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Objective Determine the pharmacokinetics (PK) and exposure–response of hydroxychloroquine (HCQ) and desethylhydroxychloroquine (DHCQ) in paediatric SLE (pSLE).Methods We conducted an exploratory phase 2, direct-to-family trial. Children enrolled in the Childhood Arthritis and Rheumatology Research Alliance (CARRA) Registry with a diagnosis of pSLE were eligible if they were receiving HCQ as standard of care for ≥3 months. Biological samples were collected at up to four visits over a 6-month period. At each visit, plasma was obtained to measure the concentrations of HCQ and DHCQ, as well as cytokines. HCQ and DHCQ plasma PK data were analysed using a population PK modelling approach.Results Twenty-five subjects provided a total of 88 plasma concentrations for PK analysis. There was a poor linear fit between HCQ concentrations and total body weight (R2=0.03). There was a decline in both interferon (IFN)-alpha and IFN-gamma with higher concentrations of HCQ and DHCQ. Volume of distribution for HCQ in plasma was higher in children compared with published values in adults (73 000 L vs 44 000 L), but clearance values in children were similar to adults.Conclusions We report the first population PK model for HCQ and DHCQ in children using data from a novel direct-to-family clinical trial. We observed high interindividual variability in HCQ PK and found that weight-based dosing for HCQ is poorly correlated with drug concentrations, suggesting the need to use therapeutic drug monitoring to individualise dosing. Furthermore, our results suggest that the current weight-based dosing paradigm for HCQ may result in suboptimal drug exposures, particularly for children with obesity. Accordingly, additional studies of HCQ are needed in pSLE to determine the optimal drug concentration and dosing to reduce disease activity and improve outcomes.Trial registration number NCT04358302.

Introduction Direct-to-family clinical trials efficiently provide data while reducing the participation burden for children and their families. Although these trials can offer significant advantages over traditional clinical trials, the process of designing and implementing direct-to-family studies is poorly defined, especially in children with rheumatic disease. This paper provides lessons learnt from the design and implementation of a self-controlled, direct-to-family pilot trial aimed to evaluate the effects of a medication management device on adherence to hydroxychloroquine in paediatric SLE.Methods Several design features accommodate a direct-to-family approach. Participants meeting eligibility criteria from across the USA were identified a priori through a disease registry, and all outcome data are collected remotely. The primary outcome (medication adherence) is evaluated using electronic medication event-monitoring, plasma drug levels, patient questionnaires and pill counts. Secondary and exploratory endpoints include (1) lupus disease activity measured by a remote SLE Disease Activity Index examination and the Systemic Lupus Activity Questionnaire; and (2) hydroxychloroquine pharmacokinetics and pharmacodynamics. Recruitment of the initial target of 20 participants was achieved within 10 days. Due to initial recruitment success, enrolment was increased to 26 participants. Additional participants who were interested were placed on a waiting list in case of dropouts during the study.Discussion and dissemination Direct-to-family trials offer several advantages but present unique challenges. Lessons learnt from the protocol development, design, and implementation of this trial will inform future direct-to-family trials for children and adults with rheumatic diseases. Additionally, the data collected remotely in this trial will provide critical information regarding the accuracy of teleresearch in lupus, the impact of adherence to hydroxychloroquine on disease activity and a pharmacokinetic analysis to inform paediatric-specific dosing of hydroxychloroquine.Trial registration number ClinicalTrials.gov Registry (NCT04358302).

Abstract Background The prognosis of children with juvenile dermatomyositis (JDM) has improved remarkably since the 1960’s with the use of corticosteroid and immunosuppressive therapy. Yet there remain a minority of children who have refractory disease. Since 2003 the sporadic use of biologics (genetically-engineered proteins that usually are derived from human genes) for inflammatory myositis has been reported. In 2011–2016 we investigated our collective experience of biologics in JDM through the Childhood Arthritis and Rheumatology Research Alliance (CARRA). Methods The JDM biologic study group developed a survey on the CARRA member experience using biologics for Juvenile DM utilizing Delphi consensus methods in 2011–2012. The survey was completed online by the CARRA members interested in JDM in 2012. A second survey was similarly developed that provided more opportunity to describe their experiences with biologics in JDM in detail and was completed by CARRA members in Feb 2013. During three CARRA meetings in 2013–2015, nominal group techniques were used for achieving consensus on the current choices of biologic drugs. A final survey was performed at the 2016 CARRA meeting. Results One hundred and five of a potential 231 pediatric rheumatologists (42%) responded to the first survey in 2012. Thirty-five of 90 had never used a biologic for Juvenile DM at that time. Fifty-five of 91 (denominators vary) had used biologics for JDM in their practice with 32%, 5%, and 4% using rituximab, etanercept, and infliximab, respectively, and 17% having used more than one of the three drugs. Ten percent used a biologic as monotherapy, 19% a biologic in combination with methotrexate (mtx), 52% a biologic in combination with mtx and corticosteroids, 42% a combination of a biologic, mtx, corticosteroids (steroids), and an immunosuppressive drug, and 43% a combination of a biologic, IVIG and mtx. The results of the second survey supported these findings in considerably more detail with multiple combinations of drugs used with biologics and supported the use of rituximab, abatacept, anti-TNFα drugs, and tocilizumab in that order. One hundred percent recommended that CARRA continue studying biologics for JDM. The CARRA meeting survey in 2016 again supported the study and use of these four biologic drug groups. Conclusions Our CARRA JDM biologic work group developed and performed three surveys demonstrating that pediatric rheumatologists in North America have been using multiple biologics for refractory JDM in numerous scenarios from 2011 to 2016. These survey results and our consensus meetings determined our choice of four biologic therapies (rituximab, abatacept, tocilizumab and anti-TNFα drugs) to consider for refractory JDM treatment when indicated and to evaluate for comparative effectiveness and safety in the future. Significance and Innovations This is the first report that provides a substantial clinical experience of a large group of pediatric rheumatologists with biologics for refractory JDM over five years. This experience with biologic therapies for refractory JDM may aid pediatric rheumatologists in the current treatment of these children and form a basis for further clinical research into the comparative effectiveness and safety of biologics for refractory JDM.

Abstract This paper presents the energy dependence of multiplicity and net-electric charge fluctuations in p+p interactions at beam momenta 20, 31, 40, 80, and 158 $$\text{ Ge }\hspace{-1.00006pt}\text{ V }\!/\!c$$ Ge V / c . Results are corrected for the experimental biases and quantified with the use of cumulants and factorial cumulants. Cumulant ratios are an essential tool in the search for the critical point of strongly interacting matter in heavy ion collisions. Measurements performed in p+p interactions provide a vital baseline estimation in these studies. The measured signals are compared with the string hadronic models Epos1.99 and FTFP-BERT.

Abstract The yields of $$K^0_S$$ K S 0 mesons have been measured in inelastic p+p interactions at incident projectile momenta of 31, 40 and 80 $$\text {Ge}\hspace{-1.00006pt}\text {V}\!/\!c$$ Ge V / c ( $$\sqrt{s_{NN}}=7.7, 8.8$$ s NN = 7.7 , 8.8 and 12.3 $$\text {Ge}\hspace{-1.00006pt}\text {V}$$ Ge V , respectively). The data were recorded by the NA61 $$/$$ / SHINE spectrometer at the CERN Super Proton Synchrotron and the $$K^0_S$$ K S 0 mesons identified via their decays into $$\pi ^{+} \pi ^{-}$$ π + π - pairs. Double-differential distributions are presented as function of transverse momentum and rapidity. The mean multiplicities of $$K^0_S$$ K S 0 mesons were determined to be $$(5.95 \pm 0.19 (stat) \pm 0.30 (sys)) \times 10^{-2}$$ ( 5.95 ± 0.19 ( s t a t ) ± 0.30 ( s y s ) ) × 10 - 2 at 31 $$\text {Ge}\hspace{-1.00006pt}\text {V}\!/\!c$$ Ge V / c , $$(7.61 \pm 0.13 (stat) \pm 0.43 (sys)) \times 10^{-2}$$ ( 7.61 ± 0.13 ( s t a t ) ± 0.43 ( s y s ) ) × 10 - 2 at 40 $$\text {Ge}\hspace{-1.00006pt}\text {V}\!/\!c$$ Ge V / c and $$(11.58 \pm 0.12 (stat) \pm 0.55 (sys)) \times 10^{-2}$$ ( 11.58 ± 0.12 ( s t a t ) ± 0.55 ( s y s ) ) × 10 - 2 at 80 $$\text {Ge}\hspace{-1.00006pt}\text {V}\!/\!c$$ Ge V / c . The results on $$K^{0}_{S}$$ K S 0 production are compared with the production of charged kaons in corresponding reactions and with model calculations (Epos1.99, SMASH 2.0 and PHSD) as well as with published data from other experiments.

Abstract The critical point of strongly interacting matter is searched for at the CERN SPS by the NA61/SHINE experiment in central $$^{40}$$ 40 Ar + $$^{45}$$ 45 Sc collisions at 13 A, 19 A, 30 A, 40 A, and 75 A GeV/c. The dependence of the second-order scaled factorial moments of proton multiplicity distributions on the number of subdivisions in transverse momentum space is measured. The intermittency analysis uses statistically independent data sets for every subdivision in transverse and cumulative-transverse momentum variables. The results obtained do not indicate the searched intermittent pattern. An upper limit on the fraction of correlated protons and the intermittency index is obtained based on a comparison with the Power-law Model.

Abstract Background The key pathological signature of ALS/ FTLD is the mis-localization of endogenous TDP-43 from the nucleus to the cytoplasm. However, TDP-43 gain of function in the cytoplasm is still poorly understood since TDP-43 animal models recapitulating mis-localization of endogenous TDP-43 from the nucleus to the cytoplasm are missing. Methods CRISPR/Cas9 technology was used to generate a zebrafish line (called CytoTDP), that mis-locates endogenous TDP-43 from the nucleus to the cytoplasm. Phenotypic characterization of motor neurons and the neuromuscular junction was performed by immunostaining, microglia were immunohistochemically localized by whole-mount tissue clearing and muscle ultrastructure was analyzed by scanning electron microscopy. Behavior was investigated by video tracking and quantitative analysis of swimming parameters. RNA sequencing was used to identify mis-regulated pathways with validation by molecular analysis. Results CytoTDP fish have early larval phenotypes resembling clinical features of ALS such as progressive motor defects, neurodegeneration and muscle atrophy. Taking advantage of zebrafish’s embryonic development that solely relys on yolk usage until 5 days post fertilization, we demonstrated that microglia proliferation and activation in the hypothalamus is independent from food intake. By comparing CytoTDP to a previously generated TDP-43 knockout line, transcriptomic analyses revealed that mis-localization of endogenous TDP-43, rather than TDP-43 nuclear loss of function, leads to early onset metabolic dysfunction. Conclusions The new TDP-43 model mimics the ALS/FTLD hallmark of progressive motor dysfunction. Our results suggest that functional deficits of the hypothalamus, the metabolic regulatory center, might be the primary cause of weight loss in ALS patients. Cytoplasmic gain of function of endogenous TDP-43 leads to metabolic dysfunction in vivo that are reminiscent of early ALS clinical non-motor metabolic alterations. Thus, the CytoTDP zebrafish model offers a unique opportunity to identify mis-regulated targets for therapeutic intervention early in disease progression.

Purpose CHEK2 c.1100delC is associated with an increased breast cancer risk in women. While this variant is prevalent in the Netherlands (1% in the general population), knowledge of aetiology and prognosis of breast cancer and other tumours in CHEK2 c.1100delC carriers is lacking. The nationwide HEreditary Breast and Ovarian cancer study the Netherlands (Hebon) cohort aims to answer study questions in families with an increased risk of breast cancer and ovarian cancer. While initially focusing on BRCA1/2-variant families, Hebon gradually expanded to include pathogenic variants in other genes associated with breast and/or ovarian cancer over time. This provides an excellent setting to establish a cohort to ultimately study the impact of CHEK2 c.1100delC on cancer risk prediction and surveillance, breast cancer treatment and prognosis.Participants We invited all heterozygous and homozygous CHEK2 c.1100delC indexes and tested female relatives. 1802 women were included, of whom 1374 were heterozygotes and 938 were breast cancer cases. Pedigrees were collected from all clinical genetic departments. Furthermore, participants completed a detailed questionnaire on hormonal and lifestyle factors, family history, cancer diagnosis and treatment.Findings to date Mean age at study inclusion was 53 years. Linkage with the Netherlands Cancer Registry showed a younger age at diagnosis in homozygotes (mean age 41.7 years) and heterozygotes (47.9 years) than non-carriers (51.2 years). Furthermore, carriers were more often diagnosed with grade 2, oestrogen receptor-positive breast cancer and more often developed contralateral breast cancer than non-carriers. Most women consumed alcohol regularly and about half never smoked.Future plans Further data linkages with the Netherlands Cancer Registry will allow prospective follow-up and breast cancer risk assessment in unaffected women at the time of genetic testing, risk of contralateral breast cancer and survival in patients with breast cancer. Also, linkage with the nationwide network and registry of histopathology and cytopathology in The Netherlands (PALGA) allows us to retrieve tumour samples to study tumourigenesis.

Abstract Chronic pain represents a significant unmet medical need, affecting one‐fifth of the U.S. population. EC5026 is a small molecule inhibitor of the enzyme soluble epoxide hydrolase (sEH) which is being developed as a novel non‐opioid, non‐NSAID analgesic. EC5026 prolongs the action of epoxy fatty acids, endogenous analgesic lipid mediators that are rapidly metabolized by sEH. We evaluated the safety and pharmacokinetic profile of EC5026 in two phase I trials, a single‐ascending dose (SAD) study and a fed‐fasted study. The SAD study evaluated EC5026 doses ranging from 0.5 to 24 mg in healthy volunteers. EC5026 was well tolerated. No treatment‐emergent adverse events were considered related to EC5026. No apparent treatment‐ or dose‐related trends in laboratory results, vital signs, physical examinations, or electrocardiograms were observed. A linear, near‐dose‐proportional increase in exposure was observed with progressive doses in the SAD study; plasma exposure was below or near the lower limit of quantification after 0.5–2 mg doses. Mean half‐lives ranged from 41.8 to 59.1 h. for doses of 8–24 mg, supporting a once‐daily dosing regimen. In the fed‐fasted study using 8 mg EC5026 tablets, higher peak concentrations (66%) and total exposures (53%) were observed under the fed condition. Plasma concentrations declined in a monoexponential manner with mean half‐lives of 59.5 h. in the fed state and 66.9 h. in the fasted state. Future clinical trials using EC5026 for the treatment of pain are justified based on the favorable outcomes from both clinical trials along with preclinical evidence of analgesic activity.

Critical or delayed bone healing in rat osteotomy (OT) models is mostly achieved through large defects or instability. We aimed to design a rat OT model for impaired bone healing based on age, gender and parity. The outcome should be controllable through variations of the haematoma in the OT including a bone morphogenetic protein (BMP) 2 guided positive control.Using external fixation to stabilise femoral a 2 mm double OT in 12 month old, female Sprague Dawley rats after a minimum of 3 litters healing was characterised following in situ haematoma formation (ISH-group)), transplantation of a BMP charged autologous blood clot (BMP-group) and the artificial blood clot only (ABC-group) into the OT-gap. In vivo micro-computer tomography (µCT) scans were performed after 2, 4 and 6 weeks. After 6 weeks specimens underwent histological analyses.In µCT examinations and histological analyses no bony bridging was observed in all but one animal in the ISH-group. In the BMP group complete bridging was achieved in all animals. The ABC-group showed less mineralised tissue formation and smaller bridging scores during the course of healing than the ISH-group.In this pilot study we introduce a model for impaired bone healing taking the major biological risk factors into account. We could show that the in situ fracture haematoma is essential for bone regeneration. Using BMP as a positive control the presented experimental setup can serve to evaluate innovative therapeutical concepts in long bone application.

The assumption that mesenchymal stromal cell (MSC)-based-therapies are capable of augmenting physiological regeneration processes has fostered intensive basic and clinical research activities. However, to achieve sustained therapeutic success in vivo, not only the biological, but also the mechanical microenvironment of MSCs during these regeneration processes needs to be taken into account. This is especially important for e.g., bone fracture repair, since MSCs present at the fracture site undergo significant biomechanical stimulation. This study has therefore investigated cellular characteristics and the functional behaviour of MSCs in response to mechanical loading.Our results demonstrated a reduced expression of MSC surface markers CD73 (ecto-5’-nucleotidase) and CD29 (integrin β1) after loading. On the functional level, loading led to a reduced migration of MSCs. Both effects persisted for a week after the removal of the loading stimulus. Specific inhibition of CD73/CD29 demonstrated their substrate dependent involvement in MSC migration after loading. These results were supported by scanning electron microscopy images and phalloidin staining of actin filaments displaying less cell spreading, lamellipodia formation and actin accumulations. Moreover, focal adhesion kinase and Src-family kinases were identified as candidate downstream targets of CD73/CD29 that might contribute to the mechanically induced decrease in MSC migration.These results suggest that MSC migration is controlled by CD73/CD29, which in turn are regulated by mechanical stimulation of cells. We therefore speculate that MSCs migrate into the fracture site, become mechanically entrapped, and thereby accumulate to fulfil their regenerative functions.

This study reports that treatment of osseous defects with different growth factors initiates distinct rates of repair. We developed a new method for monitoring the progression of repair, based upon measuring the in vivo mechanical properties of healing bone. Two different members of the bone morphogenetic protein (BMP) family were chosen to initiate defect healing: BMP-2 to induce osteogenesis, and growth-and-differentiation factor (GDF)-5 to induce chondrogenesis. To evaluate bone healing, BMPs were implanted into stabilised 5 mm bone defects in rat femurs and compared to controls. During the first two weeks, in vivo biomechanical measurements showed similar values regardless of the treatment used. However, 2 weeks after surgery, the rhBMP-2 group had a substantial increase in stiffness, which was supported by the imaging modalities. Although the rhGDF-5 group showed comparable mechanical properties at 6 weeks as the rhBMP-2 group, the temporal development of regenerating tissues appeared different with rhGDF-5, resulting in a smaller callus and delayed tissue mineralisation. Moreover, histology showed the presence of cartilage in the rhGDF-5 group whereas the rhBMP-2 group had no cartilaginous tissue.Therefore, this study shows that rhBMP-2 and rhGDF-5 treated defects, under the same conditions, use distinct rates of bone healing as shown by the tissue mechanical properties. Furthermore, results showed that in vivo biomechanical method is capable of detecting differences in healing rate by means of change in callus stiffness due to tissue mineralisation.

Abstract Malignant melanoma, the most aggressive form of skin cancer, is often incurable once metastatic dissemination of cancer cells to distant organs has occurred. We investigated the role of Transcription Factor Activating Enhancer-Binding Protein 2ε (AP2ε) in the progression of metastatic melanoma. Here, we observed that AP2ε is a potent activator of metastasis and newly revealed AP2ε to be an important player in melanoma plasticity. High levels of AP2ε lead to worsened prognosis of melanoma patients. Using a transgenic melanoma mouse model with a specific loss of AP2ε expression, we confirmed the impact of AP2ε to modulate the dynamic switch from a migratory to a proliferative phenotype. AP2ε deficient melanoma cells show a severely reduced migratory potential in vitro and reduced metastatic behavior in vivo. Consistently, we revealed increased activity of AP2ε in quiescent and migratory cells compared to heterogeneously proliferating cells in bioprinted 3D models. In conclusion, these findings disclose a yet-unknown role of AP2ε in maintaining plasticity and migration in malignant melanoma cells.

Abstract The NA61/SHINE experiment at the CERN Super Proton Synchrotron studies the onset of deconfinement in strongly interacting matter through a beam energy scan of particle production in collisions of nuclei of varied sizes. This paper presents results on inclusive double-differential spectra, transverse momentum and rapidity distributions and mean multiplicities of $$\pi ^\pm $$ π ± , $$K^\pm $$ K ± , p and $$\bar{p}$$ p ¯ produced in $$^{40}\hbox {Ar+}^{45}\hbox {Sc}$$ 40 Ar+ 45 Sc collisions at beam momenta of 13A, 19A, 30A, 40A, 75A and 150A $$\text{ Ge }\hspace{-1.00006pt}\text{ V }\!/\!c$$ Ge V / c . The analysis uses the 10% most central collisions, where the observed forward energy defines centrality. The energy dependence of the $$K^\pm $$ K ± / $$\pi ^\pm $$ π ± ratios as well as of inverse slope parameters of the $$K^\pm $$ K ± transverse mass distributions are placed in between those found in inelastic $$p+p$$ p + p and central Pb + Pb collisions. The results obtained here establish a system-size dependence of hadron production properties that so far cannot be explained either within statistical or dynamical models.

Amniogenesis, a process critical for continuation of healthy pregnancy, is triggered in a collection of pluripotent epiblast cells as the human embryo implants. Previous studies have established that bone morphogenetic protein (BMP) signaling is a major driver of this lineage specifying process, but the downstream BMP-dependent transcriptional networks that lead to successful amniogenesis remain to be identified. This is, in part, due to the current lack of a robust and reproducible model system that enables mechanistic investigations exclusively into amniogenesis. Here, we developed an improved model of early amnion specification, using a human pluripotent stem cell-based platform in which the activation of BMP signaling is controlled and synchronous. Uniform amniogenesis is seen within 48 hr after BMP activation, and the resulting cells share transcriptomic characteristics with amnion cells of a gastrulating human embryo. Using detailed time-course transcriptomic analyses, we established a previously uncharacterized BMP-dependent amniotic transcriptional cascade, and identified markers that represent five distinct stages of amnion fate specification; the expression of selected markers was validated in early post-implantation macaque embryos. Moreover, a cohort of factors that could potentially control specific stages of amniogenesis was identified, including the transcription factor TFAP2A. Functionally, we determined that, once amniogenesis is triggered by the BMP pathway, TFAP2A controls the progression of amniogenesis. This work presents a temporally resolved transcriptomic resource for several previously uncharacterized amniogenesis states and demonstrates a critical intermediate role for TFAP2A during amnion fate specification.

Although 2D cancer models have been the standard for drug development, they don't resemble in vivo properties adequately. 3D models can potentially overcome this. Bioprinting is a promising technique for more refined models to investigate central processes in tumor development such as proliferation, dormancy or metastasis.We aimed to analyze bioinks, which could mimic these different tumor stages in a cast vascularized arteriovenous loop melanoma model in vivo. It has the advantage to be a closed system with a defined microenvironment, supplied only with one vessel—ideal for metastasis research.Tested bioinks showed significant differences in composition, printability, stiffness and microscopic pore structure, which led to different tumor stages (Matrigel and Alg/HA/Gel for progression, Cellink Bioink for dormancy) and resulted in different primary tumor growth (Matrigel significantly higher than Cellink Bioink). Light-sheet fluorescence microscopy revealed differences in vascularization and hemorrhages with no additional vessels found in Cellink Bioink. Histologically, typical human melanoma with different stages was demonstrated. HMB-45-positive tumors in progression inks were infiltrated by macrophages (CD163), highly proliferative (Ki67) and metastatic (MITF/BRN2, ATX, MMP3). Stainings of lymph nodes revealed metastases even without significant primary tumor growth in Cellink Bioink.This model can be used to study tumor pathology and metastasis of different tumor stages and therapies.

To determine the changes in surveillance category by adding a polygenic risk score based on 311 breast cancer (BC)-associated variants (PRS311), questionnaire-based risk factors and breast density on personalized BC risk in unaffected women from Dutch CHEK2 c.1100delC families.In total, 117 unaffected women (58 heterozygotes and 59 non-carriers) from CHEK2 families were included. Blood-derived DNA samples were genotyped with the GSAMDv3-array to determine PRS311. Lifetime BC risk was calculated in CanRisk, which uses data from the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA). Women, were categorized into three surveillance groups.The surveillance advice was reclassified in 20 (34.5%) heterozygotes and 21 (35.6%) non-carriers after adding PRS311. Including questionnaire-based risk factors resulted in an additional change in 11 (20.0%) heterozygotes and 8 (15.1%) non-carriers; and a sub-analysis showed that adding breast density on top shifted another 9 (23.1%) heterozygotes and 5 (27.8%) non-carriers. Overall, the majority of heterozygotes were reclassified to a less intensive surveillance, while non-carriers would require intensified surveillance.The addition of PRS311, questionnaire-based risk factors and breast density to family history resulted in a more personalized BC surveillance advice in CHEK2-families, which may lead to more efficient use of surveillance.

Abstract Relationships among carnivore species are complex, potentially switching from competition to facilitation on a context-dependent basis. Negative associations are predicted to increase with latitude, due to limited resources emphasising competition and/or intra-guild predation. Accordingly, a stronger negative correlation between large- and meso-carnivore abundances should be expected at higher latitudes, with a substantial spatio-temporal partitioning favouring interspecific coexistence. Human presence may influence spatio-temporal relationships between (meso)carnivore species, as it can be perceived as a risk factor, but anthropogenic food can also provide an important additional food resource. Using camera-trap data, we studied the spatio-temporal associations between two of the most widespread carnivores in Europe, i.e., the red fox and wolf. We compared their monthly/daily spatio-temporal partitioning between two different landscapes: Białowieża Primeval Forest (Poland) and the Mediterranean Maremma Regional Park (Italy). We predicted a stronger interspecific partitioning, as well as more attraction of red foxes to humans in the northern site (Poland). Temporal activity patterns of the two carnivores overlapped in both sites, and their detection rates were positively associated, even though in weaker way in Poland. We observed a positive spatial association of red foxes with human activity in Białowieża, but not in Maremma. This association occurred only at a monthly temporal scale and disappeared at a daily scale, suggesting some disturbance in the shorter term. Our results provided partial support to our predictions and suggest that, despite the ecological differences between our study areas, only weak differences in wolf-fox relations were observed, suggesting that red fox responses to wolves may be relatively comparable over large spatial scales.

Abstract Background Due to the abundant usage of chemotherapy in young triple-negative breast cancer (TNBC) patients, the unbiased prognostic value of BRCA1-related biomarkers in this population remains unclear. In addition, whether BRCA1-related biomarkers modify the well-established prognostic value of stromal tumor-infiltrating lymphocytes (sTILs) is unknown. This study aimed to compare the outcomes of young, node-negative, chemotherapy-naïve TNBC patients according to BRCA1 status, taking sTILs into account. Methods We included 485 Dutch women diagnosed with node-negative TNBC under age 40 between 1989 and 2000. During this period, these women were considered low-risk and did not receive chemotherapy. BRCA1 status, including pathogenic germline BRCA1 mutation (gBRCA1m), somatic BRCA1 mutation (sBRCA1m), and tumor BRCA1 promoter methylation (BRCA1-PM), was assessed using DNA from formalin-fixed paraffin-embedded tissue. sTILs were assessed according to the international guideline. Patients’ outcomes were compared using Cox regression and competing risk models. Results Among the 399 patients with BRCA1 status, 26.3% had a gBRCA1m, 5.3% had a sBRCA1m, 36.6% had tumor BRCA1-PM, and 31.8% had BRCA1-non-altered tumors. Compared to BRCA1-non-alteration, gBRCA1m was associated with worse overall survival (OS) from the fourth year after diagnosis (adjusted HR, 2.11; 95% CI, 1.18–3.75), and this association attenuated after adjustment for second primary tumors. Every 10% sTIL increment was associated with 16% higher OS (adjusted HR, 0.84; 95% CI, 0.78–0.90) in gBRCA1m, sBRCA1m, or BRCA1-non-altered patients and 31% higher OS in tumor BRCA1-PM patients. Among the 66 patients with tumor BRCA1-PM and ≥ 50% sTILs, we observed excellent 15-year OS (97.0%; 95% CI, 92.9–100%). Conversely, among the 61 patients with gBRCA1m and

Future changes in suspended sediment export from deglaciating high-alpine catchments affect downstream hydropower reservoirs, flood hazard, ecosystems and water quality. Yet, quantitative projections of future sediment export have so far been hindered by the lack of process-based models that can take into account all relevant processes within the complex systems determining sediment dynamics at the catchment scale. As a promising alternative, machine-learning (ML) approaches have recently been successfully applied to modeling suspended sediment yields (SSYs). This study is the first, to our knowledge, exploring a machine-learning approach to derive sediment export projections until the year 2100. We employ quantile regression forest (QRF), which proved to be a powerful method to model past SSYs in previous studies, for two nested glaciated high-alpine catchments in the Ötztal, Austria, above gauge Vent (98.1 km2) and gauge Vernagt (11.4 km2). As predictors, we use temperature and precipitation projections (EURO-CORDEX) and discharge projections (AMUNDSEN physically based hydroclimatological and snow model) for the two gauges. We address uncertainties associated with the known limitation of QRF that underestimates can be expected if values in the projection period exceed the range represented in the training data (out-of-observation-range days, OOOR). For this, we assess the frequency and extent of these exceedances and the sensitivity of the resulting mean annual suspended sediment concentration (SSC) estimates. We examine the resulting SSY projections for trends, the estimated timing of peak sediment and changes in the seasonal distribution. Our results show that the uncertainties associated with the OOOR data points are small before 2070 (max. 3 % change in estimated mean annual SSC). Results after 2070 have to be treated more cautiously as OOOR data points occur more frequently, and glaciers are projected to have (nearly) vanished by then in some projections, which likely substantially alters sediment dynamics in the area. The resulting projections suggest decreasing sediment export at both gauges in the coming decades, regardless of the emission scenario, which implies that peak sediment has already passed or is underway. This is linked to substantial decreases in discharge volumes, especially during the glacier melt phase in late summer, as a result of increasing temperatures and thus shrinking glaciers. Nevertheless, high(er) annual yields can occur in response to heavy summer precipitation, and both developments would need to be considered in managing sediments, as well as e.g., flood hazard. While we chose the predictors to act as proxies for sediment-relevant processes, future studies are encouraged to try and include geomorphological changes more explicitly, e.g., changes in connectivity, landsliding, rockfalls or vegetation colonization, as these could improve the reliability of the projections.

Abstract PF614, a trypsin‐activated abuse protection oxycodone prodrug designed to reduce recreational drug abuse, was compared to OxyContin for safety and pharmacokinetics (PKs) of plasma oxycodone following oral administration. This study was a two‐part design including a multi‐ascending dose (part A) and a bioequivalence (BE) study (part B) in healthy volunteers. In part A, 24 subjects were randomized 3:1 to receive PF614 (50, 100, or 200 mg, n = 6/cohort) or OxyContin (20, 40, or 80 mg; n = 2/cohort) in ascending cohorts, delivered every 12 h for a total of nine doses. In part B, 60 subjects randomized in a four‐way crossover to evaluate BE, received PF614 100 mg and OxyContin 40 mg in fasted and fed (high‐fat diet) states. All subjects were naltrexone blocked prior to first study drug administration to protect against opioid‐related adverse effects; repeat doses were provided on days 1–5. In part A, PF614 was well‐tolerated following twice daily doses of up to 200 mg for 5 days. Plasma oxycodone maximal plasma concentration and area under the concentration time curve increased linearly with increasing doses. Part B showed that plasma oxycodone BE was achieved following 100 mg PF614 or 40 mg OxyContin under both fasted and fed conditions. Additionally, PF614 provided similar oxycodone exposures following both fasted and fed states. This study confirms findings from our single‐ascending dose study, showing that PF614 100 mg releases oxycodone with a PK profile comparable to 40 mg OxyContin under both fasted and fed conditions and with a similar safety profile under naltrexone‐blocked conditions.

Microbial communities in alpine environments >7,500 m.a.s.l. have not been well studied using modern cultivation-independent sequencing approaches due to the challenges and danger associated with reaching such high elevations. For this reason, we know little about the microorganisms found in sediments on Earth’s tallest mountains, how they reach these surfaces, and how they survive and remain active at such extreme elevations. Here, we explore the microbial diversity recovered from three sediment samples collected from the South Col (~7,900 m.a.s.l.) of Sagarmatha (Mount Everest) using both culturing and next generation sequencing approaches (16S rRNA gene, internal transcribed spacer [ITS] region, and 18S rRNA gene sequencing). Both approaches detected very low diversity of bacteria, protists, and fungi that included a combination of cosmopolitan taxa and specialized microorganisms often found at high elevations like those of the genera Modestobacter and Naganishia. Though we managed to grow viable cultures of many of these taxa, it remains likely that few, if any, can be active in situ at the South Col. Instead, these high-elevation surfaces may act as deep-freeze collection zones of organisms deposited from the atmosphere or left by climbers scaling the Earth’s highest mountain.

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization–Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.

Cavity optomechanics aims to establish optical control over vibrations of nanoscale mechanical systems, to heat, cool or to drive them toward coherent, or nonclassical states. This field was recently extended to encompass molecular optomechanics: the dynamics of THz molecular vibrations coupled to the optical fields of lossy cavities via Raman transitions. The molecular platform should prove suitable for demonstrating more sophisticated optomechanical effects, including engineering of nonclassical mechanical states, or inducing coherent molecular vibrations. We propose two schemes for implementing these effects, exploiting the strong intrinsic anharmonicities of molecular vibrations. First, to prepare a nonclassical mechanical state, we propose an incoherent analogue of the mechanical blockade, in which the molecular anharmonicity and optical response of hybrid cavities isolate the two lowest-energy vibrational states. Secondly, we show that for a strongly driven optomechanical system, the anharmonicity can suppress the mechanical amplification, shifting and reshaping the onset of coherent mechanical oscillations. Our estimates indicate that both effects should be within reach of existing platforms for Surface Enhanced Raman Scattering.

Pseudomonas aeruginosa (Pa), a WHO priority 1 pathogen, resulted in approximately 559,000 deaths globally in 2019. Pa has a multitude of host-immune evasion strategies that enhance Pa virulence. Most clinical isolates of Pa are infected by a phage called Pf that has the ability to misdirect the host-immune response and provide structural integrity to biofilms. Previous studies demonstrate that vaccination against the coat protein (CoaB) of Pf4 virions can assist in the clearance of Pa from the dorsal wound model in mice. Here, a consensus peptide was derived from CoaB and conjugated to cross-reacting material 197 (CRM197). This conjugate was adjuvanted with a novel synthetic Toll-like receptor agonist (TLR) 4 agonist, INI-2002, and used to vaccinate mice. Mice vaccinated with CoaB-CRM conjugate and INI-2002 developed high anti-CoaB peptide-specific IgG antibody titers. Direct binding of the peptide-specific antibodies to whole-phage virus particles was demonstrated by ELISA. Furthermore, a functional assay demonstrated that antibodies generated from vaccinated mice disrupted the replicative cycle of Pf phages. The use of an adjuvanted phage vaccine targeting Pa is an innovative vaccine strategy with the potential to become a new tool targeting multi-drug-resistant Pa infections in high-risk populations.

We use a recently derived gauge-invariant formulation of the problem of a two-level system coupled to an optical cavity to explore the transition between the weak and the ultrastrong coupling regimes of light-matter interaction. We explore this transition using the intensity correlations g^{(2)}(τ) of the emitted light, and we find strong, unbounded bunching of the emission from systems governed by the Rabi Hamiltonian. Surprisingly, this effect is observed not only in the ultrastrong coupling regime, but also for weakly coupled systems, where the Jaynes-Cummings Hamiltonian would predict the opposite, antibunched emission. This suggests that the higher-order correlations are a particularly sensitive probe of the divergence between the Jaynes-Cummings and Rabi Hamiltonians, and they can serve as an indicator of the breakdown of the rotating wave approximation. Our findings indicate also that the boundary between the weakly, strongly, and ultra-strongly-coupled dynamics is much richer than currently accepted.

Focused beams of electrons, which act as both sources and sensors of electric fields, can be used to characterize the electric response of complex photonic systems by locally probing the induced optical near fields. This functionality can be complemented by embracing the recently developed vortex electron beams (VEBs), made up of electrons with orbital angular momentum, which could, in addition, probe induced magnetic near fields. In this work we revisit the theoretical description of this technique, dubbed vortex electron energy-loss spectroscopy (v-EELS). We map the fundamental, quantum-mechanical picture of the scattering of the VEB electrons to the intuitive classical models, which treat the electron beams as a superposition of linear electric and magnetic currents. We then apply this formalism to characterize the optical response of dielectric nanoantennas with v-EELS. Our calculations reveal that VEB electrons probe electric or magnetic modes with different efficiency, which can be adjusted by changing either beam vorticity or acceleration voltage to determine the nature of the probed excitations. We also study a chirally arranged nanostructure, which in the interaction with electron vortices produces dichroism in electron-energy-loss spectra. Our theoretical work establishes VEBs as versatile probes that could provide information on optical excitations otherwise inaccessible with conventional electron beams.