Promoting COVID-19 vaccine acceptance: recommendations from the Lancet Commission on Vaccine Refusal, Acceptance, and Demand in the USA.

Since the first case of COVID-19 was identified in the USA in January, 2020, over 46 million people in the country have tested positive for SARS-CoV-2 infection. Several COVID-19 vaccines have received emergency use authorisations from the US Food and Drug Administration, with the Pfizer-BioNTech vaccine receiving full approval on Aug 23, 2021. When paired with masking, physical distancing, and ventilation, COVID-19 vaccines are the best intervention to sustainably control the pandemic. However, surveys have consistently found that a sizeable minority of US residents do not plan to get a COVID-19 vaccine. The most severe consequence of an inadequate uptake of COVID-19 vaccines has been sustained community transmission (including of the delta [B.1.617.2] variant, a surge of which began in July, 2021). Exacerbating the direct impact of the virus, a low uptake of COVID-19 vaccines will prolong the social and economic repercussions of the pandemic on families and communities, especially low-income and minority ethnic groups, into 2022, or even longer. The scale and challenges of the COVID-19 vaccination campaign are unprecedented. Therefore, through a series of recommendations, we present a coordinated, evidence-based education, communication, and behavioural intervention strategy that is likely to improve the success of COVID-19 vaccine programmes across the USA.

Vaccines Mandates and Religion: Where are We Headed with the Current Supreme Court?

This article argues that the Supreme Court should not require a religious exemption from vaccine mandates. For children, who cannot yet make autonomous religious decision, religious exemptions would allow parents to make a choice that puts the child at risk and makes the shared environment of the school unsafe - risking other people's children. For adults, there are still good reasons not to require a religious exemption, since vaccines mandates are adopted for public health reasons, not to target religion, are an area where free riding is a real risk, no religion actually prohibits vaccinating under a mandate, and policing religious exemptions is very difficult.

The influence of anisotropic membrane inclusions on curvature elastic properties of lipid membranes.

A membrane inclusion can be defined as a complex of protein or peptide and the surrounding significantly distorted lipids. We suggest a theoretical model that allows for the estimation of the influence of membrane inclusions on the curvature elastic properties of lipid membranes. Our treatment includes anisotropic inclusions whose energetics depends on their in-plane orientation within the membrane. On the basis of continuum elasticity theory, we calculate the inclusion-membrane interaction energy that reflects the protein or peptide-induced short-ranged elastic deformation of a bent lipid layer. A numerical estimate of the corresponding interaction constants indicates the ability of inclusions to sense membrane bending and to accumulate at regions of favorable curvature, matching the effective shape of the inclusions. Strongly anisotropic inclusions interact favorably with lipid layers that adopt saddlelike curvature; such structures may be stabilized energetically. We explore this possibility for the case of vesicle budding where we consider a shape sequence of closed, axisymmetric vesicles that form a (saddle-curvature adopting) membrane neck. It appears that not only isotropic but also strongly anisotropic inclusions can significantly contribute to the budding energetics, a finding that we discuss in terms of recent experiments.

Amphiphile-induced tubular budding of the bilayer membrane.

Amphiphile-induced tubular budding of the erythrocyte membrane was studied using transmission electron microscopy. No chiral patterns of the intramembraneous particles were found, either on the cylindrical buds, or on the tubular nanoexovesicles. In agreement with these observations, the tubular budding may be explained by in-plane ordering of anisotropic membrane inclusions in the buds where the difference between the principal membrane curvatures is very large. In contrast to previously reported theories, no direct external mechanical force is needed to explain tubular budding of the bilayer membrane.

Protein cofactor-dependent acquisition of novel catalytic activity by the RNase P ribonucleoprotein of E. coli.

Escherichia coli RNase P derivatives were evolved in vitro for DNA cleavage activity. Ribonucleoproteins sampled after ten generations of selection show a >400-fold increase in the first-order rate constant (k(cat)) on a DNA substrate, reflecting a significant improvement in the chemical cleavage step. This increase is offset by a reduction in substrate binding, as measured by K(M). We trace the catalytic enhancement to two ubiquitous A-->U sequence changes at positions 136 and 333 in the M1 RNA component, positions that are phylogenetically conserved in the Eubacteria. Furthermore, although the mutations are located in different folding domains of the catalytic RNA, the first in the substrate binding domain, the second near the catalytic core, their effect on catalytic activity is significantly influenced by the presence of the C5 protein. The activity of the evolved ribonucleoproteins on both pre-4.5 S RNA and on an RNA oligo substrate remain at wild-type levels. In contrast, improved DNA cleavage activity is accompanied by a 500-fold decrease in pre-tRNA cleavage efficiency (k(cat)/K(M)). The presence of the C5 component does not buffer this tradeoff in catalytic activities, despite the in vivo role played by the C5 protein in enhancing the substrate versatility of RNase P. The change at position 136, located in the J11/12 single-stranded region, likely alters the geometry of the pre-tRNA-binding cleft and may provide a functional explanation for the observed tradeoff. These results thus shed light both on structure/function relations in E. coli RNase P and on the crucial role of proteins in enhancing the catalytic repertoire of RNA.

Direct DNA sequencing of PCR products.

PCR products can be sequenced using either the dideoxy (Sanger) or chemical (Maxam-Gilbert) approaches. In the dideoxy methods presented here, the target sequence is amplified and an excess of one strand of the target sequence (relative to its complement) is then generated by "asymmetric PCR," where one primer is present in vast excess over the other. This single-stranded product serves as the template for conventional dideoxy sequencing methods. Another procedure prepares PCR products for use as templates fes for characterizing unlabeled product by genomic sequencing and chemical sequencing of end-labeled products are also presented.

cDNA amplification using one-sided (anchored) PCR.

The polymerase chain reaction (PCR) offers an opportunity to directly select, amplify, and isolate a message of interest. This unit describes anchored PCR, a modification of the standard reaction that allows amplification of full-length mRNA when only a small amount of sequence information is available. Anchored PCR can be employed when only a small region of sequence lying within the mRNA is known in advance, unlike the amplification of RNA or cDNA by conventional PCR which requires prior knowledge of the sequences flanking the region of interest to design the PCR primers.

PCR cloning of neural gene products.

Of the many proteins that are known to be involved in neuronal signaling, one family of gene products, collectively referred to as the G protein-coupled receptors (GPCRs), has received considerable attention. Within the transmembrane domains of GPCRs are clusters of amino acids that tend to be conserved among receptors that bind related ligands. Polymerase chain reaction (PCR)-based approaches to cloning novel GPCRs typically begin with the identification of these well-conserved amino acid motifs, which are then back-translated into degenerate oligonucleotide primers. These pools of degenerate oligonucleotides are the most important variables in PCR cloning of GPCRs. Although GPCRs are used as the focus of this unit, the strategies and techniques described are applicable to the cloning of a wide variety of neuronal gene products. In the first procedure in this unit, either total or poly(A)(+) purified RNA is reverse transcribed into first-strand cDNA. In subsequent steps the cDNA product serves as the template for synthesis and amplification of target receptor sequences by PCR primed with degenerate oligodeoxynucleotides. The product is ready to be cloned and screened as described. Guidelines for database searching are provided to help identify the cloned gene from the known sequence. Typically, only a portion of the receptor coding region is cloned by the above approach. Rapid amplification of cDNA ends (RACE) or anchored PCR is described in this unit and is used to obtain a full-length cDNA amenable for expression studies.

cDNA amplification using one-sided (anchored) PCR.

This unit presents a modification of PCR, called anchored PCR, that allows amplification of full-length mRNA when only a small amount of sequence information lying within the mRNA is available. Both the original and reamplifications use an oligo(dT) primer complementary either to the poly(A) tail of the mature mRNA [when amplifying downstream (3') to the known sequence] or to an enzymatically synthesized homopolymer tail added to the cDNA following first strand synthesis [when amplifying upstream (5') to the known sequence]. The two rounds of PCR amplification result in a single product that can be sequenced directly or cloned into an appropriate vector for further analysis.

Replicability and recurrence in the experimental evolution of a group I ribozyme.

In order to explore the variety of possible responses available to a ribozyme population evolving a novel phenotype, five Tetrahymena thermophila group I intron ribozyme pools were evolved in parallel for cleavage of a DNA oligonucleotide. These ribozyme populations were propagated under identical conditions and characterized when they reached apparent phenotypic plateaus; the populations that reached the highest plateau showed a near 100-fold improvement in DNA cleavage activity. A detailed characterization of the evolved response in these populations reveals at least two distinct phenotypic trajectories emerging as a result of the imposed selection. Not only do these distinct solutions exhibit differential DNA cleavage activity, but they also exhibit a very different correlation with a related, but unselected, phenotype: RNA cleavage activity. In turn, each of these trajectories is underwritten by differing genotypic profiles. This study underscores the complex network of possible trajectories through sequence space available to an evolving population and uncovers the diversity of solutions that result when the process of experimental evolution is repeated multiple times in a simple, engineered system.

Molecular evolution of odorant-binding protein genes OS-E and OS-F in Drosophila.

The Drosophila olfactory genes OS-E and OS-F are members of a family of genes that encode insect odorant-binding proteins (OBPs). OBPs are believed to transport hydrophobic odorants through the aqueous fluid within olfactory sensilla to the underlying receptor proteins. The recent discovery of a large family of olfactory receptor genes in Drosophila raises new questions about the function, diversity, regulation, and evolution of the OBP family. We have investigated the OS-E and OS-F genes in a variety of Drosophila species. These studies highlight potential regions of functional significance in the OS-E and OS-F proteins, which may include a region required for interaction with receptor proteins. Our results suggest that the two genes arose by an ancient gene duplication, and that in some lineages, one or the other gene has been lost. In D. virilis, the OS-F gene shows a different spatial pattern of expression than in D. melanogaster. One of the OS-F introns shows a striking degree of conservation between the two species, and we identify a putative regulatory sequence within this intron. Finally, a phylogenetic analysis places both OS-E and OS-F within a large family of insect OBPs and OBP-like proteins.

Acquisition of novel catalytic activity by the M1 RNA ribozyme: the cost of molecular adaptation.

The ribonucleoprotein RNase P is a critical component of metabolism in all known organisms. In Escherichia coli, RNase P processes a vast array of substrates, including precursor-tRNAs and precursor 4. 5S RNA. In order to understand how such catalytic versatility is achieved and how novel catalytic activity can be acquired, we evolve the M1 RNA ribozyme (the catalytic component of E. coli RNase P) in vitro for cleavage of a DNA substrate. In so doing, we probe the consequences of enhancing catalytic activity on a novel substrate and investigate the cost this versatile enzyme pays for molecular adaptation. A total of 25 generations of in vitro evolution yield a population showing more than a 1000-fold increase in DNA substrate cleavage efficiency (kcat/KM) relative to wild-type M1 RNA. This enhancement is accompanied by a significant reduction in the ability of evolved ribozymes to process the ptRNA class of substrates but also a contrasting increase in activity on the p4.5S RNA class of substrates. This change in the catalytic versatility of the evolved ribozymes suggests that the acquired activity comes at the cost of substrate versatility, and indicates that E. coli RNase P catalytic flexibility is maintained in vivo by selection for the processing of multiple substrates. M1 RNA derivatives enhance cleavage of the DNA substrate by accelerating the catalytic step (kcat) of DNA cleavage, although overall processing efficiency is offset by reduced substrate binding. The enhanced ability to cleave a DNA substrate cannot be readily traced to any of the predominant mutations found in the evolved population, and must instead be due to multiple sequence changes dispersed throughout the molecule. This conclusion underscores the difficulty of correlating observed mutations with changes in catalytic behavior, even in simple biological catalysts for which three-dimensional models are available.

Evolution of the HOXB6 intergenic region: motif conservation at the lateral plate mesoderm (LPM) enhancer element.

This study reports the results of a comparative sequencing study in higher primates, focusing on the intergenic region located between HOXB6 and HOXB7. We have examined an 832 bp. region, encompassing a putative Lateral Plate Mesoderm (LPM) enhancer element in a variety of anthropoid apes. The interspecific comparisons reveal extensive substitutions occurring within this region, with a marked bias in favor of C-->T transitions within the enhancer element. The pattern of these substitutions suggests that the LPM enhancer region is subject to specific sequence and compositional constraints that are only revealed through comparative sequencing. These constraints produce an enhancer signature, the CpG microisland, which may be useful in identifying additional regulatory elements located within the HOX complexes. J. Exp. Zool. (Mol. Dev. Evol.) 285:170-176, 1999.

Molecular evolution of genes controlling petal and stamen development: duplication and divergence within the APETALA3 and PISTILLATA MADS-box gene lineages.

The specification of floral organ identity in the higher dicots depends on the function of a limited set of homeotic genes, many of them members of the MADS-box gene family. Two such genes, APETALA3 (AP3) and PISTILLATA (PI), are required for petal and stamen identity in Arabidopsis; their orthologs in Antirrhinum exhibit similar functions. To understand how changes in these genes may have influenced the morphological evolution of petals and stamens, we have cloned twenty-six homologs of the AP3 and PI genes from two higher eudicot and eleven lower eudicot and magnolid dicot species. The sequences of these genes reveal the presence of characteristic PI- and AP3-specific motifs. While the PI-specific motif is found in all of the PI genes characterized to date, the lower eudicot and magnolid dicot AP3 homologs contain distinctly different motifs from those seen in the higher eudicots. An analysis of all the available AP3 and PI sequences uncovers multiple duplication events within each of the two gene lineages. A major duplication event in the AP3 lineage coincides with the base of the higher eudicot radiation and may reflect the evolution of a petal-specific AP3 function in the higher eudicot lineage.

Experimental evolution of complexity: in vitro emergence of intermolecular ribozyme interactions.

In the course of evolving variants of the Tetrahymena thermophila Group I ribozyme for improved DNA cleavage in vitro, we witnessed the unexpected emergence of a derived molecular species, capable of acting as a partner for the ribozyme, but no longer autocatalytic. This new RNA species exhibits a deletion in the catalytic core and participates in a productive intermolecular interaction with an active ribozyme, thus insuring its survival in the population. These novel RNA molecules have evolved a precise catalytic interaction with the Group I ribozyme and depend for their survival on the continued presence of active catalysts. This interaction hints at the complexity that may inevitably arise even in simple evolving systems.