In vivo bioluminescence imaging revealed the change of the time window of BDNF expression in the brain elicited by a single bout of exercise following repeated exercise.

Exercise increases the expression of brain-derived neurotrophic factor (BDNF) in the brain and contributes to cognitive and sensorimotor functions. This study aimed to elucidate how repeated exercise modifies BDNF expression elicited by a single bout of exercise in the brain using in vivo bioluminescence imaging (BLI). Bdnf-luciferase (Luc) mice with the firefly luciferase gene inserted at the translation start point of the Bdnf gene were used for BLI to monitor changes in BDNF expression in the brain. The treadmill exercise at a speed of 10 m/s for 60 min was repeated 5 days a week for 4 weeks. BLI in individual subjects was repeated four times: before the exercise intervention, on the first exercise day, and 14 and 28 days after the start of the intervention. Each BLI was performed after a single bout of exercise and monitored for 8 h after exercise. Repetitive BLI showed that the exercise regimen enhanced BDNF expression in the brain, specifically at 4-8 h after a single bout of exercise. Repeated exercise for 2 weeks accelerated the start of enhancement after a single bout of exercise, but not after 4 weeks of repeated exercise. This study showed that repeated exercise modulated the time window of exercise-enhanced BDNF expression, suggesting that repeated exercise could change the sensitivity of gene expression to a single bout of exercise. These findings can be attributed to the advantages of in vivo BLI, which allowed us to precisely measure the time course of BDNF expression after repeated exercise in individual subjects.

Analysis of the Anticipatory Behavior Formation Mechanism Induced by Methamphetamine Using a Single Hair.

While the suprachiasmatic nucleus (SCN) coordinates many daily rhythms, some circadian patterns of expression are controlled by SCN-independent systems. These include responses to daily methamphetamine (MAP) injections. Scheduled daily injections of MAP resulted in anticipatory activity, with an increase in locomotor activity immediately prior to the time of injection. The MAP-induced anticipatory behavior is associated with the induction and a phase advance in the expression rhythm of the clock gene Period1 (Per1). However, this unique formation mechanism of MAP-induced anticipatory behavior is not well understood. We recently developed a micro-photomultiplier tube (micro-PMT) system to detect a small amount of Per1 expression. In the present study, we used this system to measure the formation kinetics of MAP-induced anticipatory activity in a single whisker hair to reveal the underlying mechanism. Our results suggest that whisker hairs respond to daily MAP administration, and that Per1 expression is affected. We also found that elevated Per1 expression in a single whisker hair is associated with the occurrence of anticipatory behavior rhythm. The present results suggest that elevated Per1 expression in hairs might be a marker of anticipatory behavior formation.

Temporal dynamics of brain BDNF expression following a single bout of exercise: A bioluminescence imaging study.

Physical exercise increases brain-derived neurotrophic factor (BDNF) expression in the brain. However, the absence of non-invasive and repetitive monitoring of BDNF expression in the brains of living animals has limited the understanding of how BDNF expression changes after exercise. This study aimed to elucidate the temporal dynamics of BDNF expression in the brain after a single bout of exercise, using in vivo bioluminescence imaging. This study included Bdnf-Luc mice with a firefly Luciferase gene inserted at the translation start site of the mouse Bdnf gene. BDNF expression was evaluated based on the luminescence signal of the luciferase substrate administered to mice. Bioluminescence imaging was performed at 0, 1, 3, 6, 12, and 24 h after treadmill exercise (15 m/min for 1 h). Compared to the sedentary condition of each mouse, the luminescence signal increased by approximately 60 % between 1 and 3 h after exercise. The luminescence signal remained slightly increased by approximately 20 % even 6-24 h after exercise. This study is the first to demonstrate exercise-enhanced BDNF expression in the brains of living animals. These results provide evidence that a single bout of exercise transiently increases BDNF expression in the brain within a limited time window.

The usefulness of measuring n-butyric acid concentration as a new indicator of blood decomposition in forensic autopsy.

In forensic medicine, although various alcohols have been reported as indicators of decomposition in collected blood, no studies have examined short-chain fatty acids as indicators. In this study, the blood n-butyric acid concentration was quantified, and the association between n-butyric acid and decomposition was investigated to determine whether the detection of n-butyric acid could be a new indicator of decomposition. Among the forensic autopsies performed from 2016 to 2018 in our laboratory, the cases were divided into decomposed (n = 20) and non-decomposed (n = 20) groups based on macroscopic findings. Blood samples collected at the time of autopsy were derivatized with 3-nitrophenylhydrazine hydrochloride after solid-phase extraction. The n-butyric acid concentration was measured using liquid chromatography-tandem mass spectrometry. In addition, ethanol and n-propanol were measured using a gas chromatography-flame ionization detector. There was a significant difference (p < 0.01) in the concentrations of n-butyric acid between the decomposed and non-decomposed groups (0.343 ± 0.259 [0.030-0.973] and 0.003 ± 0.002 [0.001-0.007] mg/mL, respectively). In the decomposed group, n-butyric acid was detected at high concentrations, even in cases where n-propanol was low. These results suggest that n-butyric acid is more likely to be an indicator of blood decomposition than n-propanol.

Poly(ADP-ribose) Polymerase (PARP) is Critically Involved in Liver Ischemia/Reperfusion-injury.

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) is a DNA-repairing enzyme activated by extreme genomic stress, and therefore is potently activated in the remnant liver suffering from ischemia after surgical resection. However, the impact of PARP on post-ischemic liver injury has not been elucidated yet. MATERIALS AND METHODS: We investigated the impact of PARP on murine hepatocyte/liver injury induced by hypoxia/ischemia, respectively. RESULTS: PJ34, a specific inhibitor of PARP, markedly protected against hypoxia/reoxygenation (H/R)-induced cell death, though z-VAD-fmk, a pan-caspase inhibitor similarly showed the protective effect. PJ34 did not affect H/R-induced caspase activity or caspase-mediated cell death. z-VAD-fmk also did not affect the production of PAR (i.e., PARP activity). Therefore, PARP- and caspase-mediated cell death occurred in a mechanism independent of each other in H/R. H/R immediately induced activation of PARP and cell death afterwards, both of which were suppressed by PJ34 or Trolox, an antioxidant. This suggests that H/R-induced cell death occurred redox-dependently through PARP activation. H/R and OS induced nuclear translocation of apoptosis inducing factor (AIF, a marker of parthanatos) and RIP1-RIP3 interaction (a marker of necroptosis), both of which were suppressed by PJ34. H/R induced PARP-mediated parthanatos and necroptosis redox-dependently. In mouse experiments, PJ34 significantly reduced serum levels of AST, ALT & LDH and areas of hepatic necrosis after liver ischemia/reperfusion, similar to z-VAD-fmk or Trolox. CONCLUSION: PARP, activated by ischemic damage and/or oxidative stress, may play a critical role in post-ischemic liver injury by inducing programmed necrosis (parthanatos and necroptosis). PARP inhibition may be one of the promising strategies against post-ischemic liver injury.

Period1 gene expression in the olfactory bulb and liver of freely moving streptozotocin-treated diabetic mouse.

Clock genes express circadian rhythms in most organs. These rhythms are organized throughout the whole body, regulated by the suprachiasmatic nucleus (SCN) in the brain. Disturbance of these clock gene expression rhythms is a risk factor for diseases such as obesity. In the present study, to explore the role of clock genes in developing diabetes, we examined the effect of streptozotocin (STZ)-induced high glucose on Period1 (Per1) gene expression rhythm in the liver and the olfactory bub (OB) in the brain. We found a drastic increase of Per1 expression in both tissues after STZ injection while blood glucose content was low. After a rapid expression peak, Per1 expression showed no rhythm. Associated with an increase of glucose content, behavior became arrhythmic. Finally, we succeeded in detecting an increase of Per1 expression in mice hair follicles on day 1 after STZ administration, before the onset of symptoms. These results show that elevated Per1 expression by STZ plays an important role in the aggravation of diabetes.

Sample preparation method with ultrafiltration for whole blood thiosulfate measurement.

Quantitative analysis of thiosulfate is useful for diagnosing hydrogen sulfide poisoning. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables more rapid and sensitive measurements than previous methodologies. As simple measurements of blood thiosulfate concentration are affected by the blood matrix, blood is used as the solvent to prepare the standard solution for calibration curve generation. Thus, a large amount of blood devoid of thiosulfate is required. We developed a preparation method by incorporating an ultrafiltration step to overcome this limitation and generate a calibration curve using a standard solution prepared with pure water. We used this improved method to investigate the stability of thiosulfate in refrigerated samples. To compare the effects of refrigeration, blood samples were prepared using the following two methods: one sample was treated with a 50-kDa exclusion ultrafiltration membrane and the other was not treated. The samples were stored at 4 °C, and then measured at 0, 3, 6, 24, 48, and 96 h. The incorporation of the ultrafiltration step in the measurement procedure enabled the quantification of thiosulfate, by plotting a calibration curve using a standard of pure water; it did not require a blood standard. Additionally, the reduction in whole blood thiosulfate concentration was within 10% during 2 days of refrigeration. Thus, the need for a large amount of blood to prepare the standard solution was resolved by the ultrafiltration step in test sample preparation. This method is useful to measure thiosulfate concentration and is not hindered by sample refrigeration for a few days.

Extracts of bilberry (Vaccinium myrtillus L.) fruits improve liver steatosis and injury in mice by preventing lipid accumulation and cell death.

Bilberry has been reported to have anti-oxidant and anti-inflammatory properties. We studied the effect of bilberry (Vaccinium myrtillus L.) fruits extracts (BEs) on the pathogenesis caused by lipid accumulation in fatty liver and non-alcoholic steatohepatitis (NASH). 5 µg/ml of BEs was enough to suppress lipid accumulation in the fatty liver model of the mouse hepatic AML12 cells. BEs increased cell viability and anti-oxidant capacity, presumably by activating (phosphorylating) Akt/STAT3 and inducing MnSOD/catalase. BEs also significantly reduced Rubicon and induced p62/SQSTM1, possibly contributing to reduce cellular lipids (lipophagy). When the mice were fed supplemented with BEs (5% or 10%, w/w), hepatic steatosis, injury, and hypercholesterolemia/hyperglycemia were significantly improved. Furthermore, histological and cytokine studies indicated that BEs possibly suppress hepatic inflammation (hepatitis) and fibrosis. Therefore, BEs improved liver steatosis and injury, and potentially suppress fibrosis by suppressing inflammatory response, which therefore may prevent the progression of fatty liver to NASH.

Activation of caspase-3 during Chlamydia trachomatis-induced apoptosis at a late stage.

The obligate intracellular bacterium Chlamydia trachomatis activates the host cell apoptosis pathway at a late stage of its developmental cycle. However, whether caspase-3, which is a key enzyme of apoptosis, is activated in Chlamydia-infected cells remains unknown. Here, we established HEp-2 cells stably expressing cFluc-DEVD, which is a caspase-3 substrate sequence inserted into cyclic firefly luciferase, and then monitored the dynamics of caspase-3 activity in cells infected with Chlamydia. Transfected cells without infection showed a significant increase in luciferase activity due to stimulation with staurosporine, an inducer of apoptosis. Activation was significantly blocked by addition of caspase inhibitor z-VAD-fmk. Furthermore, as expected, Chlamydia infection caused a significant increase in luciferase activation at 36-48 h postinfection with a contrastive decrease at 24 h postinfection, which is already well known. Such activation caused by the infection was much stronger when the amount of bacteria was increased. Thus, caspase-3 activation was accurately monitored by the luciferase activity in HEp-2 cells constitutively expressing the cFluc-DEVD probe. Furthermore, our data showed that C. trachomatis activates caspase-3 in host cells at a late stage of infection.

Photo-Activatable Akt Probe: A New Tool to Study the Akt-Dependent Physiopathology of Cancer Cells.

Akt is commonly overexpressed and activated in cancer cells and plays a pivotal role in cell survival, protection, and chemoresistance. Therefore, Akt is one of the target molecules in understanding characters of cancer cells and developing anticancer drugs. Here we examined whether a newly developed photo-activatable Akt (PA-Akt) probe, based on a light-inducible protein interaction module of plant cryptochrome2 (CRY2) and cryptochrome-interacting basic helix-loop-helix (CIB1), can regulate Akt-associated cell functions. By illuminating blue light to the cells stably transfected with PA-Akt probe, CRY2-Akt (a fusion protein of CRY2 and Akt) underwent a structural change and interacted with Myr-CIBN (myristoylated N-terminal portion of CIB1), anchoring it at the cell membrane. Western blot analysis revealed that S473 and T308 of the Akt of probe-Akt were sequentially phosphorylated by intermittent and continuous light illumination. Endogenous Akt and GSK-3ß, one of the main downstream signals of Akt, were also phosphorylated, depending on light intensity. These facts indicate that photo-activation of probe-Akt can activate endogenous Akt and its downstream signals. The photo-activated Akt conferred protection against nutritional deprivation and H2O2 stresses to the cells significantly. Using the newly developed PA-Akt probe, endogenous Akt was activated easily, transiently, and repeatedly. This probe will be a unique tool in studying Akt-associated specific cellular functions in cancer cells and developing anticancer drugs.

Detection of Necroptosis in Ligand-Mediated and Hypoxia-Induced Injury of Hepatocytes Using a Novel Optic Probe-Detecting Receptor-Interacting Protein (RIP)1/RIP3 Binding.

Liver injury is often observed in various pathological conditions including posthepatectomy state and cancer chemotherapy. It occurs mainly as a consequence of the combined necrotic and apoptotic types of cell death. In order to study liver/hepatocyte injury by the necrotic type of cell death, we studied signal-regulated necrosis (necroptosis) by developing a new optic probe for detecting receptor-interacting protein kinase 1 (RIP)/RIP3 binding, an essential process for necroptosis induction. In the mouse hepatocyte cell line, TIB-73 cells, TNF-α/cycloheximide (T/C) induced RIP1/3 binding only when caspase activity was suppressed by the caspase-specific inhibitor z-VAD-fmk (zVAD). T/C/zVAD-induced RIP1/3 binding was inhibited by necrostatin-1 (Nec-1), an allosteric inhibitor of RIP1. The reduced cell survival by T/C/zVAD was improved by Nec-1. These facts indicate that T/C induces necroptosis of hepatocytes when the apoptotic pathway is inhibited/unavailable. FasL also induced cell death, which was only partially inhibited by zVAD, indicating the possible involvement of necroptosis rather than apoptosis. FasL activated caspase 3 and, similarly, induced RIP1/3 binding when the caspases were inactivated. Interestingly, FasL-induced RIP1/3 binding was significantly suppressed by the antioxidants Trolox and N-acetyl cysteine (NAC), suggesting the involvement of reactive oxygen species (ROS) in FasL-induced necroptotic cellular processes. H2O2, by itself, induced RIP1/3 binding that was suppressed by Nec-1, but not by zVAD. Hypoxia induced RIP1/3 binding after reoxygenation, which was suppressed by Nec-1 or by the antioxidants. Cell death induced by hypoxia/reoxygenation (H/R) was also improved by Nec-1. Similar to H2O2, H/R did not require caspase inhibition for RIP1/3 binding, suggesting the involvement of a caspase-independent mechanism for non-ligand-induced and/or redox-mediated necroptosis. These data indicate that ROS can induce necroptosis and mediate the FasL- and hypoxia-induced necroptosis via a molecular mechanism that differs from a conventional caspase-dependent pathway. In conclusion, necroptosis is potentially involved in liver/hepatocyte injury induced by oxidative stress and FasL in the absence of apoptosis.

Relevance of FXR-p62/SQSTM1 pathway for survival and protection of mouse hepatocytes and liver, especially with steatosis.

BACKGROUND: Liver injury and regeneration involve complicated processes and are affected by various physio-pathological conditions. Surgically, severe liver injury after surgical resection often leads to fatal liver failure, especially with some underlying pathological conditions such as steatosis. Therefore, protection from the injury of hepatocytes and liver is a serious concern in various clinical settings. METHODS: We studied the effects of the farnesoid X receptor (FXR) on cell survival and steatosis in mouse hepatocytes (AML12 mouse liver cells) and investigated their molecular mechanisms. We next studied whether or not FXR improves liver injury, regeneration and steatosis in a mouse model of partial hepatectomy (PH) with steatosis. RESULTS: An FXR-specific agonist, GW4064, induced expressions of the p62/SQSTM1 gene and protein in AML12 mouse liver cells. Because we previously reported p62/SQSTM1 as a key molecule for antioxidation and cell survival in hepatocytes, we next examined the activation of nuclear factor erythroid 2-related factor-2 (Nrf2) and induction of the antioxidant molecules by GW4064. GW4064 activated Nrf2 and subsequently induced antioxidant molecules (Nrf2, catalase, HO-1, and thioredoxin). We also examined expressions of pro-survival and cell protective molecules associated with p62/SQSTM1. Expectedly, GW4064 induced phosphorylation of Akt, expression of the anti-apoptotic molecules (Bcl-xL and Bcl-2), and reduced harmful hepatic molecules (Fas ligand and Fas). GW4064 promoted hepatocyte survival, which was cancelled by p62/SQSTM1 siRNA. These findings suggest the potential relevance of the FXR-p62/SQSTM1 pathway for the survival and protection of hepatocytes. Furthermore, GW4064 induced the expression of small heterodimer partners (SHP) and suppressed liver X receptor (LXR)-induced steatosis in hepatocytes, expecting the in vivo protective effect of FXR on liver injury especially with steatosis. In the hepatectomy model of db/db mice with fatty liver, pre-treatment by GW4064 significantly reduced post-PH liver injury (serum levels of LDH, AST & ALT and histological study) and improved steatosis. The key molecules, p62/SQSTM1, Nrf2 and SHP were upregulated in fatty liver tissue by GW4064 treatment. CONCLUSIONS: The present study is the first to demonstrate the relevance of FXR-p62/SQSTM1 and -SHP in the protection against injury of hepatocytes and post-PH liver, especially with steatosis.

Development for the measurement of serum thiosulfate using LC-MS/MS in forensic diagnosis of H2S poisoning.

Thiosulfate measurement is crucial to diagnosis of hydrogen sulfide (H2S) poisoning in forensic toxicology. Although GC-MS method is currently regarded as a standard thiosulfate measurement, it requires complicated sample preparation prior to analysis. This study presents a simple, rapid, and highly sensitive method for the quantitative analysis of serum thiosulfate by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method is based on selected reaction monitoring and has high sensitivity with a lower quantification limit of 0.5µM. Precision and accuracy of this method meet the basic requirements for quantitative analysis (intra- and inter-day tests have a relative standard deviation of ⩽10.4%; range of analytical recovery is 94.3-102.6%). On the measurements of serum thiosulfate by our developed method, a thiosulfate concentration as 57.5µM was detected clearly in the H2S poisoning case comparing to the non poisoning case in which only a trace amount of thiosulfate was observed.

Inflammatory responses increase secretion of MD-1 protein.

Radioprotective 105 (RP105) is a type I transmembrane protein, which associates with a glycoprotein, MD-1. Monoclonal antibody (mAb)-mediated ligation of RP105/MD-1 robustly activates B cells. RP105/MD-1 is structurally similar to Toll-like receptor 4 (TLR4)/MD-2. B-cell responses to TLR2 and TLR4/MD-2 ligands are impaired in the absence of RP105 or MD-1. In addition to RP105/MD-1, MD-1 alone is secreted. The structure of MD-1 shows that MD-1 has a hydrophobic cavity that directly binds to phospholipids. Little is known, however, about a ligand for MD-1 and the role of MD-1 in vivo To study the role of RP105/MD-1 and MD-1 alone, specific mAbs against MD-1 are needed. Here, we report the establishment and characterization of two anti-MD-1 mAbs (JR2G9, JR7G1). JR2G9 detects soluble MD-1, whereas JR7G1 binds both soluble MD-1 and the cell surface RP105/MD-1 complex. With these mAbs, soluble MD-1 was detected in the serum and urine. The MD-1 concentration was altered by infection, diet and reperfusion injury. Serum MD-1 was rapidly elevated by TLR ligand injection in mice. The quantitative PCR and supernatant-precipitated data indicate that macrophages are one of the sources of serum soluble MD-1. These results suggest that soluble MD-1 is a valuable biomarker for inflammatory diseases.

Long-term ex vivo and in vivo monitoring of tumor progression by using dual luciferases.

We propose a new concept of tumor progression monitoring using dual luciferases in living animals to reduce stress for small animals and the cost of luciferin. The secreted Cypridina luciferase (CLuc) was used as an ex vivo indicator to continuously monitor tumor progression. On the other hand, the non-secreted firefly luciferase was used as an in vivo indicator to analyze the spatial distribution of the tumor at suitable time points indicated by CLuc. Thus, the new monitoring systems that use dual luciferases are available, allowing long-term bioluminescence imaging under minimal stress for the experimental animals.

Mitochondrial delivery of Coenzyme Q10 via systemic administration using a MITO-Porter prevents ischemia/reperfusion injury in the mouse liver.

We herein report on a mitochondrial therapeutic effect based on the delivery of coenzyme Q10 (CoQ10), an anti-oxidant, to in vivo mitochondria using a MITO-Porter, a liposome-based mitochondrial delivery system that functions via membrane fusion. To evaluate the effects, we used a mouse liver ischemia/reperfusion injury (I/R injury) model, in which mitochondrial reactive oxygen species are overexpressed. We packaged CoQ10 in the lipid phase of a MITO-Porter and optimized the mitochondrial fusogenic activities to produce the CoQ10-MITO-Porter. A histological observation of the carriers in the liver by confocal laser scanning microscopy was done and the accumulation of the carrier labeled with a radio isotope in the liver confirmed that the CoQ10-MITO-Porter was delivered to liver mitochondria via systemic injection. These analytical results permitted us to optimize the compositions of the CoQ10-MITO-Porter so as to permit it to efficiently accumulate in mouse liver mitochondria. Finally, we applied the optimized CoQ10-MITO-Porter to mice via tail vein injection, and hepatic I/R injury was then induced, followed by measuring serum alanine aminotransferase (ALT) levels, a marker of liver injury. We confirmed that the use of the CoQ10-MITO-Porter resulted in a significant decrease in serum ALT levels, indicating that in vivo mitochondrial delivery of the CoQ10 via MITO-Porter prevents I/R injury in mice livers. This provides a demonstration of the potential use of such a delivery system in mitochondrial therapies.

Growth arrest and DNA damage-inducible 34 regulates liver regeneration in hepatic steatosis in mice.

UNLABELLED: The liver has robust regenerative potential in response to damage, but hepatic steatosis (HS) weakens this potential. We found that the enhanced integrated stress response (ISR) mediated by phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) impairs regeneration in HS and that growth arrest and DNA damage-inducible 34 (Gadd34)-dependent suppression of ISR plays a crucial role in fatty liver regeneration. Although mice fed a high-fat diet for 2 weeks developed moderate fatty liver with no increase in eIF2α phosphorylation before 70% hepatectomy, they showed impaired liver regeneration as a result of reduced proliferation and increased death of hepatocytes with increased phosphorylation of eIF2α and ISR. An increased ISR through Gadd34 knockdown induced C/EBP homologous protein (CHOP)-dependent apoptosis and receptor-interacting protein kinase 3-dependent necrosis, resulting in increased hepatocyte death during fatty liver regeneration. Furthermore, Gadd34 knockdown and increased phosphorylation of eIF2α decreased cyclin D1 protein and reduced hepatocyte proliferation. In contrast, enhancement of Gadd34 suppressed phosphorylation of eIF2α and reduced CHOP expression and hepatocyte apoptosis without affecting hepatocyte proliferation, clearly improving fatty liver regeneration. In more severe fatty liver of leptin receptor-deficient db/db mice, forced expression of hepatic Gadd34 also promoted hepatic regeneration after hepatectomy. CONCLUSION: Gadd34-mediated regulation of ISR acts as a physiological defense mechanism against impaired liver regeneration resulting from steatosis and is thus a possible therapeutic target for impaired regeneration in HS.

p62/SQSTM1 plays a protective role in oxidative injury of steatotic liver in a mouse hepatectomy model.

AIMS: Liver injury and regeneration involve complicated processes and are affected by various physio-pathological factors. We investigated the mechanisms of steatosis-associated liver injury and delayed regeneration in a mouse model of partial hepatectomy. RESULTS: Initial regeneration of the steatotic liver was significantly delayed after hepatectomy. Although hepatocyte proliferation was not significantly suppressed, severe liver injury with oxidative stress (OS) occurred immediately after hepatectomy in the steatotic liver. Fas-ligand (FasL)/Fas expression was upregulated in the steatotic liver, whereas the expression of antioxidant and anti-apoptotic molecules (catalase/MnSOD/Ref-1 and Bcl-2/Bcl-xL/FLIP, respectively) and p62/SQSTM1, a steatosis-associated protein, was downregulated. Interestingly, pro-survival Akt was not activated in response to hepatectomy, although it was sufficiently expressed even before hepatectomy. Suppression of p62/SQSTM1 increased FasL/Fas expression and reduced nuclear factor erythroid 2-related factor-2 (Nrf-2)-dependent antioxidant response elements activity and antioxidant responses in steatotic and nonsteatotic hepatocytes. Exogenously added FasL induced severe cellular OS and necrosis/apoptosis in steatotic hepatocytes, with only the necrosis being inhibited by pretreatment with antioxidants, suggesting that FasL/Fas-induced OS mainly leads to necrosis. Furthermore, p62/SQSTM1 re-expression in the steatotic liver markedly reduced liver injury and improved liver regeneration. INNOVATION: This study is the first which demonstrates that reduced expression of p62/SQSTM1 plays a crucial role in posthepatectomy acute injury and delayed regeneration of steatotic liver, mainly via redox-dependent mechanisms. CONCLUSION: In the steatotic liver, reduced expression of p62/SQSTM1 induced FasL/Fas overexpression and suppressed antioxidant genes, mainly through Nrf-2 inactivation, which, along with the hypo-responsiveness of Akt, caused posthepatectomy necrotic/apoptotic liver injury and delayed regeneration, both mainly via a redox-dependent mechanism.

Contribution of toll-like receptor 2 to the innate response against Staphylococcus aureus infection in mice.

Staphylococcus aureus is a common pathogen that causes a wide range of infectious diseases. The function of TLRs, specifically TLR2, during S. aureus infection is still debated. In this study, we investigated the extent to which TLR2 contributes to the host innate response against the bacterial infection using TLR2-deficient mice. Intravenous inoculation with S. aureus resulted in all TLR2-deficient mice dying within 4 d, along with a high bacterial burden in the livers. However, histological examination showed the same degree of macrophage and neutrophil accumulation in the livers of infected TLR2-deficient mice as that in infected wild-type (WT) mice. TLR2-deficient mouse macrophages also showed normal phagocytic activity, although they failed to express CD36 that appeared on the surface of WT mouse cells upon challenge with heat-killed S. aureus. These data indicate that TLR2, as well as CD36, does not directly affect S. aureus clearance and that CD36 expression on macrophages depends on the presence of TLR2. In vivo infection with S. aureus caused significantly elevated production of TNF-α and IL-6 in the livers and blood of TLR2-deficient mice compared with those in WT mice, while the hepatic and serum levels of IL-10 decreased in these mice. In contrast, lower expression of IL-6 and IL-10, but not of TNF-α, at both the gene and protein levels was found in TLR2-deficient mouse macrophages compared to that in WT mouse cells, in response to challenge with heat-killed S. aureus. These findings suggest that the S. aureus-induced pro-inflammatory cytokine response is not dependent on macrophages and that TLR2 deficiency results in decreased IL-10 release by macrophages, which contributes to dysregulated cytokine balance, impaired bacterial clearance, and mouse death. Therefore, TLR2 possesses a protective function during S. aureus infection by regulating pro- and anti-inflammatory cytokine responses.

Sustained accurate recording of intracellular acidification in living tissues with a photo-controllable bioluminescent protein.

Regulation of an intracellular acidic environment plays a pivotal role in biological processes and functions. However, spatiotemporal analysis of the acidification in complex tissues of living subjects persists as an important challenge. We developed a photo-inactivatable bioluminescent indicator, based on a combination of luciferase-fragment complementation and a photoreaction of a light, oxygen, and voltage domain from Avena sativa Phototropin1 (LOV2), to visualize temporally dynamic acidification in living tissue samples. Bioluminescence of the indicator diminished upon light irradiation and it recovered gradually in the dark state thereafter. The recovery rate was remarkably sensitive to pH changes but unsusceptible to fluctuation of luciferin or ATP concentrations. Bioluminescence imaging, taken as an index of the recovery rates, enabled long-time recording of acidification in apoptotic and autophagous processes in a cell population and an ischemic condition in living mice. This technology using the indicator is widely applicable to sense organelle-specific acidic changes in target biological tissues.