Purification and analysis of human muc reporter proteins

Abstract only availableExocrine mucous glycoproteins are a family of multifunctional heavily glycosylated, anionic macromolecules that typically possess a high serine/threonine content. The size and complexity of these glycoprotein molecules, while physiologically beneficial, prohibits direct analysis of small-to-moderate changes within the side-chain oligosaccharides. These polydispersed molecules have a protein core encoded by muc-genes, which possess multiple repeats within their sequences. To assist in developing a method to study these complex molecules, a reporter-DNA construct, for eventual eukaryotic expression, consisting of an IgK secretory leader sequence, two polyHis regions, two HSV and one myc antigenic sites was synthesized. This construct was then utilized by incorporating two separate muc repeat-sequence units; one consisting of muc-2/muc-2 and one possessing muc-2/muc-4 (i.e., amuc2c and amuc24c, respectively). DNA plasmids pET28-amuc2c and pET28-amuc24c were transformed into the bacterial strain Ecoli BL21DE3. Expressed proteins from transformants were isolated and purified, and then analyzed by MALDI-TOF MS. Mass+H+ (avg) of 16306.2 Da and 17062.8 Da for amuc2c and amuc24c, respectively, were observed. MS and MS/MS analysis of the tryptic digests of these expressed proteins also confirmed their respective sequences. To test the efficacy of possible coexpression of fluorescent protein transfection markers with the muc-constructs in eukaryotic cells, preliminary transfections of fluorescent DNA plasmids pEGFPc1 (green, cytoplasmic), pDSRed2-N1 (red, secretory-IgK), pEYFP-Golgi (yellow) and pECFP-Golgi (cyan) into MATLyLu cells (rat prostate cancer) are being performed. The successful conclusion of these ongoing studies will result in the expression of a small, glycated, and secreted muc-protein from transfected human intestional and respiratory cells in vitro that also are producing muc-related macromolecules. These posttranslationally modified amuc2c and amuc24c reporter proteins can then be analyzed, in detail, by contemporary methods. They will be employed as tools to help provide an insight into the changes that occur in the posttranslational modifications of macromolecular glycoproteins in human disease, such as cystic fibrosis

Chemical composition of cultivated seaweed ulva clathrata (Roth) C. Agardh

Samples of cultivated Ulva clathrata were collected from a medium scale system (MSS, 1.5 1.5 m tank), or from a large scale system (LSS, 0.8 ha earthen pond). MSS samples were dried directly while the LSS sample was washed in freshwater and pressed before drying. Crude protein content ranged 20–26%, essential amino acids accounting for 32–36% of crude protein. The main analysed monosaccharides were rhamnose (36–40%), uronic acids (27–29%), xylose (10–13%) and glucose (10–16%). Some notable variations between MSS and LSS samples were observed for total dietary fibre (26% vs 41%), saturated fatty acids (31% vs 51%), PUFAS (33% vs 13%), carotenoids (358 vs 169 mg kg1 dw) and for Ca (9 vs 19 g kg1 ), Fe (0.6 vs 4.2 g kg1 ), Cu (44 vs 14 mg kg1 ), Zn (93 vs 17 mg kg1 ) and As (2 vs 9 mg kg1 ). The chemical composition of U. clathrata indicates that it has a good potential for its use in human and animal food

Flavor-Changing Processes in Extended Technicolor

We analyze constraints on a class of extended technicolor (ETC) models from neutral flavor-changing processes induced by (dimension-six) four-fermion operators. The ETC gauge group is taken to commute with the standard-model gauge group. The models in the class are distinguished by how the left- and right-handed $(L,R)$ components of the quarks and charged leptons transform under the ETC group. We consider $K^{0} - \bar K^0$ and other pseudoscalar meson mixings, and conclude that they are adequately suppressed if the $L$ and $R$ components of the relevant quarks are assigned to the same (fundamental or conjugate-fundamental) representation of the ETC group. Models in which the $L$ and $R$ components of the down-type quarks are assigned to relatively conjugate representations, while they can lead to realistic CKM mixing and intra-family mass splittings, do not adequately suppress these mixing processes. We identify an approximate global symmetry that elucidates these behavioral differences and can be used to analyze other possible representation assignments. Flavor-changing decays, involving quarks and/or leptons, are adequately suppressed for any ETC-representation assignment of the $L$ and $R$ components of the quarks, as well as the leptons. We draw lessons for future ETC model building.Comment: 25 page

Neutrino Masses in Theories with Dynamical Electroweak Symmetry Breaking

We address the problem of accounting for light neutrino masses in theories with dynamical electroweak symmetry breaking. We discuss this in the context of a class of (extended) technicolor (ETC) models and analyze the full set of Dirac and Majorana masses that arise in such theories. As a possible solution, we propose a combination of suppressed Dirac masses and a seesaw involving dynamically generated $|\Delta L|=2$ condensates of standard-model singlet, ETC-nonsinglet fermions. We show how this can be realized in an explicit ETC model. An important feature of this proposal is that, because of the suppression of Dirac neutrino mass terms, a seesaw yielding realistic neutrino masses does not require superheavy Majorana masses; indeed, these Majorana masses are typically much smaller than the largest ETC scale.Comment: 13 pages, latex; simplified mode

Athlete, coach and practitioner knowledge and perceptions of post-exercise cold-water immersion for recovery: a qualitative and quantitative exploration

This survey sought to establish current use, knowledge and perceptions of Cold-water immersion (CWI) when used for recovery. 111 athletes, coaches & support practitioners completed the anonymous online survey, answering questions about their current CWI protocols, perceptions of benefits associated with CWI and knowledge of controlling mechanisms. Respondents were largely involved in elite sport at international, national and club level, with many having used CWI previously and finding its use beneficial for recovery. Protocols differed, with the duration of immersion one aspect that failed to align with recommendations in the scientific literature. Whilst many respondents were aware of benefits associated with CWI there remains some confusion. There also seems to be a gap in mechanistic knowledge, where respondents are aware of benefits associated with CWI but failed to identify the underlying mechanisms. This identifies the need for an improved method of knowledge transfer between scientific and applied practice communities. Moreover, data herein emphasises the important role of the ‘support practitioner’ as respondents in this role tended to favour CWI protocols more aligned to recommendations within the literature. With a significant number of respondents claiming they were made aware of CWI for recovery through a colleague, the importance of knowledge transfer and context being appropriately applied to data is as important as ever. With the firm belief that CWI is useful for recovery in sport, the focus should now be on investigating the psychophysiological interaction and correct use of this methodology

Fermion Masses and Mixing in Extended Technicolor Models

We study fermion masses and mixing angles, including the generation of a seesaw mechanism for the neutrinos, in extended technicolor (ETC) theories. We formulate an approach to these problems that relies on assigning right-handed $Q=-1/3$ quarks and charged leptons to ETC representations that are conjugates of those of the corresponding left-handed fermions. This leads to a natural suppression of these masses relative to the $Q=2/3$ quarks, as well as the generation of quark mixing angles, both long-standing challenges for ETC theories. Standard-model-singlet neutrinos are assigned to ETC representations that provide a similar suppression of neutrino Dirac masses, as well as the possibility of a realistic seesaw mechanism with no mass scale above the highest ETC scale of roughly $10^3$ TeV. A simple model based on the ETC group SU(5) is constructed and analyzed. This model leads to non-trivial, but not realistic mixing angles in the quark and lepton sectors. It can also produce sufficiently light neutrinos, although not simultaneously with a realistic quark spectrum. We discuss several aspects of the phenomenology of this class of models.Comment: 74 pages, revtex with embedded figure

Analysis of large oxygenated and nitrated polycyclic aromatic hydrocarbons formed under simulated diesel engine exhaust conditions (by compound fingerprints with SPE/LC-API-MS)

The analysis of organic compounds in combustion exhaust particles and the chemical transformation of soot by nitrogen oxides are key aspects of assessment and mitigation of the climate and health effects of aerosol emissions from fossil fuel combustion and biomass burning. In this study we present experimental and analytical techniques for efficient investigation of oxygenated and nitrated derivatives of large polycyclic aromatic hydrocarbons (PAHs), which can be regarded as well-defined soot model substances. For coronene and hexabenzocoronene exposed to nitrogen dioxide under simulated diesel exhaust conditions, several reaction products with high molecular mass could be characterized by liquid chromatography-atmospheric pressure chemical (and photo) ionization-mass spectrometry (LC-APCI-MS and LC-APPI-MS). The main products of coronene contained odd numbers of nitrogen atoms (m/z 282, 256, 338), whereas one of the main products of hexabenzocoronene exhibited an even number of nitrogen atoms (m/z 391). Various reaction products containing carbonyl and nitro groups could be tentatively identified by combining chromatographic and mass spectrometric information, and changes of their relative abundance were observed to depend on the reaction conditions. This analytical strategy should highlight a relatively young technique for the characterization of various soot-contained, semi-volatile, and semi-polar reaction products of large PAHs

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty