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The evolution of SARS-CoV-2 has led to the emergence of numerous variants of concern (VOCs), marked by changes in the viral spike glycoprotein, the primary target for neutralising antibody (nAb) responses. Emerging VOCs, particularly omicron sub-lineages, show resistance to nAbs induced by prior infection or vaccination. The precise spike protein changes contributing to this resistance remain unclear in infectious cell culture systems. In the present study, a large panel of infectious SARS-CoV-2 mutant viruses, each with spike protein changes found in VOCs, including omicron JN.1 and its derivatives KP.2 and KP.3, was generated using a reverse genetic system. The susceptibility of these viruses to antibody neutralisation was measured using plasma from convalescent and vaccinated individuals. Synergistic roles of combined substitutions in the spike receptor binding domain (RBD) were observed in neutralisation resistance. However, recombinant viruses with the entire spike protein from a specific VOC showed enhanced resistance, indicating that changes outside the RBD are also significant. In silico analyses of spike antibody epitopes suggested that changes in neutralisation could be due to altered antibody binding affinities. Assessing ACE2 usage for entry through anti-ACE2 antibody blocking and ACE2 siRNA revealed that omicron BA.2.86 and JN.1 mutant viruses were less dependent on ACE2 for entry. However, surface plasmon resonance analysis showed increased affinity for ACE2 for both BA.2.86 and JN.1 compared to the ancestral spike. This detailed analysis of specific changes in the SARS-CoV-2 spike enhances understanding of coronavirus evolution, particularly regarding neutralising antibody evasion and ACE2 entry receptor dependence.

Abstract Lymphoid malignancies are characterized by clonal cell expansion, often identifiable by unique immunoglobulin rearrangements. Heavy (IGH) and light‐chain gene usage offers diagnostic insights and enables sensitive residual disease detection via next‐generation sequencing. With its adaptable throughput and variable read lengths, Oxford Nanopore thirdgeneration sequencing now holds promise for clonotyping. This study analyzed CD138+ plasma‐cell DNA from eight multiple myeloma patients, comparing clonotyping performance between Nanopore sequencing, Illumina MiSeq, and Ion Torrent S5. We demonstrated clonotype consistency across platforms through Smith‐Waterman local alignment of nanopore reads. The mean clonal percentage of IGH V and J gene usage in the CD138+ cells was 69% for Nanopore, 67% for S5, and 76% for MiSeq. When aligned with known clonotypes, clonal cells averaged a 91% similarity, exceeding 85%. In summary, Nanopore sequencing, with its capacity for generating millions of high‐quality reads, proves effective for detecting clonal IGH rearrangements. This versatile platform offers the potential for measuring residual disease down to a sensitivity level of 10‐6 at a lower cost, marking a significant advancement in clonotyping techniques.

Niels Abildgaard,1,2 Jonatan Freilich,3,4 Pekka Anttila,5 Nawal Bent-Ennakhil,6 Yuanjun Ma,3 Mariann Lassenius,7 Sigurd Ørstavik,8 Iiro Toppila,7 Anders Waage,9 Ingemar Turesson,10 Markus Hansson11 1Hematology Research Unit, Department of Hematology, Odense University Hospital, Odense, Denmark; 2Department of Clinical Research, University of Southern Denmark, Odense, Denmark; 3Department of Access Consulting, PAREXEL International, Stockholm, Sweden; 4Department of Public Health and Clinical Medicine, Dermatology, Umeå University, Umeå, Sweden; 5Comprehensive Cancer Center, Department of Hematology, University of Helsinki, and Helsinki University Hospital, Helsinki, Finland; 6Takeda Pharmaceuticals International AG, Zurich, Switzerland; 7Medaffcon, Espoo, Finland; 8Takeda Pharmaceuticals International AG, Oslo, Norway; 9Department of Hematology, St Olav’s University Hospital, Trondheim, Norway; 10Lund University Cancer Centre, University of Lund, Skåne University Hospital, Lund, Sweden; 11Sahlgrenska Academy and Sahlgrenska University Hospital, Göteborg, SwedenCorrespondence: Niels Abildgaard, Department of Hematology, Odense University Hospital, Kloevervaenget 10, Floor 12, 5000 Odense C, Denmark, Tel +45 6541 1155, Email niels.abildgaard@rsyd.dkPurpose: Linked health-care registries and high coverage in Nordic countries lend themselves well to epidemiologic research. Given its relatively high incidence in Western Europe, complexity in diagnosis, and challenges in registration, multiple myeloma (MM) was selected to compare registries in Denmark, Finland, and Sweden.Patients and Methods: Data were obtained from four archetypal registries in each country (spanning January 2005–October 2018): National Patient Registry (NPR), Prescribed Drug Registry (PDR), Cancer Registry (CR), and Cause of Death Registry. Patients newly diagnosed with MM who received MM-specific treatment were included. PDR/NPR treatment records were used to assess incident NPR cases. The registration quality of MM-specific drugs in the PDR of each country was also evaluated.Results: In Denmark, only 6% of patients in the NPR were not registered in the CR; in Sweden, it was 16.9%. No systematic differences were identified that could explain this discrepancy. In Denmark, lenalidomide and bortezomib were registered in the NPR with high coverage, but less expensive drugs typically given in combination with bortezomib were not covered in any of the registries. In Finland and Sweden, bortezomib records were not identified in the PDR, but some were in the NPR; other drugs had good coverage in the PDR.Conclusions: The registries evaluated in this study can be used to identify the MM population; however, given the gaps in MM registration in the Finnish and Swedish CRs, Danish registries provide the most comprehensive datasets for research on treatment patterns for MM.Plain Language Summary: National patient registries collect observational data on populations of patients and are often used for research. In this study, we investigated how complete the national patient registries were for Denmark, Finland and Sweden when recording the number of patients with multiple myeloma (MM; a type of bone marrow cancer) between 2005 and 2018. We also investigated the completeness in registration of given treatments during the study period in the national registries. Overall, the Danish national patient registries had the most comprehensive information on patients with MM. Registry data from Denmark could therefore be useful for conducting further research into the characteristics, treatment patterns, and outcomes of patients with MM.Keywords: incidence, prevalence, real-world evidence

Abstract Membrane fouling significantly reduces membrane permeability, leading to higher operational expenses. In situ monitoring of membrane fouling can potentially be used to reduce operation cost by optimizing operational parameters and cleaning conditions. In this study, a platinum wire with a diameter of 20 µm was attached to the surface of a ceramic ultrafiltration membrane, and by measuring the voltage across the wire while applying an AC current, the amplitude of the third harmonic wave, the so-called 3ω signal, was obtained. Results showed increasing 3ω signals during formation of fouling layers, which correlates directly to the hydraulic resistance of the formed fouling layer in semi-dead end filtration of polymeric core shell particles and crossflow filtration of diluted milk. This is explained by the insulating effect of the fouling layers which reduces heat convection by crossflow and the different thermal conductivity in the fouling layer compared with the feed. After membrane cleaning, the permeability and the magnitude of the 3ω signal were partly restored, showing that the 3ω method can be used to monitor the effect of cleaning. The frequency of the AC current was varied so it was possible to measure the heat conductivity in the fouling layer (high frequency) and heat convection due to cross-flow (low frequency). This may potentially be used to get information of the type of fouling (heat conductivity) and thickness of the fouling layer (AC frequency where heat conductivity becomes dominating).

Healthcare employees are experiencing poor wellbeing at an increasing rate. The healthcare workforce is exposed to challenging tasks and a high work pace, a situation that worsened during and after the COVID-19 pandemic. In turn, exposure to these high demands contributes to poor health, increased turnover, reduced job satisfaction, reduced efficacy, and reduced patient satisfaction and safety. Therefore, it is imperative that we identify measures to mitigate this crisis. One piece of this puzzle is how to implement sustainable tools and processes to improve the work environment of healthcare organizations. In this paper, we present the study protocol for the outlining and piloting of a joint training for pairs of healthcare line managers and their associated health and safety representatives in a Swedish healthcare organization. The objective of the training is to aid and advance the implementation of interventions to improve the work environment at the unit level. Following recommendations in the literature, the training is based on a stepwise approach that considers the specific context and focuses on the involvement of employees in creating interventions based on their needs. A central component of the training is the development of the pairs’ collaboration in prioritizing, developing, implementing, and evaluating the interventions. The training is based on an on-the-job train-the-trainer approach in which participants are progressively trained during four workshops in the steps of a participatory intervention process. Between these workshops, the pairs follow the same progressive steps together with their employees to develop and implement interventions at their unit. The pilot will involve four pairs (i.e., eight participants) representing different parts and functions of the organization and will be conducted over a period of three months. We will use a mixed method design to evaluate preconditions, the process, and proximal transfer and implementation outcome factors of the training. The overall aim of the pilot is to appraise its feasibility and be able to adjust the training before a potential scale-up.

Blindness or vision loss due to neuroretinal and photoreceptor degeneration affects millions of individuals worldwide. In numerous neurodegenerative diseases, including age-related macular degeneration, dysregulated immune response-mediated retinal degeneration has been found to play a critical role in the disease pathogenesis. To better understand the pathogenic mechanisms underlying the retinal degeneration, we used a mouse model of systemic immune activation where we infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13. Here, we evaluated the effects of LCMV infection and present a comprehensive discovery-based proteomic investigation using tandem mass tag (TMT) labeling and high-resolution liquid chromatography–tandem mass spectrometry (LC-MS/MS). Changes in protein regulation in the posterior part of the eye, neuroretina, and RPE/choroid were compared to those in the spleen as a secondary lymphoid organ and to the kidney as a non-lymphoid but encapsulated organ at 1, 8, and 28 weeks of infection. Using bioinformatic tools, we found several proteins responsible for maintaining normal tissue homeostasis to be differentially regulated in the neuroretina and the RPE/choroid during the degenerative process. Additionally, in the organs we observed, several important protein pathways contributing to cellular homeostasis and tissue development were perturbed and associated with LCMV-mediated inflammation, promoting disease progression. Our findings suggest that the response to a systemic chronic infection differs between the neuroretina and the RPE/choroid, and the processes induced by chronic systemic infection in the RPE/choroid are not unlike those induced in non-immune-privileged organs such as the kidney and spleen. Overall, our data provide detailed insight into several molecular mechanisms of neuroretinal degeneration and highlight various novel protein pathways that further suggest that the posterior part of the eye is not an isolated immunological entity despite the existence of neuroretinal immune privilege.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.