Targeting sex determination to suppress mosquito populations.

Each year, hundreds of millions of people are infected with arboviruses such as dengue, yellow fever, chikungunya, and Zika, which are all primarily spread by the notorious mosquito Aedes aegypti. Traditional control measures have proven insufficient, necessitating innovations. In response, here we generate a next-generation CRISPR-based precision-guided sterile insect technique (pgSIT) for Ae. aegypti that disrupts genes essential for sex determination and fertility, producing predominantly sterile males that can be deployed at any life stage. Using mathematical models and empirical testing, we demonstrate that released pgSIT males can effectively compete with, suppress, and eliminate caged mosquito populations. This versatile species-specific platform has the potential for field deployment to effectively control wild populations of disease vectors.

Targeting Sex Determination to Suppress Mosquito Populations.

Each year, hundreds of millions of people are infected with arboviruses such as dengue, yellow fever, chikungunya, and Zika, which are all primarily spread by the notorious mosquito Aedes aegypti. Traditional control measures have proven insuficient, necessitating innovations. In response, here we generate a next generation CRISPR-based precision-guided sterile insect technique (pgSIT) for Aedes aegypti that disrupts genes essential for sex determination and fertility, producing predominantly sterile males that can be deployed at any life stage. Using mathematical models and empirical testing, we demonstrate that released pgSIT males can effectively compete with, suppress, and eliminate caged mosquito populations. This versatile species-specific platform has the potential for field deployment to control wild populations, safely curtailing disease transmission.

Targeting Sex Determination to Suppress Mosquito Populations.

Each year, hundreds of millions of people are infected with arboviruses such as dengue, yellow fever, chikungunya, and Zika, which are all primarily spread by the notorious mosquito Aedes aegypti. Traditional control measures have proven insufficient, necessitating innovations. In response, here we generate a next generation CRISPR-based precision-guided sterile insect technique (pgSIT) for Aedes aegypti that disrupts genes essential for sex determination and fertility, producing predominantly sterile males that can be deployed at any life stage. Using mathematical models and empirical testing, we demonstrate that released pgSIT males can effectively compete with, suppress, and eliminate caged mosquito populations. This versatile species-specific platform has the potential for field deployment to effectively control wild populations of disease vectors.

DiffNILM: A Novel Framework for Non-Intrusive Load Monitoring Based on the Conditional Diffusion Model.

Non-intrusive Load Monitoring (NILM) is a critical technology that enables detailed analysis of household energy consumption without requiring individual metering of every appliance, and has the capability to provide valuable insights into energy usage behavior, facilitate energy conservation, and optimize load management. Currently, deep learning models have been widely adopted as state-of-the-art approaches for NILM. In this study, we introduce DiffNILM, a novel energy disaggregation framework that utilizes diffusion probabilistic models to distinguish power consumption patterns of individual appliances from aggregated power. Starting from a random Gaussian noise, the target waveform is iteratively reconstructed via a sampler conditioned on the total active power and encoded temporal features. The proposed method is evaluated on two public datasets, REDD and UKDALE. The results demonstrated that DiffNILM outperforms baseline models on several key metrics on both datasets and shows a remarkable ability to effectively recreate complex load signatures. The study highlights the potential of diffusion models to advance the field of NILM and presents a promising approach for future energy disaggregation research.

CRISPR-Cas9 Methods and Key Considerations in the Production of Aedes aegypti Mutant Strains.

Since the characterization of the CRISPR-Cas9 system in prokaryotes, it has become the prime choice in gene editing because of its exceptional flexibility, ease of use, high efficiency, and superior specificity. As a result, CRISPR-Cas9-mediated gene-editing technologies have enabled researchers not only to engineer transgenic animal strains with site-directed insertions more efficiently but also to generate desired mutants for previously intractable species. One such species is the invasive yellow fever mosquito, Aedes aegypti, which is notorious for its ability to transmit many blood-borne human pathogens. Methods for developing new transgenic strains of the yellow fever mosquito may aid in the effort to control its populations and provide significant benefits for the public. Here, we provide an overview of injection and noninjection methods for generating transgenic mosquitoes and also highlight important experimental design features.

Generating Aedes aegypti Mutant Strains with Transgenic Cas9.

Here, we provide a protocol for generating Aedes aegypti mutant strains via end-joining (EJ) or homology-directed repair (HDR) mechanisms using genetically encoded Cas9.

CRISPR-Mediated Genome Engineering in Aedes aegypti.

CRISPR-mediated genome engineering technologies have been adapted to a wide variety of organisms with high efficiency and specificity. The yellow fever mosquito, Aedes aegypti , is one such organism. It is also responsible for transmitting a wide variety of deadly viruses including Dengue, Zika, Yellow fever, and Chikungunya. The key to successful CRISPR-mediated gene editing applications is the delivery of both Cas9 ribonuclease and single-guide RNA (sgRNA ) to the nucleus of desired cells. Various methods have been developed for supplying the Cas9 endonuclease, sgRNA , and donor DNA to Ae. aegypti. In this chapter, we focus on methods of direct embryo delivery of editing components, presenting detailed step-by-step CRISPR/Cas9-based genome-editing protocols for inducing desired heritable edits in mosquitoes as well as insights into successful application of these protocols. We also highlight potential opportunities for customizing these protocols to manipulate the mosquito genome for innovative in vivo gene function studies.

Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx.

CasRx, a member of the RNA-targeting Cas13 family, is a promising new addition of the CRISPR/Cas technologies in efficient gene transcript reduction with an attractive off-target profile at both cellular and organismal levels. It is recently reported that the CRISPR/CasRx system can be used to achieve ubiquitous and tissue-specific gene transcript reduction in Drosophila melanogaster. This paper details the methods from the recent work, consisting of three parts: 1) ubiquitous in vivo endogenous RNA targeting using a two-component CasRx system; 2) ubiquitous in vivo exogenous RNA targeting using a three-component CasRx system; and 3) tissue-specific in vivo RNA targeting using a three-component CasRx system. The effects of RNA targeting observed include targeted gene specific phenotypic changes, targeted RNA transcript reduction, and occasional lethality phenotypes associated with high expression of CasRx protein and collateral activity. Overall, these results showed that the CasRx system is capable of target RNA transcript reduction at the organismal level in a programmable and efficient manner, demonstrating that in vivo transcriptome targeting, and engineering is feasible and lays the foundation for future in vivo CRISPR-based RNA targeting technologies.

Programmable RNA Targeting Using CasRx in Flies.

CRISPR-Cas genome editing technologies have revolutionized the fields of functional genetics and genome engineering, but with the recent discovery and optimization of RNA-targeting Cas ribonucleases, we may soon see a similar revolution in the study of RNA function and transcriptome engineering. However, to date, successful proof of principle for Cas ribonuclease RNA targeting in eukaryotic systems has been limited. Only recently has successful modification of RNA expression by a Cas ribonuclease been demonstrated in animal embryos. This previous work, however, did not evaluate endogenous expression of Cas ribonucleases and only focused on function in early developmental stages. A more comprehensive evaluation of this technology is needed to assess its potential impact. Here we report on our efforts to develop a programmable platform for RNA targeting using a Cas ribonuclease, CasRx, in the model organism Drosophila melanogaster. By genetically encoding CasRx in flies, we demonstrate moderate transcript targeting of known phenotypic genes in addition to unexpected toxicity and lethality. We also report on the off-target effects following on-target transcript cleavage by CasRx. Taken together, our results present the current state and limitations of a genetically encoded programmable RNA-targeting Cas system in Drosophila melanogaster, paving the way for future optimization of the system.

LOC101928834, a novel lncRNA in Wnt/ß-catenin signaling pathway, promotes cell proliferation and predicts poor clinical outcome in myelodysplastic syndromes.

Long non-coding RNAs (lncRNAs) play important roles in hematological malignancies. We have previously identified several differentially expressed lncRNAs in myelodysplastic syndromes (MDS) by microarray analysis. In the present study, we explored the regulatory circuitry, potential functions, clinical and prognostic relevance of these lncRNAs in MDS by developing a lncRNA regulation network. We identified a novel lncRNA, LOC101928834, which was significantly up-regulated in the bone marrow of patients with MDS and acute myeloid leukemia (AML). We further evaluated the clinical relevance of LOC101928834 in 89 MDS and 110 AML patients and found that higher level of LOC101928834 expression was associated with higher white blood cell count, higher blast percentage, the subtype of refractory cytopenia with excess blasts (RAEB) and shorter overall survival in MDS patients. Receiver operating characteristic (ROC) curve analysis showed that LOC101928834 expression could discriminate MDS-RAEB patients from control with an area under the receiver-operating curve (AUC) of 0.9048. Moreover, functional analysis showed that LOC101928834 promoted cell proliferation and cell cycle progression, and activated Wnt/ß-catenin signaling pathway in vitro. In conclusion, LOC101928834 expression is correlated with clinical and biological features of MDS and may serve as a novel diagnostic and prognostic biomarker.

Anti-instinctive Learning Behavior Revealed by Locomotion-Triggered Mild Heat Stress in Drosophila.

Anti-instinctive learning, an ability to modify an animal's innate behaviors in ways that go against one's innate tendency, can confer great evolutionary advantages to animals and enable them to better adapt to the changing environment. Yet, our understanding of anti-instinctive learning and its underlying mechanisms is still limited. In this work, we describe a new anti-instinctive learning behavior of fruit flies. This learning paradigm requires the fruit fly to respond to a recurring, aversive, mild heat stress by modifying its innate locomotion behavior. We found that experiencing movement-triggered mild heat stress repeatedly significantly reduced walking activity in wild type fruit flies, indicating that fruit flies are capable of anti-instinctive learning. We also report that such learning ability is reduced in dopamine 1-like receptor 1 (Dop1R1) null mutant and dopamine 2-like receptor (Dop2R) null mutant flies, suggesting that these two dopamine receptors are involved in mediating anti-instinctive learning in flies.

Macronutrient variability in human milk from donors to a milk bank: Implications for feeding preterm infants.

BACKGROUND AND OBJECTIVE: The composition of human milk varies widely and impacts the ability to meet nutrient requirements for preterm infants. The purpose of this study is to use a large dataset of milk composition from donors to a milk bank to: (1) describe the macronutrient variability in human milk and how it contributes to the ability to meet the protein and calorie targets for the preterm infant using fortification with commercially available multi-nutrient fortifiers; (2) assess how temporal versus subject effects explain macronutrient variability; (3) determine how macronutrient variability contributes to the nutrient distribution in pooled donor milk. METHODS: This is a retrospective, observational study that analyzes the macronutrient data of 1,119 human milk samples from 443 individual donors to a milk bank. We test fortification strategies with potential basic, intermediate, and high protein and calorie commercial fortifiers. Additionally, we simulate the random pooling of multiple donors to model the impact of macronutrient variability on pooled donor milk. RESULTS: Fat was the most variable nutrient and accounted for 80% of the difference in calories. A subject-effect predicted more of the variability after 4 weeks postpartum in all macronutrients (R2 > = 0.50) than a time-effect (R2 < = 0.28). When pooling multiple donors, variability was reduced by increasing the number of donors randomly selected for a pool or targeted pooling based on macronutrient analysis of donor pools. Over 75% of mature milk samples fortified with a basic protein fortifier did not meet daily protein targets of 3.5 g/kg without exceeding volumes of 160 ml/kg/day. CONCLUSION: There is a strong individual signature to human milk that impacts the pooling of donor milk, and the ability to meet protein and energy requirements for the preterm infant with basic and intermediate protein and calorie fortifiers.

Applications of complementary therapies during rehabilitation for individuals with traumatic Spinal Cord Injury: Findings from the SCIRehab Project.

Objective: Evaluate the use of complementary therapies during rehabilitation for patients with traumatic spinal cord injury (SCI). Design: Secondary analyses were conducted to identify the use and associated outcomes of complementary therapies provided by occupational therapists (OTs) and physical therapists (PTs) during rehabilitation from a public dataset. Setting: Inpatient rehabilitation. Participants: A public dataset composed of 1376 patients with SCI that were enrolled in a five-year, multi-center investigation, the SCIRehab Project. Secondary analyses focused on a subset of 93 patients (47 who received complementary therapy during treatment and 46 case-matched controls who received no complementary therapy). Interventions: OTs and PTs recorded use of complementary therapies during sessions, including yoga, Pilates, tai chi, aromatherapy, relaxation techniques, imagery and other. Outcome Measures: Pain interference, pain severity, mobility, and social integration. Results: Three percent of participants received any complementary therapies. Patients who received complementary therapies showed greater reductions in pain severity from 6 months to 12 months relative to matched controls. Furthermore, the amount of time that patients received complementary therapies during physical therapy sessions was associated with reduced pain interference at 6 months and with reduced pain severity at the 6-month and 12-month follow-ups. Complementary therapy use was not associated with mobility or social integration. Conclusion: The current study provides preliminary evidence documenting the limited use of complementary therapies in rehabilitation settings and highlights the opportunity for further research, particularly regarding pain-related outcomes.

Calpain activator dibucaine induces platelet apoptosis.

Calcium-dependent calpains are a family of cysteine proteases that have been demonstrated to play key roles in both platelet glycoprotein Ibα shedding and platelet activation and altered calpain activity is associated with thrombotic thrombocytopenic purpura. Calpain activators induce apoptosis in several types of nucleated cells. However, it is not clear whether calpain activators induce platelet apoptosis. Here we show that the calpain activator dibucaine induced several platelet apoptotic events including depolarization of the mitochondrial inner transmembrane potential, up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-X(L), caspase-3 activation and phosphatidylserine exposure. Platelet apoptosis elicited by dibucaine was not affected by the broad spectrum metalloproteinase inhibitor GM6001. Furthermore, dibucaine did not induce platelet activation as detected by P-selectin expression and PAC-1 binding. However, platelet aggregation induced by ristocetin or α-thrombin, platelet adhesion and spreading on von Willebrand factor were significantly inhibited in platelets treated with dibucaine. Taken together, these data indicate that dibucaine induces platelet apoptosis and platelet dysfunction.