Optimisation of ex vivo memory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses

Background: Human memory B cells play a vital role in the long-term protection of the host from pathogenic re-challenge. In recent years the importance of a number of different memory B cell subsets that can be formed in response to vaccination or infection has started to become clear. To study memory B cell responses, cells can be cultured ex vivo, allowing for an increase in cell number and activation of these quiescent cells, providing sufficient quantities of each memory subset to enable full investigation of functionality. However, despite numerous papers being published demonstrating bulk memory B cell culture, we could find no literature on optimised conditions for the study of memory B cell subsets, such as IgM + memory B cells. Methods: Following a literature review, we carried out a large screen of memory B cell expansion conditions to identify the combination that induced the highest levels of memory B cell expansion. We subsequently used a novel Design of Experiments approach to finely tune the optimal memory B cell expansion and differentiation conditions for human memory B cell subsets. Finally, we characterised the resultant memory B cell subpopulations by IgH sequencing and flow cytometry. Results: The application of specific optimised conditions induce multiple rounds of memory B cell proliferation equally across Ig isotypes, differentiation of memory B cells to antibody secreting cells, and importantly do not alter the Ig genotype of the stimulated cells.  Conclusions: Overall, our data identify a memory B cell culture system that offers a robust platform for investigating the functionality of rare memory B cell subsets to infection and/or vaccination

Coupling models of cattle and farms with models of badgers for predicting the dynamics of bovine tuberculosis (TB)

Bovine TB is a major problem for the agricultural industry in several countries. TB can be contracted and spread by species other than cattle and this can cause a problem for disease control. In the UK and Ireland, badgers are a recognised reservoir of infection and there has been substantial discussion about potential control strategies. We present a coupling of individual based models of bovine TB in badgers and cattle, which aims to capture the key details of the natural history of the disease and of both species at approximately county scale. The model is spatially explicit it follows a very large number of cattle and badgers on a different grid size for each species and includes also winter housing. We show that the model can replicate the reported dynamics of both cattle and badger populations as well as the increasing prevalence of the disease in cattle. Parameter space used as input in simulations was swept out using Latin hypercube sampling and sensitivity analysis to model outputs was conducted using mixed effect models. By exploring a large and computationally intensive parameter space we show that of the available control strategies it is the frequency of TB testing and whether or not winter housing is practised that have the most significant effects on the number of infected cattle, with the effect of winter housing becoming stronger as farm size increases. Whether badgers were culled or not explained about 5%, while the accuracy of the test employed to detect infected cattle explained less than 3% of the variance in the number of infected cattle

Guidelines for the deployment and implementation of manufacturing scheduling systems

It has frequently been stated that there exists a gap between production scheduling theory and practice. In order to put theoretical findings into practice, advances in scheduling models and solution procedures should be embedded into a piece of software - a scheduling system - in companies. This results in a process that entails (1) determining its functional features, and (2) adopting a successful strategy for its development and deployment. In this paper we address the latter question and review the related literature in order to identify descriptions and recommendations of the main aspects to be taken into account when developing such systems. These issues are then discussed and classified, resulting in a set of guidelines that can help practitioners during the process of developing and deploying a scheduling system. 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The cancer preventative agent resveratrol is converted to the anticancer agent piceatannol by the cytochrome P450 enzyme CYP1B1

Resveratrol is a cancer preventative agent that is found in red wine. Piceatannol is a closely related stilbene that has antileukaemic activity and is also a tyrosine kinase inhibitor. Piceatannol differs from resveratrol by having an additional aromatic hydroxy group. The enzyme CYP1B1 is overexpressed in a wide variety of human tumours and catalyses aromatic hydroxylation reactions. We report here that the cancer preventative agent resveratrol undergoes metabolism by the cytochrome P450 enzyme CYP1B1 to give a metabolite which has been identified as the known antileukaemic agent piceatannol. The metabolite was identified by high performance liquid chromatography analysis using fluorescence detection and the identity of the metabolite was further confirmed by derivatisation followed by gas chromatography–mass spectrometry studies using authentic piceatannol for comparison. This observation provides a novel explanation for the cancer preventative properties of resveratrol. It demonstrates that a natural dietary cancer preventative agent can be converted to a compound with known anticancer activity by an enzyme that is found in human tumours. Importantly this result gives insight into the functional role of CYP1B1 and provides evidence for the concept that CYP1B1 in tumours may be functioning as a growth suppressor enzyme

Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP

Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post- translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B’. Results also show that unstructured post- ranslationally modified C-terminal tails are responsible for the dynamics of Sm-B/B’ and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.This work was funded by: BBSRC (OVM), BBSRC and EPSRC (HH and NM), EU Prospects (HH), European Science Foundation (NM), the Royal Society (CVR), and fellowship from JSPS and HFSP (YM and DAPK respectively)

Decreased transcription-coupled nucleotide excision repair capacity is associated with increased p53- and MLH1-independent apoptosis in response to cisplatin

Abstract Background One of the most commonly used classes of anti-cancer drugs presently in clinical practice is the platinum-based drugs, including cisplatin. The efficacy of cisplatin therapy is often limited by the emergence of resistant tumours following treatment. Cisplatin resistance is multi-factorial but can be associated with increased DNA repair capacity, mutations in p53 or loss of DNA mismatch repair capacity. Methods RNA interference (RNAi) was used to reduce the transcription-coupled nucleotide excision repair (TC-NER) capacity of several prostate and colorectal carcinoma cell lines with specific defects in p53 and/or DNA mismatch repair. The effect of small inhibitory RNAs designed to target the CSB (Cockayne syndrome group B) transcript on TC-NER and the sensitivity of cells to cisplatin-induced apoptosis was determined. Results These prostate and colon cancer cell lines were initially TC-NER proficient and RNAi against CSB significantly reduced their DNA repair capacity. Decreased TC-NER capacity was associated with an increase in the sensitivity of tumour cells to cisplatin-induced apoptosis, even in p53 null and DNA mismatch repair-deficient cell lines. Conclusion The present work indicates that CSB and TC-NER play a prominent role in determining the sensitivity of tumour cells to cisplatin even in the absence of p53 and DNA mismatch repair. These results further suggest that CSB represents a potential target for cancer therapy that may be important to overcome resistance to cisplatin in the clinic

Increasing physical activity in postpartum multiethnic women in Hawaii: results from a pilot study

<p>Abstract</p> <p>Background</p> <p>Mothers of an infant are much less likely to exercise regularly compared to other women. This study tested the efficacy of a brief tailored intervention to increase physical activity (PA) in women 3–12 months after childbirth. The study used a pretest-posttest design. Sedentary women (n = 20) were recruited from a parenting organization. Half the participants were ethnic minorities, mean age was 33 ± 3.8, infants' mean age was 6.9 ± 2.4 months, 50% were primiparas, and mean body mass index was 23.6 ± 4.2.</p> <p>Methods</p> <p>The two-month intervention included telephone counseling, pedometers, referral to community PA resources, social support, email advice on PA/pedometer goals, and newsletters.</p> <p>The primary outcome of the study was minutes per week of moderate and vigorous leisure-time physical activity measured by the Godin physical activity instrument.</p> <p>Results</p> <p>All women (100%) returned for post-test measures; thus, paired t-tests were used for pre-post increase in minutes of moderate and vigorous leisure-time physical activity and comparisons of moderate and vigorous leisure-time physical activity increases among ethnic groups. At baseline participants' reported a mean of 3 ± 13.4 minutes per week moderate and vigorous leisure-time physical activity. At post-test this significantly increased to 85.5 ± 76.4 minutes per week of moderate and vigorous leisure-time physical activity (p < .001, Cohen's d = 2.2; effect size r = 0.7). There were no differences in pre to post increases in minutes of moderate and vigorous leisure-time physical activity among races.</p> <p>Conclusion</p> <p>A telephone/email intervention tailored to meet the needs of postpartum women was effective in increasing physical activity levels. However, randomized trials comparing tailored telephone and email interventions to standard care and including long-term follow-up to determine maintenance of physical activity are warranted.</p

Association Analysis of Canonical Wnt Signalling Genes in Diabetic Nephropathy

Several studies have provided compelling evidence implicating the Wnt signalling pathway in the pathogenesis of diabetic nephropathy. Gene expression profiles associated with renal fibrosis have been attenuated through Wnt pathway modulation in model systems implicating Wnt pathway members as potential therapeutic targets for the treatment of diabetic nephropathy. We assessed tag and potentially functional single nucleotide polymorphisms (SNPs; n = 31) in four key Wnt pathway genes (CTNNB1, AXIN2, LRP5 and LRP6) for association with diabetic nephropathy using a case-control design.SNPs were genotyped using Sequenom or Taqman technologies in 1351 individuals with type 1 diabetes (651 cases with nephropathy and 700 controls without nephropathy). Cases and controls were white and recruited from the UK and Ireland. Association analyses were performed using PLINK, to compare allele and haplotype frequencies in cases and controls. Adjustment for multiple testing was performed by permutation testing.Following logistic regression analysis adjusted by collection centre, duration of T1D, and average HbA1c as covariates, a single SNP in LRP6 (rs1337791) was significantly associated with DN (OR = 0.74; CI: 0.57-0.97; P = 0.028), although this was not maintained following correction for multiple testing. Three additional SNPs (rs2075241 in LRP6; rs3736228 and rs491347 both in LRP5) were marginally associated with diabetic nephropathy, but none of the associations were replicated in an independent dataset. Haplotype and subgroup analysis (according to duration of diabetes, and end-stage renal disease) also failed to reveal an association with diabetic nephropathy.Our results suggest that analysed common variants in CTNNB1, AXIN2, LRP5 and LRP6 are not strongly associated with diabetic nephropathy in type 1 diabetes among white individuals. Our findings, however, cannot entirely exclude these genes or other members of the Wnt pathway, from involvement in the pathogenesis of diabetic nephropathy as our study had limited power to detect variants with small effect size

Genetic and Non-Genetic Influences during Pregnancy on Infant Global and Site Specific DNA Methylation: Role for Folate Gene Variants and Vitamin B12

Inter-individual variation in patterns of DNA methylation at birth can be explained by the influence of environmental, genetic and stochastic factors. This study investigates the genetic and non-genetic determinants of variation in DNA methylation in human infants. Given its central role in provision of methyl groups for DNA methylation, this study focuses on aspects of folate metabolism. Global (LUMA) and gene specific (IGF2, ZNT5, IGFBP3) DNA methylation were quantified in 430 infants by Pyrosequencing®. Seven polymorphisms in 6 genes (MTHFR, MTRR, FOLH1, CβS, RFC1, SHMT) involved in folate absorption and metabolism were analysed in DNA from both infants and mothers. Red blood cell folate and serum vitamin B12 concentrations were measured as indices of vitamin status. Relationships between DNA methylation patterns and several covariates viz. sex, gestation length, maternal and infant red cell folate, maternal and infant serum vitamin B12, maternal age, smoking and genotype were tested. Length of gestation correlated positively with IGF2 methylation (rho = 0.11, p = 0.032) and inversely with ZNT5 methylation (rho = −0.13, p = 0.017). Methylation of the IGFBP3 locus correlated inversely with infant vitamin B12 concentration (rho = −0.16, p = 0.007), whilst global DNA methylation correlated inversely with maternal vitamin B12 concentrations (rho = 0.18, p = 0.044). Analysis of common genetic variants in folate pathway genes highlighted several associations including infant MTRR 66G>A genotype with DNA methylation (χ2 = 8.82, p = 0.003) and maternal MTHFR 677C>T genotype with IGF2 methylation (χ2 = 2.77, p = 0.006). These data support the hypothesis that both environmental and genetic factors involved in one-carbon metabolism influence DNA methylation in infants. Specifically, the findings highlight the importance of vitamin B12 status, infant MTRR genotype and maternal MTHFR genotype, all of which may influence the supply of methyl groups for DNA methylation. In addition, gestational length appears to be an important determinant of infant DNA methylation patterns