Chlorisondamine (CSD) has been used to assess the neurogenic contribution to blood pressure (BP) and vasomotor sympathetic tone in animal models. It is assumed that the reduction in BP following CSD administration is associated to decreases in cardiac output (CO) and peripheral resistance, reflecting cardiac and vasomotor sympathetic tone, respectively. Surprisingly, this has not been characterized experimentally in mice, despite the extensive use of this animal model in cardiovascular research. We hypothesize that a specific dose of CSD can selectively block the sympathetic vasomotor tone. To test this hypothesis, we evaluated the effects of different doses of CSD  on BP and heart rate (HR) using telemetry, and on CO using echocardiography. BP and HR in normotensive C57Bl/6J mice reduced to a similar extent by all CSD doses tested (1–6 mg/kg). CSD at 6 mg/kg also reduced CO without affecting left ventricular stroke volume or fractional shortening. On the other hand, lower doses of CSD (1 and 2 mg/kg) produced significantly larger BP and HR reductions in DOCA‐salt–induced hypertensive mice, indicating a greater neurogenic BP response. In addition, all doses of CSD reduced CO in hypertensive mice. Our data suggest that the BP response to CSD in mice likely reflects reduced CO and vasomotor sympathetic tone. We conclude that CSD can be used to assess the neurogenic contribution to BP in mice but may not be appropriate for specifically estimating vasomotor sympathetic tone. Ganglionic blocker, chlorisondamine, can be used to assess the neurogenic contribution to BP in mice, but may not be appropriate to specifically estimate the vasomotor sympathetic tone. All mice were maintained in the animal facility at the University of Nevada, Reno, under a 12‐h light‐dark cycle and room temperature of 21–23°C with ad libitum access to a standard chow diet  and water. The effects of different doses of CSD  on BP and cardiac function were tested in normotensive mice and DOCA‐salt–induced hypertensive mice, the latter of which were produced by subcutaneously implanting WT mice with a 50 mg DOCA pellet . CSD solutions for in vivo studies were prepared in sterile 0.9% saline and administered by intraperitoneal (i.p.) injection on different days, allowing at least 48 hours for recovery; 0.9% saline was used as a vehicle. Mice in both normotensive and hypertensive groups were provided 2.22, flushed at 1 L/min for 2 min. Anesthesia was then maintained using 0.75–1.5% isoflurane. After the neck was shaved and sterilized with alcohol swabs, an incision (~1 cm) was made to separate the oblique and tracheal muscles and expose the left carotid artery. The catheter of a radio telemetry transmitter  was surgically implanted into the left carotid artery and secured. The body of the radio transmitter was embedded in a subcutaneous pocket under the right arm. Mice were allowed to recover for 14 days  were performed in both normotensive and DOCA‐salt hypertensive mice at baseline and 30 min after i.p. injection of CSD. Anesthesia was induced with 5% isoflurane (1 L/min) and maintained with constant 1.5% isoflurane (1 L/min). ECG electrodes were placed in a standard limb configuration for monitoring HR. Ultrasound 2D images of parasternal long and short axes of the left ventricle (LV) at the level of papillary muscles were obtained using a 40 MHz linear transducer. M‐mode echocardiographic images were analyzed using Vevo Lab software 3.1.1 . Measurements of CO, LV stroke volume, ejection fraction, LV end diastolic volume (LVEDV), and fractional shortening were acquired, and measurements from long and short axes (three different cardiac cycles) of the LV for each variable were averaged.2.4After 21 days of sham or DOCA‐salt treatment, mice used in the echocardiography study were euthanized by cervical dislocation. Body weight (g), heart and left kidney weight (mg), and the length of the right tibia (mm) were measured. Cardiac and renal hypertrophy were then determined by normalizing heart and kidney weights to body weight and tibia length.2.5F‐test) and Gaussian distribution (Shapiro–Wilk test). Parametric Student's t‐tests or nonparametric Mann–Whitney tests were used for the statistical comparisons. No data were removed in this study. A p‐value ≤0.05 was considered statistically significant.Data are presented as means ± standard errors (SE) and were compared using Prism 9.0.0 software (GraphPad). For the comparison of BP, HR, and cardiac function and account for possible interactions between the independent variables condition (normotensive or hypertensive) and treatment with CSD , data were compared with two‐way ANOVA followed by the Fisher's LSD post‐hoc test. Data were checked if the data pass the assumptions to perform statistical comparisons using this test. Importantly, outliers were checked using the ROUT method and data were tested for variance homogeneity (Spearman's test) and Gaussian distribution (Kolmogorov–Smirnov test). For anthropometric measurements, data were assessed for the presence of outliers (ROUT method) and tested for variance homogeneity  using a telemetric recording system. We found that all doses of CSD significantly reduced BP, decreasing mean arterial (ΔMAP) by 16.7 ± 3.3, 25.5 ± 3.3, 31.1 ± 3.4, and 31.0 ± 7.0 mmHg at 1, 2, 3, and 6 mg/kg, respectively, compared with vehicle , and producing corresponding reductions in HR of 152.7 ± 39.6 beats/min and 197.5 ± 77.5 beats/min versus a ΔHR of +46.4 ± 12.2 beats/min in vehicle‐injected (i.p.) mice  and kidney weight (KW) to body weight (BW) or tibia length (TL). As expected, DOCA‐salt hypertensive mice developed cardiac Figure  and rena4Autonomic dysfunction and sympathetic hyperactivity have been shown to play important roles in the development and maintenance of hypertension in both humans  reduced HR but not CO in normotensive mice, whereas the lower doses of CSD significantly reduced both HR and CO in hypertensive mice. Collectively, these results suggest that hypertensive mice respond more strongly or are more sensitive to ganglionic blockade—an indication of elevated sympathetic tone (Basting & Lazartigues, A limitation of this study is that BP and HR responses to CSD were assessed in awake freely moving mice using a telemetric system, whereas the CO response to CSD was evaluated in anesthetized mice using echocardiography. Currently, there is no approach available for evaluating CO in conscious freely moving mice. However, although isoflurane has been reported to alter autonomic function and BP (Constantinides et al., In summary, we report here that the ganglionic blocker CSD decreases BP and HR in association with a reduction in cardiac output, suggesting that the BP response to CSD is a combination of blockade of cardiac and vasomotor sympathetic tone in both normotensive and hypertensive mice. We conclude that CSD is appropriate for use in assessing the overall neurogenic contribution to BP in mice but may not be sufficiently accurate to assess vasomotor sympathetic tone. We propose that a ganglionic blocker that can reduce BP without affecting heart rate would be a better reagent should sympathetic vasomotor tone be the interest of investigation.The authors declare that there are no competing interests associated with the manuscript.L.A.C.S. and Y.F.E. conceived and designed the research; L.A.C.S. and S.G.C. performed experiments; L.A.C.S., S.G.C., C.J.W., P.T., and Y.F.E. analyzed data; L.A.C.S., S.G.C., C.J.W., P.T., and Y.F.E. interpreted experimental results; L.A.C.S. and Y.F.E. prepared figures; L.A.C.S. and Y.F.E. drafted the manuscript; L.A.C.S., C.J.W., W.L., F.T., L.A.C.S., S.G.C., C.J.W., P.T., and Y.F.E. edited and revised the manuscript; and L.A.C.S., S.G.C., C.J.W., P.T., and Y.F.E. approved the final version of the manuscript.


Ophiocordyceps langbianensis was collected from Lang Biang Biosphere Reserve, located in Lam Dong Province, Vietnam. It is characterized as a species of Ophiocordyceps  having the unique characteristics of a cylindrical fertile part and several branched apical appendices. Each ascospore develops as two swollen, constricted part-spores. A phylogenetic analysis of multiple genes, including nrLSU, nrSSU, Rpb1, ITS and Tef, supported its systematic position in the genus of Ophiocordyceps; it is related to O. brunneipunctata. Based on morphological and phylogenetic analyses, O. langbianensis was confirmed as a new species from Vietnam.An entomopathogenic fungus newly named  Ophiocordyceps, first established by Petch in 1931, belongs to the family Ophiocordycipitaceae, order Hypocreales, comprising approximately 250 species2. Originally, Ophiocordyceps was classified as a subgenus of Cordyceps by Kobayasi  and Mains (1958)5. In 2007, Sung et al. established a new called family Ophiocordycipitaceae, comprising Ophiocordyceps, based on morphological and phylogenetic analyses7. The distinction of the genus Ophiocordyceps from Cordyceps was done due to the darkly pigmented stromata of Ophiocordyceps, which are pliant, wiry or fibrous and tough in texture, compared to the brightly pigmented stromata of Cordyceps7. Species of Ophiocordyceps are entomopathogenic on a wide range of insects. The hosts of species of Ophiocordyceps are the larvas of Coleoptera and Lepidoptera as well as the adults of Araneae, Diptera, Hemiptera, Hymenoptera, Odonata and Orthoptera7. Although Ophiocordyceps has worldwide distribution, the tropics and subtropics are where the highest numbers of the species are recorded. Moreover, it is considered that there is an underestimation of the number of Ophicordyceps species.The genus Ophiocordyceps that parasitizes the larva of Coleoptera. We present a morphological description and phylogenetic analysis based on the phylogenetic construction of nuclear large ribosomal subunit (nrLSU), nuclear small ribosomal subunit (nrSSU) and RNA Polymerase II Subunit B1(rpb1) of species of Ophiocordyceps, including this new species.Vietnam is located in a tropical region with terrestrial ecosystems. The forests feature a rich biodiversity of both flora and fauna due to the tropical monsoon climate with high temperature and rainfall. This is a favorable environment for the development of entomopathogenic fungi. Lang Biang Biosphere Reserve is located in Lam Dong Province and comprises a vast primitive jungle with the Lang Bian Mountain at its core, one of Vietnam’s four biodiversity centers. During our expedition to discover the diversity of entomopathogenic fungi, we collected the sample DL0017. In this study, we introduce this specimen as a new species of The specimen, DL0017, used for this study was collected from Lang Biang Biosphere Reserve  in 9th August, 2016. The specimen, including the host, was extracted carefully, noted, and photographed in the field using a digital camera. The specimen was immediately wrapped in wax paper, placed in a collection bag, and taken to the laboratory.According to the identification of conidia, phialides and colony coloration, the isolate cultures were grown on YMG media, composed of 4 g/l yeast extract , 10 g/l malt extract , 4 g/l glucose , and incubated at 20 °C for a period of 20 days with PDA media .For fruit body induction, cultures were grown on millet substrate (millet/silkworm pupae powder = 20:1 (w/w)) and brown rice substrate (brown rice/silkworm pupae powder = 20:1 (w/w)) at 20 °C under 12 h light and 12 h darkness with relative humidity of over 90%.7. The macroscopic characteristics of the fresh fruit body were carefully observed, including the stipe, stroma, etc. Moreover, the color was noted according to Kornerup and Wanscher8. Additionally, the host insect was identified based on morphological characteristics, such as mandibulate mouthparts, antennae, shape of head and thorax. For the micro-morphological analysis, one or two perithecia were removed from the stroma and placed on a microscope slide in lactophenol-cotton blue to measure the sizes and shapes of the perithecia, asci and ascospores. Finally, the nomenclatural novelty and descriptions were deposited in MycoBank.Morphological observations were carried out and recorded according to the guidelines of Kobayasi and Sung et al.11. The fruiting body was incubated in a lysis buffer  at 65 °C overnight. The supernatant was collected by centrifugation, and a volume of 700 μL of phenol/chloroform/isoamyl alcohol (25:24:1) was supplemented and centrifuged. The supernatant was collected and precipitated with absolute isopropanol. Finally, the isolated genomic DNA was stored in Tris–EDTA buffer at − 20 °C for further studies.Genomic DNA was isolated by using the phenol/chloroform method (pH = 8)nrLSU, nrSSU, rpb1, ITS and Tef regions are shown in Table The primer pairs used to amplify nrLSU, nrSSU, rpb1, ITS and tef sequences were established by sequences downloaded from Genbank (NCBI) and based on the previous data published by Sung et al.7. The nrLSU, nrSSU, rpb1, ITS and tef were noted with accession number, name of taxon and locality. The amplified DNA sequences were proofread to remove ambiguous signals at both ends by different software, including Seaview 4.2.12 and Chromas Lite 2.1.1. The phylogenetic tree was constructed based on neighbor-joining (NJ), maximum parsimony (MP), and maximum likelihood (ML), using Molecular Evolutionary Genetics Analysis (MEGA) version 5. Additionally, the best evolution model was predicted using jModelTest.The data set of Ophiocordyceps langbianensis T. D. Lao, T. A. H. Le & N. B. Truong, sp. nov.Mycobank MB836716 Figs. Coleoptera in moist soil surrounded by dried leaves. Truong B.N. DL0017 .VIETNAM. Lam Dong Province, Lang Bian Biosphere Reserve, Lang Bian mountain: N 12°02′19.0″, E108°26′04.7″; elevation 1680 m; humidity: over 85%; temperature: day 20 °C–22 °C, night: 14 °C–16 °C; collected between 9h00–15h00 of the day on 9 August, 2016, from the larva of a beetle of Vietnam, only known from Lang Bian Mountain.“Langbianensis” refers to Lang Bian Mountain, Lam Dong province, Vietnam.Coleoptera. Larva: 28–32 mm long, hard-body, shiny, smooth, dark brownish yellow; body composed of 13 segments with black edges; larva with three pairs of jointed legs attached to thorax.On the larva of a beetle of Acer laurinum (Aceraceae), Baccaurea harmandii (Euphorbiaceae), Castanopsis chinensis (Fagaceae), Eriobotrya poilanei (Rosaceae), Jasminum longisepalum (Oleaceae), Phoebe petelotii (Lauraceae) and Tetrastigma lanceolarium (Vitaceae).Individuals of associated species appeared at the type locality, including pioneer species such as Stroma arising from the head of the host larva, solitary, rarely branched, 40–100 mm long; host covered with thin, tough layer of mycelium. Stipe filiform, cylindrical, 30–67 mm × 0.7–1.0 mm, pale yellow. Fertile portion, cylindrical, 7.0–14.0 mm × 1.5–2.0 mm, brownish yellow with dark brown ostiolar dots of perithecia. Apical appendices, pale yellow, 2–10 primary or secondary branches, 4.0–10.0 × 0.5 mm. Perithecia immersed, ovate or pyriform, 260–400 μm × 100–190 µm. Asci, cylindrical, 200–250 μm × 5.0–6.0 μm, with thickened cap. Ascospores filiform, multiseptate, articulated in long-chain after discharging, sometimes breaking into 1-celled part spores, cylindrical, swollen, two waist-like constrictions, 5–7.5 μm × 1.3–2 µm.Germination of ascospores after 48 h on PDA; white colony, slow growing on YMG and PDA media, 25.00 mm and 24.58 mm after 40 days (respectively); septate hyphae, branched, chlamydospores developing in intercalary or terminal cells. Aerial hyphae with divergent phialides; elliptical conidia in chains after release from phialide. Stromata without fertile part forming on cereal substrates. Minor differences in morphological characteristics of stromata developing from different substrates. Stromata, white, branched when developing on millet substrate; brownish yellow, solitary, rarely branched, when developing on brown rice substrate.nrLSU, nrSSU, rpb1, ITS and tef, were successfully amplified with corresponding primers  , with accession numbers of DQ518756, DQ522542, DQ522369, GU723777 and DQ522324. Sequences were aligned and edited using the MEGA 5.2. Gaps were excluded from the phylogenetic analysis. The dataset of representative taxa and DL0017 target gene sequence consisted of 451 bp for nrLSU, 674 bp for nrSSU, 392 bp for Rpb1, 158 bp for ITS and 790 bp for tef. The evolution model that was most fixed with nrLSU, nrSSU, Rpb1, ITS and tef were TN93 + G, K2 + G + I, T92 + G + I, K2 + G, and TN93 + G + I respectively. The phylogenetic trees were generated with Neighbor Joining (NJ), Maximum Parsimony (MP), and Maximum Likelihood (ML) methods with replication of 1000. Based on the NJ, MP, and ML phylogenetic trees, individual nrLSU, nrSSU, Rpb1, ITS, and tef of DL0017 clustered together with Ophiocordyceps brunneipunctata within separate branches with credible bootstrap (≥ 50%), suggesting that these species are related  and formed a separate, monophyletic branch. Within this monophyletic branch, DL0017 and O. brunneipunctata clustered together closely, suggesting that these species were truly associated , tough, fibrous, pliant, and rarely fleshy. Additionally, asci are usually cylindrical with thickened ascus apex. Ascospores are usually cylindrical, multiseptate, and disarticulate into part-spores or non-disarticulating7. Our specimen shares these common characteristics.Morphological analysis indicated that DL0017, named Ophiocordyceps brunneipunctata in Ophiocordycipitaceae12. However, the morphologies of these two species are different in many characteristics, including color, size of stroma, stipe, and dots in the fertile portion. The apical appendix of O. brunneipunctata lacks branching, while O. langbianensis has 2–10 branches. Additionally, the ascospores of O. brunneipunctata break into part-spores, while the ascospores of O. langbianensis stick together to form a multiseptate chain, separating into unicellular part-spores under a strong interaction force. Multiple gene sequences of the related Ophiocordyceps species were used in the phylogenetic analysis. A comparison was done among the species listed in Table Cordyceps furcicaodata have a branch-forming apical appendix. In the comparison between Cordyceps furcicaodata and O. langbianensis, Cordyceps furcicaodata was found to be smaller than O. langbianensis. The stroma of Cordyceps furcicaodata arose from the middle of the host larva, while that of O. langbianensis arose from one end of the insect larva13. As mentioned above, ascospores of O. langbianensis stick together to form a multiseptate chain, which could only be ruptured into unicellular part-spores by a strong force, while ascospores of Cordyceps furcicaodata often break into unicellular part-spores.Based on the phylogenetic analysis, the specimen DL0017 clustered with O. langbianensis consists of long and divergent phialides, elliptical conidia usually in chains considered paecilomyces-like or purpureocillium-like15. Conversely, O. bruneipunctata produced a mononematous hirsutella-like asexual morph from colonies after 3–4 weeks.The asexual morph of nrLSU, nrSSU, rpb1, ITS, and Tef, to delimit sample DL0017, collected from Lang Biang Biosphere Reserve located in Lam Dong Province, Vietnam, as a new species named Ophiocordyceps langbianensis, belonging to the genus of Ophiocordyceps .We successfully applied morphological characterization in combination with phylogenetic analysis of multiple genes, including


In Fig. 4A, there was a duplicated image in the bright-field and GFP entries for the CagA mutant strain at 1 dpi. There are no corresponding changes to the text or figure legend.Volume 9, no. 2, e00649-18, 2018,


Hitherto, however, little has been learned about how to manipulate the spin degree of freedom in thermoelectrics. Here, we report the enhanced thermoelectric performance of GeMnTe2 by controlling the spin's thermodynamic entropy. The anomalously large thermopower of GeMnTe2 is demonstrated to originate from the disordering of spin orientation under finite temperature. Based on the careful analysis of Heisenberg model, it is indicated that the spin-system entropy can be tuned by modifying the hybridization between Te-p and Mn-d orbitals. As a consequent strategy, Se doping enlarges the thermopower effectively, while neither carrier concentration nor band gap is affected. The measurement of magnetic susceptibility provides a solid evidence for the inherent relationship between the spin's thermodynamic entropy and thermopower. By further introducing Bi doing, the maximum ZT in Ge0.94Bi0.06MnTe1.94Se0.06 reaches 1.4 at 840 K, which is 45% higher than the previous report of Bi-doped GeMnTe2. This work reveals the high thermoelectric performance of GeMnTe2 and also provides an insightful understanding of the spin degree of freedom in thermoelectrics.Na ZT = α2σT/κe + κph, where α, σ, κe, κph, and T are the Seebeck coefficient, electrical conductivity, electrical thermal conductivity, lattice thermal conductivity, and absolute temperature, respectively [ZT is always a big challenge.Thermoelectrics offers a unique solution for the direct conversion from waste heat into versatile electricity –3. The cectively –14. Sinc2Si, band engineering has been proved to be a very efficient strategy for improving ZT, since it partly decouples the electrical conductivity and Seebeck coefficient [xCoO2 exhibits an anomalous Seebeck coefficient [xCoO2. It is conceivable that a system with more adjustable degrees of freedom has the larger potential to be improved. “Thermoelectrics needs a spin,” it was addressed by J. He and T. Tritt recently [In principle, a physical phenomenon in a solid can be understood on the basis of the four fundamental degrees of freedom, i.e., charge, lattice, spin, and orbital –18. Therfficient –27. The fficient , 29, whifficient . This pufficient . Owing trecently .xCoO2 has a moderate maximum ZT around 1 at 800 K [ZT in some traditional semiconductors, such as PbTe, GeTe, and SnSe, has been improved above 2 [ZT in NaxCoO2 may be owing to two reasons. First, the localized d-orbital of Co strongly reduces the carrier mobility, although it is also the origin of spin entropy. Second, the microscopic understanding of spin entropy is not insightful enough to make the further optimization. The original spin entropy explanation for NaxCoO2 is an approximation with high-temperature limit (T → +∞), i.e., the spin entropy takes its maximum, and the deduced Seebeck coefficient is temperature-independent [ZT of Li-doped MnTe reaches 1 at 900 K [Despite the significantly enhanced Seebeck coefficient, Naat 800 K , whereas above 2 –35. The ependent . For thiependent –38. The at 900 K .PF = α2σ [2 at room temperature [2 was found to have a very small band gap less than 0.05 eV, while its Seebeck coefficient reaches 200 μV/K at 700-800 K. According to the Goldsmid-Sharp relationship, Eg = 2eSmaxT(Smax) [d transition element Mn in GeMnTe2 induces a ferromagnetic phase transition below room-temperature [xCoO2 and MnTe, it is reasonable to presume that the enhancement of Seebeck coefficient is related to the spin degree of freedom. Previous studies on GeMnTe2 have treated the system as a traditional semiconductor. Bi doping and Pb doping have been applied to optimize the performance, while the peak ZT of 0.9 and 1.4 were achieved, respectively [2 is not only theoretically meaningful, and it may also promote the realistic application of this material.The IV-VI compound GeTe is a remarkable thermoelectric material characterized by high electrical conductivity and high power factor PF = α2σ , 39–45. PF = α2σ . When 50perature , 48. GeMxT(Smax) , the estectively , 48. To 2 are investigated by manipulating the spin degree of freedom. Based on the analysis of Heisenberg model, two specific strategies are designed to enhance the spin's thermodynamic entropy. Se doping and Bi doping are introduced individually to reduce the spin-spin interaction by fully filling the p-orbital of anions. The Seebeck coefficient is found to be effectively enhanced, while the measurements of magnetic susceptibility prove that the enhancements are inherently related to the variation of entropy. In Ge0.94Bi0.06Te1.94Se0.06, a high peak ZT of 1.4 at 843 K is achieved. Compared with the previous report on singly Bi-doping, the maximum ZT is increased by 45%. This work shows GeMnTe2 is a promising thermoelectric material and also provides an insightful understanding on the spin degree of freedom in thermoelectrics.In this work, the thermoelectric properties of GeMnTeμ is given by μ = −T(∂S/∂N)U,V, where entropy S = S is a function of energy U, volume V, and electron number N. Then, Seebeck coefficient α can be written asSeebeck coefficient and entropy are related via the electron chemical potential. The chemical potential of electron α = −(1/e)(∂S/∂N). The entropy S = S includes the contributions from both charge and spin degrees of freedom. If spin degree of freedom is frozen, its contribution to entropy is zero, and the given Seebeck coefficient should obey the Goldsmid-Sharp relationship [2, the estimated band gap from Goldsmid-Sharp relationship is one order higher than the measured one. This indicates that the spin degree of freedom dominates the Seebeck coefficient. Thus, the Seebeck coefficient could be further enhanced by simply increasing the spin entropy.Within high temperature limit, entropy is temperature independent, and equation becomes Heikes formula tionship . In GeMnxCoO2, the large entropy is from the high degeneracy of d-electron local states, owing to the competition between crystal field splitting and exchange splitting. It was argued that the coexistence of Co3+ and Co4+ ions plays an important role [2, the 3d element Mn only has the +2 valence state, and therefore, the degeneracy of local states should be much lower than that in NaxCoO2. Another kind of spin entropy may be related to the spin orientation. As shown in S = S is difficult, especially for a spin-correlated system. But we still can get some useful information from qualitative analysis. The interaction of a spin system can be described by Heisenberg model:J is the exchange integralIn Naant role . In GeMnJ. As for a given system, a larger ∣J∣ will lead to a higher transition temperature. The ordering of spin, i.e., entropy, depends on the competition between temperature T and exchange integral J  samples are homogenous solid solutions.GeMnTe2-xSex are displayed in 1.94Se0.06 shows a high Seebeck coefficient about 190 μV/K, compared the value of 120 μV/K for the undoped GeMnTe2. The thermoelectric properties of GeMnTe2 has been reported by Zhou et al., and they used similar synthesis method [1.94Se0.06 is found to have the largest power factor of 15 μwcm−1 K−2 at 840 K.The measured Seebeck coefficients for GeMnTes method . Compare2-xSex at room temperature. The measured carrier concentrations are all around 5 × 1021 cm−3. It is not surprising that the substitution of Se for Te does not change the carrier concentration. x = 0 and x = 0.06 show the very small band gap less than 0.05 eV. This result is consistent with the previous report [2, but failed to produce a band gap. It is not uncommon that density functional theory fails to describe the electron-correlated system. Then, the enhancement of Seebeck coefficient in Se-doped samples is neither from the variation of band gap nor from the decreasing carrier concentration.s report . We applJ. GeMnTe2 was found to have a ferromagnetic phase transition below room temperature. If J is reduced by Se doping, the magnetic transition temperature TC should decrease. To verify this purposive design, we measured the low temperature magnetic susceptibility for x = 0 and x = 0.06 samples. Using the measured magnetic susceptibility, the derivative of magnetization dM/dT can be calculated. The Curie temperature TC can be roughly determined by the minimum of dM/dT. In dM/dT is plotted as a function of temperature. As may be seen, the estimated transition temperature TC is decreased from 76 K for x = 0 to 71 K for x = 0.06. The enhanced Seebeck coefficient and decreased Curie temperature by Se doping are consistent with our experiment design, indicating the crucial role of spin's thermodynamic entropy in GeMnTe2.As we discussed, Se doping was designed to increase the spin's thermodynamic entropy by reducing the exchange integral 2 was treated as a traditional semiconductor, and Bi doping was introduced to adjust the carrier concentration [μV/K for GeMnTe2 to 140 μV/K for Ge0.9Bi0.1MnTe2, while the hole concentration is decreased from 3.6 × 1021 cm−3 to 1.0 × 1021 cm−3. Considering the high level of carrier concentration, the measured Seebeck coefficient is unusually large. For comparison, the Seebeck coefficient of GeTe is about 100 μV/K at room temperature with the carrier concentration of 1.5 × 1020 cm−3 [1.94Se0.06 sample.In the study of Zhou et al., GeMnTentration . At room020 cm−3 . The lar1-yBiyMnTe1.94Se0.06. It may be seen that the Seebeck coefficient shows a monotonous increase as Bi doping increases from y = 0 to y = 0.1. At room temperature, Seebeck coefficient is significantly enhanced from 85 μV/K for y = 0 to 175 μV/K for y = 0.1. In 0.9Bi0.1MnTe2 from Zhou et al. are also presented [1-yBiyMnTe1.94Se0.06 are presented. The measured carrier concentration continuously decreases from 5.3 × 1021 cm−3 for y = 0 to 1.2 × 1021 cm−3 for y = 0.1. The carrier concentration of Ge0.9Bi0.1MnTe1.94Se0.06 is very closed to the reported value of Ge0.9Bi0.1MnTe2 [resented . Compare0.1MnTe2 . This is2 has a ferromagnetic phase at low temperature [2, GeMnTe1.94Se0.06, and Ge0.9Bi0.1MnTe1.94Se0.06. The reciprocals of magnetic susceptibility for all the samples show the good linear dependence on temperature within a large temperature range. 2 has the largest magnetic susceptibility and the smallest Seebeck coefficient, GeMnTe1.94Se0.06 has the intermediate magnetic susceptibility and the intermediate Seebeck coefficient, while Ge0.9Bi0.1MnTe1.94Se0.06 has the smallest magnetic susceptibility and the largest Seebeck coefficient. Furthermore, the differences between magnetic susceptibility is found to be in proportion to the difference between Seebeck coefficient. It is noticeable that the variation of Seebeck coefficient between the three samples cannot be explained by Mott's expressions. The Goldsmid-sharp relationship gives the limit of the maximum of Seebeck coefficient. As we discussed, the measured small band gap indicates that the Goldsmid-sharp relationship fails to describe the thermopower of GeMnTe2. According to our discussions, Bi doping could increase the spin's thermodynamic entropy by reducing the carrier concentration. It is difficult to measure the entropy directly. The ordering of spin orientation, however, can be characterized by the magnetic susceptibility under weak field. GeMnTeperature . If the μV/K at 170 K. 1-yBiyMnTe1.94Se0.06, respectively. The lattice thermal conductivity is determined by relationship of κL = κtot − κe, where κtot is the total thermal conductivity and κe is the electrical thermal conductivity. The Wiedemann-Franz relation κe = LσT is used to calculate the electrical thermal conductivity, where L, σ, and T are the Lorenz number, the electrical conductivity, and the absolute temperature, respectively. The Lorenz number is calculated based on the SPB model. As may be seen, the total thermal conductivity decreases as Bi doping increased up to y = 0.06. At y = 0.08, the total thermal conductivity is close to the one of y = 0.06. The lattice thermal conductivity of Ge1-yBiyMnTe1.94Se0.06 shows similar tendency to the total thermal conductivity. At y = 0.06, the lattice thermal conductivity reaches a minimum 0.41 at 840 K. At y = 0.08, the lattice thermal conductivity shows an increase relative to y = 0.06. Such nonmonotonic thermal conductivity in solid solutions is not uncommon.Figures 1-yBiyMnTe1.94Se0.06. The maximum PF appears at the y = 0 sample. With Bi doping, the peak of power factor is reduced from 15 μWcm−1 K−2 at y = 0.0 to 10 μWcm−1 K−2 at y = 0.10. The reduction of PF by Bi doping is mainly due to the decrease of electrical conductivity  and MnGe1-yBiyTe1.94Se0.06 (y = 0 − 0.10) were prepared by vacuum melting combined with HP process. High purity bulk germanium , manganese , bismuth , tellurium , and selenium  were weighted according to the stoichiometric proportions to obtain the compounds. The raw materials were sealed into a vacuum quartz tube, melted and swung at 1223 K for 1 h, quenched in the cold water, and then annealed at 950 K for 72 h. The obtained ingots were crushed and hand ground into fine powders. The powders were vacuum hot pressed in a ϕ12.7 mm graphite mold at 900 K for 30 min under the uniaxial stress of 50 MPa to obtained dense bulk samples.The compounds with nominal compositions of GeMnTeα radiation and scanning electron microscope  equipped with an energy dispersive spectrometer (EDS), respectively.The phase structure and microstructure were characterized by X-ray diffraction  using Cu Kκ = λρCp, where λ is thermal diffusivity measured by the laser flash method , ρ is the sample density determined by the Achimedes method, and Cp is the heat capacity calculated by the Dulong-Petit law. The Hall coefficient (RH) at room temperature was measured using a Quantum Design physical properties measurement system  in magnet fields from -5 T to 5 T. Hall carrier coefficient (nH) and mobility (μH) were calculated from nH = 1/(eRH) and μH = σRH, where e is the electron charge.The Seebeck coefficient and electrical conductivity were measured by a ZEM-3 apparatus  under a helium atmosphere from 300 K to 853 K. The thermal conductivity was calculated from Magnetization measurements were performed by a superconducting quantum interference device magnetometer from Quantum Design, MPMS (magnetic property measurement system) under magnetic field of 200 Oe from 0 to 300 K, and the rate of heating and cooling is 1 K/min. The Seebeck coefficient of low temperature (from 50 K to 275 K) was using a physical property measurement system  from 50 to 300 K and the rate of heating and cooling is 0.5 K/min.


Schisandra chinensis fruits, has been shown to exhibit hepatoprotective, cardioprotective, renoprotective, and memory-enhancing properties. This study sought to design a sensitive and efficient HPLC-MS/MS approach to measuring Schisandrol B levels in rat plasma and tissues in order to assess the pharmacokinetics, oral bioavailability, and tissue distributions of this compound in vivo. For this analysis, bifendate was chosen as an internal standard (IS). A liquid-liquid extraction (LLE) approach was employed for the preparation of samples that were subsequently separated with an Agilent ZORBAX Eclipse XDB-C18  column with an isocratic mobile phase consisting of methanol and water containing 5 mM ammonium acetate and 0.1% formic acid . A linear calibration curve was obtained over the 5–2000 ng/mL and 1–1000 ng/mL ranges for plasma samples and tissue homogenates, respectively. This established method was then successfully applied to investigate the pharmacokinetics, oral bioavailability, and tissue distributions of Schisandrol B in Sprague-Dawley (SD) rats that were intravenously administered 2 mg/kg of Schisandrol B monomer, intragastrically administered Schisandrol B monomer (10 mg/kg), or intragastrically administered 6 mL/kg SCE . The oral absolute bioavailability of Schisandrol B following intragastric Schisandrol B monomer and SCE administration was approximately 18.73% and 68.12%, respectively. Tissue distribution studies revealed that Schisandrol B was distributed throughout several tested tissues, with particular accumulation in the liver and kidneys. Our data represent a valuable foundation for future studies of the pharmacologic and biological characteristics of Schisandrol B.Schisandrol B, a lignan isolated from dried  Schisandra chinensis is a perennial deciduous woody vine that is primarily found in China, Japan, and Korea and that is a popular Traditional Chinese Medicine (TCM) that is recorded in pharmacopeias in China, Japan, and the United States [Schisandra chinensis contains many chemical compounds including lignans, volatile oils, organic acids, vitamins, terpenoids, and polysaccharides [d States , 2. Rececharides . Of thescharides –7.Schisandra chinensis fruits and that exhibits hepatoprotective, cardioprotective, renoprotective [β1-assisted NF-κB signaling [in vivo. As such, it is vital that a sensitive and efficient approach to measuring tissue and plasma Schisandrol B levels be developed in order to facilitate such analyses in a rat model system.Schisandrol B is a lignan that can be isolated from dried otective –12, and otective . There iignaling . In miceignaling . Togethein vivo. Herein, an HPLC-MS/MS approach was established and validated for the evaluation of these pharmacokinetic parameters in male Sprague-Daley (SD) rats administered Schisandrol B. Together, our data represent a valuable foundation for future studies of the pharmacologic and biological properties of Schisandrol B.To date, several methods have been used to measure Schisandrol B levels in different sample types, including gas chromatography-mass spectrometry , HPLC wiSchisandra chinensis was obtained from the Yijishan Hospital of Wannan Medical College and was positively identified by Professor Lu Jianping of Wannan Medical College . Schisandrol B (>98% pure) was obtained from Shanghai Yuanye Biological Technology Co., Ltd. Internal standard (IS) bifendate (>98% pure) was procured from Shanghai Macklin Biochemical Co., Ltd . HPLC-grade formic acid, methanol, and methyl tert-butyl ether  were used for the present study, as was analytical grade ammonium acetate. A Milli-Q water system  was employed for the preparation of diH2O.18  column was used for all chromatographic separation. A Pro 200 Hand-Held or Post-Mounted Laboratory Homogenizer  was used to prepare tissue homogenates.A SCIEX Triple Quad 3200 HPLC-MS/MS system  and a Shimadzu LC-20AD binary pump equipped with a SIL-HTc autosampler and CTO-10ASvp column oven  were used for the present study. An Agilent ZORBAX Eclipse XDB-CμL injection volume was used for these analyses. MS detection was conducted using an ESI source in positive ion mode. Ion quantification was conducted in MRM mode, with transitions of m/z 399.4⟶368.2 for Schisandrol B and m/z 387.1⟶328.3 for bifendate (IS). For these MS analyses, settings were as follows: Gas1 (N2) 40 psi; Gas2 (N2) 40 psi; ion spray voltage 5500 V; ion source temperature 550°C; curtain gas (N2) 25 psi. Compound-specific declustering potential (DP), collision energy (CE), entrance potential (EP), and collision cell exit potential (CXP) were 45, 29, 6, and 7 V for Schisandrol B, and 76, 28, 9, and 6 V for bifendate, respectively. Acquisition and analysis of data were evaluated with the Analyst 1.6.2 software .Schisandrol B chromatographic separation was carried out with a mobile phase composed of methanol with 0.1% formic acid (A) and water containing 5 mM ammonium acetate and 0.1% formic acid  (B). Separation was conducted at a 0.5 mL/min flow rate, while the temperature of the column and autosampler was maintained at 30°C and room temperature, respectively. A 10 μg/mL. These stock solutions were diluted as appropriate to prepare working solutions. Quality control (QC) samples and calibration standards were generated by adding appropriate working solution volumes to samples of blank plasma. For analyses of plasma samples, calibration standards were prepared at 5.0, 10.0, 50.0, 100.0, 500.0, 1000.0, and 2000.0 ng/mL, while the preparation of QC samples was carried out at 15 (LQC), 150 (MQC), and 1500 (HQC) ng/mL. For analyses of tissue homogenate samples, calibration standards of seven different concentrations  were prepared, and QC samples were prepared at 2 (LQC), 40 (MQC), and 800 (HQC) ng/mL. Additionally, the preparation of a diluted solution (500 ng/mL) of IS was carried out in methanol using the stock solution. All working solutions were kept at 4°C before use.The preparation of stock solutions of Schisandrol B and IS was carried out by dissolving these standards in methanol at respective concentrations of 450 and 730 Schisandra chinensis was dried and crushed, and a 50 g sample of the resultant powder was subjected to ultrasonic extraction in 400 mL of 95% ethanol for 30 minutes. This extraction procedure was repeated twice, after which these extracts were filtered, pooled, evaporated in a rotary evaporator, and resuspended in 25 mL of normal saline. The resultant SCE preparation was equivalent to 2 g of crude Schisandra chinensis per milliliter and was stored at 4°C before use. Optimized HPLC-MS/MS conditions were used to determine that the Schisandrol B level in this prepared extract was 2.5 mg/mL.μL) were combined with 10 μL of the IS working solution, after which samples were shaken for 2 minutes. Next, protein was precipitated via the addition of 1 mL of methyl tert-butyl ether. IS and Schisandra B were then extracted from plasma samples via shaking for 5 minutes. Samples were then spun for 5 minutes at 4,000 rpm, and supernatants were dried at 45°C under nitrogen and were then resuspended in methanol (150 μL) containing formic acid (0.1%). The final solution was then shaken for 2 minutes, after which it was spun for 5 minutes at 12,000 rpm. A 10 μL volume of the resultant supernatant was then injected for HPLC-MS/MS analysis.Samples of rat plasma (100 μL of an IS solution (250 ng/mL) was combined with 100 μL of each tissue homogenate sample. These homogenates were then processed and analyzed identically to plasma samples.Tissue samples were homogenized in physiological saline , after which 10 This analytical approach was validated as per the Bioanalytical Method Validation Guidance for Industry issued by the US-FDA   concentrations of Schisandrol B or IS were analyzed in parallel with samples obtained from rats following oral SCE or Schisandrol B monomer administration in order to exclude the interference of endogenous substance.x2 as a weighting factor. LLOQ, described as the concentration of Schisandrol B with a S/N ratio >10, was evaluated based upon six replicate analyses with a percentage relative standard deviation (RSD) < 20% and a percentage relative error (RE) not exceeding ± 20%.Calibration curves for Schisandrol B in rat plasma (5–2000 ng/mL) and tissue homogenates (1–1000 ng/mL) were generated by plotting analyte to IS peak area ratios against expected analyte concentrations and were fitted via a weighted least-squares linear regression approach using 1/Intra-day accuracy and precision values were assessed by evaluating six replicate QC samples prepared at HQC, MQC, LQC, and LLOQ concentrations on a given day, whereas inter-day accuracy and precision were calculated via evaluating these same replicate samples on three subsequent days. The overall accuracy of this analytical approach was determined based upon the calculated analyte concentration as a percentage of the expected nominal concentration. Precision was defined by RSD values. The acceptable criteria were an RSD <15% (LLOQ <20%) and an accuracy within 85–115% (LLOQ: 80–120%).Schisandrol B extraction recovery was assessed by comparing peak area values for extracted LQC, MQC, and HQC samples to the peak area of extracted plasma or tissue homogenate samples that had been spiked with the corresponding analyte. The matrix eﬀect was determined by comparing the peak areas of Schisandrol B and IS in the spiked samples after extraction and a standard sample directly dissolved with a comparable concentration of solvent.Schisandrol B stability was assessed in plasma and tissue homogenate samples by assessing QC samples that had been stored under a range of conditions. To evaluate analyte benchtop stability over the course of handling, QC samples were stored at approximately 25°C for 4 h before analysis. The stability of these samples in storage was evaluated via freezing them at −80°C for 20 days, while freeze-thaw stability was assessed by passing samples through three cycles of freezing and thawing at −80°C and approximately 25°C, respectively. Autosampler stability was assessed by analyzing QC samples that had been stored for 24 h in the autosampler.Specific pathogen-free (SPF) male SD rats (180–220 g) were obtained from Qinglong Mountain Animal Breeding Farm in Jiangning District, Nanjing, Jiangsu Province, with license number: SCXK (Su) 2017–0001. The approval for this study was provided by the Animal Ethics Committee of Wannan Medical College, and these experiments were conducted in a manner consistent with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People's Republic of China. Animals were housed in a climate-controlled facility  with free food and water access, and rats were fasted with free access to water for 12 h prior to experimentation.μL) were collected from the ocular fundus vein into heparinized 2 mL tubes. For Group 1, samples were collected at 0, 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 12 h postinjection, while for Group 2 samples were obtained at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h postoral administration, and for Group 3 samples were collected at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, and 36 h postoral administration. Blood samples were spun for 5 minutes at 4500 rpm, after which plasma was collected and stored at −80°C.For pharmacokinetic analyses, SD rats were randomized into three treatment groups. Rats in Group 1 received an intravenous tail vein injection of Schisandrol B monomer (2 mg/kg). Rats in Group 2 and Group 3 were intragastrically administered a single dose of Schisandrol B monomer (10 mg/kg) or SCE , respectively. Samples of blood (250 n = 3/group). Rats were orally administered Schisandrol B monomer (10 mg/kg), after which the samples of heart, lung, kidney, liver, spleen, and brain tissues were obtained at 0.5, 1, 2, 4, 8, and 12 h postdosing. Tissue samples were washed to remove blood using normal saline, after which they were dried using filter paper, weighed, and stored at −20°C.For tissue distribution studies, 18 rats were randomized into six groups (t½) were obtained using Phoenix WinNonlin  with a noncompartment model. Following intravenous or oral administration, the maximum drug plasma concentration (Cmax) and the time to Cmax (Tmax) were determined based upon concentration-time curves. Absolute oral bioavailability (BA) was calculated with the equation F (%) = (AUCig/AUCiv) × (Doseiv/Doseig) × 100%. The concentrations of Schisandrol B in tested rat tissues were expressed in μg/g and were calculated with the equation Ct = Cs × Vs/W, where Ct represented the tissue concentration (μg/g), while Cs, Vs, and W were the concentration (ng/mL), the volume (mL), and the weight (g) of the tissue samples, respectively. Data are given as means ± SD.Plasma and tissue Schisandrol B concentrations were determined using calibration curves. Average plasma pharmacokinetic parameters including the area under the curve (AUC) for concentrations versus time and the elimination half-life  bifendate solutions at appropriate concentrations were injected directly into the mass spectrometer. Negative as well as positive ion modes were tested in this study, and the positive ion mode was found to yield a better signal-to-noise ratio. The primary precursor molecular ion peaks of Schisandrol B and bifendate were observed at m/MRM mode .We initially tested both methanol-water and acetonitrile-water mobile phases to optimize chromatographic conditions. We determined that the addition of formic acid to the aqueous phase in these analyses was sufficient to improve sensitivity, while ammonium acetate improved peak shape. Methanol was found to yield a superior peak shape relative to acetonitrile and was associated with less background noise. We therefore utilized a mobile phase containing 90% methanol and 0.1% formic acid (A) and water containing 5 mmol ammonium acetate and 0.1% formic acid  (B). This combination yielded optimal peak shape, lower background noise, higher intensity, and shorter running times for both Schisandrol B and IS.The specificity and selectivity of this analytical approach for IS and Schisandrol B were next assessed. Representative MRM chromatograms of pooled blank rat plasma and liver homogenate samples, as well as chromatograms from plasma and liver homogenate samples spiked with LLOQ Schisandrol B levels, plasma samples obtained at 1 h postdosing, and liver homogenate samples collected at 0.5 h postdosing, are shown in No endogenous substances were found to interfere with Schisandrol B or IS retention time owing to the elevated level of MRM selectivity.Calibration curves for homogenates (plasma and tissue) were found to exhibit significant linearity in the 5–2000 ng/mL and 1–1000 ng/mL ranges, respectively. The LLOQ for Schisandrol B in rat plasma and tissue homogenates was 5 ng/mL and 1 ng/mL, respectively, as depicted in The precision and accuracy of this analytical approach were next tested at four QC levels , as shown in Extraction recovery values at all three tested concentration levels were >75%, indicating that this extraction approach was reliable and efficient. The matrix effect of Schisandrol B in plasma and tissue homogenate samples ranged within 90.62%–114.08%, suggesting that matrix did not significantly interfere with Schisandrol B analyses .Stability was evaluated by assessing analyte levels in six replicate low and high concentration QC samples stored under a range of conditions. No significant differences in Schisandrol B concentrations were observed under any of these tested conditions for either QC sample type , suggestt0- (ng.h/mL) values were 456.68 ± 91.24, 426.22 ± 32.64, and 2288.87 ± 276.44 respectively, while AUC0-∞(ng.h/mL) values were 456.84 ± 91.63, 427.93 ± 33.15, and 2334.08 ± 381.07, respectively. Schisandrol B monomer was found to exhibit an oral bioavailability of 18.73%, whereas the bioavailability of Schisandrol B in SCE was as high as 68.12%. When comparing the pharmacokinetics of Schisandrol B in rats administered SCE to those in rats administered Schisandrol B monomer, we found that Schisandrol B derived from SCE exhibited a longer T1/2 (2.55 ± 0.66 vs. 1.59 ± 1.42), a longer MRT0-∞ (9.09 ± 1.51 vs. 3.31 ± 0.46), a lower CL (6.43 ± 1.83 vs. 23.37 ± 3.25), a later Tmax (2.00 ± 0.87 vs. 0.54 ± 0.40), and a higher dosage exposure and bioavailability (68.12% vs. 18.73%) relative to Schisandrol B monomer at an equivalent dosage. Previous data indicated that Schisandrol B monomer is eliminated within 12 h of absorption, whereas this period extends for 24 h following SCE administration. As such, the final collection time points in Groups 2 and 3 were 24 h and 36 h, respectively. This suggests that other components in SCE may enhance the absorption of Schisandrol B, decreasing its elimination and enhancing its bioavailability.After successfully validating this method, we assessed the pharmacokinetics of Schisandrol B in rats following intravenous Schisandrol B monomer administration (2 mg/kg), intragastric Schisandrol B monomer administration (10 mg/kg), or intragastric SCE administration . Schisandrol B mean plasma concentration-time profiles and key pharmacokinetic parameters were calculated based on these analyses , Table 5Tmax values for these tissues were ∼0.5 hours slower, confirming that the absorption of oral Schisandrol B monomer requires processing such that some amount of time is required for Schisandrol B to affect target organs. Maximal Schisandrol B concentrations were detected in the liver, followed by the kidneys, heart, lung, spleen, and brain, respectively. This suggests that Schisandrol B may be best-suited to the treatment of hepatic diseases. Given their high levels of vascularization and blood flow, it is perhaps unsurprising that the liver and kidneys exhibited the highest levels of Schisandrol B, which is likely to be primarily metabolized in the former and excreted via the latter of these organs. Given that we were also able to detect Schisandrol B in the brain following its oral administration, this suggests that it can penetrate the blood-brain barrier and may thus warrant future study as a potentially valuable pharmaceutical agent capable of targeting the central nervous system.Having successfully validated this HPLC-MS/MS approach, it was additionally used to assess Schisandrol B concentrations in heart, liver, spleen, lung, kidney, and brain homogenates prepared at 0.5, 1, 2, 4, 8, and 12 h after the single dose (10 mg/kg) intragastric administration of Schisandrol B. The tissue distribution profiles of Schisandrol B were assessed over time , revealiin vivo.Herein, we developed and validated a rapid and sensitive HPLC-MS/MS-based approach to evaluating Schisandrol B levels in rat plasma and tissue homogenates. This study is the first time to our knowledge to have assessed Schisandrol B oral bioavailability and tissue distribution patterns. We determined that the absolute oral bioavailability of Schisandrol B monomer was 18.73%, while for SCE it was 68.12%. We also found that Schisandrol B preferentially localized to the liver, kidneys, heart, and brain, providing a scientific basis for the beneficial effects of this compound on these organs. Together, our data offer a foundation for future studies of the pharmacological activity of Schisandrol B


Portulaca oleracea cultivars in response to salt stress (100 mM and 200 mM) were dramatically changed. Similarly, significant differences were found in the metabolite profiles among samples under salinity stress treatments as compared with the control. Thorough metabolic pathway analysis, 132 different metabolites in response to 28 days of particular salt stress treatments were recognized and quantified in roots and leaves of purslane, including 35 organic acids, 26 amino acids, 20 sugars, 14 sugar alcohols, 20 amines, 13 lipids and sterols, and 4 other acids. In conclusion, this study can be useful for future molecular experiments as a reference to select gene expression levels for the functional characterization of purslane.Purslane, a fleshy herbaceous plant, plays a pivotal role in various preventive and therapeutic purposes. To date, no report has documented the consequence of salt stress on metabolite accumulation in purslane. Herein, we proposed an insight into the metabolic and physiological traits of purslane under saline stress environments. The gas chromatography-mass spectrometry analysis was used to scrutinize the metabolic profiling of leaves and roots of two purslane genotypes, Tall Green (TG) and Shandong Wild (SD), under the control and saline exposures. Results revealed that the morphological and physiological traits of leaves and roots of both the tested  Portulaca oleracea) as one of the most important C4 medicinal plants, and it was named as “Global Panacea” [−1 , generating an osmotic pressure of about 0.2 MPa that substantially reduces the crop yield. In addition, salt stress causes necrosis and chlorosis due to the accumulation of Na+ that impedes many physiological developments in plants [The World Health Organization (WHO) documented purslane  due to the development of salt tolerance mechanisms . It is aSeveral studies have reported on the accumulation of metabolites from plant parts under different salt stress conditions in many crops and found that saline stress exerts differential consequences on the evolution, ion equilibrium, compatible solutes, and metabolism in leaves , 16. ManReports have shown that studying the effects of salinity in a heterogeneous split root system is more practical than by exposing whole roots to specific levels of NaCl stress , 24. Thi3 per cell) were used. The substrate used was composed of 30% perlite, 40% peat, and 30% vermiculite, and the substrate was supplemented with a sufficient amount of water during the seedling stage. After 14 days, the seedlings with an identical number of leaves and height for “TG” or “SD” were transferred into plastic hydroponic boxes (525 mm × 365 mm × 205 mm) in the greenhouse of School of Agriculture and Biology, Shanghai Jiao Tong University, China, on 23rd March 2018. Plants of both genotypes were treated with three different salt concentrations, i.e., 0 mM, 100 mM, and 200 mM NaCl. For each salt treatment, 12 plants of “TG” and “SD” were set in the same box as one replicate, and experiments were run at least four times. A 15 L quarter strength of Hoagland's solution [−1 and a pH of 5.8 was put in each plastic box, and a quarter strength of Hoagland's solutions with the same salt concentration was replaced 2 times per week. The plantlets were allowed to grow in a greenhouse with a day and night temperature of 28 ± 2°C and 16 ± 2°C, along with relative moisture and photosynthetically active radiation of 70%-80% and 400 μmol·m−2·s−1, respectively.To scrutinize the influence of saline treatment on the accumulation of metabolites, two different purslane genotypes were chosen from different geographical locations: “Tall Green” local (“TG”—American origin) and a wild variety “Shandong, China” local (“SD”) Figure . Both gesolution  with an Cinitial/Cmax [At the end of 28 days of salt treatments, the number of leaves, diameter of the stem, main stem length, and root length were recorded with a minimum number of six plantlets. Appropriately, 0.2 g samples of dried roots and leaves were taken, immediately placed in liquid nitrogen, and preserved at -80°C freezer for the physiological and metabolite identification. For Fv/Fm analysis, leaf photochemical efficiency was estimated by measuring chlorophyll fluorescence in the form of the Fv/Fm ratio, with a fluorescence induction monitor . Leaves were covered in a leaf clip to darkness for 30 min before Fv/Fm measurement. For leaf chlorophyll and carotenoid analysis, we cut 0.1 g leaves into pieces, which were placed into small centrifuge tubes with 10 mL dimethyl sulfoxide (DMSO) and saved in the dark environment for 2 to 3 days. After the designated time, the chlorophyll and carotenoid were measured at 663 nm and 645 nm, respectively, by a spectrophotometer . Electrolyte leakage was recorded as the percentage of ial/Cmax .v/v) aqueous methanol in a 10 mL centrifuge tube. The resultant mixture was centrifuged for 2 h followed by incubation at 70°C in a water bath for 15 min. Afterward, the extracts were centrifuged (at 12000 rpm) for half an hour and the supernatants were decanted into new culture tubes. Following the addition of 0.75 mL of chloroform and 1.4 mL of water, the mixture was vortexed and centrifuged (at 5000 rpm for 5 min), and 300 μL of the polar phase (methanol/water) was dried in a vacuum concentrator. The dried residue was subjected to derivatization in methoxyamine hydrochloride, and N-methyl-N-(trimethylsilyl)trifluoroacetamide, and analyzed by GC-MS [μL extract was injected into a DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm) with an inlet temperature of 260°C. After a 5 min solvent delay, the initial temperature of the GC oven was maintained at 80°C, which was raised to 280°C with 5°C min−1 after 2 min of injection and finally retained at 280°C for 13 min. Helium was employed as the carrier gas with a continuous flow rate of 1 mL min−1. The analytical measurements were ensured by using electron impact ionization (70 eV) in the full scan mode (m/z 30–550).Leaves and roots of each genotype were harvested separately after the salt treatment and kept at –80°C until further investigation. The polar metabolites were extracted by adopting the protocols as reported earlier with some modifications , 28. Froby GC-MS . The derP = 0.05 or 0.01 probability level, and the figures displayed were constructed in Microsoft Excel 2016 and SigmaPlot 10.0. For GC-MS analysis, the compounds were identified using TurboMass 4.1.1 software (PerkinElmer Inc.) with online accessible compound libraries . SAS version 8.2 was implemented for the statistical analysis of peak areas as reported earlier [The Statistical Analysis System  recorded comparisons among species and each species' responses to salt stress. Fisher's protected least significant difference (LSD) test was used to assess differences among genotypes and treatment means at the  earlier . The KyoAfter 28 days of the growth period, ANOVA was used to confirm the differences between morphological parameters. There are significant variations observed under particular salt stress in both cultivars. t-test .The metabolic variations in roots and leaves of purslane cultivars under the particular condition of salt stress were analyzed by GC-MS to understand the physiological responses and contrast strategies of purslane cultivars to saline stress. Noteworthy differentiations subsist on the metabolite profiles among samples under the salinity stress treatments and control. A total of 132 different metabolites in response to 28 days of salt stress at 0 mM, 100 mM, and 200 mM treatments were identified and quantified in roots and leaves of purslane, mainly including 35 organic acids, 26 amino acids, 20 sugars, 14 sugar alcohols, 20 amines, 13 lipids and sterols, and 4 other acids . First, α-Linolenic acid and guanosine were meaningfully decreased. In roots, a total of 33 metabolites were selected generally: 11 metabolites increased along with 2 organic acids, 4 amino acid, 2 sugars, 1 sugar alcohol, and 2 lipids and sterols; 16 had no significant change; and 6 were decreased under a salt treatment of 100 mM. Besides, 27 metabolites increased along with 10 organic acids, 7 amino acids, 6 sugar, 2 sugar alcohols, and 2 lipids and sterols at 200 mM salt concentration; three had no difference, and 3 decreased at 200 mM as compared to CK. In “Shandong Wild,” the metabolic responses are dramatically changed in both leaves and roots. In leaves, 12 metabolites including 1 organic acid, 3 amino acids, 1 sugar, 5 sugar alcohols, and 2 amines were increased significantly. In 200 mM salt stress concentration, the 12 metabolites increased including 3 organic acids, 3 amino acids, 1 sugar, and 5 sugar alcohols. Furthermore, 80 metabolites were calculated with a significant fold change in “SD” roots. In 80 filtered metabolites, 50 increased including 20 organic acids, 19 amino acids, 10 sugars, 8 sugar alcohols, 10 amines, and 3 lipids and sterols; 24 had no difference; and 6 decreased significantly at 200 mM. On the other hand, 70 metabolites showed a significant improvement together with 12 organic acids, 14 amino acids, 7 sugars, 7 sugar alcohols, 7 amines, and 3 lipids and sterols. In addition, six metabolites exhibited no changes and 4 were decreased at 100 mM salt concentration compared with the control , and the R package software was used to construct a heat map, displaying the changes in levels of metabolites between P. oleracea cultivars in roots and leaves under different salinity stress conditions , citric acid (1.54), L-norleucine (2.85), L-valine (1.38), L-isoleucine (1.59), L-serine (1.12), L-threonine (2.73), β-alanine (1.50), L-5-oxoproline (1.55), DL-phenylalanine (1.21), L-asparagine (2.05), xylitol (1.34), and cadaverine (1.45). Under 100 mM, sucrose and L-tyrosine resulted in a downregulation in the fold increment and were affected under both salinity stress conditions. Furthermore, DL-ornithine acid showed upregulation but did not demonstrate any significant effect under both salinity concentrations as compared to the control , GABA (1.07), proline (3.03), xylitol (1.61), myoinositol (1.67), and glycerol 3-phosphate (1.25) were significantly increased at 100 mM. Additionally, similar metabolites such as myoinositol (1.77), proline (1.72), glycerol 3-phosphate (1.64), and citric acid with (1.57) were increased at 200 mM. Tryptophan, implicated in the shikimic pathway, showed no change under both salinity stress conditions compared to the control. Besides this, in the “Shandong Wild” root, most of the metabolites significantly increased with upregulated fold change at a salt level of 200 mM in contrast to the control. For instance, D-gluconic (4.78), L-proline (4.49), L-lysine (6.21), L-tryptophan (5.89), and D-mannose are more upregulated, and a significant fold change was observed at 200 mM. Moreover, sucrose, citric acid, oxalic acid, butanedioic acid, proline, tryptophan, D-allose, uracil, and palmitic acid were upregulated but were not significantly affected by salt stress compared to the control (Supplementary File  control .2 can reduce the root length and disrupt the natural architecture of the root system because the cell wall of roots under salinity becomes often irregularly thickened and complex. There is a general decrease in plant growth especially in the number of leaves, reduction in root growth through osmosis, and toxic effect in plants subjected to salinity stress [In this study, the morphological, physiological, and metabolic changes of the purslane plant were compared under different saline conditions. Results showed that the morphological attributes were affected by 200 mM salt stress compared to 100 mM in “TG.” The number of leaves and roots was decreased at 200 mM than the control, whereas the diameter of the stem and length of roots was reduced at 200 mM in the “SD” wild genotype. During the salinity stress, different abiotic factors such as variations in temperature and nonexistence of Oy stress . The phoy stress , 33. The4 plants with prominent palisade layers on both sides of leaves. C4 plants exhibit high water use efficiency and CO2 adaptive strategies to make C4 photosynthesis, which directly affect chlorophyll pigments during the photosynthesis process in different abiotic stress conditions [α-ketoglutarate are increased in roots compared to leaves along with citrate at 200 mM in both genotypes. Besides this, the oxalic acid increased significantly in “SD” roots under 200 mM salt stress. Oxalic acid plays a vital role during low temperature and salt stress due to elevated antioxidant capacity in mango and pomegranate. The content of amines was not much higher compared to other metabolites. Even though 200 mM salt stress showed an increase in metabolites in leaves of genotype “TG” (Purslane is among the Cnditions , 35. Undnditions , 7. Furtnditions , 37. It nditions . In thesnditions . We founnditions . Most ofnditions . In thisnditions . The salnditions . In aminnditions . Amino anditions . Our resnditions . High lenditions , 48. Thenditions . In addinditions , 51. Sugnditions , 53. Li nditions , to mainype “TG” , leaves,ype “TG” , leaves.P. oleracea cultivars were dramatically altered following salt stress exposure at 100 and 200 mM. Likewise, significant differences subsist on the metabolite profiles among samples under the salinity stress treatments as compared with the control. Metabolic pathway analysis quantified 132 different metabolites in roots and leaves of purslane in response to particular salt stress treatments including 35 organic acids, 26 amino acids, 20 sugars, 14 sugar alcohols, 20 amines, 13 lipids and sterols, and 4 other acids. Most of the metabolites detected are involved in biochemical pathways, such as the TCA cycle, GS/GOGAT cycle, GABA, glycolysis, proline synthesis pathway, shikimic acid, and amino acid metabolic pathway. In conclusion, this study can be useful for future proteomic and molecular research as a reference to select the gene expression level for functional characterization of purslane, which in turn can be advantageous for sustainable agriculture to meet ever-increasing demands for fresh vegetables.In this study, the metabolic profiling of leaves and roots of two purslane genotypes, Tall Green “TG” and Shandong Wild “SD,” was investigated under the saline stress environments by GC-MS. The morphophysiological attributes of leaves and roots of both the tested


Slow lorises are enigmatic animal that represent the only venomous primate lineage. Their defensive secretions have received little attention. In this study we determined the full length sequence of the protein secreted by their unique brachial glands. The full length sequences displayed homology to the main allergenic protein present in cat dander. We thus compared the molecular features of the slow loris brachial gland protein and the cat dander allergen protein, showing remarkable similarities between them. Thus we postulate that allergenic proteins play a role in the slow loris defensive arsenal. These results shed light on these neglected, novel animals. The origin and evolution of animal defensive and predatory toxins has been the subject of intense research , but thaFelis catus dander allergen Fel d1. Fel d1 is a 35 kDa secretoglobin protein produced by sebaceous, salivary, perianal, and lachrymal glands as well as squamous epithelial cells, and is distributed by F. catus over its fur during grooming and licking [F. catus dander allergen Fel d1 is a heterodimer formed by three inter-chain disulphide bonds. Chain One is 70 residues while chain two is 90 or 92 residues long [F. catus dander allergen Fel d1 [Despite this interest in the field, the nature of the secretions produced by the brachial glands of slow lorises has been studied only to a limited detail from captive animals. The secretions produce a characteristic odour that has been shown to be due to a complex array of over two hundred aromatic molecules . While t licking ,18,19,20ues long ,22. It hues long . Two copn Fel d1 ,25. In pn Fel d1 . Only Chn Fel d1 ,25. ThusF. catus dander allergen Fel d1 and other proteins within the broader allergen family. In this study, we therefore elucidate the full-length sequence of both chains of the Javan slow lorises’ (Nycticebus javanicus) brachial gland secreted proteins, with samples from wild animals. We discuss our findings in the light of available information from the cat F. catus dander allergens possibly leading to insights not only on the molecular evolution of mammalian allergens but also gaining a greater understanding how to treat humans when envenomated by slow lorises.The limited sequence information is insufficient to reconstruct the molecular evolutionary history of the slow loris brachial gland secreted proteins and therefore their relationship to N. javanicus, with the full nucleotide sequence of Chain-One determined as (tgtcccgccgtagaaaaacacgctaacctcttcctgaagggaaccactgatgaatttctcaattatgcgaaaaatttcgtaaaatcctctgcagtattggaaaatgctaagcaactgaagatgtgttccgacaataaactgacagaagaggataaggataatgtccagtctgggctggacaaaatatactcaagcaatttttgt) while Chain Two was sequenced using protein sequencing  are identical.Sequence alignment of target to templates revealed that the cysteine residues are conserved indicating that disulfide bridges are conserved as well . The N. N. javanicus brachial secretion protein, the F. catus dander allergen Fel d1 structure template 2EJN was first downloaded from the protein databank (https://www.rcsb.org) [F. catus dander allergen Fel d1 heterodimer consists of two chains (A and B) linked to each other by three disulfide bonds within each heterodimer. All three disulfide bonds link together in the format of Chain A cysteine1 – Chain B cysteine3; Chain A cysteine2 – Chain B cysteine3; and Chain A cystine3 – Chain B cysteine1. Each domain consists of eight α-helices, for each to Chain One and Two, respectively. A Ca2+-binding site at the domain interface consists of I125 of Domain A and D130 of Domain B as well as N89 from both Domain A and B and three water molecules [2+-binding Sites Two and Three involved D46 and M49 in each of the domains [N. javanicus brachial gland protein, cavities were identified at the same region in each domain of F. catus dander allergen Fel d1 2EJN [3 in Domain A versus 730 Å3 in Domain B) due to the differing conformations of the eleven residue long stretch from E121 to G131 in both domains. Kaiser et al. hypothesised that these cavities may host progesterone [In order to examine the structural evolution of the csb.org)  consisti domains ,35. Of pesterone ,25. Thisesterone ,38,39,40N. javanicus brachial gland protein chains are shorter than the F. catus allergen chains, the structures shown for both proteins appear slightly different in shape, despite being superimposed and rotated the same way, but overall the structures are highly similar  is held in position by two salt bridges from K40 and K52. In F. catus dander allergen Fel d1 the homologous stabilizing residues are K42 and K54.When modelled, as the  similar , which i2+-binding site revealed that in the F. catus dander allergen Fel d1, the Ca2+-binding site 1 is located in a prominent position at the contact area between Domains A and B. Four residues form Ca2+-binding site 1: the side chain carbonyl atom of N89 and backbone carbonyl atom of I125 of Domain A as well as the side chain carbonyl atoms of N89 and D130 of Domain B. Since the N. javanicus brachial secretion protein was modelled without Ca2+-ions present, the attractive force of Ca2+ is missing, affecting the side-chain placement of T81(A), T81(B), and D123(B). Like I125 in F. catus dander allergen Fel d1, the respective T117 (A) orients its backbone carbonyl toward Ca2+-binding Site One. Despite these differences in side chain orientation, we postulate that Ca2+-binding Site One is also present in the dimerization area of the N. javanicus brachial secretion protein.Comparative examination of the CaF. catus dander allergen Fel d1 a hydrophobic core made of six residues, namely F85, G131, and L132 of both Domains A and B [N. javanicus brachial secretion protein this interdomain region is not all hydrophobic: G77 (A and B), D123 (A and B), I124 (A and B). Moreover, it is differently shaped. Where in F. catus dander allergen Fel d1 the large phenylalanine side chain reaches deep into the cavity, in the N. javanicus brachial secretion protein there is a glycine. By contrast, the position homologous to G131 of F. catus dander allergen Fel d1 is in the N. javanicus brachial secretion protein occupied by D123. We postulate that for the N. javanicus brachial secretion protein the hydrophobic core, therefore, differs from that of the allergen of F. catus.Comparative examination of the hydrophobic core revealed that the  A and B . In the F. catus dander allergen Fel d1 and that of the N. javanicus brachial secretion protein form a homodimer. The interface between the two domains consists of 21 amino acids of Domain A  and 20 residues of Domain B . Accessible surface area was calculated for both domains separately and the homodimer -binding to solvent exposed residues . IgE is F. catus ,43. The  as well ,45,46. FFuture work should investigate the potential role of immunological feedback loops that are overstimulated and may explain the festering, non-healing wounds characteristic of slow loris bites. Treatment with cat antiallergens may be a starting point.Nycticebus javanicus from wild slow lorises studied at the ecological research station in Cipaganti, Garut Regency, West Java, Indonesia (S7°6’6–7°7’0& E 107°46’0–107°46’5). We collected samples used here from April 2012 to June 2014, during which time we captured the animals for health checks associated with a radio-tracking project [We obtained brachial gland secretions of  project . After cProtein was first desalted on C8 column, and resuspended in 100 mM ammonium bicarbonate. Protein was reduced with 10 mM DTT, 95 °C, 15 min, cooled to room temperature, and alkylated with iodoacetamide  for 30 min, RT in the dark. Additional DTT  was added to quench excess iodoacetamide. The sample was split into aliquots, and digestion was performed overnight at 37 °C with either trypsin, chymotrypsin, AspN, GluC or LysC. Samples were ziptipped  before analysis by mass spectrometry.Peptides were separated using reversed-phase chromatography on a Shimadzu Prominence nanoLC system. Using a flow rate of 30 µL/min, samples were desalted on an Agilent C18 trap  for 3 min, followed by separation on a Vydac Everest C18  column at a flow rate of 1 µL/min. A gradient of 10–60% buffer B over 30 min where buffer A = 1 % ACN / 0.1% FA and buffer B = 80% ACN / 0.1% FA was used to separate peptides. Eluted peptides were directly analyzed on a TripleTof 5600 instrument (ABSciex) using a Nanospray III interface. Gas and voltage settings were adjusted as required. MS TOF scan across m/z 350–1800 was performed for 0.5 sec followed by information dependent acquisition of up to 20 peptides across m/z 40–1800 (0.05 sec per spectra). LC-MS/MS data was de novo sequenced using PeaksStudio software, with enzyme specified based on sample, mass tolerance of 20 ppm, fragment mass tolerance of 0.1 Da, and variable modifications including oxidation of methionine, and carbamidomethylation set as fixed modification. Resulting de novo sequences were manually curated and validated, with sequences BLAST searched to determine other proteins with similarity.The 3’ rapid amplification of cDNA ends (RACE) first strand cDNA was synthesized from 34 ng total RNA using the FirstChoice RLM-RACE kit (Ambion), following the manufacturer’s instructions. The resulting cDNA was used as template in a polymerase chain reaction (PCR) using a forward primer (F) designed from the partial α-chain venom peptide sequence and a reverse 3’RACE outer primer supplied in the FirstChoice RLM-RACE kit (Ambion). The known peptide sequence was reverse-translated into a nucleotide sequence for primer design. Primer sequences were F=5’-GGTGGAAAAACATGCGAAC-3’ and 3’RACE outer primer = 5’-GCGAGCACAGAATTAATACGACT-3’. The PCR reaction was performed using FastStart Taq DNA polymerase (Roche) with 3’RACE-cDNA as template under the following cycling conditions: 95 °C for 4 min, followed by 40 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min and a final elongation step at 72 °C for 7 min. PCR products were analyzed and purified after separation on a 1% agarose gel using a QIAquick Gel Extraction kit (Qiagen) and cloned into vector pCR2.1 (Invitrogen) for sequencing by the Australian Genome Research Facility using an M13 forward primer.F. catus allergen Fel d1 in Swiss PDB Viewer [F. catus allergen Fel d1 template structures 1ZKR and 2EJN [www.rcsb.org) [In order to structurally modelling of Chain 1 + 2 heterodimers and homodimers of Chain A and B, the target sequence was aligned to the template sequence of the major B Viewer . The majand 2EJN ,25 were csb.org) . It was csb.org) . The alicsb.org) . Models csb.org)  through csb.org)  and sidecsb.org) . Hydrophcsb.org)  as implecsb.org) . The cal