Detecting variable (V), diversity (D) and joining (J) gene segment recombination using a two-colour fluorescence system

<p>Abstract</p> <p>Background</p> <p>Diversity of immunoglobulins and the T cell antigen receptors is achieved via the recombination activating gene (RAG)-mediated rearrangement of variable (V), diversity (D) and joining (J) gene segments, and this underpins the efficient recognition of a seemingly limitless array of antigens. Analysis of V(D)J recombination activity is typically performed using extrachromosomal recombination substrates that are recovered from transfected cells and selected using bacterial transformation. We have developed a two-colour fluorescence-based system that simplifies detection of both deletion and inversion joining events mediated by RAG proteins.</p> <p>Results</p> <p>This system employs two fluorescent reporter genes that differentially mark unrearranged substrates and those that have undergone RAG-mediated deletion or inversion events. The recombination products bear the hallmarks of true V(D)J recombination and activity can be detected using fluorescence microscopy or flow cytometry. Recombination events can be detected without the need for cytotoxic selection of recombination products and the system allows analysis of recombination activity using substrates integrated into the genome.</p> <p>Conclusions</p> <p>This system will be useful in the analysis and exploitation of the V(D)J recombination machinery and suggests that similar approaches could be used to replace expression of one gene with another during lymphocyte development.</p

Tumour Cell Generation of Inducible Regulatory T-Cells in Multiple Myeloma Is Contact-Dependent and Antigen-Presenting Cell-Independent

Regulatory T-cells (TReg cells) are increased in patients with multiple myeloma (MM). We investigated whether MM cells could generate and/or expand TReg cells as a method of immuno-surveillance avoidance. In an in vitro model, CD4+CD25-FoxP3- T-cells co-cultured with malignant plasma cells (primary MM cells and cell lines) induced a significant generation of CD4+CD25+FoxP3+ inducible TReg cells (tTReg cells; p<0.0001), in a contact-dependent manner. tTReg cells were polyclonal, demonstrated a suppressive phenotype and phenotypically, demonstrated increased FoxP3 (p = 0.0001), increased GITR (p<0.0001), increased PD1 (p = 0.003) and decreased CD62L (p = 0.007) expression compared with naturally occurring TReg cells. FACS-sorted tTReg cells differentiated into FoxP+IL-17+ and FoxP3-IL-17+ CD4+ cells upon TCR-mediated stimulation. Blocking experiments with anti-ICOS-L MoAb resulted in a significant inhibition of tTReg cell generation whereas both IL-10 & TGFβ blockade did not. MM tumour cells can directly generate functional TReg cells in a contact-dependent manner, mediated by ICOS/ICOS-L. These features suggest that tumour generation of TReg cells may contribute to evasion of immune surveillance by the host

Relationship between DNA methylation and mutational patterns induced by a sequence selective minor groove methylating agent.

Me-lex, a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generate N3-methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions. In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis. In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results showed that: 1) more than 99% of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A's that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52%) were AT-targeted; 5) AT --TA transversions were the predominant mutations observed (13/33; 39%); 6) 13 out of 33 (39%) independent mutations involved a single lex-binding site encompassing positions A600-602 and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n = 9, p10(-6), Poisson's normal distribution). A hypothetical model for the interpretation of mutational events at this site is proposed. The present work is the first report on mutational properties of Me-lex. Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner

Influences of base excision repair defects on the lethality and mutagenicity induced by Me-lex, a sequence-selective N3-adenine methylating agent.

Due to its minor groove selectivity, Me-lex preferentially generates N3-methyladenine (3-MeA) adducts in double-stranded DNA. We undertook a genetic approach in yeast to establish the influence of base excision repair (BER) defects on the processing of Me-lex lesions on plasmid DNA that harbors the p53 cDNA as target. We constructed a panel of isogenic strains containing a reporter gene to test p53 function and the following gene deletions: deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. When compared with the wild-type strain, a decrease in survival was observed in deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. The Me-lex-induced mutation frequency increased in the following order: wild typedeltamag1deltaapn1apn2 = deltaapn1apn2mag1. A total of 77 mutants (23 in wild type, 31 in deltamag1, and 23 in deltaapn1apn2) were sequenced. Eighty-one independent mutations (24 in wild type, 34 in deltamag1, and 23 in deltaapn1apn2) were detected. The majority of base pair substitutions were AT-targeted in all strains (14/23, 61% in wild type; 20/34, 59%, in deltamag1; and 14/23, 61%, in deltaapn1apn2). The Mag1 deletion was associated with a significant decrease of GCAT transitions when compared with both the wild-type and the AP endonuclease mutants. This is the first time that the impact of Mag1 and/or AP endonuclease defects on the mutational spectra caused by 3-MeA has been determined. The results suggest that 3-MeA is critical for Me-lex cytotoxicity and that its mutagenicity is slightly elevated in the absence of Mag1 glycosylase activity but significantly higher in the absence of AP endonuclease activity

Sugar-Sweetened Beverages and Adverse Human Health Outcomes: An Umbrella Review of Meta-Analyses of Observational Studies

Our aim was to conduct an umbrella review of evidence from meta-analyses of observational studies investigating the link between sugar-sweetened beverage consumption and human health outcomes. Using predefined evidence classification criteria, we evaluated evidence from 47 meta-analyses encompassing 22,055,269 individuals. Overall, 79% of these analyses indicated direct associations between greater sugar-sweetened beverage consumption and higher risks of adverse health outcomes. Convincing evidence (class I) supported direct associations between sugar-sweetened beverage consumption and risks of depression, cardiovascular disease, nephrolithiasis, type 2 diabetes mellitus, and higher uric acid concentrations. Highly suggestive evidence (class II) supported associations with risks of nonalcoholic fatty liver disease and dental caries. Out of the remaining 40 meta-analyses, 29 were graded as suggestive or weak in the strength of evidence (classes III and IV), and 11 showed no evidence (class V). These findings inform and provide support for population-based and public health strategies aimed at reducing sugary drink consumption for improved health.</p

Functional assays to determine the significance of two common XPC 3'UTR variants found in bladder cancer patients

<p>Abstract</p> <p>Background</p> <p><it>XPC </it>is involved in the nucleotide excision repair of DNA damaged by carcinogens known to cause bladder cancer. Individuals homozygous for the variant allele of <it>XPC </it>c.1496C > T (p.Ala499Val) were shown in a large pooled analysis to have an increased bladder cancer risk, and we found two 3'UTR variants, *611T > A and c.*618A > G, to be in strong linkage disequilibrium with c.1496T. Here we determined if these two 3'UTR variants can affect mRNA stability and assessed the impact of all three variants on mRNA and protein expression.</p> <p>Methods</p> <p><it>In vitro </it>mRNA stability assays were performed and mRNA and protein expression measured both in plasmid-based assays and in lymphocytes and lymphoblastoid cell lines from bladder and breast cancer patients.</p> <p>Results</p> <p>The two 3'UTR variants were associated with reduced protein and mRNA expression in plasmid-based assays, suggesting an effect on mRNA stability and/or transcription/translation. A near-significant reduction in XPC protein expression (p = 0.058) was detected in lymphoblastoid cell lines homozygous for these alleles but no differences in mRNA stability in these lines was found or in mRNA or protein levels in lymphocytes heterozygous for these alleles.</p> <p>Conclusion</p> <p>The two 3'UTR variants may be the variants underlying the association of c.1496C > T and bladder cancer risk acting via a mechanism modulating protein expression.</p

Epidemiology, prehospital care and outcomes of patients arriving by ambulance with dyspnoea: An observational study

Background: This study aimed to determine epidemiology and outcome for patients presenting to emergency departments (ED) with shortness of breath who were transported by ambulance. Methods: This was a planned sub-study of a prospective, interrupted time series cohort study conducted at three time points in 2014 and which included consecutive adult patients presenting to the ED with dyspnoea as a main symptom. For this sub-study, additional inclusion criteria were presentation to an ED in Australia or New Zealand and transport by ambulance. The primary outcomes of interest are the epidemiology and outcome of these patients. Analysis was by descriptive statistics and comparisons of proportions. Results: One thousand seven patients met inclusion criteria. Median age was 74 years (IQR 61-68) and 46.1 % were male. There was a high rate of co-morbidity and chronic medication use. The most common ED diagnoses were lower respiratory tract infection (including pneumonia, 22.7 %), cardiac failure (20.5%) and exacerbation of chronic obstructive pulmonary disease (19.7 %). ED disposition was hospital admission (including ICU) for 76.4 %, ICU admission for 5.6 % and death in ED in 0.9 %. Overall in-hospital mortality among admitted patients was 6.5 %. Discussion: Patients transported by ambulance with shortness of breath make up a significant proportion of ambulance caseload and have high comorbidity and high hospital admission rate. In this study, >60 % were accounted for by patients with heart failure, lower respiratory tract infection or COPD, but there were a wide range of diagnoses. This has implications for service planning, models of care and paramedic training. Conclusion: This study shows that patients transported to hospital by ambulance with shortness of breath are a complex and seriously ill group with a broad range of diagnoses. Understanding the characteristics of these patients, the range of diagnoses and their outcome can help inform training and planning of services

The Vehicle, Fall 1982

Vol. 24, No. 1 Table of Contents Winter SurveillanceB.L. Davidsonpage 3 The InvitationBecky Lawsonpage 4 Check In, Check OutSteve Sandstrompage 4 On The Front Porch StepKeila Tooleypage 5 Old Greek ManDevon Flesorpage 5 Exotic PassionsBecky Lawsonpage 6 PhotographLisa Owenspage 7 Beyond The ThornsBrook Wilsonpage 8 Ritual Of HeatB.L. Davidsonpage 11 The GamerBecky Lawsonpage 12 It\u27s OverKeila Tooleypage 13 DreamJohn Stockmanpage 14 Silver DollarGina J. Grillopage 15 The DancerJessica Lewispage 16 Snapshots Of Rural IllinoisIsabel M. Parrottpage 16 The Last SeasonTheresa Whitesidepage 17 DrawingKaren Haneypage 17 Rotary LuncheonJessica Lewispage 18 Factory TourLinda Fraembspage 18 The ImmigrantsD.L. Lewispage 19 At Shedd AquariumLinda Fraembspage 20 The GuardianBecky Lewispage 20 Digital LifeEverett Tackettpage 21 Full ServiceScott Graypage 22 Dust ShowLinda A. Brownpage 23 At SixMaureen Foertschpage 24 DrawingJean Imherrpage 24 ReflectionMaggie Kennedypage 25 Cat DefiningBecky Lawsonpage 26 Ode To An Unread NewspaperLinda Fraembspage 26 GumSteve Sandstrompage 27 The DancerChrystal Clarkpage 27 PoemD.L. Lewispage 28 For LucyStacey Flanniganpage 29 An AbortionDevon Flesorpage 29 ReveriesKeila Tooleypage 30 Sunday Morning After Tequila With LemonScott Graypage 33 Staging A Living Jewel BoxMichelle Mitchellpage 34 The Other WomanStacey Flanniganpage 35 The Natural LookMichelle Mitchellpage 35 Poem To A Girl Named SandalsJohn Stockmanpage 36 PhotographLisa Owenspage 37 In The Balcony Of The Bijou On A Saturday NightScott Graypage 38 The Canadian Soccer PlayerBecky Lawsonpage 39 The HealingJohn Stockmanpage 39 AppeasedDevon Flesorpage 40 CodaJohn Stockmanpage 40https://thekeep.eiu.edu/vehicle/1040/thumbnail.jp

Biological, clinical and population relevance of 95 loci for blood lipids.

Plasma concentrations of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides are among the most important risk factors for coronary artery disease (CAD) and are targets for therapeutic intervention. We screened the genome for common variants associated with plasma lipids in &gt;100,000 individuals of European ancestry. Here we report 95 significantly associated loci (P &lt; 5 x 10(-8)), with 59 showing genome-wide significant association with lipid traits for the first time. The newly reported associations include single nucleotide polymorphisms (SNPs) near known lipid regulators (for example, CYP7A1, NPC1L1 and SCARB1) as well as in scores of loci not previously implicated in lipoprotein metabolism. The 95 loci contribute not only to normal variation in lipid traits but also to extreme lipid phenotypes and have an impact on lipid traits in three non-European populations (East Asians, South Asians and African Americans). Our results identify several novel loci associated with plasma lipids that are also associated with CAD. Finally, we validated three of the novel genes-GALNT2, PPP1R3B and TTC39B-with experiments in mouse models. Taken together, our findings provide the foundation to develop a broader biological understanding of lipoprotein metabolism and to identify new therapeutic opportunities for the prevention of CAD

Positive Psychology in Sales: Integrating Psychological Capital

As positive psychology moves into the workplace, researchers have been able to demonstrate the desirable impact of positive organizational behavior. Specifically, psychological capital (PsyCap) improves employee attitudes, behaviors, and performance. Advancing PsyCap in sales research is important given the need for a comprehensive positive approach to drive sales performance, offset the high cost of salesperson turnover, improve cross-functional sales interfaces, and enrich customer relationships. The authors provide an integrative review of PsyCap, discuss its application in sales, and advance an agenda for future research. Research prescriptions are organized according to individual-level, intra-organizational, and extra-organizational outcomes pertinent to the sales field