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Process mining uses historical executions of business processes (as recorded in an event log) to uncover and describe the process’ behaviour as a process model. The goal of conformance checking is to ensure that the model is of high quality and is a good representation of the event log, thus assuring the model is a solid foundation for subsequent analysis of the process. To date, few conformance checking approaches have considered model quality beyond what is determined by the execution order of process activities. In data-aware process models, which are generated by decision mining techniques, process activities are annotated with conditions to represent the decision-making of a process. Such models are more expressive than those that represent only process activities. With the current notions of conformance checking, it is unclear what properties determine the quality of these data-aware process models. To address this gap, we introduce desirable properties, as axioms, for conformance checking of data-aware models. Our contribution is threefold: i) we present a generalisation that abstracts from the representation of data-aware models, ii) we present nine axioms of desirable properties for data-aware conformance checking, and iii) we define two measures for model recall and precision. Using our axioms as a yardstick, we compare our proposed recall and precision measures with existing measures. Our experimental results show that existing measures exhibit limited adherence to our axioms; while, our two proposed measures exhibit high adherence to our axioms.

Extensive subtidal eelgrass (Zostera marina) meadows (~150 km2) once grew in the Dutch Wadden Sea, supporting diverse species communities, but disappeared in the 1930s and have been absent ever since. Identifying the most critical bottlenecks for eelgrass survival is a crucial first step for reintroduction through active restoration measures. Seagrasses are ecosystem engineers, inducing self-facilitating feedbacks that ameliorate stressful conditions. Consequently, once seagrass, including its self-facilitating feedbacks, is lost, reintroduction can be challenging. Therefore, we aimed to test whether 1) sediment stabilization and 2) hydrodynamic stress relief would facilitate eelgrass survival in a field experiment replicated at two sites in the Dutch Wadden Sea. We induced feedbacks using biodegradable root-mimicking structures (BESE-elements) and sandbag barriers. Root mimics had a significant positive effect, increasing the chances of short-term survival by +67% compared to controls. Contrary to our expectations, barriers decreased short-term survival probabilities by -26%, likely due to hydrodynamic turbulence created by the barrier edges, leading to high erosion rates (-14 cm). Site selection proved crucial as short-term survival was entirely negated on one of the two study sites after five weeks due to high floating and epiphytic macroalgae loads. No long-term survival occurred, as plants died at the other site two weeks later. Overall, we found that sediment stabilization by root-mimicking structures was promising, whereas manipulating hydrodynamic forces using sandbag barriers had adverse effects. A mechanistic understanding of transplant failures is required before attempting large-scale restoration. Our study indicates that for seagrass restoration in the Wadden Sea, one should carefully consider 1) the reintroduction of positive feedbacks through restoration tools, 2) donor population choice and transplantation timing, and 3) site selection based on local biotic and abiotic conditions. Optimizing these restoration facets might lower additive stress to a degree that allows long-term survival.

Foundation species that modify their habitat can facilitate other species, including other foundation species. Most studies focus solely on a single foundation species, overlooking such facilitation cascades. In this study, we investigated the interactions between the two coastal foundation species Mytilus edulis (blue mussel) and Lanice conchilega (sand mason worm). We investigated whether these species engage in facilitative interactions or if their association simply reflects a shared ecological niche on the soft-sediment intertidal flats of the Dutch Wadden Sea. To do so, we performed species distribution modeling, manipulative field experiments, and field surveys. We found a positive association between both foundation species, with a 2.45 times higher occurrence of both species compared to a random distribution. In addition, these species partially occupied the same ecological niche. We demonstrated that L. conchilega provided settlement substrate for M. edulis spat, increasing densities by 400 times compared to bare plots. Furthermore, M. edulis reefs facilitated L. conchilega occurrence in the wake of the reef. Biogenic reef development revealed that this interspecific facilitation resulted in spatial habitat heterogeneity. Therefore, we conclude that interspecific facilitation can significantly enhance the occurrence of these two important intertidal foundation species. Acknowledgment of such complex facilitation interactions has an untapped potential for improving the success of restoration and conservation programs.

Summary: Background: Neurofilament light chain (NfL) is a biomarker for axonal damage in several neurological disorders. We studied the longitudinal changes in serum NfL in patients with Guillain-Barré syndrome (GBS) in relation to disease severity, electrophysiological subtype, treatment response, and prognosis. Methods: We included patients with GBS who participated in a double-blind, randomised, placebo-controlled trial that evaluated the effects of a second course of intravenous immunoglobulin (IVIg) on clinical outcomes. Serum NfL levels were measured before initiation of treatment and at one, two, four, and twelve weeks using a Simoa HD-X Analyzer. Serum NfL dynamics were analysed using linear mixed-effects models. Logistic regression was employed to determine the associations of serum NfL with clinical outcome and the prognostic value of serum NfL after correcting for known prognostic markers included in the modified Erasmus GBS Outcome Score (mEGOS). Findings: NfL levels were tested in serum from 281 patients. Serum NfL dynamics were associated with disease severity and electrophysiological subtype. Strong associations were found between high levels of serum NfL at two weeks and inability to walk unaided at four weeks (OR = 1.74, 95% CI = 1.27–2.45), and high serum NfL levels at four weeks and inability to walk unaided at 26 weeks (OR = 2.79, 95% CI = 1.72–4.90). Baseline serum NfL had the most significant prognostic value for ability to walk, independent of predictors included in the mEGOS. The time to regain ability to walk unaided was significantly longer for patients with highest serum NfL levels at baseline (p = 0.0048) and week 2 (p

Abstract Biological trait analysis (BTA) is a valuable tool for evaluating changes in community diversity and its link to ecosystem processes as well as environmental and anthropogenic perturbations. Trait-based analytical techniques like BTA rely on standardised datasets of species traits. However, there are currently only a limited number of datasets available for marine macrobenthos that contain trait data across multiple taxonomic groups. Here, we present an open-access dataset of 16 traits for 235 macrozoobenthic species recorded throughout multiple sampling campaigns of the Dutch Wadden Sea; a dynamic soft bottom system where humans have long played a substantial role in shaping the coastal environment. The trait categories included in this dataset cover a variety of life history strategies that are tightly linked to ecosystem functioning and the resilience of communities to (anthropogenic) perturbations and can advance our understanding of environmental changes and human impacts on the functioning of soft bottom systems.

Abstract In soft‐bottom marine ecosystems, bedform variation is induced by wind‐ and tidal‐driven hydrodynamics. The resulting megaripples, sand waves and sandbanks form a spatially and temporally heterogeneous seafloor landscape. The strong physical forces imposed by the migration of these bedforms are important determinants for the occurrence of different macrozoobenthic species. Quantifying the effect of these forces can help in differentiating natural‐ and anthropogenically induced physical stressors. However, large‐scale mapping of seabed morphology at high resolution using multibeam echosounder is challenging, costly and time‐consuming, especially in shallow seas, prohibiting wide swaths. Instead, their bathymetry is typically studied using single‐beam transects that are interpolated to bathymetric grids with a relatively coarse resolution (20 m). However, this leaves out information on smaller scale (

Summary: The inherent stochasticity of metabolism raises a critical question for understanding homeostasis: are cellular processes regulated in response to internal fluctuations? Here, we show that, in E. coli cells under constant external conditions, catabolic enzyme expression continuously responds to metabolic fluctuations. The underlying regulatory feedback is enabled by the cyclic AMP (cAMP) and cAMP receptor protein (CRP) system, which controls catabolic enzyme expression based on metabolite concentrations. Using single-cell microscopy, genetic constructs in which this feedback is disabled, and mathematical modeling, we show how fluctuations circulate through the metabolic and genetic network at sub-cell-cycle timescales. Modeling identifies four noise propagation modes, including one specific to CRP regulation. Together, these modes correctly predict noise circulation at perturbed cAMP levels. The cAMP-CRP system may thus have evolved to control internal metabolic fluctuations in addition to external growth conditions. We conjecture that second messengers may more broadly function to achieve cellular homeostasis.

Introduction: Specific alleles in human leukocyte antigens (HLAs) are associated with an increased risk of developing drug hypersensitivity reactions induced by abacavir, allopurinol, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, or flucloxacillin. Transplant patients are genotyped for HLA as a routine practice to match a potential donor to a recipient. This study aims to investigate the feasibility and potential impact of repurposing these HLA genotype data from kidney transplant patients to prevent drug hypersensitivity reactions.Methods: A cohort of 1347 kidney transplant recipients has been genotyped in the Leiden University Medical Center (LUMC) using next-generation sequencing (NGS). The risk alleles HLA-A*31:01, HLA-B*15:02, HLA-B*15:11, HLA-B*57:01, and HLA-B*58:01 were retrieved from the NGS data. Medical history, medication use, and allergic reactions were obtained from the patient’s medical records. Carrier frequencies found were compared to a LUMC blood donor population.Results: A total of 13.1% of transplant cohort patients carried at least one of the five HLA risk alleles and therefore had an increased risk of drug-induced hypersensitivity for specific drugs. HLA-A*31:01, HLA-B*15:02, HLA-B*57:01, and HLA-B*58:01 were found in carrier frequencies of 4.61%, 1.19%, 4.46%, and 3.35% respectively. No HLA-B*15:11 carrier was found. In total nine HLA-B*57:01 carriers received flucloxacillin and seven HLA-B*58:01 carriers within our cohort received allopurinol.Discussion: Our study shows that repurposing HLA genotype data from transplantation patients for the assignment of HLA risk alleles associated with drug hypersensitivity is feasible. The use of these data by physicians while prescribing drugs or by the pharmacist when dispensing drugs holds the potential to prevent drug hypersensitivity reactions. The utility of this method was highlighted by 13.1% of the transplant cohort patients carrying an actionable HLA allele.

Abstract Evolution has traditionally been a historical and descriptive science, and predicting future evolutionary processes has long been considered impossible. However, evolutionary predictions are increasingly being developed and used in medicine, agriculture, biotechnology and conservation biology. Evolutionary predictions may be used for different purposes, such as to prepare for the future, to try and change the course of evolution or to determine how well we understand evolutionary processes. Similarly, the exact aspect of the evolved population that we want to predict may also differ. For example, we could try to predict which genotype will dominate, the fitness of the population or the extinction probability of a population. In addition, there are many uses of evolutionary predictions that may not always be recognized as such. The main goal of this review is to increase awareness of methods and data in different research fields by showing the breadth of situations in which evolutionary predictions are made. We describe how diverse evolutionary predictions share a common structure described by the predictive scope, time scale and precision. Then, by using examples ranging from SARS‐CoV2 and influenza to CRISPR‐based gene drives and sustainable product formation in biotechnology, we discuss the methods for predicting evolution, the factors that affect predictability and how predictions can be used to prevent evolution in undesirable directions or to promote beneficial evolution (i.e. evolutionary control). We hope that this review will stimulate collaboration between fields by establishing a common language for evolutionary predictions.

Approximately 10–12% of patients with oesophageal or gastric cancer (OGC) present with oligometastatic disease at diagnosis. It remains unclear if there is a role for radical surgery in these patients. We aimed to assess the outcomes of OGC patients who underwent simultaneous treatment for the primary tumour and synchronous liver metastases. Patients with OGC who underwent surgical treatment between 2008 and 2020 for the primary tumour and up to five synchronous liver metastases aiming for complete tumour removal or ablation (i.e., no residual tumour) were identified from four institutional databases. The primary outcome was overall survival (OS), calculated with the Kaplan–Meier method. Secondary outcomes were disease-free survival and postoperative outcomes. Thirty-one patients were included, with complete follow-up data for 30 patients. Twenty-six patients (84%) received neoadjuvant therapy followed by response evaluation. Median OS was 21 months [IQR 9–36] with 2- and 5-year survival rates of 43% and 30%, respectively. While disease recurred in 80% of patients (20 of 25 patients) after radical resection, patients with a solitary liver metastasis had a median OS of 34 months. The number of liver metastases was a prognostic factor for OS (solitary metastasis aHR 0.330; p-value = 0.025). Thirty-day mortality was zero and complications occurred in 55% of patients. Long-term survival can be achieved in well-selected patients who undergo surgical resection of the primary tumour and local treatment of synchronous liver metastases. In particular, patients with a solitary liver metastasis seem to have a favourable prognosis.

We collected data on mass mortality in Sandwich terns (Thalasseus sandvicensis) during the 2022 breeding season in the Netherlands. Mortality was associated with at least 2 variants of highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b. We report on carcass removal efforts relative to survival in colonies. Mitigation strategies urgently require structured research.

BackgroundThe promise of digital health is principally dependent on the ability to electronically capture data that can be analyzed to improve decision-making. However, the ability to effectively harness data has proven elusive, largely because of the quality of the data captured. Despite the importance of data quality (DQ), an agreed-upon DQ taxonomy evades literature. When consolidated frameworks are developed, the dimensions are often fragmented, without consideration of the interrelationships among the dimensions or their resultant impact. ObjectiveThe aim of this study was to develop a consolidated digital health DQ dimension and outcome (DQ-DO) framework to provide insights into 3 research questions: What are the dimensions of digital health DQ? How are the dimensions of digital health DQ related? and What are the impacts of digital health DQ? MethodsFollowing the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines, a developmental systematic literature review was conducted of peer-reviewed literature focusing on digital health DQ in predominately hospital settings. A total of 227 relevant articles were retrieved and inductively analyzed to identify digital health DQ dimensions and outcomes. The inductive analysis was performed through open coding, constant comparison, and card sorting with subject matter experts to identify digital health DQ dimensions and digital health DQ outcomes. Subsequently, a computer-assisted analysis was performed and verified by DQ experts to identify the interrelationships among the DQ dimensions and relationships between DQ dimensions and outcomes. The analysis resulted in the development of the DQ-DO framework. ResultsThe digital health DQ-DO framework consists of 6 dimensions of DQ, namely accessibility, accuracy, completeness, consistency, contextual validity, and currency; interrelationships among the dimensions of digital health DQ, with consistency being the most influential dimension impacting all other digital health DQ dimensions; 5 digital health DQ outcomes, namely clinical, clinician, research-related, business process, and organizational outcomes; and relationships between the digital health DQ dimensions and DQ outcomes, with the consistency and accessibility dimensions impacting all DQ outcomes. ConclusionsThe DQ-DO framework developed in this study demonstrates the complexity of digital health DQ and the necessity for reducing digital health DQ issues. The framework further provides health care executives with holistic insights into DQ issues and resultant outcomes, which can help them prioritize which DQ-related problems to tackle first.

Since the last decades, previous long-term Wadden Sea studies revealed significant changes in the abundance, biomass and spatial distribution of characteristic macrofauna communities in response to environmental changes and anthropogenic stressors. In this study, we performed statistical community analysis for the East-Frisian Wadden Sea (EFWS, southern North Sea) on two reference datasets across a period with severe climatic and environmental changes (1980s-2018). Therefore, historical macrofauna data from the Quantitative Sensitivity Mapping (1980s, SENSI 1) were reanalyzed and compared with data from the Synoptic Intertidal Benthic Survey (SIBES/SENSI 3) collected in 2018. Our results revealed significant quantitative and spatial changes in the characteristic macrofauna communities between the 1980s and 2018, most likely in response to de-eutrophication and sea level rise mediated habitat changes. Since the 1980s, the total number of taxa remained relatively stable (1980s: 90, 2018: 81), but the total abundance decreased by ca. -31% and the total biomass decreased by ca. -45%, particularly in the eastern regions of the study site probably due to de-eutrophication processes. Thereby, the mean abundances/m2 of ≥ -80% (1980s-2018) in the EFWS of several dominant species decreased: e.g. the gastropod Peringia ulvae, the polychaete Lanice conchilega and the bivalve Mya arenaria. In contrast, the mean abundance/m2 of one dominant species increased by ≥ +80% (1980s-2018): the invasive bivalve Ensis leei. The mean biomass [g/m2] of three dominant species decreased by ≥ -80% (1980s-2018): P. ulvae, L. conchilega and the amphipod Corophium volutator. In contrast, the mean biomass [g/m2] of one dominant species increased by ≥ +75% (1980s-2018): the polychaete Arenicola marina. In the western part of the EFWS, not only higher abundances of A. marina, but also of L. conchilega and P. ulvae were found in 2018, caused i.a. by sea level rise mediated decreasing mud contents and sand accretion on tidal flats. At the community level, the Oligochaeta/Heteromastus community increased in spatial distribution in the western EFWS in 2018 in the vicinity of increasing mussel/oyster beds.

Objective To combine targeted transcriptomic and proteomic data in an unsupervised hierarchical clustering method to stratify patients with childhood-onset SLE (cSLE) into similar biological phenotypes, and study the immunological cellular landscape that characterises the clusters.Methods Targeted whole blood gene expression and serum cytokines were determined in patients with cSLE, preselected on disease activity state (at diagnosis, Low Lupus Disease Activity State (LLDAS), flare). Unsupervised hierarchical clustering, agnostic to disease characteristics, was used to identify clusters with distinct biological phenotypes. Disease activity was scored by clinical SELENA-SLEDAI (Safety of Estrogens in Systemic Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index). High-dimensional 40-colour flow cytometry was used to identify immune cell subsets.Results Three unique clusters were identified, each characterised by a set of differentially expressed genes and cytokines, and by disease activity state: cluster 1 contained primarily patients in LLDAS, cluster 2 contained mainly treatment-naïve patients at diagnosis and cluster 3 contained a mixed group of patients, namely in LLDAS, at diagnosis and disease flare. The biological phenotypes did not reflect previous organ system involvement and over time, patients could move from one cluster to another. Healthy controls clustered together in cluster 1. Specific immune cell subsets, including CD11c+ B cells, conventional dendritic cells, plasmablasts and early effector CD4+ T cells, differed between the clusters.Conclusion Using a targeted multiomic approach, we clustered patients into distinct biological phenotypes that are related to disease activity state but not to organ system involvement. This supports a new concept where choice of treatment and tapering strategies are not solely based on clinical phenotype but includes measuring novel biological parameters.

The Wadden Sea is the world’s largest intertidal ecosystem and provides vital food resources for a large number of migratory bird and fish species during seasonal stopovers. Previous work using bulk stable isotope analysis of carbon found that microphytobenthos (MPB) was the dominant resource fueling the food web with particulate organic matter making up the remainder. However, this work was unable to account for the trophic structure of the food web or the considerable increase in δ15N values of bulk tissue throughout the benthic food web occurring in the Eastern regions of the Dutch Wadden Sea. Here, we combine compound-specific and bulk analytical stable isotope techniques to further resolve the trophic structure and resource use throughout the benthic food web in the Wadden Sea. Analysis of δ15N for trophic and source amino acids allowed for better identification of trophic relationships due to the integration of underlying variation in the nitrogen resources supporting the food web. Baseline-integrated trophic position estimates using glutamic acid (Glu) and phenylalanine (Phe) allow for disentanglement of baseline variations in underlying δ15N sources supporting the ecosystem and trophic shifts resulting from changes in ecological relationships. Through this application, we further confirmed the dominant ecosystem support by MPB-derived resources, although to a lesser extent than previously estimated. In addition to phytoplankton-derived particulate, organic matter and MPB supported from nutrients from the overlying water column there appears to be an additional resource supporting the benthic community. From the stable isotope mixing models, a subset of species appears to focus on MPB supported off recycled (porewater) N and/or detrital organic matter mainly driven by increased phenylalanine δ15N values. This additional resource within MPB may play a role in subsidizing the exceptional benthic productivity observed within the Wadden Sea ecosystem and reflect division in MPB support along green (herbivory) and brown (recycled/detrital) food web pathways.

Rhodopseudomonas palustris is an alphaproteobacterium with impressive metabolic versatility, capable of oxidizing ferrous iron to fix carbon dioxide using light energy. Photoferrotrophic iron oxidation is one of the most ancient metabolisms, sustained by the pio operon coding for three proteins: PioB and PioA, which form an outer-membrane porin–cytochrome complex that oxidizes iron outside of the cell and transfers the electrons to the periplasmic high potential iron–sulfur protein (HIPIP) PioC, which delivers them to the light-harvesting reaction center (LH-RC). Previous studies have shown that PioA deletion is the most detrimental for iron oxidation, while, the deletion of PioC resulted in only a partial loss. The expression of another periplasmic HiPIP, designated Rpal_4085, is strongly upregulated in photoferrotrophic conditions, making it a strong candidate for a PioC substitute. However, it is unable to reduce the LH-RC. In this work we used NMR spectroscopy to map the interactions between PioC, PioA, and the LH-RC, identifying the key amino acid residues involved. We also observed that PioA directly reduces the LH-RC, and this is the most likely substitute upon PioC deletion. By contrast, Rpal_4085 demontrated significant electronic and structural differences from PioC. These differences likely explain its inability to reduce the LH-RC and highlight its distinct functional role. Overall, this work reveals the functional resilience of the pio operon pathway and further highlights the use of paramagnetic NMR for understanding key biological processes.

Wruck et al. investigate the folding of the small zinc-finger domain ADR1a inside and at the vestibule of the ribosomal tunnel, using optical tweezers, single-molecule FRET, and molecular dynamics simulations. They find that the ribosomal tunnel accelerates folding while stabilizing the folded state like chaperonins. This study provides insights into the role of the ribosomal tunnel in the folding dynamics of nascent polypeptides.

Stem-cell derived in vitro systems, such as organoids or embryoids, hold great potential for modeling in vivo development. Full control over their initial composition, scalability, and easily measurable dynamics make those systems useful for studying specific developmental processes in isolation. Here we report the formation of gastruloids consisting of mouse embryonic stem cells (mESCs) and extraembryonic endoderm (XEN) cells. These XEN-enhanced gastruloids (XEGs) exhibit the formation of neural epithelia, which are absent in gastruloids derived from mESCs only. By single-cell RNA-seq, imaging, and differentiation experiments, we demonstrate the neural characteristics of the epithelial tissue. We further show that the mESCs induce the differentiation of the XEN cells to a visceral endoderm-like state. Finally, we demonstrate that local inhibition of WNT signaling and production of a basement membrane by the XEN cells underlie the formation of the neuroepithelial tissue. In summary, we establish XEGs to explore heterotypic cellular interactions and their developmental consequences in vitro.

Abstract While CRISPR‐Cas defence mechanisms have been studied on a population level, their temporal dynamics and variability in individual cells have remained unknown. Using a microfluidic device, time‐lapse microscopy and mathematical modelling, we studied invader clearance in Escherichia coli across multiple generations. We observed that CRISPR interference is fast with a narrow distribution of clearance times. In contrast, for invaders with escaping PAM mutations we found large cell‐to‐cell variability, which originates from primed CRISPR adaptation. Faster growth and cell division and higher levels of Cascade increase the chance of clearance by interference, while slower growth is associated with increased chances of clearance by priming. Our findings suggest that Cascade binding to the mutated invader DNA, rather than spacer integration, is the main source of priming heterogeneity. The highly stochastic nature of primed CRISPR adaptation implies that only subpopulations of bacteria are able to respond quickly to invading threats. We conjecture that CRISPR‐Cas dynamics and heterogeneity at the cellular level are crucial to understanding the strategy of bacteria in their competition with other species and phages.

Abstract Background Whereas accumulating studies on patients with coronavirus disease 2019 (COVID‐19) report high incidences of thrombotic complications, large studies on clinically relevant thrombosis in patients with other respiratory tract infections are lacking. How this high risk in COVID‐19 patients compares to those observed in hospitalized patients with other viral pneumonias such as influenza is unknown. Objectives To assess the incidence of venous and arterial thrombotic complications in hospitalized patients with influenza as opposed to that observed in hospitalized patients with COVID‐19. Methods This was a retrospective cohort study; we used data from Statistics Netherlands (study period: 2018) on thrombotic complications in hospitalized patients with influenza. In parallel, we assessed the cumulative incidence of thrombotic complications—adjusted for competing risk of death—in patients with COVID‐19 in three Dutch hospitals (February 24 to April 26, 2020). Results Of the 13 217 hospitalized patients with influenza, 437 (3.3%) were diagnosed with thrombotic complications, versus 66 (11%) of the 579 hospitalized patients with COVID‐19. The 30‐day cumulative incidence of any thrombotic complication in influenza was 11% (95% confidence interval [CI], 9.4–12) versus 25% (95% CI, 18–32) in COVID‐19. For venous thrombotic (VTC) complications and arterial thrombotic complications alone, these numbers were, respectively, 3.6% (95% CI, 2.7–4.6) and 7.5% (95% CI, 6.3–8.8) in influenza versus 23% (95% CI, 16–29) and 4.4% (95% CI, 1.9–8.8) in COVID‐19. Conclusions The incidence of thrombotic complications in hospitalized patients with influenza was lower than in hospitalized patients with COVID‐19. This difference was mainly driven by a high risk of VTC complications in the patients with COVID‐19 admitted to the Intensive Care Unit. Remarkably, patients with influenza were more often diagnosed with arterial thrombotic complications.

The development of gene therapies for central nervous system disorders is challenging because it is difficult to translate preclinical data from current in vitro and in vivo models to the clinic. Therefore, we developed induced pluripotent stem cell (iPSC)-derived cerebral organoids as a model for recombinant adeno-associated virus (rAAV) capsid selection and for testing efficacy of AAV-based gene therapy in a human context. Cerebral organoids are physiological 3D structures that better recapitulate the human brain compared with 2D cell lines. To validate the model, we compared the transduction efficiency and distribution of two commonly used AAV serotypes (rAAV5 and rAAV9). In cerebral organoids, transduction with rAAV5 led to higher levels of vector DNA, transgenic mRNA, and protein expression as compared with rAAV9. The superior transduction of rAAV5 was replicated in iPSC-derived neuronal cells. Furthermore, rAAV5-mediated delivery of a human sequence-specific engineered microRNA to cerebral organoids led to a lower expression of its target ataxin-3. Our studies provide a new tool for selecting and deselecting AAV serotypes, and for demonstrating therapeutic efficacy of transgenes in a human context. Implementing cerebral organoids during gene therapy development could reduce the usage of animal models and improve translation to the clinic.

Huntington disease (HD) is a fatal neurodegenerative genetic disorder, thought to reflect a toxic gain of function in huntingtin (Htt) protein. Adeno-associated viral vector serotype 5 (AAV5)- microRNA targeting huntingtin (miHTT) is a HD gene-therapy candidate that efficiently lowers HTT using RNAi. This study analyzed the efficacy and potential for off-target effects with AAV5-miHTT in neuronal and astrocyte cell cultures differentiated from induced pluripotent stem cells (iPSCs) from two individuals with HD (HD71 and HD180). One-time AAV5-miHTT treatment significantly reduced human HTT mRNA by 57% and Htt protein by 68% in neurons. Small RNA sequencing showed that mature miHTT was processed correctly without off-target passenger strand. No cellular microRNAs were dysregulated, indicating that endogenous RNAi machinery was unaffected by miHTT overexpression. qPCR validation of in silico-predicted off-target transcripts, next-generation sequencing, and pathway analysis confirmed absence of dysregulated genes due to sequence homology or seed-sequence activity of miHTT. Minor effects on gene expression were observed in both AAV5-miHTT and AAV5-GFP-treated samples, suggesting that they were due to viral transduction rather than miHTT. This study confirms the efficacy of AAV5-miHTT in HD patient iPSC-derived neuronal cultures and lack of off-target effects in gene expression and regulation in neuronal cells and astrocytes. Keywords: AAV5, AAV5-miHTT, gene therapy, Huntington disease, Huntingtin, microRNA, RNA-seq, iPSC, off-target, patient-derived neuronal cultures

Gene therapy for severe hemophilia B is advancing and offers sustained disease amelioration with a single treatment. We have reported the efficacy and safety of AMT-060, an investigational gene therapy comprising an adeno-associated virus serotype 5 capsid encapsidating the codon-optimized wild-type human factor IX (WT hFIX) gene with a liver-specific promoter, in patients with severe hemophilia B. Treatment with 2 × 1013 gc/kg AMT-060 showed sustained and durable FIX activity of 3%–13% and a substantial reduction in spontaneous bleeds without T cell-mediated hepatoxicity. To achieve higher FIX activity, we modified AMT-060 to encode the R338L “Padua” FIX variant that has increased specific activity (AMT-061). We report the safety and increased FIX activity of AMT-061 in non-human primates. Animals (n = 3/group) received intravenous AMT-060 (5 × 1012 gc/kg), AMT-061 (ranging from 5 × 1011 to 9 × 1013 gc/kg), or vehicle. Human FIX protein expression, FIX activity, and coagulation markers including D-dimer and thrombin-antithrombin complexes were measured. At equal doses, AMT-060 and AMT-061 resulted in similar human FIX protein expression, but FIX activity was 6.5-fold enhanced using AMT-061. Both vectors show similar safety and transduction profiles. Thus, AMT-061 holds great promise as a more potent FIX replacement gene therapy with a favorable safety profile. Keywords: AAV, AAV5, AMT-060, AMT-061, adeno-associated virus, factor IX, gene therapy, hemophilia B, non-human primate

In recent years, increasing efforts have been devoted to designing new functional stimuli-responsive supramolecular assemblies. Here, we present three isomeric supramolecular coordination complexes consisting of a Pd2L4 stoichiometry. As shown by NMR, CD and X-ray studies, as well as DFT calculations, these complexes form cage-like structures by chiral self-sorting. Photochromic ligands derived from first generation molecular motors enable light-driven interconversion between the three isomers. Two of the isomers were able to form host–guest complexes opening up new prospects toward stimuli-controlled substrate binding and release.

Of the adeno-associated viruses (AAVs), AAV9 is known for its capability to cross the blood–brain barrier (BBB) and can, therefore, be used as a noninvasive method to target the central nervous system. Furthermore, the addition of the peptide PhP.B to AAV9 increases its transduction across the BBB by 40-fold. Another neurotropic serotype, AAV5, has been shown as a gene therapeutic delivery vehicle to ameliorate several neurodegenerative diseases in preclinical models, but its administration requires invasive surgery. In this study, AAV9-PhP.B and AAV5-PhP.B were designed and produced in an insect cell–based system. To AAV9, the PhP.B peptide TLAVPFK was added, whereas in AAV5-PhP.B (AQTLAVPFKAQAQ), with AQ-AQAQ sequences used to swap with the corresponding sequence of AAV5. The addition of PhP.B to AAV5 did not affect its capacity to cross the mouse BBB, while increased transduction of liver tissue was observed. Then, intravenous (IV) and intrastriatal (IStr) delivery of AAV9-PhP.B and AAV5 were compared. For AAV9-PhP.B, similar transduction and expression levels were achieved in the striatum and cortex, irrespective of the delivery method used. IStr administration of AAV5 resulted in significantly higher amounts of vector DNA and therapeutic miRNA in the target regions such as striatum and cortex when compared with an IV administration of AAV9-PhP.B. These results illustrate the challenge in developing a vector that can be delivered noninvasively while achieving a transduction level similar to that of direct administration of AAV5. Thus, for therapeutic miRNA delivery with high local expression requirements, intraparenchymal delivery of AAV5 is preferred, whereas a humanized AAV9-PhP.B may be useful when widespread brain (and peripheral) transduction is needed.

Organoids have emerged as powerful model systems to study organ development and regeneration at the cellular level. Recently developed microscopy techniques that track individual cells through space and time hold great promise to elucidate the organizational principles of organs and organoids. Applied extensively in the past decade to embryo development and 2D cell cultures, cell tracking can reveal the cellular lineage trees, proliferation rates, and their spatial distributions, while fluorescent markers indicate differentiation events and other cellular processes. Here, we review a number of recent studies that exemplify the power of this approach, and illustrate its potential to organoid research. We will discuss promising future routes, and the key technical challenges that need to be overcome to apply cell tracking techniques to organoid biology.

Background: Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Despite the impact of CVDs, risk factors are often insufficiently controlled in patients at high risk. Recently, integrated multidisciplinary cardiovascular risk management (CVRM) programmes have been introduced in primary care. Aim: To investigate the effects of a CVRM programme on systolic blood pressure (SBP) and low-density lipoprotein (LDL)-cholesterol. Design & setting: A prospective observational study was undertaken in patients at high cardiovascular (CV) risk who were aged 40–80 years. Integrated CVRM care was compared with usual care in general practice in the Netherlands. Method: Intervention and usual care patients were matched at baseline on age, sex, and presence of CVD. During 1 year of follow-up, patients received integrated or usual CVRM care in general practice. Primary outcomes were SBP and LDL-cholesterol. Secondary outcomes included calculated 10-year CV risk, body mass index (BMI), lifestyle (smoking, physical activity, and dietary habits), medication use, patient satisfaction, healthcare consumption, morbidity, comorbidity, and mortality. Mixed-model analyses were used to assess the outcomes. Results: Totals of 372 and 317 patients were included in the intervention and usual care group, respectively. Mean age at baseline was 65.1 years and 66.2 years, respectively, and 42% were female in both groups. After 1 year, no differences were observed in: SBP (137.2 mmHg versus 139.0 mmHg in the intervention and usual care group, respectively); LDL-cholesterol (2.6 mmol/l in both groups); or in any of the secondary outcomes. Conclusion: Integrated CVRM care in general practice did not lead to a lower SBP or LDL-cholesterol in patients at high CV risk. Further research is needed to improve CVRM.

Abstract: Adeno-associated virus (AAV)–based liver gene therapy has been shown to be clinically successful. However, the presence of circulating neutralizing antibodies (NABs) against AAV vector capsids remains a major challenge as it may prevent successful transduction of the target cells. Therefore, there is a need to develop strategies that would enable AAV-mediated gene delivery to patients with preexisting anti-AAV NABs. In the current study, the feasibility of using an immunoadsorption (IA) procedure for repeated, liver-targeted gene delivery in nonhuman primates was explored. The animals were administered IV with recombinant AAV5 (rAAV5) carrying the reporter gene human secreted embryonic alkaline phosphatase (hSEAP). Seven weeks after the first rAAV treatment, all of the animals were readministered with rAAV5 carrying the therapeutic hemophilia B gene human factor IX (hFIX). Half of the animals administered with rAAV5-hSEAP underwent IA prior to the second rAAV5 exposure. The transduction efficacies of rAAV5-hSEAP and rAAV5-hFIX were assessed by measuring the levels of hSEAP and hFIX proteins. Although no hFIX was detected after rAAV5-hFIX readministration without prior IA, all animals submitted to IA showed therapeutic levels of hFIX expression, and a threshold of anti-AAV5 NAB levels compatible with successful readministration was demonstrated. In summary, our data demonstrate that the use of a clinically applicable IA procedure enables successful readministration of an rAAV5-based gene transfer in a clinically relevant animal model. Finally, the analysis of anti-AAV NAB levels in human subjects submitted to IA confirmed the safety and efficacy of the procedure to reduce anti-AAV NABs. Furthermore, clinical translation was assessed using an immunoglobulin G assay as surrogate.

Huntington disease (HD) is a fatal neurodegenerative disorder caused by an autosomal dominant CAG repeat expansion in the huntingtin (HTT) gene. The translated expanded polyglutamine repeat in the HTT protein is known to cause toxic gain of function. We showed previously that strong HTT lowering prevented neuronal dysfunction in HD rodents and minipigs after single intracranial injection of adeno-associated viral vector serotype 5 expressing a microRNA targeting human HTT (AAV5-miHTT). To evaluate long-term efficacy, AAV5-miHTT was injected into the striatum of knockin Q175 HD mice, and the mice were sacrificed 12 months post-injection. AAV5-miHTT caused a dose-dependent and sustained HTT protein reduction with subsequent suppression of mutant HTT aggregate formation in the striatum and cortex. Functional proof of concept was shown in transgenic R6/2 HD mice. Eight weeks after AAV5-miHTT treatment, a significant improvement in motor coordination on the rotarod was observed. Survival analysis showed that a single AAV5-miHTT treatment resulted in a significant 4-week increase in median survival compared with vehicle-treated R6/2 HD mice. The combination of long-term HTT lowering, reduction in aggregation, prevention of neuronal dysfunction, alleviation of HD-like symptoms, and beneficial survival observed in HD rodents treated with AAV5-miHTT supports the continued development of HTT-lowering gene therapies for HD. Keywords: Huntington disease, gene silencing, HD mouse model, gene therapy, therapeutic microRNAs, adeno-associated viral vector serotype 5

A hexanucleotide GGGGCC expansion in intron 1 of chromosome 9 open reading frame 72 (C9orf72) gene is the most frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The corresponding repeat-containing sense and antisense transcripts cause a gain of toxicity through the accumulation of RNA foci in the nucleus and deposition of dipeptide-repeat (DPR) proteins in the cytoplasm of the affected cells. We have previously reported on the potential of engineered artificial anti-C9orf72-targeting miRNAs (miC) targeting C9orf72 to reduce the gain of toxicity caused by the repeat-containing transcripts. In the current study, we tested the silencing efficacy of adeno-associated virus (AAV)5-miC in human-derived induced pluripotent stem cell (iPSC) neurons and in an ALS mouse model. We demonstrated that AAV5-miC transduces different types of neuronal cells and can reduce the accumulation of repeat-containing C9orf72 transcripts. Additionally, we demonstrated silencing of C9orf72 in both the nucleus and cytoplasm, which has an added value for the treatment of ALS and/or FTD patients. A proof of concept in an ALS mouse model demonstrated the significant reduction in repeat-containing C9orf72 transcripts and RNA foci after treatment. Taken together, these findings support the feasibility of a gene therapy for ALS and FTD based on the reduction in toxicity caused by the repeat-containing C9orf72 transcripts. Keywords: C9orf72, ALS, FTD, miRNA, gene therapy, AAV

The most common pathogenic mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic GGGGCC (G4C2) repeat in the chromosome 9 open reading frame 72 (C9orf72) gene. Cellular toxicity due to RNA foci and dipeptide repeat (DPR) proteins produced by the sense and antisense repeat-containing transcripts is thought to underlie the pathogenesis of both diseases.RNA sequencing (RNA-seq) data of C9orf72-ALS patients and controls were analyzed to better understand the sequence conservation of C9orf72 in patients. MicroRNAs were developed in conserved regions to silence C9orf72 (miC), and the feasibility of different silencing approaches was demonstrated in reporter overexpression systems. In addition, we demonstrated the feasibility of a bidirectional targeting approach by expressing two concatenated miC hairpins. The efficacy of miC was confirmed by the reduction of endogenously expressed C9orf72 mRNA, in both nucleus and cytoplasm, and an ∼50% reduction of nuclear RNA foci in (G4C2)44-expressing cells. Ultimately, two miC candidates were incorporated in adeno-associated virus vector serotype 5 (AAV5), and silencing of C9orf72 was demonstrated in HEK293T cells and induced pluripotent stem cell (iPSC)-derived neurons. These data support the feasibility of microRNA (miRNA)-based and AAV-delivered gene therapy that could alleviate the gain of toxicity seen in ALS and FTD patients. Keywords: C9orf72, ALS, FTD, gene therapy, microRNA

The crystal structures of four dimethoxybenzaldehyde (C9H10O3) isomers, namely the 2,3-, 2,4-, 2,5- and 3,5- isomers, are reported and compared to the previously reported crystal structures of 3,4-dimethoxybenzaldehyde and 2,6-dimethoxybenzaldehyde. All dimethoxybenzaldehyde molecules in the crystal structures are nearly planar. The largest deviation (1.2 Å) from the aromatic plane is found for one of the methoxy groups of 2,3-dimethoxybenzaldehyde. Upon rapid cooling of 3,4-dimethoxybenzaldehyde and 3,5-dimethoxybenzaldehyde, a metastable polymorph is formed. The crystal studied for the 3,5- isomer was refined as a two-component twin.

Time-lapse microscopy is routinely used to follow cells within organoids, allowing direct study of division and differentiation patterns. There is an increasing interest in cell tracking in organoids, which makes it possible to study their growth and homeostasis at the single-cell level. As tracking these cells by hand is prohibitively time consuming, automation using a computer program is required. Unfortunately, organoids have a high cell density and fast cell movement, which makes automated cell tracking difficult. In this work, a semi-automated cell tracker has been developed. To detect the nuclei, we use a machine learning approach based on a convolutional neural network. To form cell trajectories, we link detections at different time points together using a min-cost flow solver. The tracker raises warnings for situations with likely errors. Rapid changes in nucleus volume and position are reported for manual review, as well as cases where nuclei divide, appear and disappear. When the warning system is adjusted such that virtually error-free lineage trees can be obtained, still less than 2% of all detected nuclei positions are marked for manual analysis. This provides an enormous speed boost over manual cell tracking, while still providing tracking data of the same quality as manual tracking.

The data described in this article are related to drought occurrence in inland valleys and farmers adaptation strategies. The data were collected in 300 inland valleys distributed in 14 regions of West Africa. The data were collected in two phases. In the first phase, 300 inland valleys were identified in 14 regions and their locations were determined with handheld GPS devices. Questionnaires and informal interviews were administered to inland valleys users to collect data on physical and socio-economic characteristics, hydrology, farmers experience with drought affecting rice production in inland valleys and adaptation strategies. In the second phase, the locations of the inland valleys were imported in a GIS environment and were used to extract additional parameters on soil characteristics and water demand from the Shuttle Radar Topography Mission (SRTM), Africa Soil Information Service (africasoils.net) and POWER database (http://power.larc.nasa.gov). In total, the dataset contains 41 variables divided into seven themes: farmers’ experience with drought, adaptive management of rice farmers to drought, physical characteristics, hydrology, management practices, socio-economic characteristics and weather data of inland valleys. Keywords: Drought, Inland valleys, Rice, Africa

Unidirectional rotation in a synthetic molecular motor is typically driven by intrinsic asymmetry or sequences of chemical transformations. Here, the authors control the direction of a molecule’s rotation through supramolecular binding of a chiral guest and subsequent transfer of its chiral information.

Sign epistasis clearly constrains evolution, but its causes are difficult to decipher. Here, the authors study epistasis in a signalling cascade, and arrive at a general criterion and understanding of sign epistasis as arising from the inherent hierarchy between signalling cascade components.

HIV is an independent risk factor of cardiovascular disease (CVD); therefore, perinatally HIV-infected (PHIV) children potentially have a greater CVD risk at older age. Lipoprotein(a) (Lp(a)) is an established risk factor for CVD in the general population. To evaluate a potential increased CVD risk for PHIV children, we determined their lipid profiles including Lp(a). In the first substudy, we assessed the lipid profiles of 36 PHIV children visiting the outpatient clinic in Amsterdam between 2012 and 2020. In the second substudy, we enrolled 21 PHIV adolescents and 23 controls matched for age, sex and ethnic background on two occasions with a mean follow-up time of 4.6 years. We assessed trends of lipid profiles and their determinants, including patient and disease characteristics, using mixed models. In the first substudy, the majority of PHIV children were Black (92%) with a median age of 8.0y (5.7–10.8) at first assessment. Persistent elevated Lp(a) levels were present in 21/36 (58%) children (median: 374 mg/L (209–747); cut off = 300). In the second substudy, the median age of PHIV adolescents was 17.5y (15.5–20.7) and of matched controls 16.4y (15.8–19.5) at the second assessment. We found comparable lipid profiles between groups. In both studies, increases in LDL-cholesterol and total cholesterol were associated with higher Lp(a) levels. A majority of PHIV children and adolescents exhibited elevated Lp(a) levels, probably associated with ethnic background. Nonetheless, these elevated Lp(a) levels may additionally contribute to an increased CVD risk.

The dataset described in this data article represents four agricultural zones in West-Africa that are located in three countries: Benin, Mali and Sierra Leone. The dataset was created through a research collaboration between the Africa Rice Center (AfricaRice), Sierra Leone Agricultural Research Institute (SLARI) and the Institute for Rural Economy (IER). The dataset was compiled to investigate the potential for rice production in inland valleys of the three countries. The results of the investigation were published in Dossou-Yovo et al. (2017) and Djagba et al. (2018). The dataset describes the biophysical and socioeconomic conditions of 499 inland valleys in the four agricultural zones. In each inland valley data were collected through a focus group interview with a minimum of three farmers. In 499 interviews a total of 7496 farmers participated. The location of each inland valley was determined with handheld GPS devices. The geographic locations were used to extract additional parameters from digital maps on soils, elevation, population density, rainfall, flow accumulation, and distances to roads, market places, rice mills, chemical input stores, and settlements. The dataset contains 65 parameters in four themes (location, biophysical characteristics, socioeconomic characteristics, and inland valley land development and use). The GPS coordinates indicate the location of an inland valley, but they do not lead to the location of individual fields of farmers that were interviewed. The dataset is publicly shared as Supplementary data to this data article.

Huntington disease (HD) is a fatal, neurodegenerative genetic disorder with aggregation of mutant Huntingtin protein (mutHTT) in the brain as a key pathological mechanism. There are currently no disease modifying therapies for HD; however, HTT-lowering therapies hold promise. Recombinant adeno-associated virus serotype 5 expressing a microRNA that targets HTT mRNA (AAV5-miHTT) is in development for the treatment of HD with promising results in rodent and minipig HD models. To support a clinical trial, toxicity studies were performed in non-human primates (NHP, Macaca fascicularis) and Sprague-Dawley rats to evaluate the safety of AAV5-miHTT, the neurosurgical administration procedure, vector delivery and expression of the miHTT transgene during a 6-month observation period. For accurate delivery of AAV5-miHTT to the striatum, real-time magnetic resonance imaging (MRI) with convection-enhanced delivery (CED) was used in NHP. Catheters were successfully implanted in 24 NHP, without neurological symptoms, and resulted in tracer signal in the target areas. Widespread vector DNA and miHTT transgene distribution in the brain was found, particularly in areas associated with HD pathology. Intrastriatal administration of AAV5-miHTT was well tolerated with no clinically relevant changes in either species. These studies demonstrate the excellent safety profile of AAV5-miHTT, the reproducibility and tolerability of intrastriatal administration, and the delivery of AAV5-miHTT to the brain, which support the transition of AAV5-miHTT into clinical studies.

Since their introduction, process trees have been frequently used as a process modeling formalism in many process mining algorithms. A process tree is a (mathematical) tree-based model of a process, in which internal vertices represent behavioral control-flow relations and leaves represent process activities. Translation of a process tree into a sound workflow net is trivial. However, the reverse is not the case. Simultaneously, an algorithm that translates a WF-net into a process tree is of great interest, e.g., the explicit knowledge of the control-flow hierarchy in a WF-net allows one to reason on its behavior more easily. Hence, in this paper, we present such an algorithm, i.e., it detects whether a WF-net corresponds to a process tree, and, if so, constructs it. We prove that, if the algorithm finds a process tree, the language of the process tree is equal to the language of the original WF-net. The experiments conducted show that the algorithm’s corresponding implementation has a quadratic time complexity in the size of the WF-net. Furthermore, the experiments show strong evidence of process tree rediscoverability.

Traditional methods based on field campaigns are generally used to assess the performance of irrigation schemes in Burkina Faso, resulting in labor-intensive, time-consuming, and costly processes. Despite their extensive application for such performance assessment, remote sensing (RS)-based approaches remain very much underutilized in Burkina Faso. Using multi-temporal Landsat images within the Python module for the Surface Energy Balance Algorithm for Land model, we investigated the spatiotemporal performance patterns of the Kou Valley irrigation scheme (KVIS) during two consecutive cropping seasons. Four performance indicators (depleted fraction, relative evapotranspiration, uniformity of water consumption, and crop water productivity) for rice, maize, and sweet potato were calculated and compared against standard values. Overall, the performance of the KVIS varied depending on year, crop, and the crop’s geographical position in the irrigation scheme. A gradient of spatially varied relative evapotranspiration was observed across the scheme, with the uniformity of water consumption being fair to good. Although rice was the most cultivated, a shift to more sweet potato farming could be adopted to benefit more from irrigation, given the relatively good performance achieved by this crop. Our findings ascertain the potential of such RS-based cost-effective methodologies to serve as basis for improved irrigation water management in decision support tools.

In recent decades, there have been substantial increases in crop production in sub-Saharan Africa (SSA) as a result of higher yields, increased cropping intensity, expansion of irrigated cropping systems, and rainfed cropland expansion. Yet, to date much of the research focus of the impact of climate change on crop production in the coming decades has been on crop yield responses. In this study, we analyse the impact of climate change on the potential for increasing rainfed cropping intensity through sequential cropping and irrigation expansion in central Benin. Our approach combines hydrological modelling and scenario analysis involving two Representative Concentration Pathways (RCPs), two water-use scenarios for the watershed based on the Shared Socioeconomic Pathways (SSPs), and environmental water requirements leading to sustained streamflow. Our analyses show that in Benin, warmer temperatures will severely limit crop production increases achieved through the expansion of sequential cropping. Depending on the climate change scenario, between 50% and 95% of cultivated areas that can currently support sequential cropping or will need to revert to single cropping. The results also show that the irrigation potential of the watershed will be at least halved by mid-century in all scenario combinations. Given the urgent need to increase crop production to meet the demands of a growing population in SSA, our study outlines challenges and the need for planned development that need to be overcome to improve food security in the coming decades.