Immunotherapeutic Potential of Mollusk Hemocyanins in Murine Model of Melanoma.

The development of antitumor drugs and therapy requires new approaches and molecules, and products of natural origin provide intriguing alternatives for antitumor research. Gastropodan hemocyanins-multimeric copper-containing glycoproteins have been used in therapeutic vaccines and antitumor agents in many cancer models. MATERIALS AND METHODS: We established a murine model of melanoma by challenging C57BL/6 mice with a B16F10 cell line for solid tumor formation in experimental animals. The anticancer properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) were evaluated in this melanoma model using various schemes of therapy. Flow cytometry, ELISA, proliferation, and cytotoxicity assays, as well as histology investigations, were also performed. RESULTS: Beneficial effects on tumor growth, tumor incidence, and survival of tumor-bearing C57BL/6 mice after administration of the RtH or HaH were observed. The generation of high titers of melanoma-specific IgM antibodies, pro-inflammatory cytokines, and tumor-specific CTLs, and high levels of tumor-infiltrated M1 macrophages enhanced the immune reaction and tumor suppression. DISCUSSION: Both RtH and HaH exhibited promising properties for applications as antitumor therapeutic agents and future experiments with humans.

Contribution of parasite and host genotype to immunopathology of schistosome infections.

BACKGROUND: The role of pathogen genotype in determining disease severity and immunopathology has been studied intensively in microbial pathogens including bacteria, fungi, protozoa and viruses but is poorly understood in parasitic helminths. The medically important blood fluke Schistosoma mansoni is an excellent model system to study the impact of helminth genetic variation on immunopathology. Our laboratory has demonstrated that laboratory schistosome populations differ in sporocyst growth and cercarial production in the intermediate snail host and worm establishment and fecundity in the vertebrate host. Here, we (i) investigate the hypothesis that schistosome genotype plays a significant role in immunopathology and related parasite life history traits in the vertebrate mouse host and (ii) quantify the relative impact of parasite and host genetics on infection outcomes. METHODS: We infected BALB/c and C57BL/6 mice with four different laboratory schistosome populations from Africa and the Americas. We quantified disease progression in the vertebrate host by measuring body weight and complete blood count (CBC) with differential over a 12-week infection period. On sacrifice, we assessed parasitological (egg and worm counts, fecundity), immunopathological (organ measurements and histopathology) and immunological (CBC with differential and cytokine profiles) characteristics to determine the impact of parasite and host genetics. RESULTS: We found significant variation between parasite populations in worm numbers, fecundity, liver and intestine egg counts, liver and spleen weight, and fibrotic area but not in granuloma size. Variation in organ weight was explained by egg burden and intrinsic parasite factors independent of egg burden. We found significant variation between infected mouse lines in cytokine levels (IFN-γ, TNF-α), eosinophils, lymphocytes and monocyte counts. CONCLUSIONS: This study showed that both parasite and host genotype impact the outcome of infection. While host genotype explains most of the variation in immunological traits, parasite genotype explains most of the variation in parasitological traits, and both host and parasite genotypes impact immunopathology outcomes.

Empirically derived formulae for calculation of age- and region-related levels of iron, copper and zinc in the adult C57BL/6 mouse brain.

Metal dyshomeostasis is associated with neurodegenerative disorders, cancers and vascular disease. We report the effects of age (range: 3 to 18 months) on regional copper, iron and zinc levels in the brain of the C57BL/6 mouse, a widely used inbred strain with a permissive background allowing maximal expression of mutations in models that recapitulate these disorders. We present formulae that can be used to determine regional brain metal concentrations in the C57BL/6 mouse at any age in the range of three to eighteen months of life. Copper levels in the C57BL/6 mouse adult brain were highest in the striatum and cerebellum and increased with age, excepting the cortex and hippocampus. Regional iron levels increased linearly with age in all brain regions, while regional zinc concentrations became more homogeneous with age. Knockdown of the copper transporter Ctr1 reduced brain copper, but not iron or zinc, concentrations in a regionally-dependent manner. These findings demonstrate biometals in the brain change with age in a regionally-dependent manner. These data and associated formulae have implications for improving design and interpretation of a wide variety of studies in the C57BL/6 mouse.

Contribution of parasite and host genotype to immunopathology of schistosome infections.

Background: The role of pathogen genotype in determining disease severity and immunopathology has been studied intensively in microbial pathogens including bacteria, fungi, protozoa, and viruses, but is poorly understood in parasitic helminths. The medically important blood fluke Schistosoma mansoni is an excellent model system to study the impact of helminth genetic variation on immunopathology. Our laboratory has demonstrated that laboratory schistosome populations differ in sporocyst growth and cercarial production in the intermediate snail host and worm establishment and fecundity in the vertebrate host. Here, we (i) investigate the hypothesis that schistosome genotype plays a significant role in immunopathology and related parasite life history traits in the vertebrate mouse host and (ii) quantify the relative impact of parasite and host genetics on infection outcomes. Methods: We infected BALB/c and C57BL/6 mice with four different laboratory schistosome populations from Africa and the Americas. We quantified disease progression in the vertebrate host by measuring body weight and complete blood count (CBC) with differential over an infection period of 12 weeks. On sacrifice, we assessed parasitological (egg and worm counts, fecundity), immunopathological (organ measurements and histopathology), and immunological (CBC with differential and cytokine profiles) characteristics to determine the impact of parasite and host genetics. Results: We found significant variation between parasite populations in worm numbers, fecundity, liver and intestine egg counts, liver and spleen weight, and fibrotic area, but not in granuloma size. Variation in organ weight was explained by egg burden and by intrinsic parasite factors independent of egg burden. We found significant variation between infected mouse lines in cytokines (IFN-γ, TNF-α), eosinophil, lymphocyte, and monocyte counts. Conclusions: This study showed that both parasite and host genotype impact the outcome of infection. While host genotype explains most of the variation in immunological traits, parasite genotype explains most of the variation in parasitological traits, and both host and parasite genotype impact immunopathology outcomes.

Escherichia coli K88 activates NLRP3 inflammasome-mediated pyroptosis in vitro and in vivo.

Pyroptosis induced by lipopolysaccharide (LPS) has an obvious impact on intestinal inflammation and immune regulation. Enterotoxigenic Escherichia coli (ETEC) K88 has been proved to induce inflammatory responses in several models, but whether E. coli K88 participates in the same process of pyroptotic cell death as LPS remains to be identified. We conducted a pilot experiment to confirm that E. coli K88, instead of Escherichia coli O157 and Salmonella typhimurium, promotes the secretion of interleukin-1 beta (IL-1ß) and interleukin-18 (IL-18) in macrophages. Further experiments were carried out to dissect the molecular mechanism both in vitro and in vivo. The Enzyme-Linked Immunosorbent Assay (ELISA) results suggested that E. coli K88 treatment increased the expression of pro-inflammatory cytokines IL-18 and IL-1ß in both C57BL/6 mice and the supernatant of J774A.1 cells. Intestinal morphology observations revealed that E. coli K88 treatment mainly induced inflammation in the colon. Real-time PCR and Western blot analysis showed that the mRNA and protein expressions of pyroptosis-related factors, such as NLRP3, ASC, and Caspase1, were significantly upregulated by E. coli K88 treatment. The RNA-seq results confirmed that the effect was associated with the activation of NLRP3, ASC, Caspase1, GSDMD, IL-18, and IL-1ß, and might also be related to inflammatory bowel disease and the tumor necrosis factor pathway. The pyroptosis-activated effect of E. coli K88 was significantly blocked by NLRP3 siRNA. Our data suggested that E. coli K88 caused inflammation by triggering pyroptosis, which provides a theoretical basis for the prevention and treatment of ETEC in intestinal infection.

Contribution of parasite and host genotype to immunopathology of schistosome infections.

Background: The role of pathogen genotype in determining disease severity and immunopathology has been studied intensively in microbial pathogens including bacteria, fungi, protozoa, and viruses, but is poorly understood in parasitic helminths. The medically important blood fluke Schistosoma mansoni is an excellent model system to study the impact of helminth genetic variation on immunopathology. Our laboratory has demonstrated that laboratory schistosome populations differ in sporocyst growth and cercarial production in the intermediate snail host and worm establishment and fecundity in the vertebrate host. Here, we (i) investigate the hypothesis that schistosome genotype plays a significant role in immunopathology and related parasite life history traits in the vertebrate mouse host and (ii) quantify the relative impact of parasite and host genetics on infection outcomes. Methods: We infected BALB/c and C57BL/6 mice with four different laboratory schistosome populations from Africa and the Americas. We quantified disease progression in the vertebrate host by measuring body weight and complete blood count (CBC) with differential over an infection period of 12 weeks. On sacrifice, we assessed parasitological (egg and worm counts, fecundity), immunopathological (organ measurements and histopathology), and immunological (CBC with differential and cytokine profiles) characteristics to determine the impact of parasite and host genetics. Results: We found significant variation between parasite populations in worm numbers, fecundity, liver and intestine egg counts, liver and spleen weight, and fibrotic area, but not in granuloma size. Variation in organ weight was explained by egg burden and by intrinsic parasite factors independent of egg burden. We found significant variation between infected mouse lines in cytokines (IFN-γ, TNF-α), eosinophil, lymphocyte, and monocyte counts. Conclusions: This study showed that both parasite and host genotype impact the outcome of infection. While host genotype explains most of the variation in immunological traits, parasite genotype explains most of the variation in parasitological traits, and both host and parasite genotype impact immunopathology outcomes.

The analysis of fatty acids and their derivatives in the liver of C57BL/6 mice with long-term caloric restrictions.

Caloric restriction (CR) is a dietetic intervention based on the reduction of daily calorie intake by 10-30 %. When subjected to CR, the organism adjusts its metabolism to the changing availability of key nutrients. However, fatty acids' content in organisms subjected to long-term CR has not been evaluated. The aim of the research was to analyze the influence of long-term CR on the contents of medium- and long-chain fatty acids, as well as on the contents of fatty acid derivatives in liver. The study was performed on C57BL female (n = 12) and male (n = 12) mice subjected to lifelong 30 % calorie restriction. Fatty acids were analyzed using gas chromatography, while fatty acid derivatives were analyzed with liquid chromatography. The dynamics of change of the lipid profile of the labeled fatty acids observed in the liver tissue confirms that lipolysis actively takes place in this organ when hungry. Moreover, it is highly possible that de novo synthesis of acids takes place, with the aim to ensure energy substrates to the body. Moreover, an increase of concentration was observed for fatty acid derivatives, those with anti-inflammatory properties (resolvin, LTX A4). However, there was no increase in the concentration of pro-inflammatory eicosanoids. The results suggest that it is important to take into consideration the introduction of appropriate supplements when using CR.

Differential Protein Expression of Taenia crassiceps ORF Strain in the Murine Cysticercosis Model Using Resistant (C57BL/6) Mice.

A cysticercosis model of Taenia crassiceps ORF strain in susceptible BALB/c mice revealed a Th2 response after 4 weeks, allowing for the growth of the parasite, whereas resistant C57BL/6 mice developed a sustained Th1 response, limiting parasitic growth. However, little is known about how cysticerci respond to an immunological environment in resistant mice. Here, we show that the Th1 response, during infection in resistant C57BL/6 mice, lasted up to 8 weeks and kept parasitemia low. Proteomics analysis of parasites during this Th1 environment showed an average of 128 expressed proteins; we chose 15 proteins whose differential expression varied between 70 and 100%. A total of 11 proteins were identified that formed a group whose expression increased at 4 weeks and decreased at 8 weeks, and another group with proteins whose expression was high at 2 weeks and decreased at 8 weeks. These identified proteins participate in tissue repair, immunoregulation and parasite establishment. This suggests that T. crassiceps cysticerci in mice resistant under the Th1 environment express proteins that control damage and help to establish a parasite in the host. These proteins could be targets for drugs or vaccine development.

Peripheral Nerve Injury Induced by Japanese Encephalitis Virus in C57BL/6 Mouse.

Japanese encephalitis virus (JEV), with neurotoxic and neuroinvasive properties, is the major cause of human viral encephalitis in Asia. Although Guillain-Barré syndrome caused by JEV infections is not frequent, a few cases have been reported in recent years. To date, no existing animal model for JEV-induced peripheral nerve injury (PNI) has been established, and thus the pathogenic mechanism is not clarified. Therefore, an animal model is urgently required to clarify the correlation between JEV infection and PNI. In the present study, we used JEV GIb strain of NX1889 to establish a mouse model of JEV infection. The general neurological signs emerged on day 3 of modeling. The motor function continued to deteriorate, reaching a maximum at 8 to 13 days postinfection (dpi) and gradually recovered after 16 dpi. The injuries of 105 PFU and 106 PFU groups were the most severe. Transmission electron microscopy and immunofluorescence staining showed varying degrees of demyelination and axonal degeneration in the sciatic nerves. The electrophysiological recordings demonstrated the presence of demyelinating peripheral neuropathy with reduced nerve conduction velocity. The decreased amplitudes and the prolonged end latency revealed axonal-type motor neuropathy. Demyelination is predominant in the early stage, followed by axonal injury. The expression level of JEV-E protein and viral RNA was elevated in the injured sciatic nerves, suggesting that it may cause PNI at the early stage. Inflammatory cell infiltration and increased inflammatory cytokines indicated that neuroinflammation is involved in JEV-induced PNI. IMPORTANCE JEV is a neurotropic flavivirus belonging to the Flaviviridae family and causes high mortality and disability rates. It invades the central nervous system and induces acute inflammatory injury and neuronal death. Thus, JEV infection is a major global public health concern. Previously, motor dysfunction was mainly attributed to central nervous system damage. Our knowledge regarding JEV-induced PNI is vague and neglected. Therefore, a laboratory animal model is essential. Herein, we showed that C57BL/6 mice can be used to study JEV-induced PNI through multiple approaches. We also demonstrated that viral loads might be positively correlated with lesion severity. Therefore, inflammation and direct virus infection may be the putative mechanisms underlying JEV-induced PNI. The results of this study laid the foundation for further elucidation of the pathogenesis mechanisms of PNI caused by JEV.

A blood-brain barrier-penetrating AAV2 mutant created by a brain microvasculature endothelial cell-targeted AAV2 variant.

Upon systemic administration, adeno-associated virus serotype 9 (AAV9) and the capsid variant PHP.eB show distinct tropism for the central nervous system (CNS), whereas AAV2 and the capsid variant BR1 transduce brain microvascular endothelial cells (BMVECs) with little transcytosis. Here, we show that a single amino acid substitution (from Q to N) in the BR1 capsid at position 587 (designated BR1N) confers a significantly higher blood-brain barrier (BBB) penetration capacity to BR1. Intravenously infused BR1N showed significantly higher CNS tropism than BR1 and AAV9. BR1 and BR1N likely use the same receptor for entry into BMVECs; however, the single amino acid substitution has profound consequences on tropism. This suggests that receptor binding alone does not determine the final outcome in vivo and that further improvements of capsids within predetermined receptor usage are feasible.

Cu,Zn-Superoxide Dismutase has Minimal Effects Against Cuprizone-Induced Demyelination, Microglial Activation, and Neurogenesis Defects in the C57BL/6 Mouse Hippocampus.

Cuprizone causes consistent demyelination and oligodendrocyte damage in the mouse brain. Cu,Zn-superoxide dismutase 1 (SOD1) has neuroprotective potential against various neurological disorders, such as transient cerebral ischemia and traumatic brain injury. In this study, we investigated whether SOD1 has neuroprotective effects against cuprizone-induced demyelination and adult hippocampal neurogenesis in C57BL/6 mice, using the PEP-1-SOD1 fusion protein to facilitate the delivery of SOD1 protein into hippocampal neurons. Eight weeks feeding of cuprizone-supplemented (0.2%) diets caused a significant decrease in myelin basic protein (MBP) expression in the stratum lacunosum-moleculare of the CA1 region, the polymorphic layer of the dentate gyrus, and the corpus callosum, while ionized calcium-binding adapter molecule 1 (Iba-1)-immunoreactive microglia showed activated and phagocytic phenotypes. In addition, cuprizone treatment reduced proliferating cells and neuroblasts as shown using Ki67 and doublecortin immunostaining. Treatment with PEP-1-SOD1 to normal mice did not show any significant changes in MBP expression and Iba-1-immunoreactive microglia. However, Ki67-positive proliferating cells and doublecortin-immunoreactive neuroblasts were significantly decreased. Simultaneous treatment with PEP-1-SOD1 and cuprizone-supplemented diets did not ameliorate the MBP reduction in these regions, but mitigated the increase of Iba-1 immunoreactivity in the corpus callosum and alleviated the reduction of MBP in corpus callosum and proliferating cells, not neuroblasts, in the dentate gyrus. In conclusion, PEP-1-SOD1 treatment only has partial effects to reduce cuprizone-induced demyelination and microglial activation in the hippocampus and corpus callosum and has minimal effects on proliferating cells in the dentate gyrus.

A mouse model of naturally occurring age-related cognitive impairment.

Age-related cognitive impairment (ARCI) is a neurological condition that affects millions of older people, but little is known about the increased risk of developing more severe neurodegeneration and dementia. Preclinical research is needed to understand the mechanisms of the impairment and the neuropathology associated with it. We have characterized a model of naturally occurring ARCI in the C57BL/6J mouse strain that shows an age-dependent development of cognitive impairment. As in people, some mice have little cognitive impairment while others have more severe cognitive impairment. Therefore, mice can be categorized as resistant or susceptible and the two groups can be studied for behavioral and neuropathology differences. Preliminary observations show no difference in strength and agility test scores between ARCI resistant and susceptible mice of either sex suggesting the cognitive impairment in ARCI susceptible mice is not accompanied by impairment in daily living activities, similar to ARCI in humans. The hippocampal area of the brain from ARCI susceptible mice shows evidence of an increase in the inflammatory cytokine MCP-1 compared to ARCI resistant mice, suggesting inflammation may be associated with ARCI. These preliminary observations suggest that ARCI in C57BL/6J mice could be a high-impact model to study how resilience to brain aging may predict resilience to dementia associated with Alzheimer's disease and other age-related neurological conditions.

Metal Ion Periplasmic-Binding Protein YfeA of Glaesserella parasuis Induces the Secretion of Pro-Inflammatory Cytokines of Macrophages via MAPK and NF-κB Signaling through TLR2 and TLR4.

The YfeA gene, belonging to the well-conserved ABC (ATP-binding cassette) transport system Yfe, encodes the substrate-binding subunit of the iron, zinc, and manganese transport system in bacteria. As a potential vaccine candidate in Glaesserella parasuis, the functional mechanisms of YfeA in the infection process remain obscure. In this study, vaccination with YfeA effectively protected the C56BL6 mouse against the G. parasuis SC1401 challenge. Bioinformatics analysis suggests that YfeA is highly conserved in G. parasuis, and its metal-binding sites have been strictly conserved throughout evolution. Stimulation of RAW 264.7 macrophages with YfeA verified that toll-like receptors (TLR) 2 and 4 participated in the positive transcription and expression of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α. The activation of TLR2 and TLR4 utilized the MyD88/MAL and TRIF/TRAM pairs to initiate TLRs signaling. Furthermore, YfeA was shown to stimulate nuclear translocation of NF-κB and activated diverse mitogen-activated protein (MAP) kinase signaling cascades, which are specific to the secretion of particular cytokine(s) in murine macrophages. Separate blocking TLR2, TLR4, MAPK, and RelA (p65) pathways significantly decreased YfeA-induced pro-inflammatory cytokine production. In addition, YfeA-stimulated RAW 264.7 produces the pro-inflammatory hallmark, reactive oxygen species (ROS). In conclusion, our findings indicate that YfeA is a novel pro-inflammatory mediator in G. parasuis and induces TLR2 and TLR4-dependent pro-inflammatory activity in RAW 264.7 macrophages through P38, JNK-MAPK, and NF-κB signaling pathways.

Garland chrysanthemum consumption ameliorates age-related hearing loss in C57BL/6 mouse; model system to explore hearing loss prevention foods in a short period.

Garland chrysanthemum (Glebionis coronaria L.) is an antioxidant-rich leafy vegetable. We found that garland chrysanthemum consumption ameliorated age-related hearing loss (AHL) in C57BL/6J mice, an early onset model. We also found that AHL progression was significantly ameliorated by three of ten products. Metabolome analysis of the 10 products using nuclear magnetic resonance (NMR) spectroscopy indicated that phytosterols may be involved in the amelioration of AHL. However, the direct inhibitory effect of phytosterol mixture on mouse AHL progression was not identified. These results suggest that garland chrysanthemum consumption delays AHL development in mice and its efficiency varies depending on the source of the product. Our findings also suggest that phytosterol content in garland chrysanthemum functions as an evaluation marker for the efficiency. Furthermore, to accelerate the search for foods that prevent AHL, we have used these data to develop an automatic threshold determination method for auditory brainstem response using machine learning.

Antitumor Properties of Epitope-Specific Engineered Vaccine in Murine Model of Melanoma.

Finding new effective compounds of natural origin for composing anti-tumor vaccines is one of the main goals of antitumor research. Promising anti-cancer agents are the gastropodan hemocyanins-multimeric copper-containing glycoproteins used so far for therapy of different tumors. The properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) upon their use as carrier-proteins in conjugated vaccines, containing ganglioside mimotope GD3P4 peptide, were studied in the developed murine melanoma model. Murine melanoma cell line B16F10 was used for solid tumor establishment in C57BL/6 mice using various schemes of therapy. Protein engineering, flow cytometry, and cytotoxicity assays were also performed. The administration of the protein-engineered vaccines RtH-GD3P4 or HaH-GD3P4 under the three different regimens of therapy in the B16F10 murine melanoma model suppressed tumor growth, decreased tumor incidence, and prolonged the survival of treated animals. The immunization of experimental mice induced an infiltration of immunocompetent cells into the tumors and generated cytotoxic tumor-specific T cells in the spleen. The treatment also generates significantly higher levels of tumor-infiltrated M1 macrophages, compared to untreated tumor-bearing control mice. This study demonstrated a promising approach for cancer therapy having potential applications for cancer vaccine research.

Mitochondrial NAD(P)+ Transhydrogenase: From Molecular Features to Physiology and Disease.

Significance: Proton-translocating NAD(P)+ transhydrogenase, also known as nicotinamide nucleotide transhydrogenase (NNT), catalyzes a reversible reaction coupling the protonmotive force across the inner mitochondrial membrane and hydride (H-, a proton plus two electrons) transfer between the mitochondrial pools of NAD(H) and NADP(H). The forward NNT reaction is a source of NADPH in the mitochondrial matrix, fueling antioxidant and biosynthetic pathways with reductive potential. Despite the greater emphasis given to the net forward reaction, the reverse NNT reaction that oxidizes NADPH also occurs in physiological and pathological conditions. Recent Advances: NNT (dys)function has been linked to various metabolic pathways and disease phenotypes. Most of these findings have been based on spontaneous loss-of-function Nnt mutations found in the C57BL/6J mouse strain (NntC57BL/6J mutation) and disease-causing Nnt mutations in humans. The present review focuses on recent advances based on the mouse NntC57BL/6J mutation. Critical Issues: Most studies associating NNT function with disease phenotypes have been based on comparisons between different strains of inbred mice (with or without the NntC57BL/6J mutation), which creates uncertainties over the actual contribution of NNT in the context of other potential genetic modifiers. Future Directions: Future research might contribute to understanding the role of NNT in pathological conditions and elucidate how NNT regulates physiological signaling through its forward and reverse reactions. The importance of NNT in redox balance and tumor cell proliferation makes it a potential target of new therapeutic strategies for oxidative-stress-mediated diseases and cancer. Antioxid. Redox Signal. 36, 864-884.

Intestinal microecology in mice bearing diethylnitrosamine-induced primary hepatocellular carcinoma.

OBJECTIVE: To explore the characteristics of intestinal microecology in hepatocellular carcinoma (HCC) model mice. METHODS: C57BL/6 male mice aged 2 weeks were divided into normal control group and HCC model group. Mice in HCC model group were exposed to a single intraperitoneal injection of diethylnitrosamine (DEN) 2 weeks after birth; the surviving mice were intraperitoneally injected with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), once every 2 weeks for 8 times starting from the 4 th week after birth. Mice in each group were randomly selected and sacrificed at 10 th, 18 th and 32 nd weeks after birth, respectively, the liver tissue samples were obtained for histopathological examination. At the 32 nd week, all mice in both groups were sacrificed and the feces samples were collected under sterile conditions right before the sacrifice. The feces samples were sequenced for the V3-V4 hypervariable regions of the 16S rRNA gene, and the species abundance, flora diversity and phenotype, as well as flora correlation and functional prediction were analyzed. RESULTS: Alpha diversity analysis showed that all Good's coverage reached the maximum value of 1.00, and the differences in the Observed features, Chao1 index, Shannon index and Simpson index of the intestinal flora of mice between normal control group and HCC model group were all statistically significant (all P<0.05). Beta diversity analysis showed that PCoA based on weighted or unweighted Unifrac distances all yielded R>0, confirming that the intra-group differences of the samples were less than the inter-group differences; the trend of separation between the two groups was significant ( P<0.05). Bacteroidetes, Firmicutes, Actinobacteria and Patescibacteria were the dominant taxa at the phylum level in both normal control group and HCC model group. However, compared with normal control group, the abundance of Bacteroidetes in HCC model group was significantly decreased ( P<0.01), while the abundance of Patescibacteria was significantly increased ( P<0.05). Moreover, the dominant taxa at the genus level in normal control group mainly included Muribaculaceae_unclassified, Paramuribaculum, Muribaculum, Lachnospiraceae_NK4A 136 group, Olsenella. The dominant taxa at the genus level in HCC model group mainly included Akkermansia, Dubosiella, Muribaculaceae_unclassified, Lachnospiraceae_NK4A 136 group, Coriobacteriaceae_UCG-002. There were 30 genera with statistically significant differences in relative abundance at the genus level between the two groups (all P<0.05). LEfSe analysis of the intestinal flora of mice in the two groups revealed a total of 14 multi-level differential taxa (all P<0.05, LDA score>4.0), which were mainly enriched in Bacteroidetes. The enrichment of 10 differential taxa including Bacteroidetes, Bacteroidia, Bacteroidales, Muribaculaceae, etc. were found in normal control group, and the enrichment of 4 differential taxa including Dubosiella, Peptostreptococus, etc. were found in HCC model group. There were both positive and negative correlations between the dominant intestinal genera in normal control group (|rho|>0.5, P<0.05), while the correlations of the dominant intestinal genera in HCC model group, being less complex than that in normal control group, were all positive. The relative abundance of gram positive and mobile element containing in the intestinal flora of mice in HCC model group was significantly up-regulated compared with normal control group (both P<0.05), while that of gram negative ( P<0.05) and pathogenic potential ( P<0.05) was significantly down-regulated. The metabolic pathways of the intestinal flora in the two groups were significantly different. For instance, 18 metabolic pathways were enriched in normal control group (all P<0.005), including those related to energy metabolism, cell division, nucleotide metabolism, etc., while 12 metabolic pathways were enriched in HCC model group (all P<0.005), including those related to energy metabolism, amino acid metabolism, carbohydrate metabolism, etc. Conclusions: The amount of intestinal flora in DEN-induced primary HCC model mice decreased, and the composition, correlation, phenotype and function of the intestinal flora in mice were significantly altered. Bacteroidetes at the phylum level, as well as several microbial taxa at the genus level such as Muribaculaceae_unclassified, Muribaculum, Peptostreptococus and Dubosiella could be closely associated with DEN-induced primary HCC in mice.

One extraction tool for in vitro-in vivo extrapolation? SPME-based metabolomics of in vitro 2D, 3D, and in vivo mouse melanoma models.

Solid phase microextraction (SPME) in combination with high-resolution mass spectrometry was employed for the determination of metabolomic profile of mouse melanoma growth within in vitro 2D, in vitro 3D, and in vivo models. Such multi-model approach had never been investigated before. Due to the low-invasiveness of SPME, it was possible to perform time-course analysis, which allowed building time profile of biochemical reactions in the studied material. Such approach does not require the multiplication of samples as subsequent analyses are performed from the very same cell culture or from the same individual. SPME already reduces the number of animals required for experiment; therefore, it is with good concordance with the 3Rs rule (replacement, reduction, and refinement). Among tested models, the largest number of compounds was found within the in vitro 2D cell culture model, while in vivo and in vitro 3D models had the lowest number of detected compounds. These results may be connected with a higher metabolic rate, as well as lower integrity of the in vitro 2D model compared to the in vitro 3D model resulting in a lower number of compounds released into medium in the latter model. In terms of in vitro-in vivo extrapolation, the in vitro 2D model performed more similar to in vivo model compared to in vitro 3D model; however, it might have been due to the fact that only compounds secreted to medium were investigated. Thus, in further experiments to obtain full metabolome information, the intraspheroidal assessment or spheroid dissociation would be necessary.

Voluntary Oral Ingestion of a Sedative Prior to Euthanasia with CO2: Behavioural Responses of Mice.

Laboratory mice are commonly euthanised with carbon dioxide (CO2); however, there is ample evidence that this gas is aversive. Previous work suggests that sedation achieved via injection with benzodiazepines prior to CO2 administration could reduce aversive behaviours during euthanasia. We explored the potential of using a voluntarily ingested sedative (tiletamine-zolazepam, Zoletil®) prior to euthanasia. Male and female C57BL/6 mice were allocated into one of the five experimental groups, which differed in the dose of Zoletil: 0, 10, 20, 40, 80 or 100 mg/kg. A dose of 20 mg/kg was found to achieve mild sedation prior to euthanasia; mice which received this dose numerically reared and walked on the cage lid less, and showed ataxia, immobility and recumbency for longer than mice that received a lower dose. During euthanasia, mice that received 20 mg/kg showed fewer aversive responses to CO2. Doses of 40 to 100 mg/kg were associated with signs of moderate to severe sedation, but resulted in an incomplete intake of the sedative, which made the interpretation of the aversiveness to CO2 difficult. Voluntary oral administration of a sedative is an effective, affordable, and easy way to minimize the stress of mice to euthanasia with CO2.

Integrated Metagenomics and Metabolomics to Reveal the Effects of Policosanol on Modulating the Gut Microbiota and Lipid Metabolism in Hyperlipidemic C57BL/6 Mice.

The aim of the study was to investigate the regulatory effects of policosanol on hyperlipidemia, gut microbiota and metabolic status in a C57BL/6 mouse model. A total of 35 C57BL/6 mice were assigned to 3 groups, chow (n=12), high fat diet (HFD, n=12) and HFD+policosanol (n=11), then treated for 18 weeks. Policosanol supplementation significantly reduced serum triglycerides and total cholesterol, as well as the weight of brown adipose tissue (BAT) (p<0.05), without affecting body weight in HFD-fed mice (p>0.05). Combined 16S rRNA gene sequencing and untargeted metabolomic analysis demonstrated that policosanol had regulatory effects on gut microbiota and serum metabolism in mice. In obese mice, policosanol increased the proportion of Bacteroides, decreased the proportion of Firmicutes, and increased the ratio of Bacteroides to Firmicutes (p<0.05). Policosanol promoted lipolysis and thermogenesis process, including tricarboxylic acid (TCA) cycle and pyruvate cycle, correlated with the increasing level of Bacteroides, Parasutterella, and decreasing level of Lactobacillus and Candidatus_Saccharimonas. Moreover, policosanol decreased fatty acid synthase (FAS) in the iWAT of obese mice. Policosanol also increased peroxisome proliferators-activated receptor-γ (PPARγ), uncoupling Protein-1 (UCP-1), peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and PR domain containing 16 (PRDM16) in brown adipose tissue (BAT) obese mice (p<0.05). This study presents the new insight that policosanol may inhibit the synthesis of fatty acids, and promote lipolysis, thermogenesis related gene expression and regulate gut microbiota constituents, which provides potential for policosanol as an antihyperlipidemia functional food additive and provide new evidence for whole grain food to replace refined food.