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Objective: This article extends the compartmental model previously developed by Scribner et al. in the context of college drinking to a mathematical model of the consequences of lowering the legal drinking age. Method: Using data available from 32 U.S. campuses, the analyses separate underage and legal age drinking groups into an eight-compartment model with different alcohol availability (wetness) for the underage and legal age groups. The model evaluates the likelihood that underage students will incorrectly perceive normative drinking levels to be higher than they actually are (i.e., misperception) and adjust their drinking accordingly by varying the interaction between underage students in social and heavy episodic drinking compartments. Results: The results evaluate the total heavy episodic drinker population and its dependence on the difference in misperception, as well as its dependence on underage wetness, legal age wetness, and drinking age. Conclusions: Results suggest that an unrealistically extreme combination of high wetness and low enforcement would be needed for the policies related to lowering the drinking age to be effective. (J Stud, Alcohol Drugs. 72. 15-23, 2011), ALCOHOL USE ON COLLEGE CAMPUSES continues to be a subject of concern both in terms of health consequences and social costs (National Institute of Alcohol Abuse and Alcoholism, 2002). Alcohol-related [...]

Objective: To critically examine the evidence-base for survival benefit of pulmonary metastasectomy (PM) for osteosarcoma (OS) in the pediatric population., Background: PM for OS is recommended as the standard of care in both pediatric and adult treatment protocols. Recent results from the "Pulmonary Metastasectomy in Colorectal Cancer" trial demonstrate no survival benefit from PM in colorectal cancer in adults., Methods: A systematic review was undertaken according to "Preferred Reporting Items for Systematic Reviews and Meta-Analysis" guidelines. Medline, Embase, and 2 clinical trial registers were searched for all studies detailing pediatric patients with OS (<18 years) undergoing PM with a comparison cohort group that did not receive PM., Results: Eleven studies met inclusion criteria dating from 1984 to 2017. All studies were retrospective and none directly compared PM versus no PM in pediatric patients as its main objective(s). Three-year survival rates ranged from 0% to 54% for PM and 0% to 16% for no PM. No patients receiving PM were usually those with unresectable disease and/or considered to have a poor prognosis. All studies were at high risk of bias and there was marked heterogeneity in the patient selection., Conclusions: There is a weak evidence base (level IV) for a survival benefit of PM for OS in pediatric patients likely due to selection bias of "favorable cases." The included studies many of which detailed outdated treatment protocols were not designed in their reporting to specifically address the questions directly. A randomized controlled trial-while ethically challenging in a pediatric population-incorporating modern OS chemotherapy protocols is needed to crucially address any "survival benefit.", Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: To determine the efficacy of steroid injections for pain relief in patients with meralgia paresthetica (MP)., Methods: All the literature published until March 2023 was explored from several databases, including EBSCO, PubMed, EMBASE, Cochrane Library, Google Scholar, and Scopus. Articles investigating the change in pain status of MP patients after steroid injection were included. The primary outcomes were complete pain relief, pain scores at 15 days and one month after intervention. When compared to the baseline, the secondary outcomes for the steroid group included pain scores at the end of treatment and quality of life, which were further evaluated by two factors, namely mental and physical health., Results: The analysis of the studies validated that steroids were significantly successful in providing complete pain relief (p-value = 0.00001), and in reducing the pain score of patients with meralgia paresthetica at 15 days (p-value = 0.02), but not at one month (p-value = 0.79) as compared to the control group. The analysis did not reveal any significant subgroup differences among various steroids (P = 0.52; CI: 0.01 - 0.10; RR: 0.04; I 2 = 0%). Mental health (MD = 4.23; 95% CI = 0.42 to 8.03; p = 0.03, I2 = 0%) was significantly improved in the steroid group when compared with baseline., Conclusion: Steroids injections can play an important role in improving symptoms and complications of meralgia paresthetica, especially in the short term., Competing Interests: Conflicts of Interest: None., (Copyright: © Pakistan Journal of Medical Sciences.)

Sodium valproate and carbamazepine are two time-tested drugs for treatment of epilepsy. Individually, they are usually excellent choices in treating a broad spectrum of epileptic seizures. They are, however, not friendly with each other. Their co-administration affects the drug levels of each other by influencing the action of hepatic enzymes. This write up attempts to give an overview of the mechanism of this drug interaction, and informs readers why this combination is a bad choice. The author hopes that this will help in raising awareness among the physicians regarding the dangers of this prescription, and in putting a full stop to this practice., Competing Interests: Conflict of interest: None., (Copyright: © Pakistan Journal of Medical Sciences.)

Objectives: This study aimed to evaluate the etiology, outcomes and prognostic factors associated with status epilepticus (SE) admissions in Neurology Department of a tertiary care hospital., Methods: A retrospective review was performed on all SE admissions at Dr. Ruth K.M. Pfau Civil Hospital Karachi over a five-year period from July 2015 to June 2020. Demographic, clinical, and etiological factors were investigated for prognostic value. Statistical tests were applied to determine significant prognostic factors. A five percent significance level was used., Results: A total of 176 patients were included in the study. Mortality was reported in 22 cases (12.5%) and morbidity at six months was observed in 44 cases (25.0%). Male gender, previous history of SE, prolonged seizure duration, and late presentation to hospital were significantly associated with mortality (p<0.05). De novo cases of SE tended to be older (p=0.048) and were associated with morbidity at follow-up (p=0.000). The most common causes of epilepsy in our patients with SE were CNS infections (n=54) and Idiopathic epilepsy (n=34). Non-compliance to medicines/under-dosing was the most common provocative factor (n=68). Acute symptomatic causes of SE were more likely to be associated with greater morbidity (p=0.000). Refractory and super-refractory SE were strongly associated with higher mortality (p=0.000). A longer duration of hospital stay was associated with higher morbidity (p=0.000)., Conclusion: Male gender, poor control of seizures, CNS infections, prolonged seizures, delayed hospital arrival and refractory/super-refractory status epilepticus were key determinants of mortality in our setting. Previous history of status epilepticus, and acute and symptomatic etiologies were associated with higher morbidity., Competing Interests: Conflicts of Interest: None., (Copyright: © Pakistan Journal of Medical Sciences.)

Objective: Guillain-Barré syndrome (GBS) is an autoimmune disease characterized by weakness in limbs or cranial nerve innervated muscles. Acute inflammatory demyelinating polyradiculoneuropathy (AIDP) is the most common variant. Electrophysiologic abnormalities and elevated cerebrospinal fluid (CSF) protein are frequently present in AIDP, but the relationship between these two parameters is not well known. We aimed to fill this gap by studying this relationship. Methods: This was a prospective cross-sectional study conducted for two years in the Department of Neurology, Dr. Ruth K. M. Pfau Civil Hospital, Karachi, Pakistan. All 90 adult patients with the AIDP variant of GBS were selected. Nerve conduction studies were performed to determine the degree of demyelination through the four electrophysiologic demyelination criteria. The CSF sample was sent to lab immediately after lumbar puncture. SPSS version 20.0 was used. The CSF protein level was measured with mean ±SD. Demyelination criteria were measured in frequency and percentages. Chi-square test was applied to a number of demyelination criteria and T-test/ANOVA was applied on mean CSF protein level. Results: We found a mean CSF protein of 37.41 mg/dl (±3.69) with one demyelination criterion, 81.87 mg/dl (±17.39) with two demyelination criteria, 119.75 mg/dl (±31.42) with three demyelination criteria, and 134.00 mg/dl (±42.87) with four demyelination criteria (P-value <0.001). Conclusion: This study demonstrates a significant relationship between CSF protein levels and degree of demyelination in the AIDP variant of GBS. This is an under-researched area in GBS and this study adds favorably to limited data in this regard., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Jawaid et al.)

Introduction:  A developing body of literature suggests that the presence of a hernia sac in fetuses with congenital diaphragmatic hernia (CDH) may indicate improved prognosis. By examining a large cohort of CDH newborns admitted to a single United Kingdom specialist center, we aimed to establish if presence of hernia sac is a robust predictor of improved survival., Materials and Methods:  All CDH patients admitted to a single center were recruited. Postneonatal presentations and Morgagni hernias were excluded. Demographics, defect type, laterality, survival, and hernia recurrence were recorded., Results:  In this study, 192 CDH newborns were managed from 1997 to 2017; 39 were excluded (10 Morgagni and 29 postneonatal); 22 (14%) neonates had a hernia sac. Survival in patients with a hernia sac was 21/22 (95%) versus 107/124 (86%) in cases without hernia sac ( p  = 0.2). There was no difference in hernia sac proportion by gender (male:female 15 vs. 13.2%, p  = 0.8)., Conclusion:  In contrast to studies showing a survival advantage, albeit with smaller patient numbers, we report a statistical nonsignificant benefit of hernia sac. Better survival outcomes at this specialist center with CDH patients without a hernia sac than reported in other published studies are likely responsible for the lack of statistical significance observed, despite a larger cohort. National and international CDH registries yielding "big data" may provide further answers on the utility of a CDH hernia sac as a new prognostic scoring tool., Competing Interests: None declared., (Thieme. All rights reserved.)

Introduction Stroke is the most debilitating of neurologic diseases. The rationale of the current study was to determine the association between hyperuricemia and ischemic stroke to establish a local perspective. Methods A total of 148 patients at a tertiary care hospital in Pakistan who fulfilled the inclusion criteria were enrolled in the study and then equally distributed into two study groups consisting of cases and controls (n = 74 in each group). In this study, there were 36 (48.6%) participants in the case group with hyperuricemia and ischemic stroke and 18 (24.3%) participants in the control group with hyperuricemia. The mean and standard deviations were computed for quantitative variables such as age, body mass index (BMI), and duration of stroke. Frequencies and percentages for the qualitative variables such as gender, hypertension, type 2 diabetes (T2D), dyslipidemia, smoking status, socioeconomic status, educational level, and hyperuricemia were calculated. The chi-square test was applied to compare both groups, with p ≤ 0.05 indicating significance. The odds ratio was also calculated to determine the association between case and control. Effect modifiers were controlled through stratification of age, gender, BMI, duration of stroke, hypertension, T2D, dyslipidemia, socioeconomic status, educational level, and smoking status to determine the effect of these on outcome variables. A post-stratification chi-square test was applied, with p ≤ 0.05 indicating statistical significance. Results In our study, stratification of hyperuricemia into cases and controls was performed for age, gender, T2D, hypertension, dyslipidemia, smoking status, socioeconomic status, and educational status. The maximum results were significant, with high strength of association between both groups. In the case group, the frequency of elevated uric acid was significantly higher than that of the control group. A comparison of hyperuricemia indicated p = 0.002, with an odds ratio of 2.95, which showed that elevated uric acid could be taken as a predictor of ischemic stroke. The uric acid level was significantly higher in men than in women. Additionally, hyperuricemia was associated with dyslipidemia. In patients with ischemic stroke, there was a significant association between serum uric acid level and T2D, hypertension, and smoking. Conclusions This study showed that the prevalence of hyperuricemia in patients with ischemic stroke was significantly higher as compared to the healthy population. Hyperuricemia can be considered as a risk factor for ischemic stroke because of its high prevalence in ischemic stroke patients. Our study explored the relationship between stroke and hyperuricemia and enabled increased understanding for caregivers so that effective management plans can be formulated for patients with ischemic stroke to prevent adverse outcomes., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2020, Irfan et al.)

Introduction Epilepsy is a burdensome disorder for affected individuals and community. There is limited data available on the epidemiological aspects of seizures in Pakistan and further research is necessary. We aimed to fill this gap by studying this information in epilepsy patients presenting to our neurology department. The purpose of this study is to evaluate the causes and types of seizures among the target population. Method This is a cross-sectional study conducted at the Department of Neurology, Dr. Ruth K.M. Pfau Civil Hospital Karachi. In this study we evaluated the causes and types of seizures among patients presenting to our department during the two-year study duration (January 2018-December 2019). Informed consent was taken. Detailed history was taken including features of seizure episodes, age at first seizure, family history and comorbid conditions. Relevant investigations were carried out. The data was compiled to deduce the relevant information using SPSS v20.0. T-test and Chi-square were used for analyzing the data. Results A total of 996 patients presented during the study duration. Primary seizures were found in 58% cases while secondary seizures were found in 42% cases. This distribution was more equal in children with 49.6% primary seizures and 50.4% secondary seizures; the gap widened in adults with 64.3% primary seizures and 35.7% secondary seizures. The most common cause of secondary seizures was neonatal encephalopathy which was present in 18.7% patients, followed by traumatic head injury in 18.2% patients. Central nervous system (CNS) infection was the cause in 17.9% patients, cerebral tumors in 14.1% patients, stroke in 11.5%, metabolic encephalopathy in 7.4%, febrile seizures in 6.5% and CNS malformations in 5.7% patients. The top three causes in children were neonatal encephalopathy (28.3%), CNS infections (19.3%) and febrile seizures (12.7%). Adults with secondary seizures were diagnosed most often with head trauma (25.2%), cerebral tumors (19.9%) and stroke (18.4%) as causative factors. The most common type of seizures was generalized onset tonic-clonic seizures which was found in 73.0% patients followed by focal to bilateral tonic-clonic seizures in 8.9% patients. Other types of seizures included focal aware seizures in 5.0%, mixed seizure types in 4.2%, focal impaired awareness seizures in 3.1%, absence seizures in 2.7%, myoclonic seizures in 2.0% and atonic seizures in 1.0% patients. Seizures in children were mostly generalized onset tonic-clonic seizures (75.4%), mixed seizure types (5.7%) and focal to bilateral tonic-clonic seizures (5.2%). In adults the three most common types corresponded to the overall result: generalized onset tonic-clonic seizures (71.2%), focal to bilateral tonic-clonic seizures (11.6%) and focal aware seizures (6.6%). Conclusion We found that the most common cause of seizures overall in our study population was primary seizures, though primary and secondary seizures were more evenly present in children. Among secondary causes neonatal encephalopathy stood out as the most common cause in children; head trauma was the predominant cause in adults. Most common type of seizures overall and in adults was generalized onset tonic-clonic seizures, followed by focal to bilateral tonic-clonic and focal aware seizure types. Pediatric patients presented most often with generalized onset tonic-clonic seizures, followed by mixed seizure types and focal to bilateral tonic-clonic seizures., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2020, Jawaid et al.)

Whereas hundreds of cells in the mouse embryonic aorta transdifferentiate to hematopoietic cells, only very few establish hematopoietic stem cell (HSC) identity at a single time point. The Gata2 transcription factor is essential for HSC generation and function. In contrast to surface-marker-based cell isolation, Gata2-based enrichment provides a direct link to the internal HSC regulatory network. Here, we use iterations of index-sorting of Gata2-expressing intra-aortic hematopoietic cluster (IAHC) cells, single-cell transcriptomics, and functional analyses to connect HSC identity to specific gene expression. Gata2-expressing IAHC cells separate into 5 major transcriptomic clusters. Iterative analyses reveal refined CD31, cKit, and CD27 phenotypic parameters that associate specific molecular profiles in one cluster with distinct HSC and multipotent progenitor function. Thus, by iterations of single-cell approaches, we identify the transcriptome of the first functional HSCs as they emerge in the mouse embryo and localize them to aortic clusters containing 1-2 cells., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pluripotency is regulated by a network of transcription factors that maintain early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However, Nanog is expressed during gastrulation across all the posterior epiblast, and only later in development is its expression restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which Nanog specifically represses genes involved in anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed Pou3f1 expression to be negatively correlated with that of Nanog during the early stages of differentiation. We have functionally demonstrated Pou3f1 to be a direct target of NANOG by using a dual transgene system for the controlled expression of Nanog Use of Nanog null ES cells further demonstrated a role for Nanog in repressing a subset of anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of Pou3f1 revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of Nanog inhibiting neural regulatory networks by repressing Pou3f1 at the onset of gastrulation.This article has an associated First Person interview with the joint first authors of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The mammalian embryo's caudal lateral epiblast (CLE) harbours bipotent progenitors, called neural mesodermal progenitors (NMPs), that contribute to the spinal cord and the paraxial mesoderm throughout axial elongation. Here, we performed a single cell analysis of different in vitro NMP populations produced either from embryonic stem cells (ESCs) or epiblast stem cells (EpiSCs) and compared them with E8.25 CLE mouse embryos. In our analysis of this region, our findings challenge the notion that NMPs can be defined by the exclusive co-expression of Sox2 and T at mRNA level. We analyse the in vitro NMP-like populations using a purpose-built support vector machine (SVM) based on the embryo CLE and use it as a classification model to compare the in vivo and in vitro populations. Our results show that NMP differentiation from ESCs leads to heterogeneous progenitor populations with few NMP-like cells, as defined by the SVM algorithm, whereas starting with EpiSCs yields a high proportion of cells with the embryo NMP signature. We find that the population from which the Epi-NMPs are derived in culture contains a node-like population, which suggests that this population probably maintains the expression of T in vitro and thereby a source of NMPs. In conclusion, differentiation of EpiSCs into NMPs reproduces events in vivo and suggests a sequence of events for the emergence of the NMP population., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Progenitors of the first hematopoietic cells in the mouse arise in the early embryo from Brachyury -positive multipotent cells in the posterior-proximal region of the epiblast, but the mechanisms that specify primitive blood cells are still largely unknown. Pluripotency factors maintain uncommitted cells of the blastocyst and embryonic stem cells in the pluripotent state. However, little is known about the role played by these factors during later development, despite being expressed in the postimplantation epiblast. Using a dual transgene system for controlled expression at postimplantation stages, we found that Nanog blocks primitive hematopoiesis in the gastrulating embryo, resulting in a loss of red blood cells and downregulation of erythropoietic genes. Accordingly, Nanog -deficient embryonic stem cells are prone to erythropoietic differentiation. Moreover, Nanog expression in adults prevents the maturation of erythroid cells. By analysis of previous data for NANOG binding during stem cell differentiation and CRISPR/Cas9 genome editing, we found that Tal1 is a direct NANOG target. Our results show that Nanog regulates primitive hematopoiesis by directly repressing critical erythroid lineage specifiers., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1 -/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.

Capturing where and how multipotency is lost is crucial to understand how blood formation is controlled. Blood lineage specification is currently thought to occur downstream of multipotent haematopoietic stem cells (HSC). Here we show that, in human, the first lineage restriction events occur within the CD19 - CD34 + CD38 - CD45RA - CD49f + CD90 + (49f + ) HSC compartment to generate myelo-lymphoid committed cells with no erythroid differentiation capacity. At single-cell resolution, we observe a continuous but polarised organisation of the 49f + compartment, where transcriptional programmes and lineage potential progressively change along a gradient of opposing cell surface expression of CLEC9A and CD34. CLEC9A hi CD34 lo cells contain long-term repopulating multipotent HSCs with slow quiescence exit kinetics, whereas CLEC9A lo CD34 hi cells are restricted to myelo-lymphoid differentiation and display infrequent but durable repopulation capacity. We thus propose that human HSCs gradually transition to a discrete lymphoid-primed state, distinct from lymphoid-primed multipotent progenitors, representing the earliest entry point into lymphoid commitment.

During gastrulation, cell types from all three germ layers are specified and the basic body plan is established 1 . However, molecular analysis of this key developmental stage has been hampered by limited cell numbers and a paucity of markers. Single-cell RNA sequencing circumvents these problems, but has so far been limited to specific organ systems 2 . Here, we report single-cell transcriptomic characterization of >20,000 cells immediately following gastrulation at E8.25 of mouse development. We identify 20 major cell types, which frequently contain substructure, including three distinct signatures in early foregut cells. Pseudo-space ordering of somitic progenitor cells identifies dynamic waves of transcription and candidate regulators, which are validated by molecular characterization of spatially resolved regions of the embryo. Within the endothelial population, cells that transition from haemogenic endothelial to erythro-myeloid progenitors specifically express Alox5 and its co-factor Alox5ap, which control leukotriene production. Functional assays using mouse embryonic stem cells demonstrate that leukotrienes promote haematopoietic progenitor cell generation. Thus, this comprehensive single-cell map can be exploited to reveal previously unrecognized pathways that contribute to tissue development.

Objectives: To identify sub-populations of intestinal preproglucagon-expressing (PPG) cells producing Glucagon-like Peptide-1, and their associated expression profiles of sensory receptors, thereby enabling the discovery of therapeutic strategies that target these cell populations for the treatment of diabetes and obesity., Methods: We performed single cell RNA sequencing of PPG-cells purified by flow cytometry from the upper small intestine of 3 GLU-Venus mice. Cells from 2 mice were sequenced at low depth, and from the third mouse at high depth. High quality sequencing data from 234 PPG-cells were used to identify clusters by tSNE analysis. qPCR was performed to compare the longitudinal and crypt/villus locations of cluster-specific genes. Immunofluorescence and mass spectrometry were used to confirm protein expression., Results: PPG-cells formed 3 major clusters: a group with typical characteristics of classical L-cells, including high expression of Gcg and Pyy (comprising 51% of all PPG-cells); a cell type overlapping with Gip-expressing K-cells (14%); and a unique cluster expressing Tph1 and Pzp that was predominantly located in proximal small intestine villi and co-produced 5-HT (35%). Expression of G-protein coupled receptors differed between clusters, suggesting the cell types are differentially regulated and would be differentially targetable., Conclusions: Our findings support the emerging concept that many enteroendocrine cell populations are highly overlapping, with individual cells producing a range of peptides previously assigned to distinct cell types. Different receptor expression profiles across the clusters highlight potential drug targets to increase gut hormone secretion for the treatment of diabetes and obesity., (Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.)

In mammals, specification of the three major germ layers occurs during gastrulation, when cells ingressing through the primitive streak differentiate into the precursor cells of major organ systems. However, the molecular mechanisms underlying this process remain unclear, as numbers of gastrulating cells are very limited. In the mouse embryo at embryonic day 6.5, cells located at the junction between the extra-embryonic region and the epiblast on the posterior side of the embryo undergo an epithelial-to-mesenchymal transition and ingress through the primitive streak. Subsequently, cells migrate, either surrounding the prospective ectoderm contributing to the embryo proper, or into the extra-embryonic region to form the yolk sac, umbilical cord and placenta. Fate mapping has shown that mature tissues such as blood and heart originate from specific regions of the pre-gastrula epiblast, but the plasticity of cells within the embryo and the function of key cell-type-specific transcription factors remain unclear. Here we analyse 1,205 cells from the epiblast and nascent Flk1(+) mesoderm of gastrulating mouse embryos using single-cell RNA sequencing, representing the first transcriptome-wide in vivo view of early mesoderm formation during mammalian gastrulation. Additionally, using knockout mice, we study the function of Tal1, a key haematopoietic transcription factor, and demonstrate, contrary to previous studies performed using retrospective assays, that Tal1 knockout does not immediately bias precursor cells towards a cardiac fate., Competing Interests: statement The authors declare no competing financial interests.

A single-step lateral flow immunoassay was developed and validated to detect okadaic acid (OA) and dinophysis toxins (DTXs), which cause diarrhetic shellfish poisoning. The performance characteristics of the test were investigated, in comparison to reference methods (liquid chromatography tandem mass spectrometry and/or bioassay), using both spiked and naturally contaminated shellfish. A portable reader was used to generate a qualitative result, indicating the absence or presence of OA-group toxins, at concentrations relevant to the maximum permitted level (MPL). Sample homogenates could be screened in 20 min (including extraction and assay time) for the presence of free toxins (OA, DTX1, DTX2). DTX3 detection could be included with the addition of a hydrolysis procedure. No matrix effects were observed from the species evaluated (mussels, scallops, oysters, and clams). Results from naturally contaminated samples (n = 72) indicated no false compliant results and no false noncompliant results at <50% MPL. Thus, the development of a new low-cost but highly effective tool for monitoring a range of important phycotoxins has been demonstrated.

A single-step lateral flow immunoassay (LFIA) was developed and validated for the rapid screening of paralytic shellfish toxins (PSTs) from a variety of shellfish species, at concentrations relevant to regulatory limits of 800 μg STX-diHCl equivalents/kg shellfish meat. A simple aqueous extraction protocol was performed within several minutes from sample homogenate. The qualitative result was generated after a 5 min run time using a portable reader which removed subjectivity from data interpretation. The test was designed to generate noncompliant results with samples containing approximately 800 μg of STX-diHCl/kg. The cross-reactivities in relation to STX, expressed as mean ± SD, were as follows: NEO: 128.9% ± 29%; GTX1&4: 5.7% ± 1.5%; GTX2&3: 23.4% ± 10.4%; dcSTX: 55.6% ± 10.9%; dcNEO: 28.0% ± 8.9%; dcGTX2&3: 8.3% ± 2.7%; C1&C2: 3.1% ± 1.2%; GTX5: 23.3% ± 14.4% (n = 5 LFIA lots). There were no indications of matrix effects from the different samples evaluated (mussels, scallops, oysters, clams, cockles) nor interference from other shellfish toxins (domoic acid, okadaic acid group). Naturally contaminated sample evaluations showed no false negative results were generated from a variety of different samples and profiles (n = 23), in comparison to reference methods (MBA method 959.08, LC-FD method 2005.06). External laboratory evaluations of naturally contaminated samples (n = 39) indicated good correlation with reference methods (MBA, LC-FD). This is the first LFIA which has been shown, through rigorous validation, to have the ability to detect most major PSTs in a reliable manner and will be a huge benefit to both industry and regulators, who need to perform rapid and reliable testing to ensure shellfish are safe to eat.

Reconstruction of the molecular pathways controlling organ development has been hampered by a lack of methods to resolve embryonic progenitor cells. Here we describe a strategy to address this problem that combines gene expression profiling of large numbers of single cells with data analysis based on diffusion maps for dimensionality reduction and network synthesis from state transition graphs. Applying the approach to hematopoietic development in the mouse embryo, we map the progression of mesoderm toward blood using single-cell gene expression analysis of 3,934 cells with blood-forming potential captured at four time points between E7.0 and E8.5. Transitions between individual cellular states are then used as input to develop a single-cell network synthesis toolkit to generate a computationally executable transcriptional regulatory network model of blood development. Several model predictions concerning the roles of Sox and Hox factors are validated experimentally. Our results demonstrate that single-cell analysis of a developing organ coupled with computational approaches can reveal the transcriptional programs that underpin organogenesis.

A lateral flow immunoassay (LFIA) has been developed and fully validated to detect the primary amnesic shellfish poisoning (ASP) toxin, domoic acid (DA). The performance characteristics of two versions of the test were investigated using spiked and naturally contaminated shellfish (mussels, scallops, oysters, clams, and cockles). The tests provide a qualitative result, to indicate the absence or presence of DA in extracts of shellfish tissues, at concentrations that are relevant to regulatory limits. The new rapid assay (LFIA version 2) was designed to overcome the performance limitations identified in the first version of the assay. The improved test uses an electronic reader to remove the subjective nature of the generated results, and the positive cut-off for screening of DA in shellfish was increased from 10 ppm (version 1) to 17.5 ppm (version 2). A simple extraction and test procedure was employed, which required minimal equipment and materials; results were available 15 min after sample preparation. Stability of the aqueous extracts at room temperature (22 °C) at four time points (up to 245 min after extraction) and across a range of DA concentrations was 100.3±1.3% and 98.8±2.4% for pre- and post-buffered extracts, respectively. The assay can be used both within laboratory settings and in remote locations. The accuracy of the new assay, to indicate negative results at or below 10 ppm DA, and positive results at or above 17.5 ppm, was 99.5% (n=216 tests). Validation data were obtained from a 2-day, randomised, blind study consisting of multiple LFIA lots (n=3), readers (n=3) and operators (n=3), carrying out multiple extractions of mussel tissue (n=3) at each concentration (0, 10, 17.5, and 20 ppm). No matrix effects were observed on the performance of the assay with different species (mussels, scallops, oysters, clams, and cockles). There was no impact on accuracy or interference from other phycotoxins, glutamic acid or glutamine with various strip incubations (8, 10, and 12 min). The accuracy of the assay, using naturally contaminated samples to indicate negative results at or below 12.5 ppm and positive results at or above 17.5 ppm, was 100%. Variability between three LFIA lots across a range of DA concentrations, expressed as coefficient of variation (% CV), was 1.1±0.4% (n=2 days) based on quantitative readings from the electronic reader. During an 8 week stability study, accuracy of the method with test strips stored at various temperatures (6, 22, 37 and 50 °C) was 100%. Validation for both versions included comparisons with results obtained using reference LC-UV methods., (© 2013 Elsevier B.V. All rights reserved.)

Background: Subspecialization defined pediatric surgery using Alder Hey innovations in neonatal surgical units (Rickham) and anesthesia (Jackson-Rees). In neonatal surgery, United Kingdom subspecialization for cloacal extrophy and biliary atresia acknowledges their dependence on multidisciplinary management and the desirability of caseload for training. We phased in regional subspecialization for esophageal atresia (EA) repair and replacement surgery while trainee numbers increased nationally to reduce hours. We examined EA outcomes and training during subspecialization., Methods: We analyzed EA cases (1999-2009) treated at Alder Hey Children's Hospital in two 5-year cohorts, the first early phase of incomplete subspecialization and the later near-total or "comprehensive" subspecialization phase. These periods approximated those before and after trainee numbers rose sharply to reduce working hours., Results: Of 119 cases, 60 in the early cohort shared similar characteristics with the 59 in the later cohort. Near-complete subspecialization was achieved in the second 5 years with 97% of cases performed under a surgeon with an EA specialty interest; in the earlier cohort, 25% of surgeries were undertaken by surgeons without EA subspecialty interest. With near-complete subspecialization, pediatric intensive care unit stay fell from 5 (4-11) to 4 (2-7) days (median (IQR); P = .005), and approaches such as the Bianchi axillary repair and Bax single-stage jejunal interposition were introduced; hospital stay went from 25 (12-63) to 17 (13-28) days (P = .27), and deaths, from 6 to 3 children (P = .49). Mortality was 7.6% (9/119) compared with 14% (19/134) in our previous institutional series (χ(2) = 2.8, P = .09), and neonatal mortality fell from 6% to 0 (P = .008). Near doubling of trainee numbers accompanied an approximately 3-fold fall in repairs per trainee over the study., Conclusion: Near-complete subspecialization for EA coincided with reduced pediatric intensive care unit stay, successful introduction of cosmetic axillary approaches, and extension of our replacement service to offer all interposition types. It has not reversed the steep decline in trainee experience of EA that has been associated with the greater numbers of trainees that have been employed to reduce working hours., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Life-threatening bleeding is a hazard of major tumor excision in children. However, fatalities from inadvertent arterial ligation should not be overlooked. Sacrococcygeal teratoma is the commonest neonatal tumor. Laparotomy to ligate the median sacral artery has been used to preempt potentially fatal resectional bleeding. Use of laparoscopy to achieve the same is an evolving technique, with only 7 neonatal cases described. As such, the Idea, Development, Exploration, Assessment, Long-term study (IDEAL) guidelines on surgical innovation recommend case reports addressing proof of concept, technical factors and safety tips. Fortunately, mistaken arterial division is so far unreported during laparoscopic median sacral artery ligation. However, as uptake widens, anatomical distortion by tumor and surgeon disorientation at endosurgery are risk factors for even such inconceivable complications. We report a successful case of laparoscopic vascular control for neonatal sacrococcygeal teratoma and demonstrate an observation that serves as a useful safety check for this procedure (as well as the open alternative)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Retroperitoneal teratomas are rare, often massive tumors usually presenting in infancy; being mostly mature lesions, their treatment is surgical but may represent a formidable challenge. Major vessel displacement may not be well demonstrated on imaging: vascular injuries are well-recognized surgical complications with urgent repair, ligation, or even segmental excision of major vessels being required. However, the literature provides few suggestions to avoid these problems. In our approach, we assessed the important effaced abdominal veins on imaging and at laparotomy to allow us to electively excise the suprarenal and infrarenal vena cava (with both renal vein ostia) and thereby resect a giant retroperitoneal teratoma without inadvertent vessel injury, major bleeding, renal disturbance, or tumor recurrence. Described for renal tumors, elective cavectomy has not been reported as a technique to manage primary retroperitoneal teratomas. In selected cases, with careful preservation of renal venous collaterals, we show this can be a well-tolerated, preemptive option to reduce the high risks of surgical complications., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Hypercalcaemia is a rare life-threatening complication of paediatric cancer that is commoner in haematological than solid malignancies and associated rarely with acute renal failure. Often refractory to medical therapy, control of hypercalcaemia in children with solid tumours may necessitate excision of localised tumours or urgent chemotherapy for metastatic ones. We present a child with refractory hypercalcaemia, bulky chemosensitive metastatic tumours and acute renal failure in whom chemotherapy posed high-risk of tumour lysis syndrome (TLS). Resection of the metastatic tumours successfully normalised the hypercalcaemia and represents a practical alternative control strategy in cases at high risk of TLS., (Copyright © 2011 Elsevier Inc. All rights reserved.)

High retropharyngeal neuroblastic tumors in children have been excised and debulked transorally or cervically, often with a covering tracheostomy. Although we and others have approached high thoracic lesions thoracoscopically, the trapdoor incision (or modification thereof) is generally reserved for cervicothoracic tumors with significant vessel encasement around the thoracic inlet. We report a case of symptomatic ganglioneuroma extending from the nasopharynx, at the level of the skull base, down to the aortic arch: macroscopic clearance was achieved via an extended trapdoor incision and without recourse to tracheostomy, transoral surgery, or transfusion., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Pyloroplasty and pyloric dilatation are methods used to ameliorate the symptoms of nonobstructive delayed gastric emptying in children. Our purpose was to review the results of these methods with respect to symptomatic improvements., Methods: We retrospectively reviewed the records of children who, exclusive of fundoplications, in the 11-year period before December 31, 2005, had undergone at our institution a Heineke-Mikulicz pyloroplasty (HMP) and/or a fluoroscopically guided balloon pyloric dilatation (FGBD). We recorded evidence of either short- or long-term subjective improvement of symptoms., Results: Twenty-three children underwent HMP. Five had no prior medical treatment. Of the 23, 3 had no subjective improvement in symptoms. Of the 20 who had subjective evidence of improvement recorded, 13 (56.5%) proved to be long-term and 7 short-term improvement. Eight children had FGBD. All 8 had prior medical treatment that had failed. There were 3 who displayed no subjective evidence of improvement, whereas 5 had subjective evidence of improvement-3 (37.5%) long term and 2 short term., Conclusions: Heineke-Mikulicz pyloroplasty and FGBD in this study demonstrated only in limited numbers long-term improvement of symptoms. Efforts to understand more fully gastric emptying and to define those children who should undergo FGBD or HMP are needed.