Publications - Publikationer

Gal-3 blocks the binding between PD-1 and pembrolizumab

Greisen, S. R., Bendix, M., Nielsen, M. A., et al. — Wed, 02 Oct 2024 00:19:24 +0200

INTRODUCTION: Immune checkpoint inhibitors (ICI) have revolutionized the treatment of metastatic malignant melanoma (MM) and improved long-term survival. Despite the impressive results, some patients still have progressive disease, and the search for biomarkers predicting response to ICI treatment is ongoing. In this search, galectin-3 (Gal-3) has been suggested as a molecule of interest, both as a marker of treatment response and as a treatment target to potentiate ICI therapy. We have previously demonstrated the binding between programmed cell death 1 (PD-1) and Gal-3, and here, we investigated the interaction between PD-1, pembrolizumab, and Gal-3 in metastatic MM patients. METHODS: The binding between PD-1, pembrolizumab and Gal-3 was investigated by surface plasmon resonance (SPR) and cryogenic electron microscopy (cryo-EM). The function was studied in in vitro cultures and soluble levels of both PD-1 and Gal-3 were measured in metastatic MM patients, treated with pembrolizumab. RESULTS: By SPR, we demonstrated that Gal-3 can block the binding between PD-1 and pembrolizumab, and further visualized a steric inhibition using cryo-EM. T cells cultured with Gal-3 had reduced pro-inflammatory cytokine production, which could not be rescued by pembrolizumab. In patients with metastatic MM, high levels of Gal-3 in plasma were found in patients with a longer progression-free survival in the study period, whereas high Gal-3 expression in the tumor was seen in patients with disease progression. Soluble PD-1 levels in plasma increased after treatment with pembrolizumab and correlated with disease progression. CONCLUSION: We demonstrate that the interaction between PD-1 and Gal-3 interferes with the binding of pembrolizumab, supporting that an immune suppression induced by Gal-3 in the tumor microenvironment cannot be rescued by pembrolizumab.

Beyond basic characterization and omics

Palviainen, M., Puutio, J., Østergaard, R. H., et al. — Tue, 01 Oct 2024 00:19:24 +0200

Renowned for their role in haemostasis and thrombosis, platelets are also increasingly recognized for their contribution in innate immunity, immunothrombosis and inflammatory diseases. Platelets express a wide range of receptors, which allows them to reach a variety of activation endpoints and grants them immunomodulatory functions. Activated platelets release extracellular vesicles (PEVs), whose formation and molecular cargo has been shown to depend on receptor-mediated activation and environmental cues. This study compared the immunomodulatory profiles of PEVs generated via activation of platelets by different receptors, glycoprotein VI, C-type lectin-like receptor 2 and combining all thrombin-collagen receptors. Functional assays in vivo in zebrafish and in vitro in human macrophages highlighted distinct homing and secretory responses triggered by the PEVs. In contrast, omics analyses of protein and miRNA cargo combined with physicochemical particle characterization found only subtle differences between the activated PEV types, which were insufficient to predict their different immunomodulatory functions. In contrast, constitutively released PEVs, formed in the absence of an exogenous activator, displayed a distinct immunomodulatory profile from the receptor-induced PEVs. Our findings underscore that PEVs are tunable through receptor-mediated activation. To truly comprehend their role(s) in mediating platelet functions among immune cells, conducting functional assays is imperative.

Whole genome sequencing analysis identifies sex differences of familial pattern contributing to phenotypic diversity in autism

Kim, S. W., Lee, H., Song, D. Y., et al. — Fri, 27 Sep 2024 00:19:24 +0200

BACKGROUND: Whole-genome sequencing (WGS) analyses have found higher genetic burden in autistic females compared to males, supporting higher liability threshold in females. However, genomic evidence of sex differences has been limited to European ancestry to date and little is known about how genetic variation leads to autism-related traits within families across sex. METHODS: To address this gap, we present WGS data of Korean autism families (n = 2255) and a Korean general population sample (n = 2500), the largest WGS data of East Asian ancestry. We analyzed sex differences in genetic burden and compared with cohorts of European ancestry (n = 15,839). Further, with extensively collected family-wise Korean autism phenotype data (n = 3730), we investigated sex differences in phenotypic scores and gene-phenotype associations within family. RESULTS: We observed robust female enrichment of de novo protein-truncating variants in autistic individuals across cohorts. However, sex differences in polygenic burden varied across cohorts and we found that the differential proportion of comorbid intellectual disability and severe autism symptoms mainly drove these variations. In siblings, males of autistic females exhibited the most severe social communication deficits. Female siblings exhibited lower phenotypic severity despite the higher polygenic burden than male siblings. Mothers also showed higher tolerance for polygenic burden than fathers, supporting higher liability threshold in females. CONCLUSIONS: Our findings indicate that genetic liability in autism is both sex- and phenotype-dependent, expanding the current understanding of autism's genetic complexity. Our work further suggests that family-based assessments of sex differences can help unravel underlying sex-differential liability in autism.

Rhizobial Secretion of Truncated Exopolysaccharides Severely Impairs the Mesorhizobium-Lotus Symbiosis

Wightman, T., Muszyński, A., Kelly, S. J., et al. — Sun, 01 Sep 2024 00:19:24 +0200

The symbiosis between Mesorhizobium japonicum R7A and Lotus japonicus Gifu is an important model system for investigating the role of bacterial exopolysaccharides (EPS) in plant-microbe interactions. Previously, we showed that R7A exoB mutants that are affected at an early stage of EPS synthesis and in lipopolysaccharide (LPS) synthesis induce effective nodules on L. japonicus Gifu after a delay, whereas exoU mutants affected in the biosynthesis of the EPS side chain induce small uninfected nodule primordia and are impaired in infection. The presence of a halo around the exoU mutant when grown on Calcofluor-containing media suggested the mutant secreted a truncated version of R7A EPS. A nonpolar ΔexoA mutant defective in the addition of the first glucose residue to the EPS backbone was also severely impaired symbiotically. Here, we used a suppressor screen to show that the severe symbiotic phenotype of the exoU mutant was due to the secretion of an acetylated pentasaccharide, as both monomers and oligomers, by the same Wzx/Wzy system that transports wild-type exopolysaccharide. We also present evidence that the ΔexoA mutant secretes an oligosaccharide by the same transport system, contributing to its symbiotic phenotype. In contrast, ΔexoYF and polar exoA and exoL mutants have a similar phenotype to exoB mutants, forming effective nodules after a delay. These studies provide substantial evidence that secreted incompatible EPS is perceived by the plant, leading to abrogation of the infection process. [Formula: see text]

Chemical modification of hyaluronan oligosaccharides differentially modulates hyaluronan–hyaladherin interactions

Sun, 01 Sep 2024 00:19:24 +0200

The glycosaminoglycan hyaluronan (HA) is a ubiquitous, nonsulfated polysaccharide with diverse biological roles mediated through its interactions with HA-binding proteins (HABPs). Most HABPs belong to the Link module superfamily, including the major HA receptor, CD44, and secreted protein TSG-6, which catalyzes the covalent transfer of heavy chains from inter-α-inhibitor onto HA. The structures of the HA-binding domains (HABDs) of CD44 (HABD_CD44) and TSG-6 (Link_TSG6) have been determined and their interactions with HA extensively characterized. The mechanisms of binding are different, with Link_TSG6 interacting with HA primarily via ionic and CH−π interactions, whereas HABD_CD44 binds solely via hydrogen bonds and van der Waals forces. Here, we exploit these differences to generate HA oligosaccharides, chemically modified at their reducing ends, that bind specifically and differentially to these target HABPs. Hexasaccharides (HA₆^AN) modified with 2- or 3-aminobenzoic acid (HA₆-2AA, HA₆-3AA) or 2-amino-4-methoxybenzoic acid (HA₆-2A4MBA), had increased affinities for Link_TSG6 compared to unmodified HA₆^AN. These modifications did not increase the affinity for CD44_HABD. A model of HA₆-2AA (derived from the solution dynamic 3D structure of HA₄-2AA) was docked into the Link_TSG6 structure, providing evidence that the 2AA-carboxyl forms a salt bridge with Arginine-81. These modeling results informed a second series of chemical modifications for HA oligosaccharides, which again showed differential binding to the two proteins. Several modifications to HA₄ and HA₆ were found to convert the oligosaccharide into substrates for heavy chain transfer, whereas unmodified HA₄ and HA₆ are not. This study has generated valuable research tools to further understand HA biology.

Functional porous protein nanofibrils/polysaccharides aerogel beads for efficient dyes removal from water

Dilamian, M., Montazer, M., Yousefi, H., Otzen, D. E., Morshedi, D. — Sat, 01 Jun 2024 00:19:24 +0200

The rational design and fabrication of functional and feasible adsorbents with enhanced adsorption properties for pollutant removal remain challenging. Here, to achieve efficient adsorption of dyes, a radial-freezing technique was employed to develop freeze-dried bio-nanocomposites in the form of aerogel beads composed of cellulose nanofibers, protein nanofibers, and chitosan (CPCs). This strategy led to the formation of a spherical aerogel with a dandelion-like structure in the radial cross-section. FE-SEM micrographs of the aerogel beads revealed a highly porous morphology with a network of interconnected pores, allowing for the effective adsorption of liquids. The characterization results of the functional aerogel beads showed a remarkable ability to adsorb various cationic and anionic azo dyes. The maximum adsorption capacity of 1349.7 ± 34.36 mg g⁻¹ and removal efficiency of nearly 100% in the initial 1000 mg L⁻¹ Congo Red (CR) solution were obtained for CPCs aerogel beads. The resulting adsorption experimental data were fit by the sip isotherm and pseudo-second-order models. The porous structure of the CPCs aerogel bead enhanced the diffusion of dye molecules into the pores and inner surface. Furthermore, combined with the analysis results of FT-IR spectroscopy and XPS, multiple adsorption mechanisms (strong electrostatic interactions, hydrogen bonds, CH-π and π-π bonds) were ascribed between the CPCs composite and dye cations. It is believed that our CPCs aerogel beads can be regarded as a sustainable green bio-adsorbent for water remediation.

Adaptive closed-loop modulation of cortical theta oscillations

Sun, 01 Sep 2024 00:19:24 +0200

Navigational decision-making tasks, such as spatial working memory (SWM), rely highly on information integration from several cortical and sub-cortical regions. Performance in SWM tasks is associated with theta rhythm, including low-frequency oscillations related to movement and memory. The interaction of the ventral hippocampus (vHPC) and medial prefrontal cortex (mPFC), reflected in theta synchrony, is essential in various steps of information processing during SWM. We used a closed-loop neurofeedback (CLNF) system to upregulate theta power in the mPFC and investigate its effects on circuit dynamics and behavior in animal models. Specifically, we hypothesized that enhancing the power of the theta rhythm in the mPFC might improve SWM performance. Animals were divided into three groups: closed-loop (CL), random-loop (RL), and OFF (without stimulation). We recorded local field potential (LFP) in the mPFC while electrical reward stimulation contingent on cortical theta activity was delivered to the lateral hypothalamus (LH), which is considered one of the central reward-associated regions. We also recorded LFP in the vHPC to evaluate the related subcortical neural changes. Results revealed a sustained increase in the theta power in both mPFC and vHPC for the CL group. Our analysis also revealed an increase in mPFC-vHPC synchronization in the theta range over the stimulation sessions in the CL group, as measured by coherence and cross-correlation in the theta frequency band. The reinforcement of this circuit improved spatial decision-making performance in the subsequent behavioral results. Our findings provide direct evidence of the relationship between specific theta upregulation and SWM performance and suggest that theta oscillations are integral to cognitive processes. Overall, this study highlights the potential of adaptive CLNF systems in investigating neural dynamics in various brain circuits.

Comprehensive characterization of the rhizosphere metabolome

Salomonsen, C., Martyn, A., Quilbé, J., et al. — Sun, 01 Dec 2024 00:19:24 +0100

Plant root exudates play a pivotal role in shaping soil dynamics and the microbial community in the rhizosphere. The chemical composition of root exudates includes primary and secondary metabolites, including amino acids, organic acids, flavonoids, and small peptides. Comprehensive characterization of root exudates will allow for a better understanding of rhizosphere processes and interactions, but analysis of root exudates is hindered by complicated collection setups, time-consuming sample preparation, and a lack of definitive annotations within metabolomics. We present a method optimized for non-targeted analysis of primary and secondary metabolites in root exudate samples using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry. The method was tested on root exudates from Lotus japonicus, collected using distinct and well-established sampling methods: a hydroponic-soil-hybrid approach, as well as a modification of a soil-leaching method, thus exemplifying the versatility of the analysis method. The method allows for non-targeted screening of plant metabolites, and provides low detection limits (0.002–0.05 μg/mL) and high recoveries (78 ± 30%), though a matrix effect was observed for certain plant metabolites. Detection of a large number of features was achieved (670–2785) of which the majority could be putatively annotated at the compound class level. Of these, 14 features were putatively annotated to a specific structure with high confidence, three of which were confirmed with analytical reference standards. The method can be used for investigation of the overall change in root exudation, as well as for investigating significant changes in metabolites in response to intraenous and extraneous parameters.

CsgA gatekeeper residues control nucleation but not stability of functional amyloid

Olsen, W. P., Courtade, G., Peña-Díaz, S., et al. — Tue, 01 Oct 2024 00:19:24 +0200

Functional amyloids, beneficial to the organism producing them, are found throughout life, from bacteria to humans. While disease-related amyloids form by uncontrolled aggregation, the fibrillation of functional amyloid is regulated by complex cellular machinery and optimized sequences, including so-called gatekeeper residues such as Asp. However, the molecular basis for this regulation remains unclear. Here we investigate how the introduction of additional gatekeeper residues affects fibril formation and stability in the functional amyloid CsgA from E. coli. Step-wise introduction of additional Asp gatekeepers gradually eliminated fibrillation unless preformed fibrils were added, illustrating that gatekeepers mainly affect nucleus formation. Once formed, the mutant CsgA fibrils were just as stable as wild-type CsgA. HSQC NMR spectra confirmed that CsgA is intrinsically disordered, and that the introduction of gatekeeper residues does not alter this ensemble. NMR-based Dark-state Exchange Saturation Transfer (DEST) experiments on the different CsgA variants, however, show a decrease in transient interactions between monomeric states and the fibrils, highlighting a critical role for these interactions in the fibrillation process. We conclude that gatekeeper residues affect fibrillation kinetics without compromising structural integrity, making them useful and selective modulators of fibril properties.

Static Mechanical Allodynia is a paradoxical painful touch-evoked tactile HYPO-aesthesia

de Andrade Melo Knaut, S., Hoang, M. A., Bossard, E. K., et al. — Mon, 01 Apr 2024 00:19:24 +0200

Objective: To determine the falsifiability of the proposed etiology of static mechanical allodynia (SMA) as a paradoxical painful touch-evoked slight tactile HYPO-aesthesia caused by Aβ axonal lesions in patients treated for Complex Regional Pain Syndrome (CRPS) of the foot. Methods: This is a retrospective analysis of prospectively collected clinical data of patients with CRPS of the foot. Patients who achieved resolution of their SMA following the assessment and treatment of the method of Somatosensory Pain Rehabilitation (SPR) were included in the study. The shift from SMA to an underlying tactile HYPO-aesthesia supported the recommendation of somatosensory re-learning of the method of SPR. Allodynography and Rainbow Pain Scale were two clinical examination signs used pre-treatment to quantify the allodynic territory. The quality and intensity of pain were assessed using the McGill Pain Questionnaire (MPQ). Then, the underlying tactile HYPO-aesthesia was assessed with the 2-Point Discrimination Test (2PDT) and Pressure Perception Threshold (PPT). Results: Eighty-six patients with a CRPS of the foot were included. On admission, 43 patients had a discrete, 26 a consequential and 17 a serious SMA. After disappearance of SMA, patients had a slight and partial underlying HYPO-aesthetic territory confirmed with a PTT mean of 2.8 g. Significant improvements were noted for the MPQ, PPT and 2PDT (p < 0.001) with a large effect size. Conclusion: This study confirms that SMA is a paradoxical painful touch-evoked tactile HYPO-aesthesia in patients with CRPS. Moreover, results support the efficacy of the method of SPR to improve neuropathic pain within this population.

The pro-atherogenic enzyme PAPP-A is active in eluates from human carotid and femoral atherosclerotic plaques

Gude, M. F., Hjortebjerg, R., Bjerre, M., et al. — Sun, 01 Sep 2024 00:19:24 +0200

Background: Pregnancy-associated plasma protein-A (PAPP-A) regulates bioavailability of insulin-like growth factor 1 (IGF1) in various tissues by proteolytic cleavage of a subset of IGF-binding proteins (IGFBPs). Pre-clinical studies have established a role of PAPP-A in atherosclerosis and proposed that targeting the proteolytic activity of PAPP-A has therapeutic value. This study aimed to investigate whether human atherosclerotic plaques contain proteolytically active PAPP-A, a prerequisite for further considering PAPP-A as a therapeutic target in patients. Methods: We obtained carotid (n = 9) and femoral (n = 11) atherosclerotic plaques from patients undergoing vascular surgery and incubated freshly harvested plaque tissue in culture media for 24 h. Subsequently, conditioned media were assayed for PAPP-A, STC2, IGFBP4, and IGF1 using immunoassays. Enzymatic activity of PAPP-A was assessed by its ability to process recombinant IGFBP4-IGF1 complexes - a specific substrate of PAPP-A - by Western blotting. Results: PAPP-A and STC2 were detectable in conditioned media from both carotid and femoral plaques, with higher STC2 concentrations in eluates from carotid plaque incubations (p = 0.02). IGFBP4 and IGF1 were undetectable. Conditioned media from all 20 plaques exhibited PAPP-A proteolytic activity. However, no correlation between PAPP-A concentration and its proteolytic activity was observed, whereas the PAPP-A: STC2 molar ratio correlated with PAPP-A activity (R² = 0.25, p = 0.03). Conclusion: This study provides evidence for the presence of enzymatically active PAPP-A in atherosclerotic plaques and underscores the need for further investigating potential beneficial effects associated with targeting PAPP-A in atherosclerotic cardiovascular disease.

Experimental study on temperature difference between the interior and exterior of the vehicle transporting weaner pigs

Chen, G., Kobek-Kjeldager, C., Jensen, L. D., et al. — Fri, 01 Nov 2024 00:19:24 +0100

Transporting pigs poses a significant challenge in maintaining proper interior thermal conditions. This study conducted 36 field trials run in Denmark and collected data from a certified livestock vehicle, during journeys of 8 h and 23 h respectively. This study aims to investigate the air temperature inside a livestock vehicle during the transportation and the influence of five factors on DT (difference in air temperature between interior of the vehicle and exterior): compartment location, deck height, height of openings for natural ventilation, wind speed and vehicle speed. The compartment location was the most important influencing factor of interior air temperature. The maximum percentage of time when air temperature exceeded 30 °C was 13.6% observed in the front compartment of trailer. The maximum difference in mean DT between the front and rear compartments at the same deck was 11.0 ± 0.67 °C occurred in the lorry. The maximum differences in mean DT between the two investigated deck heights were 1.2 ± 0.39 °C in the lorry (70 vs. 90 cm) and 0.9 ± 0.26 °C in the trailer (60 vs. 80 cm), respectively. The DT decreased with increasing height of opening for natural ventilation and wind speed, while the DT was insensitive to vehicle speed. Extra sensors installed on the front partition wall during the last 4 journeys showed significant temperature variability (up to 12 °C) within compartment. Further studies identifying the efficient monitoring of thermal condition and prompt interior environmental control are needed in vehicles for pig transport.

ATAXIN-2 intermediate-length polyglutamine expansions elicit ALS-associated metabolic and immune phenotypes

Vieira de Sá, R., Sudria-Lopez, E., Cañizares Luna, M., et al. — Sun, 01 Dec 2024 00:19:24 +0100

Intermediate-length repeat expansions in ATXN-2 are the strongest genetic risk factor for ALS. Here, the authors combine patient-derived motor neurons and organoids with mouse models to dissect the pathogenic effects of ATXN2 intermediate expansions.

Markers of Mitochondrial Function and DNA Repair Associated with Physical Function in Centenarians

Sanchez-Roman, I., Ferrando, B., Myrup Holst, C., et al. — Thu, 01 Aug 2024 00:19:24 +0200

Mitochondrial dysfunction and genomic instability are key hallmarks of aging. The aim of this study was to evaluate whether maintenance of physical capacities at very old age is associated with key hallmarks of aging. To investigate this, we measured mitochondrial bioenergetics, mitochondrial DNA (mtDNA) copy number and DNA repair capacity in peripheral blood mononuclear cells from centenarians. In addition, circulating levels of NAD+/NADH, brain-derived neurotrophic factor (BDNF) and carbonylated proteins were measured in plasma and these parameters were correlated to physical capacities. Centenarians without physical disabilities had lower mitochondrial respiration values including ATP production, reserve capacity, maximal respiration and non-mitochondrial oxygen-consumption rate and had higher mtDNA copy number than centenarians with moderate and severe disabilities (p < 0.05). In centenarian females, grip strength had a positive association with mtDNA copy number (p < 0.05), and a borderline positive trend for activity of the central DNA repair enzyme, APE 1 (p = 0.075), while a negative trend was found with circulating protein carbonylation (p = 0.07) in the entire cohort. Lastly, a trend was observed for a negative association between BDNF and activity of daily living disability score (p = 0.06). Our results suggest that mechanisms involved in maintaining mitochondrial function and genomic stability may be associated with maintenance of physical function in centenarians.

The metalloproteinase PAPP-A is required for IGF-dependent chondrocyte differentiation and organization

Harboe, M., Kjaer-Sorensen, K., Füchtbauer, E. M., et al. — Thu, 29 Aug 2024 00:19:24 +0200

Insulin-like growth factor (IGF) signaling is required for proper growth and skeletal development in vertebrates. Consequently, its dysregulation may lead to abnormalities of growth or skeletal structures. IGF is involved in the regulation of cell proliferation and differentiation of chondrocytes. However, the availability of bioactive IGF may be controlled by antagonizing IGF binding proteins (IGFBPs) in the circulation and tissues. As the metalloproteinase PAPP-A specifically cleaves members of the IGFBP family, we hypothesized that PAPP-A activity liberates bioactive IGF in cartilage. In PAPP-A knockout mice, the femur length was reduced and the mice showed a disorganized columnar organization of growth plate chondrocytes. Similarly, zebrafish lacking pappaa showed reduced length of Meckel’s cartilage and disorganized chondrocytes, reminiscent of the mouse knockout phenotype. Expression of chondrocyte differentiation markers (sox9a, ihha, and col10a1) was markedly affected in Meckel’s cartilage of pappaa knockout zebrafish, indicating that differentiation of chondrocytes was compromised. Additionally, the zebrafish pappaa knockout phenotype was mimicked by pharmacological inhibition of IGF signaling, and it could be rescued by treatment with exogenous recombinant IGF-I. In conclusion, our data suggests that IGF activity in the growing cartilage, and hence IGF signaling in chondrocytes, requires the presence of PAPP-A. The absence of PAPP-A causes aberrant chondrocyte organization and compromised growth in both mice and zebrafish.

Defining, Re-defining, and Understanding the Brain and Mental Health During Ageing

Kaur, G., Rattan, S. — Mon, 01 Jan 2024 00:19:24 +0100

Brain and Mental Health in Ageing

Mon, 01 Jan 2024 00:19:24 +0100

Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites

Tue, 15 Oct 2024 00:19:24 +0200

The autoinhibited plasma membrane calcium ATPase ACA8 from A. thaliana has an N-terminal autoinhibitory domain. Binding of calcium-loaded calmodulin at two sites located at residues 42–62 and 74–96 relieves autoinhibition of ACA8 activity. Through activity studies and a yeast complementation assay we investigated wild-type (WT) and N-terminally truncated ACA8 constructs (Δ20, Δ30, Δ35, Δ37, Δ40, Δ74 and Δ100) to explore the role of conserved motifs in the N-terminal segment preceding the calmodulin binding sites. Furthermore, we purified WT, Δ20- and Δ100-ACA8, tested activity in vitro and performed structural studies of purified Δ20-ACA8 stabilized in a lipid nanodisc to explore the mechanism of autoinhibition. We show that an N-terminal segment between residues 20 and 35 including conserved Phe32, upstream of the calmodulin binding sites, is important for autoinhibition and the activation by calmodulin. Cryo-EM structure determination at 3.3 Å resolution of a beryllium fluoride inhibited E2 form, and at low resolution for an E1 state combined with AlphaFold prediction provide a model for autoinhibition, consistent with the mutational studies.

Architecture of RabL2-associated complexes at the ciliary base

Boegholm, N., Petriman, N. A., Tanvir, N. M., Lorentzen, E. — Sun, 01 Sep 2024 00:19:24 +0200

Cilia are slender, micrometer-long organelles present on the surface of eukaryotic cells. They function in signaling and locomotion and are constructed by intraflagellar transport (IFT). The assembly of IFT complexes into so-called IFT trains to initiate ciliary entry at the base of the cilium remains a matter of debate. Here, we use structural modeling to provide an architectural framework for how RabL2 is anchored at the ciliary base via CEP19 before being handed over to IFT trains for ciliary entry. Our models suggest that the N-terminal domain of CEP43 forms a homo-dimer to anchor at the subdistal appendages of cilia through a direct interaction with CEP350. A long linker region separates the N-terminal domain of CEP43 from the C-terminal domain, which captures CEP19 above the subdistal appendages and close to the distal appendages. Furthermore, we present a structural model for how RabL2-CEP19 associates with the IFT-B complex, providing insight into how RabL2 is handed over from CEP19 to the IFT complex. Interestingly, RabL2 association with the IFT-B complex appears to induce a significant conformational change in the IFT complex via a kink in the coiled-coils of the IFT81/74 proteins, which may prime the IFT machinery for entry into cilia.

Plug-and-play nucleic acid-mediated multimerization of biparatopic nanobodies for molecular imaging

Teodori, L., Kroer, S., Omer, M., et al. — Tue, 10 Sep 2024 00:19:24 +0200

In cancer molecular imaging, selecting binders with high specificity and affinity for biomarkers is paramount for achieving high-contrast imaging within clinical time frames. Nanobodies have emerged as potent candidates, surpassing antibodies in pre-clinical imaging due to their convenient production, rapid renal clearance, and deeper tissue penetration. Multimerization of nanobodies is a popular strategy to enhance their affinity and pharmacokinetics; however, traditional methods are laborious and may yield heterogeneous products. In this study, we employ a Holliday junction (HJ)-like nucleic acid-based scaffold to create homogeneous nanostructures with precise multivalent and multiparatopic nanobody displays. The plug-and-play assembly allowed the screening of several nanobody multimer configurations for the detection of the breast cancer biomarker, human epidermal growth factor receptor 2 (HER2). In vitro studies demonstrated significant improvements in binding avidity, particularly with the biparatopic construct exhibiting high sensitivity, surpassing that of traditional antibody-based cell binding. Furthermore, our HJ platform allowed for adaptation from fluorescence-based to nuclear imaging, as demonstrated in xenografted mice, thereby allowing for future in vivo applications. This work highlights the potential of nucleic acid-mediated multimerization to markedly enhance nanobody binding, by exploring synergistic combinations and offering versatility for both in vitro diagnostics and cancer molecular imaging with prospects for future theranostic applications.

From genotype to phenotype: Genetic redundancy and the maintenance of an adaptive polymorphism in the context of high gene flow

Sat, 01 Jan 2022 00:19:24 +0100

HSP60 chaperone deficiency disrupts the mitochondrial matrix proteome and dysregulates cholesterol synthesis

Cömert, C., Kjær-Sørensen, K., Hansen, J., et al. — Tue, 01 Oct 2024 00:19:24 +0200

Objective: Mitochondrial proteostasis is critical for cellular function. The molecular chaperone HSP60 is essential for cell function and dysregulation of HSP60 expression has been implicated in cancer and diabetes. The few reported patients carrying HSP60 gene variants show neurodevelopmental delay and brain hypomyelination. Hsp60 interacts with more than 260 mitochondrial proteins but the mitochondrial proteins and functions affected by HSP60 deficiency are poorly characterized. Methods: We studied two model systems for HSP60 deficiency: (1) engineered HEK cells carrying an inducible dominant negative HSP60 mutant protein, (2) zebrafish HSP60 knockout larvae. Both systems were analyzed by RNASeq, proteomics, and targeted metabolomics, and several functional assays relevant for the respective model. In addition, skin fibroblasts from patients with disease-associated HSP60 variants were analyzed by proteomics. Results: We show that HSP60 deficiency leads to a differentially downregulated mitochondrial matrix proteome, transcriptional activation of stress responses, and dysregulated cholesterol biosynthesis. This leads to lipid accumulation in zebrafish knockout larvae. Conclusions: Our data provide a compendium of the effects of HSP60 deficiency on the mitochondrial matrix proteome. We show that HSP60 is a master regulator and modulator of mitochondrial functions and metabolic pathways. HSP60 dysfunction also affects cellular metabolism and disrupts the integrated stress response. The effect on cholesterol synthesis explains the effect of HSP60 dysfunction on myelination observed in patients carrying genetic variants of HSP60.

Design of functional intrinsically disordered proteins

Garg, A., González-Foutel, N. S., Gielnik, M. B., Kjaergaard, M. — Fri, 01 Mar 2024 00:19:24 +0100

Many proteins do not fold into a fixed three-dimensional structure, but rather function in a highly disordered state. These intrinsically disordered proteins pose a unique challenge to protein engineering and design: How can proteins be designed de novo if not by tailoring their structure? Here, we will review the nascent field of design of intrinsically disordered proteins with focus on applications in biotechnology and medicine. The design goals should not necessarily be the same as for de novo design of folded proteins as disordered proteins have unique functional strengths and limitations. We focus on functions where intrinsically disordered proteins are uniquely suited including disordered linkers, desiccation chaperones, sensors of the chemical environment, delivery of pharmaceuticals, and constituents of biomolecular condensates. Design of functional intrinsically disordered proteins relies on a combination of computational tools and heuristics gleaned from sequence-function studies. There are few cases where intrinsically disordered proteins have made it into industrial applications. However, we argue that disordered proteins can perform many roles currently performed by organic polymers, and that these proteins might be more designable due to their modularity.

Optimization of Genotyping-by-Sequencing (GBS) for Germplasm Fingerprinting and Trait Mapping in Faba Bean

Zhang, H., Fechete, L., Himmelbach, A., et al. — Sun, 01 Sep 2024 00:19:24 +0200

Faba bean (Vicia faba L.) is an important pulse crop with a wide range of agroecological adaptations. The development of genomic tools and a comprehensive catalog of extant genetic diversity are crucial for developing improved faba bean cultivars. The lack of a cost-effective genotyping platform limits the characterization of large germplasm collections, understanding of genetic diversity across populations, and implementing breeder's tools like genomic selection. Genotyping-by-sequencing (GBS) offers high-resolution genotyping for both model and crop plant species, even without a reference genome sequence. The genome fragments targeted by GBS depend substantially on the restriction enzyme (RE) used for the complexity reduction step. Species with complex genomic architecture require optimization of GBS with proper RE to realize the full potential of GBS. Here, we evaluated various REs in the GBS method and identified that the combination of ApeKI/MseI proved to be the most appropriate for faba bean based on the best library quality, a high number of genomic loci spread across chromosomes, and high enrichment loci associated with the gene space. With the new optimized protocol, we constructed a genetic map using a recombinant inbred line (RIL) population and identified a QTL for seed hilum color on Chromosome 1. In addition, we also genotyped a diversity panel and performed a genome-wide association studies (GWAS) for important agronomic traits, including plant height (PH), flowering time (FT), and number of pods per plant (PPP). We identified six SNP markers significantly associated with these traits and listed potential candidate genes. The optimized faba bean-specific GBS procedure will facilitate access to the untapped genetic diversity for genetic research and breeding and may facilitate functional genomics.

The role of immune dysfunction in the pathopsychology of autism spectrum disorders

Abdallah, M.W., Hougaard, D.M., Grove, J., et al. — Sun, 01 Jan 2012 00:19:24 +0100

Circular RNAs regulate neuron size and migration of midbrain dopamine neurons during development

Rybiczka-Tešulov, M., Garritsen, O., Venø, M. T., et al. — Sun, 01 Dec 2024 00:19:24 +0100

Midbrain dopamine (mDA) neurons play an essential role in cognitive and motor behaviours and are linked to different brain disorders. However, the molecular mechanisms underlying their development, and in particular the role of non-coding RNAs (ncRNAs), remain incompletely understood. Here, we establish the transcriptomic landscape and alternative splicing patterns of circular RNAs (circRNAs) at key developmental timepoints in mouse mDA neurons in vivo using fluorescence-activated cell sorting followed by short- and long-read RNA sequencing. In situ hybridisation shows expression of several circRNAs during early mDA neuron development and post-transcriptional silencing unveils roles for different circRNAs in regulating mDA neuron morphology. Finally, in utero electroporation and time-lapse imaging implicate circRmst, a circRNA with widespread morphological effects, in the migration of developing mDA neurons in vivo. Together, these data for the first time suggest a functional role for circRNAs in developing mDA neurons and characterise poorly defined aspects of mDA neuron development.

O-GlcNAc transferase congenital disorder of glycosylation (OGT-CDG)

Mayfield, J. M., Hitefield, N. L., Czajewski, I., et al. — Sun, 01 Sep 2024 00:19:24 +0200

O-GlcNAc transferase (OGT) is the sole enzyme responsible for the post-translational modification of O-GlcNAc on thousands of target nucleocytoplasmic proteins. To date, nine variants of OGT that segregate with OGT Congenital Disorder of Glycosylation (OGT-CDG) have been reported and characterized. Numerous additional variants have been associated with OGT-CDG, some of which are currently undergoing investigation. This disorder primarily presents with global developmental delay and intellectual disability (ID), alongside other variable neurological features and subtle facial dysmorphisms in patients. Several hypotheses aim to explain the etiology of OGT-CDG, with a prominent hypothesis attributing the pathophysiology of OGT-CDG to mutations segregating with this disorder disrupting the OGT interactome. The OGT interactome consists of thousands of proteins, including substrates as well as interactors that require noncatalytic functions of OGT. A key aim in the field is to identify which interactors and substrates contribute to the primarily neural-specific phenotype of OGT-CDG. In this review, we will discuss the heterogenous phenotypic features of OGT-CDG seen clinically, the variable biochemical effects of mutations associated with OGT-CDG, and the use of animal models to understand this disorder. Furthermore, we will discuss how previously identified OGT interactors causal for ID provide mechanistic targets for investigation that could explain the dysregulated gene expression seen in OGT-CDG models. Identifying shared or unique altered pathways impacted in OGT-CDG patients will provide a better understanding of the disorder as well as potential therapeutic targets.

Crystal structure of Kunitz-type trypsin inhibitor

Mehmood, S., Thirup, S. S., Ahmed, S., et al. — Sun, 01 Dec 2024 00:19:24 +0100

Trypsin inhibitors are known to act against insect pests by inhibiting proteases of the digestive tract. In this study, we report structural and functional characterization of ∼ 19 kDa Albizia procera Kunitz-type trypsin inhibitor (ApKTI) protein with potential bio-insecticidal applications. Crystal structure of ApKTI protein has been refined to 1.42 Å and molecular structure (8HNR) showed highly beta sheeted conformation including 12 beta sheets, 15 loops and two small alpha helices. Docking between predicted model of Tribolium castaneum trypsin (TcPT) and 8HNR produced a stable complex (−11.3 kcal/mol) which reflects the inhibitory potential of ApKTI against insect gut trypsin. Significant mortality was observed in all life stages of T. castaneum including egg, larvae, pupae and adults with a 3.0 mg native ApKTI treatment in comparison to negative control. Although standard trypsin inhibitor (Glycine max trypsin inhibitors; GmKTI; 3.0 mg) produced maximum reduction against all above life stages; however, a non-significant mortality difference was observed in comparison to 3.0 mg native ApKTI. The study further explores the synthesis and characterization of Graphene (GNPs) and Zinc oxide (ZnONPs) nanoparticles, followed by the optimization of ApKTI and GmKTI loading on both nanoparticles to evaluate their enhanced insecticidal effectiveness. Encapsulated proteins showed significant mortality against T. castaneum across all concentrations, with GNPs proving more effective than ZnONPs. Additionally, encapsulated GmKTI produced significant mortality of eggs compared to loaded ApKTI treatments while other life stages were non-significantly affected by two proteins. This research highlights the importance of encapsulated ApKTI protein for eco-friendly pest management strategies.

Holistic approaches to healthy longevity.

Rattan, S., Bartke, A., Rose, M. R., et al. — Mon, 01 Jan 2024 00:19:24 +0100

Molecular Mechanisms of Temperature Tolerance Plasticity in an Arthropod

Aagaard, A., Bechsgaard, J., Sørensen, J. G., et al. — Thu, 01 Aug 2024 00:19:24 +0200

How species thrive in a wide range of environments is a major focus of evolutionary biology. For many species, limited genetic diversity or gene flow among habitats means that phenotypic plasticity must play an important role in their capacity to tolerate environmental heterogeneity and to colonize new habitats. However, we have a limited understanding of the molecular components that govern plasticity in ecologically relevant phenotypes. We examined this hypothesis in a spider species (Stegodyphus dumicola) with extremely low species-wide genetic diversity that nevertheless occupies a broad range of thermal environments. We determined phenotypic responses to temperature stress in individuals from four climatic zones using common garden acclimation experiments to disentangle phenotypic plasticity from genetic adaptations. Simultaneously, we created data sets on multiple molecular modalities: the genome, the transcriptome, the methylome, the metabolome, and the bacterial microbiome to determine associations with phenotypic responses. Analyses of phenotypic and molecular associations reveal that acclimation responses in the transcriptome and metabolome correlate with patterns of phenotypic plasticity in temperature tolerance. Surprisingly, genes whose expression seemed to be involved in plasticity in temperature tolerance were generally highly methylated contradicting the idea that DNA methylation stabilizes gene expression. This suggests that the function of DNA methylation in invertebrates varies not only among species but also among genes. The bacterial microbiome was stable across the acclimation period; combined with our previous demonstrations that the microbiome is temporally stable in wild populations, this is convincing evidence that the microbiome does not facilitate plasticity in temperature tolerance. Our results suggest that population-specific variation in temperature tolerance among acclimation temperatures appears to result from the evolution of plasticity in mainly gene expression.

Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification

Malle, M. G., Song, P., Löffler, P. M.G., et al. — Wed, 01 May 2024 00:19:24 +0200

Synthetic nanoparticles as lipid nanoparticles (LNPs) are widely used as drug delivery vesicles. However, they hold several drawbacks, including low biocompatibility and unfavorable immune responses. Naturally occurring extracellular vesicles (EVs) hold the potential as native, safe, and multifunctional nanovesicle carriers. However, loading of EVs with large biomolecules remains a challenge. Here, we present a controlled loading methodology using DNA-mediated and programmed fusion between EVs and messenger RNA (mRNA)-loaded liposomes. The fusion efficiency is characterized at the single-particle level by real-time microscopy through EV surface immobilization via lipidated biotin-DNA handles. Subsequently, fused EV−liposome particles (EVLs) can be collected by employing a DNA strand-replacement reaction. Transferring the fusion reaction to magnetic beads enables us to scale up the production of EVLs one million times. Finally, we demonstrated encapsulation of mCherry mRNA, transfection, and improved translation using the EVLs compared to liposomes or LNPs in HEK293-H cells. We envision this as an important tool for the EV-mediated delivery of RNA therapeutics.

Evidence for feminized genetic males in a flea beetle using newly identified X-linked markers

Rohlfing, K., Grewoldt, M., Cordellier, M., Dobler, S. — Thu, 01 Aug 2024 00:19:24 +0200

The equilibrium of sex ratios in sexually reproducing species is often disrupted by various environmental and genetic factors, including endosymbionts like Wolbachia. In this study, we explore the highly female-biased sex ratio observed in the flea beetle, Altica lythri, and its underlying mechanisms. Ancient hybridization events between Altica species have led to mitochondrial DNA introgression, resulting in distinct mitochondrial haplotypes that go along with different Wolbachia infections (HT1-wLytA1, HT1*- uninfected, HT2-wLytA2, and HT3-wLytB). Notably, beetles with some haplotypes exclusively produce female offspring, suggesting potential Wolbachia-induced phenomena such as feminization of genetic males. However, the observed female bias could also be a consequence of the ancient hybridization resulting in nuclear-cytoplasmic conflicts between introgressed mtDNA and nuclear genes. Through transcriptomic analysis and the program SEX-DETector, we established markers for genotypic sex differentiation for A. lythri, enabling genetic sexing via qPCR. Our findings suggest that feminization of genetic males is contributing to the skewed sex ratios, highlighting the intricate dynamics of sex determination and reproductive strategies in this flea beetle. This study provides valuable insights into the dynamics of genetic conflicts, endosymbionts, and sex ratios, revealing the novel phenomenon of genetic male feminization in the flea beetle A. lythri.

Structure of biofilm-forming functional amyloid PSMα1 from Staphylococcus aureus

Holst Hansen, K., Byeon, C. H., Liu, Q., et al. — Thu, 01 Aug 2024 00:19:24 +0200

Biofilmbeskyttet patogen Staphylococcus aureus forårsager kroniske infektioner, som er svære at behandle. En væsentlig byggesten i disse biofilm er funktionelle amyloidfibriller, der samles fra phenolopløselige moduliner (PSM'er). PSMα1 krydsfrø andre PSM'er i kryds-β amyloidfolder og er derfor et nøgleelement i initiering af biofilmdannelse. Imidlertid hindrer manglen på højopløsningsstrukturer bestræbelser på at forhindre amyloidsamling og biofilmdannelse. Her præsenterer vi et 3,5 Å opløsningstæthedskort af den store PSMα1-fibrilform, der afslører en venstrehåndet kryds-β-fibril sammensat af to C2-symmetriske U-formede protofilamenter, hvis underenheder er usædvanligt vippet ud af planet. Monomer α-helical PSMα1 er ekstremt cytotoksisk for celler på trods af den moderate toksicitet af cross-β fibrillen. Vi foreslår mekanistisk indsigt i PSM-funktionel amyloiddannelse og konformationstransformation på vejen fra monomer-til-fibrildannelse. Detaljer om PSMα1-samling og fibrilpolymorfi antyder, hvordan S. aureus anvender funktionelle amyloider til at danne biofilm og etablerer en ramme for udvikling af terapeutika mod infektion og antimikrobiel resistens.

Crystal structure of the aminoglycosides N-acetyltransferase Eis2 from Mycobacterium abscessus

Ung, K. L., Alsarraf, H. M.A.B., Olieric, V., Kremer, L., Blaise, M. — Fri, 01 Nov 2019 00:19:24 +0100

Mycobacterium abscessus is an emerging human pathogen that is notorious for being one of the most drug-resistant species of Mycobacterium. It has developed numerous strategies to overcome the antibiotic stress response, limiting treatment options and leading to frequent therapeutic failure. The panel of aminoglycosides (AG) usually used in the treatment of M. abscessus pulmonary infections is restricted by chemical modification of the drugs by the N-acetyltransferase Eis2 protein (Mabs_Eis2). This enzyme acetylates the primary amine of AGs, preventing these antibiotics from binding ribosomal RNA and thereby impairing their activity. In this study, the high-resolution crystal structures of Mabs_Eis2 in its apo- and cofactor-bound forms were solved. The structural analysis of Mabs_Eis2, supported by the kinetic characterization of the enzyme, highlights the large substrate specificity of the enzyme. Furthermore, in silico docking and biochemical approaches attest that Mabs_Eis2 modifies clinically relevant drugs such as kanamycin and amikacin, with a better efficacy for the latter. In line with previous biochemical and in vivo studies, our work suggests that Mabs_Eis2 represents an attractive pharmacological target to be further explored. The high-resolution crystal structures presented here may pave the way to the design of Eis2-specific inhibitors with the potential to counteract the intrinsic resistance levels of M. abscessus to an important class of clinically important antibiotics. Database: Structural data are available in the PDB database under the accession numbers: 6RFY, 6RFX and 6RFT.

Structural analysis of the N-acetyltransferase Eis1 from Mycobacterium abscessus reveals the molecular determinants of its incapacity to modify aminoglycosides

Ung, K. L., Kremer, L., Blaise, M. — Fri, 01 Jan 2021 00:19:24 +0100

Enhanced intracellular survival (Eis) proteins belonging to the superfamily of the GCN5-related N-acetyltransferases play important functions in mycobacterial pathogenesis. In Mycobacterium tuberculosis, Eis enhances the intracellular survival of the bacilli in macrophages by modulating the host immune response and is capable to chemically modify and inactivate aminoglycosides. In nontuberculous mycobacteria (NTM), Eis shares similar functions. However, Mycobacterium abscessus, a multidrug resistant NTM, possesses two functionally distinct Eis homologues, Eis1_Mab and Eis2_Mab. While Eis2_Mab participates in virulence and aminoglycosides resistance, this is not the case for Eis1_Mab, whose exact biological function remains to be determined. Herein, we show that overexpression of Eis1_Mab in M. abscessus fails to induce resistance to aminoglycosides. To clarify why Eis1_Mab is unable to modify this class of antibiotics, we solved its crystal structure bound to its cofactor, acetyl-CoA. The structure revealed that Eis1_Mab has a typical homohexameric Eis-like organization. The structural analysis supported by biochemical approaches demonstrated that while Eis1_Mab can acetylate small substrates, its active site is too narrow to accommodate aminoglycosides. Comparison with other Eis structures showed that an extended loop between strands 9 and 10 is blocking the access of large substrates to the active site and movement of helices 4 and 5 reduces the volume of the substrate-binding pocket to these compounds in Eis1_Mab. Overall, this study underscores the molecular determinants explaining functional differences between Eis1_Mab and Eis2_Mab, especially those inherent to their capacity to modify aminoglycosides.

The crystal structure of the mycobacterial trehalose monomycolate transport factor A, TtfA, reveals an atypical fold

Ung, K. L., Alsarraf, H. M.A.B., Kremer, L., Blaise, M. — Mon, 01 Jun 2020 00:19:24 +0200

Trehalose monomycolate (TMM) represents an essential element of the mycobacterial envelope. While synthesized in the cytoplasm, TMM is transported across the inner membrane by MmpL3 but, little is known regarding the MmpL3 partners involved in this process. Recently, the TMM transport factor A (TtfA) was found to form a complex with MmpL3 and to participate in TMM transport, although its biological role remains to be established. Herein, we report the crystal structure of the Mycobacterium smegmatis TtfA core domain. The phylogenetic distribution of TtfA homologues in non-mycolate containing bacteria suggests that TtfA may exert additional functions.

Method for Testing Etiologic Heterogeneity Among Non-Competing Diagnoses, Applied to Impact of Perinatal Exposures on Autism and Attention Deficit Hyperactivity Disorder

Kalkbrenner, A. E., Zheng, C., Yu, J., et al. — Sun, 01 Sep 2024 00:19:24 +0200

Background: Testing etiologic heterogeneity, whether a disorder subtype is more or less impacted by a risk factor, is important for understanding causal pathways and optimizing statistical power. The study of mental health disorders especially benefits from strategic subcategorization because these disorders are heterogeneous and frequently co-occur. Existing methods to quantify etiologic heterogeneity are not appropriate for noncompeting events in an open cohort of variable-length follow-up. Thus, we developed a new method. Methods: We estimated risks from urban residence, maternal smoking during pregnancy, and parental psychiatric history, with subtypes defined by the presence or absence of a codiagnosis: autism alone, attention deficit hyperactivity disorder (ADHD) alone, and joint diagnoses of autism + ADHD. To calculate the risk of a single diagnosis (e.g., autism alone), we subtracted the risk for autism + ADHD from the risk for autism overall. We tested the equivalency of average risk ratios over time, using a Wald-type test and bootstrapped standard errors. Results: Urban residence was most strongly linked with autism + ADHD and least with ADHD only; maternal smoking was associated with ADHD only but not autism only; and parental psychiatric history exhibited similar associations with all subgroups. Conclusion: Our method allowed the calculation of appropriate P values to test the strength of association, informing etiologic heterogeneity wherein two of these three risk factors exhibited different impacts across diagnostic subtypes. The method used all available data, avoided neurodevelopmental outcome misclassification, exhibited robust statistical precision, and is applicable to similar heterogeneous complex conditions using common diagnostic data with variable follow-up.

Unraveling the metabolomic architecture of autism in a large Danish population-based cohort

Ottosson, F., Russo, F., Abrahamsson, A., et al. — Sun, 01 Dec 2024 00:19:24 +0100

Background: The prevalence of autism in Denmark has been increasing, reaching 1.65% among 10-year-old children, and similar trends are seen elsewhere. Although there are several factors associated with autism, including genetic, environmental, and prenatal factors, the molecular etiology of autism is largely unknown. Here, we use untargeted metabolomics to characterize the neonatal metabolome from dried blood spots collected shortly after birth. Methods: We analyze the metabolomic profiles of a subset of a large Danish population-based cohort (iPSYCH2015) consisting of over 1400 newborns, who later are diagnosed with autism and matching controls and in two Swedish population-based cohorts comprising over 7000 adult participants. Mass spectrometry analysis was performed by a timsTOF Pro operated in QTOF mode, using data-dependent acquisition. By applying an untargeted metabolomics approach, we could reproducibly measure over 800 metabolite features. Results: We detected underlying molecular perturbations across several metabolite classes that precede autism. In particular, the cyclic dipeptide cyclo-leucine-proline (FDR-adjusted p = 0.003) and the carnitine-related 5-aminovaleric acid betaine (5-AVAB) (FDR-adjusted p = 0.03), were associated with an increased probability for autism, independently of known prenatal and genetic risk factors. Analysis of genetic and dietary data in adults revealed that 5-AVAB was associated with increased habitual dietary intake of dairy (FDR-adjusted p < 0.05) and with variants near SLC22A4 and SLC22A5 (p < 5.0e − 8), coding for a transmembrane carnitine transporter protein involved in controlling intracellular carnitine levels. Conclusions: Cyclo-leucine-proline and 5-AVAB are associated with future diagnosis of autism in Danish neonates, both representing novel early biomarkers for autism. 5-AVAB is potentially modifiable and may influence carnitine homeostasis.

TMEFF1 is a neuron-specific restriction factor for herpes simplex virus

Dai, Y., Idorn, M., Serrero, M. C., et al. — Thu, 01 Aug 2024 00:19:24 +0200

The brain is highly sensitive to damage caused by infection and inflammation^1,2. Herpes simplex virus 1 (HSV-1) is a neurotropic virus and the cause of herpes simplex encephalitis³. It is unknown whether neuron-specific antiviral factors control virus replication to prevent infection and excessive inflammatory responses, hence protecting the brain. Here we identify TMEFF1 as an HSV-1 restriction factor using genome-wide CRISPR screening. TMEFF1 is expressed specifically in neurons of the central nervous system and is not regulated by type I interferon, the best-known innate antiviral system controlling virus infections. Depletion of TMEFF1 in stem-cell-derived human neurons led to elevated viral replication and neuronal death following HSV-1 infection. TMEFF1 blocked the HSV-1 replication cycle at the level of viral entry through interactions with nectin-1 and non-muscle myosin heavy chains IIA and IIB, which are core proteins in virus–cell binding and virus–cell fusion, respectively^4–6. Notably, Tmeff1^−/− mice exhibited increased susceptibility to HSV-1 infection in the brain but not in the periphery. Within the brain, elevated viral load was observed specifically in neurons. Our study identifies TMEFF1 as a neuron-specific restriction factor essential for prevention of HSV-1 replication in the central nervous system.

Non-Hebbian plasticity transforms transient experiences into lasting memories

Faress, I., Khalil, V., Hou, W. H., et al. — Mon, 01 Jul 2024 00:19:24 +0200

The dominant models of learning and memory, such as Hebbian plasticity, propose that experiences are transformed into memories through input-specific synaptic plasticity at the time of learning. However, synaptic plasticity is neither strictly input-specific nor restricted to the time of its induction. The impact of such forms of non-Hebbian plasticity on memory has been difficult to test, and hence poorly understood. Here, we demonstrate that synaptic manipulations can deviate from the Hebbian model of learning, yet produce a lasting memory. First, we established a weak associative conditioning protocol in mice, where optogenetic stimulation of sensory thalamic input to the amygdala was paired with a footshock, but no detectable memory was formed. However, when the same input was potentiated minutes before or after, or even 24 hr later, the associative experience was converted into a lasting memory. Importantly, potentiating an independent input to the amygdala minutes but not 24 hr after the pairing produced a lasting memory. Thus, our findings suggest that the process of transformation of a transient experience into a memory is neither restricted to the time of the experience nor to the synapses triggered by it; instead, it can be influenced by past and future events.

Molecular properties and diagnostic potential of monoclonal antibodies targeting cytotoxic α-synuclein oligomers

Nielsen, J., Lauritsen, J., Pedersen, J. N., et al. — Sat, 01 Jun 2024 00:19:24 +0200

α-Synuclein (α-syn) accumulates as insoluble amyloid but also forms soluble α-syn oligomers (αSOs), thought to be even more cytotoxic than fibrils. To detect and block the unwanted activities of these αSOs, we have raised 30 monoclonal antibodies (mAbs) against different forms of αSOs, ranging from unmodified αSOs to species stabilized by lipid peroxidation products and polyphenols, αSOs formed by C-terminally truncated α-syn, and multivalent display of α-syn on capsid virus-like particles (cVLPs). While the mAbs generally show a preference for αSOs, they also bind fibrils, but to variable extents. Overall, we observe great diversity in the mAbs' relative affinities for monomers and αSOs, varied requirements for the C-terminal extension of α-syn, and only a modest effect on α-syn fibrillation. Several mAbs show several orders of magnitude preference for αSOs over monomers in in-solution studies, while the commercial antibody MJF14 only bound 10-fold more strongly to αSOs than monomeric α-syn. Gratifyingly, seven mAbs almost completely block αSO permeabilization of membrane vesicles. Five selected mAbs identified α-syn-related pathologies like Lewy bodies (LBs) and Lewy Neurites, as well as Glial Cytoplasmic Inclusions in postmortem brains from people diagnosed for PD, dementia with LBs or multiple system atrophy, although to different extents. Three mAbs were particularly useful for pathological evaluation of postmortem brain human tissue, including early stages of PD. Although there was no straightforward connection between the mAbs' biophysical and immunohistochemical properties, it is encouraging that this comprehensive collection of mAbs able to recognize different aggregated α-syn species in vitro also holds diagnostic potential.

Improving laboratory animal genetic reporting

Teboul, L., Amos-Landgraf, J., Benavides, F. J., et al. — Mon, 01 Jul 2024 00:19:24 +0200

The biomedical research community addresses reproducibility challenges in animal studies through standardized nomenclature, improved experimental design, transparent reporting, data sharing, and centralized repositories. The ARRIVE guidelines outline documentation standards for laboratory animals in experiments, but genetic information is often incomplete. To remedy this, we propose the Laboratory Animal Genetic Reporting (LAG-R) framework. LAG-R aims to document animals' genetic makeup in scientific publications, providing essential details for replication and appropriate model use. While verifying complete genetic compositions may be impractical, better reporting and validation efforts enhance reliability of research. LAG-R standardization will bolster reproducibility, peer review, and overall scientific rigor.

A suicidal and extensively disordered luciferase with a bright luminescence

Dijkema, F. M., Escarpizo-Lorenzana, M. I., Nordentoft, M. K., et al. — Thu, 01 Aug 2024 00:19:24 +0200

Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis, GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here, we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 Å and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate-binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase.

Effects of High-Dose Vitamin D Supplementation on Placental Vitamin D Metabolism and Neonatal Vitamin D Status

Mon, 01 Jul 2024 00:19:24 +0200

Vitamin D (vitD) deficiency (25-hydroxy-vitamin D < 50 nmol/L) is common in pregnancy and associated with an increased risk of adverse pregnancy outcomes. High-dose vitD supplementation is suggested to improve pregnancy health, but there is limited knowledge about the effects on placental vitD transport and metabolism and the vitD status of newborns. Comparing the current standard maternal supplementation, 10 µg/day to a 90 µg vitD supplement, we investigated placental gene expression, maternal vitD transport and neonatal vitD status. Biological material was obtained from pregnant women randomized to 10 µg or 90 µg vitD supplements from week 11–16 onwards. Possible associations between maternal exposure, neonatal vitD status and placental expression of the vitD receptor (VDR), the transporters (Cubilin, CUBN and Megalin, LRP2) and the vitD-activating and -degrading enzymes (CYP24A1, CYP27B1) were investigated. Maternal vitD-binding protein (VDBP) was determined before and after supplementation. Overall, 51% of neonates in the 10 µg vitD group were vitD-deficient in contrast to 11% in the 90 µg group. High-dose vitD supplementation did not significantly affect VDBP or placental gene expression. However, the descriptive analyses indicate that maternal obesity may lead to the differential expression of CUBN, CYP24A1 and CYP27B1 and a changed VDBP response. High-dose vitD improves neonatal vitD status without affecting placental vitD regulation.

Integrative network analysis of miRNA-mRNA expression profiles during epileptogenesis in rats reveals therapeutic targets after emergence of first spontaneous seizure

Khemka, N., Morris, G., Kazemzadeh, L., et al. — Sun, 01 Dec 2024 00:19:24 +0100

Epileptogenesis is the process by which a normal brain becomes hyperexcitable and capable of generating spontaneous recurrent seizures. The extensive dysregulation of gene expression associated with epileptogenesis is shaped, in part, by microRNAs (miRNAs) – short, non-coding RNAs that negatively regulate protein levels. Functional miRNA-mediated regulation can, however, be difficult to elucidate due to the complexity of miRNA-mRNA interactions. Here, we integrated miRNA and mRNA expression profiles sampled over multiple time-points during and after epileptogenesis in rats, and applied bi-clustering and Bayesian modelling to construct temporal miRNA-mRNA-mRNA interaction networks. Network analysis and enrichment of network inference with sequence- and human disease-specific information identified key regulatory miRNAs with the strongest influence on the mRNA landscape, and miRNA-mRNA interactions closely associated with epileptogenesis and subsequent epilepsy. Our findings underscore the complexity of miRNA-mRNA regulation, can be used to prioritise miRNA targets in specific systems, and offer insights into key regulatory processes in epileptogenesis with therapeutic potential for further investigation.

Specific inhibition of α-synuclein oligomer generation and toxicity by the chaperone domain Bri2 BRICHOS

Adam, L., Kumar, R., Arroyo-Garcia, L. E., et al. — Thu, 01 Aug 2024 00:19:24 +0200

Protein misfolding and aggregation are involved in several neurodegenerative disorders, such as α-synuclein (αSyn) implicated in Parkinson's disease, where new therapeutic approaches remain essential to combat these devastating diseases. Elucidating the microscopic nucleation mechanisms has opened new opportunities to develop therapeutics against toxic mechanisms and species. Here, we show that naturally occurring molecular chaperones, represented by the anti-amyloid Bri2 BRICHOS domain, can be used to target αSyn-associated nucleation processes and structural species related to neurotoxicity. Our findings revealed that BRICHOS predominantly suppresses the formation of new nucleation units on the fibrils surface (secondary nucleation), decreasing the oligomer generation rate. Further, BRICHOS directly binds to oligomeric αSyn species and effectively diminishes αSyn fibril-related toxicity. Hence, our studies show that molecular chaperones can be utilized as tools to target molecular processes and structural species related to αSyn neurotoxicity and have the potential as protein-based treatments against neurodegenerative disorders.

Assigning function to an unexplored Integrator module

Garland, W., Rouvière, J. O., Heick Jensen, T. — Thu, 11 Jul 2024 00:19:24 +0200

In this issue of Molecular Cell, Razew et al.¹ and Sabath et al.² assign function to an unexplored module of the Integrator (INT) complex, expanding the toolbox of this genome-wide attenuator of RNA polymerase II (RNAPII) transcription.

Mapping the Periostin splice isoforms in atopic dermatitis and an in vitro asthma model - A multi-platform analysis using mass spectrometry and RT-qPCR

Rusbjerg-Weberskov, C. E., Hollensen, A. K., Damgaard, C. K., et al. — Sun, 01 Sep 2024 00:19:24 +0200

Periostin is a matricellular protein known to be alternatively spliced to produce ten isoforms with a molecular weight of 78-91 kDa. Within the extracellular matrix, periostin attaches to cell surfaces to induce signaling via integrin-binding and actively participates in fibrillogenesis, orchestrating the arrangement of collagen in the extracellular environment. In atopic diseases such as atopic dermatitis (AD) and asthma, periostin is known to participate in driving the disease-causing type 2 inflammation. The periostin isoforms expressed in these diseases and the implication of the alternative splicing events are unknown. Here, we present two universal assays to map the expression of periostin isoforms at the mRNA (RT-qPCR) and protein (PRM-based mass spectrometry) levels. We use these assays to study the splicing profile of periostin in AD lesions as well as in in vitro models of AD and asthma. In these conditions, periostin displayed overexpression with isoforms 3 and 5 standing out as highly overexpressed. Notably, isoforms 9 and 10 exhibited a divergent pattern relative to the remaining isoforms. Isoforms 9 and 10 are often overlooked in periostin research and this paper presents the first evidence of their expression at the protein level. This underlines the necessity to include isoforms 9 and 10 in future research addressing periostin splice isoforms. The assays presented in this paper hold the potential to improve our insight into the splicing profile of periostin in tissues and diseases of interest. The application of these assays to AD lesions and in vitro models demonstrated their potential for identifying isoforms of particular significance, warranting a further in-depth investigation.

Development of a top-down MS assay for specific identification of human periostin isoforms

Mon, 01 Jan 2024 00:19:24 +0100

Periostin is a matricellular protein encoded by the POSTN gene that is alternatively spliced to produce ten different periostin isoforms with molecular weights ranging from 78 to 91 kDa. It is known to promote fibrillogenesis, organize the extracellular matrix, and bind integrin-receptors to induce cell signaling. As well as being a key component of the wound healing process, it is also known to participate in the pathogenesis of different diseases including atopic dermatitis, asthma, and cancer. In both health and disease, the functions of the different periostin isoforms are largely unknown. The ability to precisely determine the isoform profile of a given human sample is fundamental for characterizing their functional significance. Identification of periostin isoforms is most often carried out at the transcriptional level using RT-PCR based approaches, but due to high sequence homogeneity, identification on the protein level has always been challenging. Top-down proteomics, where whole proteins are measured by mass spectrometry, offers a fast and reliable method for isoform identification. Here we present a fully developed top-down mass spectrometry assay for the characterization of periostin splice isoforms at the protein level.

Characterization of a Trispecific PD-L1 Blocking Antibody That Exhibits EGFR-Conditional 4-1BB Agonist Activity

Rubio-Pérez, L., Frago, S., Compte, M., et al. — Sat, 01 Jun 2024 00:19:24 +0200

Immune checkpoint blockade has changed the treatment paradigm for advanced solid tumors, but the overall response rates are still limited. The combination of checkpoint blockade with anti-4-1BB antibodies to stimulate tumor-infiltrating T cells has shown anti-tumor activity in human trials. However, the further clinical development of these antibodies has been hampered by significant off-tumor toxicities. Here, we generated an anti-4-1BB/EGFR/PD-L1 trispecific antibody consisting of a triple-targeting tandem trimerbody (TT) fused to an engineered silent Fc region. This antibody (IgTT-4E1-S) was designed to combine the blockade of the PD-L1/PD-1 axis with conditional 4-1BB costimulation specifically confined to the tumor microenvironment (TME). The antibody demonstrated simultaneous binding to purified EGFR, PD-L1, and 4-1BB in solution, effective blockade of the PD-L1/PD1 interaction, and potent 4-1BB-mediated costimulation, but only in the presence of EGFR-expressing cells. These results demonstrate the feasibility of IgTT-4E1-S specifically blocking the PD-L1/PD-1 axis and inducing EGFR-conditional 4-1BB agonist activity.