Epithelial outgrowth through mesenchymal rings drives alveologenesis
With real-time, long-term 4-dimensional imaging of living lung slices, Negretti et al. developed a model of lung formation of epithelial ballooning through contractile mesenchymal rings. The cover image is a still frame from an 80-hour 4-dimensional live-imaging experiment of a precision cut lung slice from a postnatal day 5 mouse, at the saccular-alveolar transition. Alveolar type 2 cells are shown in cyan, and other cells are shown in magenta. Image credit: Nicholas Negretti and Jennifer Sucre.
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Research Letter
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Abstract
Authors
Jing Zeng, Derrick Zhao, Grayson Way, Andrew Fagan, Michael Fuchs, Puneet Puri, Brian C. Davis, Xuan Wang, Emily C. Gurley, Phillip B. Hylemon, Jian-Gao Fan, Masoumeh Sikaroodi, Patrick M. Gillevet, Huiping Zhou, Jasmohan S. Bajaj
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Research Articles
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Abstract
Disrupted feeding and fasting cycles as well as chronic high-fat diet–induced (HFD-induced) obesity are associated with cardiovascular disease risk factors. We designed studies that determined whether 2 weeks of time-restricted feeding (TRF) intervention in mice fed a chronic HFD would reduce cardiovascular disease risk factors. Mice were fed a normal diet (ND; 10% fat) ad libitum or HFD (45% fat) for 18 weeks ad libitum to establish diet-induced obesity. ND or HFD mice were continued on ad libitum diet or subjected to TRF (limiting food availability to 12 hours only during the dark phase) during the final 2 weeks of the feeding protocol. TRF improved whole-body metabolic diurnal rhythms without a change in body weight. HFD mice showed reduced blood pressure dipping compared with ND, which was restored by TRF. Further, TRF reduced aortic wall thickness, decreased aortic stiffness, as well as increased kidney tubular brush border integrity, decreased renal medullary fibrosis, and reduced renal medullary T cell inflammation in HFD mice. These findings indicate that TRF may be an effective intervention for improving vascular and kidney health in a model of established diet-induced obesity.
Authors
Paramita Pati, Carmen De Miguel, Jodi R. Paul, Dingguo Zhang, Jackson Colson, John Miller Allan, Claudia J. Edell, Megan K. Rhoads, Luke S. Dunaway, Sara N. Biswal, Yihan Zhong, Randee Sedaka, Telisha Millender-Swain, Shannon M. Bailey, Karen L. Gamble, David M. Pollock, Jennifer S. Pollock
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This study examined the involvement of fibroblast growth factor-23 (FGF-23) in primary aldosteronism (PA), a condition characterized by elevated aldosterone levels and hypertension. We recruited patients with unilateral PA (uPA) and observed increased levels of C-terminal FGF-23 (cFGF-23) and C-terminal to intact FGF-23 (iFGF-23) in patients with uPA compared with essential hypertension control participants. Elevated preoperative cFGF-23 levels were associated with adverse outcomes, including mortality and cardiovascular or kidney events. Plasma cFGF-23 levels demonstrated a nonlinear rise with aldosterone, but iFGF-23 levels were not correlated with plasma aldosterone concentration. Higher cFGF-23 levels independently predicted hypertension remission after adrenalectomy for patients with uPA. Patients with uPA, who exhibited elevated cFGF-23 levels, had decreased levels after adrenalectomy. In cell cultures, aldosterone enhanced cleavage of iFGF-23, leading to increased levels of cFGF-23 fragments, an effect mitigated by silencing of family with sequence similarity 20, member C (FAM20C). However, the enhancement of cFGF-23 levels remained unaffected by the furin inhibitor. The study suggests that aldosterone influences FGF-23 phosphorylation by interacting with FAM20C, with docking experiments indicating aldosterone’s binding to FAM20C. This work highlights that patients with uPA with elevated cFGF-23 levels are associated with cardiovascular risks, and adrenalectomy reduces cFGF-23. Aldosterone likely promotes cFGF-23 production through FAM20C-mediated phosphorylation of iFGF-23.
Authors
Vin-Cent Wu, Kang-Yung Peng, Tsu-I Chen, Chiao-Yin Sun, Hung-Wei Liao, Chieh-Kai Chan, Yen-Hung Lin, Hung-Hsiang Liou, Jeff S. Chueh
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Pancreatic ductal adenocarcinoma (PDAC) is a drug-resistant and lethal cancer. Identification of the genes that consistently show altered expression across patient cohorts can expose effective therapeutic targets and strategies. To identify such genes, we separately analyzed 5 human PDAC microarray datasets. We defined genes as “consistent” if upregulated or downregulated in 4 or more datasets (adjusted P < 0.05). The genes were subsequently queried in additional datasets, including single-cell RNA-sequencing data, and we analyzed their pathway enrichment, tissue specificity, essentiality for cell viability, and association with cancer features, e.g., tumor subtype, proliferation, metastasis, and poor survival outcome. We identified 2,010 consistently upregulated and 1,928 downregulated genes, of which more than 50% to our knowledge were uncharacterized in PDAC. These genes spanned multiple processes, including cell cycle, immunity, transport, metabolism, signaling, and transcriptional/epigenetic regulation — cell cycle and glycolysis being the most altered. Several upregulated genes correlated with cancer features, and their suppression impaired PDAC cell viability in prior CRISPR/Cas9 and RNA interference screens. Furthermore, the upregulated genes predicted sensitivity to bromodomain and extraterminal (epigenetic) protein inhibition, which, in combination with gemcitabine, disrupted amino acid metabolism and in vivo tumor growth. Our results highlight genes for further studies in the quest for PDAC mechanisms, therapeutic targets, and biomarkers.
Authors
Zeribe C. Nwosu, Heather M. Giza, Maya Nassif, Verodia Charlestin, Rosa E. Menjivar, Daeho Kim, Samantha B. Kemp, Peter Sajjakulnukit, Anthony Andren, Li Zhang, William K.M. Lai, Ian Loveless, Nina Steele, Jiantao Hu, Biao Hu, Shaomeng Wang, Marina Pasca di Magliano, Costas A. Lyssiotis
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Craniosynostosis (CRS) is characterized by the development of abnormal cranial suture ossification and premature fusion. Despite the identification of several associated genetic disorders, the genetic determinants of CRS remain poorly understood. In this study, we conducted integrative analyses on 225 exomes, comprising 121 CRS probands and 104 parental exomes (52 trios). These analyses encompassed de novo and pathogenic variants, and digenic combinations within haploinsufficient genes harboring rare variants. Our analysis unveils a shared molecular network between genes associated with CRS and those linked to skeletal and neurodevelopmental disorders, with a notable enrichment of deleterious variants within haploinsufficient genes. Additionally, we identified a unique digenic pair (IL6ST and TRPS1) within haploinsufficient genes that was present in 2 patients with nonsyndromic CRS but absent in parents or 1,048 population controls. In vitro experiments provided evidence that the identified missense variants were hypomorphs, and accelerated bone mineralization could result from the additive effects of diminished IL6ST and TRPS1 activities in osteoblasts. Overall, our study underscores the important role of rare variations in haploinsufficient genes and suggests that in a subset of undiagnosed patients, the CRS phenotype may arise from multiple genetic variations.
Authors
Jung Woo Yu, Jihoon G. Yoon, Chaerim Han, Shin Hye Noh, Dong Min Shin, Yu-Mi Yang, Yong Oock Kim, Kyu-Won Shim, Min Goo Lee
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Necrobiosis is a histologic term used to describe abnormal deposits of “degenerating” collagen within the skin. It can be found as an incidental finding in various granulomatous conditions, but is a hallmark of necrobiosis lipoidica (NL) and necrobiotic xanthogranuloma (NXG). There is limited prior research on necrobiosis. Here, we employed single-cell analysis of lesional and nonlesional skin to study the pathophysiology of necrobiosis. Our findings demonstrate that necrobiotic lesional skin is characterized by SPP1hi macrophages expressing MARCO; NKG7-expressing effector CD8+ T cells coexpressing CCL5, IFNG, GZMs, and PRF1; CCL5hi fibroblasts coexpressing CXCL9, diverse collagens (e.g., COL4A4, COL11A1, COL8A1), and TIMP1; and IGHM-expressing plasma cells. Integrative analysis of signaling ligands and receptor expression identified strong cell-cell communication between NKG7+ T cells, CCL5hi fibroblasts, and SPP1-expressing macrophages. In contrast, these cell populations were not dominant features of systemic sclerosis, another collagen deposition disease. Furthermore, although SPP1-expressing macrophages were detectable in sarcoidosis, IFNG-expressing T cells were a more defining feature of sarcoidosis compared with NL and NXG. From these findings, we speculate that necrobiosis results from the deposition of diverse collagens and ECM proteins through a process driven by CCL5-expressing fibroblasts and SPP1-expressing macrophages.
Authors
Stephanie T. Le, Alina I. Marusina, Alexander A. Merleev, Amanda Kirane, Olga Kruglinskaya, Andrey Kunitsyn, Nikolay Yu Kuzminykh, Xianying Xing, Sophie Y. Li, William Liakos, J. Michelle Kahlenberg, Andrea Gompers, Lauren Downing, Sahiti Marella, Allison C. Billi, Paul W. Harms, Lam C. Tsoi, Marie-Charlotte Brüggen, Iannis E. Adamopoulos, Johann E. Gudjonsson, Emanual Maverakis
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Dengue is widespread in tropical and subtropical regions globally and imposes a considerable disease burden. Annually, dengue virus (DENV) causes up to 400 million infections, of which approximately 25% present with clinical manifestations ranging from mild to fatal. Despite its significance as a growing public health concern, developing effective DENV vaccines has been challenging. One reason is the lack of comprehensive understanding of the influence exerted by prior DENV infections and immune responses with cross-reactive properties. To investigate this, we collected samples from a pediatric cohort study in dengue-endemic Managua, Nicaragua. We characterized T cell responses in 71 healthy children who had previously experienced 1 or more natural DENV infections and who, within 1 year after sample collection, had a subsequent DENV infection that was either symptomatic or inapparent. Our study investigated the effect of preexisting DENV-specific T cell responses on clinical outcomes of subsequent DENV infection. We assessed DENV-specific T cell responses using an activation-induced marker assay. Children with only 1 prior DENV infection displayed heterogeneous DENV-specific CD4+ and CD8+ T cell frequencies. In contrast, children with 2 or more prior DENV infections showed significantly higher DENV-specific CD4+ and CD8+ T cell frequencies associated with inapparent rather than symptomatic outcomes in subsequent infection. These findings demonstrate the protective role of DENV-specific T cells against symptomatic DENV infection and advance efforts to identify protective immune correlates against dengue.
Authors
Rosa Isela Gálvez, Amparo Martínez-Pérez, E. Alexandar Escarrega, Tulika Singh, José Victor Zambrana, Ángel Balmaseda, Eva Harris, Daniela Weiskopf
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CD8+ T cells are critical for immune protection against severe COVID-19 during acute infection with SARS-CoV-2. However, the induction of antiviral CD8+ T cell responses varies substantially among infected people, and a better understanding of the mechanisms that underlie such immune heterogeneity is required for pandemic preparedness and risk stratification. In this study, we analyzed SARS-CoV-2–specific CD4+ and CD8+ T cell responses in relation to age, clinical status, and inflammation among patients infected primarily during the initial wave of the pandemic in France or Japan. We found that age-related contraction of the naive lymphocyte pool and systemic inflammation were associated with suboptimal SARS-CoV-2–specific CD4+ and, even more evidently, CD8+ T cell immunity in patients with acute COVID-19. No such differences were observed for humoral immune responses targeting the spike protein of SARS-CoV-2. We also found that the proinflammatory cytokine IL-18, concentrations of which were significantly elevated among patients with severe disease, suppressed the de novo induction and memory recall of antigen-specific CD8+ T cells, including those directed against SARS-CoV-2. These results potentially explain the vulnerability of older adults to infections that elicit a profound inflammatory response, exemplified by acute COVID-19.
Authors
Gaëlle Autaa, Laura Papagno, Takuto Nogimori, Andrea Boizard-Moracchini, Daniil Korenkov, Maeva Roy, Koichiro Suzuki, Yuji Masuta, Eoghann White, Sian Llewellyn-Lacey, Yasuo Yoshioka, Francesco Nicoli, David A. Price, Julie Dechanet-Merville, Takuya Yamamoto, Isabelle Pellegrin, Victor Appay
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Thrombin promotes the proliferation and function of CD8+ T cells. To test if thrombin prevents exhaustion and sustains antiviral T cell activity during chronic viral infection, we depleted the thrombin-precursor prothrombin to 10% of normal levels in mice prior to infection with the clone 13 strain of lymphocytic choriomeningitis virus. Unexpectedly, prothrombin insufficiency resulted in 100% mortality after infection that was prevented by depletion of CD8+ T cells, suggesting that reduced availability of prothrombin enhances virus-induced immunopathology. Yet, the number, function, and apparent exhaustion of virus-specific T cells were measurably unaffected by prothrombin depletion. Histological analysis of the lung, heart, liver, kidney, spleen, intestine, and brain did not reveal any evidence of hemorrhage or increased tissue damage in mice with low levels of prothrombin that could explain mortality. Viral loads were also similar in infected mice regardless of prothrombin levels. Instead, infection of prothrombin-depleted mice resulted in a severe, T cell–dependent anemia associated with increased hemolysis. Thus, thrombin plays an unexpected protective role in preventing hemolytic anemia during virus infection, with potential implications for patients who are using direct thrombin inhibitors as an anticoagulant therapy.
Authors
Rachel Cantrell, H. Alex Feldman, Leah Rosenfeldt, Ayad Ali, Benjamin Gourley, Cassandra Sprague, Daniel Leino, Jeff Crosby, Alexey Revenko, Brett Monia, Stephen N. Waggoner, Joseph S. Palumbo
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BACKGROUND HIV-1–specific broadly neutralizing monoclonal antibodies (bNAbs) have emerged as promising interventions with the potential to effectively treat and prevent HIV-1 infections. We conducted a phase I clinical trial evaluating the potent CD4-binding site–specific (CD4bs-specific) bNAbs VRC01LS and VRC07-523LS in people with HIV-1 (PWH) not receiving antiretroviral therapy (ART).METHODS Participants received a single intravenous 40 mg/kg dose of either VRC01LS (n = 7) or VRC07-523LS (n = 9) and did not initiate ART for a minimum of 14 days. The primary study objective was to evaluate safety and tolerability; the secondary study objectives were to evaluate pharmacokinetics (PK) and the impact of administered bNAbs on viral loads (VL) and CD4+ T cell counts in the absence of ART.RESULTS This trial enrolled 16 PWH aged 20 to 57 years. Both bNAbs were safe and well tolerated. Mild local reactogenicity was only reported in participants who received VRC07-523LS, while both bNAbs were associated with mild systemic symptoms. Maximum serum concentrations (Cmax) following VRC01LS or VRC07-523LS were 1,566 ± 316 and 1,295 ± 376 μg/mL, respectively. VRC07-523LS administration significantly decreased VL in 8 out of 9 participants, with an average decline of 1.7 ± 0.8 log10 copies/mL within 14 days after administration. In contrast, VRC01LS administration resulted in a smaller average decline (0.8 ± 0.8 log10 copies/mL), and 3 out of 7 participants showedno change in VL. Postinfusion maximum decline in VL correlated with post hoc baseline in vitro viral susceptibility results for both bNAbs.CONCLUSION The results of this trial support inclusion of potent CD4bs-specific bNAbs, such as VRC07-523LS, into next-generation treatment regimens for HIV-1.TRIAL REGISTRATION ClinicalTrials.gov NCT02840474.FUNDING National Institute of Allergy and Infectious Diseases (NIAID)/NIH (grants UM1 AI068634, UM1 AI068636, UM1 AI106701, UM1AI069424, UM1AI069501, UM1AI69415, UM1AI069534, UM1AI69494); the Intramural Research Program of the NIAID/NIH; National Center for Advancing Translational Sciences/NIH (grants UM1TR004548, UL1TR001881, and UL1TR001878); and the National Cancer Institute/NIH (contract 75N91019D00024).
Authors
Myra Happe, Rebecca M. Lynch, Carl J. Fichtenbaum, Sonya L. Heath, Susan L. Koletar, Raphael J. Landovitz, Rachel M. Presti, Jorge L. Santana-Bagur, Randall L. Tressler, LaSonji A. Holman, Laura Novik, Jhoanna C. Roa, Ro Shauna Rothwell, Larisa Strom, Jing Wang, Zonghui Hu, Michelle Conan-Cibotti, Anjali M. Bhatnagar, Bridget Dwyer, Sung Hee Ko, Frida Belinky, Aryan M. Namboodiri, Janardan P. Pandey, Robin Carroll, Manjula Basappa, Leonid Serebryannyy, Sandeep R. Narpala, Bob C. Lin, Adrian B. McDermott, Eli A. Boritz, Edmund V. Capparelli, Emily E. Coates, Richard A. Koup, Julie E. Ledgerwood, John R. Mascola, Grace L. Chen, Pablo Tebas, the VRC 607/A5378 Study Team
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The impact of remdesivir on SARS-CoV-2 diversity and evolution in vivo has remained unclear. In this single-center, retrospective cohort study, we assessed SARS-CoV-2 diversification and diversity over time in a cohort of hospitalized patients who did or did not receive remdesivir. Whole-genome sequencing was performed on 98 paired specimens collected from 49 patients before and after remdesivir administration. The genetic divergence between paired specimens was not significantly different in this cohort compared with that in a control group of patients who did not receive the drug. However, when we focused on minority variants, several positions showed preferential diversification after remdesivir treatment, some of which were associated with specific variants of concern. Most notably, remdesivir administration resulted in strong selection for a nonsynonymous mutation in nsp12, G671S, previously associated with enhanced viral fitness. This same mutation was found to be enriched in a second cohort of 143 inpatients with specimens collected after remdesivir administration compared with controls. Only one other mutation previously implicated in remdesivir resistance (nsp12:V792I) was found to be preferentially selected for after remdesivir administration. These data suggest that SARS-CoV-2 variants with enhanced replicative fitness may be selected for in the presence of antiviral therapy as an indirect means to overcome this selective pressure.
Authors
Ted Ling-Hu, Lacy M. Simons, Estefany Rios-Guzman, Alexandre Machado Carvalho, Maria Francesca R. Agnes, Arghavan Alisoltanidehkordi, Egon A. Ozer, Ramon Lorenzo-Redondo, Judd F. Hultquist
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Access to the brain for treating neurological sequalae requires a craniotomy, which can be complicated by infection. Staphylococcus aureus accounts for half of craniotomy infections, increasing morbidity in a medically fragile patient population. T cells preferentially traffic to the brain during craniotomy infection; however, their functional importance is unknown. Using a mouse model of S. aureus craniotomy infection, CD4+ T cells were critical for bacterial containment, as treatment of WT animals with anti-CD4 exacerbated infection that was similar to phenotypes in Rag1–/– mice. Single-cell RNA-Seq (scRNA-Seq) revealed transcriptional heterogeneity in brain CD3+ infiltrates, with CD4+ cells most prominent that displayed Th1- and Th17-like characteristics, and adoptive transfer of either subset in Rag1–/– animals during early infection prevented S. aureus outgrowth. scRNA-Seq identified a robust IFN signature in several innate immune clusters, and examination of cell-to-cell interactions revealed extensive T cell crosstalk with monocytes/macrophages that was also observed in human craniotomy infection. A cooperative role for Th1 and Th17 responses was demonstrated by treatment of Ifng–/– mice with IL-17A neutralizing antibody that recapitulated phenotypes in Rag1–/– animals. Collectively, these findings implicate Th1- and Th17-mediated proinflammatory responses in shaping the innate immune landscape for S. aureus containment during craniotomy infection.
Authors
Gunjan Kak, Zachary Van Roy, Rachel W. Fallet, Lee E. Korshoj, Tammy Kielian
×Abstract
The hERG1 potassium channel conducts the cardiac repolarizing current, IKr. hERG1 has emerged as a therapeutic target for cardiac diseases marked by prolonged action potential duration (APD). Unfortunately, many hERG1 activators display off-target and proarrhythmic effects that limit their therapeutic potential. A Per-Arnt-Sim (PAS) domain in the hERG1 N-terminus reduces IKr by slowing channel activation and promoting inactivation. Disrupting PAS activity increases IKr and shortens APD in human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). We thus hypothesized that the hERG1 PAS domain could represent a therapeutic target to reduce arrhythmogenic potential in a long QT syndrome (LQTS) background. To test this, we measured the antiarrhythmic capacity of a PAS-disabling single-chain variable fragment antibody, scFv2.10, in a hiPSC-CM line derived from a patient with Jervell and Lange Nielsen (JLN) syndrome. JLN is a severe form of LQTS caused by autosomal recessive mutations in KCNQ1. The patient in this study carried compound heterozygous mutations in KCNQ1. Corresponding JLN hiPSC-CMs displayed prolonged APD and early afterdepolarizations (EADs). Disrupting PAS with scFv2.10 increased IKr, shortened APD, and reduced the incidence of EADs. These data demonstrate that the hERG1 PAS domain could serve as a therapeutic target to treat disorders of cardiac electrical dysfunction.
Authors
Chiamaka U. Ukachukwu, Eric N. Jimenez-Vazquez, Shreya Salwi, Matthew Goodrich, Francisco G. Sanchez-Conde, Kate M. Orland, Abhilasha Jain, Lee L. Eckhardt, Timothy J. Kamp, David K. Jones
×Abstract
Chronic lung allograft dysfunction (CLAD) substantially limits long-term survival following lung transplantation. To identify potential targets for CLAD prevention, T cells from explanted CLAD lungs and lung-draining lymph nodes, as well as diseased and nondiseased controls were isolated and single-cell RNA sequencing and TCR sequencing were performed. TCR sequencing revealed a clonally expanded population of CD8+ tissue-resident memory T cells (TRMs) with high cytotoxic potential, including upregulation of KLRK1, encoding the co-receptor NKG2D. These cytotoxic CD8+ TRMs accumulated around the CLAD airways and had a 100-fold increase in clonal overlap with lung-draining lymph nodes when compared with non-CLAD lungs. Using a murine model of orthotopic lung transplantation, we confirmed that cytotoxic CD8+ TRM accumulation was due to chronic rejection and not transplantation alone. Furthermore, blocking NKG2D in vivo attenuated the airway remodeling following transplantation and diminished airway accumulation of CD8+ T cells. Our findings support NKG2D as a potential therapeutic target for CLAD, affecting cytotoxic CD8+ TRM accumulation.
Authors
Kaveh Moghbeli, Madeline A. Lipp, Marta Bueno, Andrew Craig, Michelle Rojas, Minahal Abbas, Zachary I. Lakkis, Byron Chuan, John Sembrat, Kentaro Noda, Daniel J. Kass, Kong Chen, Li Fan, Tim Oury, Zihe Zhou, Xingan Wang, John F. McDyer, Oliver Eickelberg, Mark E. Snyder
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Obscurin is a giant protein that coordinates diverse aspects of striated muscle physiology. Obscurin immunoglobulin domains 58/59 (Ig58/59) associate with essential sarcomeric and Ca2+ cycling proteins. To explore the pathophysiological significance of Ig58/59, we generated the Obscn-ΔIg58/59 mouse model, expressing obscurin constitutively lacking Ig58/59. Males in this line develop atrial fibrillation by 6 months, with atrial and ventricular dilation by 12 months. As Obscn-ΔIg58/59 left ventricles at 6 months exhibit no deficits in sarcomeric ultrastructure or Ca2+ signaling, we hypothesized that susceptibility to arrhythmia may emanate from the atria. Ultrastructural evaluation of male Obscn-ΔIg58/59 atria uncovered prominent Z-disk streaming by 6 months and further misalignment by 12 months. Relatedly, isolated Obscn-ΔIg58/59 atrial cardiomyocytes exhibited increased Ca2+ spark frequency and age-specific alterations in Ca2+ cycling dynamics, coinciding with arrhythmia onset and progression. Quantitative analysis of the transverse-axial tubule (TAT) network using super-resolution microscopy demonstrated significant TAT depletion in Obscn-ΔIg58/59 atria. These structural and Ca2+ signaling deficits were accompanied by age-specific alterations in the expression or phosphorylation of T-cap protein, which links transverse tubules to Z-disks, and junctophilin 2, which connects transverse tubules to the sarcoplasmic reticulum. Collectively, our work establishes the Obscn-ΔIg58/59 model as a reputable genetic model for atrial cardiomyopathy and provides mechanistic insights into atrial fibrillation and remodeling.
Authors
Alyssa Grogan, Annie Brong, Humberto C. Joca, Liron Boyman, Aaron D. Kaplan, Christopher W. Ward, Maura Greiser, Aikaterini Kontrogianni-Konstantopoulos
×Abstract
Chronic wounds have emerged as a tough clinical challenge. An improved understanding of wound-healing mechanisms is paramount. Collagen XVII (COL17), a pivotal constituent of hemidesmosomes, holds considerable promise for regulating epidermal cell adhesion to the basement membrane as well as for epidermal cell motility and self-renewal of epidermal stem cells. However, the precise role of COL17 in wound repair remains elusive, and the upstream regulatory mechanisms involved have not been fully elucidated. In this study, we delineated the temporal and spatial expression patterns of COL17 at the epidermal wound edge. Subsequently, we investigated the indispensable role of COL17 in keratinocyte activation and reepithelialization during wound healing, demonstrating the restoration of the normal repair process by COL17 overexpression in diabetic wounds. Notably, we identified a key transcriptional signaling pathway for COL17, wherein pyruvate kinase isozyme M2 (PKM2) promotes phosphorylation of STAT3, leading to its activation and subsequent induction of COL17 expression upon injury. Ultimately, by manipulating this pathway using the PKM2 nuclear translocator SAICAR, we revealed a promising therapeutic strategy for enhancing the healing of chronic wounds.
Authors
Yangdan Liu, Chiakang Ho, Dongsheng Wen, Jiaming Sun, Yuxin Liu, Qingfeng Li, Yifan Zhang, Ya Gao
×Abstract
Hereditary angioedema is an autosomal dominant disorder caused by defects in C1-esterase inhibitor (C1-INH), resulting in poorly controlled activation of the kallikrein-kinin system and bradykinin overproduction. C1-INH is a heavily glycosylated protein in the serine protease inhibitor (SERPIN) family, yet the role of these glycosylation sites remains unclear. To elucidate the functional impact of N-glycosylation in the SERPIN domain of C1-INH, we engineered 4 sets consisting of 26 variants at or near the N-linked sequon (NXS/T). Among these, 6 are reported in patients with hereditary angioedema and 5 are known C1-INH variants without accessible clinical histories. We systematically evaluated their expression, structure, and functional activity with C1s̄, FXIIa, and kallikrein. Our findings showed that of the 11 reported variants, 7 were deleterious. Deleting N at the 3 naturally occurring N-linked sequons (N238, N253, and N352) resulted in pathologic consequences. Altering these sites by substituting N with A disrupted N-linked sugar attachment, but preserved protein expression and function. Furthermore, an additional N-linked sugar generated at N272 impaired C1-INH function. These findings highlight the importance of N-linked sequons in modulating the expression and function of C1-INH. Insights gained from identifying the pathological consequences of N-glycan variants should assist in defining more tailored therapy.
Authors
Zhen Ren, John Bao, Shuangxia Zhao, Nicola Pozzi, H. James Wedner, John P. Atkinson
×Abstract
Since multiple front-line immune checkpoint inhibitor–based (ICI-based) combinations are approved for metastatic renal cell carcinoma, biomarkers predicting for ICI responses are needed past clinical prognostication scores and transcriptome gene expression profiling. Circulating markers represent opportunities to assess baseline and dynamic changes in immune cell frequency and cytokine levels while on treatment. We conducted an exploratory prospective correlative study of 33 patients with metastatic clear cell renal cell carcinoma undergoing treatment with ICIs and correlated changes in circulating immune cell subsets and cytokines with clinical responses to treatment. Cell frequencies and cytokine levels were compared between responders and nonresponders using unpaired parametric t tests, using prespecified alpha level of significance of 0.05. Classical monocyte subsets (CD14+CD16–), as well as 7 cytokines (IL-12/23 p40, macrophage inflammatory protein-1a, macrophage inflammatory protein-1b, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, IL-8, and TNF-α) were higher at baseline for responding versus nonresponding patients. Dynamic changes in thymus- and activation-regulated chemokine (TARC), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) also correlated with patients with ICI response. In summary, macrophage-activating agents were observed to be important in ICI response and may highlight the importance of the innate immune response in ICI responses.
Authors
Joyce Hwang, Eda Holl, Yuan Wu, Anika Agarwal, Mark D. Starr, Marco A. Reyes Martinez, Andrew Z. Wang, Andrew J. Armstrong, Michael R. Harrison, Daniel J. George, Andrew B. Nixon, Tian Zhang
×Abstract
The nucleus accumbens-associated protein 1 (NAC1) has recently emerged as a pivotal factor in oncogenesis by promoting glycolysis. Deletion of NAC1 in regulatory T cells (Tregs) has been shown to enhance FoxP3 stability, a suppressor of glycolysis. This study delves into the intriguing dual role of NAC1, uncovering that Treg-specific deletion of NAC1 fosters metabolic fitness in Tregs, thereby promoting tumorigenesis. Our results unveil that NAC1-deficient Tregs exhibited prolonged survival and heightened function, particularly in acidic environments. Mechanistically, we find that NAC1-deficient Tregs adapted to adverse conditions by upregulating FoxP3 expression, engaging in CD36-mediated lipid metabolism, and enhancing peroxisome proliferator–activated receptor gamma coactivator 1-alpha–regulated mitochondrial function. In mouse tumor xenograft models, NAC1-deficient mice demonstrated increased susceptibility to tumor growth. Notably, Tregs lacking NAC1 not only displayed elevated lipid metabolism and mitochondrial fitness but also exhibited enhanced tumoral infiltration. Adoptive Treg transfer experiments further underscored the supportive role of NAC1-deficient Tregs in tumor growth. These findings suggest that modulating NAC1 expression in FoxP3+ Tregs could serve as a promising approach to augment antitumor immunity. Understanding the intricate interplay between NAC1 and Tregs opens avenues for potential therapeutic strategies targeting the tumor microenvironment.
Authors
Anil Kumar, Jugal Kishore Das, Hao-Yun Peng, Liqing Wang, Darby Jane Ballard, Yijie Ren, Xiaofang Xiong, Xingcong Ren, Jin-Ming Yang, Paul de Figueiredo, Jianxun Song
×Abstract
Chagas disease is a tropical disease caused by Trypanosoma cruzi with clinical presentations ranging from asymptomatic to cardiac and/or gastrointestinal complications. The mechanisms of pathogenesis are still poorly understood, but T. cruzi strain diversity may be associated with disease progression. Therefore, we evaluated the transcriptomic response of PBMCs from macaques with natural chronic infections and tested for heterogeneity in their gene signatures. Remarkably, transcriptomic response to T. cruzi infection matched parasite strain profiles, indicating that parasite diversity is a key determinant of host response. While differences in adaptive immune responses were identified, more striking alterations of innate immune processes were detected. Thus, initial innate response to T. cruzi infection may be conditioned by parasite strain diversity, resulting in different profiles of trained immunity modulating subsequent adaptive responses, allowing parasite control or its persistence during the chronic phase. These results call for further characterization of the cross-talk between innate and adaptive immunity according to parasite diversity as well as how altered trained immunity contributes to pathogenesis, as this may lead to better treatments and vaccines.
Authors
Hans Desale, Weihong Tu, Kelly Goff, Preston A. Marx, Claudia Herrera, Eric Dumonteil
×Abstract
BACKGROUND The immunogenicity of current influenza vaccines needs improvement. Inactivated influenza and COVID-19 mRNA vaccines can be coadministered, but randomized controlled trial data are lacking on whether the 2 vaccines are more immunogenic if given in the same arm or opposite arms. Murine studies suggest mRNA vaccines can adjuvant influenza vaccines when coformulated and codelivered.METHODS We randomly assigned 56 adults to receive the Afluria quadrivalent inactivated influenza and Moderna monovalent SARS-CoV-2 XBB.1.5 mRNA vaccines, either in opposite arms or both in the same arm at the same site. The primary endpoint was the difference in median combined serum hemagglutination inhibition titer to the H1, H3, and B-Vic vaccine influenza strains after vaccination.RESULTS We found no significant difference in hemagglutination inhibition antibody levels between the groups (P = 0.30), with the same-arm group having a 1.26-fold higher titer than the opposite-arm group. There were no differences in analyses of antibodies against individual influenza strains or in nasal or saliva antibody levels. While both binding and neutralizing antibody titers against SARS-CoV-2 were not significantly different between groups postvaccination, there was a higher fold-change in BA.5 and ancestral strain neutralizing antibodies in the opposite-arm group.CONCLUSION Influenza vaccination is equivalently immunogenic if given in the same arm or opposite arms as the SARS-CoV-2 vaccine, but it may be preferable to administer the SARS-CoV-2 vaccine at a different site from influenza vaccines.TRIAL REGISTRATION Australian New Zealand Clinical Trials Registry ACTRN12624000445572.FUNDING Australian National Health and Medical Research Council, Australian Medical Research Future Fund, and National Institutes of Health (UH2AI176172).
Authors
Wen Shi Lee, Kevin J. Selva, Jennifer Audsley, Helen E. Kent, Arnold Reynaldi, Timothy E. Schlub, Deborah Cromer, David S. Khoury, Heidi Peck, Malet Aban, Mai Ngoc Vu, Ming Z.M. Zheng, Amy W. Chung, Marios Koutsakos, Hyon-Xhi Tan, Adam K. Wheatley, Jennifer A. Juno, Steven Rockman, Miles P. Davenport, Ian Barr, Stephen J. Kent
×Abstract
Cerebral endothelial cell (EC) injury and blood-brain barrier (BBB) permeability contribute to neuronal injury in acute neurological disease states. Preclinical experiments have used animal models to study this phenomenon, yet the response of human cerebral ECs to BBB disruption remains unclear. In our phase I clinical trial (ClinicalTrials.gov NCT04528680), we used low-intensity pulsed ultrasound with microbubbles (LIPU/MB) to induce transient BBB disruption of peritumoral brain in patients with recurrent glioblastoma. We found radiographic evidence that BBB integrity was mostly restored within 1 hour of this procedure. Using single-cell RNA sequencing and transmission electron microscopy, we analyzed the acute response of human brain ECs to ultrasound-mediated BBB disruption. Our analysis revealed distinct EC gene expression changes after LIPU/MB, particularly in genes related to neurovascular barrier function and structure, including changes to genes involved in the basement membrane, EC cytoskeleton, and junction complexes, as well as caveolar transcytosis and various solute transporters. Ultrastructural analysis showed that LIPU/MB led to a decrease in luminal caveolae, the emergence of cytoplasmic vacuoles, and the disruption of the basement membrane and tight junctions, among other things. These findings suggested that acute BBB disruption by LIPU/MB led to specific transcriptional and ultrastructural changes and could represent a conserved mechanism of BBB repair after neurovascular injury in humans.
Authors
Andrew Gould, Yu Luan, Ye Hou, Farida V. Korobova, Li Chen, Victor A. Arrieta, Christina Amidei, Rachel Ward, Cristal Gomez, Brandyn Castro, Karl Habashy, Daniel Zhang, Mark Youngblood, Crismita Dmello, John Bebawy, Guillaume Bouchoux, Roger Stupp, Michael Canney, Feng Yue, M. Luisa Iruela-Arispe, Adam M. Sonabend
×Abstract
Determining how alveoli are formed and maintained is critical to understanding lung organogenesis and regeneration after injury. To study the cellular dynamics of this critical stage of lung development, we have used scanned oblique-plane illumination microscopy of living lung slices to observe alveologenesis in real time at high resolution over several days. Contrary to the prevailing notion that alveologenesis occurs by airspace subdivision via ingrowing septa, we found that alveoli form by ballooning epithelial outgrowth supported by contracting mesenchymal ring structures. Systematic analysis has produced a computational model of finely timed cellular structural changes that drive normal alveologenesis. With this model, we can now quantify how perturbing known regulatory intercellular signaling pathways and cell migration processes affects alveologenesis. In the future, this paradigm and platform can be leveraged for mechanistic studies and screening for therapies to promote lung regeneration.
Authors
Nicholas M. Negretti, Yeongseo Son, Philip Crooke, Erin J. Plosa, John T. Benjamin, Christopher S. Jetter, Claire Bunn, Nicholas Mignemi, John Marini, Alice N. Hackett, Meaghan Ransom, Shriya Garg, David Nichols, Susan H. Guttentag, Heather H. Pua, Timothy S. Blackwell, William Zacharias, David B. Frank, John A. Kozub, Anita Mahadevan-Jansen, Evan Krystofiak, Jonathan A. Kropski, Christopher V.E. Wright, Bryan Millis, Jennifer M.S. Sucre
×Abstract
BACKGROUND Current clinical sequencing methods cannot effectively detect DNA methylation and allele-specific variation to provide parent-of-origin information from the proband alone. Parent-of-origin effects can lead to differential disease, and the inability to assign parent of origin in de novo cases limits prognostication in the majority of affected individuals with retinoblastoma, a hereditary cancer with suspected parent-of-origin effects.METHODS To directly assign parent of origin in patients with retinoblastoma, we extracted genomic DNA from blood samples for sequencing using a programmable, targeted, single-molecule, long-read DNA genomic and epigenomic approach. This allowed germline variant calling and simultaneous haplotype-resolved CpG methylation in participants with familial (n = 7) and de novo (n = 9) retinoblastoma.RESULTS Targeted long-read sequencing allowed phasing genomic variation with a differentially methylated region in intron 2 of the retinoblastoma gene to confirm parent of origin in known familial samples. This approach allowed us to directly assign parent of origin in simple and complex de novo cases from the proband alone. The ability to assign parent of origin in all retinoblastoma cases showed that harboring disease-causing variants on the paternally inherited allele, whether arising familially or de novo, was associated with more advanced cancer staging at presentation and significantly greater risk of chemotherapy failure (P = 0.002).CONCLUSION This study demonstrates the diagnostic potential of multiomic long-read profiling to unveil the parent-of-origin effect in hereditary cancer. The approach in this work will be instrumental in assigning parent of origin to other genetic diseases using local and distant imprinting signals in the genome.FUNDING National Eye Institute, NIH; Gerber Foundation; Research to Prevent Blindness; Angie Karalis Johnson Fund; Dawn’s Light Foundation; and Mark J. Daily, MD Research Fund
Authors
Andrew W. Stacey, Kenji Nakamichi, Jennifer Huey, Jeffrey Stevens, Natalie Waligorski, Erin E. Crotty, Russell N. Van Gelder, Debarshi Mustafi
×Abstract
Lower urinary tract symptoms (LUTS) affect approximately 50% of the population over 40 years of age and are strongly associated with obesity and metabolic syndrome. Adipose tissue plays a key role in obesity/metabolic syndrome by releasing adipokines that regulate systemic energy/lipid metabolism, insulin resistance, and inflammation. Adiponectin (ADPN), the most abundant adipokine, modulates energy/metabolism homeostasis through its insulin-sensitizing and antiinflammatory effects. Human plasma ADPN levels are inversely associated with obesity and diabetes. To the best of our knowledge, the role of adipokines such as ADPN in the LUTS associated with obesity/metabolic syndrome remains unknown. We have tested such a possible role in a global ADPN-knockout (Adpn–/–) mouse model. Adpn–/– mice exhibited increased voiding frequency, small voids, and reduced bladder smooth muscle (BSM) contractility, with absence of purinergic contraction. Molecular examination indicated significantly altered metabolic and purinergic pathways. The ADPN receptor agonist AdipoRon was found to abolish acute BSM contraction. Intriguingly, both AMPK activators and inhibitors also abolished BSM purinergic contraction. These data indicate the important contribution of what we believe is a novel ADPN signaling pathway to the regulation of BSM contractility. Dysregulation of this ADPN signaling pathway might be an important mechanism leading to LUTS associated with obesity/metabolic syndrome.
Authors
Zhaobo Luo, Ali Wu, Simon Robson, Seth L. Alper, Weiqun Yu
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Abstract
Type 1 diabetes (T1D) is precipitated by the autoimmune destruction of the insulin-producing β-cells in the pancreatic islets of Langerhans. Chemokines have been identified as major conductors of islet infiltration by autoaggressive leukocytes, including antigen-presenting cells and islet autoantigen-specific T cells. We have previously generated a roadmap of gene expression in the islet microenvironment during T1D in a mouse model and found that most of the chemokine axes are chronically upregulated during T1D. The XCL1/XCR1 chemokine axis is of particular interest, since XCR1 is exclusively expressed on conventional dendritic cells type 1 (cDC1) that excel by their high capacity for T cell activation. Here we demonstrate cDC1 expressing XCR1 are present in and around the islets of patients with T1D and of islet-autoantibody positive individuals. Further, we show that XCL1 plays an important role in the attraction of highly potent dendritic cells expressing XCR1 to the islets in an inducible mouse model for T1D. XCL1-deficient mice display a diminished infiltration of XCR1+ cDC1 and subsequently a reduced magnitude and activity of islet autoantigen-specific T cells resulting in a profound decrease in T1D incidence. Interference with the XCL1/XCR1 chemokine axis might constitute a novel therapy for T1D.
Authors
Camilla Tondello, Christine Bender, Gregory J. Golden, Deborah Puppe, Elisa Blickberndt, Monika Bayer, Giulia K. Buchmann, Josef Pfeilschifter, Malte Bachmann, Edith Hintermann, Ralf P. Brandes, Michael R. Betts, Richard A. Kroczek, Urs Christen
Abstract
While immune checkpoint inhibition (CPI) has reshaped cancer treatment, the majority of cancer patients do not benefit from this approach, which can also cause immune-related adverse events. Induction of IFNγ responses is thought be necessary for anti-tumor immunity, but growing evidence also implicates IFNγ as a tumor-intrinsic mediator of CPI resistance. CPI-induced IFNγ mediates activation-induced cell death in T cells as an immune-intrinsic mechanism of resistance. In this study, we show that transient block of IFNγ signaling through administration of the JAK1 inhibitor ABT-317 enhances anti-tumor T cell responses with CPI in pre-clinical models. Importantly, sequential but not concomitant ABT-317 treatment led to significantly reduced toxicity and improved tumor efficacy. Sequential treatment reduced activation-induced T cell death and enhanced expansion of tumor-reactive T cell subsets with increased effector function in vivo and ex vivo. Only CPI in combination with ABT-317 also enhanced memory responses by protecting mice from tumor rechallenge. These results demonstrate that JAK inhibition within a discrete time window following CPI addresses an immune-intrinsic mechanism of therapeutic resistance.
Authors
Marcel Arias-Badia, PeiXi Chen, Yee May Lwin, Aahir Srinath, Aram Lyu, Zenghua Fan, Serena S. Kwek, Diamond N. Luong, Ali Setayesh, Mason Sakamoto, Matthew Clark, Averey Lea, Rachel M. Wolters, Andrew Goodearl, Fiona A. Harding, Jacob V. Gorman, Wendy Ritacco, Lawrence Fong
Abstract
Chlamydia trachomatis (CT) is the most common bacterial sexually transmitted infection globally. Understanding natural immunity to CT will inform vaccine design. This study aimed to profile immune cells and associated functional features in CT-infected women, and determine immune profiles associated with reduced risk of ascended endometrial CT infection and CT reinfection. PBMCs from CT-exposed women were profiled by mass cytometry and random forest models identified key features that distinguish outcomes. CT+ participants exhibited higher frequencies of CD4+ Th2, Th17, and Th17 DN CD4 T effector memory (TEM) cells than uninfected participants with decreased expression of T cell activation and differentiation markers. Minimal differences were detected between women with or without endometrial CT infection. Participants who remained follow-up negative (FU-) showed higher frequencies of CD4 T central memory (TCM) Th1, Th17, Th1/17, and Th17 DN but reduced CD4 TEM Th2 cells than FU+ participants. Expression of markers associated with central memory and Th17 lineage were increased on T cell subsets among FU- participants. These data indicate that peripheral T cells exhibit distinct features associated with resistance to CT reinfection. The highly plastic Th17 lineage appears to contribute to protection. Addressing these immune nuances could promote efficacy of CT vaccines.
Authors
Kacy S. Yount, Chi-Jane Chen, Avinash Kollipara, Chuwen Liu, Neha Vivek Mokashi, Xiaojing Zheng, C Bruce Bagwell, Taylor B. Poston, Harold C. Wiesenfeld, Sharon L. Hillier, Catherine M. O'Connell, Natalie Stanley, Toni Darville
Abstract
The role of mesenchymal cells during respiratory infection is not well defined, including whether, which, and how the different types of mesenchymal cells respond. We collected all mesenchymal cells from lung single-cell suspensions of mice that were naïve (after receiving only saline vehicle), pneumonic (after intratracheal instillation of pneumococcus 24 hours previously), or resolved from infection (after non-lethal pneumococcal infections 6 weeks previously) and performed single-cell RNA sequencing. Cells clustered into five well-separated groups based on their transcriptomes: matrix fibroblasts, myofibroblasts, pericytes, smooth muscle cells, and mesothelial cells. Fibroblasts were the most abundant and could be further segregated into Pdgfra+Npnt+Ces1d+Col13a1+ alveolar fibroblasts and Cd9+Pi16+Sca1+Col14a1+ adventitial fibroblasts. The cells from naïve and resolved groups overlapped in dimension reduction plots, suggesting the mesenchymal cells returned to baseline transcriptomes after resolution. During pneumonia, all mesenchymal cells responded with altered transcriptomes, revealing a core response that had been conserved across cell types as well as distinct mesenchymal cell type-specific responses. The different subsets of fibroblasts induced similar gene sets, but the alveolar fibroblasts responded more strongly than the adventitial fibroblasts. These data demonstrated diverse and specialized immune activities of lung mesenchymal cells during pneumonia.
Authors
Alicia M. Soucy, Jourdan E. Brune, Archana Jayaraman, Anukul T. Shenoy, Filiz T. Korkmaz, Neelou S. Etesami, Bradley E. Hiller, Ian M.C. Martin, Wesley N. Goltry, Catherine T. Ha, Nicholas A. Crossland, Joshua D. Campbell, Thomas G. Beach, Katrina E. Traber, Matthew R. Jones, Lee J. Quinton, Markus Bosmann, Charles W. Frevert, Joseph P. Mizgerd
Abstract
Metabolic dysfunction-associated steatotic liver disease (MASLD)—characterized by excess accumulation of fat in the liver—now affects one third of the world’s population. As MASLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply characterized cohort of patients spanning the full histopathologic spectrum of MASLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of MASLD-associated liver fibrosis.
Authors
Sebastian Steinhauser, David Estoppey, Dennis P. Buehler, Yanhua Xiong, Nicolas Pizzato, Amandine Rietsch, Fabian Wu, Nelly Leroy, Tiffany Wunderlin, Isabelle Claerr, Philipp Tropberger, Miriam Müller, Alexandra Vissieres, Lindsay M. Davison, Eric H. Farber-Eger, Quinn S. Wells, Quanhu Sheng, Sebastian Bergling, Sophia A Wild, Pierre Moulin, Jiancong Liang, Wayne J. English, Brandon Williams, Judith Knehr, Marc Altorfer, Alejandro Reyes, Johannes Voshol, Craig Mickanin, Dominic Hoepfner, Florian Nigsch, Mathias Frederiksen, Charles R. Flynn, Barna D. Fodor, Jonathan D. Brown, Christian Kolter
