The Opioid Receptor Influences Circadian Rhythms in Human Keratinocytes through the ß-Arrestin Pathway.

The recent emphasis on circadian rhythmicity in critical skin cell functions related to homeostasis, regeneration and aging has shed light on the importance of the PER2 circadian clock gene as a vital antitumor gene. Furthermore, delta-opioid receptors (DOPrs) have been identified as playing a crucial role in skin differentiation, proliferation and migration, which are not only essential for wound healing but also contribute to cancer development. In this study, we propose a significant association between cutaneous opioid receptor (OPr) activity and circadian rhythmicity. To investigate this link, we conducted a 48 h circadian rhythm experiment, during which RNA samples were collected every 5 h. We discovered that the activation of DOPr by its endogenous agonist Met-Enkephalin in N/TERT-1 keratinocytes, synchronized by dexamethasone, resulted in a statistically significant 5.6 h delay in the expression of the core clock gene PER2. Confocal microscopy further confirmed the simultaneous nuclear localization of the DOPr-ß-arrestin-1 complex. Additionally, DOPr activation not only enhanced but also induced a phase shift in the rhythmic binding of ß-arrestin-1 to the PER2 promoter. Furthermore, we observed that ß-arrestin-1 regulates the transcription of its target genes, including PER2, by facilitating histone-4 acetylation. Through the ChIP assay, we determined that Met-Enkephalin enhances ß-arrestin-1 binding to acetylated H4 in the PER2 promoter. In summary, our findings suggest that DOPr activation leads to a phase shift in PER2 expression via ß-arrestin-1-facilitated chromatin remodeling. Consequently, these results indicate that DOPr, much like its role in wound healing, may also play a part in cancer development by influencing PER2.

No NFATC2 fusion in simple bone cyst of the jaw.

AIMS: Simple Bone Cysts (SBCs) predominantly occur in long bones and 59% harbour NFATC2 rearrangements. Jaw SBC is rare and was previously referred to as traumatic bone cyst. It can rarely occur in association with cemento-osseous dysplasia (COD). To determine whether jaw SBCs represent the same entity as SBC of the long bones, or if they have a different molecular signature, we collected 48 jaw SBC cases of 47 patients to assess NFATC2 rearrangement. METHODS AND RESULTS: Out of the 48 cases, 36 could be used for fluorescence in-situ hybridization (FISH), of which nine (two of which associated with COD) were successful using an NFATC2 split probe. The remaining cases failed to show adequate FISH signals. All nine cases lacked NFATC2 rearrangement and five of these showed no detectable gene fusions using Archer FusionPlex. CONCLUSION: In our study, NFATC2 rearrangement is absent in solitary jaw SBC (n = 7) and COD-associated SBC (n = 2). Our findings suggest that SBC presenting in the jaw is molecularly different from SBC in long bones. Future molecular studies may confirm the absence of clonal molecular aberrations in SBC of the jaw which would support a non-neoplastic, reactive origin.

MCC is a centrosomal protein that relocalizes to non-centrosomal apical sites during intestinal cell differentiation.

The gene mutated in colorectal cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis and disease remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, where the Mcc protein is distinctly associated with the centrosome. Upon intestinal cellular differentiation, Mcc is redeployed to the apical domain of polarized villus cells where non-centrosomal microtubule organizing centers (ncMTOCs) are positioned. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by casein kinases 1δ and ε, which are critical modulators of WNT signaling. Together, our findings support a role for MCC in establishing and maintaining the cellular architecture of the intestinal epithelium as a component of both the centrosome and ncMTOC.

Identification of stable housekeeping genes for induced pluripotent stem cells and -derived endothelial cells for drug testing.

There are no validated housekeeping genes in induced pluripotent stem cells (iPSC) and derived endothelial iPSC (iPSC-EC). Thus a comparison of gene expression levels is less reliable, especially during drug treatments. Here, we utilized transcriptome sequencing data of iPSC and iPSC-EC with or without CRISPR-Cas9 induced translocation to identify a panel of 15 candidate housekeeping genes. For comparison, five commonly used housekeeping genes (B2M, GAPDH, GUSB, HMBS, and HPRT1) were included in the study. The panel of 20 candidate genes were investigated for their stability as reference genes. This panel was analyzed and ranked based on stability using five algorithms, delta-Ct, bestkeeper, geNorm, Normfinder, and Reffinder. Based on the comprehensive ranking of Reffinder, the stability of the top two genes-RPL36AL and TMBIM6, and the bottom two genes-UBA1 and B2M, were further studied in iPSC-EC with and without genetic manipulation, and after treatment with telatinib. Using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), it was shown that gene expression of the top two housekeeping genes, RPL36AL and TMBIM6, remained stable during drug treatment. We identified a panel of housekeeping genes that could be utilized in various conditions using iPSC and iPSC-derived endothelial cells as well as genetically modified iPSC for drug treatment.

Gene fusions in vascular tumors and their underlying molecular mechanisms.

INTRODUCTION: The group of vascular tumors contains many different entities, and is considered difficult by pathologists, as they often have overlapping histological characteristics. Chromosomal translocations have been identified in ~20% of mesenchymal tumors and are considered the drivers of tumor formation. Many translocations have been discovered over the past decade through next-generation sequencing. This technological advancement has also revealed several recurrent gene fusions in vascular tumors. AREAS COVERED: This review will discuss the various vascular tumors for which recurrent gene fusions have been identified. The gene fusions and the presumed molecular mechanisms underlying tumorigenesis are shown, and potential implications for targeted therapies discussed. The identification of these gene fusions in vascular tumors has improved diagnostic accuracy, especially since several of these fusions can be easily detected using surrogate immunohistochemical markers. EXPERT OPINION: The identification of gene fusions in a subset of vascular tumors over the past decade has improved diagnostic accuracy, and has provided the pathologists with novel diagnostic tools to accurately diagnose these often difficult tumors. Moreover, the increased understanding of the underlying molecular mechanisms can guide the development of targeted therapeutic strategies.

Expanding the Spectrum of EWSR1-NFATC2-rearranged Benign Tumors: A Common Genomic Abnormality in Vascular Malformation/Hemangioma and Simple Bone Cyst.

A simple bone cyst (SBC) is a cystic bone lesion predominantly affecting young males. The cyst is lined by a fibrous membrane and filled with serosanguinous fluid. EWSR1/FUS-NFATC2 rearrangements were recently identified in SBC. We here report exactly the same rearrangement in 3 lesions diagnosed as vascular malformations of 2 elderly patients. In total, through Archer FusionPlex, fluorescence in situ hybridization and/or reverse transcriptase-polymerase chain reaction the EWSR1-NFATC2 rearrangement was identified in 6 of 9 SBC, 3 of 12 benign vascular tumors, and none of 5 aneurysmal bone cyst lacking USP6 fusion. Using fluorescence in situ hybridization, it was apparent that amplification of the fusion, as seen in EWSR1-NFATC2 round cell sarcomas, was absent, and that in the vascular tumors the fusion was present both in the lining cells as well as in the surrounding spindle cells. Of note, not all of the spaces in the vascular malformations were lined by endothelial cells. Aggrecan was positive in all cases but was not specific. NKX2-2 and NKX3-1 staining were negative in all cases. Thus, even though the overlap between the 2 entities is limited to the presence of few thick-walled cysts lacking endothelial lining in the benign vascular malformations, the spectrum of benign tumors containing NFATC2 fusions should be expanded and contains not only SBC in the young, but also vascular malformation/hemangioma in elderly patients.

Unintended consequences of infection prevention and control measures during COVID-19 pandemic.

BACKGROUND: In the current COVID-19 pandemic, aggressive Infection Prevention and Control (IPC) measures have been adopted to prevent health care-associated transmission of COVID-19. We evaluated the impact of a multimodal IPC strategy originally designed for the containment of COVID-19 on the rates of other hospital-acquired-infections (HAIs). METHODOLOGY: From February-August 2020, a multimodal IPC strategy was implemented across a large health care campus in Singapore, comprising improved segregation of patients with respiratory symptoms, universal masking and heightened adherence to Standard Precautions. The following rates of HAI were compared pre- and postpandemic: health care-associated respiratory-viral-infection (HA-RVI), methicillin-resistant Staphylococcus aureus, and CP-CRE acquisition rates, health care-facility-associated C difficile infections and device-associated HAIs. RESULTS: Enhanced IPC measures introduced to contain COVID-19 had the unintended positive consequence of containing HA-RVI. The cumulative incidence of HA-RVI decreased from 9.69 cases per 10,000 patient-days to 0.83 cases per 10,000 patient-days (incidence-rate-ratio = 0.08; 95% confidence interval [CI] = 0.05-0.13, P< .05). Hospital-wide MRSA acquisition rates declined significantly during the pandemic (incidence-rate-ratio = 0.54, 95% CI = 0.46-0.64, P< .05), together with central-line-associated-bloodstream infection rates (incidence-rate-ratio = 0.24, 95% CI = 0.07-0.57, P< .05); likely due to increased compliance with Standard Precautions. Despite the disruption caused by the pandemic, there was no increase in CP-CRE acquisition, and rates of other HAIs remained stable. CONCLUSIONS: Multimodal IPC strategies can be implemented at scale to successfully mitigate health care-associated transmission of RVIs. Good adherence to personal-protective-equipment and hand hygiene kept other HAI rates stable even during an ongoing pandemic where respiratory infections were prioritized for interventions.

The novel zebrafish model pretzel demonstrates a central role for SH3PXD2B in defective collagen remodelling and fibrosis in Frank-Ter Haar syndrome.

Frank-Ter Haar syndrome (FTHS, MIM #249420) is a rare skeletal dysplasia within the defective collagen remodelling spectrum (DECORS), which is characterised by craniofacial abnormalities, skeletal malformations and fibrotic soft tissues changes including dermal fibrosis and joint contractures. FTHS is caused by homozygous or compound heterozygous loss-of-function mutation or deletion of SH3PXD2B (Src homology 3 and Phox homology domain-containing protein 2B; MIM #613293). SH3PXD2B encodes an adaptor protein with the same name, which is required for full functionality of podosomes, specialised membrane structures involved in extracellular matrix (ECM) remodelling. The pathogenesis of DECORS is still incompletely understood and, as a result, therapeutic options are limited. We previously generated an mmp14a/b knockout zebrafish and demonstrated that it primarily mimics the DECORS-related bone abnormalities. Here, we present a novel sh3pxd2b mutant zebrafish, pretzel, which primarily reflects the DECORS-related dermal fibrosis and contractures. In addition to relatively mild skeletal abnormalities, pretzel mutants develop dermal and musculoskeletal fibrosis, contraction of which seems to underlie grotesque deformations that include kyphoscoliosis, abdominal constriction and lateral folding. The discrepancy in phenotypes between mmp14a/b and sh3pxd2b mutants suggests that in fish, as opposed to humans, there are differences in spatiotemporal dependence of ECM remodelling on either sh3pxd2b or mmp14a/b The pretzel model presented here can be used to further delineate the underlying mechanism of the fibrosis observed in DECORS, as well as screening and subsequent development of novel drugs targeting DECORS-related fibrosis.This paper has an associated First Person interview with the first author of the article.

Microfibril-associated glycoprotein 4 (Mfap4) regulates haematopoiesis in zebrafish.

Microfibril-associated glycoprotein 4 (MFAP4) is an extracellular matrix protein belonging to the fibrinogen-related protein superfamily. MFAP4 is produced by vascular smooth muscle cells and is highly enriched in the blood vessels of the heart and lung, where it is thought to contribute to the structure and function of elastic fibers. Genetic studies in humans have implicated MFAP4 in the pathogenesis of Smith-Magenis syndrome, in which patients present with multiple congenital abnormalities and mental retardation, as well as in the severe cardiac malformation left-sided congenital heart disease. Comprehensive genetic analysis of the role of MFAP4 orthologues in model organisms during development and tissue homeostasis is however lacking. Here, we demonstrate that zebrafish mfap4 transcripts are detected embryonically, resolving to the macrophage lineage by 24 h post fertilization. mfap4 null mutant zebrafish are unexpectedly viable and fertile, without ostensible phenotypes. However, tail fin amputation assays reveal that mfap4 mutants have reduced numbers of macrophages, with a concomitant increase in neutrophilic granulocytes, although recruitment of both cell types to the site of injury was unaffected. Molecular analyses suggest that loss of Mfap4 alters the balance between myeloid and lymphoid lineages during both primitive and definitive haematopoiesis, which could significantly impact the downstream function of the immune system.

PRDM15 loss of function links NOTCH and WNT/PCP signaling to patterning defects in holoprosencephaly.

Holoprosencephaly (HPE) is a congenital forebrain defect often associated with embryonic lethality and lifelong disabilities. Currently, therapeutic and diagnostic options are limited by lack of knowledge of potential disease-causing mutations. We have identified a new mutation in the PRDM15 gene (C844Y) associated with a syndromic form of HPE in multiple families. We demonstrate that C844Y is a loss-of-function mutation impairing PRDM15 transcriptional activity. Genetic deletion of murine Prdm15 causes anterior/posterior (A/P) patterning defects and recapitulates the brain malformations observed in patients. Mechanistically, PRDM15 regulates the transcription of key effectors of the NOTCH and WNT/PCP pathways to preserve early midline structures in the developing embryo. Analysis of a large cohort of patients with HPE revealed potentially damaging mutations in several regulators of both pathways. Our findings uncover an unexpected link between NOTCH and WNT/PCP signaling and A/P patterning and set the stage for the identification of new HPE candidate genes.

Functional analysis of a hypomorphic allele shows that MMP14 catalytic activity is the prime determinant of the Winchester syndrome phenotype.

Winchester syndrome (WS, MIM #277950) is an extremely rare autosomal recessive skeletal dysplasia characterized by progressive joint destruction and osteolysis. To date, only one missense mutation in MMP14, encoding the membrane-bound matrix metalloprotease 14, has been reported in WS patients. Here, we report a novel hypomorphic MMP14 p.Arg111His (R111H) allele, associated with a mitigated form of WS. Functional analysis demonstrated that this mutation, in contrast to previously reported human and murine MMP14 mutations, does not affect MMP14's transport to the cell membrane. Instead, it partially impairs MMP14's proteolytic activity. This residual activity likely accounts for the mitigated phenotype observed in our patients. Based on our observations as well as previously published data, we hypothesize that MMP14's catalytic activity is the prime determinant of disease severity. Given the limitations of our in vitro assays in addressing the consequences of MMP14 dysfunction, we generated a novel mmp14a/b knockout zebrafish model. The fish accurately reflected key aspects of the WS phenotype including craniofacial malformations, kyphosis, short-stature and reduced bone density owing to defective collagen remodeling. Notably, the zebrafish model will be a valuable tool for developing novel therapeutic approaches to a devastating bone disorder.

MINDY1 Is a Downstream Target of the Polyamines and Promotes Embryonic Stem Cell Self-Renewal.

Embryonic stem cells have the ability to self-renew or differentiate and these processes are under tight control. We previously reported that the polyamine regulator AMD1 is critical for embryonic stem cell self-renewal. The polyamines putrescine, spermidine, and spermine are essential organic cations that play a role in a wide array of cellular processes. Here, we explore the essential role of the polyamines in the promotion of self-renewal and identify a new stem cell regulator that acts downstream of the polyamines: MINDY1. MINDY1 protein levels are high in embryonic stem cells (ESCs) and are dependent on high polyamine levels. Overexpression of MINDY1 can promote ESC self-renewal in the absence of the usually essential cytokine Leukemia Inhibitory Factor (LIF). MINDY1 protein is prenylated and this modification is required for its ability to promote self-renewal. We go on to show that Mindy1 RNA is targeted for repression by mir-710 during Neural Precursor cell differentiation. Taken together, these data demonstrate that high polyamine levels are required for ESC self-renewal and that they function, in part, through promotion of high MINDY1 levels. Stem Cells 2018;36:1170-1178.

ELABELA deficiency promotes preeclampsia and cardiovascular malformations in mice.

Preeclampsia (PE) is a gestational hypertensive syndrome affecting between 5 and 8% of all pregnancies. Although PE is the leading cause of fetal and maternal morbidity and mortality, its molecular etiology is still unclear. Here, we show that ELABELA (ELA), an endogenous ligand of the apelin receptor (APLNR, or APJ), is a circulating hormone secreted by the placenta. Elabela but not Apelin knockout pregnant mice exhibit PE-like symptoms, including proteinuria and elevated blood pressure due to defective placental angiogenesis. In mice, infusion of exogenous ELA normalizes hypertension, proteinuria, and birth weight. ELA, which is abundant in human placentas, increases the invasiveness of trophoblast-like cells, suggesting that it enhances placental development to prevent PE. The ELA-APLNR signaling axis may offer a new paradigm for the treatment of common pregnancy-related complications, including PE.

PRDM15 safeguards naive pluripotency by transcriptionally regulating WNT and MAPK-ERK signaling.

The transcriptional network acting downstream of LIF, WNT and MAPK-ERK to stabilize mouse embryonic stem cells (ESCs) in their naive state has been extensively characterized. However, the upstream factors regulating these three signaling pathways remain largely uncharted. PR-domain-containing proteins (PRDMs) are zinc-finger sequence-specific chromatin factors that have essential roles in embryonic development and cell fate decisions. Here we characterize the transcriptional regulator PRDM15, which acts independently of PRDM14 to regulate the naive state of mouse ESCs. Mechanistically, PRDM15 modulates WNT and MAPK-ERK signaling by directly promoting the expression of Rspo1 (R-spondin1) and Spry1 (Sprouty1). Consistent with these findings, CRISPR-Cas9-mediated disruption of PRDM15-binding sites in the Rspo1 and Spry1 promoters recapitulates PRDM15 depletion, both in terms of local chromatin organization and the transcriptional modulation of these genes. Collectively, our findings uncover an essential role for PRDM15 as a chromatin factor that modulates the transcription of upstream regulators of WNT and MAPK-ERK signaling to safeguard naive pluripotency.

Long-Term Culture of Self-renewing Pancreatic Progenitors Derived from Human Pluripotent Stem Cells.

Pluripotent stem cells have been proposed as an unlimited source of pancreatic ß cells for studying and treating diabetes. However, the long, multi-step differentiation protocols used to generate functional ß cells inevitably exhibit considerable variability, particularly when applied to pluripotent cells from diverse genetic backgrounds. We have developed culture conditions that support long-term self-renewal of human multipotent pancreatic progenitors, which are developmentally more proximal to the specialized cells of the adult pancreas. These cultured pancreatic progenitor (cPP) cells express key pancreatic transcription factors, including PDX1 and SOX9, and exhibit transcriptomes closely related to their in vivo counterparts. Upon exposure to differentiation cues, cPP cells give rise to pancreatic endocrine, acinar, and ductal lineages, indicating multilineage potency. Furthermore, cPP cells generate insulin+ ß-like cells in vitro and in vivo, suggesting that they offer a convenient alternative to pluripotent cells as a source of adult cell types for modeling pancreatic development and diabetes.

The PDZ domain protein Mcc is a novel effector of non-canonical Wnt signaling during convergence and extension in zebrafish.

During vertebrate gastrulation, a complex set of mass cellular rearrangements shapes the embryonic body plan and appropriately positions the organ primordia. In zebrafish and Xenopus, convergence and extension (CE) movements simultaneously narrow the body axis mediolaterally and elongate it from head to tail. This process is governed by polarized cell behaviors that are coordinated by components of the non-canonical, ß-catenin-independent Wnt signaling pathway, including Wnt5b and the transmembrane planar cell polarity (PCP) protein Vangl2. However, the intracellular events downstream of Wnt/PCP signals are not fully understood. Here, we show that zebrafish mutated in colorectal cancer (mcc), which encodes an evolutionarily conserved PDZ domain-containing putative tumor suppressor, is required for Wnt5b/Vangl2 signaling during gastrulation. Knockdown of mcc results in CE phenotypes similar to loss of vangl2 and wnt5b, whereas overexpression of mcc robustly rescues the depletion of wnt5b, vangl2 and the Wnt5b tyrosine kinase receptor ror2. Biochemical experiments establish a direct physical interaction between Mcc and the Vangl2 cytoplasmic tail. Lastly, CE defects in mcc morphants are suppressed by downstream activation of RhoA and JNK. Taken together, our results identify Mcc as a novel intracellular effector of non-canonical Wnt5b/Vangl2/Ror2 signaling during vertebrate gastrulation.