iPSC-derived healthy human astrocytes selectively load miRNAs targeting neuronal genes into extracellular vesicles.

Astrocytes are in constant communication with neurons during the establishment and maturation of functional networks in the developing brain. Astrocytes release extracellular vesicles (EVs) containing microRNA (miRNA) cargo that regulates transcript stability in recipient cells. Astrocyte released factors are thought to be involved in neurodevelopmental disorders. Healthy astrocytes partially rescue Rett Syndrome (RTT) neuron function. EVs isolated from stem cell progeny also correct aspects of RTT. EVs cross the blood-brain barrier (BBB) and their cargo is found in peripheral blood which may allow non-invasive detection of EV cargo as biomarkers produced by healthy astrocytes. Here we characterize miRNA cargo and sequence motifs in healthy human astrocyte derived EVs (ADEVs). First, human induced Pluripotent Stem Cells (iPSC) were differentiated into Neural Progenitor Cells (NPCs) and subsequently into astrocytes using a rapid differentiation protocol. iPSC derived astrocytes expressed specific markers, displayed intracellular calcium transients and secreted ADEVs. miRNAs were identified by RNA-Seq on astrocytes and ADEVs and target gene pathway analysis detected brain and immune related terms. The miRNA profile was consistent with astrocyte identity, and included approximately 80 miRNAs found in astrocytes that were relatively depleted in ADEVs suggestive of passive loading. About 120 miRNAs were relatively enriched in ADEVs and motif analysis discovered binding sites for RNA binding proteins FUS, SRSF7 and CELF5. miR-483-5p was the most significantly enriched in ADEVs. This miRNA regulates MECP2 expression in neurons and has been found differentially expressed in blood samples from RTT patients. Our results identify potential miRNA biomarkers selectively sorted into ADEVs and implicate RNA binding protein sequence dependent mechanisms for miRNA cargo loading.

Evaluating the seasonal efficacy of commonly used chemical treatments on Varroa destructor (Mesostigmata: Varroidae) population resurgence in honey bee colonies.

The purpose of this research was to determine how common chemical treatments influence Varroa destructor (Anderson and Trueman) population resurgence rates (defined as time posttreatment for mite populations to reach 3 mites/100 adult bees) in managed honey bee (Apis mellifera L.) colonies seasonally. We conducted 2 experiments that followed the same basic protocol to address this purpose. We established 6 treatment groups in Experiment 1 in the fall of 2014: untreated control, Apivar, Apistan, CheckMite+, ApiLifeVar, and Mite Away II applied to 10 colonies per treatment. In Experiment 2, we applied 8 chemical treatments to each of 4 seasonal (spring, summer, fall, and winter) cohorts of honey bee colonies to determine how mite populations are influenced by the treatments. The treatments/formulations tested were Apivar, Apistan, Apiguard, MAQS, CheckMite+, oxalic acid (dribble), oxalic acid (shop towels), and amitraz (shop towels soaked in Bovitraz). In Experiment 1, Apivar and Mite Away II were able to delay V. destructor resurgence for 2 and 6 months, respectively. In Experiment 2, Apiguard, MAQS, oxalic acid (dribble), and Bovitraz treatments were effective at delaying V. destructor resurgence for at least 2 months during winter and spring. Only the Bovitraz and MAQS treatments were effective at controlling V. destructor in the summer and fall. Of the 2 amitraz-based treatments, the off-label Bovitraz treatment was the only treatment to reduce V. destructor populations in every season. The data gathered through this study allow for the refinement of treatment recommendations for V. destructor, especially regarding the seasonal efficacy of each miticide and the temporal efficacy posttreatment.

Developing a method to rear Varroa destructor in vitro.

Varroa destructor is a significant mite pest of western honey bees (Apis mellifera). Developing a method to rear and maintain populations of V. destructor in vitro would provide year-round access to the mites, allowing scientists to study their biology, behavior, and control more rapidly. In this study, we determined the impact of various rearing parameters on V. destructor survival and reproduction in vitro. This was done by collecting V. destructor from colonies, placing them in gelatin capsules containing honey bee larvae, and manipulating the following conditions experimentally: rearing temperature, colony source of honey bee larva, behavioral/developmental stages of V. destructor and honey bee larva, and mite:bee larva ratio. Varroa destructor survival was significantly impacted by temperature, colony source of larvae and mite behavioral stage. In addition, V. destructor reproduction was significantly impacted by mite: larva ratio, larval developmental stage, colony source of larva, and temperature. The following conditions optimized mite survival and reproduction in vitro: using a 4:1 mite:larva ratio, beginning the study with late stage uncapped larvae, using mites collected from adult bees, maintaining the rearing temperature at 34.5° C, and screening larval colony source. Ultimately, this research can be used to improve V. destructor in vitro rearing programs.

Generation and characterization of a human iPSC line and gene-corrected isogenic line derived from a patient with a CELF2 gene mutation.

The identification of neurodevelopmental defects in a patient harboring a heterozygous de novo missense variant (NM_006561.4, c.1517G > A, p.Arg506His) within the CELF2 gene. Here, we describe the establishment of a patient-derived induced pluripotent stem cell (iPSC) line, alongside an isogenic gene-corrected iPSC line, achieved through CRISPR/Cas9 genome editing. These lines exhibit the expression of pluripotency markers, demonstrate differentiation potential into all three germ layers, and maintain a normal karyotype. These iPSC lines serve as valuable tools for investigating the consequences of CELF2 related neurodevelopmental disorders.

Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses.

Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.

Calcium-Dependent Hyperexcitability in Human Stem Cell-Derived Rett Syndrome Neuronal Networks.

Background: Mutations in MECP2 predominantly cause Rett syndrome and can be modeled in vitro using human stem cell-derived neurons. Patients with Rett syndrome have signs of cortical hyperexcitability, such as seizures. Human stem cell-derived MECP2 null excitatory neurons have smaller soma size and reduced synaptic connectivity but are also hyperexcitable due to higher input resistance. Paradoxically, networks of MECP2 null neurons show a decrease in the frequency of network bursts consistent with a hypoconnectivity phenotype. Here, we examine this issue. Methods: We reanalyzed multielectrode array data from 3 isogenic MECP2 cell line pairs recorded over 6 weeks (n = 144). We used a custom burst detection algorithm to analyze network events and isolated a phenomenon that we termed reverberating super bursts (RSBs). To probe potential mechanisms of RSBs, we conducted pharmacological manipulations using bicuculline, EGTA-AM, and DMSO on 1 cell line (n = 34). Results: RSBs, often misidentified as single long-duration bursts, consisted of a large-amplitude initial burst followed by several high-frequency, low-amplitude minibursts. Our analysis revealed that MECP2 null networks exhibited increased frequency of RSBs, which produced increased bursts compared with isogenic controls. Bicuculline or DMSO treatment did not affect RSBs. EGTA-AM selectively eliminated RSBs and rescued network burst dynamics. Conclusions: During early development, MECP2 null neurons are hyperexcitable and produce hyperexcitable networks. This may predispose them to the emergence of hypersynchronic states that potentially translate into seizures. Network hyperexcitability depends on asynchronous neurotransmitter release that is likely driven by presynaptic Ca2+ and can be rescued by EGTA-AM to restore typical network dynamics.

The small hive beetle's capacity to disperse over long distances by flight.

The spread of invasive species often follows a jump-dispersal pattern. While jumps are typically fostered by humans, local dispersal can occur due to the specific traits of a species, which are often poorly understood. This holds true for small hive beetles (Aethina tumida), which are parasites of social bee colonies native to sub-Saharan Africa. They have become a widespread invasive species. In 2017, a mark-release-recapture experiment was conducted in six replicates (A-F) using laboratory reared, dye-fed adults (N = 15,690). Honey bee colonies were used to attract flying small hive beetles at fixed spatial intervals from a central release point. Small hive beetles were recaptured (N = 770) at a maximum distance of 3.2 km after 24 h and 12 km after 1 week. Most small hive beetles were collected closest to the release point at 0 m (76%, replicate A) and 50 m (52%, replicates B to F). Temperature and wind deviation had significant effects on dispersal, with more small hive beetles being recaptured when temperatures were high (GLMM: slope = 0.99, SE = 0.17, Z = 5.72, P < 0.001) and confirming the role of wind for odour modulated dispersal of flying insects (GLMM: slope = - 0.39, SE = 0.14, Z = - 2.90, P = 0.004). Our findings show that the small hive beetles is capable of long-distance flights, and highlights the need to understand species specific traits to be considered for monitoring and mitigation efforts regarding invasive alien species.

Gold nanoshells for prostate cancer treatment: evidence for deposition in abdominal organs.

PURPOSE: Gold-silica nanoshell therapy [AuroShells with subsequent focal laser therapy (AuroLase)] is an emerging targeted treatment modality for prostate cancer. We reviewed pre- and post-treatment unenhanced CT imaging to assess for retained gold-silica nanoshells in the abdomen and pelvis. METHODS: This single-institution retrospective study identified patients in the AuroLase pilot who underwent pre- and post-treatment unenhanced abdominopelvic CT. The attenuation, before and after gold-silica nanoshell administration, of the liver, spleen, pancreas, kidneys, prostate, blood pool, paraspinal musculature, and abnormal lymph nodes were manually measured by two readers. After inter-reader agreement was calculated using intraclass correlation (ICC), a permutation test was used to assess pre- and post-therapy attenuation differences. RESULTS: Four patients met the inclusion criteria. Mean age was 72.3 ± 5.9 years. Median time interval between pre-treatment CT and treatment, and between treatment and post-treatment CT, was 232 days and 236.5 days, respectively. The two readers' attenuation measurements had very high agreement (ICC = 0.99, p < 0.001). The highest differences in organ attenuation between pre- and post-therapy scans were seen in all four patients in the liver and spleen (liver increased by an average of 28.9 HU, p = 0.010; spleen increased by an average of 63.7 HU, p = 0.012). A single measured lymph node increased by an average of 58.9 HU. In the remainder of the measured sites, the change in attenuation from pre- to post-therapy scans ranged from -0.1 to 3.8 HU (p > 0.05). CONCLUSION: Increased attenuation of liver and spleen at CT can be an expected finding in patients who have received gold-silica nanoshell therapy.

Myosin inhibitor reverses hypertrophic cardiomyopathy in genotypically diverse pediatric iPSC-cardiomyocytes to mirror variant correction.

Pathogenic variants in MYH7 and MYBPC3 account for the majority of hypertrophic cardiomyopathy (HCM). Targeted drugs like myosin ATPase inhibitors have not been evaluated in children. We generate patient and variant-corrected iPSC-cardiomyocytes (CMs) from pediatric HCM patients harboring single variants in MYH7 (V606M; R453C), MYBPC3 (G148R) or digenic variants (MYBPC3 P955fs, TNNI3 A157V). We also generate CMs harboring MYBPC3 mono- and biallelic variants using CRISPR editing of a healthy control. Compared with isogenic and healthy controls, variant-positive CMs show sarcomere disorganization, higher contractility, calcium transients, and ATPase activity. However, only MYH7 and biallelic MYBPC3 variant-positive CMs show stronger myosin-actin binding. Targeted myosin ATPase inhibitors show complete rescue of the phenotype in variant-positive CMs and in cardiac Biowires to mirror isogenic controls. The response is superior to verapamil or metoprolol. Myosin inhibitors can be effective in genotypically diverse HCM highlighting the need for myosin inhibitor drug trials in pediatric HCM.