Polymorphisms in K13 and Falcipain-2 Associated with Artemisinin Resistance Are Not Prevalent in Plasmodium falciparum Isolated from Ugandan Children

The emergence of resistance to artemisinin derivatives in Southeast Asia, manifested as delayed clearance of Plasmodium falciparum following treatment with artemisinins, is a major concern. Recently, the artemisinin resistance phenotype was attributed to mutations in portions of a P. falciparum gene (PF3D7_1343700) encoding kelch (K13) propeller domains, providing a molecular marker to monitor the spread of resistance. The P. falciparum cysteine protease falcipain-2 (FP2; PF3D7_1115700) has been shown to contribute to artemisinin action, as hemoglobin degradation is required for potent drug activity, and a stop mutation in the FP2 gene was identified in parasites selected for artemisinin resistance. Although delayed parasite clearance after artemisinin-based combination therapy (ACT) has not yet been noted in Uganda and ACTs remain highly efficacious, characterizing the diversity of these genes is important to assess the potential for resistance selection and to provide a baseline for future surveillance. We therefore sequenced the K13-propeller domain and FP2 gene in P. falciparum isolates from children previously treated with ACT in Uganda, including samples from 2006–7 (n = 49) and from 2010–12 (n = 175). Using 3D7 as the reference genome, we identified 5 non-synonymous polymorphisms in the K13-propeller domain (133 isolates) and 35 in FP2 (160 isolates); these did not include the polymorphisms recently associated with resistance after in vitro selection or identified in isolates from Asia. The prevalence of K13-propeller and FP2 polymorphisms did not increase over time, and was not associated with either time since prior receipt of an ACT or the persistence of parasites ≥2 days following treatment with an ACT. Thus, the K13-propeller and FP2 polymorphisms associated with artemisinin resistance are not prevalent in Uganda, and we did not see evidence for selection of polymorphisms in these genes.     

Rapid Isolation of Extracellular Vesicles from Cell Culture and Biological Fluids Using a Synthetic Peptide with Specific Affinity for Heat Shock Proteins

Recent studies indicate that extracellular vesicles are an important source material for many clinical applications, including minimally-invasive disease diagnosis. However, challenges for rapid and simple extracellular vesicle collection have hindered their application. We have developed and validated a novel class of peptides (which we named venceremin, or Vn) that exhibit nucleotide-independent specific affinity for canonical heat shock proteins. The Vn peptides were validated to specifically and efficiently capture HSP-containing extracellular vesicles from cell culture growth media, plasma, and urine by electron microscopy, atomic force microscopy, sequencing of nucleic acid cargo, proteomic profiling, immunoblotting, and nanoparticle tracking analysis. All of these analyses confirmed the material captured by the Vn peptides was comparable to those purified by the standard ultracentrifugation method. We show that the Vn peptides are a useful tool for the rapid isolation of extracellular vesicles using standard laboratory equipment. Moreover, the Vn peptides are adaptable to diverse platforms and therefore represent an excellent solution to the challenge of extracellular vesicle isolation for research and clinical applications.     

Initial Characterization of the FlgE Hook High Molecular Weight Complex of Borrelia burgdorferi

The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility.     

A MIV-150/Zinc Acetate Gel Inhibits SHIV-RT Infection in Macaque Vaginal Explants

To extend our observations that single or repeated application of a gel containing the NNRTI MIV-150 (M) and zinc acetate dihydrate (ZA) in carrageenan (CG) (MZC) inhibits vaginal transmission of simian/human immunodeficiency virus (SHIV)-RT in macaques, we evaluated safety and anti-SHIV-RT activity of MZC and related gel formulations ex vivo in macaque mucosal explants. In addition, safety was further evaluated in human ectocervical explants. The gels did not induce mucosal toxicity. A single ex vivo exposure to diluted MZC (1∶30, 1∶100) and MC (1∶30, the only dilution tested), but not to ZC gel, up to 4 days prior to viral challenge, significantly inhibited SHIV-RT infection in macaque vaginal mucosa. MZC''s activity was not affected by seminal plasma. The antiviral activity of unformulated MIV-150 was not enhanced in the presence of ZA, suggesting that the antiviral activity of MZC was mediated predominantly by MIV-150. In vivo administration of MZC and CG significantly inhibited ex vivo SHIV-RT infection (51–62% inhibition relative to baselines) of vaginal (but not cervical) mucosa collected 24 h post last gel exposure, indicating barrier effect of CG. Although the inhibitory effect of MZC (65–74%) did not significantly differ from CG (32–45%), it was within the range of protection (∼75%) against vaginal SHIV-RT challenge 24 h after gel dosing. Overall, the data suggest that evaluation of candidate microbicides in macaque explants can inform macaque efficacy and clinical studies design. The data support advancing MZC gel for clinical evaluation.     

Unbiased Identification of Patients with Disorders of Sex Development

Disorders of sex development (DSD) represent a collection of rare diseases that generate substantial controversy regarding best practices for diagnosis and treatment. A significant barrier preventing a better understanding of how patients with these conditions should be evaluated and treated, especially from a psychological standpoint, is the lack of systematic and standardized approaches to identify cases for study inclusion. Common approaches include “hand-picked” subjects already known to the practice, which could introduce bias. We implemented an informatics-based approach to identify patients with DSD from electronic health records (EHRs) at three large, academic children’s hospitals. The informatics approach involved comprehensively searching EHRs at each hospital using a combination of structured billing codes as an initial filtering strategy followed by keywords applied to the free text clinical documentation. The informatics approach was implemented to replicate the functionality of an EHR search engine (EMERSE) available at one of the hospitals. At the two hospitals that did not have EMERSE, we compared case ascertainment using the informatics method to traditional approaches employed for identifying subjects. Potential cases identified using all approaches were manually reviewed by experts in DSD to verify eligibility criteria. At the two institutions where both the informatics and traditional approaches were applied, the informatics approach identified substantially higher numbers of potential study subjects. The traditional approaches yielded 14 and 28 patients with DSD, respectively; the informatics approach yielded 226 and 77 patients, respectively. The informatics approach missed only a few cases that the traditional approaches identified, largely because those cases were known to the study team, but patient data were not in the particular children’s hospital EHR. The use of informatics approaches to search electronic documentation can result in substantially larger numbers of subjects identified for studies of rare diseases such as DSD, and these approaches can be applied across hospitals.     

Large Scale Spatial Risk and Comparative Prevalence of Borrelia miyamotoi and Borrelia burgdorferi Sensu Lato in Ixodes pacificus

Borrelia miyamotoi is a newly described emerging pathogen transmitted to people by Ixodes species ticks and found in temperate regions of North America, Europe, and Asia. There is limited understanding of large scale entomological risk patterns of B. miyamotoi and of Borreila burgdorferi sensu stricto (ss), the agent of Lyme disease, in western North America. In this study, B. miyamotoi, a relapsing fever spirochete, was detected in adult (n = 70) and nymphal (n = 36) Ixodes pacificus ticks collected from 24 of 48 California counties that were surveyed over a 13 year period. Statewide prevalence of B. burgdorferi sensu lato (sl), which includes B. burgdorferi ss, and B. miyamotoi were similar in adult I. pacificus (0.6% and 0.8%, respectively). In contrast, the prevalence of B. burgdorferi sl was almost 2.5 times higher than B. miyamotoi in nymphal I. pacificus (3.2% versus 1.4%). These results suggest similar risk of exposure to B. burgdorferi sl and B. miyamotoi from adult I. pacificus tick bites in California, but a higher risk of contracting B. burgdorferi sl than B. miyamotoi from nymphal tick bites. While regional risk of exposure to these two spirochetes varies, the highest risk for both species is found in north and central coastal California and the Sierra Nevada foothill region, and the lowest risk is in southern California; nevertheless, tick-bite avoidance measures should be implemented in all regions of California. This is the first study to comprehensively evaluate entomologic risk for B. miyamotoi and B. burgdorferi for both adult and nymphal I. pacificus, an important human biting tick in western North America.     

Bi-Epitope SPR Surfaces: A Solution to Develop Robust Immunoassays

Surface plasmon resonance (SPR)-based immunoassays have numerous applications and require high affinity reagents for sensitive and reliable measurements. We describe a quick approach to turn low affinity antibodies into appropriate capture reagents. We used antibodies recognizing human ephrin type A receptor 2 (EphA2) and a ProteOn XPR36 as a model system. We generated so-called ‘bi-epitope’ sensor surfaces by immobilizing various pairs of anti-EphA2 antibodies using standard amine coupling. The apparent binding affinities to EphA2 and EphA2 detection sensitivities of the bi-epitope and ‘single-epitope’ surfaces were then compared. For all antibody pairs tested, bi-epitope surfaces exhibited an ∼10–100-fold improvement in apparent binding affinities when compared with single-epitope ones. When pairing 2 antibodies of low intrinsic binding affinities (∼10⁻⁸ M) and fast dissociation rates (∼10⁻² s⁻¹), the apparent binding affinity and dissociation rate of the bi-epitope surface was improved up to ∼10^–10 M and 10⁻⁴ s⁻¹, respectively. This led to an ∼100–200-fold enhancement in EphA2 limit of detection in crude cell supernatants. Our results show that the use of antibody mixtures in SPR applications constitutes a powerful approach to develop sensitive immunoassays, as previously shown for non-SPR formats. As SPR-based assays have significantly expanded their reach in the last decade, such an approach promises to further accelerate their development.     

Is Matang Mangrove Forest in Malaysia Sustainably Rejuvenating after More than a Century of Conservation and Harvesting Management?

Matang Mangrove Forest Reserve (MMFR) in Peninsular Malaysia is under systematic management since 1902 and still considered as the best managed mangrove forest in the world. The present study on silvimetrics assessed the ongoing MMFR forest management, which includes a first thinning after 15 years, a second thinning after 20 years and clear-felling of 30-year old forest blocks, for its efficiency and productivity in comparison to natural mangroves. The estimated tree structural parameters (e.g. density, frequency) from three different-aged mangrove blocks of fifteen (MF15), twenty (MF20), and thirty (MF30) years old indicated that Bruguiera and Excoecaria spp. did not constitute a significant proportion of the vegetation (<5%), and hence the results focused majorly on Rhizophora apiculata. The density of R. apiculata at MF15, MF20 and MF30 was 4,331, 2,753 and 1,767 stems ha⁻¹, respectively. In relation to ongoing practices of the artificial thinnings at MMFR, the present study suggests that the first thinning could be made earlier to limit the loss of exploitable wood due to natural thinning. In fact, the initial density at MF15 was expected to drop down from 6,726 to 1,858 trees ha⁻¹ before the first thinning. Therefore the trees likely to qualify for natural thinning, though having a smaller stem diameter, should be exploited for domestic/commercial purposes at an earlier stage. The clear-felling block (MF30) with a maximum stem diameter of 30 cm was estimated to yield 372 t ha⁻¹ of the above-ground biomass and suggests that the mangrove management based on a 30-year rotation is appropriate for the MMFR. Since Matang is the only iconic site that practicing sustainable wood production, it could be an exemplary to other mangrove locations for their improved management.     

Executive Function and Mental Health in Adopted Children with a History of Recreational Drug Exposures

Adoptive children are at increased risk for problematic behaviors but the origin of these individual differences in neurobehavioral function is unclear. This investigation examined whether adopted children with prenatal exposure to a wide variety of recreational drugs exhibited higher scores (i.e. more problems) with executive function and psychiatric symptomology. Caregivers of children ages 5 to 18 completed an online survey with items about use of alcohol, nicotine, or methamphetamine during pregnancy followed by the Behavior Rating Inventory of Executive Function (BRIEF, N = 437 including 59 adoptive parents) or the Child Behavior Checklist (CBCL, N = 549 including 54 adoptive parents). Relative to a comparison group of children raised by their biological parents, adoptive children that were polysubstance exposed during prenatal development exhibited higher rates of academic difficulties and were behind their classmates in math and reading. Adoptive children had statistically and clinically significant higher BRIEF ratings and this pattern was similar for boys and girls. CBCL ratings were significantly increased in adoptive children, particularly for Externalizing and Attention problems. Adoptive children with a history of polysubstance exposures including alcohol, nicotine, and methamphetamine are at heightened risk for difficulties with executive function as well as various psychopathologies. These findings suggest that increased monitoring to identify and implement remediation strategies may be warranted for adopted children with a history of in utero drug exposures.     

Reward Associated with Singing Behavior Correlates with Opioid-Related Gene Expression in the Medial Preoptic Nucleus in Male European Starlings

Birdsong consists of species-specific learned vocal sequences that are used primarily to attract mates and to repel competitors during the breeding season. However, many birds continue to sing at times when vocal production has no immediate or obvious impact on conspecific behavior. The mechanisms that ensure that animals produce important behaviors in contexts in which the function of these behaviors is not immediate or obvious are not known. One possibility is that animals engage in such behaviors because they are associated with pleasure. Here we examined the hypothesis that male European starlings sing outside of the breeding season in part because the act of singing in this context is facilitated and/or maintained by opioid-mediated reward. We measured song-associated reward using a conditioned place preference (CPP) test in male starlings producing fall, non-breeding season-typical song. We used quantitative real time PCR to measure expression of the enkephalin opioid precursor preproenkephalin (PENK) and mu opioid receptors (MOR) in the medial preoptic nucleus (POM; a region in which opioids are implicated in both reward and starling fall song) and additionally the song control region HVC as a control. Starlings developed a strong preference for a place that had been paired previously with the act of producing fall-typical song, indicating that fall song production was associated with a positive affective state. Both PENK and MOR mRNA expression in the POM, but not HVC, correlated positively with both individual reward state (as reflected in CPP) and undirected singing behavior. These results suggest that singing induces opioid receptor and enkephalin expression in the POM and consequent reward, and/or that opioid release in the POM induced by individual or environmental factors (e.g., the presence of food, safety of a flock or the absence of predators) induces a positive affective state which then facilitates singing behavior.