Identifying the molecular basis of Laminin N-terminal domain Ca2+ binding using a hybrid approach.

Ca2+ is a highly abundant ion involved in numerous biological processes, particularly in multicellular eukaryotic organisms where it exerts many of these functions through interactions with Ca2+ binding proteins. The laminin N-terminal (LN) domain is found in members of the laminin and netrin protein families where it plays a critical role in the function of these proteins. The LN domain of laminins and netrins is a Ca2+ binding domain and in many cases requires Ca2+ to perform its biological function. Here, we conduct a detailed examination of the molecular basis of the LN domain Ca2+ interaction combining structural, computational, bioinformatics, and biophysical techniques. By combining computational and bioinformatic techniques with x-ray crystallography we explore the molecular basis of the LN domain Ca2+ interaction and identify a conserved sequence present in Ca2+ binding LN domains. These findings enable a sequence-based prediction of LN domain Ca2+ binding ability. We use thermal shift assays and isothermal titration calorimetry to explore the biophysical properties of the LN domain Ca2+ interaction. We show that the netrin-1 LN domain exhibits a high affinity and specificity for Ca2+, which structurally stabilizes the LN domain. This study elucidates the molecular foundation of the LN domain Ca2+ binding interaction and provides a detailed functional characterization of this essential interaction, advancing our understanding of protein-Ca2+ dynamics within the context of the LN domain.

Thermophilisation of communities differs between land plant lineages, land use types and elevation.

Bryophytes provide key ecosystem services at the global scale such as carbon storage and primary production in resource limited habitats, but compared to vascular plants knowledge on how these organisms face recent climate warming is fragmentary. This is particularly critical because bryophytes differ fundamentally from vascular plants in their ecophysiological and biological characteristics, so that community alterations most likely have different dynamics. In a comparative approach, we analysed thermophilisation of bryophyte and vascular plant communities in 1146 permanent plots distributed along an elevational gradient of nearly 3.000 m in Switzerland (Central Europe) that were visited in 5-years intervals between 2001 and 2021. We estimated thermophilisation from changes in unweighted mean temperature indicator values of species, compared it to expected thermophilisation rates given the shift of isotherms and addressed differences between the two lineages, major land use types (managed grasslands, forests, unmanaged open areas), life strategy types (long- and short-lived species) and in elevation. Thermophilisation of bryophyte communities was on average 2.1 times higher than of vascular plant communities and at high elevations it approximated the expected rate given the shift of isotherms. Thermophilisation of both, bryophyte and vascular plant communities was not driven by a loss of cryophilic species but by an increase in thermophilic and mesophilic species, indicating an in-filling process. Furthermore, our data show that thermophilisation is higher in managed grasslands than in forests. We suggest that the higher responsiveness of bryophytes compared to vascular plants depends on their poikilohydry and dispersal capacity and that lower thermophilisation of forests communities is related to the buffering effect of microclimatic conditions in the interior of forests. Our study emphasises the heterogeneity of climate warming effects on plants because response dynamics differ between taxonomic groups as well as between land use types and along elevational gradients.

The dynamic nature of netrin-1 and the structural basis for glycosaminoglycan fragment-induced filament formation.

Netrin-1 is a bifunctional chemotropic guidance cue that plays key roles in diverse cellular processes including axon pathfinding, cell migration, adhesion, differentiation, and survival. Here, we present a molecular understanding of netrin-1 mediated interactions with glycosaminoglycan chains of diverse heparan sulfate proteoglycans (HSPGs) and short heparin oligosaccharides. Whereas interactions with HSPGs act as platform to co-localise netrin-1 close to the cell surface, heparin oligosaccharides have a significant impact on the highly dynamic behaviour of netrin-1. Remarkably, the monomer-dimer equilibrium of netrin-1 in solution is abolished in the presence of heparin oligosaccharides and replaced with highly hierarchical and distinct super assemblies leading to unique, yet unknown netrin-1 filament formation. In our integrated approach we provide a molecular mechanism for the filament assembly which opens fresh paths towards a molecular understanding of netrin-1 functions.

Investigation of dynamic solution interactions between NET-1 and UNC-5B by multi-wavelength analytical ultracentrifugation.

NET-1 is a key chemotropic ligand that signals commissural axon migration and change in direction. NET-1 and its receptor UNC-5B switch axon growth cones from attraction to repulsion. The biophysical properties of the NET-1 + UNC-5B complex have been poorly characterized. Using multi-wavelength-AUC by adding a fluorophore to UNC-5B, we were able to separate the UNC-5B sedimentation from NET-1. Using both multi-wavelength- and single-wavelength AUC, we investigated NET-1 and UNC-5B hydrodynamic parameters and complex formation. The sedimentation velocity experiments show that NET-1 exists in a monomer-dimer equilibrium. A close study of the association shows that NET-1 forms a pH-sensitive dimer that interacts in an anti-parallel orientation. UNC-5B can form equimolar NET-1 + UNC-5B heterocomplexes with both monomeric and dimeric NET-1.

Enhanced nucleosome assembly at CpG sites containing an extended 5-methylcytosine analogue.

Methylation of cytosine to 5-methylcytosine (mC) at CpG sites is a prevalent reversible epigenetic mark in vertebrates established by DNA methyltransferases (MTases); the attached methyl groups can alter local structure of DNA and chromatin as well as binding of dedicated proteins. Nucleosome assembly on methylated DNA has been studied extensively, however little is known how the chromatin structure is affected by larger chemical variations in the major groove of DNA. Here, we studied the nucleosome formation in vitro on DNA containing an extended 5mC analog, 5-(6-azidohex-2-ynyl)cytosine (ahyC) installed at biological relevant CpG sites. We found that multiple ahyC residues on 80-Widom and Hsp70 promoter DNA fragments proved compatible with nucleosome assembly. Moreover, unlike mC, ahyC increases the affinity of histones to the DNA, partially altering nucleosome positioning, stability, and the action of chromatin remodelers. Based on molecular dynamics calculations, we suggest that these new features are due to increased DNA flexibility at ahyC-modified sites. Our findings provide new insights into the biophysical behavior of modified DNA and open new ways for directed design of synthetic nucleosomes.

Survival and mortality analysis in two nephrology centers: A prospective observational study.

BACKGROUND: The prevalence of chronic kidney disease (CKD) requiring dialysis therapy is increasing globally. Survival and mortality data of these patients in Germany are fragmentary since the nationwide registry was terminated in 2006. OBJECTIVE: The objective of this study was to analyze the survival, causes of death, and co-morbidities of dialysis patients in a German population cohort as well as to assess the factors influencing mortality in these patients. MATERIALS AND METHODS: We included adult, prevalent chronic dialysis patients from the German population who underwent hemodialysis and peritoneal dialysis at our centers between 2014 and 2018. We compared the characteristics of living and deceased patients and assessed survival. Patients with and without diabetes mellitus were also examined, and their co-morbidities were analyzed. RESULTS: Of the 425 patients included in our study (m/f: 235/190), 182 died within the observation period. Mean survival of patients with coronary artery disease (CAD) (n = 217), peripheral artery disease (PAD) (n = 128), and cardiorenal syndrome (CRS) (n = 99) was significantly reduced compared to patients without the disease (CAD: 4.2 vs. 6.4 years; PAD: 4.3 vs. 6.5 years; CRS: 3.7 vs. 7.3 years, p < 0.001, respectively). Patients with diabetes mellitus (n = 110) showed no reduced survival compared to patients without the disease (n = 315) (4.8 vs. 4.9 years, p = 0.421). Diastolic blood pressure (DBP) and C-reactive protein (CRP) levels were associated with dialysis time in linear regression analysis (DBP: R = 0.029, p < 0,001; CRP: R = 0.085, p < 0.001). CONCLUSION: Our results provide novel data regarding German CKD patients requiring dialysis and factors influencing mortality, which could serve as a useful reference for further studies.

Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor.

The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. KEY POINTS: • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality.

Bayesian Markov models improve the prediction of binding motifs beyond first order.

Transcription factors (TFs) regulate gene expression by binding to specific DNA motifs. Accurate models for predicting binding affinities are crucial for quantitatively understanding of transcriptional regulation. Motifs are commonly described by position weight matrices, which assume that each position contributes independently to the binding energy. Models that can learn dependencies between positions, for instance, induced by DNA structure preferences, have yielded markedly improved predictions for most TFs on in vivo data. However, they are more prone to overfit the data and to learn patterns merely correlated with rather than directly involved in TF binding. We present an improved, faster version of our Bayesian Markov model software, BaMMmotif2. We tested it with state-of-the-art motif discovery tools on a large collection of ChIP-seq and HT-SELEX datasets. BaMMmotif2 models of fifth-order achieved a median false-discovery-rate-averaged recall 13.6% and 12.2% higher than the next best tool on 427 ChIP-seq datasets and 164 HT-SELEX datasets, respectively, while being 8 to 1000 times faster. BaMMmotif2 models showed no signs of overtraining in cross-cell line and cross-platform tests, with similar improvements on the next-best tool. These results demonstrate that dependencies beyond first order clearly improve binding models for most TFs.

Molecular characterization of the RNA-protein complex directing -2/-1 programmed ribosomal frameshifting during arterivirus replicase expression.

Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs -1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical -1 and -2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1ß (nsp1ß) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1ß and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate -1 and -2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1ß and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1ß:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1ß within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1ß and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1ß and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.

Auxin-mediated root branching is determined by the form of available nitrogen.

To improve water and nutrient acquisition from the soil, plants can modulate their root system architecture. Despite the importance of changes in root architecture to exploit local nutrient patches occurring in heterogenous soils or after placed fertilization, mechanisms integrating external nutrient signals into the root developmental programme remain poorly understood. Here, we show that local ammonium supply stimulates the accumulation of shoot-derived auxin in the root vasculature and promotes lateral root emergence to build a highly branched root system. Activities of pH and auxin reporters indicate that ammonium uptake mediated by ammonium transporters acidifies the root apoplast, which increases pH-dependent import of protonated auxin into cortical and epidermal cells overlaying lateral root primordia, and subsequently promotes their emergence from the parental root. Thereby, ammonium-induced and H+-ATPase-mediated acidification of the apoplast allows auxin to bypass the auxin importers AUX1 and LAX3. In nitrogen-deficient plants, auxin also accumulates in the root vasculature but a more alkaline apoplast leads to retention of auxin in these tissues and prevents lateral root formation. Our study highlights the impact of externally available nitrogen forms on pH-dependent radial auxin mobility and its regulatory function in organ development.

Structural and Hydrodynamic Characterization of Dimeric Human Oligoadenylate Synthetase 2.

Oligoadenylate synthetases (OASs) are a family of interferon-inducible enzymes that require double-stranded RNA (dsRNA) as a cofactor. Upon binding dsRNA, OAS undergoes a conformational change and is activated to polymerize ATP into 2'-5'-oligoadenylate chains. The OAS family consists of several isozymes, with unique domain organizations to potentially interact with dsRNA of variable length, providing diversity in viral RNA recognition. In addition, oligomerization of OAS isozymes, potentially OAS1 and OAS2, is hypothesized to be important for 2'-5'-oligoadenylate chain building. In this study, we present the solution conformation of dimeric human OAS2 using an integrated approach involving small-angle x-ray scattering, analytical ultracentrifugation, and dynamic light scattering techniques. We also demonstrate OAS2 dimerization using immunoprecipitation approaches in human cells. Whereas mutation of a key active-site aspartic acid residue prevents OAS2 activity, a C-terminal mutation previously hypothesized to disrupt OAS self-association had only a minor effect on OAS2 activity. Finally, we also present the solution structure of OAS1 monomer and dimer, comparing their hydrodynamic properties with OAS2. In summary, our work presents the first, to our knowledge, dimeric structural models of OAS2 that enhance our understanding of the oligomerization and catalytic function of OAS enzymes.

Chemical radiation dosimetry in magnetic fields: characterization of a Fricke-type chemical detector in 6 MV photon beams and magnetic fields up to 1.42 T.

In magnetic resonance guided radiotherapy (MRgRT) radiation dose measurements needs to be performed in the presence of a magnetic field. In this study, the influence of magnetic fields on the readings of a Fricke detector, a chemical dosimeter, have been investigated in 6 MV photon beams. This type of detector has been chosen, as the Federal Office of Metrology (METAS, Switzerland) has great experience with Fricke dosimetry and since it is not expected that this detector is greatly affected by the presence of a magnetic field. Magnetic fields with field strengths between 0 T and 1.42 T were applied during the detector irradiation. In a 5 × 10 cm2 irradiation field, the Fricke readings are affected less than 0.9% by the applied magnetic fields. Taking the altered dose distribution due to the magnetic field ([Formula: see text]) into account, the magnetic field correction factors ([Formula: see text]) for the Fricke detector at 0.35 T and 1.42 T are determined to be 0.9948 and 0.9980, respectively. These small corrections hardly exceed the measurement uncertainties. Hence, we could proof that the Fricke detector is not significantly influenced by the presence of a magnetic field. The Fricke detector was also tested for the feasibility of measuring output factors in the presence of magnetic fields. For irradiation field sizes larger than the detector (>2 × 2 cm2), comparable results were obtained as for other detectors. The output factors decrease when a magnetic field is applied. This effect is more pronounce for larger magnetic field strengths and smaller irradiation fields due to shifts of the depth dose curves and asymmetry of lateral dose profiles.

HH-suite3 for fast remote homology detection and deep protein annotation.

BACKGROUND: HH-suite is a widely used open source software suite for sensitive sequence similarity searches and protein fold recognition. It is based on pairwise alignment of profile Hidden Markov models (HMMs), which represent multiple sequence alignments of homologous proteins. RESULTS: We developed a single-instruction multiple-data (SIMD) vectorized implementation of the Viterbi algorithm for profile HMM alignment and introduced various other speed-ups. These accelerated the search methods HHsearch by a factor 4 and HHblits by a factor 2 over the previous version 2.0.16. HHblits3 is ∼10× faster than PSI-BLAST and ∼20× faster than HMMER3. Jobs to perform HHsearch and HHblits searches with many query profile HMMs can be parallelized over cores and over cluster servers using OpenMP and message passing interface (MPI). The free, open-source, GPLv3-licensed software is available at https://github.com/soedinglab/hh-suite . CONCLUSION: The added functionalities and increased speed of HHsearch and HHblits should facilitate their use in large-scale protein structure and function prediction, e.g. in metagenomics and genomics projects.

Solution Structure of C. elegans UNC-6: A Nematode Paralogue of the Axon Guidance Protein Netrin-1.

UNCoordinated-6 (UNC-6) was the first member of the netrin family to be discovered in Caenorhabditis elegans. With homology to human netrin-1, it is a key signaling molecule involved in directing axon migration in nematodes. Similar to netrin-1, UNC-6 interacts with multiple receptors (UNC-5 and UNC-40, specifically) to guide axon migration in development. As a result of the distinct evolutionary path of UNC-6 compared to vertebrate netrins, we decided to employ an integrated approach to study its solution behavior and compare it to the high-resolution structure we previously published on vertebrate netrins. Dynamic light scattering and analytical ultracentrifugation on UNC-6 (with and without its C-domain) solubilized in a low-ionic strength buffer suggested that UNC-6 forms high-order oligomers. An increase in the buffer ionic strength resulted in a more homogeneous preparation of UNC-6, that was used for subsequent solution x-ray scattering experiments. Our biophysical analysis of UNC-6 ΔC solubilized in a high-ionic strength buffer suggested that it maintains a similar head-to-stalk arrangement as netrins -1 and -4. This phenomenon is thought to play a role in the signaling behavior of UNC-6 and its ability to move throughout the extracellular matrix.

The Notch repressor complex in Drosophila: in vivo analysis of Hairless mutants using overexpression experiments.

During development of higher animals, the Notch signalling pathway governs cell type specification by mediating appropriate gene expression responses. In the absence of signalling, Notch target genes are silenced by repressor complexes. In the model organism Drosophila melanogaster, the repressor complex includes the transcription factor Suppressor of Hairless [Su(H)] and Hairless (H) plus general co-repressors. Recent crystal structure analysis of the Drosophila Notch repressor revealed details of the Su(H)-H complex. They were confirmed by mutational analyses of either protein; however, only Su(H) mutants have been further studied in vivo. Here, we analyse three H variants predicted to affect Su(H) binding. To this end, amino acid replacements Phenylalanine 237, Leucines 245 and 247, as well as Tryptophan 258 to Alanine were introduced into the H protein. A cell-based reporter assay indicates substantial loss of Su(H) binding to the respective mutant proteins HFA, HLLAA and HWA. For in vivo analysis, UAS-lines HFA, HLLAA and HWA were generated to allow spatially restricted overexpression. In these assays, all three mutants resembled the HLD control, shown before to lack Su(H) binding, indicating a strong reduction of H activity. For example, the H variants were impaired in wing margin formation, but unexpectedly induced ectopic wing venation. Concurrent overexpression with Su(H), however, suggests that all mutant H protein isoforms are still able to bind Su(H) in vivo. We conclude that a weakening of the cohesion in the H-Su(H) repressor complex is sufficient for disrupting its in vivo functionality.

Sonographic Venous Velocity Index Identifies Patients with Chronic Kidney Disease and Severe Diastolic Dysfunction.

OBJECTIVE: Diagnosing cardiorenal syndrome (CRS) in patients with chronic kidney disease (CKD) continues to remain challenging in outpatient practice. In this study, we investigate whether a newly developed venous velocity ultrasound index (VVI) can differentiate between patients with CRS and patients with CKD of other cause or normal renal function (NRF). METHODS: Patients with CRS (n = 30), CKD (n=30), and NRF (n=30) were included in the study. For each patient, duplex ultrasound scans of intrarenal segmental veins were retrospectively analyzed. The VVI was calculated from the renal venous doppler curve as the ratio of the maximal positive venous velocity to the maximal negative venous velocity. Patients with CRS were compared to age-matched controls with NRF and to GFR-matched controls with CKD. RESULTS: The GFRs of patients with CRS and those with CKD were comparable (26.4±5 and 25.6±7 ml/min/m2), as was the age in patients with CRS and NRF (6 ±12 years and 68±16 years, respectively). There was no significant difference in ejection fraction between patients with CRS and those with CKD (44.2±6.2% vs. 47.4 ±7.2), but there was a significant decrease compared to those with NRF (52.6 ±5.1, p<0.01). The VVI was significantly higher in the CRS group (0.81± 0.18) compared to the CKD group (0.18± 0.17, p<0.01) or NRF group (0.22± 0.20, p<0.01). The positive predictability of CRS was 96.4% in patients with VVI values of >0.6. CONCLUSION: The newly developed VVI was useful in successfully predicting severe diastolic dysfunction (CRS) in patients with severe kidney injury in outpatient care.

Proteinuria and the Clinical Course of Dobrava-Belgrade Hantavirus Infection.

PURPOSE: Human infection with Dobrava-Belgrade virus (DOBV) in Northern Germany causes a mild form of hantavirus disease predominantly characterized by acute kidney injury due to interstitial nephritis. We evaluated the largest number of DOBV-infected patients so far regarding clinical course, proteinuria, and prognostic markers. PATIENTS AND METHODS: Patients with DOBV-associated hantavirus disease admitted to the Renal Division of the University of Lübeck (Germany) between 1997 and 2012 were included in this study. Symptoms, clinical course, laboratory parameters, and urinary protein analysis were investigated at admission (baseline, t0), 3-5 days (t3-5), 10-17 days (t10-17), and after 1 year of follow-up (t365). RESULTS: Of the 34 patients (male/female ratio: 23/11; age: 41 ± 14 years) included in the study, 4 underwent hemodialysis (HD). Glomerular filtration rate was 17 ± 14 mL/min at t0 and increased to 27 ± 26 mL/min (t3-5), 57 ± 20 mL/min (t10-17), and 84 ± 16 mL/min (t365). Albuminuria and tubular proteinuria (α1- and ß2-microglobulin) decreased during follow-up; the urinary α1-microglobulin concentration in patients who required HD was significantly higher than that in patients not requiring HD (t0: 186 ± 51 vs. 45 ± 26 mg/g creatinine; t3-5: 87 ± 14 vs. 32 ± 16 mg/g creatinine; t10-17: 63 ± 18 vs. 28 ± 12 mg/g creatinine; p < 0.001). CONCLUSIONS: DOBV infection of inpatients in Northern Germany is associated with severe kidney injury that recovers within a few weeks and normalizes within 1 year. Tubular proteinuria is associated with the severity of kidney injury and the necessity of renal replacement therapy in these DOBV-infected patients.

Structure and hydrodynamics of a DNA G-quadruplex with a cytosine bulge.

The identification of four-stranded G-quadruplexes (G4s) has highlighted the fact that DNA has additional spatial organisations at its disposal other than double-stranded helices. Recently, it became clear that the formation of G4s is not limited to the traditional G3+NL1G3+NL2G3+NL3G3+ sequence motif. Instead, the G3 triplets can be interrupted by deoxythymidylate (DNA) or uridylate (RNA) where the base forms a bulge that loops out from the G-quadruplex core. Here, we report the first high-resolution X-ray structure of a unique unimolecular DNA G4 with a cytosine bulge. The G4 forms a dimer that is stacked via its 5'-tetrads. Analytical ultracentrifugation, static light scattering and small angle X-ray scattering confirmed that the G4 adapts a predominantly dimeric structure in solution. We provide a comprehensive comparison of previously published G4 structures containing bulges and report a special γ torsion angle range preferentially populated by the G4 core guanylates adjacent to bulges. Since the penalty for introducing bulges appears to be negligible, it should be possible to functionalize G4s by introducing artificial or modified nucleotides at such positions. The presence of the bulge alters the surface of the DNA, providing an opportunity to develop drugs that can specifically target individual G4s.