miR-194-3p regulates epithelial-mesenchymal transition in embryonic epicardial cells via p120/ß-catenin signaling.

The epicardium is integral to cardiac development and facilitates endogenous heart regeneration and repair. While miR-194-3p is associated with cellular migration and invasion, its impact on epicardial cells remains uncharted. In this work we use gain-of-function and loss-of-function methodologies to investigate the function of miR-194-3p in cardiac development. We culture embryonic epicardial cells in vitro and subject them to transforming growth factor ß (TGF-ß) treatment to induce epithelial-mesenchymal transition (EMT) and monitor miR-194-3p expression. In addition, the effects of miR-194-3p mimics and inhibitors on epicardial cell development and changes in EMT are investigated. To validate the binding targets of miR-194-3p and its ability to recover the target gene-phenotype, we produce a mutant vector p120-catenin-3'UTR-MUT. In epicardial cells, TGF-ß-induced EMT results in a notable overexpression of miR-194-3p. The administration of miR-194-3p mimics promotes EMT, which is correlated with elevated levels of mesenchymal markers. Conversely, miR-194-3p inhibitor attenuates EMT. Further investigations reveal a negative correlation between miR-194-3p and p120-catenin, which influences ß-catenin level in the cell adhesion pathway. The suppression of EMT caused by the miR-194-3p inhibitor is balanced by silencing of p120-catenin. In conclusion, miR-194-3p directly targets p120-catenin and modulates its expression, which in turn alters ß-catenin expression, critically influencing the EMT process in the embryonic epicardial cells via the cell adhesion mechanism.

Wogonin improves colitis by activating the AhR pathway to regulate the plasticity of ILC3/ILC1.

BACKGROUND: Intestinal barrier dysfunction caused by the disrupted balance of group 3 innate lymphoid cells (ILC3)/group 1 innate lymphoid cells (ILC1) is a significant feature in the pathogenesis of inflammatory bowel disease (IBD). Activation of aryl hydrocarbon receptor (AhR) signaling contributes to the maintenance of ILC3/ILC1 balance. Wogonin, a natural flavonoid from Scutellaria baicalensis Georgi, can repair intestinal mucosal damage of IBD. However, it remains unclear if wogonin can exert a therapeutic effect by activating the AhR pathway to regulate the plasticity of ILC3/ILC1. PURPOSE: In this study, we investigated the immunomodulatory effects of wogonin on IBD and its potential mechanisms in vitro and in vivo. STUDY DESIGN AND METHODS: Chronic colitis was induced by four cycles of 2 % DSS treatment in mice. 20 mg kg-1/day wogonin was administrated by oral gavage and mice were treated intraperitoneally with 10 mg kg-1/2 days CH223191 to block the AhR pathway. Colon tissues were processed for histopathological examination and evaluation of the epithelial barrier function by immunohistochemistry. The activation of the AhR pathway and the plasticity of ILC3/ILC1 were determined by western blot and flow cytometry. Then, we also detected the intestinal microflora and their metabolites by 16 s sequencing and non-targeted Metabolomics analysis. Furthermore, an in vitro culture system consisting of MNK3 cells and NCM460 cells, and a CETSA assay were performed to confirm the molecular mechanism. RESULTS: Wogonin ameliorated histological severity of the colon, decreased the secretion of inflammatory factors, and increased tight junction proteins in colitis mice. These effects are associated with the tendency of conversion from ILC3 to ILC1 prevented by wogonin, which was offset by AhR antagonist CH223191. In addition, wogonin exerted the curative effect by altering gut microbiota to produce metabolites such as Kynurenic acid, and 1H-Indole-3-carboxaldehyde as AhR endogenous ligands. In vitro data further verified that wogonin as an exogenous ligand directly binds to the structural domain of AhR by CETSA. Also, the supernatant of MNK-3 cells stimulated with wogonin enhanced expression of Occludin and Claudin1 in NCM460 cells induced by LPS. CONCLUSION: Cumulatively, our study illustrated that wogonin improved the outcomes of DSS-induced chronic colitis via regulating the plasticity of ILC3/ILC1. Its specific mechanism is to binding to AhR directly, and to activate the AhR pathway indirectly by altering the tryptophan metabolisms of gut microbiota.

Determination of marker residues of quinoxaline-1,4-di-N-oxides and its prototype identification by liquid chromatography tandem mass spectrometry.

Quinoxaline-1,4-di-N-oxides (QdNOs), such as carbadox, olaquindox, mequindox, quinocetone, etc. are a class of antibacterial drugs. Prototype drugs residues can not be detected due to their rapid metabolism in animals. Quinoxaline-2-carboxylic acid (QCA) and 3-methyl-QCA (MQCA) are their common marker residues, so it has been always a challenge to trace the specific QdNOs drug used in food animal production. Herein, a liquid chromatography tandem mass spectrometry method was developed to determine QCA and MQCA, and meanwhile, the prototype drugs were identified by analyzing bis-desoxy QdNOs metabolites in single ion-pair monitoring mode. The method indicated that the average recoveries for QCA and MQCA were from 90 % to 105 % with relative standard deviations below 10 %, and the limits of quantification were 1.0 µg/kg. The limits of detection of five bis-desoxy QdNOs (qualitative markers) reached 0.5 µg/kg. This new analytical strategy can effectively solve the identification problem of QdNOs drugs in animal-derived food.

Angstrom-Scale Electrochemistry at Electrodes with Dimensions Commensurable and Smaller than Individual Reacting Species.

In nature and technologies, many chemical reactions occur at interfaces with dimensions approaching that of a single reacting species in nano- and angstrom-scale. Mechanisms governing reactions at this ultimately small spatial regime remain poorly explored because of challenges to controllably fabricate required devices and assess their performance in experiment. Here we report how efficiency of electrochemical reactions evolves for electrodes that range from just one atom in thickness to sizes comparable with and exceeding hydration diameters of reactant species. The electrodes are made by encapsulating graphene and its multilayers within insulating crystals so that only graphene edges remain exposed and partake in reactions. We find that limiting current densities characterizing electrochemical reactions exhibit a pronounced size effect if reactant's hydration diameter becomes commensurable with electrodes' thickness. An unexpected blockade effect is further revealed from electrodes smaller than reactants, where incoming reactants are blocked by those adsorbed temporarily at the atomically narrow interfaces. The demonstrated angstrom-scale electrochemistry offers a venue for studies of interfacial behaviors at the true molecular scale.

Analog signal metasurface processor supporting mathematical operator reconfiguration.

Electromagnetic wave analog computing is an effective method to overcome the bottleneck of electronic computing, which has attracted the attention of scientists. However, many spatial analog signal processing systems based on electromagnetic waves can only execute one unique mathematical operator and cannot provide multiple operators for users to choose arbitrarily. In order to enhance the function of the current spatial analog computing system, we design a coding structure with amplitude-phase decoupling modulation to realize the analog signal processor that supports the switching of mathematical operators and demonstrate the precise switching from the first-order spatial differential operator to the first-order spatial integral operator. Our design idea can be used as a paradigm for designing small reconfigurable analog computing systems, paving the way for the construction of high-speed, multifunctional, and universal signal processing systems. This idea can be extended to any other range of waves.

Exercise to prevent the negative effects of sleep deprivation: A systematic review and meta-analysis.

Ample sleep is an important basis for maintaining health, however with the pace of life accelerating in modern society, more people are using sacrificial sleep to cope with these social changes. Sleep deprivation can have negative effects on cognitive performance and psychosomatic health. It is well known that exercise, as a beneficial intervention strategy for human health, has been increasingly used in the clinic. But it's not clear if it can prevent the negative effects of sleep deprivation. In this meta-analysis, we reviewed 23 articles from PubMed and Web of Science to investigate whether moderate physical exercise can prevent the negative effects of sleep deprivation in rodents. Our findings suggest that exercise can prevent sleep deprivation-induced cognitive impairment and anxiety-like behaviors through multiple pathways. We also discuss possible molecular mechanisms involved in this protective effect, highlighting the potential of exercise as a preventive or therapeutic strategy for sleep deprivation-induced negative effects.

Associations of starchy and non-starchy vegetables with risk of metabolic syndrome: evidence from the NHANES 1999-2018.

BACKGROUND: Higher dietary quality, including increased vegetable consumption, was associated with a reduced risk of metabolic syndrome (MetS). However, specific vegetable consumption in the development of MetS remains obscure. Our study aimed to investigate the correlation between starchy and non-starchy vegetables and MetS. METHODS: Secondary data analysis from the National Health and Nutrition Examination Survey (NHANES 1999-2018). MetS was defined by National Cholesterol Education Program-Adult treatment Panel III (NCEP ATPIII) and dietary consumption was assessed by trained staff using two 24-h diet recall methods. Weighted logistic regression analysis was carried out to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Subgroup analyses and restricted cubic spline (RCS) regression were performed to further investigate specific vegetable subtypes and MetS. RESULTS: This research enrolled 24,646 individuals (11,725 females and 12,921 males), with an average age of 45.84 ± 0.23 years. Approximately 15,828(64.22%) participants were defined to be with non-MetS and 8818(35.78%) were with MetS. Both total starchy vegetables and potatoes were associated with increased MetS risk, with the corresponding OR per standard deviation (SD) (95%CI, p-trend) being 1.06(1.02-1.11, p-trend = 0.028) and 1.08(1.04-1.13, p-trend = 0.011), respectively. However, an inverse correlation was found between dark-green vegetables and MetS, and the OR per SD (95%CI, p-trend) was 0.93(0.90-0.97, p-trend = 0.010). Subgroup analyses showed that the positive associations of starchy vegetables and potatoes on MetS risk were stronger in non-Hispanic White participants (p for interaction < 0.050). CONCLUSION: Total starchy vegetables and white potatoes were both associated with an increased risk of MetS, while consumption of dark-green vegetables was negatively associated with MetS risk. These findings might provide a promising and healthy dietary strategy for preventing MetS.

Social rank-dependent effects of testosterone on huddling strategies in mice.

Huddling behavior, a typical social interaction among animals, has the benefits of obtaining social support and adapting environment. Huddling behavior is determined by social (social hierarchy), environmental factors (stress events), and the neuroendocrine system. Nevertheless, the huddling behavior of different social hierarchies and the underlying mechanisms have not been fully elucidated. In the present study, acute 2-methyl-2-thiazoline (2 MT) can induce huddling behavior and significantly increase serum levels of testosterone (T) in mice; and the increased T level was positively correlated with huddling behavior. Further, the T treatment significantly increased the huddling behavior in mice under 2 MT exposure condition. More interestingly, T can quickly promote dominant individuals to occupy safe positions when huddling together under predator odor. Collectively, T can rapidly regulate the individual's adaptive response to threats in a social rank-dependent manner, which provides a new perspective for the in-depth study of the influencing factors and underlying mechanisms of huddling behavior.

Genome-wide identification and characterization of Toll-like receptors (TLR) genes in Haliotis discus hannai, H. rufescens, and H. laevigata.

Toll-like receptors (TLRs) play essential roles in the innate immune system and have been extensively studied in mollusks. In this study, through a genome-wide search, TLR genes were identified as 29 in Haliotis discus hannai, 33 in H. rufescens, and 16 in H. laevigata. Domain analysis indicated that these TLR genes contain leucine-rich repeat (LRR) and Toll/IL-1 receptor (TIR) domains and exons ranging from 1 to 5. Polymorphism analysis showed that the TLRs in abalones did not have high diversities with 143 SNPs and no Indel in H. discus hannai, 92 SNPs and 3 Indels together with 6 missense mutations in H. rufescens, and no SNP or Indel in H. laevigata. The expression of 8 TLR genes in H. discus hannai was confirmed in the hepatopancreas, gill, hemolymph, gonads, intestine, muscle, and mantle. The expression of five TLR genes (out of 8) in gills (p < 0.05), three in hepatopancreas (p < 0.05), and three in hemolymph (p < 0.05) was upregulated separately in response to the infection caused by Vibrio parahaemolyticus. The findings in this study would contribute to a better understanding of the molecular immune mechanism of H. discus hannai against stimulation by V. parahaemolyticus and provide a basis for the study of TLRs in abalones.

Association between meteorological factors and the epidemics of influenza (sub)types in a subtropical basin of Southwest China.

BACKGROUND: The effects of climatic conditions on the prevalence of individual influenza (sub)types are not well understood in the subtropics. This study aims to evaluate the associations between meteorological factors and seasonal epidemics of A(H3N2), A(H1N1)pdm09, and type B influenza viruses, as well as to estimate the interactions between climatic variables in a subtropical basin region. METHODS: The seasonality of influenza (sub)types during 2010-2019 were characterized in Chengdu Plain Economic Zone, a densely populated and highly humid plain area in Sichuan Basin in subtropical Southwest China. Generalized additive models were adopted to assess the independent exposure-response relationship between meteorological variables and influenza prevalence. The interactions of meteorological variables were further estimated using bivariate response surface models and strata models. RESULTS: Our analyses indicated that the temperature, relative humidity, and absolute humidity have exhibited a major influence on influenza infection in Chengdu Plain Economic Zone. Low temperature was shown to promote the prevalence of A(H1N1)pdm09 and type B in winter-spring days at all levels of relative humidity. High risk of A(H3N2) infections was observed at low temperature or high temperature, and at higher relative humidity. Moreover, absolute humidity decreased or increased influenza (sub)type infections within different ranges. CONCLUSIONS: This study found different nonlinear relationships between meteorological factors and the seasonality of influenza (sub)types, as well as significant interactive effects between climatic variables, contributing to the research on the climate drivers of influenza prevalence in warm-humid basin regions in the subtropics.

Transcription factor Foxp1 stimulates angiogenesis in adult rats after myocardial infarction.

Forkhead box protein P1 (FoxP1) is essential for cardiac development and the regulation of neovascularization, but its potential for cardiac angiogenesis has not been explored. This study aims to investigate the angiogenic role of FoxP1 in a rat model of myocardial infarction (MI). Adult male rats were subjected to MI, and Foxp1 was knocked down with lentivirus FoxP1 siRNA. Endothelial cell proliferation, angiogenesis, and cardiac function were also assessed. Cell scratch assay and tubule formation analysis were used to detect the migration ability and tube formation ability of human umbilical vein endothelial cells (HUVECs). Compared with that in the sham group, results showed that the expression of FoxP1 was significantly increased in the MI group. Foxp1 knockdown decreases FoxP1 expression, reduces angiogenesis, and increases collagen deposition. When Foxp1 was knocked down in HUVECs using FoxP1 siRNA lentivirus, cell proliferation, migration, and tube formation abilities decreased significantly. Our study showed that FoxP1 elicits pleiotropic beneficial actions on angiogenesis in the post-MI heart by promoting the proliferation of endothelial cells. FoxP1 should be considered a candidate for therapeutic cardiac angiogenesis.

Identification of key genes and mechanisms of epicardial adipose tissue in patients with diabetes through bioinformatic analysis.

Background: In recent years, peri-organ fat has emerged as a diagnostic and therapeutic target in metabolic diseases, including diabetes mellitus. Here, we performed a comprehensive analysis of epicardial adipose tissue (EAT) transcriptome expression differences between diabetic and non-diabetic participants and explored the possible mechanisms using various bioinformatic tools. Methods: RNA-seq datasets GSE108971 and GSE179455 for EAT between diabetic and non-diabetic patients were obtained from the public functional genomics database Gene Expression Omnibus (GEO). The differentially expressed genes (DEGs) were identified using the R package DESeq2, then Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were analyzed. Next, a PPI (protein-protein interaction) network was constructed, and hub genes were mined using STRING and Cytoscape. Additionally, CIBERSORT was used to analyze the immune cell infiltration, and key transcription factors were predicted based on ChEA3. Results: By comparing EAT samples between diabetic and non-diabetic patients, a total of 238 DEGs were identified, including 161 upregulated genes and 77 downregulated genes. A total of 10 genes (IL-1ß, CD274, PDCD1, ITGAX, PRDM1, LAG3, TNFRSF18, CCL20, IL1RN, and SPP1) were selected as hub genes. GO and KEGG analysis showed that DEGs were mainly enriched in the inflammatory response and cytokine activity. Immune cell infiltration analysis indicated that macrophage M2 and T cells CD4 memory resting accounted for the largest proportion of these immune cells. CSRNP1, RELB, NFKB2, SNAI1, and FOSB were detected as potential transcription factors. Conclusion: Comprehensive bioinformatic analysis was used to compare the difference in EAT between diabetic and non-diabetic patients. Several hub genes, transcription factors, and immune cell infiltration were identified. Diabetic EAT is significantly different in the inflammatory response and cytokine activity. These findings may provide new targets for the diagnosis and treatment of diabetes, as well as reduce potential cardiovascular complications in diabetic patients through EAT modification.

Evolution and functional diversification of catalase genes in the green lineage.

BACKGROUND: Catalases (CATs) break down hydrogen peroxide into water and oxygen to prevent cellular oxidative damage, and play key roles in the development, biotic and abiotic stresses of plants. However, the evolutionary relationships of the plant CAT gene family have not been systematically reported. RESULTS: Here, we conducted genome-wide comparative, phylogenetic, and structural analyses of CAT orthologs from 29 out of 31 representative green lineage species to characterize the evolution and functional diversity of CATs. We found that CAT genes in land plants were derived from core chlorophytes and detected a lineage-specific loss of CAT genes in Fabaceae, suggesting that the CAT genes in this group possess divergent functions. All CAT genes were split into three major groups (group α, ß1, and ß2) based on the phylogeny. CAT genes were transferred from bacteria to core chlorophytes and charophytes by lateral gene transfer, and this led to the independent evolution of two types of CAT genes: α and ß types. Ten common motifs were detected in both α and ß groups, and ß CAT genes had five unique motifs, respectively. The findings of our study are inconsistent with two previous hypotheses proposing that (i) new CAT genes are acquired through intron loss and that (ii) the Cys-343 residue is highly conserved in plants. We found that new CAT genes in most higher plants were produced through intron acquisition and that the Cys-343 residue was only present in monocots, Brassicaceae and Pp_CatX7 in P. patens, which indicates the functional specificity of the CATs in these three lineages. Finally, our finding that CAT genes show high overall sequence identity but that individual CAT genes showed developmental stage and organ-specific expression patterns suggests that CAT genes have functionally diverged independently. CONCLUSIONS: Overall, our analyses of the CAT gene family provide new insights into their evolution and functional diversification in green lineage species.

Metformin Ameliorates Chronic Colitis-Related Intestinal Fibrosis via Inhibiting TGF-ß1/Smad3 Signaling.

Intestinal fibrosis is considered to be a chronic complication of inflammatory bowel disease (IBD) and seriously threatening human health. Effective medical therapies or preventive measures are desirable but currently unavailable. Metformin has been proved to have a satisfactory anti-inflammatory effects in ulcerative colitis (UC) patients. Whether metformin can ameliorate chronic colitis-related intestinal fibrosis and the possible mechanisms remain unclear. Here, we established colitis-related intestinal fibrosis in mice by repetitive administration of TNBS or DSS. Preventive and therapeutic administration of metformin to chronic TNBS or DSS colitis mice indicated that metformin significantly attenuated intestinal fibrosis by suppressing Smad3 phosphorylation. In vitro studies with human colon fibroblast cell line (CCD-18Co) and primary human intestinal fibroblast treated with TGF-ß1 confirmed the anti-fibrotic function of metformin for fibroblast activation, proliferation and collagen production. Mechanistically, metformin particularly inhibited phosphorylation and nuclear translocation of Smad3 by blocking the interaction of Smad3 with TßRI. These findings suggest that metformin will be an attractive anti-fibrotic drug for intestinal fibrosis in future therapies.

Multivessel vs. Culprit Vessel-Only Percutaneous Coronary Intervention for ST-Segment Elevation Myocardial Infarction in Patients With Cardiogenic Shock: An Updated Systematic Review and Meta-Analysis.

Background: The optimal revascularization strategy in patients with ST-segment elevation myocardial infarction (STEMI) complicating by cardiogenic shock (CS) remains controversial. This study aims to evaluate the clinical outcomes of multivessel percutaneous coronary intervention (MV-PCI) compared to culprit vessel-only PCI (CO-PCI) for the treatment, only in patients with STEMI with CS. Methods: A comprehensive literature search was conducted. Studies assessed the efficacy outcomes of short (in-hospital or 30 days)/long-term mortality, cardiac death, myocardial reinfarction, repeat revascularization, and safety outcomes of stroke, bleeding, acute renal failure with MV-PCI vs. CO-PCI in patients with STEMI with CS were included. The publication bias and sensitivity analysis were also performed. Results: A total of 15 studies were included in this meta-analysis. There was no significant difference in short- and long-term mortality in patients treated with MV-PCI compared to CO-PCI group [odds ratio (OR) = 1.17; 95% confidence interval (CI), 0.92-1.48; OR = 0.86; 95% CI, 0.58-1.28]. Similarly, there were no significant differences in cardiac death (OR = 0.67; 95% CI, 0.44-1.00), myocardial reinfarction (OR = 1.24; 95% CI, 0.77-2.00), repeat revascularization (OR = 0.75; 95% CI, 0.40-1.42), bleeding (OR = 1.53; 95% CI, 0.53-4.43), or stroke (OR = 1.42; 95% CI, 0.90-2.23) between the two groups. There was a higher risk in acute renal failure (OR = 1.33; 95% CI, 1.04-1.69) in patients treated with MV-PCI when compared with CO-PCI. Conclusion: This meta-analysis suggests that there may be no significant benefit for patients with STEMI complicating CS treated with MV-PCI compared with CO-PCI, and patients are at increased risk of developing acute renal failure after MV-PCI intervention.

Analysis of the Expression and Function of Key Genes in Pepper Under Low-Temperature Stress.

The mechanism of resistance of plants to cold temperatures is very complicated, and the molecular mechanism and related gene network in pepper are largely unknown. Here, during cold treatment, we used cluster analysis (k-means) to classify all expressed genes into 15 clusters, 3,680 and 2,405 differentially expressed genes (DEGs) were observed in the leaf and root, respectively. The DEGs associated with certain important basic metabolic processes, oxidoreductase activity, and overall membrane compositions were most significantly enriched. In addition, based on the homologous sequence alignment of Arabidopsis genes, we identified 14 positive and negative regulators of the ICE-CBF-COR module in pepper, including CBF and ICE, and compared their levels in different data sets. The correlation matrix constructed based on the expression patterns of whole pepper genes in leaves and roots after exposure to cold stress showed the correlation between 14 ICE-CBF-COR signaling module genes, and provided insight into the relationship between these genes in pepper. These findings not only provide valuable resources for research on cold tolerance, but also lay the foundation for the genetic modification of cold stress regulators, which would help us achieve improved crop tolerance. To our knowledge, this is the first study to demonstrate the relationship between positive and negative regulators related to the ICE-CBF-COR module, which is of great significance to the study of low-temperature adaptive mechanisms in plants.

Comparative Transcriptomics of the Northern Quahog Mercenaria mercenaria and Southern Quahog Mercenaria campechiensis in Response to Chronic Heat Stress.

The northern quahog (Mercenaria mercenaria) supports lucrative aquaculture industries in the USA. In the southeastern USA, aquacultured M. mercenaria faces increasing risks of summer die-offs from prolonged heat waves. We used a comparative transcriptomic approach to investigate the molecular responses of M. mercenaria and its southern congener, Mercenaria campechiensis, to controlled incremental heat stress over a 4-week period. Mercenaria were exposed to temperatures from 24 to 34 °C with 2.5 °C/week, after which, gill transcriptomes were de novo assembled and annotated. During the 4 weeks of chronic heat exposure, both species had the same survival rate (96%); M. mercenaria experienced body weight gain/loss depending on the originated hatcheries while M. campechiensis experienced an average net weight loss. The upregulated genes in both species included those in chaperone-mediated protein folding and regulation of cell death pathways, while the downregulated genes in both species involved in mRNA processing and splicing pathways. Compared to M. mercenaria, M. campechiensis appears to be more sensitive to prolonged heat stress as indicated by upregulating significantly more genes in coping with oxidative stress and in the protein degradation pathways, while downregulating some inhibitors of apoptosis. We discussed this finding within their ecological and evolutionary context. Our findings highlighted the potential vulnerability of the two quahogs, especially the southern quahog, to continued ocean warming.

Is catalase an effective additive to alleviate oxidative stress during cryopreservation of zebrafish sperm at the repository level?

The goal of this study was to investigate whether supplementation of cryoprotective medium with catalase (CAT), an antioxidation enzyme, is efficient for zebrafish sperm cryopreservation from the viewpoint of high-throughput genetic repository operations. Three cryoprotectants (10%, v/v), dimethylacetamide (DMA), dimethylformamide (DMF), and methanol were used. The objectives were to evaluate the effects of CAT on sperm motility, plasma membrane integrity, and concentration for: 1) fresh sperm at equilibration up to 60 min; 2) post-thaw sperm after cooling at 10, 20, and 40 °C/min), and 3) post-thaw fertilization and embryo survival rates. Catalase addition did not improve sperm motility, regardless of the cryoprotectants added. After 10-min exposure to DMA or methanol, membrane integrity was significantly decreased (70-75%) compared to controls. With catalase, sperm cells maintained membrane integrity and after 50 min equilibration, cell concentrations were maintained with CAT compared to cryoprotectant-only test groups. However, after cryopreservation and thawing, CAT did not affect the outcome of motility, membrane integrity, cell concentration, fertilization, or embryo survival assays. Analysis of cooling rates also indicated that CAT did not affect 3-hpf fertilization or 24-hpf survival rates. Overall, addition of CAT could provide some protection of sperm from oxidative stress before freezing, but not after thawing. We propose that decisions concerning routine use of CAT for repositories, especially those handling tens of thousands of frozen samples per year, would depend on whether efficient high-throughput operation, or specific research questions are programmatic goals.

Gene cloning and transcriptional regulation of the alkaline and acid phosphatase genes in Scylla paramamosain.

In crustaceans, innate immunity serves as the frontline of defense against microbes. Alkaline phosphatases (ALPs) and acid phosphatases (ACPs) are essential enzymes that play a significant role in crustaceans' immune defenses. However, the function and transcriptional regulation of the alp and acp genes in the Scylla paramamosain, an important aquaculture species in China, have not been elucidated. In this study, the full-length cDNAs of Spalp and Spacp were identified, which consist of 2,718 bp and 3,768 bp, encoding 579 and 452 amino acids, respectively. Multiple sequence alignment and phylogenetic analysis showed that these two genes were conserved among different species and shared high homology with crustaceans. The mRNA expression of Spalp and Spacp were examined in eight tested tissues, with the highest levels in the hepatopancreas. The 5'-flanking regions of Spalp and Spacp were cloned and sequenced. The core promoter region of the Spalp and Spacp was -39 bp∼+8 bp and -39 bp∼+10 bp, respectively. Potential binding sequences for SOX-2, c-fos, SP1, NF-κB, GATA-1, YY1, and AP-1 transcription factors were found in the 5'-flanking regions of Spalp and Spacp. The NF-κB binding site located between -1,223 bp and -972 bp in Spalp while SP1 and AP-1 binding sites located between -1,249 bp and -514 bp in Spacp. Mutation analysis confirmed that NF-κB negatively regulated the expression of Spalp gene, and SP1 and AP-1 positively regulated Spacp gene expression. These results provide us with essential information to elucidate the function of the Spalp and Spacp in S. paramamosain. This study is the first one to analyze the activity of Spalp and Spacp promoters.