Uncrewed aerial vehicle with onboard winch system for rapid, cost-effective, and safe oceanographic profiling in hazardous and inaccessible areas.

Interactions between coastal waters and marine-terminating glaciers in the Polar Regions play a significant role in global sea level rise fueled by a rapidly warming Arctic. The risk of glacier calving, and the abundance of ice, can make it impossible for surface vessels to access the waters near glacier termini. Alternative methods using manned aircraft are expensive. As a result, oceanographic measurements are limited near glacier termini. We present an uncrewed aerial vehicle (UAV) with an on-board winch system that allows oceanographic profiling in remote, hazardous areas using a commercial conductivity, temperature, and depth (CTD) sensor payload. The UAV is optimized for easy handling and deployment and is capable of high-speed and efficient cruise flight. An autopilot system provides pilot assistance and autonomous flight capabilities. The total weight of the UAV including payload is 6.5 kg with an endurance of 24 min. Testing of the system was conducted in South Greenland during winter conditions in March 2023 with successful profiles collected near a glacier terminus (<5 m) and in small openings in ice mélange (2.2 m). The system proved capable, reliable, and efficient. Further development of the system will allow other sensors for an even more flexible measurement suite.

A mobile observatory powered by sun and wind for near real time measurements of atmospheric, glacial, terrestrial, limnic and coastal oceanic conditions in remote off-grid areas.

Climate change is rapidly altering the Arctic environment. Although long-term environmental observations have been made at a few locations in the Arctic, the incomplete coverage from ground stations is a main limitation to observations in these remote areas. Here we present a wind and sun powered multi-purpose mobile observatory (ARC-MO) that enables near real time measurements of air, ice, land, rivers, and marine parameters in remote off-grid areas. Two test units were constructed and placed in Northeast Greenland where they have collected data from cabled and wireless instruments deployed in the environment since late summer 2021. The two units can communicate locally via WiFi (units placed 25 km apart) and transmit near-real time data globally over satellite. Data are streamed live and accessible from (https://gios.org). The cost of one mobile observatory unit is c. 304.000€. These test units demonstrate the possibility for integrative and automated environmental data collection in remote coastal areas and could serve as models for a proposed global observatory system.

Lightweight drone-deployed autonomous ocean profiler for repeated measurements in hazardous areas - Example from glacier fronts in NE Greenland.

Accelerated melting of ice in Polar Regions due to global warming increases freshwater input to coastal waters from marine terminating glaciers. Lack of measurements near the glacier terminus limits our knowledge of the mixing processes between freshwater and the underlying ocean. We present a low-cost (< € 3200) and lightweight (2.6 kg) drone-deployed, retrievable conductivity, temperature and depth (CTD) instrument for remote controlled (1 km) autonomous profiling in highly hazardous and remote areas. The instrument was deployed with a drone taking off from land and marine vessels to perform measurements near tidewater glaciers termini of the Greenland ice sheet. The free-flowing profiler is reusable due to a compact ballast based single-shot buoyancy engine and post-profiling pickup by drone. It can reach a depth of up to 250 m, and is equipped with low-cost sensors for conductivity, temperature, and depth measurements. During decent the profiler reaches a velocity of about 0.48 m/s, resulting in about 3.5 data points pr. m depth, but is designed to easily vary the velocity by changing buoyancy setup before deployment. Successful tests were conducted at marine terminating glaciers in Northeast Greenland in August 2021.

The low-density lipoprotein receptor-related protein 1 (LRP1) interactome in the human cornea.

The human cornea is responsible for approximately 70% of the eye's optical power and, together with the lens, constitutes the only transparent tissue in the human body. Low-density lipoprotein receptor-related protein 1 (LRP1), a large, multitalented endocytic receptor, is expressed throughout the human cornea, yet its role in the cornea remains unknown. More than 30 years ago, LRP1 was purified by exploiting its affinity for the activated form of the protease inhibitor alpha-2-macroblulin (A2M), and the original purification protocol is generally referred to in studies involving full-length LRP1. Here, we provide a novel and simplified LRP1 purification protocol based on LRP1's affinity for receptor-related protein (RAP) that produces significantly higher yields of authentic LRP1. Purified LRP1 was used to map its unknown interactome in the human cornea. Corneal proteins extracted under physiologically relevant conditions were subjected to LRP1 affinity pull-down, and LRP1 ligand candidates were identified by LC-MS/MS. A total of 28 LRP1 ligand candidates were found, including 22 novel ligands. The LRP1 corneal interactome suggests a novel role for LRP1 as a regulator of the corneal immune response, structure, and ultimately corneal transparency.

Unheeded SARS-CoV-2 proteins? A deep look into negative-sense RNA.

SARS-CoV-2 is a novel positive-sense single-stranded RNA virus from the Coronaviridae family (genus Betacoronavirus), which has been established as causing the COVID-19 pandemic. The genome of SARS-CoV-2 is one of the largest among known RNA viruses, comprising of at least 26 known protein-coding loci. Studies thus far have outlined the coding capacity of the positive-sense strand of the SARS-CoV-2 genome, which can be used directly for protein translation. However, it has been recently shown that transcribed negative-sense viral RNA intermediates that arise during viral genome replication from positive-sense viruses can also code for proteins. No studies have yet explored the potential for negative-sense SARS-CoV-2 RNA intermediates to contain protein-coding loci. Thus, using sequence and structure-based bioinformatics methodologies, we have investigated the presence and validity of putative negative-sense ORFs (nsORFs) in the SARS-CoV-2 genome. Nine nsORFs were discovered to contain strong eukaryotic translation initiation signals and high codon adaptability scores, and several of the nsORFs were predicted to interact with RNA-binding proteins. Evolutionary conservation analyses indicated that some of the nsORFs are deeply conserved among related coronaviruses. Three-dimensional protein modeling revealed the presence of higher order folding among all putative SARS-CoV-2 nsORFs, and subsequent structural mimicry analyses suggest similarity of the nsORFs to DNA/RNA-binding proteins and proteins involved in immune signaling pathways. Altogether, these results suggest the potential existence of still undescribed SARS-CoV-2 proteins, which may play an important role in the viral lifecycle and COVID-19 pathogenesis.

Mutation-induced dimerization of transforming growth factor-ß-induced protein may drive protein aggregation in granular corneal dystrophy.

Protein aggregation in the outermost layers of the cornea, which can lead to cloudy vision and in severe cases blindness, is linked to mutations in the extracellular matrix protein transforming growth factor-ß-induced protein (TGFBIp). Among the most frequent pathogenic mutations are R124H and R555W, both associated with granular corneal dystrophy (GCD) characterized by the early-onset formation of amorphous aggregates. The molecular mechanisms of protein aggregation in GCD are largely unknown. In this study, we determined the crystal structures of R124H, R555W, and the lattice corneal dystrophy-associated A546T. Although there were no changes in the monomeric TGFBIp structure of any mutant that would explain their propensity to aggregate, R124H and R555W demonstrated a new dimer interface in the crystal packing, which is not present in wildtype TGFBIp or A546T. This interface, as seen in both the R124H and R555W structures, involves residue 124 of the first TGFBIp molecule and 555 in the second. The interface is not permitted by the Arg124 and Arg555 residues of wildtype TGFBIp and may play a central role in the aggregation exhibited by R124H and R555W in vivo. Using cross-linking mass spectrometry and in-line size exclusion chromatography-small-angle X-ray scattering, we characterized a dimer formed by wildtype and mutant TGFBIps in solution. Dimerization in solution also involves interactions between the N- and C-terminal domains of two TGFBIp molecules but was not identical to the crystal packing dimerization. TGFBIp-targeted interventions that disrupt the R124H/R555W crystal packing dimer interface might offer new therapeutic opportunities to treat patients with GCD.

Superoxide dismutase 3 is expressed in bone tissue and required for normal bone homeostasis and mineralization.

Superoxide dismutase 3 (SOD3) is an extracellular protein with the capacity to convert superoxide into hydrogen peroxide, an important secondary messenger in redox regulation. To investigate the utility of zebrafish in functional studies of SOD3 and its relevance for redox regulation, we have characterized the zebrafish orthologues; Sod3a and Sod3b. Our analyses show that both recombinant Sod3a and Sod3b express SOD activity, however, only Sod3b is able to bind heparin. Furthermore, RT-PCR analyses reveal that sod3a and sod3b are expressed in zebrafish embryos and are present primarily in separate organs in adult zebrafish, suggesting distinct functions in vivo. Surprisingly, both RT-PCR and whole mount in situ hybridization showed specific expression of sod3b in skeletal tissue. To further investigate this observation, we compared femoral bone obtained from wild-type and SOD3-/- mice to determine whether a functional difference was apparent in healthy adult mice. Here we report, that bone from SOD3-/- mice is less mineralized and characterized by significant reduction of cortical and trabecular thickness in addition to reduced mechanical strength. These analyses show that SOD3 plays a hitherto unappreciated role in bone development and homeostasis.

Protein Composition of the Subretinal Fluid Suggests Selective Diffusion of Vitreous Proteins in Retinal Detachment.

Purpose: To study the proteome of the subretinal fluid (SRF) from rhegmatogenous retinal detachment (RRD) in search for novel markers for improved diagnosis and prognosis of RRD. Methods: Human undiluted SRF obtained during vitrectomy for primary RRD using a 41-gauge needle (n = 24) was analyzed and compared to vitreous humor from 2-day postmortem eyes (n = 20). Sample preparation underwent nanoflow liquid chromatography-tandem mass spectrometry. Label-free quantification (LFQ) using MaxQuant was used to determine differentially expressed proteins between SRF and vitreous humor. The intensity-based absolute quantification (iBAQ) was used to rank proteins according to their molar fractions within groups. Identification of proteins beyond the quantitative level was performed using the Mascot search engine. Results: The protein concentration of the control vitreous humor was lower and more consistent (1.2 ± 0.4 mg) than that of the SRF (17.9 ± 22 mg). The iBAQ analysis showed high resemblance between SRF and vitreous humor, except for crystallins solely identified in vitreous humor. The LFQ analysis found 38 protein misregulations between SRF and vitreous humor of which the blood coagulation pathway was found to be enriched using the PANTHER Classification System. Combined, the iBAQ, LFQ, and Mascot analysis found an overlap only in chitinase-3-like protein 1 and galectin-3-binding protein unique to the SRF. Conclusions: The proteome of the SRF was highly represented by proteins involved in proteolysis. Such proteins can possibly serve as targets in modulating the effects of SRF in RD. Translational Relevance: To identify potential novel biomarkers for therapeutic targeting in RD.

Modulation of Small RNA Signatures in Schwann-Cell-Derived Extracellular Vesicles by the p75 Neurotrophin Receptor and Sortilin.

Schwann cells (SCs) are the main glial cells of the peripheral nervous system (PNS) and are known to be involved in various pathophysiological processes, such as diabetic neuropathy and nerve regeneration, through neurotrophin signaling. Such glial trophic support to axons, as well as neuronal survival/death signaling, has previously been linked to the p75 neurotrophin receptor (p75NTR) and its co-receptor Sortilin. Recently, SC-derived extracellular vesicles (EVs) were shown to be important for axon growth and nerve regeneration, but cargo of these glial cell-derived EVs has not yet been well-characterized. In this study, we aimed to characterize signatures of small RNAs in EVs derived from wild-type (WT) SCs and define differentially expressed small RNAs in EVs derived from SCs with genetic deletions of p75NTR (Ngfr-/-) or Sortilin (Sort1-/-). Using RNA sequencing, we identified a total of 366 miRNAs in EVs derived from WT SCs of which the most highly expressed are linked to the regulation of axonogenesis, axon guidance and axon extension, suggesting an involvement of SC EVs in axonal homeostasis. Signaling of SC EVs to non-neuronal cells was also suggested by the presence of several miRNAs important for regulation of the endothelial cell apoptotic process. Ablated p75NTR or sortilin expression in SCs translated into a set of differentially regulated tRNAs and miRNAs, with impact in autophagy and several cellular signaling pathways such as the phosphatidylinositol signaling system. With this work, we identified the global expression profile of small RNAs present in SC-derived EVs and provided evidence for a regulatory function of these vesicles on the homeostasis of other cell types of the PNS. Differentially identified miRNAs can pave the way to a better understanding of p75NTR and sortilin roles regarding PNS homeostasis and disease.

SARS-CoV-2 evades immune detection in alveolar macrophages.

Respiratory infections, like the current COVID-19 pandemic, target epithelial cells in the respiratory tract. Alveolar macrophages (AMs) are tissue-resident macrophages located within the lung. They play a key role in the early phases of an immune response to respiratory viruses. AMs are likely the first immune cells to encounter SARS-CoV-2 during an infection, and their reaction to the virus will have a profound impact on the outcome of the infection. Interferons (IFNs) are antiviral cytokines and among the first cytokines produced upon viral infection. In this study, AMs from non-infectious donors are challenged with SARS-CoV-2. We demonstrate that challenged AMs are incapable of sensing SARS-CoV-2 and of producing an IFN response in contrast to other respiratory viruses, like influenza A virus and Sendai virus, which trigger a robust IFN response. The absence of IFN production in AMs upon challenge with SARS-CoV-2 could explain the initial asymptotic phase observed during COVID-19 and argues against AMs being the sources of pro-inflammatory cytokines later during infection.

Protein Analysis of the TGFBIR124H Mouse Model Gives Insight into Phenotype Development of Granular Corneal Dystrophy.

PURPOSE: Mutations in the transforming growth factor ß-induced protein (TGFBIp) are associated with TGFBI-linked corneal dystrophies, which manifests as protein deposits in the cornea. A total of 70 different disease-causing mutations have been reported so far including the common R124H substitution, which is associated with granular corneal dystrophy type 2 (GCD2). The disease mechanism of GCD2 is not known and the current treatments only offer temporary relief due to the reoccurrence of deposits. EXPERIMENTAL DESIGN: The corneal protein profiles of the three genotypes (wild-type (WT), heterozygotes, and homozygotes) of a GCD2 mouse model are compared using label-free quantitative LC-MS/MS. RESULTS: The mice do not display corneal protein deposits and the global protein expression between the three genotypes is highly similar. However, the expression of mutated TGFBIp is 41% of that of the WT protein. CONCLUSIONS AND CLINICAL RELEVANCE: It is proposed that the lowered expression level of mutant TGFBIp protein relative to WT protein is the direct cause of the missing development of corneal deposits in the mouse. The overall protein profiles of the corneas are not impacted by the reduced amount of TGFBIp. Altogether, this supports a partial reduction in mutated TGFBIp as a potential treatment strategy for GCD2.

Biochemical mechanisms of aggregation in TGFBI-linked corneal dystrophies.

Transforming growth factor-ß-induced protein (TGFBIp), an extracellular matrix protein, is the second most abundant protein in the corneal stroma. In this review, we summarize the current knowledge concerning the expression, molecular structure, binding partners, and functions of human TGFBIp. To date, 74 mutations in the transforming growth factor-ß-induced gene (TGFBI) are associated with amyloid and amorphous protein deposition in TGFBI-linked corneal dystrophies. We discuss the current understanding of the biochemical mechanisms of TGFBI-linked corneal dystrophies and propose that mutations leading to granular corneal dystrophy (GCD) decrease the solubility of TGFBIp and affect the interactions between TGFBIp and components of the corneal stroma, whereas mutations associated with lattice corneal dystrophy (LCD) lead to a destabilization of the protein that disrupts proteolytic turnover, especially by the serine protease HtrA1. Future research should focus on TGFBIp function in the cornea, confirmation of the biochemical mechanisms in vivo, and the development of disease models. Future therapies for TGFBI-linked corneal dystrophies might include topical agents that regulate protein aggregation or gene therapy that targets the mutant allele by CRISPR/Cas9 technology.

The serine protease HtrA1 cleaves misfolded transforming growth factor ß-induced protein (TGFBIp) and induces amyloid formation.

The serine protease high-temperature requirement protein A1 (HtrA1) is associated with protein-misfolding disorders such as Alzheimer's disease and transforming growth factor ß-induced protein (TGFBIp)-linked corneal dystrophy. In this study, using several biochemical and biophysical approaches, including recombinant protein expression, LC-MS/MS and 2DE analyses, and thioflavin T (ThT) fluorescence assays for amyloid fibril detection, and FTIR assays, we investigated the role of HtrA1 both in normal TGFBIp turnover and in corneal amyloid formation. We show that HtrA1 can cleave WT TGFBIp but prefers amyloidogenic variants. Corneal TGFBIp is extensively processed in healthy people, resulting in C-terminal degradation products spanning the FAS1-4 domain of TGFBIp. We show here that HtrA1 cleaves the WT FAS1-4 domain only inefficiently, whereas the amyloidogenic FAS1-4 mutations transform this domain into a considerably better HTRA1 substrate. Moreover, HtrA1 cleavage of the mutant FAS1-4 domains generated peptides capable of forming in vitro amyloid aggregates. Significantly, these peptides have been previously identified in amyloid deposits in vivo, supporting the idea that HtrA1 is a causative agent for TGFBIp-associated amyloidosis in corneal dystrophy. In summary, our results indicate that TGFBIp is an HtrA1 substrate and that some mutations in the gene encoding TGFBIp cause aberrant HtrA1-mediated processing that results in amyloidogenesis in corneal dystrophies.

Sortilin gates neurotensin and BDNF signaling to control peripheral neuropathic pain.

Neuropathic pain is a major incurable clinical problem resulting from peripheral nerve trauma or disease. A central mechanism is the reduced expression of the potassium chloride cotransporter 2 (KCC2) in dorsal horn neurons induced by brain-derived neurotrophic factor (BDNF), causing neuronal disinhibition within spinal nociceptive pathways. Here, we demonstrate how neurotensin receptor 2 (NTSR2) signaling impairs BDNF-induced spinal KCC2 down-regulation, showing how these two pathways converge to control the abnormal sensory response following peripheral nerve injury. We establish how sortilin regulates this convergence by scavenging neurotensin from binding to NTSR2, thus modulating its inhibitory effect on BDNF-mediated mechanical allodynia. Using sortilin-deficient mice or receptor inhibition by antibodies or a small-molecule antagonist, we lastly demonstrate that we are able to fully block BDNF-induced pain and alleviate injury-induced neuropathic pain, validating sortilin as a clinically relevant target.

Proteomic profiling of TGFBI-null mouse corneas reveals only minor changes in matrix composition supportive of TGFBI knockdown as therapy against TGFBI-linked corneal dystrophies.

TGFBIp is a constituent of the extracellular matrix in many human tissues including the cornea, where it is one of the most abundant proteins expressed. TGFBIp interacts with Type I, II, IV, VI, and XII collagens as well as several members of the integrin family, suggesting it plays an important role in maintaining structural integrity and possibly corneal transparency as well. Significantly, more than 60 point mutations within the TGFBI gene have been reported to result in aberrant TGFBIp folding and aggregation in the cornea, resulting in severe visual impairment and blindness. Several studies have focused on targeting TGFBIp in the cornea as a therapeutic approach to treat TGFBI-linked corneal dystrophies, but the effect of this approach on corneal homeostasis and matrix integrity remained unknown. In the current study, we evaluated the histological and proteomic profiles of corneas from TGFBI-deficient mice as well as potential redundant functions of the paralogous protein POSTN. The absence of TGFBIp in mouse corneas did not grossly affect the collagen scaffold, and POSTN is unable to compensate for loss of TGFBIp. Proteomic comparison of wild-type and TGFBI-/- mice revealed 11 proteins were differentially regulated, including Type VI and XII collagens. However, as these alterations did not manifest at the macroscopic and behavioral levels, these data support partial or complete TGFBI knockdown as a potential therapy against TGFBI-linked corneal dystrophies. Lastly, in situ hybridization verified TGFBI mRNA in the epithelial cells but not in other cell types, supportive of a therapy directed specifically at this lineage.

Mutation-Induced Deamidation of Corneal Dystrophy-Related Transforming Growth Factor ß-Induced Protein.

Mutations in the transforming growth factor ß-induced protein (TGFBIp) cause phenotypically diverse corneal dystrophies, where protein aggregation in the cornea leads to severe visual impairment. Previous studies have shown a relationship between mutant-specific corneal dystrophy phenotypes and the thermodynamic stability of TGFBIp. Using liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance (NMR), we investigated correlations between the structural integrity of disease-related mutants of the fourth FAS1 domain (FAS1-4) and deamidation of TGFBIp residue Asn622. We observed a high rate of Asn622 deamidation in the A546D and A546D/P551Q FAS1-4 mutants that were both largely unstructured as determined by NMR. Conversely, the more structurally organized A546T and V624M FAS1-4 mutants had reduced deamidation rates, suggesting that a folded and stable FAS1-4 domain precludes Asn622 deamidation. Wild-type, R555Q, and R555W FAS1-4 mutants displayed very slow deamidation, which agrees with their similar and ordered NMR structures, where Asn622 is in a locked conformation. We confirmed the FAS1-4 mutational effect on deamidation rates in full-length TGFBIp mutants and found a similar ranking compared to that of the FAS1-4 domain alone. Consequently, the deamidation rate of Asn622 can be used to predict the structural effect of the many destabilizing and/or stabilizing mutations reported for TGFBIp. In addition, the deamidation of Asn622 may influence the pathophysiology of TGFBIp-induced corneal dystrophies.

Structural and Functional Implications of Human Transforming Growth Factor ß-Induced Protein, TGFBIp, in Corneal Dystrophies.

A major cause of visual impairment, corneal dystrophies result from accumulation of protein deposits in the cornea. One of the proteins involved is transforming growth factor ß-induced protein (TGFBIp), an extracellular matrix component that interacts with integrins but also produces corneal deposits when mutated. Human TGFBIp is a multi-domain 683-residue protein, which contains one CROPT domain and four FAS1 domains. Its structure spans ∼120 Å and reveals that vicinal domains FAS1-1/FAS1-2 and FAS1-3/FAS1-4 tightly interact in an equivalent manner. The FAS1 domains are sandwiches of two orthogonal four-stranded ß sheets decorated with two three-helix insertions. The N-terminal FAS1 dimer forms a compact moiety with the structurally novel CROPT domain, which is a five-stranded all-ß cysteine-knot solely found in TGFBIp and periostin. The overall TGFBIp architecture discloses regions for integrin binding and that most dystrophic mutations cluster at both molecule ends, within domains FAS1-1 and FAS1-4.

An Aberrant Phosphorylation of Amyloid Precursor Protein Tyrosine Regulates Its Trafficking and the Binding to the Clathrin Endocytic Complex in Neural Stem Cells of Alzheimer's Disease Patients.

Alzheimer's disease (AD) is the most common cause of dementia and is likely caused by defective amyloid precursor protein (APP) trafficking and processing in neurons leading to amyloid plaques containing the amyloid-ß (Aß) APP peptide byproducts. Understanding how APP is targeted to selected destinations inside neurons and identifying the mechanisms responsible for the generation of Aß are thus the keys for the advancement of new therapies. We previously developed a mouse model with a mutation at tyrosine (Tyr) 682 in the C-terminus of APP. This residue is needed for APP to bind to the coating protein Clathrin and to the Clathrin adaptor protein AP2 as well as for the correct APP trafficking and sorting in neurons. By extending these findings to humans, we found that APP binding to Clathrin is decreased in neural stem cells from AD sufferers. Increased APP Tyr phosphorylation alters APP trafficking in AD neurons and it is associated to Fyn Tyr kinase activation. We show that compounds affecting Tyr kinase activity and counteracting defects in AD neurons can control APP location and compartmentalization. APP Tyr phosphorylation is thus a potential therapeutic target for AD.

Serum Amyloid P Component (SAP) Interactome in Human Plasma Containing Physiological Calcium Levels.

The pentraxin serum amyloid P component (SAP) is secreted by the liver and found in plasma at a concentration of approximately 30 mg/L. SAP is a 25 kDa homopentamer known to bind both protein and nonprotein ligands, all in a calcium-dependent manner. The function of SAP is unclear but likely involves the humoral innate immune system spanning the complement system, inflammation, and coagulation. Also, SAP is known to bind to the generic structure of amyloid deposits and possibly to protect them against proteolysis. In this study, we have characterized the SAP interactome in human plasma containing the physiological Ca2+ concentration using SAP affinity pull-down and co-immunoprecipitation experiments followed by mass spectrometry analyses. The analyses resulted in the identification of 33 proteins, of which 24 were direct or indirect interaction partners not previously reported. The SAP interactome can be divided into categories that include apolipoproteins, the complement system, coagulation, and proteolytic regulation.