Dynamic Imaging of Blood Coagulation Within the Hematoma of Patients With Acute Hemorrhagic Stroke.

BACKGROUND: The dynamics of blood clot (combination of Hb [hemoglobin], fibrin, and a higher concentration of aggregated red blood cells) formation within the hematoma of an intracerebral hemorrhage is not well understood. A quantitative neuroimaging method of localized coagulated blood volume/distribution within the hematoma might improve clinical decision-making. METHODS: The deoxyhemoglobin of aggregated red blood cells within extravasated blood exhibits a higher magnetic susceptibility due to unpaired heme iron electrons. We propose that coagulated blood, with higher aggregated red blood cell content, will exhibit (1) a higher positive susceptibility than noncoagulated blood and (2) increase in fibrin polymerization-restricted localized diffusion, which can be measured noninvasively using quantitative susceptibility mapping and diffusion tensor imaging. In this serial magnetic resonance imaging study, we enrolled 24 patients with acute intracerebral hemorrhage between October 2021 to May 2022 at a stroke center. Patients were 30 to 70 years of age and had a hematoma volume >15 cm3 and National Institutes of Health Stroke Scale score >1. The patients underwent imaging 3×: within 12 to 24 (T1), 36 to 48 (T2), and 60 to 72 (T3) hours of last seen well on a 3T magnetic resonance imaging system. Three-dimensional anatomic, multigradient echo and 2-dimensional diffusion tensor images were obtained. Hematoma and edema volumes were calculated, and the distribution of coagulation was measured by dynamic changes in the susceptibilities and fractional anisotropy within the hematoma. RESULTS: Using a coagulated blood phantom, we demonstrated a linear relationship between the percentage coagulation and susceptibility (R2=0.91) with a positive red blood cell stain of the clot. The quantitative susceptibility maps showed a significant increase in hematoma susceptibility (T1, 0.29±0.04 parts per millions; T2, 0.36±0.04 parts per millions; T3, 0.45±0.04 parts per millions; P<0.0001). A concomitant increase in fractional anisotropy was also observed with time (T1, 0.40±0.02; T2, 0.45±0.02; T3, 0.47±0.02; P<0.05). CONCLUSIONS: This quantitative neuroimaging study of coagulation within the hematoma has the potential to improve patient management, such as safe resumption of anticoagulants, the need for reversal agents, the administration of alteplase to resolve the clot, and the need for surgery.

SAMHD1 compound heterozygous rare variants associated with moyamoya and mitral valve disease in the absence of other features of Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome (AGS) is an autosomal recessive inflammatory syndrome that manifests as an early-onset encephalopathy with both neurologic and extraneurologic clinical findings. AGS has been associated with pathogenic variants in nine genes: TREX1, RNASEH2B, RNASEH2C, RNASEH2A, SAMHD1, ADAR, IFIH1, LSM11, and RNU7-1. Diagnosis is established by clinical findings (encephalopathy and acquired microcephaly, intellectual and physical impairments, dystonia, hepatosplenomegaly, sterile pyrexia, and/or chilblains), characteristic abnormalities on cranial CT (calcification of the basal ganglia and white matter) and MRI (leukodystrophic changes), or the identification of pathogenic/likely pathogenic variants in the known genes. One of the genes associated with AGS, SAMHD1, has also been associated with a spectrum of cerebrovascular diseases, including moyamoya disease (MMD). In this report, we describe a 31-year-old male referred to genetics for MMD since childhood who lacked the hallmark features of AGS patients but was found to have compound heterozygous SAMHD1 variants. He later developed mitral valve insufficiency due to recurrent chordal rupture and ultimately underwent a heart transplant at 37 years of age. Thus, these data suggest that SAMHD1 pathogenic variants can cause MMD without typical AGS symptoms and support that SAMHD1 should be assessed in MMD patients even in the absence of AGS features.

Investigating the role of lipid genes in liver disease using fatty liver models of alcohol and high fat in zebrafish (Danio rerio).

BACKGROUND: Accumulation of lipid in the liver is the first hallmark of both alcohol-related liver disease (ALD) and non-alcohol-related fatty liver disease (NAFLD). Recent studies indicate that specific mutations in lipid genes confer risk and might influence disease progression to irreversible liver cirrhosis. This study aimed to understand the function/s of lipid risk genes driving disease development in zebrafish genetic models of alcohol-related and non-alcohol-related fatty liver. METHODS: We used zebrafish larvae to investigate the effect of alcohol and high fat to model fatty liver and tested the utility of this model to study lipid risk gene functions. CRISPR/Cas9 gene editing was used to create knockdowns in 5 days post-fertilisation zebrafish larvae for the available orthologs of human cirrhosis risk genes (pnpla3, faf2, tm6sf2). To establish fatty liver models, larvae were exposed to ethanol and a high-fat diet (HFD) consisting of chicken egg yolk. Changes in morphology (imaging), survival, liver injury (biochemical tests, histopathology), gene expression (qPCR) and lipid accumulation (dye-specific live imaging) were analysed across treatment groups to test the functions of these genes. RESULTS: Exposure of 5-day post-fertilisation (dpf) WT larvae to 2% ethanol or HFD for 48 h developed measurable hepatic steatosis. CRISPR-Cas9 genome editing depleted pnpla3, faf2 and tm6sf2 gene expression in these CRISPR knockdown larvae (crispants). Depletion significantly increased the effects of ethanol and HFD toxicity by increasing hepatic steatosis and hepatic neutrophil recruitment ≥2-fold in all three crispants. Furthermore, ethanol or HFD exposure significantly altered the expression of genes associated with ethanol metabolism (cyp2y3) and lipid metabolism-related gene expression, including atgl (triglyceride hydrolysis), axox1, echs1 (fatty acid ß-oxidation), fabp10a (transport), hmgcra (metabolism), notch1 (signalling) and srebp1 (lipid synthesis), in all three pnpla3, faf2 and tm6sf2 crispants. Nile Red staining in all three crispants revealed significantly increased lipid droplet size and triglyceride accumulation in the livers following exposure to ethanol or HFD. CONCLUSIONS: We identified roles for pnpla3, faf2 and tm6sf2 genes in triglyceride accumulation and fatty acid oxidation pathways in a zebrafish larvae model of fatty liver.

Establishing a pediatric acute stroke protocol: experience of a new pediatric stroke program and predictors of acute stroke.

Introduction: Pediatric stroke is among the top 10 causes of death in pediatrics. Rapid recognition and treatment can improve outcomes in select patients, as evidenced by recent retrospective studies in pediatric thrombectomy. We established a collaborative protocol involving the vascular neurology and pediatric neurology division in our institution to rapidly diagnose and treat pediatric suspected stroke. We also prospectively collected data to attempt to identify predictors of acute stroke in pediatric patients. Methods: IRB approval was obtained to prospectively collect clinical data on pediatric code stroke activations based on timing metrics in resident-physician note templates. The protocol emphasized magnetic resonance imaging over computed tomography imaging when possible. We analyzed performance of the system with descriptive statistics. We then performed a Bayesian statistical analysis to search for predictors of pediatric stroke. Results: There were 40 pediatric code strokes over the 2.5-year study period with a median age of 10.8 years old. 12 (30%) of patients had stroke, and 28 (70%) of code stroke patients were diagnosed with a stroke mimic. Median time from code stroke activation to completion of imaging confirming or ruling out stroke was 1 h. In the Bayesian analysis, altered mental status, hemiparesis, and vasculopathy history were associated with increased odds of stroke, though credible intervals were wide due to the small sample size. Conclusion: A trainee developed and initiated pediatric acute stroke protocol quickly implemented a hospital wide change in management that led to rapid diagnosis and triage of pediatric stroke and suspected stroke. No additional personnel or resources were needed for this change, and we encourage other hospitals and emergency departments to implement similar systems. Additionally, hemiparesis and altered mental status were predictors of stroke for pediatric acute stroke activation in our Bayesian statistical analysis. However credible intervals were wide due to the small sample size. Further multicenter data collection could more definitively analyze predictors of stroke, as well as the help in the creation of diagnostic tools for clinicians in the emergency setting.

Generation of an induced pluripotent stem cell line from a patient with conduction disease and recurrent ventricular fibrillation with a sodium voltage-gated channel alpha subunit 5 (SCN5A) gene c.392 + 3A > G splice-site variant.

Variants in the sodium voltage-gated channel alpha subunit 5 gene (SCN5A) produce variable cardiac phenotypes including Brugada syndrome, conduction disease and cardiomyopathy. These phenotypes can lead to life-threatening arrhythmias, heart failure, and sudden cardiac death. Novel variants in splice-site regions of SCN5A require functional studies to characterise their pathogenicity as they are poorly understood. The generation of an induced pluripotent stem cell line provides a valuable resource to investigate the functional effects of potential splice-disrupting variants in SCN5A.

A National, Electronic Health Record-Based Study of Perinatal Hemorrhagic and Ischemic Stroke.

BACKGROUND: Perinatal stroke occurs in approximately 1 in 1100 live births. Large electronic health record (EHR) data can provide information on exposures associated with perinatal stroke in a larger number of patients than is achievable through traditional clinical studies. The objective of this study is to assess prevalence and odds ratios of known and theorized comorbidities with perinatal ischemic and hemorrhagic stroke. METHODS: The data for patients aged 0-28 days with a diagnosis of either ischemic or hemorrhagic stroke were extracted from the Cerner Health Facts Electronic Medical Record (EMR) database. Incidence of birth demographics and perinatal complications were recorded. Odds ratios were calculated against a control group. RESULTS: A total of 535 (63%) neonates were identified with ischemic stroke and 312 (37%) with hemorrhagic stroke. The most common exposures for ischemic stroke were sepsis (n = 82, 15.33%), hypoxic injury (n = 61, 11.4%), and prematurity (n = 49, 9.16%). The most common comorbidities for hemorrhagic stroke were prematurity (n = 81, 26%) and sepsis (n = 63, 20%). No perinatal ischemic stroke patients had diagnosis codes for cytomegalovirus disease. Procedure and diagnosis codes related to critical illness, including intubation and resuscitation, were prominent in both hemorrhagic (n = 46, 15%) and ischemic stroke (n = 45, 8%). CONCLUSION: This electronic health record-based study of perinatal stroke, the largest of its kind, demonstrated a wide variety of comorbid conditions with ischemic and hemorrhagic stroke. Sepsis, prematurity, and hypoxic injury are associated with perinatal hemorrhagic and ischemic stroke, though prevalence varies between types. Much of our data were similar to prior studies, which lends validity to the electronic health record database in studying perinatal stroke.

Rare variants in ANO1, encoding a calcium-activated chloride channel, predispose to moyamoya disease.

Moyamoya disease, a cerebrovascular disease leading to strokes in children and young adults, is characterized by progressive occlusion of the distal internal carotid arteries and the formation of collateral vessels. Altered genes play a prominent role in the aetiology of moyamoya disease, but a causative gene is not identified in the majority of cases. Exome sequencing data from 151 individuals from 84 unsolved families were analysed to identify further genes for moyamoya disease, then candidate genes assessed in additional cases (150 probands). Two families had the same rare variant in ANO1, which encodes a calcium-activated chloride channel, anoctamin-1. Haplotype analyses found the families were related, and ANO1 p.Met658Val segregated with moyamoya disease in the family with an LOD score of 3.3. Six additional ANO1 rare variants were identified in moyamoya disease families. The ANO1 rare variants were assessed using patch-clamp recordings, and the majority of variants, including ANO1 p.Met658Val, displayed increased sensitivity to intracellular Ca2+. Patients harbouring these gain-of-function ANO1 variants had classic features of moyamoya disease, but also had aneurysm, stenosis and/or occlusion in the posterior circulation. Our studies support that ANO1 gain-of-function pathogenic variants predispose to moyamoya disease and are associated with unique involvement of the posterior circulation.

Previsual and Early Detection of Myrtle Rust on Rose Apple Using Indices Derived from Thermal Imagery and Visible-to-Short-Infrared Spectroscopy.

Myrtle rust, caused by the fungus Austropuccinia psidii, is a serious disease, which affects many Myrtaceae species. Commercial nurseries that propagate Myrtaceae species are prone to myrtle rust and require a reliable method that allows previsual and early detection of the disease. This study uses time-series thermal imagery and visible-to-short-infrared spectroscopy measurements acquired over 10 days from 81 rose apple plants (Syzygium jambos) that were either inoculated with myrtle rust or maintained disease-free. Using these data, the objectives were to (i) quantify the accuracy of models using thermal indices and narrowband hyperspectral indices (NBHI) for previsual and early detection of myrtle rust using data from older resistant green leaves and young susceptible red leaves and (ii) identify the most important NBHI and thermal indices for disease detection. Using predictions made on a validation dataset, models using indices derived from thermal imagery were able to perfectly (F1 score = 1.0; accuracy = 100%) distinguish control from infected plants previsually one day before symptoms appeared (1 DBS) and for all stages after early symptoms appeared. Compared with control plants, plants with myrtle rust had lower and more variable normalized canopy temperature, which was associated with higher stomatal conductance and transpiration. Using NBHI derived from green leaves, excellent previsual classification was achieved 3 DBS, 2 DBS, and 1 DBS (F1 score range = 0.89 to 0.94). The accurate characterization of myrtle rust during previsual and early stages of disease development suggests that a robust detection methodology could be developed within a nursery setting. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Transitional forestry in New Zealand: re-evaluating the design and management of forest systems through the lens of forest purpose.

Forestry management worldwide has become increasingly effective at obtaining high timber yields from productive forests. In New Zealand, a focus on improving an increasingly successful and largely Pinus radiata plantation forestry model over the last 150 years has resulted in some of the most productive timber forests in the temperate zone. In contrast to this success, the full range of forested landscapes across New Zealand, including native forests, are impacted by an array of pressures from introduced pests, diseases, and a changing climate, presenting a collective risk of losses in biological, social and economic value. As the national government policies incentivise reforestation and afforestation, the social acceptability of some forms of newly planted forests is also being challenged. Here, we review relevant literature in the area of integrated forest landscape management to optimise forests as nature-based solutions, presenting 'transitional forestry' as a model design and management paradigm appropriate to a range of forest types, where forest purpose is placed at the heart of decision making. We use New Zealand as a case study region, describing how this purpose-led transitional forestry model can benefit a cross section of forest types, from industrialised forest plantations to dedicated conservation forests and a range of multiple-purpose forests in between. Transitional forestry is an ongoing multi-decade process of change from current 'business-as-usual' forest management to future systems of forest management, embedded across a continuum of forest types. This holistic framework incorporates elements to enhance efficiencies of timber production, improve overall forest landscape resilience, and reduce some potential negative environmental impacts of commercial plantation forestry, while allowing the ecosystem functioning of commercial and non-commercial forests to be maximised, with increased public and biodiversity conservation value. Implementation of transitional forestry addresses tensions that arise between meeting climate mitigation targets and improving biodiversity criteria through afforestation, alongside increasing demand for forest biomass feedstocks to meet the demands of near-term bioenergy and bioeconomy goals. As ambitious government international targets are set for reforestation and afforestation using both native and exotic species, there is an increasing opportunity to make such transitions via integrated thinking that optimises forest values across a continuum of forest types, while embracing the diversity of ways in which such targets can be reached.

Stroke in the young.

PURPOSE OF REVIEW: The purpose of this review is to review recent findings regarding stroke epidemiology, etiologies, and treatment in children and young adults. RECENT FINDINGS: Incidence in young adults is increasing, and incidence, recurrence, and survival is worse in patients with cryptogenic stroke and in developing countries. Careful consideration of patent foramen ovale closure is now recommended in young adults with cryptogenic stroke. Thrombectomy has recently been extended to carefully selected children with acute ischemic stroke, and two recent publications strongly suggest that it can be beneficial for children. Sickle cell is also an important global contributor to stroke burden, but hydroxyurea can be a cost effective medication for stroke prevention in children. Recent advances in genetic testing and treatments may improve outcomes for patients with monogenic causes of stroke, such as deficiency of adenosine deaminase 2, hemophilia, and Fabry's disease. SUMMARY: Stroke in children and young adults is a morbid disease responsible for enormous indirect societal costs and a high burden of years with disability per affected patient. Recent advances have improved access to care for children with large vessel occlusion and adults with rare causes of stroke. Future research may bring effective treatments for other monogenic causes of stroke as well as increasing access to hyperacute therapies for young stroke patients.

Consensus-Based Evaluation of Outcome Measures in Pediatric Stroke Care: A Toolkit.

Following a pediatric stroke, outcome measures selected for monitoring functional recovery and development vary widely. We sought to develop a toolkit of outcome measures that are currently available to clinicians, possess strong psychometric properties, and are feasible for use within clinical settings. A multidisciplinary group of clinicians and scientists from the International Pediatric Stroke Organization comprehensively reviewed the quality of measures in multiple domains described in pediatric stroke populations including global performance, motor and cognitive function, language, quality of life, and behavior and adaptive functioning. The quality of each measure was evaluated using guidelines focused on responsiveness and sensitivity, reliability, validity, feasibility, and predictive utility. A total of 48 outcome measures were included and were rated by experts based on the available evidence within the literature supporting the strengths of their psychometric properties and practical use. Only three measures were found to be validated for use in pediatric stroke: the Pediatric Stroke Outcome Measure, the Pediatric Stroke Recurrence and Recovery Questionnaire, and the Pediatric Stroke Quality of Life Measure. However, multiple additional measures were deemed to have good psychometric properties and acceptable utility for assessing pediatric stroke outcomes. Strengths and weaknesses of commonly used measures including feasibility are highlighted to guide evidence-based and practicable outcome measure selection. Improving the coherence of outcome assessment will facilitate comparison of studies and enhance research and clinical care in children with stroke. Further work is urgently needed to close the gap and validate measures across all clinically significant domains in the pediatric stroke population.

International Prevalence and Mechanisms of SARS-CoV-2 in Childhood Arterial Ischemic Stroke During the COVID-19 Pandemic.

BACKGROUND: Data from the early pandemic revealed that 0.62% of children hospitalized with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had an acute arterial ischemic stroke (AIS). In a larger cohort from June 2020 to December 2020, we sought to determine whether our initial point estimate was stable as the pandemic continued and to understand radiographic and laboratory data that may clarify mechanisms of pediatric AIS in the setting of SARS-CoV-2. METHODS: We surveyed international sites with pediatric stroke expertise to determine numbers of hospitalized SARS-CoV-2 patients <18 years, numbers of incident AIS cases among children (29 days to <18 years), frequency of SARS-CoV-2 testing for children with AIS, and numbers of childhood AIS cases positive for SARS-CoV-2 June 1 to December 31, 2020. Two stroke neurologists with 1 neuroradiologist determined whether SARS-CoV-2 was the main stroke risk factor, contributory, or incidental. RESULTS: Sixty-one centers from 21 countries provided AIS data. Forty-eight centers (78.7%) provided SARS-CoV-2 hospitalization data. SARS-CoV-2 testing was performed in 335/373 acute AIS cases (89.8%) compared with 99/166 (59.6%) in March to May 2020, P<0.0001. Twenty-three of 335 AIS cases tested (6.9%) were positive for SARS-CoV-2 compared with 6/99 tested (6.1%) in March to May 2020, P=0.78. Of the 22 of 23 AIS cases with SARS-CoV-2 in whom we could collect additional data, SARS-CoV-2 was the main stroke risk factor in 6 (3 with arteritis/vasculitis, 3 with focal cerebral arteriopathy), a contributory factor in 13, and incidental in 3. Elevated inflammatory markers were common, occurring in 17 (77.3%). From centers with SARS-CoV-2 hospitalization data, of 7231 pediatric patients hospitalized with SARS-CoV-2, 23 had AIS (0.32%) compared with 6/971 (0.62%) from March to May 2020, P=0.14. CONCLUSIONS: The risk of AIS among children hospitalized with SARS-CoV-2 appeared stable compared with our earlier estimate. Among children in whom SARS-CoV-2 was considered the main stroke risk factor, inflammatory arteriopathies were the stroke mechanism.

Intracellular flow cytometric lipid analysis - a multiparametric system to assess distinct lipid classes in live cells.

The lipid content of mammalian cells varies greatly between cell type. Current methods for analysing lipid components of cells are technically challenging and destructive. Here, we report a facile, inexpensive method to identify lipid content - intracellular flow cytometric lipid analysis (IFCLA). Distinct lipid classes can be distinguished by Nile Blue fluorescence, Nile Red fluorescence or violet autofluorescence. Nile Blue is fluorescent in the presence of unsaturated fatty acids with a carbon chain length greater than 16. Cis-configured fatty acids induce greater Nile Blue fluorescence than their trans-configured counterparts. In contrast, Nile Red exhibits greatest fluorescence in the presence of cholesterol, cholesteryl esters, some triglycerides and phospholipids. Multiparametric spanning-tree progression analysis for density-normalized events (SPADE) analysis of hepatic cellular lipid distribution, including vitamin A autofluorescence, is presented. This flow cytometric system allows for the rapid, inexpensive and non-destructive identification of lipid content, and highlights the differences in lipid biology between cell types by imaging and flow cytometry.

A De Novo case of autosomal dominant mitochondrial membrane protein-associated neurodegeneration.

BACKGROUND: Mitochondrial membrane protein-associated neurodegeneration (MPAN) is a genetic neurodegenerative condition previously thought to be inherited only in an autosomal recessive pattern through biallelic pathogenic variants in C19orf12. Recent evidence has proposed that MPAN can also follow autosomal dominant forms of inheritance. We present a case of a de novo pathogenic variant in C19orf12 identified in a female with clinical features consistent with a diagnosis of MPAN, adding further evidence that the disease can be inherited in an autosomal dominant fashion. METHODS: A 17-year-old Hispanic female was born to non-consanguineous healthy parents. She developed progressive muscle weakness and dystonia beginning when she was 12 years old. Trio, whole-exome sequencing with mitochondrial genome sequencing, and deletion/duplication analysis of both nuclear and mitochondrial genomes was performed in December 2019. RESULTS: Whole-exome sequencing analysis revealed a single de novo variant in C19orf12. The specific variant is c.256C>T (p.Q86X) located in exon 3. CONCLUSION: Our clinical report provides further clinical evidence that MPAN can be inherited in an autosomal dominant or recessive fashion. The patient's age of onset and clinical symptoms are very similar to the previous patient published with this specific variant as well as others with heterozygous pathogenic variants in C19orf12 in Gregory et al. 2019. Our case report highlights the importance of considering both autosomal dominant and autosomal recessive version of MPAN with all patients demonstrating clinical features suggestive of MPAN.

Association of De Novo RNF213 Variants With Childhood Onset Moyamoya Disease and Diffuse Occlusive Vasculopathy.

OBJECTIVE: To test the hypothesis that de novo genetic variants are responsible for moyamoya disease (MMD) in children with unaffected relatives, we performed exome sequencing of 28 affected children and their unaffected parents. METHODS: Exome sequencing was performed on 28 trios of affected patients with MMD and unaffected parents. RESULTS: We identified 3 novel rare de novo RNF213 variants, 1 in the RING domain and 2 in a highly conserved region distal to the RING domain (4,114-4,120). These de novo cases of MMD present at a young age with aggressive MMD and uniquely have additional occlusive vascular lesions, including renal artery stenosis. Two previously reported cases had de novo variants in the same limited region and presented young with aggressive MMD, and 1 case had narrowing of the inferior abdominal aorta. CONCLUSIONS: These results indicate a novel syndrome associated with RNF213 rare variants defined by de novo mutations disrupting highly conserved amino acids in the RING domain and a discrete region distal to the RING domain delimited by amino acids 4,114 to 4,120 leading to onset of severe MMD before 3 years of age and occlusion of other arteries, including the abdominal aorta, renal, iliac, and femoral arteries.

Thermally drawn biodegradable fibers with tailored topography for biomedical applications.

There is a growing demand for polymer fiber scaffolds for biomedical applications and tissue engineering. Biodegradable polymers such as polycaprolactone have attracted particular attention due to their applicability to tissue engineering and optical neural interfacing. Here we report on a scalable and inexpensive fiber fabrication technique, which enables the drawing of PCL fibers in a single process without the use of auxiliary cladding. We demonstrate the possibility of drawing PCL fibers of different geometries and cross-sections, including solid-core, hollow-core, and grooved fibers. The solid-core fibers of different geometries are shown to support cell growth, through successful MCF-7 breast cancer cell attachment and proliferation. We also show that the hollow-core fibers exhibit a relatively stable optical propagation loss after submersion into a biological fluid for up to 21 days with potential to be used as waveguides in optical neural interfacing. The capacity to tailor the surface morphology of biodegradable PCL fibers and their non-cytotoxicity make the proposed approach an attractive platform for biomedical applications and tissue engineering.

Detection of cell-surface phosphatidylserine using the fluorogenic probe P-IID.

The fluorogenic probe P-IID enables the detection of cell-surface phosphatidylserine (PS) using both fluorescence imaging and flow cytometry. Here we provide a detailed protocol for the use of P-IID for the qualitative detection of externalized PS in apoptotic cells using confocal microscopy, including the real-time imaging of apoptosis upon drug treatment. We also provide a detailed method for the quantitative analysis of cell death by flow cytometry, using P-IID in conjunction with the nuclear stain propidium iodide. P-IID is superior to commonly used Annexin-V fluorophore conjugates for PS detection as it provides a "turn-on" fluorescence response, displays rapid binding kinetics and can be used at low temperature (4°C), without washing and in the absence of Ca2+ ions.

Cellular dynamics of mammalian red blood cell production in the erythroblastic island niche.

Red blood cells, or erythrocytes, make up approximately a quarter of all cells in the human body with over 2 billion new erythrocytes made each day in a healthy adult human. This massive cellular production system is coupled with a set of cell biological processes unique to mammals, in particular, the elimination of all organelles, and the expulsion and destruction of the condensed erythroid nucleus. Erythrocytes from birds, reptiles, amphibians and fish possess nuclei, mitochondria and other organelles: erythrocytes from mammals lack all of these intracellular components. This review will focus on the dynamic changes that take place in developing erythroid cells that are interacting with specialized macrophages in multicellular clusters termed erythroblastic islands. Proerythroblasts enter the erythroblastic niche as large cells with active nuclei, mitochondria producing heme and energy, and attach to the central macrophage via a range of adhesion molecules. Proerythroblasts then mature into erythroblasts and, following enucleation, in reticulocytes. When reticulocytes exit the erythroblastic island, they are smaller cells, without nuclei and with few mitochondria, possess some polyribosomes and have a profoundly different surface molecule phenotype. Here, we will review, step-by-step, the biophysical mechanisms that regulate the remarkable process of erythropoiesis with a particular focus on the events taking place in the erythroblastic island niche. This is presented from the biological perspective to offer insight into the elements of red blood cell development in the erythroblastic island niche which could be further explored with biophysical modelling systems.

Differentiation of Urothelium from Mouse Embryonic Stem Cells in Chemically Defined Conditions.

The urothelium of the bladder and urethra are derived from the definitive endoderm during development. Cellular signaling molecules important to the developmental specification of the urothelium are also implicated in the dysregulation of the tissue repair mechanism characteristic of bladder disease. Hence, a complete understanding of the regulation of urothelium development is central to understanding the processes of bladder disease, and in development of simple chemically defined methods for use in regenerative medicine. Key to this is a suitable in vitro model that readily allows for the prosecution of biologically pertinent questions. Here a method for differentiating urothelium from mouse embryonic stem cells in chemically defined conditions is described. The method includes a description of flow cytometry and RT-PCR analysis of definitive endoderm markers Cxcr4, c-Kit, and FoxA2, and of terminally differentiated urothelial cell markers Upk1b and Upk2.