Increased survival of the honey bee Apis mellifera infected with the microsporidian Nosema ceranae by effective gene silencing

This study examined the control of nosemosis caused by Nosema ceranae, one of the hard-to-control diseases of honey bees, using RNA interference (RNAi) technology. Double-stranded RNA (dsRNA) for RNAi application targeted the mitosome-related genes of N. ceranae. Among the various mitosome-related genes, NCER_100882, NCER_101456, NCER_100157, and NCER_100686 exhibited relatively low homologies with the orthologs of Apis mellifera. Four gene-specific dsRNAs were prepared against the target genes and applied to the infected A. mellifera to analyze Nosema proliferation and honey bee survival. Two dsRNAs specifics to NCER_101456 and NCER_100157 showed high inhibitory effects on spore production by exhibiting only 62% and 67%, respectively, compared with the control. In addition, these dsRNA treatments significantly rescued the honey bees from the fatal nosemosis. It was confirmed that the inhibition of Nosema spore proliferation and the increase in the survival rate of honey bees were resulted from a decrease in the expression level of each target gene by dsRNA treatment. However, dsRNA mixture treatment was no more effective than single treatments in the rescue from the nosemosis. It is expected that the four newly identified mitosome-related target genes in this study can be effectively used for nosemosis control using RNAi technology.     

ParA encoded on chromosome II of Deinococcus radiodurans binds to nucleoid and inhibits cell division in Escherichia coli

Bacterial genome segregation and cell division has been studied mostly in bacteria harbouring single circular chromosome and low-copy plasmids. Deinococcus radiodurans, a radiation-resistant bacterium, harbours multipartite genome system. Chromosome I encodes majority of the functions required for normal growth while other replicons encode mostly the proteins involved in secondary functions. Here, we report the characterization of putative P-loop ATPase (ParA2) encoded on chromosome II of D. radiodurans. Recombinant ParA2 was found to be a DNA-binding ATPase. E. coli cells expressing ParA2 showed cell division inhibition and mislocalization of FtsZ-YFP and those expressing ParA2-CFP showed multiple CFP foci formation on the nucleoid. Although, in trans expression of ParA2 failed to complement SlmA loss per se, it could induce unequal cell division in slmAminCDE double mutant. These results suggested that ParA2 is a nucleoid-binding protein, which could inhibits cell division in E. coli by affecting the correct localization of FtsZ and thereby cytokinesis. Helping slmAminCDE mutant to produce minicells, a phenotype associated with mutations in the ‘Min’ proteins, further indicated the possibility of ParA2 regulating cell division by bringing nucleoid compaction at the vicinity of septum growth.     

Bioreactance Is Not Interchangeable with Thermodilution for Measuring Cardiac Output during Adult Liver Transplantation

BackgroundThermodilution technique using a pulmonary artery catheter is widely used for the assessment of cardiac output (CO) in patients undergoing liver transplantation. However, the unclearness of the risk-benefit ratio of this method has led to an interest in less invasive modalities. Thus, we evaluated whether noninvasive bioreactance CO monitoring is interchangeable with thermodilution technique.MethodsNineteen recipients undergoing adult-to-adult living donor liver transplantation were enrolled in this prospective observational study. COs were recorded automatically by the two devices and compared simultaneously at 3-minute intervals. The Bland–Altman plot was used to evaluate the agreement between bioreactance and thermodilution. Clinically acceptable agreement was defined as a percentage error of limits of agreement <30%. The four quadrant plot was used to evaluate concordance between bioreactance and thermodilution. Clinically acceptable concordance was defined as a concordance rate >92%.ResultsA total of 2640 datasets were collected. The mean CO difference between the two techniques was 0.9 l/min, and the 95% limits of agreement were -3.5 l/min and 5.4 l/min with a percentage error of 53.9%. The percentage errors in the dissection, anhepatic, and reperfusion phase were 50.6%, 56.1%, and 53.5%, respectively. The concordance rate between the two techniques was 54.8%.ConclusionBioreactance and thermodilution failed to show acceptable interchangeability in terms of both estimating CO and tracking CO changes in patients undergoing liver transplantation. Thus, the use of bioreactance as an alternative CO monitoring to thermodilution, in spite of its noninvasiveness, would be hard to recommend in these surgical patients.     

Comment on: HMGB1-dependent and -independent autophagy

HMGB1 (high mobility group box 1), a ubiquitously expressed DNA-binding nucleoprotein, has not only been attributed with important functions in the regulation of gene expression but is thought to function as an important damage-associated molecular pattern in the extracellular space. Recently, conditional Hmgb1 deletion strategies have been employed to overcome the perinatal mortality of global Hmgb1 deletion and to understand HMGB1 functions under disease conditions. From these studies, it has become evident that HMGB1 is not required for normal organ function. However, the different conditional ablation strategies have yielded contradictory results in some disease models. With nearly complete recombination in all transgenic mouse models, the main reason for opposite results is likely to lie within different targeting strategies. In summary, different targeting strategies need to be taken into account when interpreting HMGB1 functions, and further efforts need to be undertaken to compare these models side by side.We appreciate the thoughtful analysis on HMGB1-dependent and -independent autophagy by Sun and Tang.¹ However, we disagree with several statements in this review. Sun and Tang write “Mice with hepatocyte-specific deletion of Hmgb1 from Robert Schwabe''s lab are not complete conditional knockout mice; the protein level of HMGB1 in the liver is decreased by about 70%,” as well as “a major difference between Robert Schwabe''s engineered HMGB1 mice and other groups is the tissue-level expression of HMGB1 after knockout.”¹We would like to point out that livers are not solely composed of hepatocytes and that albumin-Cre mediated deletion of target genes in the liver cannot result in complete loss of hepatic mRNA or protein of target genes due to the presence of unrecombined nonparenchymal cells, unless the target gene is exclusively expressed in hepatocytes and/or cholangiocytes. The reduction of hepatic HMGB1 in our studies—reaching 90% and 72% at the mRNA and protein level, respectively—is precisely at the expected level for this conditional strategy, and similar to other studies that employed albumin-Cre for hepatocyte-specific knockout of other target genes.^2-5 Hepatocytes account only for approximately 52% of cells in the liver, with other cell types including Kupffer cells (∼18% of liver cells), hepatic stellate cells (˜8% of liver cells), endothelial cells (∼22% cells of liver cells) and cholangiocytes (<1 % of liver cells) contributing to the remainder.⁶ Accordingly, albumin-Cre-mediated reduction of mRNA and protein levels of target genes (i.e., Hmgb1 and HMGB1 in our study) in the liver cannot exceed the amount of mRNA and protein expressed by hepatocytes and cholangiocytes (which is typically about 70–90%,^2-5 due to higher mRNA and protein levels in hepatocytes than in other hepatic cell types). The high efficacy of our conditional approach is best demonstrated by almost complete loss of HMGB1 expression in the hepatocellular compartment of albumin-Cre mice—as evidenced by loss of HMGB1 expression in all HNF4α-positive cells and in isolated primary hepatocytes—whereas HMGB1 expression is retained in nonparenchymal cells, as demonstrated by costaining for Kupffer cell marker F4/80, endothelial cell marker endomucin, and hepatic stellate cell marker desmin.^7,8 The nearly perfect recombination rate in our mice was further confirmed by experiments that employed Mx1Cre for Hmgb1 deletion, which resulted in almost complete loss of hepatic Hmgb1 mRNA and HMGB1 protein.^7,8 Moreover, our transgenic mice show early postnatal mortality when bred with a germline Cre deleter,⁷ thus reproducing the phenotype of the global HMGB1 knockout.⁹In summary, our transgenic mouse model results in nearly perfect recombination efficiency with virtually complete loss of Hmgb1 mRNA and HMGB1 protein in all targeted cell types, and constitutes a valid tool for the assessment of HMGB1 functions in vivo. Findings from this model need to be taken into account for proper interpretation of the role of HMGB1 in the normal and diseased liver, and cannot be interpreted as a result of incomplete deletion efficiency. Hence, differences in targeting strategies (exons 2–4 by our approach, exons 2–3 in mice from Tang and colleagues) are likely to explain opposite findings, e.g. improvement of ischemia-reperfusion injury in our hands, but aggravation of liver damage in the study by Huang et al.^8,10 Further analysis needs to be performed to determine whether ablation of exons 2–3 versus exons 2–4 leads to complete loss of HMGB1 function.     

Prognostic Value of Tumor-Associated Macrophages According to Histologic Locations and Hormone Receptor Status in Breast Cancer

Tumor-associated macrophages (TAMs) are involved in tumor progression by promoting epithelial-mesenchymal transition (EMT), tumor cell invasion, migration and angiogenesis. However, in breast cancer, the clinical relevance of the TAM infiltration according to distinct histologic locations (intratumoral vs. stromal) and hormone receptor status is unclear. We investigated the significance of the levels of TAM infiltration in distinct histologic locations in invasive breast cancer. We also examined the relationship of the TAM levels with the clinicopathologic features of tumors, expression of EMT markers, and clinical outcomes. Finally, we analyzed the prognostic value of TAM levels according to hormone receptor status. High levels of infiltration of intratumoral, stromal and total TAMs were associated with high histologic grade, p53 overexpression, high Ki-67 proliferation index and negative hormone receptor status. Infiltration of TAMs was also correlated with overexpression of vimentin, smooth muscle actin and alteration of β-catenin. Overall, a high level of infiltration of intratumoral TAMs was associated with poor disease-free survival, and was found to be an independent prognostic factor. In subgroup analyses by hormone receptor status, a high level of infiltration of intratumoral TAM was an independent prognostic factor in the hormone receptor-positive subgroup, but not in the hormone-receptor negative subgroup. Our findings suggest that intratumoral TAMs play an important role in tumor progression in breast cancer, especially in the hormone receptor-positive group, and the level of TAM infiltration may be used as a prognostic factor and even a therapeutic target in breast cancer.     

Different Survival of Barcelona Clinic Liver Cancer Stage C Hepatocellular Carcinoma Patients by the Extent of Portal Vein Invasion and the Type of Extrahepatic Spread

Portal vein invasion (PVI) and extrahepatic spread (ES) are two tumor-related factors that define advanced stage in the Barcelona Clinic Liver Cancer (BCLC) staging system (BCLC stage C), and the recommended first line therapy in this stage is sorafenib. However, the extent of PVI and the type of ES may affect patient prognosis as well as treatment outcome. This study analyzed survival of BCLC stage C HCC patients in order to see whether sub-classification of BCLC stage C is necessary. A total of 582 treatment naïve, BCLC stage C HCC patients [age: 54.3 ± 10.8 years, males = 494 (84.9%), hepatitis B virus (458, 78.7%)], defined by PVI and/or ES, were analyzed. Extent of PVI was divided into none, type I-segmental/sectoral branches, type II-left and/or right portal vein, and type III-main portal vein trunk. Type of ES was divided into nodal and distant metastasis. The extent of PVI and type of ES were independent factors for survival. When patients were sub-classified according to the extent of PVI and type of ES, the median survival was significantly different [11.7 months, 5.7 months, 4.9 months and 2.3 months for C1 (PVI-O/I without distant ES), C2 (PVI-II/III without distant ES), C3 (PVI-0/I with distant ES), and C4 (PVI-II/III with distant ES), respectively, P = 0.01]. Patients’ survival was different according to the treatment modality in each sub-stage. Sub-classification of BCLC stage C according to the extent of PVI and type of ES resulted in a better prediction of survival. Also, different outcome was observed by treatment modalities in each sub-stage. Sub-classification of BCLC stage C is required to minimize heterogeneity within the same tumor stage, that will help better predict survival and to select optimal treatment strategies.     

Characterization of xylanase from Lentinus edodes M290 cultured on waste mushroom logs

Extracellular enzymes from Lentinus edodes M290 on normal woods (Quercus mongolica) and waste logs from oak mushroom production were comparatively investigated. Endoglucanase, cellobiohydrolase, beta-glucosidase, and xylanase activities were higher on waste mushroom logs than on normal woods after L. edodes M290 inoculation. Xylanase activity was especially different, with a three times higher activity on waste mushroom logs. When the waste mushroom logs were used as a carbon source, a new 35 kDa protein appeared. After the purification, the optimal pH and temperature for xylanase activity were determined to be 4.0 and 50 degrees C, respectively. More than 50% of the optimal xylanase activity was retained when the temperature was increased from 20 to 60 degrees C, after a 240 min reaction. At 40 degrees C, the xylanase maintained 93% of the optimal activity, after a 240 min reaction. The purified xylanase showed a very high homology to the xylanase family 10 from Aspergillus terreus by LC/MS-MS analysis. The highest Xcorr (1.737) was obtained from the peptide KWI SQGIPIDGIG SQTHLGSGGS WTVK originated from Aspergillus terreus, indicating that the 35 kDa protein was xylanase. This protein showed low homology to a previously reported L. edodes xylanase sequence.     

Aerobic training increases the expression of adiponectin receptor genes in the peripheral blood mononuclear cells of young men

Little is known about the effect of exercise training on the expression of adiponectin receptor genes in peripheral blood mononuclear cells (PBMCs). In this study, we investigated the effects of aerobic training on the expression of AdipoR1 and AidpoR2 mRNAs in PBMCs, whole body insulin sensitivity, and circulating adiponectins in men. Thirty young men were randomly assigned to either a control (n=15) or an exercise (n=15) group. Subjects assigned to the exercise group underwent a 12-week jogging and/or running programme on a motor-driven treadmill at an intensity of 60%-75% of the age-based maximum heart rate with duration of 40 minutes per session and a frequency of 5 days per week. Two-way mixed ANOVA with repeated measures was used to test any significant time-by-group interaction effects for the measured variables at p=0.05. We found significant time-by-group interaction effects for waist circumference (p=0.001), VO_2max (p<0.001), fasting insulin (p=0.016), homeostasis model assessment for insulin resistance (HOMA-IR) (p=0.010), area under the curve (AUC) for insulin response during the 75-g oral glucose tolerance test (p=0.002), high-molecular weight (HMW) adiponectin (p=0.016), and the PBMC mRNA levels of AdipoR1 (p<0.001) and AdipoR2 (p=0.001). The exercise group had significantly increased mRNA levels of AdipoR1 and AdipoR2 in PBMCs, along with increased whole body insulin sensitivity and HMW adiponectin, decreased waist circumference, and increased VO_2max compared with the control group. In summary, the current findings suggest that exercise training modulates the expression of AdipoR1 and AdipoR2 mRNAs in PBMCs, implying that manipulation of the expression of these genes could be a potential surrogate for lifestyle intervention-mediated improvements of whole body insulin sensitivity and glucose homeostasis.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.