Plasma-Membrane-Localized Transporter NREET1 is Responsible for Rare Earth Element Uptake in Hyperaccumulator Dicranopteris linearis.

Rare earth elements (REEs) are critical for numerous modern technologies, and demand is increasing globally; however, production steps are resource-intensive and environmentally damaging. Some plant species are able to hyperaccumulate REEs, and understanding the biology behind this phenomenon could play a pivotal role in developing more environmentally friendly REE recovery technologies. Here, we identified a REE transporter NRAMP REE Transporter 1 (NREET1) from the REE hyperaccumulator fern Dicranopteris linearis. Although NREET1 belongs to the natural resistance-associated macrophage protein (NRAMP) family, it shares a low similarity with other NRAMP members. When expressed in yeast, NREET1 exhibited REE transport capacity, but it could not transport divalent metals, such as zinc, nickel, manganese, or iron. NREET1 is mainly expressed in D. linearis roots and predominantly localized in the plasma membrane. Expression studies in Arabidopsis thaliana revealed that NREET1 functions as a transporter mediating REE uptake and transfer from root cell walls into the cytoplasm. Moreover, NREET1 has a higher affinity for transporting light REEs compared to heavy REEs, which is consistent to the preferential enrichment of light REEs in field-grown D. linearis. We therefore conclude that NREET1 may play an important role in the uptake and consequently hyperaccumulation of REEs in D. linearis. These findings lay the foundation for the use of synthetic biology techniques to design and produce sustainable, plant-based REE recovery systems.

Nicotinamide Riboside Enhances Endothelial Precursor Cell Function to Promote Refractory Wound Healing Through Mediating the Sirt1/AMPK Pathway.

Lack of vascularization is directly associated with refractory wound healing in diabetes mellitus (DM). Enrichment of endothelial precursor cells (EPCs) is a promising but challenging approach for the treatment of diabetic wounds. Herein, we investigate the action of nicotinamide riboside (NR) on EPC function for improved healing of diabetic wounds. Db/db mice that were treated with NR-supplemented food (400 mg/kg/d) for 12 weeks exhibited higher wound healing rates and angiogenesis than untreated db/db mice. In agreement with this phenotype, NR supplementation significantly increased the number of blood EPCs and bone marrow (BM)-derived EPCs of db/db mice, as well as the tube formation and adhesion functions of BM-EPCs. Furthermore, NR-supplemented BM-EPCs showed higher expression of sirtuin 1 (Sirt1), phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), and lower expression of acetylated peroxisome proliferator-activated receptor γ coactivator (PGC-1α) than BM-EPCs isolated from untreated db/db mice. Knockdown of Sirt1 in BM-EPCs significantly abolished the tube formation and adhesion function of NR as well as the expression of p-AMPK and deacetylated PGC-1a. Inhibition of AMPK abolished the NR-regulated EPC function but had no effect on Sirt1 expression, demonstrating that NR enhances EPC function through the Sirt1-AMPK pathway. Overall, this study demonstrates that the oral uptake of NR enhances the EPC function to promote diabetic wound healing, indicating that NR supplementation might be a promising strategy to prevent the progression of diabetic complications.

Mutation of the chloroplast-localized phosphate transporter OsPHT2;1 reduces flavonoid accumulation and UV tolerance in rice.

Phosphorus (P) is an essential macronutrient required for plant development and production. The mechanisms regulating phosphate (Pi) uptake are well established, but the function of chloroplast Pi homeostasis is poorly understood in Oryza sativa (rice). PHT2;1 is one of the transporters/translocators mediating Pi import into chloroplasts. In this study, to gain insight into the role of OsPHT2;1-mediated stroma Pi, we analyzed OsPHT2;1 function in Pi utilization and photoprotection. Our results showed that OsPHT2;1 was induced by Pi starvation and light exposure. Cell-based assays showed that OsPHT2;1 localized to the chloroplast envelope and functioned as a low-affinity Pi transporter. The ospht2;1 had reduced Pi accumulation, plant growth and photosynthetic rates. Metabolite profiling revealed that 52.6% of the decreased metabolites in ospht2;1 plants were flavonoids, which was further confirmed by 40% lower content of total flavonoids compared with the wild type. As a consequence, ospht2;1 plants were more sensitive to UV-B irradiation. Moreover, the content of phenylalanine, the precursor of flavonoids, was also reduced, and was largely associated with the repressed expression of ADT1/MTR1. Furthermore, the ospht2;1 plants showed decreased grain yields at relatively high levels of UV-B irradiance. In summary, OsPHT2;1 functions as a chloroplast-localized low-affinity Pi transporter that mediates UV tolerance and rice yields at different latitudes.

Structural characterization and anti-alcoholic liver injury activity of a polysaccharide from Coriolus versicolor mycelia.

Polysaccharopeptide (PSP) from the medicinal mushroom Coriolus versicolor has been widely used in Asia as an adjunctive immunotherapy for treating cancers and liver diseases. However, the composition and structure of bioactive components in PSP remain elusive. Herein, we purified a hepatoprotective polysaccharide (PSP-1b1) with a molecular weight of 21.7 kDa from C. versicolor mycelia in submerged culture. PSP-1b1 consists of fucose, galactose, xylose, mannose, glucuronic acid and glucose at a relative molar ratio of 0.16:0.60:0.02:0.55:0.04:1.00. Structural features were investigated by methylation and gas chromatography-mass spectrometry, Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. The PSP-1b1 backbone consists of →4)-α-Galp-(1 → 4)-α-Galp-(1 → 2)-α-Manp-(1 → 4)-α-Galp-(1 → 2)-α-Manp-(1 → 4)-α-Galp-(1 → 4)-α-Galp-(1 → 2)-α-Manp-(1 → 4)-α-Galp-(1 → 2)-α-Manp-(1 → 4)→, with branches of α-1,6-Manp, ß-1,6-Glcp, ß-1,3,6-Glcp, α-1,3-Manp, α-1,6-Galp, α-1,3-Fucp, T-α-Glcp and T-α-Galp on the O-6 position of α-Manp of the main chain, and secondary branches linked to the O-6 position of ß-Glcp of the major branch. Treatment with PSK-1b1 (80 and 160 mg/kg/day) resulted in hepatoprotective effects against alcohol-induced liver injury in mice by reducing oxidative stress and modulating immunity.

Binding of cellular nucleolin with the viral core RNA G-quadruplex structure suppresses HCV replication.

Hepatitis C virus (HCV) infection is a major cause of human chronic liver disease and hepatocellular carcinoma. G-quadruplex (G4) is an important four-stranded secondary structure of nucleic acids. Recently, we discovered that the core gene of HCV contains a G4 RNA structure; however, the interaction between the HCV core RNA G4 and host cellular proteins, and the roles of the HCV core RNA G4 in HCV infection and pathogenesis remain elusive. Here, we identified a cellular protein, nucleolin (NCL), which bound and stabilized the HCV core RNA G4 structure. We demonstrated the direct interaction and colocalization between NCL and wild-type core RNA G4 at both in vitro and in cell physiological conditions of the alive virus; however no significant interaction was found between NCL and G4-modified core RNA. NCL is also associated with HCV particles. HCV infection induced NCL mRNA and protein expression, while NCL suppressed wild-type viral replication and expression, but not G4-modified virus. Silencing of NCL greatly enhanced viral RNA replication. Our findings provide new insights that NCL may act as a host factor for anti-viral innate immunity, and binding of cellular NCL with the viral core RNA G4 structure is involved in suppressing HCV replication.

OsPHT1;3 Mediates Uptake, Translocation, and Remobilization of Phosphate under Extremely Low Phosphate Regimes.

Plant roots rely on inorganic orthophosphate (Pi) transporters to acquire soluble Pi from soil solutions that exists at micromolar levels in natural ecosystems. Here, we functionally characterized a rice (Oryza sativa) Pi transporter, Os Phosphate Transporter-1;3 (OsPHT1;3), that mediates Pi uptake, translocation, and remobilization. OsPHT1;3 was directly regulated by Os Phosphate Starvation Response-2 and, in response to Pi starvation, showed enhanced expression in young leaf blades and shoot basal regions and even more so in roots and old leaf blades. OsPHT1;3 was able to complement a yeast mutant strain defective in five Pi transporters and mediate Pi influx in Xenopus laevis oocytes. Overexpression of OsPHT1;3 led to increased Pi concentration both in roots and shoots. However, unlike that reported for other known OsPHT1 members that facilitate Pi uptake at relatively higher Pi levels, mutation of OsPHT1;3 impaired Pi uptake and root-to-shoot Pi translocation only when external Pi concentration was below 5 µm Moreover, in basal nodes, the expression of OsPHT1;3 was restricted to the phloem of regular vascular bundles and enlarged vascular bundles. An isotope labeling experiment with 32P showed that ospht1;3 mutant lines were impaired in remobilization of Pi from source to sink leaves. Furthermore, overexpression and mutation of OsPHT1;3 led to reciprocal alteration in the expression of OsPHT1;2 and several other OsPHT1 genes. Yeast-two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays all demonstrated a physical interaction between OsPHT1;3 and OsPHT1;2. Taken together, our results indicate that OsPHT1;3 acts as a crucial factor for Pi acquisition, root-to-shoot Pi translocation, and redistribution of phosphorus in plants growing in environments with extremely low Pi levels.

Therapeutic Effect and Potential Mechanisms of Lion's Mane Medicinal Mushroom, Hericium erinaceus (Agaricomycetes), Mycelia in Submerged Culture on Ethanol-Induced Chronic Gastric Injury.

Hericium erinaceus (HE) is an edible and medicinal mushroom traditionally used for the treatment of gastric injury in clinical practice. However, scientific evidence of its pharmacological activities has not yet been revealed. This study was designed to investigate the therapeutic effect of HE mycelia in submerged culture on ethanol-induced chronic gastric injury (ECGI) in mice. Gastric injury model was induced by ethanol with chronic and binge ethanol feeding in mice, and then mice were treated with HE mycelia. The stomachs were removed for histopathological examination and inflammatory cytokines measurement. Meanwhile, total proteins of gastric tissue were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling analysis to quantitatively identify differentially expressed proteins (DEPs) in three groups of animals. Bioinformatics analysis of DEPs was conducted through clustering analysis, Venn analysis, Gene Ontology (GO) annotation enrichment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment. The histopathologic characteristics and biochemical data showed that HE mycelia (0.5 and 1.0 g/kg) exhibited therapeutic effects on the ECGI mice. Based on the results of iTRAQ analysis, a total of 308 proteins were differentially expressed in the ethanol group when compared with the control group; 205 DEPs in the high dose of HE (HEH) group when compared with control group; and 230 DEPs in HE group (1.0 g/kg) when compared with ethanol group. KEGG analysis showed that the p53 signaling pathway was closely related to the therapeutic effect of HE mycelia on ECGI. Furthermore, the expression levels of several DEPs, including keratin (KRT) 16, KRT6b and transglutaminase E (TGE), were verified by quantitative real-time polymerase chain reaction (qRT-PCR). In conclusion, H. erinaceus mycelia could relieve ethanol-induced chronic gastric injury in mice by ameliorating inflammation as well as regulating epidermal differentiation.

Recent Progress in the Construction of Functional Artificial Bone by Cytokine-Controlled Strategies.

OBJECTIVE: Combining artificial scaffolds with stimulatory factors to reconstruct lost bone tissues is one of the hottest research directions. The purpose of this review was to conduct a retrospective survey on the latest reports on artificial bone fabrication with functional cytokines. DATA SOURCES: The status of related scientific research from the year 2005 to 2018 was analyzed through the mode of literature retrieval in PubMed and VIP Database. The retrieval words are as follows: "bone tissue engineering," "angiogenesis," "cytokines," "osteogenesis," "biomimetic bone marrow," "sol-gel," "delivery system," and the corresponding Chinese words. STUDY SELECTION: After reading through the title and abstract for early screening, the full text of relevant studies was evaluated and those not related with this review had been ruled out. RESULTS: According to the literature retrospective survey, there were three key points for the successful construction of functional artificial bones: (1) the continuous supply of relatively low concentration of cytokines during the required period; (2) the delivery of two or more cytokines essential to the process and ensure the relatively spatial independence to reduce the unnecessary interference; and (3) supporting the early-stage angiogenesis and late-stage osteogenesis, respectively, regulating and balancing the crosslinking of both to avoid the surface ossification that would probably block the osteogenesis inside. CONCLUSIONS: The synergistic effect of both angiogenic factors and osteogenic factors applied in bone regeneration is a key point in the combined functional artificial bone. Through analysis, comparison, and summary of the current strategies, we proposed that the most promising one is to mimic the natural bone marrow function to facilitate the regeneration process and ensure the efficient repair of large weight-bearing bone defect.

The Cucurbit[7]Uril-Based Supramolecular Chemistry for Reversible B/Z-DNA Transition.

As a left-handed helical structure, Z-DNA is biologically active and it may be correlated with transcription and genome stability. Until recently, it remained a significant challenge to control the B/Z-DNA transition under physiological conditions. The current study represents the first to reversibly control B/Z-DNA transition using cucurbit[7]uril-based supramolecular approach. It is demonstrated that cucurbit[7]uril can encapsulate the central butanediamine moiety [HN(CH2)4NH] and reverses Z-DNA caused by spermine back to B-DNA. The subsequent treatment with 1-adamantanamine disassembles the cucurbit[7]uril/spermine complex and readily induces reconversion of B- into Z-DNA. The DNA conformational change is unequivocally demonstrated using different independent methods. Direct evidence for supramolecular interactions involved in DNA conformational changes is further provided. These findings can therefore open a new route to control DNA helical structure in a reversible way.

Polysaccharide of Hericium erinaceus attenuates colitis in C57BL/6 mice via regulation of oxidative stress, inflammation-related signaling pathways and modulating the composition of the gut microbiota.

Inflammatory bowel disease (IBD) is a disease caused by a dysregulated immune with unknown etiology. Hericium erinaceus (H. erinaceus) is a Chinese medicinal fungus, with the effect of prevention and treatment of gastrointestinal disorders. In this study, we have tested the anti-inflammatory effect of polysaccharide of H. erinaceus (HECP, Mw: 86.67 kDa) in the model of dextran sulfate sodium (DSS)-induced colitis in C57BL/6 mice. Our data indicated that HECP could improve clinical symptoms and down-regulate key markers of oxidative stresses, including nitric oxide (NO), malondialdehyde (MDA), total superoxide dismutase (T-SOD), and myeloperoxidase (MPO). HECP also suppressed the secretion of interleukin (IL)-6, interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and decreased the expression of related mRNA. Meanwhile, HECP blocked phosphorylation of nuclear factor-κB (NF-κB) p65, NF-κB inhibitor alpha (IκB-α), mitogen-activated protein kinases (MAPK) and Protein kinase B (Akt) in DSS-treated mice. Moreover, HECP reversed DSS-induced gut dysbiosis and maintained intestinal barrier integrity. In conclusion, HECP ameliorates DSS-induced intestinal injury in mice, which suggests that HECP can serve as a protective dietary nutrient against IBD.

Chemical Targeting of a G-Quadruplex RNA in the Ebola Virus L Gene.

In the present study, our bioinformatics analysis first reveals the existence of a conserved guanine-rich sequence within the Zaire ebolavirus L gene. Using various methods, we show that this sequence tends to fold into G-quadruplex RNA. TMPyP4 treatment evidently inhibits L gene expression at the RNA level. Moreover, the mini-replicon assay demonstrates that TMPyP4 effectively inhibits the artificial Zaire ebolavirus mini-genome and is a more potent inhibitor than ribavirin. Although TMPyP4 treatment reduced the replication of the mutant mini-genome when G-quadruplex formation was abolished in the L gene, its inhibitory effect was significantly alleviated compared with wild-type. Our findings thus provide the first evidence that G-quadruplex RNA is present in a negative-sense RNA virus. Finally, G-quadruplex RNA stabilization may represent a new therapeutic strategy against Ebola virus disease.

A highly conserved G-rich consensus sequence in hepatitis C virus core gene represents a new anti-hepatitis C target.

G-quadruplex (G4) is one of the most important secondary structures in nucleic acids. Until recently, G4 RNAs have not been reported in any ribovirus, such as the hepatitis C virus. Our bioinformatics analysis reveals highly conserved guanine-rich consensus sequences within the core gene of hepatitis C despite the high genetic variability of this ribovirus; we further show using various methods that such consensus sequences can fold into unimolecular G4 RNA structures, both in vitro and under physiological conditions. Furthermore, we provide direct evidences that small molecules specifically targeting G4 can stabilize this structure to reduce RNA replication and inhibit protein translation of intracellular hepatitis C. Ultimately, the stabilization of G4 RNA in the genome of hepatitis C represents a promising new strategy for anti-hepatitis C drug development.

[Present fertilization and the potential of high yield on watermelon in Jiangsu Province, China.] / 江苏省西瓜施肥现状及高产潜力.

Watermelon fertilization in main planting regions of Jiangsu Province was investigated in order to evaluate the characteristics of efficient fertilization and improve the yield and quality of watermelon. The investigation was performed in Dongtai, Dafeng, Sihong, Pizhou and Donghai. The results showed that the average yield of watermelon in Jiangsu Province was 48325 kg·hm-2, and the highest yield was 97500 kg·hm-2. The watermelon yield was improved by straw return of 14-15 t·hm-2. The watermelon yield was increased with the increase of organic manure application under the quantity of 30 t·hm-2, while it trended to decrease when the manure application exceeded 30 t·hm-2. The peasant households of efficient high yield and inefficient high yield on organic manure were 16.5% and 29.1%, respectively. The peasant households of efficient high yield and inefficient high yield on total nitrogen fertilizer were 14.9% and 26.3%, while those on chemical nitrogen fertilizer were 19.6% and 22.4%, respectively. The peasant households of efficient high yield and inefficient high yield on total phosphorus fertilizer were 20.9% and 21.8%, while those on chemical phosphorus fertilizer were 13.7% and 29.5%, respectively. The peasant households of efficient high yield and inefficient high yield on total potassium fertilizer were 14.9% and 26.3%, while those on chemical potassium fertilizer were 29.6% and 12.2%, respectively. Compared to basic application, basic and top application combination enhanced the watermelon yield, so did the foliar fertilization. It was suggested that the peasant households of inefficient high yield and inefficient low yield should decrease the fertilization quantity to improve fertilizer efficiency, while those of efficient low yield should regulate nutrient components and fertilization stage.

Surgical Incision and Approach in Thoracolumbar Extreme Lateral Interbody Fusion Surgery: An Anatomic Study of the Diaphragmatic Attachments.

STUDY DESIGN: Cadaveric study. OBJECTIVE: To provide anatomical basis for deciding the surgical approach and skin incision in thoracolumbar extreme lateral interbody fusion (XLIF) by delineating the attachment points of diaphragm. SUMMARY OF BACKGROUND DATA: Although the general anatomy of the thoracic diaphragm is well described, the specific attachment points of diaphragm concerned with the XLIF approach is yet to be elaborated. METHODS: Dissections were performed on 21 cases of formalin fixed specimens (12 males, 9 females, a total of 42 sets of data). Special attention was paid to the attachment points of diaphragm on both sides at the midaxillary line (MAL point) and the vertebral level parallel to the MAL point (VL-MAL). The attachment points of diaphragm on the front and back edge of the spinal column (FES point and BES point) were also described. RESULTS: The MAL point of diaphragm muscle lied between the inferior edge of the 10th rib and the superior edge of the 12th rib (20 out of 21 on left, 21 out of 21 on right). VL-MAL lied between L1 and L2 vertebrae level (20 out of 21 on left, 18 out of 21 on right). The attachments on both sides of the vertebral column mainly located between the upper edge of T12 vertebrae and L1-L2 disc (38 out of 42). CONCLUSION: A transthoracic approach should be considered when the target level was above T12 vertebrae, whereas a retroperitoneal approach should be chosen when target level was below L1-L2 disc. If the target level is located between T12 and L1-L2 disc, whether via transthoracic, retropleural, or retroperitoneal approach should be determined according to the conditions of patients and the skill and experience of the surgeon. Incision should be made above the 10th rib for the transthoracic approach and below the 12th rib for the retroperitoneal approach. LEVEL OF EVIDENCE: 4.

[Research progress of self-assembled monolayer in biomedical metallic materials].

Because of the excellent mechanical properties, biocompatibility and reasonable prices, biomedical metallic materials are widely used in the manufacture of vascular stents, heart valve membrane, artificial joints and other body implants. However, the physiological environment in the body is very complex, the long-term embedding of the metal implants may result in corrosion or some nonspecific effects. The properties of medical metal surfaces may decay, which can cause serious injury to human body. By means of the self-assembled monolayer(SAM) technology, the physical and chemical properties of the medical metal surfaces can be modified, and through the SAM medium, some functional materials can be grafted on the metal surfaces, which can largely improve the stability and compatibility of implants in the body, and find wide applications in promoting cell adhesion, improving hemocompatibility, inhibiting bacteria growth, and constructing drug delivery coatings. This paper reviews the progress in the application of SAM in biomedical metallic materials.

A potent anticancer agent of shikonin derivative targeting tubulin.

In this study, a shikonin ester derivative, compound , was selected to evaluate its anticancer activities and we found that compound exhibited better antitubulin activities against the human HepG2 cell line with an IC50 value of 1.097 µM. Furthermore, the inhibition of tubulin polymerization results indicated that compound demonstrated the most potent antitubulin activity (IC50 = 13.88), which was compared with shikonin and colchicine as positive controls (IC50 = 25.28 µM and 22.56 µM), respectively. Compound was simulated to have good binding site with tubulin and arrested the cell cycle at G2/M phase, which also induces apoptosis in HepG2 cells, in which P53 and members of Bcl-2 protein family were both involved in the progress of apoptosis revealed by western blot. Confocal microscopy observations revealed compound targeted tubulin and altered its polymerization by interfering with microtubule organization. Based on these results, compound functions as a potent anticancer agent targeting tubulin.

Clinical characteristics and neurologic recovery of patients with cervical spinal tuberculosis: should conservative treatment be preferred? A retrospective follow-up study of 115 cases.

OBJECTIVE: To present the clinical characteristics and prognostic factors of neurologic recovery in patients with cervical spinal tuberculosis (CST). METHODS: General description and multivariate analysis were used to detect possible predictors of the outcome of patients with neurologic deficit. Follow-up data were used to generate a Kaplan-Meier curve of neurologic recovery. RESULTS: Protective factors in neurologic recovery included less involved vertebrae, surgery, and higher Japanese Orthopaedic Association score before treatment; not shorter symptom duration was not a protective factor. Normal neurologic function was present in 30% of patients 6 months after treatment, in 56% of patients 12 months after treatment, and in 93% of patients 28 months after treatment. The cumulative complete neurologic recovery rates at 6 months, 12 months, and 28 months were 44%, 68%, and 91.7% in the surgery group and 16.7%, 38.8%, and 94.4% in the nonsurgery group. CONCLUSIONS: Surgery and Japanese Orthopaedic Association score before treatment are important predictors of neurologic recovery in patients with CST. A neurologic recovery curve could predict neurologic recovery in patients with CST and indicate in patients with neurologic deficit how long it will take to achieve complete neurologic recovery. The effect of surgery is quick, and the effect of conservative treatment is slower, but no difference in neurologic recovery was found in the long-term. Conservative treatment should be tried in every patient with CST with no obvious indication for surgery. In contrast to patients with tuberculosis without cervical spine involvement but with more complications, comprehensive conservative therapy should be preferred for patients with neurologic deficit to avoid unnecessary surgery and overtreatment and to conserve medical resources. Indications for surgical intervention for CST should be carefully selected.

Stimulated production of steroids in Inonotus obliquus by host factors from birch.

Steroids was considered as one of the bioactive components in Inonotus obliquus, while this kind of secondary metabolites are less accumulated in cultured mycelia. In this study, effect of extracts from bark and core of host-related species, birch (Betula platyphylla Suk.), on steroid production of I. obliquus in submerged culture were evaluated. The results showed that all dosages (0.01 and 0.1 g/L) of aqueous extracts and methanol extracts from birch bark and birch core possessed significantly stimulatory effect on steroid production of I. obliquus (P < 0.05). Among the eight extracts, the aqueous extract (0.01 g/L) from birch bark gave the highest steroid production (225.5 ± 8.7 mg/L), which is 97.3% higher than that of the control group. The aqueous extract (0.01 and 0.1 g/L) from birch bark could simultaneously stimulated mycelial growth and steroid content, while the methanol extract from birch bark only elevated the steroid content. High performance liquid chromatography analysis showed that productions of betulin, ergosterol, cholesterol, lanosterol, stigmasterol, and sitosterol in I. obliquus simultaneously increased in the presence of aqueous extract and methanol extract from birch bark. The results presented herein indicate that extracts from birch bark could act as an inducer for steroid biosynthesis of I. obliquus.

Hydrogen gas protects against serum and glucose deprivation­induced myocardial injury in H9c2 cells through activation of the NF­E2­related factor 2/heme oxygenase 1 signaling pathway.

Ischemia or hypoxia­induced myocardial injury is closely associated with oxidative stress. Scavenging free radicals and/or enhancing endogenous antioxidative defense systems may be beneficial for the impediment of myocardial ischemic injury. Hydrogen (H2) gas, as a water­ and lipid­soluble small molecule, is not only able to selectively eliminate hydroxyl (·OH) free radicals, but also to enhance endogenous antioxidative defense systems in rat lungs and arabidopsis plants. However, thus far, it has remained elusive whether H2 gas protects cardiomyocytes through enhancement of endogenous antioxidative defense systems. In the present study, the cardioprotective effect of H2 gas against ischemic or hypoxic injury was investigated, along with the underlying molecular mechanisms. H9c2 cardiomyoblasts (H9c2 cells) were treated in vitro with a chemical hypoxia inducer, cobalt chloride (CoCl2), to imitate hypoxia, or by serum and glucose deprivation (SGD) to imitate ischemia. Cell viability and intracellular ·OH free radicals were assessed. The role of an endogenous antioxidative defense system, the NF­E2­related factor 2 (Nrf2)/heme oxygenase 1 (HO­1) signaling pathway, was evaluated. The findings revealed that treatment with CoCl2 or SGD markedly reduced cell viability in H9c2 cells. H2 gas­rich medium protected against cell injury induced by SGD, but not that induced by CoCl2. When the cells were exposed to SGD, levels of intracellular ·OH free radicals were markedly increased; this was mitigated by H2 gas­rich medium. Exposure of the cells to SGD also resulted in significant increases in HO­1 expression and nuclear Nrf2 levels, and the HO­1 inhibitor ZnPP IX and the Nrf2 inhibitor brusatol aggravated SGD­induced cellular injury. H2 gas­rich medium enhanced SGD­induced upregulation of HO­1 and Nrf2, and the HO­1 or Nrf2 inhibition partially suppressed H2 gas­induced cardioprotection. Furthermore, following genetic silencing of Nrf2 by RNA interference, the effects of H2 gas on the induction of HO­1 and cardioprotection were markedly reduced. In conclusion, H2 gas protected cardiomyocytes from ischemia­induced myocardial injury through elimination of ·OH free radicals and also through activation of the Nrf2/HO­1 signaling pathway.