Synthesis of Anti-Sense Phosphorothioate Analogues for Pharmacokinetic and Pre-Clinical Studies

Abstract

Nudease-resistant oligonucleotides (11 to 28-mers) containing stereorandom phosphorothioate linkages have been recently reported to exhibit potent anti-HIV III effects and sequence-specific inhibition of protein synthesis. Relatively large amounts (100 mg 1g) of these analogues. which are needed for further biological testing and initial pharmacokinetic and pre-clinical studies, were readily obtained by automated hydrogen phosphonate chemistry followed by reversed-phase HPLC and further processing. This chemistry features 1 -adamanetanecarbonyl chloride as the activator, capping with isopropyl phosphite, and more complete sulfurization in only one-step following chain assembly. An automated, quantitative. picomole method for analysis of the analogues in blood samples has been developed.     

Natural Infection of Burkholderia pseudomallei in an Imported Pigtail Macaque (Macaca nemestrina) and Management of the Exposed Colony

Identification of the select agent Burkholderia pseudomallei in macaques imported into the United States is rare. A purpose-bred, 4.5-y-old pigtail macaque (Macaca nemestrina) imported from Southeast Asia was received from a commercial vendor at our facility in March 2012. After the initial acclimation period of 5 to 7 d, physical examination of the macaque revealed a subcutaneous abscess that surrounded the right stifle joint. The wound was treated and resolved over 3 mo. In August 2012, 2 mo after the stifle joint wound resolved, the macaque exhibited neurologic clinical signs. Postmortem microbiologic analysis revealed that the macaque was infected with B. pseudomallei. This case report describes the clinical evaluation of a B. pseudomallei-infected macaque, management and care of the potentially exposed colony of animals, and protocols established for the animal care staff that worked with the infected macaque and potentially exposed colony. This article also provides relevant information on addressing matters related to regulatory issues and risk management of potentially exposed animals and animal care staff.Abbreviations: CDC, Centers for Disease Control and Prevention; IHA, indirect hemagglutination assay; PEP, postexposure prophylacticBurkholderia pseudomallei, formerly known as Pseudomonas pseudomallei, is a gram-negative, aerobic, bipolar, motile, rod-shaped bacterium. B. pseudomallei infections (melioidosis) can be severe and even fatal in both humans and animals. This environmental saprophyte is endemic to Southeast Asia and northern Australia, but it has also been found in other tropical and subtropical areas of the world.^7,22,32,42 The bacterium is usually found in soil and water in endemic areas and is transmitted to humans and animals primarily through percutaneous inoculation, ingestion, or inhalation of a contaminated source.^{8, 22,28,32,42} Human-to-human, animal-to-animal, and animal-to-human spread are rare.^8,32 In December 2012, the National Select Agent Registry designated B. pseudomallei as a Tier 1 overlap select agent.³⁹ Organisms classified as Tier 1 agents present the highest risk of deliberate misuse, with the most significant potential for mass casualties or devastating effects to the economy, critical infrastructure, or public confidence. Select agents with this status have the potential to pose a severe threat to human and animal health or safety or the ability to be used as a biologic weapon.³⁹Melioidosis in humans can be challenging to diagnose and treat because the organism can remain latent for years and is resistant to many antibiotics.^12,37,41 B. pseudomallei can survive in phagocytic cells, a phenomenon that may be associated with latent infections.^19,38 The incubation period in naturally infected animals ranges from 1 d to many years, but symptoms typically appear 2 to 4 wk after exposure.^13,17,35,38 Disease generally presents in 1 of 2 forms: localized infection or septicemia.²² Multiple methods are used to diagnose melioidosis, including immunofluorescence, serology, and PCR analysis, but isolation of the bacteria from blood, urine, sputum, throat swabs, abscesses, skin, or tissue lesions remains the ‘gold standard.’^9,22,40,42 The prognosis varies based on presentation, time to diagnosis, initiation of appropriate antimicrobial treatment, and underlying comorbidities.^7,28,42 Currently, there is no licensed vaccine to prevent melioidosis.There are several published reports of naturally occurring melioidosis in a variety of nonhuman primates (NHP; 2,10,13,17,25,30,31,35 The first reported case of melioidosis in monkeys was recorded in 1932, and the first published case in a macaque species was in 1966.³⁰ In the United States, there have only been 7 documented cases of NHP with B. pseudomallei infection.^2,13,17 All of these cases occurred prior to the classification of B. pseudomallei as a select agent. Clinical signs in NHP range from subclinical or subacute illness to acute septicemia, localized infection, and chronic infection. NHP with melioidosis can be asymptomatic or exhibit clinical signs such as anorexia, wasting, purulent drainage, subcutaneous abscesses, and other soft tissue lesions. Lymphadenitis, lameness, osteomyelitis, paralysis and other CNS signs have also been reported.^{2,7,10,22,28,32} In comparison, human''s clinical signs range from abscesses, skin ulceration, fever, headache, joint pain, and muscle tenderness to abdominal pain, anorexia, respiratory distress, seizures, and septicemia.^7,9,21,22

Table 1.

Summary of reported cases of naturally occurring Burkholderia pseudomalleiinfections in nonhuman primates

Multi-dimensional Co-separation Analysis Reveals Protein–Protein Interactions Defining Plasma Lipoprotein Subspecies

The distribution of circulating lipoprotein particles affects the risk for cardiovascular disease (CVD) in humans. Lipoproteins are historically defined by their density, with low-density lipoproteins positively and high-density lipoproteins (HDLs) negatively associated with CVD risk in large populations. However, these broad definitions tend to obscure the remarkable heterogeneity within each class. Evidence indicates that each class is composed of physically (size, density, charge) and compositionally (protein and lipid) distinct subclasses exhibiting unique functionalities and differing effects on disease. HDLs in particular contain upward of 85 proteins of widely varying function that are differentially distributed across a broad range of particle diameters. We hypothesized that the plasma lipoproteins, particularly HDL, represent a continuum of phospholipid platforms that facilitate specific protein–protein interactions. To test this idea, we separated normal human plasma using three techniques that exploit different lipoprotein physicochemical properties (gel filtration chromatography, ionic exchange chromatography, and preparative isoelectric focusing). We then tracked the co-separation of 76 lipid-associated proteins via mass spectrometry and applied a summed correlation analysis to identify protein pairs that may co-reside on individual lipoproteins. The analysis produced 2701 pairing scores, with the top hits representing previously known protein–protein interactions as well as numerous unknown pairings. A network analysis revealed clusters of proteins with related functions, particularly lipid transport and complement regulation. The specific co-separation of protein pairs or clusters suggests the existence of stable lipoprotein subspecies that may carry out distinct functions. Further characterization of the composition and function of these subspecies may point to better targeted therapeutics aimed at CVD or other diseases.Lipoproteins are circulating emulsions of protein and lipid that play important roles, both positive and negative, in cardiovascular disease (CVD).¹ Historically defined by their density as separated by ultracentrifugation, the major lipoprotein classes include the neutral lipid ester-rich very low-density and low-density lipoproteins (VLDLs and LDLs, respectively), which function to transport triglyceride and cholesterol from the liver to the peripheral tissues. Significant epidemiological evidence, in vitro studies, animal experiments, and human clinical trials have shown that high-LDL cholesterol is a bona fide causative factor in CVD (1). In contrast, protein- and phospholipid-rich high-density lipoproteins (HDLs) are thought to mediate the reverse transport of cholesterol from the periphery to the liver for catabolism and to perform anti-oxidative and anti-inflammatory functions (reviewed in Refs. 2 and 3). A host of human epidemiology and animal studies indicate that HDLs are atheroprotective (4). However, recent clinical trials of therapeutics that generically raise HDL, at least as measured by its cholesterol levels, have failed to confer the expected CVD protections (5–7).Although these traditional density-centric definitions have been used for nearly 40 years, accumulating evidence indicates that they are not particularly reflective of lipoprotein compositional and functional complexity. With respect to most physical traits (size, charge, lipid content, protein content, etc.), one can demonstrate significant heterogeneity within each density class. This suggests that particle subspecies exist with unique functions and effects on disease. For example, LDL can be resolved into large, buoyant and small, dense forms (8), with subjects carrying more cholesterol in the small, dense LDL exhibiting a greater CVD risk (9). HDL is particularly noted for heterogeneity, as it can be separated into numerous subfractions by density (10), diameter (11), charge (12), and major apolipoprotein content (13). Most strikingly, recent applications of soft-ionization mass spectrometry (MS) have identified upward of 85 HDL proteins with functions that go well beyond the structural apolipoproteins, lipid transport proteins, and lipid-modifying enzymes known from previous biochemical studies (14, 15). Many of these proteins imply functions as diverse as complement regulation, acute phase response, protease inhibition, and innate immunity (16). Individual HDL subspecies can apparently draw from this palette of proteins to produce distinct particles of distinct function. One well-defined HDL subfraction, termed trypanosome lytic factor, contains apolipoprotein apoA-I, haptoglobin-related protein, and apoL-I. Working together, these proteins enter the trypanosome brucei brucei and kill it via lysosomal disruption (17). There are numerous other instances of on-particle protein cooperation in HDL related to CVD (reviewed in Ref. 15). Furthermore, two-dimensional electrophoresis studies by Asztalos and colleagues (18), as well as our own work (11, 19), strongly support the concept that certain apolipoproteins segregate among different HDL particles. These observations present the intriguing possibility that the phospholipids of HDLs act as an organizing platform that facilitates the assembly of specific protein complexes (20). Such subspecies could have important functional implications in the context of CVD protection, inflammation, or even innate immune function. Furthermore, this subspeciation may explain why therapeutics that raise HDL cholesterol levels across the board have not yet shown promise with regard to CVD.To address this hypothesis, we began to think of lipoproteins as a continuum of phospholipid platforms that support the assembly of specific protein complexes analogous to those in cells that perform coordinated biological functions (i.e. ribosomes, centrosomes, etc.). Two common methods for characterizing protein complexes are tandem affinity purification (21) and immunoprecipitation. Both rely on the specific pull-down of a target protein (by either an introduced affinity tag or an antibody) followed by the identification of co-precipitated proteins via MS. Unfortunately, tandem affinity purification strategies are impractical in humans, and we have found that immunoprecipitation experiments with human plasma lipoproteins result in a high false-positive rate due to the low abundance of most of these proteins, particularly those in HDLs. Therefore, we took an alternative approach called co-separation analysis, a method based on the principle that stable protein complexes can be identified by tracking their co-migration as they undergo biochemical separation by multiple orthogonal approaches (22). Native proteins are analyzed in an unbiased manner without affinity tags or antibodies, and purification to homogeneity is not necessary for the identification of putative protein complexes.Most current studies of the lipoprotein proteome utilize samples isolated via density ultracentrifugation because contaminating lipid-unassociated lipoproteins, which can be highly abundant and obscure the identification of targeted lipid-associated proteins, are thus removed prior to the analysis. In previous work, we characterized the use of a calcium silica hydrate (CSH) resin that allowed the specific isolation of phospholipid-associated proteins and their subsequent MS identification without ultracentrifugation (11). This advance enabled the use of a variety of non-density-based separation methods for the study of plasma lipoproteins. Here, we take advantage of this to analyze the proteome of human plasma lipoproteins separated via three separation techniques that exploit different physicochemical properties: (i) gel filtration chromatography (size), (ii) anion exchange chromatography (charge interaction), and (iii) isoelectric focusing. By tracking the co-migration of specific proteins across these separations (Fig. 1), we identified a host of putative protein pairings, including the previously known trypanosome lytic factor HDL fraction, for further biochemical verification and characterization.Open in a separate windowFig. 1.Overview of the multi-dimensional separation co-migration analysis used in this study (see “Experimental Procedures” for details).     

System-wide Analysis Reveals Intrinsically Disordered Proteins Are Prone to Ubiquitylation after Misfolding Stress

Damaged and misfolded proteins that are no longer functional in the cell need to be eliminated. Failure to do so might lead to their accumulation and aggregation, a hallmark of many neurodegenerative diseases. Protein quality control pathways play a major role in the degradation of these proteins, which is mediated mainly by the ubiquitin proteasome system. Despite significant focus on identifying ubiquitin ligases involved in these pathways, along with their substrates, a systems-level understanding of these pathways has been lacking. For instance, as misfolded proteins are rapidly ubiquitylated, unconjugated ubiquitin is rapidly depleted from the cell upon misfolding stress; yet it is unknown whether certain targets compete more efficiently to be ubiquitylated. Using a system-wide approach, we applied statistical and computational methods to identify characteristics enriched among proteins that are further ubiquitylated after heat shock. We discovered that distinct populations of structured and, surprisingly, intrinsically disordered proteins are prone to ubiquitylation. Proteomic analysis revealed that abundant and highly structured proteins constitute the bulk of proteins in the low-solubility fraction after heat shock, but only a portion is ubiquitylated. In contrast, ubiquitylated, intrinsically disordered proteins are enriched in the low-solubility fraction after heat shock. These proteins have a very low abundance in the cell, are rarely encoded by essential genes, and are enriched in binding motifs. In additional experiments, we confirmed that several of the identified intrinsically disordered proteins were ubiquitylated after heat shock and demonstrated for two of them that their disordered regions are important for ubiquitylation after heat shock. We propose that intrinsically disordered regions may be recognized by the protein quality control machinery and thereby facilitate the ubiquitylation of proteins after heat shock.Cells face the constant threat of protein misfolding and aggregation, and thus protein quality control pathways are important in selectively targeting damaged and misfolded proteins for degradation (1, 2). The ubiquitin proteasome system serves as a major mediator of this pathway by conjugating the small protein ubiquitin onto substrates through the E1-E2-E3 (ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin ligase, respectively) cascade for their recognition and degradation by the proteasome (3, 4). It is known that the activity of the ubiquitin-proteasome system is associated with many neurodegenerative diseases. For instance, ubiquitin is found enriched in protein inclusions associated with these diseases (5). Furthermore, proteasome activity has been shown to decrease with age in a large variety of organisms (6), leading to increased proteotoxicity in the cell.Because of the importance of maintaining protein homeostasis, numerous ubiquitin ligases in different cellular compartments function in protein quality control pathways to target misfolded or damaged proteins for degradation via the proteasome. For instance, the conserved Hrd1 ubiquitin ligase is involved in the endoplasmic-reticulum-associated degradation pathway that targets endoplasmic reticulum proteins for retro-translocation to the cytoplasm and proteasome degradation (7). A major question is what features are recognized by ubiquitin ligases that allow them to selectively target terminally misfolded proteins for degradation, given that the folding rates and physicochemical properties vary largely from protein to protein. Several E3 ubiquitin ligases involved in cytosolic protein quality control target their substrates via their interactions with chaperone proteins. For instance, the CHIP ubiquitin ligase can directly bind to Hsp70 and Hsp90 proteins (8), which may hand over client proteins that are not successfully folded. Understanding which features are recognized by these degradation quality-control pathways might help us understand how certain misfolded proteins evade this system, leading to their accumulation and aggregation in the cell.Many studies investigating degradation protein quality control have employed model substrates (e.g. mutated proteins that misfold) to reveal which components are involved in a given quality control machinery. However, these approaches do not typically reveal the whole spectrum of substrates for these pathways. Thus, alternative system-wide approaches are also needed to provide a bigger picture. Heat shock (HS)¹ induces general misfolding at the proteome level by increasing thermal energy and was shown to cause an increase in ubiquitylation levels in the cell over 25 years ago (9, 10). However, the exact mechanism and pathways that target misfolded proteins have remained uncharacterized for a long time. We recently showed that the Hul5 ubiquitin ligase plays a major role in this heat stress response that mainly affects cytosolic proteins (11). Absence of Hul5 averts the ubiquitylation in the cytoplasm of several misfolded targets after HS, as well as low-solubility proteins in unstressed cells. Other E3 ubiquitin ligases are likely involved in this pathway (12). Interestingly, as ubiquitin constitutes about only 1% of the proteome, free unconjugated ubiquitin is rapidly depleted under stress conditions (13, 14). Given the limited amount of this protein, how does the cell triage ubiquitin among an excess of misfolded proteins? In order to gain systems-level insight, we sought to identify characteristics enriched among proteins ubiquitylated after HS using a combination of statistical and computational analysis, and we conducted additional proteomics and biochemical experiments to support our hypotheses. We discovered an unexpected susceptibility of intrinsically disordered proteins for ubiquitylation after misfolding stress.     

Projected climate impacts to South African maize and wheat production in 2055: a comparison of empirical and mechanistic modeling approaches

Crop model‐specific biases are a key uncertainty affecting our understanding of climate change impacts to agriculture. There is increasing research focus on intermodel variation, but comparisons between mechanistic (MMs) and empirical models (EMs) are rare despite both being used widely in this field. We combined MMs and EMs to project future (2055) changes in the potential distribution (suitability) and productivity of maize and spring wheat in South Africa under 18 downscaled climate scenarios (9 models run under 2 emissions scenarios). EMs projected larger yield losses or smaller gains than MMs. The EMs’ median‐projected maize and wheat yield changes were ?3.6% and 6.2%, respectively, compared to 6.5% and 15.2% for the MM. The EM projected a 10% reduction in the potential maize growing area, where the MM projected a 9% gain. Both models showed increases in the potential spring wheat production region (EM = 48%, MM = 20%), but these results were more equivocal because both models (particularly the EM) substantially overestimated the extent of current suitability. The substantial water‐use efficiency gains simulated by the MMs under elevated CO₂ accounted for much of the EM?MM difference, but EMs may have more accurately represented crop temperature sensitivities. Our results align with earlier studies showing that EMs may show larger climate change losses than MMs. Crop forecasting efforts should expand to include EM?MM comparisons to provide a fuller picture of crop–climate response uncertainties.     

Simulated warming does not impair seedling survival and growth of Nothofagus pumilio in the southern Andes

It has been predicted that subalpine forests will be negatively affected by global warming; however, direct responses to experimental warming have been scarcely examined in these systems. In this study we evaluated the effects of higher temperatures with and without water addition on the survival and growth of recently emerged (small) and large seedlings of the widely distributed species Nothofagus pumilio in subalpine forests of the southern Chilean Andes. We also examined the variations in seedling traits related to carbon balance in order to infer the causal mechanisms of survival and growth responses. Treatments of open top chambers (OTCs) were combined with watering in two locations with differing climates: Antillanca (40°S, humid) and Cerro Castillo (46°S, drier). OTCs increased mean and maximum air temperatures by 0.6 °C and 2–3 °C, respectively, and decreased soil humidity by 56% in Antillanca and 30% in Cerro Castillo, fulfilling methodological expectations and climate model predictions. After two complete growing seasons, the survival, relative growth rate (RGR), biomass, and a suite of seedling traits were measured and analyzed using mixed-effects models. Warming and warming in combination with watering significantly increased large seedling survival in Cerro Castillo. In Antillanca, warmer conditions increased the height, biomass, and leaf area of small seedlings, and the RGR of large seedlings. In this location, warming also caused lower leaf carbon isotopic composition in both age classes and higher specific leaf area in small seedlings, suggesting whole-plant carbon gain improvements; warming did not produce any drought effects. Our results indicate that warming produces positive effects on the seedling establishment of N. pumilio in the southern Andes, highlighting the importance of site-specific effects in response to climate change in widespread species. Site-specific effects can most likely explain the discrepancies between the results of this study and the predictions outlined by previous studies for these forests.     

Of mast and mean: differential‐temperature cue makes mast seeding insensitive to climate change

Mast‐seeding plants often produce high seed crops the year after a warm spring or summer, but the warm‐temperature model has inconsistent predictive ability. Here, we show for 26 long‐term data sets from five plant families that the temperature difference between the two previous summers (ΔT) better predicts seed crops. This discovery explains how masting species tailor their flowering patterns to sites across altitudinal temperature gradients; predicts that masting will be unaffected by increasing mean temperatures under climate change; improves prediction of impacts on seed consumers; demonstrates that strongly masting species are hypersensitive to climate; explains the rarity of consecutive high‐seed years without invoking resource constraints; and generates hypotheses about physiological mechanisms in plants and insect seed predators. For plants, ΔT has many attributes of an ideal cue. This temperature‐difference model clarifies our understanding of mast seeding under environmental change, and could also be applied to other cues, such as rainfall.     

Rapid Expectation Adaptation during Syntactic Comprehension

When we read or listen to language, we are faced with the challenge of inferring intended messages from noisy input. This challenge is exacerbated by considerable variability between and within speakers. Focusing on syntactic processing (parsing), we test the hypothesis that language comprehenders rapidly adapt to the syntactic statistics of novel linguistic environments (e.g., speakers or genres). Two self-paced reading experiments investigate changes in readers’ syntactic expectations based on repeated exposure to sentences with temporary syntactic ambiguities (so-called “garden path sentences”). These sentences typically lead to a clear expectation violation signature when the temporary ambiguity is resolved to an a priori less expected structure (e.g., based on the statistics of the lexical context). We find that comprehenders rapidly adapt their syntactic expectations to converge towards the local statistics of novel environments. Specifically, repeated exposure to a priori unexpected structures can reduce, and even completely undo, their processing disadvantage (Experiment 1). The opposite is also observed: a priori expected structures become less expected (even eliciting garden paths) in environments where they are hardly ever observed (Experiment 2). Our findings suggest that, when changes in syntactic statistics are to be expected (e.g., when entering a novel environment), comprehenders can rapidly adapt their expectations, thereby overcoming the processing disadvantage that mistaken expectations would otherwise cause. Our findings take a step towards unifying insights from research in expectation-based models of language processing, syntactic priming, and statistical learning.