An update and review of arthropod vector sensory systems: Potential targets for behavioural manipulation by parasites and other disease agents.

For over a century, vector ecology has been a mainstay of vector-borne disease control. Much of this research has focused on the sensory ecology of blood-feeding arthropods (black flies, mosquitoes, ticks, etc.) with terrestrial vertebrate hosts. Of particular interest are the cues and sensory systems that drive host seeking and host feeding behaviours as they are critical for a vector to locate and feed from a host. An important yet overlooked component of arthropod vector ecology are the phenotypic changes observed in infected vectors that increase disease transmission. While our fundamental understanding of sensory mechanisms in disease vectors has drastically increased due to recent advances in genome engineering, for example, the advent of CRISPR-Cas9, and high-throughput "big data" approaches (genomics, proteomics, transcriptomics, etc.), we still do not know if and how parasites manipulate vector behaviour. Here, we review the latest research on arthropod vector sensory systems and propose key mechanisms that disease agents may alter to increase transmission.

The DDHD2-STXBP1 interaction mediates long-term memory via generation of saturated free fatty acids.

The phospholipid and free fatty acid (FFA) composition of neuronal membranes plays a crucial role in learning and memory, but the mechanisms through which neuronal activity affects the brain's lipid landscape remain largely unexplored. The levels of saturated FFAs, particularly of myristic acid (C14:0), strongly increase during neuronal stimulation and memory acquisition, suggesting the involvement of phospholipase A1 (PLA1) activity in synaptic plasticity. Here, we show that genetic ablation of the PLA1 isoform DDHD2 in mice dramatically reduces saturated FFA responses to memory acquisition across the brain. Furthermore, DDHD2 loss also decreases memory performance in reward-based learning and spatial memory models prior to the development of neuromuscular deficits that mirror human spastic paraplegia. Via pulldown-mass spectrometry analyses, we find that DDHD2 binds to the key synaptic protein STXBP1. Using STXBP1/2 knockout neurosecretory cells and a haploinsufficient STXBP1+/- mouse model of human early infantile encephalopathy associated with intellectual disability and motor dysfunction, we show that STXBP1 controls targeting of DDHD2 to the plasma membrane and generation of saturated FFAs in the brain. These findings suggest key roles for DDHD2 and STXBP1 in lipid metabolism and in the processes of synaptic plasticity, learning, and memory.

Automated Egg-Counting Approaches for Aedes aegypti Oviposition Experiments.

Egg-laying preferences of mosquitoes can reveal key neurosensory mechanisms informing the decision-making process for this critical behavior. A single blood meal results in a gravid female Aedes aegypti mosquito laying more than 100 eggs. Therefore, egg counting represents a potentially time-consuming and laborious component to behavioral assays, such as those that measure oviposition preference or fecundity. Automated algorithms that count eggs from images can dramatically reduce the time required for this step of data processing and analysis and increase reproducibility associated with having multiple human observers count the eggs. Here, we present two distinct approaches for counting melanized Ae. aegypti eggs laid on white filter paper.

Evaluating Egg-Laying Preference of Individual Aedes aegypti Mosquitoes.

Egg-laying behavior in insects has a dramatic impact on their fitness, controlling their extent and distribution across ecosystems. Thus, understanding the cues that guide mosquito egg laying is an important goal. Measuring mosquito egg-laying preference is critically important for understanding the sensory and neural mechanisms guiding adaptive breeding decisions. Here, we present a method for ascertaining the egg-laying preference of individual Aedes aegypti mosquitoes in small chambers with two distinct oviposition substrate choices. This assay is best suited for medium- to high-throughput analysis of egg-laying preference to liquids containing distinct contact chemosensory cues, such as differing salinities. We discuss all steps of the process from assay construction and setup to data acquisition and analysis.

Measuring Oviposition Preference in Aedes aegypti Mosquitoes.

The female mosquito is endowed with the responsibility of gene transfer from one generation to another through careful delivery of her eggs to an appropriate environment. To guarantee a successful reproduction process, female mosquitoes use an evaluative approach to select an oviposition site that will ensure that their progeny successfully hatch and develop from larvae to pupa to adult. Specific conditions must be met during oviposition site selection before gravid females deposit eggs, including but not limited to appropriate temperature, salinity, and pH; the presence of nutrition; and a low risk of predation. Mosquito species exhibit a remarkable diversity in their oviposition site selection behaviors. For instance, Aedes aegypti, which is a vector for Zika, dengue, yellow fever, and chikungunya viruses, prefers to lay its eggs in natural depressions (e.g., tree holes) or artificial containers. In contrast, other mosquito species, like Anopheles, gravitate toward more open water bodies. Understanding the oviposition preference behavior of the Ae. aegypti mosquito will facilitate the development of surveillance and control efforts to hinder the successful progression of this vector. Here, we present information on some of the known behaviors and preferences of Ae. aegypti during oviposition site selection, techniques to measure these preferences, and some open questions.

Atmospheric turbulence characterization with simultaneous measurement of phase, angle of arrival, and intensity in a retroreflected optical link.

Free-space optical transmission through the Earth's atmosphere is applicable to high-speed data transmission and optical clock comparisons, among other uses. Fluctuations in the refractive index of the atmosphere limit the performance of atmospheric optical transmission by inducing phase noise, angle-of-arrival variation, and scintillation. The statistics of these deleterious effects are predicted by models for the spatial spectrum of the atmospheric refractive index structure. We present measurements of phase fluctuations, angle-of-arrival variations, and scintillation, taken concurrently and compared with models for the atmospheric refractive index structure. The measurements are also cross-compared by deriving independent estimates of the turbulence structure constant $C_n^2$. We find agreement within an order of magnitude for derived $C_n^2$ values for all three metrics.

Design and Validation of Guide RNAs for CRISPR-Cas9 Genome Editing in Mosquitoes.

CRISPR-Cas9 has revolutionized gene editing for traditional and nontraditional model organisms alike. This tool has opened the door to new mechanistic studies of basic mosquito biology as well as the development of novel vector control strategies based on CRISPR-Cas9, including gene drives that spread genetic elements in the population. Although the promise of the specificity, flexibility, and ease of deployment CRISPR is real, its implementation still requires empirical optimization for each new species of interest, as well as to each genomic target within a given species. Here, we provide an overview of designing and testing single-guide RNAs for the use of CRISPR-based gene editing tools.

Validating Single-Guide RNA for Aedes aegypti Gene Editing.

Creating transgenic mosquitoes allows for mechanistic studies of basic mosquito biology and the development of novel vector control strategies. CRISPR-Cas9 gene editing has revolutionized gene editing, including in mosquitoes. This protocol details part of the gene editing process of Aedes aegypti mosquitoes via CRISPR-Cas9, through testing and validating single-guide RNAs (sgRNAs). Gene editing activity varies depending on the sequence of sgRNAs used, so validation of sgRNA activity should be done before large-scale generation of mutants or transgenics. sgRNA is designed using online tools and synthesized in <1 h. Once mutants or transgenics are generated via embryo microinjection, sgRNA activity is validated by quick genotyping polymerase chain reaction (PCR) and DNA sequencing.

High-bandwidth coherent optical communication over 10.3 km of turbulent air.

We demonstrate 111.8 Gb/s coherent optical communication throughput over a 10.3 km folded free-space laser range. Folded links are low complexity to establish and provide a high uptime for testing equipment. The communication signals were sourced from an un-modified commercial off-the-shelf transceiver intended for long-haul fiber networks. Wavelength dependence was explored by testing 52 optical C-band channels over the course of an evening. In the future, such high-bandwidth communications will be used in feeder links from satellites in geosynchronous orbit. Optical power measurements of the received signal are compared with atmospheric theory to determine the turbulence strength exhibited and therefore the applicability of the laser range to space-to-ground links. We show that the high-uptime, 10.3 km laser range is suitable for testing high-bandwidth space-to-ground optical communication systems intended for links to geosynchronous orbit at 20°-50° elevation.

Lipids and Secretory Vesicle Exocytosis.

In recent years, the number of studies implicating lipids in the regulation of synaptic vesicle exocytosis has risen considerably. It has become increasingly clear that lipids such as phosphoinositides, lysophospholipids, cholesterol, arachidonic acid and myristic acid play critical regulatory roles in the processes leading up to exocytosis. Lipids may affect membrane fusion reactions by altering the physical properties of the membrane, recruiting key regulatory proteins, concentrating proteins into exocytic "hotspots" or by modulating protein functions allosterically. Discrete changes in phosphoinositides concentration are involved in multiple trafficking events including exocytosis and endocytosis. Lipid-modifying enzymes such as the DDHD2 isoform of phospholipase A1 were recently shown to contribute to memory acquisition via dynamic modifications of the brain lipid landscape. Considering the increasing reports on neurodegenerative disorders associated with aberrant intracellular trafficking, an improved understanding of the control of lipid pathways is physiologically and clinically significant and will afford unique insights into mechanisms and therapeutic methods for neurodegenerative diseases. Consequently, this chapter will discuss the different classes of lipids, phospholipase enzymes, the evidence linking them to synaptic neurotransmitter release and how they act to regulate key steps in the multi-step process leading to neuronal communication and memory acquisition.

Stabilized free space optical frequency transfer using digitally enhanced heterodyne interferometry.

Free-space continuous-wave laser interferometry using folded links has applications in precision measurement for velocimetry, vibrometry, optical communications, and verification of frequency transfer for metrology. However, prompt reflections from the transceiver optics degrade the performance of these systems, especially when the power of the returning signal is equal to or less than the power of the prompt reflections. We demonstrate phase stabilized free-space continuous-wave optical frequency transfer that exploits the auto-correlation properties of pseudo-random binary sequences to filter out prompt reflections. We show that this system significantly improves the stability and robustness of optical frequency transfer over a 750 m turbulent free-space channel, achieving a best fractional frequency stability of 8 × 10-20 at an integration time of τ = 512 s, and cycle-slip-free periods up to 162 min.

Towards optical frequency geopotential difference measurements via a flying drone.

Geopotential and orthometric height differences between distant points can be measured via timescale comparisons between atomic clocks. Modern optical atomic clocks achieve statistical uncertainties on the order of 10-18, allowing height differences of around 1 cm to be measured. Frequency transfer via free-space optical links will be needed for measurements where linking the clocks via optical fiber is not possible, but requires line of sight between the clock locations, which is not always practical due to local terrain or over long distances. We present an active optical terminal, phase stabilization system, and phase compensation processing method robust enough to enable optical frequency transfer via a flying drone, greatly increasing the flexibility of free-space optical clock comparisons. We demonstrate a statistical uncertainty of 2.5×10-18 after 3 s of integration, corresponding to a height difference of 2.3 cm, suitable for applications in geodesy, geology, and fundamental physics experiments.

PR1P, a VEGF-stabilizing peptide, reduces injury and inflammation in acute lung injury and ulcerative colitis animal models.

Acute Respiratory Distress Syndrome (ARDS) and Ulcerative Colitis (UC) are each characterized by tissue damage and uncontrolled inflammation. Neutrophils and other inflammatory cells play a primary role in disease progression by acutely responding to direct and indirect insults to tissue injury and by promoting inflammation through secretion of inflammatory cytokines and proteases. Vascular Endothelial Growth Factor (VEGF) is a ubiquitous signaling molecule that plays a key role in maintaining and promoting cell and tissue health, and is dysregulated in both ARDS and UC. Recent evidence suggests a role for VEGF in mediating inflammation, however, the molecular mechanism by which this occurs is not well understood. We recently showed that PR1P, a 12-amino acid peptide that binds to and upregulates VEGF, stabilizes VEGF from degradation by inflammatory proteases such as elastase and plasmin thereby limiting the production of VEGF degradation products (fragmented VEGF (fVEGF)). Here we show that fVEGF is a neutrophil chemoattractant in vitro and that PR1P can be used to reduce neutrophil migration in vitro by preventing the production of fVEGF during VEGF proteolysis. In addition, inhaled PR1P reduced neutrophil migration into airways following injury in three separate murine acute lung injury models including from lipopolysaccharide (LPS), bleomycin and acid. Reduced presence of neutrophils in the airways was associated with decreased pro-inflammatory cytokines (including TNF-α, IL-1ß, IL-6) and Myeloperoxidase (MPO) in broncho-alveolar lavage fluid (BALF). Finally, PR1P prevented weight loss and tissue injury and reduced plasma levels of key inflammatory cytokines IL-1ß and IL-6 in a rat TNBS-induced colitis model. Taken together, our data demonstrate that VEGF and fVEGF may each play separate and pivotal roles in mediating inflammation in ARDS and UC, and that PR1P, by preventing proteolytic degradation of VEGF and the production of fVEGF may represent a novel therapeutic approach to preserve VEGF signaling and inhibit inflammation in acute and chronic inflammatory diseases.

Postmenopausal mixed gestational trophoblastic neoplasia with complete response to immunotherapy - A case report.

•Mixed GTN.•GTN in a post-menopausal woman.•Treatment of GTN with immunotherapy.

GPR183 antagonism reduces macrophage infiltration in influenza and SARS-CoV-2 infection.

RATIONALE: Severe viral respiratory infections are often characterised by extensive myeloid cell infiltration and activation and persistent lung tissue injury. However, the immunological mechanisms driving excessive inflammation in the lung remain poorly understood. OBJECTIVES: To identify the mechanisms that drive immune cell recruitment in the lung during viral respiratory infections and identify novel drug targets to reduce inflammation and disease severity. METHODS: Preclinical murine models of influenza A virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. RESULTS: Oxidised cholesterols and the oxysterol-sensing receptor GPR183 were identified as drivers of monocyte/macrophage infiltration to the lung during influenza A virus (IAV) and SARS-CoV-2 infection. Both IAV and SARS-CoV-2 infection upregulated the enzymes cholesterol 25-hydroxylase (CH25H) and cytochrome P450 family 7 subfamily member B1 (CYP7B1) in the lung, resulting in local production of the oxidised cholesterols 25-hydroxycholesterol (25-OHC) and 7α,25-dihydroxycholesterol (7α,25-OHC). Loss-of-function mutation of Gpr183 or treatment with a GPR183 antagonist reduced macrophage infiltration and inflammatory cytokine production in the lungs of IAV- or SARS-CoV-2-infected mice. The GPR183 antagonist significantly attenuated the severity of SARS-CoV-2 infection and viral loads. Analysis of single-cell RNA-sequencing data on bronchoalveolar lavage samples from healthy controls and COVID-19 patients with moderate and severe disease revealed that CH25H, CYP7B1 and GPR183 are significantly upregulated in macrophages during COVID-19. CONCLUSION: This study demonstrates that oxysterols drive inflammation in the lung via GPR183 and provides the first preclinical evidence for the therapeutic benefit of targeting GPR183 during severe viral respiratory infections.

Demonstration of 100 Gbps coherent free-space optical communications at LEO tracking rates.

Free-space optical communications are poised to alleviate the data-flow bottleneck experienced by spacecraft as traditional radio frequencies reach their practical limit. While enabling orders-of-magnitude gains in data rates, optical signals impose much stricter pointing requirements and are strongly affected by atmospheric turbulence. Coherent detection methods, which capitalize fully on the available degrees of freedom to maximize data capacity, have the added complication of needing to couple the received signal into single-mode fiber. In this paper we present results from a coherent 1550 nm link across turbulent atmosphere between a deployable optical terminal and a drone-mounted retroreflector. Through 10 Hz machine vision optical tracking with nested 200 Hz tip/tilt adaptive optics stabilisation, we corrected for pointing errors and atmospheric turbulence to maintain robust single mode fiber coupling, resulting in an uninterrupted 100 Gbps optical data link while tracking at angular rates of up to 1.5 deg/s, equivalent to that of spacecraft in low earth orbit. With the greater data capacity of coherent communications and compatibility with extant fiber-based technologies being demonstrated across static links, ground-to-low earth orbit links of Terabits per second can ultimately be achieved with capable ground stations.

Non-canonical odor coding in the mosquito.

Aedes aegypti mosquitoes are a persistent human foe, transmitting arboviruses including dengue when they feed on human blood. Mosquitoes are intensely attracted to body odor and carbon dioxide, which they detect using ionotropic chemosensory receptors encoded by three large multi-gene families. Genetic mutations that disrupt the olfactory system have modest effects on human attraction, suggesting redundancy in odor coding. The canonical view is that olfactory sensory neurons each express a single chemosensory receptor that defines its ligand selectivity. We discovered that Ae. aegypti uses a different organizational principle, with many neurons co-expressing multiple chemosensory receptor genes. In vivo electrophysiology demonstrates that the broad ligand-sensitivity of mosquito olfactory neurons depends on this non-canonical co-expression. The redundancy afforded by an olfactory system in which neurons co-express multiple chemosensory receptors may increase the robustness of the mosquito olfactory system and explain our long-standing inability to disrupt the detection of humans by mosquitoes.

The Role of Sentinel Lymph Node Mapping in High-grade Endometrial Cancer.

OPINION STATEMENT: Total hysterectomy with lymph node assessment is the current standard-of-care for surgical staging in apparent early-stage endometrial cancer. Compared to the traditional complete pelvic lymphadenectomy with or without para-aortic lymphadenectomy, sentinel lymph node (SLN) mapping results in fewer surgical complications, decreased operative time, and lower rates of chronic lymphedema. The technique is endorsed by the National Comprehensive Cancer Network and the Society of Gynecologic Oncology guidelines, and over the past two decades the majority of gynecologic oncologists worldwide have adopted SLN mapping into their practice. However, as the results of the initial SLN studies were mostly based on low-grade tumors, adoption of the technique in high-grade tumors has been slower and more controversial. In this review, we discuss the most recent studies evaluating the SLN mapping in high-grade endometrial cancers. The results of these studies suggest that the SLN detection rate is acceptably high and the negative predictive value is sufficiently low to support the use of SLN mapping in high-grade endometrial tumors to replace complete lymphadenectomy. Validity of SLN mapping techniques does, however, require following a standard algorithm, and success depends also on surgeon expertise. Moreover, the impact of SLN mapping on overall survival in high-grade tumors requires future prospective randomized studies. Finally, a transition toward near-universal SLN mapping techniques for endometrial cancers could significantly impact on the adequacy of gynecologic oncology fellows' surgical training and competency in lymphadenectomy.