Poria cocos (Schw.) Wolf, a Traditional Chinese Edible Medicinal Herb, Promotes Neuronal Differentiation, and the Morphological Maturation of Newborn Neurons in Neural Stem/Progenitor Cells.

Neurogenesis in the adult brain comprises the entire set of events of neuronal development. It begins with the division of precursor cells to form a mature, integrated, and functioning neuronal network. Adult neurogenesis is believed to play an important role in animals' cognitive abilities, including learning and memory. In the present study, significant neuronal differentiation-promoting activity of 80% (v/v) ethanol extract of P. cocos (EEPC) was found in Neuro-2a cells and mouse cortical neural stem/progenitor cells (NSPCs). Subsequently, a total of 97 compounds in EEPC were identified by UHPLC-Q-Exactive-MS/MS. Among them, four major compounds-Adenosine; Choline; Ethyl palmitoleate; and L-(-)-arabinitol-were further studied for their neuronal differentiation-promoting activity. Of which, choline has the most significant neuronal differentiation-promoting activity, indicating that choline, as the main bioactive compound in P. cocos, may have a positive effect on learning and memory functions. Compared with similar research literature, this is the first time that the neuronal differentiation-promoting effects of P. cocos extract have been studied.

Transcriptome analysis reveals the peptide toxins diversity of Macrothele palpator venom.

Spider venom is a large pharmacological repertoire of different bioactive peptide toxins. However, obtaining crude venom from some spiders is challenging. Thus, studying individual toxins through venom purification is a daunting task. In this study, we constructed the cDNA library and transcriptomic sequencing from the Macrothele palpator venom glands. Subsequently, 718 high-quality expressed sequence tags (ESTs) were identified, and grouped into three categories, including 449 toxin-like (62.53 %), 136 cellular component (18.94 %) and 133 non-matched (18.52 %) based on the gene function annotation. Additionally, 112 non-redundant toxin-like peptides were classified into 13 families (families A-M) based on their sequence homology and cysteine framework. Bioinformatics analysis revealed a high sequence similarity between families A-J and the toxins from Macrothele gigas in the NR database. In contrast, families K-M had a generally low sequence homology with known spider peptide toxins and unpredictable biological functions. Taken together, this study adds many new members to the spider toxin superfamily and provides a basis for identifying various potential biological tools in M. palpator venom.

Untargeted metabolomics identifies potential serum biomarkers associated with Crohn's disease.

Crohn's disease (CD) is well characterized by chronic inflammation of the gastrointestinal tract. The diagnose of CD relays on the comprehensive evaluation of patient symptoms, laboratory examination, radiology, and endoscopy. There is lack of biomarkers or simple test for CD detection. Serum samples from healthy subjects (n = 16) and CD patients (n = 16) were collected and prepared for untargeted metabolomics analysis using the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) method. The alterations of serum metabolites and the potential biomarkers were profiled by statistical analysis. And the associated metabolic pathway was analyzed based on Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The performance of potential biomarkers was assessed by receiver operating characteristic (ROC) analysis. A complete separation between HS and CD groups was seen in OPLS-DA. A total of 108 and 131 significantly altered metabolites in positive and negative ion mode, respectively, were identified, and most of them belong to several pathways ranging from lipid metabolism to amino acid metabolism and energy homeostasis. KEGG analysis revealed that lipid metabolism enriched most significantly. Further, ceramide, phosphatidylethanolamine (PE), and taurochenodeoxycholic acid (TCDCA) presented the highest predictive accuracy of the patients with CD as analyzed by ROC. The current study demonstrated that lipid metabolism is mostly related to CD pathogenesis. Further investigations are indicated to examine the use of lipid-related metabolites of ceramide, PE, and TCDCA as potential biomarkers for CD diagnosis.

A molecular link between autophagy and circadian rhythm in plants.

Extremely high or low autophagy levels disrupt plant survival under nutrient starvation. Recently, autophagy has been reported to display rhythms in animals. However, the mechanism of circadian regulation of autophagy is still unclear. Here, we observed that autophagy has a robust rhythm and that various autophagy-related genes (ATGs) are rhythmically expressed in Arabidopsis. Chromatin immunoprecipitation (ChIP) and dual-luciferase (LUC) analyses showed that the core oscillator gene TIMING OF CAB EXPRESSION 1 (TOC1) directly binds to the promoters of ATG (ATG1a, ATG2, and ATG8d) and negatively regulates autophagy activities under nutritional stress. Furthermore, autophagy defects might affect endogenous rhythms by reducing the rhythm amplitude of TOC1 and shortening the rhythm period of CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1). Autophagy is essential for the circadian clock pattern in seedling development and plant sensitivity to nutritional deficiencies. Taken together, our studies reveal a plant strategy in which the TOC1-ATG axis involved in autophagy-rhythm crosstalk to fine-tune the intensity of autophagy.

Transcriptomic Analysis of the Spider Venom Gland Reveals Venom Diversity and Species Consanguinity.

Selenocosmia jiafu (S. jiafu) has been recently identified as a new species of spider in China. It lives in the same habitat as various other venomous spiders, including Chilobrachys jingzhao (C. jingzhao), Selenocosmia huwena (S. huwena), and Macrothele raveni (M. raveni). The venom from these different species of spiders exhibits some similarities and some differences in terms of their biochemical and electrophysiological properties. With the objective to illustrate the diversity in venom peptide toxins and to establish the evolutionary relationship between different spider species, we first performed transcriptomic analysis on a cDNA library from the venom gland of S. jiafu. We identified 146 novel toxin-like sequences, which were classified into eighteen different superfamilies. This transcriptome was then compared with that of C. jingzhao, which revealed that the putative toxins from both spider venoms may have originated from the same ancestor, although novel toxins evolved independently in the two species. A BLAST search and pharmacological analysis revealed that the two venoms have similar sodium channel modulation activity. This study provides insights into the venom of two closely related species of spider, which will prove useful towards understanding the structure and function of their toxins.

A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel.

Voltage-gated sodium channels (NaVs) are activated by transiting the voltage sensor from the deactivated to the activated state. The crystal structures of several bacterial NaVs have captured the voltage sensor module (VSM) in an activated state, but structure of the deactivated voltage sensor remains elusive. In this study, we sought to identify peptide toxins stabilizing the deactivated VSM of bacterial NaVs. We screened fractions from several venoms and characterized a cystine knot toxin called JZTx-27 from the venom of tarantula Chilobrachys jingzhao as a high-affinity antagonist of the prokaryotic NaVs NsVBa (nonselective voltage-gated Bacillus alcalophilus) and NaChBac (bacterial sodium channel from Bacillus halodurans) (IC50 = 112 nM and 30 nM, respectively). JZTx-27 was more efficacious at weaker depolarizing voltages and significantly slowed the activation but accelerated the deactivation of NsVBa, whereas the local anesthetic drug lidocaine was shown to antagonize NsVBa without affecting channel gating. Mutation analysis confirmed that JZTx-27 bound to S3-4 linker of NsVBa, with F98 being the critical residue in determining toxin affinity. All electrophysiological data and in silico analysis suggested that JZTx-27 trapped VSM of NsVBa in one of the deactivated states. In mammalian NaVs, JZTx-27 preferably inhibited the inactivation of NaV1.5 by targeting the fourth transmembrane domain. To our knowledge, this is the first report of peptide antagonist for prokaryotic NaVs. More important, we proposed that JZTx-27 stabilized the NsVBa VSM in the deactivated state and may be used as a probe to determine the structure of the deactivated VSM of NaVs.-Tang, C., Zhou, X., Nguyen, P. T., Zhang, Y., Hu, Z., Zhang, C., Yarov-Yarovoy, V., DeCaen, P. G., Liang, S., Liu, Z. A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel.

Characterization of ion channels on subesophageal ganglion neurons from Chinese tarantula Ornithoctonus huwena: Exploring the myth of the spider insensitive to its venom.

Chinese tarantula Ornithoctonus huwena is one of the most venomous spiders distributing in the hilly areas of southern China. In this study, using whole-cell patch-clamp technique we investigated electrophysiological and pharmacological properties of ion channels from tarantula subesophageal ganglion neurons. It was found that the neurons express multiple kinds of ion channels at least including voltage-gated calcium channels, TTX-sensitive sodium channels and two types of potassium channels. They exhibit pharmacological properties similar to mammalian subtypes. Spider calcium channels were sensitive to ω-conotoxin GVIA and diltiazem, two well-known inhibitors of mammalian neuronal high-voltage-activated (HVA) subtypes. 4-Aminopyridine and tetraethylammonium could inhibit spider outward transient and delayed-rectifier potassium channels, respectively. Huwentoxin-I and huwentoxin-IV are two abundant toxic components in the venom of Ornithoctonus huwena. Interestingly, although in our previous work they inhibit HVA calcium channels and TTX-sensitive sodium channels from mammalian sensory neurons, respectively, they fail to affect the subtypes from spider neurons. Moreover, the crude venom has no effect on delayed-rectifier potassium channels and only slightly reduces transient outward potassium channels with an IC50 value of ∼51.3 mg/L. Therefore, our findings provide important evidence for ion channels from spiders having an evolution as self-defense and prey mechanism.

[Effects of the extracts from Ajuga decumbens on anti-fatigue in mice].

OBJECTIVE: To investigate the effects of extracts from Ajuga decumbens on anti-fatigue in mice. METHODS: One hundred and twenty female Kunming (KM) mice were randomly divided into quiet control group, sport control group, positive control group and 3 experimental groups which were the low, medium and high dose group given the extracts from Ajuga decumbens. The low, medium and high dose group were given the extracts with 100 mg/kg, 200 mg/kg, 400 mg/kg by body weight of mice for 30 d, respectively, but the positive control group was given American ginseng granules, while the quiet control group and the sport control groups were treated with saline. After this, the exhausting time, the physio-biochemical indexes (including lactic acid, blood urea nitrogen, blood sugar, total cholesterol and triglyceride) in serum, the contents of muscle and liver glycogen, and the antioxidative indexes (including glutathione peroxidase, superoxide dismutase, catalase and malondialdehyde) of organs in mice were investigated. RESULTS: The exhausting time, the number of red blood cell, the contents of hemoglobin and blood sugar, the contents of muscle and liver glycogen, and the activities of glutathione peroxidase, superoxide dismutase and catalase in organs of mice in the medium dose group and the high dose group were significantly more than those of the sport control group, but the contents of blood lactic acid, blood urea nitrogen and that of triglyceride and total cholesterol in serum, and the content of malondialdehyde in organs of mice in the medium dose group and the high dose group were significantly lower than those of the sport control group. And the effect of medium dose extracts from Ajuga decumbens on anti-fatigue was better than that of American ginseng granules. CONCLUSIONS: The extracts from Ajuga decumbens has significant anti-fatigue effect in mice.

[Determination of peptide and protein diversity in venom of the spider Selenocosmia jiafu by high performance liquid chromatography and mass spectrometry].

Selenocosmia jiafu (S. jiafu) is recently identified as a new species of spider in P. R. China. These medium bodied venomous spiders are distributed mainly in the hilly areas of southwest of China, mostly at Yunnan and Guangxi Provinces. In order to understand the composition of the S. jiafu venom, we performed a preliminary analysis of this venom using reversed-phase high performance liquid chromatography (RP-HPLC), matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The S. jiafu venom was separated by RP-HPLC in an analytical C18 column (phenomenex 100 A, 250 mm x 4.6 mm, 5 µm) equilibrated with solution A (distilled water with 0.1% trifluoroacetic acid), using a gradient from 0% to 50% of solution B (acetonitrile with 0.1% trifluoroacetic acid) over 50 min with a flow rate of 1 mL/min. The isolated venom proteins were treated with in-gel digestion separated by SDS- PAGE and then identified by liquid chromatography-electrospray ionization quadrupole-time of flight mass spectrometry (LC-ESI-QTOF-MS) techniques. The results show that more than 40 fractions eluted were monitored at 215 nm in the RP-HPLC chromatogram of the venom of the spider S. jiafu. Most of the fractions were eluted with retention times of 5-15 min and 25-40 min, corresponding to 5%-15% and 25%-40% acetonitrile, respectively. The venom contains 238 peptides that follow a bimodal distribution, with about 62.5% of the peptides having a relative molecular mass of 3,000-4,500 and about 33.2% of the peptides having a relative molecular mass of 1,000-3,000. This distribution model is rather different from those of peptides from other tarantula spider venoms analyzed. To explore the relative molecular mass distribution of the venom proteins, the venom was analyzed by SDS-PAGE using standard protocols. Except for peptides with relative molecular mass lower than 10,000, the SDS-PAGE electrophoresis revealed three more obvious bands around 50, 72 and 90 kD respectively. Further MS analysis indicated that there are mainly hemocyanin, potassium ion channel protein, calcium protease and so on. Altogether, this study not only indicated there are many peptides and proteins in the S. jiafu venom, but also provided a basis for further case-by-case investigation of peptide toxins from this venom.

A Comparative Analysis of the Venom Gland Transcriptomes of the Fishing Spiders Dolomedes mizhoanus and Dolomedes sulfurous.

Dolomedes sulfurous and Dolomedes mizhoanus are predaceous arthropods catching and feeding on small fish. They live in the same area and have similar habits. Their venoms exhibit some similarities and differences in biochemical and electrophysiological properties. In the present work, we first performed a transcriptomic analysis by constructing the venom gland cDNA library of D. sulfurous and 127 novel putative toxin sequences were consequently identified, which were classified into eight families. This venom gland transcriptome was then compared with that of D. mizhoanus, which revealed that the putative toxins from both spider venoms might have originated from the same gene ancestors although novel toxins were evolved independently in the two spiders. The putative toxins from both spiders contain 6-12 cysteine residues forming seven cysteine patterns. As revealed by blast search, the two venoms are rich in neurotoxins targeting ion channels with pharmacological and therapeutic significance. This study provides insight into the venoms of two closely related species of spider, which will be of use for future investigations into the structure and function of their toxins.

[Effects of Lonicera Japonica flavone on immunomodulation in mice].

OBJECTIVE: To study immunomodulating activity of Lonicera Japonica flavone by investigating immune enzymatic activity of serum and antoxidized activity of lymphoid organs in mice. METHODS: Fifty KM mice were randomly divided into control group, model group, low dose group, middle dose group and high dose group(n = 10), respectively. And low dose group, middle dose group and high dose group were given Lonicera Japonica flavone with 100 mg/kg, 200 mg/kg and 400 mg/kg every day, respectively, while control group and model group were administered with NS. After continuously giving drug 7 weeks, other groups were injected with Dexamethasome (Dex: 25 mg /kg) for 3 days by subcutaneous injection, but the control group were treated with NS. And after giving Lonicera Japonica flavone 1 week simultaneously, organ indexes , the activity of acid phosphatase (ACP), alkaline phosphatase (AKP) and lysozyme (LSZ) in serum , and the content of monoamine oxidase (MAO), total antioxidant capacity (T-AOC), total superoxide dismutase (SOD) and malondialdehyde (MDA) in lymphoid organs in mice were tested, respectively. RESULTS: Lonicera Japonica flavone could significantly improve the organ indexes, and significantly improve the activity of ACP, AKP and LSZ in serum, and significantly improve the contents of T-AOC and SOD, but reduce that of MAO and MDA in lymphoid organs in immunosuppressed mice. CONCLUSION: Ionicera Japonica flavone can significantly improve the activity of immune enzyme in serum and the antioxidized activity of lymphoid organs in mice. It suggests that Ionicera Japonica flavone has a good immunomodulatory effects.

Synergetic action of domain II and IV underlies persistent current generation in Nav1.3 as revealed by a tarantula toxin.

The persistent current (INaP) through voltage-gated sodium channels enhances neuronal excitability by causing prolonged depolarization of membranes. Nav1.3 intrinsically generates a small INaP, although the mechanism underlying its generation remains unclear. In this study, the involvement of the four domains of Nav1.3 in INaP generation was investigated using the tarantula toxin α-hexatoxin-MrVII (RTX-VII). RTX-VII activated Nav1.3 and induced a large INaP. A pre-activated state binding model was proposed to explain the kinetics of toxin-channel interaction. Of the four domains of Nav1.3, both domain II and IV might play important roles in the toxin-induced INaP. Domain IV constructed the binding site for RTX-VII, while domain II might not participate in interacting with RTX-VII but could determine the efficacy of RTX-VII. Our results based on the use of RTX-VII as a probe suggest that domain II and IV cooperatively contribute to the generation of INaP in Nav1.3.

The tarantula toxin jingzhaotoxin-XI (κ-theraphotoxin-Cj1a) regulates the activation and inactivation of the voltage-gated sodium channel Nav1.5.

Specific peptide toxins interact with voltage-gated sodium channels by regulating the activation or inactivation of targeted channels. However, few toxins possessing dual effects have been identified. In the present study, we showed that jingzhaotoxin-XI/κ-theraphotoxin-Cj1a (JZTX-XI), a 34-residue peptide from the venom of the Chinese spider Chilobrachys jingzhao, inhibits the sodium conductance (IC50 = 124 ± 26 nM) and slows the fast inactivation (EC50 = 1.18 ± 0.2 µM) of Nav1.5 expressed in Chinese hamster ovary (CHO-K1) cells. JZTX-XI significantly shifted the activation to more depolarized voltages and decreased the deactivation of Nav1.5 currents upon extreme depolarization, but only slightly affected voltage-dependence of steady-state inactivation. In addition, JZTX-XI caused an approximately five-fold decrease in the rate of recovery from inactivation and an approximately 1.9-fold reduction in the closed-state inactivation rate. Our data suggest that JZTX-XI integrates the functions of site 3 toxins (α-scorpion toxins) with site 4 toxins (ß-scorpion and spider toxins) by targeting multiple sites on Nav1.5. The unique properties displayed by JZTX-XI in its inhibitory activity on Nav1.5 suggest that its mechanism of action is distinct from those of site 3 and site 4 toxins, making JZTX-XI a useful probe for investigating the gating mechanism of Nav1.5 and toxin-channel interactions.

The venom of the spider Selenocosmia jiafu contains various neurotoxins acting on voltage-gated ion channels in rat dorsal root ganglion neurons.

Selenocosmia jiafu is a medium-sized theraphosid spider and an attractive source of venom, because it can be bred in captivity and it produces large amounts of venom. We performed reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses and showed that S. jiafu venom contains hundreds of peptides with a predominant mass of 3000-4500 Da. Patch clamp analyses indicated that the venom could inhibit voltage-gated Na+, K+ and Ca2+ channels in rat dorsal root ganglion (DRG) neurons. The venom exhibited inhibitory effects on tetrodotoxin-resistant (TTX-R) Na+ currents and T-type Ca2+ currents, suggesting the presence of antagonists to both channel types and providing a valuable tool for the investigation of these channels and for drug development. Intra-abdominal injection of the venom had severe toxic effects on cockroaches and caused death at higher concentrations. The LD50 was 84.24 µg/g of body weight in the cockroach. However, no visible symptoms or behavioral changes were detected after intraperitoneal injection of the venom into mice even at doses up to 10 mg/kg body weight. Our results provide a basis for further case-by-case investigations of peptide toxins from this venom.

Isolation technique and proteomic analysis of the erythrocyte ghosts of red-eared turtle (Trachemys scripta).

To proceed proteomic analysis of erythrocyte of the red-eared turtle Trachemys scripta, a method for obtaining turtle erythrocyte ghosts (TEG) was first developed. After hypotonic lysis using a special buffer, forcing the erythrocyte through the syringe with an "N"-shaped needle, applying low speed homogenizing and differential centrifugation, highly purified TEG fractions were obtained. The isolated TEG proteins were treated with in-gel digestion separated by SDS-PAGE or in-solution digestion followed by HPLC predissociation, and then identified by nano-ESI-LC MS/MS techniques. A total of 169 TEG proteins was identified, validated, and categorized. Among these proteins, tubulins, and cell-surface-located F-type ATP synthase revealed important information into the super tolerance of Trachemys scripta in anoxia and low temperature exposure. Altogether, this study not only provided a method to isolate high quality TEG and a dataset of comprehensive characterization of TEG proteins, but also provides a tool for proteomic research of all nucleated red blood cells, and thus opened a new research field for exploring the mechanisms of super tolerance of turtles in harsh environment.