Development and validation of an approach to produce large‐scale quantities of CpG‐methylated plasmid DNA

The prokaryotic CpG‐specific DNA methylase from Spiroplasma, SssI methylase, has been extensively used to methylate plasmid DNA in vitro to investigate the effects of methylation in vertebrate systems. Currently available methods to produce CpG‐methylated plasmid DNA have certain limitations and cannot generate large quantities of methylated DNA without cost or problems of purity. Here we describe an approach in which the SssI methylase gene has been introduced into the Escherichia coli bacterial genome under the control of an inducible promoter. Plasmid DNA propagated in this bacterium under conditions which induce the methylase gene result in significant (> 90%) CpG methylation. Methylated DNA produced by this approach behaves similarly to methylated DNA produced in vitro using the purified methylase. The approach is scalable allowing for the production of milligram quantities of methylated plasmid DNA.     

Cell-free co-expression of functional membrane proteins and apolipoprotein, forming soluble nanolipoprotein particles

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Delta1-49 apolipoprotein A-I fragment (Delta49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR --> M transition. Importantly the functional bR was solubilized in discoidal bR.NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Delta49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.     

The inhibitory role of stromal cell mesenchyme on human embryonic stem cell hepatocyte differentiation is overcome by Wnt3a treatment

Pluripotent stem cells are derived from the inner cell mass of preimplantation embryos, and display the ability of the embryonic founder cells by forming all three germ lineages in vitro. It is well established that the cellular niche plays an important role in stem cell maintenance and differentiation. Stem cells generally have limited function without the specialized microenvironment of the niche that provides key cell-cell contact, soluble mediators, and extracellular matrices. We were interested in the role that Wnt signaling, in particular Wnt3a, played in human embryonic stem cell (hESC) differentiation to hepatic endoderm in vitro. hESC differentiation to hepatic endoderm was efficient in pure stem cell populations. However, in younger hESC lines, generating stromal cell mesenchyme, our model was very inefficient. The negative effect of stroma could be reversed by pretreating hESCs with Wnt3a prior to the onset of hepatocyte differentiation. Wnt3a pretreatment reinstated efficient hESC differentiation to hepatic endoderm. These studies represent an important step in understanding hepatocyte differentiation from hESCs and the role played by the cellular niche in vitro.     

Vulnerability to winter mortality in elderly people in Britain: population based study

Objective To examine the determinants of vulnerability to winter mortality in elderly British people.Design Population based cohort study (119 389 person years of follow up).Setting 106 general practices from the Medical Research Council trial of assessment and management of older people in Britain.Participants People aged ≥ 75 years.Main outcome measures Mortality (10 123 deaths) determined by follow up through the Office for National Statistics.Results Month to month variation accounted for 17% of annual all cause mortality, but only 7.8% after adjustment for temperature. The overall winter:non-winter rate ratio was 1.31 (95% confidence interval 1.26 to 1.36). There was little evidence that this ratio varied by geographical region, age, or any of the personal, socioeconomic, or clinical factors examined, with two exceptions: after adjustment for all major covariates the winter:non-winter ratio in women compared with men was 1.11 (1.00 to 1.23), and those with a self reported history of respiratory illness had a winter:non-winter ratio of 1.20 (1.08 to 1.34) times that of people without a history of respiratory illness. There was no evidence that socioeconomic deprivation or self reported financial worries were predictive of winter death.Conclusion Except for female sex and pre-existing respiratory illness, there was little evidence for vulnerability to winter death associated with factors thought to lead to vulnerability. The lack of socioeconomic gradient suggests that policies aimed at relief of fuel poverty may need to be supplemented by additional measures to tackle the burden of excess winter deaths in elderly people.     

Effect of influenza vaccination on excess deaths occurring during periods of high circulation of influenza: cohort study in elderly people

Objective To estimate the protection against death provided by vaccination against influenza.Design Prospective cohort follow up supplemented by weekly national counts of influenza confirmed in the community.Setting Primary care.Participants 24 535 patients aged over 75 years from 73 general practices in Great Britain.Main outcome measure Death.Results In unvaccinated members of the cohort daily all cause mortality was strongly associated with an index of influenza circulating in the population (mortality ratio 1.16, 95% confidence interval 1.04 to 1.29 at 90th centile of circulating influenza). The association was strongest for respiratory deaths but was also present for cardiovascular deaths. In contrast, in vaccinated people mortality from any cause was not associated with circulating influenza. The difference in patterns between vaccinated and unvaccinated people could not easily be due to chance (P = 0.02, all causes).Conclusions This study, using a novel and robust approach to control for confounding, provides robust evidence of a protective effect on mortality of vaccination against influenza.     

Sleep and circadian rhythms in spousally bereaved seniors

A laboratory study of sleep and circadian rhythms was undertaken in 28 spousally bereaved seniors (> or =60 yrs) at least four months after the loss event. Measures taken included two nights of polysomnography (second night used), approximately 36 h of continuous core body temperature monitoring, and four assessments of mood and alertness throughout a day. Preceding the laboratory study, two-week diaries were completed, allowing the assessment of lifestyle regularity using the 17-item Social Rhythm Metric (SRM) and the timing of sleep using the Pittsburgh Sleep Diary (PghSD). Also completed were questionnaires assessing level of grief (Texas Revised Inventory of Grief [TRIG] and Index of Complicated Grief [ICG]), subjective sleep quality (Pittsburgh Sleep Quality Index [PSQI]), morningness-eveningness (Composite Scale of Morningness [CSM]), and clinical interview yielding a Hamilton Depression Rating Scale (HDRS) score. Grief was still present, as indicated by an average TRIG score of about 60. On average, the bereaved seniors habitually slept between approximately 23:00 and approximately 06:40 h, achieving approximately 6 h of sleep with a sleep efficiency of approximately 80%. They took about 30 min to fall asleep, and had their first REM episode after 75 min. About 20% of their sleep was in Stage REM, and about 3% in Stages 3 or 4 (slow wave sleep). Their mean PSQI score was 6.4. Their circadian temperature rhythms showed the usual classic shape with a trough at approximately 01:00 h, a fairly steep rise through the morning hours, and a more gradual rise to mid-evening, with an amplitude of approximately 0.8 degrees C. In terms of lifestyle regularity, the mean regularity (SRM) score was 3.65 (slightly lower than that usually seen in seniors). Mood and alertness showed time-of-day variation with peak alertness in the late morning and peak mood in the afternoon. Correlations between outcome sleep/circadian variables and level of grief (TRIG score) were calculated; there was a slight trend for higher grief to be associated with less time spent asleep (p=0.07) and reduced alertness at 20:00 h (p=0.05). Depression score was not correlated with TRIG score (p>0.20). When subjects were divided into groups by the nature of their late spouse's death (expected/after a long-term chronic illness [n=18] versus unexpected [n=10]), no differences emerged in any of the variables. In conclusion, when studied at least four months after the loss event, there appears to be some sleep disruption in spousally bereaved seniors. However, this disruption does not appear to be due to bereavement-related disruptions in the circadian system.     

The dichotomy in the DNA-binding behaviour of ruthenium(II) complexes bearing benzoxazole and benzothiazole groups

The substituted tris(bipyridine)ruthenium(II) complexes {[Ru(bpy)(2)(4,4'-bbob)](2+) and [Ru(bpy)(2)(5,5'-bbob)](2+) [where bpy=2,2'-bipyridine and bbob=bis(benzoxazol-2-yl)-2,2'-bipyridine] have been prepared and compared to the previously studied complex [Ru(bpy)(2)(4,4'-bbtb)](2+) [where bbtb=bis(benzothiazol-2-yl)-2,2'-bipyridine]. From the UV/VIS titration studies, Delta-[Ru(bpy)(2)(4,4'-bbob)](2+) displays a stronger association than the Lambda-isomer with calf-thymus DNA (ct-DNA). For [Ru(bpy)(2)(5,5'-bbob)](2+), there appears to be minimal interaction with ct-DNA. The results of fluorescence titration studies suggest that [Ru(bpy)(2)(4,4'-bbob)](2+) gives an increase in emission intensity with increasing ct-DNA concentrations, with an enantiopreference for the Delta isomer, confirmed by membrane dialysis studies. The fluorescent intercalation displacement studies revealed that [Ru(bpy)(2)(4,4'-bbob)](2+) and [Ru(bpy)(2)(5,5'-bbob)](2+) display a preference for more open DNA structures such as bulge and hairpin sequences. While Lambda-[Ru(bpy)(2)(4,4'-bbtb)](2+) has shown the most significant affinity for all the oligonucleotides sequences screened in previous studies, it is the Delta isomer of the comparable benzoxazole ruthenium(II) complex (Delta-[Ru(bpy)(2)(4,4'-bbob)](2+)) that preferentially binds to DNA.     

Effects of vibratory microscreening on proximate composition and recovery of poultry processing wastewater particulate matter

Experiments were conducted to compare the effects of tertiary microscreen gap size on the proximate composition and rate of recovery of particulate matter from poultry processing wastewater (PPW). A high-speed vibratory screen was installed within the wastewater treatment area of a southeast US broiler slaughter plant after the existing primary and secondary mechanical rotary screens. Microscreen panels with nominal gap size openings of 212, 106 and 45mum were investigated. The particulate matter samples recovered were subjected to proximate analysis to determine percent moisture, fat, protein, crude fiber and ash. The average percent wet weight moisture (%WW) content for all samples was 79.1. The average percent dry matter (%DM) fat, protein, crude fiber and ash were 63.5, 17.5, 4.8 and 1.5, respectively. The mean concentration of total solids (TS) recovered from all microscreen runs was 668mg/L, which represents a potential additional daily offal recovery rate of 12.1metric tons (MT) per 3.78 million L (1.0 million gallons US) of PPW. There was no significant difference in the performance of the three microscreen gap sizes with regard to proximate composition or mass of particulate matter recovered.