamon e glasses . The effx)LiBO2–xLi2SO4 (LiBSO) (x = 0.4–0.8) amorphous solid electrolytes thin films and reported that the ionic conductivity of the electrolyte increased with x and was the highest (~2.5 × 10−6 S cm−1) when x = 0.7 at room temperature. In addition, they noted that at x > 0.7 the conductivity values slowly decreased owing to the partial crystallization of the electrolytes. Furthermore, Schwenzel et al. [2 thin film battery and Notten et al. [Ea of the Lix3+SixPx1−O4 (LiSiPON) thin films obtained via RF sputtering under Ar and N2 atmospheres were 2.06 × 10−5 S cm−1 and 0.45 eV, respectively, and these values were one order of magnitude higher than those of LiPON thin films. Furthermore, Clancy and Rohan [Joo et al.  studied l et al.  studied n et al.  fabricatn et al.  reportednd Rohan  conducte+ ion conductivity values of Li2ZrO3, Li4ZrO4 and LiScO2 were 3.3 × 10−5, 3.0 × 10−4, and 4.2 × 10−7 S m−1, respectively, at 300 °C. Although these materials presented low room-temperature conductivity, their chemical stability when paired with Li metal anodes was good. Furthermore, few studies focused on Zr-based fast ion conductors, such as bare and Ta-, Nb-, Y-, and In-doped Li6Zr2O7 [6Zr2O7 oxide was reported to be 1 × 10−3 S cm−1 at 300 °C [In 1981, Hellstrom and Van Gool  reportedLi6Zr2O7 ,534,535.t 300 °C .3PS4 and LiPS5Cl have been the most studied sulfide electrolytes. The AASBs formed when these electrolytes were paired with Ni-rich NMC cathodes achieved high energy densities. Although the room-temperature ionic conductivity of sulfide electrodes is good and these electrolytes can be easily fabricated, their stability should be further improved to expand their large-scale applications. To fabricate batteries with good electrochemical performance, the sulfide electrolyte should be paired with cathodes that are coated with protective layers of LiNbO3, Li3PO4, Li2ZrO3 or other metal oxides. Moreover, the surface protection of the cathodes involves additional costs, and therefore, a cost-effective novel approach for the large-scale manufacturing of ASSBs is needed. Sulfide electrolytes present a few other shortcomings, including short cycle life, low stability, narrow electrochemical voltage window, suboptimal electrode/electrolyte interface, and low stability in air.In this review article, we summarized the recent advances and challenges of ASSBs with sulfides and oxide electrolyte systems. Owing to their excellent ionic conductivities, Li7La3Zr2O12, and NASICON-type LATP and LAGP have been studied in depth for ASSBs, owing to their good conductivity. Only few studies have been conducted on ASSBs with Ta-doped LLZO electrolytes, owing to their better stability when paired with Li metal anodes. Most oxide-based electrolytes use 15–25 wt.% inorganic superionic conductors  in polymer composites with combination of ionic liquid electrolytes. However, the progress in this field has been rather slow, mainly owing to the high cell resistance, which was attributed to the high-temperature sintering process required for better particle-to-particle contact between composite cathodes and electrolyte layers. Most of the best-reported garnet-based electrolyte used high content of Ta dopant (0.5–0.6 mol%) for large-scale application, which can be further reduced below 0.25 mol%. LATP, LAGP, and LLTO contain Ti and Ge, which undergo electrochemical reactions with Li metal, and thus, further improving the surface protection of the electrolytes is needed for large-scale applications and to reduce the cost associated with the use of Ge.Among all oxide-based electrolytes, garnet-based oxides, Ta-, Ga-, and Al-doped LiThe most common shortcoming of ASSBs with sulfide and oxide electrolyte is their low electrochemical cycling performance at high charge–discharge rates, which is attributed to the poorly conducting decomposition products and small contact areas or space-charge between electrode and electrolyte layers. In addition, the roles of the microstructure adhesion and mechanical and surface interfacial properties of both Li metal and solid electrolytes should be further elucidated. Furthermore, the reactivities of Li metal, solid electrolytes, and cathodes should be further investigated. Currently, it is difficult to access the electrolyte/electrode interface using conventional post-mortem techniques without creating artifacts, and thus, further advances should be made on developing in situ analysis techniques. Moreover, the search for highly stable conductive electrolytes should continue. Lastly, an important aspect related to the fabrication of ASBB would be the cooperation between scientists and engineers, which could facilitate the fabrication of large-area cells and address the current transportation technology challenges.


Plantago major, a species that accumulates the polyol sorbitol to high concentrations. We grew P. major plants on soil substrate adjusted to low-, medium-, or high-potassium conditions. We found that biomass, seed yield, and leaf tissue K contents increased in a soil K-dependent manner. K gradually increased the photosynthetic efficiency and decreased the non-photochemical quenching. Concomitantly, sorbitol levels and sorbitol to sucrose ratio in leaves and phloem sap increased in a K-dependent manner. K supply also fostered plant cold acclimation. High soil K levels mitigated loss of water from leaves in the cold and supported cold-dependent sugar and sorbitol accumulation. We hypothesize that with increased K nutrition, P. major preferentially channels photosynthesis-derived electrons into sorbitol biosynthesis and that this increased sorbitol is supportive for sink development and as a protective solute, during abiotic stress.Potassium (K) is essential for the processes critical for plant performance, including photosynthesis, carbon assimilation, and response to stress. K also influences translocation of sugars in the phloem and regulates sucrose metabolism. Several plant species synthesize polyols and transport these sugar alcohols from source to sink tissues. Limited knowledge exists about the involvement of K in the above processes in polyol-translocating plants. We, therefore, studied K effects in  In contrast to other highly abundant nutrients like phosphate, sulfate, or nitrogen, K does not become part of a certain cellular structure or molecule. Nevertheless, K serves as an essential ion required for a wide number of different processes that are critical for plant development and performance. K has crucial functions in enzyme activation, osmotic adjustment, regulation of turgor pressure, and membrane electric potential , non-photochemical quenching [Y(NPQ)], and non-regulated energy dissipation [Y(NO)] . ETR incA) increased with increasing K level in the soil substrate and was twice as high in the K3 plants, in comparison to the K1 plants  as well as PmSUC2 as the phloem sucrose loader. In addition, the expression levels of sorbitol dehydrogenase (SDH) that regulates sorbitol accumulation was measured , sweet potato (Ipomoea batatas L.), and barley (Hordeum vulgare L.) [P. major also responded positively to application of K, where growth and development, as well as seed yield, increased with higher doses of K  ,46,47. Ises of K . In the ses of K . ETR, Y. In bean (Vicia faba L.), continued K-deficiency resulted in decreased sugar in phloem exudates and higher leaf sugar contents, suggesting impaired phloem loading and backlogging of sugars in the leaf mesophyll or the apoplasm, under low K [akt2/3 mutants impaired in phloem K-loading transported lower amounts of both, K and sucrose in the phloem, as determined by aphid stylectomy. Comparing P. major whole leaf accumulation , we observed cold-induced wilting and water loss of leaves almost in all K conditions, but the severity of the water loss was dependent on the K soil level , KH2PO4 (40 g·L−1), K2SO4 (6 g·L−1), MgSO4 (20 g·L−1), H3BO3 (1.4 g·L−1), FeNa-EDTA (0.7 g·L−1), CuSO4·5H2O (0.8 g·L−1), ZnSO4·7H2O (0.9 g·L−1), MnCl2·4H2O (1.8 g·L−1), Na2MoO4 (7.5 mg·L−1). Per 20 kg unfertilized soil substrate, 200 mL of each of the stock solutions were added. A total of 1 L suspensions of each CaO (60 g·L−1) and CaCO3 (80 g·L−1) were directly mixed into 20 kg soil substrate. The soil substrate per nutrient mixtures were homogenized by repeated shoveling and dispersing of the mixtures. For the K-deficient K1 substrate, KH2PO4 was replaced with NaH2PO4 (40 g·L−1). For the K over-fertilization K3 substrate, KCl (70 g·L−1) was applied. The final calculated elemental concentrations in the soil substrates were as following: N (200 mg·kg−1), S (55 mg·kg−1), Mg (40 mg·kg−1), Ca (3978 mg·kg−1), P (93 mg·kg−1), K , B (2.5 mg·kg−1) Fe (0.92 mg·kg−1), Cu (2 mg·kg−1), Zn (2 mg·kg−1), Mn (5.2 mg·kg−1), and Mo (0.03 mg·kg−1).Plants were grown in pots (7.5 cm diameter) filled with a peat substrate, with an extremely low nutritional value . This substrate contained background levels of K lower than 0.1 g·kg-1 . Three dPlants were initially grown for 8 weeks in growth chambers, under short-day conditions (10 h light/14 h dark), at 110 µE (tubular fluorescent lamp light) and 21 °C. Plants were then transferred to a neighboring growth chamber with identical light conditions, and a temperature of 4 °C. Immediately prior to transfer and at one, two, and four days after transfer, whole shoots of six plants from each K soil substrate condition were harvested and the FW, DW, and water content were determined. Plants were harvested in the cold and the shoots were enclosed in pre-weighted 25 mL scintillation flasks. FW was determined and the plants were immediately stored at −80 °C. Plants were then freeze-dried for four days and used for DW and water-content determination. The freeze-dried material was ground in a ball mill and the powder used for metabolite and gene expression analysis.M-Series system , as described previously [−2 s−1) 0 to PAR 726, in 14 steps. The dark interval between each light pulse was 20 s. Recorded fluorescence was used for calculation of the effective quantum yield of PSII, as described [Photosynthetic activity was measured using an Imaging-PAM eviously . Measureescribed ,41.2-assimilation rate, respiration, leaf CO2 concentration, and stomatal conductance were recorded. Temperature, humidity, and CO2 concentrations of the cuvette were set to the conditions that the plants were grown in. Light respiration was measured at PAR 125 and dark respiration at PAR 0, over a time of 1 min for each plant. The CO2 concentration of the gas exchange chamber was set to 500 ppm, for both light and dark recordings. Each plant was measured three times with 30 s intervals between measurement, to allow the leaves to return to the stabilized value.Plants were grown as described above for 6 weeks. Gas-exchange-related parameters were analyzed with a GFS-3000 system . Measurements were performed with four to five plants per condition and each plant was measured three times . Individual plants were placed in a whole plant gas-exchange cuvette and the COExudates were collected, as described in Pommerrenig et al. . Exudateg and 700 µL of the supernatants transferred to new 2 mL reaction tubes. Another 600 µL of 80% ethanol p.a. were added and the samples were extracted for 30 min at 50 °C, on a heating block, while shaking. The samples were again centrifuged as above and with 500 µL of the supernatant, combined with the already collected supernatants. Ethanol was removed in a vacuum concentrator  and the pellets re-suspended in 500 µL ddH2O. Glucose, fructose, sucrose, sorbitol, and maltose were measured with ion chromatography, using an 871 Advanced Biosensor, 818 IC Pump, 837 De-gaser configuration from Metrohm . A Metrosep Carb2 −250/4.0 column was used in combination with a Metrosep Carb2 Guard/4.0 guard column . A gold working electrode  was used for amperometric detection. The eluent was 10 mM NaOAc and 100 mM NaOH. The flow rate was 0.6 mL min−1.Freeze-dried plant material was ground to a fine powder using a ball mill . About 15 mg of ground tissue were weighed into 2 mL screw-lid reaction tubes containing a steel ball and extracted with 900 µL of 80% ethanol p.a., on an overhead shaker, by inverting samples overnight at RT. Samples were centrifuged for 5 min at 11,000× g and 700 µL of the supernatants transferred to new 2 mL reaction tubes. Another 600 µL of 80% ethanol p.a. were added and the samples were extracted for 30 min at 50 °C, on a heating block, while shaking. The samples were again centrifuged as above and with 500 µL of the supernatant, combined with the already collected supernatants. Ethanol was removed in a vacuum concentrator  and the pellets re-suspended in 500 µL ddH2O. Glucose, fructose, sucrose, sorbitol, and maltose were measured with ion chromatography, using an 871 Advanced Biosensor, 818 IC Pump, 837 De-gaser configuration from Metrohm . A Metrosep Carb2 −250/4.0 column was used in combination with a Metrosep Carb2 Guard/4.0 guard column . A gold working electrode  was used for amperometric detection. The eluent was 10 mM NaOAc and 100 mM NaOH. The flow rate was 0.6 mL min−1.Freeze-dried plant material was ground to a fine powder using a ball mill . About 15 mg of ground tissue were weighed into 2 mL screw-lid reaction tubes containing a steel ball and extracted with 900 µL of 80% ethanol p.a., on an overhead shaker, by inverting samples overnight at RT. Samples were centrifuged for 5 min at 11,000× a and b were measured by the absorbance of the supernatant at 649 nm and 665 nm. The chlorophyll content was calculated using the formulas (Equations (1) and (2)):Photosynthetic pigments were extracted from ground leave tissue, with 90% acetone/10% 0.2 M Tris/HCl pH 7.5, for 48 h, at 4 °C in the dark. Chlorophyll Violaxanthin, Antheraxanthin, and Zeaxanthin were analyzed through reverse-phase high-performance liquid chromatography, as described .p = 0.05 was set for defining the significant differences. The testing was performed using the “One-Way ANOVA  with post-hoc Tukey HSD (Honestly Significant Difference) Test Calculator for comparing multiple treatments” (https://astatsa.com/OneWay_Anova_with_TukeyHSD/). Boxplots were generated using BoxPlotR [Statistical analysis was performed by one-way ANOVA with post-hoc Tukey HSD test. A threshold of BoxPlotR .P. major responded to K nutrition and performed better under low temperature, due to the regulation of carbohydrate metabolism, particularly sorbitol accumulation. Sorbitol might have a dual role in the cold acclimation process. First, sorbitol biosynthesis functions as electron sink via regeneration of NADPH/H+ to NADP+, during stress, alleviating high electron pressure from photosystems. Second, sorbitol, like other sugars might act as an ROS-quenching compound itself, due to the numerous hydroxyl groups and could be beneficial for absorption of free electrons during stress conditions. High K conditions promote such sorbitol accumulation. Our data link beneficial K nutritional effects for cold acclimation to sugar and sorbitol metabolism and offers a possible explanation of the physiological mode of action of K in abiotic stress response.The present study provided evidence that


Haemophilus parasuis in piglets.Gamithromycin is a macrolide approved for the treatment of bovine and swine respiratory diseases. Our study aims to establish the clinical breakpoint and optimum dose regimen for gamithromycin against H. parasuis isolates ranged from 0.008 to 128 mg/L and the epidemiological cutoff (ECOFF) was calculated as 1.0 mg/L. A large potentiation effect of serum on in vitro susceptibility of gamithromycin was observed for H. parasuis, with broth/serum ratios of 8.93 for MICs and 4.46 for MBCs, respectively. The postantibiotic effects were 1.5 h (1 × MIC) and 2.4 h (4 × MIC), and the postantibiotic sub-MIC effects ranged from 2.7 to 4.3 h. Gamithromycin had rapid and concentration-dependent killing against H. parasuis, and the AUC24h/MIC ratio correlated well with ex vivo efficacy (R2 = 0.97). The AUC24h/MIC targets in serum associated with bacteriostatic, bactericidal and eradication activities were 15.8, 30.3 and 41.2, respectively. The PK/PD-based population dose prediction indicated a probability of target attainment (PTA) for the current marketed dose (6 mg/kg) of 88.9% against H. parasuis. The calculated gamithromycin dose for a PTA ≥ 90% was 6.55 mg/kg. Based on Monte Carlo simulations, the PK/PD cutoff (COPD) was determined to be 0.25 mg/L.Gamithromycin was well absorbed and fully bioavailable (87.2–101%) after intramuscular and subcutaneous administrations. The MICs of gamithromycin for 192 clinical The determined cutoffs and PK/PD-based dose prediction will be of great importance in gamithromycin resistance surveillance and serve as an important step in the establishment of optimum dose regimen and clinical breakpoints. Haemophilus parasuis is the etiological agent of Glässer’s disease that causes inflammatory infections such as fibrinous polyserositis, meningitis, arthritis and bronchopneumonia in weaned piglets  and PK/PD cutoff value  to assist the selection of a CBP for gamithromycin against H. parasuis.In this study, we described ex vivo PK/PD relationships of gamithromycin in porcine serum against max; 0.99 and 0.61 mg/L) at 0.36 and 1.59 h after intramuscular (IM) and subcutaneous (SC) routes, respectively (Table 1/2) ranged from 25.4 to 29.4 h after IV, IM and SC administrations of gamithromycin. Piglets received the drug IM and SC resulted in AUC0-∞ of 6.63 and 5.72 mg·h/L that were comparable to the IV AUC0-∞ at the same dose. This indicated a high bioavailability (87.2–101.1%) for SC and IM injections of gamithromycin (Table We observed gamithromycin peak levels in piglets (CH. parasuis LM15 in Haemophilus test medium (HTM) broth (0.25 mg/L) was 8-fold greater than that in serum (0.031 mg/L), indicating a large potentiation effect by serum. To further confirm the serum effects, MICs and MBCs of gamithromycin were determined in both HTM and serum against 19 selected H. parasuis isolates. Interestingly, the geometric means of the MICs and MBCs were significantly different between HTM and serum, resulting in HTM/serum ratios of 8.93 for MICs and 4.46 for MBCs , Log2 SD (1.73), n = 175] using the Kolmogorov-Smirnov (K-S) normality test at P > 0.1. The fitted MIC distribution contained > 95% that possessed gamithromycin MICs between 0.008 and 1 mg/L, and the ECOFF value was therefore calculated to be 1 mg/L  produced a visible growth inhibition whereas > 3.0 log10 cfu/mL) after 24 h exposure. Compared with killing pattern in HTM, a more rapid and greater reduction was achieved for serums collected at 12 h and 24 h after IM administration that contained the mean drug levels of 0.121 and 0.058 mg/L . In serum collected up to 12 h after IM injection, bacterial densities were reduced rapidly by ~ 4 log10 cfu/mL after 6 h exposure . The dose-response curve was steep, with the highest decrease from the initial density of 4.68 log10 cfu/mL . The MIC distribution was divided by a scaling factor of 8.93 to bridge HTM and serum. Based on Monte Carlo simulation, the calculated dose of gamithromycin for a PTA ≥ 90% was 6.55 mg/kg. In view that the current recommended dose is 6.0 mg/kg, the corresponding PTA was calculated at 88.9%  obtained by dividing the target value of AUCH. parasuis infections. The PTAs of the current dose (6.0 mg/kg) for the typical AUC24h/MIC targets at each possible MIC in HTM were thus calculated to obtain the COPD value. At a MIC of ≤0.25 mg/L in HTM, the PTA achieving an AUC24h/MIC target of 30.3  was 96.7%  pathogens such as eumoniae . In studectively . Similarefficacy , 10. It efficacy , 12. Thiefficacy , 13. In h medium . More reH. parasuis to macrolides in serum matrix is likely related to the downregulation of oprM gene (encoding an active efflux pump) and increased outer-membrane permeability in biological fluids  and the Institutional Animal Ethical Committee of South China Agricultural University (SCAU 2018A014).Blood samples (5 mL) were collected from the jugular veins into vacutainers without anticoagulant before (0 h) and at 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72 and 96 h after administration of gamithromycin. Serum was separated by centrifugation of blood samples at 3000×g for 10 min and stored at − 80 °C until analysis.2 > 0.996), with limits of quantification (LOQ) of 0.0005 mg/L. All samples with drug levels > 0.5 mg/L were diluted proportionally with control serum prior to extraction with acetonitrile. Recoveries of gamithromycin from serum ranged from 95.9 to 106.2%, and both the intraday and interday variations were < 9.27% (data not shown).Gamithromycin concentrations in serum were determined by a validated HPLC-MS/MS method using an Agilent 1200 HPLC system linked to an API 4000 triple quadrupole mass spectrometer as previously reported , 31. In All PK parameters were measured using a non-compartmental model in WinNonlin software Version 5.2.1 . The absolute bioavailability (F) of gamithromycin in piglets was calculated from the following equation , 32:\doH. parasuis isolates were determined using broth dilution in accordance with CLSI guidelines  was the MIC that best describes the end of the wild-type distribution . All PK parameters were assumed to be normally distributed. SF is a scaling factor of 8.93 to bridge the MICs between HTM and serum. The COPD was defined as the MIC in HTM broth at which the PTA for bactericidal effect reached 90% [A 10,000-subject Monte Carlo simulation was used to calculate the CO follows : AUC24h/ched 90% , 29.Additional file 1: Figure S1. Standard curve constructed by regression of the viable bacterial counts and optical density (OD600nm) of H. parasuis cultured in liquid medium. The solid points represent the observed data and the line represents the best fitting curve as follows: y = 0.7138 ln(x) + 10.722 (R2=0.9857).


Pseudomonas microorganisms are used for bioremediation of soils contaminated with petroleum hydrocarbons. The overall remediation efficiency is largely dependent on the presence of macro- and micronutrients. Widely varying concentrations of available nitrogen and iron (Fe) in soils were shown to affect residual hydrocarbons in the course of biodegradation. The regulatory mechanisms of expression of hydrocarbon catabolic genes in low nitrogen/low iron conditions remain unclear. The catabolism of naphthalene, a two-ring polycyclic aromatic hydrocarbon, has been well studied in pseudomonads in terms of the involvement of specific transcriptional activators, thus making it useful in revealing additional regulatory control of the adaptation of hydrocarbon destructors to a low level of the essential nutrients. The Pseudomonas putida strain BS3701 is a component of the “MicroBak” preparation for soil remediation. Previously, this strain was shown to contain genes encoding the key enzymes for naphthalene catabolism: naphthalene 1,2-dioxygenase, salicylate hydroxylase, catechol 2,3-dioxygenase, and catechol 1,2-dioxygenase. Our study aimed to clarify whether the naphthalene catabolic gene expression is dependent on the amount of nitrogen and iron in the growth culture medium, and if so, at exactly which stages the expression is regulated. We cultivated the strain in low nitrogen/low iron conditions with the concurrent evaluation of the activity of the key enzymes and the mRNA level of genes encoding these enzymes. We are the first to report that naphthalene catabolic genes are subject not only to transcriptional but also post-transcriptional regulation. Pseudomonas are used for bioremediation of soils contaminated with petroleum hydrocarbons. The overall remediation efficiency is largely dependent on the presence of macro- and micronutrients. Widely varying concentrations of available nitrogen and iron (Fe) in soils (Microorganisms of the genus in soils  were shoin soils . The regPseudomonas putida PpG1 (P. putida strain G7 (pNAH7), where the naphthalene conversion proceeded via salicylate  and catechol further oxidized by the meta-pathway . Activation of the nah- and sal-operons occurred with the assistance of the transcriptional regulator NahR  in the presence of the salicylate ion  acts as an activator in the presence of toluene and meta-benzyl alcohol  or sodium salicylate (1 g/l) as the sole source of carbon and energy. The modified Evans mineral medium contained (per liter) 50 mM K2HPO4, 5 mM NH4Cl, 0.1 mM Na2SO4, 0.0625 mM MgCl2, 0.018 mM FeCl3, 0.01 mM MnCl2, 0.005 mM ZnO, 0.002 mM CoCl2, 0.001 mM CaCl2, 0.97 μM CuCl2, 0.97 μM H3BO3, and 0.005 μM (NH4)6Mo7O24  as the sole source of carbon. The low iron growth medium contained 100 μM 2,2′-bipyridyl  and was supplemented with sodium salicylate (1 g/l) as the sole source of carbon. BS3701 grown on modified Evans with glucose (10 g/l) for 1 day was further used for inoculation (1 ml per 100 ml of medium). The harvested cells were centrifuged at 10,000 rpm for 10 min at 4°C and re-suspended in appropriate modified Evans at the final concentration of 3 × 108 CFU/ml. The 750-ml flasks used in the experiments contained the appropriate mineral medium and substrate (100 ml each). The growth period lasted until the end of the exponential growth phase. The time of cultivation on all used media was 31 h, the end of exponential growth phase was 21 h.The )6Mo7O24 . The medThe oxidative activity (OA) of microorganisms was determined from sodium salicylate degradation vs. control (microorganism-free medium):k is sodium salicylate concentration (mg/l) in the microorganism-free medium and Xi is that in the inoculated flask (mg/l). Residual concentration of sodium salicylate (20 μl sample) was analyzed by liquid chromatography using a silica gel-packed Agilent (1260 infinity) column  grafted with hexadecyl groups. The used wavelength was 310 nm. The mobile phase was (A) water (0.1% CH3COOH); (B) acetonitrile (0.1% CH3COOH). The gradient mode was as follows: up to 3 min – 20% of eluent B, by 12 min – 50% of eluent B, by 14 min – 100% of eluent B. The eluent flow rate was 1 ml/min. System stabilization took 5 min; the analysis time was 19 min (where Xs 19 min . The colg and 4°C. The supernatant was used as a cell-free extract in measuring enzymatic activity.The biomass was washed twice with chilled 0.05 M phosphate buffer (pH 7.0) and re-suspended in 0.02 M phosphate buffer (pH 7.5). Cell suspension (5 ml sample) was subjected to ultrasonic disintegration (using an MSE150 disintegrator) for 1.5 min (3 × 30 s) at 4°C. Cell debris was removed by centrifugation  for 20 min at 32,000 × Activities of the enzymes were determined as previously described for naphthalene 1,2-dioxygenase in 1 . The relative amount of transcripts was quantified using the delta-delta Cp method, with amplification efficiency taken into account:Specific primers were constructed using the Primer-BLAST tool1 . A kit cstKeeper , the most−test for data comparison, p-value < 0.05. All results are derived from three independent replicates and presented as a mean score ± confidence interval.where «E» is the amplification efficiency, «Cp» is the crossing point. We used unpaired Student’s MN442422.1, MN413629.1, MN442423.1, MN442424.1, MN442425.1, and MN442426.1.The nucleotide sequences of the naphthalene catabolic genes have been deposited in GenBank under accession nos. P. putida strain BS3701, the transformation of naphthalene to salicylate is assisted by the plasmid-localized gene cluster nahAaAbAcAdBFCQED. BS3701 cultivation in salicylate-supplemented (vs. glucose) Evans medium (nahAa mRNA level [in contrast to the case of P. putida G7 (pNAH7)] but showed an ∼1.5-fold decrease in naphthalene 1,2-dioxygenase activity (NDO) (from 0.016 ± 0.003 to 0.0107 ± 0.0008 μmol/min∗mg of protein).In the s medium  did not nahAa mRNA level remained unchanged, while the NDO enzyme activity changed (4Cl instead of 5 mM NH4Cl) retained the same nahAa mRNA level, but the NDO activity increased by ∼3.4-fold (from 0.0107 ± 0.0008 to 0.036 ± 0.006 μmol/min∗mg of protein). Previously, the literature offered no reports on the stimulation of NDO synthesis by low nitrogen.Under nitrogen limitation, the  changed . Evans-cnahAa mRNA level and the NDO enzyme activity changed .Under iron limitation, both the  changed . Evans-cP. putida BS3701 chromosome was found to carry the functional nahU gene encoding salicylate hydroxylase (SH). BS3701 cultivation in Evans medium supplemented with salicylate (vs. glucose) (∗mg of protein).The glucose)  increasenahU mRNA level and the SH enzyme activity changed (nahU mRNA level by ∼9.6-fold and in the SH activity by ∼3.3-fold (from 0.077 ± 0.013 to 0.023 ± 0.005 μmol/min∗mg of protein). The extent of salicylate degradation was also reduced . However, similar to cultivation in 1 mM NH4Cl Evans, the residual content of salicylate was 4.3 ± 0.6 mg/L.Under nitrogen limitation, both the  changed . Evans-c reduced . The ininahU mRNA level and the SH enzyme activity changed (nahU mRNA decrease with ∼15.4-fold lower activity of SH (from 0.077 ± 0.013 to 0.0050 ± 0.0010 μmol/min∗mg of protein). At the end of the exponential growth phase, the residual salicylate content was 69 ± 18 mg/L .Under iron limitation, both the  changed . Evans-c 18 mg/L . The lityochelin  and promyochelin . As revePseudomonas putida BS3701 was found to contain a plasmid-localized gene encoding catechol 2,3-dioxygenase (C23O). During BS3701 cultivation in the presence of salicylate used as a source of carbon .f carbon , the nahnahH mRNA level remained unchanged, while the C23O enzyme activity changed (nahH mRNA level but the C23O decreased by ∼2-fold (from 0.057 ± 0.007 to 0.028 ± 0.003 μmol/min∗mg of protein). To date, the literature presents no data on the regulatory mechanisms of expression of genes encoding aromatic ring-hydroxylating dioxygenases in low nitrogen conditions. Under iron limitation, both the nahH mRNA level and the C23O enzyme activity changed (nahH mRNA level by ∼3.7-fold and C23O activity by ∼3.3-fold (from 0.057 ± 0.007 to 0.0175 ± 0.0009 μmol/min∗mg of protein).Under nitrogen limitation, the  changed . Evans-c changed . Evans-cP. putida BS3701 genome contains three copies of the gene encoding catechol 1,2-dioxygenase (C12O) that are localized to the chromosome and denoted as catA(0), catA(2), and catA(8). Although the catA(2) gene is in the immediate vicinity of nahU (the distance between the nucleotide A of the catA(2) start codon ATG and the nucleotide A of the nahU stop-codon TGA is 74 bp), it apparently has its own promoter. In BS3701 grown on Evans supplemented with salicylate (vs. glucose) (catA(0) mRNA remained unchanged, while the content of catA(2)- and catA(8) mRNA increased by ∼32-fold (from 0.39 ± 0.15 to 12.46 ± 3.11) and ∼8-fold (from 4.8 ± 0.6 to 38.2 ± 28.4), respectively. C12O cultivated in the presence of glucose showed no activity which was observed only with salicylate used as a source of carbon (0.0186 ± 0.0018 μmol/min∗mg of protein).The glucose) , the levcatA mRNA level remained unchanged, while the C12O enzyme activity changed (catA copies: for catA(0) it was an approximately 2-fold decrease (from 0.58 ± 0.19 to 0.29 ± 0.01), for catA(2) ∼33-fold (from 12.46 ± 3.11 to 0.38 ± 0.07), and for catA(8) ∼6-fold (from 38.2 ± 28.4 to 6.5 ± 2.8). Though the decrease extents were different, the tendency was the same, reflecting the regulation of catechol 1,2-dioxygenase expression as early as at the level of the varying mRNA content. The activity of C12O was ∼3 times lower (from 0.0186 ± 0.0018 to 0.0064 ± 0.0016 μmol/min∗mg of protein).Under nitrogen limitation, the  changed . BS3701 catA mRNA level changed for 2 out of 3 copies, and the C23O enzyme activity changed as well (catA(0) mRNA level and showed a decrease in catA(2)- and catA(8) mRNAs by ∼13.5- (from 12.46 ± 3.11 to 0.92 ± 0.15) and ∼33-fold (from 38.2 ± 28.4 to 11.5 ± 0.9), respectively. The activity of C12O decreased by ∼1.5-fold (from 0.0186 ± 0.0018 to 0.012 ± 0.003 μmol/min∗mg of protein).Under iron limitation, the  as well . Evans-cnahAaAbAcAdBFCQED operon, the nahU gene, and nahH-containing gene clusters were identified using BPROM, a SoftBerry prediction program. Localization of the putative promoter of a catA(0)-containing gene cluster was defined using the literature data. Upstream of the benA gene [which probably shares the sole operon with the catA(0) gene] resides a sequence that is 98% identical to the regulatory region adjacent to +1 point of the benABCDKEF operon in P. putida PRS2000 (catA(2) gene exhibit homology, though extremely low, to the −10 and −35 boxes of the sigma-70-dependent promoter. Since the catA(2) mRNA content was observed to increase in cells grown in the presence of salicylate, it cannot be ruled out that the transcription activation is mediated by a specific transcription factor that enables RNA polymerase (RNAP) to recognize such a weak promoter. The promoter of the catA(8)-containing catBCA operon is identical to that identified for P. putida catBCA ( PRS2000 . The rega catBCA . Using tP. putida BS3701 genome contains one copy of genes coding for Fis (factor for inversion stimulation), Lrp (leucine responsive protein), and IciA (inhibitor of chromosome initiation), five copies of the gene coding for H-NS , as well as one copy of genes coding for alpha- and beta-subunits of IHF (integration host factor) (unpublished data).The rpoD encoding RNAP σ70-subunit , and catA(8), leaving unchanged rpoD mRNA  and catA  were down-regulated at decreasing NH4Cl concentration from 5 to 1 mM, the rpoD mRNA level remained unchanged, but the ihfA mRNA level altered. Although IHF has been reported to stimulate the RNAP interaction with specific transcriptional regulators  and nahH  remained unchanged, while their enzyme activity changed. Naphthalene 1,2-dioxygenase and catechol 2,3-dioxygenase possibly undergo the posttranscriptional regulation by inhibition/stimulation of translation. The activity of catechol 2,3-dioxygenase decreased. We are the first to report this observation. In contrast, low nitrogen conditions resulted in a higher enzyme activity of naphthalene 1,2-dioxygenase. This effect has not been mentioned in the literature either. Only one fact was found in support of the interconnection between naphthalene 1,2-dioxygenase and nitrogen metabolism. NDO can use indole (a heterocyclic nitrogen-containing compound) as a substrate, having oxidized it to 2,3 dihydroxyindole and further to the salicylate  or in terms of catabolite repression (in part). Here, the activity of key enzymes of the naphthalene catabolic pathway was studied along with the mRNA level of genes encoding these enzymes in  of glnA  and rhlAnA , and catA(8), though that of rpoD and NAPs remained unchanged. We assume that the mRNA level undergoes post-transcriptional regulation by changing the mRNA degradation rate. Low iron conditions can produce a critical effect on the functioning of dioxygenases with iron-sulfur clusters . As shown previously .Fur (Ferric uptake regulator) acts as a repressor under iron-rich conditions , but wittability . Our stueviously , these ceviously . The expyochelin  and promyochelin ]. AnnotaPseudomonas, naphthalene catabolism has been best studied in terms of the effects of specific transcription activators. We propose that under low nitrogen and low iron conditions the naphthalene catabolic genes are subject not only to transcriptional but also post-transcriptional regulation. These results form a basis for studying the mechanisms of additional regulatory control of genes involved in aromatic hydrocarbon catabolism.In https://www.ncbi.nlm.nih.gov/genbank/, MN442422.1; https://www.ncbi.nlm.nih. gov/genbank/, MN413629.1; https://www.ncbi.nlm.nih.gov/gen bank/, MN442423.1; https://www.ncbi.nlm.nih.gov/genbank/, MN44P2424.1; https://www.ncbi.nlm.nih.gov/genbank/, MN44 2425.1; https://www.ncbi.nlm.nih.gov/genbank/, MN442426.1.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found here: IP-F, KP, AV, and MZ contributed to the experiment design. IP-F, KP, AV, AF, and RS conducted all experimental work and analyses. IP wrote the manuscript with critical review and inputs from KP, AV, and MZ.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Patients experience moderate-high intensity postoperative pain after cesarean section (CS). The aim of this study was to investigate the optimal concentrations of ropivacaine and sufentanil for use in controlling pain after CS.Methods: One hundred and seventy-four women undergoing elective CS were randomly allocated to four groups. Epidural analgesia was administered with 0.1% ropivacaine in the R1 group, 0.15% ropivacaine in the R2 group, a combination of 0.1% ropivacaine and 0.5 μg/ml of sufentanil in the R1S group, and a combination of 0.15% ropivacaine and 0.5 μg/ml of sufentanil in the R2S group . Pain scores  at rest (NRS-R), during movement (NRS-M), and when massaging the uterus (NRS-U) were documented at 6 and 24 h. We also recorded patient satisfaction scores, time to first flatus, motor deficits, and adverse drug reactions.Results: NRS  scores in the R2S group  were lower than in the R1 group   at 6 h; and patient satisfaction (9 [8–10]) was improved compared to the R1 group (8 [6–8]) (p < 0.01). The time to first flatus (18.7 ± 11.8 h) was reduced relative to the R1 group (25.9 ± 12.0 h) (p < 0.05). The time to first ambulation was not delayed (p > 0.05). However, the incidence of pruritus (4 [9.3%]) was increased compared to the R2 group (0 [0]) (p < 0.05) at 6 h, and the incidence of numbness  was increased compared to the R1 group   at both 6 and 24 h.Conclusions: Although we observed a higher incidence of pruritus and numbness, co-administration of 0.15% ropivacaine and 0.5 μg/ml of sufentanil administered epidurally optimized pain relief after CS, with treated subjects exhibiting lower NRS scores, shorter time to first flatus, and higher patient-satisfaction scores. A surfeit of cesarean sections are performed annually worldwide. In 2016, the cesarean section (CS) rate was 31.9%  and 41.1et al. found that patient-controlled epidural analgesia (PCEA) provided significantly superior analgesia compared with intravenous patient-controlled analgesia (PCIA)  . Epiduraa (PCIA) , but cana (PCIA) . These ablockade .Ropivacaine has been used in labor for epidural analgesia and post-cesarean analgesia because of its lower potential for accumulation, greater sensorimotor-differential block, and increased cardiovascular safety profile . The breThis trial was conducted at the Department of Anesthesiology, Xuanwu Hospital of Capital Medical University in China. Ethics approval (ea1/185/10) was obtained from the ethics committee of the Xuanwu Hospital of Capital Medical University. The trial was registered with the Chinese clinical trial registry (ChiCTR 1900 021740), and written informed consent was obtained on the day before surgery.Our primary aim was to compare the analgesic efficacy of the four groups at the first 24 h after CS by a prospective, randomized, double-blinded clinical trial. Our secondary objective was to compare patient satisfaction, gastrointestinal symptoms, postsurgical recovery, and ADRs among the four groups.This trial included 174 women who were enrolled from March 2019 to September 2019. Women were eligible for inclusion if they were 21–45 years of age after 32 weeks of gestation  and at the American Society of Anesthesiology physical status classification I–II , and werAfter pre-hydration with 500 ml of lactated Ringer’s solution, women underwent combined spinal-epidural anesthesia (CSEA) at the L2–L3 or L3–L4 spinal interspace in the right lateral position, receiving an injection containing between 7.5 and 10 mg of 0.5% hypobaric bupivacaine , depending upon their height . After intrathecal injection, the epidural catheter was inserted up to 3 cm into the epidural space, and then patients were placed in a supine position in a left-lateral tilt. Once a bilateral sensory block to the T4 dermatome was achieved (which we tested by pinprick), the operation began. If sensory block was not achieved to the T4 level, 2% lidocaine  was administered into the epidural space to achieve adequate anesthesia, and the patients were excluded from further analysis.1 group, 0.1% ropivacaine ; a R2 group, 0.15% ropivacaine; a R1S group, 0.1% ropivacaine +0.5 μg/ml of sufentanil ; and a R2S group, 0.15% ropivacaine +0.5 μg/ml of sufentanil. Dosage was a 4-ml/hour background infusion with a bolus dosage of 4 ml/20 min as needed, and a maximal dosage of 16 ml/h.Before skin closure, a 3-ml 2% lidocaine solution was administered into the epidural space to confirm adequate placement of the epidural catheter, and a CADD™ Administration set, Model 6,300 , was then connected to each patient. Patients were randomized to the receipt of a continuous epidural infusion with PCEA of one of the four solutions using the sealed-envelope technique: a RA blinded observer evaluated the patients. The primary outcome was pain scores as measured using a numerical rating scale (NRS) : NRS pain scores at rest (NRS-R), NRS pain scores during movement (NRS-M), and NRS pain scores when massaging the uterus (NRS-U)  at 6 andIn this study, analgesic efficacy was the primary outcome. A power analysis showed that 42 patients in each group would provide 80% of the power needed to detect differences among the four groups.p = 0.05 (2-sided). A Shapiro-Wilk test, Q-Q plot, and p-p plot were used to confirm normality for continuous variables. All normally distributed continuous variables are reported as means ± SD, and non-normally distributed continuous variables are described as medians (25–75% interquartile range [IQR]). One-way ANOVA or Kruskal-Wallis methods were used to compare the differences between/among groups, and Tukey’s and Dscf tests were used to perform the post-hoc analyses. The Chi-squared test was used to compare the differences between categorical variables, and data are expressed as N (%) patients.We performed all statistical analyses with SPSS 25.0 software, and statistical significance was set at 1 group, 45 in the R2 group, 43 in the R1S group, and 43 in the R2S group .Baseline characteristics for the four groups are shown in 1S (3 [2–3]) and R2S (2 [1–3]) groups were lower than those in either the R1 (3 [3–4])  or R2 groups (3 [2–4]) . At 24 h after skin closure, there were no statistically significant differences in NRS-R scores among the four groups (p > 0.05)  .1S group (4 [3–5]) were lower than those in the R2 group (5 [4–6]) (p < 0.05), and NRS-M scores in the R2S group (4 [3–5]) were lower than in the R1 (5 [4–6]) (p < 0.05) and R2 (5 [4–6]) (p < 0.01) groups. By 24 h after CS, there were no longer any statistically significant differences in NRS-M scores among the four groups (p > 0.05)  .2S group (6 [5–6]) were lower than those in the R1 group (7 [6–8]) (p < 0.01), but there was no statistically significant differences between R1S and R1 groups (p > 0.05). By 24 h after CS, there were no statistically significant differences in scores among the four groups (p > 0.05)  .2S group (9 [8–10]) relative to the R1 group (8 [6–8]) (p < 0.01). There were no statistically significant differences between any of other two groups  (Patient satisfaction scores were significantly higher in the R > 0.05) .2S group (18.7 ± 11.8 h) compared to the R1 group (25.9 ± 12.0 h) (p < 0.05) (The time to patient’s first flatus was shorter in the R < 0.05) .2S group (0 [0–2]) was less than in the R1 (1 [0–3]) (p < 0.05) and R2 groups (1 [0–4]) (p < 0.05), and that was less in the R1S group (0 [0–2]) than in the R2 group (1 [0–4]) (p < 0.05) at 6 h, while there were no statistically significant differences among the groups at 24 h (p > 0.05). There were no statistically significant differences among the groups regarding the time when highest pain scores were recorded (p > 0.05) or the number of patients who requested supplementary analgesia, either at 6 or 24 h   in the R > 0.05) .2S group (24.8 [22.5–27.5 h]) than in the R1S group (27.3 [25.0–28.5 h]) (p < 0.05). There were no statistically significant differences between any of other two groups  (The time to patient’s first ambulation was shorter in the R > 0.05) .p > 0.05)  .1S group (4 [9.3%]) had a higher incidence of pruritus at 6 h compared to the R1 (0 [0]) (p < 0.05) and R2 groups (0 [0]) (p < 0.05), while patients in the R2S group (4 [9.3%]) exhibited a higher incidence of pruritus at 6 h compared to the R2 group (0 [0]) (p < 0.05). The incidence of numbness in the R2  and R2S groups  was greater than in the R1 group  at both 6 and 24 h. There were no statistically significant differences among the groups regarding other ADRs, including nausea and vomiting, headache, dizziness, hypotension, or respiratory depression  (Patients in the R > 0.05) .Postoperative analgesia after CS is challenging since we need not only to consider maternal comfort, but the anesthetic technique should also have no ADRs on the newborn. Epidural morphine is commonly used for analgesia after CS , but theThe present study indicated that co-administration 0.15% ropivacaine with 0.5 μg/ml of sufentanil epidurally alleviated CS pain at rest, during movement, and when massaging the uterus, and it reduced the number of self-administered doses of the PCA compared to 0.1% ropivacaine or 0.15% ropivacaine at 6 h. Moreover, it improved overall patient satisfaction scores and reduced the time to first flatus compared to 0.1% ropivacaine. The time to first ambulation was not delayed. However, co-administered with sufentanil increased the incidence of pruritus compared to ropivacaine only at 6 h. Higher concentration of ropivacaine increased the incidence of numbness compared to lower concentration at both 6 and 24 h, whether or not it was co-administered with sufentanil. We believe that although a higher incidence of pruritus and numbness was demonstrated, co-administration of 0.15% ropivacaine with 0.5 μg/ml of sufentanil epidurally may be the better choice for pain relief relative to the other groups due to the lower NRS-R, NRS-M, and NRS-U scores, shorter time to first flatus, and higher patient-satisfaction scores.Published guidelines advocate for fentanyl and sufentanil for epidural analgesia . The aut2S group relative to the other groups, the analgesic effect was not satisfactory in any group when massage the uterus. Carvalho et al. found that patients tolerated moderate pain rather than exposing their baby to any possible harmful effects of analgesics . Other investigators also reported similar results, Liu et al. found that 0.2% ropivacaine induced a 30% incidence in motor block, but not in those individuals who received 0.05% or 0.1% ropivacaine  increased numbness.Complications of neuraxial drugs are the results of drug absorption into the blood circulation or presence in cerebrospinal fluid, or both. The principal ADRs observed in the present study were numbness and pruritus. The incidence of numbness was higher in the R2S group relative to the R2 group, and that was higher in the R1S group compared to the R1 and R2 groups at 6 h. But the incidence of pruritus in the present study was overall low . By contrast, the incidence of epidural morphine (2.5 mg)-induced pruritus was 44% , which may be due to the lower concentration of sufentanil (0.3 μg/ml) the authors used  may be the better choice for post-cesarean analgesia compared to the other three regimens investigated in this study.Although the ADRs (pruritus and numbness) were greater in the R


Schistosoma japonicum HSP40 (Sjp40) and HSP90α (Sjp90α) in this study. Western blot analysis revealed both are present in soluble egg antigens and egg secretory proteins, implicating them in triggering the host immune response after secretion from eggs into host tissues. These observations were confirmed by immunolocalization showing both HSPs are located in the Reynolds’ layer within mature eggs, suggesting they are secreted by miracidia and accumulate between the envelope and the eggshell. Both HSPs are present in the musculature and parenchyma of adult males and in the vitelline cells of females; only Sjp90α is present on the tegument of adults. Sjp40 was able to enhance the expression of macrophages, dendritic cells, and eosinophilic cells in mouse liver non-parenchymal cells, whereas rSjp90α only stimulated the expression of dendritic cells. T helper 1 (Th1), Th2, and Th17 responses were increased upon rSjp40 stimulation in vitro, but rSjp90 only stimulated an increased Th17 response. Sjp40 has an important role in reducing the expression of fibrogenic gene markers in hepatic stellate cells in vitro. Overall, these findings provide new information on HSPs in S. japonicum, improving our understanding of the pathological roles they play in their interaction with host immune cells.We characterized  Schistosoma japonicum, is still a major public health problem that threatens many millions of people in the People’s Republic of China [+ T cells and other immune cells [Schistosomiasis, caused by of China ,2. The mof China . The patne cells . DetermiS. japonicum eggs has been shown to induce strong immunomodulatory effects, including immunostimulatory and immunosuppressive reactivities [S. japonicum is the egg-derived heat shock protein 40 , which is able to restrain the activation of hepatic stellate cells (HSCs) through the STAT3/p53/p21 pathway [Heat-shock proteins (HSPs) constitute the first line of protection for cells exposed to stressful conditions. They constitute a large group of molecular chaperones, which have been classified into several families, including HSP27, HSP40, HSP60, HSP70, HSP90, and HSP110, based on their molecular weight and sequence homology ,6. Secretivities . One of  pathway , indicatS. japonicum, named heat shock protein 90 (Sjp90), has been identified in adult worms [S. japonicum, implying a central role in immunomodulation in the host–parasite relationship [Another abundant HSP derived from lt worms . Expresslt worms , probabllt worms . In additionship . Furthertionship . HoweverS. japonicum. In addition, we used real-time PCR to measure the transcription levels of Sjp40 and Sjp90α in different life cycle stages. Immunolocalization studies of Sjp40 and Sjp90α were also undertaken in the eggs and worms. Furthermore, the potential effects of Sjp40 and Sjp90α on inflammatory cells, including macrophages, neutrophils, eosinophils, and T cells subsets, were analyzed by flow cytometry. In this study, we cloned and characterized Sjp40 and Sjp90α from S. japonicum HSP40  comprises an open reading frame (ORF) of 1065 bp encoding 354 amino acids. Multiple alignments showed that Sjp40 shares 69% amino acid sequence identity with Schistosoma mansoni HSP40 (Smp_302290), 64% amino acid sequence identity with S. haematobium, and 31% amino acid identity with Homo sapiens. S. japonicum HSP90α  comprises an open reading frame (ORF) of 780 bp encoding 259 amino acids. Sjp90α shares 82% amino acid sequence identity with S. mansoni HSP90 (Smp_072330), 43% amino acid sequence identity with S. haematobium, and 61% amino acid identity with Homo sapiens. The sequence of The three-dimensional (3D) structure of the two proteins was predicted by homology modeling using the online SWISS-MODEL tool . For theS. mansoni, Homo sapiens, and Mus musculus. Using the Scan Prosite of ExPASy analysis tools (https://prosite.expasy.org), 23 motif hits (by six distinct patterns) were identified in Sjp40 , which has been validated as a standard reference gene in transcriptomic analysis of S. japonicum in a number of studies [S. japonicum  = 47.47, Least Significant Difference (LSD) post-hoc test: p < 0.0001)  = 124.1, all LSD: p < 0.0001)  a, wherea 0.0001) b.Immunolocalization of eggs trapped in infected mouse livers using either HRP labeling or immunofluorescence showed that native Sjp40 and Sjp90α were expressed inside immature eggs a, with bS. japonicum eggs, Western blot analysis was undertaken using mouse anti-rSjp40 or rSjp90α serum to probe egg secreted proteins (ESP) and soluble egg antigens (SEA) from purified eggs   = 4.705, LSD: p = 0.0136) (− CD11c+)  = 6.027, LSD: p = 0.0113) (+ CD11c−)  = 30.25, LSD: p < 0.0001) (− CD11c+)  = 6.027, LSD: p = 0.0166) (+CD11c−)  a,b, dend 0.0113) a,c, and  0.0001) a,d were  0.0166) a,c. Howe+CD11c−) e.+ Th cells play essential roles in the initiation and regulation of the hepatic immune response and pathology during schistosome infection [S. japonicum-infected mice with rSjp40 and rSjp90α resulted in an increase in the proportion of CD4+ cells (CD90.2+CD4+)  = 115.0, both LSD: p < 0.0001) (+CD4+ IFN-γ+)  = 3.551, LSD: p = 0.0405), Th2 cells (CD90.2+CD4+ IL-4+)  = 5.226, LSD: p = 0.0159), Th17 cells (CD90.2+CD4+ IL-17+)  = 6.623, LSD: p = 0.0031), but not Treg cells (CD90.2+CD4+Foxp3+), whereas rSjp90α predominantly induced the expression of Th17 cells  = 6.623, LSD: p = 0.0168), but not Th1/2 or Tregs  = 115.0, LSD: p < 0.0001), Th2  = 5.226, LSD: p = 0.0118), and Treg cells  = 4.373, LSD: p = 0.0171)  = 29.35, both LSD: p < 0.0001)  = 4.489, LSD: p = 0.0159) and CD90.2−CD4+IL-17+ cells  = 4.089, LSD: p = 0.0349)  = 3.551, LSD: p = 0.0272) and CD90.2−CD4+ IL-17+ cells  = 4.089, LSD: p = 0.0223) (CD4nfection . We foun 0.0001) a,b; thes+CD4+)  a–f. Of n4+ cells e–i, whic 0.0001) g. In add 0.0349) h,j, wher 0.0223) i,j.F = 7.918, LSD: p = 0.0118) and COL1A1 = 8.438, LSD: p = 0.0011), but in contrast, rSjp90α had no significant effect on the expression of these two components.We investigated the interaction between Sjp40 and Sjp90α with hepatic stellate cells (HSCs), which are the key cellular players in the development of hepatic fibrosis, by stimulating LX-2 cells with rSjp40 and rSjp90α. As noted in S. japonicum, expressed them in Escherichia coli (E. coli), and obtained purified soluble recombinant proteins to study their expression and their preliminary characterization. In addition, we report for the first time, an investigation on the roles of Sjp40 and Sjp90α on the regulation of hepatic immune cells. HSPs, such as HSP40, HSP60, HSP70, and HSP90, are known to be widely expressed by parasites and they play critical roles in the activation and regulation of host innate and antigen-specific adaptive immune responses, the study of which might potentially reveal new anti-parasite vaccine candidates ,35,36,37S. japonicum and its effect on mouse hepatic immune cells and hepatic stellate cells following a previous investigation of its diagnostic potential [S. japonicum worms, but no further characterization had been undertaken on this HSP. Accordingly, we isolated Sjp90α from S. japonicum and used the recombinant protein for more functional exploits. Our immunolocalization analysis showed Sjp90α was not only expressed throughout the tissues (parenchyma or vitelline cells) of adult worms but was also present in the worm tegument. Given the high homology evident between host HSP90 and Sjp90α, the tegumental location of Sjp90α in adult S. japonicum raises concerns whether the signaling we observed by immunolocalization is indeed specifically representing the location of Sjp90α itself rather than host HSp90. Regarding this point, during the immunolocalization procedure, goat serum (1% in blocking buffer) was used to block the worm sections, thereby removing the non-specific background before reacting with primary antibody. In addition, freshly perfused adult worms were washed with perfusion buffer (at least three times) before fixing and the host HSP90 attached to the surface of worms was likely removed. This was reinforced with our observations of negative control worms when normal mouse serum was used as primary antibody  , a type + T cell populations do not express CD90.2 (CD90.2−CD4+ cells), which is highly expressed in gut-associated Peyer’s patches [−CD4+ cell subsets response appeared to increase in hepatic immune cells after they had been in vitro stimulated by either rSjp40 or rSjp90α, suggesting that the growth of these cell subsets may be associated with the process of immune regulation during S. japonicum infection. In addition, we found that the expression of fibrogenic gene markers (α-SMA and COL1A1) was decreased in human HSC after being incubated with rSjp40 but not with rSjp90α. This suggests Sjp40 might play a pivotal role in the suppression of liver fibrosis, whereas Sjp90α appears to play no part in this process.It is well recognized that the dominant Th1 response is induced in mice at the early stage (at 3–5 weeks post-infection) of a schistosome infection. With the sexual maturation of adult worms in the host and a large number of eggs released by mature females (at 5–6 weeks post-infection), the host immune response shifts from Th1 to a strong Th2/Th17 response [ patches  and in t patches . Our resThe conduct and procedures involving animal experimentation were approved by the Animal Ethics Committee of QIMR Berghofer Medical Research Institute (project number 288 and ethics ID A0108-054). This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the USA National Institutes of Health.S. japonicum adult worms were collected by perfusion of ARC Swiss mice infected percutaneously with 60 cercariae of S. japonicum , shed from Oncomelania hupensis snails, which were transported to the QIMR Berghofer Medical Research Institute in Australia. Female Swiss mice were percutaneously infected with S. japonicum cercariae. Adult worms were collected from infected mice 6 weeks post infection. Mice were perfused with perfusion buffer [v/v) penicillin/streptomycin  overnight at 37 ℃. The digested eggs were then centrifuged at 500× g for 5 min, followed by washing with ice-cold PBS 3 times. Then the eggs were collected by passing through 250 μm and 150 μm cell strainers, separately, and then purified using a Percoll  gradient.n buffer  to removn buffer . Brieflyhttps://parasite.wormbase.org). The BioEdit software was used for multiple sequence alignments. The motif domains of Sjp40 and Sjp90α were analyzed using the ScanProsite of ExPASy analysis tools (https://prosite.expasy.org). Sjp40 and Sjp90α homologous sequences were analyzed by the neighbor-joining method for phylogenetic analysis and the tree was plotted using MEGA-X software [https://swissmodel.expasy.org) [http://biomine.cs.vcu.edu/servers/NsitePred/) [BLAST of Sjp40 (Sjp_0021240) and Sjp90α (Sjp_0044660) was performed in the Wormbase (software . The demasy.org) . ATP-bintePred/) .S. japonicum were prepared as described [g for 1 h, the supernatant (SEA) was collected. Residual endotoxin in the SEA extracts was determined using a Pierce LAL Chromogenic Endotoxin Quantitation Kit  according to the manufacturer’s instructions.Soluble egg antigen (SEA) of escribed  with somv/v) penicillin/streptomycin at 37 °C. Culture supernatant was collected twice with a three-day interval and replaced by 1 mL fresh media. The mixture of supernatants was then filtered through a 20 µm strainer and concentrated using a 3–5 KDa MW filter.For the preparation of excretory secretory protein (ESP) antigens from eggs, approximately 30,000 eggs/well were cultured in a 24-well plate with 1 mL RPMI1640 and 1% (E. coli BL21 (DE3) cells (Invitrogen) were transformed with the reconstructed plasmids for protein expression as described [E. coli lysates using Ni-NTA affinity chromatography . Given previous studies showing the 6 × His tag did not stimulate any immunogenic activity [E. coli) Standards kits  were used subsequently to confirm there was no Endotoxin contamination in either protein.A 1065 bp fragment of cDNA encoding Sjp40 and a 780 bp fragment of cDNA encoding Sjp90α were amplified by PCR and inserted into the pET28b vector . escribed . The expactivity ,53,54,55activity  and EndoSjp40 and Sjp90α antiserum were prepared in Swiss mice. Recombinant Sjp40 or Sjp90α were formulated with either Complete Freund’s Adjuvant  (primary injection) or Incomplete Freund’s Adjuvant  (Sigma-Aldrich) and the preparations were subcutaneously injected into the mice. Mouse bloods were collected 2 weeks after the last injection using cardiac puncture and sera obtained. Antibody titres were determined using an enzyme-linked immunosorbent assay (ELISA) against rSjp40  or rSjp90α  as described .® PVDF membranes . After blocking in Odyssey Blocking Buffer for 1 h at room temperature, membranes were incubated at 4 °C overnight with primary antibodies: mouse anti-rSjp40 or mouse anti-rSjp90α, respectively. After washing with PBS plus 0.1% Tween 20 with gentle shaking, the membranes were incubated at room temperature for 1 h with IRDye® secondary antibody  in Odyssey Blocking Buffer plus 0.1–0.2% Tween 20. After washing, the blots were developed using the Odyssey Imaging System.For Western blot analysis, equivalent amounts of the Sjp40 and Sjp90α recombinant proteins (~10 μg) or protein (~200 μg) from SEA or ESP were loaded into each lane for separation by SDS-PAGE and electrophoresed proteins were then transferred onto Immu-BlotS. japonicum or adult worms perfused from infected mice with perfusion buffer (0.85% (w/v) NaCl plus 1% (w/v) trisodium citrate) were fixed in 10% formalin, embedded in paraffin and sectioned at 3 µm. Following antigen retrieval by boiling in 0.01 M sodium citrate, pH 6, for 20 min in a water bath, endogenous peroxidase activity was blocked by incubation with 3% (v/v) H2O2 for 20 min at room temperature. Slides were washed three times with PBS and blocked with 5% bovine serum albumin and 1% (w/v) goat serum in tris buffered saline (TBS) for 30 min at room temperature. Tissue sections were then probed with 1:100 diluted mouse anti-rSjp40 or rSjp90α sera for 1 h at 37 °C . Slides were then washed three times with 1 × PBS, and HRP conjugated secondary antibody (Sigma) was added as secondary antibody at a dilution of 1:1000 and incubated for 10 min at room temperature. Sections were examined with an Aperio Scanscope XT scanner  and analyzed using ImageScope software .Liver tissue from mice infected with w/v) bovine serum albumin and 1% (w/v) goat serum in TBS for 60 min at 4 °C, the liver tissue or worm sections were stained with mouse anti-mouse Sjp40 or Sjp90α overnight at 4 °C as described [v/v) Tween 20 in TBS). Then the liver tissue or sections were incubated with Alexa-Fluor 647 goat anti-mouse IgG (1:500)  at 37 °C for 1 h. Diamidino-2-phenylindole (DAPI) gold (Invitrogen) was used to stain for nuclei and the samples were analyzed using a Zeiss 780 NLO laser .To confirm the integrity of the HRP labelling of Sjp40 and Sjp90α, we replaced the HRP conjugated secondary antibody with Alexa-Fluor 647 conjugated antibody for the immunolocalisation analysis. Briefly, the fixed liver tissue or adult worm sections were treated with Revealt A solution  at 120 °C for 5 min for antigen retrieval. After the non-specific binding sites were blocked with 1%  using RNeasy Mini Kits . First strand cDNA was synthesized using a Sensiscript Reverse Transcription for First strand cDNA synthesis Kit (Qiagen) and was subsequently used as template in qPCR to determine the expression levels of Sjp40 and Sjp90α in different life cycle stages and the α-SMA and COL1A1 expression levels in hepatic stellate cells. S. japonicum PSMD4 (26S proteasome non-ATPase regulatory subunit 4) [−ΔΔCt) [S. japonicum stage were normalized to the data obtained with female worms, based on their suitably expressed CT values. The sequences of primers used in this analysis are shown in Total RNA was extracted from human hepatic stellate or different life cycle stages of bunit 4)  or the hbunit 4)  was used [−ΔΔCt) . Then, mS. japonicum infection were cut into small pieces and digested in 20 mg Collagenase B at 37 ℃ for 45 min. The digested liver tissue was then mashed through a 100 µm filter, followed by centrifugation at 400 g for 6 min. The cell pellets were purified using Percoll gradients  by centrifuging at 690× g for 12 min at room temperature. After discarding the upper layers carefully, the cell pellets were collected and resuspended in red cell lysis buffer (Sigma-Aldrich) and then washed in 10 mL complete RPMI1640  containing 10% (v/v) FBS (Gibco) and 1% (v/v) penicillin/streptomycin (Gibco). Then, the single cell suspension was collected through the Pre-Separation Filters  for further study.Liver nonparenchymal cells (NPCs) were isolated following an established method with some modifications . Briefly6 of single cell suspensions were cultured in complete RPMI 1640 medium and stimulated with rSjp40, rSjp90α, or SEA, at 20 μg/mL for 44 h; then the cells were mixed with 25 ng/mL PMA (Sigma) and 1 μg/mL ionomycin (Sigma) in the presence of 0.66 μL/mL Golgistop  at 37 °C in 5% CO2 for 4 h. Cells were blocked with Fc receptor (BD Bioscience) and stained with the Live/Dead-Horizon stain  for 15 min, surface-stained with CD90.2 (Thy1.2)-PEcy7 and CD4-BV785 antibodies  for 30 min, then cells were fixed and permeabilized with BD Cytofix/Cytoperm buffer (BD Bioscience) as per the manufacturer’s protocol and intracellularly stained with IFN-γ-Percp/cy5.5, IL-4-PE-CF594, and IL-17A-AF647  for 30 min.For Th1/Th2/Th17 cells analysis, 2 × 106 of single cell suspensions were incubated with rSjp40 (20 μg/mL), rSjp90α (20 μg/mL), or SEA (20 μg/mL) for 48 h, followed by staining with the Live/Dead-Horizon stain  for 15 min, and then surface-stained with CD90.2 (Thy-1.2)-PEcy7 and CD4-BV785  for 30 min. Then, cells were then fixed and permeabilized with fixation/permeabilization buffers (BD Bioscience) for 50 min and then blocked with Fc receptor (BD Bioscience). Finally, cells were stained with PE-conjugated anti-Foxp3 antibodies  for 30 min.For Treg cells analysis, 2 × 106 of single cell suspensions were stimulated with rSjp40, rSjp90α, or SEA, at 20 μg/mL for 48 h and stained with the Live/Dead-Horizon stain (Thermo Fisher Scientific) for 15 min and then surface-stained with Gr-1-AF700, CD11c-APC/cy7, F4/80-BV421, and Siglecf-BV605  for 30 min. All the stained cells were collected using Fortessa 4A and FACS Diva software (BD Bioscience). Flow cytometry analysis was performed using FlowJo version 10 software .For other hepatic immune cells analysis, 2 × 105 of LX-2 cells/well were cultured in six-well plates containing DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin, at 37 ℃ in a humidified atmosphere containing 5% CO2. LX-2 cells were stimulated with PBS, rSjp40 (20 μg/mL) or rSjp90 (20 μg/mL) for 48 h, respectively; then the cells were collected by centrifugation at 500 g for 5 min. Total RNA was extracted from these cells and used as template to synthesize cDNA which was used for RT-PCR analysis to determine the expression level of fibrosis-related genes (α-SMA and COL1A1) as described above.A total of 5 × 10F values and p values were calculated by GraphPad software. p values < 0.05 were considered significant. The F distribution has two parameters: Degrees of freedom numerator (dfn) and degree of freedom denominator (dfd).All analyses were carried out with GraphPad software (Version 7.02). Data are shown as means ± SEM. The significance of the difference between two groups was determined using Student’s t test. Multiple comparisons were performed by one-way ANOVA, followed by LSD post-testing for comparisons between two groups. Dfn = the number of groups−1; dfd = the total number of subjects in the experiment-dfn.S. japonicum. Immunolocalisation of these proteins in eggs and in adult S. japonicum and their contribution in the development of hepatic immunopathology, potentially provides new insights into the disease of schistosomiasis. In addition, in contrast to Sjp40, Sjp90α being located on the tegument of adult S. japonicum may play an important role in helping the parasite adapt to the host immune microenvironment, as it switches from an immune-sensitive to an immune-tolerance state.To better understand the roles of HSPs in schistosomes, we isolated and characterized two HSPs, Sjp40 and Sjp90α, from


Bacillus velezensis GJ11.Molasses is a wildly used feedstock for fermentation, but it also poses a severe wastewater-disposal problem worldwide. Recently, the wastewater from yeast molasses fermentation is being processed into fulvic acid (FA) powder as a fertilizer for crops, but it consequently induces a problem of soil acidification after being directly applied into soil. In this study, the low-cost FA powder was bioconverted into a value-added product of γ-PGA by a glutamate-independent producer of 2+, Mn2+, Ca2+, and Fe3+ in the fermentation medium for producing γ-PGA. In the optimized medium with FA powder, the γ-PGA was produced at 42.55 g/L with a productivity of 1.15 g/(L·h), while only 2.87 g/L was produced in the medium without FA powder. Hydrolyzed γ-PGA could trigger induced systemic resistance (ISR), e.g., H2O2 accumulation and callose deposition, against the pathogen’s infection in plants. Further investigations found that the ISR triggered by γ-PGA hydrolysates was dependent on the ethylene (ET) signaling and nonexpressor of pathogenesis-related proteins 1 (NPR1).FA power could partially substitute the high-cost substrates such as sodium glutamate and citrate sodium for producing γ-PGA. With FA powder in the fermentation medium, the amount of sodium glutamate and citrate sodium used for producing γ-PGA were both decreased around one-third. Moreover, FA powder could completely substitute MgTo our knowledge, this is the first report to use the industry waste, FA powder, as a sustainable substrate for microbial synthesis of γ-PGA. This bioprocess can not only develop a new way to use FA powder as a cheap feedstock for producing γ-PGA, but also help to reduce pollution from the wastewater of yeast molasses fermentation. Due to the petroleum crisis, renewable sources, such as bioethanol, have been explored and used as alternative energy in recent years . MolasseIn China, the wastewater produced during molasses fermentation can be processed into a biological product, fulvic acid (FA) powder, by dry-spraying. This technology can produce approximately 200 thousands of tons of FA powder every year. Thereby, processing wastewater into FA powder is favorable for reducing the pollution from molasses fermentation. Moreover, the FA powder is rich in FA (> 45%) as well as other nutrients, such as N (> 3%), P (> 0.5%), K (> 12%), and amino acids (> 6%), so it can be used as a fertilizer for crops via promoting plant growth and microorganism activity, controlling plant diseases, and protecting plants against abiotic stresses –19. HoweBacillus is ubiquitously distributed in the natural environment. Many Bacillus species, such as B. subtilis, B. licheniformis, B. amyloliquefaciens, and B. velezensis, are generally used as hosts for producing fermentation products including poly-γ-glutamic acid (γ-PGA). γ-PGA is a natural anionic polymer of d/l-glutamic acids linked together via amide bonds between the α-amino group and the γ-carboxylic acid group, resulting in numerous properties, such as holding water, biodegradability, and non-toxicity . As shown in Table 3, sodium glutamate, and citrate sodium improved the γ-PGA production, while FA powder was negative for the γ-PGA production. According to R value obtained from the orthogonal tests, we found that γ-PGA production was successively affected by sodium glutamate, NaNO3, citrate sodium, FA powder, glucose, and KH2PO4. The optimal combination of medium was A3B3C2D1E3F3, corresponding to 80 g/L glucose, 20 g/L NaNO3, 0.7 g/L KH2PO4, 20 g/L FA powder, 20 g/L sodium glutamate, and 20 g/L citrate sodium, which resulted in the highest γ-PGA production of 35.54 g/L. On the other hand, glucose and FA powder were favorable, while NaNO3, sodium glutamate, citrate sodium, and KH2PO4 were unfavorable, for the bacterial growth. On the basis of R value, we found that the biomass was successively affected by glucose, NaNO3, citrate sodium, FA powder, sodium glutamate, and KH2PO4. The bacterial growth showed a negative correlation with the γ-PGA production of GJ11. Here, we mainly focused on the γ-PGA production, so the optimized medium formula was selected as 80 g/L glucose, 20 g/L NaNO3, 0.5 g/L KH2PO4, 20 g/L FA powder, 20 g/L sodium glutamate, and 20 g/L citrate sodium. Compared to the original fermentation medium, the cost of sodium glutamate and citrate sodium were both decreased around one-third due to an addition of FA powder in the medium. Moreover, FA powder could be a substitute for NH4Cl, MgSO4, MnSO4, CaCl2, and FeCl3 in the original fermentation medium.On the basis of above results, we further optimized the fermentation medium formula with orthogonal test. Glucose, citrate sodium, sodium glutamate, NaNOWith the optimized fermentation medium formula, we further detected the effect of pH on γ-PGA production. As shown in Fig. We also investigated the effect of liquid volume on γ-PGA production, and found the γ-PGA production was decreased when the liquid volume was increased in the flask Fig. b. The biWe cultured GJ11 in the optimized fermentation medium and conditions, and found glucose in the medium was not obviously consumed by GJ11 during the first 12 h, corresponding to the result that no γ-PGA was accumulated in this period. During 12—36 h, the biomass was dramatically increased, corresponding to a rapid decrease of residual glucose in the broth. Consistently, γ-PGA was rapidly biosynthesized in this period, with a maximum production of 41.47 g/L and a high productivity of 1.15 g/(L·h). During 36—48 h, the biomass, residual glucose, and γ-PGA production had no obvious change. During 48—96 h, the biomass was increased again, accompanied by a decrease of residual glucose in the broth. In this period, γ-PGA production was gradually decreased  infection  infection  accumulation and callose deposition , as listed in Table On the basis of above optimization, the orthogonal test was set up for further optimizing the fermentation medium. Glucose, citrate sodium, sodium glutamate, NaNOOn the basis of the orthogonal experiment, we further studied the effect of culture conditions on γ-PGA production. To study the impact of pH on γ-PGA production, the initial pH value of medium was set at 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. To study the impact of ventilation on γ-PGA production, several 250 mL flasks containing 25, 50, 75, 100, and 125 mL medium, respectively, were prepared for culturing GJ11. The amounts of inoculation were set at 1%, 3%, 5%, 7%, and 10% (v/v), respectively, for culturing GJ11 to produce γ-PGA. All other factors were held constantly.600 value of broth. The γ-PGA production was determined by the cetyltrimethylammonium bromide (CTAB) turbidimetry method, and the SDS-PAGE analysis with methylene blue staining [4 oxidation method. After dissolution, the content of fulvic acid was valued by titration with ferrous ammonium sulfate and N-phenyl anthranilic acid, which could indicate the end point [The biomass of GJ11 was assayed by detecting the ODstaining , 32, 34.staining . The pH nd point , 36. Thend point .Pseudomonas syringae pv. tomato DC3000  was used for infecting tobacco plants by spraying the leaves evenly. The leaves were collected 3 days after the infection, and then sterilized and homogenized for spreading plates. After incubation at 37 °C for 48 h, the bacterial colonies were counted for calculating the Pst DC3000 content per gram of fresh leaf (cfu g−1) [γ-PGA was recovered from the GJ11 broth . 20 μL ocfu g−1) .Pst DC3000 infection as above.The γ-PGA solution (5 g/L) was adjusted to pH 2.0, and then incubated at 80 °C for 0, 1, 2, 3, 4, 5, 6, 7, and 8 h, respectively. After digestion, the pH value of solution was adjusted to 7.0 with 6 M NaOH, and then, the samples were collected for analysis of the hydrolysates using SDS-PAGE . ThereafA. thaliana seedlings , including Col-0, NahG, npr1, jar1-1, and ein2, were irrigated with 5 mL of the γ-PGA hydrolysates (5 g/L of γ-PGA was hydrolyzed at pH 2.0 and 80 °C for 5 h), and then infected with Pst DC3000 by spraying the leaves as above. After infection for 12 and 24 h, the leaves were collected and stained with DAB to detect the accumulation of H2O2, a kind of reactive oxygen species (ROS), in plants [A. thaliana were fixed with 1% (v/v) glutaraldehyde solution overnight, treated with ethanol at 90 C for 15 min, and stained with 0.1% (w/v) aniline blue for 12 h, and then observed by fluorescence microscope after being extensively washed by 50% (v/v) ethanol solution.Different lines of t test.All experiments were repeated in triplicates. The data between two groups were compared using Student's Additional file 1: Figure S1. Compatibility of GJ11 to FA power. a Influence of FA power on the growth of GJ11. b Influence of sterilization on pH of the medium with different concentrations of FA power with a natural pH. c Influence of sterilization on pH of the medium with different concentrations of FA power with an original pH of 7.0. d Effect of FA power on cell biomass and γ-PGA production.