Expanding Global Health Engagement through Fogarty Fellowship Programs.

Training the next generation of global health researchers is vital for sustainable research partnerships and global health equity. The Fogarty International Center (National Institutes of Health) supports postdoctoral fellows and professional/graduate students in long-term, hands-on mentored research in low- and middle-income countries (LMICs). We surveyed 627 alumni (58% from the United States, 42% from LMICs) from three sequential Fogarty-sponsored global health research training programs (response rate: N = 257, 41%). Publications in the Index Medicus were used to ascertain scholarly output. Most alumni (63%) reported remaining engaged in LMICs and/or worked in academic/research careers (70%). Since completing their Fogarty fellowship, 144 alumni (56%) had received 438 new grants as principal investigator (PI), co-/multi-PI, or site PI. The 257 responding alumni had 5,318 publications during and since their Fogarty fellowships; 2,083 (39%) listed the Fogarty trainee as the first or senior author. These global health training programs highlight the value of LMIC research experience in nurturing the global health research workforce.

REVIEW: Assessing North Atlantic right whale health: threats, and development of tools critical for conservation of the species.

Whaling has decimated North Atlantic right whales Eubalaena glacialis (NARW) since the 11th century and southern right whales E. australis (SRW) since the 19th century. Today, NARWs are Critically Endangered and decreasing, whereas SRWs are recovering. We review NARW health assessment literature, NARW Consortium databases, and efforts and limitations to monitor individual and species health, survival, and fecundity. Photographs are used to track individual movement and external signs of health such as evidence of vessel and entanglement trauma. Post-mortem examinations establish cause of death and determine organ pathology. Photogrammetry is used to assess growth rates and body condition. Samples of blow, skin, blubber, baleen and feces quantify hormones that provide information on stress, reproduction, and nutrition, identify microbiome changes, and assess evidence of infection. We also discuss models of the population consequences of multiple stressors, including the connection between human activities (e.g. entanglement) and health. Lethal and sublethal vessel and entanglement trauma have been identified as major threats to the species. There is a clear and immediate need for expanding trauma reduction measures. Beyond these major concerns, further study is needed to evaluate the impact of other stressors, such as pathogens, microbiome changes, and algal and industrial toxins, on NARW reproductive success and health. Current and new health assessment tools should be developed and used to monitor the effectiveness of management measures and will help determine whether they are sufficient for a substantive species recovery.

Building evidence around ghost gear: Global trends and analysis for sustainable solutions at scale.

Abandoned, lost or discarded fishing gear (ALDFG) comprises a significant amount of global marine debris, with diverse impacts to marine environments, wildlife, and the fishing industry. Building evidence on ALDFG is critical to holistically understand the marine debris issue, and to inform the development of solutions that reduce amounts of ALDFG sources and recover existing gear. Substantial work has been and continues to be undertaken around the world to collect data on ALDFG, much of which remains unpublished. To provide a global picture of data on ALDFG, we organized a technical session that brought together seven ALDFG leaders to share their expertise in data collection, retrieval, and awareness-raising. This paper summarizes the technical session to highlight: 1) case studies that feature innovative approaches to ALDFG data collection and retrieval; 2) examples of opportunities to fill data gaps and improve our understanding of wildlife ingestion of and entanglement in ALDFG; and 3) awareness-raising through the development of a publicly accessible global ALDFG database.

Predicting lethal entanglements as a consequence of drag from fishing gear.

Large whales are frequently entangled in fishing gear and sometimes swim while carrying gear for days to years. Entangled whales are subject to additional drag forces requiring increased thrust power and energy expenditure over time. To classify entanglement cases and aid potential disentanglement efforts, it is useful to know how long an entangled whale might survive, given the unique configurations of the gear they are towing. This study establishes an approach to predict drag forces on fishing gear that entangles whales, and applies this method to ten North Atlantic right whale cases to estimate the resulting increase in energy expenditure and the critical entanglement duration that could lead to death. Estimated gear drag ranged 11-275N. Most entanglements were resolved before critical entanglement durations (mean±SD 216±260days) were reached. These estimates can assist real-time development of disentanglement action plans and U.S. Federal Serious Injury assessments required for protected species.

Assessment of management to mitigate anthropogenic effects on large whales.

United States and Canadian governments have responded to legal requirements to reduce human-induced whale mortality via vessel strikes and entanglement in fishing gear by implementing a suite of regulatory actions. We analyzed the spatial and temporal patterns of mortality of large whales in the Northwest Atlantic (23.5°N to 48.0°N), 1970 through 2009, in the context of management changes. We used a multinomial logistic model fitted by maximum likelihood to detect trends in cause-specific mortalities with time. We compared the number of human-caused mortalities with U.S. federally established levels of potential biological removal (i.e., species-specific sustainable human-caused mortality). From 1970 through 2009, 1762 mortalities (all known) and serious injuries (likely fatal) involved 8 species of large whales. We determined cause of death for 43% of all mortalities; of those, 67% (502) resulted from human interactions. Entanglement in fishing gear was the primary cause of death across all species (n = 323), followed by natural causes (n = 248) and vessel strikes (n = 171). Established sustainable levels of mortality were consistently exceeded in 2 species by up to 650%. Probabilities of entanglement and vessel-strike mortality increased significantly from 1990 through 2009. There was no significant change in the local intensity of all or vessel-strike mortalities before and after 2003, the year after which numerous mitigation efforts were enacted. So far, regulatory efforts have not reduced the lethal effects of human activities to large whales on a population-range basis, although we do not exclude the possibility of success of targeted measures for specific local habitats that were not within the resolution of our analyses. It is unclear how shortfalls in management design or compliance relate to our findings. Analyses such as the one we conducted are crucial in critically evaluating wildlife-management decisions. The results of these analyses can provide managers with direction for modifying regulated measures and can be applied globally to mortality-driven conservation issues.

Efforts toward expansion of the genetic alphabet: structure and replication of unnatural base pairs.

Expansion of the genetic alphabet has been a long-time goal of chemical biology. A third DNA base pair that is stable and replicable would have a great number of practical applications and would also lay the foundation for a semisynthetic organism. We have reported that DNA base pairs formed between deoxyribonucleotides with large aromatic, predominantly hydrophobic nucleobase analogues, such as propynylisocarbostyril (dPICS), are stable and efficiently synthesized by DNA polymerases. However, once incorporated into the primer, these analogues inhibit continued primer elongation. More recently, we have found that DNA base pairs formed between nucleobase analogues that have minimal aromatic surface area in addition to little or no hydrogen-bonding potential, such as 3-fluorobenzene (d3FB), are synthesized and extended by DNA polymerases with greatly increased efficiency. Here we show that the rate of synthesis and extension of the self-pair formed between two d3FB analogues is sufficient for in vitro DNA replication. To better understand the origins of efficient replication, we examined the structure of DNA duplexes containing either the d3FB or dPICS self-pairs. We find that the large aromatic rings of dPICS pair in an intercalative manner within duplex DNA, while the d3FB nucleobases interact in an edge-on manner, much closer in structure to natural base pairs. We also synthesized duplexes containing the 5-methyl-substituted derivatives of d3FB (d5Me3FB) paired opposite d3FB or the unsubstituted analogue (dBEN). In all, the data suggest that the structure, electrostatics, and dynamics can all contribute to the extension of unnatural primer termini. The results also help explain the replication properties of many previously examined unnatural base pairs and should help design unnatural base pairs that are better replicated.

Optimization of unnatural base pair packing for polymerase recognition.

As part of an effort to expand the genetic alphabet, we have been examining the ability of predominately hydrophobic nucleobase analogues to pair in duplex DNA and during polymerase-mediated replication. We previously reported the synthesis and thermal stability of unnatural base pairs formed between nucleotides bearing simple methyl-substituted phenyl ring nucleobase analogues. Several of these pairs are virtually as stable and selective as natural base pairs in the same sequence context. Here, we report the characterization of polymerase-mediated replication of the same unnatural base pairs. We find that every facet of replication, including correct and incorrect base pair synthesis, as well as continued primer extension beyond the unnatural base pair, is sensitive to the specific methyl substitution pattern of the nucleobase analogue. The results demonstrate that neither hydrogen bonding nor large aromatic surface area is required for polymerase recognition, and that interstrand interactions between small aromatic rings may be optimized for replication. Combined with our previous results, these studies suggest that appropriately derivatized phenyl nucleobase analogues represent a promising approach toward developing a third base pair and expanding the genetic alphabet.

The evolution of DNA polymerases with novel activities.

DNA and RNA polymerases have evolved in nature to function in specific environments with specific substrates. Thus, although the commercial availability of these enzymes has revolutionized the biotechnology industry, their applications are limited. The availability of polymerases that have unnatural properties would be of even greater utility. Towards this goal, several activity-based screening and selection approaches have been developed. Using these techniques, polymerases that synthesize a variety of different polymers, including those containing 2'-O-methyl-modified nucleotides or unnatural base pairs, have been evolved. These results suggest that polymerases tailored for any specific application could soon be available.

Directed evolution of novel polymerases.

DNA and RNA polymerases evolved to function in specific environments with specific substrates to propagate genetic information in all living organisms. The commercial availability of these polymerases has revolutionized the biotechnology industry, but for many applications native polymerases are limited by their stability or substrate recognition. Thus, there is great interest in the directed evolution of DNA and RNA polymerases to generate enzymes with novel, desired properties, such as thermal stability, resistance to inhibitors, and altered substrate specificity. Several screening and selection approaches have been developed, both in vivo and in vitro, and have been used to evolve polymerases with a variety of important activities. Both the techniques and the evolved polymerases are reviewed here, along with a comparison of the in vivo and in vitro approaches.

Preliminary characterization of light harvesting in E. coli DNA photolyase.

E. coli DNA photolyase is a monomeric light-harvesting enzyme that utilizes a methenyltetrahydrofolate (MTHF) antenna cofactor to harvest light energy for the repair of thymine dimers in DNA. For this purpose, the enzyme evolved to bind the cofactor and red-shift its absorption maximum by 25 nm. Using the crystal structure as a guide, we mutated each protein residue that contacts the cofactor in an effort to identify the interactions responsible for this selective stabilization of the cofactor's excited state. Hydrogen bonding, packing, and electrostatic interactions were examined. Remarkably, a single residue, Glu109, appears to play an important, if not exclusive, role in inducing the observed red-shift. Thus, this protein, the simplest light-harvesting system known, appears to have evolved a remarkably simple mechanism to tune the photophysical properties of the antenna cofactor appropriately for biological function.

Efforts to expand the genetic alphabet: identification of a replicable unnatural DNA self-pair.

Six unnatural nucleotides featuring fluorine-substituted phenyl nucleobase analogues have been synthesized, incorporated into DNA, and characterized in terms of the structure and replication properties of the self-pairs they form. Each unnatural self-pair is accommodated in B-form DNA without detectable structural perturbation, and all are thermally stable and selective to roughly the same degree. Furthermore, the efficiency of polymerase-mediated mispair synthesis is similar for each unnatural nucleotide in the template. In contrast, the efficiency of polymerase-mediated self-pair extension is highly dependent on the specific fluorine substitution pattern. The most promising unnatural base pair candidate of this series is the 3-fluorobenzene self-pair, which is replicated with reasonable efficiency and selectivity.

Expanding the substrate repertoire of a DNA polymerase by directed evolution.

Nucleic acid polymerases are the most important reagents in biotechnology. Unfortunately, their high substrate specificity severely limits their applications. Polymerases with tailored substrate repertoires would significantly expand their potential and allow enzymatic synthesis of unnatural polymers for in vivo and in vitro applications. For example, the ability to synthesize 2'-O-methyl-modified polymers would provide access to materials possessing properties that make them attractive for biotechnology and therapeutic applications, but unfortunately, no known polymerases are capable of efficiently accepting these modified substrates. To evolve such enzymes, we have developed an activity-based selection method which isolates polymerase mutants with the desired property from libraries of the enzyme displayed on phage. In this report, mutants that could efficiently synthesize an unnatural polymer from 2'-O-methyl ribonucleoside triphosphates were immobilized and isolated by means of their activity-dependent modification of a DNA oligonucleotide primer attached to the same phage particle. In each case, directed evolution resulted in relocating a critical side chain to a different position in the polypeptide, thus re-engineering the overall active site while preserving critical protein-DNA interactions. Remarkably, one evolved polymerase is shown to incorporate the modified substrates with an efficiency and fidelity equivalent to that of the wild-type enzyme with natural substrates.

Beyond A, C, G and T: augmenting nature's alphabet.

Efforts to expand the genetic alphabet are predicated upon a stable and replicable third base pair. Recent progress has resulted in the development of several candidates that are both stable in duplex DNA and replicated by DNA polymerases with various degrees of efficiency and fidelity. The candidate base pairs draw upon unnatural hydrogen-bonding topologies as well as upon shape complementarity and hydrophobic forces. This review provides a critical comparison of the third base pair candidates and discusses the further work required to expand the genetic alphabet.

Determinants of unnatural nucleobase stability and polymerase recognition.

Six new unnatural nucleobases have been synthesized and characterized in terms of stability and selectivity of self-pairing in duplex DNA and efficiency and fidelity of self-pairing during polymerase-mediated replication. Each nucleobase has a conserved ring structure but differs from the others in its specific pattern of substitution with oxygen and sulfur atoms. Heteroatom derivatization within the conserved scaffold is shown to have only moderate effects on unnatural self-pair synthesis by the polymerase; larger effects were observed on the thermal stability and polymerase-mediated extension of the self-pairs. The largest effects of heteroatom substitution were on the stability and synthesis of mispairs between the unnatural and natural bases. Certain heteroatom substitutions were found to have a general effect while others were found to have effects that were specific for a particular unnatural or natural base. The data are useful for designing stable and replicable third base pairs and for understanding the contributions of nucleobase shape, polarity, and polarizability to the stability and replication of DNA.

The effect of minor-groove hydrogen-bond acceptors and donors on the stability and replication of four unnatural base pairs.

The stability and replication of DNA containing self-pairs formed between unnatural nucleotides bearing benzofuran, benzothiophene, indole, and benzotriazole nucleobases are reported. These nucleobase analogues are based on a similar scaffold but have different hydrogen-bond donor/acceptor groups that are expected to be oriented in the duplex minor groove. The unnatural base pairs do not appear to induce major structural distortions and are accommodated within the constraints of a B-form duplex. The differences between these unnatural base pairs are manifest only in the polymerase-mediated extension step, not in base-pair stability or synthesis. The benzotriazole self-pair is extended with an efficiency that is only 200-fold less than a correct natural base pair. The data are discussed in terms of available polymerase crystal structures and imply that further modifications may result in unnatural base pairs that can be both efficiently synthesized and extended, resulting in an expanded genetic alphabet.

Stability and selectivity of unnatural DNA with five-membered-ring nucleobase analogues.

In an effort to develop an orthogonal third base pair for the storage of genetic information, thiophene and furan heterocycles have been examined as nucleobase analogues. The stability of the unnatural bases was evaluated in duplex DNA paired opposite other unnatural bases as well as opposite the natural bases. Several unnatural base pairs are identified that are both reasonably stable and strongly selective against mispairing with native bases. These results expand the potential nucleobase analogues with which the genetic alphabet may be expanded to include five-membered-ring heterocycles.

Development of a universal nucleobase and modified nucleobases for expanding the genetic code.

This unit presents protocols for the synthesis and characterization of nucleosides with unnatural bases in order to develop bases for the expansion of the genetic alphabet or for nonselective pairing opposite natural bases. Protocols describe the design, synthesis, and characterization of unnatural base pairs involving 1-beta-D-2-deoxyribosyl-N- and -C-nucleosides. Determination of the thermodynamic and kinetic parameters of unnatural nucleosides is accomplished by incorporation into oligonucleotides and subsequent evaluation as described herein.