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Induction of fetal hemoglobin (HbF) has been shown to be a viable therapeutic approach to treating sickle cell disease and potentially other β-hemoglobinopathies. To identify targets and target-modulating small molecules that enhance HbF expression, we engineered a human umbilical-derived erythroid progenitor reporter cell line (HUDEP2_HBG1_HiBiT) by genetically tagging a HiBiT peptide to the carboxyl (C)-terminus of the endogenous HBG1 gene locus, which codes for γ-globin protein, a component of HbF. Employing this reporter cell line, we performed a chemogenomic screen of approximately 5000 compounds annotated with known targets or mechanisms that have achieved clinical stage or approval by the US Food and Drug Administration (FDA). Among them, 10 compounds were confirmed for their ability to induce HbF in the HUDEP2 cell line. These include several known HbF inducers, such as pomalidomide, lenalidomide, decitabine, idoxuridine, and azacytidine, which validate the translational nature of this screening platform. We identified avadomide, autophinib, triciribine, and R574 as novel HbF inducers from these screens. We orthogonally confirmed HbF induction activities of the top hits in both parental HUDEP2 cells as well as in human primary CD34+ hematopoietic stem and progenitor cells (HSPCs). Further, we demonstrated that pomalidomide and avadomide, but not idoxuridine, induced HbF expression through downregulation of several transcriptional repressors such as BCL11A, ZBTB7A, and IKZF1. These studies demonstrate a robust phenotypic screening workflow that can be applied to large-scale small molecule profiling campaigns for the discovery of targets and pathways, as well as novel therapeutics for sickle cell disease and other β-hemoglobinopathies., Competing Interests: Takeda Development Center Americas, Inc. provided sponsorship and financial support for this study. All the authors are employees of Takeda Pharmaceutical Industries, Ltd., and had equity ownership with Takeda Pharmaceutical Industries, Ltd. The Takeda commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare., (Copyright: © 2024 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

COVID-19 is linked to endotheliopathy and coagulopathy, which can result in multi-organ failure. The mechanisms causing endothelial damage due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain elusive. Here, we developed an infection-competent human vascular organoid from pluripotent stem cells for modeling endotheliopathy. Longitudinal serum proteome analysis identified aberrant complement signature in critically ill patients driven by the amplification cycle regulated by complement factor B and D (CFD). This deviant complement pattern initiates endothelial damage, neutrophil activation, and thrombosis specific to organoid-derived human blood vessels, as verified through intravital imaging. We examined a new long-acting, pH-sensitive (acid-switched) antibody targeting CFD. In both human and macaque COVID-19 models, this long-acting anti-CFD monoclonal antibody mitigated abnormal complement activation, protected endothelial cells, and curtailed the innate immune response post-viral exposure. Collectively, our findings suggest that the complement alternative pathway exacerbates endothelial injury and inflammation. This underscores the potential of CFD-targeted therapeutics against severe viral-induced inflammathrombotic outcomes., Competing Interests: Declaration of interests N.S. and T.T. are patent holders associated with the technology described in this project., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: Several clinical phenotypes including fetal hydrops, central conducting lymphatic anomaly or capillary malformations with arteriovenous malformations 2 (CM-AVM2) have been associated with EPHB4 (Ephrin type B receptor 4) variants, demanding new approaches for deciphering pathogenesis of novel variants of uncertain significance (VUS) identified in EPHB4, and for the identification of differentiated disease mechanisms at the molecular level., Methods: Ten index cases with various phenotypes, either fetal hydrops, CM-AVM2, or peripheral lower limb lymphedema, whose distinct clinical phenotypes are described in detail in this study, presented with a variant in EPHB4. In vitro functional studies were performed to confirm pathogenicity., Results: Pathogenicity was demonstrated for six of the seven novel EPHB4 VUS investigated. A heterogeneity of molecular disease mechanisms was identified, from loss of protein production or aberrant subcellular localization to total reduction of the phosphorylation capability of the receptor. There was some phenotype-genotype correlation; however, previously unreported intrafamilial overlapping phenotypes such as lymphatic-related fetal hydrops (LRFH) and CM-AVM2 in the same family were observed., Conclusion: This study highlights the usefulness of protein expression and subcellular localization studies to predict EPHB4 variant pathogenesis. Our accurate clinical phenotyping expands our interpretation of the Janus-faced spectrum of EPHB4-related disorders, introducing the discovery of cases with overlapping phenotypes.

1p36 deletion syndrome is a well-described condition with a recognizable phenotype, including cognitive impairment, seizures, and structural brain anomalies such as periventricular leukomalacia (PVL). In a large series of these individuals by Battaglia et al., "birth history was notable in 50% of the cases for varying degrees of perinatal distress." Given the potential for perinatal distress, seizures and PVL, we questioned if this disorder has clinical overlap with hypoxic ischemic encephalopathy (HIE). We reviewed the medical records of 69 individuals with 1p36 deletion to clarify the perinatal phenotype of this disorder and determine if there is evidence of perinatal distress and/or hypoxic injury. Our data provides evidence that these babies have signs of perinatal distress. The majority (59% term; 75% preterm) needed resuscitation and approximately 18% had cardiac arrest. Most had abnormal brain imaging (84% term; 73% preterm) with abnormal white matter findings in over half of patients. PVL or suggestion of "hypoxic insult" was present in 18% of term and 45% of preterm patients. In conclusion, individuals with 1p36 deletion have evidence of perinatal distress, white matter changes, and seizures, which can mimic HIE but are likely related to their underlying chromosome disorder., (© 2019 Wiley Periodicals, Inc.)

Alport syndrome (AS) is a genetic disease characterized by haematuric glomerulopathy variably associated with hearing loss and anterior lenticonus. It is caused by mutations in the COL4A3, COL4A4 or COL4A5 genes encoding the α3α4α5(IV) collagen heterotrimer. AS is rare, but it accounts for >1% of patients receiving renal replacement therapy. Angiotensin-converting enzyme inhibition slows, but does not stop, the progression to renal failure; therefore, there is an urgent requirement to expand and intensify research towards discovering new therapeutic targets and new therapies. The 2015 International Workshop on Alport Syndrome targeted unmet needs in basic science, genetics and diagnosis, clinical research and current clinical care. In three intensive days, more than 100 international experts including physicians, geneticists, researchers from academia and industry, and patient representatives from all over the world participated in panel discussions and breakout groups. This report summarizes the most important priority areas including (i) understanding the crucial role of podocyte protection and regeneration, (ii) targeting mutations by new molecular techniques for new animal models and potential gene therapy, (iii) creating optimal interaction between nephrologists and geneticists for early diagnosis, (iv) establishing standards for mutation screening and databases, (v) improving widespread accessibility to current standards of clinical care, (vi) improving collaboration with the pharmaceutical/biotech industry to investigate new therapies, (vii) research in hearing loss as a huge unmet need in Alport patients and (viii) the need to evaluate the risk and benefit of novel (including 'repurposing') therapies on an international basis., (© The Author 2016. Published by Oxford University Press on behalf of ERAEDTA.)

Array comparative genomic hybridization (aCGH) is a powerful genetic tool that has enabled the identification of novel imbalances in individuals with intellectual disability (ID), autistic disorders and congenital malformations. Here we report a 'genotype first' approach using aCGH on 13 unrelated patients with 19p13.3 submicroscopic rearrangement (11 deletions and 2 duplications) and review cases in the literature and in public databases. Shared phenotypic features suggest that these patients represent an interstitial microdeletion/microduplication syndrome at 19p13.3. Common features consist of abnormal head circumference in most patients (macrocephaly with the deletions and microcephaly with the duplications), ID with developmental delay (DD), hypotonia, speech delay and common dysmorphic features. The phenotype is associated with at least a ~0.113 Mb critical region harboring three strong candidate genes probably associated with DD, ID, speech delay and other dysmorphic features: MAP2K2, ZBTB7A and PIAS4, an E3 ubiquitin ligase involved in the ubiquitin signaling pathways, which we hypothesize for the first time to be associated with head size in humans.

Chromosome 17p13.3 is a gene rich region that when deleted is associated with the well-known Miller-Dieker syndrome. A recently described duplication syndrome involving this region has been associated with intellectual impairment, autism and occasional brain MRI abnormalities. We report 34 additional patients from 21 families to further delineate the clinical, neurological, behavioral, and brain imaging findings. We found a highly diverse phenotype with inter- and intrafamilial variability, especially in cognitive development. The most specific phenotype occurred in individuals with large duplications that include both the YWHAE and LIS1 genes. These patients had a relatively distinct facial phenotype and frequent structural brain abnormalities involving the corpus callosum, cerebellar vermis, and cranial base. Autism spectrum disorders were seen in a third of duplication probands, most commonly in those with duplications of YWHAE and flanking genes such as CRK. The typical neurobehavioral phenotype was usually seen in those with the larger duplications. We did not confirm the association of early overgrowth with involvement of YWHAE and CRK, or growth failure with duplications of LIS1. Older patients were often overweight. Three variant phenotypes included cleft lip/palate (CLP), split hand/foot with long bone deficiency (SHFLD), and a connective tissue phenotype resembling Marfan syndrome. The duplications in patients with clefts appear to disrupt ABR, while the SHFLD phenotype was associated with duplication of BHLHA9 as noted in two recent reports. The connective tissue phenotype did not have a convincing critical region. Our experience with this large cohort expands knowledge of this diverse duplication syndrome., (Copyright © 2013 Wiley Periodicals, Inc.)

Currently, products containing interferon beta (IFNβ) are injected either intramuscularly or subcutaneously. To avoid the necessity of injection, we developed a novel monomeric Fc fusion protein of IFNβ (IFNβFc) that is absorbed via an immunoglobulin transport system present in the upper and central airways upon administration of the drug as an inhaled aerosol. The systemic absorption of IFNβFc through the lung in non-human primates, at deposited doses of 1, 3, and 10 μg/kg, was compared to the absorption of a single 3 μg/kg dose of IFNβ-1a (Avonex®) subcutaneously administered. IFNβFc was well absorbed through the lung, displaying dose proportional increases in serum concentrations, and was biologically active, as shown by increases in plasma neopterin levels. The circulating half-life of IFNβFc was ∼3 times longer (∼30 h) than that of IFNβ-1a, (8-9 h). At approximately equimolar doses of IFNβFc (10 μg/kg) and IFNβ-1a (3 μg/kg), the stimulation of neopterin over background levels was approximately equivalent, demonstrating that the longer half-life of IFNβFc compensated for the lower relative specific antiviral activity of IFNβFc measured in vitro. In conclusion, IFNβFc was efficiently absorbed after pulmonary delivery in non-human primates, retained its biological activity, and may offer a convenient alternative to injectable IFNβ.

The effects of missense changes and small in-frame deletions and insertions on protein function are not easy to predict, and the identification of such variants in individuals at risk of a genetic disease can complicate genetic counselling. One option is to perform functional tests to assess whether the variants affect protein function. We have used this strategy to characterize variants identified in the TSC1 and TSC2 genes in individuals with, or suspected of having, Tuberous Sclerosis Complex (TSC). Here we present an overview of our functional studies on 45 TSC1 and 107 TSC2 variants. Using a standardized protocol we classified 16 TSC1 variants and 70 TSC2 variants as pathogenic. In addition we identified eight putative splice site mutations (five TSC1 and three TSC2). The remaining 24 TSC1 and 34 TSC2 variants were classified as probably neutral., (© 2011 Wiley-Liss, Inc.)

As described for a long time, carcinoma-derived Caco-2 cells form a polarized epithelium in culture, whereas HT29-D4 cells are nonpolarized and undifferentiated but can form a polarized monolayer when cultured in a galactose-supplemented medium. Using NF-kappaB translocation and IL-8 and ICAM-1 gene activation as an index, we have studied the relationship between the differentiation state and the cell response to cytokines. We found that differentiated Caco-2 and HT29-D4 cells were responsive to both cytokines TNFalpha- and IL-1beta-mediated activation of NF-kappaB but that undifferentiated HT29-D4 cells were unresponsive to IL-1beta. However, the expression of endogenous ICAM-1 and IL-8 genes was upregulated by these cytokines in either cell lines differentiated or not. Upregulation of ICAM-1 gene occurred when IL-1beta or TNFalpha was added to the basal, but not apical surface of the differentiated epithelia. Finally, it appeared that in polarized HT29-D4 cells, the IL-1beta-induced translocation of NF-kappaB was connected to PKCdelta translocation.

IkappaBbeta, one of the major IkappaB proteins, is only partially degraded in response to most extracellular signals. However, the molecular mechanism of this event is unknown. We show here that IkappaBbeta exists in at least two different forms: one that is bound to the NF-kappaB dimer and the other bound to both NF-kappaB and kappaB-Ras, a Ras-like small G protein. Removal of cellular kappaB-Ras enhances whereas excess kappaB-Ras blocks induced IkappaBbeta degradation. Remarkably, kappaB-Ras functions in both GDP- and GTP-bound states, and mutations of the conserved guanine-binding residues of kappaB-Ras abrogate its ability to block degradation of IkappaBbeta. kappaB-Ras also directly blocks the in vitro phosphorylation of IkappaBbeta by IKKbeta. These observations suggest that IkappaBbeta in the ternary complex is resistant to degradation by most signals. We suggest that specific signals, in addition to those that activate only IKK, are essential for the complete degradation of IkappaBbeta.

We showed previously that microtubule disassembly by vinblastine induces the proto-oncogene c-myc in epithelial mammary HBL100 cells. In this study, we demonstrate that vinblastine treatment in these cells, in contrast to what was observed with the colon adenocarcinoma cell line HT29-D4, activated the transcription factor NFkappaB, which has been involved in c-myc regulation. The microtubule disassembly also induced IkappaB degradation. Using transient transfection analysis, we show that the trans-activation of c-myc by vinblastine was decreased when NFkappaB binding sites on c-myc promoter were mutated. Additionally, we demonstrate that microtubule dissolution trans-activated a thymidine kinase-CAT construct containing an NFkappaB binding site at -1180 to -1080 bp relative to the P1 promoter. Thus, vinblastine up-regulates the enhancer activity of the NFkappaB binding site. These results suggest that microtubule disassembly induced by vinblastine can trans-activate the c-myc oncogene through NFkappaB. Taking into consideration the paradoxical roles of both c-myc and NFkappaB in proliferation or apoptosis, this data reveals the complex action mechanism of this microtubule interfering agent.

We have studied the role of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) on salt appetite developed by deoxycorticosterone acetate (DOCA) treated rats. To this end, we measured the effects of DOCA given on alternate days on (1) salt intake; (2) MR and GR in hippocampus (HIPPO), amygdala (AMYG), and hypothalamus (HT); (3) the activity of ornithine decarboxylase (ODC), a GR-mediated response, and (4) the salt intake after treatment with the antiglucocorticoid RU 486 or the antimineralocorticoid ZK 91587. First, we demonstrated that 10 but not 1 mg DOCA induced natriogenesis. Forty-eight hours after adrenalectomy and 24 h after the last DOCA injection, 10 but not 1 mg hormone reduced binding to GR in HIPPO, AMYG, and HT. Both doses of DOCA also reduced the binding to MR in HIPPO, without changes in AMYG; in HT the 1-mg dose was without effect, but the natriogenic dose (10 mg) highly increased binding of [3H]-corticosterone to MR. Scatchard analysis demonstrated increased Bmax and Kd values in the HT of DOCA-treated rats. Occupation of GR by DOCA did not stimulate the ODC activity, in contrast to the four-fold increment effected by the glucocorticoid dexamethasone. Also, administration of RU 486 did not inhibit the sale intake promoted by DOCA, in contrast to ZK 91587 which partly delayed the natriogenic effect of DOCA. It is suggested that brain MR are involved in the natriogenic effect of DOCA, whereas the role of GR is inconclusive.(ABSTRACT TRUNCATED AT 400 WORDS)

Type I corticosteroid receptors were determined in cytosol from hippocampus (HIPPO) and amygdala (AMYG), using [3H]aldosterone (ALDO), [3H]dexamethasone (DEX) or the mineralocorticoid antagonist [3H]ZK 91587 as ligands. Incubations with the first two compounds also contained the pure glucocorticoid RU 28362 to block type II receptors. Binding of the three ligands was comparable in cytosol from HIPPO and it was slightly higher for [3H]DEX in AMYG. However, after heat-induced receptor transformation, binding to DNA-cellulose was observed for [3H]ALDO-receptor complex obtained from HIPPO or AMYG, whereas it was negligible for [3H]ZK 91587. Receptors charged with [3H]DEX or [3H]ALDO showed similar retention on DNA-cellulose columns in the case of the AMYG, while binding to the polynucleotide was higher for [3H]ALDO in the HIPPO. Finally, only [3H]ALDO was taken up to a significant extent in purified cell nuclei prepared from slices of HIPPO and AMYG previously incubated with the three ligands. It is concluded that binding of a natural agonist steroid may be a prerequisite for type I receptor transformation and translocation from the cytoplasm into the nuclear fraction. DEX binding to type I receptors resembles a partial agonist with antagonist properties, whereas antagonists such as ZK 91587 are bound and retained in cytoplasm, without further translocation.

1. We have used in situ hybridization techniques to determine the mRNA for (Na + K)ATPase in 20 brain regions from control rats and rats treated with high doses of deoxycorticosterone (DOC). 2. DOC-treated rats developed a salt appetite following the second hormone administration on alternate days and were used after the fourth DOC administration. 3. DOC treatment did not change the number of silver grains/cell deposited in cells from Ca1, CA2, CA3, and CA4 hippocampal subfields, dentate gyrus, cerebral cortex, medial preoptic area (POA), substantia nigra, and periventricular gray matter. 4. Nonsignificant reductions were detected in lateral POA, medial and lateral septum, caudate-putamen, and three amygdaloid nuclei (cortical, basolateral, and central) from DOC-treated rats. 5. Significant reductions were obtained, after DOC administration, in arcuate and ventromedial hypothalamic nuclei and medial and lateral amygdala. 6. The results suggested that regulation of the beta-subunit mRNA of (Na + K)-ATPase may be related to the central actions of mineralocorticoids in the control of salt intake.

We have studied the binding of the synthetic antimineralocorticoid [3H]ZK 91587 to soluble receptors in brain of adrenalectomized rats. It was observed that [3H]ZK 91587 labeled a single receptor class with high affinity (Kd 1.3 nM) and low capacity (51.1 fmol/mg prot.) in cytosol of hippocampus (HIPPO). The ligand was efficiently displaced in vitro from the receptor by aldosterone (IC50 2.0 nM) and corticosterone (2.3), while dexamethasone showed less potency (IC50 5.1 nM) and the pure antiglucocorticoid RU 28362 competed weakly (161 nM). Furthermore, there was a widespread distribution of binding sites all over the brain for this compound, but with CA1 and CA3 regions of HIPPO, some amygdaloid nuclei and lateral septum containing most of the binding sites, as revealed by binding assays employing 16 different microdissected brain regions. Finally, the receptor labeled with [3H]ZK 91587 was readily displaced by administration of aldosterone in vivo in physiological amounts, from 5 whole brain regions examined, but preferentially from preoptic area, amygdala and HIPPO. It is concluded that [3H]ZK 91587 is a useful ligand for further studies on putative mineralocorticoid responsive cells in brain, due to its high affinity, stability and lack of cross reactivity with glucocorticoid receptors. Its brain distribution is similar to that previously obtained using [3H]aldosterone in the presence of RU 28362 to block ligand binding to the glucocorticoid receptor.