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Abstract Neutron imaging has gained significant importance as a material characterisation technique and is particularly useful to visualise hydrogenous materials in objects opaque to other radiations. Fields of application include investigations of hydrogen in metals as well as metal corrosion, thanks to the fact that neutrons can penetrate metals better than e.g. X-rays and are highly sensitive to hydrogen. However, at interfaces refraction effects sometimes obscure the attenuation image, which is used for hydrogen quantification. Refraction, a differential phase effect, diverts the neutron beam away from the interface in the image leading to intensity gain and intensity loss regions, which are superimposed to the attenuation image, thus obscuring the interface region and hindering quantitative analyses of e.g. hydrogen content in the vicinity of the interface. For corresponding effects in X-ray imaging, a phase filter approach was developed and is generally based on transport-of-intensity considerations. Here, we compare such an approach, that has been adapted to neutrons, with another simulation-based assessment using the ray-tracing software McStas. The latter appears superior and promising for future extensions which enable fitting forward models via simulations in order to separate phase and attenuation effects and thus pave the way for overcoming quantitative limitations at refracting interfaces.

Abstract Intransitive gestures are expressive and symbolic, whereas pantomimes are object-related actions. These gestures convey different meanings depending on whether they are directed toward (TB) or away from the body (AB). TB gestures express mental states (intransitive) or hygiene/nutritional activities (pantomime), while AB gestures modify the behaviour of the observer (intransitive) or demonstrate tool use with an object (pantomime). A substantial body of literature suggests that females exhibit stronger social cue processing compared to males. Considering the social significance of gestures, this study aimed to explore the physiological gender differences in the observation of AB and TB gestures. Pupil dilation and High Frequency Heart Rate Variability (HF-HRV) were measured in 54 participants (27 female) while observing TB and AB gestures. The Interpersonal Reactivity Index (IRI) and the Vicarious Distress Questionnaire (VDQ) were used to assess social-emotional processes. Results showed greater pupil dilation in females for TB gestures, but no significant gender differences for HF-HRV. Males showed a significant correlation between increased pupil dilation to both TB and AB gestures and empathy levels (IRI). The support scale of the VDQ correlated significantly with TB gestures in males. These findings provide insight into the neurobiological basis of gender differences in perceiving social gestures.

Abstract Postoperative fluid overload (FO) after cardiac surgery is common and affects recovery. Predicting FO could help optimize fluid management. This post-hoc analysis of the HERACLES randomized controlled trial evaluated the predictive value of MR-proADM for FO post-cardiac surgery. MR-proADM levels were measured at four different timepoints in 33 patients undergoing elective cardiac surgery. Patients were divided into FO (> 5% weight gain) and no-FO at ICU discharge. The primary outcome was the predictive power of MR-proADM at ICU admission for FO at discharge. Secondary outcomes included the predictive value of MR-proADM for FO on day 6 post-surgery and changes over time. The association between MR-proADM and FO at ICU discharge or day 6 post-surgery was not significant (crude odds ratio (cOR): 4.3 (95% CI 0.5–40.9, p = 0.201) and cOR 1.1 (95% CI 0.04–28.3, p = 0.954)). MR-proADM levels over time did not differ significantly between patients with and without FO at ICU discharge (p = 0.803). MR-proADM at ICU admission was not associated with fluid overload at ICU discharge in patients undergoing elective cardiac surgery. MR-proADM levels over time were not significantly different between groups, although elevated levels were observed in patients with FO.

Abstract Within the brain, the connections between neurons are constantly changing in response to environmental stimuli. A prime environmental regulator of neuronal activity is diet, and previous work has highlighted changes in hypothalamic connections in response to diets high in dietary fat and elevated sucrose. We sought to determine if the change in hypothalamic neuronal connections was driven primarily by an elevation in dietary fat alone. Analysis was performed in both male and female animals. We measured Agouti-related peptide (AgRP) neuropeptide and Synaptophysin markers in the paraventricular nucleus of the hypothalamus (PVH) in response to an acute 48 h high fat diet challenge. Using two image analysis methods described in previous studies, an effect of a high fat diet on AgRP neuronal projections in the PVH of male or female mice was not identified. These results suggest that it may not be dietary fat alone that is responsible for the previously published alterations in hypothalamic connections. Future work should focus on deciphering the role of individual macronutrients on neuroanatomical and functional changes.

Abstract As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor (ACE2-OE) that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. Interferon-lambda was upregulated in cells with low-level infection while the NF-kB inhibitor alpha gene (encoding IkBa) was consistently upregulated in infected cells, and its expression positively correlated with infection levels. Confocal microscopy showed more IkBa expression in infected than bystander cells, but found concurrent nuclear translocation of NF-kB that IkBa usually prevents. Overexpressing a nondegradable IkBa mutant reduced NF-kB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and underscore that the strength of the NF-kB feedback loop in infected cells controls viral replication.

Abstract In advanced drug delivery, versatile liposomal formulations are commonly employed for safer and more accurate therapies. Here we report a method that allows a straightforward production of synthetic monodisperse (~ 100 μm) giant unilamellar vesicles (GUVs) using a microfluidic system. The stability analysis based on the microscopy imaging showed that at ambient conditions the produced GUVs had a half-life of 61 ± 2 h. However, it was observed that ~ 90% of the calcein dye that was loaded into GUVs was transported into a surrounding medium in 24 h, thus indicating that the GUVs may release these small dye molecules without distinguishable membrane disruption. We further demonstrated the feasibility of our method by loading GUVs with larger and very different cargo objects; small soluble fluorescent proteins and larger magnetic microparticles in a suspension. Compared to previously reported microfluidics-based production techniques, the obtained results indicate that our simplified method could be equally harnessed in creating GUVs with less cost, effort and time, which could further benefit studying closed membrane systems.

Abstract Next-generation sequencing (NGS) is emerging as a powerful tool for molecular diagnostics but remains limited by cumbersome and inefficient sample preparation. We present an innovative automated NGS library preparation system with a simplified mechanical design that exploits both macro- and microfluidic properties for optimizing heat transfer, reaction kinetics, mass transfer, fluid mechanics, adsorption–desorption rates, and molecular thermodynamics. Our approach introduces a unique two-cannula cylindrical capillary system connected to a programmable syringe pump and a Peltier heating element able to execute all steps with high efficiency. Automatic reagent movement, mixing, and magnetic bead-based washing with capillary-based thermal cycling (capillary-PCR) are completely integrated into a single platform. The manual 3-h library preparation process is reduced to less than 15 min of hands-on time via optimally pre-plated reagent plates, followed by less than 6 h of instrument run time during which no user interaction is required. We applied this method to two library preparation assays with different DNA fragmentation requirements (mechanical vs. enzymatic fragmentation), sufficiently limiting consumable use to one cartridge and one 384 well-plate per run. Our platform successfully prepared eight libraries in parallel, generating sequencing data for both human and Escherichia coli DNA libraries with negligible coverage bias compared to positive controls. All sequencing data from our libraries attained Phred (Q) scores > 30, mapping to reference genomes at 99% confidence. The method achieved final library concentrations and size distributions comparable with the conventional manual approach, demonstrating compatibility with downstream sequencing and subsequent data analysis. Our engineering design offers repeatability and consistency in the quality of sequence-able libraries, asserting the importance of mechanical design considerations that employ and optimize fundamental fluid mechanics and heat transfer properties. Furthermore in this work, we provide unique insights into the mechanisms of sample loss within NGS library preparation assays compared with automated adaptations and pinpoint areas in which the principles of thermodynamics, fluid mechanics, and heat transfer can improve future mechanical design iterations.

Abstract The thalamic reticular nucleus (TRN) is a brain region that influences vital neurobehavioral processes, including executive functioning and the generation of sleep rhythms. TRN dysfunction underlies hyperactivity, attention deficits, and sleep disturbances observed across various neurodevelopmental disorders. A specialized sarco-endoplasmic reticulum calcium (Ca2+) ATPase 2 (SERCA2)-dependent Ca2+ signaling network operates in the dendrites of TRN neurons to regulate their bursting activity. Phospholamban (PLN) is a prominent regulator of SERCA2 with an established role in myocardial Ca2 +-cycling. Our findings suggest that the role of PLN extends beyond the cardiovascular system to impact brain function. Specifically, we found PLN to be expressed in TRN neurons of the adult mouse brain, and utilized global constitutive and innovative conditional genetic knockout mouse models in concert with electroencephalography (EEG)-based somnography and the 5-choice serial reaction time task (5-CSRTT) to investigate the role of PLN in sleep and executive functioning, two complex behaviors that map onto thalamic reticular circuits. The results of the present study indicate that perturbed PLN function in the TRN results in aberrant TRN-dependent phenotypes in mice (i.e., hyperactivity, impulsivity and sleep deficits) and support a novel role for PLN as a critical regulator of SERCA2 in the TRN neurocircuitry.

We report a method for the phase reconstruction of an ultrashort laser pulse based on the deep learning of the nonlinear spectral changes induce by self-phase modulation. The neural networks were trained on simulated pulses with random initial phases and spectra, with pulse durations between 8.5 and 65 fs. The reconstruction is valid with moderate spectral resolution, and is robust to noise. The method was validated on experimental data produced from an ultrafast laser system, where near real-time phase reconstructions were performed. This method can be used in systems with known linear and nonlinear responses, even when the fluence is not known, making this method ideal for difficult to measure beams such as the high energy, large aperture beams produced in petawatt systems.

The novel SARS-CoV-2 virus emerged in December 2019 and has few effective treatments. We applied a computational drug repositioning pipeline to SARS-CoV-2 differential gene expression signatures derived from publicly available data. We utilized three independent published studies to acquire or generate lists of differentially expressed genes between control and SARS-CoV-2-infected samples. Using a rank-based pattern matching strategy based on the Kolmogorov-Smirnov Statistic, the signatures were queried against drug profiles from Connectivity Map (CMap). We validated 16 of our top predicted hits in live SARS-CoV-2 antiviral assays in either Calu-3 or 293T-ACE2 cells. Validation experiments in human cell lines showed that 11 of the 16 compounds tested to date (including clofazimine, haloperidol and others) had measurable antiviral activity against SARS-CoV-2. These initial results are encouraging as we continue to work towards a further analysis of these predicted drugs as potential therapeutics for the treatment of COVID-19.

Abstract Zinc inhibits intestinal copper uptake, an effect utilized for treating Wilson’s disease (WD). We used copper-64 (64Cu) PET/CT to examine how much four weeks of treatment with different zinc regimens reduced the hepatic 64Cu content after oral 64Cu administration and test if alternative regimens were noninferior to the standard regimen of zinc acetate 50 mg × 3 daily. Forty healthy persons were randomized to four different zinc protocols. The WD standard treatment zinc acetate 50 mg × 3 reduced the hepatic 64Cu content from 26.9 ± 7.5% to 13.3 ± 5.6% of the administered 64Cu. Zinc gluconate 50 mg × 3 was noninferior (P = 0.02) (35.8 ± 9.0% to 17.4 ± 7.5%). Zinc acetate 150 mg × 1 (33.1 ± 9.9% to 17.4 ± 7.5%) and zinc gluconate 150 mg × 1 (28.1 ± 6.7% to 22.0 ± 6.7%) were less effective. These effects were intra- and inter-individually highly variable, and 14% had no effect of any zinc regimen, which may explain disparities in zinc treatment efficacy in WD patients.

The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson's disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn. Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson's disease.

Abstract Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), also known as Rhodanese, was initially discovered as a cyanide detoxification enzyme. However, it was recently also found to be a genetic predictor of resistance to obesity-related type 2 diabetes. Diabetes type 2 is characterized by progressive loss of adequate β-cell insulin secretion and onset of insulin resistance with increased insulin demand, which contributes to the development of hyperglycemia. Diabetic complications have been replicated in adult hyperglycemic zebrafish, including retinopathy, nephropathy, impaired wound healing, metabolic memory, and sensory axonal degeneration. Pancreatic and duodenal homeobox 1 (Pdx1) is a key component in pancreas development and mature beta cell function and survival. Pdx1 knockdown or knockout in zebrafish induces hyperglycemia and is accompanied by organ alterations similar to clinical diabetic retinopathy and diabetic nephropathy. Here we show that pdx1-knockdown zebrafish embryos and larvae survived after incubation with thiosulfate and no obvious morphological alterations were observed. Importantly, incubation with hTST and thiosulfate rescued the hyperglycemic phenotype in pdx1-knockdown zebrafish pronephros. Activation of the mitochondrial TST pathway might be a promising option for therapeutic intervention in diabetes and its organ complications.

Abstract We report a method for the phase reconstruction of an ultrashort laser pulse based on the deep learning of the nonlinear spectral changes induce by self-phase modulation. The neural networks were trained on simulated pulses with random initial phases and spectra, with pulse durations between 8.5 and 65 fs. The reconstruction is valid with moderate spectral resolution, and is robust to noise. The method was validated on experimental data produced from an ultrafast laser system, where near real-time phase reconstructions were performed. This method can be used in systems with known linear and nonlinear responses, even when the fluence is not known, making this method ideal for difficult to measure beams such as the high energy, large aperture beams produced in petawatt systems.

Abstract Climate change and disturbance can alter invasion success of clonal plants by differentially affecting the clonal traits influencing their establishment as young plants. Clonal traits related to the vegetative reproduction of native Pascopyrum smithii and non-native Bromus inermis grass seedlings were evaluated under altered precipitation frequencies and a single grazing event. Pascopyrum smithii maintained similar vegetative reproduction under three simulated precipitation frequencies whereas B. inermis vegetative reproduction declined as precipitation became more intermittent. Vegetative reproduction of the non-native B. inermis was greater than the native P. smithii under all simulated precipitation frequencies except the most intermittent scenario. A single grazing event did not affect either species’ response to intra-annual precipitation variability but did slightly reduce their clonal growth and increase their bud dormancy. In young plants, clonal traits of the invasive grass favored its superior expansion and population growth compared to the native grass except under the most severe climate change scenario. Grassland restoration using native P. smithii seeds would be successful in most years due to its resilient clonal growth in a changing climate. Clonal infrastructure development in young plants is critical to clonal plant establishment and persistence in a changing climate and under disturbed conditions.

Postoperative fluid overload (FO) after cardiac surgery is common and affects recovery. Predicting FO could help optimize fluid management. This post-hoc analysis of the HERACLES randomized controlled trial evaluated the predictive value of MR-proADM for FO post-cardiac surgery. MR-proADM levels were measured at four different timepoints in 33 patients undergoing elective cardiac surgery. Patients were divided into FO (> 5% weight gain) and no-FO at ICU discharge. The primary outcome was the predictive power of MR-proADM at ICU admission for FO at discharge. Secondary outcomes included the predictive value of MR-proADM for FO on day 6 post-surgery and changes over time. The association between MR-proADM and FO at ICU discharge or day 6 post-surgery was not significant (crude odds ratio (cOR): 4.3 (95% CI 0.5–40.9, p = 0.201) and cOR 1.1 (95% CI 0.04–28.3, p = 0.954)). MR-proADM levels over time did not differ significantly between patients with and without FO at ICU discharge (p = 0.803). MR-proADM at ICU admission was not associated with fluid overload at ICU discharge in patients undergoing elective cardiac surgery. MR-proADM levels over time were not significantly different between groups, although elevated levels were observed in patients with FO. [ABSTRACT FROM AUTHOR]

Abstract A clinical implementation of cell-based bone regeneration in combination with scaffold materials requires the development of efficient, controlled and reproducible seeding procedures and a tailor-made bioreactor design. A perfusion system for efficient, homogeneous, and rapid seeding with human adipogenic stem cells in bone substitute scaffolds was designed. Variants concerning medium inlet and outlet port geometry, i.e. cylindrical or conical diffuser, cell concentration, perfusion mode and perfusion rates were simulated in silico. Cell distribution during perfusion was monitored by dynamic [18F]FDG micro-PET/CT and validated by laser scanning microscopy with three-dimensional image reconstruction. By iterative feedback of the in silico and in vitro experiments, the homogeneity of cell distribution throughout the scaffold was optimized with adjustment of flow rates, cell density and perfusion properties. Finally, a bioreactor with a conical diffusor geometry was developed, that allows a homogeneous cell seeding (hoover coefficient: 0.24) in less than 60 min with an oscillating perfusion mode. During this short period of time, the cells initially adhere within the entire scaffold and stay viable. After two weeks, the formation of several cell layers was observed, which was associated with an osteogenic differentiation process. This newly designed bioreactor may be considered as a prototype for chairside application.

Abstract A non-invasive and sensitive blood test has long been a goal for early stage disease diagnosis and treatment for Alzheimer’s disease (AD) and other proteinopathy diseases. We previously reported that preeclampsia (PE), a severe pregnancy complication, is another proteinopathy disorder with impaired autophagy. We hypothesized that induced autophagy deficiency would promote accumulation of pathologic protein aggregates. Here, we describe a novel, sensitive assay that detects serum protein aggregates from patients with PE (n = 33 early onset and 33 late onset) and gestational age-matched controls (n = 77) as well as AD in both dementia and prodromal mild cognitive impairment (MCI, n = 24) stages with age-matched controls (n = 19). The assay employs exposure of genetically engineered, autophagy-deficient human trophoblasts (ADTs) to serum from patients. The aggregated protein complexes and their individual components, including transthyretin, amyloid β-42, α-synuclein, and phosphorylated tau231, can be detected and quantified by co-staining with ProteoStat, a rotor dye with affinity to aggregated proteins, and respective antibodies. Detection of protein aggregates in ADTs was not dependent on transcriptional upregulation of these biomarkers. The ROC curve analysis validated the robustness of the assay for its specificity and sensitivity (PE; AUC: 1, CI: 0.949–1.00; AD; AUC: 0.986, CI: 0.832–1.00). In conclusion, we have developed a novel, noninvasive diagnostic and predictive assay for AD, MCI and PE.

Abstract Cilia are protrusions of the cell surface and composed of hundreds of proteins many of which are evolutionary and functionally well conserved. In cells assembling motile cilia the expression of numerous ciliary components is under the control of the transcription factor FOXJ1. Here, we analyse the evolutionary conserved FOXJ1 target CFAP161 in Xenopus and mouse. In both species Cfap161 expression correlates with the presence of motile cilia and depends on FOXJ1. Tagged CFAP161 localises to the basal bodies of multiciliated cells of the Xenopus larval epidermis, and in mice CFAP161 protein localises to the axoneme. Surprisingly, disruption of the Cfap161 gene in both species did not lead to motile cilia-related phenotypes, which contrasts with the conserved expression in cells carrying motile cilia and high sequence conservation. In mice mutation of Cfap161 stabilised the mutant mRNA making genetic compensation triggered by mRNA decay unlikely. However, genes related to microtubules and cilia, microtubule motor activity and inner dyneins were dysregulated, which might buffer the Cfap161 mutation.

Abstract The novel SARS-CoV-2 virus emerged in December 2019 and has few effective treatments. We applied a computational drug repositioning pipeline to SARS-CoV-2 differential gene expression signatures derived from publicly available data. We utilized three independent published studies to acquire or generate lists of differentially expressed genes between control and SARS-CoV-2-infected samples. Using a rank-based pattern matching strategy based on the Kolmogorov–Smirnov Statistic, the signatures were queried against drug profiles from Connectivity Map (CMap). We validated 16 of our top predicted hits in live SARS-CoV-2 antiviral assays in either Calu-3 or 293T-ACE2 cells. Validation experiments in human cell lines showed that 11 of the 16 compounds tested to date (including clofazimine, haloperidol and others) had measurable antiviral activity against SARS-CoV-2. These initial results are encouraging as we continue to work towards a further analysis of these predicted drugs as potential therapeutics for the treatment of COVID-19.

Abstract Atom Probe Tomography (APT) is currently a well-established technique to analyse the composition of solid materials including metals, semiconductors and ceramics with up to near-atomic resolution. Using an aqueous glucose solution, we now extended the technique to frozen solutions. While the mass signals of the common glucose fragments C x H y and C x O y H z overlap with (H2O) n H from water, we achieved stoichiometrically correct values via signal deconvolution. Density functional theory (DFT) calculations were performed to investigate the stability of the detected pyranose fragments. This paper demonstrates APT’s capabilities to achieve sub-nanometre resolution in tracing whole glucose molecules in a frozen solution by using cryogenic workflows. We use a solution of defined concentration to investigate the chemical resolution capabilities as a step toward the measurement of biological molecules. Due to the evaporation of nearly intact glucose molecules, their position within the measured 3D volume of the solution can be determined with sub-nanometre resolution. Our analyses take analytical techniques to a new level, since chemical characterization methods for cryogenically-frozen solutions or biological materials are limited.

Abstract There are no clinical studies that have investigated the differences in blood serum metabolome between obstructive sleep apnea (OSA) patients and controls. In a single-center prospective observational study, we compared metabolomic profiles in the serum of OSA patients with apnea–hypopnea index (AHI) ≥ 15/h and control individuals. Peripheral blood was obtained at 3 different time points overnight: 9:00 p.m.; 5:00 a.m. and 7:00 a.m. We used a targeted approach for detecting amino acids and biogenic amines and analyzed the data with ranked general linear model for repeated measures. We recruited 31 patients with moderate-to-severe OSA and 32 controls. Significant elevations in median concentrations of alanine, proline and kynurenine in OSA patients compared to controls were detected. Significant changes in the overnight dynamics of serum concentrations occurred in OSA: glutamine, serine, threonine, tryptophan, kynurenine and glycine levels increased, whereas a fall occurred in the same biomarker levels in controls. Phenylalanine and proline levels decreased slightly, compared to a steeper fall in controls. The study indicates that serum profiles of amino acid and biogenic amines are significantly altered in patients with OSA referring to vast pathophysiologic shifts reflected in the systemic metabolism.

Abstract The pig is an important model organism for biomedical research, mainly due to its extensive genetic, physiological and anatomical similarities with humans. Until date, direct conversion of somatic cells into hepatocyte-like cells (iHeps) has only been achieved in rodents and human cells. Here, we employed lentiviral vectors to screen a panel of 12 hepatic transcription factors (TF) for their potential to convert porcine fibroblasts into hepatocyte-like cells. We demonstrate for the first time, hepatic conversion of porcine somatic cells by over-expression of CEBPα, FOXA1 and HNF4α2 (3TF-piHeps). Reprogrammed 3TF-piHeps display a hepatocyte-like morphology and show functional characteristics of hepatic cells, including albumin secretion, Dil-AcLDL uptake, storage of lipids and glycogen and activity of cytochrome P450 enzymes CYP1A2 and CYP2C33 (CYP2C9 in humans). Moreover, we show that markers of mature hepatocytes are highly expressed in 3TF-piHeps, while fibroblastic markers are reduced. We envision piHeps as useful cell sources for future studies on drug metabolism and toxicity as well as in vitro models for investigation of pig-to-human infectious diseases.

Abstract New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients’ anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.

Abstract One major bottleneck in the ongoing COVID-19 pandemic is the limited number of critical care beds. Due to the dynamic development of infections and the time lag between when patients are infected and when a proportion of them enters an intensive care unit (ICU), the need for future intensive care can easily be underestimated. To infer future ICU load from reported infections, we suggest a simple statistical model that (1) accounts for time lags and (2) allows for making predictions depending on different future growth of infections. We have evaluated our model for three heavily affected regions in Europe, namely Berlin (Germany), Lombardy (Italy), and Madrid (Spain). Before extensive containment measures made an impact, we first estimate the region-specific model parameters, namely ICU rate, time lag between infection, and ICU admission as well as length of stay in ICU. Whereas for Berlin, an ICU rate of 6%, a time lag of 6 days, and a stay of 12 days in ICU provide the best fit of the data, for Lombardy and Madrid the ICU rate was higher (18% and 15%) and the time lag (0 and 3 days) and the stay in ICU (3 and 8 days) shorter. The region-specific models are then used to predict future ICU load assuming either a continued exponential phase with varying growth rates (0–15%) or linear growth. By keeping the growth rates flexible, this model allows for taking into account the potential effect of diverse containment measures. Thus, the model can help to predict a potential exceedance of ICU capacity depending on future growth. A sensitivity analysis for an extended time period shows that the proposed model is particularly useful for exponential phases of the disease.

Abstract Finger-tapping tasks are classically used to investigate sensorimotor synchronization in relation to neutral auditory cues, such as metronomes. However, music is more commonly associated with an entrained bodily response, such as toe tapping, or dancing. Here we report an experimental procedure that was designed to bridge the gap between timing and intervention studies by directly comparing the effects of metronome and musical cue types on motor timing abilities across the three naturalistic voluntary actions of finger tapping, toe tapping, and stepping on the spot as a simplified case of whole body movement. Both pacing cues were presented at slow, medium, and fast tempi. The findings suggested that the task of stepping on the spot enabled better timing performances than tapping both in younger and older adults (75+). Timing performances followed an inverse U shape with best performances observed in the medium tempi that were set close to the spontaneous motor tempo in each movement type. Finally, music provided an entrainment effect in addition to pace setting that enabled better motor timing and greater stability than classically reported using a metronome. By applying time-stamp analyses to kinetic data, we demonstrate that tapping and stepping engage different timing modes. This work details the importance of translational research for a better understanding of motor timing. It offers a simple procedure that strengthens the validity of applying academic work and contributes in knowledge towards a wide range of therapeutic interventions.

Abstract LINE-1 hypomethylation of cell-free DNA has been described as an epigenetic biomarker of human aging. However, in the past, insufficient differentiation between cellular and cell-free DNA may have confounded analyses of genome-wide methylation levels in aging cells. Here we present a new methodological strategy to properly and unambiguously extract DNA methylation patterns of repetitive, as well as single genetic loci from pure cell-free DNA from peripheral blood. Since this nucleic acid fraction originates mainly in apoptotic, senescent and cancerous cells, this approach allows efficient analysis of aged and cancerous cell-specific DNA methylation patterns for diagnostic and prognostic purposes. Using this methodology, we observe a significant age-associated erosion of LINE-1 methylation in cfDNA suggesting that the threshold of hypomethylation sufficient for relevant LINE-1 activation and consequential harmful retrotransposition might be reached at higher age. We speculate that this process might contribute to making aging the main risk factor for many cancers.

Abstract Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations.