Human herpesvirus 6 and 7 are biomarkers for fatigue, which distinguish between physiological fatigue and pathological fatigue.

Fatigue reduces productivity and is a risk factor for lifestyle diseases and mental disorders. Everyone experiences physiological fatigue and recovers with rest. Pathological fatigue, however, greatly reduces quality of life and requires therapeutic interventions. It is therefore necessary to distinguish between the two but there has been no biomarker for this. We report on the measurement of salivary human herpesvirus (HHV-) 6 and HHV-7 as biomarkers for quantifying physiological fatigue. They increased with military training and work and rapidly decreased with rest. Our results suggested that macrophage activation and differentiation were necessary for virus reactivation. However, HHV-6 and HHV-7 did not increase in obstructive sleep apnea syndrome (OSAS), chronic fatigue syndrome (CFS) and major depressive disorder (MDD), which are thought to cause pathological fatigue. Thus, HHV-6 and HHV-7 would be useful biomarkers for distinguishing between physiological and pathological fatigue. Our findings suggest a fundamentally new approach to evaluating fatigue and preventing fatigue-related diseases.

Mobile and accurate detection system for infection by the 2009 pandemic influenza A (H1N1) virus with a pocket-warmer reverse-transcriptase loop-mediated isothermal amplification.

The 2009 pandemic H1N1 influenza A virus spread quickly worldwide in 2009. Since most of the fatal cases were reported in developing countries, rapid and accurate diagnosis methods that are usable in poorly equipped laboratories are necessary. In this study, a mobile detection system for the 2009 H1N1 influenza A virus was developed using a reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) kit with a disposable pocket-warmer as a heating device (designated as pwRT-LAMP). The pwRT-LAMP can detect as few as 100 copies of the virus--which is nearly as sensitive as real-time reverse-transcription polymerase chain reaction (RT-PCR)--and does not cross-react with RNA of seasonal influenza viruses. To evaluate the usefulness of the pwRT-LAMP system, nasal swab samples were collected from 56 patients with flu-like symptoms and were tested. Real-time RT-PCR confirmed that the 2009 H1N1 influenza A virus was present in 27 of the 56 samples. Of these 27 positive samples, QuickVue Influenza A+B immunochromatography detected the virus in only 11 samples (11/27; 40.7%), whereas the pwRT-LAMP system detected the virus in 26 of the 56 samples (26/27 of the positive samples; 96.3%). These findings indicate that the mobile pwRT-LAMP system is an accurate diagnostic system for the 2009 H1N1 influenza A virus, and has great potential utility in diagnosing future influenza pandemics.

Axonal damage and behavioral deficits in rats with repetitive exposure of the brain to laser-induced shock waves: Effects of inter-exposure time.

Much attention has been given to effects of repeated exposure to a shock wave as a possible factor causing severe higher brain dysfunction and post-traumatic stress disorder (PTSD)-like symptoms in patients with mild to moderate blast-induced traumatic brain injury (bTBI). However, it is unclear how the repeated exposure and the inter-exposure time affect the brain. In this study, we topically applied low-impulse (∼54 Pa·s) laser-induced shock waves (LISWs; peak pressure, ∼75.7 MPa) to the rat brain once or twice with the different inter-exposure times (15 min, 1 h, 3 h, 24 h and 7 days) and examined anxiety-related behavior and motor dysfunction in the rats as well as expression of ß-amyloid precursor protein (APP) as an axonal damage marker in the brains of the rats. The averaged APP expression scores for the rat brains doubly-exposed to LISWs with inter-exposure times from 15 min to 24 h were significantly higher than those for rats with a single exposure (P < 0.0001). The rats with double exposure to LISWs showed significantly more frequent anxiety-related behavior (P < 0.05) and poorer motor function (P < 0.01) than those of rats with a single exposure. When the inter-exposure time was extended to 7 days, however, the rats showed no significant differences either in axonal damage score or level of motor dysfunction. The results suggest that the cumulative effects of shock wave-related brain injury can be avoided with an appropriate inter-exposure time. However, clinical bTBI occurs in much more complex environments than those in our model. Further study considering other factors, such as the effects of acceleration, is needed to know the clinically-relevant, necessary inter-exposure time.

Treatment of irradiated mice with high-dose ascorbic acid reduced lethality.

Ascorbic acid is an effective antioxidant and free radical scavenger. Therefore, it is expected that ascorbic acid should act as a radioprotectant. We investigated the effects of post-radiation treatment with ascorbic acid on mouse survival. Mice received whole body irradiation (WBI) followed by intraperitoneal administration of ascorbic acid. Administration of 3 g/kg of ascorbic acid immediately after exposure significantly increased mouse survival after WBI at 7 to 8 Gy. However, administration of less than 3 g/kg of ascorbic acid was ineffective, and 4 or more g/kg was harmful to the mice. Post-exposure treatment with 3 g/kg of ascorbic acid reduced radiation-induced apoptosis in bone marrow cells and restored hematopoietic function. Treatment with ascorbic acid (3 g/kg) up to 24 h (1, 6, 12, or 24 h) after WBI at 7.5 Gy effectively improved mouse survival; however, treatments beyond 36 h were ineffective. Two treatments with ascorbic acid (1.5 g/kg × 2, immediately and 24 h after radiation, 3 g/kg in total) also improved mouse survival after WBI at 7.5 Gy, accompanied with suppression of radiation-induced free radical metabolites. In conclusion, administration of high-dose ascorbic acid might reduce radiation lethality in mice even after exposure.

Decreased plasma brain-derived neurotrophic factor and vascular endothelial growth factor concentrations during military training.

Decreased concentrations of plasma brain-derived neurotrophic factor (BDNF) and serum BDNF have been proposed to be a state marker of depression and a biological indicator of loaded psychosocial stress. Stress evaluations of participants in military mission are critically important and appropriate objective biological parameters that evaluate stress are needed. In military circumstances, there are several problems to adopt plasma BDNF concentration as a stress biomarker. First, in addition to psychosocial stress, military missions inevitably involve physical exercise that increases plasma BDNF concentrations. Second, most participants in the mission do not have adequate quality or quantity of sleep, and sleep deprivation has also been reported to increase plasma BDNF concentration. We evaluated plasma BDNF concentrations in 52 participants on a 9-week military mission. The present study revealed that plasma BDNF concentration significantly decreased despite elevated serum enzymes that escaped from muscle and decreased quantity and quality of sleep, as detected by a wearable watch-type sensor. In addition, we observed a significant decrease in plasma vascular endothelial growth factor (VEGF) during the mission. VEGF is also neurotrophic and its expression in the brain has been reported to be up-regulated by antidepressive treatments and down-regulated by stress. This is the first report of decreased plasma VEGF concentrations by stress. We conclude that decreased plasma concentrations of neurotrophins can be candidates for mental stress indicators in actual stressful environments that include physical exercise and limited sleep.

Correlation between liver metastasis of the colocalization of actin-related protein 2 and 3 complex and WAVE2 in colorectal carcinoma.

Directed movement of normal cells occurs when actin-related protein 2 and 3 complex (Arp2/3 complex) triggers the actin polymerization that forms lamellipodia immediately after binding to WAVE2. In order to determine whether the same mechanism correlates with liver metastasis from colorectal cancer, paired mirror sections of 154 cancer specimens (29 cases with liver metastasis and 125 cases without liver metastasis in which T factor, gender, primary tumor site, and age at operation were matched) were examined immunohistochemically for the localization of Arp2 and WAVE2. Expression of both Arp2 and WAVE2 was detected in the same cancer cells in 55 (35.7%) of the 154 cases, but not detected in the normal colonic epithelial cells. Univariate analysis showed that the colocalization was significantly predictive of liver metastasis (risk ratio [RR] 8.760. Likewise, histological grade (RR 2.46), lymphatic invasion (RR 9.95), and tumor budding (RR 4.00) were significant predictors. Among these, colocalization and lymphatic invasion were shown to be independent risk factors by multivariate analysis. Another 59 colorectal specimens were examined for mRNA expression of Arp2 by real time polymerase chain reaction. High mRNA levels of Arp2, that in situ hybridization revealed to be expressed by the cancer cells, were significantly associated with liver metastasis. However, its effect was absorbed by the influence of risk of the colocalization that is closely related to high expression of Arp2. These results indicate that the colocalization of Arp2 and WAVE2 is an independent risk factor for liver metastasis of colorectal carcinoma.

Involvement of Arp2/3 complex in MCP-1-induced chemotaxis.

The migrating monocyte shows dynamic actin polymerization in response to MCP-1. We investigated the involvement of the actin-related protein 2 and 3 complex (Arp2/3 complex) during chemotaxis of a human monocyte cell line (THP-1). To clarify whether the Arp2/3 complex directly polymerizes actin in response to MCP-1 stimulation, THP-1 cells were transfected with complementary DNA constructs encoding ScarWA. In ScarWA-transfected cells, neither recruitment of Arp2/3 complex at the leading edge nor actin polymerization was detected. Indeed, migration induced by MCP-1 was almost completely blocked. At the same time, transfection also interfered with the recruitment of integrin beta-1 at the leading edge and reduced affinity binding to fibronectin. Immunoprecipitation with an anti-Arp2 antibody showed that integrin beta-1 and WASP were co-precipitated under the condition of MCP-1 stimulation. These results indicate that interaction between the Arp2/3 complex and WASP stimulates actin polymerization and integrin beta-1-mediated adhesion during MCP-1-induced chemotaxis of THP-1 cells.

Interferon-beta-induced activation of c-Jun NH2-terminal kinase mediates apoptosis through up-regulation of CD95 in CH31 B lymphoma cells.

Type I interferon (IFN)-induced antitumor action is due in part to apoptosis, but the molecular mechanisms underlying IFN-induced apoptosis remain largely unresolved. In the present study, we demonstrate that IFN-beta induced apoptosis and the loss of mitochondrial membrane potential (delta psi m) in the murine CH31 B lymphoma cell line, and this was accompanied by the up-regulation of CD95, but not CD95-ligand (CD95-L), tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL). Pretreatment with anti-CD95-L mAb partially prevented the IFN-beta-induced loss of delta psi m, suggesting that the interaction of IFN-beta-up-regulated CD95 with CD95-L plays a crucial role in the induction of fratricide. IFN-beta induced a sustained activation of c-Jun NH2-terminal kinase 1 (JNK1), but not extracellular signal-regulated kinases (ERKs). The IFN-beta-induced apoptosis and loss of delta psi m were substantially compromised in cells overexpressing a dominant-negative form of JNK1 (dnJNK1), and it was slightly enhanced in cells carrying a constitutively active JNK construct, MKK7-JNK1 fusion protein. The IFN-beta-induced up-regulation of CD95 together with caspase-8 activation was also abrogated in the dnJNK1 cells while it was further enhanced in the MKK7-JNK1 cells. The levels of cellular FLIP (c-FLIP), competitively interacting with caspase-8, were down-regulated by stimulation with IFN-beta but were reversed by the proteasome inhibitor lactacystin. Collectively, the IFN-beta-induced sustained activation of JNK mediates apoptosis, at least in part, through up-regulation of CD95 protein in combination with down-regulation of c-FLIP protein.

Ubiquitin-immunoreactive degradation products of cytokeratin 8/18 correlate with aggressive breast cancer.

Decreased amounts of cytokeratin (CK) 8/18 in the cytoplasm of breast cancer cells correlate with a poor prognosis. Although such decreases have been attributed to suppressed gene expression, accelerated protein degradation may also be responsible. In order to investigate whether selective degradation via the ubiquitin (Ub)-dependent proteasome pathway occurs in breast cancer, one- and two-dimensional (1-D and 2-D) immunoblot analysis was performed on cancerous and normal breast tissue from 50 breast cancer patients using the anti-Ub monoclonal antibodies (mAbs) KM691 and KM690. On 1-D gel electrophoresis, one broad band or two bands were detected at about 43 kDa; these were detected only in cancer tissue. Immunoreactive bands at 43 kDa were significantly associated with aggressive morphology (P = 0.011), nuclear p53 accumulation (P = 0.015) and overexpression of Her2 / neu protein (P = 0.012). On 2-D gel electrophoresis, these bands were fractionated into a group of several spots that formed a staircase pattern at 40-45 kDa. Partial amino acid sequencing analysis demonstrated that these Ub-immunoreactive spots corresponded to CK8 and CK18; however, since they did not have an amino-terminal domain, and were located at lower molecular weight positions than intact CK8 and CK18 on the 2-D gel, they were regarded as degradation products. CK18 degradation was confirmed by confocal microscopy as loss of the frame-like network that forms the luminal structure. These results indicate that CK 8/18 degradation products are detected specifically in breast cancer and may determine its aggressiveness.

Involvement of Arp2/3 complex in the process of colorectal carcinogenesis.

Increased motility is one of the characteristics of cancer cells, and actin polymerization and disassembly are essential for cellular motility. Since actin-related protein (Arp) 2/3 complex acts as a nucleus for actin polymerization, in this study, we immunohistochemically investigated the expression of Arp2 and Arp3 in 175 colorectal tumors in various stages of neoplastic progression. Arp2 and Arp3 showed identical expression patterns, and both were expressed in the stromal cells around neoplastic tubules or glands and in the tumor cells themselves. The frequency of expression of Arp2 and Arp3 (Arp2 and 3) by the stromal cells increased with the atypia of the colorectal neoplasms, from 5.5% (3/55) in adenoma with mild or moderate atypia, to 11.8% (2/17) in adenoma with severe atypia, 53.3% (16/30) in intramucosal carcinoma, and 91.8% (67/73) in invasive carcinoma (P<0.0001). The frequency of expression of Arp2 and 3 in the tumor cells was similar and was 1.8% (1/55) in adenoma with mild or moderate atypia, 23.5% (4/17) in adenoma with severe atypia, 23.5% (7/30) in intramucosal carcinoma, and 32.9% (24/73) in invasive carcinoma. Expression of Arp2 and 3 by the stromal cells was significantly correlated with nuclear accumulation of p53 in the tumor cells and stromal expression of CD10. These results suggest that formation of Arp2/3 complex by both neoplastic and stromal cells contributes to the increased motility of both cell types and thus provides suitable conditions for invasion.