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The most devastating disease affecting the global citrus industry is Huanglongbing (HLB), caused by the pathogenCandidatus Liberibacter asiaticus. HLB is primarily spread by the insect vectorDiaphorina citri(Asian Citrus Psyllid). To counteract the rapid spread of HLB byD. citri, traditional vector control strategies such as insecticide sprays, the release of natural predators, and mass introductions of natural parasitoids are used. However, these methods alone have not managed to contain the spread of disease. To further expand the available tools forD. citricontrol via generating specific modifications of theD. citrigenome, we have developed protocols for CRISPR/Cas9-based genetic modification. Until now, genome editing inD. citrihas been challenging due to the general fragility and size ofD.citrieggs. Here we present optimized methods for collecting and preparing eggs to introduce the Cas9 ribonucleoprotein (RNP) into early embryos and alternative methods (ReMOT Control) for injecting RNP into the hemocoel of adult females for ovarian transduction. Using these methods, we have generated visible somatic mutations, indicating their suitability for gene editing inD. citri. These methods represent the first steps towards advancingD. citriresearch in preparation for future genetic-based systems for controlling HLB.

Escalating vector disease burdens pose significant global health risks, so innovative tools for targeting mosquitoes are critical. We engineered an antiviral strategy termed REAPER (vRNA Expression Activates Poisonous Effector Ribonuclease) that leverages the programmable RNA-targeting capabilities of CRISPR Cas13 and its potent collateral activity. Akin to a stealthy Trojan Horse hiding in stealth awaiting the presence of its enemy, REAPER remains concealed within the mosquito until an infectious blood meal is uptaken. Upon target viral RNA infection, REAPER activates, triggering programmed destruction of its target arbovirus such as chikungunya. Consequently, Cas13 mediated RNA targeting significantly reduces viral replication and its promiscuous collateral activity can even kill infected mosquitoes. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.

As a major insect vector of multiple arboviruses, Aedes aegypti poses a significant global health and economic burden. A number of genetic engineering tools have been exploited to understand its biology with the goal of reducing its impact. For example, current tools have focused on knocking-down RNA transcripts, inducing loss-of-function mutations, or expressing exogenous DNA. However, methods for transactivating endogenous genes have not been developed. To fill this void, here we developed a CRISPR activation (CRISPRa) system in Ae. aegypti to transactivate target gene expression. Gene expression is activated through pairing a catalytically-inactive (‘dead’) Cas9 (dCas9) with a highly-active tripartite activator, VP64-p65-Rta (VPR) and synthetic guide RNA (sgRNA) complementary to a user defined target-gene promoter region. As a proof of concept, we demonstrate that engineered Ae. aegypti mosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes, even-skipped (eve) and hedgehog (hh), resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known as AaRel1, which resulted in a significant suppression of dengue virus serotype 2 (DENV2) titers in the mosquito. This system provides a versatile tool for research pathways not previously possible in Ae. aegypti, such as programmed overexpression of endogenous genes, and may aid in gene characterization studies and the development of innovative vector control tools.

The mosquito Aedes aegypti is the principal vector for arboviruses including dengue/yellow fever, chikungunya, and Zika virus, infecting hundreds of millions of people annually. Unfortunately, traditional control methodologies are insufficient, so innovative control methods are needed. To complement existing measures, here we develop a molecular genetic control system termed precision-guided sterile insect technique (pgSIT) in Aedes aegypti. PgSIT uses a simple CRISPR-based approach to generate flightless females and sterile males that are deployable at any life stage. Supported by mathematical models, we empirically demonstrate that released pgSIT males can compete, suppress, and even eliminate mosquito populations. This platform technology could be used in the field, and adapted to many vectors, for controlling wild populations to curtail disease in a safe, confinable, and reversible manner.

INTRODUCTION AND OBJECTIVE:International surgical collaboration presents an opportunity to address the need for specialized urologic care and teaching in low-middle income countries (LMICs) and pro...

Aedes aegypti have a wide variety of sensory pathways that have supported success as a species as well as a highly competent vector of numerous debilitating infectious pathogens. Investigations into mosquito sensory systems and their effects on behavior are valuable resources for the advancement of mosquito control strategies. Numerous studies have elucidated key aspects of mosquito sensory systems, however there remains critical gaps within the field. In particular, compared to that of the adult form, there has been a lack of studies directed towards the immature life stages. Additionally, although numerous studies have pinpointed specific sensory receptors as well as relevant response behaviors, there has been a lack of studies able to monitor both concurrently. To begin filling aforementioned gaps, here we engineered Ae. aegypti to ubiquitously express a genetically encoded calcium indicator, GCaMP6s. Using this strain, combined with advanced confocal microscopy, we were able to simultaneously measure live stimulus-evoked calcium responses in both neuronal and muscle cells with a wide spatial range and resolution. Moreover, by coupling in vivo calcium imaging with behavioral assays we were able to gain functional insights into how stimulus-evoked neural and muscle activities are represented, modulated, and transformed in mosquito larvae enabling us to elucidate mosquito sensorimotor properties important for life-history-specific foraging strategies.Significance StatementUnderstanding mosquito sensory systems and resulting behavior has been a major factor in the advancement of mosquito control innovations. Aedes aegypti larvae offer an effective life stage for further elucidating information on mosquito sensory systems. Due to their relatively simplified nervous system, mosquito larvae are ideal for studying neural signal transduction, coding, and behavior. Moreover, a better understanding of the larval sensory system may enable the development of novel control methodologies able to target mosquitoes before they reach a vector-competent stage. Here we engineer Ae. aegypti to ubiquitously express a genetically encoded calcium indicator, GCaMP6s and use this tool to observe links between sensorimotor responses and behavior by exploiting live calcium imaging as well as live tracking based behavioral assays.

The mosquito Aedes aegypti is the principal vector for arboviruses including dengue/yellow fever, chikungunya, and zika, infecting hundreds of millions of people annually. Unfortunately, traditional control methodologies are insufficient, so innovative control methods are needed. To complement existing measures, here we develop a molecular genetic control system termed precision guided sterile insect technique (pgSIT) in Aedes aegypti. PgSIT uses a simple CRISPR-based approach to generate sterile males that are deployable at any life stage. Supported by mathematical models, we empirically demonstrate that released pgSIT males can compete, suppress, and eliminate mosquitoes in multigenerational population cages. This platform technology could be used in the field, and adapted to many vectors, for controlling wild populations to curtail disease in a safe, confinable, and reversible manner.

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.

This chapter uses clinical vignettes and multiple-choice questions to explore core and advanced principles of airway and ENT ultrasound. The cases assess image acquisition pearls and pitfalls, image interpretation, false positive and negative findings, and integration into patient management. Clinical vignettes include stable and unstable patients presenting with undifferentiated symptoms such as neck pain, shortness of breath, and neck swelling. Each case includes detailed explanations for correct and incorrect answer choices, incorporating landmark studies as well as the latest literature that clinical sonographers will find of interest. Relevant references and sources for each case are also included, as well as keywords for each section.

Aedes aegypti is the principal mosquito vector for many arboviruses that increasingly infect millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has sparked significant enthusiasm for genetic control of mosquitoes; however, no such system has been developed in Ae. aegypti. To fill this void, here we develop several CRISPR-based split gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, mathematical models predict that these systems could spread anti-pathogen effector genes into wild populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effector-linked gene drives that could safely control wild populations of Ae. aegypti to combat local pathogen transmission.

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Author(s): Becker, Brent A; Lahham, Shadi; Gonzales, Mark A; Nomura, Jason T; Bui, Michelle K; Truong, Taylor A; Stahlman, Barbara A; Fox, John C; Kehrl, Thompson | Abstract: ObjectiveThe main objective of this study was to evaluate the accuracy of emergency physician-performed point-of-care ultrasound (POCUS) for the diagnosis of small-bowel obstruction (SBO) compared to computed tomography (CT).MethodsWe performed a prospective, multicenter, observational study examining a convenience sample of adult patients with potential SBO presenting to the emergency department (ED) between July 2014 and May 2017. Each POCUS was interpreted at the bedside by the performing emergency physician and retrospectively by an expert reviewer. Test characteristics were calculated for POCUS, blinded expert interpretation, and specific POCUS parameters.ResultsA total of 217 subjects were included in the primary analysis with an overall SBO prevalence of 42.9%. For the diagnosis of SBO, POCUS demonstrated an overall sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio of 0.88 (95% confidence interval [CI]n= 0.80 to 0.94), 0.54 (95% CIn= 0.45 to 0.63), 1.92 (95% CIn= 1.56 to 2.35), and 0.22 (95% CIn= 0.12 to 0.39), respectively. Expert review yielded a similar sensitivity (0.89 [95% CIn= 0.81 to 0.95]) with a significantly higher specificity (0.82 [95% CIn= 0.74 to 0.88]). The more sensitive sonographic parameters for both POC sonographers and expert reviewers were small-bowel dilation ≥ 25 mm (0.87 [95% CIn= 0.79 to 0.93], 0.87 [95% CIn= 0.79 to 0.93]) and abnormal peristalsis (0.82 [95% CIn= 0.72 to 0.89], 0.85 [95% CIn= 0.76 to 0.87]). The more specific parameters for both groups were transition point (0.82 [95% CIn= 0.74 to 0.89], 0.98 [95% CIn= 0.94 to 1.00]), intraperitoneal free fluid (0.82 [95% CIn= 0.74 to 0.89], 0.93 [95% CIn= 0.87 to 0.97]), and bowel wall edema (0.76 [95% CIn= 0.67 to 0.83], 0.93 [95% CIn= 0.87 to 0.97]).ConclusionPOCUS is moderately sensitive for SBO, although less specific, when performed by a diverse group of emergency physicians across multiple EDs. Interpretation of acquired POCUS images is significantly more accurate when performed by physicians with prior emergency ultrasound fellowship training and familiarity with the sonographic appearance of SBO.

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology-directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we engineered, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a step toward the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives.

Significance Aedes aegypti is the principal vector of multiple arboviruses that significantly affect human health, including dengue, chikungunya, and zika. Development of tools for efficient genome engineering in this mosquito will not only lay the foundation for the application of genetic control strategies, but will also accelerate basic research on key biological processes involved in disease transmission. Here, we report the development of a transgenic CRISPR approach for rapid gene disruption in this organism. Given their high editing efficiencies, the Cas9 strains we developed can be used to quickly generate genome modifications, allowing for high-throughput gene targeting, and can possibly facilitate the development of gene drives, thereby accelerating comprehensive functional annotation and development of innovative population control strategies., The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti. Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology-directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we engineered, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a step toward the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives.

The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general,in vivogermline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vectorAedes aegypti. Here, we describe the generation of multiple stable, transgenicAe. aegyptistrains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we built, they can be used for high-throughput reverse genetic screens to help functionally annotate theAe. aegyptigenome. Additionally, these strains represent a first step towards the development of novel population control technologies targetingAe. aegyptithat rely on Cas9-based gene drives.Significance StatementAedes aegyptiis the principal vector of multiple arboviruses that significantly affect human health including dengue, chikungunya, and zika. Development of tools for efficient genome engineering in this mosquito will not only lay the foundation for the application of novel genetic control strategies that do not rely on insecticides, but will also accelerate basic research on key biological processes involved in disease transmission. Here, we report the development of a transgenic CRISPR approach for rapid gene disruption in this organism. Given their high editing efficiencies, the Cas9 strains we developed can be used to quickly generate novel genome modifications allowing for high-throughput gene targeting, and can possibly facilitate the development of gene drives, thereby accelerating comprehensive functional annotation and development of innovative population control strategies forAe. aegypti.

The S-phase kinase-associated protein 2 (Skp2) is overexpressed in prostate cancer and associated with tumor stage, disease recurrence, and worse patient survival. Studies also have demonstrated that Skp2 overexpression plays a critical role in development and progression of prostate cancer in several prostate cancer models, including PTEN knockout and TRAMP carcinogenesis and CWR22 xenograft models. Therefore, Skp2 is a promising target for developing new therapeutic and preventive approaches for prostate cancer. Here, we have found that gartanin, 4-prenylated xanthone from the Garcinia mangostana fruit (mangosteen), promoted Skp2 degradation in a proteasome-dependent manner in prostate cancer cells. In addition, gartanin induced the expression of FBXW2, a novel E3 ligase of SKP2 for targeted degradation, and downregulated the expression of NEDD8, leading to reduced neddylation levels of UBC12 and Cullin1. Molecular modelling analysis predicts that gartanin reasonably docks onto the regulatory subunit of NEDD8 activating enzyme E1 (NAE1) and next to the NEDD8 binding complex. More interestingly, gartanin selectively inhibits the growth of Skp2 overexpressing PC3 cells compared to parental PC cells (IC50 values for PC3/Skp2 vs. PC3 cells are approximately 4 μM vs. 14 μM). Together, our data indicate that gartanin is a novel natural agent for targeting Skp2 degradation via induction of FBXW2 expression and inhibition of neddylation. Gartanin deserves further investigation for its usefulness in prostate cancer prevention and treatment. Citation Format: Victor Pham, Michelle Bui, Raymond Rendon, Ericka Agredano, Thanh Le, Liankun Song, Xiaolin Zi. Gartanin, a 4-prenylated xanthone in the mangosteen fruit, inhibits the growth of prostate cancer cells associated with Skp2 degradation via neddylation inhibition and FBXW2 induction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5865.