Moving out of the shadows: accomplishing bisexual motherhood

Our qualitative study explored the ways in which bisexual mothers came to identify as such and how they structured their relationships and parenting within hetero-patriarchal society. The experiences of seven self-identified White bisexual women (aged from 28 to 56-years-old) from across England and the Republic of Ireland were investigated through semi-structured interviews. Participants’ children were aged 8 months to 28 years old at the time of their interviews. A thematic narrative analysis highlighted the following issues that participants had encountered in constructing their self-identity: prioritizing children; connecting and disconnecting with others and finessing self-definition; questioning societal relationship expectations. Nevertheless, participants varied considerably in how each of the themes identified were reflected in their lives, in particular depending upon each participant’s interpretation of her local social context. Both motherhood and self-identifying as bisexual gave a sense of meaning and purpose to participants’ life stories, although participants sometimes foregrounded their commitment to their children even at a personal cost to their bisexual identity. Using three different theoretical perspectives from feminist theory, queer theory and life course theory, the narratives analysed revealed ways in which bisexual motherhood not only had been influenced both intentionally and unintentionally by heteronormative expectations but also had directly and indirectly challenged these expectations

Interaction of short modified peptides deriving from glycoprotein gp36 of feline immunodeficiency virus with phospholipid membranes

A tryptophan-rich octapeptide, C8 (Ac-Trp-Glu-Asp-Trp-Val-Gly-Trp-Ile-NH2), modelled on the membrane-proximal external region of the feline immunodeficiency virus (FIV) gp36 glycoprotein ectodomain, exhibits potent antiviral activity against FIV. A mechanism has been proposed by which the peptide, being positioned on the surface of the cell membrane, inhibits its fusion with the virus. In the present work, peptide–lipid interactions of C8 with dimyristoyl phosphatidylcholine liposomes are investigated using electron spin resonance spectroscopy of spin-labelled lipids. Three other peptides, obtained from modifications of C8, have also been investigated, in an attempt to clarify the essential molecular features of the interactions involving the tryptophan residues. The results show that C8 adsorbs strongly on the bilayer surface. Membrane binding requires not only the presence of the Trp residues in the sequence, but also their common orientation on one side of the peptide that is engendered by the WX2 WX2 W motif. Membrane interaction correlates closely with peptide antiviral activity, indicating that the membrane is essential in stabilizing the peptide conformation that will be able to inhibit viral infection

Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus

Commercial cultivation of the mushroom fungus, Agaricus bisporus, utilizes a substrate consisting of a lower layer of compost and upper layer of peat. Typically, the two layers are seeded with individual mycelial inoculants representing a single genotype of A. bisporus. Studies aimed at examining the potential of this fungal species as a heterologous protein expression system have revealed unexpected contributions of the mycelial inoculants in the morphogenesis of the fruiting body. These contributions were elucidated using a dual-inoculant method whereby the two layers were differientially inoculated with transgenic β-glucuronidase (GUS) and wild-type (WT) lines. Surprisingly, use of a transgenic GUS line in the lower substrate and a WT line in the upper substrate yielded fruiting bodies expressing GUS activity while lacking the GUS transgene. Results of PCR and RT-PCR analyses for the GUS transgene and RNA transcript, respectively, suggested translocation of the GUS protein from the transgenic mycelium colonizing the lower layer into the fruiting body that developed exclusively from WT mycelium colonizing the upper layer. Effective translocation of the GUS protein depended on the use of a transgenic line in the lower layer in which the GUS gene was controlled by a vegetative mycelium-active promoter (laccase 2 and β-actin), rather than a fruiting body-active promoter (hydrophobin A). GUS-expressing fruiting bodies lacking the GUS gene had a bonafide WT genotype, confirmed by the absence of stably inherited GUS and hygromycin phosphotransferase selectable marker activities in their derived basidiospores and mycelial tissue cultures. Differientially inoculating the two substrate layers with individual lines carrying the GUS gene controlled by different tissue-preferred promoters resulted in up to a ∼3.5-fold increase in GUS activity over that obtained with a single inoculant. Our findings support the existence of a previously undescribed phenomenon of long-distance protein translocation in A. bisporus that has potential application in recombinant protein expression and biotechnological approaches for crop improvement

Impact of Host Genes and Strand Selection on miRNA and miRNA* Expression

Dysregulation of miRNAs expression plays a critical role in the pathogenesis of genetic, multifactorial disorders and in human cancers. We exploited sequence, genomic and expression information to investigate two main aspects of post-transcriptional regulation in miRNA biogenesis, namely strand selection regulation and expression relationships between intragenic miRNAs and host genes. We considered miRNAs expression profiles, measured in five sizeable microarray datasets, including samples from different normal cell types and tissues, as well as different tumours and disease states. First, the study of expression profiles of “sister” miRNA pairs (miRNA/miRNA*, 5′ and 3′ strands of the same hairpin precursor) showed that the strand selection is highly regulated since it shows tissue-/cell-/condition-specific modulation. We used information about the direction and the strength of the strand selection bias to perform an unsupervised cluster analysis for the sample classification evidencing that is able to distinguish among different tissues, and sometimes between normal and malignant cells. Then, considering a minimum expression threshold, in few miRNA pairs only one mature miRNA is always present in all considered cell types, whereas the majority of pairs were concurrently expressed in some cell types and alternatively in others. In a significant fraction of concurrently expressed pairs, the major and the minor forms found at comparable levels may contribute to post-transcriptional gene silencing, possibly in a coordinate way. In the second part of the study, the behaved tendency to co-expression of intragenic miRNAs and their “host” mRNA genes was confuted by expression profiles examination, suggesting that the expression profile of a given host gene can hardly be a good estimator of co-transcribed miRNA(s) for post-transcriptional regulatory networks inference. Our results point out the regulatory importance of post-transcriptional phases of miRNAs biogenesis, reinforcing the role of such layer of miRNA biogenesis in miRNA-based regulation of cell activities

The evolution of multiple active site configurations in a designed enzyme

Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis

Elevated miR-499 Levels Blunt the Cardiac Stress Response

The heart responds to myriad stresses by well-described transcriptional responses that involve long-term changes in gene expression as well as more immediate, transient adaptations. MicroRNAs quantitatively regulate mRNAs and thus may affect the cardiac transcriptional output and cardiac function. Here we investigate miR-499, a microRNA embedded within a ventricular-specific myosin heavy chain gene, which is expressed in heart and skeletal muscle.We assessed miR-499 expression in human tissue to confirm its potential relevance to human cardiac gene regulation. Using a transgenic mouse model, we found that elevated miR-499 levels caused cellular hypertrophy and cardiac dysfunction in a dose-dependent manner. Global gene expression profiling revealed altered levels of the immediate early stress response genes (Egr1, Egr2 and Fos), ß-myosin heavy chain (Myh7), and skeletal muscle actin (Acta1). We verified the effect of miR-499 on the immediate early response genes by miR-499 gain- and loss-of-function in vitro. Consistent with a role for miR-499 in blunting the response to cardiac stress, asymptomatic miR-499-expressing mice had an impaired response to pressure overload and accentuated cardiac dysfunction.Elevated miR-499 levels affect cardiac gene expression and predispose to cardiac stress-induced dysfunction. miR-499 may titrate the cardiac response to stress in part by regulating the immediate early gene response

Heavy and light roles: myosin in the morphogenesis of the heart

Myosin is an essential component of cardiac muscle, from the onset of cardiogenesis through to the adult heart. Although traditionally known for its role in energy transduction and force development, recent studies suggest that both myosin heavy-chain and myosin lightchain proteins are required for a correctly formed heart. Myosins are structural proteins that are not only expressed from early stages of heart development, but when mutated in humans they may give rise to congenital heart defects. This review will discuss the roles of myosin, specifically with regards to the developing heart. The expression of each myosin protein will be described, and the effects that altering expression has on the heart in embryogenesis in different animal models will be discussed. The human molecular genetics of the myosins will also be reviewed