Optimizing platelet inhibition in peripheral artery disease: A comparison of mono-antiplatelet therapy and dual-antiplatelet therapy using thromboelastography.

BACKGROUND: Antiplatelet therapy is used to prevent thrombosis in patients with peripheral artery disease (PAD) following revascularization. However, the current standard of care for these patients remains at the physician's discretion, varying from mono-antiplatelet therapy (MAPT) to dual-antiplatelet therapy (DAPT). Viscoelastic assays such as Thromboelastography with Platelet Mapping (TEG-PM) provide insight into individual coagulation profiles and measure real-time platelet function. This prospective, observational study looks at the differences in platelet function for patients on MAPT versus DAPT using TEG-PM. METHODS: Patients with PAD undergoing revascularization were prospectively evaluated between December 2020 and June 2023. TEG-PM analysis compared platelet function for patients prescribed MAPT (aspirin or clopidogrel) at the initial encounter and DAPT (aspirin and clopidogrel) at the next visit. Platelet function measured in percent inhibition was evaluated at these visits, and within-group t-tests were performed. RESULTS: Of the 195 patients enrolled, 486 samples were analyzed by TEG-PM. Sixty-four patients met the study criteria. At the initial visit, 52 patients had been prescribed aspirin, and 12 patients had been prescribed clopidogrel. For patients initially prescribed aspirin MAPT, an increase of 96.8%in the mean ADP platelet inhibition was exhibited when transitioning to DAPT [22.0% vs. 43.3%, p < .01], as well as an increase of 34.6%in the mean AA platelet inhibition when transitioning to DAPT [60.9% vs. 82.0%, p < .01]. For patients prescribed initial clopidogrel MAPT, an increase of 100% in AA platelet inhibition was exhibited on DAPT compared to the MAPT state [42.3% vs. 84.6%, p < .01]. CONCLUSIONS: Patients on DAPT showed a significant increase in platelet inhibition when compared to initial aspirin MAPT. A significant difference in AA %platelet inhibition was shown for patients on DAPT when compared to initial clopidogrel MAPT. The results show that patients may benefit from DAPT post-revascularization. Personalizing antiplatelet therapy with objective viscoelastic testing to confirm adequate treatment may be the next step in optimizing patient outcomes to reduce thrombosis in PAD patients.

Thromboelastography with Platelet Mapping Identifies High Platelet Reactivity is Associated with Obesity, Diabetes, and Thrombotic Events.

BACKGROUND: Metabolic comorbidities such as diabetes and obesity are considered pro-inflammatory states which theoretically increase the risk of perioperative thrombotic events across many surgical disciplines. Currently, there is a paucity of objective metrics to determine such risk and ideal pharmacologic targets. Thromboelastography with Platelet Mapping (TEG-PM) provides a comprehensive profile of coagulation and may provide insight into clot dysregulation. METHODS: Patients undergoing lower extremity revascularization underwent serial TEG-PM analysis. The relationship between the TEG-PM metrics and thrombosis was evaluated. Preoperative TEG-PM samples of patients with body mass index (BMI)≥25 were compared to those of patients with a normal BMI, and between patients with diabetes mellitus (DM) and those without. RESULTS: 218 TEG-PM samples from 202 patients were analyzed. The BMI≥25 cohort showed significantly greater platelet aggregation [81.9% (±20.9) vs. 68.6% (±27.7), P < 0.01]. Patients with DM were more frequently on full-dose anticoagulation [47.7% vs. 29.7% P = 0.01] yet demonstrated increased clot strength, or adenosine diphosphate (ADP)-Maximum Clot Amplitude (MA) [49.1 (±16.1) vs. 41.5 (±17.1) and 37.7 (±19.6) vs. 31.6 (±17.4) P < 0.01]. 49 patients experienced thrombosis and exhibited greater platelet aggregation [76.6% (±17.8) vs. 66.8% (±30.4) P = 0.03] and greater ADP/arachidonic acid MA [47.1 (±16.6) vs. 41.9 (±18.8) and 38.2 (±17.8) vs. 32.5 (±19.9) both P = 0.05]. Patients who thrombosed were more often diabetic [69.5% versus 51.0% P = 0.03] and on full-dose anticoagulation [75.0% vs. 56.8% P = 0.02]. CONCLUSIONS: Patients with a BMI≥ 25 and those with diabetes demonstrated TEG-PM profiles similar to patients with thrombosis. Diabetes was independently associated with thrombosis, and full-dose anticoagulation was not protective. This suggests the potential utility of TEG-PM for thrombotic risk stratification based on metabolic factors and suggests antiplatelet agents may be effective at prevention of thrombotic events in this population.

The impact of sex on platelet responses to aspirin in patients with peripheral artery disease.

Women with peripheral artery disease (PAD) have poorer limb salvage outcomes in spite of having lower risk factors for vascular disease than their male counterparts. Mono antiplatelet therapy with aspirin is the cornerstone of medical treatment for PAD to reduce the risk of arterial thrombosis, but platelets in women may have a variable response to this standard of care compared to men. Viscoelastic assays, such as thromboelastography with platelet mapping (TEG-PM), have been utilized to identify prothrombotic states and may provide insight into a patient's real-time coagulation profile and their response to specific antiplatelet medications. The aim of this prospective, observational study was to delineate the sex differences in platelet function using TEG-PM in patients with PAD on aspirin post-revascularization for PAD. All patients with PAD undergoing revascularization on aspirin monotherapy were prospectively enrolled between December 2020 and September 2023. The cohort was divided by sex, demographics, medications, procedure type, and outcomes were documented. Serial perioperative TEG-PM assays (1, 3, and 6 months) were performed up to 6 months postoperatively and platelet function was evaluated in both groups. Statistical analysis between women and men was performed to identify sex-specific differences in platelet function. Over the study period, a total of 303 patients were enrolled. Of this cohort, 149 patients met the study criteria and 266 samples were analyzed; 52 (34.89%) were women and 97 (65.11%) were men. In the platelet mapping assay, women showed significantly greater MAActF and MAAA, than men (16.66 vs. 14.94, p < .03 and 37.26 vs. 32.38, p < .01, respectively) indicating stronger thrombotic propensity. Additionally, platelet inhibition was significantly lower in women compared to men (52.95% vs. 61.65%, p < .05). In clinical outcomes reported as thrombotic events, women showed significantly higher occlusion in the area of intervention than men (4 vs. 1, p < .05). There is a growing awareness of the variations in the natural course, underlying mechanisms, and resulting outcomes of cardiovascular conditions, including PAD, in relation to sex. In this study, women did not achieve the same levels of platelet inhibition and displayed a procoagulant tendency in comparison to men when administered aspirin. Overall, aspirin monotherapy may be potentially sufficient for men, but women may require increased doses and/or additional antiplatelet medications to achieve an equivalent therapeutic effect.

Cytochrome oxidase I DNA barcodes of crocodilians meat selling in Hong Kong.

The crocodilians include true crocodiles, alligators, caimans, and gharial, and the trade of crocodilian products is regulated in accordance with the Convention of Wild Fauna and Flora (CITES). Hong Kong does not have her own wild crocodilians; thus, all crocodilians meat available is presumably imported with proper license. Here, we obtained a dataset of cytochrome oxidase I (COI) gene markers of 114 crocodilian meat samples (including frozen and dried crocodilian meat products) available in the contemporary market. We have also validated these barcodes in a phylogenetic approach with other data deposited on the GenBank, and detected 112 samples belonging to four crocodile species Crocodylus siamensis, C. porosus, C. niloticus and Alligator mississippiensis, and 2 samples belonging to snake Malayopython reticulatus. The dataset generated in this study will be useful for further studies including meat inspection, illegal trading, and enhancement of international and local legislations on illegal reptile importation.

Platelet function testing and clinical outcomes in peripheral arterial disease: Systematic review and narrative synthesis.

OBJECTIVE: This systematic review aims to comprehensively assess the contemporary literature on platelet function testing (PFT) in individuals undergoing revascularization therapy for peripheral arterial disease (PAD). The goal is to identify whether PFT can aid in detecting antiplatelet resistance, predicting post-procedural thrombotic complications, and informing tailored treatment strategies. METHODS: Following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, a literature review was conducted using PubMed databases. Search terms included relevant medical subject headings (MeSH) terms. Eligible articles published in English between 1990 and 2023 were analyzed. Studies that examined PFT outcomes in patients with PAD after lower extremity revascularization were included. RESULTS: Ten studies met the inclusion criteria. Various PFT methods were used, including thromboelastography with platelet mapping, multiplate analyzer, Cytochrome P450 2C19 testing, VerifyNow, corrected whole blood aggregometry, platelet function analyzer-100, and light transmission aggregometry. PFT identified individuals who were resistant or non-sensitive to antiplatelet therapy, with such patients facing increased risks of graft/stent thrombosis, amputation, and reintervention. However, substantial heterogeneity in surgical procedures, drug regimens, and testing methods was observed among the studies. CONCLUSIONS: PFTs can play a crucial role in detecting resistance and non-sensitivity to antiplatelet drugs in patients with PAD post-revascularization. However, heterogeneity of data and methods underlines the need for standardized protocols and consensus-building among PFTs. Enhancing clinical utility and reliability could help optimize antiplatelet thromboprophylaxis, minimize thrombotic complications, and improve treatment strategies in vascular surgery. Further research is necessary to solidify the role of PFTs in guiding antiplatelet therapy post-revascularization in patients with PAD.

Impact of Factor Xa Inhibition on Coagulation, Platelet Reactivity, and Thrombosis in Patients with Peripheral Artery Disease.

BACKGROUND: The role of thrombin in vascular pathology is a focus of investigation. The incorporation of direct Factor Xa inhibition into practice patterns is based on its theoretical dual-pathway attenuation of both thrombin generation and platelet aggregation. However, quantification of the effect of direct anti-Xa medications on platelet function is not established. Thromboelastography with platelet mapping (TEG-PM) leverages dual-pathway metrics to provide comprehensive coagulation profiles. We evaluated the effects of direct oral anticoagulants (DOACs) on coagulation and platelet function profiles and correlate these data with postoperative major adverse limb events (MALEs) in patients with PAD. METHODS: We conducted a prospective study of patients undergoing lower extremity revascularization with serial perioperative TEG-PM analysis. Patients on DOACs were compared to those not on DOACs, and stratified by concurrent mono-antiplatelet or dual-antiplatelet regimens (MAPT/DAPT). Postoperative MALE was recorded and difference in antithrombotic regimens and TEG-PM analysis compared between groups. RESULTS: Four hundred seventy-one samples from 141 patients were analyzed. Twenty-nine point five percent were reflective of circulating DOAC therapy. Compared to MAPT alone, patients on DOAC + MAPT exhibited longer time to clot formation (R-time) [7.4 (±2.4) vs. 6.7 (±2.7); P < 0.02], but less platelet inhibition. Patients on DAPT exhibited greater platelet inhibition compared to either group [23.7 (±26.9) vs. 31.0 (±28.3) vs. 42.2 (±31.2); P < 0.01]. Patients who experienced MALE were more likely to be on DOAC therapy [43.8% vs. 22.0% P = 0.02]. Thromboelastography with platelet mapping analysis from patients who experienced MALE also demonstrated longer R-time [8.6 (±3.9 vs. 7.3 (±3.0); P = 0.05] and increased maximum clot amplitude (MA) [66.7 (±4.2) vs. 61.8 (±8.2); P = 0.001]. CONCLUSIONS: Direct oral anticoagulant therapy resulted in a prolonged R-time but had no impact on platelet inhibition. Patients who experienced MALE were more often on DOACs and demonstrated an increased R-time, but also showed greater platelet reactivity evident by increased MA, suggesting DOACs may not be effective at protecting against MALE. Further research comparing DOAC therapy to a DAPT approach may add clarity to emerging multimodal antithrombotic recommendations.

The genome and sex-dependent responses to temperature in the common yellow butterfly, Eurema hecabe.

BACKGROUND: Lepidoptera (butterflies and moths) is one of the most geographically widespread insect orders in the world, and its species play important and diverse ecological and applied roles. Climate change is one of the biggest challenges to biodiversity this century, and lepidopterans are vulnerable to climate change. Temperature-dependent gene expression differences are of relevance under the ongoing climate crisis. However, little is known about how climate affects gene expression in lepidopterans and the ecological consequences of this, particularly with respect to genes with biased expression in one of the sexes. The common yellow butterfly, Eurema hecabe (Family Pieridae), is one of the most geographically widespread lepidopterans that can be found in Asia, Africa, and Australia. Nevertheless, what temperature-dependent effects there may be and whether the effects differ between the sexes remain largely unexplored. RESULTS: Here, we generated high-quality genomic resources for E. hecabe along with transcriptomes from eight developmental stages. Male and female butterflies were subjected to varying temperatures to assess sex-specific gene expression responses through mRNA and microRNA transcriptomics. We find that there are more temperature-dependent sex-biased genes in females than males, including genes that are involved in a range of biologically important functions, highlighting potential ecological impacts of increased temperatures. Further, by considering available butterfly data on sex-biased gene expression in a comparative genomic framework, we find that the pattern of sex-biased gene expression identified in E. hecabe is highly species-specific, rather than conserved across butterfly species, suggesting that sex-biased gene expression responses to climate change are complex in butterflies. CONCLUSIONS: Our study lays the foundation for further understanding of differential responses to environmental stress in a widespread lepidopteran model and demonstrates the potential complexity of sex-specific responses of lepidopterans to climate change.

Atorvastatin Effect on Clopidogrel Efficacy in Patients with Peripheral Artery Disease.

BACKGROUND: Both clopidogrel and atorvastatin metabolism are rooted in hepatic cytochrome p450 activation. There are published reports of atorvastatin interfering with clopidogrel metabolism by inhibiting the activation of clopidogrel. This in turn would decrease the therapeutic effect of clopidogrel potentially resulting in an increase in thrombotic events in patients who are taking both medications. The emergence of viscoelastic assays, such as Thromboelastography with platelet mapping (TEG-PM), has been utilized to identify prothrombotic states and may provide insight into a patient's microvascular coagulation profile. The aim of this prospective, observational study was to delineate the differences in platelet function between patients on clopidogrel alone versus those on clopidogrel and atorvastatin in patients that are undergoing peripheral revascularization. METHODS: All patients undergoing revascularization between December 2020 and August 2022 were prospectively evaluated. Patients on clopidogrel and atorvastatin were compared to those on clopidogrel alone. Serial perioperative TEG-PM analysis was performed up to 6 months postoperatively and the platelet function in terms of percent inhibition was evaluated in both groups. Statistical analysis was performed using unpaired t-test to identify differences in platelet function. RESULTS: Over the study period, a total of 182 patients were enrolled. Of this cohort 72 patients met study criteria. 87 samples from the 72 patients were analyzed. 31 (43.05%) patients were on clopidogrel alone and 41 (56.94%) were on clopidogrel and atorvastatin. Patients on clopidogrel alone showed significantly greater platelet inhibition compared to those on clopidogrel and atorvastatin [49.01% vs. 34.54%, P = 0.03]. There was no statistical difference in platelet inhibition between groups in terms of aspirin use alone versus aspirin and atorvastatin. CONCLUSIONS: Patients on clopidogrel and atorvastatin showed significantly less platelet inhibition compared to those on clopidogrel alone, supporting the concept that atorvastatin may interfere with the therapeutic effect of clopidogrel. Patients taking atorvastatin may require an alternative antiplatelet therapy regimen that does not include clopidogrel to achieve adequate thromboprophylaxis.

Innovating on innovation training with the Virtual Magic Wand (VMW) program: a qualitative study.

To identify and solve unmet needs and bring new therapies to patients, clinicians at all levels must engage in innovation. The Magic Wand Initiative, a program based at Massachusetts General Hospital-Wellman Center for Photomedicine, created a 10-months course called the Virtual Magic Wand (VMW) program that is a curriculum that teaches the biomedical innovation pathway to dermatologists and engages them in this creative process. This study aims to identify the impact of the VMW program on participants and consider the potential benefits of an innovation curriculum. The authors conducted semi-structured interviews in which alumni of the VMW program were asked about their experiences with innovation before, during, and after the program. Using grounded theory methodology, data were analyzed using deductive coding methods. The most cited benefit of the program was the opportunity to network (n = 12, 100%)-specifically, the mentorship opportunities (n = 10, 83%) and specialty-specific peer groups (n = 9, 75%). Other benefits included a change in mindset regarding their clinical work (n = 11, 92%) and learning the process of innovation (83%). Among barriers, lack of time (n = 7, 58%), knowledge (n = 6, 50%), and resources (n = 5, 42%), were the most mentioned. All alumni interviewed have stayed engaged in the field of biomedical innovation after their completion of the VMW program. These findings show that the VMW program positively impacted the lives and careers of participants. This study identified some of the systemic reasons that deter physicians from regularly engaging in innovation and provides guidance for how to design other innovation programs and further support the advancement of medicine.

Single-Cell Atlas of the Drosophila Leg Disc Identifies a Long Non-Coding RNA in Late Development.

The Drosophila imaginal disc has been an excellent model for the study of developmental gene regulation. In particular, long non-coding RNAs (lncRNAs) have gained widespread attention in recent years due to their important role in gene regulation. Their specific spatiotemporal expressions further support their role in developmental processes and diseases. In this study, we explored the role of a novel lncRNA in Drosophila leg development by dissecting and dissociating w1118 third-instar larval third leg (L3) discs into single cells and single nuclei, and performing single-cell RNA-sequencing (scRNA-seq) and single-cell assays for transposase-accessible chromatin (scATAC-seq). Single-cell transcriptomics analysis of the L3 discs across three developmental timepoints revealed different cell types and identified lncRNA:CR33938 as a distal specific gene with high expression in late development. This was further validated by fluorescence in-situ hybridization (FISH). The scATAC-seq results reproduced the single-cell transcriptomics landscape and elucidated the distal cell functions at different timepoints. Furthermore, overexpression of lncRNA:CR33938 in the S2 cell line increased the expression of leg development genes, further elucidating its potential role in development.

Dermoscopy practice guidelines for use in telemedicine.

Teledermoscopy, or the utilization of dermatoscopic images in telemedicine, can help diagnose dermatologic disease remotely, triage lesions of concern (i.e., determine whether in-person consultation with a dermatologist is necessary, biopsy, or reassure the patient), and monitor dermatologic lesions over time. Handheld dermatoscopes, a magnifying apparatus, have become a commonly utilized tool for providers in many healthcare settings and professions and allows users to view microstructures of the epidermis and dermis. This Dermoscopy Practice Guideline reflects current knowledge in the field of telemedicine to demonstrate the correct capture, usage, and incorporation of dermoscopic images into everyday practice.

Evaluation of Teledermatology Practice Guidelines and Recommendations for Improvement.

Background: The current coronavirus disease 2019 (COVID-19) pandemic has forced dermatology practices to rapidly adopt or expand teledermatology services. To date, teledermatology clinical practice guidelines (CPGs) have not been evaluated for quality. This may hinder the implementation and sustainability of high-quality teledermatology services. The objective of this study is to evaluate teledermatology CPGs by using a comprehensive, validated appraisal tool and provide actionable recommendations for improvement for future guideline developers. Materials and Methods: A systematic literature search was performed to identify all CPGs published in teledermatology. Identified guidelines meeting inclusion criteria were scored for guideline quality by three researchers using the Appraisal of Guidelines and Research and Evaluation tool (AGREE II). Results: Three guidelines met inclusion criteria, including guidelines published by the American Telemedicine Association, the British Association of Dermatologists, and the Australian College of Dermatologists and the University of Queensland. Each of these guidelines exhibited unique strengths and areas for improvement. Areas of strength include clear objective statements, stakeholder involvement, and clarity of presentation. Areas for improvement include rigor of guideline development, applicability, and editorial independence. Conclusions: All guidelines were determined to be of sufficient quality for adoption based on AGREE scoring. Adoption of the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) evidence appraisal framework would improve the rigor of development of each guideline. Administering and publishing questionnaires to assess financial competing interests and external review processes would shed more light on the presence and/or extent of author bias. One inherent limitation of CPG selection criteria is that large providers of teledermatology services, such as the United States Veterans Affairs, were not examined in this review. However, the results of this study provide actionable targets to improve the quality of the teledermatology CPGs examined in this study.

Individual Variation in Functional Brain Network Topography is Linked to Schizophrenia Symptomatology.

Resting-state fMRI (rsfMRI) demonstrates that the brain is organized into distributed networks. Numerous studies have examined links between psychiatric symptomatology and network functional connectivity. Traditional rsfMRI analyses assume that the spatial organization of networks is invariant between individuals. This dogma has recently been overturned by the demonstration that networks show significant variation between individuals. We tested the hypothesis that previously observed relationships between schizophrenia-negative symptom severity and network connectivity are actually due to individual differences in network spatial organization. Forty-four participants diagnosed with schizophrenia underwent rsfMRI scans and clinical assessments. A multivariate pattern analysis determined how whole-brain functional connectivity correlates with negative symptom severity at the individual voxel level. Brain connectivity to a region of the right dorsolateral prefrontal cortex correlates with negative symptom severity. This finding results from individual differences in the topographic distribution of 2 networks: the default mode network (DMN) and the task-positive network (TPN). Both networks demonstrate strong (r = ~0.49) and significant (P < .001) relationships between topography and symptom severity. For individuals with low symptom severity, this critical region is part of the DMN. In highly symptomatic individuals, this region is part of the TPN. Previously overlooked individual variation in brain organization is tightly linked to differences in schizophrenia symptom severity. Recognizing critical links between network topography and pathological symptomology may identify key circuits that underlie cognitive and behavioral phenotypes. Individual variation in network topography likely guides different responses to clinical interventions that rely on anatomical targeting (eg, transcranial magnetic stimulation [TMS]).

Innovation interest within dermatology: a needs assessment for novel thought processes.

Medical innovation is crucial to advancing our field, and physicians have the potential to play a leading role due to their daily patient care experiences. The objective of this study was to evaluate the interest in, and barriers to participating in innovation. Two surveys were conducted; the first cross-sectional survey was conducted among attendees of the Advancing Innovation in Dermatology Forum in Feburary 2019. The second survey was conducted among trainees (resident/fellows) and faculty dermatologists at Brown, Emory, Iowa, Stanford, and Vanderbilt Universities between June and November 2019. Demographic data were collected, as well as factors involved with identifying problems, developing solutions, training in innovation, and perceived barriers to innovation. In the first survey, the greatest perceived benefits include bringing joy to one's work and increasing professional fulfillment with work. Innovation was also perceived to decrease burnout. In the second survey of academic centers, faculty more commonly expressed interest in identifying problems (p = 0.04), and was also more confident in their ability to generate solutions to these problems as compared to trainees (p < 0.01). Major barriers to participating in innovation processes included lack of time and lack of training or education in innovation. Both trainees and faculty groups noted a lack of knowledge in creating prototypes, understanding regulatory approval for medical products, and inexperience with pitching to investors or obtaining funding. These cross-sectional needs assessment surveys found a strong interest in innovation coupled with a lack of education in innovation processes. These findings suggest an urgent need and opportunity for providing formal training to empower dermatologists with the tools to lead innovation within our field.

Terpenes and Terpenoids in Plants: Interactions with Environment and Insects.

The interactions of plants with environment and insects are bi-directional and dynamic. Consequently, a myriad of mechanisms has evolved to engage organisms in different types of interactions. These interactions can be mediated by allelochemicals known as volatile organic compounds (VOCs) which include volatile terpenes (VTs). The emission of VTs provides a way for plants to communicate with the environment, including neighboring plants, beneficiaries (e.g., pollinators, seed dispersers), predators, parasitoids, and herbivores, by sending enticing or deterring signals. Understanding terpenoid distribution, biogenesis, and function provides an opportunity for the design and implementation of effective and efficient environmental calamity and pest management strategies. This review provides an overview of plant-environment and plant-insect interactions in the context of terpenes and terpenoids as important chemical mediators of these abiotic and biotic interactions.

Patient perspectives on frame versus mask immobilization for gamma knife stereotactic radiosurgery.

PURPOSE: To assess patient experiences and perspectives following Gamma Knife (GK) stereotactic radiosurgery (SRS) using frame versus mask immobilization. METHODS: Patients who received GK-SRS using both frame and mask immobilization were included in this study. One-on-one semi-structured interviews, led by a third-party expert, were used to gain insight into the patient experience. To reduce memory bias of either immobilization device, patients underwent the interview at their follow-up appointment. Initial assessment of patient transcriptions was completed by one study staff; a second member reviewed transcripts for thematic saturation. All interviews were independently coded for themes to minimize interpretation bias. RESULTS: Fifteen patients were consented; 12 were successfully interviewed (3 lost due to deteriorating health status). Interviews ranged from 30 to 60 min in duration. The most common patient concern regarding the frame was pain (9 patients), while the primary concerns with the mask system were the ability to remain still (6 patients) and claustrophobia (4 patients). Eleven patients chose the mask as their preferred choice in terms of their overall experience. Two themes emerged during the interviews that spoke to patient satisfaction with each process: unexpected pain with frame placement; and tightness experienced while wearing the mask during treatment. CONCLUSIONS: From the patient perspective there was overwhelming agreement that the mask was the preferred choice for GK-SRS. The patient experience could be improved by enhanced education to better prepare patients on what to expect during the frame placement and mask treatment processes.

Infection patterns of dengue, Zika and endosymbiont Wolbachia in the mosquito Aedes albopictus in Hong Kong.

BACKGROUND: The mosquito Aedes albopictus is a vector of dengue and Zika viruses. Insecticide-resistant mosquito populations have evolved in recent decades, suggesting that new control strategies are needed. Hong Kong has a monsoon-influenced humid subtropical climate, which favours the spread of mosquitoes. However, baseline information on the composition and dynamics of the occurrence of endosymbiont Wolbachia in local Ae. albopictus is lacking, hindering the development of scientifically-informed control measures. This study identifies the presence and absence of dengue and Zika viruses, and Wolbachia infection in Aedes albopictus in Hong Kong. METHODS: Oviposition traps were set at 57 areas in Hong Kong, and both immature and adult mosquitoes were collected on a monthly basis between April 2018 and April 2019 as the study sample. Each individual mosquito in this sample was processed and screened for the presence of the dengue and Zika viruses and the endosymbionts Wolbachia wAlbA and wAlbB with PCR. RESULTS: Totals of 967 and 984 mosquitoes were tested respectively for the presence of dengue and Zika viruses, and no trace of either infection was found in these samples. The presence of wAlbA and wAlbB was also tested in 1582 individuals. Over 80% of these individuals were found to be stably infected with Wolbachia throughout the thirteen-month collection period (~ 47% singly-infected; ~ 36.8% doubly infected with both wAlbA and wAlbB). CONCLUSIONS: The high degree of Wolbachia wAlbA and wAlbB infection in Ae. albopictus mosquitoes in Hong Kong, coupled with the absence of any signs of infection by dengue and Zika viruses, contrasts significantly with the pattern of mosquito infection in other parts of Asia. Further studies of the infection pattern in local mosquitoes are warranted before mosquito control strategies used in other regions are implemented in Hong Kong.

Micro-RNA Clusters Integrate Evolutionary Constraints on Expression and Target Affinities: The miR-6/5/4/286/3/309 Cluster in Drosophila.

A striking feature of micro-RNAs is that they are often clustered in the genomes of animals. The functional and evolutionary consequences of this clustering remain obscure. Here, we investigated a micro-RNA cluster miR-6/5/4/286/3/309 that is conserved across drosophilid lineages. Small RNA sequencing revealed expression of this micro-RNA cluster in Drosophila melanogaster leg discs, and conditional overexpression of the whole cluster resulted in leg appendage shortening. Transgenic overexpression lines expressing different combinations of micro-RNA cluster members were also constructed. Expression of individual micro-RNAs from the cluster resulted in a normal wild-type phenotype, but either the expression of several ancient micro-RNAs together (miR-5/4/286/3/309) or more recently evolved clustered micro-RNAs (miR-6-1/2/3) can recapitulate the phenotypes generated by the whole-cluster overexpression. Screening of transgenic fly lines revealed downregulation of leg-patterning gene cassettes in generation of the leg-shortening phenotype. Furthermore, cell transfection with different combinations of micro-RNA cluster members revealed a suite of downstream genes targeted by all cluster members, as well as complements of targets that are unique for distinct micro-RNAs. Considered together, the micro-RNA targets and the evolutionary ages of each micro-RNA in the cluster demonstrate the importance of micro-RNA clustering, where new members can reinforce and modify the selection forces on both the cluster regulation and the gene regulatory network of existing micro-RNAs. Key words: micro-RNA, cluster, evolution.