Activation of APE1 modulates Nrf2 protected against acute liver injury by inhibit hepatocyte ferroptosis and promote hepatocyte autophagy.

BACKGROUND: Apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) plays a crucial role in DNA base excision repair, cell apoptosis, cell signaling, and the regulation of transcription factors through redox modulation and the control of reactive oxygen species (ROS). However, the connection between APE1 and acute liver injury (ALI) remains enigmatic. This study aims to unravel the molecular mechanisms underlying ALI and shed light on the role of APE1 in this context. METHOD: We induced acute liver injury (ALI) in mice by lipopolysaccharide/D-galactosamine (LPS/GalN) and intervened with the APE1 inhibitor E3330. We examined the expression of APE1 in ALI mice and ALI patient tissues after E3330 intervention, Additionally, we measured hepatic oxidative stress, ferroptosis, and autophagy marker proteins and genes. In establishing an AML-12 liver cell injury model, we utilized the Nrf2 activator tert-butylhydroquinone (TBHQ) as an intervention and examined APE1, Nrf2, ferroptosis-related proteins, and autophagy marker proteins and mRNA. RESULTS: Both ALI patients and ALI mice exhibited reduced APE1 expression levels. After E3330 intervention, there was a significant exacerbation of liver injury, oxidative stress, and a reduction in the expression of proteins, including GPX4, X-CT, ATG3, ATG5, and LC3 (LC3I/II). Consistent results were also observed in AML-12 cells. With TBHQ intervention, Nrf2 expression increased, along with the expression of proteins associated with iron death and autophagy. Mechanistically, APE1 activation regulates Nrf2 to inhibit ferroptosis and promote autophagy in hepatocytes. CONCLUSION: The data suggest that APE1 is a pivotal player in ALI, closely linked to its regulation of Nrf2. Strategies involving APE1 activation to modulate Nrf2, thereby inhibiting hepatocyte ferroptosis and promoting autophagy, may represent innovative therapeutic approaches for ALI. Additionally, tert-butylhydroquinone (TBHQ) holds significant promise in the treatment of acute liver injury.

Stigmasterol attenuates hepatic steatosis in rats by strengthening the intestinal barrier and improving bile acid metabolism.

Stigmasterol (ST) has been shown to improve both lipid and bile acid (BA) metabolism. However, the mechanism(s) by which ST prevents dyslipidemia via BA metabolism, and the potential involvement of other regulatory mechanisms, remains unclear. Here, we found that ST treatment effectively alleviates lipid metabolism disorder induced by a high-fat diet (HFD). Moreover, we also show that fecal microbiota transplantation from ST-treated rats displays similar protective effects in rats fed on an HFD. Our data confirm that the gut microbiota plays a key role in attenuating HFD-induced fat deposition and metabolic disorders. In particular, ST reverses HFD-induced gut microbiota dysbiosis in rats by reducing the relative abundance of Erysipelotrichaceae and Allobaculum bacteria in the gut. In addition, ST treatment also modifies the serum and fecal BA metabolome profiles in rats, especially in CYP7A1 mediated BA metabolic pathways. Furthermore, chenodeoxycholic acid combined with ST improves the therapeutic effects in HFD-induced dyslipidemia and hepatic steatosis. In addition, this treatment strategy also alters BA metabolism profiles via the CYP7A1 pathway and gut microbiota. Taken together, ST exerts beneficial effects against HFD-induced hyperlipidemia and obesity with the underlying mechanism being partially related to both the reprogramming of the intestinal microbiota and metabolism of BAs in enterohepatic circulation. This study provides a theoretical basis for further study of the anti-obesity effects of ST and consideration of the gut microbiota as a potential target for the treatment of HFD-induced dyslipidemia.

The complete mitochondrial genome sequence of the Metschnikowia bicuspidata (Metschnikoff, 1884), an emerging pathogen of 'milky disease' in Chinese mitten crab.

The outbreak of milky disease of Chinese mitten crab caused by M. bicuspidata seriously restricted the development of the crab industry. In this study, the mitochondrial genome sequence of M. bicuspidata was assembled, annotated, and further analyzed. The results indicated that the complete mitochondrial genome of M. bicuspidata was 75,095 bp, which contained two rRNAs, 23 tRNAs, and 13 protein-coding genes. The phylogenetic tree of 13 yeasts based on the complete mitochondrial genome was constructed which showed that M. bicuspidata (accession number OK514652) and M. bicuspidata (accession number MW147605.1) were clustered in a clade. To sum up, our research results would further provide essential data for the systematics and evolution study of M. bicuspidata.

[Effect of Gegen Qinlian Decoction on cardiac diastolic function of diabetic mice with damp-heat syndrome].

This study was designed to explore the effect and mechanism of Gegen Qinlian Decoction(GQD) on cardiac function of diabetic mice with damp-heat syndrome. The db/db diabetic mice were exposed to the damp-heat environment test chamber for inducing the damp-heat syndrome. Forty-eight six-week-old db/db mice were randomly divided into six groups, namely the db/db diabetic model group, db/db diabetic mouse with damp-heat syndrome(db/db-dh) group, db/db diabetic mouse with damp-heat syndrome treated with low-dose GQD(db/db-dh+GQD-L) group, db/db-dh+GQD-M(medium-dose) group, db/db-dh+GQD-H(high-dose) group, and db/db-dh+lipro(liprostatin-1, the inhibitor of ferroptosis) group, with eight six-week-old db/m mice classified into the control group. The results showed that mice presented with the damp-heat syndrome after exposure to the "high-fat diet" and "damp-heat environment", manifested as the elevated fasting blood glucose, reduced food intake, low urine output, diarrhea, listlessness, loose and coarse hair, and dark yellow and lusterless fur. However, the intragastric administration of the high-dose GQD for 10 weeks ameliorated the above-mentioned symptoms, inhibited myocardial hypertrophy and fibrosis, and improved the cardiac diastolic function of db/db-dh mice. qPCR suggested that GQD regulated the expression of ferroptosis-related genes, weakened the lipid peroxidation in the myocardium, and up-regulated glutathione peroxidase 4(GPX4) expression in comparison with those in the db/db-dh group. At the same time, the ferroptosis inhibitor liprostatin-1 significantly improved the cardiac function and reversed the cardiac remodeling of db/db-dh mice. It can be concluded that the damp-heat syndrome may aggravate myocardial ferroptosis and accelerate cardiac remodeling of db/db mice, thus leading to diastolic dysfunction. GQD is able to improve cardiac remodeling and diastolic function in diabetic mice with damp-heat syndrome, which may be related to its inhibition of myocardial ferroptosis.

First case of Coxiella burnetii infection in Palaemonetes sinensis cultured in Liaoning, China.

The Palaemonetes sinensis aquaculture industry in Panjin City, Liaoning Province, China, experienced heavy losses in October 2018. Morbidity of cultured shrimp reached 50% and was characterized by cloudiness of muscle and the gradual spread of disease within the population. When the infection was mild, histopathological examinations revealed that the muscle cells contained a considerable number of microorganisms. In extreme cases, the structure of the hepatopancreatic glandular and muscle fiber was obscured or even vanished. Electron microscope observations revealed the presence of granular cytoplasmic inclusions in cells from hepatopancreas and muscle tissues. The 16S rDNA sequence of the intracytoplasmic organism was 94.7% identity to that of Coxiella burnetii. This is the first report of infection by C. burnetii in P. sinensis.

Validation of radiographic quality of simulation software - ImaSim.

BACKGROUND: Virtual radiographic simulation has been found educationally effective for students to practice their clinical examinations remotely or online. A free available virtual simulator-ImaSim has received particular attention for radiographic science education because of its portability, free of charge and no constrain of location and physical facility. However, it lacks evidence to validate this virtual simulation software to faithfully reproduce radiographs comparable to that taken from a real X-ray machine to date. OBJECTIVE: To evaluate image quality of the virtual radiographs produced by the ImaSim. Thus, the deployment of this radiographic simulation software for teaching and experimental studying of radiography can be justified. METHODS: A real medical X-ray examination machine is employed to scan three standard QC phantoms to produce radiographs for comparing to the corresponding virtual radiographs generated by ImaSim software. The high and low range of radiographic contrast and comprehensive contrast-detail performance are considered to characterize the radiographic quality of the virtual simulation software. RESULTS: ImaSim software can generate radiographs with a contrast ranging from 30% to 0.8% and a spatial resolution as low as 0.6mm under the selected exposure setting condition. The characteristics of contrast and spatial resolution of virtual simulation generally agree with that of real medical X-ray examination machine. CONCLUSION: ImaSim software can be used to simulate a radiographic imaging process to generate radiographs with contrast and detail detectability comparable to those produced by a real X-ray imaging machine. Therefore, it can be adopted as a flexible educational tool for proof of concept and experimental design in radiography.

Gabapentin increases expression of δ subunit-containing GABAA receptors.

BACKGROUND: Gabapentin is a structural analog of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Its anticonvulsant, analgesic and anxiolytic properties suggest that it increases GABAergic inhibition; however, the molecular basis for these effects is unknown as gabapentin does not directly modify GABA type A (GABAA) receptor function, nor does it modify synaptic inhibition. Here, we postulated that gabapentin increases expression of δ subunit-containing GABAA (δGABAA) receptors that generate a tonic inhibitory conductance in multiple brain regions including the cerebellum and hippocampus. METHODS: Cell-surface biotinylation, Western blotting, electrophysiologic recordings, behavioral assays, high-performance liquid chromatography and gas chromatography-mass spectrometry studies were performed using mouse models. FINDINGS: Gabapentin enhanced expression of δGABAA receptors and increased a tonic inhibitory conductance in neurons. This increased expression likely contributes to GABAergic effects as gabapentin caused ataxia and anxiolysis in wild-type mice but not δ subunit null-mutant mice. In contrast, the antinociceptive properties of gabapentin were observed in both genotypes. Levels of GABAA receptor agonists and neurosteroids in the brain were not altered by gabapentin. INTERPRETATION: These results provide compelling evidence to account for the GABAergic properties of gabapentin. Since reduced expression of δGABAA receptor occurs in several disorders, gabapentin may have much broader therapeutic applications than is currently recognized. FUND: Supported by a Foundation Grant (FDN-154312) from the Canadian Institutes of Health Research (to B.A.O.); a NSERC Discovery Grant (RGPIN-2016-05538), a Canada Research Chair in Sensory Plasticity and Reconsolidation, and funding from the University of Toronto Centre for the Study of Pain (to R.P.B.).

Fungal Systematics and Evolution: FUSE 3.

The present study introduces seven new species, one new combination, one new variety and several interesting taxonomical notes and/or geographical records. Most of the new taxa are Ascomycetes, but the study also includes a new variety of a Basidiomycete. Novel species include Gyromitra khanspurensis (Discinaceae, Pezizales, Pezizomycetes) from Pakistan growing near Cedrus deoadara and Paramyrothecium guiyangense and Paramyrothecium verruridum (Stachybotriaceae, Hypocreales, Sordariomycetes) both isolated from soil in China. New species from South Africa are Sclerostagonospora elegiae on culm litter of Elegia equisetacea, Sclerostagonospora fusiformis on culm litter of Thamnochortus spicigerus, Sclerostagonospora pinguis on culm litter of Cannomois virgata and Sclerostagonospora sulcata on culm litter of Ischyrolepis subverticellata (Phaeosphaeriaceae, Pleosporales, Dothideomycetes). Hapalocystis berkeleyi var. kickxii with its basionym Hypoxylon kickxii is shown to be a taxon on species level and thus recombined as Hapalocystis kickxii (Sydowiellaceae, Diaporthales, Sordariomycetes), and it is lecto- and epitypified. The new variety Pluteus romellii var. luteoalbus (Pluteaceae, Agaricales, Agaricomycetes) growing on a mossy fallen stem of a deciduous tree is described from Czech Republic. Cortinarius scaurocaninus (Cortinariaceae, Agaricales, Agaricomycetes) is new for Austria, Humicola grisea (Chaetomiaceae, Sordariales, Sordariomycetes) is an interesting new record for Chile. Two taxa are reported as new for Turkey: the lichenicolous fungus Opegrapha parasitica (Opegraphaceae, Arthoniales, Arthoniomycetes) growing partly immersed in the thallus of Aspicilia and the lichen Rinodina zwackhiana (Physciaceae, Teloschistales, Lecanoromycetes) from calcareous rock. Finally, Xerula strigosa (Physalacriaceae, Agaricales, Agaricomycetes), described from China, is confirmed to be present also in Pakistan.

High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli.

Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

Altered expression of δGABAA receptors in health and disease.

γ-Aminobutyric acid type A receptors that contain the δ subunit (δGABAA receptors) are expressed in multiple types of neurons throughout the central nervous system, where they generate a tonic conductance that shapes neuronal excitability and synaptic plasticity. These receptors regulate a variety of important behavioral functions, including memory, nociception and anxiety, and may also modulate neurogenesis. Given their functional significance, δGABAA receptors are considered to be novel therapeutic targets for the treatment of memory dysfunction, pain, insomnia and mood disorders. These receptors are highly responsive to sedative-hypnotic drugs, general anesthetics and neuroactive steroids. A further remarkable feature of δGABAA receptors is that their expression levels are highly dynamic and fluctuate substantially during development and in response to physiological changes including stress and the reproductive cycle. Furthermore, the expression of these receptors varies in pathological conditions such as alcoholism, fragile X syndrome, epilepsy, depression, schizophrenia, mood disorders and traumatic brain injury. Such fluctuations in receptor expression have significant consequences for behavior and may alter responsiveness to therapeutic drugs. This review considers the alterations in the expression of δGABAA receptors associated with various states of health and disease and the implications of these changes.

Sustained increase in α5GABAA receptor function impairs memory after anesthesia.

Many patients who undergo general anesthesia and surgery experience cognitive dysfunction, particularly memory deficits that can persist for days to months. The mechanisms underlying this postoperative cognitive dysfunction in the adult brain remain poorly understood. Depression of brain function during anesthesia is attributed primarily to increased activity of γ-aminobutyric acid type A receptors (GABA(A)Rs), and it is assumed that once the anesthetic drug is eliminated, the activity of GABA(A)Rs rapidly returns to baseline and these receptors no longer impair memory. Here, using a murine model, we found that a single in vivo treatment with the injectable anesthetic etomidate increased a tonic inhibitory current generated by α5 subunit-containing GABA(A)Rs (α5GABA(A)Rs) and cell-surface expression of α5GABA(A)Rs for at least 1 week. The sustained increase in α5GABA(A)R activity impaired memory performance and synaptic plasticity in the hippocampus. Inhibition of α5GABA(A)Rs completely reversed the memory deficits after anesthesia. Similarly, the inhaled anesthetic isoflurane triggered a persistent increase in tonic current and cell-surface expression of α5GABA(A)Rs. Thus, α5GABA(A)R function does not return to baseline after the anesthetic is eliminated, suggesting a mechanism to account for persistent memory deficits after general anesthesia.

Hydrogen peroxide increases GABAA receptor-mediated tonic current in hippocampal neurons.

Hydrogen peroxide (H2O2), a key reactive oxygen species, is produced at low levels during normal cellular metabolism and at higher concentrations under pathological conditions such as ischemia-reperfusion injury. The mechanisms by which H2O2 contributes to physiological and pathological processes in the brain remain poorly understood. Inhibitory GABA type A (GABAA) receptors critically regulate brain function by generating tonic and synaptic currents; however, it remains unknown whether H2O2 directly modulates GABAA receptor function. Here, we performed patch-clamp recordings, together with pharmacological and genetic approaches, to investigate the effects of H2O2 on GABAA receptor-mediated tonic and synaptic currents recorded in cultured mouse hippocampal neurons and CA1 pyramidal neurons in hippocampal slices. We found that H2O2 caused a dramatic increase in tonic current, whereas synaptic currents were unaffected. This increase in tonic current resulted from an extracellular oxidative reaction, which increased the potency of GABA, but only when GABAA receptors were activated by low concentrations of GABA. Oxygen-glucose deprivation, which produces high endogenous levels of H2O2, similarly increased the tonic current. These results suggest that GABAA receptor-mediated tonic current, which is potentiated by H2O2, might contribute to H2O2-induced brain dysfunction.

Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABA(A) receptor δ subunit in cerebellar granule neurons and delays motor development in rats.

Exposure to ethanol (EtOH) during fetal development can lead to long-lasting alterations, including deficits in fine motor skills and motor learning. Studies suggest that these are, in part, a consequence of cerebellar damage. Cerebellar granule neurons (CGNs) are the gateway of information into the cerebellar cortex. Functionally, CGNs are heavily regulated by phasic and tonic GABAergic inhibition from Golgi cell interneurons; however, the effect of EtOH exposure on the development of GABAergic transmission in immature CGNs has not been investigated. To model EtOH exposure during the 3rd trimester-equivalent of human pregnancy, neonatal pups were exposed intermittently to high levels of vaporized EtOH from postnatal day (P) 2 to P12. This exposure gradually increased pup serum EtOH concentrations (SECs) to ∼60 mM (∼0.28 g/dl) during the 4 h of exposure. EtOH levels gradually decreased to baseline 8 h after the end of exposure. Surprisingly, basal tonic and phasic GABAergic currents in CGNs were not significantly affected by postnatal alcohol exposure (PAE). However, PAE increased δ subunit expression at P28 as detected by immunohistochemical and western blot analyses. Also, electrophysiological studies with an agonist that is highly selective for δ-containing GABA(A) receptors, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol (THIP), showed an increase in THIP-induced tonic current. Behavioral studies of PAE rats did not reveal any deficits in motor coordination, except for a delay in the acquisition of the mid-air righting reflex that was apparent at P15 to P18. These findings demonstrate that repeated intermittent exposure to high levels of EtOH during the equivalent of the last trimester of human pregnancy has significant but relatively subtle effects on motor coordination and GABAergic transmission in CGNs in rats.

Hyperpolarization-activated current (In) is reduced in hippocampal neurons from Gabra5-/- mice.

Changes in the expression of γ-aminobutyric acid type A (GABAA) receptors can either drive or mediate homeostatic alterations in neuronal excitability. A homeostatic relationship between α5 subunit-containing GABAA (α5GABAA) receptors that generate a tonic inhibitory conductance, and HCN channels that generate a hyperpolarization-activated cation current (Ih) was recently described for cortical neurons, where a reduction in Ih was accompanied by a reciprocal increase in the expression of α5GABAA receptors resulting in the preservation of dendritosomatic synaptic function. Here, we report that in mice that lack the α5 subunit gene (Gabra5-/-), cultured embryonic hippocampal pyramidal neurons and ex vivo CA1 hippocampal neurons unexpectedly exhibited a decrease in Ih current density (by 40% and 28%, respectively), compared with neurons from wild-type (WT) mice. The resting membrane potential and membrane hyperpolarization induced by blockade of Ih with ZD-7288 were similar in cultured WT and Gabra5-/- neurons. In contrast, membrane hyperpolarization measured after a train of action potentials was lower in Gabra5-/- neurons than in WT neurons. Also, membrane impedance measured in response to low frequency stimulation was greater in cultured Gabra5-/- neurons. Finally, the expression of HCN1 protein that generates Ih was reduced by 41% in the hippocampus of Gabra5-/- mice. These data indicate that loss of a tonic GABAergic inhibitory conductance was followed by a compensatory reduction in Ih. The results further suggest that the maintenance of resting membrane potential is preferentially maintained in mature and immature hippocampal neurons through the homeostatic co-regulation of structurally and biophysically distinct cation and anion channels.

Memory deficits induced by inflammation are regulated by α5-subunit-containing GABAA receptors.

Systemic inflammation causes learning and memory deficits through mechanisms that remain poorly understood. Here, we studied the pathogenesis of memory loss associated with inflammation and found that we could reverse memory deficits by pharmacologically inhibiting α5-subunit-containing γ-aminobutyric acid type A (α5GABA(A)) receptors and deleting the gene associated with the α5 subunit. Acute inflammation reduces long-term potentiation, a synaptic correlate of memory, in hippocampal slices from wild-type mice, and this reduction was reversed by inhibition of α5GABA(A) receptor function. A tonic inhibitory current generated by α5GABA(A) receptors in hippocampal neurons was increased by the key proinflammatory cytokine interleukin-1ß through a p38 mitogen-activated protein kinase signaling pathway. Interleukin-1ß also increased the surface expression of α5GABA(A) receptors in the hippocampus. Collectively, these results show that α5GABA(A) receptor activity increases during inflammation and that this increase is critical for inflammation-induced memory deficits.

Regional differences in nerve terminal Na+ channel subtype expression and Na+ channel-dependent glutamate and GABA release in rat CNS.

We tested the hypothesis that expression of pre-synaptic voltage-gated sodium channel (Na(v)) subtypes coupled to neurotransmitter release differs between transmitter types and CNS regions in a nerve terminal-specific manner. Na(v) coupling to transmitter release was determined by measuring the sensitivity of 4-aminopyridine (4AP)-evoked [(3)H]glutamate and [(14)C]GABA release to the specific Na(v) blocker tetrodotoxin (TTX) for nerve terminals isolated from rat cerebral cortex, hippocampus, striatum and spinal cord. Expression of various Na(v) subtypes was measured by immunoblotting using subtype-specific antibodies. Potencies of TTX for inhibition of glutamate and GABA release varied between CNS regions. However, the efficacies of TTX for inhibition of 4AP-evoked glutamate release were greater than for inhibition of GABA release in all regions except spinal cord. The relative nerve terminal expression of total Na(v) subtypes as well as of specific subtypes varied considerably between CNS regions. The region-specific potencies of TTX for inhibition of 4AP-evoked glutamate release correlated with greater relative expression of total nerve terminal Na(v) and Na(v)1.2. Nerve terminal-specific differences in the expression of specific Na(v) subtypes contribute to transmitter-specific and regional differences in pharmacological sensitivities of transmitter release.

[Design of a high-voltage insulation testing system of X-ray high frequency generators].

In this paper, we analyze the transformer of X-ray high-voltage high-frequency generators and, have designed and implemented a high-voltage insulation testing system for its oil tank using full-bridge series resonant soft switching PFM DC-DC converter.