AsIII Selectively Induces a Disorder-to-Order Transition in the Metalloid Binding Region of the AfArsR Protein.

Arsenic is highly toxic and a significant threat to human health, but certain bacteria have developed defense mechanisms initiated by AsIII binding to AsIII-sensing proteins of the ArsR family. The transcriptional regulator AfArsR responds to AsIII and SbIII by coordinating the metalloids with three cysteines, located in a short sequence of the same monomer chain. Here, we characterize the binding of AsIII and HgII to a model peptide encompassing this fragment of the protein via solution equilibrium and spectroscopic/spectrometric techniques (pH potentiometry, UV, CD, NMR, PAC, EXAFS, and ESI-MS) combined with DFT calculations and MD simulations. Coordination of AsIII changes the peptide structure from a random-coil to a well-defined structure of the complex. A trigonal pyramidal AsS3 binding site is formed with almost exactly the same structure as observed in the crystal structure of the native protein, implying that the peptide possesses all of the features required to mimic the AsIII recognition and response selectivity of AfArsR. Contrary to this, binding of HgII to the peptide does not lead to a well-defined structure of the peptide, and the atoms near the metal binding site are displaced and reoriented in the HgII model. Our model study suggests that structural organization of the metal site by the inducer ion is a key element in the mechanism of the metalloid-selective recognition of this protein.

Identifying interactions in omics data for clinical biomarker discovery using symbolic regression.

MOTIVATION: The identification of predictive biomarker signatures from omics and multi-omics data for clinical applications is an active area of research. Recent developments in assay technologies and machine learning (ML) methods have led to significant improvements in predictive performance. However, most high-performing ML methods suffer from complex architectures and lack interpretability. RESULTS: We present the application of a novel symbolic-regression-based algorithm, the QLattice, on a selection of clinical omics datasets. This approach generates parsimonious high-performing models that can both predict disease outcomes and reveal putative disease mechanisms, demonstrating the importance of selecting maximally relevant and minimally redundant features in omics-based machine-learning applications. The simplicity and high-predictive power of these biomarker signatures make them attractive tools for high-stakes applications in areas such as primary care, clinical decision-making and patient stratification. AVAILABILITY AND IMPLEMENTATION: The QLattice is available as part of a python package (feyn), which is available at the Python Package Index (https://pypi.org/project/feyn/) and can be installed via pip. The documentation provides guides, tutorials and the API reference (https://docs.abzu.ai/). All code and data used to generate the models and plots discussed in this work can be found in https://github.com/abzu-ai/QLattice-clinical-omics. SUPPLEMENTARY INFORMATION: Supplementary material is available at Bioinformatics online.

Ca2+-Responsive Glyco-insulin.

Chemical modification of peptides and proteins, such as PEGylation and lipidation, creates conjugates with new properties. However, they are typically not dynamic or stimuli-responsive. Self-assembly controlled by a stimulus will allow adjusting properties directly. Here, we report that conjugates of oligogalacturonic acids (OGAs), isolated from plant-derived pectin, are Ca2+-responsive. We report the conjugation of OGA to human insulin (HI) to create new glyco-insulins. In addition, we coupled OGA to model peptides. We studied their self-assembly by dynamic light scattering, small-angle X-ray scattering, and circular dichroism, which showed that the self-assembly to form nanostructures depended on the length of the OGA sequence and Zn2+ and Ca2+ concentrations. Subcutaneous administration of OGA12-HI with Zn2+ showed a stable decrease in blood glucose over a longer period of time compared to HI, despite the lower receptor binding affinity.

Tying Up a Loose End: On the Role of the C-Terminal CCHHRAG Fragment of the Metalloregulator CueR.

The transcriptional regulator CueR is activated by the binding of CuI , AgI , or AuI to two cysteinates in a near-linear fashion. The C-terminal CCHHRAG sequence in Escherichia coli CueR present potential additional metal binding ligands and here we explore the effect of deleting this fragment on the binding of AgI to CueR. CD spectroscopic and ESI-MS data indicate that the high AgI -binding affinity of WT-CueR is significantly reduced in Δ7C-CueR.[111 Ag PAC spectroscopy demonstrates that the WT-CueR metal site structure (AgS2 ) is conserved, but less populated in the truncated variant. Thus, the function of the C-terminal fragment may be to stabilize the two-coordinate metal site for cognate monovalent metal ions. In a broader perspective this is an example of residues beyond the second coordination sphere affecting metal site physicochemical properties while leaving the structure unperturbed.

Polyamine-Functionalized 2'-Amino-LNA in Oligonucleotides: Facile Synthesis of New Monomers and High-Affinity Binding towards ssDNA and dsDNA.

Attachment of cationic moieties to oligonucleotides (ONs) promises not only to increase the binding affinity of antisense ONs by reducing charge repulsion between the two negatively charged strands of a duplex, but also to augment their in vivo stability against nucleases. In this study, polyamine functionality was introduced into ONs by means of 2'-amino-LNA scaffolds. The resulting ONs exhibited efficient binding towards ssDNA, ssRNA and dsDNA targets, and the 2'-amino-LNA analogue carrying a triaminated linker showed the most pronounced duplex- and triplex-stabilizing effect. Molecular modelling revealed that favourable conformational and electrostatic effects led to salt-bridge formation between positively charged polyamine moieties and the Watson-Hoogsteen groove of the dsDNA targets, resulting in the observed triplex stabilization. All the investigated monomers showed increased resistance against 3'-nucleolytic digestion relative to the non-functionalized controls.

Self-Assembly of DNA-Peptide Supermolecules: Coiled-Coil Peptide Structures Templated by d-DNA and l-DNA Triplexes Exhibit Chirality-Independent but Orientation-Dependent Stabilizing Cooperativity.

DNA nanostructures have been designed and used in many different applications. However, the use of nucleic acid scaffolds to promote the self-assembly of artificial protein mimics is only starting to emerge. Herein five coiled-coil peptide structures were templated by the hybridization of a d-DNA triplex or its mirror-image counterpart, an l-DNA triplex. The self-assembly of the desired trimeric structures in solution was confirmed by gel electrophoresis and small-angle X-ray scattering, and the stabilizing synergy between the two domains was found to be chirality-independent but orientation-dependent. This is the first example of using a nucleic acid scaffold of l-DNA to template the formation of artificial protein mimics. The results may advance the emerging POC-based nanotechnology field by adding two extra dimensions, that is, chirality and polarity, to provide innovative molecular tools for rational design and bottom-up construction of artificial protein mimics, programmable materials and responsive nanodevices.

Flexibility of the CueR Metal Site Probed by Instantaneous Change of Element and Oxidation State from AgI to CdII.

Selectivity for monovalent metal ions is an important facet of the function of the metalloregulatory protein CueR. 111 Ag perturbed angular correlation of γ-rays (PAC) spectroscopy probes the metal site structure and the relaxation accompanying the instantaneous change from AgI to CdII upon 111 Ag radioactive decay. That is, a change from AgI , which activates transcription, to CdII , which does not. In the frozen state (-196 °C) two nuclear quadrupole interactions (NQIs) are observed; one (NQI1 ) agrees well with two coordinating thiolates and an additional longer contact to the S77 backbone carbonyl, and the other (NQI2 ) reflects that CdII has attracted additional ligand(s). At 1 °C only NQI2 is observed, demonstrating that relaxation to this structure occurs within ≈10 ns of the decay of 111 Ag. Thus, transformation from AgI to CdII rapidly disrupts the functional linear bis(thiolato)AgI metal site structure. This inherent metal site flexibility may be central to CueR function, leading to remodelling into a non-functional structure upon binding of non-cognate metal ions. In a broader perspective, 111 Ag PAC spectroscopy may be applied to probe the flexibility of protein metal sites.

Comorbidity and early death in Danish stage I lung cancer patients - an individualised approach.

Background: Comorbidity is an important prognostic marker and a treatment indicator for lung cancer patients. Register-based studies often describe the burden of comorbidity by the Charlson comorbidity index (CCI) based on hospital discharge data. We assessed the association between somatic and psychiatric comorbidity and death within one year in early lung cancer and, furthermore, the burden of comorbidity according to treatment type.Material and methods: We conducted a population-based matched case-control study of stage I lung cancer identifying all treated patients who died (all-cause) within one year after diagnosis (early death group, cases). On the basis of data from the Danish Lung Cancer Registry these patients were then matched with two controls who survived more than one year (survivors). Through a review of the medical records, we validated inclusion criteria and collected data on somatic and psychiatric comorbidity. We assessed the association between comorbidity and early death with multivariate conditional logistic regression.Results: We included 221 cases and 410 controls. The mean CCI score in the early death group was 2.3 vs. 1.3 in the survivor group (p < .001). Still, 22% vs. 30% had a CCI score of zero (p = .04) with an average number of comorbidities among these patients of 1.63 vs. 1.06 respectively (p = .006). Among women, 23% in the early death group had depression vs. 13% in the survivor group, corresponding to an unadjusted odds ratio (OR) of 2.0 (CI 95% 1.0-3.7). However, in an adjusted analysis (incl. somatic comorbidities) the OR was 1.7 (CI 95% 0.8-3.5). Patients undergoing oncological therapy were older and tended to have more somatic comorbidities than the surgically treated patients.Conclusion: Comorbidity remains a significant prognostic marker even for stage I lung cancer patients with a CCI score of zero. The suggested association between early death and depression among women needs to be studied further.

Formation and Structure of Fluorescent Silver Nanoclusters at Interfacial Binding Sites Facilitating Oligomerization of DNA Hairpins.

Fluorescent, DNA-stabilized silver nanoclusters (DNA-AgNCs) are applied in a range of applications within nanoscience and nanotechnology. However, their diverse optical properties, mechanism of formation, and aspects of their composition remain unexplored, making the rational design of nanocluster probes challenging. Herein, a synthetic procedure is described for obtaining a high yield of emissive DNA-AgNCs with a C-loop hairpin DNA sequence, with subsequent purification by size-exclusion chromatography (SEC). Through a combination of optical spectroscopy, gel electrophoresis, inductively coupled plasma mass spectrometry (ICP-MS), and small-angle X-ray scattering (SAXS) in conjunction with the systematic study of various DNA sequences, the low-resolution structure and mechanism of the formation of AgNCs were investigated. Data indicate that fluorescent DNA-AgNCs self-assemble by a head-to-head binding of two DNA hairpins, bridged by a silver nanocluster, resulting in the modelling of a dimeric structure harboring an Ag12 cluster.

Glycoconjugate Oxime Formation Catalyzed at Neutral pH: Mechanistic Insights and Applications of 1,4-Diaminobenzene as a Superior Catalyst for Complex Carbohydrates.

The reaction of unprotected carbohydrates with aminooxy reagents to provide oximes is a key method for the construction of glycoconjugates. Aniline and derivatives serve as organocatalysts for the formation of oximes from simple aldehydes, and we have previously reported that aniline also catalyzes the formation of oximes from the more complex aldehydes, carbohydrates. Here, we present a comprehensive study of the effect of aniline analogues on the formation of carbohydrate oximes and related glycoconjugates depending on organocatalyst structure, pH, nucleophile, and carbohydrate, covering more than 150 different reaction conditions. The observed superiority of the 1,4-diaminobenzene (PDA) catalyst at neutral pH is rationalized by NMR analyses and DFT studies of reaction intermediates. Carbohydrate oxime formation at pH 7 is demonstrated by the formation of a bioactive glycoconjugate from a labile, decorated octasaccharide originating from exopolysaccharides of the soil bacterium Mesorhizobium loti. This study of glycoconjugate formation includes the first direct comparison of aniline-catalyzed reaction rates and equilibrium constants for different classes of nucleophiles, including primary oxyamines, secondary N-alkyl oxyamines, as well as aryl and arylsulfonyl hydrazides. We identified 1,4-diaminobenzene as a superior catalyst for the construction of oxime-linked glycoconjugates under mild conditions.

Synergy of Two Highly Specific Biomolecular Recognition Events: Aligning an AT-Hook Peptide in DNA Minor Grooves via Covalent Conjugation to 2'-Amino-LNA.

Two highly specific biomolecular recognition events, nucleic acid duplex hybridization and DNA-peptide recognition in the minor groove, were coalesced in a miniature ensemble for the first time by covalently attaching a natural AT-hook peptide motif to nucleic acid duplexes via a 2'-amino-LNA scaffold. A combination of molecular dynamics simulations and ultraviolet thermal denaturation studies revealed high sequence-specific affinity of the peptide-oligonucleotide conjugates (POCs) when binding to complementary DNA strands, leveraging the bioinformation encrypted in the minor groove of DNA duplexes. The significant cooperative DNA duplex stabilization may pave the way toward further development of POCs with enhanced affinity and selectivity toward target sequences carrying peptide-binding genetic islands.

Early death in Danish stage I lung cancer patients: a population-based case study.

OBJECTIVE: Clinical stage (c-stage) at diagnosis is the most significant prognostic marker for patients with cancer, where 1- and 5-year survival rates as main landmarks when assessing outcomes. This is a population-based case study of Danish c-stage I lung cancer patients who were considered candidates for curative therapy and then died within 1 year after diagnosis (cases). Cases were identified in the Danish Lung Cancer Register (DLCR), and medical records were used to retrieve treatment details and cause of death (CoD). Our aims were, if possible, to identify and describe clusters of patients, in terms of CoD and treatment modality at risk for an adverse short-term outcome. RESULTS: Patients who died early were more frequently male, older, had squamous-cell histology, were less frequently surgically treated and generally had a higher burden of comorbidity. In terms of CoD, 29% died of lung cancer with distant recurrence (DR) as the most common type of recurrence (55%). Death from co-morbidity occurred for 23%, where the largest proportion (36%) died from another cancer. Nineteen percentage died from treatment complications, with the majority being male (p < .001). The remainder died of unknown or other causes. CONCLUSIONS: Lung cancer with DR remains the most common CoD. Identifying and accordingly treating patients at risk for DR could potentially improve outcomes. Further studies of the predominantly male subgroup of patients who die of treatment complications are needed. Death from co-morbidity especially in patients with another cancer is a significant CoD and when assessing the quality of lung cancer care a competing event.

Assessing the pattern of recurrence in Danish stage I lung cancer patients in relation to the follow-up program: are we failing to identify patients with cerebral recurrence?

BACKGROUND: There is paucity of evidence regarding the optimal follow-up (FU) regimen for lung cancer. Consequently, FU is organized differently across countries. The Danish FU regimen has short FU intervals with a computed tomography (CT) scan of the chest and upper abdomen every three months in the early phase (first 2 years), then every six months in the late phase of FU (3rd, 5th year). Characterizing recurrences missed by the FU program in terms of site, tumor histology, department, and phase of FU, could improve the FU program. MATERIAL AND METHOD: A case-control study of curatively treated stage I lung cancer patients who attended the Danish FU-program and had recurrence identified through the follow-up program (controls, FU group) or outside FU program (cases, symptomatic group). RESULTS: Of 233 included patients with recurrence, the FU group constituted 85% (n = 197). Among the 15% (n = 36) in the symptomatic group, 53% had involvement of the central nervous system compared with 3% in the FU group. The unadjusted odds ratio (OR) for having an isolated brain recurrence (IBR) in the symptomatic group was 52.3 (95%CI: 15.1-181.4) as compared with the FU group. The OR for having a symptomatic recurrence in the early phase of FU was 2.5 (95%CI: 0.7-8.7) compared with the late phase. CONCLUSIONS: The FU program did not identify the majority of patients with IBR. Including cerebral imaging in the FU program may result in an earlier detection of brain metastases. These matters should be studied in a prospective setting.

Oligonucleotides Containing Aminated 2'-Amino-LNA Nucleotides: Synthesis and Strong Binding to Complementary DNA and RNA.

Mono- and diaminated 2'-amino-LNA monomers were synthesized and introduced into oligonucleotides. Each modification imparts significant stabilization of nucleic acid duplexes and triplexes, excellent sequence selectivity, and significant nuclease resistance. Molecular modeling suggested that structural stabilization occurs via intrastrand electrostatic attraction between the protonated amino groups of the aminated 2'-amino-LNA monomers and the host oligonucleotide backbone.

Folding Topology of a Short Coiled-Coil Peptide Structure Templated by an Oligonucleotide Triplex.

The rational design of a well-defined protein-like tertiary structure formed by small peptide building blocks is still a formidable challenge. By using peptide-oligonucleotide conjugates (POC) as building blocks, we present the self-assembly of miniature coiled-coil α-helical peptides guided by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex under physiological pH, and to be capable of templating the three peptide sequences to constitute a small coiled-coil motif displaying remarkable α-helicity. The formed trimeric complex was characterized by ultraviolet thermal denaturation, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12 nm) in solution was revealed to be independent of concentration. The topological folding of the peptide moiety differed strongly from those of the individual POC strands and the unconjugated peptide, exclusively adopting the designed triple helical structure.

Lung cancer guidelines in Sweden, Denmark, Norway and Finland: a comparison.

BACKGROUND: The Nordic countries are similar in terms of demographics and health care organization. Yet there are marked differences in lung cancer mortality, for which Denmark historically has had the poorest outcome. One of several possible reasons for these differences could have to do with how lung cancer is diagnosed and treated in the different Nordic countries. However, among the four most populous Nordic countries: Sweden, Denmark, Norway and Finland, there is a paucity of knowledge about differences and similarities in recommendations in the national guidelines for non-small cell lung cancer (NSCLC) and the methodology by which the guidelines are developed. METHODS: We identified and evaluated the development and content of the available clinical care guidelines for NSCLC in the four countries. Moreover, we compared the integrated cancer pathways in these countries. We have used case examples to illustrate areas with clear differences in clinical care recommendations. RESULTS: There are notable differences in the methodology by which the guidelines are developed, published and updated to comply with international recommendations. The Norwegian guidelines are developed and updated according to the most rigorous methodology and have so far been updated most frequently. We found that on the basis of recommendations patients with NSCLC are treated differently with regard to bevacizumab therapy and radiation dosing regimens. Cerebral imaging practices in patients with locally advanced NSCLC also differ. There is, moreover, a marked difference with regard to efforts to help patients to quit smoking. All except Finland have integrated cancer pathways for fast track diagnosis and treatment. Guidelines for follow-up of lung cancer patients also differ, with the Danish follow-up regimen as the most comprehensive. To obtain consensus on optimal clinical care, areas with differences in recommendations or where recommendations are based on a low level of evidence should be subjected to further studies.

Comparison of non-parametric methods for ungrouping coarsely aggregated data.

BACKGROUND: Histograms are a common tool to estimate densities non-parametrically. They are extensively encountered in health sciences to summarize data in a compact format. Examples are age-specific distributions of death or onset of diseases grouped in 5-years age classes with an open-ended age group at the highest ages. When histogram intervals are too coarse, information is lost and comparison between histograms with different boundaries is arduous. In these cases it is useful to estimate detailed distributions from grouped data. METHODS: From an extensive literature search we identify five methods for ungrouping count data. We compare the performance of two spline interpolation methods, two kernel density estimators and a penalized composite link model first via a simulation study and then with empirical data obtained from the NORDCAN Database. All methods analyzed can be used to estimate differently shaped distributions; can handle unequal interval length; and allow stretches of 0 counts. RESULTS: The methods show similar performance when the grouping scheme is relatively narrow, i.e. 5-years age classes. With coarser age intervals, i.e. in the presence of open-ended age groups, the penalized composite link model performs the best. CONCLUSION: We give an overview and test different methods to estimate detailed distributions from grouped count data. Health researchers can benefit from these versatile methods, which are ready for use in the statistical software R. We recommend using the penalized composite link model when data are grouped in wide age classes.

Noninvasive particle sizing using camera-based diffuse reflectance spectroscopy.

Diffuse reflectance measurements are useful for noninvasive inspection of optical properties such as reduced scattering and absorption coefficients. Spectroscopic analysis of these optical properties can be used for particle sizing. Systems based on optical fiber probes are commonly employed, but their low spatial resolution limits their validity ranges for the coefficients. To cover a wider range of coefficients, we use camera-based spectroscopic oblique incidence reflectometry. We develop a noninvasive technique for acquisition of apparent particle size distributions based on this approach. Our technique is validated using stable oil-in-water emulsions with a wide range of known particle size distributions. We also measure the apparent particle size distributions of complex dairy products. These results show that our tool, in contrast to those based on fiber probes, can deal with a range of optical properties wide enough to track apparent particle size distributions in a typical industrial process.

Construction of Insulin 18-mer Nanoassemblies Driven by Coordination to Iron(II) and Zinc(II) Ions at Distinct Sites.

Controlled self-assembly (SA) of proteins offers the possibility to tune their properties or to create new materials. Herein, we present the synthesis of a modified human insulin (HI) with two distinct metal-ion binding sites, one native, the other abiotic, enabling hierarchical SA through coordination with two different metal ions. Selective attachment of an abiotic 2,2'-bipyridine (bipy) ligand to HI, yielding HI-bipy, enabled Zn(II)-binding hexamers to SA into trimers of hexamers, [[HI-bipy]6]3, driven by octahedral coordination to a Fe(II)  ion. The structures were studied in solution by small-angle X-ray scattering and on surfaces with AFM. The abiotic metal ligand had a higher affinity for Fe(II) than Zn(II)  ions, enabling control of the hexamer formation with Zn(II) and the formation of trimers of hexamers with Fe(II)  ions. This precise control of protein SA to give oligomers of oligomers provides nanoscale structures with potential applications in nanomedicine.

Fluid shifts, vasodilatation and ambulatory blood pressure reduction during long duration spaceflight.

KEY POINTS: Weightlessness in space induces initially an increase in stroke volume and cardiac output, accompanied by unchanged or slightly reduced blood pressure.It is unclear whether these changes persist throughout months of flight.Here, we show that cardiac output and stroke volume increase by 35­41% between 3 and 6 months on the International Space Station, which is more than during shorter flights.Twenty-four hour ambulatory brachial blood pressure is reduced by 8­10 mmHg by a decrease in systemic vascular resistance of 39%, which is not a result of the suppression of sympathetic nervous activity, and the nightly dip is maintained in space.It remains a challenge to explore what causes the systemic vasodilatation leading to a reduction in blood pressure in space, and whether the unexpectedly high stroke volume and cardiac output can explain some vision acuity problems encountered by astronauts on the International Space Station. ABSTRACT: Acute weightlessness in space induces a fluid shift leading to central volume expansion. Simultaneously, blood pressure is either unchanged or decreased slightly. Whether these effects persist for months in space is unclear. Twenty-four hour ambulatory brachial arterial pressures were automatically recorded at 1­2 h intervals with portable equipment in eight male astronauts: once before launch, once between 85 and 192 days in space on the International Space Station and, finally, once at least 2 months after flight. During the same 24 h, cardiac output (rebreathing method) was measured two to five times (on the ground seated), and venous blood was sampled once (also seated on the ground) for determination of plasma catecholamine concentrations. The 24 h average systolic, diastolic and mean arterial pressures (mean ± se) in space were reduced by 8 ± 2 mmHg (P = 0.01; ANOVA), 9 ± 2 mmHg (P < 0.001) and 10 ± 3 mmHg (P = 0.006), respectively. The nightly blood pressure dip of 8 ± 3 mmHg (P = 0.015) was maintained. Cardiac stroke volume and output increased by 35 ± 10% and 41 ± 9% (P < 0.001); heart rate and catecholamine concentrations were unchanged; and systemic vascular resistance was reduced by 39 ± 4% (P < 0.001). The increase in cardiac stroke volume and output is more than previously observed during short duration flights and might be a precipitator for some of the vision problems encountered by the astronauts. The spaceflight vasodilatation mechanism needs to be explored further.