Andrew H. Lee

mirWIP: microRNA target prediction based on microRNA-containing ribonucleoprotein-enriched transcripts

Target prediction for animal microRNAs (miRNAs) has been hindered by the small number of verified targets available to evaluate the accuracy of predicted miRNA-target interactions. Recently, a dataset of 3,404 miRNA-associated mRNA transcripts was identified by immunoprecipitation of the RNA-induced silencing complex components AIN-1 and AIN-2. Our analysis of this AIN-IP dataset revealed enrichment for defining characteristics of functional miRNA-target interactions, including structural accessibility of target sequences, total free energy of miRNA-target hybridization and topology of base-pairing to the 5′ seed region of the miRNA. We used these enriched characteristics as the basis for a quantitative miRNA target prediction method, miRNA targets by weighting immunoprecipitation-enriched parameters (mirWIP), which optimizes sensitivity to verified miRNA-target interactions and specificity to the AIN-IP dataset. MirWIP can be used to capture all known conserved miRNA-mRNA target relationships in Caenorhabditis elegans at a lower false-positive rate than can the current standard methods.

Site-specific genome editing in Plasmodium falciparum using engineered zinc-finger nucleases

Malaria afflicts over 200 million people worldwide, and its most lethal etiologic agent, Plasmodium falciparum, is evolving to resist even the latest-generation therapeutics. Efficient tools for genome-directed investigations of P. falciparum-induced pathogenesis, including drug-resistance mechanisms, are clearly required. Here we report rapid and targeted genetic engineering of this parasite using zinc-finger nucleases (ZFNs) that produce a double-strand break in a user-defined locus and trigger homology-directed repair. Targeting an integrated egfp locus, we obtained gene-deletion parasites with unprecedented speed (2 weeks), both with and without direct selection. ZFNs engineered against the parasite gene pfcrt, responsible for escape under chloroquine treatment, rapidly produced parasites that carried either an allelic replacement or a panel of specified point mutations. This method will enable a diverse array of genome-editing approaches to interrogate this human pathogen.

DNA Repair Mechanisms and Their Biological Roles in the Malaria Parasite Plasmodium falciparum

SUMMARY Research into the complex genetic underpinnings of the malaria parasite Plasmodium falciparum is entering a new era with the arrival of site-specific genome engineering. Previously restricted only to model systems but now expanded to most laboratory organisms, and even to humans for experimental gene therapy studies, this technology allows researchers to rapidly generate previously unattainable genetic modifications. This technological advance is dependent on DNA double-strand break repair (DSBR), specifically homologous recombination in the case of Plasmodium. Our understanding of DSBR in malaria parasites, however, is based largely on assumptions and knowledge taken from other model systems, which do not always hold true in Plasmodium. Here we describe the causes of double-strand breaks, the mechanisms of DSBR, and the differences between model systems and P. falciparum. These mechanisms drive basic parasite functions, such as meiosis, antigen diversification, and copy number variation, and allow the parasite to continually evolve in the contexts of host immune pressure and drug selection. Finally, we discuss the new technologies that leverage DSBR mechanisms to accelerate genetic investigations into this global infectious pathogen.

Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite’s food vacuole and alter drug sensitivities

Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs.

Soft tissue ossification and condylar cartilage degeneration following TMJ disc perforation in a rabbit pilot study

OBJECTIVE There are limited clinical treatments for temporomandibular joint (TMJ) pathologies, including degenerative disease, disc perforation and heterotopic ossification (HO). One barrier hindering the development of new therapies is that animal models recapitulating TMJ diseases are poorly established. The objective of this study was to develop an animal model for TMJ cartilage degeneration and disc pathology, including disc perforation and soft tissue HO. METHODS New Zealand white rabbits (n = 9 rabbits) underwent unilateral TMJ disc perforation surgery and sham surgery on the contralateral side. A 2.5 mm defect was created using a punch biopsy in rabbit TMJ disc. The TMJ condyles and discs were evaluated macroscopically and histologically after 4, 8 and 12 weeks. Condyles were blindly scored by four independent observers using OARSI recommendations for macroscopic and histopathological scoring of osteoarthritis (OA) in rabbit tissues. RESULTS Histological evidence of TMJ condylar cartilage degeneration was apparent in experimental condyles following disc perforation relative to sham controls after 4 and 8 weeks, including surface fissures and loss of Safranin O staining. At 12 weeks, OARSI scores indicated experimental condylar cartilage erosion into the subchondral bone. Most strikingly, HO occurred within the TMJ disc upon perforation injury in six rabbits after 8 and 12 weeks. CONCLUSION We report for the first time a rabbit TMJ injury model that demonstrates condylar cartilage degeneration and disc ossification, which is indispensible for testing the efficacy of potential TMJ therapies.

Evolution of Fitness Cost-Neutral Mutant PfCRT Conferring P . falciparum 4-Aminoquinoline Drug Resistance Is Accompanied by Altered Parasite Metabolism and Digestive Vacuole Physiology

Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pressures on the subcontinent have recurrently produced several allelic variants of parasite drug resistance genes, including the P. falciparum chloroquine resistance transporter (pfcrt). Despite significant reductions in the deployment of the 4-aminoquinoline drug chloroquine (CQ), which selected for the mutant pfcrt alleles that halted CQ efficacy decades ago, the parasite pfcrt locus is continuously evolving. This is highlighted by the presence of a highly mutated allele, Cam734 pfcrt, which has acquired the singular ability to confer parasite CQ resistance without an associated fitness cost. Here, we used pfcrt-specific zinc-finger nucleases to genetically dissect this allele in the pathogenic setting of asexual blood-stage infection. Comparative analysis of drug resistance and growth profiles of recombinant parasites that express Cam734 or variants thereof, Dd2 (the most common Southeast Asian variant), or wild-type pfcrt, revealed previously unknown roles for PfCRT mutations in modulating parasite susceptibility to multiple antimalarial agents. These results were generated in the GC03 strain, used in multiple earlier pfcrt studies, and might differ in natural isolates harboring this allele. Results presented herein show that Cam734-mediated CQ resistance is dependent on the rare A144F mutation that has not been observed beyond Southeast Asia, and reveal distinct impacts of this and other Cam734-specific mutations on CQ resistance and parasite growth rates. Biochemical assays revealed a broad impact of mutant PfCRT isoforms on parasite metabolism, including nucleoside triphosphate levels, hemoglobin catabolism and disposition of heme, as well as digestive vacuole volume and pH. Results from our study provide new insights into the complex molecular basis and physiological impact of PfCRT-mediated antimalarial drug resistance, and inform ongoing efforts to characterize novel pfcrt alleles that can undermine the efficacy of first-line antimalarial drug regimens.

Evidence of a Mild Mutator Phenotype in Cambodian Plasmodium falciparum Malaria Parasites

Malaria control efforts have been continuously stymied by drug-resistant strains of Plasmodium falciparum, which typically originate in Southeast Asia prior to spreading into high-transmission settings in Africa. One earlier proposed explanation for Southeast Asia being a hotbed of resistance has been the hypermutability or “Accelerated Resistance to Multiple Drugs” (ARMD) phenotype, whereby multidrug-resistant Southeast Asian parasites were reported to exhibit 1,000-fold higher rates of resistance to unrelated antimalarial agents when compared to drug-sensitive parasites. However, three recent studies do not recapitulate this hypermutability phenotype. Intriguingly, genome sequencing of recently derived multidrug-resistant Cambodian isolates has identified a high proportion of DNA repair gene mutations in multidrug-resistant parasites, suggesting their potential role in shaping local parasite evolution. By adapting fluctuation assays for use in P. falciparum, we have examined the in vitro mutation rates of five recent Cambodian isolates and three reference laboratory strains. For these studies we also generated a knockout parasite line lacking the DNA repair factor Exonuclease I. In these assays, parasites were typed for their ability to acquire resistance to KAE609, currently in advanced clinical trials, yielding 13 novel mutations in the Na+/H+-ATPase PfATP4, the primary resistance determinant. We observed no evidence of hypermutability. Instead, we found evidence of a mild mutator (up to a 3.4-fold increase in mutation rate) phenotype in two artemisinin-resistant Cambodian isolates, which carry DNA repair gene mutations. We observed that one such mutation in the Mismatch Repair protein Mlh1 contributes to the mild mutator phenotype when modeled in yeast (scmlh1-P157S). Compared to basal rates of mutation, a mild mutator phenotype may provide a greater overall benefit for parasites in Southeast Asia in terms of generating drug resistance without incurring detrimental fitness costs.

Are e‐cigarettes effective in smoking cessation?

BACKGROUND Electronic nicotine delivery systems (ENDS; commonly known as electronic cigarettes or e-cigarettes) have been commercially available in the United States since 2007, yet recently have generated substantial controversy as their use and availability expands. ENDS are battery-operated devices designed to resemble traditional cigarettes that heat and vaporize nicotinecontaining solutions. Each device contains a microelectrical circuit, activated by inhaling through a mouthpiece that vaporizes a propylene glycol–nicotine solution and delivers it to the user, an act often termed vaping. They are ubiquitously available commercially, sold from vendors ranging from the local corner gas station and drug store to the Internet marketplace. ENDS do not depend on combustion, meaning that the user and bystanders are theoretically not exposed to many of the harmful compounds and particulate matter produced by traditional cigarettes. Recently, there has been a drastic increase in usage and awareness of ENDS, largely as a result of shrewd, aggressive marketing. Although ENDS are marketed as a safer and healthier alternative to conventional cigarettes, much remains unknown. The longterm safety data on ENDS is scant and inconsistent, and there is a lack of internationally certified manufacturing sites. Whereas some public health officials have embraced ENDS as a potential pathway to smoking reduction or cessation, other experts are concerned that ENDS could undermine a decades-long public health campaign to denormalize and stigmatize smoking. These experts are concerned that ENDS could maintain nicotine addiction by deterring smokers from utilizing proven cessation tools, thus acting as a gateway to future smoking and increasing nicotine addiction among youth. As physicians primarily involved in the treatment of head and neck malignancies, otolaryngologists have a particular responsibility in guiding patients toward effective methods of tobacco cessation. Furthermore, evidence-based recommendations on the efficacy of ENDS in smoking cessation will be paramount for sound policy development.

CPAS: Surgical approach with combined sublabial bone resection and inferior turbinate reduction without stents

Congenital pyriform aperture stenosis (CPAS) is a form of nasal obstruction caused by congenital narrowing of the maxilla at the medial processes. Traditionally, surgical correction involves a sublabial approach with subperiosteal dissection, widening of the aperture by drilling, and the use of nasal stents postoperatively. Although this approach may lead to symptomatic improvement, it alone may fail to provide a patent airway secondary to unaddressed posterior narrowing. Additionally, the use of stents is problematic because they are prone to clogging and can cause internal nasal scarring and septal or alar necrosis. We present the surgical management of this condition in six patients using a novel approach that aims to correct these limitations by including both the traditional sublabial procedure and an endonasal reduction of the inferior turbinates, without the use of stents postoperatively.

Evidence for Regulation of Hemoglobin Metabolism and Intracellular Ionic Flux by the Plasmodium falciparum Chloroquine Resistance Transporter

Plasmodium falciparum multidrug resistance constitutes a major obstacle to the global malaria elimination campaign. Specific mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) mediate resistance to the 4-aminoquinoline drug chloroquine and impact parasite susceptibility to several partner agents used in current artemisinin-based combination therapies, including amodiaquine. By examining gene-edited parasites, we report that the ability of the wide-spread Dd2 PfCRT isoform to mediate chloroquine and amodiaquine resistance is substantially reduced by the addition of the PfCRT L272F mutation, which arose under blasticidin selection. We also provide evidence that L272F confers a significant fitness cost to asexual blood stage parasites. Studies with amino acid-restricted media identify this mutant as a methionine auxotroph. Metabolomic analysis also reveals an accumulation of short, hemoglobin-derived peptides in the Dd2 + L272F and Dd2 isoforms, compared with parasites expressing wild-type PfCRT. Physiologic studies with the ionophores monensin and nigericin support an impact of PfCRT isoforms on Ca2+ release, with substantially reduced Ca2+ levels observed in Dd2 + L272F parasites. Our data reveal a central role for PfCRT in regulating hemoglobin catabolism, amino acid availability, and ionic balance in P. falciparum, in addition to its role in determining parasite susceptibility to heme-binding 4-aminoquinoline drugs.