Polystyrene nanoplastics exert cardiotoxicity through the Notch and Wnt pathways in zebrafish (Danio rerio).

The ubiquity of micro(nano)plastics has raised significant concerns among people. Their accumulation in the cardiovascular system necessitates attention to their cardiotoxicity. However, research on the cardiotoxicity of micro(nano)plastics remains scarce. Our study exposed zebrafish embryos to four different concentrations (0, 1, 10, 100 µg/mL) of polystyrene nanoplastics (PSNPs) for a period of 7 days. The results indicated that PSNPs noticeably decreased the hatching and survival rates of zebrafish and also induced cardiac developmental abnormalities. The mRNA level analysis revealed significant upregulations of heart development-related genes nkx2.5, cmlc-2, and myh-7 in response to PSNPs. Additionally, PSNPs significantly up-regulated the mRNA level associated with the Notch signaling pathway (notch-1a, jag-1a, and her-7) while remarkably suppressing the expression of the Wnt signaling pathway gene (wnt-3a). Further research showed that PSNPs significantly increased the expression of endoplasmic reticulum stress genes atf-6 and chop, while noticeably inhibiting mitochondrial copy numbers. Moreover, PSNPs were found to decrease calcium ion level and superoxide dismutase (SOD) activity in zebrafish larvae. Additionally, prolonged exposure to PSNPs for 7 days exacerbated abnormalities in various indicators compared to a 4-day exposure. In conclusion, our study demonstrates that PSNPs induce oxidative stress in zebrafish larvae, thereby activating endoplasmic reticulum stress and inhibiting mitochondrial activity, ultimately disrupting the Notch and Wnt signaling pathways. These disruptions result in abnormalities in cardiac developmental genes, ultimately leading to cardiac developmental abnormalities in zebrafish. The present research contributes to a novel understanding of the cardiotoxicity of PSNPs.

Strategies on biosynthesis and production of bioactive compounds in medicinal plants.

Medicinal plants are a valuable source of essential medicines and herbal products for healthcare and disease therapy. Compared with chemical synthesis and extraction, the biosynthesis of natural products is a very promising alternative for the successful conservation of medicinal plants, and its rapid development will greatly facilitate the conservation and sustainable utilization of medicinal plants. Here, we summarize the advances in strategies and methods concerning the biosynthesis and production of natural products of medicinal plants. The strategies and methods mainly include genetic engineering, plant cell culture engineering, metabolic engineering, and synthetic biology based on multiple "OMICS" technologies, with paradigms for the biosynthesis of terpenoids and alkaloids. We also highlight the biosynthetic approaches and discuss progress in the production of some valuable natural products, exemplifying compounds such as vindoline (alkaloid), artemisinin and paclitaxel (terpenoids), to illustrate the power of biotechnology in medicinal plants.

Enhanced poly-γ-glutamic acid synthesis in Corynebacterium glutamicum by reconstituting PgsBCA complex and fermentation optimization.

Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.

Time-resolved fluorescence microspheres-antibody-penicillin-binding protein assisted construction of immunochromatographic assay for sensitive detection of 22 ß-lactams in milk.

A sensitive immunochromatographic assay (ICA) using time-resolved fluorescence microspheres (TRFMs) coupled with an indirect-labeling mode was developed for simultaneously determining 22 kinds of ß-lactams in milk samples. The TRFMs labeled anti-receptor monoclonal antibodies (mAbs) conjugated to penicillin-binding proteins (PBPs) as ternary TRFMs-mAb-PBPs (TMP) nanoscaffolds provide excellent solubility, brightness, and stability. Thanks to the fact that they not only fully expose the binding sites of PBPs, thereby enhancing the biological affinity of PBPs towards the target, but also generated superb fluorescence signals, the versatile TMP manifested unique possibilities as efficient probes for ICA with remarkable enhancement in sensitivity in ß-lactams screening. The results showed that the standard curves of the 22 varying ß-lactams displayed linearity in their respective concentration ranges (R2 > 0.98), with the cutoff values of 1-100 ng/mL. The constructed TMP-ICA was successfully applied to the analysis of real milk, with consistent results compared with liquid chromatography-tandem mass spectrometry (LC-MS), providing an effective method for sensing ß-lactams in food matrices.

Construction of Marigold-like Poly(vinyl alcohol) Microspheres for Catalytic Microreactors.

It has long been pursued to develop polymer microspheres with various special morphologies and structures for better results in applications such as catalysis, drug delivery, and bioscaffolds. However, it remains a challenge to develop a facile method to produce poly(vinyl alcohol) (PVA) microspheres with special morphologies. Herein, a micron-sized marigold-like poly(vinyl alcohol) (CE-PVATPA) microsphere was engineered and fabricated by a feasible strategy, that is, emulsification-cross-linking, freeze-drying, and secondary acetal reaction steps. The morphological evolution of microspheres was systematically investigated under different conditions, and the procedure of constructing PVA microspheres with stabilizing marigold-like structures was proposed. More importantly, a specially structured PVA microsphere microreactor synergistically loading palladium metal nanoparticles (CE-PVATPA@Pd) for the heterogeneous catalyst 4-nitrophenol (4-NP) could be further demonstrated, which indicated high catalytic activity and excellent recyclability. The resultant stabilized fabricating method is promising to provide valuable guidance for the design and fabrication of a high-performance PVA microsphere microreactor.

Impacts of Climate Changes on Geographic Distribution of Primula filchnerae, an Endangered Herb in China.

Primula filchnerae, an endangered plant endemic to China, has drawn people's attention in recent years due to its ornamental value in flower. It was rarely recorded since being described in 1902, but it was rediscovered in 2009 and is now known from a limited number of sites located in Hubei and Shaanxi Provinces. Since the species is still poorly known, a number of unanswered questions arise related to it: How has P. filchnerae responded to past climate change and how might it respond in the future? Why was P. filchmerae so rarely collected during the past century? We assembled geographic coordinates for P. filchnerae through the field surveys and website searches, and then used a maximum entropy model (MaxEnt) to simulate its potential suitable distribution in six periods with varied carbon emission levels by combining bioclimatic and environmental factors. MaxEnt showed that Min Temperature of the Coldest Month (bio6) and Precipitation of the Coldest Quarter (bio19) affected P. filchnerae's distribution most, with an aggregate contribution >60% and suitable ranges above -5 °C and below 40 mm, respectively. We also analyzed potential habitat distribution in various periods with differing impacts of climate change compared to today's suitable habitats, and in most cases, Shaanxi and Sichuan remained the most stable areas and with possible expansion to the north under various carbon emission scenarios, but the 2050s SSP5-8.5 scenario may be an exception. Moreover, we used MaxEnt to evaluate population shifts, with various scenarios indicating that geometric center would be concentrated in Sichuan Province in China. Finally, conservation strategies are suggested, including the creation of protected areas, long-term monitoring, raising public awareness of plant conservation, situ conservation measures, assisted migration, and species introduction. This study demonstrates how P. filchnerae may have adapted to changes in different periods and provides a scientific basis for germplasm conservation and management.

Exposure to polystyrene nanoplastics and PCB77 induced oxidative stress, histopathological damage and intestinal microbiota disruption in white hard clam Meretrix lyrata.

The toxic effects of organic pollutants and nanoplastics on fish have been extensively studied, but there is limited research available on their combined toxicity to bivalves. This research aimed to investigate the accumulation and ecotoxicological impacts such as antioxidant capacity, histopathology and intestinal microbiota in white hard clam Meretrix lyrata, resulting from 7 days of single and mixture exposure to 3,3',4,4'-tetrachlorobiphenyl (PCB77, 0.1 mg/L) and polystyrene nanoplastics (PS-NPs, 80 nm, 1 mg/L). Our findings revealed that PS-NPs accumulated in various tissues such as the intestine, gill, mantle, foot, and siphon. And when compared to the PCB-PSNPs (PP) co-exposure group, the intestinal fluorescence intensity mediated by plastic particles in the PS-NPs (PS group) was significantly higher. The gill, digestive gland, and intestine were all damaged to varying extent by single exposure to PS-NPs or PCB77, according to histopathological analysis, which was aggravated by PP group. Moreover, the co-exposure induced a higher level of oxidative stress, which reflected by increase of activities of superoxide dismutase, catalase, glutamate oxaloacetate transaminase and glutamic-pyruvic transaminase and malondialdehyde content. In addition, the intestine microbial composition was dramatically altered by the combined exposure, reducing the abundance of probiotics such as Firmicutes, thereby posing a great threat to the health and metabolism of M. lyrata. In conclusion, our findings showed that PS-NPs and PCB77 co-exposure induced a higher toxicity to M. lyrata, including histopathological changes, altered antioxidant capacity and intestinal microbiota disruption. This study provides novel insights into PCB77 and PS-NPs' combined toxicity to marine organisms and its underlying molecular mechanisms of ecotoxicological effects.

Single and combined toxicity of polystyrene nanoplastics and PCB-52 to the aquatic duckweed Spirodela polyrhiza.

As nanoplastics and persistent organic pollutants are broadly distributed in aquatic ecosystems and pose a potential threat to ecosystem, most pertinent studies have focused on aquatic animals, while studies on freshwater plants have been rarely reported. Therefore, we analyzed the single and combined toxicological impacts of various concentrations of 80 nm polystyrene nanoplastics (PS-NPs) including 0.5, 5, 10, and 20 mg/L and polychlorinated biphenyl-52 (PCB-52, 2,2',5,5'- tetrachlorobiphenyl) at 0.1 mg/L on the aquatic plant Spirodela polyrhiza (S. polyrhiza) after a 10-day hydroponic experiment. Laser confocal scanning microscopy (LCSM) showed the accumulation of PS-NPs mainly in the root surface and the lower epidermis of leaves, and the enrichment of PS-NPs was aggravated by the presence of PCB-52. PS-NPs at 10 mg/L and 20 mg/L alone or in combination with PCB-52 notably inhibited the growth of S. polyrhiza, reduced the synthesis of chlorophylls a and b, and increased the activities of superoxide dismutase (SOD) and peroxidase (POD) as well as malondialdehyde (MDA) levels, and induced osmotic imbalance (soluble protein and soluble sugar contents) (p < 0.05). However, a single treatment with low levels of PS-NPs had positive effects on the growth (0.5 mg/L) and photosynthetic systems (0.5, 5 mg/L) of S. polyrhiza, while co-exposure exacerbated the damaging impacts of PS-NPs on the antioxidant defense system of S. polyrhiza, which was more pronounced in the roots. Furthermore, correlation analysis revealed that plant growth parameters were positively correlated with chlorophyll a and b content and negatively correlated with soluble sugars, antioxidant enzymes, lipid peroxidation, and carotenoid content (p < 0.05). These results provide data to improve the understanding of the single and combined ecotoxicological effects of nanoplastics and polychlorinated biphenyls (PCBs) in aquatic plants and their application in phytoremediation measures.

The Distribution Pattern and Species Richness of Scorpionflies (Mecoptera: Panorpidae).

The uneven distribution of species diversity on earth, with mountainous regions housing half of the high species diversity areas, makes mountain ecosystems vital to biodiversity conservation. The Panorpidae are ecological indicators, ideal for studying the impact of climate change on potential insect distribution. This study examines the impact of environmental factors on the distribution of the Panorpidae and analyzes how their distribution has changed over three historical periods, the Last Interglacial (LIG), the Last Glacial Maximum (LGM), and Current. The MaxEnt model is used to predict the potential distribution area of Panorpidae based on global distribution data. The results show that precipitation and elevation are the primary factors affecting species richness, and the suitable areas for Panorpidae are distributed in southeastern North America, Europe, and southeastern Asia. Throughout the three historical periods, there was an initial increase followed by a decrease in the area of suitable habitats. During the LGM period, there was a maximum range of suitable habitats for cool-adapted insects, such as scorpionflies. Under the scenarios of global warming, the suitable habitats for Panorpidae would shrink, posing a challenge to the conservation of biodiversity. The study provides insights into the potential geographic range of Panorpidae and helps understand the impact of climate change on their distribution.

Morphology, morphogenesis and molecular phylogeny of Lamtostyla granulifera sinensis subsp. nov. (Ciliophora, Hypotrichia) from a wetland in China.

The morphology, morphogenesis and molecular phylogeny of a hypotrichous ciliate, Lamtostyla granulifera sinensis subsp. nov., isolated from northern China, were investigated. This population appeared highly similar in morphology to L. granulifera Foissner, 1997. However, on detailed investigation some non-overlapping features were identified, i.e., the body shape and the arrangement of the cortical granules. These differences suggested the separation at subspecies level. Furthermore, the morphogenesis of the new subspecies is described, which is characterized by: (1) the posterior part of the parental adoral zone of membranelles is renewed; (2) the amphisiellid median cirral row is formed from two anlagen; and (3) the frontoventral-transverse cirral anlagen II to VI generate one transverse cirrus each. Phylogenetic analyses based on SSU rDNA sequence data show that Lamtostyla species are scattered in different clades. The monophyly of the genus Lamtostyla is also rejected by the AU test in this study.

Involvement of the DNA Phosphorothioation System in TorR Binding and Anaerobic TMAO Respiration in Salmonella enterica.

Although the phosphorothioate (PT) modification, in which the nonbridging oxygen in the DNA sugar-phosphate backbone is replaced by sulfur, has been reported to play versatile roles in multiple cellular processes, very little data have been obtained to define the role of PT in epigenetic regulation. In this study, we report that the PT system in Salmonella enterica serovar Cerro 87 is involved in the transcriptional regulation of the torCAD operon encoding the trimethylamine N-oxide (TMAO) respiration machinery that enables the use of TMAO as a terminal electron acceptor for respiration when oxygen is not available. In vitro, PT enhanced the binding of the transcriptional activator of the torCAD operon, namely, TorR, to its DNA substrate (tor boxes). However, in vivo, the PT modification protein complex DndCDE downregulated torCAD transcription through competing with the binding of TorR to the tor boxes. The altered expression of torCAD caused by PT modification proteins affected cell growth that relied on TMAO respiration. To our knowledge, this is the first report supporting that PT proteins participate in transcriptional regulation, showing a new function of PT systems. IMPORTANCE Since the initial discovery of DNA phosphorothioate (PT) modification systems in Streptomyces lividans in the 1980s, explorations of the biological functions of DNA PT systems have advanced and yielded a number of important findings. However, the functions of PT systems, especially in genetic regulation, remain largely unknown. In this study, we report a case in which the PT system participates in the transcriptional regulation of the torCAD operon in Salmonella enterica serovar Cerro 87. While the PT modification enhanced the binding of TorR, the torCAD operon transcriptional activator, to its DNA substrate in vitro, we found that the PT modification protein complex DndCDE directly competed with TorR binding in vivo and subsequently repressed the expression of torCAD and attenuated cell growth that relied on TMAO respiration. These findings provide a deeper understanding of the characteristics of the PT chemical structure and broaden our understanding of the mechanisms by which PT regulates gene expression.

An ultrasensitive, homogeneous fluorescence quenching immunoassay integrating separation and detection of aflatoxin M1 based on magnetic graphene composites.

A homogeneous fluorescence quenching immunoassay is described for simultaneous separation and detection of aflatoxin M1 (AFM1) in milk. The novel assay relies on monoclonal antibody (mAb) functionalized Fe3O4 decorated reduced-graphene oxide (rGO-Fe3O4-mAb) as both capture probe and energy acceptor, combined with tetramethylrhodamine cadaverine-labeled aflatoxin B1 (AFB1-TRCA) as the energy donor. In the assay, AFB1-TRCA binds to rGO-Fe3O4-mAb in the absence of AFM1, quenching the fluorescence of TRCA by resonance energy transfer. Significantly, the immunoassay integrates sample preparation and detection into a single step, by using magnetic graphene composites to avoid washing and centrifugation steps, and the assay can be completed within 10 min. Under optimized conditions, the visual and quantitative detection limits of the assay for AFM1 were 50 and 3.8 ng L-1, respectively, which were significantly lower than those obtained by fluorescence polarization immunoassay using the same immunoreagents. Owing to its operation and highly sensitivity, the proposed assay provides a powerful tool for the detection of AFM1.

An investigation of bacillary dysentery outbreaks in three schools in Ankang / 中国学校卫生

Objective@#To investigate risk factors and epidemiological characteristics of bacillary dysentery outbreaks in three schools, and to provide scientific basis for the prevention and control of the epidemic in the future.@*Methods@#Case definition was established. All suspected, possible and confirmed cases of all students and faculty members from 3 schools (A, B, C) were selected for epidemiological investigation. Control group was used for case-control analysis, and relevant samples were collected for laboratory testing.@*Results@#A total of 132 cases were found in 3 schools, all of which were from students, with the incidence rate of 17.74%. The morbidity in kindergarten A was 20.00%, in center primary school B it was 21.74%, and in junior middle school C it was 11.61%. Cohort studies and casecontrol studies suggested that schools are exposed places and that washing hands with raw water in schools was possible risk factor [OR(95%CI) =4.50(1.01-20.11)]. Nine stool samples were tested in laboratory, among which 8 were positive for Shigella(88.99%), and Shigella was detected in the end nodes of school s pipeline network, the water samples from canteen bucket, and the floor drains of sewer pipe.@*Conclusion@#The bacillary dysentery outbreaks in 3 schools was caused by Shigella, which may be due to fecal contamination of domestic water in 3 schools before the start of the school year. It is suggested to strengthen the management of centralized water supply and construction in rural areas, intensify the supervision at all levels, and sanitation and disinfection before school opens at all levels.

The Wolfiporia cocos Genome and Transcriptome Shed Light on the Formation of Its Edible and Medicinal Sclerotium.

Wolfiporia cocos (F. A. Wolf) has been praised as a food delicacy and medicine for centuries in China. Here, we present the genome and transcriptome of the Chinese strain CGMCC5.78 of W. cocos. High-confidence functional prediction was made for 9277 genes among the 10,908 total predicted gene models in the W. cocos genome. Up to 2838 differentially expressed genes (DEGs) were identified to be related to sclerotial development by comparing the transcriptomes of mycelial and sclerotial tissues. These DEGs are involved in mating processes, differentiation of fruiting body tissues, and metabolic pathways. A number of genes encoding enzymes and regulatory factors related to polysaccharide and triterpenoid production were strikingly regulated. A potential triterpenoid gene cluster including the signature lanosterol synthase (LSS) gene and its modified components were annotated. In addition, five nonribosomal peptide synthase (NRPS)-like gene clusters, eight polyketide synthase (PKS) gene clusters, and 15 terpene gene clusters were discovered in the genome. The differential expression of the velevt family proteins, transcription factors, carbohydrate-active enzymes, and signaling components indicated their essential roles in the regulation of fungal development and secondary metabolism in W. cocos. These genomic and transcriptomic resources will be valuable for further investigations of the molecular mechanisms controlling sclerotial formation and for its improved medicinal applications.

bHLH transcription factor SmbHLH92 negatively regulates biosynthesis of phenolic acids and tanshinones in Salvia miltiorrhiza.

Objective: Salvia miltiorrhiza is a valuable herbal medicine with tanshinone and phenolic acid as the main biological active ingredients. The biosynthetic regulation of these bioactive compounds is controlled by a set of transcription factors (TFs). The basic helix-loop-helix (bHLH) transcription factor plays an important role in various physiological and biochemical processes in plants. However, research on bHLH TFs regulating phenolic acid or tanshinone biosynthesis in S. miltiorrhiza is limited. Methods: qRT-PCR was used for gene expression analysis. The subcellular localization of SmbHLH92 was detected by SmbHLH92-GFP transient transformation into tobacco leaves, and its fluorescence was observed using a confocal laser scanning microscope. The transcriptional activity of SmbHLH92 was confirmed in the AH109 yeast strain. RNA interference hairy roots of SmbHLH92-RNAi transgenic lines were obtained through Agrobacterium-mediated genetic transformation. Ultra performance liquid chromatography (UPLC) was used to detect the changes of phenolic acids and tanshinones. Results: SmbHLH92 is a bHLH transcription factor that is highly expressed in the root and phloem of S. miltiorrhiza. The subcellular localization and transcriptional activity of SmbHLH92 indicated that SmbHLH92 was located in the nucleus and may be a transcription factor. RNA interference (RNAi) of SmbHLH92 in hairy roots of S. miltiorrhiza significantly increased the accumulation of phenolic acid and tanshinone. Quantitative RT-PCR (RT-qPCR) analysis showed the transcription level of genes encoding the key enzymes involved in the phenolic acid and tanshinone biosynthetic pathways was increased in the hairy roots of the SmbHLH92-RNAi transgenic line, comparing with the control line. Conclusion: These data indicate that SmbHLH92 is a negative regulator involved in the regulation of phenolic acid and tanshinone biosynthesis in S. miltiorrhiza.

Microcystin-LR promotes zebrafish (Danio rerio) oocyte (in vivo) maturation by activating ERK1/2-MPF signaling pathways, and cAMP is involved in this process.

Cyanobacterial blooms and their secondary metabolites, microcystins (MCs), are not only toxic to aquatic organisms, but also to humans. MCs exert reproductive toxicity in female fish by affecting the oocyte development. However, the mechanism behind MC-LR interference in oocyte development remains largely unknown. In our study, adult female zebrafish were exposed to MC-LR (0, 1, 5, 20 µg/L) for 30 d. After exposure to MC-LR for 30 d, fertilized eggs from the treated females and healthy males were collected and cultured in water without MC-LR. Histomorphological observations showed pathological damage in the ovary after MC-LR exposure, which was mainly characterized by enlarged intercellular spaces, detachment of follicular cells from oocytes, and vacuolation of parenchymal tissues. The 20 µg/L MC-LR treatment caused a remarkable increase in the rate of the zebrafish oocytes germinal vesicle breakdown (GVBD) and a significant decrease in the levels of cyclic adenosine monophosphate (cAMP) and vitellogenin (VTG). In addition, the phosphorylation levels of the extracellular signal-regulated kinases (ERK) were elevated in ovaries from zebrafish exposed to 5 and 20 µg/L MC-LR, and cyclinB phosphorylation levels were also upregulated notably in the 20 µg/L MC-LR group. However, MC-LR exposure did not cause any change in the levels of cAMP-dependent protein kinase (PKA) protein and cdc2 phosphorylation in all the treatments. All the doses of MC-LR reduced the number of eggs, prematurely hatched the fertilized eggs and increased the abnormal rate of offspring generation. In summary, the present study demonstrates that MC-LR promotes oocyte maturation by activating the ERK1/2 and MPF signaling pathways, and cAMP is involved in this process.

Microcystin-LR triggers different endoplasmic reticulum stress pathways in the liver, ovary, and offspring of zebrafish (Danio rerio).

The existence of microcystins (MCs), the secondary metabolite of cyanobacteria, has become a growing public health concern. Previous researches have proved that MCs can trigger endoplasmic reticulum stress (ERS), but the underlying mechanisms remain unclear. In the present study, adult female zebrafish were exposed to MC-LR (0, 1, 5 and 20 µg/L) for 30 d, and the offspring derived from the treated females and healthy males were cultured in water without MC-LR until 96 h post fertilization (hpf). Our data suggested that MC-LR causes a significant increase in the eif2s1a, atf4, and eif2ak3 transcription levels in the liver and ovary. The mRNA levels of atf4, atf6, bcl-2, hspa5, eif2s1a and eif2ak3 upregulated notably in the offspring. JNK phosphorylation level and cleaved-caspase3 protein expression elevated obviously in the liver and ovary, but had no remarkable change in the offspring. Furthermore, TUNEL results showed that MC-LR significantly induced apoptosis in the liver and ovary, while acridine orange (AO) staining indicated that MC-LR did not cause abnormal apoptosis in offspring. Concisely, the present study indicated that MC-LR leads to apoptosis through different ERS pathways in the liver, ovary and offspring, and also provides a new perspective for understanding the apoptosis caused by MC-LR.

Quantitation of cytidine-5'-monophospho-N-acetylneuraminic acid in human leukocytes using LC-MS/MS: method development and validation.

The biosynthesis of sialic acid (Neu5Ac) leads to the intracellular production of cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), the active sialic acid donor to nascent glycans (glycoproteins and glycolipids) in the Golgi. UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase myopathy is a rare autosomal recessive muscular disease characterized by progressive muscle weakness and atrophy. To quantify the intracellular levels of CMP-Neu5Ac as well as N-acetylmannosamine (ManNAc) and Neu5Ac in human leukocytes, we developed and validated robust liquid chromatography-tandem mass spectrometry methods. A fit-for-purpose approach was implemented for method validation. Hydrophilic interaction chromatography was used to retain three hydrophilic analytes. The human leukocyte pellets were lysed and extracted in a methanol-water mixture and the leukocyte extract was used for LC-MS/MS analysis. The lower limits of quantitation for ManNAc, Neu5Ac and CMP-Neu5Ac were 25.0, 25.0 and 10.0 ng/ml, respectively. These validated methods were applied to a clinical study.

Microcystin-LR influences the in vitro oocyte maturation of zebrafish by activating the MAPK pathway.

Harmful cyanobacteria and their production of microcystins (MCs) exert significant toxicity on reproduction of fish, especially the process of oogenesis. Our previous studies demonstrated that MCs have negative impacts on the quantity and quality of mature oocytes in female zebrafish. However, the underlying mechanisms of MCs disrupting oocyte maturation (OM) have been rarely reported. In the present study, in vitro oocytes (immature) were separated from zebrafish and treated with 1, 10, 100 µg/L MC-LR. The serine/threonine protein phosphatase 2A (PP2A) activity was downregulated significantly in oocytes exposed to 10 and 100 µg/L MC-LR for both 2 and 4 h. The phosphorylation levels of mitogen-activated protein kinase (MAPK) were detected without noticeable change in all oocytes treated with MC-LR for 2 h, whereas the activated levels of MAPK subtypes (ERK, p38 and JNK) increased remarkably in the 100 µg/L MC-LR treatment of 4 h. In the oocytes exposed to 100 µg/L MC-LR for 4 h, germinal vesicle breakdown (GVBD) rates changed abnormally and maturation-promoting factor (MPF) activity increased significantly, in accordance with the upregulation of Cyclin B protein levels. Moreover, the MAPK inhibitors (10 µM) were applied to explore the role of MAPK subtypes during MC-LR influencing OM and results showed that ERK inhibitor U0126 and p38 inhibitor SB203580 mitigated the effects of 100 µg/L MC-LR-induced MAPK hyper-phosphorylation and elevated GVBD in the oocytes. In conclusion, the present study indicates that microcystins disrupt the meiotic maturation by the pathway of MC-PP2A-MAPK-OM due to the phosphorylation disorder in oocytes.

Recovery of reproductive function of female zebrafish from the toxic effects of microcystin-LR exposure.

Fish has a strong resistance to microcystins (MCs), cyclic heptapeptide cyanotoxins, known as endocrine disrupting chemicals (EDCs) which are released during cyanobacterial blooms and many laboratory and field studies have found the hepatic recovery of fish from the MCs exposure. The aim of the present study was to investigate the recovery mechanisms of reproductive function of adult zebrafish (Danio rerio) from microcystin-LR (MC-LR) exposure. Therefore, adult female zebrafish were exposed to 0, 1 or 50 µg/L of MC-LR for 21days and transferred to MC free water for another 21 days to investigate the recovery. After MC-LR exposure, marked histological lesions in the gonads, decreased the percentage of mature oocytes, decreased number of spawned eggs, decreased fertilization and hatching rates were observed. MC-LR exposure increased the concentration of 17ß-estradiol (E2), testosterone (T) and vitellogenin (VTG) in female zebrafish. Some gene transcriptions of the hypothalamic-pituitary-gonad (HPG) axis significantly changed. The protein levels of 17ßhsd and cyp19a remarkably increased in the MC-LR exposure groups. However, our laboratory observation also indicates that zebrafish transferred from microcystin exposure to toxin-free water and reared for 21 days exhibited a nearly complete recovery of reproductive functions, including histological structure, increased the percentage of matured oocytes and spawned eggs, stable hormone levels, well-balanced transcriptional and translational levels. These results indicate that after MC-LR exposure, the reproductive impairments in zebrafish are also reversible likewise hepatic recovery seen by different studies in fish. Future studies should be conducted to explore a better understanding of the recovery mechanisms of fish from microcystins exposure.