Subgenomic RNA identification in SARS-CoV-2 genomic sequencing data.

We have developed periscope, a tool for the detection and quantification of subgenomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed "subgenomic RNAs." sgRNAs are produced through discontinuous transcription, which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L, which is located in the 5' UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5' end of all sgRNA. We applied periscope to 1155 SARS-CoV-2 genomes from Sheffield, United Kingdom, and validated our findings using orthogonal data sets and in vitro cell systems. By using a simple local alignment to detect reads that contain the leader sequence, we were able to identify and quantify reads arising from canonical and noncanonical sgRNA. We were able to detect all canonical sgRNAs at the expected abundances, with the exception of ORF10. A number of recurrent noncanonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/- cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing data sets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.

Determination of sensed and paced atrial-ventricular delay in cardiac resynchronization therapy.

BACKGROUND: Optimization of atrial-ventricular delay (AVD) during atrial sensing (SAVD) and pacing (PAVD) provides the most effective cardiac resynchronization therapy (CRT). We demonstrate a novel electrocardiographic methodology for quantifying electrical synchrony and optimizing SAVD/PAVD. METHODS: We studied 40 CRT patients with LV activation delay. Atrial-sensed to RV-sensed (As-RVs) and atrial-paced to RV-sensed (Ap-RVs) intervals were measured from intracardiac electrograms (IEGM). LV-only pacing was performed over a range of SAVD/PAVD settings. Electrical dyssynchrony (cardiac resynchronization index; CRI) was measured at each setting using a multilead ECG system placed over the anterior and posterior torso. Biventricular pacing, which included multiple interventricular delays, was also conducted in a subset of 10 patients. RESULTS: When paced LV-only, peak CRI was similar (93 ± 5% vs. 92 ± 5%) during atrial sensing or pacing but optimal PAVD was 61 ± 31 ms greater than optimal SAVD. The difference between As-RVs and Ap-RVs intervals on IEGMs (62 ± 31 ms) was nearly identical. The slope of the correlation line (0.98) and the correlation coefficient r (0.99) comparing the 2 methods of assessing SAVD-PAVD offset were nearly 1 and the y-intercept (0.63 ms) was near 0. During simultaneous biventricular (BiV) pacing at short AVD, SAVD and PAVD programming did not affect CRI, but CRI was significantly (p < .05) lower during atrial sensing at long AVD. CONCLUSIONS: A novel methodology for measuring electrical dyssynchrony was used to determine electrically optimal SAVD/PAVD during LV-only pacing. When BiV pacing, shorter AVDs produce better electrical synchrony.

Investigation of CRISPR-Independent Phage Resistance Mechanisms Reveals a Role for FtsH in Phage Adsorption to Streptococcus thermophilus.

Prokaryotes are under constant pressure from phage infection and thus have evolved multiple means of defense or evasion. While CRISPR-Cas constitutes a robust immune system and appears to be the predominant means of survival for Streptococcus thermophilus when facing lytic phage infection, other forms of phage resistance coexist in this species. Here, we show that S. thermophilus strains with deleted CRISPR-Cas loci can still give rise to phage-resistant clones following lytic phage challenge. Notably, non-CRISPR phage-resistant survivors had multiple mutations which would truncate or recode a membrane-anchored host protease, FtsH. Phage adsorption was dramatically reduced in FtsH mutants, implicating this protein in phage attachment. Phages were isolated which could bypass FtsH-based resistance through mutations predicted to alter tape measure protein translation. Together, these results identify key components in phage propagation that are subject to mutation in the molecular arms race between phage and host cell. IMPORTANCE Streptococcus thermophilus is an important organism for production of cultured dairy foods, but it is susceptible to lytic phages which can lead to failed products. Consequently, mechanisms for phage resistance are an active area of research. One such mechanism is CRISPR-Cas, and S. thermophilus is a model organism for the study of this form of adaptive immunity. Here, we expand on known mechanisms with our finding that spontaneous mutations in ftsH, a gene encoding a membrane-anchored protease, protected against phage infection by disrupting phage adsorption. In turn, mutations in phage tail protein genes allowed phages to overcome ftsH-based resistance. Our results identified components in phage propagation that are subject to mutation in the molecular arms race between phage and host.

Person-first language in diabetes and obesity scientific publications.

AIMS: We aimed to quantify the use of person-first language (PFL) among scholarly articles focusing on diabetes or obesity. METHODS: PFL and condition-first language (CFL) terms for diabetes and obesity (e.g. diabetic, obese) were identified from existing guidelines and a review of the literature. Exact phrase literature searches were conducted between 2011 and 2020 and results were categorised as PFL, CFL or both. RESULTS: Among diabetes articles, 43% used PFL, 40% used CFL and 17% contained both. Among obesity articles, 0.5% used PFL, 99% used CFL and 0.2% used both. The use of PFL increased by 3% per year for diabetes articles, compared to 117% for obesity articles. The rate of adoption of PFL in diabetes articles was unchanged in 2018-2020 compared to the 3 years prior. CONCLUSIONS: While the use of person-first language in diabetes articles had increased over the review period, its rate of adoption has started to slow. Conversely, the use of PFL in obesity articles is nascent and increasing.

Accidental and Abusive Mandible Fractures in Infants and Toddlers.

OBJECTIVE: Mandible fractures are uncommon injuries in infants and young children and may raise concern for nonaccidental trauma. Our study describes several children with mandible fractures to identify features that might differentiate abuse from accident. METHODS: Records and imaging were reviewed for children aged 24 months and younger who were diagnosed with mandible fractures at 2 tertiary pediatric care centers. Twenty-one cases were included, 8 of whom had formal child abuse consultations. Cases were reviewed for mechanisms of injury, physical examination findings, and occult injuries identified, as well as the final abuse determination. RESULTS: Among children with child abuse consultations, 5 injuries (62.5%) were determined to be accidental, 1 (12.5%) was abusive, and 2 were indeterminate for abuse or accident (25%). In each accidentally injured child, the reported mechanism of injury was a short fall with evidence of facial impact. No accidentally injured child had unexpected occult injuries or noncraniofacial cutaneous injuries. CONCLUSIONS: Infants and young children can sometimes sustain mandible fractures accidentally after well-described short falls with evidence of facial impact. Abuse remains in the differential diagnosis, and children should be evaluated accordingly. We propose that accidental injury be considered when a well-evaluated child with an isolated mandible fracture has a history of a short fall.

Clinical utility of vinblastine therapeutic drug monitoring for the treatment of infantile myofibroma patients: A case series.

Infantile myofibroma is a rare, benign tumour of infancy typically managed surgically. In a minority of cases, more aggressive disease is seen and chemotherapy with vinblastine and methotrexate may be used, although evidence for this is limited. Chemotherapy dosing in infants is challenging, and vinblastine disposition in infants is unknown. We describe the use of vinblastine therapeutic drug monitoring in four cases of infantile myofibroma. Marked inter- and intrapatient variability was observed, highlighting the poorly understood pharmacokinetics of vinblastine in children, the challenges inherent in treating neonates, and the role of adaptive dosing in optimising drug exposure in challenging situations.

Diagnosis of SARS-CoV-2 Infection with LamPORE, a High-Throughput Platform Combining Loop-Mediated Isothermal Amplification and Nanopore Sequencing.

LamPORE is a novel diagnostic platform for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA combining loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyze thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against reverse transcriptase PCR (RT-PCR) using RNA extracted from spiked respiratory samples and stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 10 genome copies/µl of extracted RNA, which is above the limit achievable by RT-PCR, but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among them, LamPORE had a diagnostic sensitivity of 99.1% (226/228; 95% confidence interval [CI], 96.9% to 99.9%). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279; 98.0% to 100.0%). Overall, 1.4% (7/514; 0.5% to 2.9%) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494; 94.8% to 98.1%). LamPORE has a similar performance as RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients and offers a promising approach to high-throughput testing.

Parental neurotic excoriation injury of children: A case series of hidden physical abuse.

BACKGROUND: Recognizable skin findings of child physical abuse include bruises, abrasions, lacerations, bite marks, burns, and oral injuries. Self-induced skin picking, or neurotic excoriation, can correspond to emotional stress, underlying psychiatric illness, or substance abuse. Parental neurotic excoriation injury of children has not been reported previously as a form of physical abuse. METHODS: We present a case series of five children abused via parental excoriation. All affected children were three years of age or younger and otherwise healthy. Each child presented with wounds determined to be consistent with chronic picking. Patient age, injury location, and in some cases, witness accounts confirmed the lesions were not self-inflicted. RESULTS: In three cases, caregivers reported methamphetamine use. In these cases, caregivers repeatedly picked or wiped the infants' skin. In two cases, the caregiver demonstrated personal neurotic excoriation behavior, which was imposed upon her children resulting in similar lesions. One affected child died at 14 days of age from abusive head trauma, while the other four children were placed in foster care by Child Protective Services. CONCLUSION: Excoriation injury places children at risk for significant scarring and other long-term effects. We report examples demonstrating that repetitive skin injury by caregivers is a diagnostic consideration for abuse in young children.

Palm Bruising in Infants: A Recognizable Pattern of Abuse.

BACKGROUND: Bruising in an infant is an important sentinel injury that should raise concern for child physical abuse, and should prompt a medical evaluation for occult injury. Hyperflexion during forceful squeezing of an infant's hand results in a distinct pattern of bruising along the palmar and interdigital creases, as well as the palmar eminences. Self-inflicted injury by the infant or injury resulting from benign handling should not be accepted as plausible explanations for this injury. The presence of concurrent occult injuries is common, and further supports concerns for abuse. However, when this distinct pattern of palm bruising is identified in an infant, the absence of occult injuries should not prevent the recognition of child physical abuse. CASE REPORT: We report 11 cases depicting this distinct pattern of palm bruising in infants. Additionally, we include a perpetrator's documented confession. To the best of our knowledge, this pattern of bruising has not been previously described in the medical literature. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Emergency physicians play a crucial role in recognizing and addressing child physical abuse. Prompt recognition of this finding can aid in the identification of child physical abuse, even in the absence of underlying occult injury. This, in turn, can potentially prevent further injury, and even death, of an infant.

Understanding Displacements of the Gel Liner for Below Knee Prosthetic Users.

Many people with amputation utilize a prosthetic device to maintain function and ambulation. During the use of the prosthetic device, their residual limbs can develop wounds called pressure ulcers. The formation of these wounds has been linked to deformation and loading conditions of the skin and deeper tissues. Our research objective was to develop a complete profile of displacements on the gel liner at the interface with the socket during walking in transtibial amputees. Displacements for seven regions along the limb were quantified in addition to six calculations of displacement and three rotations relative to the prosthetic socket. The largest displacements were observed in the distal region of the gel liner, near the pin locking mechanism on the gel liner. Displacements were uneven throughout the liner with distal regions showing higher displacements. This mechanics-based information, combined with clinical information, will allow us to understand the local skin and muscle displacements, and will provide insights regarding localized tissue breakdown. Knowledge of how the liner displaces within the prosthetic socket can also help prosthetists modify designs to reduce these displacements, and reduce the potential for shear on the skin and in deeper tissues.

Effect of Intranasal Vasoconstrictors on Blood Pressure: A Randomized, Double-Blind, Placebo-Controlled Trial.

BACKGROUND: Treatment for epistaxis includes application of intranasal vasoconstrictors. These medications have a precaution against use in patients with hypertension. Given that many patients who present with epistaxis are hypertensive, these warnings are commonly overridden by clinical necessity. OBJECTIVE: Our aim was to determine the effects of intranasal vasoconstrictors on blood pressure. METHODS: We conducted a single-center, randomized, double-blind, placebo-controlled trial from November 2014 through July 2016. Adult patients being discharged from the emergency department (ED) at Mayo Clinic (Rochester, Minnesota) were recruited. Patients were ineligible if they had a contraindication to study medications, had a history of hypertension, were currently taking antihypertensive or antidysrhythmic medications, or had nasal abnormalities, such as epistaxis. Subjects were randomized to one of four study arms (phenylephrine 0.25%; oxymetazoline 0.05%; lidocaine 1% with epinephrine 1:100,000; or bacteriostatic 0.9% sodium chloride [saline]). Blood pressure and heart rate were measured every 5 min for 30 min. RESULTS: Sixty-eight patients were enrolled in the study; of these, 63 patients completed the study (oxymetazoline, n = 15; phenylephrine, n = 20; lidocaine with epinephrine, n = 11; saline, n = 17). We did not observe any significant differences in mean arterial pressure over time between phenylephrine and saline, oxymetazoline and saline, or lidocaine with epinephrine and saline. The mean greatest increases from baseline in mean arterial pressure, systolic and diastolic blood pressure, and heart rate for each treatment group were also not significantly different from the saline group. CONCLUSIONS: Intranasal vasoconstrictors did not significantly increase blood pressure in patients without a history of hypertension. Our findings reinforce the practice of administering these medications to patients who present to the ED with epistaxis, regardless of high blood pressure.

Parents' adverse childhood experiences and mental health screening using home visiting programs: A pilot study.

BACKGROUND: Adverse childhood experiences (ACEs) are associated with adult high-risk behaviors and diseases. There is value in screening parents for ACEs given the repercussions parental ACEs may have on parenting behaviors and child development. The primary aim of this study was to assess the feasibility of parental ACE screening in the home setting. A secondary aim was to evaluate whether or not maternal ACEs correlated with maternal mental health measures. METHODS: Two home visiting programs that support early childhood development and conduct parental mental health screening implemented ACE screening for parents of infants <1 year of age. Descriptive statistics were produced for population surveillance of ACEs as well as standard practice screens for depression, anxiety, substance use, and intimate partner violence. Logistic models were used to examine associations between ACE score and mental health measures. RESULTS: A total of 110 parents completed the ACE screen. All possible ACE score outcomes were represented (0-10). A trend toward association of positive prenatal maternal depression screen with ACE score was identified (p = .05). CONCLUSION: This novel prospective home-based screening program for parental ACEs was feasible and identified a trend toward increasing ACE score association with positive prenatal maternal depression screen.

In good company. The effect of an eating companion's appearance on food intake.

The purpose of this study was to examine whether or not the presence of an overweight eating companion influences healthy and unhealthy eating behavior, and to determine if the effect is moderated by how the companion serves herself. A professional actress either wore an overweight prosthesis (i.e., "fatsuit") or did not wear one, and served herself either healthily (i.e., a small amount of pasta and a large amount of salad) or unhealthily (i.e., a large amount of pasta and a small amount of salad) for lunch. After observing her, male and female participants were asked to serve themselves pasta and salad to eat. Results demonstrated that regardless of how the confederate served, participants served and ate a larger amount of pasta when she was wearing the prosthesis than when she was not. In addition, when the confederate served herself healthily, participants served and ate a smaller amount of salad when she was wearing the prosthesis than when she was not. Consistent with the "lower health commitment" hypothesis, these results demonstrated that people may eat larger portions of unhealthy food and smaller portions of healthy food when eating with an overweight person, probably because the health commitment goal is less activated. More generally, this study provides evidence that the body type of an eating companion, as well as whether she serves herself healthily or unhealthily, influences the quantity of food intake.

Patient and parent perspectives on paediatric cancer multidisciplinary team working and national advisory panels in the UK: a qualitative research study.

OBJECTIVE: The concept of patient-centred care is central to the role of cancer multidisciplinary teams (MDTs) and particularly pertinent with the recent rise in number of virtual national advisory panels (NAPs) for childhood cancer in the UK. We sought to explore patient and caregiver views regarding MDT working and NAPs. METHODS: Three focus groups were undertaken between March 2019 and January 2020. RESULTS: Sixteen participants attended. All regarded MDTs and NAPs highly, while highlighting patient involvement in decision-making should not be diluted by this process. The importance of personalised consultations was stressed, acknowledging that information-sharing preferences may change with circumstance and time. Most participants felt they had not been actively involved in decisions, including those made following MDT or NAP discussions. Group suggestions to improve patient-centred care included a clinician knowing them presenting their case, referral proformas to include family-related factors and an advocate attending meetings to represent the patient/family view. CONCLUSION: Several changes have been driven forward by this work, including the modification of NAP referral proformas to include additional information. Patient and parent perspectives are now embedded into a best practice model for the NAPs to promote personalised recommendations at national level.

Reduced insulin/IGF-1 signalling upregulates two anti-viral immune pathways, decreases viral load and increases survival under viral infection in C. elegans.

Reduced insulin/IGF-1 signalling (rIIS) improves survival across diverse taxa and there is a growing interest in its role in regulating immune function. Whilst rIIS can improve anti-bacterial resistance, the consequences for anti-viral immunity are yet to be systematically examined. Here, we show that rIIS in adult Caenorhabditis elegans increases the expression of key genes in two different anti-viral immunity pathways, whilst reducing viral load in old age, increasing survival and reducing rate-of-senescence under infection by naturally occurring positive-sense single-stranded RNA Orsay virus. We found that both drh-1 in the anti-viral RNA interference (RNAi) pathway and cde-1 in the terminal uridylation-based degradation of viral RNA pathway were upregulated in early adulthood under rIIS and increased anti-viral resistance was not associated with reproductive costs. Remarkably, rIIS increased anti-viral gene expression only in infected worms, potentially to curb the costs of constitutively upregulated immunity. RNA viruses are found across taxa from plants to mammals and we demonstrate a novel role for rIIS in regulating resistance to viral infection. We therefore highlight this evolutionarily conserved signalling pathway as a promising therapeutic target to improve anti-viral immunity.

Validity evidence for a novel multiple-choice question bank in the field of child abuse pediatrics.

BACKGROUND: Though child abuse pediatrics has been a board-certified subspecialty for 15 years, there are few formalized board preparation resources available. OBJECTIVE: The purpose of this project was to establish a multiple-choice question bank with sufficient validity evidence for use in preparation for the child abuse pediatrics board examination. PARTICIPANTS AND SETTING: The question bank was distributed via an electronic child abuse pediatrics mailing list. Participants completing the entire question bank included 27 board-certified child abuse pediatricians (CAPs), 19 board-eligible CAPs, and 18 CAP fellows. METHODS: We used Messick's framework to conduct the validity investigation, which includes five components: content evidence, response process, internal structure, relation to other variables, and consequences. Item analyses included difficulty index, discrimination index, and distractor analysis. We used Cronbach's alpha to estimate internal consistency reliability. We conducted linear regressions of scores on the question bank compared to in-training exam scores and career stage. RESULTS: Eighty-four participants completed part of the question bank, and 64 completed the entire question bank. Of the original 117 questions ("items"), 94 met inclusion criteria. The mean score among board-certified CAPs was 80 %, and among participants reporting passing third-year ITE scores was 81 %. Correlation coefficient of scores on this question bank by career stage was r = 0.94, and by year of fellowship was r = 0.99. Cronbach's alpha for internal consistency reliability was 0.83. CONCLUSIONS: This multiple-choice question bank is the first question bank with a robust validity investigation for use by child abuse pediatrics trainees.

Desert diversity: genome sequence of Gordonia rubripertincta cluster DJ phage Mossy and cluster DV phage Erutan.

Lytic bacteriophages Mossy and Erutan were directly isolated from desert soil on Gordonia rubripertincta and characterized by their morphologies and genomes. Mossy, part of the DJ cluster of Actinobacteriophage, has a genome of 61,183 bp. The genome of Erutan, part of the DV cluster, is 66,957 bp.

Use of micro-emulsion technology for the directed evolution of antibodies.

Affinity reagents, such as antibodies, are needed to study protein expression patterns, sub-cellular localization, and post-translational modifications in complex mixtures and tissues. Phage Emulsion, Secretion, and Capture (ESCape) is a novel micro-emulsion technology that utilizes water-in-oil (W/O) emulsions for the identification and isolation of cells secreting phage particles that display desirable antibodies. Using this method, a large library of antibody-displaying phage will bind to beads in individual compartments. Rather than using biopanning on a large mixed population, phage micro-emulsion technology allows us to individually query clonal populations of amplified phage against the antigen. The use of emulsions to generate microdroplets has the promise of accelerating phage selection experiments by permitting fine discrimination of kinetic parameters for binding to targets. In this study, we demonstrate the ability of phage micro-emulsion technology to distinguish two scFvs with a 300-fold difference in binding affinities (100nM and 300pM, respectively). In addition, we describe the application of phage micro-emulsion technology for the selection of scFvs that are resistant to elevated temperatures.

Communicating about the social determinants of health: development of a local brand.

BACKGROUND: Successful comprehensive population-based approaches to chronic disease prevention leverage mass media to amplify messages and support a culture of health. We report on a community-engaged formative evaluation to segment audiences and identify major themes to guide campaign message development for a transformative health communication campaign. METHODS: Four key phases of campaign development: (I) Formative evaluation to identify priorities, guiding themes, and audience segments (interviews/focus groups with residents, N = 85; representatives of community-based partner organizations, N = 10); (II) Brand development (focus groups and closed-ended surveys; N = 56); (III) Message testing approaches to verbal and visual appeals (N = 50 resident intercept interviews); (IV) Workshop (N = 26 participants representing 15 organizations). RESULTS: Residents were engaged throughout campaign development and the resulting campaign materials, including the campaign name and visual aesthetic (logo, color schemes, overall look and feel) reflect the diversity of the community and were accepted and valued by diverse groups in the community. Campaign materials featuring photos of county residents were created in English, Spanish, and Hmong. Plain language messages on social determinants of health resonate with residents. The county was described as a sort of idyllic environment burdened by inequality and structural challenges. Residents demonstrated enthusiasm for the campaign and provided specific suggestions for content (education about disease risks, prevention, management; information about accessing resources; testimonials from similar people) and tone. CONCLUSIONS: Communication to support a policy, systems, and environmental change approach to chronic disease prevention must carefully match messages with appropriate audiences. We discuss challenges in such messaging and effectiveness across multiple, diverse audiences.