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.

Excess Translation of Epigenetic Regulators Contributes to Fragile X Syndrome and Is Alleviated by Brd4 Inhibition.

Fragile X syndrome (FXS) is a leading genetic cause of intellectual disability and autism. FXS results from the loss of function of fragile X mental retardation protein (FMRP), which represses translation of target transcripts. Most of the well-characterized target transcripts of FMRP are synaptic proteins, yet targeting these proteins has not provided effective treatments. We examined a group of FMRP targets that encode transcriptional regulators, particularly chromatin-associated proteins. Loss of FMRP in mice results in widespread changes in chromatin regulation and aberrant gene expression. To determine if targeting epigenetic factors could reverse phenotypes associated with the disorder, we focused on Brd4, a BET protein and chromatin reader targeted by FMRP. Inhibition of Brd4 function alleviated many of the phenotypes associated with FXS. We conclude that loss of FMRP results in significant epigenetic misregulation and that targeting transcription via epigenetic regulators like Brd4 may provide new treatments for FXS.

Improved reference genome of Aedes aegypti informs arbovirus vector control.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

Discrepancies in Conflict-of-Interest Disclosures Among Physicians Receiving Compensation for Monoclonal Antibody Drugs.

BACKGROUND: Monoclonal antibody drugs are widely used, highly marketed, expensive compounds. Relationships between these drug manufacturers and physicians may increase the potential for bias in relevant studies. OBJECTIVE: The aim of this study is to determine the rate of disclosures among physicians receiving compensations for monoclonal antibody drugs (MAbDs). DESIGN: This is a retrospective, population-based, cross-sectional study. PARTICIPANTS: The 50 physicians who received the highest financial compensation for selected MAbDs from 2016 to 2020 were included. MAIN MEASURES: Payment data were obtained from the Open Payments Database, bibliometric data were obtained from SCOPUS, and disclosure data were obtained from relevant publications found in PubMed. The primary outcome was rate of disclosure concordance between self-declared conflict-of-interest and industry-reported payments documented in the Open Payments Database. KEY RESULTS: Of the 50 physicians examined, 74% (N = 37) had publications examined. A cumulative 6170 payments totaling $18,484,228 were analyzed. A total of 418 relevant papers were reviewed. The rate of full disclosure (all relevant financial relationships disclosed) was 39.5%, partial disclosure (some but not all financial relationships disclosed) was 28.0%, and no disclosure was 26.3%. 6.2% did not require disclosure. Publications authored by dermatologists had the highest rate of full disclosure at 49.3%. There was no association between h-index and disclosure rate. Practice guidelines had the highest rate of full disclosure at 69.2% while basic science papers had the lowest (0%). Lastly, substantial variations in specific journal disclosure policies were found. CONCLUSIONS: Substantial inconsistencies were found between self-reported disclosures and the Open Payments Database among physicians receiving high compensation for MAbDs. A policy of full disclosure for all publications should be adopted.

T cells targeting a neuronal paraneoplastic antigen mediate tumor rejection and trigger CNS autoimmunity with humoral activation.

Paraneoplastic neurologic diseases (PND) involving immune responses directed toward intracellular antigens are poorly understood. Here, we examine immunity to the PND antigen Nova2, which is expressed exclusively in central nervous system (CNS) neurons. We hypothesized that ectopic expression of neuronal antigen in the periphery could incite PND. In our C57BL/6 mouse model, CNS antigen expression limits antigen-specific CD4+ and CD8+ T-cell expansion. Chimera experiments demonstrate that this tolerance is mediated by antigen expression in nonhematopoietic cells. CNS antigen expression does not limit tumor rejection by adoptively transferred transgenic T cells but does limit the generation of a memory population that can be expanded upon secondary challenge in vivo. Despite mediating cancer rejection, adoptively transferred transgenic T cells do not lead to paraneoplastic neuronal targeting. Preliminary experiments suggest an additional requirement for humoral activation to induce CNS autoimmunity. This work provides evidence that the requirements for cancer immunity and neuronal autoimmunity are uncoupled. Since humoral immunity was not required for tumor rejection, B-cell targeting therapy, such as rituximab, may be a rational treatment option for PND that does not hamper tumor immunity.

RNA In Situ Hybridization of Whole-Mount Mosquito Olfactory Tissues.

This protocol presents a method for RNA in situ hybridization of whole-mounted preparations of mosquito antennae and maxillary palps. It can be adapted for other peripheral appendages such as the legs or proboscis.

Immunostaining Whole-Mount Olfactory Tissues of Mosquitoes.

Mosquito olfactory tissues are covered in fine hair-like sensory structures called sensilla that house olfactory sensory neurons. This protocol for immunostaining whole-mounted mosquito antennae and maxillary palps enables the visualization of these neurons.

RNA In Situ Hybridization and Immunohistochemistry to Visualize Gene Expression in Peripheral Chemosensory Tissues of Mosquitoes.

Mosquito-borne diseases such as malaria, Zika virus, and dengue virus are a menace to the human population. Although many mosquito species are not attracted to humans and do not feed on blood, human-biting female mosquitoes are strongly attracted to people and use chemosensory cues to identify a suitable host for a blood meal. Mosquitoes need blood components to reproduce, rendering them excellent vectors for blood-borne diseases. The three genera (Culex, Anopheles, and Aedes) responsible for most of these diseases find hosts by using their peripheral sensory organs. These organs include the antennae, maxillary palps, and proboscis. All three contain diverse populations of highly sensitive neurons that express sensory receptors that can detect odorants, temperature, chemicals, and tastants. Although these organs are essential to the host-seeking behavior that results in biting, their small size and thick outer cuticle can hinder typical histochemical analyses. Here, we briefly review the role the peripheral sensory organs play in mosquito behavior. Then, we introduce how to investigate their gene expression profiles using immunohistochemical and RNA in situ approaches for both whole-mount and frozen-section preparations.

Two novel, tightly linked, and rapidly evolving genes underlie Aedes aegypti mosquito reproductive resilience during drought.

Female Aedes aegypti mosquitoes impose a severe global public health burden as vectors of multiple viral pathogens. Under optimal environmental conditions, Aedes aegypti females have access to human hosts that provide blood proteins for egg development, conspecific males that provide sperm for fertilization, and freshwater that serves as an egg-laying substrate suitable for offspring survival. As global temperatures rise, Aedes aegypti females are faced with climate challenges like intense droughts and intermittent precipitation, which create unpredictable, suboptimal conditions for egg-laying. Here, we show that under drought-like conditions simulated in the laboratory, females retain mature eggs in their ovaries for extended periods, while maintaining the viability of these eggs until they can be laid in freshwater. Using transcriptomic and proteomic profiling of Aedes aegypti ovaries, we identify two previously uncharacterized genes named tweedledee and tweedledum, each encoding a small, secreted protein that both show ovary-enriched, temporally-restricted expression during egg retention. These genes are mosquito-specific, linked within a syntenic locus, and rapidly evolving under positive selection, raising the possibility that they serve an adaptive function. CRISPR-Cas9 deletion of both tweedledee and tweedledum demonstrates that they are specifically required for extended retention of viable eggs. These results highlight an elegant example of taxon-restricted genes at the heart of an important adaptation that equips Aedes aegypti females with 'insurance' to flexibly extend their reproductive schedule without losing reproductive capacity, thus allowing this species to exploit unpredictable habitats in a changing world.

Assessment of Medical Industry Compensation to US Physicians by Gender.

Importance: It has been well established that female physicians earn less than their male counterparts in all specialties and ranks despite controlling for confounding variables. Objective: To investigate payments made from highest-grossing medical industry companies to female and male physicians and to assess compensation and engagement disparities based on gender. Design, Setting, and Participants: This retrospective, population-based cross-sectional study used data from the Open Payments database for the 5 female and 5 male physicians who received the most financial compensation from each of the 15 highest-grossing medical supply companies in the US from January 2013 to January 2019. Main Outcomes and Measures: The primary outcome was total general payments received by female and male physicians from medical industry over time and across industries. The secondary outcome was trends in industry payment to female and male physicians from 2013 to 2019. Results: Among the 1050 payments sampled, 1017 (96.9%) of the 5 highest earners were men and 33 (3.1%) were women. Female physicians were paid a mean (SD) of $41 320 ($88 695), and male physicians were paid a mean (SD) of $1 226 377 ($3 377 957) (P < .001). On multivariate analysis, male gender was significantly associated with higher payment after adjusting for rank, h-index, and specialty (mean [SD], $1 025 413 [$162 578]; P < .001). From 2013 to 2019, the payment gap between female and male physicians increased from $54 343 to $166 778 (P < .001). Conclusions and Relevance: This study found that male physicians received significantly higher payments from the highest-grossing medical industry companies compared with female physicians. This disparity persisted across all medical specialties and academic ranks. The health care industry gender payment gap continued to increase from 2013 to 2019, with a wider compensation gap in 2019.

Fruitless mutant male mosquitoes gain attraction to human odor.

The Aedesaegypti mosquito shows extreme sexual dimorphism in feeding. Only females are attracted to and obtain a blood-meal from humans, which they use to stimulate egg production. The fruitless gene is sex-specifically spliced and encodes a BTB zinc-finger transcription factor proposed to be a master regulator of male courtship and mating behavior across insects. We generated fruitless mutant mosquitoes and showed that males failed to mate, confirming the ancestral function of this gene in male sexual behavior. Remarkably, fruitless males also gain strong attraction to a live human host, a behavior that wild-type males never display, suggesting that male mosquitoes possess the central or peripheral neural circuits required to host-seek and that removing fruitless reveals this latent behavior in males. Our results highlight an unexpected repurposing of a master regulator of male-specific sexual behavior to control one module of female-specific blood-feeding behavior in a deadly vector of infectious diseases.

Sexual dimorphism is a phenomenon among animals, insects and plants where the two sexes of a species show differences in body size, physical features or colors. The bushy mane of a male lion, for example, is nowhere to be seen on a female lioness, and only male peacocks have extravagant tails. Most examples of sexual dimorphism, such as elaborate visual displays or courtship behaviors, are linked to mating. However, there are a few species where behavioral differences between the sexes are not connected to mating. Mosquitoes are an example: while female mosquitoes feed on humans, and are attracted to a person's body heat and odor, male mosquitoes have little interest in biting humans for their blood. Therefore, female mosquitoes are the ones responsible for transmitting the viruses that cause certain blood-borne diseases such as dengue fever or Zika. Determining which genes are linked to feeding behaviors in mosquitoes could allow researchers to genetically engineer females so they no longer bite people, thus stopping the spread of these diseases. Unfortunately, the genes that control mosquito feeding behaviors have not been well studied. In other insects, some of the genes that control mating behaviors that depend on sex have been identified. For example, a gene called fruitless controls courtship behaviors in male flies and silkworms, and is thought to be the 'master regulator' of male sexual behavior across insects. Yet it remains to be seen whether the fruitless gene has any effect in mosquitoes, where sex differences relate to feeding habits. To investigate this, Basrur et al. removed the fruitless gene from Aedes aegypti mosquitoes. The genetically altered male mosquitoes became unable to mate successfully, but ­ similar to unmodified males ­ still preferred sugar water over blood when feeding. Unlike unmodified males, however, the male mosquitoes lacking fruitless were attracted to the body odor of a person's arm (like females). These results reveal that fruitless, a gene that controls sex-specific mating behaviors in other insects, controls a sex-specific feeding behavior in mosquitoes. The fruitless gene, Basrur et al. speculate, likely gained this role controlling mosquito feeding behavior in the course of evolution. More research is required to fully understand the effects of the fruitless gene in male and female mosquitoes.

The chromatin landscape of neuronal plasticity.

Examining the links between neuronal activity, transcriptional output, and synaptic function offers unique insights into how neurons adapt to changing environments and form memories. Epigenetic markers, such as DNA methylation and histone modifications, have been implicated in the formation of not only cellular memories such as cell fate, but also memories of experience at the organismal level. Here, we review recent advances in chromatin regulation that contribute to synaptic plasticity and drive adaptive behaviors through dynamic and precise regulation of transcription output in neurons. We discuss chromatin-associated proteins, histone variant proteins, the contribution of cis-regulatory elements and their interaction with histone modifications, and how these mechanisms are integrated into distinct behavior and environmental response paradigms.

HITS-CLIP and integrative modeling define the Rbfox splicing-regulatory network linked to brain development and autism.

The RNA binding proteins Rbfox1/2/3 regulate alternative splicing in the nervous system, and disruption of Rbfox1 has been implicated in autism. However, comprehensive identification of functional Rbfox targets has been challenging. Here, we perform HITS-CLIP for all three Rbfox family members in order to globally map, at a single-nucleotide resolution, their in vivo RNA interaction sites in the mouse brain. We find that the two guanines in the Rbfox binding motif UGCAUG are critical for protein-RNA interactions and crosslinking. Using integrative modeling, these interaction sites, combined with additional datasets, define 1,059 direct Rbfox target alternative splicing events. Over half of the quantifiable targets show dynamic changes during brain development. Of particular interest are 111 events from 48 candidate autism-susceptibility genes, including syndromic autism genes Shank3, Cacna1c, and Tsc2. Alteration of Rbfox targets in some autistic brains is correlated with downregulation of all three Rbfox proteins, supporting the potential clinical relevance of the splicing-regulatory network.

Neuronal Elav-like (Hu) proteins regulate RNA splicing and abundance to control glutamate levels and neuronal excitability.

The paraneoplastic neurologic disorders target several families of neuron-specific RNA binding proteins (RNABPs), revealing that there are unique aspects of gene expression regulation in the mammalian brain. Here, we used HITS-CLIP to determine robust binding sites targeted by the neuronal Elav-like (nElavl) RNABPs. Surprisingly, nElav protein binds preferentially to GU-rich sequences in vivo and in vitro, with secondary binding to AU-rich sequences. nElavl null mice were used to validate the consequence of these binding events in the brain, demonstrating that they bind intronic sequences in a position dependent manner to regulate alternative splicing and to 3'UTR sequences to regulate mRNA levels. These controls converge on the glutamate synthesis pathway in neurons; nElavl proteins are required to maintain neurotransmitter glutamate levels, and the lack of nElavl leads to spontaneous epileptic seizure activity. The genome-wide analysis of nElavl targets reveals that one function of neuron-specific RNABPs is to control excitation-inhibition balance in the brain.