Local and systemic responses to SARS-CoV-2 infection in children and adults.

It is not fully understood why COVID-19 is typically milder in children1-3. Here, to examine the differences between children and adults in their response to SARS-CoV-2 infection, we analysed paediatric and adult patients with COVID-19 as well as healthy control individuals (total n = 93) using single-cell multi-omic profiling of matched nasal, tracheal, bronchial and blood samples. In the airways of healthy paediatric individuals, we observed cells that were already in an interferon-activated state, which after SARS-CoV-2 infection was further induced especially in airway immune cells. We postulate that higher paediatric innate interferon responses restrict viral replication and disease progression. The systemic response in children was characterized by increases in naive lymphocytes and a depletion of natural killer cells, whereas, in adults, cytotoxic T cells and interferon-stimulated subpopulations were significantly increased. We provide evidence that dendritic cells initiate interferon signalling in early infection, and identify epithelial cell states associated with COVID-19 and age. Our matching nasal and blood data show a strong interferon response in the airways with the induction of systemic interferon-stimulated populations, which were substantially reduced in paediatric patients. Together, we provide several mechanisms that explain the milder clinical syndrome observed in children.

APJ+ cells in the SHF contribute to the cells of aorta and pulmonary trunk through APJ signaling.

Outflow tract (OFT) develops from cardiac progenitor cells in the second heart field (SHF) domain. APJ, a G-Protein Coupled Receptor, is expressed by cardiac progenitors in the SHF. By lineage tracing APJ+SHF cells, we show that these cardiac progenitors contribute to the cells of OFT, which eventually give rise to aorta and pulmonary trunk/artery upon its morphogenesis. Furthermore, we show that early APJ â€‹+ â€‹cells give rise to both aorta and pulmonary cells but late APJ â€‹+ â€‹cells predominantly give rise to pulmonary cells. APJ is expressed by the outflow tract progenitors in the SHF but its role is unclear. We performed knockout studies to determine the role of APJ in SHF cell proliferation and survival. Our data suggested that APJ knockout in the SHF reduced the proliferation of SHF progenitors, while there was no significant impact on survival. In addition, we show that ectopic overexpression of WNT in these cells disrupted aorta and pulmonary morphogenesis from OFT. Overall, our study has identified APJ â€‹+ â€‹progenitor population within the SHF that give rise to aorta and pulmonary trunk/artery cells. Furthermore, we show that APJ signaling stimulates proliferation of these cells in the SHF.

Anatomy of an extensively drug-resistant Klebsiella pneumoniae outbreak in Tuscany, Italy.

A protracted outbreak of New Delhi metallo-ß-lactamase (NDM)-producing carbapenem-resistant Klebsiella pneumoniae started in Tuscany, Italy, in November 2018 and continued in 2020 and through 2021. To understand the regional emergence and transmission dynamics over time, we collected and sequenced the genomes of 117 extensively drug-resistant, NDM-producing K. pneumoniae isolates cultured over a 20-mo period from 76 patients at several healthcare facilities in southeast Tuscany. All isolates belonged to high-risk clone ST-147 and were typically nonsusceptible to all first-line antibiotics. Albeit sporadic, resistances to colistin, tigecycline, and fosfomycin were also observed as a result of repeated, independent mutations. Genomic analysis revealed that ST-147 isolates circulating in Tuscany were monophyletic and highly genetically related (including a network of 42 patients from the same hospital and sharing nearly identical isolates), and shared a recent ancestor with clinical isolates from the Middle East. While the blaNDM-1 gene was carried by an IncFIB-type plasmid, our investigations revealed that the ST-147 lineage from Italy also acquired a hybrid IncFIB/IncHIB-type plasmid carrying the 16S methyltransferase armA gene as well as key virulence biomarkers often found in hypervirulent isolates. This plasmid shared extensive homologies with mosaic plasmids circulating globally including from ST-11 and ST-307 convergent lineages. Phenotypically, the carriage of this hybrid plasmid resulted in increased siderophore production but did not confer virulence to the level of an archetypical, hypervirulent K. pneumoniae in a subcutaneous model of infection with immunocompetent CD1 mice. Our findings highlight the importance of performing genomic surveillance to identify emerging threats.

Six Extensively Drug-Resistant Bacteria in an Injured Soldier, Ukraine.

Blood and surveillance cultures from an injured service member from Ukraine grew Acinetobacter baumannii, Klebsiella pneumoniae, Enterococcus faecium, and 3 distinct Pseudomonas aeruginosa strains. Isolates were nonsusceptible to most antibiotics and carried an array of antibiotic resistant genes, including carbapenemases (blaIMP-1, blaNDM-1, blaOXA-23, blaOXA-48, blaOXA-72) and 16S methyltransferases (armA and rmtB4).

Generation of human gastric assembloids from primary fetal organoids.

PURPOSE: Understanding human gastric epithelium homeostasis remains partial, motivating the exploration of innovative in vitro models. Recent literature showcases the potential of fetal stem cell-derived organoids in developmental and disease modelling and translational therapies. To scale the complexity of the model, we propose to generate assembloids, aiming to increase gastric maturation to provide new structural and functional insights. METHODS: Human fetal gastric organoids (fGOs) were expanded in 3D Matrigel cultures. Confluent organoid cultures were released from the Matrigel dome and resuspended in a collagen I hydrogel. Subsequently, the organoid mixture was seeded in a ring shape within a 24-well plate and allowed to gelate. The structure was lifted in the medium and cultured in floating conditions, allowing for organoid self-assembling into a gastric assembloid. After 10 days of maturation, the assembloids were characterized by immunostaining and RT-PCR, comparing different fetal developmental stages. RESULTS: Successful generation of human fetal gastric assembloids (fGAs) was achieved using spontaneous self-aggregation within the collagen I hydrogel. Immunostaining analysis of early and late fGAs showed the establishment of apico-basal cell polarity, secretion of gastric mucins, and the presence of chromogranin A in both samples. Transcriptional markers analysis revealed distinct disparities in markers associated with mature cell types between late and early fetal stages. CONCLUSIONS: fGOs can reliably be generated from human fetal samples. This pioneering assembloid approach paves the way for advancing our comprehension of human gastric epithelium homeostasis and its perturbation, offering a better in vitro platform for the study of gastric epithelial development and therapeutic translation.

Patient-level costing analysis of paediatric short bowel syndrome care in a specialist tertiary centre.

PURPOSE: To undertake a pilot study estimating patient-level costs of care for paediatric short bowel syndrome (SBS) from the healthcare provider perspective. METHODS: A pilot group of patients with anatomical SBS was selected at a single specialist tertiary centre in the United Kingdom. The Patient Level Information and Costing System (PLICS) was used to extract costing data for all hospital-based activities related to SBS, from the implementation of PLICS in 2016 to April 2021. Patient-specific and pooled data were reported descriptively in per patient-year terms. RESULTS: Five patients had full PLICS data available for the 5-year study period and 2 patients had 4 years of data. The median cost for hospital care of SBS was £52,834 per patient-year (range £1804-£331,489). The key cost drivers were inpatient beds, pharmacy, and staffing costs, which made up > 60% of annual costs. In the first 3 years following index admission (n = 2), there was a steady decline in the annual cost of care to a level comparable with patients with established SBS. CONCLUSION: Patient-level cost of care analysis for SBS is feasible using PLICS. Hospital-related costs vary widely between and within individual patients over time. Key drivers of cost are related to complications of SBS.

Paediatric gastric organoids as a tool for disease modelling and clinical translation.

PURPOSE: Knowledge of gastric epithelial homeostasis remains incomplete, lacking human-specific models for study. This study establishes a protocol for deriving gastric epithelial organoids from paediatric gastric biopsies, providing a platform for modelling disease and developing translational therapies. METHODS: Full-thickness surgical samples and endoscopic mucosal biopsies were obtained from six patients. Gastric glands were isolated by a chemical chelation protocol and then plated in 3D culture in Matrigel® droplets in chemically defined medium. After formation, organoids were passaged by single cell dissociation or manual disaggregation. Cell composition and epithelial polarity of organoids were assessed by bright field microscopy and immunofluorescence analysis, comparing them to native paediatric gastric tissue. RESULTS: Gastric glands were successfully isolated from all six patients who were aged 4 months to 16 years. Gastric glands from all patients sealed to form spherical gastric organoids. These organoids could be passaged by manual disaggregation or single cell dissociation, remaining proliferative up to 1 year in culture. Organoids retained normal epithelial cell polarity, with the apical surface orientated towards the central lumen. Organoids expressed markers of mature gastric epithelial cell types, except for parietal cells. CONCLUSION: Gastric organoids can be reliably generated from paediatric biopsies and are a representative in vitro model for studying gastric epithelium.

Renal tumours in Australian children: 30 years of incidence, outcome and second primary malignancy data from the Australian Childhood Cancer Registry.

AIM: This paper describes the incidence and outcomes of childhood renal malignancies in Australia using national population-based data from the Australian Childhood Cancer Registry. METHODS: De-identified data for children (0-14 years) diagnosed with renal malignancies from 1983 to 2015 inclusive were extracted. Cause-specific (CSS) and event-free survival up to 20 years from diagnosis were estimated using the cohort method. Adjusted excess mortality hazard ratios were calculated using a multivariable flexible parametric survival model. Details relating to second primary malignancies (SPMs) were also examined. RESULTS: There were 1046 children diagnosed with renal malignancies in Australia between 1983 and 2015 (91% nephroblastoma), generating an annual age-standardised incidence rate of 8 per million children, which remained constant over the study period. CSS was 89% (95% confidence interval = 87-91%) and 88% (86-90%) at 5 and 20 years, respectively, and 5-year event-free survival was 82% (80-84%). Five-year CSS did not change over the study period and was highest for nephroblastoma (91%). Of the 94% of patients achieving remission, 15% relapsed and subsequent 5-year CSS was 49% (40%-58%). Eleven children were diagnosed with SPM (standardised incidence ratio = 2.9, 95% confidence interval = 1.6-5.3, P < 0.001), and five of them (45%) died within 5 years of the second diagnosis. CONCLUSIONS: Children treated for renal malignancies in Australia have excellent long-term survival, which is unchanged since 1983. SPMs are uncommon following treatment for childhood renal cancer but carry a poor prognosis. Relapse carries a similarly poor prognosis to SPM but is more common. These data are comparable to registry outcomes in similarly developed nations.

Incidence and outcomes of neuroblastoma in Australian children: A population-based study (1983-2015).

AIM: Neuroblastoma predominantly affects younger children and exhibits heterogeneous behaviour. This study describes incidence and outcomes for neuroblastoma using national population-based data from the Australian Childhood Cancer Registry. METHODS: Deidentified data for all children (0-14 years) diagnosed with neuroblastoma and ganglioneuroblastoma from 1983 to 2015 were extracted. Cause-specific (CSS) and event-free survival were estimated using the cohort method. Adjusted hazard ratios were calculated using a multivariable flexible parametric survival model. Other outcomes investigated included recurrence and second primary malignancies (SPMs). RESULTS: The study cohort comprised 1269 patients. Age-standardised incidence rates remained steady across the study period at approximately 9.5 per million children per year. The proportion of patients with metastatic disease at diagnosis decreased from 63% in 1983-1995 to 42% by 2006-2015 (P < 0.001). CSS and event-free survival both improved significantly over time and reached 75% (95% confidence interval (CI) = 71-79%) and 71% (95% CI = 66-75%) at 5 years post-diagnosis, respectively, for children diagnosed between 2004 and 2013. Of patients achieving full remission, 28% relapsed with subsequent 5-year CSS of only 20%. Although SPMs were rare, neuroblastoma survivors carried a fivefold increased risk compared to cancer rates in the general population (standardised incidence ratio = 5.18, 95% CI = 3.01-8.91), with 7 of the 13 patients (54%) who were diagnosed with an SPM dying within 5 years. CONCLUSIONS: CSS for childhood neuroblastoma has improved substantially over time in Australia, but still remains lower than for most other types of childhood cancer. SPMs are uncommon and carry a better prognosis than relapse of the primary tumour.

A Method-Comparison Study Highlighting the Disparity between Osseous- and Skin-Based Measures of Foot Mobility.

PURPOSE: This study examined the validity of standard clinical measures of arch height mobility, midfoot width mobility (MWM), and foot mobility magnitude (FMM) relative to skin-based and osseous measures derived from radiographs. METHODS: Skin-based clinical indices of foot mobility were calculated from standard, caliper-based measures of foot length, midfoot width, and dorsal arch height of the left limb of 20 healthy participants (8-71 yr) during non-weight-bearing and weight-bearing. Skin-based radiographic and osseous indices were derived from concurrent anteroposterior and lateral radiographs. Agreement between skin-based clinical and skin-based radiographic measures of foot mobility with those of osseous measures was investigated using the Bland and Altman approach. RESULTS: Foot mobility indices derived from clinical measures were significantly higher (20%-50%) than skin-based radiographic measures ( P < 0.01), which were, in turn, significantly higher (200%-250%) than osseous measures ( P < 0.01). Clinical measures demonstrated significant levels of proportional bias compared with radiographic measures of foot mobility ( P < 0.01). The contribution of osseous movement to skin-based clinical measures of mobility was highly variable between individuals, ranging between 19% and 81% for arch height mobility, between 4% and 87% for MWM, and between 14% and 75% for FMM. The limits of tolerance for clinical measures of foot mobility ranged from ±3.2 mm for MWM to ±6.6 mm for measures of FMM. The limits of tolerance for skin-based clinical and skin-based radiographic measures were generally larger than osseous movement with weight-bearing. CONCLUSIONS: Skin-based measures of foot mobility, whether clinical or radiographic methods, are not interchangeable and are poor indicators of osseous mobility. Although further research regarding the utility of osseous measures is warranted, these findings strongly caution against the use of skin-based clinical measures of foot mobility in clinical and research settings.

Treatment of a contained rupture of a splenic artery pseudoaneurysm with direct thrombin injection.

Splenic artery pseudoaneurysm is a rare and potentially fatal condition. In the present report, we describe the case of a 50-year-old woman with chronic pancreatitis who presented with worsening abdominal pain. Computed tomography demonstrated a 3.5-cm splenic artery pseudoaneurysm of the mid-splenic artery. The patient underwent attempted endovascular repair of the pseudoaneurysm that was unsuccessful. Open conversion revealed an inaccessible splenic artery due to chronic pancreatitis that resulted in dense retroperitoneal fibrosis, and repair was achieved via direct thrombin injection under ultrasound guidance of the pseudoaneurysm and splenectomy. The patient recovered well, and computed tomography at 3 days postoperatively revealed complete thrombosis of the pseudoaneurysm.

Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids.

Isolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages.

Hypoxia regulate developmental coronary angiogenesis potentially through VEGFR2- and SOX17-mediated signaling.

Background: Coronary vessels in embryonic mouse heart arises from multiple progenitor population including sinus venosus (SV), endocardium, and proepicardium. ELA/APJ signaling is shown to regulate coronary growth from SV pathway within the subepicardium, whereas VEGF-A/VEGF-R2 pathways is implicated to regulate coronary growth from endocardium pathway. Our previous study show hypoxia as a potential signaling cue to stimulate overall coronary growth and expansion within the myocardium. However, the role of hypoxia and its downstream signaling pathways in the regulation of coronary vessel development is not known. In this study, we investigated the role of hypoxia in coronary vessel development and have identified SOX17- and VEGF-R2-mediated signaling as a potential downstream pathway of hypoxia in the regulation of coronary vessel development. Results: We show that hypoxia gain-of-function in the myocardium through upregulation of HIF-1α disrupts the normal pattern of coronary angiogenesis in developing mouse hearts and displays phenotype that is reminiscent of accelerated coronary growth. We show that VEGF-R2 expression is increased in coronary endothelial cells under hypoxia gain-of-function in vivo and in vitro . Furthermore, we show that SOX17 expression is upregulated in developing mouse heart under hypoxia gain-of-function conditions, whereas SOX17 expression is repressed under hypoxia loss-of-function conditions. Furthermore, our results show that SOX17 loss-of-function disrupts normal pattern of coronary growth. Conclusion: Collectively, our data provide strong phenotypic evidence to show that hypoxia might regulate coronary growth in the developing mouse heart potentially through VEGF-R2- and SOX17-mediated downstream signaling pathways.

Long-term urethral function measured by uroflowmetry after hypospadias surgery: comparison with an age matched control.

PURPOSE: Long-term outcomes of hypospadias surgery, particularly urinary function, have not been examined thoroughly. Uroflowmetry can be used to evaluate long-term functional outcomes by assessing flow shape and the rate of micturition. We assessed urethral function using uroflowmetry in adolescents after undergoing hypospadias repair in infancy and compared this with age matched controls. MATERIALS AND METHODS: After human research ethics committee approval (HREC 28111A), 17 boys 13 to 15 years old with no history of urological or neurological disorders underwent uroflowmetry (65 separate voids) to determine standard values for boys of this age. Then 60 boys age 13 to 15 years who had undergone treatment for hypospadias in early infancy were seen for long-term followup and underwent uroflow assessment. RESULTS: Standard urinary flow rates were established in the 13 to 15-year-old control group and represented on a nomogram. In the boys who underwent hypospadias surgery the urine flow rates were significantly lower compared to the control nomogram (p <0.0001), with half the patients having uroflow rates below 1 SD from the control mean but without symptoms. Boys with significant preoperative chordee were more likely to have poorer urinary flow (p <0.04). A poor urinary flow rate also was significantly associated with post-void residual bladder volume (p <0.03). There was no correlation with original meatal location, number of operations, presence of postoperative complications, current anatomy and lower urinary tract symptoms (eg post-void dribble, hesitancy, incontinence). CONCLUSIONS: At long-term followup after hypospadias surgery urinary flow rates were significantly lower compared to age matched controls but still fell within the normal range. In the hypospadias cohort there was no significant association with lower urinary tract symptoms and poor urinary flow. Detection of poor urinary flow may indicate incomplete bladder emptying. The presence of severe chordee preoperatively is a significant risk factor for poor urinary flow rates on long-term followup.

Regenerative medicine for childhood gastrointestinal diseases.

Several paediatric gastrointestinal diseases result in life-shortening organ failure. For many of these conditions, current therapeutic options are suboptimal and may not offer a cure. Regenerative medicine is an inter-disciplinary field involving biologists, engineers, and clinicians that aims to produce cell and tissue-based therapies to overcome organ failure. Exciting advances in stem cell biology, materials science, and bioengineering bring engineered gastrointestinal cell and tissue therapies to the verge of clinical trial. In this review, we summarise the requirements for bioengineered therapies, the possible sources of the various cellular and non-cellular components, and the progress towards clinical translation of oesophageal and intestinal tissue engineering to date.

Building gut from scratch - progress and update of intestinal tissue engineering.

Short bowel syndrome (SBS), a condition defined by insufficient absorptive intestinal epithelium, is a rare disease, with an estimated prevalence up to 0.4 in 10,000 people. However, it has substantial morbidity and mortality for affected patients. The mainstay of treatment in SBS is supportive, in the form of intravenous parenteral nutrition, with the aim of achieving intestinal autonomy. The lack of a definitive curative therapy has led to attempts to harness innate developmental and regenerative mechanisms to engineer neo-intestine as an alternative approach to addressing this unmet clinical need. Exciting advances have been made in the field of intestinal tissue engineering (ITE) over the past decade, making a review in this field timely. In this Review, we discuss the latest advances in the components required to engineer intestinal grafts and summarize the progress of ITE. We also explore some key factors to consider and challenges to overcome when transitioning tissue-engineered intestine towards clinical translation, and provide the future outlook of ITE in therapeutic applications and beyond.

Oil spill causes mass mortality of sea snakes in the Gulf of Oman.

Oil spills in the marine environment inflict significant impacts on a wide diversity of marine fauna. Despite the abundance of literature describing these impacts on numerous species, no studies describe the impacts on sea snakes. In this study we report, for the first time, details of an oil spill which caused mass mortality of sea snakes. In this study, 39 sea snake mortalities from the Gulf of Oman, in particular, the coast of Kalba, Sharjah, UAE, were examined. The investigated sea snakes belong to four different species (Hydrophis platurus, H. lapemoides, H. spiralis and H. ornatus). The majority (84.6%) of sea snakes were observed to have oil covering 75-100% of their bodies. The majority (91.4%) of sea snakes were also observed with oil covering their snouts and eyes. A large proportion (25.8, 41.4 and 34.5%) of sea snakes were observed with oil in their mouth, esophagus and stomach.

The Population Reference Sample, POPRES: a resource for population, disease, and pharmacological genetics research.

Technological and scientific advances, stemming in large part from the Human Genome and HapMap projects, have made large-scale, genome-wide investigations feasible and cost effective. These advances have the potential to dramatically impact drug discovery and development by identifying genetic factors that contribute to variation in disease risk as well as drug pharmacokinetics, treatment efficacy, and adverse drug reactions. In spite of the technological advancements, successful application in biomedical research would be limited without access to suitable sample collections. To facilitate exploratory genetics research, we have assembled a DNA resource from a large number of subjects participating in multiple studies throughout the world. This growing resource was initially genotyped with a commercially available genome-wide 500,000 single-nucleotide polymorphism panel. This project includes nearly 6,000 subjects of African-American, East Asian, South Asian, Mexican, and European origin. Seven informative axes of variation identified via principal-component analysis (PCA) of these data confirm the overall integrity of the data and highlight important features of the genetic structure of diverse populations. The potential value of such extensively genotyped collections is illustrated by selection of genetically matched population controls in a genome-wide analysis of abacavir-associated hypersensitivity reaction. We find that matching based on country of origin, identity-by-state distance, and multidimensional PCA do similarly well to control the type I error rate. The genotype and demographic data from this reference sample are freely available through the NCBI database of Genotypes and Phenotypes (dbGaP).

Microsatellite markers in the Australian desert plant, Solanum centrale (Solanaceae).

PREMISE OF THE STUDY: The first microsatellite primers were developed for Solanum centrale, a native Australian species used in the bush foods industry. Existing markers developed for other Solanum species were also amplified. METHODS AND RESULTS: Using an enrichment cloning protocol, seven novel markers were developed, and 48 existing markers from other Solanum species were tested, resulting in the characterization of a set of six highly polymorphic co-dominant loci for use in S. centrale. Microsatellite screening revealed polyploidy. Among the six highly polymorphic loci, allelic diversity ranged from 7 to 14. CONCLUSIONS: These markers will be useful for investigating genetic diversity and as a simple way of estimating ploidy of wild populations.

The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure.

Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79). In Saccharomyces cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA damage response. The biological function of H3K79 methylation in mammals, however, remains poorly understood. Using gene targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES) cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres. These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.