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

Transcriptomic, lipid, and histological profiles suggest changes in health in fish from a pesticide hot spot.

Barramundi (Lates calcarifer) were collected at the beginning (1st sampling) and end (2nd sampling) of the wet season from Sandy Creek, an agriculturally impacted catchment in the Mackay Whitsundays region of the Great Barrier Reef catchment area, and from Repulse Creek, located approximately 100 km north in Conway National Park, to assess the impacts of pesticide exposure. Gill and liver histology, lipid class composition in muscle, and the hepatic transcriptome were examined. The first sample of Repulse Creek fish showed little tissue damage and low transcript levels of xenobiotic metabolism enzymes. Sandy Creek fish showed altered transcriptomic patterns, including those that regulate lipid metabolism, xenobiotic metabolism, and immune response; gross histological alterations including lipidosis; and differences in some lipid classes. The second sampling of Repulse Creek fish showed similar alterations in hepatic transcriptome and tissue structure as fish from Sandy Creek. These changes may indicate a decrease in health of pesticide exposed fish

Enhancing Lay Counselor Capacity to Improve Patient Outcomes with Multimedia Technology

Multimedia technologies offer powerful tools to increase capacity of health workers to deliver standardized, effective, and engaging antiretroviral medication adherence counseling. Masivukeni—is an innovative multimedia-based, computer-driven, lay counselor-delivered intervention designed to help people living with HIV in resource-limited settings achieve optimal adherence. This pilot study examined medication adherence and key psychosocial outcomes among 55 non-adherent South African HIV+ patients, on antiretroviral therapy (ART) for at least 6 months, who were randomized to receive either Masivukeni or standard of care (SOC) counseling for ART non-adherence. At baseline, there were no significant differences between the SOC and Masivukeni groups on any outcome variables. At post-intervention (approximately 5–6 weeks after baseline), -clinic-based pill count adherence data available for 20 participants (10 per intervention arm) showed a 10 % improvement for—participants and a decrease of 8 % for SOC participants. Masivukeni participants reported significantly more positive attitudes towards disclosure and medication social support, less social rejection, and better clinic–patient relationships than did SOC participants. Masivukeni shows promise to promote optimal adherence and provides preliminary evidence that multimedia, computer-based technology can help lay counselors offer better adherence counseling than standard approaches

Masivukeni: Development of a Multimedia Based Antiretroviral Therapy Adherence Intervention for Counselors and Patients in South Africa

Effective medical treatment for HIV/AIDS requires patients’ optimal adherence to antiretroviral therapy (ART). In resource-constrained settings, lack of adequate standardized counseling for patients on ART remains a significant barrier to adherence. Masivukeni (“Let’s Wake Up” in Xhosa) is an innovative multimedia-based intervention designed to help people living with HIV in resource-limited settings achieve and maintain high levels of ART adherence. Adapted from a couples-based intervention tested in the United States (US), Masivukeni was developed through community-based participatory research with US and South African partners and informed by Ewart’s Social Action Theory. Innovative computer-based multimedia strategies were used to translate a labor- and training-intensive intervention into one that could be readily and widely used by lay counselors with relatively little training with low-literacy patients. In this paper, we describe the foundations of this new intervention, the process of its development, and the evidence of its high acceptability and feasibility

Using Sequence Similarity Networks for Visualization of Relationships Across Diverse Protein Superfamilies

The dramatic increase in heterogeneous types of biological data—in particular, the abundance of new protein sequences—requires fast and user-friendly methods for organizing this information in a way that enables functional inference. The most widely used strategy to link sequence or structure to function, homology-based function prediction, relies on the fundamental assumption that sequence or structural similarity implies functional similarity. New tools that extend this approach are still urgently needed to associate sequence data with biological information in ways that accommodate the real complexity of the problem, while being accessible to experimental as well as computational biologists. To address this, we have examined the application of sequence similarity networks for visualizing functional trends across protein superfamilies from the context of sequence similarity. Using three large groups of homologous proteins of varying types of structural and functional diversity—GPCRs and kinases from humans, and the crotonase superfamily of enzymes—we show that overlaying networks with orthogonal information is a powerful approach for observing functional themes and revealing outliers. In comparison to other primary methods, networks provide both a good representation of group-wise sequence similarity relationships and a strong visual and quantitative correlation with phylogenetic trees, while enabling analysis and visualization of much larger sets of sequences than trees or multiple sequence alignments can easily accommodate. We also define important limitations and caveats in the application of these networks. As a broadly accessible and effective tool for the exploration of protein superfamilies, sequence similarity networks show great potential for generating testable hypotheses about protein structure-function relationships

Disorders of sex development : insights from targeted gene sequencing of a large international patient cohort

Background: Disorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously. Results: We analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46, XY DSD and 48 with 46, XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46, XY DSD. In patients with 46, XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management. Conclusions: Our massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field