A sensitive switch for visualizing natural gene silencing in single cells

RNA interference is a natural gene expression silencing system that appears throughout the tree of life. As the list of cellular processes linked to RNAi grows, so does the demand for tools to accurately measure RNAi dynamics in living cells. We engineered a synthetic RNAi sensor that converts this negative regulatory signal into a positive output in living mammalian cells thereby allowing increased sensitivity and activation. Furthermore, the circuit's modular design allows potentially any microRNA of interest to be detected. We demonstrated that the circuit responds to an artificial microRNA and becomes activated when the RNAi target is replaced by a natural microRNA target (miR-34) in U2OS osteosarcoma cells. Our studies extend the application of rationally designed synthetic switches to RNAi, providing a sensitive way to visualize the dynamics of RNAi activity rather than just the presence of miRNA molecules.     

Can physical activity improve the mental health of older adults?

The world population is aging rapidly. Whilst this dramatic demographic change is a desirable and welcome phenomenon, particularly in view of people's increasing longevity, it's social, financial and health consequences can not be ignored. In addition to an increase of many age related physical illnesses, this demographic change will also lead to an increase of a number of mental health problems in older adults and in particular of dementia and depression. Therefore, any health promotion approach that could facilitate introduction of effective primary, secondary and even tertiary prevention strategies in old age psychiatry would be of significant importance. This paper explores physical activity as one of possible health promotion strategies and evaluates the existing evidence that supports its positive effect on cognitive impairment and depression in later life.     

Improving the health behaviours of elderly people: randomised controlled trial of a general practice education programme

ObjectivesTo establish the effect of an educational intervention for general practitioners on the health behaviours and wellbeing of elderly patients.DesignRandomised controlled trial with 1 year follow up.SettingMetropolitan general practices in Melbourne, Australia.Subjects42 general practitioners and 267 of their patients aged over 65 years.InterventionEducational and clinical practice audit programme for general practitioners on health promotion for elderly people.ResultsPatients in the intervention group had increased (a) walking by an average of 88 minutes per fortnight, (b) frequency of pleasurable activities, and (c) self rated health compared with the control group. No change was seen in drug usage, rate of influenza vaccination, functional status, or psychological wellbeing as a result of the intervention. Extrapolations of the known effect of these changes in behaviour suggest mortality could be reduced by 22% if activity was sustained for 5 years.ConclusionsEducation of the general practitioners had a positive effect on health outcomes of their elderly patients. General practitioners may have considerable public health impact in promotion of health for elderly patients.

Key messages

• Few educational interventions for doctors have shown benefit to the health of patients
• Elderly people were identified in the UK health initiatives as in need of additional attention, and levels of health protective behaviours were low in community surveys
• A multifaceted educational intervention for general practitioners was effective in improving walking behaviour, self rated health status, and the frequency of social contacts in elderly patients
• General practitioners are effective in improving health and health behaviours in their elderly patients

     

Association between depression and hospital outcomes among older men

Background:

Studies that have investigated the relation between depression and the type, nature, extent and outcome of general hospital admissions have been limited by their retrospective designs and focus on specific clinical populations. We explored this relation prospectively in a large, community-based sample of older men.

Methods:

A cohort of 5411 men aged 69 years and older enrolled in the Health in Men Study was assessed at baseline for depressive symptoms, defined as a score of 7 or higher on the 15-item Geriatric Depression Scale. Participants were followed for 2 years for occurrence and number of hospital admissions, type of hospital admission, length of hospital stay and inpatient death as recorded in the Western Australian Data Linkage System.

Results:

Of 339 men with depressive symptoms, 152 (44.8%) had at least 1 emergency hospital admission, compared with 1164 of 5072 (22.9%) nondepressed men (p < 0.001). In multivariate analyses, the presence of depressive symptoms was a significant independent predictor of hospital admission (hazard ratio 1.67, 95% confidence interval [CI] 1.38–2.01), number of hospital admissions (incidence rate ratio [IRR] 1.22, 95% CI 1.07–1.39) and total length of hospital stay (IRR 1.65, 95% CI 1.36–2.01).

Interpretation:

Participants with depressive symptoms were at higher risk of hospital admission for nonpsychiatric conditions and were more likely to have longer hospital stays and worse hospital outcomes, compared with nondepressed participants. These results highlight the potential to target this high-risk group to reduce the burden of health care costs in an aging population.Older people are the most frequent users of health services, and the progressive aging of the world’s population may lead to a saturation of available services. Therefore, we must find ways to reduce preventable admissions to hospital and uncover the factors associated with potentially preventable use of health services. An association between depression and hospital admission for nonpsychiatric conditions has been postulated, although the data have been limited to specific clinical populations and the interpretation of the results hampered by the retrospective study design and the use of self-reported outcomes.^1–8 Consequently, these findings cannot be easily generalized or used to develop data-driven interventions.We addressed this gap in the literature by using a community-based population survey with prospective data linkage to measure important health-related outcomes. Our main objective was to investigate whether community-dwelling older men with depressive symptoms were more likely than nondepressed men to be admitted to general hospitals. Our other aims were to determine whether the long-term clinical outcomes of these 2 groups differed in relation to the number of future hospital admissions, length of hospital stay and inpatient deaths.     

Sensitive high-throughput screening for the detection of reducing sugars

The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 μM. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8%. Furthermore, the assay was combined with a microscale cultivation procedure for the activity-based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei β-mannanase, or T. reesei cellobiohydrolase 2.     

MMP-9, TIMP-1 and inflammatory cells in sputum from COPD patients during exacerbation

Background

Irreversible airflow obstruction in Chronic Obstructive Pulmonary Disease (COPD) is thought to result from airway remodelling associated with aberrant inflammation. Patients who experience frequent episodes of acute deterioration in symptoms and lung function, termed exacerbations, experience a faster decline in their lung function, and thus over time greater disease severity However the mechanisms by which these episodes may contribute to decreased lung function are poorly understood.This study has prospectively examined changes in sputum levels of inflammatory cells, MMP-9 and TIMP-1 during exacerbations comparing with paired samples taken prior to exacerbation.

Methods

Nineteen COPD patients ((median, [IQR]) age 69 [63 to 74], forced expiratory volume in one second (FEV1) 1.0 [0.9 to1.2], FEV1% predicted 37.6 [27.3 to 46.2]) provided sputa at exacerbation. Of these, 12 were paired with a samples collected when the patient was stable, a median 4 months [2 to 8 months] beforehand.

Results

MMP-9 levels increased from 10.5 μg/g [1.2 to 21.1] prior to exacerbation to 17.1 μg/g [9.3 to 48.7] during exacerbation (P < 0.01). TIMP-1 levels decreased from 3.5 μg/g [0.6 to 7.8] to 1.5 μg/g [0.3 to 4.9] (P = 0.16). MMP-9/TIMP-1 Molar ratio significantly increased from 0.6 [0.2 to 1.1] to 3.6 [2.0 to 25.3] (P < 0.05). Neutrophil, eosinophil and lymphocyte counts all showed significant increase during exacerbation compared to before (P < 0.05). Macrophage numbers remained level. MMP-9 levels during exacerbation showed highly significant correlation with both neutrophil and lymphocyte counts (Rho = 0.7, P < 0.01).

Conclusion

During exacerbation, increased inflammatory burden coincides with an imbalance of the proteinase MMP-9 and its cognate inhibitor TIMP-1. This may suggest a pathway connecting frequent exacerbations with lung function decline.     

Immunochemical identity of peroxisomal enoyl-CoA hydratase with the peroxisome-proliferation -associated 80,000 mol wt polypeptide in rat liver

Peroxisome proliferators, which induce proliferation of hepatic peroxisomes, have been shown previously to cause a marked increase in an 80,000 mol wt polypeptide predominantly in the light mitochondrial and microsomal fractions of liver of rodents. We now present evidence to show that this hepatic peroxisome-proliferation-associated polypeptide, referred to as polypeptide PPA-80, is immunochemically identical with the multifunctional peroxisome protein displaying heat-labile enoyl-CoA hydratase activity. This conclusion is based on the following observations: (a) the purified polypeptide PPA-80 and the heat- labile enoyl-CoA hydratase from livers of rats treated with the peroxisome proliferators Wy-14,643 {[4-chloro-6(2,3-xylidino)-2-pyrimidinylthio]acetic acid} exhibit identical minimum molecular weights of approximately 80,000 on SDS polyacrylamide gel electrophoresis; (b) these two proteins are immunochemically identical on the basis of ouchterlony double diffusion, immunotitration, rocket immunoelectrophoresis, and crossed immunoelectrophoresis analysis; and (c) the immunoprecipitates formed by antibodies to polypeptide PPA-80 when dissociated on a sephadex G-200 column yield enoyl-CoA hydratase activity. Whether the polypeptide PPA-80 exhibits the activity of other enzyme(s) of the peroxisomal β-oxidation system such as fatty acyl-CoA oxidase activity or displays immunochemical identity with such enzymes remains to be determined. The availability of antibodies to polypeptide PPA-80 and enoyl-CoA hydratase facilitated immunofluorescent and immunocytochemical localization of the polypeptide PPA- 80 and enoyl-CoA hydratase in the rat liver. The indirect immunofluorescent studies with these antibodies provided direct visual evidence for the marked induction of polypeptide PPA-80 and enoyl-CoA hydratase in the livers of rats treated with Wy-14,643. The present studies also provide immunocytochemical evidence for the localization of polypeptide PPA- 80 and the heat-labile enoyl-CoA hydratase in the peroxisome, but not in the mitochondria, of hepatic parenchymal cells. These studies, therefore, provide morphological evidence for the existence of fatty acyl-CoA oxidizing system in peroxisomes. An increase of polypeptide PPA-80 on SDS polyacrylamide gel electrophoretic analysis of the subcellular fractions of liver of rodents treated with lipid-lowering drugs should serve as a reliable and sensitive indicator of enhanced peroxisomal β- oxidation system.     

A change in the twist of the actin-containing filaments occurs during the extension of the acrosomal process in Limulus sperm

The spectacular extension of the acrosomal process in Limulus sperm is effected by a bundle of actin-containing filaments with apparently no contribution from myosin. The bundle is coiled about the base of the sperm and, upon reaction, unwinds and extends out of the anterior end of the sperm with a screwing motion. We have analyzed the structure of the bundle in the coil and following its discharge. Optical diffraction studies of electron micrographs show a difference in the twist of the filaments in the two forms. The filaments in the coil have a twist of 0.23 ° per subunit more than that in the true discharge. As the signal to extend moves down the coil, the filaments change their twist and the bundle straightens. The coupling of these two movements produces the screwing motion. In the coil, the filaments wind around the axis of the bundle. As the filaments change their twist, the winding is undone. From freeze-fracture replicas we determined the hand of the winding of filaments in the coil and, in thin sections, we were able to determine the number of turns the filaments make for each loop of the coil. From these data we were able to predict the hand and amount of rotation during the discharge. From movie film sequences we could determine only the amount of rotation and found it to be 0.25 ° ± 0.05 ° per subunit discharged. This is in reasonable agreement with the expected value of 0.23 ° ± 0.05 ° per subunit. We propose that it is the change in twist of the actin filaments themselves that is responsible for the generation of force for the extension of the acrosomal process.