Toxic effects of sodium dodecyl sulfate on planarian Dugesia japonica.

Sodium dodecyl sulfate (SDS) is an anionic surfactant, which is widely used in various fields in human life. However, SDS discharged into the water environment has a certain impact on aquatic organisms. In this study, planarian Dugesia japonica (D. japonica) was used to identify the toxic effects of SDS. A series of SDS solutions with different concentrations were used to treat planarians for the acute toxicity test , and the results showed that the semi-lethal concentration (LC50) of SDS to D. japonica at 24 h, 48 h, 72 h, and 96 h were 4.29 mg/L, 3.76 mg/L, 3.45 mg/L, and 3.20 mg/L respectively. After the planarians were exposed to 0.5 mg/L and 1.0 mg/L SDS solutions for 1, 3, and 5 days, the activities of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) content were measured to detect the oxidative stress and lipid peroxidation in planarians. Random amplified polymorphic DNA (RAPD) analysis was performed to detect the genotoxicity caused by SDS to planarians. The results showed that the activities of SOD, CAT, and MDA content increased after the treatment, indicating that SDS induced oxidative stress in planarians. RAPD analysis showed that the genomic template stability (GTS) values of planarians treated by 0.5 mg/L and 1.0 mg/L SDS for 1, 3, and 5 days were 67.86%, 64.29%, 58.93%, and 64.29%, 60.71%, 48.21%, respectively. GTS values decreased with the increasing of SDS concentration and exposure time, indicating that SDS had genotoxicity to planarians in a time and dose-related manner. Fluorescent quantitative PCR (qPCR) was used to investigate the effects of SDS on gene expression of planarians. After the planarians were exposed to 1.0 mg/L SDS solution for 1, 3, and 5 days, the expression of caspase3 was upregulated, and that of piwiA, piwiB, PCNA, cyclinB, and RAD51 were downregulated. These results suggested that SDS might induce apoptosis, affect cell proliferation, differentiation, and DNA repair ability of planarian cells and cause toxic effects on planarian D. japonica.

Genotoxicity Evaluation of Metformin in Freshwater Planarian Dugesia japonica by the Comet Assay and RAPD Analysis.

Objective: To investigate the genotoxicity of metformin on planarian with different concentrations and exposure times. Methods: The planarians were treated, respectively, with 10 mmol/L and 50 mmol/L metformin for 1, 3, and 5 days, and then, the comet assay and random amplified polymorphic DNA (RAPD) analysis were performed. 13 random primers were used for PCR amplification with the genomic DNAs as templates. Planarians cultured in clear water were used as the control. Genomic template stability (GTS) was calculated by comparing and analyzing the RAPD patterns of the control group and the treatment groups. Results: In the comet assay, DNA damage of planarians treated with 10 mmol/L metformin for 1, 3, and 5 days was 10.2%, 25.4%, and 36.8%, respectively, and that of planarians treated with 50 mmol/L metformin was 40.6%, 62.8%, and 65.4%, respectively. GTS values of planarians exposed to 10 mmol/L metformin for 1, 3, and 5 days were 64.1%, 62.8%, and 52.6%, respectively, and those of planarians exposed to 50 mmol/L metformin for 1, 3, and 5 days were 52.6%, 51.3%, and 50%, respectively. DNA damage increased and GTS values decreased with the increasing metformin exposure concentration and exposure time. Conclusion: Metformin has certain genotoxicity on planarian in a dose- and time-related manner. The comet assay and RAPD analysis are highly sensitive methods for detecting genotoxicity with drugs.

Sodium Benzoate Delays the Development of Drosophila melanogaster Larvae and Alters Commensal Microbiota in Adult Flies.

Sodium benzoate (SB), the sodium salt of benzoic acid, is widely used as a preservative in foods and drinks. The toxicity of SB to the human body attracted people's attention due to the excessive use of preservatives and the increased consumption of processed and fast foods in modern society. The SB can inhibit the growth of bacteria, fungi, and yeast. However, less is known of the effect of SB on host commensal microbial community compositions and their functions. In this study, we investigated the effect of SB on the growth and development of Drosophila melanogaster larvae and whether SB affects the commensal microbial compositions and functions. We also attempted to clarify the interaction between SB, commensal microbiota and host development by detecting the response of commensal microbiota after the intervention. The results show that SB significantly retarded the development of D. melanogaster larvae, shortened the life span, and changed the commensal microbial community. In addition, SB changed the transcription level of endocrine coding genes such as ERR and DmJHAMT. These results indicate that the slow down in D. melanogaster larvae developmental timing and shortened life span of adult flies caused by SB intake may result from the changes in endocrine hormone levels and commensal microbiota. This study provided experimental data that indicate SB could affect host growth and development of D. melanogaster through altering endocrine hormone levels and commensal microbial composition.

Exposure to polystyrene microplastics reduces regeneration and growth in planarians.

The potential adverse effects of microplastics (MPs) on ecosystems and human health have received much attention in recent years. However, only limited data are available on the mechanisms for the uptake, distribution, and effects of MPs in freshwater organisms, especially with respect to tissue repair, regeneration and impairment of stem cell functions. To address this knowledge gap, we conducted exposure experiments in which planarians (Dugesia japonica) were exposed to polystyrene (PS)-MPs mixed in liver homogenate and examined the tissue growth and regeneration, stem cell functions, and oxidative stress. The body and blastema areas decreased upon exposure to PS-MPs, indicating that the growth and regeneration of planarians were delayed. The proliferation and differentiation processes of stem cells were inhibited, and the proportion of mitotic stem cells decreased, which may be related to the activation of the TGFß/SMAD4 and Notch signaling pathways. The enhancement of antioxidant enzyme activities and malondialdehyde on the first day of exposure to PS-MPs confirmed the oxidative stress response of planarians to PS-MPs. The present study demonstrated the likelihood of biotoxicity induced by PS-MPs. These results will provide clues for further investigations into the potential risks of PS-MPs to human stem cells.

Plasma Glucosylsphingosine in GBA1 Mutation Carriers with and without Parkinson's Disease.

BACKGROUND: Biallelic mutations in the GBA1 gene encoding glucocerebrosidase cause Gaucher's disease, whereas heterozygous carriers are at risk for Parkinson's disease (PD). Glucosylsphingosine is a clinically meaningful biomarker of Gaucher's disease but could not be assayed previously in heterozygous GBA1 carriers. OBJECTIVE: The aim of this study was to assess plasma glucosylsphingosine levels in GBA1 N370S carriers with and without PD. METHODS: Glucosylsphingosine, glucosylceramide, and four other lipids were quantified in plasma from N370S heterozygotes with (n = 20) or without (n = 20) PD, healthy controls (n = 20), idiopathic PD (n = 20), and four N370S homozygotes (positive controls; Gaucher's/PD) using quantitative ultra-performance liquid chromatography tandem mass spectrometry. RESULTS: Plasma glucosylsphingosine was significantly higher in N370S heterozygotes compared with noncarriers, independent of disease status. As expected, Gaucher's/PD cases showed increases in both glucocerebrosidase substrates, glucosylsphingosine and glucosylceramide. CONCLUSIONS: Plasma glucosylsphingosine accumulation in N370S heterozygotes shown in this study opens up its future assessment as a clinically meaningful biomarker of GBA1-PD. © 2021 International Parkinson and Movement Disorder Society.

Unsupervised Reconstruction of Analyte-Specific Mass Spectra Based on Time-Domain Morphology with a Modified Cross-Correlation Approach.

Concomitant species that appear at the same or very similar times in a mass-spectral analysis can clutter a spectrum because of the coexistence of many analyte-related ions (e.g., molecular ions, adducts, fragments). One method to extract ions stemming from the same origin is to exploit the chemical information encoded in the time domain, where the individual temporal appearances inside the complex structures of chronograms or chromatograms differ with respect to analytes. By grouping ions with very similar or identical time-domain structures, single-component mass spectra can be reconstructed, which are much easier to interpret and are library-searchable. While many other approaches address similar objectives through the Pearson's correlation coefficient, we explore an alternative method based on a modified cross-correlation algorithm to compute a metric that describes the degree of similarity between features inside any two ion chronograms. Furthermore, an automatic workflow was devised to be capable of categorizing thousands of mass-spectral peaks into different groups within a few seconds. This approach was tested with direct mass-spectrometric analyses as well as with a simple, fast, and poorly resolved LC-MS analysis. Single-component mass spectra were extracted in both cases and were identified based on accurate mass and a mass-spectral library search.

IN SITU MASS SPECTROMETERS FOR APPLICATIONS IN SPACE.

Mass spectrometry (MS) has played a remarkable role in exploring the chemical make-up of our solar system. In situ probes were historically developed to analyze inorganic/elemental compositions while leveraging native ions or harsh ionization methods to aid in exploring astrophysics applications (e.g., heliophysics). The part played by MS is demonstrated in a majority of scientific payloads focused on exploration, particularly at the turn of the century with missions including Cassini-Huygens, Rosetta, and now Mars Science Laboratory. Plasma mass spectrometers have grown more sophisticated to interrogate fundamental inorganic analysis (e.g., solar wind and magnetospheres) including both native ions and neutrals. Cosmic dust floating in-between and orbiting planetary bodies has been targeted by unique sampling via impact ionization. More complex systems rely on landed planetary instrumentation with lessons learned from pioneering missions in the 1970s and 1980s to near neighbors Mars and Venus. Modern probes have expanded applicable target chemicals by recognizing the needs to provide for molecular analyses, extended mass range, and high resolution to provide unequivocal detection and identification. Notably, as the field surrounding astrobiology has gained momentum, so has the in situ detection of complex molecular chemistry including the chemical evolution of organic molecules. Mission context often includes long term timelines from spacecraft launch to arrival and additionally the diverse target environments across various planets. Therefore, customized experimental designs for space MS have been born of necessity. To this point, the development of MS instrumentation on Earth has now far outpaced development for experiments in space. Therefore, exciting developments lie ahead among various international space agencies conducting current and future mission planning with increasingly enhanced instrumentation. For instance, near-neighbor Mars has entertained considerable attention with complex MS instrumentation with laser desorption ionization aboard the Mars Organic Molecule Analyzer instrument. To study comets, the Rosetta mission employs a secondary ionization mechanism. Meanwhile, the various moons of Jupiter and Saturn have intriguing surface and subsurface properties that warrant more advanced analyzer systems. Instrumentation design will continue to evolve as requirements develop and this review serves as a reflection of the contribution of in situ MS to space exploration in the past 20 years and the anticipated contribution yet to come. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.

Inexpensive Ultrasonic Nebulization Coupled with Direct Current Corona Discharge Ionization Mass Spectrometry for Liquid Samples and Its Fundamental Investigations.

The concept of direct mass-spectrometric analysis, especially exploited by ambient desorption/ionization (ADI) methods, provides numerous means for convenient sample analysis. While many simple and versatile ionization sources have been developed, challenges lay in achieving efficient sample introduction. In previous work, a sample introduction method employing direct current corona discharge (CD) coupled to a surface acoustic wave nebulization (SAWN) device enhanced sampling performance for both polar and nonpolar analytes by up to 4 orders of magnitude. In fact, the SAWN-CD method generated a multiply charged peptide ion signal comparable to that of conventional ESI. Unfortunately, the high cost of the SAWN devices themselves limits their accessibility. Herein, we report on an analogous implementation of CD with an inexpensive ultrasonic nebulizer (USN) on the basis of a commercial room humidifier demonstrating equivalent exemplary performance. We subsequently compare the two methods of SAWN-CD and USN-CD in a screening application of milk for the detection of two antibiotic drugs, ciprofloxacin and ampicillin. Finally, we further investigate the relative softness of these CD-coupled acoustic nebulization methods in comparison to that of ESI using a survival yield study of the thermometer ion nitrobenzylpyridinium.

Molecular cloning and characterization of DjRac1, a novel small G protein gene from planarian Dugesia japonica.

Rac proteins are classified as a subfamily of the Rho family of small G proteins. They are important molecular switches which act as key signal transducers regulating a wide variety of processes in the cell. DjRac1, a novel Rac gene from planarian Dugesia japonica was cloned by RACE method and characterized. This cDNA contains 851 bp with a putative open reading frame of 190 amino acids. It has a predicted molecular mass of 21.12 kDa and an isoelectric point of 8.42. Whole-mount in situ hybridization and relative quantitative real-time PCR were used to study the spatial and temporal expression pattern of DjRac1 from 1 to 7 days in the regenerating planarians. Results showed that the expression of DjRac1 was concentrated in the blastema and the transcription level of DjRac1 was significantly upregulated after amputation within three days, suggesting DjRac1 might participate in the process of regeneration in planarian.

Effects of Fe3+ on Acute Toxicity and Regeneration of Planarian (Dugesia japonica) at Different Temperatures.

OBJECTIVE: To investigate the effects of different concentrations of Fe3+ on the acute toxicity and regeneration of planarian at different temperatures. METHOD: The planarians were treated with 40 mg/l, 50 mg/l, 60 mg/l, and 70 mg/l Fe3+ solution and placed in 15°C, 20°C, and 25°C, respectively, to observe the mortality and the poisoning pattern of the planarian. In addition, the planarians were cut into three parts of head, trunk, and tail, then placed in Fe3+ solution at concentrations of 10 mg/l, 15 mg/l, 20 mg/l, and 30 mg/l, and placed in 15°C, 20°C, and 25°C respectively, and the regeneration rate of the planarian was investigated. RESULTS: At the same temperature, in the concentration of Fe3+ from 40 mg/l to 70 mg/l, the mortality of the planarian increased with the increasing of the concentration of Fe3+; at the same concentration and different temperatures, the death speed of the planarian is the fastest at 20°C, the next at 25°C, and the lowest at 15°C, indicating that the toxic effect of Fe3+ can be accelerated at a suitable temperature of 20°C. At the same temperature, in the low concentration of Fe3+ from 10 mg/l to 30 mg/l, the regeneration rate of the planarian gradually decreased with the increasing of the concentration of Fe3+; at the same concentration and different temperature, the regeneration rate of planarian was faster at 20°C and 25°C, but the difference between 20°C and 25°C was small, and the slowest at 15°C, indicating that the low temperature significantly affects the planarian regeneration speed. The study also found the regeneration rates of the head, trunk, and tail of the planarian were different; the head regeneration was the fastest, the trunk was the second, and the tail was the slowest. CONCLUSION: Fe3+ had obvious toxic effects on the survival and regeneration of planarian; the planarian is sensitive to Fe3+ and may be used to detect Fe3+ water pollution; in addition, temperature can affect the toxic effects of Fe3+ and thus affect the survival and regeneration of the planarian. Therefore, the temperature should be taken into consideration when detecting water Fe3+ pollution.

Surface Acoustic Wave Nebulization with Atmospheric-Pressure Chemical Ionization for Enhanced Ion Signal.

Many ambient desorption/ionization mass spectrometry (ADI-MS) techniques rely critically on thermal desorption. Meanwhile, the analyte classes that are successfully studied by any particular ADI-MS methods are strongly dependent on the type of ionization source. Generally, spray-based ionization sources favor polar analytes, whereas plasma-based sources can be used for more hydrophobic analytes and are more suitable for molecules with small molar masses. In the present work, classic atmospheric-pressure chemical ionization (APCI) is used. To provide improved desorption performance for APCI, a surface acoustic wave nebulization (SAWN) device was implemented to convert liquid analytes into fine airborne particles. Compared to conventional SAWN that is used solely as an ionization source for liquid samples, the coupling of SAWN and APCI significantly improves ion signal by up to 4 orders of magnitude, reaching comparable ion abundances to those of electrospray ionization (ESI). Additionally, this coupling also extends the applicable mass range of an APCI source, conventionally known for the ionization of small molecules <500 Da. Herein, we discuss cursory evidence of this applicability to a variety of analytes including both polar and nonpolar small molecules and novel peptides that mimic biomolecules upward of 1000 Da. Observed species are similar to ESI-derived ions including doubly charged analyte ions despite presumably different charging mechanisms. SAWN-APCI coupling may thus involve more nuanced ionization pathways in comparison to other ADI approaches.

Molecular cloning and characterization of a novel Y-box gene from planarian Dugesia japonica.

Y-box binding protein (YB protein) is an ancient conserved multifunctional DNA/RNA-binding protein. A novel YB protein DjY2 gene from planarian Dugesia japonica was cloned by RACE method and characterized. This cDNA contains 689 bp with a putative open reading frame of 197 amino acids. It has a predicted molecular mass of 22.14 kDa and an isoelectric point of 9.67. Whole-mount in situ hybridization and relative quantitative real-time PCR were used to study the spatial and temporal expression pattern of DjY2 in the process of planarian regeneration. Results showed that DjY2 was expressed in many parts of the body in intact planarian, but the expression level was low in head and pharynx. The transcripts of DjY2 was significantly increased both at the head parts and the tail parts after amputation, especially at the site of cutting. The spatial expression gradually recovered to the state of intact planarian with the time of regeneration. Our results indicated that DjY2 might participate in the process of regeneration in planarian.

Upregulation of CX3CL1 mediated by NF-κB activation in dorsal root ganglion contributes to peripheral sensitization and chronic pain induced by oxaliplatin administration.

Painful peripheral neuropathy is a severe side effect in oxaliplatin therapy that compromises cancer patients' quality of life. However, its underlying pathogenic mechanisms remain largely unknown. Here, we found that intraperitoneal consecutive administration of oxaliplatin significantly increased excitability of small diameter dorsal root ganglion neurons and induced thermal hyperalgesia in rats. Furthermore, the CX3CL1 expression was significantly increased after oxaliplatin treatment, and intrathecal injection of a neutralizing antibody against CX3CL1 markedly attenuated the enhanced excitability of dorsal root ganglion neurons and thermal hyperalgesia. Importantly, the upregulated CX3CL1 is mediated by the NF-κB signaling pathway, as inhibition of NF-κB p65 activation with pyrrolidine dithiocarbamate or p65 siRNA inhibited the upregulation of CX3CL1, the enhanced excitability of dorsal root ganglion neurons, and thermal hyperalgesia induced by oxaliplatin. Further studies with chromatin immunoprecipitation found that oxaliplatin treatment increased the recruitment of NF-κB p65 to the CX3Cl1 promoter region. Our results suggest that upregulation of CX3CL1 in dorsal root ganglion mediated by NF-κB activation contributes to the peripheral sensitization and chronic pain induced by oxaliplatin administration.

Deep sequencing identifies regulated small RNAs in Dugesia japonica.

MicroRNAs (miRNAs), which are 18 ~ 24 nucleotides length, play important roles in regulating the expression of gene at the post-transcription level. Dugesia japonica is a branch of planarian organism. It is a model organism for studying the role of miRNAs in stem cell function. Next generation sequencing technology was used to identify the miRNAs of D. japonica. Bioinformatic analysis showed that 262 miRNA and miRNA* sequences were discovered, of which 102 miRNAs were the same as Schmidtea mediterranea and 160 miRNAs were related to other animals. There were 21 miRNAs expressed differentially after amputation. Results also revealed that some key miRNAs might play essential roles in the regeneration progress and some miRNAs might take part in the regulation progress of polarity regeneration in D. japonica.

Molecular cloning and characterization of a phosphoglycerate mutase gene from Clonorchis sinensis.

Phosphoglycerate mutase (PGM) is a widely distributed glycolytic enzyme. Two known distinct classes of PGM enzymes were identified, a cofactor-dependent one (dPGM) and a cofactor-independent one (iPGM). A complementary DNA (cDNA) encoding a PGM was cloned from a Clonorchis sinensis cDNA library by large-scale sequencing. This new cDNA contains 955 bp with a putative open reading frame of 256 amino acids, which has a high homology with dPGMs from a number of species. The putative peptide was produced in E. coli and was purified to electrophoretic homogeneity. Enzymatic assays showed that the product of this gene could catalyze the conversion of 3-phosphoglycerate to 2-phosphoglycerate when the cofactor was present and the enzyme activities could be inhibited by vanadate.

Clonorchis sinensis: molecular cloning and functional expression of novel cytosolic malate dehydrogenase.

The NAD-dependent cytosolic malate dehydrogenase (cMDH, EC 1.1.1.37) plays a pivotal role in the malate-aspartate shuttle pathway that operates in a metabolic coordination between cytosol and mitochondria, and thus is crucial for the survival and pathogenicity of the parasite. In the high throughput sequencing of the cDNA library constructed from the adult stage of Clonorchis sinensis, a cDNA clone containing 1152bp insert was identified to encode a putative peptide of 329 amino acids possessing more than 50% amino acid sequence identities with the cMDHs from other organisms such as fish, plant, and mammal. But low sequence similarities have been found between this cMDH and mitochondrial malate dehydrogenase as well as glyoxysomal malate dehydrogenase from other organisms. Northern blot analysis showed the size of the C. sinensis cMDH mRNA was 1.2 kb. The cMDH was expressed in Escherichia coli M15 as a His-tag fusion protein and purified by BD TALON metal affinity column. The recombinant cMDH showed high MDH activity of 241 U mg(-1), without lactate dehydrogenase and NADP(H) selectivity. It provides a model for the structure, function analysis, and drug screening on cMDH.

Molecular cloning and characterization of a novel adenylate kinase 3 gene from Clonorchis sinensis.

Adenylate kinase (AK) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in living cells. AK catalyzes reversible high energy phosphoryl transfer reactions between ATP (or GTP) and AMP to generate ADP (or GDP). From a Clonorchis sinensis adult worm cDNA library, we isolated a cDNA clone encoding a novel AK3 isozyme. The 956 bp cDNA encodes a putative protein of 228 amino acids with a predicted molecular mass of 26.2 kDa. The recombinant CsAK3 protein produced in Escherichia coli can be refolded into a functional protein with AK3 activity. The optimum pH and temperature for the enzyme are 8.5 and 40 degrees C, respectively. The calculated activation energy is 56.04 kJ mol-1. The Km of the CsAK3 for AMP and GTP are 118 microM and 359 microM, respectively. CsAK3 is inhibited by Ap5A (>70% inhibition by 2.0 mM AP5A). Ap5A may be a potential lead compound acting on C. sinensis in which AK3 as a drug target.

Molecular cloning and characterization of cDNA encoding a ubiquitin-conjugating enzyme from Clonorchis sinensis.

The ubiquitin-proteasome system is an essential mechanism for protein degradation in eukaryotes. Protein ubiquitination is composed of a series of enzymatic reactions. The ubiquitin-conjugating enzyme (E2) is one of the important enzymes involved in the process. A cDNA encoding an E2 enzyme was cloned from a Clonorchis sinensis cDNA library by large-scale sequencing. This new cDNA contains 862 bp with a putative open reading frame of 156 amino acids. The deduced amino acid sequence is 77% identical to the human E2, HHR6A and HHR6B. The coding region of this cDNA was expressed in E. coli as a GST-tagged protein, and was purified to electrophoretic homogeneity. Enzymatic assays showed that this E2 had the capacity to form a thiolester linkage, and could conjugate ubiquitin to histone H2A in an E3-independent manner in vitro, which indicated that the expressed protein was functionally active. The nucleotide sequence reported in this paper has been submitted to the Genbank Database with accession number AY632078.