The human fetal γ-globin gene is repressed in the adult stage through complex regulatory mechanisms involving transcription factors and epigenetic modifiers. Reversing γ-globin repression, or maintaining its expression by manipulating regulatory mechanisms, has become a major clinical goal in the treatment of β-hemoglobinopathies. Here, we identify the orphan nuclear receptor Coup-TFII (NR2F2/ARP-1) as an embryonic/fetal stage activator of γ-globin expression. We show that Coup-TFII is expressed in early erythropoiesis of yolk sac origin, together with embryonic/fetal globins. When overexpressed in adult cells (including peripheral blood cells from human healthy donors and β039 thalassemic patients) Coup-TFII activates the embryonic/fetal globins genes, overcoming the repression imposed by the adult erythroid environment. Conversely, the knock-out of Coup-TFII increases the β/γ+β globin ratio. Molecular analysis indicates that Coup-TFII binds in vivo to the β-locus and contributes to its conformation. Overall, our data identify Coup-TFII as a specific activator of the γ-globin gene.

von Willebrand factor (VWF) plays an important role in ischemic stroke. However, the exact mechanism by which VWF mediates progression of ischemic stroke brain damage is not completely understood. Using flow cytometric analysis of single cell suspensions prepared from brain tissue and immunohistochemistry, we investigated the potential inflammatory mechanisms by which VWF contributes to ischemic stroke brain damage in a mouse model of cerebral ischemia/reperfusion injury. Twenty-four hours after stroke, flow cytometric analysis of brain tissue revealed that overall white blood cell recruitment in the ipsilesional brain hemisphere of VWF KO mice was 2 times lower than WT mice. More detailed analysis showed a specific reduction of proinflammatory monocytes, neutrophils and T-cells in the ischemic brain of VWF KO mice compared to WT mice. Interestingly, histological analysis revealed a substantial number of neutrophils and T-cells still within the microcirculation of the stroke brain, potentially contributing to the no-reflow phenomenon. Specific therapeutic targeting of the VWF A1 domain in WT mice resulted in reduced immune cell numbers in the affected brain and protected mice from ischemic stroke brain damage. More specifically, recruitment of proinflammatory monocytes was reduced two-fold, neutrophil recruitment was reduced five-fold and T-cell recruitment was reduced two-fold in mice treated with a VWF A1-targeting nanobody compared to mice receiving a control nanobody. In conclusion, our data identify a potential role for VWF in the recruitment of proinflammatory monocytes, neutrophils and T-cells to the ischemic brain via a mechanism that is mediated by its A1 domain.

During red blood cell (RBC) lysis hemoglobin and heme leak out of the cells and cause damage to the endothelium and nearby tissue. Protective mechanisms exist; however, these systems are not sufficient in diseases with increased extravascular hemolysis e.g. hemolytic anemia. α1-microglobulin (A1M) is a ubiquitous reductase and radical- and heme-binding protein with antioxidation properties. Although present in the circulation in micromolar concentrations, its function in blood is unclear. Here, we show that A1M provides RBC stability. A1M-/- mice display abnormal RBC morphology, reminiscent of macrocytic anemia conditions, i.e. fewer, larger and more heterogeneous cells. Recombinant human A1M (rA1M) reduced in vitro hemolysis of murine RBC against spontaneous, osmotic and heme-induced stress. Moreover, A1M is taken up by human RBCs both in vitro and in vivo. Similarly, rA1M also protected human RBCs against in vitro spontaneous, osmotic, heme- and radical-induced hemolysis as shown by significantly reduced leakage of hemoglobin and LDH. Addition of rA1M resulted in decreased hemolysis compared to addition of the heme-binding protein hemopexin and the radical-scavenging and reducing agents ascorbic acid and Trolox (vitamin E). Furthermore, rA1M significantly reduced spontaneous and heme-induced fetal RBC cell death. Addition of A1M to human whole blood resulted in a significant reduction of hemolysis, whereas removal of A1M from whole blood resulted in increased hemolysis. We conclude that A1M has a protective function in reducing hemolysis which is neither specific to the origin of hemolytic insult, nor species specific.

Alzheimer's disease (AD) is a neurodegenerative disorder, considered a disconnection syndrome with regional molecular pattern abnormalities quantifiable by the aid of PET imaging. Solutions for accurate quantification of network dysfunction are scarce. We evaluate the extent to which PET molecular markers reflect quantifiable network metrics derived through the graph theory framework and how partial volume effects (PVE)-correction (PVEc) affects these PET-derived metrics 75 AD patients and 126 cognitively normal older subjects (CN). Therefore our goal is twofold: 1) to evaluate the differential patterns of [18F]FDG- and [18F]AV45-PET data to depict AD pathology; and ii) to analyse the effects of PVEc on global uptake measures of [18F]FDG- and [18F]AV45-PET data and their derived covariance network reconstructions for differentiating between patients and normal older subjects. Network organization patterns were assessed using graph theory in terms of "degree", "modularity", and "efficiency". PVEc evidenced effects on global uptake measures that are specific to either [18F]FDG- or [18F]AV45-PET, leading to increased statistical differences between the groups. PVEc was further shown to influence the topological characterization of PET-derived covariance brain networks, leading to an optimised characterization of network efficiency and modularisation. Partial-volume effects correction improves the interpretability of PET data in AD and leads to optimised characterization of network properties for organisation or disconnection.

Pleuroparenchymal fibroelastosis (PPFE) is a type of rare idiopathic interstitial pneumonia that is characterized by predominantly upper lobe involvement with pleural fibrosis and subjacent parenchymal fibrosis. This study aimed to determine the clinico-radiologic-pathologic features and prognosis of Korean patients with PPFE.

Wiskott-Aldrich syndrome (WAS) and osteopetrosis are 2 different, rare hereditary diseases. Here we report clinical and molecular genetics investigations on an infant patient with persistent thrombocytopenia and prolonged fever. He was clinical diagnosed as osteopetrosis according to clinical presentation, radiologic skeletal features, and bone biopsy results. Gene sequencing demonstrated a de novo homozygous mutation in 5'-untranslated region of TNFRSF11A, c.-45A>G, which is relating to osteopetrosis. Meanwhile, a hemizygous transition mutation in WAS gene, c.400G>A diagnosed the infant with WAS. This is the first clinical report for the diagnosis of osteopetrosis coinheritance with WAS in a single patient.

A comprehensive characterization of the tumour microenvironment is lacking in neuroendocrine tumours (NETs), where programmed cell death-1 receptor-ligand (PD-1/PD-L1) inhibitors are undergoing efficacy testing.

Psychiatric disorders are a collection of heterogeneous mental disorders arising from a contribution of genetic and environmental insults, many of which molecularly converge on transcriptional dysregulation, resulting in altered synaptic functions. The underlying mechanisms linking the genetic lesion and functional phenotypes remain largely unknown. Patient iPSC-derived neurons with a rare frameshift DISC1 (Disrupted-in-schizophrenia 1) mutation have previously been shown to exhibit aberrant gene expression and deficits in synaptic functions. How DISC1 regulates gene expression is largely unknown. Here we show that Activating Transcription Factor 4 (ATF4), a DISC1 binding partner, is more abundant in the nucleus of DISC1 mutant human neurons and exhibits enhanced binding to a collection of dysregulated genes. Functionally, overexpressing ATF4 in control neurons recapitulates deficits seen in DISC1 mutant neurons, whereas transcriptional and synaptic deficits are rescued in DISC1 mutant neurons with CRISPR-mediated heterozygous ATF4 knockout. By solving the high-resolution atomic structure of the DISC1-ATF4 complex, we show that mechanistically, the mutation of DISC1 disrupts normal DISC1-ATF4 interaction, and results in excessive ATF4 binding to DNA targets and deregulated gene expression. Together, our study identifies the molecular and structural basis of an DISC1-ATF4 interaction underlying transcriptional and synaptic dysregulation in an iPSC model of mental disorders.

Genome-wide association studies (GWAS) of complex traits, such as alcohol use disorders (AUD), usually identify variants in non-coding regions and cannot by themselves distinguish whether the associated variants are functional or in linkage disequilibrium with the functional variants. Transcriptome studies can identify genes whose expression differs between alcoholics and controls. To test which variants associated with AUD may cause expression differences, we integrated data from deep RNA-seq and GWAS of four postmortem brain regions from 30 subjects with AUD and 30 controls to analyze allele-specific expression (ASE). We identified 88 genes with differential ASE in subjects with AUD compared to controls. Next, to test one potential mechanism contributing to the differential ASE, we analyzed single nucleotide polymorphisms (SNPs) in the 3' untranslated regions (3'UTR) of these genes. Of the 88 genes with differential ASE, 61 genes contained 437 SNPs in the 3'UTR with at least one heterozygote among the subjects studied. Using a modified PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) assay, we identified 25 SNPs that affected RNA levels in a consistent manner in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Many of these SNPs are in binding sites of miRNAs and RNA-binding proteins, indicating that these SNPs are likely causal variants of AUD-associated differential ASE. In sum, we demonstrate that a combination of computational and experimental approaches provides a powerful strategy to uncover functionally relevant variants associated with the risk for AUD.

Lonicerae Japonicae Flos (LJF) and Lonicerae Flos (LF) belong to different genera of Caprifoliaceae. They have been historically utilised as herbal medicine to treat various diseases. However, the comprehensive assessment of them still remains a challenge.

The extensive use of amphetamines to treat attention deficit hyperactivity disorders in children provides a compelling rationale for understanding the mechanisms of action of amphetamines and amphetamine-related drugs. We have previously shown that acute amphetamine (AMPH) regulates the trafficking of both dopamine and glutamate transporters in dopamine neurons by increasing activation of the small GTPase RhoA and of protein kinase A. Here we demonstrate that these downstream signaling events depend upon the direct activation of a trace amine-associated receptor, TAAR1, an intracellular G-protein coupled receptor (GPCR) that can be activated by amphetamines, trace amines, and biogenic amine metabolites. Using cell lines and mouse lines in which TAAR1 expression has been disrupted, we demonstrate that TAAR1 mediates the effects of AMPH on both RhoA and cAMP signaling. Inhibition of different Gα signaling pathways in cell lines and in vivo using small cell-permeable peptides confirms that the endogenous intracellular TAAR1 couples to G13 and to GS α-subunits to increase RhoA and PKA activity, respectively. Results from experiments with RhoA- and PKA-FRET sensors targeted to different subcellular compartments indicate that AMPH-elicited PKA activation occurs throughout the cell, whereas G13-mediated RhoA activation is concentrated near the endoplasmic reticulum. These observations define TAAR1 as an obligate intracellular target for amphetamines in dopamine neurons and support a model in which distinct pools of TAAR1 mediate the activation of signaling pathways in different compartments to regulate excitatory and dopaminergic neurotransmission.

The Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signalling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs. Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42, which will be of great value for the field of vascular biology.

A 2017 update of the resuscitation guideline indicated the use of cardiopulmonary resuscitation (CPR) feedback devices as a resuscitation teaching method. The aim of the study was to compare the influence of two techniques of CPR teaching on the quality of resuscitation performed by medical students.

Objectives: Alzheimer's disease (AD) is a growing global health crisis exacerbated by increasing life span and an aging demographic. Convergent lines of evidence, including genome-wide association studies, strongly implicate neuroinflammation in the pathogenesis of AD. Several dietary agents, including phenolic-rich foods, show promise for the prevention and/or management of AD, which in large part, has been attributed to their anti-inflammatory effects. We previously reported that a food-grade phenolic-enriched maple syrup extract (MSX) inhibited neuroinflammation in vitro but whether these effects are translatable in vivo remain unknown. Herein, we assessed MSX's ability to attenuate early neuroinflammation in a transgenic mouse model of AD.Methods: The effects of MSX on AD-related neuroinflammation was evaluated by orally administering MSX (100 and 200 mg/kg/day for 30 days) to the 3xTg-AD mouse model of AD. The expression of inflammatory markers in mouse brains were analyzed with LC-MS/MS with SWATH acquisition.Results: 3xTg-AD mice dosed orally with MSX have decreased expression of several inflammatory proteins, including, most notably, the AD risk-associated protein 'triggering receptor expressed on myeloid cells-2' (TREM2), and stimulator of interferon genes TMEM173, and suppressor of cytokine signaling-6 (SOCS6). However, this decrease in inflammation did not coincide with a decrease in pathogenic amyloid generation or lipid peroxidation.Discussion: These data demonstrate that oral administration of this maple syrup derived natural product reduces key neuroinflammatory indices of AD in the 3xTg-AD model of AD. Therefore, further studies to investigate MSX's potential as a dietary intervention strategy for AD prevention and/or management are warranted.

Among tetrapods, only salamanders can regenerate their limbs and tails throughout life. This amazing regenerative ability has attracted the attention of scientists for hundreds of years. Now that large, salamander genomes are beginning to be sequenced for the first time, omics tools and approaches can be used to integrate new perspectives into the study of tissue regeneration. Here we argue the need to move beyond the primary salamander models to investigate regeneration in other species. Salamanders at first glance come across as a phylogenetically conservative group that has not diverged greatly from their ancestors. While salamanders do present ancestral characteristics of basal tetrapods, including the ability to regenerate limbs, data from fossils and data from studies that have tested for species differences suggest there may be considerable variation in how salamanders develop and regenerate their limbs. We review the case for expanded studies of salamander tissue regeneration and identify questions and approaches that are most likely to reveal commonalities and differences in regeneration among species. We also address challenges that confront such an initiative, some of which are regulatory and not scientific. The time is right to gain evolutionary perspective about mechanisms of tissue regeneration from comparative studies of salamander species.

Long-term hematopoietic output is dependent on hematopoietic stem cell (HSC) homeostasis which is maintained by a complex molecular network. Among these, microRNAs play crucial roles, while the underlying molecular basis has not been fully elucidated. Here, we show that miR-21 is enriched in murine HSCs, and mice with conditional knockout of miR-21 exhibit an obvious perturbation in normal hematopoiesis. Moreover, significant loss of HSC quiescence and long-term reconstituting ability are observed in the absence of miR-21. Further studies reveal that miR-21 deficiency markedly decreases the NF-κB pathway, accompanied by increased expression of PDCD4, a direct target of miR-21, in HSCs. Interestingly, overexpression of PDCD4 in wild-type HSCs generates similar phenotypes as those of miR-21-deficient HSCs. More importantly, knockdown of PDCD4 can significantly rescue the attenuation of NF-κB activity, thereby improving the defects in miR-21-null HSCs. On the other hand, we find that miR-21 is capable of preventing HSCs from ionizing radiation-induced DNA damage via activation of the NF-κB pathway. Collectively, our data demonstrate that miR-21 is involved in maintaining HSC homeostasis and function, at least in part, by regulating the PDCD4-mediated NF-κB pathway and provide a new insight into the radioprotection of HSCs.

During infection neuraminidase desialylates platelets and induces their rapid clearance from circulation. The underlying molecular basis, particularly the role of platelet glycoprotein (GP)Ibα therein, is not clear. Utilizing genetically altered mice we report that the extracellular domain of GPIbα, but neither von Willebrand factor nor ADAM17 (a disintegrin and metalloprotease 17), is required for platelet clearance induced by intravenous injection of neuraminidase. Lectin binding to platelets following neuraminidase injection over time revealed that the extent of desialylation of O-glycans correlates with the decrease of platelet count in mice. Injection of α2,3-neuraminidase reduces platelet counts in wild-type but not in transgenic mice expressing only a chimeric GPIbα that misses most of its extracellular domain. Neuraminidase treatment induces unfolding of the O-glycosylated mechanosensory domain in GPIbα as monitored by single-molecule force spectroscopy, increases the exposure of the ADAM17 shedding cleavage site in the mechanosensory domain on the platelet surface, and induces ligand-independent GPIb-IX signaling in human and murine platelets. These results suggest that desialylation of O-glycans of GPIbα induces unfolding of the mechanosensory domain, subsequent GPIb-IX signaling including amplified desialylation of N-glycans, and eventually rapid platelet clearance. This new molecular mechanism of GPIbα-facilitated clearance could potentially resolve many puzzling and seemingly contradicting observations associated with clearance of desialylated or hyposialylated platelets.

Aberrant expression of long non-coding RNAs (lncRNAs) has been detected in several types of cancer, including acute lymphoblastic leukemia (ALL), but lncRNA mapped on transcribed ultraconserved regions (T-UCRs) are little explored. The T-UCRs uc.112, uc.122, uc.160 and uc.262 were evaluated by quantitative real-time PCR in bone marrow samples from children with T-ALL (n=32) and common-ALL/pre-B ALL (n=30). In pediatric ALL, higher expression levels of uc.112 were found in patients with T-ALL, compared to patients with B-ALL. T-cells did not differ significantly from B-cells regarding uc.112 expression in non-tumor precursors from public data. Additionally, among B-ALL patients, uc.112 was also found to be increased in patients with hyperdiploidy, compared to other karyotype results. The uc.122, uc.160, and uc.262 were not associated with biological or clinical features. These findings suggest a potential role of uc.112 in pediatric ALL and emphasize the need for further investigation of T-UCR in pediatric ALL.

Schizophrenia occurs in about one in four individuals with 22q11.2 deletion syndrome (22q11.2DS). The aim of this International Brain and Behavior 22q11.2DS Consortium (IBBC) study was to identify genetic factors that contribute to schizophrenia, in addition to the ~20-fold increased risk conveyed by the 22q11.2 deletion. Using whole-genome sequencing data from 519 unrelated individuals with 22q11.2DS, we conducted genome-wide comparisons of common and rare variants between those with schizophrenia and those with no psychotic disorder at age ≥25 years. Available microarray data enabled direct comparison of polygenic risk for schizophrenia between 22q11.2DS and independent population samples with no 22q11.2 deletion, with and without schizophrenia (total n = 35,182). Polygenic risk for schizophrenia within 22q11.2DS was significantly greater for those with schizophrenia (padj = 6.73 × 10-6). Novel reciprocal case-control comparisons between the 22q11.2DS and population-based cohorts showed that polygenic risk score was significantly greater in individuals with psychotic illness, regardless of the presence of the 22q11.2 deletion. Within the 22q11.2DS cohort, results of gene-set analyses showed some support for rare variants affecting synaptic genes. No common or rare variants within the 22q11.2 deletion region were significantly associated with schizophrenia. These findings suggest that in addition to the deletion conferring a greatly increased risk to schizophrenia, the risk is higher when the 22q11.2 deletion and common polygenic risk factors that contribute to schizophrenia in the general population are both present.

Mitochondria operate as a central hub for many metabolic processes by sensing and responding to the cellular environment. Developmental cues from the environment have been implicated in selective autophagy, or mitophagy, of mitochondria during cell differentiation and tissue development. Mitophagy occurring in this context, termed programmed mitophagy, responds to cell state rather than mitochondrial damage and is often accompanied by a metabolic transition. However, little is known about the mechanisms that engage and execute mitophagy under physiological or developmental conditions. As the mammary gland undergoes post-natal development and lactation challenges mitochondrial homeostasis, we investigated the contribution of mitochondria to differentiation of mammary epithelial cells (MECs). Using lactogenic differentiation of the HC11 mouse MEC line, we demonstrated that HC11 cells transition to a highly energetic state during differentiation by engaging both oxidative phosphorylation and glycolysis. Interestingly, this transition was lost when autophagy was inhibited with bafilomycin A1 or knockdown of Atg7 (autophagy related 7). To evaluate the specific targeting of mitochondria, we traced mitochondrial oxidation and turnover in vitro with the fluorescent probe, pMitoTimer. Indeed, we found that differentiation engaged mitophagy. To further evaluate the requirement of mitophagy during differentiation, we knocked down the expression of Prkn/parkin in HC11 cells. We found that MEC differentiation was impaired in shPrkn cells, implying that PRKN is required for MEC differentiation. These studies suggest a novel regulation of MEC differentiation through programmed mitophagy and provide a foundation for future studies of development and disease associated with mitochondrial function in the mammary gland.Abbreviations: AA: antimycin A; ATG5: autophagy related 5; BAF: bafilomycin A1; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; COX8A: cytochrome c oxidase subunit 8A; CQ: chloroquine; CSN2: casein beta; ECAR: extracellular acidification rate; FCCP: trifluoromethoxy carbonylcyanide phenylhydrazone; FUNDC1: FUN14 domain containing 1; HIF1A: hypoxia inducible factor 1 subunit alpha; L1: lactation day 1; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEC: mammary epithelial cell; mitoQ: mitoquinol; mROS: mitochondrial reactive oxygen species; OCR: oxygen consumption rate; P: priming; P16: pregnancy day 16; PARP1: poly(ADP-ribose) polymerase 1; PINK1: PTEN induced kinase 1; PPARGC1A: PPARG coactivator 1 alpha; PRKN: parkin RBR E3 ubiquitin protein ligase; shNT: short hairpin non-targeting control; SQSTM1: sequestosome 1; STAT3: signal transducer and activator of transcription 3; TEM: transmission electron microscopy; TFAM: transcription factor A, mitochondrial; U: undifferentiated.