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Proposed genetic approaches for reducing human malaria include population modification, which introduces genes into vector mosquitoes to reduce or prevent parasite transmission. We demonstrate the potential of Cas9/guide RNA (gRNA)-based gene-drive systems linked to dual antiparasite effector genes to spread rapidly through mosquito populations. Two strains have an autonomous gene-drive system coupled to dual anti-Plasmodium falciparum effector genes comprising single-chain variable fragment monoclonal antibodies targeting parasite ookinetes and sporozoites in the African malaria mosquitoes Anopheles gambiae (AgTP13) and Anopheles coluzzii (AcTP13). The gene-drive systems achieved full introduction within 3 to 6 mo after release in small cage trials. Life-table analyses revealed no fitness loads affecting AcTP13 gene-drive dynamics but AgTP13 males were less competitive than wild types. The effector molecules reduced significantly both parasite prevalence and infection intensities. These data supported transmission modeling of conceptual field releases in an island setting that shows meaningful epidemiological impacts at different sporozoite threshold levels (2.5 to 10 k) for human infection by reducing malaria incidence in optimal simulations by 50 to 90% within as few as 1 to 2 mo after a series of releases, and by ≥90% within 3 mo. Modeling outcomes for low sporozoite thresholds are sensitive to gene-drive system fitness loads, gametocytemia infection intensities during parasite challenges, and the formation of potentially drive-resistant genome target sites, extending the predicted times to achieve reduced incidence. TP13-based strains could be effective for malaria control strategies following validation of sporozoite transmission threshold numbers and testing field-derived parasite strains. These or similar strains are viable candidates for future field trials in a malaria-endemic region.

The field-testing and eventual adoption of genetically-engineered mosquitoes (GEMs) to control vector-borne pathogen transmission will require them meeting safety criteria specified by regulatory authorities in regions where the technology is being considered for use and other locales that might be impacted. Preliminary risk considerations by researchers and developers may be useful for planning the baseline data collection and field research used to address the anticipated safety concerns. Part of this process is to identify potential hazards (defined as the inherent ability of an entity to cause harm) and their harms, and then chart the pathways to harm and evaluate their probability as part of a risk assessment. The University of California Malaria Initiative (UCMI) participated in a series of workshops held to identify potential hazards specific to mosquito population modification strains carrying gene-drive systems coupled to anti-parasite effector genes and their use in a hypothetical island field trial. The hazards identified were placed within the broader context of previous efforts discussed in the scientific literature. Five risk areas were considered i) pathogens, infections and diseases, and the impacts of GEMs on human and animal health, ii) invasiveness and persistence of GEMs, and interactions of GEMs with target organisms, iii) interactions of GEMs with non-target organisms including horizontal gene transfer, iv) impacts of techniques used for the management of GEMs and v) evolutionary and stability considerations. A preliminary hazards list (PHL) was developed and is made available here. This PHL is useful for internal project risk evaluation and is available to regulators at prospective field sites. UCMI project scientists affirm that the subsequent processes associated with the comprehensive risk assessment for the application of this technology should be driven by the stakeholders at the proposed field site and areas that could be affected by this intervention strategy.

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 transition of new technologies for public health from laboratory to field is accompanied by a broadening scope of engagement challenges. Recent developments of vector control strategies involving genetically engineered mosquitoes with gene drives to assist in the eradication of malaria have drawn significant attention. Notably, questions have arisen surrounding community and regulatory engagement activities and of the need for examples of models or frameworks that can be applied to guide engagement. A relationship-based model (RBM) provides a framework that places stakeholders and community members at the center of decision-making processes, rather than as recipients of predetermined strategies, methods, and definitions. Successful RBM application in the transformation of healthcare delivery has demonstrated the importance of open dialogue and relationship development in establishing an environment where individuals are actively engaged in decision-making processes regarding their health. Although guidelines and recommendations for engagement for gene drives have recently been described, we argue here that communities and stakeholders should lead the planning, development, and implementation phases of engagement. The RBM provides a new approach to the development of ethical, transparent, and effective engagement strategies for malaria control programs.

The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect's innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers selfproperties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanizationbased immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only latestage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes frommelanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization. [ABSTRACT FROM AUTHOR]

Immunocompromised patients with hematologic diseases may experience life-threatening infections with rather uncommon manifestations. Laryngitis has been described as a potential infection in such vulnerable patients and may result in major complications, ranging from impending airway obstruction to total laryngeal necrosis. Immediate laryngoscopy is of paramount importance, as it provides quantification of laryngeal edema and evidence of necrosis. Documentation of the causative pathogen is usually feasible through tissue culture. In the literature, 14 cases of necrotizing laryngitis have already been published. Here, we present the case of a 38-year-old male with a recent diagnosis of multiple myeloma, who received the first cycle of therapy a few days before admission. The patient presented with neutropenic fever, diarrhea, and multiple organ dysfunction. His course was complicated with hemophagocytic lymphohistiocytosis and stridor. A diagnosis of necrotizing laryngitis attributed to Acinetobacter baumannii invasion of the larynx was established. This manuscript highlights that the management of patients with hematologic disease and necrotizing laryngitis should be coordinated in highly specialized centers and clinicians should have a high level of clinical suspicion and act promptly. [ABSTRACT FROM AUTHOR]

Osmotically dehydrated cherry tomatoes and spinach leaves were incorporated into Greek salad-type (including OD-treated and air-dried feta cheese trimmings and air-dried olive rings) and green salad-type (including OD-treated and air-dried feta cheese trimmings and roasted ground peanuts) ready-to-eat (RTE) product prototypes, respectively. The osmotic dehydration of cherry tomatoes and spinach leaves was conducted in a pilot scale setting (100 L) in a 60% glycerol-based solution at 35 °C and 25 °C for 180 min and 60 min, respectively. To quantify the moisture transfer between the three ingredients of different moisture content (and water activity), the moisture equilibrium curves for each ingredient of the RTE product were determined. The equilibrium water activity of RTE products was 0.86 and 0.76, respectively. The quality of the RTE products (more specifically, tomato and spinach color and texture, instrumentally measured and sensorially perceived, sensory characteristics) was evaluated. The shelf life of the prototypes (from 4 °C to 20 °C) was kinetically modeled based on sensory deterioration and microbial growth, using the zero-order kinetic model and the Gompertz model, respectively. In the case of the tomato-based product, a shelf life of 54 days (based on sensory deterioration) was achieved at 4 °C, a shelf-life extension of 40 days compared to untreated, fresh-cut tomato. The shelf life of the spinach-based product (based on sensory deterioration) was 36 days at 4 °C, 30 days longer when compared to untreated spinach. Our results indicate that osmotic dehydration was successful in significantly extending the shelf life of such products, contributing to the increased temperature resilience of their keeping quality and allowing for their distribution and storage in a variable cold chain. [ABSTRACT FROM AUTHOR]

This study focused on the osmotic dehydration (OD) of ready-to-eat spinach leaves combined with the pulsed electric field (PEF) pre-treatment. Untreated and PEF-treated (0.6 kV/cm, 0–200 pulses) spinach leaves were osmotically dehydrated at room temperature for up to 120 min. The application of PEF (0.6 kV/20 pulses) prior to OD (60% glycerol, 25 °C, 60 min) lowered water activity (aw = 0.891) while achieving satisfactory product acceptability (total sensory hedonic scoring of 8). During the storage of the product (at 4, 8, 12, and 20 °C for up to 30 d), a significant reduction in total microbial count evolution was observed (9.7 logCFU/g for the untreated samples vs. 5.1 logCFU/g for the PEF-OD-treated samples after 13 d of storage at 4 °C). The selection of these PEF and OD treatment conditions enabled the extension of the product shelf life by up to 33 d under chilled storage. Osmotically treated spinach could find application in ready-to-eat salad products with an extended shelf life, which is currently not possible due to the high perishability of the specific plant tissue. [ABSTRACT FROM AUTHOR]