Determinant factors for first-line treatment choice and effectiveness in pediatric eosinophilic esophagitis: an analysis of the EUREOS EoE CONNECT registry.

This study compared short-term effectiveness of proton pump inhibitors (PPI), swallowed topical corticosteroids (STC), and dietary therapies in reversing clinical and histological features in pediatric patients with eosinophilic esophagitits (EoE). Determinants for treatment choice and PPI therapy effectiveness were also assessed.  A cross-sectional study analysis of patients under 18 years old recruited onto the multicenter EoE CONNECT registry was performed. Clinico-histological response was defined as symptomatic improvement plus a peak eosinophil count below 15 per high-power field after treatment. Effectiveness of first-line options used in monotherapy was compared. Overall, 393 patients (64% adolescents) receiving PPI, STC, or dietary monotherapy to induce EoE remission were identified. PPI was the preferred option (71.5%), despite STC providing the highest clinico-histological response rates (66%) compared to PPI (44%) and diet (42%). Logistic regression identified fibrotic features and recruitment at Italian sites independently associated to first-line STC treatment; age under 12 associated to dietary therapy over other options. Analysis of 262 patients in whom PPI effectiveness was evaluated after median (IQR) 96 (70-145) days showed that this effectiveness was significantly associated with management at pediatric facilities and use of high PPI doses. Among PPI responders, decrease in rings and structures in endoscopy from baseline was documented, with EREFS fibrotic subscore for rings also decreasing among responders (0.27 ± 0.63 vs. 0.05 ± 0.22, p < 0.001). Conclusion: Initial therapy choice for EoE depends on endoscopic phenotype, patient's age, and patients' origin. High PPI doses and treatment in pediatric facilities significantly determined effectiveness, and reversed fibrotic endoscopic features among responders. What is Known: • Proton pump inhibitors are widely used to induce and maintain remission in EoE in real practice, despite other first-line alternative therapies possibly providing higher effectiveness. What is New: • Proton pump inhibitors represent up to two-thirds of first-line monotherapies used to induce EoE remission in pediatric and adolescent patients with EoE. The choice of STC as first-line treatment for EoE was significantly associated with fibrotic features at baseline endoscopy and recruitment in Italian centers; age less than 12 years was associated with dietary therapy. • PPI effectiveness was found to be determined by use of high doses, attendance at pediatric facilities, presenting inflammatory instead of fibrotic or mixed phenotypes, and younger age. Among responders, PPI therapy reversed both inflammatory and fibrotic features of EoE after short-term treatment.

CRISPR-AsCas12a Efficiently Corrects a GPR143 Intronic Mutation in Induced Pluripotent Stem Cells from an Ocular Albinism Patient.

Mutations in the GPR143 gene cause X-linked ocular albinism type 1 (OA1), a disease that severely impairs vision. We recently generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of an OA1 patient carrying a point mutation in intron 7 of GPR143. This mutation activates a new splice site causing the incorporation of a pseudoexon. In this study, we present a high-performance CRISPR-Cas ribonucleoprotein strategy to permanently correct the GPR143 mutation in these patient-derived iPSCs. Interestingly, the two single-guide RNAs available for SpCas9 did not allow the cleavage of the target region. In contrast, the cleavage achieved with the CRISPR-AsCas12a system promoted homology-directed repair at a high rate. The CRISPR-AsCas12a-mediated correction did not alter iPSC pluripotency or genetic stability, nor did it result in off-target events. Moreover, we highlight that the disruption of the pathological splice site caused by CRISPR-AsCas12a-mediated insertions/deletions also rescued the normal splicing of GPR143 and its expression level.

Evolutionary plasticity in the requirement for force exerted by ligand endocytosis to activate C. elegans Notch proteins.

The conserved transmembrane receptor Notch has diverse and profound roles in controlling cell fate during animal development. In the absence of ligand, a negative regulatory region (NRR) in the Notch ectodomain adopts an autoinhibited confirmation, masking an ADAM protease cleavage site;1,2 ligand binding induces cleavage of the NRR, leading to Notch ectodomain shedding as the first step of signal transduction.3,4 In Drosophila and vertebrates, recruitment of transmembrane Delta/Serrate/LAG-2 (DSL) ligands by the endocytic adaptor Epsin, and their subsequent internalization by Clathrin-mediated endocytosis, exerts a "pulling force" on Notch that is essential to expose the cleavage site in the NRR.4-6 Here, we show that Epsin-mediated endocytosis of transmembrane ligands is not essential to activate the two C. elegans Notch proteins, LIN-12 and GLP-1. Using an in vivo force sensing assay in Drosophila,6 we present evidence (1) that the LIN-12 and GLP-1 NRRs are tuned to lower force thresholds than the NRR of Drosophila Notch, and (2) that this difference depends on the absence of a "leucine plug" that occludes the cleavage site in the Drosophila and vertebrate Notch NRRs.1,2 Our results thus establish an unexpected evolutionary plasticity in the force-dependent mechanism of Notch activation and implicate a specific structural element, the leucine plug, as a determinant.

Establishment and characterization of two iPSC lines derived from healthy controls.

We have generated two iPSC lines from skin biopsies of two healthy individuals. Skin fibroblasts were derived and reprogrammed using a Sendai virus-based approach. The resulting iPSC lines have normal karyotype, express stemness markers and can generate endoderm, mesoderm and ectoderm in vitro. These iPSC lines can be used as healthy controls in differentiation paradigms as well as backbone for gene editing experiments.

Standardized Reporter Systems for Purification and Imaging of Human Pluripotent Stem Cell-derived Motor Neurons and Other Cholinergic Cells.

Reliable and consistent pluripotent stem cell reporter systems for efficient purification and visualization of motor neurons are essential reagents for the study of normal motor neuron biology and for effective disease modeling. To overcome the inherent noisiness of transgene-based reporters, we developed a new series of human induced pluripotent stem cell lines by knocking in tdTomato, Cre, or CreERT2 recombinase into the HB9 (MNX1) or VACHT (SLC18A3) genomic loci. The new lines were validated by directed differentiation into spinal motor neurons and immunostaining for motor neuron markers HB9 and ISL1. To facilitate efficient purification of spinal motor neurons, we further engineered the VACHT-Cre cell line with a validated, conditional CD14-GFP construct that allows for both fluorescence-based identification of motor neurons, as well as magnetic-activated cell sorting (MACS) to isolate differentiated motor neurons at scale. The targeting strategies developed here offer a standardized platform for reproducible comparison of motor neurons across independently derived pluripotent cell lines.

Development of MAP4 Kinase Inhibitors as Motor Neuron-Protecting Agents.

Disease-causing mutations in many neurodegenerative disorders lead to proteinopathies that trigger endoplasmic reticulum (ER) stress. However, few therapeutic options exist for patients with these diseases. Using an in vitro screening platform to identify compounds that protect human motor neurons from ER stress-mediated degeneration, we discovered that compounds targeting the mitogen-activated protein kinase kinase kinase kinase (MAP4K) family are neuroprotective. The kinase inhibitor URMC-099 (compound 1) stood out as a promising lead compound for further optimization. We coupled structure-based compound design with functional activity testing in neurons subjected to ER stress to develop a series of analogs with improved MAP4K inhibition and concomitant increases in potency and efficacy. Further structural modifications were performed to enhance the pharmacokinetic profiles of the compound 1 derivatives. Prostetin/12k emerged as an exceptionally potent, metabolically stable, and blood-brain barrier-penetrant compound that is well suited for future testing in animal models of neurodegeneration.

DPP-4 Levels Are Increased in Subjects with Poor Sleep Quality.

Background: Up to 30% of the population has sleep disturbances, generating a negative health impact, a situation that is often not known and no medical attention is sought. It has been observed that after a total deprivation of sleep, the levels of dipeptidyl peptidase 4 (DPP-4) tend to increase. The aim of this study was to compare serum levels of DPP-4 in healthy subjects, with adequate and poor-quality sleep needing medical/pharmacological treatment. Materials and Methods: Cross-sectional study of subjects scheduled for elective surgery with low cardiometabolic risk. Subjects between 18-70 years of age were included, without previous diagnosed pathology (diabetes mellitus type 2; neoplasm; nephropathy; and liver disease) and major amputations, and who signed informed consent. The study protocol was aproved in the Local Committee for Ethics and Research, number 45-16. Anthropometry was performed (% body fat; waist and neck circumferences), and sleep quality assessment (Pittsburgh Sleep Quality Index [PSQI]) to classify them as worthy or not worthy of medical/pharmacological care. Serum DPP-4 was determined by Enzime Linked Immunosorbent Assay (ELISA). The statistical analysis was done in RStudio Software. Results: Fifty seven subjects (2017-2018) were included, with a combined frequency of overweight/obesity of 66.6% and with abdominal circumference values of 93.2 ± 13.6, higher than that proposed by the International Diabetes Federation. The PSQI was 8.3 ± 4.1, and 56.1% were classified as worthy of medical/pharmacological attention. When comparing the levels of DPP-4, these were higher in this group 2385.0 ± 2082.0 versus not worthy 1716.7 ± 1261.7 pg/mL, being statistically significant (P = 0.035). Conclusions: The elevated levels of DPP-4 in person with poor quality sleep worthy of medical/pharmacological treatment could be an early indicator of metabolic disorders, which need to be evaluated in depth.

Assessment of reliability and validity of the Spanish version of the Nursing Students' Perception of Instructor Caring (S-NSPIC).

The care that clinical instructors demonstrate to students is essential for their education, considering the strong impact it has on their future relationships with patients, relatives, and other health professionals. Nursing Students' Perceptions of Instructor Caring (NSPIC) is an instrument designed to assess nursing students' perceptions of instructor's caring behaviors. A trans-cultural, conceptual, and psychometric validation study was conducted with 315 nursing students at the University of Seville during their clinical practices in three regional hospitals. The NSPIC was translated and adapted to Spanish. The content validity was established by a panel of experts. To assess concurrent validity the culturally adapted Spanish version of the Clinical Placement Evaluation Tool (CPET) was used as a gold standard. The construct validity was determined by an exploratory factor analysis to identify the internal structure of the NSPIC-S. The internal consistency was established by Cronbach's α and the intra-observer reliability for each item was established by test-retest. The content validity index varied between 0.53 and 0.93 and the correlation to the CPET was moderate. The factor analysis revealed a structure of five factors, one of which differed from the original scale. The value of Cronbach's α was 0.95 and intraclass correlation coefficients varied between 0.5 and 0.89. Our study provided a culturally adapted version of the NSPIC, valid and reliable to be used in the Spanish context, the NSPIC-S.

Generation of a human Ocular Albinism type 1 iPSC line, SEIi001-A, with a mutation in GPR143.

Ocular albinism type 1 is a genetic eye disease caused by mutations in the GPR143 gene. Little is known about the molecular pathways involved in this disease and no therapeutic candidate has been identified as yet. Here we report the generation of an iPSC line from the skin fibroblasts of a patient with a mutation in the GPR143 gene using Sendai Virus vectors. This new iPSC line will allow a better understanding of the Ocular Albinism type 1 disease and to screen for potential therapeutic candidates.

Kelch Domain of Gigaxonin Interacts with Intermediate Filament Proteins Affected in Giant Axonal Neuropathy.

Patients with giant axonal neuropathy (GAN) show progressive loss of motor and sensory function starting in childhood and typically live for less than 30 years. GAN is caused by autosomal recessive mutations leading to low levels of gigaxonin (GIG), a ubiquitously-expressed BTB/Kelch cytoplasmic protein believed to be an E3 ligase substrate adaptor. GAN pathology is characterized by aggregates of intermediate filaments (IFs) in multiple tissues. To delineate the molecular pathway between GIG deficiency and IF pathology, we undertook a proteomic screen to identify the normal binding partners of GIG. Prominent among them were several classes of IFs, including the neurofilament subunits whose accumulation leads to the axonal swellings for which GAN is named. We showed these interactions were dependent on the Kelch domain of GIG. Furthermore, we identified the E3 ligase MYCBP2 and the heat shock proteins HSP90AA1/AB1 as interactors with the BTB domain that may result in the ubiquitination and subsequent degradation of intermediate filaments. Our open-ended proteomic screen provides support to GIG's role as an adaptor protein, linking IF proteins through its Kelch domain to the ubiquitin pathway proteins via its BTB domain, and points to future approaches for reversing the phenotype in human patients.

Neurotrophic requirements of human motor neurons defined using amplified and purified stem cell-derived cultures.

Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC(50) 1-2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.

Intracellular bacteria encode inhibitory SNARE-like proteins.

Pathogens use diverse molecular machines to penetrate host cells and manipulate intracellular vesicular trafficking. Viruses employ glycoproteins, functionally and structurally similar to the SNARE proteins, to induce eukaryotic membrane fusion. Intracellular pathogens, on the other hand, need to block fusion of their infectious phagosomes with various endocytic compartments to escape from the degradative pathway. The molecular details concerning the mechanisms underlying this process are lacking. Using both an in vitro liposome fusion assay and a cellular assay, we showed that SNARE-like bacterial proteins block membrane fusion in eukaryotic cells by directly inhibiting SNARE-mediated membrane fusion. More specifically, we showed that IncA and IcmG/DotF, two SNARE-like proteins respectively expressed by Chlamydia and Legionella, inhibit the endocytic SNARE machinery. Furthermore, we identified that the SNARE-like motif present in these bacterial proteins encodes the inhibitory function. This finding suggests that SNARE-like motifs are capable of specifically manipulating membrane fusion in a wide variety of biological environments. Ultimately, this motif may have been selected during evolution because it is an efficient structural motif for modifying eukaryotic membrane fusion and thus contribute to pathogen survival.

Alternative zippering as an on-off switch for SNARE-mediated fusion.

Membrane fusion between vesicles and target membranes involves the zippering of a four-helix bundle generated by constituent helices derived from target- and vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). In neurons, the protein complexin clamps otherwise spontaneous fusion by SNARE proteins, allowing neurotransmitters and other mediators to be secreted when and where they are needed as this clamp is released. The membrane-proximal accessory helix of complexin is necessary for clamping, but its mechanism of action is unknown. Here, we present experiments using a reconstituted fusion system that suggest a simple model in which the complexin accessory helix forms an alternative four-helix bundle with the target-SNARE near the membrane, preventing the vesicle-SNARE from completing its zippering.

Distinct domains of complexins bind SNARE complexes and clamp fusion in vitro.

In regulated exocytosis, the core membrane fusion machinery proteins, the SNARE proteins, are assisted by a group of regulatory factors in order to couple membrane fusion to an increase of intracellular calcium ion (Ca(2+)) concentration. Complexin-I and synaptotagmin-I have been shown to be key elements for this tightly regulated process. Many studies suggest that complexin-I can arrest the fusion reaction and that synaptotagmin-I can release the complexin-I blockage in a calcium-dependent manner. Although the actual molecular mechanism by which they exert their function is still unknown, recent in vivo experiments postulate that domains of complexin-I produce different effects on neurotransmitter release. Herein, by using an in vitro flipped SNARE cell fusion assay, we have identified and characterized the minimal functional domains of complexin-I necessary to couple calcium and synaptotagmin-I to membrane fusion. Moreover, we provide evidence that other isoforms of complexin, complexin-II, -III, and -IV, can also be functionally coupled to synaptotagmin-I and calcium. These correspond closely to results from in vivo experiments, providing further validation of the physiological relevance of the flipped SNARE system.

Functional mapping of disease susceptibility loci using cell biology.

In most genome-wide linkage studies, implication of a causative disease gene often requires years of expanding the study to more families and finer mapping of the initially described region. Even after such efforts, unobtainable sample sizes can be required to make statistically meaningful conclusions about a single gene. Here we demonstrate that by adding a layer of functional biology to statistical genetic results, this process can be accelerated. The diabetes susceptibility locus (chromosome 18p11) was systematically dissected by using a cell-based secretion assay and RNA interference, and we identified laminin alpha1 to have a role in pancreatic beta cell secretion. The screen was extended to identify laminin receptor 1 as a functional partner in regards to beta cell function. Our approach can potentially be widely used in the setting of high-throughput cellular screening of other loci to identify candidate genes.