Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene.

The amplification refractory mutation system (ARMS) is a simple, rapid and reliable method for the detection of any mutation involving single base changes or small deletions. We have applied ARMS methodology to the detection of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Single ARMS tests have been developed for 11 CFTR mutations found in the northwest of England. ARMS reactions for the most common mutations have been multiplexed to give a test which will detect the presence of the delta F508, G551D, G542X, and 621 + 1G----T mutations in a DNA sample. The multiplex test has been validated by the analysis of over 500 previously genotyped samples and has been found to be completely accurate. The rapid detection of the most common mutations has enabled early molecular confirmation of suspected cystic fibrosis in neonates, rapid typing of cystic fibrosis patients and their relatives, and testing of sperm and egg donors.     

The topoisomerase II inhibitor teniposide (VM-26) induces apoptosis in unstimulated mature murine lymphocytes.

This study shows that not only concanavalin A-stimulated proliferating lymphocytes but also unstimulated mouse splenic lymphocytes are sensitive to the topoisomerase II (topo II) inhibitor teniposide (VM-26). When unstimulated lymphocytes are pretreated with VM-26 for a 2-h period and are then incubated in drug-free medium, cell viability, as determined by trypan blue exclusion, decreases to 40% of the control by 6 h. The drug-treated cultures show two to three times the level of detergent soluble DNA than the control cultures and agarose gel electrophoresis of the soluble DNA shows the presence of oligonucleosomal-sized fragments, a feature considered to be a hallmark of apoptosis. Phase contrast microscopy, Hoechst staining for DNA, and immunofluorescence microscopy of various nuclear and cytoplasmic antigens (nucleolar fibrillarin, snRNP, ubiquitin, vimentin, tubulin) in the VM-26-treated cells characterize the morphological changes during apoptosis of these cells. The role of topo II as the mediator of the VM-26 effects is supported by pulsed field gel electrophoresis, which shows the typical topo II-induced cleavage of supercoiled DNA into loop-sized 300- and 50-kbp fragments. We conclude that the cancer chemotherapeutic agent VM-26 interacts with topo II and induces apoptosis in unstimulated lymphocytes.     

In vitro fertilisation in a small unit in the NHS

In vitro fertilisation is one of the most effective new treatments for infertility, but financial restrictions have made it impossible for it to be widely carried out in the National Health Service. We report on the establishment of a small, largely self funded, unit that was set up with the help of the local health service management. All cycles are programmed so that most work is carried out during the working week; oocyte recoveries are performed as outpatient procedures without general anaesthesia and guided by ultrasound. Roughly a tenth of treatment cycles and roughly a fifth of embryo transfers resulted in a clinical pregnancy.     

Whole-Catchment Manipulations of Internal and External Loading Reveal the Sensitivity of a Century-Old Reservoir to Hypoxia

Climate change is predicted to have widespread impacts on freshwater lake and reservoir nutrient budgets by altering both hypolimnetic hypoxia and runoff, which will in turn alter the magnitude of internal and external nutrient loads. To examine the effects of these potential climate scenarios on nitrogen (N) and phosphorus (P) budgets, we conducted a whole-catchment manipulation of hypolimnetic oxygen conditions and external loads to Falling Creek Reservoir (FCR), an old, eutrophic reservoir in a reforested catchment with a history of agricultural land use. Throughout 2 years of monitoring, internal N and P loading during hypoxic conditions dominated the hypolimnetic mass of nutrients in FCR, regardless of changes in external loading. FCR commonly functioned as a net sink of N and P, except during hypoxic conditions, when the reservoir was a net source of ammonium (\( {\text{NH}}_{4}^{ + } \)) to downstream. We observed extremely high nitrate–nitrite (\( {\text{NO}}_{3}^{ - } {-}{\text{NO}}_{2}^{ - } \)), soluble reactive P (SRP), total nitrogen (TN), and total phosphorus (TP) retention rates, indicating that the reservoir served as a sink for greater than 70% of \( {\text{NO}}_{3}^{ - } {-}{\text{NO}}_{2}^{ - } \) inputs and greater than 30% of SRP, TN, and TP inputs, on average. Our study is notable in the length of time since reforestation (>80 years) that a reservoir is still exhibiting high N and P internal loading during hypoxia, potentially as a result of the considerable store of accumulated nutrients in its sediment from historical agricultural runoff. Our whole-catchment manipulations highlight the importance of understanding how multiple aspects of global change, waterbody and catchment characteristics, and land use history will interact to alter nutrient budgets in the future.     

Technological Analysis of the World’s Earliest Shamanic Costume: A Multi-Scalar,Experimental Study of a Red Deer Headdress from the Early Holocene Site of Star Carr,North Yorkshire,UK

Shamanic belief systems represent the first form of religious practice visible within the global archaeological record. Here we report on the earliest known evidence of shamanic costume: modified red deer crania headdresses from the Early Holocene site of Star Carr (c. 11 kya). More than 90% of the examples from prehistoric Europe come from this one site, establishing it as a place of outstanding shamanistic/cosmological significance. Our work, involving a programme of experimental replication, analysis of macroscopic traces, organic residue analysis and 3D image acquisition, metrology and visualisation, represents the first attempt to understand the manufacturing processes used to create these artefacts. The results produced were unexpected—rather than being carefully crafted objects, elements of their production can only be described as expedient.     

Identification of Molecular Markers of Delayed Graft Function Based on the Regulation of Biological Ageing

IntroductionDelayed graft function is a prevalent clinical problem in renal transplantation for which there is no objective system to predict occurrence in advance. It can result in a significant increase in the necessity for hospitalisation post-transplant and is a significant risk factor for other post-transplant complications.MethodologyThe importance of microRNAs (miRNAs), a specific subclass of small RNA, have been clearly demonstrated to influence many pathways in health and disease. To investigate the influence of miRNAs on renal allograft performance post-transplant, the expression of a panel of miRNAs in pre-transplant renal biopsies was measured using qPCR. Expression was then related to clinical parameters and outcomes in two independent renal transplant cohorts.ResultsHere we demonstrate, in two independent cohorts of pre-implantation human renal allograft biopsies, that a novel pre-transplant renal performance scoring system (GRPSS), can determine the occurrence of DGF with a high sensitivity (>90%) and specificity (>60%) for donor allografts pre-transplant, using just three senescence associated microRNAs combined with donor age and type of organ donation.ConclusionThese results demonstrate a relationship between pre-transplant microRNA expression levels, cellular biological ageing pathways and clinical outcomes for renal transplantation. They provide for a simple, rapid quantitative molecular pre-transplant assay to determine post-transplant allograft function and scope for future intervention. Furthermore, these results demonstrate the involvement of senescence pathways in ischaemic injury during the organ transplantation process and an indication of accelerated bio-ageing as a consequence of both warm and cold ischaemia.     

The Relationship between Body Mass Index and Post-Cessation Weight Gain in the Year after Quitting Smoking: A Cross-Sectional Study

Introduction

There is wide variability in the amount of weight gained when quitting smoking, but little is known about key predictors of weight gain. We examined the impact of body mass index (BMI) category and sociodemographic variables on post-cessation weight gain.

Materials and Methods

We utilized National Health and Nutrition Examination Survey data from five consecutive cycles of data collection from 2003–2004 to 2011–2012 to estimate post-cessation weight gain by BMI category among recent quitters (n = 654). We analyzed data on their “current weight” and their “past year weight”. We also compared the recent quitters with current smokers, in order to estimate the amount of weight that could be attributed to quitting smoking.

Results

Recent quitters gained 1.4 kg (95% CI: 0.8 to 2.0), while current smokers had a non-significant weight change (-0.01 kg (95% CI: -0.3 to 0.2). Weight gain was significant for those in the normal weight (3.1 kg, 95% CI: 2.3 to 3.9) and overweight BMI categories (2.2 kg, 95% CI: 1.1 to 3.2).

Conclusions

BMI category is a key factor in the extent of post-cessation weight gain, with normal and overweight recent quitters gaining significant amounts of weight.