Additional Properties Research Articles

Durability assessment of fly ash, GGBS, and silica fume based geopolymer concrete with recycled aggregates against acid and sulfate attack

There is a constant drive to develop high-performance concrete with enhanced mechanical and durability characteristics while incorporating environment-friendly materials. The study investigates the synergistic effects of fly ash (FA), ground granulated blast furnace slag (GGBS), and silica fume (SF) on the durability characteristics of 100 % recycled aggregate (RA) based geopolymer concrete (GPC) against acid and sulfate attacks. Moreover, its relative performance compared to control samples of ordinary Portland cement (OPC) concrete with natural and recycled aggregates was also assessed. The acid and sulfate resistance were determined by submerging the concrete specimens in the 5 % sulphuric acid (H2SO4), 5 % sodium sulfate (Na2SO4), and 5 % magnesium sulfate (MgSO4) for up to 180 days of exposure duration. The specimens' resistance to chemical attack was assessed by visual observation, changes in weight, and the percentage of compressive strength loss at different time intervals. In addition, the changes in the mineralogical and microstructures of the GPC matrix were also examined using X-ray diffraction (XRD), scanning electron microscope (SEM), and mercury intrusion porosimetry (MIP) tests. Results indicated that an increased proportion of GGBS and SF positively influenced the durability of GPC, as revealed by reduced mass loss and compressive strength degradation. In addition, the microstructural, mineralogical, and morphological properties depicted enhanced densification and compactness of the GPC matrix due to the formation of CSH, CASH, and NASH gels. GPC-MG15 (FA:GGBS:SF – 35:50:15) yielded the highest strength and durability performance, compensating for the trade-offs of using 100 % RAs in conventional OPC concrete. So, optimized use of these sustainable materials has depicted promising results in aggressive environments while reducing the need for conventional OPC and natural aggregates.

Investigating Rules and Parameters of Reservoir Computing with Elementary Cellular Automata, with a Criticism of Rule 90 and the Five-Bit Memory Benchmark

Reservoir computing with cellular automata (ReCAs) is a promising concept by virtue of its potential for effective hardware implementation. In this paper, we explore elementary cellular automata rules in the context of ReCAs and the 5-bit memory benchmark. We combine elementary cellular automaton theory with our results and use them to identify and explain some of the patterns found. Furthermore, we use these findings to expose weaknesses in the 5-bit memory benchmark as it is typically applied in ReCAs, such as pointing out what features it selects for or solving it using random vectors. We look deeply into previously successful rules in ReCAs such as rule 90 and explain some of the consequences of its additive properties as well as the correlation between grid size and performance. Additionally, we present results from exhaustively exploring ReCAs on key parameters such as distractor period, iterations and grid size. The findings of this paper should motivate the ReCAs community to move away from using the 5-bit memory benchmark as it is being applied today.

Three-dimensional topology-based analysis segments volumetric and spatiotemporal fluorescence microscopy.

Image analysis techniques provide objective and reproducible statistics for interpreting microscopy data. At higher dimensions, three-dimensional (3D) volumetric and spatiotemporal data highlight additional properties and behaviors beyond the static 2D focal plane. However, increased dimensionality carries increased complexity, and existing techniques for general segmentation of 3D data are either primitive, or highly specialized to specific biological structures. Borrowing from the principles of 2D topological data analysis (TDA), we formulate a 3D segmentation algorithm that implements persistent homology to identify variations in image intensity. From this, we derive two separate variants applicable to spatial and spatiotemporal data, respectively. We demonstrate that this analysis yields both sensitive and specific results on simulated data and can distinguish prominent biological structures in fluorescence microscopy images, regardless of their shape. Furthermore, we highlight the efficacy of temporal TDA in tracking cell lineage and the frequency of cell and organelle replication.

Electrochemical Sensing Based on Nanofibers Modified Electrodes for Application in Diagnostic, Food and Waste Water Samples

AbstractElectrochemical sensors and biosensors are today important analytical and monitoring tools in various fields, from agriculture and the food industry to environmental and biomedical/pharmaceutical applications. In particular, the integration of nanotechnology with electrochemical sensors and biosensors to develop a new generation of sensor platforms has made enormous progress in recent years. The outstanding properties of one‐dimensional (1D) nanofibers (NFs), such as high porosity, superior mechanical properties and high specific surface area have made them attractive electrocatalysts, support materials for the immobilization of biomolecules as well as mimetic materials for sensing and biosensing applications. Moreover, the possibility of fabricating multifunctional composites based on NFs increases (bio)sensing capabilities through synergistic effects and additive properties. This review describes the progress made over the last decade in the use of multifunctional NFs‐based composites as modified electrodes for the sensing of various analytes in biomedical, food, and wastewater treatment applications. The aim of this review is to provide a comprehensive overview and a guide for researchers from different disciplines to fabricate and improve their selective NFs‐based (bio)sensor platforms for the detection of desired analytes or multi‐analytes.

Cadmium accumulation in tropical island paddy soils: From environment and health risk assessment to model prediction

Cultivated soil quality is crucial because it directly affects food safety and human health, and rice is of primary concern because of its centrality to global food networks. However, a detailed understanding of cadmium (Cd) geochemical cycling in paddy soils is complicated by the multiple influencing factors present in many rice-growing areas that overlap with industrial centers. This study analyzed the pollution characteristics and health risks of Cd in paddy soils across Hainan Island and identified key influencing factors based on multi-source environmental data and prediction models. Approximately 27.07% of the soil samples exceeded the risk control standard screening value for Cd in China, posing an uncontaminated to moderate contamination risk. Cd concentration and exposure duration contributed the most to non-carcinogenic and carcinogenic risks to children, teens, and adults through ingestion. Among the nine prediction models tested, Extreme Gradient Boosting (XGBoost) exhibited the best performance for Cd prediction with soil properties having the highest importance, followed by climatic variables and topographic attributes. In summary, XGBoost reliably predicted the soil Cd concentrations on tropical islands. Further research should incorporate additional soil properties and environmental variables for more accurate predictions and to comprehensively identify their driving factors and corresponding contribution rates.

Sweet Bell Pepper: A Focus on Its Nutritional Qualities and Illness-Alleviated Properties.

Sweet bell pepper (SBP, Capsicum annuum L.) can be employed as a spice in many dishes and may also be eaten as a delicious fruit. These two nutritional attributes are owing to the strong, deep taste of many SBP phytochemicals. This fruit has many additional beneficial properties because it contains high concentrations of minerals and vitamins that distinguish it from other kinds of fruits. Almost every part of the SBP is thought to be an excellent source of bioactive substances that are health supporters, such as flavonoids, polyphenols, and various aromatic substances. The ability of SBP-phytochemicals to work as antioxidants, reducing the harmful effects of oxidative stress and consequently preventing many chronic illnesses, is one of their main biomedical characteristics. These phytochemicals have good antibacterial properties, mostly against gram-positive pathogenic microbes, in addition to their anti-carcinogenic and cardio-preventive effects. So, this review aims to highlight the nutritional qualities of SBP-derived phytochemicals and their illness-alleviated characteristics. Antioxidant, anti-inflammatory, antitumor, antidiabetic, and analgesic properties are some of the ones discussed.

Extensions and reductions of squarefree words

A word is squarefree if it does not contain a nonempty word of the form XX as a factor. A famous 1906 result of Thue asserts that there exist arbitrarily long squarefree words over a 3-letter alphabet. We study squarefree words with additional properties involving single-letter deletions and extensions of words.A squarefree word is steady if it remains squarefree after deletion of any single letter. We prove that there exist infinitely many steady words over a 4-letter alphabet. We also demonstrate that one may construct steady words of any length by picking letters from arbitrary alphabets of size 7 assigned to the positions of the constructed word. We conjecture that both bounds can be lowered to 4, which is best possible.In the opposite direction, we consider squarefree words that remain squarefree after insertion of a single (suitably chosen) letter at every possible position in the word. We call them bifurcate. We prove a somewhat surprising fact, that over a fixed alphabet with at least three letters, every steady word is bifurcate. We also consider families of bifurcate words possessing a natural tree structure. In particular, we prove that there exists an infinite tree of doubly-infinite bifurcate words over an alphabet of size 12.

Multidisciplinary optimization of automotive mega-castings merging classical structural optimization with response-surface-based optimization enhanced by machine learning

Large high pressure die castings (HPDC), recently referred to as mega-castings, can replace plenty of steel metal sheets usually employed for body-in-white (BIW) structures. They can save manufacturing expense and unleash additional lightweight potential thanks to additional design freedom and material properties. The BIW plays a major role in automotive design since it must fulfill numerous structural targets ranging from stiffness for vehicle dynamics, dynamic responses for NVH (noise, vibration, harshness), driving comfort standards and several passive safety requirements. The use of mega-casting structures leads to additional requirements with respect to castability and material quality. Achieving a lightweight design considering requirements related to crash or castability is a challenge on its own, due to the high computational cost of related simulation techniques. Considering multiple requirements simultaneously, therefore often leads to non-weight-optimal structures. To exploit the full lightweight potential, we present a generative multidisciplinary optimization pipeline for the structural design of automotive mega-casting parts in this paper. The approach combines established methods in automotive industry such as topology optimization and response-surface-based (RSM) optimization and enhances the latter by machine learning (ML) based clustering and classification. In a first step topology optimization is employed to derive optimal load-paths for multidisciplinary loading conditions. For this purpose, casting manufacturing constraints as well as more than hundred linearized loads are used to incorporate NVH and passive safety requirements. In a next step the optimal thickness distribution and rib orientation of the structure is achieved using RSM optimization algorithms for the computationally expensive nonlinear crash and casting simulations. Performance indicators are treated by unsupervised learning based on clustering. This enables classification constraints based on simulation field results from hundreds of samples to be included into RSM optimization. It resolves a typical risk of pure scalar, regression-type targets, where supposed optimal results fail when domain experts examine the full field result of the corresponding simulation. It is shown how this approach is superior in achieving a weight-optimal design and turnaround time compared to a design workflow classically used for BIW structures.

INVESTIGATION OF PROBIOTICS-SORPTION PROPERTIES OF THE COMPOSITION BASED ON POMACE

The use of enterosorbents to relieve intoxication and to increase the content of prebiotics in the body is a topical problem of every second metropolis resident. The pharmaceutical market offers a huge number of enterosorbents, some of which can cause negative consequences (for example, mechanical damage, constipation etc.). The others that are more compatible with the body, for example, pectins, are expensive due to the energy-intensive process of their isolation. We offer a preparation based on pomace powder (60%) with the addition of starch, kaolin and pectin from sea grass eelgrass (Zostera marina) (6%). The powder is obtained by a previously developed and patented method and has an established sufficiently high sorption activity. At the same time, apple pomace also contains pectin, which is a substrate for the growth of beneficial microorganisms living in the colon.
 The article presents the data of a comparative analysis of the sorption activity of the developed powder and a number of enterosorbents offered by the pharmaceutical market in relation to standard test substances: methylene blue and pharmaceutical gelatin. It is shown that the composite enterosorbent obtains effective sorption properties and equals to common sorbents. When dissolved in water, the powder acquires a gel-like consistency that gives it additional therapeutic and preventive properties. The use of apple pomace as a basis enables to reduce the cost of producing expensive pectin and make it more affordable for the population.

Water productivity of barley crop under laser land leveling technique and minimum tillage

ABSTRACTLaser land leveling is an important method that helps to improve the spatial distribution of irrigation water and fertilizers applications, subsequently reduce water, nutrient, and energy inputs to agriculture and contributes to increasing productivity. Thus, the aim of the study was to improve the productivity of the barley crop grown in sandy lands under conditions of water scarcity and the negative impact of climate change in Egypt by using laser leveling and the minimum tillage method. Two experiments were conducted during the 2020/2021 and 2021/2022 seasons at the Nubariya farm, Buhaira Governorate, Egypt to study the effects of laser leveling and minimum tillage (zero – tillage, 10 cm, 20 cm, and 30 cm) affecting the distribution of soil moisture, water stress, effectiveness of water application, yield characteristics, water productivity, and some quality parameters of the barley crop. The statistical analysis’ findings revealed a considerable influence of both laser soil leveling and minimum depth of plowing on productivity, water productivity and quality properties of barley crop. Laser leveling with a plowing depth of 10 cm gave the most favorable values of the soil moisture content at the root-zone as well as better grain yield and water productivity in addition to improve the quality properties of barley. The grain yield has improved by 12.65% and 10.41%, while water productivity has increased by 12.75% and 10.06% during the seasons 2020/2021 and 2021/2022, respectively. This increase is likely the result of improving soil moisture distribution and increasing irrigation application efficiency, which resulted in less water stress in the root zone and subsequently increased yield, water productivity and quality properties of barley during the two growing seasons. Generally, the application of laser land leveling as eco-friendly practice will help in sustaining barley productivity in Egypt particularly in the sandy soil regions. According to this study, laser-assisted precision field leveling has the potential to improve grain yield and crop establishment, water productivity and barley quality properties in addition to achievement of the highest net income for farmers.

Labeling Circulant Graphs: Distance Two Condition

Given n≥6, D={1,2,…,⌊n2⌋}, and a generating set S⊆D, the circulant graph Cn(S) has Zn as a vertex set in which two distinct vertices i and j are adjacent if and only if |i−j|n∈S, where |x|n=min(|x|,n−|x|) is the circular distance modulo n. In this paper, we determine the L(2,1)-labeling number of Cn(D∖X), referred to as λ(Cn(D∖X)), for X={⌊n2⌋}, X={a}, X={a,b}, and in the general case when |X|<⌊n2⌋−⌈n4⌉, where a,b∈D. Furthermore, we demonstrate that for all n≥6 and any given set S, λ(Cn(S))=n+gcd(n,S¯)−2 if and only if gcd(n,S¯)≥2, and λ(Cn(S))≤n−1 if and only if gcd(n,S¯)=1. Additionally, we establish that when the diameter of Cn(S) equals 2, λ(Cn(S))=n−1. This observation motivated us to investigate the properties of S that lead to a diameter of Cn(S) equal to 2. Then, we introduce a highly distinctive family, denoted as An, that generates a large number of generating sets. For each value of n, we acquire a circulant graph Cn(An) with a diameter of 2, λ(Cn(An))=n−1, and various additional interesting properties.

Investigation of Na7V3(P2O7)4/carbon composite as cathode material for sodium-ion battery: Influences of carbon additives and voltage windows

A sequentially processed method is developed to prepare a high-voltage cathode material sodium vanadium diphosphate Na7V3(P2O7)4 for Na-ion battery. We find that the growth of Na7V3(P2O7)4 generally introduces growth orientation due to their hybrid ionic and covalent bonding. The crystal growth of the diphosphate prefers to grow perpendicular to [002] direction, leading to an increase of the diffusion length of Na+ ion. To regulate the growth of crystal, three kinds of carbon additives, graphene oxide, Cabot carbon black and Ketjen carbon black are adopted to tune the morphologies of Na7V3(P2O7)4 to further improve its electrochemical performances. The morphologies and physicochemical properties of carbon additives and corresponding prepared Na7V3(P2O7)4 are both well characterized to clarify the effects of carbon additives on the growth process. We find that the Cabot carbon black can dramatically weaken the oriented growth direction and produce fine morphology. By adding Cabot carbon black to the sequential process, the reversible capacity of Na7V3(P2O7)4 can reach to 76 mAh g−1 with good cyclability within the voltage window of 2–4.5 V. An average voltage of 3.8 V can be obtained due to the strong inductive effect of diphosphate group.

Exploring consumer beliefs about novel fortified foods: A focus group study with UK-based older and younger adult consumers

Consuming ‘nutritionally-enhanced’ food products (including those that are fortified or enriched to deliver nutritional and functional properties) may help to improve overall diet quality and combat risks associated with malnutrition. However, fortification can negatively impact consumer acceptance, particularly where expected sensory properties of ‘delivery’ foods are affected by target ingredients. Here, we explored factors influencing consumer acceptability for six novel food products that had been fortified, including both savoury and sweet meal components (e.g., high protein dumplings, probiotic yoghurt drink). In person focus groups (25 consumers aged between 22 and 76 years old) were conducted with two stages; firstly, participants completed a blind taste test of products without awareness of fortification. Secondly, participants discussed products with awareness of additional ingredients and food properties. Reflexive thematic analysis showed that liking of sensory properties differed between foods, but informing participants about the fortification of products highlighted potential trade-offs between taste, health, price, and familiarity. Though taste and texture were generally prioritised by participants, positive perceptions of health benefits increased consumer willingness to buy, whilst both cost and uncertainty about product use were potential barriers. Trust of information was a key concern for labelling and product claims. These results highlight product features that may be optimised to support the success of fortified foods. Greater transparency when building product brands and improving consumer knowledge of fortification may also be important for longer-term consumer acceptance.

Development of TDP‐43 immunotherapy blocking transmission of seeding‐competent species from ALS/FTD

AbstractBackgroundAccumulation of pathological transactive response DNA binding protein 43 (TDP‐43) into intracellular inclusions underlies frontotemporal lobar degeneration with TDP‐43 pathology (FTLD‐TDP), amyotrophic lateral sclerosis (ALS) and limbic‐predominant age‐related TDP‐43 encephalopathy (LATE). TDP‐43 pathology is present as a co‐pathology in other neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and chronic traumatic encephalopathy (CTE). Multiple mechanisms contribute to spreading of TDP‐43 pathology including transmission across synaptic terminals, exosomes and release of misfolded TDP‐43 from dying neurons. In fact, cerebrospinal fluid (CSF) obtained from ALS/FTLD‐TDP patients contains seeding‐competent TDP‐43 species. Therefore, antibody‐mediated clearance of pathological TDP‐43 represents an attractive therapeutic strategy. Our approach is to develop a TDP‐43‐specific immunotherapy that exploits Fcγ receptor‐mediated removal mechanisms to limit neuronal damage while maintaining physiological TDP‐43 function.MethodHigh affinity monoclonal antibodies (mAbs) were generated against various regions of TDP‐43 using AC Immune’s SupraAntigen® platform and evaluated in mechanistic assays in vitro and in vivo models of TDP‐43 proteinopathies. A real‐time quaking‐induced conversion (RT‐QuIC) assay was established using patient’s CSF to evaluate the inhibitory properties of the lead therapeutic mAb on the seeding mechanism. Studies to evaluate the pharmacology and safety of the antibody were carried out in non‐human primates.ResultTargeting the C‐terminal domain of TDP‐43 but not the RNA recognition motifs reduced TDP‐43 pathology and prevented neuronal loss in vivo. This rescue was dependent on Fcγ receptor‐mediated immune complex uptake by microglia. The latter also promoted phagocytic capacity of ALS patient‐derived microglia. Importantly, the antibody efficiently neutralized seeding‐competent pathological TDP‐43 species in CSF from ALS patients. These beneficial effects were achieved while preserving physiological TDP‐43 function demonstrating safety of targeting TDP‐43 by immunotherapy. Modelling from non‐clinical pharmacokinetic studies of the antibody predicted a half‐life supporting target saturation in humans when administered monthly.ConclusionOur studies confirm the presence of extracellular seeding‐competent TDP‐43 species in the CSF of patients with ALS/FTLD‐TDP patients, providing an explanation for the spread of pathology including the non‐contiguous pattern of clinical manifestations observed in ALS. In addition, the binding and inhibitory properties of our lead therapeutic mAb in multiple patho‐mechanistic assays support clinical development in TDP‐43‐driven neurodegenerative diseases.

Artificial grass that changes colors with the seasons

Condor Grass introduces a reportedly world-first with Colorshift technology. Years of research and extensive testing have led to a type of artificial grass that reacts to outdoor temperatures and changes colors with the seasons. In doing so, the Hasselt-based company adds additional natural properties to artificial grass. Colorshift technology will be available from early 2024 and was launched at the FSB show in Cologne.

Development of the risk analysis methodology given the current safety challenges for public health in the Russian Federation: Vital issues and prospects

The contemporary socioeconomic situation creates multiple safety challenges in the Russian Federation including public health safety. These challenges cannot be ignored when formulating strategic tasks of health risk analysis within predictions of the socioeconomic development of the Russian Federation for the period up to 2030. Targeted development of the risk analysis methodology in the Russian Federation has provided solid grounds for successful solution of primary tasks related to providing sanitary-epidemiological wellbeing of the country population. It has created a platform for further improvement of activities performed by the Rospotrebnadzor in various spheres. This includes development and greater detailing of results obtained by examining how health risks occur under exposure to heterogeneous environmental factors and work-related ones considering their combined effects. Another significant trend is proactive development of methods for assessing and managing health risks associated with potentially hazardous factors of new technologies and products (nanotechnologies, new foods, etc.). Several fundamental aspects of risk analysis can be considered vital at the moment. This includes investigating mechanisms of risk occurrence; establishing regularities of risks of negative health outcomes with various severities under ongoing integration of body systems dysfunctions and considering capabilities of the body to recover its proper functioning and its adaptive resources; finding solutions to an issue associated with assessing additive properties of effects produced by risk factors including heterogeneous ones. Fundamental aspects of health risks analysis development are closely connected with an issue associated with formulating fundamentals of an information platform eligible for this methodology. It is impossible to achieve any development in investigating mechanisms of health risks occurrence under exposure to heterogeneous environmental factors and work-related ones without applying up-to-date investigation techniques, digital ones included. The existing safety challenges for public health in the Russian Federation include the necessity to protect public health in order to extend a period of economic activity of the population and to develop labor and economic potential of the country; high levels of adverse impacts on the public health exerted by chemical, physical, and biological environmental factors; insufficient ‘digital maturity’ of methods employed to predict threats and provide sanitary-epidemiological wellbeing of the population. Given all the aforementioned, it seems advisable to outline the following promising trends in the development of the health risk analysis methodology: developing the fundamentals the methodology is based on; enlarging practical use of health risk assessment results in everyday activities of the Rospotrebnadzor; creating an information platform and digitalizing health risk analysis; developing a methodology for substantiating effective preventive programs aimed at health risk management.

Flexible Lévy-Based Models for Time Series of Count Data with Zero-Inflation, Overdispersion, and Heavy Tails

The explosion of time series count data with diverse characteristics and features in recent years has led to a proliferation of new analysis models and methods. Significant efforts have been devoted to achieving flexibility capable of handling complex dependence structures, capturing multiple distributional characteristics simultaneously, and addressing nonstationary patterns such as trends, seasonality, or change points. However, it remains a challenge when considering them in the context of long-range dependence. The Lévy-based modeling framework offers a promising tool to meet the requirements of modern data analysis. It enables the modeling of both short-range and long-range serial correlation structures by selecting the kernel set accordingly and accommodates various marginal distributions within the class of infinitely divisible laws. We propose an extension of the basic stationary framework to capture additional marginal properties, such as heavy-tailedness, in both short-term and long-term dependencies, as well as overdispersion and zero inflation in simultaneous modeling. Statistical inference is based on composite pairwise likelihood. The model’s flexibility is illustrated through applications to rainfall data in Guinea from 2008 to 2023, and the number of NSF funding awarded to academic institutions. The proposed model demonstrates remarkable flexibility and versatility, capable of simultaneously capturing overdispersion, zero inflation, and heavy-tailedness in count time series data.

State estimation for sound environment system by using Bayesian filter based on fuzzy observation

The specific signal in a sound environment is measured in decibel scale and often contains the fuzziness due to several causes. Furthermore, there exists usually a background noise in addition to the specific signal. In this study, a Bayesian filter for estimating the specific signal, based on the observed data containing the fuzziness and the effects of a background noise with non-Gaussian type, is proposed. More specifically, the energy variables satisfying the additive property of the specific signal and background noise are first considered. Next, after introducing an expansion expression of the probability density function based on a log-normal distribution and a new type of membership function, which are suitable for the nonlinear relationship between the energy variable and the measurement in decibel scale, by applying probability measure of fuzzy events, a state estimation method is theoretically derived. The proposed theory is applied to the actual observation data in a sound environment, and its usefulness is experimentally confirmed.

Chicken combs as a raw material in the manufacturing of chopped semi-finished horse meat products

Food safety stands at the forefront of social policies across nations. Secondary meat raw materials present a potential source for meat production. Our research objective was to analyze the nutritional value and functional-technological properties of horsemeat and protein additives of animal origin, particularly secondary meat raw materials such as chicken combs. We also sought to empirically determine their optimal quantities for developing chopped semi-finished horsemeat products, which are protein-enriched and have high consumer appeal. We aimed to establish efficient methods for utilizing collagen-rich materials like chicken combs. Additionally, we assessed the feasibility of using slaughter by-products and the potential of minced meat and pastry products as valuable raw material sources. Based on our findings, we deduce that chicken combs, a lower-value secondary raw material, possess notable physical, chemical, and safety properties. These traits underline its high biological worth and environmental benignity, qualifying it as a viable ingredient for protein enrichment and as an additive in minced meat and paste product manufacturing.

Interferon-Alpha Impairs Neoplastic Vasculogenesis in a 3D iPSC-Based Model of JAK2V617F+ Myeloproliferative Neoplasms

Overt Myelofibrosis (MF) is an end-stage subtype of BCR-ABL1 negative myeloproliferative neoplasms (MPN), which is characterized by progressive bone marrow (BM) fibrosis resulting in loss of normal hematopoietic support as well as inflammatory and vascular complications. While aberrant neoangiogenesis is known to participate in the pathogenesis of MF, Erba et al. (Am J Pathol, 2017) suggested the occurrence of endothelial-to-mesenchymal transition (EndMT) in BM and spleen of MF patients (pts). Given the relevance of endothelial cells (EC) for MF development and the occurrence of JAK2V617F-mutated EC in MPN pts, targeting of the vascular niche has emerged as a promising therapeutic strategy. To untangle the impact of the JAK2V617F mutation on human vasculogenesis, we leveraged high-purity and clonal patient-specific induced pluripotent stem cell (iPSC)-derived EC (iEC) harboring either the JAK2V617F mutation in hetero- (JAK2V617F HET) or homozygosis (JAK2V617F HOM). JAK2 WT and CRISPR-repaired isogenic JAK2 WT (both referred to as JAK2 WT) were used as controls. In a first set of in vitro experiments, JAK2V617F mutated iEC showed a significantly increased proliferation rate in comparison to JAK2 WT iEC. We next evaluated the effect of selected drugs on iEC viability to assess their vascular toxicity. While treatment with 1 µM each of fedratinib, anagrelide or venetoclax led to a decrease in iEC viability (all **p ≤ .01), no significant alterations were observed with 1 µM ruxolitinib (RUX) or 1000 U/ml interferon-alpha (IFNa). Overall, no differences between the 3 genotypes were observed, pointing towards a genotype-independent mode of action. Given the favorable vascular toxicity profile of IFNa, as shown above, its anti-angiogenic potential was then assessed via endothelial tube formation assays. Pre-incubation with 1000 U/ml IFNa prior to seeding of the cells onto a reconstituted basement membrane matrix led to a significant inhibition of capillary-like structures formation in JAK2 WT and JAK2V617F HET or HOM iEC. One of the limitations of 2D culture assays is the lack of 3D homotypic cellular organization, compromising its translational value. Therefore, we established an adaptive 3D fibrin-based model to recapitulate, in vitro, the critical EC-pericyte crosstalk required for formation and maturation of blood vessels. Recent studies highlighted the capacity of BM mesenchymal stem cells (MSC) to differentiate into pericytes. Therefore, we seeded JAK2 WT and JAK2V617F HETor HOM iEC with or without MSC in fibrin hydrogels. MSC and iEC were visualized using different PKH dyes as fluorescent cell linkers to enable live tracking of vascular tree formation. Although self-assembly of JAK2 WT and JAK2V617F HET or HOM iEC into vessel-like structures was observed in the generated monocultures, the presence of MSC in the cocultures led to a significant increase of the formed network area (Fig. 1), hence recapitulating the critical support of pericytes. While acute IFNa exposure did not alter vascular tree formation (Fig. 1), a separate pre-treatment of iEC and MSC prior to cocultivation, significantly impaired neoplastic vasculogenesis (Fig. 2). Interestingly, interactive microscopy image analysis revealed an increased MSC/iEC interaction in JAK2V617F HOM pre-treated cocultures (*p ≤ .05). Bulk RNA-sequencing of JAK2 WT and JAK2V617F HET or HOM iEC treated for 24h with 1000 U/ml showed a strong response to IFNa. Future analysis may unravel a JAK2V617F specific transcription signature. Moreover, we induced EndMT in our 3D iEC-MSC coculture model by treatment with pro-inflammatory cytokines, reflected by the loss of EC markers (e.g. Claudin 5) and upregulation of mesenchymal markers (e.g. aSMA) by immunofluorescence. We are currently evaluating the capacity of RUX vs IFNa to inhibit EndMT. In summary, we show that IFNa impairs neoplastic vasculogenesis in a robust in vitro 3D coculture system that enables precise control of cellularity and genotype. Our model is a valuable tool to decipher the poorly investigated impact of IFNa on the BM microenvironment. In addition, its translational and scalable properties (e.g. by integrating primary or iPSC-derived hematopoietic cells) will allow us to study the impact of additional drugs on MPN-associated fibrotic transformation of the BM.