Post-injury immunosuppression and secondary infections are caused by an AIM2 inflammasome-driven signaling cascade.

Loss of lymphocytes, particularly T cell apoptosis, is a central pathological event after severe tissue injury that is associated with increased susceptibility for life-threatening infections. The precise immunological mechanisms leading to T cell death after acute injury are largely unknown. Here, we identified a monocyte-T cell interaction driving bystander cell death of T cells in ischemic stroke and burn injury. Specifically, we found that stroke induced a FasL-expressing monocyte population, which led to extrinsic T cell apoptosis. This phenomenon was driven by AIM2 inflammasome-dependent interleukin-1ß (IL-1ß) secretion after sensing cell-free DNA. Pharmacological inhibition of this pathway improved T cell survival and reduced post-stroke bacterial infections. As such, this study describes inflammasome-dependent monocyte activation as a previously unstudied cause of T cell death after injury and challenges the current paradigms of post-injury lymphopenia.

Physiological and transcriptomic responses of silkworms to graphene oxide exposure.

The growing use of nanomaterials has sparked significant interest in assessing the insect toxicities of nanoparticles. The silkworm, as an economically important insect, serves as a promising model for studying how insects respond to harmful substances. Here, we conducted a comprehensive investigation on the impact of graphene oxide (GO) on silkworms using a combination of physiological and transcriptome analyses. GO can enter the midguts and posterior silk glands of silkworms. High GO concentrations (> 25 mg/L) significantly (P < 0.01) inhibited larval growth. Additionally, GO (> 5 mg/L) significantly reduced the cocooning rate, and GO (> 15 mg/L) hindered oviduct development and egg laying in silkworms. GO increased the reactive oxygen species content and regulated catalase activity, suggesting that it may affect insect growth by regulating reactive oxygen detoxification. The transcriptome data analysis showed that 35 metabolism-related genes and 20 ribosome biogenesis-related genes were differentially expressed in response to GO, and their expression levels were highly correlated. Finally, we propose that a Ribosome biogenesis-Metabolic signaling network is involved in responses to GO. The research provides a new perspective on the molecular responses of insects to GO.

A Red-Emission Fluorescent Probe for Intracellular Biothiols and Hydrogen Sulfide Imaging in Living Cells.

This research centers on the development and synthesis of a longwave fluorescence probe, labeled as 60T, designed for the simultaneous detection of hydrogen sulfide, cysteine/homocysteine, and glutathione. The probe showcases a swift response, good linearity range, and heightened sensitivity, boasting that the detection limits of the probe for Cys, Hcy, GSH and H2S were 0.140, 0.202, 0.259 and 0.396 µM, respectively. Notably, its efficacy in monitoring thiol status changes in live MCF-7 cells is underscored by a substantial decrease in fluorescence intensity upon exposure to the thiol trapping reagent, N-ethyl maleimide (NEM). With an impressive red emission signal at 630 nm and a substantial Stokes shift of 80 nm, this probe exhibits remarkable sensitivity and selectivity for biothiols and H2S, indicating promising applications in the diagnosis and surgical navigation of relevant cancers.

Cellular and Intravital Imaging of NAD(P)H by a Red-Emitting Quinolinium-Based Fluorescent Probe that Features a Shift of Its Product from Mitochondria to the Nucleus.

NAD(P)H is a vital hydrogen donor and electron carrier involved in numerous biological processes. The development of small-molecule tools for intravital imaging of NAD(P)H is significant for further exploring their pathophysiological roles. Herein, we rationally designed a fluorescent probe NADH-R by a simple graft of pyridiniumylbutenenitrile on a 1-methylquinolinium moiety in the 3-position. Benefited from the reduction of quinolinium by NAD(P)H, this probe releases the free push-pull fluorophore NADH-RH, allowing a turn-on red-emitting fluorescence response together with an ultralow detection limit of 12 nM. Under the assistance of the probe, we first monitored exogenous and endogenous generation of NAD(P)H in living cells, subsequently observed dynamic changes of NAD(P)H levels in living cells under different metabolic perturbations, and finally visualized the declined NAD(P)H levels in live mouse brain in a stroke model. Unexpectedly, the time-dependent colocalization experiment revealed that the probe reacts with mitochondrial NAD(P)H, followed by a shift of its reduced product NADH-RH from mitochondria to the nucleus, highlighting that NADH-RH is a novel nucleus-directed dye scaffold, which would facilitate the development of nucleus-targeting fluorescent probes and drugs.

Low-dose dexamethasone promotes osteoblast viability by activating autophagy via the SGK1/FOXO3a signaling pathway.

Autophagy contributes to bone homeostasis and development under physiological conditions. Although previous studies have demonstrated the induction of the autophagy machinery by endogenous glucocorticoids (GCs), the precise mechanisms involved have not yet been clarified. The current study aimed to explore the effect of a low dose of GC (10-8 M dexamethasone, Dex) on autophagy in mouse embryonic osteoblastic precursor cells (MC3T3-E1 cells) and the potential mechanisms. The results showed that 10-8 M Dex induced significant time-dependent increases in the expression and activation of serum- and glucocorticoid-induced kinase-1 (SGK1) in MC3T3-E1 cells and that these effects were accompanied by increased cell viability and decreased apoptosis. The autophagy inhibitor 3-MA significantly inhibited Dex-mediated promotion of viability. Moreover, Dex increased LC3II and Beclin-1 levels and decreased SQSTM/p62 levels in a time-dependent manner, and these effects were attenuated by pretreatment with 3-MA. Transfection of Dex-treated MC3T3-E1 cells with shRNA-SGK1 resulted in a significant reduction in cell viability and an increase in apoptosis. 3-MA further exacerbated these effects of SGK1 inhibition. Knocking down SGK1 before Dex exposure significantly reduced the phosphorylated forkhead box O3a (p-FOXO3a)/FOXO3 ratio, suppressed LC3II and Beclin-1 levels, and increased SQSTM/p62 levels in MC3T3-E1 cells, and these effects were amplified by 3-MA. In conclusion, the results revealed that low-dose GC treatment increased osteoblast viability by activating autophagy via the SGK1/FOXO3a pathway.

Astragaloside IV ameliorates radiation-induced nerve cell damage by activating the BDNF/TrkB signaling pathway.

Radiation can induce nerve cell damage. Synapse connectivity and functionality are thought to be the essential foundation of all cognitive functions. Therefore, treating and preventing damage to synaptic structure and function is an urgent challenge. Astragaloside IV (AS-IV) is a glycoside extracted from Astragalus membranaceus (Fisch.). Bunge is a widely used traditional Chinese medicine in China with various pharmacological properties, including protective effects on the central nervous system (CNS). In this study, the effect of AS-IV on synapse damage and BDNF/TrkB signaling pathway in radiated C57BL/6 mice with X-rays was investigated. PC12 cells and primary cortical neurons were exposed to UVA in vitro. Open field test and rotarod test were used to observe the effects of AS-IV on the motor and explore the abilities of radiated mice. The pathological changes in the brain were observed by hematoxylin and eosin and Nissl staining. Immunofluorescence analysis was used to detect the synapse damage. The expressions of the BDNF/TrkB pathway and neuroprotection-related molecules were detected by Western blotting and Quantitative-RTPCR, respectively. The results showed that AS-IV could improve the motor and explore abilities of radiated mice, reduce pathological damage to the cortex, enhance neuroprotection functions, and activate BDNF/TrkB pathway. In conclusion, AS-IV could relieve radiation-induced synapse damage, at least partly through the BDNF/TrkB pathway.

Optimization of Chemical Extraction Conditions of Dietary Fiber from Cistanche deserticola Residues and Its Structural Characteristics and Physicochemical and Functional Properties.

Cistanche deserticola residues are by-products of the industrial production of Cistanche deserticola, which are currently often discarded, resulting in the waste of resources. In order to achieve the efficient utilization of Cistanche deserticola, dietary fiber from Cistanche deserticola residues was extracted chemically and the optimization of the extraction conditions was performed, using the response surface methodology to study the effects of the NaOH concentration, extraction temperature, extraction time, and solid-liquid ratio on the yield of water-soluble dietary fiber (SDF). The structural, physicochemical, and functional properties of the dietary fiber were also investigated. The results showed that the optimal conditions were as follows: NaOH concentration of 3.7%, extraction temperature of 71.7 °C, extraction time of 89.5 min, and solid-liquid ratio of 1:34. The average yield of SDF was 19.56%, which was close to the predicted value of 19.66%. The two dietary fiber types had typical polysaccharide absorption peaks and typical type I cellulose crystal structures, and the surface microstructures of the two dietary fiber types were different, with the surface of SDF being looser and more porous. Both dietary fiber types had good functional properties, with SDF having the strongest water-holding capacity and the strongest adsorption capacity for nitrite, cholesterol, sodium cholate, and glucose, while IDF had a better oil-holding capacity. These results suggest that Cistanche deserticola residues are a good source of dietary fiber and have promising applications in the functional food processing industry.

Fast Imaging of Mitochondrial Thioredoxin Reductase Using a Styrylpyridinium-Based Two-Photon Ratiometric Fluorescent Probe.

Thioredoxin reductase (TrxR) is a pivotal antioxidant enzyme, but there remains a challenge for its fast imaging. This work describes the combination of a hydroxyl styrylpyridinium scaffold as the push-pull fluorophore with a carbonate-bridged 1,2-dithiolane unit as the reaction site to develop a fast mitochondrial TrxR2 probe, DSMP. It manifested a plethora of excellent properties including a rapid specific response (12 min), large Stokes shift (170 nm), ratiometric two-photon imaging, favorable binding with TrxR (Km = 12.5 ± 0.2 µM), and the ability to cross the blood-brain barrier. With the aid of DSMP, we visualized the increased mitochondrial TrxR2 activity in cancer cells compared to normal cells. This offers the direct imaging evidence of the connection between the increased TrxR2 activity and the development of cancer. Additionally, the probe allowed the visualization of the loss in TrxR2 activity in a cellular Parkinson's disease model and, more importantly, in mouse brain tissues of a middle cerebral artery occlusion model for ischemic stroke.

Caffeine-Induced Sleep Restriction Alters the Gut Microbiome and Fecal Metabolic Profiles in Mice.

Insufficient sleep is becoming increasingly common and contributes to many health issues. To combat sleepiness, caffeine is consumed daily worldwide. Thus, caffeine consumption and sleep restriction often occur in succession. The gut microbiome can be rapidly affected by either one's sleep status or caffeine intake, whereas the synergistic effects of a persistent caffeine-induced sleep restriction remain unclear. In this study, we investigated the impact of a chronic caffeine-induced sleep restriction on the gut microbiome and its metabolic profiles in mice. Our results revealed that the proportion of Firmicutes and Bacteroidetes was not altered, while the abundance of Proteobacteria and Actinobacteria was significantly decreased. In addition, the content of the lipids was abundant and significantly increased. A pathway analysis of the differential metabolites suggested that numerous metabolic pathways were affected, and the glycerophospholipid metabolism was most significantly altered. Combined analysis revealed that the metabolism was significantly affected by variations in the abundance and function of the intestinal microorganisms and was closely relevant to Proteobacteria and Actinobacteria. In conclusion, a long-term caffeine-induced sleep restriction affected the diversity and composition of the intestinal microbiota in mice, and substantially altered the metabolic profiles of the gut microbiome. This may represent a novel mechanism by which an unhealthy lifestyle such as mistimed coffee breaks lead to or exacerbates disease.

Wnt/ß-Catenin Signaling Pathway Is Strongly Implicated in Cadmium-Induced Developmental Neurotoxicity and Neuroinflammation: Clues from Zebrafish Neurobehavior and In Vivo Neuroimaging.

Cadmium (Cd) is a toxic heavy metal and worldwide environmental pollutant which seriously threatens human health and ecosystems. It is easy to be adsorbed and deposited in organisms, exerting adverse effects on various organs including the brain. In a very recent study, making full use of a zebrafish model in both high-throughput behavioral tracking and live neuroimaging, we explored the potential developmental neurotoxicity of Cd2+ at environmentally relevant levels and identified multiple connections between Cd2+ exposure and neurodevelopmental disorders as well as microglia-mediated neuroinflammation, whereas the underlying neurotoxic mechanisms remained unclear. The canonical Wnt/ß-catenin signaling pathway plays crucial roles in many biological processes including neurodevelopment, cell survival, and cell cycle regulation, as well as microglial activation, thereby potentially presenting one of the key targets of Cd2+ neurotoxicity. Therefore, in this follow-up study, we investigated the implication of the Wnt/ß-catenin signaling pathway in Cd2+-induced developmental disorders and neuroinflammation and revealed that environmental Cd2+ exposure significantly affected the expression of key factors in the zebrafish Wnt/ß-catenin signaling pathway. In addition, pharmacological intervention of this pathway via TWS119, which can increase the protein level of ß-catenin and act as a classical activator of the Wnt signaling pathway, could significantly repress the Cd2+-induced cell cycle arrest and apoptosis, thereby attenuating the inhibitory effects of Cd2+ on the early development, behavior, and activity, as well as neurodevelopment of zebrafish larvae to a certain degree. Furthermore, activation and proliferation of microglia, as well as the altered expression profiles of genes associated with neuroimmune homeostasis triggered by Cd2+ exposure could also be significantly alleviated by the activation of the Wnt/ß-catenin signaling pathway. Thus, this study provided novel insights into the cellular and molecular mechanisms of Cd2+ toxicity on the vertebrate central nervous system (CNS), which might be helpful in developing pharmacotherapies to mitigate the neurological disorders resulting from exposure to Cd2+ and many other environmental heavy metals.

Transcriptome analyses reveal the expression profile of genes related to lignan biosynthesis in Anthriscus sylvestris L. Hoffm. Gen.

Anthriscus sylvestris L. Hoffm. Gen (A. sylvestris) is a perennial herb widely used for antitussive and diuretic purposes in traditional Korean and Chinese medicine. Lignans are critical secondary metabolites with widely pharmacological activities in A. sylvestris. Using transcriptome data of A. sylvestris, we identified genes related to lignan biosynthesis. In all, 123,852 unigenes were obtained from the flowers, leaves, roots, and stems of A. sylvestris with the Illumina HiSeq 4000 platform. The average length of unigenes was 1,123 bp and 91,217 (73.65%) of them were annotated in public databases. Differentially expressed genes and root-specific genes were analyzed between roots and the other three tissue types by comparing gene expression profiles. Specifically, the key enzyme genes involved in lignan biosynthesis were identified and analyzed. The expression levels of some of these genes were highest in the roots, consistent with the accumulation of deoxypodophyllotoxin. These expression levels were experimentally verified via quantitative real-time polymerase chain reaction (qRT-PCR). This research provides valuable information on the transcriptome data of A. sylvestris and the identification of candidate genes associated with the biosynthesis of lignans, laying the foundation for further research on genomics in A. sylvestris and related species. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01156-w.

Developing Push-Pull Hydroxylphenylpolyenylpyridinium Chromophores as Ratiometric Two-Photon Fluorescent Probes for Cellular and Intravital Imaging of Mitochondrial NQO1.

This work highlights the use of push-pull hydroxylphenylpolyenylpyridinium fluorophores coupled with trimethyl lock quinone to engineer the ratiometric two-photon probes for cellular and intravital imaging of mitochondrial NAD(P)H:quinone oxidoreductase 1 (NQO1), a critical antioxidant enzyme responsible for detoxifying quinones. As a typical representative, QBMP showed favorable binding with NQO1 with a Michaelis constant of 12.74 µM and exhibited a suite of superior properties, including rapid response (4 min), large Stokes shift (162 nm), ultralow detection limit (0.9 nM), favorable two-photon cross section for the released fluorophore (70.5 GM), and deep tissue penetration (225 µm) in fixed brain tissues. More importantly, this probe was successfully applied for distinguishing different NQO1-expressing cancer and normal cells, revealing decreased NQO1 activity in a cellular Parkinson's disease model, screening NQO1 inducers as neuroprotective agents, and imaging of NQO1 in live mouse brain.

Specific depletion of resident microglia in the early stage of stroke reduces cerebral ischemic damage.

BACKGROUND: Ischemia can induce rapid activation of microglia in the brain. As key immunocompetent cells, reactive microglia play an important role in pathological development of ischemic stroke. However, the role of activated microglia during the development of ischemia remains controversial. Thus, we aimed to investigate the function of reactive microglia in the early stage of ischemic stroke. METHODS: A Rose Bengal photothrombosis model was applied to induce targeted ischemic stroke in mice. CX3CR1CreER:R26iDTR mice were used to specifically deplete resident microglia through intragastric administration of tamoxifen (Ta) and intraperitoneal injection of diphtheria toxin (DT). At day 3 after ischemic stroke, behavioral tests were performed. After that, mouse brains were collected for further histological analysis and detection of mRNA expression of inflammatory factors. RESULTS: The results showed that specific depletion of microglia resulted in a significant decrease in ischemic infarct volume and improved performance in motor ability 3 days after stroke. Microglial depletion caused a remarkable reduction in the densities of degenerating neurons and inducible nitric oxide synthase positive (iNOS+) cells. Importantly, depleting microglia induced a significant increase in the mRNA expression level of anti-inflammatory factors TGF-ß1, Arg1, IL-10, IL-4, and Ym1 as well as a significant decline of pro-inflammatory factors TNF-α, iNOS, and IL-1ß 3 days after stroke. CONCLUSIONS: These results suggest that activated microglia is an important modulator of the brain's inflammatory response in stroke, contributing to neurological deficit and infarct expansion. Modulation of the inflammatory response through the elimination of microglia at a precise time point may be a promising therapeutic approach for the treatment of cerebral ischemia.

Unraveling the innate immune responses of Bombyx mori hemolymph, fat body, and midgut to Bombyx mori nucleopolyhedrovirus oral infection by metabolomic analysis.

Bombyx mori nucleopolyhedrovirus (BmNPV) infection causes a series of physiological and pathological changes in Bombyx mori (B. mori). Here, a metabolomic study of the innate immunity organs including hemolymph, fat body, and midgut of the silkworm strain Dazao following BmNPV challenge was conducted to reveal the metabolic variations in B. mori. Compared to the control, 4964 and 4942 features with 4077 and 4327 high-quality features were generated under positive and negative modes, respectively, from BmNPV-infected larvae. The principal component analysis and supervised learning method using partial least squares discrimination analysis demonstrated good analytical stability and experimental reproducibility of the metabolic profiles. Based on database annotations, a total of 296, 108, and 215 differential expressed metabolites (DEMs) were identified from BmNPV-infected group of hemolymph, fat body, and midgut, respectively, which were all mainly grouped into carboxylic acids and derivatives, fatty acyls, and glycerophospholipids. Kyoto Encyclopedia of Genes and Genomes Database enrichment analysis of the DEMs showed that amino acid metabolism was increased at 24 h after BmNPV infection. BmNPV induction was adopted to significantly alter a series of immune-related pathways including phospholipase D signaling pathway, FoxO signaling pathway, metabolism of xenobiotics by cytochrome P450, melanogenesis, membrane transport, carbohydrate metabolism, and lipid metabolism. The different levels of expression of several DEMs including l-glutamate, naphthalene, 3-succinoylpyridine 1-acyl-sn-glycerol 3-phosphate, and l-tyrosine which were involved in those pathways exhibited the immune responses of B. mori to BmNPV infection. Our findings are valuable for a better understanding of the antiviral mechanism of B. mori underlying the interaction between the silkworm and BmNPV.

Bombyx mori can synthesize ascorbic acid through the l-gulose pathway to varying degrees depending on developmental stage.

Vitamin C (VC) is an essential nutrient for many animals. However, whether insects, including Bombyx mori, can synthesize VC remains unclear. In this article, the optimized HPLC method was used to determine the content of l-ascorbic acid (AsA) in silkworm eggs, larvae and pupae, and the activity of l-gulono-1,4-lactone oxidase (GULO), a key enzyme in VC synthesis. The RNA interference method was used to determine the effect of the BmGulo-like gene on embryonic development and GULO activity in the pupal fat body. The AsA content increased significantly during E144 h-E168 h in the late embryonic stage and P48 h-P144 h in the middle-late pupal stage, in which exogenous VC was not ingested. Furthermore, the body AsA content in larvae fed VC-free feed also increased with larval stage. The GULO enzymatic activity was present in eggs and the fat bodies of larvae and pupae, even when the larvae were reared with fresh mulberry leaves. Moreover, the activity was higher in the later embryonic stages (E144 h-E168 h) and the early pupal stage (before P24 h). The GULO activity in the pupal fat body dramatically decreased when the screened BmGulo-like gene (BGIBMGA005735) was knocked down with small interfering RNA; in addition, the survival rate and hatching rate of eggs significantly decreased 21% and 44%, respectively, and embryonic development was delayed. Thus, Bombyx mori can synthesize AsA through the l-gulose pathway, albeit with low activity, and this synthesis ability varies with developmental stages.

Transcriptome analysis of Clinopodium gracile (Benth.) Matsum and identification of genes related to Triterpenoid Saponin biosynthesis.

BACKGROUND: Clinopodium gracile (Benth.) Matsum (C. gracile) is an annual herb with pharmacological properties effective in the treatment of various diseases, including hepatic carcinoma. Triterpenoid saponins are crucial bioactive compounds in C. gracile. However, the molecular understanding of the triterpenoid saponin biosynthesis pathway remains unclear. RESULTS: In this study, we performed RNA sequencing (RNA-Seq) analysis of the flowers, leaves, roots, and stems of C. gracile plants using the BGISEQ-500 platform. The assembly of transcripts from all four types of tissues generated 128,856 unigenes, of which 99,020 were mapped to several public databases for functional annotation. Differentially expressed genes (DEGs) were identified via the comparison of gene expression levels between leaves and other tissues (flowers, roots, and stems). Multiple genes encoding pivotal enzymes, such as squalene synthase (SS), or transcription factors (TFs) related to triterpenoid saponin biosynthesis were identified and further analyzed. The expression levels of unigenes encoding important enzymes were verified by quantitative real-time PCR (qRT-PCR). Different chemical constituents of triterpenoid saponins were identified by Ultra-Performance Liquid Chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). CONCLUSIONS: Our results greatly extend the public transcriptome dataset of C. gracile and provide valuable information for the identification of candidate genes involved in the biosynthesis of triterpenoid saponins and other important secondary metabolites.

Cooperation of ESIPT and ICT Processes in the Designed 2-(2'-Hydroxyphenyl)benzothiazole Derivative: A Near-Infrared Two-Photon Fluorescent Probe with a Large Stokes Shift for the Detection of Cysteine and Its Application in Biological Environments.

A rationally designed near-infrared two-photon fluorescent probe (SDP-A) for selectively detecting cysteine (Cys) has been developed based on a newly designed conjugation-enhanced 2-(2'-hydroxyphenyl)benzothiazole derivative as the fluorophore, an acrylate moiety as the Cys reaction site, and an N-methylpyridinium scaffold as both the unit of organelle targeting and improving water solubility. The probe SDP-A alone essentially emitted no fluorescence, whereas it achieved a superb near-infrared fluorescence emission (713 nm) enhancement within 15 min with a significant Stokes shift (302 nm) in the presence of Cys. The photoluminescence mechanism of the probe SDP-A toward Cys was modulated by excited-state intramolecular proton transfer (ESIPT) and intramolecular charge transfer (ICT) processes. It exhibited high selectivity and sensitivity (LOD = 102 nM) for monitoring Cys over other analytes such as Hcy/GSH/H2S owing to a specific conjugate addition-cyclization reaction between Cys and the acrylate moiety. More importantly, the released fluorophore SDP exhibits elevated quantum yields (1.52-18.17%) in different solvents and strong two-photon excited fluorescence with a sizeable two-photon action cross-section (Φ) of 213.5 GM at 820 nm in acetonitrile-PBS medium, which is highly desirable for two-photon fluorescence imaging of the living samples. Therefore, SDP-A was successfully applied to the imaging of Cys in live cells, zebrafish, mouse brain, and abdominal cavity down to a depth of more than 200 µm using a one/two-photon fluorescence microscope.

[Identification of key enzyme genes involved in biosynthesis of steroidal saponins and analysis of biosynthesis pathway in Polygonatum cyrtonema].

Steroidal saponins, which are the characteristic and main active constituents of Polygonatum, exhibit a broad range of pharmacological functions, such as regulating blood sugar, preventing cardiovascular and cerebrovascular diseases and anti-tumor. In this study, we performed RNA sequencing(RNA-Seq) analysis for the flowers, leaves, roots, and rhizomes of Polygonatum cyrtonema using the BGISEQ-500 platform to understand the biosynthesis pathway of steroidal saponins and study their key enzyme genes. The assembly of transcripts for four tissues generated 129 989 unigenes, of which 88 958 were mapped to several public databases for functional annotation, 22 813 unigenes were assigned to 53 subcategories and 64 877 unigenes were annotated to 136 pathways in KEGG database. Furthermore, 502 unigenes involved in the biosynthesis pathway of steroidal saponins were identified, of which 97 unigenes encoding 12 key enzymes. Cycloartenol synthase, the first key enzyme in the pathway of phytosterol biosynthesis, showed conserved catalytic domain and substrate binding domain based on sequence analysis and homology modeling. Differentially expressed genes(DEGs) were identified in rhizomes as compared to other tissues(flowers, leaves or roots).The 2 437 unigenes annotated by KEGG showed rhizome-specific expression, of which 35 unigenes involved in the biosynthesis of steroidal saponins. Our results greatly extend the public transcriptome dataset of Polygonatum and provide valuable information for the identification of candidate genes involved in the biosynthesis of steroidal saponins and other important secondary metabolites.

Transcriptome Analysis of Clinopodium chinense (Benth.) O. Kuntze and Identification of Genes Involved in Triterpenoid Saponin Biosynthesis.

Clinopodium chinense (Benth.) O. Kuntze (C. chinense) is an important herb in traditional Chinese medicine. Triterpenoid saponins are a major class of active compounds in C. chinense with broad pharmacological activities and hemostatic, antitumor, and anti-hyperglycemic effects. To identify genes involved in triterpenoid saponin biosynthesis, transcriptomic analyses of leaves, stems, and roots from C. chinense were performed. A total of 135,968 unigenes were obtained by assembling the leaf, stem, and root transcripts, of which 102,154 were annotated in public databases. Differentially expressed genes were determined based on expression profile analysis and analyzed for differential expression of unique genes related to triterpenoid saponin biosynthesis. Multiple unigenes encoding crucial enzymes or transcription factors involved in triterpenoid saponin synthesis were identified and analyzed. The expression levels of unigenes encoding enzymes were experimentally validated using quantitative real-time PCR. This study greatly broadens the public transcriptome database for this species and provides a valuable resource for identifying candidate genes involved in the biosynthesis of triterpenoid saponins and other secondary metabolites.

[Transcriptome analysis of venom gland and identification of functional genes for snake venom protein in Agkistrodon acutus].

Agkistrodon acutus is a traditional Chinese herb medicine which has immunological regulation,anti-tumor,anti-inflammatory and analgesic effects,which is mainly used for the treatment of rheumatoid arthritis,ankylosing spondylitis,sjogren's syndrome and tumors. In order to excavate more important functional genes from A. acutus,the transcriptome of the venom gland was sequenced by the Illumina Hi Seq 4000,and 32 862 unigenes were assembled. Among them,26 589 unigenes were mapped to least one public database. 2 695 unigenes were annotated and assigned to 62 TF families,and 5 920 SSR loci were identified. The majority of mapped unigenes was from Protobothrops mucrosquamatus in the NR database,which revealed their closest homology. Three secretory phospholipase A_2 with different amino acid sequences showed similar spatial structures and all had well-conserved active sites. The 3 D structural models of C-type lectin showed conserved glycosylation binding sites( Asn45). This study will lay the foundation for the further study of the function of snake venom protein,and promoting the development and utilization of genome resources from A. acutus.