Incidence, mortality, and predictive factors associated with acute respiratory distress syndrome in multiple trauma patients living in high-altitude areas: a retrospective study in Shigatse.

Background: Acute respiratory distress syndrome (ARDS) is a severe complication that can lead to fatalities in multiple trauma patients. Nevertheless, the incidence rate and early prediction of ARDS among multiple trauma patients residing in high-altitude areas remain unknown. Methods: This study included a total of 168 multiple trauma patients who received treatment at Shigatse People's Hospital Intensive Care Unit (ICU) between January 1, 2019 and December 31, 2021. The clinical characteristics of the patients and the incidence rate of ARDS were assessed. Univariable and multivariable logistic regression models were employed to identify potential risk factors for ARDS, and the predictive effects of these risk factors were analyzed. Results: In the high-altitude area, the incidence of ARDS among multiple trauma patients was 37.5% (63/168), with a hospital mortality rate of 16.1% (27/168). Injury Severity Score (ISS) and thoracic injuries were identified as significant predictors for ARDS using the logistic regression model, with an area under the curve (AUC) of 0.75 and 0.75, respectively. Furthermore, a novel predictive risk score combining ISS and thoracic injuries demonstrated improved predictive ability, achieving an AUC of 0.82. Conclusions: This study presents the incidence of ARDS in multiple trauma patients residing in the Tibetan region, and identifies two critical predictive factors along with a risk score for early prediction of ARDS. These findings have the potential to enhance clinicians' ability to accurately assess the risk of ARDS and proactively prevent its onset.

m 6 A-mediated gluconeogenic enzyme PCK1 upregulation protects against hepatic ischemia-reperfusion injury.

BACKGROUND AND AIMS: Ischemia-reperfusion (I/R) injury frequently occurs during liver surgery, representing a major reason for liver failure and graft dysfunction after operation. The metabolic shift from oxidative phosphorylation to glycolysis during ischemia increased glucose consumption and accelerated lactate production. We speculate that donor livers will initiate gluconeogenesis, the reverse process of glycolysis in theory, to convert noncarbohydrate carbon substrates (including lactate) to glucose to reduce the loss of hepatocellular energy and foster glycogen storage for use in the early postoperative period, thus improving post-transplant graft function. APPROACH AND RESULTS: By analyzing human liver specimens before and after hepatic I/R injury, we found that the rate-limiting enzyme of gluconeogenesis, PCK1, was significantly induced during liver I/R injury. Mouse models with liver I/R operation and hepatocytes treated with hypoxia/reoxygenation confirmed upregulation of PCK1 during I/R stimulation. Notably, high PCK1 level in human post-I/R liver specimens was closely correlated with better outcomes of liver transplantation. However, blocking gluconeogenesis with PCK1 inhibitor aggravated hepatic I/R injury by decreasing glucose level and deepening lactate accumulation, while overexpressing PCK1 did the opposite. Further mechanistic study showed that methyltransferase 3-mediated RNA N6-methyladinosine modification contributes to PCK1 upregulation during hepatic I/R injury, and hepatic-specific knockout of methyltransferase 3 deteriorates liver I/R injury through reducing the N6-methyladinosine deposition on PCK1 transcript and decreasing PCK1 mRNA export and expression level. CONCLUSIONS: Our study found that activation of the methyltransferase 3/N6-methyladinosine-PCK1-gluconeogenesis axis is required to protect against hepatic I/R injury, providing potential intervention approaches for alleviating hepatic I/R injury during liver surgery.

Neighbored-attention U-net (NAU-net) for diabetic retinopathy image segmentation.

Background: Diabetic retinopathy-related (DR-related) diseases are posing an increasing threat to eye health as the number of patients with diabetes mellitus that are young increases significantly. The automatic diagnosis of DR-related diseases has benefited from the rapid development of image semantic segmentation and other deep learning technology. Methods: Inspired by the architecture of U-Net family, a neighbored attention U-Net (NAU-Net) is designed to balance the identification performance and computational cost for DR fundus image segmentation. In the new network, only the neighboring high- and low-dimensional feature maps of the encoder and decoder are fused by using four attention gates. With the help of this improvement, the common target features in the high-dimensional feature maps of encoder are enhanced, and they are also fused with the low-dimensional feature map of decoder. Moreover, this network fuses only neighboring layers and does not include the inner layers commonly used in U-Net++. Consequently, the proposed network incurs a better identification performance with a lower computational cost. Results: The experimental results of three open datasets of DR fundus images, including DRIVE, HRF, and CHASEDB, indicate that the NAU-Net outperforms FCN, SegNet, attention U-Net, and U-Net++ in terms of Dice score, IoU, accuracy, and precision, while its computation cost is between attention U-Net and U-Net++. Conclusion: The proposed NAU-Net exhibits better performance at a relatively low computational cost and provides an efficient novel approach for DR fundus image segmentation and a new automatic tool for DR-related eye disease diagnosis.

[Effects of Different Planting Years of Dendrocalamus brandisii on Soil Fungal Community].

To explore the effect of soil fungal community under different planting years in Dendrocalamus brandisii, the soil samples from D. brandisii with different planting years (5, 10, 20, and 40 a) were taken as the research object. The soil fungal community structure, diversity, and its functional groups of different planting years were analyzed using high-throughput sequencing technology and the FUNGuild fungal function prediction tool, and the main soil environmental factors influencing the variations in soil fungal community were examined. The results showed that the dominant fungal communities at the phylum level were Ascomycota, Basidiomycota, Mortierellomycota, and Mucoromycota. The relative abundance of Mortierellomycota decreased and then increased with the increase in planting years, and there was a significant difference among different planting years (P<0.05). The dominant fungal communities at the class level were Sordariomycetes, Agaricomycetes, Eurotiomycetes, and Mortierellomycetes. The relative abundance of Sordariomycetes and Dothideomycetes decreased and then increased with the increase in planting years, and there were significant differences among different planting years (P<0.01). The Richness index and Shannon index of soil fungi increased and then decreased with the increase in planting years, and the Richness index and Shannon index in 10 a were significantly higher than those of other planting years. Non-metric multidimensional scaling (NMDS) and analysis of similarities (ANOSIM) showed that there were significant differences in soil fungal community structure with different planting years. The functional prediction with FUNGuild showed that the main functional trophic types of soil fungi in D. brandisii were pathotroph, symbiotroph, and saprotroph, and the most dominant functional group was endophyte-litter saprotroph-soil saprotroph-undefined saprotroph. The relative abundance of endophytes gradually increased with the increase in planting years. Correlation analysis showed that pH, total potassium (TK), and nitrate nitrogen (NO-3-N) were the main soil environmental factors affecting the change in fungal community. In summary, the planting year of D. brandisii has changed soil environmental factors and has thus changed the structure, diversity, and functional groups of soil fungal communities.

Functional analysis of hepatic long non-coding ribonucleic acids during liver transplantation in humans.

BACKGROUND: The potential function of long non-coding RNAs (lncRNAs) in human hepatic ischemia-reperfusion injury (HIRI) remains to be clarified. METHODS: Clinical samples of transplanted liver tissues from 26 patients undergoing liver transplantation (LT) and normal liver tissues from seven patients undergoing hepatic hemangiomactomy (Con) were collected. Typical samples were subjected to whole transcriptome sequencing (RNA-seq). Differentially expressed genes between groups were identified by DEGseq and were analyzed by enrichment analysis including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and gene set enrichment analysis. Transcription of five lncRNAs including NONHSAG039942, NONHSAG071405, NONHSAG027516, LXLOC_058190, and LXLOC_024376 that presented significant difference in RNA-sequencing were validated by a quantitative real-time PCR (qRT-PCR), for which the subcellular localization and the binding ability to known human RNA-binding proteins (RBPs) were respectively predicted by LncLocator and catRAPID genomics v2.1. RESULTS: We identified 2917 lncRNAs and 2811 mRNAs that were differentially expressed (p < 0.05 and log2 fold change > 1 or < -1) between groups (LT vs. Con). NONHSAG039942, NONHSAG071405, LXLOC_058190, and LXLOC_024376 were validated by qRT-PCR to be significantly increased in the LT group, and were all predicted to be localized in cytoplasm or cytosol. NONHSAG039942, NONHSAG071405, and LXLOC_058190 held an RBP interaction propensity score of 98.07%, 76.95%, and 152.99%, respectively, with heterogeneous-nuclear ribonucleoprotein U (HNRNPU). Pathways significantly activated in transplant livers that involved HNRNPU as a core enrichment gene included hypoxia, ACE2 expression, apoptosis, spliceosome formation, etc. CONCLUSIONS: NONHSAG039942, NONHSAG071405, and LXLOC_058190 were significantly increased in transplant livers after reperfusion and their role in HIRI may be associated with HNRNPU, a core protein that participates in hypoxia and chromatin accessibility.

Characteristics, sources, and risk assessment of thallium and associated with metal(loid)s in the Yarlung Tsangpo River Basin, southern Tibetan Plateau.

The Tibetan Plateau (TP) is known as the water tower of Asia, and the water quality has long been a focus of public concern, especially in the Yarlung Tsangpo River Basin (YTRB), a unique area that is climate-sensitive, geologically complex, eco-fragile, and densely populated. Thallium (Tl) is a typical metal that is more toxic than Pb, Cd, and As and often occurs in sulfide minerals. Although large-scale polymetallic sulfide mineralization developed in the YTRB, the geochemical dispersion and potential risk of Tl in aquatic environments of the YTRB remain poorly understood. In this study, the concentration, distribution, source, and health risk of Tl and associated metal(loid)s in the hot springs and surface water in the YTRB were systematically analyzed. The results showed that the trace elements (Cd, Cr, Zn, Cu, Al, Sr, Ni, Co, Mn, Pb) in water environments are within the recommended limits, except for Tl and As. Principal component analysis (PCA) and correlation analysis (CA) showed that the elements of Tl and As were positively related to each other in either both hot spring water and surface water, indicating their common origin. Spatial variations suggested that high levels of Tl and As observed in the north YTRB, which may be relevant to the reduction-dissolution of Tl (As)-bearing minerals and the magmatic hydrothermal system formed in the shallow part of the northern YTRB. Furthermore, source apportionment identified natural sources of Cu, Ni, Cr, Co, Mn, Zn, and Cd and anthropogenic inputs of Al and Pb. Exposure assessment studies have found that ingestion is the primary route of As and Tl exposure to local population, and balneological and bathing purposes do not constitute a human health concern. This study offers valuable insights into the risk of naturally occurring Tl enrichment being hidden in As-rich hydrosphere in the YTRB and other regions with similar geoenvironmental contexts.

The m6A methyltransferase Mettl3 deficiency attenuates hepatic stellate cell activation and liver fibrosis.

Activation of hepatic stellate cells (HSCs) is a central driver of liver fibrosis. Previous investigations have identified various altered epigenetic landscapes during the cellular progression of HSC activation. N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic cells and is dynamically regulated under various physiological and pathophysiological conditions. However, the functional role of Mettl3-mediated m6A in liver fibrosis remains elusive. Here, we found that the HSC-specific knockout of m6A methyltransferase Mettl3 suppressed HSC activation and significantly alleviated liver fibrosis. Multi-omics analysis of HSCs showed that Mettl3 depletion reduced m6A deposition on mRNA transcripts of Lats2 (a central player of the Hippo/YAP signaling pathway) and slowed down their degradation. Elevated Lats2 increased phosphorylation of the downstream transcription factor YAP, suppressed YAP nuclear translocation, and decreased pro-fibrotic gene expression. Overexpressing YAP mutant resistant to phosphorylation by Lats2 partially rescued the activation and pro-fibrotic gene expression of Mettl3-deficient HSCs. Our study revealed that disruption of Mettl3 in HSCs mitigated liver fibrosis by controlling the Hippo/YAP signaling pathway, providing potential therapeutic strategies to alleviate liver fibrosis by targeting epitranscriptomic machinery.

AIFM1, negatively regulated by miR-145-5p, aggravates hypoxia-induced cardiomyocyte injury.

BACKGROUND: Hypoxia-induced apoptosis is linked to the pathogenesis of myocardial infarction. The role of apoptosis-inducing factor mitochondria associated 1 (AIFM1) in cardiomyocyte injury remains unclear. This study was aimed at probing into the role and the underlying regulatory mechanism of AIFM1 in myocardial injury. METHODS: H9c2 cardiomyocytes and C57BL/6 mice were used for myocardial hypoxic/ischemic injury and myocardial infarction animal models. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate the expression levels of AIFM1 mRNA and miR-145-5p. Western blot was used for examining the expression levels of AIFM1, caspase-3, cleaved caspase-3, p-53, and γ-H2AX. Cell viability was examined by cell counting kit-8 (CCK-8) assay and BrdU assay. Interaction between AIFM1 and miR-145-5p was determined by bioinformatics analysis, qRT-PCR, Western blot, and dual-luciferase reporter assay. RESULTS: AIFM1 expression was markedly highly elevated, while miR-145-5p expression was significantly down-regulated in the myocardial infarction animal model and H9c2 cells under hypoxia. Augmentation of AIFM1 led to a dramatic decrease of cell viability, accompanied by an increase of the secretion of the inflammatory cytokines IL-1ß, TNF-α, IL-6, and the expression of cleaved caspase-3. Furthermore, AIFM1 was identified as a target of miR-145-5p. In addition, miR-145-5p/AIFM1 axis regulated the expression of p53. CONCLUSION: AIFM1 may exacerbate myocardial ischemic injury by promoting inflammation and the injury of cardiomyocytes, and its up-regulation may be partly due to the down-regulation of miR-145-5p.

A randomised trial on the therapeutic effectiveness of bronchoalveolar lavage under fiberoptic bronchoscopy in patients with severe lung infection living in the Tibetan plateau area.

BACKGROUND: People living in plateau areas are prone to upper respiratory tract infections and secondary lung infections. The current study aimed to explore the effects of bronchoalveolar lavage under fiberoptic bronchoscope for the treatment of patients with severe pulmonary infection living in plateau areas. METHODS: 148 patients with severe lung infection admitted to the intensive care unit of Shigatse People's Hospital (Shigatse, Tibet Autonomous Region, China) between July 2019 and January 2021 were enrolled. Patients were randomly assigned to the observational group or the control group. For all patients, basic clinical data including sex, age, body mass index (BMI), blood pressure, diabetes history, stroke history, presence or absence of chronic obstructive pulmonary disease, lung infection (gram-positive bacterial infection, gram-negative bacterial infection, fungal infection, acute lung abscess), surgical history, and postoperative inhalation injury. were collected. The control group received conventional treatment, and the observational group received bronchoalveolar lavage under fiberoptic bronchoscopy. Pearson's correlation was used to analyze the correlations between bronchoalveolar lavage under fiberoptic bronchoscopy and inflammatory factors levels. Logistic regression was used to investigate the correlation between bronchoalveolar lavage under fiberoptic bronchoscopy and the effectiveness of the treatment. RESULTS: Before treatment, no significant difference existed in the basic data of the observational group and the control group. After treatment, the parameters of respiratory mechanics and inflammatory factors in the 2 groups were significantly improved compared with those before treatment (P<0.05). At the same time, in the observational group, the clinical parameters were significantly higher than those of the control group, and the levels of inflammatory factors were significantly lower than those of the control group (all P<0.05). After full adjustment for age, sex, BMI, gram-negative infection, diabetes, and acute lung abscess, compared with the control group, the therapeutic effectiveness in the observational group was increased by 23% (OR =1.23, 95% CI: 1.09-1.51, P=0.007). CONCLUSIONS: For patients with severe lung infection who are resident in high altitude areas, compared with conventional treatments, bronchoalveolar lavage under fiberoptic bronchoscopy can significantly improve chest, lung, and total dynamic compliance, as well as reduce the levels of the inflammatory factors procalcitonin (PCT) and transforming growth factor-ß, thus increasing the effectiveness of the treatment.

Tetraphenylpyrazine-Based Luminescent Metal-Organic Framework for Chemical Sensing of Carcinoids Biomarkers.

A new non-interpenetrated three-dimensional (3D) pillared-layered TPP-based LMOF [Zn3(TPyTPP)0.5(BDC)3]·8DMF (denoted as Zn-MOF 1) was successfully prepared (TPyTPP = tetrakis(4-(pyridin-4-yl)phenyl)pyrazine and H2BDC = 1,4-benzenedicarboxylic acid). Zn-MOF 1 was characterized by single-crystal X-ray diffraction, PXRD, IR, N2 adsorption, thermogravimetric analysis, and luminescent spectrum. Impressively, luminescent sensing studies reveal that activated Zn-MOF 1 not only displays excellent luminescence-quenching efficiency with the values of high Ksv and low LODs toward 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA), respectively, but also possesses outstanding sensing characteristics in terms of fast response, high sensitivity, and specific selectivity. Zn-MOF 1 performs as efficient sensing of carcinoid biomarkers to provide a fresh detection platform for the diagnosis of carcinoids. In addition, the sensing mechanism was also explored on the basis of ultraviolet-visible (UV-vis) absorption, DFT calculations, and structural analysis.

[Effects of biochar addition on soil moisture infiltration process of the yellow soil in Karst area]. / ç”Ÿç‰©è´¨ç‚­æ·»åŠ å¯¹å–€æ–¯ç‰¹åœ°åŒºå¡è€•åœ°é»„å£¤æ°´åˆ†å ¥æ¸—è¿‡ç¨‹çš„å½±å“.

To investigate the effects of biochar addition on soil moisture infiltration characteristics of sloping farmland in Karst area, we used soil column simulation to study the variation characteristics of cumulative infiltration volume, infiltration rate, and wetting peak process under the different biochar addition amount (0, 1% and 2%) and different particle sizes (<0.25, 0.25-1 and >1 mm), and simulated the infiltration process in yellow soil on slope farmland. The results showed that soil infiltration process after biochar addition was significantly inhibited under the condition of constant bulk density. The cumulative infiltration amount and infiltration rate under biochar addition were significantly lower than those without biochar addition. There was no significant difference in the cumulative infiltration amount and infiltration rate of the soil with 1% and 2% biochar addition. The cumulative infiltration amount of the soil with different particle sizes followed an order of <0.25, 0.25-1 and > 1 mm after biochar addition. When the addition amount was 1%, the cumulative infiltration amount of 300 min had decreased by 20.9%, 35.2% and 45.0% compared with CK. When the addition amount was 2%, the decrease rate was 21.5%, 37.5% and 44.2%, indicating that the inhibition effect of large particle size biochar on soil infiltration being stronger than that of small particle size biochar. The change trend of soil wetting peak process to biochar addition of different contents and different particle sizes was consistent with the change trend of cumulative infiltration volume. Horton model and Kostiakov model could be used to simulate soil moisture infiltration process. The Horton model had higher fitting accuracy, the largest R2 (between 0.91 and 0.98), and the smallest RMSE (between 0.14 and 0.21). The initial infiltration rate obtained by Kostiakov model was closer to the measured result. Our results could provide scientific basis for the rational application of biochar and provide a useful reference for soil improvement and soil and water conservation in slope farmland of Karst area.

Direct Assembly of 3D-BCN Microspheres as a Microsupercapacitor Electrode for Wearable Energy Storage.

Scalable and cost-effective fabrication of three-dimensional (3D) boron carbon nitride (BCN) microspheres was first demonstrated by hydrothermal and annealing methods. In particular, the specific surface area of 3D-BCN-4 reached 1390.12 m2 g-1 and had a high hierarchical pore structure. An all-printed solid-state flexible microsupercapacitor (MSC) based on 3D-BCN-4 microspheres as an electrode material was fabricated for the first time by a screen printing method, which also provided efficacious properties. The single MSC areal capacitance reached 41.6 mF cm-2. Furthermore, the remarkable mechanical flexibility was also achieved for the device with evidence that no obvious capacitance loss occurred even upon bending to 180°, and the device had a 93.3% capacitance retention after 1000 cycles. In addition, the maximum energy density reached 0.00832 mW h cm-2, and the highest power density was 2 mW cm-2. These results show the synthesis of 3D-BCN by a facile and effective method with excellent electrochemical performance, which should provide a promising direction to wearable energy storage devices.

Regional variations in medical trainee diet and nutrition counseling competencies: Machine learning-augmented propensity score analysis of a prospective multi-site cohort study.

BACKGROUND: Medical professionals and students are inadequately trained to respond to rising global obesity and nutrition-related chronic disease epidemics, primarily focusing on cardiovascular disease. Yet, there are no multi-site studies testing evidence-based nutrition education for medical students in preventive cardiology, let alone establishing student dietary and competency patterns. METHODS: Cooking for Health Optimization with Patients (CHOP; NIH NCT03443635) was the first multi-national cohort study using hands-on cooking and nutrition education as preventive cardiology, monitoring and improving student diets and competencies in patient nutrition education. Propensity-score adjusted multivariable regression was augmented by 43 supervised machine learning algorithms to assess students outcomes from UT Health versus the remaining study sites. RESULTS: 3,248 medical trainees from 20 medical centers and colleges met study criteria from 1 August 2012 to 31 December 2017 with 60 (1.49%) being from UTHealth. Compared to the other study sites, trainees from UTHealth were more likely to consume vegetables daily (OR 1.82, 95%CI 1.04-3.17, p=0.035), strongly agree that nutrition assessment should be routine clinical practice (OR 2.43, 95%CI 1.45-4.05, p=0.001), and that providers can improve patients' health with nutrition education (OR 1.73, 95%CI 1.03-2.91, p=0.038). UTHealth trainees were more likely to have mastered 12 of the 25 competency topics, with the top three being moderate alcohol intake (OR 1.74, 95%CI 0.97-3.11, p=0.062), dietary fats (OR 1.26, 95%CI 0.57-2.80, p=0.568), and calories (OR 1.26, 95%CI 0.70-2.28, p=0.446). CONCLUSION: This machine learning-augmented causal inference analysis provides the first results that compare medical students nationally in their diets and competencies in nutrition education, highlighting the results from UTHealth. Additional studies are required to determine which factors in the hands-on cooking and nutrition curriculum for UTHealth and other sites produce optimal student - and, eventually, preventive cardiology - outcomes when they educate patients in those classes.

Fish Scale Valorization by Hydrothermal Pretreatment Followed by Enzymatic Hydrolysis for Gelatin Hydrolysate Production.

Protein hydrolysates from fish by-products have good process suitability and bioavailability in the food industry. The objective of this work was to develop a method for protein recovery from fish scales and evaluate the hydrolysis of the scale protein. The effect of the hydrothermal process on protein recovery, degree of hydrolysis (DH) and structural properties of the hydrolysates was investigated. Results showed that hydrothermal treatment could enhance protein recovery of tilapia scales without demineralization and dramatically improve the DH of the hydrolysates. The hydrothermal treated scales showed a better protein recovery (84.81%) and DH (12.88%) and released peptides more efficiently than that of the conventional treated samples. The obtained gelatin hydrolysates mainly distributed in the range of 200-2000 Da with an angiotensin I-converting enzyme (ACE) IC50 value of 0.73 mg/mL. The ACE inhibitory activity of gelatin hydrolysates was stable under high temperature, pH and gastrointestinal proteases. Hydrothermal treatment followed by enzymatic hydrolysis offers a potential solution for preparation of gelatin hydrolysates for food ingredients from fish processing by-products.

A recursive ensemble organ segmentation (REOS) framework: application in brain radiotherapy.

The aim of this work is to develop a novel recursive ensemble OARs segmentation (REOS) framework for accurate organs-at-risk (OARs) automatic segmentation. The REOS recursively segment individual OARs by ensembling images features extracted from an organ localization module and a contour detection module. Both modules are based on a 3D U-Net architecture. The organ localization module is trained for rough segmentation to localize a region of interest (ROI) that encompasses the to-be-delineated OAR, while the contour detection module is trained to segment the OAR within the identified ROI. In this study, the developed REOS framework is applied for brain radiotherapy on segmenting six OARs including the eyes, the brainstem (BS), the optical nerves and the chiasm. Eighty T1-weighted magnetic resonance images (MRI) from 80 brain cancer patients' cases with OARs' gold standard contours were collected for training and testing REOS. On 20 testing cases, the REOS achieve a high segmentation accuracy with Dice similarity coefficient (DSC) mean and standard deviation of 93.9% ± 1.4%, 94.5% ± 2.0%, 90.6% ± 2.7%, on the left and right eyes and the BS, respectively. On small and segmentation-challenging organs, the left and right optical nerves and the chiasm, the REOS achieves DSC of 78.0% ± 10.5%, 82.2% ± 5.9% and 71.1% ± 9.1%. The satisfactory performances demonstrated the effectiveness of the REOS in OARs segmentation.

Effects of both cold and heat stress on the liver of the giant spiny frog (Quasipaa spinosa): stress response and histological changes.

Ambient temperature-associated stress can affect normal physiological functions in ectotherms. To assess the effects of cold or heat stress on amphibians, giant spiny frogs (Quasipaa spinosa) were acclimated at 22°C followed by exposure to 5°C or 30°C for 0, 3, 6, 12, 24 and 48 h, respectively. Histological alterations, apoptotic index, generation of mitochondrial reactive oxygen species (ROS), antioxidant activity indices and stress-response gene expression in frog livers were subsequently determined. Results showed that many fat droplets appeared after 12 h of heat stress and the percentage of melanomacrophage centres significantly changed after 48 h at both stress conditions. Furthermore, the mitochondrial ROS levels were elevated in a time-dependent manner up to 6 h and 12 h in the cold and heat stress groups, respectively. The activities of superoxide dismutase, glutathione peroxidase and catalase were successively increased with increasing periods of cold or heat exposure, and their gene expression levels showed similar changes in both stress conditions. Most tested heat shock protein (HSP) genes were sensitive to temperature exposure, and the expression profiles of most apoptosis-related genes was significantly upregulated at 3 and 48 h under cold and heat stress, respectively. Apoptotic index at 48 h under cold stress was significantly higher than that under heat stress. Notably, lipid droplets, HSP30, HSP70 and HSP110 might be suitable bioindicators of heat stress. The results of these alterations at physiological, biochemical and molecular levels might contribute to a better understanding of the stress response of Q. spinosa, and perhaps amphibians more generally, under thermal stress.

Identification and functional analysis of inhibitor of NF-κB kinase (IKK) from Scylla paramamosain: The first evidence of three IKKs in crab species and their expression profiles under biotic and abiotic stresses.

IKK (inhibitor of NF-κB kinase) is the critical regulator for NF-κB (nuclear factor-κB) pathway against pathogenic invasion in vertebrates or invertebrates. However, the IKK from crab species has not yet been identified. In the present study, three full-length cDNA sequences of IKKs from mud crab Scylla paramamosain, designated as SpIKKß, SpIKKε1 and SpIKKε2, were firstly cloned through RT-PCR and RACE methods. This is also the first report about the identification of two IKKε genes in mud crab and even in crustaceans. The SpIKKß cDNA was 2824 bp in length with an open reading frame (ORF) of 2382 bp, which encoded a putative protein of 793 amino acids (aa). The ORF of two SpIKKε isoforms, SpIKKε1 and SpIKKε2, were 2400 bp and 2331 bp in length encoding 799 aa and 776 aa, respectively. The crucial conserved residues and functional domains, including the kinase domains (KDs) and leucine zipper (LZ), were identified in all SpIKKs. Phylogenetic analysis suggested that SpIKKß was classified into the IKKs class while SpIKKεs could be grouped into the IKK-related kinases class. The qRT-PCR analysis showed that three SpIKKs were constitutively expressed in all tested tissues and the highest expression levels of SpIKKß and SpIKKεs were all in hemocyte. The gene expression profiles of SpIKKs were distinct when crabs suffered biotic and abiotic stresses including the exposures of Vibrio alginolyticus, poly (I:C), cadmium and air exposure, suggesting that the SpIKKs might play different roles in response to pathogens infections, heavy metal and air exposure. Moreover, IKKs from mud crab can significantly activate mammalian NF-κB pathway, suggesting the function of IKKs might be evolutionally well-conserved. Results of the RNAi experiments suggested that SpIKKs might regulate the immune signaling pathway when hemocytes were challenged with V. parahemolyticus or virus-analog poly (I:C). All of these results indicated that the obtained SpIKKs might be involved in stress responses against biotic or abiotic stresses, and it also highlighted their functional conservation in the innate immune system from crustaceans to mammals.

Hemocytes of the mud crab Scylla paramamosain: Cytometric, morphological characterization and involvement in immune responses.

Hemocytes play essential roles in the innate immune system of crustaceans. Characterization of hemocytes from estuary mud crab Scylla paramamosain was performed by flow cytometry and morphological studies such as cytochemical staining and electron microscopy. The hemocyte subsets were further separated using a modified Percoll density gradient centrifugation method. Based on the morphological characteristics of the cells, three distinct categories of hemocytes were identified: granulocytes with abundant large granularity representing 5.27 ± 0.42%, semigranulocytes with small or less granularity representing 76.03 ± 3.34%, and hyalinocytes (18.70 ± 3.92%) which were almost no granularity. The total hemocyte cell count and the percentage of hemocyte subsets varied after pathogen infection, including Vibrio alginolyticus and the viral double-stranded RNA analog Poly (I:C). The phagocytic process is of fundamental importance for crustaceans' cellular immune response as well as development and survival. The results of the in vitro phagocytosis assays analyzed by flow cytometry demonstrated that granulocytes and semigranulocytes had significantly higher phagocytic ability than hyalinocytes. A primary culture system, L-15 medium supplemented with 5-10% fetal bovine serum, was developed to further investigate the immune function of hemocytes. Furthermore, adenovirus can be utilized to effectively transfer GFP gene into hemocytes. Overall, three hemocyte sub-populations of S. paramamosain were successfully discriminated, moreover, their response to pathogen infections, phagocytic activity and adenovirus mediated transfection were also investigated for the first time. This study may contribute to a better understanding of the innate immune system of estuary crabs.

Effect of cadmium exposure on hepatopancreas and gills of the estuary mud crab (Scylla paramamosain): Histopathological changes and expression characterization of stress response genes.

Cadmium (Cd) is a heavy metal that accumulates easily in organisms and causes several detrimental effects, including tissue damage. Cd contamination from anthropogenic terrestrial sources flows into rivers, and through estuaries to the ocean. To evaluate the toxic effects of Cd on estuary crustaceans, we exposed the mud crab Scylla paramamosain to various Cd concentrations (0, 10.0, 20.0, and 40.0mg/L) for 24h. We also exposed mud crabs to a fixed Cd concentration (20.0mg/L) for various periods of time (0, 6, 12, 24, 48, and 72h). We observed that after exposure to Cd, the surfaces of the gill lamellae were wrinkled, and the morphologies of the nuclei and mitochondria in the hepatopancreas were altered. We analyzed the expression profiles of 36 stress-related genes after Cd exposure, including those encoding metallothioneins, heat shock proteins, apoptosis-related proteins, and antioxidant proteins, with quantitative reverse transcription PCR. We found that exposure to Cd altered gene expression, and that some genes might be suitable bioindicators of Cd stress. Gene expression profiles were organ-, duration-, and concentration-dependent, suggesting that stress-response genes might be involved in an innate defense system for handling heavy metal exposure. To the best of our knowledge, this study is the first one of histopathology and stress-response gene expression pattern of Scylla paramamosain after Cd exposure. Our work could increase our understanding of the effect of environmental toxins on estuary crustaceans.

Identification and characterization of six peroxiredoxin transcripts from mud crab Scylla paramamosain: The first evidence of peroxiredoxin gene family in crustacean and their expression profiles under biotic and abiotic stresses.

The peroxiredoxins (Prxs) define a novel and evolutionarily conserved superfamily of peroxidases able to protect cells from oxidative damage by catalyzing the reduction of a wide range of cellular peroxides. Prxs have been identified in prokaryotes as well as in eukaryotes, however, the composition and number of Prxs family members vary in different species. In this study, six Prxs were firstly identified from the mud crab Scylla paramamosain by RT-PCR and RACE methods. Six SpPrxs can be subdivided into three classes: (a) three typical 2-Cys enzymes denominated as Prx1/2, 3, 4, (b) two atypical 2-Cys enzymes known as Prx5-1 and Prx5-2, and (c) a 1-Cys isoform named Prx6. The evolutionarily conserved signatures of peroxiredoxin catalytic center were identified in all six SpPrxs. Phylogenetic analysis revealed that SpPrx3, SpPrx4, SpPrx5s and SpPrx6 were clearly classified into Prx3-6 subclasses, respectively. Although SpPrx1/2 could not be grouped into any known Prx subclasses, SpPrx1/2 clustered together with other arthropods Prx1 or unclassified Prx and could be classified into the typical 2-Cys class. The comparative and evolutionary analysis of the Prx gene family in invertebrates and vertebrates were also conducted for the first time. Tissue-specific expression analysis revealed that these six SpPrxs were expressed in different transcription patterns while the highest expression levels were almost all in the hepatopancreas. Quantitative RT-PCR analysis exhibited that the gene expression profiles of six SpPrxs were distinct when crabs suffered biotic and abiotic stresses including the exposures of Vibrio alginolyticus, poly (I:C), cadmium and hypoosmotic salinity, suggesting that the SpPrxs might play different roles in response to various stresses. The recombinant proteins including the SpPrx1/2, SpPrx4, SpPrx5-1 and SpPrx6 were purified and the peroxidase activity assays indicated that all these proteins can reduce H2O2 in a typical DTT-dependent manner. To our knowledge, this is the first study about the comprehensive characterization of Prx gene family in Scylla paramamosain and even in crustaceans. These results would broaden the current knowledge of the whole Prx family as well as be helpful to understand and clarify the evolutionary pattern of Prx family in invertebrate and vertebrate taxa.