Citrobacter freundii induced pathogenicity towards fish: an insight investigation through serum biochemistry and gene expression analysis in Labeo rohita

BackgroundSince the emerging of COVID-19 pandemic, numerous nucleic-acid therapeutics have been approved for clinical use. Distinguishing themselves from conventional protein-targeted therapy, nucleic acid therapeutics offer the potential for sustained effects...

To investigate effects of IV administered carprofen on indices of renal function and results of serum biochemical and hematologic analyses in dogs anesthetized with acepromazine-thiopentone-isoflurane that had low blood pressure during anesthesia. 6 healthy Beagles. A randomized crossover study was conducted, using the following treatments: saline (0.9% NaCl solution)-saline, saline-carprofen, and carprofen-saline. Saline (0.08 ml/kg) and carprofen (4 mg/kg) were administered IV. The first treatment was administered 30 minutes before induction of anesthesia and immediately before administration of acepromazine (0.1 mg/kg, IM). Anesthesia was induced with thiopentone (25 mg/ml, IV) and maintained with inspired isoflurane (2% in oxygen). The second treatment was administered 30 minutes after onset of inhalation anesthesia. Blood gases, circulation, and ventilation were monitored. Renal function was assessed by glomerular filtration rate (GFR), using scintigraphy, serum biochemical analyses, and urinalysis. Hematologic analysis was performed. Statistical analysis was conducted, using ANOVA or Friedman ANOVA. Values did not differ significantly among the 3 treatments. For all treatments, sedation and anesthesia caused changes in results of serum biochemical and hematologic analyses, a decrease in mean arterial blood pressure to 65 mm Hg, an increase of 115 pmol/L in angiotensin II concentration, and an increase of 100 seconds in time required to reach maximum activity counts during scintigraphy. Carprofen administered IV before or during anesthesia did not cause detectable significant adverse effects on renal function or results of serum biochemical and hematologic analyses in healthy Beagles with low blood pressure during anesthesia.

IntroductionMicrocystins (MCs) are alarming aquatic contaminants having extensive health implications in fish. Despite growing concern, comprehensive studies on long term effects of MCs remain unexplored in Labeo rohita (rohu). This study aims to bridge the gap by investigating the pathophysiological effects of long-term sublethal exposure to microcystin-LR (MC-LR), the most toxic congener.MethodsHealthy rohu juveniles (mean weight 25 ± 2.1 g), sourced from institute farm were acclimatized for 2 weeks. The sublethal experimental study consisted of two treatments (control group: T0 and one-tenth 96 h-LD50 dose group: T1) in three replications (10 fish per tank). The toxic effects were examined after 90 days by analysing histomorphology, ultrastructure, oxidative stress level, serum biochemistry, and the gene expression levels of antioxidant enzyme [superoxide dismutase (SOD) and catalase], immune-related (lysozyme, and immunoglobulin M), pro-inflammatory cytokine (interleukin-1β), apoptosis (caspase 9) and detoxification enzyme [phase I: CYP1A and CYP3A; phase II: glutathione-S-transferase (GST)] genes following standard analytical methods. Statistical analysis was performed using SPSS v. 22.0, IBM software. Parameters were analysed using an unpaired t-test. The results were expressed as mean ± standard error (SE).ResultsMC-LR induced significant histological and ultrastructural alterations including vacuolation, hepatocyte degeneration, disintegration of heterochromatin, loss of nucleolus and mitochondrial swelling. It significantly (p-value <0.05) altered the immune and serum biochemical indices. Interestingly, the modulation in the expression of SOD, catalase, GST, CYP1A and CYP3A genes in different organs indicated their involvement in the antioxidant and detoxification process. A significant upregulation of GST expression in all organs signifies its potential as a prominent biomarker other than phase I enzymes.DiscussionBased on these findings, it is deduced that even sublethal levels of MC-LR can disrupt intrinsic antioxidant defences, immune responses, and detoxification mechanisms in rohu, potentially compromising fish health in natural ecosystems. This is the first report to detail long-term impacts in rohu, elucidating the mechanism of damage induced by MC-LR and also providing valuable insights for environmental monitoring and toxin management.

Although hypoalbuminaemia after injury may result from increased vascular permeability, dilution secondary to crystalloid infusions may contribute significantly. In this double-blind crossover study, the effects of bolus infusions of crystalloids on serum albumin, haematocrit, serum and urinary biochemistry and bioelectrical impedance analysis were measured in healthy subjects. Ten male volunteers received 2-litre infusions of 0.9% (w/v) saline or 5% (w/v) dextrose over 1 h; infusions were carried out on separate occasions, in random order. Weight, haemoglobin, serum albumin, serum and urinary biochemistry and bioelectrical impedance were measured pre-infusion and hourly for 6 h. The serum albumin concentration fell in all subjects (20% after saline; 16% after dextrose) by more than could be explained by dilution alone. This fall lasted more than 6 h after saline infusion, but values had returned to baseline 1 h after the end of the dextrose infusion. Changes in haematocrit and haemoglobin were less pronounced (7.5% after saline; 6.5% after dextrose). Whereas all the water from dextrose was excreted by 2 h after completion of the infusion, only one-third of the sodium and water from the saline had been excreted by 6 h, explaining its persistent diluting effect. Impedances rose after dextrose and fell after saline (P<0.001). Subjects voided more urine (means 1663 and 563 ml respectively) of lower osmolality (means 129 and 630 mOsm/kg respectively) and sodium content (means 26 and 95 mmol respectively) after dextrose than after saline (P<0.001). While an excess water load is excreted rapidly, an excess sodium load is excreted very slowly, even in normal subjects, and causes persistent dilution of haematocrit and serum albumin. The greater than expected change in serum albumin concentration when compared with that of haemoglobin suggests that, while dilution is responsible for the latter, redistribution also has a role in the former. Changes in bioelectrical impedance may reflect the electrolyte content rather than the volume of the infusate, and may be unreliable for clinical purposes.

Although hypoalbuminaemia after injury may result from increased vascular permeability, dilution secondary to crystalloid infusions may contribute significantly. In this double-blind crossover study, the effects of bolus infusions of crystalloids on serum albumin, haematocrit, serum and urinary biochemistry and bioelectrical impedance analysis were measured in healthy subjects. Ten male volunteers received 2-litre infusions of 0.9% (w/v) saline or 5% (w/v) dextrose over 1 h; infusions were carried out on separate occasions, in random order. Weight, haemoglobin, serum albumin, serum and urinary biochemistry and bioelectrical impedance were measured pre-infusion and hourly for 6 h. The serum albumin concentration fell in all subjects (20% after saline; 16% after dextrose) by more than could be explained by dilution alone. This fall lasted more than 6 h after saline infusion, but values had returned to baseline 1 h after the end of the dextrose infusion. Changes in haematocrit and haemoglobin were less pronounced (7.5% after saline; 6.5% after dextrose). Whereas all the water from dextrose was excreted by 2 h after completion of the infusion, only one-third of the sodium and water from the saline had been excreted by 6 h, explaining its persistent diluting effect. Impedances rose after dextrose and fell after saline (P &lt; 0.001). Subjects voided more urine (means 1663 and 563 ml respectively) of lower osmolality (means 129 and 630 mOsm/kg respectively) and sodium content (means 26 and 95 mmol respectively) after dextrose than after saline (P &lt; 0.001). While an excess water load is excreted rapidly, an excess sodium load is excreted very slowly, even in normal subjects, and causes persistent dilution of haematocrit and serum albumin. The greater than expected change in serum albumin concentration when compared with that of haemoglobin suggests that, while dilution is responsible for the latter, redistribution also has a role in the former. Changes in bioelectrical impedance may reflect the electrolyte content rather than the volume of the infusate, and may be unreliable for clinical purposes.

Alterations in innate immune parameters and oxidative stress-related gene expression in an Indian major carp, Labeo rohita (Hamilton, 1822), infected with Citrobacter freundii.

Extracellular vesicles from induced pluripotent stem cell-derived mesenchymal stem cells enhance the recovery of acute kidney injury

Diethyl ether (ether) had been widely used in Japan for anesthesia, despite its explosive properties and toxicity to both humans and animals. We also had used ether as an anesthetic for euthanizing rats for research in the Toxicogenomics Project (TGP). Because the use of ether for these purposes will likely cease, it is required to select an alternative anesthetic which is validated for consistency with existing TGP data acquired under ether anesthesia. We therefore compared two alternative anesthetic candidates, isoflurane and pentobarbital, with ether in terms of hematological findings, serum biochemical parameters, and gene expressions. As a result, few differences among the three agents were observed. In hematological and serum biochemistry analysis, no significant changes were found. In gene expression analysis, four known genes were extracted as differentially expressed genes in the liver of rats anesthetized with ether, isoflurane, or pentobarbital. However, no significant relationships were detected using gene ontology, pathway, or gene enrichment analyses by DAVID and TargetMine. Surprisingly, although it was expected that the lung would be affected by administration via inhalation, only one differentially expressed gene was extracted in the lung. Taken together, our data indicate that there are no significant differences among ether, isoflurane, and pentobarbital with respect to effects on hematological parameters, serum biochemistry parameters, and gene expression. Based on its smallest affect to existing data and its safety profile for humans and animals, we suggest isoflurane as a suitable alternative anesthetic for use in rat euthanasia in toxicogenomics analysis.

Obesity, a serious epidemic affecting much of our pet population, increases the risk of developing numerous diseases. It has been demonstrated that obesity increases oxidative stress in obese children, cats and other species. Oxidative stress can result in DNA damage with subsequent alterations in gene expression, cell signaling, mutations, cell death or cell transformation. These effects of oxidative damage predispose animals and humans to numerous disease processes and cancer. The objective of the study was to demonstrate that obese dogs are under oxidative stress resulting in DNA damage and decreased endogenous antioxidant protection measured by serum glutathione levels and the ratio of reduced (GSH) to oxidized (GSSG) glutathione. In this case–control study, 10 obese dogs were compared with aged-matched healthy control dogs. Dogs with BCS of 7 or greater (9 pt scale) were considered obese. Dogs were evaluated by history, physical exam, body condition score, CBC, serum biochemical analysis and total T4, with both groups showing no significant differences in CBC, serum biochemical or T4 analysis. Single-cell gel electrophoresis (Comet assay) was used to measure DNA damage, and high performance liquid chromatography was used to measure serum glutathione. Reduced glutathione levels were significantly higher in the obese group (p = 0.012). The results of this pilot study suggest that obesity is associated with an increase in antioxidant potential, therefore justifying a larger study with antioxidant supplementation to determine how antioxidants in weight loss diets effects endogenous antioxidant capabilities.

We showed recently that diabetic and mildly obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats were more susceptible to azoxymethane-induced carcinogenesis than control Long-Evans Tokushima Otsuka (LETO) rats, and carcinogenesis was inhibited by 20% caloric restriction (CR) and enhanced by high-fat diet (HFD, control diet with 10% safflower oil). To elucidate the mechanism of cancer susceptibility, we performed serum biochemical analysis and microarray analysis of the liver and colonic mucosa. Male LETO and OLETF rats were given control diet, 20% CR diet or HFD for 5 weeks, and euthanized at 24 weeks of age (n=5, respectively). In both strains, blood glucose, serum triglyceride, total cholesterol, insulin and leptin levels decreased by 20% CR, and increased by HFD. Steatosis of hepatocytes in OLETF rats was ameliorated by 20% CR and enhanced by HFD. Microarray analysis of the liver showed that 20% CR up-regulated metallothionein 1A gene, and down-regulated heat shock 105kDa/110kDa protein 1 (Hsph1) and heat shock 70kDa protein 1B (Hspa1b) genes in both strains. There were no histological differences in the colonic mucosa in both strains and the effect of the dietary difference was not found. Microarray analysis of the colonic mucosa showed that 20% CR down-regulated Hsph1 and Hspa1b genes, and HFD up-regulated fatty acid binding protein 2 in both strains. These data suggest that hyperinsulinemia, hyperlipidemia and oxidative stress by overnutrition exert significant role in cancer susceptibility in OLETF rats. Citation Format: Keisuke Izumi, Chie Takasu, Tetsuyuki Takahashi, Hisanori Uehara. Serum biochemistry and microarray analysis of liver and colonic mucosa in LETO and diabetic OLETF rats treated with 20% caloric restriction and high-fat diet. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1279. doi:10.1158/1538-7445.AM2013-1279

Acute diarrhea (AD) has a complex etiology and may lead to life-threatening conditions. Hematological and serum biochemistry analyses can be useful for a differential diagnosis and for determining the severity of diarrhoea. Dogs with AD (n=72) were divided into Isospora (n=18), Toxocara (n=18), Parvoviral Enteritis (n=18), and Dietary Diarrhea (n=18) subgroups following clinical and laboratory examinations. The study aimed to evaluate the diagnostic value of certain hematological and serum biochemistry parameters. Clinical examinations, rapid diagnostic tests, complete blood count (CBC), and biochemical analyses were performed. White blood cell count (WBC), granulocyte, and mean hemoglobin concentration (MCH) levels were lower in the Parvoviral Enteritis Group compared with the other groups (p&lt;0.01). Isospora, Parvoviral Enteritis, and Toxocara groups had lower glucose and total protein, and higher creatinine levels than those of the Control and Dietary Diarrhea groups (p&lt;0.0001). The albumin level of the Dietary Diarrhea Group was higher compared with the other groups (p&lt;0.0001). Parvoviral Enteritis and Isospora groups had higher ALP levels than those of the other groups (p&lt;0.0001). Blood urea nitrogen (BUN), alanine aminotransferase (ALT), C-reactive protein (CRP), and cholesterol levels were determined to be highest in the Parvoviral Enteritis Group (p&lt;0.0001). The total bilirubin level was higher in Parvoviral Enteritis and Toxocara groups compared with the Control, Isospora, and Dietary Diarrhea groups (p&lt;0.0001). As a result, it was concluded that in cases of AD due to parvoviral enteritis and Toxocara canis, serum biochemistry abnormalities may be more severe, can provide more clinical information than CBC, and can be useful in forming a differential diagnosis list, especially in triage.

To investigate the preventative activity of benzyl isothiocyante and S-carvone against high-fat diet-induced obesity and metabolic complications. Ten-week-old C57BL/6 male mice were fed a high-fat diet and injected intraperitoneally twice per week with benzyl isothiocyante, S-carvone, or vehicle for 8weeks. The body weight, food intake, and body composition were monitored, and glucose tolerance and insulin tolerance tests were performed at the end of the experiment. Serum and tissue samples were studied using serum biochemistry, histological, and gene expression analysis to define the effects of benzyl isothiocyante and S-carvone treatments on lipid and glucose metabolism and inflammatory responses. Benzyl isothiocyante and S-carvone blocked high-fat diet-induced weight gain, fat accumulation in the liver, and insulin resistance. The beneficial effects were found to be associated with an improvement of expression of macrophage marker genes in white adipose tissue, including F4/80, Cd11b, Cd11c, Cd206, and Tnf-α, and reduced expression of genes (Pparγ2, Scd1, Cd36) responsible for lipid synthesis and transport in the liver. Benzyl isothiocyante and S-carvone block high-fat diet-induced obesity and metabolism disorders and can be considered for management of the obesity epidemic that affects approximately 36% of adults and 17% of children in the USA.

BackgroundAcute kidney injury (AKI) has a complex pathophysiology and imposes serious health concerns worldwide. Extracellular vesicles (EVs) derived from induced mesenchymal stem cells (iMSCs) have been recognized as novel cell-free therapeutics for various inflammatory and degenerative disorders. In this study, we investigated whether iMSCs stimulated with a pan-peroxisome proliferator-activated receptor (PPAR) agonist could enhance the therapeutic efficacy of EVs against AKI.MethodsHuman iMSCs were primed with or without lanifibranor, a PPAR agonist for 24 h, and EVs were collected after an additional 24 h. The basic characteristics of EVs were evaluated using cryo-transmission electron microscopy imaging, immunoblot detection of EV markers, nanoparticle tracking analysis, and localization in AKI kidneys. In vitro, the potential of the EVs to promote the growth and survival of HK-2 cells undergoing cisplatin-induced apoptosis and anti-inflammatory effects in M1-polarized THP-1 was compared. Subsequently, AKI was induced in BALB/c mice using cisplatin. After 8 and 24 h of cisplatin treatment, iMSC-EVs or pan-PPAR-iMSC-EVs were injected intravascularly. At 96 h after cisplatin administration, the renoprotective effects of iMSC-EVs or pan-PPAR-iMSC-EVs in inhibiting inflammation and apoptosis were compared using serum biochemistry, histology, immunohistochemistry, and gene expression analysis by qPCR.ResultsBoth EV types expressed EV markers and had typical EV morphology, and their localization in the renal tissue was confirmed. The proliferation and survival of HK-2 cells were higher in pan-PPAR-iMSC-EVs than those in iMSC-EVs. In M1-polarized THP-1 cells, the reduction in the mRNA expression of inflammatory cytokines was more significant in pan-PPAR-iMSC-EVs than that in iMSC-EVs. In the mouse model of cisplatin-induced AKI, pan-PPAR-iMSC-EVs markedly enhanced renoprotective effects compared to iMSC-EVs. Specifically, pan-PPAR-iMSC-EVs reduced tissue inflammation, immune cell infiltration, and apoptosis. Pan-PPAR-iMSC-EVs also increased renal capillary density.ConclusionPriming iMSCs with a PPAR agonist significantly improved the therapeutic potential of EVs by reducing inflammation and apoptosis. The reported strategy may contribute to the development of a novel cell-free option for AKI treatment.Trial registration: Not applicable.

BackgroundCaspase-8, a member of the family of conserved cysteine proteases, plays a crucial role in the initiation phase of the apoptotic death-signaling cascade and thereby attracts interest for its study across the animal species including fish. In India, rohu (Labeo rohita) is an important freshwater fish species; thus, this study on caspase-8 was undertaken to investigate its role during pathogenic invasion.ResultsThe complete cDNA sequence of Labeo rohita caspase-8 (Lrcasp8) consisted of 1746 bp nucleotides (nt) having an ORF of 1440 nt encoding a polypeptide of 480 amino acid (aa) residues with the molecular mass of ∼ 54.8 kDa. Structurally, Lrcasp8 comprised two DED domains (DED11-77aa and DED297-174aa) and one CASc domain230-476aa. Within the CASc domain, various putative motifs, viz., a large subunit (p20237-360aa), a small subunit (p10389-474aa), and a penta-peptide (QACQG354-358aa) active site, were identified. The secondary structure of Lrcasp8 protein comprised seventeen α-helices, eleven β-strands, and twenty-nine coils. Phylogenetically, it is closely related to common carp caspase-8 and exhibits significant (p < 0.05) similarity (88.3%) and identity (78.7 %) in their amino acid sequence. The tissue-specific expression of Lrcasp8 has been analyzed by quantitative real-time PCR assay, and it revealed the highest expression (~ 23-fold) in the blood and lowest in the spleen. In Aeromonas hydrophila and Edwardsiella tarda infection and rhabdovirus vaccination, caspase-8 gene expression in rohu fingerlings was significantly (p <0.05) induced in various organs/tissues. Infection of the Labeo rohita gill cells with A. hydrophila resulted in apoptosis and cell death with the induction of caspase-8 gene expression.ConclusionThis is the first report on the identification and structural characterization of caspase-8 cDNA and predicted protein and the analysis of caspase-8 gene expression in Labeo rohita following Aeromonas hydrophila and Edwardsiella tarda infections and rhabdovirus vaccinations. The data in this article together suggest the critical role of caspase-8 during infection and apoptosis in Labeo rohita.

Breast cancer is the most diagnosed cancer among women, and a leading cause of death worldwide. Santolina chamaecyparissus L. is a plant with multiple health benefits, including anticancer and anti-diabetic properties. This study aimed to assess the chemopreventive effects of S. chamaecyparissus aqueous extract (SCE) in an animal model of mammary cancer. A total of 28 four-week-old female Wistar rats were divided into four groups: control, MNU-induced (IND), SCE-supplemented (SCE), and SCE+IND. SCE was added to drinking water (12.72 mg/kg body weight) ad libitum. MNU was administered via the intraperitoneal route at 50 days of age. Weekly monitoring of body weight, food/drink intake, humane endpoints, and number of mammary tumours were recorded. Twenty weeks after MNU administration, animals were sacrificed by anaesthetic overdose and a necropsy was performed. Blood samples were used to determine blood count and serum biochemistry analysis, while kidney and liver samples were analysed for oxidative stress. Tumour samples were collected for gene expression and histology studies. SCE chemical composition was analysed by LC-MS and contained 19 phenolic compounds, with the most abundant being myricetin-O-glucuronide and 1,3-O-dicaffeoylquinic acid. Two animals in the IND group were sacrificed due to exceeding the humane endpoint limits. SCE supplementation delayed mammary tumour development, reducing its volume and weight. SCE had a positive impact on haematological parameters, particularly the neutrophil-lymphocyte ratio (P=0.026). No significant differences were observed in serum biochemistry, except for creatinine kinase MB, or in oxidative stress markers. Gene expression analysis showed significantly reduced VEGF expression levels (P=0.0158) in tumours from SCE+IND. These findings suggest that SCE is deserving of further study to identify the individual compounds and to understand its influence on animal models during cancer development.