Potent activity of the HIV-1 maturation inhibitor bevirimat in SCID-hu Thy/Liv mice

Background

The HIV-1 maturation inhibitor, 3-O-(3′,3′-dimethylsuccinyl) betulinic acid (bevirimat, PA-457) is a promising drug candidate with 10 nM in vitro antiviral activity against multiple wild-type (WT) and drug-resistant HIV-1 isolates. Bevirimat has a novel mechanism of action, specifically inhibiting cleavage of spacer peptide 1 (SP1) from the C-terminus of capsid which results in defective core condensation.

Methods and Findings

Oral administration of bevirimat to HIV-1-infected SCID-hu Thy/Liv mice reduced viral RNA by >2 log₁₀ and protected immature and mature T cells from virus-mediated depletion. This activity was observed at plasma concentrations that are achievable in humans after oral dosing, and bevirimat was active up to 3 days after inoculation with both WT HIV-1 and an AZT-resistant HIV-1 clinical isolate. Consistent with its mechanism of action, bevirimat caused a dose-dependent inhibition of capsid-SP1 cleavage in HIV-1-infected human thymocytes obtained from these mice. HIV-1 NL4-3 with an alanine-to-valine substitution at the N-terminus of SP1 (SP1/A1V), which is resistant to bevirimat in vitro, was also resistant to bevirimat treatment in the mice, and SP1/AIV had replication and thymocyte kinetics similar to that of WT NL4-3 with no evidence of fitness impairment in in vivo competition assays. Interestingly, protease inhibitor-resistant HIV-1 with impaired capsid-SP1 cleavage was hypersensitive to bevirimat in vitro with a 50% inhibitory concentration 140 times lower than for WT HIV-1.

Conclusions

These results support further clinical development of this first-in-class maturation inhibitor and confirm the usefulness of the SCID-hu Thy/Liv model for evaluation of in vivo antiretroviral efficacy, drug resistance, and viral fitness.     

The Hologenome Concept: Helpful or Hollow?

With the increasing appreciation for the crucial roles that microbial symbionts play in the development and fitness of plant and animal hosts, there has been a recent push to interpret evolution through the lens of the “hologenome”—the collective genomic content of a host and its microbiome. But how symbionts evolve and, particularly, whether they undergo natural selection to benefit hosts are complex issues that are associated with several misconceptions about evolutionary processes in host-associated microbial communities. Microorganisms can have intimate, ancient, and/or mutualistic associations with hosts without having undergone natural selection to benefit hosts. Likewise, observing host-specific microbial community composition or greater community similarity among more closely related hosts does not imply that symbionts have coevolved with hosts, let alone that they have evolved for the benefit of the host. Although selection at the level of the symbiotic community, or hologenome, occurs in some cases, it should not be accepted as the null hypothesis for explaining features of host–symbiont associations.The ubiquity and importance of microorganisms in the lives of plants and animals are ever more apparent, and increasingly investigated by biologists. Suddenly, we have the aspiration and tools to open up a new, complicated world, and we must confront the realization that almost everything about larger organisms has been shaped by their history of evolving from, then with, microorganisms [1]. This development represents a dramatic shift in perspective—arguably a revolution—in modern biology.Do we need to revamp basic tenets of evolutionary theory to understand how hosts evolve with associated microorganisms? Some scientists have suggested that we do [2], and the recently introduced terms “holobiont” and “hologenome” encapsulate what has been described as an “emerging postmodern synthesis” [3]. Holobiont was initially used to refer to a host and a single inherited symbiont [4] but was later extended to a host and its community of associated microorganisms, specifically for the case of corals [5]. The idea of the holobiont is that a host and its associated microorganisms must be considered as an integrated unit in order to understand many biological and ecological features.The later introduction of the term hologenome [2,6,7] sought to describe a holobiont by its genetic composition. The term has been used in different ways by different authors, but in most contexts a hologenome is considered a genetic unit that represents the combined genomes of a host and its associated microorganisms [8]. This non-controversial definition of hologenome is linked to the idea that this entity has a role in evolution. For example, Gordon et al. [1,9] state, "The genome of a holobiont, termed the hologenome, is the sum of the genomes of all constituents, all of which can evolve within that context." That last phrase is sufficiently general that it can be interpreted in any number of ways. Like physical conditions, associated organisms can be considered as part of the environment and thus can be sources of natural selection, affecting evolution in each lineage.But a more sweeping and problematic proposal is given by originators of the term, which is that "the holobiont with its hologenome should be considered as the unit of natural selection in evolution" [2,7] or by others, that “an organism’s genetics and fitness are inclusive of its microbiome” [3,4]. The implication is that differential success of holobionts influences evolution of participating organisms, such that their observed features cannot be fully understood without considering selection at the holobiont level. Another formulation of this concept is the proposal that the evolution of host–microbe systems is “most easily understood by equating a gene in the nuclear genome to a microbe in the microbiome” [8]. Under this view, interactions between host and microbial genotypes should be considered as genetic epistasis (interactions among alleles at different loci in a genome) rather than as interactions between the host’s genotype and its environment.While biologists would agree that microorganisms have important roles in host evolution, this statement is a far cry from the claim that they are fused with hosts to form the primary units of selection, or that hosts and microorganisms provide different portions of a unified genome. Broadly, the hologenome concept contends, first, that participating lineages within a holobiont affect each other’s evolution, and, second, that that the holobiont is a primary unit of selection. Our aim in this essay is to clarify what kinds of evidence are needed for each of these claims and to argue that neither should be assumed without evidence. We point out that some observations that superficially appear to support the concept of the hologenome have spawned confusion about real biological issues (Box 1).

Box 1. Misconceptions Related to the Hologenome Concept

Misconception #1: Similarities in microbiomes between related host species result from codiversification. Reality: Related species tend to be similar in most traits. Because microbiome composition is a trait that involves living organisms, it is tempting to assume that these similarities reflect a shared evolutionary history of host and symbionts. This has been shown to be the case for some symbioses (e.g., ancient maternally inherited endosymbionts in insects). But for many interactions (e.g., gut microbiota), related hosts may have similar effects on community assembly without any history of codiversification between the host and individual microbial species (Fig 1B).Open in a separate windowFig 1Alternative evolutionary processes can result in related host species harboring similar symbiont communities.Left panel: Individual symbiont lineages retain fidelity to evolving host lineages, through co-inheritance or other mechanisms, with some gain and loss of symbiont lineages over evolutionary time. Right panel: As host lineages evolve, they shift their selectivity of environmental microbes, which are not evolving in response and which may not even have been present during host diversification. In both cases, measures of community divergence will likely be smaller for more closely related hosts, but they reflect processes with very different implications for hologenome evolution. Image credit: Nancy Moran and Kim Hammond, University of Texas at Austin. Misconception #2: Parallel phylogenies of host and symbiont, or intimacy of host and symbiont associations, reflect coevolution. Reality: Coevolution is defined by a history of reciprocal selection between parties. While coevolution can generate parallel phylogenies or intimate associations, these can also result from many other mechanisms. Misconception #3: Highly intimate associations of host and symbionts, involving exchange of cellular metabolites and specific patterns of colonization, result from a history of selection favoring mutualistic traits. Reality: The adaptive basis of a specific trait is difficult to infer even when the trait involves a single lineage, and it is even more daunting when multiple lineages contribute. But complexity or intimacy of an interaction does not always imply a long history of coevolution nor does it imply that the nature of the interaction involves mutual benefit. Misconception #4: The essential roles that microbial species/communities play in host development are adaptations resulting from selection on the symbionts to contribute to holobiont function. Reality: Hosts may adapt to the reliable presence of symbionts in the same way that they adapt to abiotic components of the environment, and little or no selection on symbiont populations need be involved. Misconception #5: Because of the extreme importance of symbionts in essential functions of their hosts, the integrated holobiont represents the primary unit of selection. Reality: The strength of natural selection at different levels of biological organization is a central issue in evolutionary biology and the focus of much empirical and theoretical research. But insofar as there is a primary unit of selection common to diverse biological systems, it is unlikely to be at the level of the holobiont. In particular cases, evolutionary interests of host and symbionts can be sufficiently aligned such that the predominant effect of natural selection on genetic variation in each party is to increase the reproductive success of the holobiont. But in most host–symbiont relationships, contrasting modes of genetic transmission will decouple selection pressures.     

Regulation of hindbrain Pyy expression by acute food deprivation, prolonged caloric restriction, and weight loss surgery in mice

PYY is a gut-derived putative satiety signal released in response to nutrient ingestion and is implicated in the regulation of energy homeostasis. Pyy-expressing neurons have been identified in the hindbrain of river lamprey, rodents, and primates. Despite this high evolutionary conservation, little is known about central PYY neurons. Using in situ hybridization, PYY-Cre;ROSA-EYFP mice, and immunohistochemistry, we identified PYY cell bodies in the gigantocellular reticular nucleus region of the hindbrain. PYY projections were present in the dorsal vagal complex and hypoglossal nucleus. In the hindbrain, Pyy mRNA was present at E9.5, and expression peaked at P2 and then decreased significantly by 70% at adulthood. We found that, in contrast to the circulation, PYY-(1-36) is the predominant isoform in mouse brainstem extracts in the ad libitum-fed state. However, following a 24-h fast, the relative amounts of PYY-(1-36) and PYY-(3-36) isoforms were similar. Interestingly, central Pyy expression showed nutritional regulation and decreased significantly by acute starvation, prolonged caloric restriction, and bariatric surgery (enterogastroanastomosis). Central Pyy expression correlated with body weight loss and circulating leptin and PYY concentrations. Central regulation of energy metabolism is not limited to the hypothalamus but also includes the midbrain and the brainstem. Our findings suggest a role for hindbrain PYY in the regulation of energy homeostasis and provide a starting point for further research on gigantocellular reticular nucleus PYY neurons, which will increase our understanding of the brain stem pathways in the integrated control of appetite and energy metabolism.     

“Evolution for Everyone”: A Course that Expands Evolutionary Theory Beyond the Biological Sciences

A firm grasp of evolution is invaluable for understanding our own species in addition to the rest of the biological world; however, not only does much of the American public reject evolution, but many thinkers within the scientific community resist its application to their own disciplines. In an attempt to overcome these challenges through education, the Evolutionary Studies (EvoS) program at Binghamton University (B.U.) strives to present evolution as a theory relevant to all human-related subjects. Here, we present the cornerstone of this program, “Evolution for Everyone,” an introductory-level, general education course with an academically diverse student population. The curriculum delves into Darwin’s theory and uses it to illustrate not only classical biology but how the same ideas pertain to the full range of human-related disciplines. This material is framed in terms of scientific inquiry, including direct participation in research. An evaluation of the course has demonstrated that it increases both understanding and acceptance of evolution and its relevance to human-related academic disciplines and everyday life. The EvoS program has received National Science Foundation funding to expand into a nationwide consortium, providing a strong infrastructure for the development of similar courses at other institutions.     

Multi-strain co-cultures surpass blends for broad spectrum biological control of maladies of potatoes in storage

Pseudomonas fluorescens strains S11:P:12, P22:Y:05, and S22:T:04 and Enterobacter cloacae strain S11:T:07 have been documented to suppress four important storage potato maladies – dry rot, late blight, pink rot, and sprouting. This research investigates the efficacy and consistency of strain mixtures produced by co-culturing strains together in one vessel or by blending them together after separate cultivations in pure cultures. Pure and co-cultures were produced in flask or fermentor cultures, viable cell concentrations were assessed using a nutrient-based selective plating method to identify and enumerate strains, and the efficacy of treatments was assessed with respect to dry rot, pink rot, late blight or sprout suppression. Experiments were designed to analyze dry rot suppression versus all strain combinations and the combination method (co-culture or blend). Results of a two-way analysis of variance of disease with strain composition and combination method showed that significantly better dry rot suppression was obtained by co-cultures (30.3±2.4% relative disease) than by similar strain blends of pure cultures (41.3±2.4%) (P<0.001). During a 3-year study, both biocontrol efficacy and consistency were assessed in 16 laboratory and small pilot trials simulating commercial storages. Three-strain co-culture had a lower mean disease rating than the blend in 9 of 16 experiments examining control of the three diseases and sprouting. The co-culture led other treatments in incidences of significant malady reduction relative to the control: 14 of 16 attempts for co-culture, 11 of 16 attempts for blend, 10 of 13 attempts for pure S11:P:12, 8 of 13 attempts for S22:T:04, and 9 of 13 attempts for P22:Y:05. Using relative performance indices to rank treatment performance across all experiments, the co-culture treatment ranked significantly higher than the blend. A synergy analysis suggested that co-culturing strains stimulated inter-strain activities to boost biocontrol efficacy and consistency, a feature not developed in strains grown separately and mixed just prior to addition to potatoes.