DNA extraction protocol for rapid PCR detection of pathogenic bacteria

Twelve reagents were evaluated to develop a direct DNA extraction method suitable for PCR detection of foodborne bacterial pathogens. Many reagents exhibited strong PCR inhibition, requiring significant dilution of the extract with a corresponding reduction in sensitivity. Most reagents also exhibited much lower recovery of DNA from the gram-positive test organism (Listeria monocytogenes) than from the gram-negative organism (Escherichia coli O157:H7), preventing unbiased detection and quantitation of both organisms. The 5× HotSHOT + Tween reagent exhibited minimal inhibition and high extraction efficiency for both test organisms, providing a 15-min single-tube DNA-extraction protocol suitable for highly sensitive quantitative PCR assays.     

All That Glitters Isn't Gold: A Survey on Acknowledgment of Limitations in Biomedical Studies

Background

Acknowledgment of all serious limitations to research evidence is important for patient care and scientific progress. Formal research on how biomedical authors acknowledge limitations is scarce.

Objectives

To assess the extent to which limitations are acknowledged in biomedical publications explicitly, and implicitly by investigating the use of phrases that express uncertainty, so-called hedges; to assess the association between industry support and the extent of hedging.

Design

We analyzed reporting of limitations and use of hedges in 300 biomedical publications published in 30 high and medium -ranked journals in 2007. Hedges were assessed using linguistic software that assigned weights between 1 and 5 to each expression of uncertainty.

Results

Twenty-seven percent of publications (81/300) did not mention any limitations, while 73% acknowledged a median of 3 (range 1–8) limitations. Five percent mentioned a limitation in the abstract. After controlling for confounders, publications on industry-supported studies used significantly fewer hedges than publications not so supported (p = 0.028).

Limitations

Detection and classification of limitations was – to some extent – subjective. The weighting scheme used by the hedging detection software has subjective elements.

Conclusions

Reporting of limitations in biomedical publications is probably very incomplete. Transparent reporting of limitations may protect clinicians and guideline committees against overly confident beliefs and decisions and support scientific progress through better design, conduct or analysis of new studies.     

The selector gene cut represses a neural cell fate that is specified independently of the Achaete-Scute-Complex and atonal.

The peripheral nervous system (PNS) of Drosophila offers a powerful system to precisely identify individual cells and dissect their genetic pathways of development. The mode of specification of a subset of larval PNS cells, the multiple dendritic (md) neurons (or type II neurons), is complex and still poorly understood. Within the dorsal thoracic and abdominal segments, two md neurons, dbd and dda1, apparently require the proneural gene amos but not atonal (ato) or Achaete-Scute-Complex (ASC) genes. ASC normally acts via the neural selector gene cut to specify appropriate sensory organ identities. Here, we show that dbd- and dda1-type differentiation is suppressed by cut in dorsal ASC-dependent md neurons. Thus, cut is not only required to promote an ASC-dependent mode of differentiation, but also represses an ASC- and ato-independent fate that leads to dbd and dda1 differentiation.     

SPR biosensor for the detection of L. monocytogenes using phage-displayed antibody

Whole cells of Listeria monocytogenes were detected with a compact, surface plasmon resonance (SPR) sensor using a phage-displayed scFv antibody to the virulence factor actin polymerization protein (ActA) for biorecognition. Phage Lm P4:A8, expressing the scFv antibody fused to the pIII surface protein was immobilized to the sensor surface through physical adsorption. A locally constructed fluidics system was used to deliver solutions to the compact, two-channel SPREETA sensor. Specificity of the sensor was tested using common food-borne bacteria and a control phage, M13K07 lacking the scFv fusion on its coat protein. The detection limit for L. monocytogenes whole cells was estimated to be 2 x 10(6)cfu/ml. The sensor was also used to determine the dissociation constant (Kd) for the interaction of phage-displayed scFv and soluble ActA in solution as 4.5 nM.     

Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil

Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae. Copyright © 2013 John Wiley & Sons, Ltd.     

Hyperpolarization-activated cation current Ih of dentate gyrus granule cells is upregulated in human and rat temporal lobe epilepsy

Phosphorylation of α-synuclein at Ser-129 is of crucial relevance to Parkinson's disease and related synucleinopathies. Here we provide biochemical evidence that PLK2 and to a lesser extent PLK3 are superior over CK2, as catalysts of Ser-129 phosphorylation both in full length α-synuclein and in a peptide reproducing the C-terminal segment of the protein. By using substituted peptides we also show that the sequence surrounding Ser-129 is optimally shaped for undergoing phosphorylation by PLK2, with special reference to the two acidic residues at positions n-3 (Glu-126) and n+2 (Glu-131) whose replacement with alanine abrogates phosphorylation.     

Impact of the UK colorectal cancer screening pilot studies on incidence, stage distribution and mortality trends

Objective: To assess the impact of the UK colorectal cancer guaiac faecal occult blood test screening pilot studies on incidence trends, stage distribution and mortality trends. Design: Ecological study. Setting: Scotland and the West Midlands. Data: We extracted anonymised colorectal cancer (ICD-10 C18–C20) registration (1982–2006) and death records (1982–2007), along with corresponding mid-year population estimates. Intervention: Residents of the screening pilot areas, in the age group 50–69 years, were offered biennial guaiac faecal occult blood test screening from 2000 onwards. Screening was not offered routinely in non-pilot areas until the start of the roll-out of the national screening programmes in England and in Scotland in 2006 and 2007, respectively. Main outcome measures: We analysed trends in age-specific incidence and mortality rates, and Dukes’ stage distribution. Within each country/region, we compared the screening pilot areas to non-screening pilot (‘control’) areas using Chi square tests and Poisson regression modelling. Results: Following the start of the screening pilots, as expected in the prevalent round of a new screening programme, in the pilot areas there was a short-lived increase in incidence of colorectal cancer among 50–69 year olds except for females in the West Midlands. A trend towards earlier stage and less advanced disease was also observed, with males showing significant increases in Dukes’ A and corresponding decreases in Dukes’ C in the screening pilot areas (all P < 0.03). With the exception of females in the West Midlands, mortality rates for colorectal cancer decreased significantly and at a faster rate in the populations invited for screening. Conclusion: The existence of a natural control population not yet invited for screening provided a unique opportunity to assess whether the benefits of colorectal cancer screening, beyond the setting of a randomised controlled trial, could be detected using routinely collected statistics. Our analysis suggests that screening will fulfil its aim of reducing mortality from colorectal cancer.     

Socioeconomic inequalities in incidence of lung and upper aero-digestive tract cancer by age, tumour subtype and sex: a population-based study in Scotland (2000-2007)

BackgroundLung and upper aero-digestive tract (UADT) cancer risk is associated with socioeconomic inequality (SEI) but the degree of socioeconomic burden by age, tumour subtype, and sex is not known.MethodsWe reviewed 216,305 cases excluding non melanoma skin cancer (All Cancer) comprising 37,274 lung; 8216 head and neck; and 6534 oesophageal cancers from 2000 to 2007 classified into anatomical or morphology subtypes. Deprivation was measured using the Scottish Index of Multiple Deprivation and SEI was measured using the Slope Index of Inequality and the Relative Index of Inequality (RII). Analyses were partitioned by 5-year age group and sex. RII was adapted to rank tumour type contribution to All Cancer SEI and to examine subtype by age and sex simultaneously. Rank was defined as proportion of All Cancer SEI.ResultsAll Cancer SEI was greater for males (RII = 0.366; female RII = 0.279); the combination of lung and UADT SEI contributed 91% and 81% respectively to All Cancer SEI. For both sexes lung and UADT subtypes showed significant SEI (P < 0.001) except oesophageal adenocarcinoma in males (P = 0.193); for females, SEI was borderline significant (P = 0.048). Although RII rank differed by sex, all lung and larynx subtypes contributed most to All Cancer SEI with RII rank for oral cavity, oesophagus-squamous cell, and oropharynx following. For males 40–44 years, SEI increased abruptly peaking at 55–59 years. For females, SEI gradually peaked 10 years later. In both sexes, the SEI peak preceded peak incidence.ConclusionSEI in lung and UADT cancers vary greatly by age, tumour subtype and sex; these variations are likely to largely reflect differences between the sexes in risk behaviours which vary by birth cohort and are socioeconomically patterned.     

Activity-dependent heteromerization of the hyperpolarization-activated, cyclic-nucleotide gated (HCN) channels: role of N-linked glycosylation

Formation of heteromeric complexes of ion channels via co-assembly of different subunit isoforms provides an important mechanism for enhanced channel diversity. We have previously demonstrated co-association of the hyperpolarization activated cyclic-nucleotide gated (HCN1/HCN2) channel isoforms that was regulated by network (seizure) activity in developing hippocampus. However, the mechanisms that underlie this augmented expression of heteromeric complexes have remained unknown. Glycosylation of the HCN channels has been implicated in the stabilization and membrane expression of heteromeric HCN1/HCN2 constructs in heterologous systems. Therefore, we used in vivo and in vitro systems to test the hypothesis that activity modifies HCN1/HCN2 heteromerization in neurons by modulating the glycosylation state of the channel molecules. Seizure-like activity (SA) increased HCN1/HCN2 heteromerization in hippocampus in vivo as well as in hippocampal organotypic slice cultures. This activity increased the abundance of glycosylated HCN1 but not HCN2-channel molecules. In addition, glycosylated HCN1 channels were preferentially co-immunoprecipitated with the HCN2 isoforms. Provoking SA in vitro in the presence of the N-linked glycosylation blocker tunicamycin abrogated the activity-dependent increase of HCN1/HCN2 heteromerization. Thus, hippocampal HCN1 molecules have a significantly higher probability of being glycosylated after SA, and this might promote a stable heteromerization with HCN2.     

Two variable active site residues modulate response regulator phosphoryl group stability

Many signal transduction networks control their output by switching regulatory elements on or off. To synchronize biological response with environmental stimulus, switching kinetics must be faster than changes in input. Two-component regulatory systems (used for signal transduction by bacteria, archaea and eukaryotes) switch via phosphorylation or dephosphorylation of the receiver domain in response regulator proteins. Although receiver domains share conserved active site residues and similar three-dimensional structures, rates of self-catalysed dephosphorylation span a >or= 40,000-fold range in response regulators that control diverse biological processes. For example, autodephosphorylation of the chemotaxis response regulator CheY is 640-fold faster than Spo0F, which controls sporulation. Here we demonstrate that substitutions at two variable active site positions decreased CheY autodephosphorylation up to 40-fold and increased the Spo0F rate up to 110-fold. Particular amino acids had qualitatively similar effects in different response regulators. However, mutant proteins matched to other response regulators at the two key variable positions did not always exhibit similar autodephosphorylation kinetics. Therefore, unknown factors also influence absolute rates. Understanding the effects that particular active site amino acid compositions have on autodephosphorylation rate may allow manipulation of phosphoryl group stability for useful purposes, as well as prediction of signal transduction kinetics from amino acid sequence.