Improving the PCR protocol to amplify a repetitive DNA sequence

Although PCR-based techniques have become an essential tool in the field of molecular and genetic research, the amplification of repetitive DNA sequences is limited. This is due to the truncated nature of the amplified sequences, which are also prone to errors during DNA polymerase-based amplification. The complex structure of repetitive DNA can form hairpin loops, which promote dissociation of the polymerase from the template, impairing complete amplification, and leading to the formation of incomplete fragments that serve as megaprimers. These megaprimers anneal with other sequences, generating unexpected fragments in each PCR cycle. Our gene model, MaSp1, is 1037-bp long, with 68% GC content, and its amino acid sequence is characterized by poly-alanine-glycine motifs, which represent the repetitive codon consensus. We describe the amplification of the MaSp1 gene through minor changes in the PCR program. The results show that a denaturation temperature of 98°C is the key determinant in the amplification of the MaSp1 partial gene sequence

Relating smoking, obesity, insulin resistance, and ovarian biomarker changes to the final menstrual period

To determine if smoking, obesity, and insulin resistance mediated age at final menstrual period (FMP), we examined anti-Müllerian hormone (AMH), inhibin B, and follicle-stimulating hormone (FSH) as biomarkers of changing follicle status and ovarian aging. We performed a longitudinal data analysis from a cohort of premenopausal women followed to their FMP. Our results found that smokers had an earlier age at FMP ( P < 0.003) and a more rapid decline in their AMH slope relative to age at FMP ( P < 0.002). Smokers had a lower baseline inhibin B level relative to age at the FMP than nonsmokers ( P = 0.002). Increasing insulin resistance was associated with a shorter time to FMP ( P < 0.003) and associations of obesity and time to FMP were observed ( P = 0.004, in model with FSH). Change in ovarian biomarkers did not mediate the time to FMP. We found that smoking was associated with age at FMP and modified associations of AMH and inhibin B with age at FMP. Insulin resistance was associated with shorter time to FMP independent of the biomarkers. Interventions targeting smoking and insulin resistance could curtail the undue advancement of reproductive aging.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79293/1/j.1749-6632.2010.05523.x.pd

Variable strength of forest stand attributes and weather conditions on the questing activity of Ixodes ricinus ticks over years in managed forests

Given the ever-increasing human impact through land use and climate change on the environment, we crucially need to achieve a better understanding of those factors that influence the questing activity of ixodid ticks, a major disease-transmitting vector in temperate forests. We investigated variation in the relative questing nymph densities of Ixodes ricinus in differently managed forest types for three years (2008–2010) in SW Germany by drag sampling. We used a hierarchical Bayesian modeling approach to examine the relative effects of habitat and weather and to consider possible nested structures of habitat and climate forces. The questing activity of nymphs was considerably larger in young forest successional stages of thicket compared with pole wood and timber stages. Questing nymph density increased markedly with milder winter temperatures. Generally, the relative strength of the various environmental forces on questing nymph density differed across years. In particular, winter temperature had a negative effect on tick activity across sites in 2008 in contrast to the overall effect of temperature across years. Our results suggest that forest management practices have important impacts on questing nymph density. Variable weather conditions, however, might override the effects of forest management practices on the fluctuations and dynamics of tick populations and activity over years, in particular, the preceding winter temperatures. Therefore, robust predictions and the detection of possible interactions and nested structures of habitat and climate forces can only be quantified through the collection of long-term data. Such data are particularly important with regard to future scenarios of forest management and climate warming

Commentary: "Consistent Superiority of Selective Serotonin Reuptake Inhibitors Over Placebo in Reducing Depressed Mood in Patients with Major Depression"

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142781/1/Fried-Nesse-SerotoninInhibitors-FrontPsychiatry-2015.pd

Combined TLR and CD40 Triggering Induces Potent CD8+ T Cell Expansion with Variable Dependence on Type I IFN

Toll-like receptors are important in the activation of innate immunity, and CD40 is a molecule critical for many T and B cell responses. Whereas agonists for either pathway have been used as vaccine adjuvants, we show that a combination of Toll-like receptor (TLR)7 and CD40 agonists synergize to stimulate CD8+ T cell responses 10–20-fold greater than the use of either agonist alone. Antigen-specific CD8+ T cells elicited from combination CD40/TLR7 treatment demonstrated both lytic activities and interferon (IFN)γ production and an enhanced secondary response to antigenic challenge. Agonists for TLRs 2/6, 3, 4, and 9 also synergized with CD40 stimulation, demonstrating that synergy with the CD40 pathway is a property of TLR-derived stimuli in general. The CD8+ T cell expansion induced by CD40/TLR7 triggering was independent of CD4+ T cells, IFNγ, and IL-12 but dependent on B7-mediated costimulation and surprisingly on type I IFN. These studies provide the rational basis for the use of TLR and CD40 agonists together as essential adjuvants to optimize vaccines designed to elicit protective or therapeutic immunity

Behaviour of piles driven in chalk

Driving resistance is difficult to predict in chalk strata, with both pile free-fall self-weight ‘runs’ and refusals being reported. Axial capacity is also highly uncertain after driving. This paper reviews recent research that has explored these topics. Programmes of onshore tests and novel, high-value offshore, experiments involving static, dynamic and cyclic loading are described. The key findings form the basis of the Chalk ICP-18 approach for estimating the driving resistance and axial capacity of piles driven in low-to medium-density chalk. The new approach is presented and the highly significant impact of set-up after driving is emphasised. It is shown that Chalk ICP-18 overcomes the main limitations of the industry’s current design guidelines by addressing the underlying physical mechanisms. While further tests are required to enlarge the available test database, the new approach is able to provide better predictions for tests available from suitably characterised sites. A new Joint Industry Project is outlined that extends the research to cover further axial, lateral, static and cyclic loading cases

The value of follicle-stimulating hormone concentration and clinical findings as markers of the late menopausal transition

CONTEXT: The Stages of Reproductive Aging Workshop proposed bleeding and hormonal criteria for the menopausal transition, but operational definitions of hormone parameters were not specified. OBJECTIVE: This paper investigates the longitudinal relationship of annual serum FSH levels with four proposed bleeding criteria for the late menopausal transition in two cohort studies. The goal is to provide empirically based guidance regarding application of hormonal criteria that may be optimal for widespread application in clinical and research settings for assessing menopausal stage. DESIGN/SETTING: Prospective menstrual calendar and annual serum FSH data were collected from two population-based cohort studies: the Melbourne Women\u27s Midlife Health Project and the Study of Women\u27s Health Across the Nation. PARTICIPANTS: Participants in the study were 193 Melbourne Women\u27s Midlife Health Project and 2223 Study of Women\u27s Health Across the Nation women aged 42-57 yr at baseline who contributed 10 or more menstrual cycles and at least one annual serum FSH value. MAIN OUTCOME MEASURE(S): Association between bleeding criteria for the late menopausal transition and FSH was a main outcome measure. Associations of bleeding criteria, FSH, and hot flashes with the final menstrual period were also measured. RESULTS: A single FSH measure is an independent marker of the late menopausal transition, but FSH concentrations are less predictive of menopausal stage than any of four proposed bleeding criteria. Criterion FSH values for the late transition are similar across both studies. Experience of hot flashes adds no information in the presence of hormonal and bleeding criteria. CONCLUSIONS: An annual serum FSH concentration of 40 IU/liter could be incorporated, in conjunction with bleeding markers, into the Stages of Reproductive Aging Workshop paradigm for markers of the late menopausal transition