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.

Transforming Growth Factor Beta and Alveolar Rhabdomyosarcoma: A Challenge of Tumor Differentiation and Chemotherapy Response.

Alveolar rhabdomyosarcoma (ARMS), an invasive subtype of rhabdomyosarcoma (RMS), is associated with chromosomal translocation events resulting in one of two oncogenic fusion genes, PAX3-FOXO1 or PAX7-FOXO1. ARMS patients exhibit an overexpression of the pleiotropic cytokine transforming growth factor beta (TGF-ß). This overexpression of TGF-ß1 causes an increased expression of a downstream transcription factor called SNAIL, which promotes epithelial to mesenchymal transition (EMT). Overexpression of TGF-ß also inhibits myogenic differentiation, making ARMS patients highly resistant to chemotherapy. In this review, we first describe different types of RMS and then focus on ARMS and the impact of TGF-ß in this tumor type. We next highlight current chemotherapy strategies, including a combination of the FDA-approved drugs vincristine, actinomycin D, and cyclophosphamide (VAC); cabozantinib; bortezomib; vinorelbine; AZD 1775; and cisplatin. Lastly, we discuss chemotherapy agents that target the differentiation of tumor cells in ARMS, which include all-trans retinoic acid (ATRA) and 5-Azacytidine. Improving our understanding of the role of signaling pathways, such as TGF-ß1, in the development of ARMS tumor cells differentiation will help inform more tailored drug administration in the future.

Estimation of Seasonal Influenza Attack Rates and Antibody Dynamics in Children Using Cross-Sectional Serological Data.

Directly measuring evidence of influenza infections is difficult, especially in low-surveillance settings such as sub-Saharan Africa. Using a Bayesian model, we estimated unobserved infection times and underlying antibody responses to influenza A/H3N2, using cross-sectional serum antibody responses to 4 strains in children aged 24-60 months. Among the 242 individuals, we estimated a variable seasonal attack rate and found that most children had ≥1 infection before 2 years of age. Our results are consistent with previously published high attack rates in children. The modeling approach highlights how cross-sectional serological data can be used to estimate epidemiological dynamics.

Level of Experience Does Not Influence the Accuracy of Radiographic and Ultrasound Measurements of Magnetically Controlled Growing Rod Distractions.

BACKGROUND: Magnetically controlled growing rods (MCGR) have become a popular surgical option for the treatment of early-onset scoliosis. Both radiographs and ultrasound are currently used to measure the amount of length achieved when MCGRs are distracted. Previous studies have investigated the intraobserver and interobserver reliability of radiographic and ultrasound measurements of MCGR distraction. Some authors have reported that there is a "learning curve" in measuring MCGR lengthening with ultrasound, suggesting that new users require several months of experience before they can accurately perform the measurements. The goal of this study was to determine whether surgical experience of the rater is associated with the accuracy of radiographic and ultrasound measurements of MCGR distraction. METHODS: Six raters evaluated 29 deidentified radiographs and 30 ultrasound images from early-onset scoliosis patients with MCGR. Raters had varying levels of experience, ranging from a senior fellowship-trained pediatric orthopaedic surgeon to a junior orthopaedic surgery resident. Raters measured the amount of rod distraction in 2 sessions spaced 2 weeks apart. All raters were provided with a document demonstrating the radiographic and ultrasound measurement techniques before the first round of measurements. Intraclass correlation coefficients were calculated. RESULTS: Excellent intraobserver and interobserver agreement was achieved for both radiographic and ultrasound measurements of MCGR distraction. Subanalysis based on experience level showed that excellent intraobserver agreement was maintained with no evidence of decreased reliability in raters with less experience. CONCLUSIONS: Excellent intraobserver and interobserver agreement was obtained with radiographic and ultrasound measurements of MCGR distraction, regardless of the experience level of the rater. Posting a document with the radiographic and ultrasound measurement techniques in the orthopaedic surgery clinic, and perhaps also the radiology reading room may help avoid inaccurate measurements of distraction length secondary to a learning curve. LEVEL OF EVIDENCE: Level III-diagnostic.

Sensory-specific modulation of adult neurogenesis in sensory structures is associated with the type of stem cell present in the neurogenic niche of the zebrafish brain.

Teleost fishes retain populations of adult stem/progenitor cells within multiple primary sensory processing structures of the mature brain. Though it has commonly been thought that their ability to give rise to adult-born neurons is mainly associated with continuous growth throughout life, whether a relationship exists between the processing function of these structures and the addition of new neurons remains unexplored. We investigated the ultrastructural organisation and modality-specific neurogenic plasticity of niches located in chemosensory (olfactory bulb, vagal lobe) and visual processing (periventricular grey zone, torus longitudinalis) structures of the adult zebrafish (Danio rerio) brain. Transmission electron microscopy showed that the cytoarchitecture of sensory niches includes many of the same cellular morphologies described in forebrain niches. We demonstrate that cells with a radial-glial phenotype are present in chemosensory niches, while the niche of the caudal tectum contains putative neuroepithelial-like cells instead. This was supported by immunohistochemical evidence showing an absence of glial markers, including glial fibrillary acidic protein, glutamine synthetase, and S100ß in the tectum. By exposing animals to sensory assays we further illustrate that stem/progenitor cells and their neuronal progeny within sensory structures respond to modality-specific stimulation at distinct stages in the process of adult neurogenesis - chemosensory niches at the level of neuronal survival and visual niches in the size of the stem/progenitor population. Our data suggest that the adult brain has the capacity for sensory-specific modulation of adult neurogenesis and that this property may be associated with the type of stem cell present in the niche.

High SARS-CoV-2 incidence and asymptomatic fraction during Delta and Omicron BA.1 waves in The Gambia.

Little is known about SARS-CoV-2 infection risk in African countries with high levels of infection-driven immunity and low vaccine coverage. We conducted a prospective cohort study of 349 participants from 52 households in The Gambia between March 2021 and June 2022, with routine weekly SARS-CoV-2 RT-PCR and 6-monthly SARS-CoV-2 serology. Attack rates of 45% and 57% were seen during Delta and Omicron BA.1 waves respectively. Eighty-four percent of RT-PCR-positive infections were asymptomatic. Children under 5-years had a lower incidence of infection than 18-49-year-olds. One prior SARS-CoV-2 infection reduced infection risk during the Delta wave only, with immunity from ≥2 prior infections required to reduce the risk of infection with early Omicron lineage viruses. In an African population with high levels of infection-driven immunity and low vaccine coverage, we find high attack rates during SARS-CoV-2 waves, with a high proportion of asymptomatic infections and young children remaining relatively protected from infection.

Maternal immunization as a strategy to decrease susceptibility to infection in newborn infants.

PURPOSE OF REVIEW: Following on from the success of maternal tetanus vaccination, recent research has shown that other vaccines given in pregnancy can protect against vaccine-preventable infections in early infancy. This review will outline these recent developments and highlight the impact on current clinical practice. RECENT FINDINGS: Maternal immunization provides protection to the newborn through the transfer of vaccine-induced IgG across the placenta, a process that is affected by multiple variables. The safety of newly recommended maternal vaccines has been further tested in recent studies. Maternal vaccination against influenza and pertussis is recommended in the United Kingdom and United States, with new studies indicating their efficacy. A number of additional maternal vaccines are also in the pipeline, which could be used to combat neonatal infection. Recent research findings have highlighted some of the reasons for the poor uptake of current recommendations among pregnant women. SUMMARY: Tetanus, influenza and pertussis vaccines are now recommended for use during pregnancy, with new vaccines, such as group B streptococcus and respiratory syncytial virus, being developed to prevent important neonatal infections in the future.

C1q is essential for myelination in the central nervous system (CNS).

Myelin sheath in the central nervous system (CNS) is essential for efficient action potential conduction. Microglia, the macrophages in the CNS, are suggested to regulate myelin development. However, the specific involvement of microglia in initial myelination is yet to be elucidated. Here, first, by culturing neural stem cells, we demonstrated that myelin sheath formation only occurred in the presence of a microglia-conditioned medium. Furthermore, the absence of C1q, a microglia-derived factor, resulted in myelination failure in the neural stem cell culture. Additionally, adding native human C1q protein was sufficient to induce myelination in vitro. Finally, in the C1q conditional knockout mouse model (C1qaFL/FL: Cx3cr1CreER), C1q deficiency prior to the onset of myelination led to reduced myelin thickness and elevated g-ratio during initial myelination. This study uncovers the pivotal role of microglia-derived C1q in developmental myelination and could potentially pave the way for new therapeutic strategies for treating demyelinating diseases.

Rhabdomyosarcoma: Current Therapy, Challenges, and Future Approaches to Treatment Strategies.

Rhabdomyosarcoma is a rare cancer arising in skeletal muscle that typically impacts children and young adults. It is a worldwide challenge in child health as treatment outcomes for metastatic and recurrent disease still pose a major concern for both basic and clinical scientists. The treatment strategies for rhabdomyosarcoma include multi-agent chemotherapies after surgical resection with or without ionization radiotherapy. In this comprehensive review, we first provide a detailed clinical understanding of rhabdomyosarcoma including its classification and subtypes, diagnosis, and treatment strategies. Later, we focus on chemotherapy strategies for this childhood sarcoma and discuss the impact of three mechanisms that are involved in the chemotherapy response including apoptosis, macro-autophagy, and the unfolded protein response. Finally, we discuss in vivo mouse and zebrafish models and in vitro three-dimensional bioengineering models of rhabdomyosarcoma to screen future therapeutic approaches and promote muscle regeneration.

Uncovering the spectrum of adult zebrafish neural stem cell cycle regulators.

Adult neural stem and progenitor cells (aNSPCs) persist lifelong in teleost models in diverse stem cell niches of the brain and spinal cord. Fish maintain developmental stem cell populations throughout life, including both neuro-epithelial cells (NECs) and radial-glial cells (RGCs). Within stem cell domains of the brain, RGCs persist in a cycling or quiescent state, whereas NECs continuously divide. Heterogeneous populations of RGCs also sit adjacent the central canal of the spinal cord, showing infrequent proliferative activity under homeostasis. With the rise of the zebrafish (Danio rerio) model to study adult neurogenesis and neuroregeneration in the central nervous system (CNS), it has become evident that aNSPC proliferation is regulated by a wealth of stimuli that may be coupled with biological function. Growing evidence suggests that aNSPCs are sensitive to environmental cues, social interactions, nutrient availability, and neurotrauma for example, and that distinct stem and progenitor cell populations alter their cell cycle activity accordingly. Such stimuli appear to act as triggers to either turn on normally dormant aNSPCs or modulate constitutive rates of niche-specific cell cycle behaviour. Defining the various forms of stimuli that influence RGC and NEC proliferation, and identifying the molecular regulators responsible, will strengthen our understanding of the connection between aNSPC activity and their biological significance. In this review, we aim to bring together the current state of knowledge on aNSPCs from studies investigating the zebrafish CNS, while highlighting emerging cell cycle regulators and outstanding questions that will help to advance this fascinating field of stem cell biology.

A Direct Posterior Approach for Subtalar Distraction Arthrodesis.

SUMMARY: Because of soft tissue constraints, definitive management of hindfoot trauma occasionally requires a direct posterior approach via a coronal split of the Achilles tendon. This article and the accompanying video demonstrate a direct posterior approach for subtalar distraction arthrodesis and review treatment decisions involved with management of these complex cases.

Medial Ankle Instability: Review of Anatomy, Evaluation, and Treatment.

The medial ankle ligamentous complex, which includes the deltoid, talocalcaneal, and calcaneonavicular ligaments, functions to provide stability to the medial ankle. Injuries to the deltoid ligament can lead to medial-sided ankle pain, subsequent instability, and posttraumatic osteoarthritis given the altered biomechanics of the ankle joint. After completing a thorough physical examination, imaging modalities such as stress radiographs and magnetic resonance imaging can be used to confirm the diagnosis. Acute injuries to the deltoid ligament should be managed conservatively with a short course of immobilization. For patients with continued pain and instability following a regimen of nonoperative management, surgical intervention can be considered. Primary repair using suture anchor fixation to the medial malleolus can be utilized if sufficient tissue remains. However, if reconstruction is necessitated, autograft or allograft can be utilized in several described techniques.Levels of Evidence: Therapeutic.

Erratum: Prior upregulation of interferon pathways in the nasopharynx impacts viral shedding following live attenuated influenza vaccine challenge in children.

[This corrects the article DOI: 10.1016/j.xcrm.2021.100465.].

Characterising within-hospitalSARS-CoV-2 transmission events using epidemiological and viral genomic data across two pandemic waves.

Hospital outbreaks of COVID19 result in considerable mortality and disruption to healthcare services and yet little is known about transmission within this setting. We characterise within hospital transmission by combining viral genomic and epidemiological data using Bayesian modelling amongst 2181 patients and healthcare workers from a large UK NHS Trust. Transmission events were compared between Wave 1 (1st March to 25th J'uly 2020) and Wave 2 (30th November 2020 to 24th January 2021). We show that staff-to-staff transmissions reduced from 31.6% to 12.9% of all infections. Patient-to-patient transmissions increased from 27.1% to 52.1%. 40%-50% of hospital-onset patient cases resulted in onward transmission compared to 4% of community-acquired cases. Control measures introduced during the pandemic likely reduced transmissions between healthcare workers but were insufficient to prevent increasing numbers of patient-to-patient transmissions. As hospital-acquired cases drive most onward transmission, earlier identification of nosocomial cases will be required to break hospital transmission chains.

Altered subgenomic RNA abundance provides unique insight into SARS-CoV-2 B.1.1.7/Alpha variant infections.

B.1.1.7 lineage SARS-CoV-2 is more transmissible, leads to greater clinical severity, and results in modest reductions in antibody neutralization. Subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome. Applying our tool (periscope) to ARTIC Network Oxford Nanopore Technologies genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA is significantly increased in B.1.1.7 (alpha) infections (n = 879). This increase is seen over the previous dominant lineage in the UK, B.1.177 (n = 943), which is independent of genomic reads, E cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median sgRNA abundance. We demonstrate that ORF9b protein levels are increased 6-fold in B.1.1.7 compared to a B lineage virus in vitro. We hypothesise that increased ORF9b in B.1.1.7 is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT > CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3' of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles to evaluate emerging potential variants of concern.

TEM, SEM, and STEM-based immuno-CLEM workflows offer complementary advantages.

Identifying endogenous tissue stem cells remains a key challenge in developmental and regenerative biology. To distinguish and molecularly characterise stem cell populations in large heterogeneous tissues, the combination of cytochemical cell markers with ultrastructural morphology is highly beneficial. Here, we realise this through workflows of multi-resolution immuno-correlative light and electron microscopy (iCLEM) methodologies. Taking advantage of the antigenicity preservation of the Tokuyasu technique, we have established robust protocols and workflows and provide a side-by-side comparison of iCLEM used in combination with scanning EM (SEM), scanning TEM (STEM), or transmission EM (TEM). Evaluation of the applications and advantages of each method highlights their practicality for the identification, quantification, and characterization of heterogeneous cell populations in small organisms, organs, or tissues in healthy and diseased states. The iCLEM techniques are broadly applicable and can use either genetically encoded or cytochemical markers on plant, animal and human tissues. We demonstrate how these protocols are particularly suited for investigating neural stem and progenitor cell populations of the vertebrate nervous system.

Percutaneous Fixation of a Fifth Metatarsal Base Fracture.

SUMMARY: Owing to a vascular watershed, zone II and III fifth metatarsal base fractures commonly progress to nonunion without operative intervention. This article and the accompanying video demonstrate the use of intramedullary screw fixation for a fifth metatarsal base fracture and review treatment decisions involved with management of these injuries.

Genomic and Epidemiological Analysis of SARS-CoV-2 Viruses in Sri Lanka.

Background: In order to understand the molecular epidemiology of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Sri Lanka, since March 2020, we carried out genomic sequencing overlaid on available epidemiological data until April 2021. Methods: Whole genome sequencing was carried out on diagnostic sputum or nasopharyngeal swabs from 373 patients with COVID-19. Molecular clock phylogenetic analysis was undertaken to further explore dominant lineages. Results: The B.1.411 lineage was most prevalent, which was established in Sri Lanka and caused outbreaks throughout the country until March 2021. The estimated time of the most recent common ancestor (tMRCA) of this lineage was June 1, 2020 (with 95% lower and upper bounds March 30 to July 27) suggesting cryptic transmission may have occurred, prior to a large epidemic starting in October 2020. Returning travellers were identified with infections caused by lineage B.1.258, as well as the more transmissible B.1.1.7 lineage, which has replaced B.1.411 to fuel the ongoing large outbreak in the country. Conclusions: The large outbreak that started in early October, is due to spread of a single virus lineage, B.1.411 until the end of March 2021, when B.1.1.7 emerged and became the dominant lineage.

Rapid feedback on hospital onset SARS-CoV-2 infections combining epidemiological and sequencing data.

Background: Rapid identification and investigation of healthcare-associated infections (HCAIs) is important for suppression of SARS-CoV-2, but the infection source for hospital onset COVID-19 infections (HOCIs) cannot always be readily identified based only on epidemiological data. Viral sequencing data provides additional information regarding potential transmission clusters, but the low mutation rate of SARS-CoV-2 can make interpretation using standard phylogenetic methods difficult. Methods: We developed a novel statistical method and sequence reporting tool (SRT) that combines epidemiological and sequence data in order to provide a rapid assessment of the probability of HCAI among HOCI cases (defined as first positive test >48 hr following admission) and to identify infections that could plausibly constitute outbreak events. The method is designed for prospective use, but was validated using retrospective datasets from hospitals in Glasgow and Sheffield collected February-May 2020. Results: We analysed data from 326 HOCIs. Among HOCIs with time from admission ≥8 days, the SRT algorithm identified close sequence matches from the same ward for 160/244 (65.6%) and in the remainder 68/84 (81.0%) had at least one similar sequence elsewhere in the hospital, resulting in high estimated probabilities of within-ward and within-hospital transmission. For HOCIs with time from admission 3-7 days, the SRT probability of healthcare acquisition was >0.5 in 33/82 (40.2%). Conclusions: The methodology developed can provide rapid feedback on HOCIs that could be useful for infection prevention and control teams, and warrants further prospective evaluation. The integration of epidemiological and sequence data is important given the low mutation rate of SARS-CoV-2 and its variable incubation period. Funding: COG-UK HOCI funded by COG-UK consortium, supported by funding from UK Research and Innovation, National Institute of Health Research and Wellcome Sanger Institute.