Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support

The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs). Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40–70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β₁-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β₁-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM) micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes.     

On the Use of Biomineral Oxygen Isotope Data to Identify Human Migrants in the Archaeological Record: Intra-Sample Variation,Statistical Methods and Geographical Considerations

Oxygen isotope analysis of archaeological skeletal remains is an increasingly popular tool to study past human migrations. It is based on the assumption that human body chemistry preserves the δ¹⁸O of precipitation in such a way as to be a useful technique for identifying migrants and, potentially, their homelands. In this study, the first such global survey, we draw on published human tooth enamel and bone bioapatite data to explore the validity of using oxygen isotope analyses to identify migrants in the archaeological record. We use human δ¹⁸O results to show that there are large variations in human oxygen isotope values within a population sample. This may relate to physiological factors influencing the preservation of the primary isotope signal, or due to human activities (such as brewing, boiling, stewing, differential access to water sources and so on) causing variation in ingested water and food isotope values. We compare the number of outliers identified using various statistical methods. We determine that the most appropriate method for identifying migrants is dependent on the data but is likely to be the IQR or median absolute deviation from the median under most archaeological circumstances. Finally, through a spatial assessment of the dataset, we show that the degree of overlap in human isotope values from different locations across Europe is such that identifying individuals’ homelands on the basis of oxygen isotope analysis alone is not possible for the regions analysed to date. Oxygen isotope analysis is a valid method for identifying first-generation migrants from an archaeological site when used appropriately, however it is difficult to identify migrants using statistical methods for a sample size of less than c. 25 individuals. In the absence of local previous analyses, each sample should be treated as an individual dataset and statistical techniques can be used to identify migrants, but in most cases pinpointing a specific homeland should not be attempted.     

Using bioacoustics to examine shifts in songbird phenology

Monitoring patterns in biodiversity and phenology have become increasingly important given accelerating levels of anthropogenic change. Long‐term monitoring programs have reported earlier occurrence of spring activity, reflecting species response to climate change. Although tracking shifts in spring migration represents a valuable approach to monitoring community‐level consequences of climate change, robust long‐term observations are challenging and costly. Audio recordings and metrics of bioacoustic activity could provide an effective method for monitoring changes in songbird activity and broader biotic interactions. We used 3 years of spring and fall recordings at six sites in Glacier Bay National Park, Alaska, an area experiencing rapid warming and glacial retreat, to examine the utility of bioacoustics to detect changes in songbird phenology. We calculated the Acoustic Complexity Index (ACI), an algorithm representing an index of bird community complexity. Abrupt changes in ACI values from winter to spring corresponded to spring transition, suggesting that ACI may be an effective, albeit coarse metric to detect the arrival of migrating songbirds. The first peak in ACI shifted from April 16 to April 11 from 2012 to 2014. Changes in ACI were less abrupt in the fall due to weather events, suggesting spring recordings are better suited to indicate phenology. To ensure changes in ACI values were detecting real changes in songbird activity, we explored the relationship between ACI and song of three species: varied thrush   (Ixoreus naevius), Pacific wren (Troglodytes pacificus), and ruby‐crowned kinglet (Regulus calendula). ACI was positively related to counts of all species, but most markedly with song of the varied thrush, the most common species in our recordings and a known indicator of forest ecosystem health. We conclude that acoustic recordings paired with bioacoustic indices may be a useful method of monitoring shifts in songbird communities due to climate change and other sources of anthropogenic disturbance.     

Mitigating the Impact of Bats in Historic Churches: The Response of Natterer’s Bats Myotis nattereri to Artificial Roosts and Deterrence

Bats frequently roost in historic churches, and these colonies are of considerable conservation value. Inside churches, bat droppings and urine can cause damage to the historic fabric of the building and to items of cultural significance. In extreme cases, large quantities of droppings can restrict the use of a church for worship and/or other community functions. In the United Kingdom, bats and their roosts are protected by law, and striking a balance between conserving the natural and cultural heritage can be a significant challenge. We investigated mitigation strategies that could be employed in churches and other historic buildings to alleviate problems caused by bats without adversely affecting their welfare or conservation status. We used a combination of artificial roost provision and deterrence at churches in Norfolk, England, where significant maternity colonies of Natterer’s bats Myotis nattereri damage church features. Radio-tracking data and population modelling showed that excluding M. nattereri from churches is likely to have a negative impact on their welfare and conservation status, but that judicious use of deterrents, especially high intensity ultrasound, can mitigate problems caused by bats. We show that deterrence can be used to move bats humanely from specific roosting sites within a church and limit the spread of droppings and urine so that problems to congregations and damage to cultural heritage can be much reduced. In addition, construction of bespoke roost spaces within churches can allow bats to continue to roost within the fabric of the building without flying in the church interior. We highlight that deterrence has the potential to cause serious harm to M. nattereri populations if not used judiciously, and so the effects of deterrents will need careful monitoring, and their use needs strict regulation.     

Rift Valley Fever Vaccine Virus Clone 13 Is Able to Cross the Ovine Placental Barrier Associated with Foetal Infections,Malformations, and Stillbirths

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that affects domesticated ruminants and occasionally humans. Classical RVF vaccines are based on formalin-inactivated virus or the live-attenuated Smithburn strain. The inactivated vaccine is highly safe but requires multiple administrations and yearly re-vaccinations. Although the Smithburn vaccine provides solid protection after a single vaccination, this vaccine is not safe for pregnant animals. An alternative live-attenuated vaccine, named Clone 13, carries a large natural deletion in the NSs gene which encodes the major virulence factor of the virus. The Clone 13 vaccine was previously shown to be safe for young lambs and calves. Moreover, a study in pregnant ewes suggested that the vaccine could also be applied safely during gestation. To anticipate on a possible future incursion of RVFV in Europe, we have evaluated the safety of Clone 13 for young lambs and pregnant ewes. In line with the guidelines from the World Organisation for Animal health (Office International des Epizooties, OIE) and regulations of the European Pharmacopeia (EP), these studies were performed with an overdose. Our studies with lambs showed that Clone 13 dissemination within vaccinated animals is very limited. Moreover, the Clone 13 vaccine virus was not shed nor spread to in-contact sentinels and did not revert to virulence upon animal-to-animal passage. Importantly, a large experiment with pregnant ewes demonstrated that the Clone 13 virus is able to spread to the fetus, resulting in malformations and stillbirths. Altogether, our results suggest that Clone 13 can be applied safely in lambs, but that caution should be taken when Clone 13 is used in pregnant animals, particularly during the first trimester of gestation.     

Cardiac Valve Replacement: The Rationale for Earlier Operation

Cardiac valve replacement (single or double) was carried out in 138 consecutive patients with valvular heart disease over a six-year period at the Wadsworth Veterans Administration Hospital. All but a few had functional class III or IV disease. Hospital mortality was 3.6 percent.Normothermia, coronary perfusion and beating of the heart were maintained throughout the procedure. All patients were followed at least one year and the average follow-up was four and a half years. The late mortality was 27 percent. Seventy-five percent of the late deaths were due to progressive cardiac disease. The mortality due to prosthetic valve dysfunction was 4 percent.Subjective evaluation of survivors revealed that a high percentage remained symptomatic. Only 50 percent of the total group of patients were significantly improved.The high incidence of late mortality and poor long-term functional results are contrasted to the low operative risk and improved reliability of prosthetic valves. The rationale of delaying operation until functional class III limitation develops is questioned. Earlier operation is recommended before irreversible myocardial hypertrophy and fibrosis develop.     

Assessment of physiological integrity of sonicated retinal rod membranes

As the size of rod membrane fragments was reduced by sonication or by addition of the detergent Emulphogene, the intensity of the circular dichroism (CD) bands (210 and 221 nm) increased progressively with a blue shift in position. The intensity of the visible CD bands (340 and 495 nm) was also increased by sonication. Since the intensity increase of the CD bands was related to a reduction in turbidity, the anomalous CD features of intact membranes could be attributed to optical artifacts caused by the particulate nature of the material. Because the magnitude of the CD bands at 221 nm and 340 nm was essentially identical for the sonic suspension and detergent-clarified solution, the adequacy of sonic suspensions can be assured by checking whether detergent affects the intensity of these bands. Suspensions of sonicated rod membranes, purified on Agarose, contained vesicles of 112 nm in average diameter. The morphology and size of the vesicles did not change upon photobleaching of rhodopsin. The vesicles retained such rod membrane properties as conformational insensitivity to photobleaching of the retinal chromophore, thermal stability, and pigment regenerability. Thus, the physiological integrity of rod membranes was maintained by the sonicated vesicles. From the most reliable estimate of the molecular ellipticity at 221 nm, the helical content of membrane-bound rhodopsin was determined to be approximately 47%.     

Resistance to Nocardia brasiliensis infection in mice immunized with either Nocardia or BCG

Different vaccination procedures to increase the mecha nisms of host resistance to Nocardia brasiliensis were studied in mice. When mice were challenged in the footpad, 2×10⁸ N. brasiliensis 20 days after footpad inoculation with either viable or killed N. brasiliensis, the mice demonstrated significant resistance to infection when compared with noninfected and nonimmunized mice. The degree of resistance seems to be correlated with the delayed-type hypersensitivity response in the vaccinated animals. Vaccination with another acid-fast bacilli, BCG, afforded both a mild protection and low DTH reactivity. Antibody levels to Nocardia were similar in either Nocardia- or BCG- treated groups indicating that they do not play an important role in resistance to infection by N. brasiliensis.