Sacrococcygeal teratomas: midline reconstruction improves cosmesis without compromising outcomes.

AIM: Several studies have addressed the long-term functional, psychosexual and psychosocial outcomes following sacrococcygeal teratoma (SCT) excision. It is well reported that the classical chevron incision and reconstruction can leave a cosmetically unsatisfactory result; however, there is little in the literature focussed on improving this outcome. In our institution the preference is to perform a midline reconstruction, where possible, this is felt to improve appearance without compromising the oncological or functional outcome. The aim of this study was to evaluate patient-perceived cosmetic outcomes of the midline reconstruction. METHODS: All patients undergoing surgery for SCT between 2007 and 2020 were included in the study. Patient demographics, operation type, functional outcome and recurrence were all recorded. The primary outcome measure was patient/parent satisfaction with the cosmetic appearance. This was assessed using both qualitative and quantitative methodologies. Following ethical approval parents were asked questions from two existing validated patient outcome questionnaires: "Patient and Observer Scar Assessment Scale" (POSAS) v2.0 and the "Patient Scar Assessment Questionnaire". RESULTS: Thirty-two patients underwent surgery at our institution for SCT during the study period. Twenty-four had a posterior approach with midline reconstruction, two laparotomy and excision (excluded from this study) and six had a combined approach. Median follow-up was 35 months (8.5-96 months). There were no recurrences. 4/30 (13%) have persistent urological symptoms, and 1/30 (3%) has constipation requiring bowel management. Questionnaires were sent to 26/30 families with a 77% return rate. Median total score was 11 (7.4-17.5) on a 60-point scale (6, as normal skin, 60, worst imaginable scar). Twenty (95%) reported that the scar never affects the child's activities and 15 (71%) said they are "not at all" conscious of the scar. CONCLUSION: Scars can lead to an array of cosmetic, functional, and psychological consequences and as such consideration needs to be given to scarring following surgery for sacrococcygeal teratomas. This study demonstrates that a midline reconstruction produces a cosmetically favourable outcome. We, therefore, recommend where appropriate a midline reconstruction should be considered for SCT.

Esophageal atresia and tracheoesophageal fistula associated with tetralogy of Fallot: a review of mortality.

INTRODUCTION: Oesophageal atresia ± tracheoesophageal fistula (EA/TEF) associated with congenital heart disease (CHD) carries a worse prognosis than EA/TEF alone. Though the Spitz classification takes major CHD into account, there are no data regarding survival with the specific combination of EA/TEF and Tetralogy of Fallot (TOF). With advances in postnatal care, we hypothesised that, survival is improving in these complex patients. This study reports morbidity and mortality outcomes of newborns with oesophageal atresia and TOF cardiac malformations METHODS: All patients with EA/TEF and TOF treated at Alder Hey Children's Hospital between the years 2000-2020, were identified. Data sets regarding gestation, birth weight, associated anomalies, operative intervention, morbidity, and mortality were analysed. RESULTS: Of a total of 350, EA/TEF patients 9 (2.6%) cases had EA/TEF associated with TOF (M:F 4:5). The median gestational age was 35/40 (range 28-41 weeks) with a median birth weight of 1790 g (range 1060-3350 g). Overall survival was 56% (5/9 cases) and all survivors remain under follow up (range 37-4458 days). Surgical strategies for managing EA/TEF with Fallot's tetralogy included 6/9 primary repairs and 3/9 cases with TEF ligation only (+ gastrostomy ± oesophagostomy). CONCLUSIONS: This study reports outcome data from one of the largest series of EA TEF patients with Fallot's tetralogy. Whilst outcomes may be challenging for this unique patient cohort, survival metrics provide important prognostic information that can be widely shared with health care teams and parents.

OSCI: standardized stem cell ontology representation and use cases for stem cell investigation.

BACKGROUND: Stem cells and stem cell lines are widely used in biomedical research. The Cell Ontology (CL) and Cell Line Ontology (CLO) are two community-based OBO Foundry ontologies in the domains of in vivo cells and in vitro cell line cells, respectively. RESULTS: To support standardized stem cell investigations, we have developed an Ontology for Stem Cell Investigations (OSCI). OSCI imports stem cell and cell line terms from CL and CLO, and investigation-related terms from existing ontologies. A novel focus of OSCI is its application in representing metadata types associated with various stem cell investigations. We also applied OSCI to systematically categorize experimental variables in an induced pluripotent stem cell line cell study related to bipolar disorder. In addition, we used a semi-automated literature mining approach to identify over 200 stem cell gene markers. The relations between these genes and stem cells are modeled and represented in OSCI. CONCLUSIONS: OSCI standardizes stem cells found in vivo and in vitro and in various stem cell investigation processes and entities. The presented use cases demonstrate the utility of OSCI in iPSC studies and literature mining related to bipolar disorder.

Genetic resistance determinants to fusidic acid and chlorhexidine in variably susceptible staphylococci from dogs.

BACKGROUND: Concern exists that frequent use of topically-applied fusidic acid (FA) and chlorhexidine (CHX) for canine pyoderma is driving clinically relevant resistance, despite rare description of FA and CHX genetic resistance determinants in canine-derived staphylococci. This study aimed to determine minimum inhibitory concentrations (MICs) and investigate presence of putative resistance determinants for FA and CHX in canine-derived methicillin-resistant (MR) and -susceptible (MS) staphylococci. Plasmid-mediated resistance genes (fusB, fusC, fusD, qacA/B, smr; PCR) and MICs (agar dilution) of FA and CHX were investigated in 578 staphylococci (50 MR S. aureus [SA], 50 MSSA, 259 MR S. pseudintermedius [SP], 219 MSSP) from Finland, U.S.A., North (NUK) and South-East U.K. (SEUK) and Germany. In all isolates with FA MIC ≥64 mg/L (n = 27) fusA and fusE were amplified and sequenced. RESULTS: FA resistance determinants (fusA mutations n = 24, fusB n = 2, fusC n = 36) were found in isolates from all countries bar U.S.A. and correlated with higher MICs (≥1 mg/L), although 4 SP isolates had MICs of 0.06 mg/L despite carrying fusC. CHX MICs did not correlate with qacA/B (n = 2) and smr (n = 5), which were found in SEUK SA, and SP from NUK and U.S.A. CONCLUSIONS: Increased FA MICs were frequently associated with fusA mutations and fusC, and this is the first account of fusB in SP. Despite novel description of qacA/B in SP, gene presence did not correlate with CHX MIC. Selection pressure from clinical use might increase prevalence of these genetic determinants, but clinical significance remains uncertain in relation to high skin concentrations achieved by topical therapy.

Evaluation of crystallographic strain, rotation and defects in functional oxides by the moiré effect in scanning transmission electron microscopy.

Moiré patterns in scanning transmission electron microscopy (STEM) images of epitaxial perovskite oxides are used to assess strain and defect densities over fields of view extending over several hundred nanometers. The patterns arise from the geometric overlap of the rastered STEM electron beam and the samples' crystal periodicities and we explore the emergence and application of these moiré fringes for rapid strain analysis. Using the epitaxial functional oxide perovskites BiFeO3 and Pr1-x Ca x MnO3, we discuss the impact of large degrees of strain on the quantification of STEM moiré patterns, identify defects in the fringe patterns and quantify strain and lattice rotation. Such a wide-area analysis of crystallographic strain and defects is crucial for developing structure-function relations of functional oxides and we find the STEM moiré technique to be an attractive means of structural assessment that can be readily applied to low dose studies of damage sensitive crystalline materials.

Neurodevelopmental origins of bipolar disorder: iPSC models.

Bipolar disorder (BP) is a chronic neuropsychiatric condition characterized by pathological fluctuations in mood from mania to depression. Adoption, twin and family studies have consistently identified a significant hereditary component to BP, yet there is no clear genetic event or consistent neuropathology. BP has been suggested to have a developmental origin, although this hypothesis has been difficult to test since there are no viable neurons or glial cells to analyze, and research has relied largely on postmortem brain, behavioral and imaging studies, or has examined proxy tissues including saliva, olfactory epithelium and blood cells. Neurodevelopmental factors, particularly pathways related to nervous system development, cell migration, extracellular matrix, H3K4 methylation, and calcium signaling have been identified in large gene expression and GWAS studies as altered in BP. Recent advances in stem cell biology, particularly the ability to reprogram adult somatic tissues to a pluripotent state, now make it possible to interrogate these pathways in viable cell models. A number of induced pluripotent stem cell (iPSC) lines from BP patient and healthy control (C) individuals have been derived in several laboratories, and their ability to form cortical neurons examined. Early studies suggest differences in activity, calcium signaling, blocks to neuronal differentiation, and changes in neuronal, and possibly glial, lineage specification. Initial observations suggest that differentiation of BP patient-derived neurons to dorsal telencephalic derivatives may be impaired, possibly due to alterations in WNT, Hedgehog or Nodal pathway signaling. These investigations strongly support a developmental contribution to BP and identify novel pathways, mechanisms and opportunities for improved treatments.

Epigenetic silencing of engineered L1 retrotransposition events in human embryonic carcinoma cells.

Long interspersed element-1 (LINE-1 or L1) retrotransposition continues to affect human genome evolution. L1s can retrotranspose in the germline, during early development and in select somatic cells; however, the host response to L1 retrotransposition remains largely unexplored. Here we show that reporter genes introduced into the genome of various human embryonic carcinoma-derived cell lines (ECs) by L1 retrotransposition are rapidly and efficiently silenced either during or immediately after their integration. Treating ECs with histone deacetylase inhibitors rapidly reverses this silencing, and chromatin immunoprecipitation experiments revealed that reactivation of the reporter gene was correlated with changes in chromatin status at the L1 integration site. Under our assay conditions, rapid silencing was also observed when reporter genes were delivered into ECs by mouse L1s and a zebrafish LINE-2 element, but not when similar reporter genes were delivered into ECs by Moloney murine leukaemia virus or human immunodeficiency virus, suggesting that these integration events are silenced by distinct mechanisms. Finally, we demonstrate that subjecting ECs to culture conditions that promote differentiation attenuates the silencing of reporter genes delivered by L1 retrotransposition, but that differentiation, in itself, is not sufficient to reactivate previously silenced reporter genes. Thus, our data indicate that ECs differ from many differentiated cells in their ability to silence reporter genes delivered by L1 retrotransposition.

Mammalian COPII coat component SEC24C is required for embryonic development in mice.

COPII-coated vesicles mediate the transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi. SEC24 is the COPII component primarily responsible for recruitment of protein cargoes into nascent vesicles. There are four Sec24 paralogs in mammals, with mice deficient in SEC24A, -B, and -D exhibiting a wide range of phenotypes. We now report the characterization of mice with deficiency in the fourth Sec24 paralog, SEC24C. Although mice haploinsufficient for Sec24c exhibit no apparent abnormalities, homozygous deficiency results in embryonic lethality at approximately embryonic day 7. Tissue-specific deletion of Sec24c in hepatocytes, pancreatic cells, smooth muscle cells, and intestinal epithelial cells results in phenotypically normal mice. Thus, SEC24C is required in early mammalian development but is dispensable in a number of tissues, likely as a result of compensation by other Sec24 paralogs. The embryonic lethality resulting from loss of SEC24C occurs considerably later than the lethality previously observed in SEC24D deficiency; it is clearly distinct from the restricted neural tube phenotype of Sec24b null embryos and the mild hypocholesterolemic phenotype of adult Sec24a null mice. Taken together, these results demonstrate that the four Sec24 paralogs have developed unique functions over the course of vertebrate evolution.

L1 retrotransposition in human neural progenitor cells.

Long interspersed element 1 (LINE-1 or L1) retrotransposons have markedly affected the human genome. L1s must retrotranspose in the germ line or during early development to ensure their evolutionary success, yet the extent to which this process affects somatic cells is poorly understood. We previously demonstrated that engineered human L1s can retrotranspose in adult rat hippocampus progenitor cells in vitro and in the mouse brain in vivo. Here we demonstrate that neural progenitor cells isolated from human fetal brain and derived from human embryonic stem cells support the retrotransposition of engineered human L1s in vitro. Furthermore, we developed a quantitative multiplex polymerase chain reaction that detected an increase in the copy number of endogenous L1s in the hippocampus, and in several regions of adult human brains, when compared to the copy number of endogenous L1s in heart or liver genomic DNAs from the same donor. These data suggest that de novo L1 retrotransposition events may occur in the human brain and, in principle, have the potential to contribute to individual somatic mosaicism.

Expression of Neurogenin 1 in mouse embryonic stem cells directs the differentiation of neuronal precursors and identifies unique patterns of down-stream gene expression.

BACKGROUND: Delineating the cascades of growth and transcription factor expression that shape the developing nervous system will improve our understanding of its molecular histogenesis and suggest strategies for cell replacement therapies. In the current investigation, we examined the ability of the proneural gene, Neurogenin1 (Neurog1; also Ngn1, Neurod3), to drive differentiation of pluripotent embryonic stem cells (ESC). RESULTS: Transient expression of Neurog1 in ESC was sufficient to initiate neuronal differentiation, and produced neuronal subtypes reflecting its expression pattern in vivo. To begin to address the molecular mechanisms involved, we used microarray analysis to identify potential down-stream targets of Neurog1 expressed at sequential stages of neuronal differentiation. CONCLUSIONS: ESC expressing Neurogenin1 begin to withdraw from cycle and form precursors that differentiate exclusively into neurons. This work identifies unique patterns of gene expression following expression of Neurog1, including genes and signaling pathways involved in process outgrowth and cell migration, regional differentiation of the nervous system, and cell cycle.

Human-Induced Pluripotent Stem Cell (iPSC)-Derived GABAergic Neuron Differentiation in Bipolar Disorder.

Bipolar disorder (BP) is a recurring psychiatric condition characterized by alternating episodes of low energy (depressions) followed by manias (high energy). Cortical network activity produced by GABAergic interneurons may be critical in maintaining the balance in excitatory/inhibitory activity in the brain during development. Initially, GABAergic signaling is excitatory; with maturation, these cells undergo a functional switch that converts GABAA channels from depolarizing (excitatory) to hyperpolarizing (inhibitory), which is controlled by the intracellular concentration of two chloride transporters. The earliest, NKCC1, promotes chloride entry into the cell and depolarization, while the second (KCC2) stimulates movement of chloride from the neuron, hyperpolarizing it. Perturbations in the timing or expression of NKCC1/KCC2 may affect essential morphogenetic events including cell proliferation, migration, synaptogenesis and plasticity, and thereby the structure and function of the cortex. We derived induced pluripotent stem cells (iPSC) from BP patients and undiagnosed control (C) individuals, then modified a differentiation protocol to form GABAergic interneurons, harvesting cells at sequential stages of differentiation. qRT-PCR and RNA sequencing indicated that after six weeks of differentiation, controls transiently expressed high levels of NKCC1. Using multi-electrode array (MEA) analysis, we observed that BP neurons exhibit increased firing, network bursting and decreased synchrony compared to C. Understanding GABA signaling in differentiation may identify novel approaches and new targets for treatment of neuropsychiatric disorders such as BP.

Gene expression alterations in bipolar disorder postmortem brains.

OBJECTIVES: Bipolar disorder (BD) is a mental illness of unknown neuropathology and has several genetic associations. Antipsychotics are effective for the treatment of acute mania, psychosis, or mixed states in individuals with BD. We aimed to identify gene transcripts differentially expressed in postmortem brains from antipsychotics-exposed individuals with BD (hereafter the 'exposed' group), non-exposed individuals with BD (hereafter the 'non-exposed' group), and controls. METHODS: We quantified the abundance of gene transcripts in postmortem brains from seven exposed individuals, seven non-exposed individuals, and 12 controls with the Affymetrix U133P2 GeneChip microarrays and technologies. We applied a q-value of ≤0.005 to identify statistically significant transcripts with mean abundance differences between the exposed, non-exposed and control groups. RESULTS: We identified 2191 unique genes with significantly altered expression levels in non-exposed brains compared to those in the control and exposed groups. The expression levels of these genes were not significantly different between exposed brains and controls, suggesting a normalization effect of antipsychotics on the expression of these genes. Gene ontology (GO) enrichment analysis showed significant (Bonferroni p ≤ 0.05) clustering of subgroups of the 2191 genes under many GO terms; notably, the protein products of genes enriched are critical to the function of synapses, affecting, for example, intracellular trafficking and synaptic vesicle biogenesis, transport, release and recycling, as well as organization and stabilization of the node of Ranvier. CONCLUSIONS: These results support a hypothesis of synaptic and intercellular communication impairment in BD. The apparent normalization of expression patterns with exposure to antipsychotic medication may represent a physiological process that relates both to etiology and improvement patterns of the disorder.

Nestin-expressing cells in the developing, mature and noise-exposed cochlear epithelium.

The auditory sensory epithelium in non-mammalian vertebrates can replace lost hair cells by transdifferentiation of supporting cells, but this regenerative ability is lost in the mammalian cochlea. Future cell-based treatment of hearing loss may depend on stem cell transplantation or on transdifferentiation of endogenous cells in the cochlea. For both approaches, identification of cells with stem cell features within the mature cochlea may be useful. Here we use a Nestin-ß-gal mouse to examine the presence of Nestin positive cells in the mature auditory epithelium, and determine how overstimulation of the ear impacts these cells. Nestin positive cells were found in the apical turn of the cochlea lateral to the outer hair cell area. This pattern of expression persisted into mature age. The area of Nestin positive cells was increased after the noise lesion. This increase in area coincided with an increase in expression of the Nestin mRNA. The data suggest that cells with potential stem cell features remain in the mature mammalian cochlea, restricted to the apical turn, and that an additional set of signals is necessary to trigger their contribution to cell replacement therapy in the ear. As such, this population of cells could serve to generate cochlear stem cells for research and potential therapy, and may be a target for treatments based on induced transdifferentiation of endogenous cochlear cells.

Dynamic changes in Wnt signaling are required for neuronal differentiation of mouse embryonic stem cells.

Embryonic stem cells (ESC) and the epiblast share a similar gene expression profile and an attenuated cell cycle, making them an accessible and tractable model system to study lineage choice at gastrulation. Differentiation of the epiblast and ESC to the mesendodermal lineage has been shown to rely on Wnt/ß-catenin signaling; which counterintuitively, is also required to inhibit differentiation and maintain pluripotency. To examine these seemingly contradictory roles, we developed a mouse ESC (ESC) line that inducibly expresses a dominant negative Tcf4 (dnTcf4) protein to block canonical Wnt signaling. Cells expressing the dnTcf4 protein differentiated largely to Sox3 positive neural precursors but were unable to progress to ßIII tubulin positive neurons unless Wnt signaling was derepressed, demonstrating a sequential requirement for Wnt signaling in lineage differentiation. To determine if Wnt/ß-catenin signaling is similarily required at sequential stages of neural differentiation in the intact embryo, we delivered shRNA targeting ß-catenin to pregnant mice on E5.5 of development. Blocking canonical Wnt signaling during post-implantation development increased the number of neural precursors which failed to differentiate to mature neurons, and produced defects of embryonic axis elongation, neurulation and neural tube closure that phenocopy the ß-catenin null embryo. These results demonstrate that lineage differentiation relies on sequential repression and derepression of critical signaling pathways involved in maintaining pluripotency versus differentiation.

Role of adiponectin in the development of high fat diet-induced metabolic abnormalities in mice.

The adipokine adiponectin is decreased in severe obesity and is inversely associated with adipose mass. Adiponectin is associated with insulin sensitivity and cardioprotection. Obesity frequently results in the development of a "cardiometabolic syndrome" characterized by increased circulating insulin and leptin, and cardiac hypertrophy and dysfunction. This study examined if adiponectin-deficiency affects the development of metabolic and cardiac abnormalities in response to modest obesity. Mice were studied under normal conditions and with mild cardiac pressure-overload induced by abdominal aortic banding. After surgery, wild type and adiponectin-deficient mice were fed a high-fat diet for 8 weeks (45% energy from fat vs. 10%). In wild type mice the high-fat diet increased fat and whole body mass, which corresponded with elevated circulating insulin and leptin and a decrease the glucose/insulin ratio. On the other hand, in adiponectin-deficient mice the high-fat diet had less impact on body mass and no effect on fat mass, insulin, leptin, or glucose/insulin. There was modest cardiac hypertrophy with aortic banding, but no cardiac dysfunction or effects of adiponectin deficiency or diet. The results suggest that the increase in adipose mass, leptin and insulin induced by a high fat diet is dependent on adiponectin. The lack of accelerated cardiac hypertrophy and dysfunction in the adiponectin-deficient mice subjected to aortic banding and the high-fat diet suggest that adiponectin may not play a major role in protecting the heart during the early stages of diet-induced obesity.

DNAzyme-Based Lithium-Selective Imaging Reveals Higher Lithium Accumulation in Bipolar Disorder Patient-Derived Neurons.

Lithium has been a drug for bipolar disorders (BD) for over 70 years; however, its usage has been limited by its narrow therapeutic window (between 0.6 and 1.2 mM). Understanding the cellular distribution of lithium ions (Li+) in patient cells will offer deep insight into this limitation, but selective imaging of Li+ in living cells under biomedically relevant concentration ranges has not been achieved. Herein, we report in vitro selection and development of a Li+-specific DNAzyme fluorescent sensor with >100-fold selectivity over other biorelevant metal ions. This sensor allows comparative Li+ visualization in HeLa cells, human neuronal progenitor cells (NPCs), and neurons derived from BD patients and healthy controls. Strikingly, we detected enhanced accumulation of Li+ in cells derived from BD patients compared with healthy controls in differentiated neurons but not NPCs. These results establish the DNAzyme-based sensor as a novel platform for biomedical research into BD and related areas using lithium drugs.

Murine SEC24D can substitute functionally for SEC24C during embryonic development.

The COPII component SEC24 mediates the recruitment of transmembrane cargos or cargo adaptors into newly forming COPII vesicles on the ER membrane. Mammalian genomes encode four Sec24 paralogs (Sec24a-d), with two subfamilies based on sequence homology (SEC24A/B and C/D), though little is known about their comparative functions and cargo-specificities. Complete deficiency for Sec24d results in very early embryonic lethality in mice (before the 8 cell stage), with later embryonic lethality (E7.5) observed in Sec24c null mice. To test the potential overlap in function between SEC24C/D, we employed dual recombinase mediated cassette exchange to generate a Sec24cc-d allele, in which the C-terminal 90% of SEC24C has been replaced by SEC24D coding sequence. In contrast to the embryonic lethality at E7.5 of SEC24C-deficiency, Sec24cc-d/c-d pups survive to term, though dying shortly after birth. Sec24cc-d/c-d pups are smaller in size, but exhibit no other obvious developmental abnormality by pathologic evaluation. These results suggest that tissue-specific and/or stage-specific expression of the Sec24c/d genes rather than differences in cargo export function explain the early embryonic requirements for SEC24C and SEC24D.

Uniform cell seeding and generation of overlapping gradient profiles in a multiplexed microchamber device with normally-closed valves.

Generation of stable soluble-factor gradients in microfluidic devices enables studies of various cellular events such as chemotaxis and differentiation. However, many gradient devices directly expose cells to constant fluid flow and that can induce undesired responses from cells due to shear stress and/or wash out of cell-secreted molecules. Although there have been devices with flow-free gradients, they typically generate only a single condition and/or have a decaying gradient profile that does not accommodate long-term experiments. Here we describe a microdevice that generates several chemical gradient conditions on a single platform in flow-free microchambers which facilitates steady-state gradient profiles. The device contains embedded normally-closed valves that enable fast and uniform seeding of cells to all microchambers simultaneously. A network of microchannels distributes desired solutions from easy-access open reservoirs to a single output port, enabling a simple setup for inducing flow in the device. Embedded porous filters, sandwiched between the microchannel networks and cell microchambers, enable diffusion of biomolecules but inhibit any bulk flow over the cells.