Biology subjects

Boehm, M.

Publications and source records attributed to Boehm, M..

18 recordsLinked to original sources

bioRxiv2026

Physiological basis of photosynthetic hydrogen production in the cyanobacterium Synechocystis

Photosynthetic hydrogen (photoH2) production by the cyanobacterium Synechocystis sp. PCC 6803 is an attractive means for storing solar energy. However, photoH2 yields remain limited by competing electron flux pathways. Recent in vitro characterization suggests that photoH2 production requires electrons from both carbohydrate oxidation and photosynthesis. Engineered fusions between photosystem I (PSI) and hydrogenase (PSI-H2ase) aim to divert electrons toward H2 production and rely exclusively on photosynthesis. Thus, photoH2 production differs fundamentally between wildtype (WT) and PSI-H2ase fusion mutants. Here, we show that photoH2 production in WT is enhanced by supplemented glucose, consistent with the recently reported confurcating nature of HoxEFUYH H2ases. PhotoH2 production was further studied in the new psaE-hoxUYH mutant by simultaneously monitoring electron flux through PSI alongside with turnover rates of O2, CO2 and H2. PsaE-hoxUYH achieved the highest photoH2 yield and longest production period among the currently available PSI-H2ase mutants in Synechocystis, prolonged by removing O2. Upon illumination, psaE-hoxUYH exhibited high initial photoH2 production rates, which decreased in parallel with CO2 fixation and ceased immediately in the presence of O2. In absence of O2, photoH2 production still declined slowly. Therefore, in addition to CO2 fixation and O2, other yet unknown factors might limit photoH2 production under these conditions. Moreover, we traced a previously observed high H2 production phase of unclear origin in psaD-hoxYH cultures to contaminating [FeFe]-H2ases from Clostridium intestinale rather than genuine photoH2 production by the mutant. Together, these findings indicate a complex metabolic interplay tuning photoH2 production in Synechocystis WT and PSI-H2ase fusion mutants.

microbiology↗

bioRxiv2026

Electron confurcation drives photosynthetic H2 production in cyanobacteria

Cyanobacteria are major contributors to global photosynthesis and are intensively studied for sustainable green H2 production. Central to this process is the bidirectional [NiFe]-hydrogenase HoxEFUYH, yet its physiological redox partners have remained unresolved. Ferredoxin, NAD(H), and NADP(H) have been proposed as partners, but the lack of active enzyme preparations has prevented a definitive assignment. Here, we purified the intact HoxEFUYH complex from Synechocystis sp. PCC 6803 under strictly anaerobic conditions and reveal its function as both a bifurcating and confurcating hydrogenase. During H2 uptake, HoxEFUYH utilizes NAD+ and oxidized ferredoxin, whereas H2 production strictly requires both NADH and reduced ferredoxin; NADPH does not support either reaction. Combining high-resolution cryo-electron microscopy with biochemical and spectroscopic analyses, our data reveal that an flavin-containing reductase module is electronically connected to the catalytic [NiFe]-hydrogenase core through an extended chain of iron-sulfur clusters, defining the structural basis for bifurcating and confurcating electron flow. These findings fundamentally revise the physiological role of HoxEFUYH by showing that photosynthetic H2 production does not rely solely on photosynthetic electrons but instead couples reduced ferredoxin from the light reaction with NADH derived from "dark" carbohydrate oxidation. This requires reassessment of current strategies for green H2 production in cyanobacteria.

biochemistry↗

bioRxiv2026

Comprehensive study on ferredoxin isoforms in the cyanobacterium Synechocystis sp. PCC 6803

Ferredoxins are central to cellular metabolism by mediating electron flow in energy conversion reactions. The focus of this study was to systematically examine twelve ferredoxin and ferredoxin-like proteins from Synechocystis sp. PCC 6803 to identify their properties, activities, and functions in electron transfer. Using electron paramagnetic resonance spectroscopy, we detected cluster types consistent with major ferredoxin families including plant-type [2Fe-2S], adrenodoxin, thioredoxin, and bacterial-type [4Fe- 4S] ferredoxins. In addition, we found that the ssr3184 ferredoxin-like protein exchanged between a [3Fe-4S] or a [4Fe-4S] cluster, pointing to a possible functional change in response to changes in oxygen or cellular redox poise. Electrochemical measurements demonstrated that these ferredoxins constitute a broad potential window, from -243 mV to -520 mV vs SHE. Investigations on their capacity to support electron-transfer focused on reactions with two major redox hubs: Photosystem I and pyruvate:ferredoxin oxidoreductase and included testing of binding interactions with nitrite reductase. Expression profiling under multiple environmental conditions was also used to predict function and revealed distinct regulatory patterns. Collectively, these findings identified a group of core ferredoxins that directly support photosynthetic electron transfer, and more specialized ones that may serve other functions. In summary, Synechocystis utilizes a suite of ferredoxins to maintain cellular redox homeostasis under dynamic environmental conditions.

biochemistry↗

bioRxiv2026

Easy-to-use whole-genome sequencing workflows and standardized practices to uncover hidden genetic variation in Synechocystis PCC 6803 wild-type and knock-out strains

Knock-out mutants are often used to study gene function by disrupting a specific gene and comparing the mutant to a wild-type strain. Reliable interpretation, however, requires that the two strains differ only by the intended mutation and that the observed phenotype is caused specifically by the deleted gene. In the highly polyploid cyanobacterium Synechocystis sp. PCC 6803, this is particularly challenging because incomplete segregation can mask genetic heterogeneity or secondary suppressor mutations. The genetic variation among laboratory wild-type lines can further confound phenotypic analyses. We show that these challenges can be addressed by routine strain validation via whole-genome sequencing (WGS). To this end, we developed and tested user friendly workflows for short-read (Illumina), long-read (Oxford Nanopore Technologies; ONT), and hybrid data, providing standardized quality control, variant calling, and structural variant detection. We benchmarked their performance in detecting single-nucleotide polymorphisms (SNPs), small indels, and structural variants using simulated datasets across different coverages and mixed populations. Applying the workflows to three Synechocystis sp. PCC 6803 wild-type lines revealed multiple sequence and structural differences relative to the reference genome, including previously undescribed genetic variants, underscoring the importance of documenting the strain background and the value of long-read sequencing. Characterization of two independent 6-phosphogluconate dehydrogenase (gnd) knock-out mutants and their complemented strains highlighted how a failed rescue can reveal a phenotype unrelated to the intended knock-out. An automated literature analysis revealed that only a minority of the investigated Synechocystis studies that used knock-out mutants included complementation as a control (39%), whereas this practice is more common in studies involving Escherichia coli (63%) and Saccharomyces cerevisiae (55%). Based on these results, we propose a practical guide for standardizing knock-out phenotyping in Synechocystis PCC 6803. Combined with accessible workflows for routine whole-genome validation, this framework aims to support more robust and reproducible knock-out studies in the future.

microbiology↗

bioRxiv2026

Story about honest mistakes: The cyanobacterium Synechocystis has a promiscuous Entner-Doudoroff (ED) aldolase but no functional ED pathway.

In 2016, the glycolytic Entner-Doudoroff (ED) pathway was reported in cyanobacteria and plants (1). The claim was based on the biochemical characterization of its key enzyme the 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase also named ED aldolase (EDA), on protein sequence alignments, physiological data from cyanobacterial mutants, and the in vivo detection of an ED pathway specific metabolite (1). However, two enzymes 6-phosphogluconate (6PG) dehydratase (EDD) and EDA are unique to this route. A recent study suggests that EDD (Slr0452) from Synechocystis sp. PCC 6803 most likely encodes an enzyme involved exclusively in amino acid synthesis, indicating that a complete ED pathway would be missing (2). To address the presence or absence of the ED pathway in Synechocystis, we conducted extended biochemical and physiological studies, revisited old data and resolved contradictions. These investigations reveal that Synechocystis lacks both an ED pathway and a glucose dehydrogenase/glucokinase (GDH/GK) bypass but contains a promiscuous aldolase EDA. EDA prefers KDPG as substrate but also decarboxylates oxaloacetate (OAA) and cleaves 2-keto-4-hydroxyglutarate (KHG). Synthesis of KDPG from pyruvate and glyceraldehyde 3-phosphate (GAP) is catalyzed with very low efficiency. These in vitro data suggest that EDA might be involved in the phosphoenolpyruvate (PEP)-pyruvate-OAA node and proline catabolism, which requires further clarification. The previous misconception was based on missing enzymatic characterizations, the oversight of a secondary mutation in a deletion strain, and an outdated view on carbohydrate fluxes. We conclude with a list of lessons and provide a solid foundation for future investigations into the role of EDA in cyanobacteria and other photoautotrophs. Significance statementThis study provides a retrospective on why, for many years, it was mistakenly assumed that the glycolytic Entner-Doudoroff (ED) pathway exists in the cyanobacterium Synechocystis sp. PCC 6803. It shows that the first enzyme of this pathway, ED dehydratase EDD is absent, while the second enzyme, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase EDA, is present but is promiscuous, cleaving KDPG in addition to 2-keto-4-hydroxyglutarate (KHG) and decarboxylating oxaloacetate (OAA) in vitro. Finally, valuable lessons are drawn from prior misconceptions and experimental limitations. This study provides a solid foundation for future studies on the role of the ED aldolase in absence of the ED pathway in cyanobacteria and other photoautotrophs.

microbiology↗

bioRxiv2026

STING-STAT3-SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis

A high prevalence of early-onset interstitial lung disease, including pulmonary fibrosis, in pediatric patients with Stimulator of interferon genes (STING)-Associated Vasculopathy with onset in infancy (SAVI) suggests a critical role for the cGAS-STING pathway in the pathogenesis of pulmonary fibrosis. We identified an endothelial-to-mesenchymal transition (EndMT) signature in lesional lung biopsies from SAVI patients, marked by a loss of endothelial and acquisition of mesenchymal markers. Consistently, induced pluripotent stem cell-derived endothelial cells (iECs) from SAVI patients harboring gain-of-function STING1 mutations spontaneously undergo EndMT, a process rescued in isogenic-correction. In endothelial cells, STING activation induces IRF3-independent STAT3 phosphorylation, initiating a SLUG-dependent mesenchymal transcriptional program while repressing SOX18 and an epigenetically-regulated endothelial maintenance network. Our studies define a non-canonical cGAS-STING-STAT3 signaling axis that couples a mesenchymal transcriptional program with epigenetic silencing of an endothelial maintenance program, promoting TGF{beta}-independent STING-mediated EndMT and endothelial dysfunction, and suggesting STING as a therapeutic target for inflammatory pulmonary fibrosis.

cell biology↗

bioRxiv2026

Longitudinal in vivo human wound healing model defines key role for smooth muscle cells in ECM remodeling

BackgroundEffective skin wound healing is essential for restoring tissue integrity following injury. Repair proceeds through phases of hemostasis, inflammation, proliferation, and remodeling, but molecular mechanisms governing these stages remain poorly defined. Vascular niche cells (VNCs)-including endothelial cells, vascular smooth muscle cells (SMCs), and fibroblasts-are central regulators of healing, but the lack of longitudinal in vivo human data has limited identification of VNC-derived signals that distinguish effective repair from pathological healing such as ulcers. Thus, defining the regulation of VNCs in wound healing addresses a critical knowledge gap. MethodsWe developed a protocol for wound healing using dermal forearm punch biopsies to track longitudinal repair in healthy volunteers. Single-cell and spatial transcriptomics were performed to identify and validate signaling activities within VNCs. ResultsWe spatiotemporally defined the inflammation, proliferation, and remodeling phases of human skin wound healing with a focus on VNCs. Spatial analysis localized this activity for VNCs and immune cells within a heterogenous granulation zone that later led to re-epithelializion. Angiogenesis was dominated by Vegf, Egf and Hif1 signaling. Extracellular matrix (ECM) remodeling occurred through Collagen, Laminin, Thrombospondin, and Fibronectin. SMCs emerged as dominant drivers of injury-induced remodeling including basement membrane and interstitial ECM components compared to fibroblasts. This SMC-led program was further defined by robust induction of TIMP1, an inhibitor of matrix degradation, which localized to granulation tissue and correlated with re-epithelialization and wound resolution. Lastly, we compared remodeling factors between healing and non-healing human diabetic foot ulcers (DFUs). SMCs in non-healing DFUs had deficient expression for core remodeling factors, including TIMP1, indicating SMC activity is needed for effective healing. ConclusionWe identified an SMC-driven model of wound repair in which TIMP1-dependent activity underpins granulation zone formation. Failure of this program defined a mechanistic basis for impaired healing in ulcers, identifying SMCs and TIMP1 as therapeutic targets.

molecular biology↗

bioRxiv2026

Hepatitis C virus replication fitness as a determinant of antiviral therapy outcome

Background and AimsHepatitis C virus (HCV) infections were previously treated with interferon (IFN) but today direct acting antivirals (DAAs) with cure rates >95% are available. DAA treatment failure is primarily attributed to resistance associated mutations (RAMs), often imposing a fitness cost. Interferon treatment outcome was shown to be associated with the interferon sensitivity determining region (ISDR), which is part of the replication enhancing domain (ReED) in non-structural protein (NS) 5A. We found that accumulation of mutations in the ReED was indicative of elevated viral genome replication fitness. This study investigates the impact of HCV replication fitness on antiviral treatment outcomes. MethodsWe utilized chimeric HCV subgenomic replicons containing RAMs and ReED sequences from patients after interferon treatment or DAA failure to assess replication fitness of patient isolates in presence and absence of inhibitors. ResultsReplication fitness did not impact on IFN sensitivity in cell culture but resulted in higher remaining antigen levels for highly replicating variants at a given IFN concentration. Furthermore, we identified ReED variants substantially increasing HCV replication in several patients who failed DAA therapy across different genotypes. High replicator ReEDs rescued the fitness loss caused by RAMs like Y93C/H (NS5A) and S282T (NS5B). While high replication fitness did not intrinsically increase drug sensitivity (IC50), it allowed the virus to sustain robust replication despite antiviral pressure. ConclusionsElevated replication fitness might support interferon treatment due to increased antigen presentation, facilitating adaptive immune responses. Furthermore, ReED mediated increase in replication fitness could contribute to DAA treatment failure by preserving higher replication upon treatment and compensating for RAM associated fitness costs. Thus, patients failing DAA treatment should be monitored for RAMs and ReED mutations.

microbiology↗

bioRxiv2025

Geno2pheno: Interpretable and accurate HIV antibody resistance prediction

BackgroundAntiretroviral therapy (ART) is a life saving option for people living with HIV-1 (PLWH) and is effective against many viral strains. The most common ARTs involve combinations of drugs targeting viral or cellular proteins. Most of these drugs have to be taken daily. An alternative to ARTs with established inhibitors comprises broadly neutralizing antibodies (bNAbs). However, bNAbs share the problem of viral resistance with protein inhibitors. We developed a web service geno2pheno[bNAbs] that allows users to upload viral genotypes and estimates the respective resistance to many common bNAbs. The service uses trained statistical models to classify the virus into sensitive and resistant, respectively or to regress the IC50. MethodsWe used two linear models as well as two neural nets for each task and multi-task (MT) learning to train both models for IC50 prediction and classification simultaneously. During multi-task learning we penalize divergence of class and IC50 score in addition to the loss individual to each of the models. FindingsWe compared the linear models of geno2pheno[bNAbs] to other state-of-the-art methods like recurrent neural nets and self-attention, and found them to be competitive in regard to accuracy and have the benefit of fast computation and being easily interpretable in regard to features, i.e., positions on the envelope. InterpretationWe developed a web service for the prediction of antibody resistance (geno2pheno[bNAbs]) to HIV-1, which is free to use and can be extended to other viruses, like Sars-Cov2, in the future.

bioinformatics↗

bioRxiv2025

Proteasome mutations associated with CANDLE syndrome cause altered neuronal development by dysregulating polyamine synthesis

Genetic mutations affecting proteasome function can result in multi-organ diseases, such as Chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) syndrome. Neurological symptoms associated with CANDLE suggest that proteasomal mutations may impact neuronal development and/or function. We generated cerebral organoids (COs) from CANDLE patient induced pluripotent stem cells (iPSCs), which exhibited impaired neuronal development when compared to COs from healthy control iPSCs. Impaired neuronal maturation in CANDLE COs was correlated with increased polyamines, which were also elevated in CANDLE patient CSF. The proteasome-regulated Ornithine decarboxylase (ODC), a rate limiting enzyme for polyamines, was elevated in CANDLE neurons. Inhibition of ODC reversed polyamine overproduction and repaired neuronal maturation in CANDLE COs, suggesting a potential therapeutic avenue for intervention. These findings demonstrate that dysfunction of the proteasome affects neuronal development through overproduction of polyamines via dysregulation of ODC and offer insight into potential therapeutic strategies for CNS-related proteasomal dysfunction.

neuroscience↗

bioRxiv2025

Fructose-1,6-bisphosphatase (FBPase) fine-tunes heterotrophic growth in cyanobacteria

Cyanobacteria switch their carbon metabolism between photoautotrophy and heterotrophy during diurnal cycles. In cyanobacteria, the classical glycolytic control point is characterized by two catabolic phosphofructokinases (PFKs) and a bifunctional anabolic fructose-1,6-biphosphatase/sedoheptulose-1,7-biphosphatase (F/SBPase; slr2094) catalyzing two key reactions in the Calvin-Benson-Bassham (CBB) cycle. In addition, Synechocystis possesses a fructose-1,6-bisphosphatase (FBPase; slr0952) with yet unknown physiological function and biochemical properties. Hence, our aim was to investigate the FBPase and the interplay of the four enzymes in photoautotrophic and heterotrophic carbon metabolism. We discovered that FBPase is specific for FBP, showing no SBPase activity, and unlike F/SBPase does not exhibit any biochemical regulatory properties. Growth studies with deletion mutants revealed that FBPase and PFKs play a major role under heterotrophic conditions. In contrast to F/SBPase, FBPase is not involved in the CBB cycle, but instead fine-tunes heterotrophic growth. Transaldolase cannot replace the function of SBPase in the CBB cycle. In conclusion, the classical Embden-Meyerhoff-Parnass pathway control point, which is known to be mediated by the antagonistic enzyme pair PFK and FBPase in heterotrophic bacteria and eukaryotes, is also present in Synechocystis. We found redox-insensitive FBPases from plant chloroplasts to be closely related to Synechocystis FBPase, indicating that they might serve a similar function. HighlightSynechocystis fructose-1,6-bisphosphatase (slr0952) is unlike fructose-1,6-biphosphatase/sedoheptulose-1,7-biphosphatase (slr2094) monofunctional, not redox-regulated, and supports heterotrophy in darkness, despite catalyzing an anabolic reaction. Thus, presumably Slr2094 alone drives two Calvin-Benson-Bassham cycle key reactions.

plant biology↗

bioRxiv2024

Structural basis of the allosteric regulation of cyanobacterial glucose-6-phosphate dehydrogenase by the redox sensor OpcA

The oxidative pentose phosphate (OPP) pathway is a fundamental carbon catabolic route for generating reducing power and metabolic intermediates for biosynthetic processes. In addition, its first two reactions form the OPP shunt, which replenishes the Calvin-Bassham cycle under certain conditions. Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and rate-limiting reaction of this metabolic route. In photosynthetic organisms, G6PDH is redox-regulated to allow fine-tuning and to prevent futile cycles while carbon is being fixed. In cyanobacteria, regulation of G6PDH requires the redox protein OpcA, but the underlying molecular mechanisms behind this allosteric activation remain elusive. Here, we used enzymatic assays and in vivo interaction analyses to show that OpcA binds G6PDH under different environmental conditions. However, complex formation enhances G6PDH activity when OpcA is oxidized and inhibits it when OpcA is reduced. To understand the molecular basis of this regulation, we used cryogenic electron microscopy to determine the structure of cyanobacterial G6PDH and the G6PDH-OpcA complex. OpcA binds the G6PDH tetramer and induces conformational changes in the active site of G6PDH. The redox sensitivity of OpcA is achieved by intramolecular disulfide bridge formation, which influences the allosteric regulation of G6PDH. In vitro assays reveal that the level of G6PDH activation depends on the number of bound OpcA molecules, which implies that this mechanism allows delicate fine-tuning. Our findings unveil a novel and unique molecular mechanism governing the regulation of the OPP pathway in cyanobacteria.

molecular biology↗

bioRxiv2023

Proton motive force dissipation drives flavodiiron proteins to the thylakoid membrane for ferredoxin-powered O2 photoreduction

Flavodiiron proteins (FDPs) catalyse light-dependent reduction of oxygen to water in photosynthetic organisms, creating an electron sink on the acceptor side of Photosystem I that protects the photosynthetic apparatus. However, the identity of the electron donor(s) and the molecular mechanisms regulating FDP activity have remained elusive. To elucidate these issues, we employed spectroscopic and gas flux analysis of photosynthetic electron transport, bimolecular fluorescence complementation assays for in vivo protein-protein interactions in the model cyanobacterium Synechocystis sp. PCC 6803, as well as in silico surface charge modelling. We demonstrated that Ferredoxin-1 interacts with Flv1, Flv2, and Flv3, and is the main electron donor to FDP heterooligomers, which are responsible for the photoreduction of oxygen. Moreover, we revealed that association of FDP heterooligomers with thylakoid membranes is promoted by dissipation of the trans-thylakoid proton motive force, providing the first in vivo evidence of a self-regulatory feedback mechanism allowing dynamic control of FDP activity and maintenance of photosynthetic redox balance in fluctuating environments. Our findings have direct implications for rationally directing electron flux toward desired reactions in biotechnological applications.

plant biology↗

bioRxiv2021

Pyruvate:ferredoxin oxidoreductase and low abundant ferredoxins support aerobic photomixotrophic growth in cyanobacteria

The decarboxylation of pyruvate is a central reaction in the carbon metabolism of all organisms. Both the pyruvate:ferredoxin oxidoreductase (PFOR) and the pyruvate dehydrogenase (PDH) complex catalyze this reaction. Whereas PFOR reduces ferredoxin, the PDH complex utilizes NAD+. Anaerobes rely on PFOR, which was replaced during evolution by the PDH complex found in aerobes. Cyanobacteria possess both. Our data challenge the view that PFOR is exclusively utilized for fermentation. Instead, we show, that the cyanobacterial PFOR is stable in the presence of oxygen in vitro and is required for optimal photomixotrophic growth under aerobic conditions while the PDH complex is inactivated under the same conditions. We found that cells rely on a general shift from utilizing NAD(H)-dependent to ferredoxin-dependent enzymes under these conditions. The utilization of ferredoxins instead of NAD(H) saves a greater share of the Gibbs free energy, instead of wasting it as heat. This obviously simultaneously decelerates metabolic reactions as they operate closer to their thermodynamic equilibrium. It is common thought that during evolution, ferredoxins were replaced by NAD(P)H due to their higher stability in an oxidizing atmosphere. However, utilization of NAD(P)H could also have been favored due to a higher competitiveness because of an accelerated metabolism.

evolutionary biology↗

bioRxiv2021

Autoantibodies against Progranulin and IL-1 receptor antagonist in critically ill COVID-19

STRUCTURED ABSTRACTHyperinflammation is frequently observed in patients with severe COVID-19. Inadequate and defective IFN type I responses against SARS-CoV-2, associated with autoantibodies in a proportion of patients, lead to severe courses of disease. In addition, hyperactive responses of the humoral immune system have been described. In the current study we investigated a possible role of neutralizing autoantibodies against antiinflammatory mediators. Plasma from adult patients with severe and critical COVID-19 was screened by ELISA for antibodies against PGRN, IL-1-Ra, IL-10, IL-18BP, IL-22BP, IL-36-Ra, CD40, IFN-2, IFN-{gamma}, IFN-{omega} and serpinB1. Autoantibodies were characterized and the antigens were analyzed for immunogenic alterations. In a discovery cohort with severe to critical COVID-19 high titers of PGRN-autoantibodies were detected in 11 of 30 (36.7%), and of IL-1-Ra-autoantibodies in 14 of 30 (46.7%) patients. In a validation cohort of 64 patients with critical COVID-19 high-titer PGRN-Abs were detected in 25 (39%) and IL-1-Ra-Abs in 32 of 64 patients (50%). PGRN-Abs and IL-1-Ra-Abs belonged to IgM and several IgG subclasses. In separate cohorts with non-critical COVID-19, PGRN-Abs and IL-1-Ra-Abs were detected in low frequency (i.e. in < 5% of patients) and at low titers. Neither PGRN-nor IL-1-Ra-Abs were found in 40 healthy controls vaccinated against SARS-CoV-2 or 188 unvaccinated healthy controls. PGRN-Abs were not cross-reactive against SARS-CoV-2 structural proteins nor against IL-1-Ra. Plasma levels of both free PGRN and free IL-1-Ra were significantly decreased in autoantibody-positive patients compared to Ab-negative and non-COVID-19 controls. In vitro PGRN-Abs from patients functionally reduced PGRN-dependent inhibition of TNF- signaling, and IL-1-Ra-Abs from patients reduced IL-1-Ra- or anakinra-dependent inhibition of IL-1{beta} signaling. The pSer81 hyperphosphorylated PGRN isoform was exclusively detected in patients with high-titer PGRN-Abs; likewise, a hyperphosphorylated IL-1-Ra isoform was only found in patients with high-titer IL-1-Ra-Abs. Thr111 was identified as the hyperphophorylated amino acid of IL-1-Ra. In longitudinally collected samples hyperphosphorylated isoforms of both PGRN and IL-1-Ra emerged transiently, and preceded the appearance of autoantibodies. In hospitalized patients, the presence of IL-1-Ra-Abs or IL-1-Ra-Abs in combination with PGRN-Abs was associated with a higher morbidity and mortality. To conclude, neutralizing autoantibodies to IL-1-Ra and PGRN occur in a significant portion of patients with critical COVID-19, with a concomitant decrease in circulating free PGRN and IL-1-Ra, indicative of a misdirected, proinflammatory autoimmune response. The break of self-tolerance is likely caused by atypical hyperphosphorylated isoforms of both antigens, whose appearances precede autoantibody induction. Our data suggest that these immunogenic secondary modifications are induced by the SARS-CoV-2-infection itself or the inflammatory environment evoked by the infection and predispose for a critical course of COVID-19.

immunology↗

bioRxiv2019

Robust generation of erythroid and multilineage hematopoietic progenitors from human iPSCs using a scalable monolayer culture system

One of the most promising objectives of clinical hematology is to derive engraftable autologous hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (iPSCs). Progress in translating iPSC technologies to the clinic relies on the availability of scalable differentiation methodologies. In this study, human iPSCs were differentiated for 21 days using STEMdiff, a monolayer-based approach for hematopoietic differentiation of human iPSCs that requires no replating, co-culture or embryoid body formation. Both monolayer and suspension cells were functionally characterized throughout differentiation. In the supernatant fraction, an early transient population of primitive CD235a+ erythroid cells first emerged, followed by hematopoietic progenitors with multilineage differentiation activity in vitro but no long-term engraftment potential in vivo. In later stages of differentiation, a nearly exclusive production of definitive erythroid progenitors was observed. In the adherent monolayer, we identified a prevalent population of mesenchymal stromal cells and limited arterial vascular endothelium (VE), suggesting that the cellular constitution of the monolayer may be inadequate to support the generation of HSCs with durable repopulating potential. Quantitative modulation of WNT/{beta}-catenin and activin/nodal/TGF{beta} signaling pathways with CHIR/SB molecules during differentiation enhanced formation of arterial VE, definitive multilineage and erythroid progenitors, but was insufficient to orchestrate the generation of engrafting HSCs. Overall, STEMdiff provides a clinically-relevant and readily adaptable platform for the generation of erythroid and multilineage hematopoietic progenitors from human pluripotent stem cells.\n\nHighlightsO_LIRobust, scalable and clinically-relevant monolayer-based culture system for hematopoietic differentiation of human iPSCs.\nC_LIO_LISuccessive emergence of primitive erythroid cells, definitive multilineage HSPCs and erythroid progenitors in the culture supernatant.\nC_LIO_LIAbundant mesenchymal cells and limited arterial vascular endothelium in the culture monolayer.\nC_LIO_LICHIR/SB molecules increase arterial vascular endothelium formation, suppress primitive hematopoiesis and promote definitive multilineage and erythroid progenitors.\nC_LI

developmental biology↗

bioRxiv2019

Adaptation and latitudinal gradients in species interactions: nest predation in birds

The "biotic interactions" hypothesis--that stronger interspecific interactions in the tropics drive faster evolution and speciation, giving rise to the latitudinal diversity gradient--has inspired many tests of whether certain biotic interactions are indeed stronger in the tropics. However, the possibility that populations have adapted to latitudinal differences in species interactions, blunting effects on evolutionary rates, has been largely ignored. Here we show that mean rates of nest predation experienced by land birds vary minimally with latitude in the Western Hemisphere. This result is surprising because nest predation in birds is a canonical example of a strong tropical biotic interaction. We explain our finding by demonstrating that (1) rates of nest predation are in fact higher in the tropics, but only when controlling for the length of the nesting period, (2) long nesting periods are associated with reduced predation rates, and (3) tropical birds have evolved particularly long nesting periods. We suggest this is a case example of how adaptation to a biotic interaction can alter observed latitudinal gradients in interaction strength, potentially equalizing evolutionary rates among latitudes. More broadly, we advocate for tests of the biotic interactions hypothesis to consider both latitudinal patterns in interaction strength and evolutionary responses to these interactions.

evolutionary biology↗

bioRxiv2018

Chemogenetic ligands for translational neurotheranostics

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for precision medicine-based clinical theranostics. DREADD ligands developed to date are not appropriate for such translational applications. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine. The second-generation DREADD agonist, Compound 21 (C21), was developed to overcome these limitations. We found that C21 has low brain penetrance, weak affinity, and low in vivo DREADD occupancy. To address these drawbacks, we developed two new DREADD agonists, JHU37152 and JHU37160, and the first dedicated positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons and at their long-range projections, enabling for the first time, noninvasive and longitudinal neuronal projection mapping and potential for neurotheranostic applications.

neuroscience↗