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This article offers a new account of the moral substance of shame. Through careful reflection on the motives and intentional structure of shame, I defend the claim that shame is an egocentric and morally blind emotion. I argue that shame is rooted in our desire for social affirmation and constituted by our ability to sense how we appear to others. What makes shame egocentric is that in shame we are essentially concerned about our own social worth and pained by the perception of our self as socially worthless. In itself, shame entails no morally pertinent concern about others or understanding of what is morally significant. I contrast shame with the possibility of relating to others—and to oneself—with love and care. Indeed, I propose that love is essential for moral understanding and motivation. The argument of the article unfolds through critical appraisals of the main strategies for defending the moral value of shame. First, against the claim that shame entails respect for others, I argue that shame's sensitivity to the opinions of others is motivated by egocentric self-concern. Second, against the view that shame over failures to live up to moral values is morally valuable, I argue that regardless of whether the values guiding our shame are moral or not, the perspective of shame is oblivious to their moral meaning. Third, against the claim that shame is crucial for self-understanding, I argue that the desire for affirmation that drives shame is a powerful source of self-deception.

Label-free characterization of single biomolecules aims to complement fluorescence microscopy in situations where labeling compromises data interpretation, is technically challenging or even impossible. However, existing methods require the investigated species to bind to a surface to be visible, thereby leaving a large fraction of analytes undetected. Here, we present nanofluidic scattering microscopy (NSM), which overcomes these limitations by enabling label-free, real-time imaging of single biomolecules diffusing inside a nanofluidic channel. NSM facilitates accurate determination of molecular weight from the measured optical contrast and of the hydrodynamic radius from the measured diffusivity, from which information about the conformational state can be inferred. Furthermore, we demonstrate its applicability to the analysis of a complex biofluid, using conditioned cell culture medium containing extracellular vesicles as an example. We foresee the application of NSM to monitor conformational changes, aggregation and interactions of single biomolecules, and to analyze single-cell secretomes.

DNA-damaging agents, such as radiation and chemotherapy, are common in cancer treatment, but the dosing has proven to be challenging, leading to severe side effects in some patients. Hence, to be able to personalize DNA-damaging chemotherapy, it is important to develop fast and reliable methods to measure the resulting DNA damage in patient cells. Here, we demonstrate how single DNA molecule imaging using fluorescence microscopy can quantify DNA-damage caused by the topoisomerase II (TopoII) poison etoposide. The assay uses an enzyme cocktail consisting of base excision repair (BER) enzymes to repair the DNA damage caused by etoposide and label the sites using a DNA polymerase and fluorescently labeled nucleotides. Using this DNA-damage detection assay we find a large variation in etoposide induced DNA-damage after in vitro treatment of blood cells from healthy individuals. We furthermore used the TopoII inhibitor ICRF-193 to show that the etoposide-induced damage in DNA was TopoII dependent. We discuss how our results support a potential future use of the assay for personalized dosing of chemotherapy.

Objectives Colistin is a last-resort antibiotic, but there has been a rapid increase in colistin resistance, threatening its use in the treatment of infections with carbapenem-resistant Enterobacterales (CRE). Plasmid-mediated colistin resistance, in particular the mcr-1 gene, has been identified and WGS is the go-to method in identifying plasmids carrying mcr-1 genes. The goal of this study is to demonstrate the use of optical DNA mapping (ODM), a fast, efficient and amplification-free technique, to characterize plasmids carrying mcr-1. Methods ODM is a single-molecule technique, which we have demonstrated can be used for identifying plasmids harbouring antibiotic resistance genes. We here applied the technique to plasmids isolated from 12 clinical Enterobacterales isolates from patients at a major hospital in Thailand and verified our results using Nanopore long-read sequencing. Results We successfully identified plasmids encoding the mcr-1 gene and, for the first time, demonstrated the ability of ODM to identify resistance gene sites in small (∼30 kb) plasmids. We further identified blaCTX-M genes in different plasmids than the ones encoding mcr-1 in three of the isolates studied. Finally, we propose a cut-and-stretch assay, based on similar principles, but performed using surface-functionalized cover slips for DNA immobilization and an inexpensive microscope with basic functionalities, to identify the mcr-1 gene in a plasmid sample. Conclusions Both ODM and the cut-and-stretch assay developed could be very useful in identifying plasmids encoding antibiotic resistance in hospitals and healthcare facilities. The cut-and-stretch assay is particularly useful in low- and middle-income countries, where existing techniques are limited.

Nanofluidic structures have over the last two decades emerged as a powerful platform for detailed analysis of DNA on the kilobase pair length scale. When DNA is confined to a nanochannel, the combination of excluded volume and DNA stiffness leads to the DNA being stretched to near its full contour length. Importantly, this stretching takes place at equilibrium, without any chemical modifications to the DNA. As a result, any DNA can be analyzed, such as DNA extracted from cells or circular DNA, and it is straight-forward to study reactions on the ends of linear DNA. In this comprehensive review, we first give a thorough description of the current understanding of the polymer physics of DNA and how that leads to stretching in nanochannels. We then describe how the versatility of nanofabrication can be used to design devices specifically tailored for the problem at hand, either by controlling the degree of confinement or enabling facile exchange of reagents to measure DNA–protein reaction kinetics. The remainder of the review focuses on two important applications of confining DNA in nanochannels. The first is optical DNA mapping, which provides the genomic sequence of intact DNA molecules in excess of 100 kilobase pairs in size, with kilobase pair resolution, through labeling strategies that are suitable for fluorescence microscopy. In this section, we highlight solutions to the technical aspects of genomic mapping, including the use of enzyme-based labeling and affinity-based labeling to produce the genomic maps, rather than recent applications in human genetics. The second is DNA–protein interactions, and several recent examples of such studies on DNA compaction, filamentous protein complexes, and reactions with DNA ends are presented. Taken together, these two applications demonstrate the power of DNA confinement and nanofluidics in genomics, molecular biology, and biophysics.

Catalyst activity can depend distinctly on nanoparticle size and shape. Therefore, understanding the structure sensitivity of catalytic reactions is of fundamental and technical importance. Experiments with single-particle resolution, where ensemble-averaging is eliminated, are required to study it. Here, we implement the selective trapping of individual spherical, cubic, and octahedral colloidal Au nanocrystals in 100 parallel nanofluidic channels to determine their activity for fluorescein reduction by sodium borohydride using fluorescence microscopy. As the main result, we identify distinct structure sensitivity of the rate-limiting borohydride oxidation step originating from different edge site abundance on the three particle types, as confirmed by first-principles calculations. This advertises nanofluidic reactors for the study of structure-function correlations in catalysis and identifies nanoparticle shape as a key factor in borohydride-mediated catalytic reactions., This work was supported by the Knut and Alice Wallenberg Foundation project 2015.0055, the Swedish Foundation for Strategic Research project RMA15-0052, and the Swedish Research Council project 2018-00329., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).

Small-molecule DNA-binding drugs have shown promising results in clinical use against many types of cancer. Understanding the molecular mechanisms of DNA binding for such small molecules can be critical in advancing future drug designs. We have been exploring the interactions of ruthenium-based small molecules and their DNA-binding properties that are highly relevant in the development of novel metal-based drugs. Previously we have studied the effects of the right-handed binuclear ruthenium threading intercalator ΔΔ-[μ-bidppz(phen)

Objectives: The carbapenemase-encoding gene blaNDM-1 has been reported in Vietnam during the last 10 years, and blaNDM-producing Enterobacteriaceae are now silently and rapidly spreading. A key factor behind dissemination of blaNDM-1 is plasmids, mobile genetic elements that commonly carry antibiotic resistance genes and spread via conjugation. The diversity of blaNDM-1-encoding plasmids from neonates at a large Vietnamese hospital was characterized in this study. Methods: 18 fecal Klebsiella pneumoniae and Klebsiella quasipneumoniae isolates collected from 16 neonates at a large pediatric hospital in Vietnam were studied using optical DNA mapping (ODM) and next-generation sequencing (NGS). Plasmids carrying the blaNDM-1 gene were identified by combining ODM with Cas9 restriction. The plasmids in the isolates were compared to investigate whether the same plasmid was present in different patients. Results: Although the same plasmid was found in some isolates, ODM confirmed that there were at least 10 different plasmids encoding blaNDM-1 among the 18 isolates, thus indicating wide plasmid diversity. The ODM results concur with the NGS data. Interestingly, some isolates had two distinct plasmids encoding blaNDM-1 that could be readily identified with ODM. The coexistence of different plasmids carrying the same blaNDM-1 gene in a single isolate has rarely been reported, probably because of limitations in plasmid characterization techniques. Conclusions: The plasmids encoding the blaNDM-1 gene in this study cohort were diverse and may represent a similar picture in Vietnamese society. The study highlights important aspects of the usefulness of ODM for plasmid analysis. Funding: Erling-Persson foundation; Swedish Research Council (VR) via the Southeast Asia-Europe joint funding scheme for research and innovation; Swedish Research Council (VR) [330-2014-6356]; EU Marie Curie; Swedish Foundation for International Cooperation in Research and Higher Education (STINT) [SG 2015-5972]; Region Ostergotland; ALF-grants [LIO-899871]; SIDA

Optical DNA mapping (ODM) has developed into an important technique for DNA analysis, where single DNA molecules are sequence-specifically labeled and stretched, for example, in nanofluidic channels. We have developed an ODM assay to analyze bacterial plasmids—circular extrachromosomal DNA that often carry genes that make bacteria resistant to antibiotics. As for most techniques, the next important step is to increase throughput and automation. In this work, we designed and fabricated a nanofluidic device that, together with a simple automation routine, allows parallel analysis of up to 10 samples at the same time. Using plasmids encoding extended-spectrum beta-lactamases (ESBL), isolated from Escherichia coli and Klebsiella pneumoniae, we demonstrate the multiplexing capabilities of the device when it comes to both many samples in parallel and different resistance genes. As a final example, we combined the device with a novel protocol for rapid cultivation and extraction of plasmids from fecal samples collected from patients. This combined protocol will make it possible to analyze many patient samples in one device already on the day the sample is collected, which is an important step forward for the ODM analysis of plasmids in clinical diagnostics.

Quantification of the DNA damage induced by chemotherapy in patient cells may aid in personalization of the dose used. However, assays to evaluate individual patient response to chemotherapy are not available today. Here, we present an assay that quantifies single-stranded lesions caused by the chemotherapeutic drug Bleomycin (BLM) in peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals. We use base excision repair (BER) enzymes to process the DNA damage induced by BLM and then extend the processed sites with fluorescent nucleotides using a DNA polymerase. The fluorescent patches are quantified on single DNA molecules using fluorescence microscopy. Using the assay, we observe a significant variation in the in vitro induced BLM damage and its repair for different individuals. Treatment of the cells with the BER inhibitor CRT0044876 leads to a lower level of repair of BLM-induced damage, indicating the ability of the assay to detect a compromised DNA repair in patients. Overall, the data suggest that our assay could be used to sensitively detect the variation in BLM-induced DNA damage and repair in patients and can potentially be able to aid in personalizing patient doses.