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Untreated obstructive sleep apnea (OSA) is associated with excessive daytime sleepiness, decreased quality of life, and cardiovascular disease. Positive airway pressure is the first-line therapy for OSA; however, adherence is difficult. Upper airway stimulation is a Food and Drug Administration-approved treatment of OSA. The objective of this study was to evaluate for a difference in treatment efficacy and adherence of upper airway stimulation therapy for OSA between individuals who are White and non-White using data from the ADHERE registry.ADHERE registry is a multicenter prospective study of real-world experience of upper airway stimulation for treatment of OSA in the United States and Europe. Propensity score matching was used to create a balanced dataset between the White and non-White groups.There were 2,755 participants of the ADHERE registry: 27 were excluded due to not having a race identified, 125 participants identified as non-White, 2,603 identify as White, and 27 did not provide race information. Propensity score matching was used to select 110 participants, with 55 White and 55 non-White for the noninferiority analysis. We did not find a difference in adherence, treatment apnea-hypopnea index, changes in Epworth Sleepiness Scale score, or clinical global impression after intervention score between White and non-White individuals.Our study found that there was no statistically significant difference in adherence or efficacy with upper airway stimulation therapy between White and non-White individuals. However, the percent of non-White people implanted is low, which suggests a need to expand access to this therapy for non-White populations with OSA who cannot tolerate positive airway pressure therapy.Khan M, Stone A, Soose RJ, et al. Does race-ethnicity affect upper airway stimulation adherence and treatment outcome of obstructive sleep apnea?

Upper airway stimulation (UAS) is an alternative treatment for obstructive sleep apnea that must be activated nightly. Although the implanted device offsets the mask- or pressure-related side effects often associated with continuous positive airway pressure therapy, some UAS recipients do not use the therapy consistently. This study qualitatively explored factors associated with UAS usage in obstructive sleep apnea patients.Semistructured interviews were conducted with 24 obstructive sleep apnea patients who received UAS treatment. Twelve patients were categorized as high users with mean usage of ≥ 4 hours/night and 12 were categorized as low users with4 hours/night or nonuse. Interviews explored patients' experiences regarding barriers and facilitators to UAS use and their advice for new UAS recipients. Demographic and clinical data including the Insomnia Severity Index and Generalized Anxiety Disorder Scale were collected.Compared to high users, low users had higher levels of insomnia (mean Insomnia Severity Index: 3.6 vs 15.2, respectively) and anxiety (mean Generalized Anxiety Disorder Scale: 3.4 vs 6.9). High users reported more positive experiences with UAS treatment, such as improvements in symptoms and convenience of treatment, as facilitators of use. Low users tended to focus on the negative aspects of treatment, particularly stimulation-related discomfort and associated sleep disturbance.Insomnia with or without anxiety contributes to differing patient-reported experiences in high vs low user groups, with increased insomnia symptoms among low users. Improved understanding of the specific barrier and facilitators of UAS adherence may drive better long-term use and more personalized management strategies, including concomitant insomnia treatment.Registry: ClinicalTrials.gov; Name: Stimulation Therapy for Apnea: Reporting Thoughts (START); URL: https://clinicaltrials.gov/ct2/show/NCT04768543; Identifier: NCT04768543.Luyster FS, Ni Q, Lee K, et al. Factors affecting obstructive sleep apnea patients' use of upper airway stimulation treatment.

STUDY OBJECTIVES: Hypoglossal nerve stimulation (HGNS) is an effective alternative treatment for obstructive sleep apnea that acts by opening the airway via selective stimulation of nerve fibers that innervate tongue muscles that protrude (genioglossus) and stiffen the tongue (transverse and vertical) while avoiding nerve fibers that innervate tongue muscles that retract the tongue (styloglossus and hyoglossus). There remains a subset of postoperative patients who fail to adequately respond to HGNS, in some cases due to mixed activation of muscles that simultaneously protrude and retract the tongue. This study aims to characterize the relationship between neurophysiological data from individual tongue muscle activation during intraoperative electromyographic recordings and postoperative apnea-hypopnea index responses to HGNS. METHODS: A single-institution review of 46 patients undergoing unilateral HGNS implantation for obstructive sleep apnea. Patients were separated into responders and nonresponders through comparison of pre and postoperative apnea-hypopnea index. Neurophysiological data included electromyographic responses of the genioglossus, styloglossus/hyoglossus, intrinsic/vertical, and hyoglossus (neck) muscles to intraoperative stimulation using unipolar (– to – and o to o) and bipolar (+ to +) settings. RESULTS: The overall treatment success rate was 61% as determined by a postoperative apnea-hypopnea index < 20 events/h with a greater than 50% AHI reduction. We observed no statistically significant relationships between treatment response and individual muscle responses. However, we did note that increasing body mass index was correlated with worse postoperative responses. CONCLUSIONS: Although we noted a significant subgroup of clinical nonresponders to HGNS postoperatively, these patients were not found to exhibit significant inclusion of tongue retractors intraoperatively on neurophysiological analysis. Further research is needed to delineate additional phenotypic factors that may contribute to HGNS treatment responses. CITATION: Wang D, Modik O, Sturm JJ, et al. Neurophysiological profiles of responders and nonresponders to hypoglossal nerve stimulation: a single-institution study. J Clin Sleep Med. 2022;18(5):1327–1333.

Overexpression is one of the classical approaches to study pleiotropic functions of genes of interest. To achieve overexpression, we often increase the transcription by introducing genes on exogenous vectors or by using the CRISPR/dCas9-based transcriptional activation system. To date, the most efficient CRISPR/dCas9-based transcriptional activator is the Synergistic Activation Mediator (SAM) system whereby three different transcriptional activation domains are directly fused to dCas9 and MS2 phage Coat Protein (MCP), respectively, and the system in Drosophila is named flySAM. Here we describe the effective and convenient transcriptional activation system, flySAM, starting from vector construction, microinjection, and transgenic fly selection to the phenotypic analysis.

The flySAM/CRISPRa system has recently emerged as a powerful tool for gain-of-function studies in Drosophila melanogaster. This system includes Gal4/UAS-driven dCas9 activators and U6 promoter-controlled sgRNA. Having established dCas9 activators superior to other combinations, to further enhance the efficiency of the targeting activators we systematically optimized the parameters of the sgRNA. Interestingly, the most efficient sgRNAs were found to accumulate in the region from -150bp to -450bp upstream of the transcription start site (TSS), and the activation efficiency showed a strong positive correlation with the GC content of the sgRNA targeting sequence. In addition, the target region is dominant to the GC content, as sgRNAs targeting areas beyond -600bp from the TSS lose efficiency even when containing 75% GC. Surprisingly, when comparing the activities of sgRNAs targeting to either DNA strand, sgRNAs targeting to the non-template strand outperform those complementary to the template strand, both in cells and in vivo. In summary, we define criteria for sgRNA design which will greatly facilitate the application of CRISPRa in gain-of-function studies.

Objective: To screen abnormally expressed lncRNAs in peripheral blood mononuclear cells from patients with COPD. Methods:The peripheral blood of 3 COPD patients and 3 normal controls were collected from our hospital, mononuclear cells were isolated, RNA was extracted and then transcriptome sequencing was performed. The expression difference between the two groups of samples was calculated based on p 1.Plot the difference lncRNA heat map and volcano map. The Lncpro database may predict mRNAs regulated by differential lncRNA, and perform the GO function and KEGG signaling clustering. Results: There were 67 lncRNAs between the COPD group and the control group that met the difference of p 1, of which 33 were up-regulated and 34 were down-regulated. Between the two groups. The target genes are mainly enriched in GO functions: regulatory functions of multicellular biological processes, regulatory functions of development processes, structured morphogenesis functions, system development functions, and development process functions. Target genes are mainly enriched in KEGG signaling pathways: multi-species apoptotic pathway, TGF-β signaling pathway, complement and coagulation cascade pathway, colorectal cancer pathway and apoptosis pathway. Conclusion: Our results provide general information and possible regulatory functions and pathways of lncRNA expression changes in peripheral blood mononuclear cells of COPD, which may help clarify the underlying mechanism of COPD.

Study Objectives Upper airway stimulation (UAS) therapy is effective for a subset of obstructive sleep apnea (OSA) patients with continuous positive airway pressure (CPAP) intolerance. While overall adherence is high, some patients have suboptimal adherence, which limits efficacy. Our goal was to identify therapy usage patterns during the first 3 months of therapy to enable targeted strategies for improved adherence. Methods Therapy data was retrieved from 2098 patients for three months after device activation. Data included mean and standard deviation (SD) of hours of use, therapy pauses, hours from midnight the therapy was turned ON and OFF, percentage of missing days, and stimulation amplitude. Cluster analysis was performed using Gaussian mixture models that categorized patients into six main groups. Results The six groups and their prevalence can be summarized as Cluster 1A: Excellent Use (34%); Cluster 1B: Excellent Use with variable timing (23%); Cluster 2A: Good Use with missing days and late therapy ON (16%), Cluster 2B: Good Use with missing days, late therapy ON, and early therapy OFF (12%); Cluster 3A: Variable Use with frequent missing days (8%); Cluster 3B: Variable Use with frequent pauses (7%). Most patients (85%) are excellent or good users with mean therapy use >6 hours per night. Conclusions Cluster analysis of early UAS usage patterns identified six distinct groups that may enable personalized interventions for improved long-term management. Differentiation of the patient clusters may have clinical implications with regard to sleep hygiene education, therapy discomfort, comorbid insomnia, and other conditions that impact adherence.

Objective: To analyze the relationship between plasma vascular endothelial growth factor and patients with chronic obstructive pulmonary disease in Hainan Li and Han nationality, and the correlation between VEGFA and COPD after different stratification, to explore the significance of VEGFA in the diagnosis and treatment of Li people with chronic obstructive pulmonary disease. Methods: 249 patients with COPD were recruited in Hainan (109 cases of Li and 140 cases of Han), and 246 cases in the control group (89 cases of Li; 157 cases of Han ) From July 2014 to March 2015, We measured plasma vascular endothelial growth factor protein levels ,and determined the expression difference of plasma VEGFA in patients with chronic obstructive pulmonary disease and healthy controls according to different races, genders, smoking status, etc., and analyzed the correlation between the expression of VEGFA in patients with chronic obstructive pulmonary disease in Li and Han in Hainan. Conclusion: (1) In the Li population, the expression of VEGFA of COPD patients and healthy controls was lower than that of Han (P

Gene expression regulation, including loss-of-function and gain-of-function assays, is a powerful method to study developmental and disease mechanisms. Drosophila melanogaster is an ideal model system particularly well-equipped with many genetic tools. In this review, we describe and discuss the gene expression regulation techniques recently developed and their applications, including the CRISPR/Cas9-triggered heritable mutation system, CRISPR/dCas9-based transcriptional activation (CRISPRa) system, and CRISPR/dCas9-based transcriptional repression (CRISPRi) system, as well as the next-generation transgenic RNAi system. The main purpose of this review is to provide the fly research community with an updated summary of newly developed gene expression regulation techniques and help the community to select appropriate methods and optimize the research strategy.

Introduction Upper airway stimulation (UAS) therapy is effective for a subset of obstructive sleep apnea (OSA) patients with CPAP intolerance. While overall adherence is high, some patients have suboptimal adherence to UAS, which limits effectiveness. Our goal was to identify UAS therapy usage patterns during the first three months of therapy that affect adherence. Methods We retrieved anonymized UAS therapy usage data from 2,091 individuals stored in a cloud-based monitoring system during the first three months after device activation. We aggregated adherence data including mean and standard deviation (SD) of nightly hours of use, therapy pauses, hours from midnight when the therapy was turned ON and OFF, and percentage of missing days. We computed the difference of the stimulation amplitude between the first and last day. We performed cluster analysis with Gaussian mixture models and computed the centroids of each cluster highlighting their main differences. Results We identified six distinct clusters of UAS usage patterns. Clusters 1A (34% of the total cohort) and 1B (23%) had excellent therapy usage with 7.23h and 7.14h on days of use, respectively; with 1B distinguished by increased night-to-night variability. Clusters 2A (16%) and 2B (12%) had good mean therapy use of 6.63h and 6.21h, respectively, but their usage patterns were distinguished by a higher percentage of missing days (8% missing days in 2A and 23% in 2B) and less favorable therapy timing with an average therapy ON time after midnight. Clusters 3A (8%) and 3B (7%) were characterized by the lowest nightly use at 6.16h and 5.50h, respectively, and the highest night-to-night variability. 3A was further distinguished by the highest percentage of missing days (34%) while 3B was characterized by the frequent therapy pauses (mean 4.1 pauses per night) and the least increase in stimulation amplitude across the first 90 days. Conclusion Cluster analysis of UAS usage patterns identified six distinct groups that may enable custom interventions for improved long-term management. Differentiation of these groups may have clinical implications on conditions (e.g. therapy discomfort, comorbid insomnia, poor sleep hygiene) that impact adherence. Support (If Any)

Notch signaling and epigenetic factors are known to play critical roles in regulating tissue homeostasis in most multicellular organisms, but how Notch signaling coordinates with epigenetic modulators to control differentiation remains poorly understood. Here, we identify heterochromatin protein 1c (HP1c) as an essential epigenetic regulator of gut homeostasis in Drosophila. Specifically, we observe that HP1c loss-of-function phenotypes resemble those observed after Notch signaling perturbation and that HP1c interacts genetically with components of the Notch pathway. HP1c represses the transcription of Notch target genes by directly interacting with Suppressor of Hairless (Su(H)), the key transcription factor of Notch signaling. Moreover, phenotypes caused by depletion of HP1c in Drosophila can be rescued by expressing human HP1γ, suggesting that HP1γ functions similar to HP1c in Drosophila. Taken together, our findings reveal an essential role of HP1c in normal development and gut homeostasis by suppressing Notch signaling.

In mechanoreceptor cells, specialized cytoskeletons are formed at the site of mechanotransduction. Sun et al. reconstruct the microtubule-based cytoskeleton in fly campaniform mechanosensory cilia using electron microscopic tomography and establish dual functions for katanin p60-like 1 in shaping the cytoskeleton., Mechanoreceptor cells develop a specialized cytoskeleton that plays structural and sensory roles at the site of mechanotransduction. However, little is known about how the cytoskeleton is organized and formed. Using electron tomography and live-cell imaging, we resolve the 3D structure and dynamics of the microtubule-based cytoskeleton in fly campaniform mechanosensory cilia. Investigating the formation of the cytoskeleton, we find that katanin p60-like 1 (kat-60L1), a neuronal type of microtubule-severing enzyme, serves two functions. First, it amplifies the mass of microtubules to form the dense microtubule arrays inside the sensory cilia. Second, it generates short microtubules that are required to build the nanoscopic cytoskeleton at the mechanotransduction site. Additional analyses further reveal the functional roles of Patronin and other potential factors in the local regulatory network. In all, our results characterize the specialized cytoskeleton in fly external mechanosensory cilia at near-molecular resolution and provide mechanistic insights into how it is formed.

Study Objectives Individuals with obstructive sleep apnea (OSA), characterized by frequent sleep disruptions from tongue muscle relaxation and airway blockage, are known to benefit from on-demand electrical stimulation of the hypoglossal nerve. Hypoglossal nerve stimulation (HNS) therapy, which activates the protrusor muscles of the tongue during inspiration, has been established in multiple clinical studies as safe and effective, but the mechanistic understanding for why some stimulation parameters work better than others has not been thoroughly investigated. Methods In this study, we developed a detailed biophysical model that can predict the spatial recruitment of hypoglossal nerve fascicles and axons within these fascicles during stimulation through nerve cuff electrodes. Using this model, three HNS programming scenarios were investigated including grouped cathode (---), single cathode (o-o), and guarded cathode bipolar (+-+) electrode configurations. Results Regardless of electrode configuration, nearly all hypoglossal nerve axons circumscribed by the nerve cuff were recruited for stimulation amplitudes Conclusions The computational models predicted that monopolar HNS provided the lowest threshold and the least sensitivity to rotational angle of the nerve cuff around the hypoglossal nerve; however, this setting also increased the likelihood for current leakage outside the nerve cuff, which could potentially activate axons in unintended branches of the hypoglossal nerve. Clinical Trial Registration NCT01161420.

Spermatogenesis‐associated protein 4 (SPATA4) is conserved across multiple species. However, the function of this gene remains largely unknown. In this study, we generated Spata4 transgenic mice to explore tissue‐specific function of SPATA4. Spata4 overexpression mice displayed increased subcutaneous fat tissue compared with wild‐type littermates at an old age, while this difference was not observed in younger mice. Aging‐induced ectopic fat distribution, inflammation, and insulin resistance were also significantly attenuated by SPATA4. In vitro, SPATA4 promoted preadipocyte differentiation through activation of the ERK1/2 and C/EBPβ pathway and increased the expression of adipokines. These data suggest SPATA4 can regulate lipid accumulation in a tissue‐specific manner and improve aging‐induced dysmetabolic syndromes. Clarifying the mechanism of SPATA4 functioning in lipid metabolism might provide novel therapeutic targets for disease interventions., SPATA4 promotes preadipocyte differentiation through activation of ERK1/2 and C/EBPβ pathway, attenuates aging‐induced ectopic distribution of fat, and improves metabolic homeostasis.

Dysregulation of CDK8 (Cyclin-Dependent Kinase 8) and its regulatory partner CycC (Cyclin C), two subunits of the conserved Mediator (MED) complex, have been linked to diverse human diseases such as cancer. Thus, it is essential to understand the regulatory network modulating the CDK8-CycC complex in both normal development and tumorigenesis. To identify upstream regulators or downstream effectors of CDK8, we performed a dominant modifier genetic screen in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in developing wing imaginal discs. We identified 26 genomic loci whose haploinsufficiency can modify these CDK8- or CycC-specific phenotypes. Further analysis of two overlapping deficiency lines and mutant alleles led us to identify genetic interactions between the CDK8-CycC pair and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGFβ, or Transforming Growth Factor-β) signaling pathway. We observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGFβ signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 (Mad homology 2) transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the MED complex have revealed six additional subunits that are required for Mad-dependent transcription in the wing discs: Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, our analyses confirmed the positive roles of CDK9 and Yorkie in regulating Mad-dependent gene expression in vivo. These results suggest that CDK8 and CycC, together with a few other subunits of the MED complex, may coordinate with other transcription cofactors in regulating Mad-dependent transcription during wing development in Drosophila., Author summary CDK8 and its dedicated partner CycC are conserved subunits of the Mediator complex that bridges transcription factors with RNA Polymerase II in eukaryotes. Here we explore the function and regulation of the CDK8-CycC pair in Drosophila by performing a dominant modifier genetic screen based on wing vein patterning defects caused by specific alteration of CDK8-CycC activities. We have observed that multiple components of the Dpp/TGFβ signaling pathway genetically interact with CDK8-CycC. CDK8 and CycC positively regulate gene expression activated by Mad, the key transcription factor downstream of Dpp/TGFβ signaling, and CDK8 can directly interact with the linker region of the Mad protein. We also identify additional, but not all, subunits of the Mediator complex that play positive roles in regulating Mad-dependent gene expression. Given the fundamental role of Dpp/TGFβ signaling in regulating development and its misregulation in a variety of diseases, understanding how Mad/Smad interacts with the Mediator complex may have broad implications in understanding the pathogenesis of these diseases.

SUMMARY Adenosine-to-inosine RNA editing, catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes, alters RNA sequences from those encoded by DNA. These editing events are dynamically regulated, but few trans regulators of ADARs are known in vivo. Here, we screen RNA-binding proteins for roles in editing regulation with knockdown experiments in the Drosophila brain. We identify zinc-finger protein at 72D (Zn72D) as a regulator of editing levels at a majority of editing sites in the brain. Zn72D both regulates ADAR protein levels and interacts with ADAR in an RNA-dependent fashion, and similar to ADAR, Zn72D is necessary to maintain proper neuromuscular junction architecture and fly mobility. Furthermore, Zn72D’s regulatory role in RNA editing is conserved because the mammalian homolog of Zn72D, Zfr, regulates editing in mouse primary neurons. The broad and conserved regulation of ADAR editing by Zn72D in neurons sustains critically important editing events., Graphical Abstract, In Brief Sapiro et al. identify Drosophila Zn72D as an influential regulator of neuronal A-to-I RNA editing and synaptic morphology. Zn72D regulates ADAR levels and editing at a large subset of editing sites, providing insight into the maintenance of critical tissue-specific RNA editing events.

Upper-Airway Stimulation (UAS) therapy is an innovative alternative to Continuous Positive Airway Pressure (CPAP) treatment for patients with obstructive sleep apnea (OSA) and CPAP intolerance. Patients who have implanted a UAS device are responsible for activating and managing the therapy at home before sleep. Consistent nightly use is required for a reduced OSA burden, measured by the apnea-hypopnea index. Thus, understanding patient behavior and possible challenges to nightly use are crucial to therapy success. In this work, we present two novel visualizations to monitor telemetry data recorded by the UAS sleep remote. They provide doctors and sleep clinicians with detailed information to easily classify therapy use and sleep patterns. We also present how to show daily metrics such as hours of average usage, therapy intensity, and duration of therapy pauses, to identify optimal therapy settings and measure the long-term effectiveness of interventions.

Mammalian sirtuin 6 (SIRT6) is involved in the regulation of many essential processes, especially metabolic homeostasis. SIRT6 knockout mice undergo premature aging and die at age ~4 weeks. Severe glycometabolic disorders have been found in SIRT6 knockout mice, and whether a dietary intervention can rescue SIRT6 knockout mice remains unknown. In our study, we found that at the same calorie intake, a high‐fat diet dramatically increased the lifespan of SIRT6 knockout mice to 26 weeks (males) and 37 weeks (females), reversed multi‐organ atrophy, and reduced body weight, hypoglycemia, and premature aging. Furthermore, the high‐fat diet partially but significantly normalized the global gene expression profile in SIRT6 knockout mice. Regarding the mechanism, excessive glucose uptake and glycolysis induced by the SIRT6 deficiency were attenuated in skeletal muscle through inhibition of insulin and IGF1 signaling by the high‐fat diet. Similarly, fatty acids but not ketone bodies inhibited glucose uptake, glycolysis, and senescence in SIRT6 knockout fibroblasts, whereas PI3K inhibition antagonized the effects of a high‐fatty‐acid medium in vitro. Overall, the high‐fat diet dramatically reverses numerous consequences of SIRT6 deficiency through modulation of insulin and IGF1 signaling, providing a new basis for elucidation of SIRT6 and fatty‐acid functions and supporting novel therapeutic approaches against metabolic disorders and aging‐related diseases., SIRT6 deficiency induces hyper activation of insulin and IGF1 signaling, accompanied with excessive anaerobic glycolysis and premature aging phenotypes. High‐fat diet effectively extends the lifespan and rescues mutant phenotypes in SIRT6 knockout mice. Fatty acid but not ketone bodies inhibit glucose uptake and anaerobic glycolysis induced by SIRT6 deficiency.

Being relatively simple and practical, Drosophila transgenic RNAi is the technique of top priority choice to quickly study genes with pleiotropic functions. However, drawbacks have emerged over time, such as high level of false positive and negative results. To overcome these shortcomings and increase efficiency, specificity and versatility, we develop a next generation transgenic RNAi system. With this system, the leaky expression of the basal promoter is significantly reduced, as well as the heterozygous ratio of transgenic RNAi flies. In addition, it has been first achieved to precisely and efficiently modulate highly expressed genes. Furthermore, we increase versatility which can simultaneously knock down multiple genes in one step. A case illustration is provided of how this system can be used to study the synthetic developmental effect of histone acetyltransferases. Finally, we have generated a collection of transgenic RNAi lines for those genes that are highly homologous to human disease genes., Drosophila transgenic RNAi can have drawbacks such as false positives and negative results. Here the authors develop a next generation RNAi system with reduced leakiness of expression and simultaneous knockdown.

Despite their essential function in terminating translation, readthrough of stop codons occurs more frequently than previously supposed. However, little is known about the regulation of stop codon readthrough by anatomical site and over the life cycle of animals. Here, we developed a set of reporters to measure readthrough in Drosophila melanogaster. A focused RNAi screen in whole animals identified upf1 as a mediator of readthrough, suggesting that the stop codons in the reporters were recognized as premature termination codons (PTCs). We found readthrough rates of PTCs varied significantly throughout the life cycle of flies, being highest in older adult flies. Furthermore, readthrough rates varied dramatically by tissue and, intriguingly, were highest in fly brains, specifically neurons and not glia. This was not due to differences in reporter abundance or nonsense-mediated mRNA decay (NMD) surveillance between these tissues. Readthrough rates also varied within neurons, with cholinergic neurons having highest readthrough compared with lowest readthrough rates in dopaminergic neurons. Overall, our data reveal temporal and spatial variation of PTC-mediated readthrough in animals, and suggest that readthrough may be a potential rescue mechanism for PTC-harboring transcripts when the NMD surveillance pathway is inhibited.

SUMMARY Phagocytic clearance of degenerating dendrites or axons is critical for maintaining tissue homeostasis and preventing neuroinflammation. Externalized phosphatidylserine (PS) has been postulated to be an ‘‘eat-me’’ signal allowing recognition of degenerating neurites by phagocytes. Here we show that in Drosophila, PS is dynamically exposed on degenerating dendrites during developmental pruning and after physical injury, but PS exposure is suppressed when dendrite degeneration is genetically blocked. Ectopic PS exposure via phospholipid flippase knockout and scramblase overexpression induced PS exposure preferentially at distal dendrites and caused distinct modes of neurite loss that differ in larval sensory dendrites and in adult olfactory axons. Surprisingly, extracellular lactadherin that lacks the integrin-interaction domain induced phagocyte-dependent degeneration of PS-exposing dendrites, revealing an unidentified bridging function that potentiates phagocytes. Our findings establish a direct causal relationship between PS exposure and neurite degeneration in vivo., Graphical Abstract, In Brief Using in vivo phosphatidylserine (PS) sensors, Sapar et al. reveal dynamic patterns of PS exposure on degenerating dendrites in Drosophila. Flippase knockout and scramblase overexpression lead to ectopic PS exposure on distal dendrites and context-dependent neurite degeneration. Lactadherin potentiates phagocytes to destruct PS-exposing dendrites, independent of its integrin-interaction domain.

The Transgenic RNAi Project (TRiP), a Drosophila melanogaster functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNA interference (RNAi) fly stocks. To date, it has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express single guide RNAs targeting upstream of a gene transcription start site. Gene activation is triggered by coexpression of catalytically dead Cas9 fused to an activator domain, either VP64-p65-Rta or Synergistic Activation Mediator. TRiP-KO stocks express one or two single guide RNAs targeting the coding sequence of a gene or genes. Cutting is triggered by coexpression of Cas9, allowing for generation of indels in both germline and somatic tissue. To date, we have generated >5000 TRiP-OE or TRiP-KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.

CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in Drosophila melanogaster. We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental use of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic overexpression resource, TRiP-OE.

Enormous economic losses and reported cases of bird-to-human transmission alert us the importance of AIVs surveillance in natural foci. However, the limited multiplexing capacity of PCR or real-time PCR method made AIVs surveillance insufficient, costly and labor intensive. In this study, we developed a multi-analyte suspension assay (MASA) to detect AIVs subtypes based on Luminex xMAP technology. With this MASA, 7 HA and 5 NA subtypes could be identified in a single reaction. The evaluation data showed that these subtype-specific amplified primers had high specificity and the coverage of each primer pairs ranged from 82% to 97%. Our studies provide a rapid and economical method for AIVs surveillance of natural foci.

While several large-scale resources are available for in vivo loss-of-function studies in Drosophila, an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator. Next, we demonstrate that this approach is scalable and has a high success rate, as >75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo.

In recent years, great progress has been made in the research of genome editing systems, one of which is the CRISPR-Cas9 system, a powerful technology that is applied to edit animal genome. Here, we describe a CRISPR-Cas9 mediated mutation protocol for efficiently and specifically editing genes in Drosophila. In this optimized system, the mutant progeny can be generated by only injecting a DNA plasmid encoding synthetic guide RNA (sgRNA) under the control of the U6b promoter into transgenic fly embryos in which Cas9 is specifically expressed in the progenitor cells, thus the gene of interest can be edited by the CRISPR in germ cells, with high rate of heritable mutations and few side effects.

The niche controls stem cell self-renewal and differentiation in animal tissues. Although the exocyst is known to be important for protein membrane trafficking and secretion, its role in stem cells and niches has never been reported. Here, this study shows that the exocyst functions in the niche to promote germline stem cell (GSC) progeny differentiation in the Drosophila ovary by directly regulating EGFR membrane trafficking and signaling. Inactivating exocyst components in inner germarial sheath cells, which form the differentiation niche, causes a severe GSC differentiation defect. The exocyst is required for maintaining niche cells and preventing BMP signaling in GSC progeny by promoting EGFR membrane targeting and signaling through direct association with EGFR. Finally, it is also required for EGFR membrane targeting, recycling and signaling in human cells. Therefore, this study has revealed a novel function of the exocyst in niche cells to promote stem cell progeny differentiation by directly controlling EGFR membrane trafficking and signaling in vivo, and has also provided important insight into how the niche controls stem cell progeny differentiation at the molecular level.

Powerful and general methods that can enhance gene expression are useful to systematically study gene function. To date, compared with the methods in generating loss-of-function mutants, methods to achieve gain-of-function are limited. The entire field in Drosophila has relied heavily on the Gal4/UAS:cDNA overexpression system developed over two decades ago. It is laborious and expensive to clone the coding DNA sequence (CDS) of a gene, especially those of large size. In addition, side effects of this method are often observed because of the ectopic expression. Also, simultaneous activation of two genes with the traditional method is often time-consuming, and few are achievable for three or more genes. In this protocol, we describe how to build an effective and convenient targeting activator system, flySAM, to activate endogenous genes in Drosophila melanogaster based on the structure-guided engineering of CRISPR-Cas9 complex.