Prediction of efficient virological response to pegylated interferon/ribavirin combination therapy by NS5A sequences of hepatitis C virus and anti-NS5A antibodies in pre-treatment sera

A considerable number of patients infected with Hepatitis C virus subtype 1b (HCV-1b) do not respond to pegylated interferon/ribavirin combination therapy. In this study we explored a useful factor(s) to predict treatment outcome. A total of 47 HCV-1b-infected patients were treated with pegylated interferon/ ribavirin for 48 weeks. Sera of the patients were examined for the entire NS5A sequence of the HCV genome, HCV RNA titers and anti-NS5A antibodies. According to their responses, the patients were divided into two groups, early viral responders who cleared the virus by week 16 (EVR[16w]) and those who did not (Non-EVR[16w]). The mean number of mutations in the V3 region (aa 2356 to 2379) or that in the V3 region plus its N-terminally flanking region, which we refer to as interferon/ribavirin resistancedetermining region (IRRDR; aa 2334 to 2379), of NS5A obtained from the pretreatment sera was signifi-cantly larger for EVR(16w) compared with Non-EVR(16w). Moreover, HCV-1b isolates with > or =5 mutations in V3 or those with > or =6 mutations in IRRDR were almost exclusively found in EVR(16w). Also, the presence of detectable levels of anti-NS5A antibodies in the pretreatment sera was closely associated with EVR(16w). In conclusion, a high degree of sequence variation in V3 (> or =5) or IRRDR (> or =6) and the presence of detectable levels of anti-NS5A antibodies in the pretreatment sera would be useful factors to predict EVR(16w). On the other hand, a less diverse sequence in V3 (< or =4) or IRRDR (< or =5) together with the absence of detectable anti-NS5A antibodies could be a predictive factor for Non-EVR(16w).     

Isolation of pathogen-induced Chinese cabbage genes by subtractive hybridization employing selective adaptor ligation

We have developed a subtractive cloning method in which target sequences are effectively enriched by selective adaptor ligation and PCR after hybridization. In this method both tester and driver DNAs are digested with RsaI, ligated with the linker DNA containing a KpnI recognition site, and amplified by PCR. The tester DNA samples are divided into two aliquots, each digested with either RsaI or KpnI. The two DNA samples are then combined and hybridized with an excess of the driver DNA retaining the linker. After hybridization, the DNA mixture is ligated to a new adaptor compatible only with double-stranded tester/tester DNAs. Therefore, only the tester/tester is selectively amplified in subsequent PCR. This also leads to complete elimination of the tester DNA hybridized with driver DNA from the tester DNA population. Although our protocol employs enzymatic treatments, the efficiency of the enzymatic treatments does not affect the subtraction efficiency. This new subtractive enrichment method was applied to isolate Chinese cabbage defense-related genes induced by Pseudomonas syringae pv. tomato (Pst), which elicits a hypersensitive response in Chinese cabbage. After two or three rounds of subtractive hybridization, the sequences of enriched DNAs were determined and examined by BLAST analysis. Northern blot hybridization showed that 12 of the 19 genes analyzed were strongly induced by Pst treatment. Among the 12 Pst-induced genes five represent pathogenesis-related genes encoding PR1a, two chitinases, a thaumatin-like protein, and a PR4 protein. Other Pst-induced genes include two cytochrome P450 genes responsible for glucosinolate biosynthesis, a disease resistance gene homolog, and several genes encoding proteins with unknown functions.     

Proteomic analysis of differential protein expression in atherosclerosis

Although recent studies have shown that several pro-inflammatory proteins can be used as biomarkers for atherosclerosis, the mechanism of atherogenesis is unclear and little information is available regarding proteins involved in development of the disease. Atherosclerotic tissue samples were collected from patients in order to identify the proteins involved in atherogenesis. The protein expression profile of atherosclerosis patients was analysed using two-dimensional electrophoresis-based proteomics. Thirty-nine proteins were detected that were differentially expressed in the atherosclerotic aorta compared with the normal aorta. Twenty-seven of these proteins were identified in the MS-FIT database. They are involved in a number of biological processes, including calcium-mediated processes, migration of vascular smooth muscle cells, matrix metalloproteinase activation and regulation of pro-inflammatory cytokines. Confirmation of differential protein expression was performed by Western blot analysis. Potential applications of the results include the identification and characterization of signalling pathways involved in atherogenesis, and further exploration of the role of selected identified proteins in atherosclerosis.     

Changes of net photosynthesis,antioxidant enzyme activities,and antioxidant contents of <Emphasis Type="Italic">Liriodendron tulipifera</Emphasis> under elevated ozone

Liriodendron tulipifera was exposed to gradually elevated ozone concentrations of 100–300 μg kg⁻¹ in the naturally irradiated environment chamber. During 15 d of exposure to O₃, net photosynthetic rate (P _N) decreased and there was large difference between the control (C) and treatment with ozone (OT), while there was no significant difference in water use efficiency. Total chlorophyll content as well as the value of fluorescence parameter F_v/F_m decreased, while antioxidant enzyme activities related to ascorbate-glutathione cycle increased after 15 d of OT. Unchanged contents of ascorbate and glutathione indirectly suggest that the species hastened the antioxidant’s oxidization/reduction cycle using enzymes instead of expanding their pool against oxidative stress.     

Differences in Clinical Outcomes According to Weaning Classifications in Medical Intensive Care Units

Background

Although the weaning classification based on the difficulty and duration of the weaning process has been evaluated in the different type of intensive care units (ICUs), little is known about clinical outcomes and validity among the three groups in medical ICU. The objectives of this study were to evaluate the clinical relevance of weaning classification and its association with hospital mortality in a medical ICU with a protocol-based weaning program.

Methods

All consecutive patients admitted to the medical ICU and requiring mechanical ventilation (MV) for more than 24 hours were prospectively registered and screened for weaning readiness by a standardized weaning program between July 2010 and June 2013. Baseline characteristics and outcomes were compared across weaning classifications.

Results

During the study period, a total of 680 patients were weaned according to the standardized weaning protocol. Of these, 457 (67%) were classified as simple weaning, 136 (20%) as difficult weaning, and 87 (13%) as prolonged weaning. Ventilator-free days within 28 days decreased significantly from simple to difficult to prolonged weaning groups (P < 0.001, test for trends). In addition, reintubation within 48 hours after extubation (P < 0.001) and need for tracheostomy during the weaning process (P < 0.001) increased significantly across weaning groups. Finally, ICU (P < 0.001), post-ICU (P = 0.001), and hospital (P < 0.001) mortalities significantly increased across weaning groups. In a multiple logistic regression model, prolonged weaning but not difficult weaning was still independently associated with ICU (adjusted OR 8.265, 95% CI 3.484–19.605, P < 0.001), and post-ICU (adjusted OR 3.180, 95% CI 1.349–7.497, P = 0.005), and hospital (adjusted OR 5.528, 95% CI 2.801–10.910, P < 0.001) mortalities.

Conclusions

Weaning classification based on the difficulty and duration of the weaning process may provide prognostic information for mechanically ventilated patients who undergo the weaning process.     

Anterior Chamber Depth and Refractive Change in Late Postoperative Capsular Bag Distension Syndrome: A Retrospective Analysis

Purpose

To assess the characteristic findings and effects of laser capsulotomy in patients with late postoperative capsular bag distension syndrome (CBDS).

Methods

Twenty patients diagnosed with late postoperative CBDS between July 2010 and August 2013 were retrospectively reviewed. Before and 1 week after capsulotomy, changes in the anterior chamber depth (ACD) were assessed using ultrasound biomicroscopy. Changes in the refractive status and uncorrected visual acuity (UCVA) were also measured 1 week and 1 month after capsulotomy. For patients who received bilateral cataract surgery, preoperative ACD and axial length measured by IOLMaster were compared between the two eyes.

Results

Twenty-two eyes from 20 patients who had undergone laser capsulotomy showed a mean UCVA improvement of 0.27 ± 0.24 logMAR (range, 0.00–0.90). ACD was increased by an average of +0.04 mm (95% confidence interval, +0.01 to +0.06 mm, p = 0.034), equivalent to predicted refractive change of +0.10 D. The discrepancy between actual (+1.33 D) and predicted refractive change after capsulotomy suggests that refractive change may not be generated from IOL displacement in late postoperative CBDS. Preoperative ACD was deeper in the eye with late postoperative CBDS in all bilaterally pseudophakic patients (mean, 3.68 mm vs. 3.44 mm in the fellow eye, p = 0.068).

Conclusions

Late postoperative CBDS showed refractive changes that were resolved successfully after laser capsulotomy. The convex lens effects of opalescent material in the distended capsular bag may play a major role in myopic shift. A larger preoperative ACD is possibly associated with the development of late postoperative CBDS.     

Therapeutic factors secreted by mesenchymal stromal cells and tissue repair

Systemic administration of MSCs resulted in remarkable functional improvements in injured tissues without either long-term engraftment or differentiation in many clinical and experimental situations. Emerging evidence suggest that most of the beneficial effects of MSCs could be explained by secretion of soluble factors that have multiple effects including modulation of inflammatory and immune reactions, protection from cell death, and stimulation of endogenous progenitor cells. In this review, we focus on the therapeutic factors that account for the beneficial effects of MSCs in animal models of human diseases.     

Prevention of salt-induced renal injury by activation of NAD(P)H:quinone oxidoreductase 1, associated with NADPH oxidase

NADPH oxidase (NOX) is a predominant source of reactive oxygen species (ROS), and the activity of NOX, which uses NADPH as a common rate-limiting substrate, is upregulated by prolonged dietary salt intake. β-Lapachone (βL), a well-known substrate of NAD(P)H:quinone oxidoreductase 1 (NQO1), decreases the cellular NAD(P)H/NAD(P)(+) ratio via activation of NQO1. In this study, we evaluated whether NQO1 activation by βL modulates salt-induced renal injury associated with NOX-derived ROS regulation in an animal model. Dahl salt-sensitive (DS) rats fed a high-salt (HS) diet were used to investigate the renoprotective effect of NQO1 activation. βL treatment significantly lowered the cellular NAD(P)H:NAD(P)(+) ratio and dramatically reduced NOX activity in the kidneys of HS diet-fed DS rats. In accordance with this, total ROS production and expression of oxidative adducts also decreased in the βL-treated group. Furthermore, HS diet-induced proteinuria and glomerular damage were markedly suppressed, and inflammation, fibrosis, and apoptotic cell death were significantly diminished by βL treatment. This study is the first to demonstrate that activation of NQO1 has a renoprotective effect that is mediated by NOX activity via modulation of the cellular NAD(P)H:NAD(P)(+) ratio. These results provide strong evidence that NQO1 might be a new therapeutic target for the prevention of salt-induced renal injury.     

CRIF1 Is Essential for the Synthesis and Insertion of Oxidative Phosphorylation Polypeptides in the Mammalian Mitochondrial Membrane

Although substantial progress has been made in understanding the mechanisms underlying the expression of mtDNA-encoded polypeptides, the regulatory factors involved in mitoribosome-mediated synthesis and simultaneous insertion of mitochondrial oxidative phosphorylation (OXPHOS) polypeptides into the inner membrane of mitochondria are still unclear. In the present study, disruption of the mouse Crif1 gene, which encodes a mitochondrial protein, resulted in a profound deficiency in OXPHOS caused by the disappearance of OXPHOS subunits and complexes in?vivo. CRIF1 was associated with large mitoribosomal subunits that were located close to the polypeptide exit tunnel, and the elimination of CRIF1 led to both aberrant synthesis and defective insertion of mtDNA-encoded nascent OXPHOS polypeptides into the inner membrane. CRIF1 interacted with nascent OXPHOS polypeptides and molecular chaperones, e.g., Tid1. Taken together, these results suggest that CRIF1 plays a critical role in the integration of OXPHOS polypeptides into the mitochondrial membrane in mammals.     

Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue.

Human mesenchymal stem cells (MSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle and neuron. This differentiation potential makes MSC excellent candidates for cell-based tissue engineering. In this study, we have examined phenotypes and gene expression profile of the human adipose tissue-derived stromal cells (ATSC) in the undifferentiated states, and compared with that of bone marrow stromal cells (BMSC). ATSC were enzymatically released from adipose tissues from adult human donors and were expanded in monolayer with serial passages at confluence. BMSC were harvested from the metaphysis of adult human femur. Flowcytometric analysis showed that ATSC have a marker expression that is similar to that of BMSC. ATSC expressed CD29, CD44, CD90, CD105 and were absent for HLA-DR and c-kit expression. Under appropriate culture conditions, MSC were induced to differentiate to the osteoblast, adipocyte, and chondrogenic lineages. ATSC were superior to BMSC in respect to maintenance of proliferating ability, and microarray analysis of gene expression revealed differentially expressed genes between ATSC and BMSC. The proliferating ability and differentiation potential of ATSC were variable according to the culture condition. The similarities of the phenotypes and the gene expression profiles between ATSC and BMSC could have broad implications for human tissue engineering.