Exploiting spatiotemporal regulation of FZD5 during neural patterning for efficient ventral midbrain specification.

The Wnt/ß-catenin signaling governs anterior-posterior neural patterning during development. Current human pluripotent stem cell (hPSC) differentiation protocols use a GSK3 inhibitor to activate Wnt signaling to promote posterior neural fate specification. However, GSK3 is a pleiotropic kinase involved in multiple signaling pathways and, as GSK3 inhibition occurs downstream in the signaling cascade, it bypasses potential opportunities for achieving specificity or regulation at the receptor level. Additionally, the specific roles of individual FZD receptors in anterior-posterior patterning are poorly understood. Here, we have characterized the cell surface expression of FZD receptors in neural progenitor cells with different regional identity. Our data reveal unique upregulation of FZD5 expression in anterior neural progenitors, and this expression is downregulated as cells adopt a posterior fate. This spatial regulation of FZD expression constitutes a previously unreported regulatory mechanism that adjusts the levels of ß-catenin signaling along the anterior-posterior axis and possibly contributes to midbrain-hindbrain boundary formation. Stimulation of Wnt/ß-catenin signaling in hPSCs, using a tetravalent antibody that selectively triggers FZD5 and LRP6 clustering, leads to midbrain progenitor differentiation and gives rise to functional dopaminergic neurons in vitro and in vivo.

A Norrin/Wnt surrogate antibody stimulates endothelial cell barrier function and rescues retinopathy.

The FZD4:LRP5:TSPAN12 receptor complex is activated by the secreted protein Norrin in retinal endothelial cells and leads to ßcatenin-dependent formation of the blood-retina-barrier during development and its homeostasis in adults. Mutations disrupting Norrin signaling have been identified in several congenital diseases leading to hypovascularization of the retina and blindness. Here, we developed F4L5.13, a tetravalent antibody designed to induce FZD4 and LRP5 proximity in such a way as to trigger ßcatenin signaling. Treatment of cultured endothelial cells with F4L5.13 rescued permeability induced by VEGF in part by promoting surface expression of junction proteins. Treatment of Tspan12-/- mice with F4L5.13 restored retinal angiogenesis and barrier function. F4L5.13 treatment also significantly normalized neovascularization in an oxygen-induced retinopathy model revealing a novel therapeutic strategy for diseases characterized by abnormal angiogenesis and/or barrier dysfunction.

Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells.

Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5+ population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2ß1, integrin ß4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP+ cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5+ ISCs. Considering the key roles Lgr5+ ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy).

Incidence and Management of Thrombotic and Thromboembolic Complications Following the Superior Cavopulmonary Anastomosis Procedure: A Literature Review.

The objective of this literature review was to estimate the incidence of thrombosis and thromboembolism associated with the superior cavopulmonary anastomosis (SCPA) procedure and its variants and to examine current thromboprophylaxis regimens utilized. MEDLINE and EMBASE were searched from inception to August 2017 for all prospective and retrospective cohort studies explicitly reporting incidence of thrombosis, thromboembolism, or shunt occlusion in neonates, infants, and children undergoing 1 or more variants of the SCPA procedure. End points included thrombotic events and thromboembolic events (strokes and pulmonary embolisms) as primary outcomes, and overall mortality as a secondary outcome, at the last available follow-up time point. Of 1303 unique references identified, 13 cohort studies were deemed eligible. Reported incidence of thrombosis and thromboembolic events ranged from 0% to 28.0% and from 0% to 12.5%, respectively. Reported incidence of major bleeding events ranged from 0% to 2.9%. Reported overall mortality ranged from 2.5% to 50.5% across studies. Thromboprophylaxis protocols varied across institutions and studies, most commonly involving unfractionated heparin (UFH), warfarin, enoxaparin, acetylsalicylic acid (ASA), or combinations of ASA and warfarin, ASA and low-molecular-weight heparin (LMWH), UFH and LMWH, and UFH and ASA; several studies did not specify a protocol. Due to substantial variability in reported event rates, no clear correlation was identified between prophylaxis protocols and postoperative thrombotic complications. Despite guidance recommending postoperative UFH as standard practice, thromboprophylaxis protocols varied across institutions and studies. More robust trials evaluating different thromboprophylaxis regimens for the management of these patients are warranted.

Lymphoepithelioma-like, a variant of urothelial carcinoma of the urinary bladder: a case report and systematic review for optimal treatment modality for disease-free survival.

BACKGROUND: Lymphoepithelioma-like carcinoma (LELC) is a rare high-grade carcinoma that resembles nasopharyngeal lymphoepithelioma and can occur throughout the body. First reported in 1991, bladder LELC has an incidence of about 1% of all bladder carcinomas. Due to its rare occurrence, prognoses and ideal treatment guidelines have not been clearly defined. METHODS: A PubMed search was performed using two terms, "lymphoepithelioma-like carcinoma" and "bladder." Review articles, articles in foreign languages, expression studies, and studies not performed in the bladder were excluded. We report a case of LELC of the bladder including treatment and outcome and performed a systematic review of all 36 available English literatures from 1991 to 2016 including the present case to identify factors affecting disease-free survival. RESULTS: One hundred forty cases of bladder LELC were analyzed. The mean age of the patients was 70.1 years ranging from 43 to 90 years with 72% males and 28% females. Pure LELC occurs most often at 46% followed by mixed LELC 28% and predominant LELC 26%. EBV testing was negative in all cases tested. Mean follow-up length for all cases was 33.8 months with no evidence of disease in 62.2%, while 11.1% died of disease, 10.4% alive with metastasis, and 8.2% died without disease. 5.0% of cases had recurrence at an average of 31.3 months. Prognosis is significantly favorable for patients presenting with pure or predominant forms of LELC compared to mixed type (p < 0.0001). The treatment significantly associated with the highest disease mortality and lowest disease-free survival was TURBT alone when compared to any multi-modality treatment (p < 0.01). CONCLUSION: We conclude that the best treatment modality associated with the highest disease-free survival is multi-modal treatment including radical cystectomy.

Incidence and Management of Thrombotic and Thromboembolic Complications Following the Norwood Procedure: A Systematic Review.

BACKGROUND: The stage 1 Norwood procedure and its variants represent the first step of palliation for hypoplastic left heart syndrome. Although appropriate postoperative thromboprophylaxis is integral, significant variance remains across institutional practices. The purpose of this systematic review is to estimate the incidence of thrombosis and thromboembolism following the Norwood or modified Blalock-Taussig shunt procedure and examine current thromboprophylaxis regimens. METHODS: Ovid MEDLINE and Embase were searched from January 2000 to June 2016 for primary studies explicitly reporting incidence of thrombosis, thromboembolism (strokes and pulmonary embolisms), or shunt occlusion in neonates, infants, and children undergoing the Norwood procedure or any variant. All-cause mortality was a secondary outcome of interest. RESULTS: Of 887 identified articles, 15 cohort studies were deemed eligible, the majority including modified Blalock-Taussig shunt patients. Reported incidence of thrombosis ranged from 0% to 40%; thromboembolic events were rarely reported. Overall mortality ranged from 4.5% to 31.3% across studies. Although most studies involved the long-term acetylsalicylic acid use, thromboprophylaxis strategies varied across studies. Due to substantial variability in event rates, no correlation was identified with thrombotic complications. DISCUSSION: Clinical practice guidelines recommend that patients receive intraoperative unfractionated heparin therapy and either aspirin or no antithrombotic therapy postoperatively. Our findings suggest a substantial risk of thrombosis and thromboembolism and demonstrate substantial variation in thromboprophylaxis practices. CONCLUSION: Although postoperative thromboprophylaxis seems optimal, it remains controversial whether the long-term aspirin use is most effective. Our findings highlight the lack of a gold-standard thromboprophylaxis strategy and emphasize the need for more consistency.

Lunar Phases and Emergency Department Visits for Renal Colic Due to Ureteral Calculus.

BACKGROUND: Urolithiasis affects an estimated 5% of the population and the lifetime risk of passing a stone in the urinary tract is estimated to be 8-10%. Urinary calculus formation is highly variable and while certain risk factors such as age, gender, seasonality, anatomic abnormality, and metabolic diseases have been identified, not much is known regarding the association of environmental factors such as lunar phases on renal colic. We conducted a retrospective study to test the hypothesis that full moon phase is an environmental factor associated for increased emergency department (ED) visits for renal colic due to ureteral calculus. METHODS: We analyzed 559 renal colic diagnoses by the ED at the University of Nebraska Medical Center in a 24-month period and compared them with corresponding lunar phases as well as supermoon events. The lunar phases were defined as full moon ± two days, new moon ± two days, and the days in-between as normal days according to the lunar calendar. Supermoon event dates were obtained from NASA. RESULTS: 90 cases (16.1%) were diagnosed during full moon phase, 89 cases (15.9%) were diagnosed during new moon phase, and 380 cases (68.0%) were diagnosed during normal days. The incidence of renal colic showed no statistically significant association with lunar phases or supermoon events. CONCLUSION: In this retrospective longitudinal study with adequate power, neither full moon phase nor supermoon event exhibited an association with increased renal colic diagnoses due to ureteral calculus by the ED at the University of Nebraska Medical Center.

Deletion of Inpp5a causes ataxia and cerebellar degeneration in mice.

The progressive and permanent loss of cerebellar Purkinje cells (PC) is a hallmark of many inherited ataxias. Mutations in several genes involved in the regulation of Ca(2+) release from intracellular stores by the second messenger IP3 have been associated with PC dysfunction or death. While much is known about the defects in production and response to IP3, less is known about the defects in breakdown of the IP3 second messenger. A mutation in Inpp4a of the pathway is associated with a severe, early-onset PC degeneration in the mouse model weeble. The step preceding the removal of the 4-phosphate is the removal of the 5-phosphate by Inpp5a. Gene expression analysis was performed on an Inpp5a (Gt(OST50073)Lex) mouse generated by gene trap insertion using quantitative real-time PCR (qRT-PCR), immunohistochemistry, and Western blot. Phenotypic analyses were performed using rotarod, ß-galactosidase staining, and phosphatase activity assay. Statistical significance was calculated. The deletion of Inpp5a causes an early-onset yet slowly progressive PC degeneration and ataxia. Homozygous mutants (90%) exhibit perinatal lethality; surviving homozygotes show locomotor instability at P16. A consistent pattern of PC loss in the cerebellum is initially detectable by weaning and widespread by P60. Phosphatase activity toward phosphoinositol substrates is reduced in the mutant relative to littermates. The ataxic phenotype and characteristics neurodegeneration of the Inpp5a (Gt(OST50073)Lex) mouse indicate a crucial role for Inpp5a in PC survival. The identification of the molecular basis of the selective PC survival will be important in defining a neuroprotective gene applicable to establishing a disease mechanism.

Prediction of regulatory gene pairs using dynamic time warping and gene ontology.

Selecting informative genes is the most important task for data analysis on microarray gene expression data. In this work, we aim at identifying regulatory gene pairs from microarray gene expression data. However, microarray data often contain multiple missing expression values. Missing value imputation is thus needed before further processing for regulatory gene pairs becomes possible. We develop a novel approach to first impute missing values in microarray time series data by combining k-Nearest Neighbour (KNN), Dynamic Time Warping (DTW) and Gene Ontology (GO). After missing values are imputed, we then perform gene regulation prediction based on our proposed DTW-GO distance measurement of gene pairs. Experimental results show that our approach is more accurate when compared with existing missing value imputation methods on real microarray data sets. Furthermore, our approach can also discover more regulatory gene pairs that are known in the literature than other methods.

The mouse mutants recoil wobbler and nmf373 represent a series of Grm1 mutations.

The identification of novel mutant alleles is important for understanding critical functional domains of a protein and establishing genotype:phenotype correlations. The recoil wobbler (rcw) allelic series of spontaneous ataxic mutants and the ENU-induced mutant nmf373 genetically mapped to a shared region of chromosome 10. Their mutant phenotypes are strikingly similar; all have an ataxic phenotype that is recessive, early-onset, and is not associated with neurodegeneration. In this study we used complementation tests to show that these series of mutants are allelic to a knockout mutant of Grm1. Subsequently, a duplication of exon 4 and three missense mutations were identified in Grm1: I160T, E292D, and G337E. All mutations occurred within the ligand-binding region and changed conserved amino acids. In the rcw mutant, the Grm1 gene is expressed and the protein product is properly localized to the molecular layer of the cerebellar cortex. Grm1 is responsible for the generation of inositol 1,4,5-trisphosphate (IP(3)). The inositol second messenger system is the central mechanism for calcium release from intracellular stores in cerebellar Purkinje cells. Several of the genes involved in this pathway are mutated in mouse ataxic disorders. The novel rcw mutants represent a resource that will have utility for further studies of inositol second-messenger-system defects in neurogenetic disorders.

A null mutation in VAMP1/synaptobrevin is associated with neurological defects and prewean mortality in the lethal-wasting mouse mutant.

The soluble N-ethylmaleimide sensitive factor attachment receptors are a large family of membrane-associated proteins that are critical for Ca(2+)-mediated synaptic vesicle release. This family includes the VAMP, synaptosomal-associated protein, and syntaxin proteins. In this report, we describe a mutation in vesicle-associated membrane protein 1(VAMP1)/synaptobrevin in the mouse neurological mutant lethal-wasting (lew). The lethal-wasting mutant phenotype is characterized by a general lack of movement and wasting, eventually leading to death before weaning. Mutants are visibly immobile and lay on their side by postnatal day 10 (P10). Before this stage, mutants can be identified by a failure to attempt to right themselves. Affected mice die on average at P15. We used a positional cloning strategy to identify the mutation associated with this neurological phenotype. Lethal wasting had previously been linked to chromosome 6. We further narrowed the genetic disease interval and selected a small number of candidate genes for mutation screening. Genes were evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) to detect differences in their expression levels between control and mutant brain ribonucleic acid (RNA) samples. VAMP1 mRNA was found to be significantly downregulated in the lethal-wasting brain compared to wild-type littermates. Subsequently, a nonsense mutation was identified in the coding region of the gene. This mutation is predicted to truncate approximately half of the protein; however, Western blot analysis showed that no protein is detectable in the mutant. VAMP1 is selectively expressed in the retina and in discrete areas of the brain including the zona incerta and rostral periolivary region, although no gross histological abnormalities were observed in these tissues. Taken together, these data indicate that VAMP1 has a vital role in a subset of central nervous system tissues.