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Abstract High-amplitude quasi-stationary atmospheric Rossby waves with zonal wave numbers 6–8 associated with the phenomenon of quasi-resonant amplification (QRA) have been linked to persistent summer extreme weather events in the Northern Hemisphere. QRA is not well-resolved in current generation climate models, therefore, necessitating an alternative approach to assessing their behavior. Using a previously-developed fingerprint-based semi-empirical approach, we project future occurrence of QRA events based on a QRA index derived from the zonally averaged surface temperature field, comparing results from CMIP 5 and 6 (Coupled Model Intercomparison Project). There is a general agreement among models, with most simulations projecting substantial increase in QRA index. Larger increases are found among CMIP6-SSP5-8.5 (42 models, 46 realizations), with 85% of models displaying a positive trend, as compared with 60% of CMIP5-RCP8.5 (33 models, 75 realizations), with a reduced spread among CMIP6-SSP5-8.5 models. CMIP6-SSP3-7.0 (23 models, 26 realizations) simulations display qualitatively similar behavior to CMIP6-SSP5-8.5, indicating a substantial increase in QRA events under business-as-usual emissions scenarios, and the results hold regardless of the increase in climate sensitivity in CMIP6. Projected aerosol reductions in CMIP6-SSP3-7.0-lowNTCF (5 models, 16 realizations) lead to halting effect in QRA index and Arctic Amplification during the 1st half of the twenty-first century. Our analysis suggests that anthropogenic warming will likely lead to an even more substantial increase in QRA events (and associated summer weather extremes) than indicated by past analyses.

Abstract Motoneurons critically depend on precise spatial and temporal control of translation for axon growth and the establishment and maintenance of neuromuscular connections. While defects in local translation have been implicated in the pathogenesis of motoneuron disorders, little is known about the mechanisms regulating axonal protein synthesis. Here, we report that motoneurons derived from Hnrnpr knockout mice show reduced axon growth accompanied by lowered synthesis of cytoskeletal and synaptic components in axons. Mutant mice display denervated neuromuscular junctions and impaired motor behavior. In axons, hnRNP R is a component of translation initiation complexes and, through interaction with O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (Ogt), modulates O-GlcNAcylation of eIF4G. Restoring axonal O-GlcNAc levels rescued local protein synthesis and axon growth defects of hnRNP R knockout motoneurons. Together, these findings demonstrate a function of hnRNP R in controlling the local production of key factors required for axon growth and formation of neuromuscular innervations.

Abstract The European rabbit has invaded numerous ecosystems worldwide, but rarely steppes. Since its various introduction attempts into the ecosystems of the Magallanes/Fuegian region, the rabbit has become a key player, interacting with species at different trophic levels and generating impacts on ecosystems. To better understand the role of the rabbit in steppe and scrub ecosystems, we characterised the food web in the Magallanes/Fuegian region to understand the identity of their interacting species, the mechanisms and complexities of their interactions to demonstrate that rabbit management may become more complex than just controlling a single species. Based on a bibliographic review and wildlife specialists’ opinions, we built the Magellanic/Fuegian food web, evaluated their topological properties and performed a rabbit extinction simulation to assess the possible short-term ecological mechanisms operating in the community. We found that the network had 206 nodes (64% native, 13% exotic, and 22% mixed) and 535 links among nodes. The European rabbit was the most connected node of the food web, had the second largest dietary breadth, and ranked as the seventh prey item with more predators. A rabbit extinction simulation shows a possible release of herbivory pressure on plants, including that on several native plants (e.g., Gunnera tinctoria, Pratia repens, Gavilea lutea, Tetroncium magellanicus), and a possible release of competition for some herbivores that share resources with the rabbit (e.g., Ovis aries, Lama guanicoe, Bos taurus). Although rabbit predators have a broad and generalist diet, some such as the native Galicitis cuja, could face a 20% reduction in their trophic width and could intensify predation on alternative prey. These results show that the European rabbit is strongly embedded in the Magellanic/Fuegian ecosystem and linked to several native species. Therefore, rabbit management should consider ecosystem approaches accompanied by monitoring programs on native fauna and experimental pilot studies on native flora to conserve the Chilean Patagonia community.

Abstract Eastern equine encephalitis virus (EEEV) is the most virulent alphavirus that infects humans, and many survivors develop neurological sequelae, including paralysis and intellectual disability. Alphavirus spike proteins comprise trimers of heterodimers of glycoproteins E2 and E1 that mediate binding to cellular receptors and fusion of virus and host cell membranes during entry. We recently identified very-low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) as cellular receptors for EEEV and a distantly related alphavirus, Semliki Forest virus (SFV). Here, we use single-particle cryo-electron microscopy (cryo-EM) to determine structures of the EEEV and SFV spike glycoproteins bound to the VLDLR ligand-binding domain and found that EEEV and SFV interact with the same cellular receptor through divergent binding modes. Our studies suggest that the ability of LDLR-related proteins to interact with viral spike proteins through very small footprints with flexible binding modes results in a low evolutionary barrier to the acquisition of LDLR-related proteins as cellular receptors for diverse sets of viruses.

Abstract This study is the first to investigate the presence and movement of the novel Liberibacter species ‘Candidatus Liberibacter brunswickensis’ (CLbr) in eggplant, Solanum melongena. The psyllid, Acizzia solanicola can transmit CLbr to eggplant and CLbr can be acquired by CLbr-negative A. solanicola individuals from CLbr-positive eggplants. In planta, CLbr can replicate, move and persist. Investigation into the early development of eggplants showed that CLbr titres had increased at the inoculation site at 14 days post inoculation access period (DPIAP). CLbr had become systemic in the majority of plants tested by 28 DPIAP. The highest bacterial titres were recorded at 35 DPIAP in all samples of the inoculated leaf, the roots, stems and the midrib and petiole samples of the newest leaf (the top leaf). This finding strongly suggests that CLbr movement in planta follows the source to sink relationship as previously described for ‘Ca. Liberibacter asiaticus’ (CLas) and ‘Ca. Liberibacter solanacearum’ (CLso). No symptoms consistent with Liberibacter-associated diseases were noted for plants colonised by CLbr during this study, consistent with the hypothesis that CLbr does not cause disease of eggplant during the early stages of host colonisation. In addition, no significant differences in biomass were found between eggplant colonised with CLbr, compared to those that were exposed to CLbr-negative A. solanicola, and to control plants.

Abstract Dental calculus is a microbial biofilm that contains biomolecules from oral commensals and pathogens, including those potentially related to cause of death (CoD). To assess the utility of calculus as a diagnostically informative substrate, in conjunction with paleopathological analysis, calculus samples from 39 individuals in the Smithsonian Institution’s Robert J. Terry Collection with CoDs of either syphilis or tuberculosis were assessed via shotgun metagenomic sequencing for the presence of Treponema pallidum subsp. pallidum and Mycobacterium tuberculosis complex (MTBC) DNA. Paleopathological analysis revealed that frequencies of skeletal lesions associated with these diseases were partially inconsistent with diagnostic criteria. Although recovery of T. p. pallidum DNA from individuals with a syphilis CoD was elusive, MTBC DNA was identified in at least one individual with a tuberculosis CoD. The authenticity of MTBC DNA was confirmed using targeted quantitative PCR assays, MTBC genome enrichment, and in silico bioinformatic analyses; however, the lineage of the MTBC strain present could not be determined. Overall, our study highlights the utility of dental calculus for molecular detection of tuberculosis in the archaeological record and underscores the effect of museum preparation techniques and extensive handling on pathogen DNA preservation in skeletal collections.

Abstract Imputation techniques provide means to replace missing measurements with a value and are used in almost all downstream analysis of mass spectrometry (MS) based proteomics data using label-free quantification (LFQ). Here we demonstrate how collaborative filtering, denoising autoencoders, and variational autoencoders can impute missing values in the context of LFQ at different levels. We applied our method, proteomics imputation modeling mass spectrometry (PIMMS), to an alcohol-related liver disease (ALD) cohort with blood plasma proteomics data available for 358 individuals. Removing 20 percent of the intensities we were able to recover 15 out of 17 significant abundant protein groups using PIMMS-VAE imputations. When analyzing the full dataset we identified 30 additional proteins (+13.2%) that were significantly differentially abundant across disease stages compared to no imputation and found that some of these were predictive of ALD progression in machine learning models. We, therefore, suggest the use of deep learning approaches for imputing missing values in MS-based proteomics on larger datasets and provide workflows for these.

Abstract Due to their exceptional solubility and stability, nanobodies have emerged as powerful building blocks for research tools and therapeutics. However, their generation in llamas is cumbersome and costly. Here, by inserting an engineered llama immunoglobulin heavy chain (IgH) locus into IgH-deficient mice, we generate a transgenic mouse line, which we refer to as ‘LamaMouse’. We demonstrate that LamaMice solely express llama IgH molecules without association to Igκ or λ light chains. Immunization of LamaMice with AAV8, the receptor-binding domain of the SARS-CoV-2 spike protein, IgE, IgG2c, and CLEC9A enabled us to readily select respective target-specific nanobodies using classical hybridoma and phage display technologies, single B cell screening, and direct cloning of the nanobody-repertoire into a mammalian expression vector. Our work shows that the LamaMouse represents a flexible and broadly applicable platform for a facilitated selection of target-specific nanobodies.

Meteoric ablation in the Earth's atmosphere produces particles of nanometer size and larger. These particles can become charged and influence the charge balance in the D region (60–90 km) and the incoherent scatter observed with radar from there. Radar studies have shown that, if enough dust particles are charged, they can influence the received radar spectrum below 100 km, provided the electron density is sufficiently high (>109 m3). Here, we study an observation made with the EISCAT VHF radar on 9 January 2014 during strong particle precipitation so that incoherent scatter was observed down to almost 60 km altitude. We found that the measured spectra were too narrow in comparison to the calculated spectra. Adjusting the collision frequency provided a better fit in the frequency range of ± 10–30 Hz. However, this did not lead to the best fit in all cases, especially not for the central part of the spectra in the narrow frequency range of ±10 Hz. By including a negatively charged dust component, we obtained a better fit for spectra observed at altitudes of 75–85 km, indicating that dust influences the incoherent-scatter spectrum at D-region altitudes. The observations at lower altitudes were limited by the small number of free electrons, and observations at higher altitudes were limited by the height resolution of the observations. Inferred dust number densities range from a few particles up to 104 cm−3, and average sizes range from approximately 0.6 to 1 nm. We find an acceptable agreement with the dust profiles calculated with the WACCM-CARMA (Whole Atmosphere Community Climate Model-Community Aerosol Radiation Model for Atmospheres) model. However, these do not include charging, which is also based on models.

Summary: Decidualization denotes the differentiation of endometrial stromal cells into specialized decidual cells, essential for embryo implantation and pregnancy. The process requires coordination of progesterone and cAMP signaling, which converge on downstream transcription factors. PGE2 and relaxin, acting, respectively, through Gαs-coupled GPCRs EP2 and RXFP1, are putative candidates for generating cAMP in differentiating stromal cells. Here, we show that PGE2 is less efficacious than relaxin in elevating intracellular cAMP levels in primary stromal cells but more effective at driving the expression of decidual genes. PGE2-and relaxin-induced cAMP generation involves receptor internalization, but EP2 is endocytosed into very early endosomes (VEEs). Perturbation of VEE machinery through depletion of key trafficking proteins; APPL1 and GIPC, dysregulates PGE2-dependent cAMP profiles and disrupts key decidual signaling pathways, resulting in a disordered differentiation response. We demonstrate that regulation of EP2 via internalization is essential for coordinated activation of the downstream signaling cascades that govern decidualization.

Understanding sexual communication requires assessing the behaviour of both the sender and the receiver. Receiver responses to sexual displays carry relevant information, but such signals or cues may be subtle and therefore technically challenging to investigate. Here, we focus on receiver body movements in response to high-intensity courtship in spotted bowerbirds (Ptilonorhynchus maculatus). Male bowerbirds perform a vigorous courtship choreography on dedicated display structures—bowers. Bower owners tolerate other non-territorial males at their bowers, yet the courtship displays of these so-called ‘subordinate’ males rarely result in successful copulations. Males that display at high intensity are preferred by females in this species, yet excessively aggressive displays may be threatening, hence scaring prospective mates away. In this study, we hypothesized that bower owners are better able to exhibit high-intensity movements without startling their audience compared with subordinate males. To address this question, we used a combination of behavioural coding and AI-based tracking of body movements, which allows precise spatial and temporal resolution for the study of subtle behavioural responses. Contrary to our predictions, we found that bower owners evoked stronger startle responses than subordinate males. We discuss these unexpected results and suggest further experimental approaches for future investigations.

Summary: Colorectal adenomas (CRAs) are potential precursor lesions to adenocarcinomas, currently classified by morphological features. We aimed to establish a molecular feature-based risk allocation framework toward improved patient stratification. Deep visual proteomics (DVP) is an approach that combines image-based artificial intelligence with automated microdissection and ultra-high sensitive mass spectrometry. Here, we used DVP on formalin-fixed, paraffin-embedded (FFPE) CRA tissues from nine male patients, immunohistologically stained for caudal-type homeobox 2 (CDX2), a protein implicated in colorectal cancer, enabling the characterization of cellular heterogeneity within distinct tissue regions and across patients. DVP identified DMBT1, MARCKS, and CD99 as protein markers linked to recurrence, suggesting their potential for risk assessment. It also detected a metabolic shift to anaerobic glycolysis in cells with high CDX2 expression. Our findings underscore the potential of spatial proteomics to refine early stage detection and contribute to personalized patient management strategies and provided novel insights into metabolic reprogramming.

There is no more consequential scientific matter today than global warming. The societal and policy implications, however, hinge upon the attribution of that warming to human activity and, specifically, continued societal reliance on the burning of fossil fuels. It was recently suggested that this warming could be explained by the non-anthropogenic factor of seismic activity. If that is the case, it would have profound implications. We have assessed the validity of the claim using a statistical technique (the method of conditional dispersion) that evaluates the existence of causal connections between variables, finding no evidence for any causal relationship between seismic activity and global warming.

Abstract Deep learning neural networks are often described as black boxes, as it is difficult to trace model outputs back to model inputs due to a lack of clarity over the internal mechanisms. This is even true for those neural networks designed to emulate mechanistic models, which simply learn a mapping between the inputs and outputs of mechanistic models, ignoring the underlying processes. Using a mechanistic model studying the pharmacological interaction between opioids and naloxone as a proof-of-concept example, we demonstrated that by reorganizing the neural networks’ layers to mimic the structure of the mechanistic model, it is possible to achieve better training rates and prediction accuracy relative to the previously proposed black-box neural networks, while maintaining the interpretability of the mechanistic simulations. Our framework can be used to emulate mechanistic models in a large parameter space and offers an example on the utility of increasing the interpretability of deep learning networks.

Solar Orbiter is equipped with electrical antennas performing fast measurements of the surrounding electric field. The antennas register high-velocity dust impacts through the electrical signatures of impact ionization. Although the basic principle of the detection has been known for decades, the understanding of the underlying process is not complete, due to the unique mechanical and electrical design of each spacecraft and the variability of the process. We present a study of electrical signatures of dust impacts on Solar Orbiter's body, as measured with the Radio and Plasma Waves electrical suite. A large proportion of the signatures present double-peak electrical waveforms in addition to the fast pre-spike due to electron motion, which are systematically observed for the first time. We believe this is due to Solar Orbiter's unique antenna design and a high temporal resolution of the measurements. The double peaks are explained as being due to two distinct processes. Qualitative and quantitative features of both peaks are described. The process for producing the primary peak has been studied extensively before, and the process for producing the secondary peak has been proposed before (Pantellini et al., 2012a) for Solar Terrestrial Relations Observatory (STEREO), although the corresponding delay of 100–300 µs between the primary and the secondary peak has not been observed until now. Based on this study, we conclude that the primary peak's amplitude is the better measure of the impact-produced charge, for which we find a typical value of around 8 pC. Therefore, the primary peak should be used to derive the impact-generated charge rather than the maximum. The observed asymmetry between the primary peaks measured with individual antennas is quantitatively explained as electrostatic induction. A relationship between the amplitude of the primary and the secondary peak is found to be non-linear, and the relation is partially explained with a model for electrical interaction through the antennas' photoelectron sheath.

Abstract The flat bands in magic-angle twisted bilayer graphene (MATBG) provide an especially rich arena to investigate interaction-driven ground states. While progress has been made in identifying the correlated insulators and their excitations at commensurate moiré filling factors, the spin-valley polarizations of the topological states that emerge at high magnetic field remain unknown. Here we introduce a technique based on twist-decoupled van der Waals layers that enables measurement of their electronic band structure and–by studying the backscattering between counter-propagating edge states–the determination of the relative spin polarization of their edge modes. We find that the symmetry-broken quantum Hall states that extend from the charge neutrality point in MATBG are spin unpolarized at even integer filling factors. The measurements also indicate that the correlated Chern insulator emerging from half filling of the flat valence band is spin unpolarized and suggest that its conduction band counterpart may be spin polarized.

Abstract An improved understanding of the underlying physicochemical properties of respiratory aerosol that influence viral infectivity may open new avenues to mitigate the transmission of respiratory diseases such as COVID-19. Previous studies have shown that an increase in the pH of respiratory aerosols following generation due to changes in the gas-particle partitioning of pH buffering bicarbonate ions and carbon dioxide is a significant factor in reducing SARS-CoV-2 infectivity. We show here that a significant increase in SARS-CoV-2 aerostability results from a moderate increase in the atmospheric carbon dioxide concentration (e.g. 800 ppm), an effect that is more marked than that observed for changes in relative humidity. We model the likelihood of COVID-19 transmission on the ambient concentration of CO2, concluding that even this moderate increase in CO2 concentration results in a significant increase in overall risk. These observations confirm the critical importance of ventilation and maintaining low CO2 concentrations in indoor environments for mitigating disease transmission. Moreover, the correlation of increased CO2 concentration with viral aerostability need to be better understood when considering the consequences of increases in ambient CO2 levels in our atmosphere.

Abstract In common with other omics technologies, mass spectrometry (MS)-based proteomics produces ever-increasing amounts of raw data, making efficient analysis a principal challenge. A plethora of different computational tools can process the MS data to derive peptide and protein identification and quantification. However, during the last years there has been dramatic progress in computer science, including collaboration tools that have transformed research and industry. To leverage these advances, we develop AlphaPept, a Python-based open-source framework for efficient processing of large high-resolution MS data sets. Numba for just-in-time compilation on CPU and GPU achieves hundred-fold speed improvements. AlphaPept uses the Python scientific stack of highly optimized packages, reducing the code base to domain-specific tasks while accessing the latest advances. We provide an easy on-ramp for community contributions through the concept of literate programming, implemented in Jupyter Notebooks. Large datasets can rapidly be processed as shown by the analysis of hundreds of proteomes in minutes per file, many-fold faster than acquisition. AlphaPept can be used to build automated processing pipelines with web-serving functionality and compatibility with downstream analysis tools. It provides easy access via one-click installation, a modular Python library for advanced users, and via an open GitHub repository for developers.

Abstract Almost all Glioblastoma (GBM) are either intrinsically resistant to the chemotherapeutical drug temozolomide (TMZ) or acquire therapy-induced mutations that cause chemoresistance and recurrence. The genome maintenance mechanisms responsible for GBM chemoresistance and hypermutation are unknown. We show that the E3 ubiquitin ligase RAD18 (a proximal regulator of TLS) is activated in a Mismatch repair (MMR)-dependent manner in TMZ-treated GBM cells, promoting post-replicative gap-filling and survival. An unbiased CRISPR screen provides an aerial map of RAD18-interacting DNA damage response (DDR) pathways deployed by GBM to tolerate TMZ genotoxicity. Analysis of mutation signatures from TMZ-treated GBM reveals a role for RAD18 in error-free bypass of O6mG (the most toxic TMZ-induced lesion), and error-prone bypass of other TMZ-induced lesions. Our analyses of recurrent GBM patient samples establishes a correlation between low RAD18 expression and hypermutation. Taken together we define molecular underpinnings for the hallmark tumorigenic phenotypes of TMZ-treated GBM.

Background: The development of obesity-associated comorbidities such as type 2 diabetes (T2D) and hepatic steatosis has been linked to selected microRNAs in individual studies; however, an unbiased genome-wide approach to map T2D induced changes in the miRNAs landscape in human liver samples, and a subsequent robust identification and validation of target genes are still missing. Methods: Liver biopsies from age- and gender-matched obese individuals with (n=20) or without (n=20) T2D were used for microRNA microarray analysis. The candidate microRNA and target genes were validated in 85 human liver samples, and subsequently mechanistically characterized in hepatic cells as well as by dietary interventions and hepatic overexpression in mice. Results: Here, we present the human hepatic microRNA transcriptome of type 2 diabetes in liver biopsies and use a novel seed prediction tool to robustly identify microRNA target genes, which were then validated in a unique cohort of 85 human livers. Subsequent mouse studies identified a distinct signature of T2D-associated miRNAs, partly conserved in both species. Of those, human-murine miR-182–5 p was the most associated with whole-body glucose homeostasis and hepatic lipid metabolism. Its target gene LRP6 was consistently lower expressed in livers of obese T2D humans and mice as well as under conditions of miR-182–5 p overexpression. Weight loss in obese mice decreased hepatic miR-182–5 p and restored Lrp6 expression and other miR-182–5 p target genes. Hepatic overexpression of miR-182–5 p in mice rapidly decreased LRP6 protein levels and increased liver triglycerides and fasting insulin under obesogenic conditions after only seven days. Conclusions: By mapping the hepatic miRNA-transcriptome of type 2 diabetic obese subjects, validating conserved miRNAs in diet-induced mice, and establishing a novel miRNA prediction tool, we provide a robust and unique resource that will pave the way for future studies in the field. As proof of concept, we revealed that the repression of LRP6 by miR-182–5 p, which promotes lipogenesis and impairs glucose homeostasis, provides a novel mechanistic link between T2D and non-alcoholic fatty liver disease, and demonstrate in vivo that miR-182–5 p can serve as a future drug target for the treatment of obesity-driven hepatic steatosis. Funding: This work was supported by research funding from the Deutsche Forschungsgemeinschaft (KI 1887/2-1, KI 1887/2-2, KI 1887/3-1 and CRC-TR296), the European Research Council (ERC, CoG Yoyo LepReSens no. 101002247; PTP), the Helmholtz Association (Initiative and Networking Fund International Helmholtz Research School for Diabetes; MB) and the German Center for Diabetes Research (DZD Next Grant 82DZD09D1G).

Abstract Innovation in healthcare and biomedicine is in decline, yet there exist no widely-known alternatives to traditional brainstorming that can be employed for innovative idea generation. McCaffrey's Innovation Enhancing Techniques (IETs) were developed to enhance creative problem-solving by helping the solver to overcome common psychological obstacles to generating innovative ideas. These techniques were devised for engineering and design problems, which involve solving practical goals using physical materials. Healthcare and science problems however often involve solving abstract goals using intangible resources. Here we adapt two of McCaffrey’s IETs, BrainSwarming and the Generic Parts Technique, to effectively enhance idea generation for such problems. To demonstrate their potential, we apply these techniques to a case study involving the use of blockchain technologies to facilitate ethical goals in biomedicine, and successfully identify 100 potential solutions to this problem. Being simple to understand and easy to implement, these and other IETs have significant potential to improve innovation and idea generation in healthcare, scientific, and technological contexts. By catalysing idea generation in problem-solving, these techniques may be used to target the innovative stagnation currently facing the scientific world.

Abstract RNA splicing is an important biological process associated with cancer initiation and progression. However, the contribution of alternative splicing to pancreatic cancer (PDAC) development is not well understood. Here, we identify an enrichment of RNA binding proteins (RBPs) involved in splicing regulation linked to PDAC progression from a forward genetic screen using Sleeping Beauty insertional mutagenesis in a mouse model of pancreatic cancer. We demonstrate downregulation of RBFOX2, an RBP of the FOX family, promotes pancreatic cancer progression and liver metastasis. Specifically, we show RBFOX2 regulates exon splicing events in transcripts encoding proteins involved in cytoskeletal remodeling programs. These exons are differentially spliced in PDAC patients, with enhanced exon skipping in the classical subtype for several RBFOX2 targets. RBFOX2 mediated splicing of ABI1, encoding the Abelson-interactor 1 adapter protein, controls the abundance and localization of ABI1 protein isoforms in pancreatic cancer cells and promotes the relocalization of ABI1 from the cytoplasm to the periphery of migrating cells. Using splice-switching antisense oligonucleotides (AONs) we demonstrate the ABI1 ∆Ex9 isoform enhances cell migration. Together, our data identify a role for RBFOX2 in promoting PDAC progression through alternative splicing regulation.

Abstract Direct, site-specific methods of protein functionalization are highly desirable for biotechnology. However, such methods are challenging due to the difficulty of chemically differentiating a single site within a large protein. Herein, we propose “metal binding targeting” strategy and develop a Copper Assisted Sequence-specific conjugation Tag (CAST) method to achieve rapid (second order rate 8.1 M−1 s−1), site-specific protein backbone chemical modification with pinpoint accuracy. We demonstrate the versatility of CAST conjugation by preparing various on-demand modified recombinant proteins, including a homogeneous antibody-drug conjugate with high plasma stability and potent efficacy in vitro and in vivo. Thus, CAST provides an efficient and quantitative method to site-specifically attach payloads on large, native proteins.

Abstract Autophagy-related protein 18 (Atg18) participates in the elongation of early autophagosomal structures in concert with Atg2 and Atg9 complexes. How Atg18 contributes to the structural coordination of Atg2 and Atg9 at the isolation membrane remains to be understood. Here, we determined the cryo-EM structures of Atg18 organized in helical tubes, Atg18 oligomers in solution as well as on lipid membrane scaffolds. The helical assembly is composed of Atg18 tetramers forming a lozenge cylindrical lattice with remarkable structural similarity to the COPII outer coat. When reconstituted with lipid membranes, using subtomogram averaging we determined tilted Atg18 dimer structures bridging two juxtaposed lipid membranes spaced apart by 80 Å. Moreover, lipid reconstitution experiments further delineate the contributions of Atg18’s FRRG motif and the amphipathic helical extension in membrane interaction. The observed structural plasticity of Atg18’s oligomeric organization and membrane binding properties provide a molecular framework for the positioning of downstream components of the autophagy machinery.

The bird cherry-oat aphid, Rhopalosiphum padi (L.), is an economically significant pest of pasture grasses, the latter being capable of hosting several fungal endophyte–perennial ryegrass symbiota rich in alkaloids and toxic to vertebrates and invertebrates. Measuring aphid feeding behaviour can provide insights into the effectiveness and mode of action of different fungal endophytes. This study investigated the effects of different Epichloë–perennial ryegrass symbiota on the feeding behaviour of R. padi using the electrical penetration graph technique while also assessing the aphid life history. In most cases, endophytes had significant feeding deterrence and paired fecundity and mortality effects. But, in some instances, endophytes with the highest aphid mortality did not significantly deter feeding, suggesting a more complicated scenario of interactions between the relative concentration of metabolites, e.g., host plant defence response metabolites and alkaloids, and/or physical changes to leaf morphology. Overall, this study sheds light on the mode of action of Epichloë endophytes against aphids and highlights the importance of Epichloë–perennial ryegrass symbiota in the management of insect pests such as aphids in pasture-based grazing systems.

Abstract The gall wasp, Leptocybe invasa, poses a significant global threat to Eucalyptus cultivation, by causing substantial economic losses. The objective of this study was to differentiate between resistant and susceptible genotypes by morphological characteristics using image analysis based on the damage caused by the gall wasp. In addition, consensus sequences derived from transposable elements (TEs) and the genome of Eucalyptus spp. Were identified by in silico analysis. Furthermore, another objective was to discriminate Eucalyptus genotypes in response to Leptocybe invasa by conducting molecular analyses involving transposable elements and inter simple sequence markers. For image analysis, the GroundEye ® system was used to collect images of 60 leaves from six genotypes, three of which were resistant and three susceptible. Eucalyptus spp. sequences were obtained from the GenBank database by in silico analysis and pairwise alignments with TE sequences were conducted using BLASTN. Multiple sequence alignment was performed with Clustal Omega, followed by the identification of conserved regions in Jalview. A motif signature was generated using Weblogo. For molecular characterization using ISSR markers and TEs, samples of young leaves were obtained from a total of 80 Eucalyptus seedlings, of which 50 were classified as resistant and 30 as susceptible to L. invasa. It was possible to distinguish gall wasp susceptible and resistant genotypes by image analysis. In silico analysis enabled the identification of conserved regions in the Eucalyptus spp. genome, which were associated with proteins involved in secondary metabolite production, e.g., terpenes, which play a role in the response to L. invasa. The discrimination capacity of TEs and ISSR primers was demonstrated and bands were generated that could be used to identify resistant genotypes. However, increasing the number of markers required to discriminate genotypes in both cases is suggested.

Abstract Lipid metabolism and signaling play pivotal functions in biology and disease development. Despite this, currently available optical techniques are limited in their ability to directly visualize the lipidome in tissues. In this study, opto‐lipidomics, a new approach to optical molecular tissue imaging is introduced. The capability of vibrational Raman spectroscopy is expanded to identify individual lipids in complex tissue matrices through correlation with desorption electrospray ionization (DESI) – mass spectrometry (MS) imaging in an integrated instrument. A computational pipeline of inter‐modality analysis is established to infer lipidomic information from optical vibrational spectra. Opto‐lipidomic imaging of transient cerebral ischemia‐reperfusion injury in a murine model of ischemic stroke demonstrates the visualization and identification of lipids in disease with high molecular specificity using Raman scattered light. Furthermore, opto‐lipidomics in a handheld fiber‐optic Raman probe is deployed and demonstrates real‐time classification of bulk brain tissues based on specific lipid abundances. Opto‐lipidomics opens a host of new opportunities to study lipid biomarkers for diagnostics, prognostics, and novel therapeutic targets.

Abstract Topological insulators have recently received attention in optoelectronic devices because of their high mobility and broadband absorption resulting from their topological surface states. In particular, theoretical and experimental studies have emerged that can improve the spin generation efficiency in a topological insulator‐based p–n junction structure called a TPNJ, drawing attention in optospintronics. Recently, research on implementing the TPNJ structure is conducted; however, studies on the device characteristics of the TPNJ structure are still insufficient. In this study, the TPNJ structure is effectively implemented without intermixing by controlling the annealing temperature, and the photocharacteristics appearing in the TPNJ structure are investigated using a cross‐pattern that can compare the characteristics in a single device. Enhanced photo characteristics are observed for the TPNJ structure. An optical pump Terahertz probe and a physical property measurement system are used to confirm the cause of improved photoresponsivity. Consequently, the photocharacteristics are improved owing to the change in the absorption mechanism and surface transport channel caused by the Fermi level shift in the TPNJ structure.

Summary: Behavior in controlled laboratory studies is not always representative of what people do in daily life. This has prompted a recent shift toward conducting studies in natural settings. We wondered whether expectations raised by how the task is presented should also be considered. To find out, we studied gaze when walking down and up a staircase. Gaze was often directed at steps before stepping on them, but most participants did not look at every step. Importantly, participants fixated more steps and looked around less when asked to navigate the staircase than when navigating the same staircase but asked to walk outside. Presumably, expecting the staircase to be important made participants direct their gaze at more steps, despite the identical requirements when on the staircase. This illustrates that behavior can be influenced by expectations, such as expectations resulting from task instructions, even when studies are conducted in natural settings.

Abstract Sporadic Parkinson’s Disease (sPD) is a progressive neurodegenerative disorder caused by multiple genetic and environmental factors. Mitochondrial dysfunction is one contributing factor, but its role at different stages of disease progression is not fully understood. Here, we showed that neural precursor cells and dopaminergic neurons derived from induced pluripotent stem cells (hiPSCs) from sPD patients exhibited a hypometabolism. Further analysis based on transcriptomics, proteomics, and metabolomics identified the citric acid cycle, specifically the α-ketoglutarate dehydrogenase complex (OGDHC), as bottleneck in sPD metabolism. A follow-up study of the patients approximately 10 years after initial biopsy demonstrated a correlation between OGDHC activity in our cellular model and the disease progression. In addition, the alterations in cellular metabolism observed in our cellular model were restored by interfering with the enhanced SHH signal transduction in sPD. Thus, inhibiting overactive SHH signaling may have potential as neuroprotective therapy during early stages of sPD.

Abstract Type I interferon (IFN) signalling is tightly controlled. Upon recognition of DNA by cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING) translocates along the endoplasmic reticulum (ER)-Golgi axis to induce IFN signalling. Termination is achieved through autophagic degradation or recycling of STING by retrograde Golgi-to-ER transport. Here, we identify the GTPase ADP-ribosylation factor 1 (ARF1) as a crucial negative regulator of cGAS-STING signalling. Heterozygous ARF1 missense mutations cause a previously unrecognized type I interferonopathy associated with enhanced IFN-stimulated gene expression. Disease-associated, GTPase-defective ARF1 increases cGAS-STING dependent type I IFN signalling in cell lines and primary patient cells. Mechanistically, mutated ARF1 perturbs mitochondrial morphology, causing cGAS activation by aberrant mitochondrial DNA release, and leads to accumulation of active STING at the Golgi/ERGIC due to defective retrograde transport. Our data show an unexpected dual role of ARF1 in maintaining cGAS-STING homeostasis, through promotion of mitochondrial integrity and STING recycling.

Abstract Recent advances in genomic technologies expand the scope and efficiency of preimplantation genetic testing (PGT). We previously developed Haploseek, a clinically-validated, variant-agnostic comprehensive PGT solution. Haploseek is based on microarray genotyping of the embryo’s parents and relatives, combined with low-pass sequencing of the embryos. Here, to increase throughput and versatility, we aimed to develop a sequencing-only implementation of Haploseek. Accordingly, we developed SHaploseek, a universal PGT method to determine genome-wide haplotypes of each embryo based on low-pass (≤ 5x) sequencing of the parents and relative(s) along with ultra-low-pass (0.2–0.4x) sequencing of the embryos. We used SHaploseek to analyze five single lymphoblast cells and 31 embryos. We validated the genome-wide haplotype predictions against either bulk DNA, Haploseek, or, at focal genomic sites, PCR-based PGT results. SHaploseek achieved > 99% concordance with bulk DNA in two families from which single cells were derived from grown-up children. In embryos from 12 PGT families, all of SHaploseek’s focal site haplotype predictions were concordant with clinical PCR-based PGT results. Genome-wide, there was > 99% median concordance between Haploseek and SHaploseek’s haplotype predictions. Concordance remained high at all assayed sequencing depths ≥ 2x, as well as with only 1ng of parental DNA input. In subtelomeric regions, significantly more haplotype predictions were high-confidence in SHaploseek compared to Haploseek. In summary, SHaploseek constitutes a single-platform, accurate, and cost-effective comprehensive PGT solution.

Atmospheric gravity waves and traveling ionospheric disturbances can be observed in the neutral atmosphere and the ionosphere at a wide range of spatial and temporal scales. Especially at medium scales, these oscillations are often not resolved in general circulation models and are parameterized. We show that ionospheric disturbances forced by upward-propagating atmospheric gravity waves can be simultaneously observed with the EISCAT very high frequency incoherent scatter radar and the Nordic Meteor Radar Cluster. From combined multi-static measurements, both vertical and horizontal wave parameters can be determined by applying a specially developed Fourier filter analysis method. This method is demonstrated using the example of a strongly pronounced wave mode that occurred during the EISCAT experiment on 7 July 2020. Leveraging the developed technique, we show that the wave characteristics of traveling ionospheric disturbances are notably impacted by the fall transition of the mesosphere and lower thermosphere. We also demonstrate the application of using the determined wave parameters to infer the thermospheric neutral wind velocities. Applying the dissipative anelastic gravity wave dispersion relation, we obtain vertical wind profiles in the lower thermosphere.

Abstract Long-acting injectable medications, such as atovaquone, offer the prospect of a “chemical vaccine” for malaria, combining drug efficacy with vaccine durability. However, selection and transmission of drug-resistant parasites is of concern. Laboratory studies have indicated that atovaquone resistance disadvantages parasites in mosquitoes, but lack of data on clinically relevant Plasmodium falciparum has hampered integration of these variable findings into drug development decisions. Here we generate atovaquone-resistant parasites that differ from wild type parent by only a Y268S mutation in cytochrome b, a modification associated with atovaquone treatment failure in humans. Relative to wild type, Y268S parasites evidence multiple defects, most marked in their development in mosquitoes, whether from Southeast Asia (Anopheles stephensi) or Africa (An. gambiae). Growth of asexual Y268S P. falciparum in human red cells is impaired, but parasite loss in the mosquito is progressive, from reduced gametocyte exflagellation, to smaller number and size of oocysts, and finally to absence of sporozoites. The Y268S mutant fails to transmit from mosquitoes to mice engrafted with human liver cells and erythrocytes. The severe-to-lethal fitness cost of clinically relevant atovaquone resistance to P. falciparum in the mosquito substantially lessens the likelihood of its transmission in the field.

Abstract Male-pattern baldness (MPB) is related to dysregulation of androgens such as testosterone. A previously observed relationship between MPB and skin cancer may be due to greater exposure to ultraviolet radiation or indicate a role for androgenic pathways in the pathogenesis of skin cancers. We dissected this relationship via Mendelian randomization (MR) analyses, using genetic data from recent male-only meta-analyses of cutaneous melanoma (12,232 cases; 20,566 controls) and keratinocyte cancers (KCs) (up to 17,512 cases; >100,000 controls), followed by stratified MR analysis by body-sites. We found strong associations between MPB and the risk of KC, but not with androgens, and multivariable models revealed that this relationship was heavily confounded by MPB single nucleotide polymorphisms involved in pigmentation pathways. Site-stratified MR analyses revealed strong associations between MPB with head and neck squamous cell carcinoma and melanoma, suggesting that sun exposure on the scalp, rather than androgens, is the main driver. Men with less hair covering likely explains, at least in part, the higher incidence of melanoma in men residing in countries with high ambient UV.

Abstract Determination of per- and polyfluoroalkyl substances (PFAS) in drinking water at the low levels set by regulatory officials has been a major focus for sensor developing researchers. However, it is becoming more apparent that detection of these contaminants in soils, foods and consumer products is relevant and necessary at part per billion and even part per million levels. Here, a fluorescent biosensor for the rapid detection of PFOA was engineered based on human liver fatty acid binding protein (hLFABP). By conjugating circularly permuted green fluorescent protein (cp.GFP) to a split hLFABP construct, the biosensor was able to detect perfluorooctanoic acid PFOA in PBS as well as environmental water samples with LODs of 236 and 330 ppb respectively. Furthermore, E. coli cells cytosolically expressing the protein-based sensor were demonstrated to quickly detect PFOA, demonstrating feasibility of whole-cell sensing. Overall, this work demonstrates a platform technology utilizing a circularly permuted GFP and split hLFABP conjugate as a label-free optical biosensor for PFOA.

Abstract Food packaging reinforced with Zn-doped TiO2 nanoparticles with enhanced prerequisite film-forming and biodegradable traits was prepared to augment fresh food storage. Pure and tailored metal (Zinc, Copper, and Selenium) doped TiO2 nanoparticles were synthesized and analyzed through multiple characterization techniques (optical spectra, XRD patterns (X-Ray Diffraction), Dynamic Light Scattering, and Scanning Electron Microscopy). The synthesized nanoparticles were tested for their Minimum Inhibitory Concentrations, antimicrobial potential against common lethal food pathogens, and cytotoxicity. Compared to Cu- and Se-doped nanoparticles, Zn-doped TiO2 nanoparticles displayed the most potent antimicrobial activity with insignificant cytotoxicity and were incorporated into the food packaging materials. The developed nano-reinforced food packaging efficaciously augmented the freshness of plums (Oemleria cerasiformis) for 16 days (42 ± 2 °C). The physicomechanical characterization of the nano-reinforced packaging establishes its utility in food packaging applications. The developed biodegradable packaging undergoes complete decomposition within 12 days of storage in natural soil.

Abstract The microvasculature plays a key role in tissue perfusion and exchange of gases and metabolites. In this study we use human blood vessel organoids (BVOs) as a model of the microvasculature. BVOs fully recapitulate key features of the human microvasculature, including the reliance of mature endothelial cells on glycolytic metabolism, as concluded from metabolic flux assays and mass spectrometry-based metabolomics using stable tracing of 13C-glucose. Pharmacological targeting of PFKFB3, an activator of glycolysis, using two chemical inhibitors results in rapid BVO restructuring, vessel regression with reduced pericyte coverage. PFKFB3 mutant BVOs also display similar structural remodelling. Proteomic analysis of the BVO secretome reveal remodelling of the extracellular matrix and differential expression of paracrine mediators such as CTGF. Treatment with recombinant CTGF recovers microvessel structure. In this work we demonstrate that BVOs rapidly undergo restructuring in response to metabolic changes and identify CTGF as a critical paracrine regulator of microvascular integrity.

One of the oldest problems in physics is that of calculating the motion of N particles under a specified mutual force: the N-body problem. Much is known about this problem if the specified force is non-relativistic gravity, and considerable progress has been made by considering the problem in one spatial dimension. Here, I review what is known about the relativistic gravitational N-body problem. Reduction to one spatial dimension has the feature of the absence of gravitational radiation, thereby allowing for a clear comparison between the physics of one-dimensional relativistic and non-relativistic self-gravitating systems. After describing how to obtain a relativistic theory of gravity coupled to N point particles, I discuss in turn the two-body, three-body, four-body, and N-body problems. Quite general exact solutions can be obtained for the two-body problem, unlike the situation in general relativity in three spatial dimensions for which only highly specified solutions exist. The three-body problem exhibits mild forms of chaos, and provides one of the first theoretical settings in which relativistic chaos can be studied. For N≥4, other interesting features emerge. Relativistic self-gravitating systems have a number of interesting problems awaiting further investigation, providing us with a new frontier for exploring relativistic many-body systems.

With the advent of robust and high-throughput mass spectrometric technologies and bioinformatics tools to analyze large data sets, proteomics has penetrated broadly into basic and translational life sciences research. More than 95% of FDA-approved drugs currently target proteins, and most diagnostic tests are protein-based. The introduction of proteomics to the clinic, for instance to guide patient stratification and treatment, is already ongoing. Importantly, ethical challenges come with this success, which must also be adequately addressed by the proteomics and medical communities. Consortium members of the H2020 European Union-funded proteomics initiative: European Proteomics Infrastructure Consortium-providing access (EPIC-XS) met at the Core Technologies for Life Sciences (CTLS) conference to discuss the emerging role and implementation of proteomics in the clinic. The discussion, involving leaders in the field, focused on the current status, related challenges, and future efforts required to make proteomics a more mainstream technology for translational and clinical research. Here we report on that discussion and provide an expert update concerning the feasibility of clinical proteomics, the ethical implications of generating and analyzing large-scale proteomics clinical data, and recommendations to ensure both ethical and effective implementation in real-world applications.

Abstract The cytoplasmic droplet is a conserved dilated area of cytoplasm situated at the neck of the sperm flagellum. Viewed as residual cytoplasm inherited from late spermatids, the cytoplasmic droplet contains numerous saccular elements as its key content. However, the origin of these saccules and the function of the cytoplasmic droplet have long been speculative. Here, we identify the molecular origin of these cytoplasmic droplet components by uncovering a vesicle pathway essential for formation and sequestration of saccules within the cytoplasmic droplet. This process is governed by a transmembrane protein SYPL1 and its interaction with VAMP3. Genetic ablation of SYPL1 in mice reveals that SYPL1 dictates the formation and accumulation of saccular elements in the forming cytoplasmic droplet. Derived from the Golgi, SYPL1 vesicles are critical for segregation of key metabolic enzymes within the forming cytoplasmic droplet of late spermatids and epididymal sperm, which are required for sperm development and male fertility. Our results uncover a mechanism to actively form and segregate saccules within the cytoplasmic droplet to promote sperm fertility.

Abstract Systemic pan-tumor analyses may reveal the significance of common features implicated in cancer immunogenicity and patient survival. Here, we provide a comprehensive multi-omics data set for 32 patients across 25 tumor types for proteogenomic-based discovery of neoantigens. By using an optimized computational approach, we discover a large number of tumor-specific and tumor-associated antigens. To create a pipeline for the identification of neoantigens in our cohort, we combine DNA and RNA sequencing with MS-based immunopeptidomics of tumor specimens, followed by the assessment of their immunogenicity and an in-depth validation process. We detect a broad variety of non-canonical HLA-binding peptides in the majority of patients demonstrating partially immunogenicity. Our validation process allows for the selection of 32 potential neoantigen candidates. The majority of neoantigen candidates originates from variants identified in the RNA data set, illustrating the relevance of RNA as a still understudied source of cancer antigens. This study underlines the importance of RNA-centered variant detection for the identification of shared biomarkers and potentially relevant neoantigen candidates.

Abstract The neuronal RNA-binding protein Ptbp2 regulates neuronal differentiation by modulating alternative splicing programs in the nucleus. Such programs contribute to axonogenesis by adjusting the levels of protein isoforms involved in axon growth and branching. While its functions in alternative splicing have been described in detail, cytosolic roles of Ptbp2 for axon growth have remained elusive. Here, we show that Ptbp2 is located in the cytosol including axons and growth cones of motoneurons, and that depletion of cytosolic Ptbp2 affects axon growth. We identify Ptbp2 as a major interactor of the 3’ UTR of Hnrnpr mRNA encoding the RNA-binding protein hnRNP R. Axonal localization of Hnrnpr mRNA and local synthesis of hnRNP R protein are strongly reduced when Ptbp2 is depleted, leading to defective axon growth. Ptbp2 regulates hnRNP R translation by mediating the association of Hnrnpr with ribosomes in a manner dependent on the translation factor eIF5A2. Our data thus suggest a mechanism whereby cytosolic Ptbp2 modulates axon growth by fine-tuning the mRNA transport and local synthesis of an RNA-binding protein.

Abstract Metabolic dysfunction underlies several chronic diseases. Dietary interventions can reverse metabolic declines and slow aging but remaining compliant is difficult. 17α-estradiol (17α-E2) treatment improves metabolic parameters and slows aging in male mice without inducing significant feminization. We recently reported that estrogen receptor α is required for the majority of 17α-E2-mediated benefits in male mice, but that 17α-E2 also attenuates fibrogenesis in liver, which is regulated by estrogen receptor β (ERβ)-expressing hepatic stellate cells (HSC). The current studies sought to determine if 17α-E2-mediated benefits on systemic and hepatic metabolism are ERβ-dependent. We found that 17α-E2 treatment reversed obesity and related systemic metabolic sequela in both male and female mice, but this was partially blocked in female, but not male, ERβKO mice. ERβ ablation in male mice attenuated 17α-E2-mediated benefits on hepatic stearoyl-coenyzme A desaturase 1 (SCD1) and transforming growth factor β1 (TGF-β1) production, which play critical roles in HSC activation and liver fibrosis. We also found that 17α-E2 treatment suppresses SCD1 production in cultured hepatocytes and hepatic stellate cells, indicating that 17α-E2 directly signals in both cell-types to suppress drivers of steatosis and fibrosis. We conclude that ERβ partially controls 17α-E2-mediated benefits on systemic metabolic regulation in female, but not male, mice, and that 17α-E2 likely signals through ERβ in HSCs to attenuate pro-fibrotic mechanisms.

Numerous studies from Europe and North America have documented sexual orientation-based health disparities, but due to data limitations, very little is known about the health of sexual minorities (i.e., lesbians, gay men, bisexual individuals, and other non-heterosexual populations) in developing countries. This research note uses newly available nationally representative data from the Chilean Socio-Economic Characterization Survey (CASEN) to explore sexual orientation-based disparities in self-rated health, health insurance coverage, and healthcare utilization in Chile. Our findings indicate that sexual minority respondents report worse self-rated health and greater health care utilization, and that sexual minority men are more likely to have private health insurance relative to heterosexual men. These findings are important in facilitating continued efforts to reduce health disparities in Latin America.

BackgroundAlthough DBT is the standard initial imaging modality for women with focal breast symptoms, the importance of ultrasound has grown rapidly in the past decades. Therefore, the Breast UltraSound Trial (BUST) focused on assessing the diagnostic value of ultrasound and digital breast tomosynthesis (DBT) for the evaluation of breast symptoms by reversing the order of breast imaging; first performing ultrasound followed by DBT. This side-study of the BUST evaluates patients' perceptions of ultrasound and DBT in a reversed setting.MethodsAfter imaging, 1181/1276 BUST participants completed a survey consisting of open and closed questions regarding both exams (mean age 47.2, ±11.74). Additionally, a different subset of BUST participants (n = 29) participated in six focus group interviews 18-24 months after imaging to analyze their imaging experiences in depth.ResultsA total of 55.3% of women reported reluctance to undergoing DBT, primarily due of pain, while the vast majority also find bilateral DBT reassuring (87.3%). Thematic analysis identified themes related to 1) imaging reluctance (pain/burden, result, and breast harm) and 2) ultrasound and DBT perceptions. Regarding the latter, the theme comfort underscores DBT as burdensome and painful, while ultrasound is largely perceived as non-burdensome. Ultrasound is also particularly valued for its interactive nature, as highlighted in the theme interaction. Perceived effectiveness reflects women's interest in bilateral breast evaluation with DBT and the visibility of lesions, while they express more uncertainty about the reliability of ultrasound. Emotional impact portrays DBT as reassuring for many women, whereas opinions on the reassurance provided by ultrasound are more diverse. Additional themes include costs, protocols and privacy.ConclusionsUltrasound is highly tolerated, and particularly valued is the interaction with the radiologist. Nearly half of women express reluctance towards DBT; nevertheless, a large portion report feeling more confident after undergoing bilateral DBT, reassuring them of the absence of abnormalities. Understanding patients' perceptions of breast imaging examinations is of great value when optimizing diagnostic pathways.