In Situ Surface-Enhanced Raman Spectroscopy on Organic Mixed Ionic-Electronic Conductors: Tracking Dynamic Doping in Light-Emitting Electrochemical Cells.

In the domain of organic mixed ionic-electronic conductors (OMIECs), simultaneous transport and coupling of ionic and electronic charges are crucial for the function of electrochemical devices in organic electronics. Understanding conduction mechanisms and chemical reactions in operational devices is pivotal for performance enhancement and is necessary for the informed and systematic development of more promising materials. Surface-enhanced Raman spectroscopy (SERS) is a potent tool for monitoring electrochemical evolution and dynamic doping in operational devices, offering enhanced sensitivity to subtle spectral changes. We demonstrate the utility of SERS for in situ tracking of doping in OMIECs in an organic light-emitting electrochemical cell (LEC) containing a conjugated polymer (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]; MEH-PPV), a molecular anion (lithium triflate), and an electrolyte network (poly(ethylene oxide); PEO). SERS enhancement is achieved via an interleaved layer of gold particles formed by spontaneous breakup of a deposited thin gold film. The results successfully highlight the ability of SERS to unveil time-resolved MEH-PPV doping and polaron formation, elucidating the effects of triflate ion transfer in the operating device and validating the electrochemical doping model in LECs.

Contractile properties and magnetic resonance imaging-assessed fat replacement of muscles in myotonia congenita.

BACKGROUND AND PURPOSE: Myotonia congenita (MC) is a muscle channelopathy in which pathogenic variants in a key sarcolemmal chloride channel Gene (CLCN1) cause myotonia. This study used muscle magnetic resonance imaging (MRI) to quantify contractile properties and fat replacement of muscles in a Danish cohort of MC patients. METHODS: Individuals with the Thomsen (dominant) and Becker (recessive) variants of MC were studied. Isometric muscle strength, whole-body MRI, and clinical data were collected. The degree of muscle fat replacement of thigh, calf, and forearm muscles was quantitively calculated on Dixon MRI as fat fractions (FFs). Contractility was evaluated as the muscle strength per contractile muscle cross-sectional area (PT/CCSA). Muscle contractility was compared with clinical data. RESULTS: Intramuscular FF was increased and contractility reduced in calf and in forearm muscles compared with controls (FF = 7.0-14.3% vs. 5.3-9.6%, PT/CCSA = 1.1-4.9 Nm/cm2 vs. 1.9-5.8 Nm/cm2 [p < 0.05]). Becker individuals also showed increased intramuscular FF and reduced contractility of thigh muscles (FF = 11.9% vs. 9.2%, PT/CCSA = 1.9 Nm/cm2 vs. 3.2 Nm/cm2 [p < 0.05]). Individual muscle analysis showed that increased FF was limited to seven of 18 examined muscles (p < 0.05). There was a weak correlation between reduced contractility and severity of symptoms. CONCLUSIONS: Individuals with MC have increased fat replacement and reduced contractile properties of muscles. Nonetheless, changes were small and likely did not impact clinically on their myotonic symptoms.

Impact of Synergy Partner Cel7B on Cel7A Binding Rates: Insights from Single-Molecule Data.

Enzymatic degradation of cellulosic biomass is a well-established route for the sustainable production of biofuels, chemicals, and materials. A strategy employed by nature and industry to achieve an efficient degradation of cellulose is that cellobiohydrolases (or exocellulases), such as Cel7A, work synergistically with endoglucanases, such as Cel7B, to achieve the complete degradation of cellulose. However, a complete mechanistic understanding of this exo-endo synergy is still lacking. Here, we used single-molecule fluorescence microscopy to quantify the binding kinetics of Cel7A on cellulose when it is acting alone on the cellulose fibrils and in the presence of its synergy partner, the endoglucanase Cel7B. To this end, we used a fluorescently tagged Cel7A and studied its binding in the presence of the unlabeled Cel7B. This provided the single-molecule data necessary for the estimation of the rate constants of association kON and dissociation kOFF of Cel7A for the substrate. We show that the presence of Cel7B does not impact the dissociation rate constant, kOFF. But, the association rate of Cel7A decreases by a factor of 2 when Cel7B is present at a molar proportion of 10:1. This ratio has previously been shown to lead to synergy. This decrease in association rate is observed in a wide range of total enzyme concentrations, from sub nM to µM concentrations. This decrease in kON is consistent with the formation of cellulase clusters recently observed by others using atomic force microscopy.

Hypokalemic periodic paralysis: a 3-year follow-up study.

BACKGROUND AND OBJECTIVES: Primary hypokalemic periodic paralysis (HypoPP) is an inherited channelopathy most commonly caused by mutations in CACNA1S. HypoPP can present with different phenotypes: periodic paralysis (PP), permanent muscle weakness (PW), and mixed weakness (MW) with both periodic and permanent weakness. Little is known about the natural history of HypoPP. METHODS: In this 3-year follow-up study, we used the MRC scale for manual muscle strength testing and whole-body muscle MRI (Mercuri score) to assess disease progression in individuals with HypoPP-causing mutations in CACNA1S. RESULTS: We included 25 men (mean age 43 years, range 18-76 years) and 12 women (mean age 42 years, range 18-76 years). Two participants were asymptomatic, 21 had PP, 12 MW, and two PW. The median number of months between baseline and follow-up was 42 (range 26-52). Muscle strength declined in 11 patients during follow-up. Four of the patients with a decline in muscle strength had no attacks of paralysis during follow-up, and two of these patients had never had attacks of paralysis. Fat replacement of muscles increased in 27 patients during follow-up. Eight of the patients with increased fat replacement had no attacks of paralysis during follow-up, and two of these patients had never had attacks of paralysis. DISCUSSION: The study demonstrates that HypoPP can be a progressive myopathy in both patients with and without attacks of paralysis.

Reconstitution and post-thaw storage of cryopreserved human mesenchymal stromal cells: Pitfalls and optimizations for clinically compatible formulants.

Introduction: The regenerative and immunomodulatory properties of multipotent mesenchymal stromal cells (MSCs) make them an intriguing asset for therapeutic applications. An off-the-shelf approach, using pre-expanded cryopreserved allogenic MSCs, bypasses many practical difficulties of cellular therapy. Reconstitution of a MSC product away from cytotoxic cryoprotectants towards a preferred administration solution might be favorable for several indications. Variations in MSC handling accompanied by a non-standardized use of reconstitution solutions complicate a general clinical standardization of MSC cellular therapies. In this study, we aimed to identify a simple and clinically compatible approach for thawing, reconstitution, and post-thaw storage of cryopreserved MSCs. Methods: Human adipose tissue-derived MSCs were expanded in human platelet lysate (hPL) supplemented culture medium and cryopreserved using a dimethyl sulfoxide (DMSO)-based cryoprotectant. Isotonic solutions (saline, Ringer's acetate and phosphate buffered saline (PBS)) with or without 2% human serum albumin (HSA) were used as thawing, reconstitution, and storage solutions. MSCs were reconstituted to 5 × 106 MSCs/mL for evaluating MSC stability. Total MSC numbers and viability were determined using 7-aminoactinomycin D (7-AAD) and flow cytometry. Results: For thawing cryopreserved MSCs the presence of protein was proven to be essential. Up to 50% of MSCs were lost when protein-free thawing solutions were used. Reconstitution and post-thaw storage of MSCs in culture medium and widely used PBS demonstrated poor MSC stability (>40% cell loss) and viability (<80%) after 1 h of storage at room temperature. Reconstitution in simple isotonic saline appeared to be a good alternative for post-thaw storage, ensuring >90% viability with no observed cell loss for at least 4 h. Reconstitution of MSCs to low concentrations was identified as critical. Diluting MSCs to <105/mL in protein-free vehicles resulted in instant cell loss (>40% cell loss) and lower viability (<80%). Addition of clinical grade HSA could prevent cell loss during thawing and dilution. Conclusion: This study identified a clinically compatible method for MSC thawing and reconstitution that ensures high MSC yield, viability, and stability. The strength of the method lies within the simplicity of implementation which offers an accessible way to streamline MSC therapies across different laboratories and clinical trials, improving standardization in this field.

Quantifying DNA-mediated liposome fusion kinetics with a fluidic trap.

Self-assembly of synthetic lipid vesicles via lipid membrane fusion is a versatile tool for creating biomimetic nano- and micron-sized particles. These so-called liposomes are used in the development of biosensing platforms, design of drug delivery schemes, and for investigating protein-mediated fusion of biological membranes. This work demonstrates DNA-induced liposome fusion in a nanofluidic trap where the reaction occurs in a 15 femtoliter volume at homogeneous mixing. In contrast to current methods for fusion in bulk, we show that the fusion reaction follows second-order kinetics with a fusion rate of (170 ± 30)/(M-1s-1) times the square number of DNA molecules per liposome. The nanofluidic trapping gives a full characterization of the size and charge of the liposomes before and after fusion. The chip-based approach limits the amount of sample (down to 440 vesicles) and can be parallelized for systematic studies in synthetic biology, diagnostics, and drug delivery.

Carbon Dioxide-Mediated Desalination.

Conventional desalination membrane technologies, although offer portable drinking water, are still energy-intensive processes. This paper proposes a potentially new approach for performing water desalination and purification by utilizing the reversible interaction of carbon dioxide (CO2) with nucleophilic amines─reminiscent of the Solvay process. Based on our model studies with small molecules, CO2-responsive amphiphilic insoluble diamines were prepared, characterized, and applied in the formation of soda and ammonium chloride upon exposure to ambient CO2 (1 atm), thus removing chloride ions from model and real seawater. This ion-exchange process and separation of chloride from the aqueous phase are spontaneous in the presence of CO2 without the need for external energy sources. We demonstrate a flow system to envisage energy-efficient CO2-mediated desalination and simultaneous carbon capture and sequestration.

Muscle fat replacement and contractility in patients with skeletal muscle sodium channel disorders.

Skeletal muscle sodium channel disorders give rise to episodic symptoms such as myotonia and/or periodic paralysis. Chronic symptoms with permanent weakness are not considered characteristic of the phenotypes. Muscle fat replacement represents irreversible damage that inevitably will impact on muscle strength. This study investigates muscle fat replacement and contractility in patients with pathogenic SCN4A variants compared to healthy controls. T1-weighted and 2-point Dixon MRI of the legs were conducted to assess fat replacement. Stationary dynamometry was used to assess muscle strength. Contractility was determined by maximal muscle contraction divided by cross-sectional muscle area. The average cross-sectional intramuscular fat fraction was greater in patients compared with controls by 2.5% in the calves (95% CI 0.74-4.29%, p = 0.007) and by 2.0% in the thighs (95% CI 0.75-3.2%, p = 0.003). Muscle contractility was less in patients vs. controls by 14-27% (p < 0.05). Despite greater fat fraction and less contractility, absolute strength was not significantly less. This study quantitatively documents greater fat fraction and additionally describes difference in muscle contractility in a large cohort of patients with skeletal muscle sodium channel disorders. The clinical impact of these abnormal findings is likely limited as muscle hypertrophy in the patients served to preserve absolute muscle strength. Subgroup analysis indicated significant difference in phenotype by genotype, however these findings lack statistical significance and serve as inspiration for future researchers to probe into the geno- phenotype relationship in these disorders.Trial registration: The study was registered at http://clinicaltrials.gov (identifier: NCT04808388).

ß-Galactosidase deficiency in the GLB1 spectrum of lysosomal storage disease can present with severe muscle weakness and atrophy.

Deficiency of the enzyme ß-galactosidase due to variants in the GLB1-gene is associated with metabolic disorders: Morquio B and GM1-gangliosidosis. Here, we report a case compound heterozygous for variants in the GLB1-gene and a severe muscular phenotype. Full body T1-w MRI was conducted for muscular involvement. Biopsy was stained with hematoxylin and eosin for histopathological evaluation. EDTA blood-sample was subjected to whole exome sequencing. Metabolic analysis included residual enzyme activity and evaluation urinary substrate secretion. Additionally, electroneurography, echocardiography, forced volume capacity and biochemistry were evaluated. Examination showed severe proximal weakness (MRC: hip flexion 2, hip extension 2, and shoulder rotation 2), Gower's sign, no extrapyramidal symptoms and normal creatine kinase levels. MRI showed severe muscle wasting of the thigh and shoulder girdle. Muscle biopsy showed mild myopathic changes. ß-galactosidase activity was reduced to 28%-34%. Urinary glycosaminoglycan was elevated by 5.9-8.6 mg/mmol (ref.:0-5.1 mg/mmol). Electrophoresis indicated excess keratan sulfate. Exome sequencing revealed two missense variants in the GLB1 gene. Clinical features, genetic testing and laboratory findings indicate a case of ß-galactosidase-deficiency with a muscular phenotype.

Resonant Laser Printing of Optical Metasurfaces.

One of the challenges for metasurface research is upscaling. The conventional methods for fabrication of metasurfaces, such as electron-beam or focused ion beam lithography, are not scalable. The use of ultraviolet steppers or nanoimprinting still requires large-size masks or stamps, which are costly and challenging in further handling. This work demonstrates a cost-effective and lithography-free method for printing optical metasurfaces. It is based on resonant absorption of laser light in an optical cavity formed by a multilayer structure of ultrathin metal and dielectric coatings. A nearly perfect light absorption is obtained via interferometric control of absorption and operating around a critical coupling condition. Controlled by the laser power, the surface undergoes a structural transition from random, semiperiodic, and periodic to amorphous patterns with nanoscale precision. The reliability, upscaling, and subwavelength resolution of this approach are demonstrated by realizing metasurfaces for structural colors, optical holograms, and diffractive optical elements.

Objective Outcome Measures Continue to Improve from 6 to 12 Months after Conservatively Treated Distal Radius Fractures in the Elderly-A Prospective Evaluation of 50 Patients.

Distal radius fractures (DRF) in the elderly population above 65 years represent 18% of all fractures and are thereby the second most frequent fracture in the elderly. Fracture dislocation and comminution are often used to determine whether non-operative or operative treatment is indicated. The purpose of this prospective case series of minimally displaced DRF treated with a dorsal cast was to assess the complication rate and patient-reported outcome measures. This single-centre, single-blinded, prospective case series followed 50 conservatively treated DRF patients for one year. Primary outcomes were complications and Quick Disability of Arm Shoulder and Hand (qDASH) score. Secondary outcomes were range of motion (ROM), grip strength and pain, and Patient-Rated Wrist/Hand Evaluation (PRWHE). Results showed only minor complications with a return to prior ROM, qDASH, and pain after 12 months and improvement in outcomes after 6-12 months. In conclusion, the majority of DRF patients who were treated non-operatively with five-week dorsal casting recover fully after minimally displaced DRF. This standard approach is thus considered safe, and the present results provide a reference for other studies.

Label-Free Sensing of Biorecognition on Liposomes.

Nanometer-sized liposomes decorated with macromolecules are increasingly used as drug delivery vehicles due to their long lifetimes and target cell specificity, but surface characterization methods often change their properties, which leads to incorrect results. Ligand binding is commonly applied for characterizing these surface modifications. Here, we use a nanofluidic-based label-free sensor for real-time sensing of ligands binding to liposomes. The liposomes are trapped in a nanochannel with a salt concentration gradient, and as the trapping position depends on the liposomes' zeta potential, it changes when charged ligands bind to the liposomes. Our sensing method does not require immobilization of the liposomes or labeling of the ligands with fluorophores, which may both affect the sensing. The zeta potential sensing is demonstrated by measuring hybridization of DNA targets with complementary DNA probes on liposome surfaces. DNA hybridization is monitored for both ensembles and individual liposomes, the latter allows for analysis of ensemble heterogeneity, and we demonstrate sensitivity to changes in surface charge down to 1.5%. DNA hybridization is used to demonstrate label-free sensing, but the method also has potential applications within exosome characterization, where biorecognition of, e.g., surface DNA, proteins, and antibodies is a promising candidate for early stage cancer diagnostics.

Pulmonary vein isolation using Ablation Index improves outcome in patients with atrial fibrillation.

AIMS: To evaluate the clinical outcome of pulmonary vein isolation (PVI) in radiofrequency ablation of atrial fibrillation (AF) comparing a strategy using Ablation Index (AI) and lesion contiguity with Contact Force (CF) only. METHODS: In a single-center retrospective design, we included 479 patients with AF (n=341 (71.2%) paroxysmal AF (PAF) and n=138 (28.8%) persistent AF (PeAF)) treated with first time radiofrequency ablation. In 2015, 210 patients underwent PVI based on a drag-and-ablate technique using CF only. In 2017, 269 patients underwent point-by-point PVI using AI and a maximum inter-lesion distance of 6 mm ensuring contiguity. Follow-up was performed after 12 months. Outcome was freedom from documented AF/atrial tachycardia (AT) after single procedure without use of anti-arrhythmic drugs at follow-up. RESULTS: There was no significant difference in baseline characteristics between the groups. The median procedure time and mean ablation time were significantly longer in the AI-group compared to the CF-group (131.5[113;156] min vs. 120.0[97;140] min, P < 0.01) and (44.1±10.0 min vs. 37.1±13.3 min, P < 0.01), respectively. Freedom from documented AF/AT was significantly higher in the AI-group compared to the conventional CF -group (71.0% vs. 62.4%, P = 0.046). The improvement in clinical outcome in the AI group is mainly driven by the outcome in patients with PeAF (64.9% vs. 50.0%, P = 0.078) and not PAF. CONCLUSIONS: An ablation strategy combining AI and lesion contiguity improves the clinical outcome after first time PVI in patients with AF compared to a strategy using CF only.

Reconstruction of corrupted datasets from ammonium-ISE sensors at WRRFs through merging with daily composite samples.

Long-term, continuous datasets of high quality are important for instrumentation, control, and automation efforts of wastewater resources recovery facility (WRRFs). This study presents a methodology to increase the reliability of measurements from ammonium ion-selective electrodes (ISEs). This is done by correcting corrupted ISE data with a data source that often is available at WRRFs (volume-proportional composite samples). A yearlong measurement campaign showed that the existing standard protocols for sensor maintenance might still create corrupted dataset, with poor sensor recalibrations responsible for abrupt and unrealistic jumps in the measurements. The proposed automatic correction methodology removes both recalibration jumps and signal drift by using information from composite samples that already are taken for reporting to legal authorities. Results showed that the developed methodology provided a continuous, high-quality time series without the major data quality issues of the original signal. In fact, the signal was improved for 87% of days when a reference sample was available. The effect of correcting the data before use in a data-driven software sensor was also investigated. The corrected dataset led to noticeably smaller day-to-day variations in estimated NH4+ loads, and to large improvements on both median estimates and prediction bounds. The long time series allowed for an investigation of how much training data that is required to fit a software sensor, which provides estimates that are representative for the entire study period. The results showed that 8 weeks of data allowed for a good median estimate, while 16 weeks are required for obtaining good 80% prediction bounds. Overall, the proposed method can increase the applicability of relatively cheaper ISE sensors for ICA application within WRRFs.

Size and surface charge characterization of nanoparticles with a salt gradient.

Exosomes are nanometer-sized lipid vesicles present in liquid biopsies and used as biomarkers for several diseases including cancer, Alzheimer's, and central nervous system diseases. Purification and subsequent size and surface characterization are essential to exosome-based diagnostics. Sample purification is, however, time consuming and potentially damaging, and no current method gives the size and zeta potential from a single measurement. Here, we concentrate exosomes from a dilute solution and measure their size and zeta potential in a one-step measurement with a salt gradient in a capillary channel. The salt gradient causes oppositely directed particle and fluid transport that trap particles. Within minutes, the particle concentration increases more than two orders of magnitude. A fit to the spatial distribution of a single or an ensemble of exosomes returns both their size and surface charge. Our method is applicable for other types of nanoparticles. The capillary is fabricated in a low-cost polymer device.

Evaluating the performance of a simple phenomenological model for online forecasting of ammonium concentrations at WWTP inlets.

A simple model for online forecasting of ammonium (NH4 +) concentrations in sewer systems is proposed. The forecast model utilizes a simple representation of daily NH4 + profiles and the dilution approach combined with information from online NH4 + and flow sensors. The method utilizes an ensemble approach based on past observations to create model prediction bounds. The forecast model was tested against observations collected at the inlet of two wastewater treatment plants (WWTPs) over an 11-month period. NH4 + data were collected with ion-selective sensors. The model performance evaluation focused on applications in relation to online control strategies. The results of the monitoring campaigns highlighted a high variability in daily NH4 + profiles, stressing the importance of an uncertainty-based modelling approach. The maintenance of the NH4 + sensors resulted in important variations of the sensor signal, affecting the evaluation of the model structure and its performance. The forecast model succeeded in providing outputs that potentially can be used for integrated control of wastewater systems. This study provides insights on full scale application of online water quality forecasting models in sewer systems. It also highlights several research gaps which - if further investigated - can lead to better forecasts and more effective real-time operations of sewer and WWTP systems.

A protocol for a single-center, single-blinded randomized-controlled trial investigating volar plating versus conservative treatment of unstable distal radius fractures in patients older than 65 years.

BACKGROUND: Distal radius fractures (DRF) are very common in elderly patients, who present at the Emergency Department. Surgical treatment with open reduction and internal fixation using volar locking plates is widely prevalent despite the lack of evidence proving its superiority to conservative treatment with closed reduction and plaster immobilization. The purpose of this study is to investigate whether conservative treatment is superior to volar plating in terms of number of complications and results in a comparable or superior functional outcome in patients ≥65 years. METHODS: In this single-center, single-blinded randomized-controlled trial, patients ≥65 years with distal radius fractures will be invited to participate. A total of 50 patients per treatment arm is required to provide 80% statistical power at a 5% alpha level assuming a difference of 20% in complication rate between operatively and conservatively treated patients. Primary outcome measures will be complication rate, Quick DASH score (Quick Disabilities of the Arm, Shoulder and Hand), PRWE (Patient rated Wrist evaluation), and range of motion of the wrist. Secondary outcome measures will be grip strength, pinch gauge, pain, use of pain medication EQ5D score (European Quality of life - 5 dimensions), standardized radiographs. One year of follow-up is planned with data collection at the day of injury, after 2 weeks, after 5 weeks, after 6 months, and after 12 months. An intention-totreat and per-protocol analysis will be performed. DISCUSSION: This prospective trial helps to clarify the best treatment strategy for displaced DRF patients ≥65 years. TRIAL REGISTRATION: This trial is approved by the Danish Scientific Ethical Committee (ID: 1-10-72-420-17) and registered at Clinicaltrials.gov (Trial registration number NCT03716661 ).

Predictive validity of the CriSTAL tool for short-term mortality in older people presenting at Emergency Departments: a prospective study.

ABSTRACT: To determine the validity of the Australian clinical prediction tool Criteria for Screening and Triaging to Appropriate aLternative care (CRISTAL) based on objective clinical criteria to accurately identify risk of death within 3 months of admission among older patients. METHODS: Prospective study of ≥ 65 year-olds presenting at emergency departments in five Australian (Aus) and four Danish (DK) hospitals. Logistic regression analysis was used to model factors for death prediction; Sensitivity, specificity, area under the ROC curve and calibration with bootstrapping techniques were used to describe predictive accuracy. RESULTS: 2493 patients, with median age 78-80 years (DK-Aus). The deceased had significantly higher mean CriSTAL with Australian mean of 8.1 (95% CI 7.7-8.6 vs. 5.8 95% CI 5.6-5.9) and Danish mean 7.1 (95% CI 6.6-7.5 vs. 5.5 95% CI 5.4-5.6). The model with Fried Frailty score was optimal for the Australian cohort but prediction with the Clinical Frailty Scale (CFS) was also good (AUROC 0.825 and 0.81, respectively). Values for the Danish cohort were AUROC 0.764 with Fried and 0.794 using CFS. The most significant independent predictors of short-term death in both cohorts were advanced malignancy, frailty, male gender and advanced age. CriSTAL's accuracy was only modest for in-hospital death prediction in either setting. CONCLUSIONS: The modified CriSTAL tool (with CFS instead of Fried's frailty instrument) has good discriminant power to improve prognostic certainty of short-term mortality for ED physicians in both health systems. This shows promise in enhancing clinician's confidence in initiating earlier end-of-life discussions.

Single-molecule DNA-mapping and whole-genome sequencing of individual cells.

To elucidate cellular diversity and clonal evolution in tissues and tumors, one must resolve genomic heterogeneity in single cells. To this end, we have developed low-cost, mass-producible micro-/nanofluidic chips for DNA extraction from individual cells. These chips have modules that collect genomic DNA for sequencing or map genomic structure directly, on-chip, with denaturation-renaturation (D-R) optical mapping [Marie R, et al. (2013) Proc Natl Acad Sci USA 110:4893-4898]. Processing of single cells from the LS174T colorectal cancer cell line showed that D-R mapping of single molecules can reveal structural variation (SV) in the genome of single cells. In one experiment, we processed 17 fragments covering 19.8 Mb of the cell's genome. One megabase-large fragment aligned well to chromosome 19 with half its length, while the other half showed variable alignment. Paired-end single-cell sequencing supported this finding, revealing a region of complexity and a 50-kb deletion. Sequencing struggled, however, to detect a 20-kb gap that D-R mapping showed clearly in a megabase fragment that otherwise mapped well to the reference at the pericentromeric region of chromosome 4. Pericentromeric regions are complex and show substantial sequence homology between different chromosomes, making mapping of sequence reads ambiguous. Thus, D-R mapping directly, from a single molecule, revealed characteristics of the single-cell genome that were challenging for short-read sequencing.