Conformational Space and Stability of ETD Charge Reduction Products of Ubiquitin

Owing to its versatility, electron transfer dissociation (ETD) has become one of the most commonly utilized fragmentation techniques in both native and non-native top-down mass spectrometry. However, several competing reactions—primarily different forms of charge reduction—occur under ETD conditions, as evidenced by the distorted isotope patterns usually observed. In this work, we analyze these isotope patterns to compare the stability of nondissociative electron transfer (ETnoD) products, specifically noncovalent c/z fragment complexes, across a range of ubiquitin conformational states. Using ion mobility, we find that more extended states are more prone to fragment release. We obtain evidence that for a given charge state, populations of ubiquitin ions formed either directly by electrospray ionization or through collapse of more extended states upon charge reduction, span a similar range of collision cross-sections. Products of gas-phase collapse are, however, less stabilized towards unfolding than the native conformation, indicating that the ions retain a memory of previous conformational states. Furthermore, this collapse of charge-reduced ions is promoted if the ions are ‘preheated’ using collisional activation, with possible implications for the kinetics of gas-phase compaction

In vitro activity of tigecycline against methicillin-resistant Staphylococcus aureus, including livestock-associated strains

The in vitro activity of tigecycline was determined using a well-defined collection of methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 202), including 33 livestock-associated strains. Susceptibility testing was performed using the Etest system. Among the 202 MRSA strains, three (1.5%) had a minimum inhibitory concentration (MIC) value for tigecycline greater than 0.5 mg/l, which are considered to be resistant. When these strains were tested using Iso-Sensitest medium, the MICs were substantially lower and no resistance was found. This discrepancy warrants further investigations into the preferred test conditions for tigecycline. In conclusion, tigecycline showed good activity against MRSA strains in vitro

Whole blood microRNA levels associate with glycemic status and correlate with target mRNAs in pathways important to type 2 diabetes

We analyzed the associations between whole blood microRNA profiles and the indices of glucose metabolism and impaired fasting glucose and examined whether the discovered microRNAs correlate with the expression of their mRNA targets. MicroRNA and gene expression profiling were performed for the Young Finns Study participants (n= 871). Glucose, insulin, and glycated hemoglobin (HbA1c) levels were measured, the insulin resistance index (HOMA2-IR) was calculated, and the glycemic status (normoglycemic [n = 534]/impaired fasting glucose [IFG] [n = 252]/type 2 diabetes [T2D] [n = 24]) determined. Levels of hsa-miR-144-5p, -122-5p, -148a-3p, -589-5p, and hsa-let-7a-5p associated with glycemic status. hsa-miR-144-5p and -148a-3p associated with glucose levels, while hsa-miR-144-5p, -122-5p, -184, and -339-3p associated with insulin levels and HOMA2-IR, and hsa-miR-148a-3p, -15b-3p, -93-3p, -146b-5p, -221-3p, -18a-3p, -642a-5p, and -181-2-3p associated with HbA1c levels. The targets of hsa-miR-146b-5p that correlated with its levels were enriched in inflammatory pathways, and the targets of hsa-miR-221-3p were enriched in insulin signaling and T2D pathways. These pathways showed indications of co-regulation by HbA1c-associated miRNAs. There were significant differences in the microRNA profiles associated with glucose, insulin, or HOMA-IR compared to those associated with HbA1c. The HbA1c-associated miRNAs also correlated with the expression of target mRNAs in pathways important to the development ofT2D

MRSA in Conventional and Alternative Retail Pork Products

In order to examine the prevalence of Staphylococcus aureus on retail pork, three hundred ninety-five pork samples were collected from a total of 36 stores in Iowa, Minnesota, and New Jersey. S. aureus was isolated from 256 samples (64.8%, 95% confidence interval [CI] 59.9%–69.5%). S. aureus was isolated from 67.3% (202/300) of conventional pork samples and from 56.8% (54/95) of alternative pork samples (labeled “raised without antibiotics” or “raised without antibiotic growth promotants”). Two hundred and thirty samples (58.2%, 95% CI 53.2%–63.1%) were found to carry methicillin-sensitive S. aureus (MSSA). MSSA was isolated from 61.0% (183/300) of conventional samples and from 49.5% (47/95) of alternative samples. Twenty-six pork samples (6.6%, 95% CI 4.3%–9.5%) carried methicillin-resistant S. aureus (MRSA). No statistically significant differences were observed for the prevalence of S. aureus in general, or MSSA or MRSA specifically, when comparing pork products from conventionally raised swine and swine raised without antibiotics, a finding that contrasts with a prior study from the Netherlands examining both conventional and “biologic” meat products. In our study spa types associated with “livestock-associated” ST398 (t034, t011) were found in 26.9% of the MRSA isolates, while 46.2% were spa types t002 and t008—common human types of MRSA that also have been found in live swine. The study represents the largest sampling of raw meat products for MRSA contamination to date in the U.S. MRSA prevalence on pork products was higher than in previous U.S.-conducted studies, although similar to that in Canadian studies

Blood hsa-miR-122-5p and hsa-miR-885-5p levels associate with fatty liver and related lipoprotein metabolism-The Young Finns Study

MicroRNAs are involved in disease development and may be utilized as biomarkers. We investigated the association of blood miRNA levels and a) fatty liver (FL), b) lipoprotein and lipid pathways involved in liver lipid accumulation and c) levels of predicted mRNA targets in general population based cohort. Blood microRNA profiling (TaqMan OpenArray), genome-wide gene expression arrays and nuclear magnetic resonance metabolomics were performed for Young Finns Study participants aged 34-49 years (n = 871). Liver fat status was assessed ultrasonographically. Levels of hsa-miR-122-5p and -885-5p were up-regulated in individuals with FL (fold change (FC) = 1.55, p = 1.36 * 10(-14) and FC = 1.25, p = 4.86 * 10(-4), respectively). In regression model adjusted with age, sex and BMI, hsa-miR122-5p and -885-5p predicted FL (OR = 2.07, p = 1.29 * 10(-8) and OR = 1.41, p = 0.002, respectively). Together hsa-miR-122-5p and -885-5p slightly improved the detection of FL beyond established risk factors. These miRNAs may be associated with FL formation through the regulation of lipoprotein metabolism as hsa-miR-122-5p levels associated with small VLDL, IDL, and large LDL lipoprotein subclass components, while hsa-miR-885-5p levels associated inversely with XL HDL cholesterol levels. Hsa-miR-885-5p levels correlated inversely with oxysterol-binding protein 2 (OSBPL2) expression (r = -0.143, p = 1.00 * 10(-4)) and suppressing the expression of this lipid receptor and sterol transporter could link hsa-miR-885-5p with HDL cholesterol levels

Comparative Membranome Expression Analysis in Primary Tumors and Derived Cell Lines

Despite the wide use of cell lines in cancer research, the extent to which their surface properties correspond to those of primary tumors is poorly characterized. The present study addresses this problem from a transcriptional standpoint, analyzing the expression of membrane protein genes - the Membranome – in primary tumors and immortalized in-vitro cultured tumor cells. 409 human samples, deriving from ten independent studies, were analyzed. These comprise normal tissues, primary tumors and tumor derived cell lines deriving from eight different tissues: brain, breast, colon, kidney, leukemia, lung, melanoma, and ovary. We demonstrated that the Membranome has greater power than the remainder of the transcriptome when used as input for the automatic classification of tumor samples. This feature is maintained in tumor derived cell lines. In most cases primary tumors show maximal similarity in Membranome expression with cell lines of same tissue origin. Differences in Membranome expression between tumors and cell lines were analyzed also at the pathway level and biological themes were identified that were differentially regulated in the two settings. Moreover, by including normal samples in the analysis, we quantified the degree to which cell lines retain the Membranome up- and down- regulations observed in primary tumors with respect to their normal counterparts. We showed that most of the Membranome up-regulations observed in primary tumors are lost in the in-vitro cultured cells. Conversely, the majority of Membranome genes down-regulated upon tumor transformation maintain lower expression levels also in the cell lines. This study points towards a central role of Membranome genes in the definition of the tumor phenotype. The comparative analysis of primary tumors and cell lines identifies the limits of cell lines as a model for the study of cancer-related processes mediated by the cell surface. Results presented allow for a more rational use of the cell lines as a model of cancer

Effectiveness of joint mobilisation after cast immobilisation for ankle fracture: a protocol for a randomised controlled trial [ACTRN012605000143628]

BACKGROUND: Passive joint mobilisation is a technique frequently used by physiotherapists to reduce pain, improve joint movement and facilitate a return to activities after injury, but its use after ankle fracture is currently based on limited evidence. The primary aim of this trial is to determine if adding joint mobilisation to a standard exercise programme is effective and cost-effective after cast immobilisation for ankle fracture in adults. METHODS/DESIGN: Ninety participants will be recruited from the physiotherapy departments of three teaching hospitals and randomly allocated to treatment or control groups using a concealed procedure. All participants will perform an exercise programme. Participants in the treatment group will also receive joint mobilisation twice a week for four weeks. Blinded follow-up assessments will be conducted four, 12 and 24 weeks after randomisation. The primary outcome measures will be the Lower Extremity Functional Scale and the Assessment of Quality of Life. Secondary outcomes will include measures of impairments, activity limitation and participation. Data on the use of physiotherapy services and participants' out-of-pocket costs will be collected for the cost-effective and cost-utility analyses. To test the effects of treatment, between-group differences will be examined with analysis of covariance using a regression approach. The primary conclusions will be based on the four-week follow-up data. DISCUSSION: This trial incorporates features known to minimise bias. It uses a pragmatic design to reflect clinical practice and maximise generalisability. Results from this trial will contribute to an evidence-based approach for rehabilitation after ankle fracture

Retromer and Its Role in Regulating Signaling at Endosomes.

The retromer complex is a key element of the endosomal protein sorting machinery being involved in trafficking of proteins from endosomes to the Golgi and also endosomes to the cell surface. There is now accumulating evidence that retromer also has a prominent role in regulating the activity of many diverse signaling proteins that traffic through endosomes and this activity has profound implications for the functioning of many different cell and tissue types from neuronal cells to cells of the immune system to specialized polarized epithelial cells of the retina. In this review, the protein composition of the retromer complex will be described along with many of the accessory factors that facilitate retromer-mediated endosomal protein sorting to detail how retromer activity contributes to the regulation of several distinct signaling pathways

HtrA2/Omi Terminates Cytomegalovirus Infection and Is Controlled by the Viral Mitochondrial Inhibitor of Apoptosis (vMIA)

Viruses encode suppressors of cell death to block intrinsic and extrinsic host-initiated death pathways that reduce viral yield as well as control the termination of infection. Cytomegalovirus (CMV) infection terminates by a caspase-independent cell fragmentation process after an extended period of continuous virus production. The viral mitochondria-localized inhibitor of apoptosis (vMIA; a product of the UL37x1 gene) controls this fragmentation process. UL37x1 mutant virus-infected cells fragment three to four days earlier than cells infected with wt virus. Here, we demonstrate that infected cell death is dependent on serine proteases. We identify mitochondrial serine protease HtrA2/Omi as the initiator of this caspase-independent death pathway. Infected fibroblasts develop susceptibility to death as levels of mitochondria-resident HtrA2/Omi protease increase. Cell death is suppressed by the serine protease inhibitor TLCK as well as by the HtrA2-specific inhibitor UCF-101. Experimental overexpression of HtrA2/Omi, but not a catalytic site mutant of the enzyme, sensitizes infected cells to death that can be blocked by vMIA or protease inhibitors. Uninfected cells are completely resistant to HtrA2/Omi induced death. Thus, in addition to suppression of apoptosis and autophagy, vMIA naturally controls a novel serine protease-dependent CMV-infected cell-specific programmed cell death (cmvPCD) pathway that terminates the CMV replication cycle