A Revised Molecular Model of Ovarian Cancer Biomarker CA125 (MUC16) Enabled by Long-read Sequencing.

The biomarker CA125, a peptide epitope located in several tandem repeats of the mucin MUC16, is the gold standard for monitoring regression and recurrence of high-grade serous ovarian cancer in response to therapy. However, the CA125 epitope along with several structural features of the MUC16 molecule are ill defined. One central aspect still unresolved is the number of tandem repeats in MUC16 and how many of these repeats contain the CA125 epitope. Studies from the early 2000s assembled short DNA reads to estimate that MUC16 contained 63 repeats.Here, we conduct Nanopore long-read sequencing of MUC16 transcripts from three primary ovarian tumors and established cell lines (OVCAR3, OVCAR5, and Kuramochi) for a more exhaustive and accurate estimation and sequencing of the MUC16 tandem repeats.The consensus sequence derived from these six sources was confirmed by proteomics validation and agrees with recent additions to the NCBI database. We propose a model of MUC16 containing 19-not 63-tandem repeats. In addition, we predict the structure of the tandem repeat domain using the deep learning algorithm, AlphaFold.The predicted structure displays an SEA domain and unstructured linker region rich in proline, serine, and threonine residues in all 19 tandem repeats. These studies now pave the way for a detailed characterization of the CA125 epitope. Sequencing and modeling of the MUC16 tandem repeats along with their glycoproteomic characterization, currently underway in our laboratories, will help identify novel epitopes in the MUC16 molecule that improve on the sensitivity and clinical utility of the current CA125 assay. SIGNIFICANCE: Despite its crucial role in clinical management of ovarian cancer, the exact molecular sequence and structure of the biomarker, CA125, are not defined. Here, we combine long-read sequencing, mass spectrometry, and in silico modeling to provide the foundational dataset for a more complete characterization of the CA125 epitope.

Strategies for Mitigating Commercial Sensor Chip Variability with Experimental Design Controls.

Surface plasmon resonance (SPR) is a popular real-time technique for the measurement of binding affinity and kinetics, and bench-top instruments combine affordability and ease of use with other benefits of the technique. Biomolecular ligands labeled with the 6xHis tag can be immobilized onto sensing surfaces presenting the Ni2+-nitrilotriacetic acid (NTA) functional group. While Ni-NTA immobilization offers many advantages, including the ability to regenerate and reuse the sensors, its use can lead to signal variability between experimental replicates. We report here a study of factors contributing to this variability using the Nicoya OpenSPR as a model system and suggest ways to control for those factors, increasing the reproducibility and rigor of the data. Our model ligand/analyte pairs were two ovarian cancer biomarker proteins (MUC16 and HE4) and their corresponding monoclonal antibodies. We observed a broad range of non-specific binding across multiple NTA chips. Experiments run on the same chips had more consistent results in ligand immobilization and analyte binding than experiments run on different chips. Further assessment showed that different chips demonstrated different maximum immobilizations for the same concentration of injected protein. We also show a variety of relationships between ligand immobilization level and analyte response, which we attribute to steric crowding at high ligand concentrations. Using this calibration to inform experimental design, researchers can choose protein concentrations for immobilization corresponding to the linear range of analyte response. We are the first to demonstrate calibration and normalization as a strategy to increase reproducibility and data quality of these chips. Our study assesses a variety of factors affecting chip variability, addressing a gap in knowledge about commercially available sensor chips. Controlling for these factors in the process of experimental design will minimize variability in analyte signal when using these important sensing platforms.

Individual recombinant repeats of MUC16 display variable binding to CA125 antibodies.

BACKGROUND: Despite its importance in the clinical management of ovarian cancer, the CA125 biomarker - located on the mucin protein MUC16 - is still not completely understood. Questions remain about MUC16's function and structure, specifically the identity and location of the CA125 epitopes. OBJECTIVE: The goal of this study was to characterize the interaction of individual recombinant repeats from the tandem repeat domain of MUC16 with antibodies used in the clinical CA125 II test. METHODS: Using E. coli expression, we isolated nine repeats from the putative antigenic domain of CA125. Amino acid composition of recombinant repeats was confirmed by high-resolution mass spectrometry. We characterized the binding of four antibodies - OC125, M11, "OC125-like," and "M11-like" - to nine recombinant repeats using Western blotting, indirect enzyme-linked immunosorbent assay (ELISA), and localized surface plasmon resonance (SPR) spectroscopy. RESULTS: Each recombinant repeat was recognized by a different combination of CA125 antibodies. OC125 and "OC125-like" antibodies did not bind the same set of recombinant repeats, nor did M11 and "M11-like" antibodies. CONCLUSIONS: Characterization of the interactions between MUC16 recombinant repeats and CA125 antibodies will contribute to ongoing efforts to identify the CA125 epitopes and improve our understanding of this important biomarker.

Can a Deep Learning Algorithm Improve Detection of Occult Scaphoid Fractures in Plain Radiographs? A Clinical Validation Study.

BACKGROUND: Occult scaphoid fractures on initial radiographs of an injury are a diagnostic challenge to physicians. Although artificial intelligence models based on the principles of deep convolutional neural networks (CNN) offer a potential method of detection, it is unknown how such models perform in the clinical setting. QUESTIONS/PURPOSES: (1) Does CNN-assisted image interpretation improve interobserver agreement for scaphoid fractures? (2) What is the sensitivity and specificity of image interpretation performed with and without CNN assistance (as stratified by type: normal scaphoid, occult fracture, and apparent fracture)? (3) Does CNN assistance improve time to diagnosis and physician confidence level? METHODS: This survey-based experiment presented 15 scaphoid radiographs (five normal, five apparent fractures, and five occult fractures) with and without CNN assistance to physicians in a variety of practice settings across the United States and Taiwan. Occult fractures were identified by follow-up CT scans or MRI. Participants met the following criteria: Postgraduate Year 3 or above resident physician in plastic surgery, orthopaedic surgery, or emergency medicine; hand fellows; and attending physicians. Among the 176 invited participants, 120 completed the survey and met the inclusion criteria. Of the participants, 31% (37 of 120) were fellowship-trained hand surgeons, 43% (52 of 120) were plastic surgeons, and 69% (83 of 120) were attending physicians. Most participants (73% [88 of 120]) worked in academic centers, whereas the remainder worked in large, urban private practice hospitals. Recruitment occurred between February 2022 and March 2022. Radiographs with CNN assistance were accompanied by predictions of fracture presence and gradient-weighted class activation mapping of the predicted fracture site. Sensitivity and specificity of the CNN-assisted physician diagnoses were calculated to assess diagnostic performance. We calculated interobserver agreement with the Gwet agreement coefficient (AC1). Physician diagnostic confidence was estimated using a self-assessment Likert scale, and the time to arrive at a diagnosis for each case was measured. RESULTS: Interobserver agreement among physicians for occult scaphoid radiographs was higher with CNN assistance than without (AC1 0.42 [95% CI 0.17 to 0.68] versus 0.06 [95% CI 0.00 to 0.17], respectively). No clinically relevant differences were observed in time to arrive at a diagnosis (18 ± 12 seconds versus 30 ± 27 seconds, mean difference 12 seconds [95% CI 6 to 17]; p < 0.001) or diagnostic confidence levels (7.2 ± 1.7 seconds versus 6.2 ± 1.6 seconds; mean difference 1 second [95% CI 0.5 to 1.3]; p < 0.001) for occult fractures. CONCLUSION: CNN assistance improves physician diagnostic sensitivity and specificity as well as interobserver agreement for the diagnosis of occult scaphoid fractures. The differences observed in diagnostic speed and confidence is likely not clinically relevant. Despite these improvements in clinical diagnoses of scaphoid fractures with the CNN, it is unknown whether development and implementation of such models is cost effective. LEVEL OF EVIDENCE: Level II, diagnostic study.

Simultaneous N-Deglycosylation and Digestion of Complex Samples on S-Traps Enables Efficient Glycosite Hypothesis Generation.

N-linked glycosylation is an important post-translational modification that is difficult to identify and quantify in traditional bottom-up proteomics experiments. Enzymatic deglycosylation of proteins by peptide:N-glycosidase F (PNGase F) prior to digestion and subsequent mass spectrometry analysis has been shown to improve coverage of various N-linked glycopeptides, but the inclusion of this step may add up to a day to an already lengthy sample preparation process. An efficient way to integrate deglycosylation with bottom-up proteomics would be a valuable contribution to the glycoproteomics field. Here, we demonstrate a proteomics workflow in which deglycosylation and proteolytic digestion of samples occur simultaneously using suspension trapping (S-Trap). This approach adds no time to standard digestion protocols. Applying this sample preparation strategy to a human serum sample, we demonstrate improved identification of potential N-glycosylated peptides in deglycosylated samples compared with non-deglycosylated samples, identifying 156 unique peptides that contain the N-glycosylation motif (asparagine-X-serine/threonine), the deamidation modification characteristic of PNGase F, and an increase in peptide intensity over a control sample. We expect that this rapid sample preparation strategy will assist in the identification and quantification of both known and potential glycoproteins. Data are available via ProteomeXchange with the identifier PXD037921.

Individual recombinant repeats of MUC16 display variable binding to CA125 antibodies.

BACKGROUND: Despite its importance in the clinical management of ovarian cancer, the CA125 biomarker-located on the mucin protein MUC16-is still not completely understood. Questions remain about MUC16's function and structure, specifically the identity and location of the CA125 epitopes. OBJECTIVE: The goal of this study was to characterize the interaction of individual recombinant repeats from the tandem repeat domain of MUC16 with antibodies used in the clinical CA125 II test. METHODS: Using E. coli expression, we isolated nine repeats from the putative antigenic domain of CA125. Amino acid composition of recombinant repeats was confirmed by high-resolution mass spectrometry. We characterized the binding of four antibodies-OC125, M11, "OC125-like," and "M11-like"-to nine recombinant repeats using Western blotting, indirect enzyme-linked immunosorbent assay (ELISA), and localized surface plasmon resonance (SPR) spectroscopy. RESULTS: Each recombinant repeat was recognized by a different combination of CA125 antibodies. OC125 and "OC125-like" antibodies did not bind the same set of recombinant repeats, nor did M11 and "M11-like" antibodies. CONCLUSIONS: Characterization of the interactions between MUC16 recombinant repeats and CA125 antibodies will contribute to ongoing efforts to identify the CA125 epitopes and improve our understanding of this important biomarker.

Rectal Injury during Penile Inversion Vaginoplasty: An Algorithmic Approach to Prevention and Management.

BACKGROUND: Penile inversion vaginoplasty (PIV) is a common procedure for transfeminine patients, with the goal of creating a functional vaginal canal and clitoris and a natural-appearing vulva. Creation of the neovagina requires opening of the prerectal space, most commonly from a perineal approach, and the reported rates of rectal perforation during this dissection range from 3% to 5%. METHODS: Adult patients who underwent PIV at the authors' institution were identified retrospectively. Demographics, operative information, and postoperative clinical outcomes were extracted from the electronic medical record. RESULTS: Ten of 146 patients (6.8%) experienced a rectal injury. All patients underwent an immediate repair (two-layer repair in eight patients, and three-layer repair in two), with two patients subsequently requiring temporary fecal diversion and two requiring muscle flaps (1.4% each). Literature review identified 18 relevant publications, with scarce in-depth analysis of management of initial rectal injuries. CONCLUSION: The authors' algorithmic approach to rectal injury during PIV is designed to facilitate decision-making based on preoperative preparation, consistent intraoperative monitoring, feasibility of primary repair of the rectum, and a multidisciplinary approach to longitudinal postoperative care. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Are Observational Studies on Distal Radius Fracture Treatment Robust? An E-value Approach to Analysis.

BACKGROUND: Reported complication frequencies after distal radius fracture (DRF) treatment vary widely in the literature and are based mostly on observational evidence. Whether that evidence is sufficiently robust to use in practice is controversial. The E-value is an innovative sensitivity analysis that quantitates the robustness of observational evidence against unmeasured confounders, whereby a greater E-value usually implies more robust evidence and vice versa; with DRF complications, this approach can help guide readers to a more confident interpretation of the available evidence. QUESTIONS/PURPOSES: In this study, we sought (1) to compare the complication frequencies among different DRF treatment modalities, and (2) to evaluate the robustness of these observational studies using the E-value as an index for unmeasured confounding. METHODS: We searched PubMed, Embase, and SCOPUS for observational studies on the management of DRFs that were published from January 2001 to July 2021 with the last database search performed on July 31, 2021. All articles that compared different DRF treatment modalities with reported complication frequencies were included to accurately capture the quality of the observational studies in research about DRF. Risk ratios (RRs) of the overall complication and major complication risks were calculated for each subgroup comparison: volar plating versus dorsal plating, casting, external fixation, and percutaneous K-wire fixation. The RRs and their corresponding lower limits of the 95% confidence intervals (CIs) were used to derive the E-values. E-values can have a minimum possible value of 1, which signifies that the treatment-outcome association is not strong and can readily be overturned by unmeasured confounders. By contrast, a large E-value means that the observed treatment-outcome association is robust against unmeasured confounders. We averaged RRs and E-values for the effect estimates and lower limits of CIs across studies in each treatment comparison group. We identified 36 comparative observational studies that met the inclusion criteria. Seven studies compared volar with dorsal plating techniques. Volar plating was also compared with casting (eight studies), external fixation (15 studies), and percutaneous K-wire fixation (six studies). RESULTS: Total and major complication risks did not differ among different DRF treatments. The mean RRs for total and major complications were 1.2 (95% CI 0.4 to 3.9; p = 0.74) and 1.8 (95% CI 0.4 to 11.4; p = 0.52) for the volar versus dorsal plating group; 1.2 (95% CI 0.3 to 11.2; p = 0.87) and 1.5 (95% CI 0.3 to 14.9; p = 0.74) for the volar plating versus casting group; 0.6 (95% CI 0.2 to 2.2; p = 0.33) and 0.8 (95% CI 0.2 to 6.7; p = 0.86) for the volar plating versus external fixation group; and 0.6 (95% CI 0.2 to 2.6; p = 0.47) and 0.7 (95% CI 0.2 to 4.0; p = 0.67) for the volar plating versus K-wire fixation group. The mean E-values for total and major complication frequencies for the between-group comparison ranged from 3.1 to 5.8; these were relatively large in the context of a known complication risk factor, such as high-energy impact (RR 3.2), suggesting a reasonable level of robustness against unmeasured confounding. However, the E-values for lower limits of CIs remained close to 1, which indicates the observed complication frequencies in these studies were likely to have been influenced by unmeasured confounders. CONCLUSION: Complication frequencies did not differ among different DRF treatment modalities, but the observed complication frequencies from most comparative observational studies were less robust against potential unmeasured confounders. The E-value method, or another type of sensitivity analysis, should be implemented in observational hand surgery research at the individual-study level to facilitate assessment of robustness against potential unmeasured confounders. LEVEL OF EVIDENCE: Level III, therapeutic study.

Factors Associated With Quality Care Among Adults With Rheumatoid Arthritis.

Importance: Although quality care markers exist for patients with rheumatoid arthritis (RA), the predictors of meeting these markers are unclear. Objective: To explore factors associated with quality care among patients with RA. Design, Setting, and Participants: A retrospective cohort study using insurance claims from 2009 to 2017 was conducted, and 6 sequential logistic regression models were built to evaluate quality care markers. Quality care markers were measured at 1 year post-RA diagnosis for each patient. The MarketScan Research Database, which contains commercial and Medicare Advantage administrative claims data from more than 100 million individuals in the US, was used to identify patients aged 18 to 64 years with a diagnosis claim for RA. Patients with conditions presenting similar to RA and missing demographic characteristics were excluded. Data analysis occurred between February 18 and May 5, 2022. Exposures: Success or failure to meet selected RA quality care markers within 1 year after RA diagnosis. Main Outcomes and Measures: Prevalence of meeting successive quality care markers for RA. Results: Among 581 770 patients, 430 843 (74.1%) were women and the mean (SD) age was 48.9 (11.3) years. Most patients (236 285 [40.6%]) resided in the South and had an income less than or equal to $45 200 (490 366 [84.3%]). Of the total study population, 399 862 individuals (68.7%) met at least 1 quality care marker and 181 908 (31.3%) met 0 markers. Most commonly, patients met annual laboratory testing (299 323 [51.5%]) and referral to a rheumatologist (256 765 [44.1%]) markers. The least met marker was receiving hepatitis B screening prior to initiation of disease-modifying antirheumatic drug (DMARD) therapy (18 548 [3.2%]). Women were most likely to meet all quality care markers except receiving DMARDs with hepatitis B screening (odds ratio, 1.14; 95% CI, 1.12-1.16). Individuals with lower median household income had lower odds of receiving a rheumatologist referral, an annual physical examination, or annual laboratory testing, but greater odds of receiving the other quality care markers. Patients with Medicare and those with comorbidities were generally less likely to meet quality care markers. Conclusions and Relevance: In this cohort study of patients with RA, findings indicated downstream associations with rheumatologist referral and receiving DMARDs and varied associations between meeting quality care markers and patient characteristics. These findings suggest that prioritizing early care, especially for vulnerable patients, will ensure that quality care continues.

Correction: Mysore et al. A Broad-Based Mosquito Yeast Interfering RNA Pesticide Targeting Rbfox1 Represses Notch Signaling and Kills Both Larvae and Adult Mosquitoes. Pathogens 2021, 10, 1251.

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Innovation in Delivering a Live, Virtual Cadaver Dissection Course.

SUMMARY: The availability of advanced telecommunication technology and the social restrictions introduced by a global pandemic have compelled the medical community to explore new avenues of surgical education. Although cadaver courses have long been a fundamental method for learning surgical anatomy and improving operative preparedness, the COVID-19 pandemic has made traditional dissections less practical. The need for quality virtual learning experiences motivated the authors to design and assess the feasibility of organizing a live, virtual upper extremity peripheral nerve cadaver dissection course. Three phases were critical when developing the course: preplanning, planning, and execution. The success of the live, virtual cadaver dissection depended not only on a detailed curriculum, but the technological audio-video-internet needs to effectively communicate and interact with the viewers. Virtual learning mitigates the risks of in-person dissection courses during a global pandemic and can be enhanced with interactive media (e.g., illustrations and schematics) to augment learning experiences.

Common Revisions after Penile Inversion Vaginoplasty: Techniques and Clinical Outcomes.

SUMMARY: Up to one-third of patients are reported to undergo secondary surgical revision to address functional and aesthetic concerns after penile inversion vaginoplasty. The most commonly performed revisions are posterior introital web release, clitoroplasty, labiaplasty, and urethroplasty. To illustrate effective strategies for each of these revisions, this Video Plus article reviews the case of a 32-year-old transgender woman undergoing revision surgery to correct functionally limiting posterior introital webbing and to improve clitoral and labial appearance. Intraoperative steps and postoperative considerations are detailed in the accompanying videos.

A Yeast RNA-Interference Pesticide Targeting the Irx Gene Functions as a Broad-Based Mosquito Larvicide and Adulticide.

Concerns for widespread insecticide resistance and the unintended impacts of insecticides on nontarget organisms have generated a pressing need for mosquito control innovations. A yeast RNAi-based insecticide that targets a conserved site in mosquito Irx family genes, but which has not yet been identified in the genomes of nontarget organisms, was developed and characterized. Saccharomyces cerevisiae constructed to express short hairpin RNA (shRNA) matching the target site induced significant Aedes aegypti larval death in both lab trials and outdoor semi-field evaluations. The yeast also induced high levels of mortality in adult females, which readily consumed yeast incorporated into an attractive targeted sugar bait (ATSB) during simulated field trials. A conserved requirement for Irx function as a regulator of proneural gene expression was observed in the mosquito brain, suggesting a possible mode of action. The larvicidal and adulticidal properties of the yeast were also verified in Aedes albopictus, Anopheles gambiae, and Culexquinquefasciatus mosquitoes, but the yeast larvicide was not toxic to other nontarget arthropods. These results indicate that further development and evaluation of this technology as an ecofriendly control intervention is warranted, and that ATSBs, an emerging mosquito control paradigm, could potentially be enriched through the use of yeast-based RNAi technology.

A Broad-Based Mosquito Yeast Interfering RNA Pesticide Targeting Rbfox1 Represses Notch Signaling and Kills Both Larvae and Adult Mosquitoes.

Prevention of mosquito-borne infectious diseases will require new classes of environmentally safe insecticides and novel mosquito control technologies. Saccharomyces cerevisiae was engineered to express short hairpin RNA (shRNA) corresponding to mosquito Rbfox1 genes. The yeast induced target gene silencing, resulting in larval death that was observed in both laboratory and outdoor semi-field trials conducted on Aedes aegypti. High levels of mortality were also observed during simulated field trials in which adult females consumed yeast delivered through a sugar bait. Mortality correlated with defects in the mosquito brain, in which a role for Rbfox1 as a positive regulator of Notch signaling was identified. The larvicidal and adulticidal activities of the yeast were subsequently confirmed in trials conducted on Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus, yet the yeast had no impact on survival of select non-target arthropods. These studies indicate that yeast RNAi pesticides targeting Rbfox1 could be further developed as broad-based mosquito larvicides and adulticides for deployment in integrated biorational mosquito control programs. These findings also suggest that the species-specificity of attractive targeted sugar baits, a new paradigm for vector control, could potentially be enhanced through RNAi technology, and specifically through the use of yeast-based interfering RNA pesticides.

Magnetic Nanoplatforms for Covalent Protein Immobilization Based on Spy Chemistry.

Immobilization of proteins on magnetic nanoparticles (MNPs) is an effective approach to improve protein stability and facilitate separation of immobilized proteins for repeated use. Herein, we exploited the efficient SpyTag-SpyCatcher chemistry for conjugation of functional proteins onto MNPs and established a robust magnetic-responsive nanoparticle platform for protein immobilization. To maximize the loading capacity and achieve outstanding water dispersity, the SpyTag peptide was incorporated into the surface-charged polymers of MNPs, which provided abundant active sites for Spy chemistry while maintaining excellent colloidal stability in buffer solution. Conjugation between enhanced green fluorescence protein (EGFP)-SpyCatcher-fused proteins and SpyTag-functionalized MNPs was efficient at ambient conditions without adding enzymes or chemical cross-linkers. Benefiting from the excellent water dispersity and interface compatibility, the surface Spy reaction has fast kinetics, which is comparable to that of the solution Spy reaction. No activity loss was observed on EGFP after conjugation due to the site-selective nature of Spy chemistry. The immobilization process of EGFP on MNPs was highly specific and robust, which was not affected by the presence of other proteins and detergents, such as bovine serum albumin and Tween 20. The MNP platform was demonstrated to be protective to the conjugated EGFP and significantly improved the shelf life of immobilized proteins. In addition, experiments confirmed the retained magnetophoresis of the MNP after protein loading, demonstrating fast MNP recovery under an external magnetic field. This MNP is expected to provide a versatile and modular platform to achieve effective and specific immobilization of other functional proteins, enabling easy reuse and storage.

A Whole-Cell Biosensor for Point-of-Care Detection of Waterborne Bacterial Pathogens.

Water contamination by pathogenic bacteria is a major public health concern globally. Monitoring bacterial contamination in water is critically important to protect human health, but this remains a critical challenge. Engineered whole-cell biosensors created through synthetic biology hold great promise for rapid and cost-effective detection of waterborne pathogens. In this study, we created a novel whole-cell biosensor to detect water contamination by Pseudomonas aeruginosa and Burkholderia pseudomallei, which are two critical bacterial pathogens and are recognized as common causative agents for waterborne diseases. The biosensor detects the target bacterial pathogens by responding to the relevant quorum sensing signal molecules. Particularly, this study constructed and characterized the biosensor on the basis of the QscR quorum sensing signal system for the first time. We first designed and constructed a QscR on the basis of the sensing module in the E. coli host cell and integrated the QscR sensing module with a reporting module that expressed an enhanced green fluorescent protein (EGFP). The results demonstrated that the biosensor had high sensitivity in response to the quorum sensing signals of the target bacterial pathogens. We further engineered a biosensor that expressed a red pigment lycopene in the reporting module to produce a visible signal readout for the pathogen detection. Additionally, we investigated the feasibility of a paper-based assay by immobilizing the lycopene-based whole-cell biosensor on paper with the aim to build a prototype for developing portable detection devices. The biosensor would provide a simple and inexpensive alternative for timely and point-of-care detection of water contamination and protect human health.

Characterization of a dual-action adulticidal and larvicidal interfering RNA pesticide targeting the Shaker gene of multiple disease vector mosquitoes.

The existing mosquito pesticide repertoire faces great challenges to sustainability, and new classes of pesticides are vitally needed to address established and emerging mosquito-borne infectious diseases. RNA interference- (RNAi-) based pesticides are emerging as a promising new biorational mosquito control strategy. In this investigation, we describe characterization of an interfering RNA pesticide (IRP) corresponding to the mosquito Shaker (Sh) gene, which encodes an evolutionarily conserved voltage-gated potassium channel subunit. Delivery of the IRP to Aedes aegypti adult mosquitoes in the form of siRNA that was injected or provided as an attractive toxic sugar bait (ATSB) led to Sh gene silencing that resulted in severe neural and behavioral defects and high levels of adult mortality. Likewise, when provided to A. aegypti larvae in the form of short hairpin RNA (shRNA) expressed in Saccharomyces cerevisiae (baker's yeast) that had been formulated into a dried inactivated yeast tablet, the yeast IRP induced neural defects and larval death. Although the Sh IRP lacks a known target site in humans or other non-target organisms, conservation of the target site in the Sh genes of multiple mosquito species suggested that it may function as a biorational broad-range mosquito insecticide. In support of this, the Sh IRP induced both adult and larval mortality in treated Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus mosquitoes, but was not toxic to non-target arthropods. These studies indicated that IRPs targeting Sh could one day be used in integrated biorational mosquito control programs for the prevention of multiple mosquito-borne illnesses. The results of this investigation also suggest that the species-specificity of ATSB technology, a new paradigm for vector control, could be enhanced through the use of RNAi-based pesticides.

Characterization of an adulticidal and larvicidal interfering RNA pesticide that targets a conserved sequence in mosquito G protein-coupled dopamine 1 receptor genes.

G protein-coupled receptors (GPCRs), key regulators of a variety of critical biological processes, are attractive targets for insecticide development. Given the importance of these receptors in many organisms, including humans, it is critical that novel pesticides directed against GPCRs are designed to be species-specific. Here, we present characterization of an interfering RNA pesticide (IRP) targeting the mosquito GPCR-encoding dopamine 1 receptor (dop1) genes. A small interfering RNA corresponding to dop1 was identified in a screen for IRPs that kill Aedes aegypti during both the adult and larval stages. The 25 bp sequence targeted by this IRP is conserved in the dop1 genes of multiple mosquito species, but not in non-target organisms, indicating that it could function as a biorational mosquito insecticide. Aedes aegypti adults treated through microinjection or attractive toxic sugar bait delivery of small interfering RNA corresponding to the target site exhibited severe neural and behavioral defects and high levels of adult mortality. Likewise, A. aegypti larval consumption of dried inactivated yeast tablets prepared from a Saccharomyces cerevisiae strain engineered to express short hairpin RNA corresponding to the dop1 target site resulted in severe neural defects and larval mortality. Aedes albopictus and Anopheles gambiae adult and larval mortality was also observed following treatment with dop1 IRPs, which were not toxic to non-target arthropods. The results of this investigation indicate that dop1 IRPs can be used for species-specific targeting of dop1 GPCRs and may represent a new biorational strategy for control of both adult and larval mosquitoes.

Characterization of a yeast interfering RNA larvicide with a target site conserved in the synaptotagmin gene of multiple disease vector mosquitoes.

New mosquito control strategies are vitally needed to address established and emerging arthropod-borne infectious diseases. Here we describe the characterization of a yeast interfering RNA larvicide that was developed through the genetic engineering of Saccharomyces cerevisiae (baker's yeast) to express a short hairpin RNA targeting the Aedes aegypti synaptotagmin (Aae syt) gene. The larvicide effectively silences the Aae syt gene, causes defects at the larval neural synapse, and induces high rates of A. aegypti larval mortality in laboratory, simulated-field, and semi-field trials. Conservation of the interfering RNA target site in multiple mosquito species, but not in humans or other non-target species, suggested that it may function as a broad-range mosquito larvicide. In support of this, consumption of the yeast interfering RNA larvicide was also found to induce high rates of larval mortality in Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus mosquito larvae. The results of these studies suggest that this biorational yeast interfering RNA larvicide may represent a new intervention that can be used to combat multiple mosquito vectors of human diseases.