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Renert-Yuval Y, da Rosa JC, Garcet S, Pavel AB, Bares J, Chima M, Hawkes JE, Gilleaudeau P, Sullivan-Whalen M, Singer GK, Krueger JG, Guttman-Yassky E
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Analysis of alopecia areata surveys suggests a threshold for improved patient-reported outcomes

Background Although alopecia areata (AA) greatly impacts patients' quality of life (QoL), there is no adequate validation of AA-targeted QoL surveys in clinical trials, hindering sufficient representation of patient-reported outcomes. Objectives Better understanding of patient-reported outcomes may guide treatment goals and future clinical trials. Methods In a recent randomized controlled trial testing dupilumab in AA, patients were administered the Alopecia Areata Quality of Life Index (AA-QLI) and the Alopecia Areata Symptom Impact Scale (AASIS) surveys, specifically evaluating QoL in patients with AA. An in-depth analysis was performed to assess the utility of these questionnaires in this patient population, both at baseline and after treatment, and to determine a threshold for improved patient-reported outcomes. Results While AASIS correlated with baseline Severity of Alopecia Tool (SALT) scores and with therapeutic response, AA-QLI showed no correlation with AA severity before or after treatment. Itch strongly correlated with serum IgE levels across both surveys. Using various approaches to estimate a discriminative threshold for decreased impact of AA on QoL (by AASIS) following treatment, a SALT score of 20 points or less post-treatment was associated with improved patient-reported outcomes, including both AA-related symptoms and items within the daily activities/feelings domain such as 'feeling sad' and 'feeling anxious or worry'. Conclusions AASIS is better than AA-QLI to assess patient-reported outcomes. SALT <= 20 following treatment should be considered as a threshold for meaningful therapeutic outcome and as a clinical endpoint in future clinical trials for AA. What is already known about this topic? Alopecia areata greatly compromises quality of life, and affected patients have increased prevalences of depression, anxiety and social phobia. Despite the significant negative impact of the disease on patients' wellbeing, validation of targeted questionnaires in alopecia areata is lacking, and a therapeutic response threshold for improved patient-reported outcomes is unknown. What does this study add? This study investigated the utility of two different alopecia areata-targeted questionnaires - Alopecia Areata Quality of Life Index and Alopecia Areata Symptom Impact Scale (AASIS) - in a clinical trial setting. AASIS was found to correlate strongly with alopecia areata severity and clinical response. What are the clinical implications of this work? Patients with <= 20% scalp hair loss after treatment reported improvement in multiple quality-of-life items, suggesting this as a meaningful therapeutic outcome that may guide clinicians and improve the development of future clinical trials.
Yoney A, Bai L, Brivanlou AH, Siggia ED
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Mechanisms underlying WNT-mediated priming of human embryonic stem cells

DEVELOPMENT 2022 OCT; 149(20):? Article dev200335
Embryogenesis is guided by a limited set of signaling pathways dynamically expressed in different places. How a context-dependent signaling response is generated has been a central question of developmental biology, which can now be addressed with in vitro models of human embryos that are derived from embryonic stem cells (hESCs). Our previous work demonstrated that during early stages of hESC differentiation, cells chronicle signaling hierarchy. Only cells that have been exposed (primed) by WNT signaling can respond to subsequent activin exposure and differentiate to mesendodermal (ME) fates. Here, we show that WNT priming does not alter SMAD2 binding nor its chromatin opening but, instead, acts by inducing the expression of the SMAD2 co-factor EOMES. Expression of EOMES is sufficient to replace WNT upstream of activin-mediated ME differentiation, thus unveiling the mechanistic basis for priming and cellular memory in early development.
Li LL, Zhou AD, Wei YJ, Liu F, Li P, Fang RP, Ma L, Zhang SC, Wang LQ, Liu JZ, Richard HT, Chen YW, Wang HB, Huang SY
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Critical role of lncEPAT in coupling dysregulated EGFR pathway and histone H2A deubiquitination during glioblastoma tumorigenesis

SCIENCE ADVANCES 2022 OCT 7; 8(40):? Article eabn2571
Histone 2A (H2A) monoubiquitination is a fundamental epigenetics mechanism of gene expression, which plays a critical role in regulating cell fate. However, it is unknown if H2A ubiquitination is involved in EGFR-driven tumorigenesis. In the current study, we have characterized a previously unidentified oncogenic lncRNA (lncEPAT) that mediates the integration of the dysregulated EGFR pathway with H2A deubiquitination in tumorigenesis. LncEPAT was induced by the EGFR pathway, and high-level lncEPAT expression positively correlated with the glioma grade and predicted poor survival of glioma patients. Mass spectrometry analyses revealed that lncEPAT specifically interacted with deubiquitinase USP16. LncEPAT inhibited USP16's recruitment to chromatin, thereby blocking USP16-mediated H2A deubiquitination and repressing target gene expression, including CDKN1A and CLUSTERIN. Depletion of lncEPAT promoted USP16-induced cell cycle arrest and cellular senescence, and then repressed GBM cell tumorigenesis. Thus, the EGFR-lncEPAT-ubH2A coupling represents a previously unidentified mechanism for epigenetic gene regulation and senescence resistance during GBM tumorigenesis.
Okazaki A, Ott J
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Machine learning approaches to explore digenic inheritance

TRENDS IN GENETICS 2022 OCT; 38(10):1013-1018
Some rare genetic disorders, such as retinitis pigmentosa or Alport syndrome, are caused by the co-inheritance of DNA variants at two different genetic loci (digenic inheritance). To capture the effects of these disease-causing variants and their possible interactive effects, various statistical methods have been developed in human genetics. Analogous developments have taken place in the field of machine learning, particularly for the field that is now called Big Data. In the past, these two areas have grown independently and have started to converge only in recent years. We discuss an overview of each of the two fields, paying special attention to machine learning methods for uncovering the combined effects of pairs of variants on human disease.
Wang X, Simon SM, Coffino P
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Single molecule microscopy reveals diverse actions of substrate sequences that impair ClpX AAA plus ATPase function

JOURNAL OF BIOLOGICAL CHEMISTRY 2022 OCT; 298(10):? Article 102457
AAA+ (ATPases Associated with diverse cellular Activities) proteases unfold substrate proteins by pulling the substrate polypeptide through a narrow pore. To overcome the barrier to unfolding, substrates may require extended association with the ATPase. Failed unfolding attempts can lead to a slip of grip, which may result in substrate dissociation, but how substrate sequence affects slippage is unresolved. Here, we measured single molecule dwell time using total internal reflection fluo-rescence microscopy, scoring time-dependent dissociation of engaged substrates from bacterial AAA+ ATPase unfoldase/ translocase ClpX. Substrates comprising a stable domain resistant to unfolding and a C-terminal unstructured tail, tag-ged with a degron for initiating translocase insertion, were used to determine dwell time in relation to tail length and compo-sition. We found greater tail length promoted substrate retention during futile unfolding. Additionally, we tested two tail compositions known to frustrate unfolding. A poly-glycine tract (polyG) promoted release, but only when adjacent to the folded domain, whereas glycine-alanine repeats (GAr) did not promote release. A high complexity motif containing polar and charged residues also promoted release. We further investi-gated the impact of these and related motifs on substrate degradation rates and ATP consumption, using the unfoldase??? protease complex ClpXP. Here, substrate domain stability modulates the effects of substrate tail sequences. polyG and GAr are both inhibitory for unfolding, but act in different ways. GAr motifs only negatively affected degradation of highly sta-ble substrates, which is accompanied by reduced ClpXP ATPase activity. Together, our results specify substrate char-acteristics that affect unfolding and degradation by ClpXP.
Hashimoto H, Ramirez DH, Lautier O, Pawlak N, Blobel G, Palancade B, Debler EW
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Structure of the pre-mRNA leakage 39-kDa protein reveals a single domain of integrated zf-C3HC and Rsm1 modules

SCIENTIFIC REPORTS 2022 OCT 21; 12(1):? Article 17691
In Saccharomyces cerevisiae, the pre-mRNA leakage 39-kDa protein (ScPml39) was reported to retain unspliced pre-mRNA prior to export through nuclear pore complexes (NPCs). Pml39 homologs outside the Saccharomycetaceae family are currently unknown, and mechanistic insight into Pml39 function is lacking. Here we determined the crystal structure of ScPml39 at 2.5 angstrom resolution to facilitate the discovery of orthologs beyond Saccharomycetaceae, e.g. in Schizosaccharomyces pombe or human. The crystal structure revealed integrated zf-C3HC and Rsm1 modules, which are tightly associated through a hydrophobic interface to form a single domain. Both zf-C3HC and Rsm1 modules belong to the Zn-containing BIR (Baculovirus IAP repeat)-like super family, with key residues of the canonical BIR domain being conserved. Features unique to the Pml39 modules refer to the spacing between the Zn-coordinating residues, giving rise to a substantially tilted helix alpha C in the zf-C3HC and Rsm1 modules, and an extra helix alpha ' in the Rsm1 module. Conservation of key residues responsible for its distinct features identifies S. pombe Rsm1 and Homo sapiens NIPA/ZC3HC1 as structural orthologs of ScPml39. Based on the recent functional characterization of NIPA/ZC3HC1 as a scaffold protein that stabilizes the nuclear basket of the NPC, our data suggest an analogous function of ScPml39 in S. cerevisiae.
Pattwell SS, Arora S, Nuechterlein N, Zager M, Loeb KR, Cimino PJ, Holland NC, Reche-Ley N, Bolouri H, Bonnin DAA, Szulzewsky F, Phadnis VV, Ozawa T, Wagner MJ, Haffner MC, Cao JY, Shendure J, Holland EC
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Oncogenic role of a developmentally regulated NTRK2 splice variant

SCIENCE ADVANCES 2022 OCT 7; 8(40):? Article eabo6789
Temporally regulated alternative splicing choices are vital for proper development, yet the wrong splice choice may be detrimental. Here, we highlight a previously unidentified role for the neurotrophin receptor splice variant TrkB.T1 in neurodevelopment, embryogenesis, transformation, and oncogenesis across multiple tumor types in humans and mice. TrkB.T1 is the predominant NTRK2 isoform across embryonic organogenesis, and forced over expression of this embryonic pattern causes multiple solid and nonsolid tumors in mice in the context of tumor suppressor loss. TrkB.T1 also emerges as the predominant NTRK isoform expressed in a wide range of adult and pediatric tumors, including those harboring tropomyosin receptor kinase fusions. Affinity purification-mass spectrometry proteomic analysis reveals distinct interactors with known developmental and oncogenic signaling pathways such as Wnt, transforming growth factor-0, Sonic Hedgehog, and Ras. From alterations in splicing factors to changes in gene expression, the discovery of isoform specific oncogenes with embryonic ancestry has the potential to shape the way we think about developmental systems and oncology.
Akagbosu B, Tayyebi Z, Shibu G, Iza YAP, Deep D, Parisotto YF, Fisher L, Pasolli HA, Thevin V, Elmentaite R, Knott M, Hemmers S, Jahn L, Friedrich C, Verter J, Wang ZM, van den Brink M, Gasteiger G, Grunewald TGP, Marie JC, Leslie C, Rudensky AY, Brown CC
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Novel antigen-presenting cell imparts T-reg-dependent tolerance to gut microbiota

NATURE 2022 OCT 27; 610(7933):752-+
Establishing and maintaining tolerance to self-antigens or innocuous foreign antigens is vital for the preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (AIRE) have a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (T-reg) cell development(1-4). Within weeks of birth, a separate wave of T-reg cell differentiation occurs in the periphery upon exposure to antigens derived from the diet and commensal microbiota(5-8), yet the cell types responsible for the generation of peripheral T-reg (pT(reg)) cells have not been identified. Here we describe the identification of a class of ROR gamma t(+) antigen-presenting cells called Thetis cells, with transcriptional features of both mTECs and dendritic cells, comprising four major sub-groups (TC I-TC IV). We uncover a developmental wave of Thetis cells within intestinal lymph nodes during a critical window in early life, coinciding with the wave of pT(reg) cell differentiation. Whereas TC I and TC III expressed the signature mTEC nuclear factor AIRE, TC IV lacked AIRE expression and was enriched for molecules required for pT(reg) generation, including the TGF-beta-activating integrin alpha v beta 8. Loss of either major histocompatibility complex class II (MHCII) or ITGB8 by Thetis cells led to a profound impairment in intestinal pT(reg) differentiation, with ensuing colitis. By contrast, MHCII expression by ROR gamma t(+) group 3 innate lymphoid cells (ILC3) and classical dendritic cells was neither sufficient nor required for pT(reg) generation, further implicating TC IV as the tolerogenic ROR gamma t(+) antigen-presenting cell with an essential function in early life. Our studies reveal parallel pathways for the establishment of tolerance to self and foreign antigens in the thymus and periphery, respectively, marked by the involvement of shared cellular and transcriptional programmes.
Capparelli EV, Ajibola G, Maswabi K, Holme MP, Bennett K, Powis KM, Moyo S, Mohammed T, Maphorisa C, Hughes MD, Seaton KE, Tomaras GD, Mosher S, Taylor A, O'Connell S, Narpala S, Mcdermott A, Caskey M, Gama L, Lockman S, Jean-Philippe P, Makhema J, Kuritzkes DR, Lichterfeld M, Shapiro RL
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Safety and Pharmacokinetics of Intravenous 10-1074 and VRC01LS in Young Children

Background: Broadly neutralizing monoclonal antibodies (bNAbs) suppress HIV-1 RNA and may deplete residual viral reservoirs. We evaluated the safety and pharmacokinetics (PK) of dual intravenous VRC01LS and 10-1074 in very early-treated children with HIV-1 on suppressive antiretroviral treatment (ART). Setting: Botswana. Methods: Children with HIV-1 (median age 3.1 years) on ART from <7 days old were enrolled. In phase A, 6 children received 10-1074 (30 mg/kg at day 0, 28, and 56) and 6 children received VRC01LS (30 mg/kg at day 0, 10 mg/kg at days 28 and 56) by intravenous infusion. In phase B, 6 children received the 2 bNAbs combined (with higher VRC01LS maintenance dose, 15 mg/kg) every 4 weeks for 32 weeks with PK evaluations over 8 weeks. Population PK models were developed to predict steady-state concentrations. Results: BNAb infusions were well tolerated. There were no infusion reactions nor any bNAb-related grade 3 or 4 events. The median (range) first dose Cmax and trough (day 28) combined from both phases were 1405 (876-1999) mu g/mL and 133 (84-319) mu g/mL for 10-1074 and 776 (559-846) mu g/mL and 230 (158-294) mu g/mL for VRC01LS. No large differences in bNAb clearances were observed when given in combination. The estimated VRC01LS half-life was shorter than in adults. Predicted steady-state troughs [median (90% prediction interval)] were 261 (95-565) and 266 (191-366) mu g/mL for 10-1074 and VRC01LS, respectively, when given in combination. Conclusions: 10-1074 and VRC01LS were safe and well-tolerated among children receiving ART. Troughs exceeded minimal targets with every 4-week administration of 10-1074 at 30 mg/kg and VRC01LS at 15 mg/kg.
Cheshenko N, Bonanno JB, Hoffmann HH, Jangra RK, Chandran K, Rice CM, Almo SC, Herold BC
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Cell-impermeable staurosporine analog targets extracellular kinases to inhibit HSV and SARS-CoV-2

COMMUNICATIONS BIOLOGY 2022 OCT 16; 5(1):? Article 1096
Herpes simplex virus (HSV) receptor engagement activates phospholipid scramblase triggering Akt translocation to the outer leaflet of the plasma membrane where its subsequent phosphorylation promotes viral entry. We hypothesize that this previously unrecognized outside-inside signaling pathway is employed by other viruses and that cell-impermeable kinase inhibitors could provide novel antivirals. We synthesized a cell-impermeable analog of staurosporine, CIMSS, which inhibited outer membrane HSV-induced Akt phosphorylation and blocked viral entry without inducing apoptosis. CIMSS also blocked the phosphorylation of 3-phosphoinositide dependent protein kinase 1 and phospholipase C gamma, which were both detected at the outer leaflet following HSV exposure. Moreover, vesicular stomatitis virus pseudotyped with SARS-CoV-2 spike protein (VSV-S), but not native VSV or VSV pseudotyped with Ebola virus glycoprotein, triggered this scramblase-Akt outer membrane signaling pathway. VSV-S and native SARS-CoV-2 infection were inhibited by CIMSS. Thus, CIMSS uncovered unique extracellular kinase processes linked to HSV and SARS-CoV-2 entry. A cell-impermeable analog of staurosporine blocks phosphorylation events at the outer leaflet of the plasma membrane, which prevents herpes simplex virus and SARS-CoV-2 entry without inducing apoptosis.