Environment-dependent metabolic investments in the mixotrophic chrysophyte Ochromonas.

Mixotrophic protists combine photosynthesis and phagotrophy to obtain energy and nutrients. Because mixotrophs can act as either primary producers or consumers, they have a complex role in marine food webs and biogeochemical cycles. Many mixotrophs are also phenotypically plastic and can adjust their metabolic investments in response to resource availability. Thus, a single species's ecological role may vary with environmental conditions. Here, we quantified how light and food availability impacted the growth rates, energy acquisition rates, and metabolic investment strategies of eight strains of the mixotrophic chrysophyte, Ochromonas. All eight Ochromonas strains photoacclimated by decreasing chlorophyll content as light intensity increased. Some strains were obligate phototrophs that required light for growth, while other strains showed stronger metabolic responses to prey availability. When prey availability was high, all eight strains exhibited accelerated growth rates and decreased their investments in both photosynthesis and phagotrophy. Photosynthesis and phagotrophy generally produced additive benefits: In low-prey environments, Ochromonas growth rates increased to maximum, light-saturated rates with increasing light but increased further with the addition of abundant bacterial prey. The additive benefits observed between photosynthesis and phagotrophy in Ochromonas suggest that the two metabolic modes provide nonsubstitutable resources, which may explain why a tradeoff between phagotrophic and phototrophic investments emerged in some but not all strains.

Engineered Antiviral Sensor Targets Infected Mosquitoes.

Escalating vector disease burdens pose significant global health risks, as such innovative tools for targeting mosquitoes are critical. CRISPR-Cas technologies have played a crucial role in developing powerful tools for genome manipulation in various eukaryotic organisms. Although considerable efforts have focused on utilizing class II type II CRISPR-Cas9 systems for DNA targeting, these modalities are unable to target RNA molecules, limiting their utility against RNA viruses. Recently, the Cas13 family has emerged as an efficient tool for RNA targeting; however, the application of this technique in mosquitoes, particularly Aedes aegypti, has yet to be fully realized. In this study, 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. 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 viral prevalence of infection, and its promiscuous collateral activity can even kill infected mosquitoes within a few days. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.

Space weather disrupts nocturnal bird migration.

Space weather, including solar storms, can impact Earth by disturbing the geomagnetic field. Despite the known dependence of birds and other animals on geomagnetic cues for successful seasonal migrations, the potential effects of space weather on organisms that use Earth's magnetic field for navigation have received little study. We tested whether space weather geomagnetic disturbances are associated with disruptions to bird migration at a macroecological scale. We leveraged long-term radar data to characterize the nightly migration dynamics of the nocturnally migrating North American avifauna over 22 y. We then used concurrent magnetometer data to develop a local magnetic disturbance index associated with each radar station (ΔBmax), facilitating spatiotemporally explicit analyses of the relationship between migration and geomagnetic disturbance. After controlling for effects of atmospheric weather and spatiotemporal patterns, we found a 9 to 17% decrease in migration intensity in both spring and fall during severe space weather events. During fall migration, we also found evidence for decreases in effort flying against the wind, which may represent a depression of active navigation such that birds drift more with the wind during geomagnetic disturbances. Effort flying against the wind in the fall was most reduced under both overcast conditions and high geomagnetic disturbance, suggesting that a combination of obscured celestial cues and magnetic disturbance may disrupt navigation. Collectively, our results provide evidence for community-wide avifaunal responses to geomagnetic disturbances driven by space weather during nocturnal migration.

Training and implementation of handheld ultrasound technology at Georgetown Public Hospital Corporation in Guyana: a virtual learning cohort study

A virtual point-of-care ultrasound (POCUS) education program was initiated to introduce handheld ultrasound technology to Georgetown Public Hospital Corporation in Guyana, a low-resource setting. We studied ultrasound competency and participant satisfaction in a cohort of 20 physicians-in-training through the urology clinic. The program consisted of a training phase, where they learned how to use the Butterfly iQ ultrasound, and a mentored implementation phase, where they applied their skills in the clinic. The assessment was through written exams and an objective structured clinical exam (OSCE). Fourteen students completed the program. The written exam scores were 3.36/5 in the training phase and 3.57/5 in the mentored implementation phase, and all students earned 100% on the OSCE. Students expressed satisfaction with the program. Our POCUS education program demonstrates the potential to teach clinical skills in low-resource settings and the value of virtual global health partnerships in advancing POCUS and minimally invasive diagnostics.

Engineered Antiviral Sensor Targets Infected Mosquitoes.

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 up taken. 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.

CRISPR mediated transactivation in the human disease vector Aedes aegypti.

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.

Evidence for evolutionary adaptation of mixotrophic nanoflagellates to warmer temperatures.

Mixotrophs, organisms that combine photosynthesis and heterotrophy to gain energy, play an important role in global biogeochemical cycles. Metabolic theory predicts that mixotrophs will become more heterotrophic with rising temperatures, potentially creating a positive feedback loop that accelerates carbon dioxide accumulation in the atmosphere. Studies testing this theory have focused on phenotypically plastic (short-term, non-evolutionary) thermal responses of mixotrophs. However, as small organisms with short generation times and large population sizes, mixotrophs may rapidly evolve in response to climate change. Here, we present data from a 3-year experiment quantifying the evolutionary response of two mixotrophic nanoflagellates to temperature. We found evidence for adaptive evolution (increased growth rates in evolved relative to acclimated lineages) in the obligately phototrophic strain, but not in the facultative phototroph. All lineages showed trends of increased carbon use efficiency, flattening of thermal reaction norms, and a return to homeostatic gene expression. Generally, mixotrophs evolved reduced photosynthesis and higher grazing with increased temperatures, suggesting that evolution may act to exacerbate mixotrophs' effects on global carbon cycling.

Suppressing mosquito populations with precision guided sterile males.

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.

Prey type constrains growth and photosynthetic capacity of the kleptoplastidic ciliate Mesodinium chamaeleon (Ciliophora).

Kleptoplastidic, or chloroplast-stealing, lineages offer insight into the process of acquiring photosynthesis. By quantifying the ability of these organisms to retain and use photosynthetic machinery from their prey, we can understand how intermediaries on the endosymbiosis pathway might have evolved regulatory and maintenance mechanisms. Here, we focus on a mixotrophic kleptoplastidic ciliate, Mesodinium chamaeleon, noteworthy for its ability to retain functional chloroplasts from at least half a dozen cryptophyte algal genera. We contrasted the performance of kleptoplastids from blue-green and red cryptophyte prey as a function of light level and feeding history. Our experiments showed that starved M. chamaeleon cells are able to maintain photosynthetic function for at least 2 weeks and that M. chamaeleon containing red plastids lost chlorophyll and electron transport capacity faster than those containing blue-green plastids. However, likely due to increased pigment content and photosynthetic rates in red plastids, M. chamaeleon had higher growth rates and more prolonged growth when feeding on red cryptophytes. For example, M. chamaeleon grew rapidly and extensively when fed the blue-green cryptophyte Chroomonas mesostigmatica, but this growth appeared to hinge on high levels of feeding supporting photosynthetic activity. In contrast, even starved M. chamaeleon containing red plastids from Rhodomonas salina could achieve high photosynthetic rates and extensive growth. Our findings show that plastid origin impacts the maintenance and magnitude of photosynthetic activity, though whether this is due to variation in ciliate control or gradual loss of plastid function in ingested prey cells remains unknown.

Soil fungal community composition and functional similarity shift across distinct climatic conditions.

A large part of ecosystem function in woodland systems depends on soil fungal communities. However, global climate change has the potential to fundamentally alter these communities as fungal species are filtered with changing environmental conditions. In this study, we examined the potential effects of climate on host-associated (i.e. tree-associated) soil fungal communities at climatically distinct sites in the Tehachapi Mountains in California, where more arid conditions represent likely regional climate futures. We found that soil fungal community composition changes strongly across sites, with species richness and diversity being highest at the most arid site. However, host association may buffer the effects of climate on community composition, as host-associated fungal communities are more similar to each other across climatically distinct sites than the whole fungal community. Lastly, an examination of functional traits for ectomycorrhizal fungi, a well-studied guild of fungal mutualist species, showed that stress-tolerant traits were more abundant at arid sites than mesic sites, providing a mechanistic understanding of these community patterns. Taken together, our results indicate that fungal community composition will likely shift with future climate change but that host association may buffer these effects, with shifts in functional traits having implications for future ecosystem function.

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus.

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.

Development of a confinable gene drive system in the human disease vector Aedes aegypti.

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.

Live calcium imaging of Aedes aegypti neuronal tissues reveals differential importance of chemosensory systems for life-history-specific foraging strategies.

BACKGROUND: The mosquito Aedes aegypti has a wide variety of sensory pathways that have supported its 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 responding motor outputs, there has been a lack of studies able to monitor both concurrently. RESULTS: 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 microscopy, we simultaneously measured live stimulus-evoked calcium responses in both neuronal and muscle cells with a wide spatial range and resolution. CONCLUSIONS: By coupling in vivo live 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.

A Prospective, Multicenter Evaluation of Point-of-care Ultrasound for Small-bowel Obstruction in the Emergency Department.

OBJECTIVE: The 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). METHODS: We 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. RESULTS: A 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] = 0.80 to 0.94), 0.54 (95% CI = 0.45 to 0.63), 1.92 (95% CI = 1.56 to 2.35), and 0.22 (95% CI = 0.12 to 0.39), respectively. Expert review yielded a similar sensitivity (0.89 [95% CI = 0.81 to 0.95]) with a significantly higher specificity (0.82 [95% CI = 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% CI = 0.79 to 0.93], 0.87 [95% CI = 0.79 to 0.93]) and abnormal peristalsis (0.82 [95% CI = 0.72 to 0.89], 0.85 [95% CI = 0.76 to 0.87]). The more specific parameters for both groups were transition point (0.82 [95% CI = 0.74 to 0.89], 0.98 [95% CI = 0.94 to 1.00]), intraperitoneal free fluid (0.82 [95% CI = 0.74 to 0.89], 0.93 [95% CI = 0.87 to 0.97]), and bowel wall edema (0.76 [95% CI = 0.67 to 0.83], 0.93 [95% CI = 0.87 to 0.97]). CONCLUSION: POCUS 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.

Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector, Aedes aegypti.

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.

Rhodiola rosea L.: an herb with anti-stress, anti-aging, and immunostimulating properties for cancer chemoprevention.

PURPOSE OF REVIEW: Rhodiola rosea extracts have been used as a dietary supplement in healthy populations, including athletes, to non-specifically enhance the natural resistance of the body to both physical and behavior stresses for fighting fatigue and depression. We summarize the information with respect to the new pharmacological activities of Rhodiola rosea extracts and its underlying molecular mechanisms in this review article. RECENT FINDINGS: In addition to its multiplex stress-protective activity, Rhodiola rosea extracts have recently demonstrated its anti-aging, anti-inflammation, immunostimulating, DNA repair and anti-cancer effects in different model systems. Molecular mechanisms of Rhodiola rosea extracts's action have been studied mainly along with one of its bioactive compounds, salidroside. Both Rhodiola rosea extracts and salidroside have contrast molecular mechanisms on cancer and normal physiological functions. For cancer, Rhodiola rosea extracts and salidroside inhibit the mTOR pathway and reduce angiogenesis through down-regulation of the expression of HIF-1α/HIF-2α. For normal physiological functions, Rhodiola rosea extracts and salidroside activate the mTOR pathway, stimulate paracrine function and promote neovascularization by inhibiting PHD3 and stabilizing HIF-1α proteins in skeletal muscles. In contrast to many natural compounds, salidroside is water-soluble and highly bioavailable via oral administration and concentrated in urine by kidney excretion. SUMMARY: Rhodiola rosea extracts and salidroside can impose cellular and systemic benefits similar to the effect of positive lifestyle interventions to normal physiological functions and for anti-cancer. The unique pharmacological properties of Rhodiola rosea extracts or salidroside deserve further investigation for cancer chemoprevention, in particular for human urinary bladder cancer.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation.

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 effects of phenoseason and storm characteristics on throughfall solute washoff and leaching dynamics from a temperate deciduous forest canopy.

Seasonal variations in the washoff and leaching dynamics of throughfall ionic fluxes represent a significant process affecting the biogeochemical cycling of forested ecosystems-particularly for temperate deciduous forests with distinct phenological seasons (or "phenoseasons"). Most studies on temperate deciduous forests aggregate seasonal throughfall fluxes to the leafed (growing) and leafless (dormant) periods, yet the phenological conditions controlling seasonality demand finer-scale demarcations that include the transitional phenoseasons (leaf senescence and emergence). To fill these gaps our study examines the washoff and leaching dynamics of Na(+), Mg(2+), K(+), Ca(2+), Cl(-), SO(4)(2-), and NO(3)(-) throughfall derived from bulk and sequentially sampled rain events across leafed, leafless and both transitional phenoseasons over a 3-year period (2008-2010). As throughfall washoff and leached solute fluxes are also closely-coupled to rainfall conditions, we further examine the effects of storm characteristics on phenoseasonal washoff-dominated (Na(+) and Cl(-)) and leaching-dominated (K(+), Ca(2+), Mg(2+)) fluxes through intrastorm event comparison plots and factorial MANOVA. Highly significant differences in leached and washoff solute fluxes were found across meteorological conditions (p<0.001) nested within phenoseasonal divisions (p<0.00001). Phenoseasonal washoff Na(+) and Cl(-) fluxes seemed to be more closely related to leaf area; whereas, leaching flux and canopy exchange of all solutes to correspond more with major phenological changes (when the canopies tend to be most metabolically active). The greatest differences in leached Mg(2+), K(+), Ca(2+), and SO(4)(2-) fluxes were not between the full leafed and leafless phenoseasons (33-80% difference), but between the transitional periods (80 to 200 fold greater during leaf senescence than leaf emergence). Intrastorm average canopy NO(3)(-) leaching, however, ranged from low losses (1 µmol(c)m(-2)h(-1)) to canopy uptake (-2 µmol(c)m(-2)h(-1)) during both transitional phenoseasons. K(+), Ca(2+), Mg(2+) were all markedly more exchangeable during senescence, with Ca(2+) and Mg(2+) being more tightly held by the canopy. Leaching rates and fluxes for all measured solutes were negligible to negative during emergence, except for K(+) and SO(4)(2-). Our results indicate that much of the variance in timing and magnitude of throughfall solute fluxes to forest soils within temperate deciduous ecosystems may be ascribed to phenologically-delineated seasons and storm conditions.