Pulmonary diseases in patients with classical Hodgkin lymphoma relative to a matched background population: A Danish national cohort study.

Late toxicities can impact survivorship in patients with classical Hodgkin lymphoma (cHL) with pulmonary toxicity after bleomycin-containing chemotherapy being a concern. The incidence of pulmonary diseases was examined in this Danish population-based study. A total of 1474 adult patients with cHL treated with ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) or BEACOPP (bleomycin, vincristine, etoposide, doxorubicin, cyclophosphamide, procarbazine and prednisone) between 2000 and 2018 were included along with 7370 age- and sex-matched comparators from the background population. Median follow-up was 8.6 years for the patients. Patients with cHL had increased risk of incident pulmonary diseases (HR 2.91 [95% CI 2.30-3.68]), with a 10-year cumulative risk of 7.4% versus 2.9% for comparators. Excess risks were observed for interstitial lung diseases (HR 15.84 [95% CI 9.35-26.84]) and chronic obstructive pulmonary disease (HR 1.99 [95% CI 1.43-2.76]), with a 10-year cumulative risk of 4.1% and 3.5% respectively for patients. No excess risk was observed for asthma (HR 0.82 [95% CI 0.43-1.56]). Risk factors for interstitial lung diseases were age ≥60 years, the presence of B-symptoms and low albumin. These findings document a significant burden of pulmonary diseases among patients with cHL and emphasize the importance of diagnostic work-up of pulmonary symptoms.

Positional nystagmus is observed in the vast majority of healthy individuals.

INTRODUCTION: Benign paroxysmal positional vertigo (BPPV) is a vestibular disease characterized by brief  positional vertigo. When examined, characteristic patterns of positional nystagmus (PN) are found with specific head position changes. Previous studies have shown a high prevalence of PN among vestibular healthy subjects. Considering the current diagnostic criteria of BPPV and the potentially high prevalence of PN in healthy individuals, this raises the question of potential over diagnosing BPPV, if diagnostics are based exclusively upon objective findings. This study aims to determine the prevalence of PN within a healthy, adult population and furthermore include a characterization of the PN observed. METHODS: This is a prospective cross-sectional study. 78 subjects were included. The subjects underwent four standardized positional tests for BPPV in a mechanical rotational chair while using a VNG-goggle to monitor and record eye movements. RESULTS: Positional nystagmus was recorded in 70.5% (55/78) of the subjects. Of the 55 subjects, who presented with PN, 81.8% (45/55) had upbeating PN. The 95th percentile of the maximum a-SPV was found to be 10.4 degrees per second, with a median of 4. Five subjects (6.4%) in total presented with PN mimicking BPPV. CONCLUSION: This study found PN to be a common finding within a healthy, adult population based on the high prevalence of PN in the study population. Upbeating PN mimicking posterior canalolithiasis was found in numerous subjects. The authors recommend a cautious approach when diagnosing BPPV, especially in cases of purely vertical PN (without a torsional component) and if no vertiginous symptoms are present during Dix-Hallpike and Supine Roll Test examinations.

Metabolic Syndrome and Morbid Obesity are Not Risk Factors for Revision Surgery in Patients Undergoing Hip and Knee Arthroplasty.

BACKGROUND: The effect of metabolic syndrome (MetS) on the risk of revision after hip and knee arthroplasty is debated. The aim of our study was to investigate the risk of short-term (minimum 2.7 years) revision due to periprosthetic joint infection (PJI) after hip and knee arthroplasty. Secondly, we aimed to investigate the risk of revision due to any cause and mortality. METHODS: During May 2017 to November 2019, a cohort of 2,901 patients undergoing a total of 3,024 hip and knee arthroplasties was established. In the cohort, 62.1% met the criteria for MetS. Data from national registries and a local database were used to determine the presence of MetS and revision surgeries, with a follow-up of at least two years and eight months. Cox regression was applied to the present hazard ratio (HR), associated 95% confidence intervals, and P values. Survival analyses were presented in a Kaplan-Meier plot. RESULTS: The risk of PJI (HR 1.6 (0.5 to 4.9), P = .380), any revision (HR 0.8 (0.4 to 1.3), P = .295), and death (HR 1.3 (0.8 to 2.1), P = .282) was not increased in patients suffering from MetS compared with patients who did not have MetS. There was no PJI in patients not having MetS and receiving a knee arthroplasty. The risk of death was increased in the MetS group receiving a knee arthroplasty (HR 2.7 (1.3 to 5.9), P = .010), but not different from the MetS group receiving a hip arthroplasty. There was no elevated risk of PJI when analyzing morbid obesity (body mass index over 40), men, or diabetes as the exposures. CONCLUSIONS: Patients suffering from MetS do not have an increased risk of revision caused by PJI. In general, performing hip and knee arthroplasty in patients suffering from MetS is without increased risk of revision surgery.

Metabolic syndrome increases the length of stay and medical complications after hip and knee arthroplasty: results from a prospective cohort study of 2,901 patients.

BACKGROUND AND PURPOSE:  Metabolic syndrome (MetS) affects more than 60% of the patients having a hip or knee arthroplasty due to osteoarthritis. As it is debated whether metabolic syndrome increases the risk of complications, we aimed to investigate the length of stay (LOS) and risk of readmission at 30 and 90 days after surgery, including causes of readmission. METHODS:  We conducted a prospective cohort study of 2,901 patients undergoing hip and knee arthroplasty from May 2017 to November 2019. Physical examination, blood samples, and medical history from national registries determined the diagnosis of metabolic syndrome from the International Diabetes Federation definition. We used multivariate linear regression to investigate differences in LOS according to MetS, and binary regression to investigate the risk and causes of readmission within 30 and 90 days, including 95% confidence intervals (CI) and P values. RESULTS:  Patients with MetS showed a slightly longer LOS (0.20 days, CI 0.10-0.29) and had an increased risk of readmission within 90 days (adjusted relative risk [RR] 1.2, CI 1.0-1.4; P = 0.02), but not within 30 days (adjusted RR 1.1, CI 0.9-1.4; P = 0.3) after surgery. Cardiovascular disease was the dominant cause of readmission. CONCLUSION: Although patients with MetS do not experience a clinically relevant longer LOS after hip and knee arthroplasty, they have an increased risk of 90-day readmission mainly due to cardiovascular complications, which should be considered when planning surgical care in this group of patients.

Late recurrence of lymphoid malignancies after initial treatment for Hodgkin lymphoma - A study from the Danish Lymphoma Registry.

We analysed a large cohort of Hodgkin lymphoma (HL) patients in order to characterize: (1) the pattern of late recurrence of lymphoid malignancies (LR) after initial treatment for HL over a 35-year period; (2) the clinicopathological parameters influencing the risk of LR; and (3) the outcome of patients experiencing LR. We reviewed data of 3350 HL patients diagnosed in Denmark between 1982 and 2018 and registered in the Danish National Lymphoma Registry (LYFO). LR was defined as a recurrence of lymphoid malignancy at least five years after initial diagnosis. LR occurred in 58 patients, with a cumulative incidence at 10, 15 and 20 years of 2.7%, 4.0% and 5.4% respectively. LR was more frequently observed in patients with nodular lymphocyte-predominant HL (NLPHL) [hazard ratio (HR) 4.5; 95% confidence interval (CI): 2.4-8.4, p < 0.001]. In classical HL (cHL) patients, older age and lymphocytopenia were risk factors for LR with HRs of 1.04 per additional year (95% CI: 1.02-1.06) and 5.6 (95% CI: 2.7-11.5) respectively. Mixed cellularity histological subtype was a risk factor for LR, but only in females, with a HR of 5.4 (95% CI: 1.4-20.4, p = 0.014). In contrast to what was observed in NLPHL, LR in cHL was associated with an almost threefold increased risk of death compared with patients in continuous complete remission. Approximately one fifth (22.4%) of patients with LR experienced a second relapse.

Mental health among patients with non-Hodgkin lymphoma: A Danish nationwide study of psychotropic drug use in 8750 patients and 43 750 matched comparators.

Psychological distress following cancer diagnosis may lead to mental health complications including depression and anxiety. Non-Hodgkin lymphomas (NHLs) include indolent and aggressive subtypes for which treatment and prognosis differ widely. Incident use of psychotropic drugs (PDs-antidepressants, antipsychotics, and anxiolytics) and its correlation to lymphoma types can give insights into the psychological distress these patients endure. In this prospective matched cohort study, we used nationwide population-based registries to investigate the cumulative risk of PD use in NHL patients compared to a sex- and age-matched cohort from the Danish background population. In addition, contact patterns to psychiatric departments and incident intentional self-harm or completed suicide were explored. In total, 8750 NHL patients and 43 750 matched comparators were included (median age 68; male:female ratio 1.6). Median follow-up was 7.1 years. Two-year cumulative risk of PD use was higher in NHL patients (16.4%) as compared to the matched comparators (5.1%, p < .01); patients with aggressive NHL subtypes had the highest incidence. Prescription rates were higher in the first years after diagnosis but approached the rate of the matched population 5 years into survivorship in aggressive NHLs, whereas patients with indolent subtypes continued to be at higher risk. NHL patients had a slightly higher two-year risk of suicide/intentional self-harm (0.3%) as compared to the matched comparators (0.2%, p = .01). These results demonstrate that mental health complications among NHL patients are frequent. Routine assessment for symptoms of depression and anxiety should be consider as part of standard follow-up of NHL patients.

Stimulation history affects vasomotor responses in rat mesenteric arterioles.

Resistance vessels regulate blood flow by continuously adjusting activity of the wall smooth muscle cells. These cells integrate a variety of stimuli from blood, endothelium, autonomic nerves, and surrounding tissues. Each stimulus elicits an intracellular signaling cascade that eventually influences activation of the contractile machinery. The characteristic time scale of each cascade and the sharing of specific reactions between cascades provide for complex behavior when a vessel receives multiple stimuli. Here, we apply sequential stimulation with invariant concentrations of vasoconstrictor (norepinephrine/methoxamine) and vasodilator (SNAP/carbacol) to rat mesenteric vessels in the wire myograph to show that (1) time elapsed between addition of two vasoactive drugs and (2) the sequence of addition may significantly affect final force development. Furthermore, force oscillations (vasomotion) often appear upon norepinephrine administration. Using computational modeling in combination with nitric oxide (NO) inhibition/NO addition experiments, we show that (3) amplitude and number of oscillating vessels increase over time, (4) the ability of NO to induce vasomotion depends on whether it is applied before or after norepinephrine, and (5) emergence of vasomotion depends on the prior dynamical state of the system; in simulations, this phenomenon appears as "hysteresis." These findings underscore the time-dependent nature of vascular tone generation which must be considered when evaluating the vasomotor effects of multiple, simultaneous stimuli in vitro or in vivo.

Tungsten Iodide Clusters as Singlet Oxygen Photosensitizers: Exploring the Domain of Resonant Energy Transfer at 1 eV.

The photophysics of selected tungsten iodide clusters was examined with respect to their role as a photosensitizer for the production of singlet oxygen, O2(a1Δg). We examined all-iodo octahedral clusters, [W6I8(I6)]2-, and ligand-substituted octahedral clusters, [W6I8(L6)]2-, in which the ligand, L, occupies the outer apical positions surrounding the cluster core. We also examined a square-pyramidal cluster, [W5I8(I5)]-, in which the tungsten core was presumably more accessible to diffusional encounter with ground state oxygen, O2(X3Σg-). For the compounds examined, we find pronounced cluster-dependent changes in the yield of photosensitized O2(a1Δg) production. In particular, although the iodine-encased octahedral cluster, [W6I8(I6)]2-, is an efficient O2(a1Δg) sensitizer, the pyramidal cluster, [W5I8(I5)]-, does not make O2(a1Δg) at all. The latter provides fundamental insight into the important case where the sensitizer triplet state is nearly degenerate with the O2(X3Σg-)-O2(a1Δg) transition energy at 1 eV. Our data indicate that even with near resonance, energy transfer to form O2(a1Δg) will not occur within the 3sensitizer-O2(X3Σg-) encounter pair if other more efficient channels for energy dissipation are available.

The association between smoking and knee osteoarthritis in a cohort of Danish patients undergoing knee arthroscopy.

BACKGROUND: It has been suggested that smoking is associated with reduced risk of knee osteoarthritis (OA). However, supplementary studies are needed to further investigate any such potential association. Thus, our aim was to examine the relationship between smoking and early or more established knee OA in a cohort of relatively young patients with meniscal tears. METHODS: This cross-sectional study included 620 participants from the Knee Arthroscopy Cohort Southern Denmark (KACS) undergoing knee arthroscopy for a meniscal tear (mean age 49.2 (18.0-76.8) years). Recruitment of patients was performed between February 1, 2013, and January 31, 2015, at four different hospitals in Denmark. We defined early or more established knee OA as the combination of patient-reported frequent knee pain, degenerative meniscal tissue and presence of cartilage defects assessed by the operating surgeons. The relationship between smoking status and knee OA was examined by risk ratio (RR) with a 95% confidence interval (CI), estimated from logistic regression adjusted for age, sex, BMI, education, work status and level of physical activity. RESULTS: The prevalence of early or more established knee OA was 37.7% in current smokers and 45.0% in non-smokers. We found no statistically significant association between current smoking and knee OA (adjusted RR 1.09, 95% CI 0.91-1.30). CONCLUSIONS: This study found no relationship between current smoking and early or more established knee OA in a cohort of patients undergoing arthroscopic meniscal surgery. Thus, the inverse association between smoking and knee OA that has been suggested by previous studies was not confirmed.

Intracellular Delivery of a Planar DNA Origami Structure by the Transferrin-Receptor Internalization Pathway.

DNA origami provides rapid access to easily functionalized, nanometer-sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA-based delivery platforms. Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA-directed, site-selective labeling technique to retain ligand functionality. A combination of confocal fluorescence microscopy and quantitative (qPCR) techniques shows up to 22-fold increased cytoplasmic uptake compared to unmodified structures and with an efficiency that correlates to the number of transferrin molecules on the origami surface.

Solvent and Heavy-Atom Effects on the O2(X3Σg-) → O2(b1Σg+) Absorption Transition.

The effect of 16 liquid solvents on both the spectrum and molar absorption coefficient of the X3Σg- → b1Σg+ transition in molecular oxygen has been examined. The ability to monitor this weak transition using air or oxygen saturated samples at atmospheric pressure was facilitated by the rapid and efficient O2(b1Σg+) → O2(a1Δg) transition, which allowed the use of O2(a1Δg) phosphorescence as a sensitive probe of O2(b1Σg+) production. The results of these O2(a1Δg) phosphorescence experiments are consistent with the results of independent experiments in which the O2(a1Δg) thus produced was "trapped" via a chemical reaction. The data recorded were used to calculate rate constants for the O2(b1Σg+) → O2(X3Σg-) radiative transition, a parameter that is otherwise difficult to directly obtain from such a wide range of solvents using O2(b1Σg+) → O2(X3Σg-) phosphorescence. The data show that the response of the O2(b1Σg+) → O2(X3Σg-) radiative transition to solvent is not the same as that of the O2(b1Σg+) → O2(a1Δg) and O2(a1Δg) → O2(X3Σg-) radiative transitions, both of which have been extensively examined over the years. However, our data are consistent with a theoretical model proposed by Minaev for the effect of solvent on radiative transitions in oxygen and, as such, arguably provide one of the final chapters in describing a system that has challenged the scientific community for years.

Quantification of cellular uptake of DNA nanostructures by qPCR.

DNA nanostructures facilitating drug delivery are likely soon to be realized. In the past few decades programmed self-assembly of DNA building blocks have successfully been employed to construct sophisticated nanoscale objects. By conjugating functionalities to DNA, other molecules such as peptides, proteins and polymers can be precisely positioned on DNA nanostructures. This exceptional ability to produce modular nanoscale devices with tunable and controlled behavior has initiated an interest in employing DNA nanostructures for drug delivery. However, to obtain this the relationship between cellular interactions and structural and functional features of the DNA delivery device must be thoroughly investigated. Here, we present a rapid and robust method for the precise quantification of the component materials of DNA origami structures capable of entering cells in vitro. The quantification is performed by quantitative polymerase chain reaction, allowing a linear dynamic range of detection of five orders of magnitude. We demonstrate the use of this method for high-throughput screening, which could prove efficient to identify key features of DNA nanostructures enabling cell penetration. The method described here is suitable for quantification of in vitro uptake studies but should easily be extended to quantify DNA nanostructures in blood or tissue samples.

Impact of forestry on total and methyl-mercury in surface waters: distinguishing effects of logging and site preparation.

Forestry operations can increase the export of mercury (both total and methyl) to surface waters. However, little is known about the relative contribution of different forestry practices. We address this question using a paired-catchment study that distinguishes the effects of site preparation from the antecedent logging. Runoff water from three catchments, two harvested and one untreated control, was sampled biweekly during one year prior to logging, two years after logging, and three years after site preparation. The logging alone did not significantly increase the concentrations of either total or methyl-mercury in runoff, but export increased by 50-70% in one of the harvested catchments as a consequence of increased runoff volume. The combined effects of logging and site preparation increased total and methyl-mercury concentrations by 30-50% relative to preharvest conditions in both treated catchments. The more pronounced concentration effect after site preparation compared to logging could be related to site preparation being conducted during summer. This caused more soil disturbance than logging, which was done during winter with snow covering the ground. The results suggest that the cumulative impact of forest harvest on catchment mercury outputs depends on when and how forestry operations are implemented.

Muscle strength in sport climbing: a systematic review and meta-analysis.

INTRODUCTION: This study aimed to synthesize the literature comparing muscle strength and endurance characteristics between 1) sport climbers and non-climbing controls; 2) sport climbers at different performance levels; and 3) boulderers and lead climbers. EVIDENCE ACQUISITION: A systematic literature search (PubMed, Embase and SportDiscus) was performed. Inclusion criteria involved participants aged ≥18, muscular performance measurements and comparisons of either: climbers and non-climbers, boulder climbers and lead-climbers, or climbers of different levels. Meta-analyses comparing grip strength and muscle endurance of the forearm between sport climbers and non-climbers, and finger strength between boulders and lead climbers were conducted providing the standardized mean difference (SMD). EVIDENCE SYNTHESIS: A total of 960 climbers and 301 non-climbers were included in the study. The data showed: 1) Compared to non-climbers, climbers showed significantly higher grip strength: SMD 1.82 (95% CI 1.23; 2.41, P<0.001) and underarm endurance: SMD 0.70 (95% CI 0.17; 1.24, P=0.01); 2) compared to lead-climbers, boulder climbers showed significantly higher finger strength: SMD 1.08 (95% CI 0.54; 1.62, P<0.001); 3) higher-level climbers showed better finger strength, grip strength, forearm endurance and powerslap when compared to lower-level climbers. CONCLUSIONS: Climbers had superior grip strength and forearm endurance compared to non-climbers. High-level climbers exhibited better finger strength, grip strength, forearm endurance and powerslap, when compared to lower-level climbers. Finally, boulder climbers exhibited greater finger strength than lead-climbers. These findings expand our understandings of climbers' physical attributes across disciplines and levels.

Metabolic syndrome and patient-reported outcome two years after hip and knee arthroplasty.

Aims: The influence of metabolic syndrome (MetS) on the outcome after hip and knee arthroplasty is debated. We aimed to investigate the change in patient-reported outcome measure (PROM) scores after hip and knee arthroplasty, comparing patients with and without MetS. Methods: From 1 May 2017 to 30 November 2019, a prospective cohort of 2,586 patients undergoing elective unilateral hip and knee arthroplasty was established in Denmark. Data from national registries and a local database were used to determine the presence of MetS. Patients' scores on Oxford Hip Score (OHS) or Oxford Knee Score (OKS), EuroQol five-dimension five-level questionnaire (EQ-5D-5L), University of California, Los Angeles (UCLA) Activity Scale, and Forgotten Joint Score (FJS) at baseline, three, 12, and 24 months after surgery were collected. Primary outcome was the difference between groups from baseline to 12 months in OHS and OKS. Secondary outcomes were scores of OHS and OKS at three and 24 months and EQ-5D-5L, UCLA Activity Scale, and FJS at three, 12, and 24 months after surgery. Generalized linear mixed model was applied, adjusting for age, sex, Charlson Comorbidity Index, and smoking to present marginal mean and associated 95% CIs. Results: A total of 62.3% (1,611/2,586) of the cohort met the criteria for MetS. Both groups showed similar increase in mean OHS (MetS group 22.5 (95% CI 21.8 to 23.1), non-MetS group 22.1 (21.3 to 22.8); p = 0.477) and mean OKS (MetS group 18.0 (17.4 to 18.6), non-MetS group 17.8 (17.0 to 18.7); p = 0.722) at 12 months' follow-up. Between groups, similar improvements were seen for OHS and OKS at three and 24 months postoperatively and for the mean EQ-5D-5L, EuroQol-visual analogue scale (EQ-VAS), UCLA Activity Scale, and FJS at every timepoint. Conclusion: Patients meeting the criteria for MetS obtain the same improvement in PROM scores as individuals without MetS up to 24 months after hip and knee arthroplasty. This is important for the clinician to take into account when assessing and advising patients with MetS.

Deconvoluting the Effect of Cell-Penetrating Peptides for Enhanced and Controlled Insertion of Large-Scale DNA Nanopores.

DNA nanopores have emerged as powerful tools for molecular sensing, but the efficient insertion of large DNA nanopores into lipid membranes remains challenging. In this study, we investigate the potential of cell-penetrating peptides (CPPs), specifically SynB1 and GALA, to enhance the insertion efficiency of large DNA nanopores. We constructed SynB1- or GALA-functionalized DNA nanopores with an 11 nm inner diameter and visualized and quantified their membrane insertion using a TIRF microscopy-based single-liposome assay. The results demonstrated that incorporating an increasing number of SynB1 or GALA peptides into the DNA nanopore significantly enhanced the membrane perforation. Kinetic analysis revealed that the DNA nanopore scaffold played a role in prearranging the CPPs, which facilitated membrane interaction and pore formation. Notably, the use of pH-responsive GALA peptides allowed highly efficient and pH-controlled insertion of large DNA pores. Furthermore, single-channel recording elucidated that the insertion process of single GALA-modified nanopores into planar lipid bilayers was dynamic, likely forming transient large toroidal pores. Overall, our study highlights the potential of CPPs as insertion enhancers for DNA nanopores, which opens avenues for improved molecule sensing and the controlled release of cargo molecules.

Machine Learning-Based Survival Prediction Models for Progression-Free and Overall Survival in Advanced-Stage Hodgkin Lymphoma.

PURPOSE: Patients diagnosed with advanced-stage Hodgkin lymphoma (aHL) have historically been risk-stratified using the International Prognostic Score (IPS). This study investigated if a machine learning (ML) approach could outperform existing models when it comes to predicting overall survival (OS) and progression-free survival (PFS). PATIENTS AND METHODS: This study used patient data from the Danish National Lymphoma Register for model development (development cohort). The ML model was developed using stacking, which combines several predictive survival models (Cox proportional hazard, flexible parametric model, IPS, principal component, penalized regression) into a single model, and was compared with two versions of IPS (IPS-3 and IPS-7) and the newly developed aHL international prognostic index (A-HIPI). Internal model validation was performed using nested cross-validation, and external validation was performed using patient data from the Swedish Lymphoma Register and Cancer Registry of Norway (validation cohort). RESULTS: In total, 707 and 760 patients with aHL were included in the development and validation cohorts, respectively. Examining model performance for OS in the development cohort, the concordance index (C-index) for the ML model, IPS-7, IPS-3, and A-HIPI was found to be 0.789, 0.608, 0.650, and 0.768, respectively. The corresponding estimates in the validation cohort were 0.749, 0.700, 0.663, and 0.741. For PFS, the ML model achieved the highest C-index in both cohorts (0.665 in the development cohort and 0.691 in the validation cohort). The time-varying AUCs for both the ML model and the A-HIPI were consistently higher in both cohorts compared with the IPS models within the first 5 years after diagnosis. CONCLUSION: The new prognostic model for aHL on the basis of ML techniques demonstrated a substantial improvement compared with the IPS models, but yielded a limited improvement in predictive performance compared with the A-HIPI.

World Health Organization-defined classification of myeloproliferative neoplasms: morphological reproducibility and clinical correlations--the Danish experience.

We examined inter- and intraobserver reproducibility and concordance between histological diagnosis and independently collected clinical findings in a large series of patients with the major subtypes of myeloproliferative neoplasms (MPNs) and controls. Seven hematopathologists reviewed 272 bone marrow biopsies including 43 controls. Diagnoses were determined according to the 2008 criteria of the World Health Organization (WHO). The participants were blinded to all clinical data except patient age. After initial evaluation all hematopathologists participated in a 3-day meeting with a leading clinician chaired by an expert hematopathologists. In cases with lack of consensus on fiber grading (n = 57), a new evaluation was performed. In cases with discordance on morphological diagnosis (n = 129), an additional nonblinded evaluation taking clinical data into consideration was carried out. For remaining cases with a lack of concordance between morphological diagnosis and clinical diagnosis (n = 33), a similar nonblinded evaluation was performed. Consensus on final histological diagnosis and concordance with clinical diagnosis were determined. Blinded histological evaluation resulted in a 53% consensus rate. After re-evaluation of fiber content, consensus was reached in 60% of cases. Adding clinical data increased the histological consensus to 83%. For cases with a histological consensus, we found a concordance of 71% with the clinician's diagnoses. This is the first study to present a larger cohort of MPN patients mimicking the diagnostic challenges that hematopathologists face in their daily practice. The results support the postulates of the WHO that both morphological and clinical findings are essential for a valid diagnosis

Parallel Functionalization of DNA Origami.

DNA origami enables the creation of large supramolecular structures, with precisely defined features at the nanoscale. The concept thus naturally lends itself to the concept of molecular patterning, i.e., the positioning of molecular moieties and functional features. Creation of nanoscale patterns was already disseminated by Rothemund in 2006, in which DNA hairpins were used to produce nanoscale patterns on the flat origami canvases (Rothemund PWK, Nature 440(7082):297-302, 2006). For this type of application, it is often desired to produce multiple different patterns using the same origami canvas by reusing existing origami staple strands, rather than ordering new, custom oligonucleotides for each unique pattern. This chapter presents a method where the enzyme terminal deoxynucleotidyl transferase (TdT) is used in a parallelized reaction to add functional moieties to the end of a selected pool of unmodified staple strand oligonucleotides, which are then incorporated at precisely defined positions in the DNA origami canvas. Introducing arrays of functional features using this enzymatic functionalization of origami staple strands offers a very high degree of flexibility, versatility, and ease of use and can often be obtained faster than custom synthesis. For small synthesis scales, typically employed during initial functional screening of many different molecular patterns, the method also offers a significant advantage in terms of cost. During the past years, we have utilized this to incorporate a large variety of molecules including bulky proteins (Sørensen RS, Okholm AH, Schaffert D, Kodal ALB, Gothelf KV, Kjems J, ACS Nano 7:8098-8104, 2013) in designed patterns from modified nucleotide triphosphate (NTP) building blocks (Jahn K, Tørring T, Voigt NV, Sørensen RS, Kodal ALB, Andersen ES, Bioconjug Chem 22:819-823, 2011). The near-quantitative yields obtained by enzymatic functionalization allow synthesis of a large set of oligonucleotides in a one-pot reaction from commercial starting materials without the need for individual post-purification. Based on the chosen subset of staple strand, it is possible to create any designed functionality, array, or pattern. Here we describe the process going from an idea/design of a DNA origami-specific molecular pattern to nucleotide synthesis and subsequent parallel functionalization of the DNA origami, assembly, and the final characterization.

The Role of Nanoscale Distribution of Fibronectin in the Adhesion of Staphylococcus aureus Studied by Protein Patterning and DNA-PAINT.

Staphylococcus aureus is a widespread and highly virulent pathogen that can cause superficial and invasive infections. Interactions between S. aureus surface receptors and the extracellular matrix protein fibronectin mediate the bacterial invasion of host cells and is implicated in the colonization of medical implant surfaces. In this study, we investigate the role of distribution of both fibronectin and cellular receptors on the adhesion of S. aureus to interfaces as a model for primary adhesion at tissue interfaces or biomaterials. We present fibronectin in patches of systematically varied size (100-1000 nm) in a background of protein and bacteria rejecting chemistry based on PLL-g-PEG and studied S. aureus adhesion under flow. We developed a single molecule imaging assay for localizing fibronectin binding receptors on the surface of S. aureus via the super-resolution DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique. Our results indicate that S. aureus adhesion to fibronectin biointerfaces is regulated by the size of available ligand patterns, with an adhesion threshold of 300 nm and larger. DNA-PAINT was used to visualize fibronectin binding receptor organization in situ at ∼7 nm localization precision and with a surface density of 38-46 µm-2, revealing that the engagement of two or more receptors is required for strong S. aureus adhesion to fibronectin biointerfaces.