Novel players in organogenesis and flavonoid biosynthesis in cucumber glandular trichomes.

Glandular trichomes (GTs) are outgrowths of plant epidermal cells that secrete and store specialized secondary metabolites that protect plants against biotic and abiotic stresses and have economic importance for human use. While extensive work has been done to understand the molecular mechanisms of trichome organogenesis in Arabidopsis (Arabidopsis thaliana), which forms unicellular, nonglandular trichomes (NGTs), little is known about the mechanisms of GT development or regulation of secondary metabolites in plants with multicellular GTs. Here, we identified and functionally characterized genes associated with GT organogenesis and secondary metabolism in GTs of cucumber (Cucumis sativus). We developed a method for effective separation and isolation of cucumber GTs and NGTs. Transcriptomic and metabolomic analyses showed that flavonoid accumulation in cucumber GTs is positively associated with increased expression of related biosynthesis genes. We identified 67 GT development-related genes, the functions of 7 of which were validated by virus-induced gene silencing. We further validated the role of cucumber ECERIFERUM1 (CsCER1) in GT organogenesis by overexpression and RNA interference transgenic approaches. We further show that the transcription factor TINY BRANCHED HAIR (CsTBH) serves as a central regulator of flavonoid biosynthesis in cucumber GTs. Work from this study provides insight into the development of secondary metabolite biosynthesis in multicellular GTs.

Green Approach for Simultaneous Quantitative Analysis of Fluoroquinolones in the Environment using Three-Dimensional Fluorescence Coupled with Second-Order Calibration Method.

In this study, a straightforward and quick analytical technique based on the self-weighted alternating trilinear decomposition (SWATLD) algorithm in conjunction with excitation-emission matrix (EEM) fluorescence for the simultaneous determination of the antibiotics levofloxacin (LVFX) and ciprofloxacin (CIP) in environmental waters and sediments was developed. This approach completely utilizes the "second-order advantage" and inherits the great sensitivity of classic fluorescence. It replaces or improves the conventional "physical/chemical separation" with "mathematical separation", enabling direct and quick quantification of the target analytes even in the presence of unknown interferences, greatly streamlining sample preparation procedures, consuming less solvent, and speeding up analysis time, and allows successful and environmentally friendly solution of overlapping fluorescence spectra of multiple components in complicated environmental matrices without cumbersome pretreatment steps and complex and expensive instrumentation. The limits of detection varied between 0.34 and 0.67 ng mL- 1, and the average spiking recoveries of LVFX and CIP in water and sediment ranged from 97.6 to 107.7% with relative standard deviations lower than 6.6%. The developed method shows the reliability of the technology and the ability to quickly detect trace antibiotics in lake water even in the presence of unidentified interferents.

Different effects of seasonal impoundment and land use change on microbiome in a tributary sediment of the three gorgers reservoir.

Anthropogenic activities significantly impact river ecosystem nutrient fluxes and microbial metabolism. Here, we examined the seasonal and spatial variation of sediments physicochemical parameters and the associated microbiome in the Pengxi river, a representative tributary of Three Gorges Reservoir, in response to seasonal impoundment and land use change by human activities. Results revealed that seasonal impoundment and land use change enhanced total organic carbon (TOC), total nitrogen (TN) and ammonium nitrogen (NH4+-N) concentration in the sediment, but have different effects on sediment microbiome. Sediment microbiota showed higher similarity during the seasonal high-water level (HWL) in consecutive two years. The abundant phyla Acidobacteria, Gemmatimonadetes, Cyanobacteria, Actinobacteria and Planctomycetes significantly increased as water level increased. Along the changes in bacterial taxa, we also observed changes in predicted carbon fixation functions and nitrogen-related functions, including the significantly higher levels of Calvin cycle, 4HB/3HP cycle, 3HP cycle and assimilatory nitrate reduction, while significantly lower level of denitrification. Though land use change significantly increased TOC, TN and NH4+-N concentration, its effects on spatial variation of bacterial community composition and predicted functions was not significant. The finding indicates that TGR hydrologic changes and land use change have different influences on the carbon and nitrogen fluxes and their associated microbiome in TGR sediments. A focus of future research will be on assessing on carbon and nitrogen flux balance and the associated carbon and nitrogen microbial cycling in TGR sediment.

Viral community distribution, assembly mechanism, and associated hosts in an industrial park wastewater treatment plant.

Viruses manipulate bacterial community composition and impact wastewater treatment efficiency. Some viruses pose threats to the environment and human populations through infection. Improving the efficiency of wastewater treatment and ensuring the health of the effluent and receptor pools requires an understanding of how viral communities assemble and interact with hosts in wastewater treatment plants (WWTPs). We used metagenomic analysis to study the distribution, assembly mechanism, and sensitive hosts for the viral communities in raw water, anaerobic tanks, and returned activated sludge units of a large-scale industrial park WWTP. Uroviricota (53.42% ± 0.14%) and Nucleocytoviricota (26.1% ± 0.19%) were dominant in all units. Viral community composition significantly differed between units, as measured by ß diversity (P = 0.005). Compared to raw water, the relative viral abundance decreased by 29.8% in the anaerobic tank but increased by 9.9% in the activated sludge. Viral community assembly in raw water and anaerobic tanks was predominantly driven by deterministic processes (MST <0.5) versus stochastic processes (MST >0.5) in the activated sludge, indicating that differences in diffusion limits may fundamentally alter the assembly mechanisms of viral communities between the solid and liquid-phase environments. Acidobacteria was identified as the sensitive host contributing to viral abundance, exhibiting strong interactions and a mutual dependence (degree = 59). These results demonstrate the occurrence and prevalence of viruses in WWTPs, their different assembly mechanism, and sensitive hosts. These observations require further study of the mechanisms of viral community succession, ecological function, and roles in the successive wastewater treatment units.

Recent advance of microbial mercury methylation in the environment.

Methylmercury formation is mainly driven by microbial-mediated process. The mechanism of microbial mercury methylation has become a crucial research topic for understanding methylation in the environment. Pioneering studies of microbial mercury methylation are focusing on functional strain isolation, microbial community composition characterization, and mechanism elucidation in various environments. Therefore, the functional genes of microbial mercury methylation, global isolations of Hg methylation strains, and their methylation potential were systematically analyzed, and methylators in typical environments were extensively reviewed. The main drivers (key physicochemical factors and microbiota) of microbial mercury methylation were summarized and discussed. Though significant progress on the mechanism of the Hg microbial methylation has been explored in recent decade, it is still limited in several aspects, including (1) molecular biology techniques for identifying methylators; (2) characterization methods for mercury methylation potential; and (3) complex environmental properties (environmental factors, complex communities, etc.). Accordingly, strategies for studying the Hg microbial methylation mechanism were proposed. These strategies include the following: (1) the development of new molecular biology methods to characterize methylation potential; (2) treating the environment as a micro-ecosystem and studying them from a holistic perspective to clearly understand mercury methylation; (3) a more reasonable and sensitive inhibition test needs to be considered. KEY POINTS: • Global Hg microbial methylation is phylogenetically and functionally discussed. • The main drivers of microbial methylation are compared in various condition. • Future study of Hg microbial methylation is proposed.

Green Synthesis and Antifungal Activities of Novel N-Aryl Carbamate Derivatives.

Carbamate is a key structural motif in the development of fungicidal compounds, which is still promising and robust in the discovery of green pesticides. Herein, we report the synthesis and evaluation of the fungicidal activity of 35 carbamate derivatives, among which 19 compounds were synthesized in our previous report. These derivatives were synthesized from aromatic amides in a single step, which was a green oxidation process for Hofmann rearrangement using oxone, KCl and NaOH. Their chemical structures were characterized by 1H NMR, 13C NMR and high-resolution mass spectrometry. Their antifungal activity was tested against seven plant fungal pathogens. Many of the compounds exhibited good antifungal activity in vitro (inhibitory rate > 60% at 50 µg/mL). Compound 1ag exhibited excellent broad-spectrum antifungal activities with inhibition rates close to or higher than 70% at 50 µg/mL. Notably, compound 1af demonstrated the most potent inhibition against F. graminearum, with an EC50 value of 12.50 µg/mL, while compound 1z was the most promising candidate fungicide against F. oxysporum (EC50 = 16.65 µg/mL). The structure-activity relationships are also discussed in this paper. These results suggest that the N-aryl carbamate derivatives secured by our green protocol warrant further investigation as potential lead compounds for novel antifungal agents.

Two Green Protocols for Halogenative Semipinacol Rearrangement.

Semipinacol rearrangement is a special type of Wagner-Meerwein rearrangement that involves carbocation 1,2-rearrangement to provide carbonyl compounds with an α-quaternary carbon center. It has been strategically used for natural product synthesis and construction of highly congested quaternary carbons. Herein, we report a safe and green protocol that uses oxone/halide and Fenton bromide to achieve halogenative semipinacol rearrangement. The key feature of this method is the green in situ generation of reactive halogenating species from oxidation of halide with oxone or H2O2, which produces a nontoxic byproduct (potassium sulfate or water). Easy operation (insensitive to air and moisture) at room temperature without using special equipment adds additional advantage over previous methods.

Microbial Mediation of Carbon, Nitrogen, and Sulfur Cycles During Solid Waste Decomposition.

Landfills are a unique "terrestrial ecosystem" and serve as a significant carbon sink. Microorganisms convert biodegradable substances in municipal solid waste (MSW) to CH4, CO2, and microbial biomass, consisting of the carbon cycling in landfills. Microbial-mediated N and S cycles are also the important biogeochemical process during MSW decomposition, resulting in N2O and H2S emission, respectively. Meanwhile, microbial-mediated N and S cycles affect carbon cycling. How microbial community structure and function respond to C, N, and S cycling during solid waste decomposition, however, are not well-characterized. Here, we show the response of bacterial and archaeal community structure and functions to C, N, and S cycling during solid waste decomposition in a long-term (265 days) operation laboratory-scale bioreactor through 16S rRNA-based pyrosequencing and metagenomics analysis. Bacterial and archaeal community composition varied during solid waste decomposition. Aerobic respiration was the main pathway for CO2 emission, while anaerobic C fixation was the main pathway in carbon fixation. Methanogenesis and denitrification increased during solid waste decomposition, suggesting increasing CH4 and N2O emission. In contract, fermentation decreased along solid waste decomposition. Interestingly, Clostridiales were abundant and showed potential for several pathways in C, N, and S cycling. Archaea were involved in many pathways of C and N cycles. There is a shift between bacteria and archaea involvement in N2 fixation along solid waste decomposition that bacteria Clostridiales and Bacteroidales were initially dominant and then Methanosarcinales increased and became dominant in methanogenic phase. These results provide extensive microbial mediation of C, N, and S cycling profiles during solid waste decomposition.

Response of denitrification microbiome to the nitrogen flux in three Gorges reservoir (TGR) sediments during two seasonal water fluctuation events.

Three Gorges Reservoir (TGR) water fluctuation creates high water level (HWL) and low water level (LWL) condition in TGR aquatic ecosystem. HWL fluies significant nutrients, mainly introducing carbon and nitrogen into the ecosystem. The nitrogen input is a concern for water quality management of TGR since the possible eutrophication caused by nitrogen spike. Sediment denitrification is widely recognized as the dominant nitrogen removal process in freshwater ecosystem. Therefore, the response of TGR sediments microbiome to the input nitrogen flucatution is crucial for both nitrogen balance and the eutrophication status of the ecosystem. Using high throughout sequencing of 16S rRNA gene and the predicted denitrification enzyme, and qualitative PCR of denitrification functional genes, we investigated how TGR sediments denitrification microbiome respond to the input nitrogen flux during two seasonal water fluctuation events. Concomitant to expected input carbon and nitrogen, we observed distinct microbial community structure and denitrification microbiota in HWL and LWL, and also in seasonal sampling events. Sediments pH, total nitrogen and nitrate were the significant impact factors in shaping the microbial community structure. Important denitrification microbiota (e.g., Saprospiraceae, Gemmatimonadaceae, Pseudomonas) are the main taxa of the microbial community and also showed water level and seasonal variation. The relative abundance of denitrification enzyme (nar, nir, nor, nos) and function genes (nirS, nirK, nosZ) were higher in LWL than HWL. Denitrification enzyme were significantly (p < 0.05) correlated with the nitrate concentration. In addition, the relative abundance of denitrification enzyme and function genes increased during the transition from 2014 HWL to 2015 LWL. Results suggested that TGR sediments denitrification is nitrate concentration dependent. The denitrification microbiome is initially inhibited due to high nitrate input, then they developed denitrification ability in response to high nitrate concentration.

Triglyceride-glucose index and short-term functional outcome and in-hospital mortality in patients with ischemic stroke.

BACKGROUND AND AIMS: The triglyceride-glucose (TyG) index has been demonstrated as an independent marker of ischemic stroke. Whether TyG index predicts short-term outcomes in patients with ischemic stroke remains uncertain. The aim of the study was to investigate the early prognosis value of TyG index in ischemic stroke patients. METHODS AND RESULTS: A total of 3216 acute ischemic stroke patients from 22 hospitals were included in this analysis. The TyG index was calculated as ln (fasting triglyceride [mg/dL] × fasting glucose [mg/dL]/2). Logistic regression model was performed to estimate the relationship between TyG index and unfavorable functional outcome of death or disability (modified Rankin Scale score of 4-6) at discharge. Risk reclassification with TyG index to predict unfavorable functional outcome was analyzed. During hospitalization, 748 patients (23.3%) experienced poor functional outcome and 105 patients (3.3%) died from all causes. The multivariable adjusted odds ratios for the highest versus lowest quartile of TyG index was 1.62 (95% CI 1.15-2.29) for unfavorable functional outcome at discharge. The addition of TyG index to the conventional model improved the risk reclassification (net reclassification improvement 10.37%; integrated discrimination improvement 0.27%; both p < 0.05) for poor functional outcome. Moreover, TyG index was associated with an odds ratio (95% CI) of 1.26 (1.02-1.55) for an ordinal shift in mRS score and 2.49 (1.21-5.12) for in-hospital mortality. CONCLUSIONS: Higher TyG index was associated with higher risk of unfavorable functional outcome at discharge and in-hospital mortality, implicating the significant short-term prognostic effect of TyG index in patients with ischemic stroke.

Novel plant-derived exosome-like nanovesicles from Catharanthus roseus: preparation, characterization, and immunostimulatory effect via TNF-α/NF-κB/PU.1 axis.

BACKGROUND: Plant-derived exosomes-like nanovesicles (PDENs) have been found to be advantageous in disease treatment and drug delivery, but research on their biogenesis, compositional analysis, and key marker proteins is still in its infancy, which limits the standardized production of PDENs. Efficient preparation of PDENs continues to be a major challenge. RESULTS: Novel PDENs-based chemotherapeutic immune modulators, Catharanthus roseus (L.) Don leaves-derived exosome-like nanovesicles (CLDENs) were isolated from apoplastic fluid. CLDENs were membrane structured vesicles with a particle size of 75.51 ± 10.19 nm and a surface charge of -21.8 mV. CLDENs exhibited excellent stability, tolerating multiple enzymatic digestions, resisting extreme pH environments, and remaining stable in the gastrointestinal simulating fluid. Biodistribution experiments showed that CLDENs could be internalized by immune cells, and targeted at immune organs after intraperitoneal injection. The lipidomic analysis revealed CLDENs' special lipid composition, which contained 36.5% ether-phospholipids. Differential proteomics supported the origin of CLDENs in multivesicular bodies, and six marker proteins of CLDENs were identified for the first time. 60 ~ 240 µg/ml of CLDENs promoted the polarization and phagocytosis of macrophages as well as lymphocyte proliferation in vitro. Administration of 20 mg/kg and 60 mg/kg of CLDENs alleviated white blood cell reduction and bone marrow cell cycle arrest in immunosuppressive mice induced by cyclophosphamide. CLDENs strongly stimulated the secretion of TNF-α, activated NF-κB signal pathway and increased the expression of the hematopoietic function-related transcription factor PU.1 both in vitro and in vivo. To ensure a steady supply of CLDENs, plant cell culture systems of C. roseus were established to provide CLDENs-like nanovesicles which had similar physical properties and biological activities. Gram-level nanovesicles were successfully obtained from the culture medium, and the yield was three times as high as the original. CONCLUSIONS: Our research supports the use of CLDENs as a nano-biomaterial with excellent stability and biocompatibility, and for post-chemotherapy immune adjuvant therapy applications.

Design, Synthesis and Antifungal Activities of Novel Pyrazole Analogues Containing the Aryl Trifluoromethoxy Group.

On the basis of the three-component synthetic methodology developed by us, a total of twenty-six pyrazole compounds bearing aryl OCF3 were designed and synthesized. Their chemical structures were characterized by 1H and 13C nuclear magnetic resonance and high-resolution mass spectrometry. These compounds were evaluated systematically for antifungal activities in vitro against six plant pathogenic fungi by the mycelium growth rate method. Most of the compounds showed some activity against each of the fungi at 100 µg/mL. Compounds 1t and 1v exhibited higher activity against all the tested fungi, and 1v displayed the highest activity against F. graminearum with an EC50 value of 0.0530 µM, which was comparable with commercial pyraclostrobin. Structure-activity relationship analysis showed that, with respect to the R1 substituent, the straight chain or cycloalkyl ring moiety was a key structural moiety for the activity, and the R2 substituent on the pyrazole ring could have significant effects on the activity. Simple and readily available pyrazoles with potent antifungal activity were obtained, which are ready for further elaboration to serve as a pharmacophore in new potential antifungal agents.

Triglyceride-glucose index and common carotid artery intima-media thickness in patients with ischemic stroke.

BACKGROUND: Triglyceride glucose (TyG) index was recently reported to be associated with an increased risk of the development and recurrence of cardiovascular events, and atherosclerosis is a main speculative mechanism. However, data on the relationship between TyG index and atherosclerosis, especially in the setting of ischemic stroke, is rare. We aimed to explore the association between TyG index and carotid atherosclerosis in patients with ischemic stroke. METHODS: A total of 1523 ischemic stroke patients with TyG index and carotid artery imaging data were enrolled in this analysis. The TyG index was calculated as ln [fasting triglyceride (mg/dL) × fasting glucose (mg/dL)/2]. Carotid atherosclerosis was measured by common carotid artery intima-media thickness (cIMT), and abnormal cIMT was defined as a mean cIMT and maximum cIMT value ≥ 1 mm. Multivariable logistic regression models and restricted cubic spline models were used to assess the relationships between TyG index and abnormal cIMT. Risk reclassification and calibration of models with TyG index were analyzed. RESULTS: The multivariable-adjusted odds ratios (95% CIs) in quartile 4 versus quartile 1 of TyG index were 1.56 (1.06-2.28) for abnormal mean cIMT and 1.46 (1.02-2.08) for abnormal maximum cIMT, respectively. There were linear relationships between TyG index and abnormal mean cIMT (P for linearity = 0.005) and abnormal maximum cIMT (P for linearity = 0.027). In addition, the TyG index provided incremental predictive capacity beyond established risk factors, shown by an increase in net reclassification improvement and integrated discrimination improvement (all P < 0.05). CONCLUSIONS: A higher TyG index was associated with carotid atherosclerosis measured by cIMT in patients with ischemic stroke, suggesting that TyG could be a promising atherosclerotic marker.

Environmentally Benign and User-Friendly In Situ Generation of Nitrile Imines from Hydrazones for 1,3-Dipolar Cycloaddition.

Nitrile imines are highly reactive and versatile dipoles and conventionally generated in situ from unstable hydrazonyl halides under basic conditions. Herein, we report the first green and user-friendly protocol for in situ generation of nitrile imines from Oxone-KBr oxidation of hydrazones and base-promoted dehydrobromination. The nitrile imines were demonstrated for 1,3-dipolar cycloaddition with various dipolarophiles, including alkene and alkyne groups. With its green nature, ease of operation, and air and moisture tolerance, we expect our method will find wide applications in organic synthesis.

Eukaryotic community composition and dynamics during solid waste decomposition.

Consortia of microbial community are involved in organic waste decomposition in municipal solid waste (MSW) landfill via competition, syntropy, and predation. Bacterial and archaeal community structure and function have been extensively studied in this process, whereas the eukaryotic community structure and function are largely unidentified. This gap stands for one of the fundamental researches of microbial ecology, that is, "what is the importance of variation in eukaryotic community structure and function to solid waste decomposition? The main idea of this work is to characterize changes in eukaryotic community composition and the associated driver during solid waste decomposition. Using high throughput sequencing targeting 18S rRNA genes, community composition and dynamics of eukaryotic during solid wasted decomposition were studied, as well as the differences with solid waste and leachate physiochemical parameters. Concomitant to the expected changes in physiochemical factors, eukaryotic community composition and diversity changed along solid waste decomposition indicated by aerobic phase (AP), anaerobic acid phase (ACP), and methanogenic phase (MP) and the structure was shaped by the nutrients (BOD5, total phosphorus, and nitrate) in leachate. Ascomycota, the predominant eukaryote, showed significant (p < 0.05) different structure among AP, ACP, and MP in phylum, genera, and species levels. Abundant Freshwater Opisthokonta was present in MP, suggesting a methane carbon cycling via grazing methane oxidation microorganism. Amoebozoa, Alveolata, Rhizaria, and Stramenopiles showed successional pattern during solid waste decomposition, indicating a short food chain establishment. Characterization on eukaryotic community composition and dynamics during solid waste decomposition are crucial for understanding of microbial consortia ecological function on solid waste decomposition and are also helpful for MSW management.Key points• Abundant Ascomycota significantly differed at AP, ACP, and MP.• Eukaryotic succession indicated a short food chain establishment.• Entire eukaryotic community structure was associated to nutrients in leachate.

A Novel Peptide Derived from Arca inflata Induces Apoptosis in Colorectal Cancer Cells through Mitochondria and the p38 MAPK Pathway.

Colorectal carcinoma (CRC) is one of the major causes of cancer-related incidence and deaths. Here, we identified a novel antitumor peptide, P6, with a molecular weight of 2794.8 Da from a marine Chinese medicine, Arca inflata Reeve. The full amino acid sequence and secondary structure of P6 were determined by tandem mass de novo sequencing and circular dichroism spectroscopy, respectively. P6 markedly inhibited cell proliferation and colony formation, and induced apoptosis in CRC cells. Mechanistically, transcriptomics analysis and a serial functional evaluation showed that P6 induced colon cancer cell apoptosis through the activation of the p38-MAPK signaling pathway. Moreover, it was demonstrated that P6 exhibited antitumor effects in a tumor xenograft model, and induced cell cycle arrest in CRC cells in a concentration-dependent mode. These findings provide the first line of indication that P6 could be a potential therapeutic agent for CRC treatment.

Arca subcrenata Polypeptides Inhibit Human Colorectal Cancer HT-29 Cells Growth via Suppression of IGF-1R/Akt/mTOR Signaling and ATP Production.

Arca subcrenata Lischke, widely scattering offshore at neritic regions, is very popular on dining table due to its edible and medical functional meatball. This study aims to investigate the suppression of a polypeptide fraction from A. subcrenata (PAS) on human colorectal cancer HT-29 cells, and its underlying mechanism. The results showed that PAS inhibited the growth of HT-29 cells with an IC50 value of 117 µg/ml after 48 h treatment, and significantly suppressed the tumor growth in nude mice bearing-xenografted HT-29 cells at the dosage of 63 mg/kg, with little influence on normal colon cells and normal colonic mucosa. PAS was then inspiringly found to induce apoptosis and G2/M phase arrest in HT-29 cells. The effect mechanism was involved in the inhibition of IGF-1/IGF-1R signaling activation, which was responsible for inactivating downstream Akt/mTOR pathway. Immunofluorescence assay also showed that PAS could reduce phosphorylation of IGF-1R (Tyr1165/1166). IGF-1, an IGF-1R activator, could reverse the suppression of PAS on IGF-1R phosphorylation. Furthermore, PAS significantly inhibited ATP production of HT-29 cells both in vitro and in vivo. Our results provide positive evidence that A. subcrenata has the potential to be a candidate for the treatment of colorectal cancer.

Purification and Characterization of a New CRISP-Related Protein from Scapharca broughtonii and Its Immunomodulatory Activity.

More and more attention has been paid to bioactive compounds isolated from marine organisms or microorganisms in recent years. At the present study, a new protein coded as HPCG2, was purified from Scapharca broughtonii by stepwise chromatography methods. The molecular weight of HPCG2 was determined to be 30.71 kDa by MALDI-TOF-MS. The complete amino acid sequence of HPCG2 was obtained by tandem mass spectrometry combined with transcriptome database analysis, and its secondary structure was analyzed using circular dichroism. HPCG2 comprised 251 amino acids and contained 28.4% α-helix, 26% ß-sheet, 18.6% ß-turn, and 29.9% random coil. HPCG2 was predicted to be a cysteine-rich secretory protein-related (CRISP-related) protein by domain prediction. Moreover, HPCG2 was proved to possess the immunomodulatory effect on the murine immune cells. MTT assay showed that HPCG2 promoted the proliferation of splenic lymphocytes and the cytotoxicity of NK cells against YAC-1 cells. Flow cytometry test revealed that HPCG2 enhanced the phagocytic function of macrophages and polarized them into M1 type in RAW264.7 cells. In particular, Western blot analysis indicated that the immunomodulatory mechanism of HPCG2 was associated with the regulation on TLR4/JNK/ERK and STAT3 signaling pathways in RAW 264.7 cells. These results suggested that HPCG2 might be developed as a potential immunomodulatory agent or new functional product from marine organisms.

Recent Advances in the Reactions of Cyclic Carbynes.

The acyclic organic alkynes and carbyne bonds exhibit linear shapes. Metallabenzynes and metallapentalynes are six- or five-membered metallacycles containing carbynes, whose carbine-carbon bond angles are less than 180°. Such distortion results in considerable ring strain, resulting in the unprecedented reactivity compared with acyclic carbynes. Meanwhile, the aromaticity of these metallacycles would stabilize the ring system. The fascinating combination of ring strain and aromaticity would lead to interesting reactivities. This mini review summarized recent findings on the reactivity of the metal-carbon triple bonds and the aromatic ring system. In the case of metallabenzynes, aromaticity would prevail over ring strain. The reactions are similar to those of organic aromatics, especially in electrophilic reactions. Meanwhile, fragmentation of metallacarbynes might be observed via migratory insertion if the aromaticity of metallacarbynes is strongly affected. In the case of metallapentalynes, the extremely small bond angle would result in high reactivity of the carbyne moiety, which would undergo typical reactions for organic alkynes, including interaction with coinage metal complexes, electrophilic reactions, nucleophilic reactions and cycloaddition reactions, whereas the strong aromaticity ensured the integrity of the bicyclic framework of metallapentalynes throughout all reported reaction conditions.

Identification and Characterization of a Novel Protein ASP-3 Purified from Arca subcrenata and Its Antitumor Mechanism.

Diverse bioactive substances derived from marine organisms have been attracting growing attention. Besides small molecules and polypeptides, numerous studies have shown that marine proteins also exhibit antitumor activities. Small anticancer proteins can be expressed in vivo by viral vectors to exert local and long-term anticancer effects. Herein, we purified and characterized a novel protein (ASP-3) with unique antitumor activity from Arca subcrenata Lischke. The ASP-3 contains 179 amino acids with a molecular weight of 20.6 kDa. The spectral characterization of ASP-3 was elucidated using Fourier Transform infrared spectroscopy (FTIR) and Circular Dichroism (CD) spectroscopy. Being identified as a sarcoplasmic calcium-binding protein, ASP-3 exhibited strong inhibitory effects on the proliferation of Human hepatocellular carcinoma (HepG2) cells with an IC50 value of 171.18 ± 18.59 µg/mL, measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The RNA-seq analysis showed that ASP-3 regulated the vascular endothelial growth factor receptor (VEGFR) signaling pathway in HepG2 cells. Immunofluorescence results indicated that ASP-3 effectively reduced VEGFR2 phosphorylation in HepG2 cells and affected the downstream components of VEGF signaling pathways. The surface plasmon resonance (SPR) analysis further demonstrated that ASP-3 direct interacted with VEGFR2. More importantly, the therapeutic potential of ASP-3 as an anti-angiogenesis agent was further confirmed by an in vitro model using VEGF-induced tube formation assay of human umbilical vein endothelial cells (HUVECs), as well as an in vivo model using transgenic zebrafish model. Taken together, the ASP-3 provides a good framework for the development of even more potent anticancer proteins and provides important weapon for cancer treatment using novel approaches such as gene therapy.