Rapid, high-throughput, cost-effective whole-genome sequencing of SARS-CoV-2 using a condensed library preparation of the Illumina DNA Prep kit.

The ongoing COVID-19 pandemic necessitates cost-effective, high-throughput, and timely whole-genome sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses for outbreak investigations, identifying variants of concern (VoC), characterizing vaccine breakthrough infections, and public health surveillance. In addition, the enormous demand for WGS on supply chains and the resulting shortages of laboratory supplies necessitated the use of low-reagent and low-consumable methods. Here, we report an optimized library preparation method (the BCCDC cutdown method) that can be used in a high-throughput scenario, where one technologist can perform 576 library preparations (6 plates of 96 samples) over the course of one 8-hour shift. The same protocol can also be used in a rapid turnaround time scenario, from primary samples (up to 96 samples) to loading on a sequencer in an 8-hour shift. This new method uses Freed et al.'s 1,200 bp primer sets (Biol Methods Protoc 5:bpaa014, 2020, https://doi.org/10.1093/biomethods/bpaa014) and a modified and condensed Illumina DNA Prep workflow (Illumina, CA, USA). Compared to the original protocol, the application of this new method using hundreds of clinical specimens demonstrated equivalent results to the full-length DNA Prep workflow at 45% of the cost, 15% of consumables required (such as pipet tips), 25% of manual hands-on time, and 15% of on-instrument time if performing on a liquid handler, with no compromise in sequence quality. Results demonstrate that this new method is a rapid, simple, cost-effective, and high-quality SARS-CoV-2 WGS protocol. IMPORTANCE: Sequencing has played an invaluable role in the response to the COVID-19 pandemic. Ongoing work in this area, however, demands optimization of laboratory workflow to increase sequencing capacity, improve turnaround time, and reduce cost without compromising sequence quality. This report describes an optimized DNA library preparation method for improved whole-genome sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen. The workflow advantages summarized here include significant time, cost, and consumable savings, which suggest that this new method is an efficient, scalable, and pragmatic alternative for SARS-CoV-2 whole-genome sequencing.

Prospective, clinical comparison of self-collected throat-bilateral nares swabs and saline gargle compared to health care provider collected nasopharyngeal swabs among symptomatic outpatients with potential SARS-CoV-2 infection.

Background: In British Columbia (BC), self-collected saline gargle (SG) is the only alternative to health care provider (HCP)-collected nasopharyngeal (NP) swabs to detect SARS-CoV-2 in an outpatient setting by polymerase chain reaction (PCR). However, some individuals cannot perform a SG. Our study aimed to assess combined throat-bilateral nares (TN) swabbing as a swab-based alternative. Methods: Symptomatic individuals greater than 12 years of age seeking a COVID-19 PCR test at one of two COVID-19 collection centres in Metro Vancouver were asked to participate in this study. Participants provided a HCP-collected NP sample and a self-collected SG and TN sample for PCR testing, which were either HCP observed or unobserved. Results: Three-hundred and eleven individuals underwent all three collections. Compared against HCP-NP, SG was 99% sensitive and 98% specific (kappa 0.97) and TN was 99% sensitive and 99% specific (kappa 0.98). Using the final clinical test interpretation as the reference standard, NP was 98% sensitive and 100% specific (kappa 0.98), and both SG and TN were 99% sensitive and 100% specific (both kappa 0.99). Mean cycle threshold values for each viral target were higher in SG specimens compared to the other sample types; however, this did not significantly impact the clinical performance, because the positivity rates were similar. The clinical performance of all specimen types was comparable within the first 7 days of symptom onset, regardless of the observation method. SG self-collections were rated the most acceptable, followed by TN. Conclusions: TN provides another less invasive self-collection modality for symptomatic outpatient SARS-CoV-2 PCR testing.

Historique: En Colombie-Britannique (C.-B.), l'autoprélèvement de gargarisme d'eau saline (GS) est la seule alternative aux écouvillons nasopharyngés (NP) prélevés par un professionnel de la santé (PdS) pour déceler le SRAS-CoV-2 par test PCR en milieu ambulatoire. Cependant, certaines personnes ne peuvent pas effectuer de GS. La présente étude visait évaluer l'écouvillonnage de la gorge et des deux narines (GN) pour remplacer le GS. Méthodologie: Les personnes symptomatiques de plus de 12 ans qui demandaient un test PCR de la COVID-19 à l'un des deux centres de dépistage de la COVID-19 du Grand Vancouver ont été invitées à participer à la présente étude. Les participants ont fourni un prélèvement NP recueilli par un PdS ainsi qu'un autoprélèvement de GS et GN en vue d'un test PCR, observés ou non par un PdS. Résultats: Au total, 311 personnes ont participé aux trois prélèvements. Par rapport au prélèvement NP-PdS, le GS avait une sensibilité de 99 % et une spécificité de 98 % (kappa 0,97) et le prélèvement GN, une sensibilité de 99 % et une spécificité de 99 % (kappa 0, 98). À l'aide de l'interprétation définitive du test clinique comme norme de référence, le prélèvement NP avait une sensibilité de 98 % et une spécificité de 100 % (kappa 0,98) et tant le GS que le prélèvement GN avaient une sensibilité de 99 % et une spécificité de 100 % (deux kappa 0,99). Les valeurs seuils du cycle moyen de chaque cible virale étaient plus élevées dans les échantillons de GS quand dans les autres types d'échantillons, mais n'avaient pas d'effet significatif sur le rendement clinique, puisque les taux de positivité étaient semblables. Le rendement clinique de tous les types d'échantillons était comparable dans les sept premiers jours suivant l'apparition de la maladie, quel que soit le mode d'observation. L'autoprélèvement de GS a été classé comme le plus acceptable, suivi du prélèvement GN. Conclusions: Le prélèvement GN est un autre mode d'autoprélèvement moins invasif chez les patients ambulatoires symptomatiques qui effectuent un test PCR du SRAS-CoV-2.

Brn3b regulates the formation of fear-related midbrain circuits and defensive responses to visual threat.

Defensive responses to visually threatening stimuli represent an essential fear-related survival instinct, widely detected across species. The neural circuitry mediating visually triggered defensive responses has been delineated in the midbrain. However, the molecular mechanisms regulating the development and function of these circuits remain unresolved. Here, we show that midbrain-specific deletion of the transcription factor Brn3b causes a loss of neurons projecting to the lateral posterior nucleus of the thalamus. Brn3b deletion also down-regulates the expression of the neuropeptide tachykinin 2 (Tac2). Furthermore, Brn3b mutant mice display impaired defensive freezing responses to visual threat precipitated by social isolation. This behavioral phenotype could be ameliorated by overexpressing Tac2, suggesting that Tac2 acts downstream of Brn3b in regulating defensive responses to threat. Together, our experiments identify specific genetic components critical for the functional organization of midbrain fear-related visual circuits. Similar mechanisms may contribute to the development and function of additional long-range brain circuits underlying fear-associated behavior.

Factors affecting perception of laypeople and dental professionals toward different smile esthetics.

Abstract Background: /purpose: Several factors such as identity, income, and age potentially associated with smile perceptions. This study aimed to identify the factors affecting the smile esthetic perception in different identities (layperson, general dentist and orthodontist) and to detect the extent of their association with smile perception. Materials and methods: Extraoral photographs in frontal, lateral, and three-quarter views were shot and adjusted on Adobe Photoshop into 95 smile photographs with different smile patterns. Based on these photographs, the investigators were asked to fill the online questionnaire. Pearson chi-square test and logistic regression were used for statistical analyses. Results: Identity, gender, age, and treatment experience were noted to affect smile esthetic perception. In addition, the perception of smile esthetics was significantly different among frontal, lateral, and three-quarters views regarding the arc ratio, most posterior teeth exposure, upper teeth exposure, and lower teeth exposure. Conclusion: Identity, gender, age, and treatment experience influence the smile esthetics perception, with a significant difference in the results of the esthetic perception based on the 3 smile views. Of all demographic factors, identity had a strong relation to the perception of smile attractiveness. Nevertheless, additional studies are needed to realize how the demographic factors influence people's perception of smile esthetics, particularly in the three-quarter and lateral views.

Perception and attitude of Mongolians on malocclusion.

Background/purpose: This study assessed the perception and attitude of Mongolians on malocclusion and compared the perceptions with researchers' perceptions on malocclusion, and this study aimed to determine factors influencing the self-perception of laypeople. Materials and methods: In addition, we evaluated the correlation between the subjective and objective perceptions of participants. In a random sample of 133 people (39.8% men and 60.2% women) aged 18-55 years, subjective perceptions were assessed using a questionnaire (oral aesthetic subjective impact scale), and objective perceptions were assessed using 10 intraoral frontal photographs of the aesthetic component of index of orthodontic treatment need (AC-IOTN) and six intraoral lateral photographs of the aesthetic component of lateral occlusion (AC-LO). Results: A significant association was observed between participants' self-perception and researchers' ratings (P < 0.001). The appearance of teeth was significantly correlated with participants' self-rating by using AC-IOTN and AC-LO. Most people (74%) believed that well-aligned teeth improve the overall facial appearance. Sex, monthly income, and education level significantly influenced the self-perception of participants on malocclusion. Conclusion: Patients' perception and attitude cannot be overlooked because they are some of the crucial aspects of the entire treatment.

How Can We Do Citizen Science Better? A Case Study Evaluating Grizzly Bear Citizen Science Using Principles of Good Practice in Alberta, Canada.

Citizen science offers an excellent opportunity to engage the public in scientific data collection, educational opportunities, and applied management. However, the practicalities of developing and implementing citizen science programming are often more complex than considered. Some challenges to effective citizen science include scientists' skepticism about the ability of public participants to rigorously collect quality data; a lack of clarity on or confidence in the utility of data; scientists' hesitancy in engaging the public in projects; limited financial commitments; and challenges associated with the temporal and geographic scales of projects. To address these challenges, and provide a foundation upon which practitioners, scientists, and the public can credibly engage in citizen science, the Government of Alberta developed a set of citizen science principles. These principles offer a framework for planning, designing, implementing, and evaluating citizen science projects that extend beyond Alberta. Here, we present a case study using these principles to evaluate GrizzTracker, a citizen science program developed to help inform provincial species-at-risk recovery efforts. While we found that GrizzTracker applied each of the six principles in some way, including successful public engagement, strengthened relationships, and raising public awareness about northwest Alberta's grizzly bears, we also identified a number of challenges. These included ongoing skepticism from the traditional scientific community about the utility of citizen science and governance challenges related to program leadership, staff capacity, and funding. By using the principles as a guideline, we provide policy recommendations for future citizen science efforts, including considerations for program design, implementation, and evaluation.

Extensive sampling of Saccharomyces cerevisiae in Taiwan reveals ecology and evolution of predomesticated lineages.

The ecology and genetic diversity of the model yeast Saccharomyces cerevisiae before human domestication remain poorly understood. Taiwan is regarded as part of this yeast's geographic birthplace, where the most divergent natural lineage was discovered. Here, we extensively sampled the broadleaf forests across this continental island to probe the ancestral species' diversity. We found that S. cerevisiae is distributed ubiquitously at low abundance in the forests. Whole-genome sequencing of 121 isolates revealed nine distinct lineages that diverged from Asian lineages during the Pleistocene, when a transient continental shelf land bridge connected Taiwan to other major landmasses. Three lineages are endemic to Taiwan and six are widespread in Asia, making this region a focal biodiversity hotspot. Both ancient and recent admixture events were detected between the natural lineages, and a genetic ancestry component associated with isolates from fruits was detected in most admixed isolates. Collectively, Taiwanese isolates harbor genetic diversity comparable to that of the whole Asia continent, and different lineages have coexisted at a fine spatial scale even on the same tree. Patterns of variations within each lineage revealed that S. cerevisiae is highly clonal and predominantly reproduces asexually in nature. We identified different selection patterns shaping the coding sequences of natural lineages and found fewer gene family expansion and contractions that contrast with domesticated lineages. This study establishes that S. cerevisiae has rich natural diversity sheltered from human influences, making it a powerful model system in microbial ecology.

Survey of state conjunctivitis policies for school-age students.

PURPOSE: To examine state policies regarding school-age students with conjunctivitis. METHODS: Analysis included the following evidence: publicly available policies for disposition of affected students; indications for exclusion and return to classroom; and completeness of information, including mention of different etiologies of conjunctivitis; signs and symptoms of viral versus bacterial conjunctivitis; student disposition and treatment based on etiology; internally consistent recommendations; reference to credible resources; and mention of the possibility of a conjunctivitis outbreak. RESULTS: Fifteen of 50 states have no policies. Ten states allow students to remain in school, 5 allow return 24 hours after initiation of antibiotic treatment, and 5 require physician approval. Seventeen states and Washington, DC, offer little detail or internally inconsistent recommendations, such as choice of antibiotic use or provider note. Twelve policies are thoroughly presented. Twenty-three states refer to sources like the Centers for Disease Control and Prevention and the American Academy of Pediatrics. No policy references the American Academy of Ophthalmology. CONCLUSIONS: State policies on conjunctivitis in students vary widely. Antibiotic use as a prerequisite for return to school has drawbacks of cost to parents, increasing antibiotic resistance, and lack of efficacy against nonbacterial etiologies, for example, viral conjunctivitis. Publicly available information and guidelines could be improved, aiming for fewer absentee days, reduced outbreak risk, and reduced risk of antibiotic resistance.

Daily experiences of type 1 diabetes stress across adulthood.

AIMS: Daily diabetes stressful events take a toll on individuals with type 1 diabetes, and these experiences may look different across adulthood. The aims of the current study were to understand the nature of daily diabetes stress across adulthood and explore whether these experiences differed by age. METHODS: In this qualitative study, adults with T1D (N = 199, Mage  = 46.81 years) described the most stressful event related to their diabetes each evening as part of a 14-day diary. Using a grounded theory approach, diabetes stressful events were coded for where they occurred, the source of stress (i.e. interpersonal or not), and content (e.g. sleep; blood glucose checking; frustration). RESULTS: Participants reported having a diabetes-related stressful event on 58% (M = 0.58, SD = (0.25)) of days. Daily stressful events included issues of diabetes management, diabetes-related interference to or from other areas of life, and negative impact on psychological well-being, but rarely included a social component. Older adults were less likely to report having a diabetes-related stressful event, but were more likely to report that stressful events occurred at home, compared to younger adults. CONCLUSION: The lived experience of diabetes-related stress appears similar across ages, with individuals continuing to experience generally the same types of diabetes-related events in similar frequencies. Interventions to help improve diabetes outcomes or well-being may benefit from targeting the most commonly experienced areas of stress, which includes reducing the interference of daily activities to and by diabetes management.

Mutations in emerging variant of concern lineages disrupt genomic sequencing of SARS-CoV-2 clinical specimens.

Mutations in emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages can interfere with laboratory methods used to generate viral genome sequences for public health surveillance. We identified 20 mutations that are widespread in variant of concern lineages and affect widely used sequencing protocols by the ARTIC network and Freed et al. Three of these mutations disrupted sequencing of P.1 lineage specimens during a recent outbreak in British Columbia, Canada. We provide laboratory validation of protocol modifications that restored sequencing performance. The study findings indicate that genomic sequencing protocols require immediate updating to address emerging mutations. This work also suggests that routine monitoring and protocol updates will be necessary as SARS-CoV-2 continues to evolve. The bioinformatic and laboratory approaches used here provide guidance for this kind of assay maintenance.

Effect of anterior overbite malocclusion on smile esthetics in adult patients.

OBJECTIVE: To investigate whether overbite affects smile esthetics. METHODS: This study involved 106 patients with complete pretreatment records. Lateral cephalometric tracings were used to measure hard tissue variables. Frontal smile and upper occlusal photographs were used to measure nine smile esthetic variables: arc ratio, number of teeth, upper incisor exposure, upper midline, buccal corridor ratio, smile index, archform index, lower teeth exposure, and interlabial gap. The patients were classified into three groups according to their overbites (B1: 0-4 mm, B2: >4 mm, and B3: <0 mm). Analysis of variance was performed to compare 14 cephalometric measurements and the 9 smile esthetic variables. Multiple linear regression analysis was performed to determine the influencing cephalometric factors. RESULTS: Only upper incisor exposure was significantly different among the groups. In the multiple linear regression analysis, upper incisor exposure was positively associated with the distance from the upper incisor to the palatal plane in Group B2. No significant correlations were observed between cephalometric measurements and smile variables in Groups B1 and B3. CONCLUSIONS: Smile variables were not significantly different among patients with various overbite malocclusions with the exception of upper incisor exposure. Overbite malocclusions do not appear to influence smile esthetics in adult patients.

Rapid Increase in SARS-CoV-2 P.1 Lineage Leading to Codominance with B.1.1.7 Lineage, British Columbia, Canada, January-April 2021.

Several severe acute respiratory syndrome coronavirus 2 variants of concern (VOCs) emerged in late 2020; lineage B.1.1.7 initially dominated globally. However, lineages B.1.351 and P.1 represent potentially greater risk for transmission and immune escape. In British Columbia, Canada, B.1.1.7 and B.1.351 were first identified in December 2020 and P.1 in February 2021. We combined quantitative PCR and whole-genome sequencing to assess relative contribution of VOCs in nearly 67,000 infections during the first 16 weeks of 2021 in British Columbia. B.1.1.7 accounted for <10% of screened or sequenced specimens early on, increasing to >50% by week 8. P.1 accounted for <10% until week 10, increased rapidly to peak at week 12, and by week 13 codominated within 10% of rates of B.1.1.7. B.1.351 was a minority throughout. This rapid expansion of P.1 but suppression of B.1.351 expands our understanding of population-level VOC patterns and might provide clues to fitness determinants for emerging VOCs.

The relationship between dentofacial morphology and smile characteristics in lateral and oblique views.

BACKGROUND/PURPOSE: The purposes of this study were to develop smile measurements for lateral and oblique view photographs to help in orthodontic analysis and treatment planning, to quantitatively evaluate the relationship between smile esthetics and different types of malocclusion using lateral and oblique view photographs, to identify the cephalometric factors affecting smile measurements. MATERIALS AND METHODS: Patients who came to orthodontic department of a university hospital from 2014 to 2017 and met the inclusion criteria were included and divided into three groups according to Angle's classification. Thirteen variables were measured for cephalometric analysis. Twenty-one variables were developed and measured on pretreatment photographs for lateral and oblique smile analysis. ANOVA and Scheffe post hoc test were used to compare cephalometric and smile variables among three groups. Multiple linear regression analysis was performed to identify cephalometric factors affecting smile measurements. RESULTS: Three-hundred and ninety patients (287 females, 103 males) with mean age of 24.5 ±â€¯7.6 years reached the criteria. All cephalometric variables differed significantly among three groups. Except for maxillary teeth exposure number, visible maxillary width, and lip thickness ratio, all smile variables differed significantly. Smile characteristics had significant correlation with some cephalometric measurements. CONCLUSION: Smile patterns on the lateral and oblique view photographs can be affected by different types of malocclusion. Therefore, we suggest to include lateral and oblique smile view photographs in the data collection for orthodontic treatment planning.

Evidence of Extensive Intraspecific Noncoding Reshuffling in a 169-kb Mitochondrial Genome of a Basidiomycetous Fungus.

Comparative genomics of fungal mitochondrial genomes (mitogenomes) have revealed a remarkable pattern of rearrangement between and within major phyla owing to horizontal gene transfer and recombination. The role of recombination was exemplified at a finer evolutionary time scale in basidiomycetes group of fungi as they display a diversity of mitochondrial DNA inheritance patterns. Here, we assembled mitogenomes of six species from the Hymenochaetales order of basidiomycetes and examined 59 mitogenomes from 2 genetic lineages of Phellinus noxius. Gene order is largely collinear, while intergene regions are major determinants of mitogenome size variation. Substantial sequence divergence was found in shared introns consistent with high horizontal gene transfer frequency observed in yeasts, but we also identified a rare case where an intron was retained in five species since speciation. In contrast to the hyperdiversity observed in nuclear genomes of Phellinus noxius, mitogenomes' intraspecific polymorphisms at protein-coding sequences are extremely low. Phylogeny network based on introns revealed turnover as well as exchange of introns between two lineages. Strikingly, some strains harbor a mosaic origin of introns from both lineages. Analysis of intergenic sequence indicated substantial differences between and within lineages, and an expansion may be ongoing as a result of exchange between distal intergenes. These findings suggest that the evolution in mitochondrial DNAs is usually lineage specific but chimeric mitotypes are frequently observed, thus capturing the possible evolutionary processes shaping mitogenomes in a basidiomycete. The large mitogenome sizes reported in various basidiomycetes appear to be a result of interspecific reshuffling of intergenes.

Traumatic Hallux Varus in Association with Agenesis of the Fibular Sesamoid: A Case Study.

Hallux varus is most commonly seen iatrogenically following overaggressive lateral release, removal of the fibular sesamoid, or overaggressive removal of the medial eminence. There are several reported cases of traumatic hallux varus, although this is much less common. We present a case of traumatic hallux varus in a patient who was later found to have bilateral absence of her fibular sesamoids. We postulated that lack of her fibular sesamoid led to weakness of her lateral capsular ligaments, thereby making her more susceptible to this injury. We performed a repair using a split extensor hallucis longus tendon transfer that was transected proximally, rerouted the tendon under the deep transverse intermetatarsal ligament, and secured it to the first metatarsal with a Bio-Tenodesis (Arthrex, Inc, Naples, Florida) screw. At 22 months postoperatively, she has demonstrated maintenance of correction and has resumed use of normal shoe gear and participation in activities. Our goal was to demonstrate a repair for this condition that successfully maintained correction over time while still allowing for functionality of the first metatarsophalangeal joint.

Parental Involvement and Executive Function in Emerging Adults with Type 1 Diabetes.

OBJECTIVE: To examine (a) changes in parental involvement across early emerging adulthood, (b) whether yearly fluctuations in parental involvement were associated with adherence and glycated hemoglobin (HbA1c) over time, and (c) whether higher involvement was more beneficial for those with poorer executive function (EF). METHODS: A total of 228 high school seniors (M age = 17.76) with type 1 diabetes reported on mothers' and fathers' acceptance, knowledge of diabetes activities, disclosure to mothers and fathers regarding diabetes, and adherence at four yearly time points. At baseline, participants completed performance-based measures of EF. HbA1c was collected from assay kits. RESULTS: Growth curve models revealed significant declines in disclosure to fathers and mothers' and fathers' knowledge of diabetes activities; no changes were found in mothers' or fathers' acceptance nor disclosure to mothers. Multilevel models indicated significant between-person effects for nearly all aspects of parental involvement with more acceptance, knowledge, and disclosure associated with better HbA1c and adherence. Within-person effects for disclosure to fathers, and mothers' and fathers' knowledge indicated that in years when emerging adults perceived higher amounts of these types of involvement (compared with their own average), HbA1c was lower. Within-person effects were found for acceptance to mothers, disclosure to mothers and fathers, and mothers' diabetes knowledge for adherence. Disclosure to fathers and mothers' knowledge of diabetes activities were especially beneficial for HbA1c for those with poorer EF performance. CONCLUSIONS: Parental involvement in diabetes management remains important during the high-risk time of emerging adulthood, especially for those with poorer EF.