The genotypes having high iron and zinc showed differential gene expression during different stages. Of which, 155 were up-regulated and 251 were down-regulated while during flowering stage and milking stage 349 and 378 transcripts were differentially expressed, respectively. Gene annotation and GO term showed the presence of transcripts involved in metabolic activities associated with uptake and transport of iron and zinc. Information generated will help in gaining insights into iron and zinc metabolism and develop genotypes with high yield, grain iron and zinc content.Pearl millet is an important staple food crop of poor people and excels all other cereals due to its unique features of resilience to adverse climatic conditions. It is rich in micronutrients like iron and zinc and amenable for focused breeding for these micronutrients along with high yield. Hence, this is a key to alleviate malnutrition and ensure nutritional security. This study was conducted to identify and validate candidate genes governing grain iron and zinc content enabling the desired modifications in the genotypes. Transcriptome sequencing using ION S5 Next Generation Sequencer generated 43.5 million sequence reads resulting in 83,721 transcripts with N Pennisetum glaucum (L.) R. Br.) is a highly nutritious and multipurpose cereal crop primarily cultivated on more than 27 million ha in the arid and semi-arid tropical regions of Africa and Asia. It is a chief source of dietary energy for the rural people in these areas. It is well adapted to the adverse conditions like drought, high salinity, low pH, low soil fertility and high temperature making it an excellent climate-resilient crop and put forward good security against crop failure in developing countries2. It is nutritionally better than rice and wheat due to presence of higher quantity of proteins, dietary fibers, iron, zinc, phosphorus, calcium, potassium, vitamin B and essential amino acids and delivers 80–90% of the calories to several millions of poor people across the world3. It supplies 19 to 63% of the total iron (Fe) intake and 16 to 56% of the total zinc (Zn) intake from pearl millet based foods in Maharashtra, Gujarat and Rajasthan states of India4. In comparison to other cereals and vegetables, it is cheaper and more economical source of Fe and Zn. It has enormous genetic variability for the key mineral elements- iron and zinc when compared to other cereal crops5.Pearl millet  leading to ill health, high mortality, lower work productivity, learning disabilities in children and meager national economic developmentiniadi’ pearl millet germplasm is the main basis of breeding lines, improved populations, hybrid parents and composites containing high Zn and Fe densities. Research on pearl millet revealed that it has huge genetic variation for Zn and Fe densities with majorly additive gene action with better parent heterosis. This enormous genetic variation (30–140 mg/kg Fe and 20–90 mg/kg Zn) can be efficiently exploited for developing high yielding cultivars with enhanced Zn and Fe densities14. Pearl millet also possesses high bioavailability (absorption) of iron which can fulfill > 50% daily requirement of children or adult males. Approximately 50–100% of the daily allowance of iron is provided by one meal of biofortified high iron variety of pearl millet and proved helpful in eliminating iron deficiency in women, men and children15.Pearl millet exhibits higher Fe density in comparison to the other major cereals and ‘16. Development of next-generation sequencing has provided the opportunity for high-throughput sequencing and used as a valuable tool with vast potential and applications in plant biology including genome sequencing and transcriptome analysis17. The next-generation sequencing (NGS) technology is a boon as it overcomes the different inadequacies of microarrays making it most preferable method of transcriptome profiling in recent years.Morphological and physiological responses are controlled by numerous genes and significantly prejudiced by the environment. Transcriptomics and metabolomics can prove useful to identify gene function and increase production of important compounds in pearl millet by understanding gene-to-metabolite pathways23. A comprehensive transcriptome analysis for drought stress response has also been carried out in pearl millet24. The molecular mechanisms underlying salinity tolerance, physiological analyses and a comparative transcriptome analysis were revealed using salinity tolerant (ICMB 01222) and salinity susceptible (ICMB 081) lines under control and salinity conditions and around 11,627 differentially expressed genes (DEGs) were reported in both lines25.Transcriptomic studies in pearl millet have been used to reveal the functions of salinity and heat stress-responsive genes such as PgDREB2A, PgNHX1, PgDHN, PgVDAC, Pghsp 16.97In the present experiment, comparative transcriptome profiling of stage-specific spikes from pearl millet with high and low Fe and Zn was carried out to know about the genes that are expressed during developing spike and the transcripts whose expression is associated with Fe and Zn.. This study provides valuable candidate genes that can be genetically targeted for yield along with improvement for Fe and Zn content in pearl millet.kharif 2018. A total of four inbred lines viz. PPMI 953, PPMI 1108, PPMI 627, 5540 B were sown in field conditions following recommended agricultural practices. The four inbred lines were grouped in two categories- high and low, based on Fe and Zn content in the grains of the genotypes as measured by Atomic Absorption Spectrophotometer (AAS). Out of the four inbred lines, the two lines namely PPMI 953 and PPMI 1108 were having high Fe & Zn content and the other two viz., PPMI 627 and 5540 B were having low Fe & Zn content and were considered as control. The samples were collected at three stages i.e., panicle initiation [28 Days After Sowing (DAS)], flowering stage (36 DAS) and milking stage (52 DAS) in 3 replicates for transcriptomic analysis. The samples were named as 1A for the sample collected at panicle initiation from high Fe and Zn containing genotype PPMI 953, 1B for the sample collected at flowering and 1C for the sample collected at milking stage from the genotype PPMI 953. Similarly, the other samples were named as 2A, 2B and 2C for genotype PPMI 1108; 3A, 3B and 3C for genotype PPMI 627 and 4A, 4B and 4C for the genotype 5540 B . The quality of RNA was analyzed on 1.2% agarose gel electrophoresis and concentration was measured using a Qubit RNA HS Assay Kit (Invitrogen) along with the Qubit 2.0 Fluorometer (Invitrogen). mRNA was extracted using Dynabeads mRNA DIRECT Kit (Thermofisher Scientific) from ~ 1 μg of total RNA input. After purification, mRNA was fragmented by RNase enzyme at 37 °C in RNase buffer provided in cDNA library preparation kit Total RNA-Seq Kit v2 (Thermofisher Scientific). These fragmented mRNA were hybridized and ligated with adapters followed by SPRI cleanup (Solid Phase Reversible Immobilization). The fragments were reverse transcribed using random hexamers and superscript II reverse transcriptase (Life Technologies) and further cleaned using Beckman colter agencourt ampure XP SPRI beads. The cDNA library was amplified using PCR for the enrichment of the adapter-ligated fragments. The individual libraries were measured using a Qubit 2.0 Fluorometer and validated for quality in E-Gel 2% Agarose (Invitrogen). Subsequently, these libraries of each sample were diluted up to 100 pM concentration and subjected to emulsion PCR . Later, enriched template positive ion sphere particles were loaded on 540 chip and were sequenced in ION S5 next generation sequencer platform (Life technologies).Total RNA was extracted from each replicate of the 12 samples  were removed from each raw data for accuracy of results using CLC genomics tool kit27 to find out the percentage match. BlastX of assembled transcripts was done with the close relative of pearl millet i.e. Foxtail millet protein database with an e-value cut-off of 1E-6, as the protein database of pearl millet was not available. Sequence annotation was carried out with the software BLAST2GO. Based on these results, gene ontology (GO) terms were assigned to the sequences28.The reads were processed for sequencing adapter removal, further trimmed for quality and ambiguity. The de novo assembly was conducted using default parameters in the CLC Genomics workbench 20.01  to develop contigs/transcripts for all the samples. The transcript reads were further matched to the draft genome of pearl millet29. Assembler provides the annotated transcripts and their annotated length and coverage values for each sample. The resulting files of aligned reads were input in Differential Expression (DE) analysis, a tool for quantifying the abundances of a set of target sequences from sampled subsequence based on a model (GLM model) using the negative binomial distribution30. The differential genes were estimated among the different stages of panicle development of low and high Fe and Zn containing genotypes i.e. panicle initiation stage of low Fe and Zn containing genotypes were compared with panicle initiation stage of high Fe and Zn containing genotypes. Similarly, flowering and milking stages were also compared. Gene expression levels were estimated by RPKM and FDR p values. Genes with RPKM fold changes > 2 or < − 2, and FDR-corrected p values < 0.05 were regarded as Differentially Expressed Genes (DEGs). DEGs transcripts were annotated for pathway analysis and analyzed in MapMan tool for data visualization32.Two groups were generated by combining the reads of High Fe and Zn containing Pearl millet Genotype (HFZPG) and Low Fe and Zn containing Pearl millet Genotype (LFZPG). For HFZPG, the reads of sample number 1A, 1B, 1C, 2A, 2B and 2C were combined and for LFZPG the reads of sample number 3A, 3B, 3C, 4A, 4B and 4C were combined. The reads were aligned back to the assembled main assembly to quantify the abundance of transcripts assembled and to determine the number of reads and reads per kilo million (rpkm) corresponding to each transcript. CLC RNA-Seq analysis was used for aligning the reads of each sample onto the assembled transcriptsAll the experiments conducted under this study are relevant and according to institutional, national and international guidelines and legislation.33. Ample work has been carried to enhance iron and zinc content in pearl millet41, however little or negligible work has been carried out to understand the mechanism or pathways involved in high Fe and Zn containing genotypes.The Fe and Zn concentration in the grains of four pearl millet genotypes PPMI 953, PPMI 1108, PPMI 627 and 5540 B was analyzed by atomic absorption spectrophotometer. Fe concentration in high Fe containing genotypes PPMI 953and PPMI 1108 was found to be 103.23 ppm and 93.71 ppm, respectively which was significantly higher than low Fe containing genotype PPMI 627 (50.33 ppm) and 5540 B (32.31 ppm). Similarly, the Zn concentration in PPMI 953, PPMI 1108, PPMI 627 and 5540 B was found to be 77.53 ppm, 56.95 ppm, 30.51 ppm and 26.62 ppm, respectively (Table 50 of 597 bp (Table The samples for RNA-sequencing were collected from four pearl millet genotypes (two genotypes for high Fe and Zn while two for low Fe and Zn) at three different stages i.e. panicle initiation stage, flowering stage and milking stage. The samples were collected in three replicates for each stage. The total RNA was isolated from each replicate and were pooled before sequencing to make a total of 12 samples. The sequencing was carried out in Ion S5 next generation sequencing platform with a chemistry of 200 bp. The total number of raw reads varied from approximately 9- 20 million reads for the samples of different stages. The raw reads were further subjected to adaptor trimming and bases quality check. The percentage of high quality (HQ) reads varied from 76.6% to 91.44% and the number of HQ reads varied from ~ 7 to 15 million  were defined as the fold change of the normalized (RPKM) expression values of at least 2 transcripts/contigs in both direction of log2 ratio ≥ 1 and When the up-regulated genes were characterized, it was found that there were 32 transcripts which were up-regulated during all the three stages of panicle development. However, 68 transcripts up-regulated only during panicle initiation stage and 120 and 122 transcripts up-regulated only during flowering and milking stages  GO terms like ion binding, small molecule binding, amide binding, lipid binding, transporter activity, transferase activity, transmembrane transporter activity, etc. were highly enriched in up-regulated genes. The further classification of ion binding components of MF indicated up-regulation of metal binding, zinc ion binding, iron ion binding, calcium ion binding etc. which may have contributed significantly in the uptake and transport of iron, zinc and other metals  pathwayspes Fig. .Figure 4., ABC transporter family proteins, Multicopper oxidase family, cytochrome P450 family and Peroxidase family, while one protein family was up-regulated only during flowering stage i.e. terpene synthase family, while 7 protein families were up-regulated only during milking stage. Protein families like catalase family, major facilitator superfamily, E3 ubiquitin-protein ligase RZFP34 and ZC3H15/TMA46 family were up-regulated during all the three stages of investigation.Transportation and accumulation of minerals  by plants from soil involves many complex regulatory processes directed by group of genes responsible for enhancing mineral uptake by the roots and their transportation to the most suitable place of accumulation. Different types of protein families involved in transportation of minerals from root/shoot to the other parts of the plant have been identified. In the present study, different protein families have been identified which are involved in different processes of mineral uptake at various stages of panicle development. During different stages of panicle development i.e. panicle initiation, flowering and milking different protein families responsible of transportation and uptake of minerals were either up-regulated or down-regulated in the HFZPG Table  and S8. 47. Functional annotation of high Fe and Zn containing genotypes with foxtail millet database revealed transcripts belonging to different families of genes to be involved in higher Fe and Zn accumulation 48.Micronutrient malnutrition due to iron and zinc deficiencies is a serious public health problem in developing countries. Globally malnutrition has been in the prime focus of researchers across the world. The development of cereal crops rich in micronutrients is one of the major approaches to overcome this problem. In India alone, about 74% of children and 80% of the pregnant women suffer from iron and zinc deficiency. At present, knowledge of the genes involved in transportation and accumulation of Fe and Zn in the seeds of pearl millet is not known and hence a study on genes responsible for Fe and Zn has become important. Various strategies for iron uptake, iron transport and iron storage in many agricultural crops mainly in rice, wheat, common bean, maize reported earlier by many researchers. Movement of iron from soil to its destination place is a complex mechanism and require transportation system which is performed by different genes that regulate the mineral uptake49. It has the potential to identify important secondary metabolic pathways, various transcripts associated with diseases and helpful for discovering novel genes50 and developing molecular markers51. RNA sequencing (RNA-Seq) is widely used among the different transcriptome analysis methods as it can efficiently detect unknown genes and novel transcripts and has much potential to study gene expression and their regulating pathways52. This approach has been successfully applied in many important crops, including sesame53, chickpea54, finger millet55, foxtail millet56, peanut57, maize58 and cotton60.Transcriptome analysis using Next Generation Sequencing (NGS) technologies is a robust and efficient method for exploring the pattern of gene expression in host. Transcriptome sequencing or RNA-seq surpasses cloning and is helpful in exploring the complexity of transcriptome by allowing RNA analysis through cDNA sequencing at massive scaleIn this study, we used transcriptome sequencing for identification of transcripts involved in Fe and Zn accumulation in the pearl millet genotypes. Two sets of pearl millet genotypes having high and low Fe and Zn in their grains were taken. The samples were collected at three stages of spike development i.e. panicle initiation, flowering stage and milking stage. mRNA was isolated and was subjected to sequencing using Ion S5 next generation sequencer. The analysis of the transcripts was carried out individually for each stage and later combining all the transcripts into two group i.e. high Fe and Zn containing pearl millet genotype (HFZPG) and low Fe and Zn containing pearl millet genotype (LFZPG).50 of 597 bp. When matched with the pearl millet de novo assembly, 84.35% of transcripts matched with the genome assembly. Further, when the differentially expressed gene were analyzed, it was found that the maximum number of genes were differentially expressed during the panicle initiation stage of panicle development indicating that the accumulation and transportation of Fe and Zn might be taking place during the panicle initiation stage of panicle development. The group of genotypes having high Fe and Zn showed that during panicle initiation stage a total of 406 transcripts were differentially expressed of which 155 were up-regulated and 251were down-regulated while during flowering stage and milking stage 349 and 378 transcripts were differentially expressed, respectively. The gene annotation and GO term assigning of up-regulated genes showed the presence of transcripts involved in metal ion binding, ATP binding, DNA binding, heme binding, iron ion binding, zinc ion binding, catalytic activity, transferase activity, transferring acyl groups, oxidoreductase activity, peroxidase activity, calcium ion binding, copper ion binding, nutrient reservoir activity, etc. indicating their role in uptake and transport of iron, zinc and other metals. The genes like multicopper oxidase family, major facilitator superfamily, cytochrome P450 family, Zinc-containing alcohol dehydrogenase family, ferredoxin–NADP reductase type 1 family and CONSTANS family having activities related to iron and zinc accumulation were also found to be up-regulated in the transcripts of high Fe and Zn containing genotypes.The pearl millet developing spikes transcriptome sequence generated 43 million sequence reads resulting in 83,721 transcripts with N61. Enzymes such as proton ATPases, ferric reductases intervene with the solubilization of iron hydroxides in the soil. Further, Fe is transported from the apoplast to the symplast and numerous transporters are needed to distribute it within the plant via xylem and phloem. Specific transporter proteins are required for its transportation across intracellular membranes and biosynthetic enzymes helps in integrating it into heme or iron-sulfur clusters. Later, iron is stored in ferritin in the plastids or in the vacuoles. Hence, any of these proteins/ genes can be utilized as biofortification targets and iron uptake entails many genes63. But, only a few them have been identified as biofortification targets. Plants possess two main approaches for iron uptake –chelate based approach in grasses and reductive approach in non-grasses. Iron concentration was observed to increase by 1.7-fold in rice leaves while it was 1.1-fold in grains due to over expression of IRT1, a divalent metal transporter which is central to reductive iron uptake64. Thus, iron accumulates in the vegetative tissues in the absence of extra sink capacity in seeds. Infact, iron concentration increased in the endosperm up to fourfold in polished rice when IRT1 is overexpressed along with PvFER165. Similarly, collective over expression of Arabidopsis IRT1 and FER1 leads to a 5.5-fold in cassava66. During reductive iron uptake mechanism, small molecules such as coumarin-derivatives are secreted in some species while flavins in others63, but till now it has not been confirmed if biosynthesis genes may be used for biofortification. Derivatives of NA-mugineic acid and deoxymugineic acid (DMA) are secreted in the rhizosphere in grasses and cereals and chelate Fe3+. The Fe-chelator complexes are transported into the cell by YSL15 in rice and YS1 in maize67. But, overexpression of IDS3  and YSL15 by increasing DMA resulted into modest increases in iron concentration in rice seeds69.The iron we consume from plant-based foods undergoes several processing steps with the help of many plant proteins before it forms the final product in form metal cofactor or in storage formWe successfully used transcriptome sequencing for identification of transcripts involved in Fe and Zn accumulation in the pearl millet genotypes and it is the first report for identification of metabolic pathways involved for Fe and Zn content in pearl millet. This will be further useful in developing hybrids/varieties rich in Fe and Zn content in pearl millet which will aid in mitigating hidden hunger and enhance nutritional security.Supplementary Tables.Supplementary Tables.


Methods: Within a period of 18 months, patients at the medical intensive care unit of a tertiary university hospital with newly developed MODS  were included in our study. The VOT occlusion slope (OS) and recovery slope (RS) were recorded in addition to unprovoked StO2. StO2 was determined non-invasively in the area of the thenar muscles using a bedside NIRS device. The VOT was carried out by inflating a blood pressure cuff on the upper arm. AKI stages were determined by the changes in creatinine levels, urinary output, and/or the need for RRT according to KDIGO. Results: 56 patients with MODS were included in the study . Incidences of the different AKI stages were: no AKI, 16.1% (n = 9); AKI stage I, 19.6% (n = 11); AKI stage II, 25% (n = 14); AKI stage III, 39.3% (n = 22). Thus, 39.3% of the patients (n = 22) developed the need for renal replacement therapy (AKI stage III). These patients had a significantly higher mortality over 28 days  vs. no RRT, 44% (n = 15/34); p = 0.03). The mean unprovoked StO2 of all patients at baseline was 81.7 ± 11.1%, and did not differ between patients with or without the need for RRT. Patients with RRT showed significantly weaker negative values of the OS  and lower values for the RS  compared to non-dialysis patients. Consistent with these results, weaker negative values of the OS were found in higher AKI stages . Unprovoked StO2 did not contain prognostic information regarding the AKI stages. Conclusions: The weaker negative values of the VOT parameter OS are associated with an increased risk of developing AKI and RRT, and increased mortality in the early phase of MODS, while unprovoked StO2 does not contain prognostic information in that regard.Background: Acute kidney injury (AKI) is associated with an increased mortality in critically ill patients, especially in patients with multiorgan dysfunction syndrome (MODS). In daily clinical practice, the grading of AKI follows the Kidney Disease: Improving Global Outcomes (KDIGO) criteria. In most cases, a relevant delay occurs frequently between the onset of AKI and detectable changes in creatinine levels as well as clinical symptoms. The aim of the present study was to examine whether a near infrared spectroscopy (NIRS)-based, non-invasive ischemia–reperfusion test (vascular occlusion test (VOT)) together with unprovoked (under resting conditions) tissue oxygen saturation (StO Acute kidney injury (AKI) is a life-threatening complication of multiorgan dysfunction syndrome (MODS), and remains a major diagnostic and therapeutic challenge ,2,3. It 2, even from an adequate systemic O2 supply [As a main pathophysiological feature in MODS patients, the impaired tissue metabolism is unable to extract sufficient levels of O2 supply , leading2) measured non-invasively via near infrared spectroscopy (NIRS), has gained growing interest. This technology has been used to introduce and characterize new diagnostic parameters. NIRS is an established and reproducible method that allows the continuous monitoring of StO2 in real time, and two conditions for measuring StO2 have been established: measurements are taken under resting conditions (unprovoked), and using a ischemia–reperfusion test (vascular occlusion test (VOT)) [2 oscillations. It seems that the analysis of changes in StO2 during this circulatory stress test may be more useful for the quantification of MODS-induced microvascular dysfunction [In the context of detecting these changes in tissue oxygenation, the concept of tissue oxygen saturation (StOt (VOT)) ,11. The function ; thus fuStudies examining the role of tissue oxygen saturation with respect to renal oxygenation and clinical AKI exist, in particular for infants, but are limited for the adult population. A previously published study on adult cardiac surgery patients by Sakaki et al. demonstrated that tissue oxygen saturation measured in the thumb region has the potential to aid the detection of local renal ischemia .2, unprovoked and by means of a VOT, in MODS patients at the time of ICU admission, regarding the need for RRT in AKI.It is important to note that the initial symptoms of AKI are clinically unspecific. Current conventional risk stratification is mainly based on functional blood and urinary markers to determine or estimate kidney injury ,15. HavifY trial population (EudraCT-Nr.: 2009-015499-88) [fY trial was approved by the ethics committee of the Martin Luther University of Halle , Germany. Critically ill patients with newly diagnosed MODS (APACHE II score ≥20) were included in the MODIfY-trial if they had a study-independent indication for invasive hemodynamic monitoring, a sinus rhythm with a heart rate ≥90/min, pre-existing contraindications to beta-blockers, and a signed declaration of informed consent [The actual VOT population was part of the prospective, randomized, controlled MODI5499-88) . The stu consent . ExclusiThe clinical endpoint of this post hoc analysis is the need for RRT within 28 days after ICU admission.0.725) × (Weight (kg)0.425) [The extent of AKI was determined post hoc by the changes in serum creatinine level, urinary output, and/or the need for RRT in accordance with Kidney Disease: Improving Global Outcomes (KDIGO) criteria . Urinaryg)0.425) ,19.©, Fresenius Medical Care, St. Wendel, Germany), using citrate anticoagulation was the mode of renal replacement therapy used for all patients. Initiation and management of CVVHD was subject to the discretion of the medical ICU staff. For vascular access, a double or triple lumen catheter, inserted either in the internal jugular or femoral vein, was used. The medical staff was unaware of the results of the VOT parameters, and thus did not make decisions regarding the initiation of RRT upon them.Continuous venovenous hemodiafiltration (CVVHD)  at the time of ICU admission (baseline) and after 96 h (96 h). StO2 measurements of any kind, at our intensive care unit, are not part of routine medical care parameters for the treatment of sepsis and/or patients in shock.The determination of StO2 system consists of a recording and display monitor, a NIRS glass fiber probe, and a connector. The system is not associated with any relevant burden or hazard to the patient, can be performed at bedside, has a low interobserver variability, and the infrared light used does not cause any physical or chemical changes in the human tissue [2 of the patient at intervals of 2 s automatically.The InSpectra StOn tissue ,12. The 2 is based on spectrophotometric principles that use light absorption to calculate chemical concentrations (Lambert–Beer law). The known specific absorption spectra of oxygenated and deoxygenated hemoglobin allow conclusions to be drawn regarding the oxygen saturation in the tissue. StO2 was calculated as follows:The probe emits light with a wavelength of 680–850 nm and a penetration depth of 14–15 mm ,12,20,212 was obtained immediately after attaching the probe to the patient’s thenar muscle. Values below 75% were considered as hypoperfusion, as recommended by previous studies [Unprovoked measurement of StO studies ,23,24.The VOT was performed non-invasively by inflating a blood pressure cuff on the upper arm to 30 mmHg above the systolic blood pressure for 5 min, and deflating immediately after. The parameter describing the phase of desaturation, during this VOT, is referred to as the occlusion slope (OS), and the parameter for the resaturation phase is known as the recovery slope (RS).2 consumption under cuff inflation, and steepens with increasing tissue O2 metabolism [2 during the VOT from cuff inflation to cuff deflation, and follows a curve with a negative slope. Therefore, the obtained values for OS are always negative. The recovery slope (RS) (%/s) is caused by the immediate return of the blood flow after the relief of the arterial occlusion, and by reactive vasodilatation [2 [2, and is measured immediately after the deflation of the pressure cuff until baseline StO2 is restored. Reported cut-off values for the OS and RS VOT parameters are −10%/min and 2.7%/s, respectively [2 typically take place slowly during cuff inflation (OS), and rapidly after cuff deflation (RS), different units of OS (%/min) and RS (%/s) are used.The OS reflects the cellular Otabolism . The OS latation . The steation [2 . The staectively ,27,28. D2 and the VOT parameters OS and RS. Receiver operating characteristic (ROC) curves were generated, and their respective area under the curve (AUC) values described. To analyze a possibly significant association between the dependent variables, as well as between the mean values of the StO2 parameters within the AKI groups, an analysis of variance (ANOVA) was used. To test for multiple hypotheses, a comparison between the individual AKI groups was based on a post hoc Bonferroni correction. The basis for the test decision was a significance level of p < 0.05.Statistical data analysis was performed using SPSS Statistics . Normal distribution was analyzed by Kolmogorov–Smirnov test. Differences in frequency of nominally scaled parameters were compared using Pearson‘s chi-squared test. Metric variables are expressed as mean ± standard deviation, tests for differences between independent and dependent variables were performed using Student’s t-test. Survival was analyzed by Kaplan–Meier statistics, together with a log-rank test. Univariate (unadjusted) as well as multivariate (adjusted) logistic regression models were used to quantify the predictive value of unprovoked StO2 parameters were analyzed in 56 critically ill patients. 2 at baseline was 81.7 ± 11.1% for all patients. Only nine patients (16.1%) had a pathological, unprovoked StO2 below 75%. A total of 52 patients (92.9%) were intubated and received an invasive mechanical ventilation, with an average FiO2 of 70.6 ± 20.3% and mean arterial pO2 of 15.1 ± 6.9 kPa, with no difference between the two groups (StOo groups .n = 17) starting dialysis within 48 h. All patients receiving RRT were included in AKI stage III. These patients had a higher mortality within the following 28 days  vs. no RRT, 44% (n = 15/34); p = 0.03). The decision for the initiation of RRT was based on the discretion of the medical ICU staff, involving multifactorial clinical considerations. Altered and/or decreased urine output was the first and major signal of renal function loss. Urine output for patients receiving RRT in this collective was variable, ranging from anuria to normal, or even above normal levels. The most common reasons for urinary output being compromised were anuria , oliguria , and polyuria . Five patients (22.7%) with normal urinary output receiving RRT were admitted to the ICU with relevantly elevated renal retention markers .Regarding changes in kidney function, we observed the frequencies displayed in 2 and VOT parameters according to RRT status. Patients not receiving RRT had significantly better (more negative) OS values and better (more positive) RS values at baseline compared to dialysis patients. After 96 h, these differences can still be observed, albeit not reaching statistical significance. In general, these values improved under standard ICU therapy within 96 h, regardless of RRT status. For unprovoked StO2, no differences could be observed in this regard; unprovoked StO2 tended to remain almost unchanged throughout the ICU stay. RRT rates did not differ between patients with a pathologically low unprovoked StO2, i.e., below 75% ) and patients with an unprovoked StO2, i.e., within normal range of above 75% ; p = 0.51).2 remained stable. The proportion of patients with dialysis requiring AKI was significantly greater in the groups with weaker occlusion and recovery slopes . Serum creatinine and blood urea nitrogen levels at baseline showed no significant AUCs, and only urinary output was the strongest clinical predictor for RRT  did not require RRT more often than patients with a baseline StO2 (>75%). Observing the ROC curve and binary logistic regression, unprovoked StO2 remains unsuitable as a prognostic marker for predicting the necessity for RRT, according to our data. We conclude that no prognostic relevance for unprovoked StO2 could be determined in the present work for patients in the early stages of MODS.Critically ill patients with a pathological baseline StO2 can be used to supplement AKI prediction, although these studies were not conducted in MODS patients.Regardless of our own findings, there are data available in the literature, that suggest that unprovoked StO2 for the prediction of postoperative AKI using transcutaneous NIRS . They used the technique intraoperatively, rather than postoperatively, in 95 cardiac surgery patients directly with transcutaneous measurements taken in the area of the kidneys. Of these patients, 35.8% postoperatively developed AKI. The onset of postoperative AKI was significantly predicted by pathologic StO2-values measured intraoperatively prior to a relevant increase in serum creatinine. In this regard, a study by Owens et al. [2 measured at the forehead using NIRS  appears to correlate with renal dysfunction (AKI), decreased systemic oxygen delivery, and the overall postoperative course. A previously mentioned study by Sakaki et al. [2 measuring set up using NIRS  that included sensors placed on three different regions, such as the patients’ forehead, abdomen, and the lateral side of the thigh, during the induction of anesthesia. StO2 measurements derived from these regions revealed that an impaired regional oxygen saturation (unprovoked) at the thigh region of ≤67% was predictive of acute kidney injury within 24 h after surgery.Choi et al.  assesseds et al.  on pediai et al.  involved2 measurements in neonates, these authors established the notion that the use of direct renal tissue oxygenation monitoring could be used to improve renal outcomes. It holds significant promise to function as a real time, early indicator of poor renal perfusion, which may help with the development of specific treatment protocols to prevent or decrease the severity of AKI [2, on the other hand, may reflect an earlier time period when renal ischemia is still reversible and responsive to fluid management, transfusions, inotropic support, or medication administration that may resolve ongoing injury [In a recent publication by Harer et al.  on conduy of AKI . The cury of AKI . Changesg injury .2 into patient monitoring, but also the importance of research concerning the location of measured tissue oxygen saturations for estimating function vs. dysfunction. However, we support an additive benefit of NIRS-derived StO2 to traditional renal function parameters, and it must be highlighted in this regard that local measurements of StO2 in the regions of the kidneys have to appreciate the heterogeneity in the metabolic requirements within the kidneys, with the medulla being relatively hypoxic compared to the cortex [These findings suggest not only the possibility of integrating StOe cortex .p < 0.05). The presented data show that especially the VOT occlusion slope could help in the prediction of the need for RRT. Generally, data for occlusion slope values predicting RRT are limited in the literature; therefore, at present, only analogies can be drawn.In the present study, 39.3% of patients with MODS developed an AKI requiring RRT. These patients had a significantly higher mortality rate over 28 days %/min, similar to our baseline occlusion slope of −9.1 (±3.7)%/min of patients that receive RRT. The baseline recovery slope of their study showed a median of 1.6 (1.2–3.1)%/s, which is also similar to the baseline recovery slope of our RRT collective of 1.7 (±0.9)%/s. The assessment of near infrared spectroscopy-derived parameters immediately before, and 1, 4, and 24 h after the initiation of CVVHD showed an improvement in the VOT parameters, although only a slight tendency could be inferred. These findings suggest higher oxygen consumption during the first 24 h of RRT, as minimum StO2 during the VOT is thought to be an indicator of the extent of ischemia [The results of our study, that VOT-derived parameters of StOs et al. , also usischemia ,39.Since there have been no further studies for patients that focus specifically on AKI and additionally using a VOT, our results cannot be compared or evaluated with broader pre-existing research.Due to the design of the MODIfY study, there are no control patients that would allow a comparison between MODS patients and ICU patients without MODS or even healthy controls. All data were derived from a single-center study, and the decision to initiate RRT was not based on a specific protocol, but left to the clinical judgement of the treating physicians. According to our data, impaired urine output was the main reason for RRT in the analysis, which is part of the KDIGO criteria. The need for RRT initiation can develop from multiple medical problems that evolve during the ICU stay and impact kidney function, and must not be related to the onset of MODS, but can also be the result of, for example, co-medication with nephrotoxic potential .Creatinine level (μmol/L) should have a strong predictability to develop AKI, although the mere requirement of RRT is the primary outcome of our study. Comparing the predictability of RRT, we observed that relevant changes in the occlusion slope (>−10%/min) at the time of ICU admission are superior to creatinine level. However, this is not surprising, as there is a diagnostic window between onset of kidney injury and subsequent changes in serum creatinine. The VOT as a functional test, however, reveals information, in real time, on oxygen consumption and microvasculature reactivity as a means to assess microvascular reserve and tissue integrity in just a few minutes; correlations of abnormal VOT parameters with creatinine are not available in literature yet ,41. HoweOverall, we included various MODS patients; in this regard, the mechanism of development of AKI must be considered multifactorial. Our methods might be associated with tissue perfusion and oxygen delivery; however, some may assume that this may be the surrogate of severity of illness, thus will not be associated with the risk of AKI independently. However, as this is a small study, there is not enough power to assess the independent associations with adjusting multiple factors, not just APACHE II score.2 via NIRS have become commercially available over the last decade. So far, there is no general agreement regarding a standard for vascular occlusion testing, which might limit the comparability of our results with other studies. For this work, VOT was performed with a five minute cuff compression, as such an approach with a fixed compression time has been preferred in the past [NIRS may be affected by the amount of adipose tissue, and by the presence of edema at the assessment site, which is a common finding in ICU patients . Furtherthe past ,46.2) allows risk stratification of patients in the early phase of MODS, and is associated with acute kidney injury and increased mortality within 28 days. Therefore, StO2 together with a VOT could be considered as a non-invasive method to supplement the monitoring of MODS in critically ill patients in order to close the therapeutic window, thus reversing or preventing further kidney injury. Unprovoked StO2 does not provide any relevant implications regarding a foreseeable RRT.The present study shows that vascular occlusion testing using tissue oxygen saturation (StO


HAAO , NADSYN1 (NAD+ Synthetase-1) and KYNU (Kynureninase) have been identified in a handful of affected individuals. All three genes encode for enzymes essential for the NAD+ de novo synthesis pathway. Using Trio-Exome analysis and CGH array analysis in combination with long range PCR, we have identified a novel homozygous copy number variant (CNV) encompassing exon 5 of KYNU in an individual presenting with overlapping features of VCRL and Catel–Manzke Syndrome. Interestingly, only the mother, not the father carried the small deletion in a heterozygous state. High-resolution SNP array analysis subsequently delineated a maternal isodisomy of chromosome 2 (UPD2). Increased xanthurenic acid excretion in the urine confirmed the genetic diagnosis. Our findings confirm the clinical, genetic and metabolic phenotype of VCRL1, adding a novel functionally tested disease allele. We also describe the first patient with NAD+ deficiency disorder resulting from a UPD. Furthermore, we provide a comprehensive review of the current literature covering the genetic basis and pathomechanisms for VCRL and Catel–Manzke Syndrome, including possible phenotype/genotype correlations as well as genetic causes of hypoplastic left heart syndrome.Vertebral, Cardiac, Renal and Limb Defect Syndrome (VCRL), is a very rare congenital malformation syndrome. Pathogenic variants in  KYNU and HAAO gene to be causative for this malformation syndrome . Recently, Szot et al. identified pathogenic variants in NADSYN1 and hereby the third causative gene for the syndrome  [HAAO as well as KYNU were found to cause reduced NAD+ levels in affected individuals, potentially resulting in the distinct malformations observed in VCRL [Vertebral, Cardiac, Renal and Limb Defect Syndrome (VCRL) is a rare autosomal-recessively inherited condition associated with cardiac malformations, including hypoplastic left heart syndrome, short stature, dysmorphic facial features such as microcephaly, low set ears and a flat nasal bridge, skeletal malformations as vertebral segmentation defects, rhizomelic shortening of the limbs, finger hyperphalangism and kidney malformations. Possible additional features include sensorineural hearing loss and developmental delay ,2,3. TheTGDS gene that encodes for TDP-glucose 4,6-dehydratase. CMS is clinically characterized by skeletal anomalies, namely bilateral hyperphalangy causing ulnar clinodactyly, radial deviation and shortening of the index finger as well as Pierre Robin sequence which can include a cleft palate [The phenotype of patients with VCRL is overlapping with that of CATMANS  patients which is caused by mutations in the t palate ,8,9,10.KYNU gene causing a phenotype overlapping VCRL and CMS. We further provide a review of the current literature.Here, we describe the identification of a novel homozygous CNV resulting from a maternal chromosome 2 isodisomy in the Written consent of the probands/legal guardians was obtained and Whole Exome Sequencing (WES) for the patient and the unaffected parents was performed using the Agilent Sure Select Human whole exome kit  for enrichment. Sequencing was undertaken on a Nextseq 2000 machine . Read alignment and variant calling were performed with GATK  using default parameters with the human genome assembly hg19 (GRCh37) as reference. Variant filtering was performed focusing on protein coding changes and splice site regions based on a minor allele frequency of <1% in public databases, including the Exome Aggregation Consortium, Genome Aggregation Database, dbSNP, the 1000 Genomes Project and gnomAD. CNV calling was performed using SeqPilot software .CGH array was performed using Agilent Sure Print G3 CGH Mircoarry 4 × 180 k kit according to the manufacturers protocol .SNP array was performed using Sure Print G3 CGH Micorarry + SNP 4 × 180 k kit according to the manufacturers protocol .Long range polymerase chain reactions was performed using the Qiagen LongRange PCR kit  using 100 ng of genomic DNA. PCR was performed as suggested by the manufacturer . Gel analysis was performed using 2.5 V/cm for 5 h and 0.8% agarose gel. Primer sequences are available upon request.Polymerase chain reactions (PCR) were performed using 50 ng of genomic DNA using a standard touchdown PCR protocol with 34 cycles. PCR protocol and primer sequences are available upon request.m/z 33–400.Urine samples  were spiked with internal standard, acidified and organic acids were extracted with ethyl acetate. The extracts were concentrated and the organic acids were derivatized using diazomethane. Di- and tri-methylated derivatives of xanthurenic acid were formed. Methylated residues were dissolved in methanol and analyzed on a 7890 A gas chromatograph coupled to a 5975 C mass spectrometer . Separation was performed on a CP-Wax 58 column  over 65 min using a temperature gradient from 50 to 260 °C. The mass spectrometer was operated in electron ionization and full scan mode, acquiring spectra in the range The affected individual has been treated in our institution since birth. Phenotypic hallmarks are shown in 2, Schwartz formula, CKD stage 2) and without signs of proteinuria or hematuria.Postnatal ultrasound examination also revealed bilateral hypoplastic kidneys. At the age of two months, the right kidney measured 3.4 cm (<P3) and the left kidney was 3.0 cm (<P3). Blood creatinine was 0.40 mg/dL, indicative of a mild impairment of renal function. At the age of one year, a mild pathological increase of kidney echogenicity was observed on ultrasound examination. At the age of 5 years, kidney sizes and volumes remained significantly below the average: the right kidney length was 5.4 cm (<P3) and the volume 27 mL (<P3), the left kidney length was 5.0 cm (P3) and the volume 25 mL (<P3), Additionally, multiple skeletal anomalies were identified, including bilateral clinodactyly of digit IV and V, a radial deviation and membranous polydactyly on the right hand with doubling of the proximal phalanges of the right digitus II, IV and V. The feet showed a bilateral shortening of the metatarsalgia IV and hallux valgus a–e. FurtIn summary, the patient presented with an overlapping phenotype of VCRL and Catel–Manzke Syndrome.TGDS gene, which is known to be causative for CMS [KYNU gene (NM_00119924.1) in the patient  gene panel analysis for ciliopathy causing genes  did not reveal any causative variants. Likewise, no causative deletions or duplication were detected by CGH array analysis. We therefore proceeded to perform Trio-Exome analysis of the patient and both parents, including CNV analysis. Trio-Exome analysis again did not reveal any pathogenic SNVs or indels fitting with the phenotype of the patient. In particular, there were no pathogenic variants in the  for CMS ,7,8,10 n patient a. Exon 5KYNU in the father, we performed Sanger sequencing of the corresponding area of the gene [SeqPilot Software detected the CNV in a heterozygous state in the mother b but unethe gene  confirmiKYNU CNV detected homozygously in the patient and heterozygously in the mother was missed by the previously performed CGH array as no CGH probe located to the deleted exon. However, a probe at the intronic position chr2:143,705,606 preceeding the deleted exon was still visible, allowing us to predict the first breakpoint to lie within an approximately 8000 bp measuring DNA stretch between this intronic probe and exon 6 and the second breakpoint in the small intron 6/7. Long range PCR revealed a slightly shorter product in the patient compared to the father as well as a healthy unrelated control  to 3-hydroxanthranilic acid (3-HAA). KYNU dysfunctions has been previously shown to result in elevated urine xanthurenic acid levels [KYNU causing VCRL with homozygosity in the patient resulting from a maternal isodisomy of chromosome 2 (UPD2). Reports in the literature show that a unipaternal iso- or heterodisomy of chromosome 2 does not lead to phenotypic anomalies of affected individuals, suggesting that there are no imprinted genes located on chromosome 2 [Here, we have identified a novel CNV in mosome 2 ,12,13,14KYNU exon 5 CNV is important for genetic counselling of our family as in this case, the recurrence risk in the family is considerably lower in comparison to a recessively inherited disorder.Identification of maternal UPD2 causing homozygosity of the KYNU encodes the enzyme kynureninase, which plays a role in the tryptophan catabolic pathway and is needed for the biosynthesis of NAD+. NAD+ is either produced by the “NAD+ de novo synthesis pathway” from dietary tryptophan, requiring among other enzymes KYNU, HAAO and NADSYN1 or by the “NAD+ salvage pathway” from dietary niacin independent from those enzymes. Disturbances of the de novo pathway by pathogenic mutations in critical enzymes can cause VCRL , presenting with hydroxykynureninuria and xanthurenic aciduria but without congenital malformations [Christensen et al. described two brothers with a missense variant in rmations . A residnge p.Thr98Ala, prWhy some of the patients with NAD+ deficiency syndrome show a more severe cardiac phenotype in comparison to others is speculative. Presuming that the amount of NAD+ in the embryonic development is an essential factor, it is imaginable that patients with less severe malformations have either a higher residual enzyme activity or have higher NAD+ level due to environmental factors such as higher nutritional niacin intake of the mother during pregnancy . On the KYNU suggests VCRL as diagnosis. Bilateral hyperphalangy with an accessory bone inserted between the second metacarpal and the phalanx, resulting in radial deviation of the index finger, has been described as a cardinal feature for CMS, however only nine out of twelve reported patients with TGDS mutations show this phenotype and also four out of twelve VCRL patients with pathological variants in KYNU, HAAO or NADYSN1 show this malformation. All reported CMS patients with TGDS mutations were diagnosed with Pierre Robin sequence while none of the patients with VCRL were. On the other hand, (mild) developmental delay more often occurs in patients with VCRL. Especially the cardiac and renal phenotype of patients with VCRL seems to be more severe in comparison to those with CMS. All screened patients with VCRL were diagnosed with congenital heart defect, including five patients with HLHS, one patient with double outlet right ventricle (DORV), one patient with tetralogy of fallot (TOF), one patient with combined atrial septal defect (ASD) and ventricular septal defect (VSD), one patient with isolated ASD and one patient with patent ductus arteriosus. Observed cardiac malformations of patients with VCRL are shown in Our patient was clinically diagnosed with Catel–Manzke Syndome (CMS)/Manzke Dysostosis, a phenotype overlapping with VCRL. Genetically, the identified CNV in Interestingly, HLHS is the most frequent congenital heart defect observed in VCRL. HLHS is a rare condition with a prevalence of 1.6 per 10,000 live births . The exaKYNU−/− mice embryos by dietary supplementation of niacin of the pregnant mice [Future research is needed to determine the exact NAD+ dependent processes responsible for the disruption of certain stages of organogenesis especially affecting heart, kidneys and skeleton in VCRL patients. Shi et al. showed a rescue of the clinical phenotype in ant mice . It is pTGDS.In summary, we describe a case of NAD+ deficiency syndrome due to a homozygous CNV on the basis of a maternal UPD2. Our findings confirm that patients with NAD+ deficiency syndromes can display characteristics previously described for CMS and therefore mutations in enzymes of the tryptophan catabolic pathway should be excluded in patients diagnosed with CMS not carrying mutations in


Individuals tend to approach positive stimuli and avoid negative stimuli. Furthermore, emotions influence whether individuals freeze or move more. These two kinds of motivated behavior refer to the approach/avoidance behavior and behavioral freezing/activation. Previous studies examined  whether individuals' behavior depends on stimulus' valence; however, the results were mixed. Thus, we aimed to test whether emotions' effects on spontaneous whole-body behavior of standing individuals also occur in the seated position. We used a computer vision method to measure the head sway in video recordings that offers ease of use, replicability, and unobtrusiveness for the seated research participant. We analyzed behavior recorded in the laboratory during emotion manipulations across five studies totaling 932 participants. We observed that individuals leaned more forward and moved more when watching positive stimuli than when watching negative stimuli. However, individuals did not behave differently when watching positive or negative stimuli than in the neutral condition. Our results indicate that head movements extracted from seated individuals' video recordings can be useful in detecting robust differences in emotional behavior (positive vs. negative emotions). Studying body sway has contributed to several applied contexts. For instance, body sway in response to affective stimuli was used to study posttraumatic stress6, aggression8, social threat9, and Parkinson's disease10. Most previous studies focused on standing participants. However, relatively less is known whether behavior of seated participants reveals the same emotional responses. Thus, our aim was to examine whether effects of valence on body sway also show up in head movement among seated participants. We focused on leaning forward vs. away and freezing vs. activation, dependent on whether individuals face positive (leaning forward and activation) or negative (leaning backward and freeze) stimuli. Moreover, whereas most previous studies used force-platforms12, we focused on video recordings to capitalize on more recent data analysis methods.Individuals tend to lean and move towards positively valenced stimuli (approach behavior)13. For direction, the biphasic approach to emotions suggests that emotion is organized around two motivational systems: approach oriented or appetitive and avoidance oriented or defensive13. For instance, individuals display avoidance behavior to pictures of mutilated bodies3, dangerous animals4, and guns1. In contrast, individuals display approach behavior to pictures of smiling individuals2, attractive individuals, beautiful landscapes, and delicious food1. Furthermore, for emotional behavior intensity, individuals decrease their body mobility facing negatively evaluated stimuli—response operationalized as behavioral freezing15. Freezing in response to aversive stimuli results from the adaptation to avoid threats17. Individuals reduce body movement when facing unpleasant stimuli, including social threat9, mutilation images16, anticipating electrical shock18, standing at height19, and recalling sad memories20. In contrast, individuals facing pleasant stimuli move more24. Activation in response to positive stimuli reflects the readiness to explore, play, or loosen up and decrease tension25. For instance, individuals activate and increase body movement when facing erotic scenes24, amusing traffic situations23, or watching pictures of offspring22.Body sway is a component of emotional behavior and is organized by its direction and intensity24 and between negative and neutral conditions24 in their influence on approach/avoidance behavior. However, in some, there was a significant difference between positive vs. negative emotions24. Moreover, for behavioral freezing/activation, some research indicated no differences between positive and neutral conditions24, between negative and neutral conditions24, and between negative and positive conditions24. These mixed findings indicate that the relationship between emotional behavior and valence is complex and easier to detect between extreme values (positive vs negative) than between extreme and middle values 26.Although most studies support the difference between negative and positive emotions, some studies did not find a differential influence of positive and negative emotions on behavior. For instance, some studies revealed no differences between positive and neutral conditions9 or during unipedal stance11. The research on body sway in a seated position is sparse27. Advancing methods of testing body sway in sitting individuals is important because individuals spend increasingly more time sitting, e.g., at work and leisure time28. Moreover, an increasing number of research focuses on new forms of activities that individuals perform while sitting . Thus, testing whether emotions' effects on standing individuals' behavior also occur in the more restricted seated position increases ecological validity and evidence diversity in the body sway research.The majority of research on body sway tested standing individuals  where shifts in center of pressure are most pronounced in the anterior–posterior direction during bipedal stance6. Video recordings allow assessing the amount of spontaneous head and body sway of standing individuals with a reliability comparable to stabilometric force platform24. The advantage of using data extracted from video recordings comes from its low-cost and ease of use. Video data can be easily collected in laboratory experiments or ambulatory settings, including real-life scenarios.Furthermore, we focused on head movements as a commonly available movement index that can be extracted from video recordings. The head position relative to a stimulus is considered a critical output of seated postural responses33. We focused on dimensional conceptualizations of emotion (positive/negative) to address research investigating emotional experiences and their effects upon motor activity34. Thus, we focused on responses to positive, negative, and neutral stimuli. Moreover, we focused on two aspects of spontaneous emotional behaviors: direction (approach and avoidance behavior) and intensity . The approach behavior is indicated by leaning forward and towards the stimulus, whereas avoidance behavior as moving away and reclining backward35. Behavioral freezing is defined as the reduction of body movement, whereas behavioral activation as an increase of body movement. Of note, these two features of behavior, i.e., direction and intensity, are relatively independent from each other. For instance, individuals facing a threat could recline backward and reduce their movement (avoidance and freezing behavior). In response to a neutral stimulus, individuals might neither recline nor lean forward but perform spontaneous nondirectional movements. Finally, to the positive stimuli, individuals might lean forward and perform more nondirectional movements. Building upon the previous research24, we expected that negative stimuli would influence individuals to move backward and move less (avoidance and freezing), whereas positive stimuli would influence individuals to lean forward and move more (approach and activation).In the present study, we reanalyzed five data sets totaling 931 participants . Of participants, 372 (40%) were female. The most frequently studied valence category was positive emotions , then negative emotions , and neutral conditions . The regression models fit data well for approach/avoidance behavior, PPp = 0.480, 95% CI , and for freezing/activation behavior PPp = 0.471, 95% CI .We accounted for five studies, presenting behavioral reactivity to 24 affective stimuli obtained from 931 participants with age ranging from 18 to 35  than while watching positive stimuli  did not differ from the behavior to negative stimuli, β = − 0.045, 95% CI , p = 0.030, and positive stimuli, β = 0.006, 95% CI , p = 0.403.We found that the approach/avoidance behavior differs between negative and positive stimuli, Δβ = − 0.050, 95% CI , p = 0.016. Individuals moved less while watching negative stimuli  than while watching positive stimuli   did not differ from negative stimuli, β = − 0.010, 95% CI , p = 0.327, and positive emotions β = 0.031, 95% CI , p = 0.068.We found that the behavioral freezing/activation differed between negative and positive stimuli, Δβ = − 0.041, 95% CI , 24. It supports the biphasic approach to emotions, which suggests that emotions are organized around two motivational systems: approach oriented or appetitive and avoidance oriented or defensive systems13. Second, individuals moved less while facing negatively evaluated stimuli relative to positively evaluated stimuli. Again, this finding is consistent with the majority of the previous studies23. It supports that the emotional behavior is organized by its direction as well as intensity13. Thus, our findings provide evidence that corroborates previous research using relatively novel methods24. However, the observed effects should be interpreted as subtle as the effect sizes were small.We demonstrated how emotions impact the spontaneous head sway in seated individuals. We observed differences between responses to positive and negative stimuli. First, the head distance was shorter relative to positive stimuli than to negative stimuli. These findings are in line with the majority of previous studies24. This, again, indicates that it is easier to differentiate between extreme values than between extreme and intermediate values26.We did not find the differences between positive or negative stimuli relative to neutral stimuli. Some previous studies also showed no differences for approach/avoidance behavior and freezing/activation behavior24. We aimed to examine previous findings derived from stabilometric force platforms with using head movements as a commonly available, low-cost movement index that can be determined from the video recordings. We observed that the head measures were sensitive to experimental manipulation, as indicated by differences between positive and negative emotions. In Study 5, we included leg movements as an additional measure of behavioral freezing/activation. Due to our knowledge, it was the first attempt to account for lower limb spontaneous mobility as the measure of emotional behavior. While head movement might be restricted by chair headrest, armrest, or attached additional apparatus , the leg activity might be the easily available index of the behavioral reactivity.Seeking to advance understanding of the emotional influence on complex human behavior, we used two novel methodological approaches. First, we used head movements as a reliable and ecologically valid index of the amount of spontaneous body sway during emotion elicitation in standing individuals17. We argue that the stiffening is not limited to ankle joints and would also occur in the upper body 36, and should be visible in the seated postural responses. Studies accounting for muscle activity and spontaneous movements will be necessary to confirm this hypothesis. Since adults spend more than half of their day sedentary28, further studying seated postural responses would ecologically valid.Second, studies within the whole-body movement paradigm mainly reported data of participants standing on a force platform, whereas we focused on the body sway of seated participants. During quiet upright standing, the human body exhibits a small amount of spontaneous postural fluctuation, which can even be reduced in response to aversive stimuli. The reduction occurs because of contraction or stiffening of the muscles around the ankle joint24, we assumed that seated individuals' video recordings would suffice to determine the influence of valence on emotional behavior. However, seated individuals are in a more restricted position than standing individuals and have fewer degrees of freedom in motion. Participants in our studies sat on the chair with a backrest that might limit their anterior–posterior body sway and an armrest that might limit their medial–lateral body sway. Thus, the subtle effect of emotions on spontaneous whole-body behavior might not be detectable while measured in a restricted body position. For instance, researchers found the effects of emotions on spontaneous behavior only in unipedal stance, but not bipedal stance, which indicates that the degrees of freedom of spontaneous motion matters11. This might explain why we found differences between the valence dimension's extremes—positive and negative emotions, but not from the neutral condition. More studies that compare whole-body standing movement and seated postural responses might support this hypothesis. Future studies may also use an experimental setup with the sitting ball without arm- and backrests to allow for additional movements while sitting. Second, we based our conclusions on data from video recordings instead of using the force platform that is the state-of-the-art method in behavioral literature. Future studies may validate the initial findings on the comparability of both methods24. Third, in our studies, we did not control for eye movements that would inform whether individuals focused on specific parts of the screen while watching the positive and negative stimuli. For instance, head movements might be stronger for stimuli where individuals had to switch their focus between different parts of the scene, i.e., to understand the social interaction between several individuals. In contrast, some negative stimuli might produce a stronger focus on one part of the screen . Thus, the complexity of the stimuli might interact with the effects of valence. Future studies might account for this limitation by controlling for eye movements or by selecting positive and negative stimuli that produce similar eye movements. Fourth, we compared positive, negative emotions, and neutral conditions, whereas the discrete emotion approach could yield more insight. Recent work has demonstrated that discrete emotions often differentially impact cognition and physiology even when of the same valence38. Furthermore, we used multiple film clips to elicit single emotion, which provides additional variation in the manipulation strength. However, this approach also generalizes the impact of emotional valence and limits the effect of using specific stimuli. Researchers might use our approach to re-examine findings from other laboratory experiments on discrete emotions, in which behavioral reactivity was not the main focus. Given that behavioral reactivity  gained relatively low popularity in affective science, reexaminations of existing data may result in an effective way to increase the number of examined emotion.The study has several limitations. First, based on the previous study on standing individualsThis study's strength is in the use of a novel method of movement analysis with a large number of participants. We evidenced that the analysis of head sway among participants in a seated position can reflect affective influences. As recording videos of sitting participants is affordable and easy, studies within the field of affective science might increasingly use this method to expand the scope of emotional behavior analysis.Participants' characteristics are presented in Table 26, participants in our study sat on a chair with the back and armrests throughout all studies.All five experiments began with a 5-min resting baseline. Participants were asked to wait for five minutes without doing any unnecessary actions. During the experiments, we elicited emotions Table , present42. Each clip lasted two minutes. Within the sessions, clips were presented in a counterbalanced order. Table We used stimuli from the emotion-eliciting video clip databases to elicit emotions, with prior evidence of reliability and validityA fish called Wanda ; (2) The visitors (The visitors destroy the postman's car); (3) When Harry met Sally ; (4) The Dead Poets Society (Students climb on their desks to manifest their solidarity with their teacher); (5) Life is beautiful ; (6) Benny & Joone (Benny plays the fool in a coffee shop); and (7) Summer Olympic Games (montage of moments showing athletes successful performance and their joyful reactions). We used films 1–6 in Study 1, films 1–3 in Study 2, and film 1 and 7 in Study 5.For positive emotions, we used validated movie clips: (1) American History X (A neo-nazi smashes a Black man's head on the curb and killing him); (2) Man bites dog ; (3) In the name of the father (Scene of interrogation); (4) The Blair Witch Project (the characters die in an abandoned house); (5) Seven (the police find the decaying corpse); (6) Dangerous minds (the teacher tells the class that one of their classmates is dead); and (7) The Champ (boy cries at the death of his father). We used films 1–6 in Study 1, films 1–3 in Study 2, and film 1 & 7 in Study 5.For negative emotions, we used validated movie clips (1) Blue 1 ; (2) Blue 2 ; (3) Blue 3 ; (4) Emperor 1 ; (5) Emperor 2 (scenes from the city life); (6) The lover ; (7) Twin Peaks: Fire Walk with Me (the character sweeps the floor in the bar). We used films 1–5 & 7 in Study 1, films 1, 4 & 5 in Study 2, and film 2 in Study 5.For neutral conditions, we used validated movie clips: (1) 31 from the Nencki Affective Pictures System43. We selected three groups of items: high-approach positive affect , low-approach positive affect , and neutral . In Study 3 & 4 we used the same set of pictures.In Study 3 and 4, we used validated pictures45, which uses computer vision algorithms to detect and determine the head positions in a three-dimensional space for each frame. Data was recorded with the sampling rate of 15 FPS in Study 1 and Study 2, 10 FPS in Study 3 and Study 4, and 24 FPS in Study 5. We also included leg movements in Study 5. We used four accelerometers  attached to left and right knee and left and right ankle to record physical activity continuously and unobtrusively.We recorded the head movements of each participant continuously using an HD camera mounted on the top of the PC screen. The distance from the camera to participants’ head was approximately 65 cm Fig. . The vid40. This is a validated approach in affective sciences in studies that compare responses to two different stimuli of different durations47. The head sway in the anterior direction (the positive difference between baseline and affective stimuli) indicates approach behavior, whereas the head sway in the posterior direction (the negative difference between baseline and affective stimuli) indicates avoidance behavior.We measured the head position along the anterior–posterior axis, as the distance of the head from the camera. We calculated the mean distance of the head from the camera from two moments in our studies: (1) the last two minutes of resting baseline to account for the part of the baseline that was the most proximal to the study, and (2) the last 2 min of emotion manipulation. To operationalize changes of avoidance/approach behavior, we used reactivity scores corrected for the baseline levels; thus, we subtracted the levels of the last 120 s of baseline from the level of the last 120 s of the emotion manipulation. We did this due to differential lengths of presentation of the pictures (3 min) and film clips (2 min) and to account for the part of manipulation that is usually more affected by emotions elicitationX, Y, Z axis, based on the difference in head position in consecutive video frames. Second, we summed up the magnitude of head sway from two moments in our studies: last 2 min of baseline and last two minutes of emotion elicitation. To operationalize behavioral change of head sway to emotional stimuli, we used reactivity scores corrected for baseline levels. The decreased total body sway (the negative difference between baseline and affective stimuli) indicates behavioral freezing, whereas the increased total head sway (the positive difference between baseline and affective stimuli) indicates behavioral activation. In Study 5, we also calculated the magnitude of legs movement as the sum of all four joints sways in X, Y, Z-axis. The internal reliability for four joints movements was satisfactory, α = 0.84. To test the impact of emotions on behavioral freezing/activation collected from two movement sources (head and legs), we converted the data from each source of behavioral activity into the z-scores, and we merged them into a single variable.We measured behavioral freezing/activation with the total movement of head sway. First, we calculated the total movement of the head in 48. We ran the three-level regression models to account for dependency within-person (level 2) and within a study (level 3). Furthermore, we used three-level regression models to determine global trends across studies to account for dependency between the studies . We regressed the approach/avoidance behavior and freezing/activation behavior on the experimental condition . We dummy-coded the experimental conditions such that significant differences in the model accounted for differences relative to the neutral condition. Furthermore, the behavioral reactivity to emotion category (positive emotions vs. negative emotions) was considered as significantly different if the 95% confidence intervals of the difference between regression coefficients in subgroups did not include zero. We ran the analysis with the Bayesian correction estimator (Bayes), and we used the Bayesian Posterior Predictive (PPp) to evaluate model fit. A well-fitting model should have a PPp value around 0.50 in combination with symmetric 95% credibility intervals centering on zero50. We removed outliers above z-scores higher than 3.2951, resulting in 721 data points (4.90%) removed from the initial data sample.To summarize data from our multiple studies showing how emotions influence behavioral reactivity, we used three-level regression models using mPlus 8.0Supplementary Information.


PBA isone of the polymers present in the structure of ASA, which increasesthe compatibility between the cork particles and the polymer matrix.This is evidenced by evaluating the mechanical properties of the compositesand examining their fracture surface by scanning electron microscopy.The analysis of the thermal properties shows that the developed compositesalso present enhanced insulating properties.Corkpowder received as a byproduct from local industries is valorizedthrough the development of composite materials suitable for fuseddeposition modeling (FDM). For this purpose, a polymeric matrix ofacrylonitrile–styrene–butyl acrylate (ASA) is used dueto its good mechanical resistance and weather resistance properties.Prior to the manufacturing of the composites, the cork particles arecharacterized and modified by surface polymerization, creating a layerof poly(butyl acrylate) (PBA). Then, filaments for FDM are preparedby solvent casting and extrusion from ASA and composites with unmodifiedcork (ASA + C) and PBA-modified cork (ASA + C In this regard, in the frame of circular economy, compositematerials fabricated from waste or byproducts from other industriessuppose an important strategy of valorization, which allow the creationof new materials with new structural or functional properties.7 For instance, cork is used in sandwich structures as a structuralmaterial to minimize the weight of panels and provide good insulatingproperties. These panels can tolerate loads with a high impact, competingeven with glass fibers, being a more sustainable alternative, whichmakes them attractive in many different applications ranging fromcivil to aerospace engineering, the automotive sector, or defense.Its acoustic and anti-vibration properties also make it attractivefor the design of cabins for planes or turbines or joints in submarines.10Cork is a renewable and sustainableraw material that can be extractedfrom the bark of oaks that naturally grow in Mediterranean regionssuch as Italy, Spain, or Portugal. Cork has been used for many centuriesin different applications due to its unique properties such as extremelylow density, high acoustic and thermal insulation behavior, high frictioncoefficient, high resilience and a Poisson coefficient of virtually0.11 For instance, cork powder canbe added to concrete in the construction sector to provide thermaland acoustic insulation, while it contributes to the creation of amore lightweight material, which has enormous consequences in termsof energy saving.13However, the cork powder generated at the industrial scaleis consideredas a residue, and it is generally burnt or disposed in landfills.During these processes, up to 30 wt % of the raw material is convertedinto powder, which may result in a good opportunity to develop newproducts.15 as well as with biodegradablepolymeric matrixes such as acid polylactic acid (PLA),16 polycaprolactone (PCL),17 or poly(PHBV).18 Most of these composites areprocessed via classical manufacturing techniques, typically by pultrusion,injection, or compression molding. However, there is an increasinginterest in the development of composite materials suitable for additivemanufacturing.19Cork powder-based compositesusing a polymer matrix have also beendeveloped in order to obtain more environmentally friendly materials.There are different studies with polyolefins such as polyethylene(PE) or polypropylene (PP),20 The most used thermoplastic polymersare PLA and poly(acrylonitrile–butadiene–styrene) (ABS).PLA is vastly used for domestic applications or sectors that do notrequire working outdoors or at temperatures above 60 °C. Thisis why for most of the engineering applications, ABS is preferredsince it has similar mechanical properties to PLA but can tolerateworking temperatures up to 100 °C.21 In the last few years, poly(acrylonitrile–styrene–butylacrylate) (ASA) has come up as an interesting alternative to ABS.Replacing the butadiene residues with butyl acrylate gives ASA greaterenvironmental resistance.22Among the differentadditive manufacturing techniques, fused depositionmodeling (FDM) is the most widespread technique employed for polymericmaterials and polymer-based composites. Briefly, in this technology,a polymeric material in the form of a filament or pellets is extrudedthrough a preheated nozzle and is deposited on a platform layer bylayer.23 However,if these composites are not adequately compounded, they may presentheterogeneities, which lead to cracks and delamination, causing theembrittlement of the material.25 In the case of corkparticles, their shape, size and microstructure have a great effecton the rheology of the material during the processing and on the mechanicalproperties of the manufactured composites.26 Moreover, the modification of its wetting properties is also criticalto enhance the interfacial adhesion with a hydrophobic polymer matrix.The strategies adopted to overcome these drawbacks include addingplasticizers, copolymers or even lignin and suberin during the compounding,27 as well as the chemical modification of either the polymer matrixor the cork to chemically modify their surface properties.28 For instance, previous reports indicate an enhancementof the adhesion properties for PLA-based composites when maleic anhydrideis grafted onto the surface of PLA26 orthe cork particles surface is acetylated, increasing their hydrophobicity.29 Other authors modified the surface of the corkparticles through a controlled polymerization reaction via atom-transferradical polymerization. For this purpose, the cork particles werefirst brominated to be able to perform in a second step the polymerizationof methyl methacrylate (MMA) selectively in their surface followinga “grafting from” approach. This conferred the corkparticles the same surface chemistry as the PMMA matrix used in thecomposite, effectively enhancing their compatibility.30Recentadvances have reported the use of biodegradable materialssuch as flax, coffee beans or tree bark as reinforcing materials forfilaments suitable for FDM.In this paper, a series of cork-based compositessuitable for 3Dprinting by FDM are developed. Taking into account the low compatibilityof cork with the ASA polymer matrix, due to their different surfacechemistries, a modification of the cork particles is explored viasurface polymerization of poly(butyl acrylate) (PBA) before manufacturingthe composites. Then, filaments valid for FDM are prepared by solventcasting and extrusion, and different specimens are printed. The cork-basedcomposites exhibited higher strength and stiffness after surface modificationwith PBA, compared to composites prepared with cork as received. Furthermore,these composites showed significantly lower thermal conductivitiesthan ASA, demonstrating that they are good candidates for lightweight,insulating materials that can be mass-produced by additive manufacturingtechnologies.3 weresupplied by the company Corchos del Estrecho, obtained from cork dustresidues from the sanding processes during the manufacture of corkstoppers. ASA pellets were purchased from LG Chem. Acetic anhydride, pyridine, and sodiumhydroxide were purchased from Sigma-Aldrich. Acryloyl chloride (96%),triethylamine (TEA), n-butyl acrylate  and 2,2′-azobis(2-methylpropionitrile) (AIBN) werepurchased from Alfa Aesar. Toluene, dimethylformamide (DMF), dichloromethane(DCM), and isopropanol were purchased from Scharlau.Cork particles with a diameter of 63–125μm and an average density of ρ = 200 kg/mSupporting Information.The hydroxyl number(OHN) is defined as the amount of available −OH groups on thecork surface per gram of cork. It was calculated by adapting the ASTMD1957 standard to estimate the OHN of fatty acids. In our case, 15mL of acetic anhydride and 110 mL of pyridine were used per 1 g ofcork particles (C). The mixture was added to a round flask under aninert atmosphere at 100 °C for 2 h. Then, 10 mL of distilledwater was added to hydrolyze the non-reactant anhydride, and the amountof acetic acid produced was titrated using a 0.5 M NaOH solution andphenolphthalein as an indicator. This was done three times to ensurethe reproducibility of the results. For each repeat, a control inthe absence of cork particles was done. The exact details of the procedureare described in the m was carried out in two steps. First, 3.72g of C was dispersed in 241 mL of DMF and 6.93 mL of TEA by magneticstirring in a round flask under an inert atmosphere at 0 °C.Then, 2.12 mL of acryloyl chloride was added dropwise. After 3 h,the obtained product  was purifiedby filtered under vacuum washing with DMF and dried. Then, 3.25 gof Cf was dispersed in 195 mL of toluene by magnetic stirringin a round flask and 42.7 mL of nBA (1.5 M) was slowlyadded to the mixture. Then, the flask was closed with a septum andnitrogen was bubbled into the mixture. Finally, 0.192 g of AIBN (0.006M) was added and the mixture was heated up to 70 °C to startthe polymerization. The reaction was stopped after 24 h, and the product(Cm) was washed with toluene, isopropanol, and water toremove any excess of the unreacted monomer. After this, the productwas filtered and dried under vacuum. Characterization of C, Cf, and Cm was done by infrared spectroscopy viaattenuated total reflectance (ATR) in a Bruker Alpha spectrometer.The morphology of the particles was examined by scanning electronmicroscopy (SEM) in an FEI Nova NanoSEM 450 microscope equipped witha field-emission gun. The particles were previously sputtered witha few nm layer of Au in a Balzers SCD 004 Sputter Coater.Thesynthesis of Cm) was added and dispersed into the solution.The blend was mechanically stirred to ensure the formation of a homogeneouscomposite with 5 wt % of cork. Depending on the cork particles used,these composites are labeled as ASA + C or ASA + Cm. Themixture was placed on aluminum trays, and the solvent was removedby heating at 60 °C for at least 24 h. Then, the films formedwere cut into pellets of approximately 3 mm in size. These pelletswere introduced afterward in an oven at 60 °C for at least 3h prior to extrusion to remove any moist traces in the material. Then,the pellets were extruded in a Noztek single-screw extruder (26:1L/D) at 60 rpm and 260 °C and a filament of ca. 1.75 mm was obtained.This process was also done with pure ASA in the absence of cork asa control. Produced filaments are shown in , tensile strength(σmax), elongation at break, specific modulus (E/ρ), and specific tensile strength (σmax/ρ) values were dissected for each one of the measured specimens.The results were averaged, and standard deviations were presentedas error bars. Analysis of variance (ANOVA) with a significance levelof α = 0.05 and Tukey's test were performed to determineifthere were statistically significant differences between the results.SEM analyses were carried out using an FEI Nova NanoSEM 450 microscopeequipped with a field-emission gun. Samples were previously sputteredwith a few nm layer of Au in a Balzers SCD 004 Sputter Coater. ImageJsoftware was used to calculate the size of the cork particles. Thethermal conductivity measurements were done in a TA Instruments DTC-25conductivity meter according to the ASTM E1530 standard. At leastthree different samples were measured for each material. The resultswere averaged, and the standard deviations were presented as errorbars.Tensile testing ofthe printed specimens was performed using a universal testing machine(Shimadzu) at a constant speed of 1 mm/min according to ASTM D638.At least five specimens were tested for each material. The densityof the materials was measured by weighing extruded filaments of ca.30 cm length and 3D-printed objects with a known volume. The resultswere compared with the theoretical calculations by applying the ruleof mixtures using the density values of ASA and cork given by thesuppliers. Young’s modulus  at1040 cm–1. These signals have similar intensityfor C. However, the peak at 1160 cm–1 increasesgradually in a significant manner for Cf and Cm, while the peak at 1040 cm–1 does not vary itsintensity. A similar trend can be observed for the C=O stretchingat 1730 cm–1 for Cf and Cm, indicating the successful immobilization of acryloyl chloride andPBA, respectively, in the surface of the cork particles.29 Moreover, a small peak at 810 cm–1 correspondingto the vinyl group of nBA can be observed for Cf, indicating that the monomer is immobilized in the surfaceof the cork particles after the first reaction. This signal disappearsfor Cm, indicating that the polymerization and purificationwere carried out adequately.31 Furtherproof of the successful modification of C was the highly hydrophobicbehavior of Cm, which floated rapidly to the surface whenimmersed in water. This effect was not observed for C particles . The morphology of the particles wasalso observed before and after the surface polymerization. Once the amount of hydroxyl groups availablefor functionalizationis known, the cork particles are chemically modified. articles S1. The mm) were manufactured via solvent casting to obtain ASA + Cand ASA + Cm. This strategy does not require the use ofhigh temperatures during the compounding process, as in twin screwextrusion, avoiding the degradation of the cork particles. A controlwas also done with ASA only by solvent casting for comparative purposes.The pellets obtained are well dried under vacuum and at 60 °Cto ensure that all the traces of DCM occluded and moist are removed.The ASA, ASA + C, and ASA + Cm pellets were then extrudedat 260 °C to obtain a filament with a homogeneous diameter of1.70 ± 0.5 mm, suitable for FDM. The filaments produced alsoshowed a very uniform color, implying that the cork particles arehomogeneously distributed in the composite material. Interestingly,ASA + C filaments exhibit a darker color than ASA + Cm,which does not vary after the specimens are printed at 245 °Cby FDM, as depicted in 32 However, previous reports indicate that irreversible thermal degradationof cork in an air atmosphere starts at 258 °C, evidencing thatcork powder can be processed at temperatures below 250 °C withoutsignificant changes in the chemical composition, even if there arecolor changes.33 In our case, the extrusion temperaturewas slightly higher, 260 °C, since lower temperatures causedclogging in the screw and made it difficult to form a suitable filamentfor FDM. However, given the low residence time of the composite insidethe extruder (less than 1 min), it was assumed that the degradationin this step was practically negligible. The color differences betweenASA + C and ASA + Cm seem to indicate that the PBA coatinglayer on the Cm particles acts as a protective barrierthat prevents, or at least delays, the degradation of the particles.Then,the composites containing 5 wt % of cork ,including statistical analysis, are summarized in the form of boxplots in 4a–c.The27 PP, or PE.34 All these studiesconclude that the use of compatibilizing agents contributes to improvethe adhesion of cork to the polymeric matrix. However, due to theproperties of the cork, the mechanical performance of the compositegenerally decreases when compared to the unmodified polymer.7 The addition of cork may increase the strengthof the material only if the original polymeric matrix used is ratherweak, as reported for PMMA composites, which undergo an increase ofthe tensile strength from 3 to 6 MPa, approximately.30 When the cork content is fixed, the enhancement of thecork-matrix compatibility can increase the mechanical properties upto 40% when compared to composites containing unmodified cork.34In fact, cork cannot be used as a reinforcing agent since its mechanicalproperties are lower than those of ASA. Similar results were previouslyreported for other cork composites based on PLA,m was done by applying the rule of mixturesusing the density values provided by the manufacturers. In particular,the upper and lower bounds of the Young’s modulus were predictedfor ASA composites with cork particles (see the Supporting Information for more details). It was found thatASA + Cm exhibits practically the maximum Young’smodulus predicted by the rule of mixtures, which indicates a goodadhesion between Cm and ASA. On the other hand, the corkparticles may be added to lighten the weight of the material. In thisregard, it is also interesting to compare the specific properties(defined by the ratio between the measured property and the densityof the composite) of the materials, which are presented in Table S2). Statistical analysis viaANOVA and Tukey’s tests indicate that the specific modulus(E/ρ) and specific strength (σmax/ρ) values of ASA + Cm are similar  to those of ASA. However, these specific properties arestatistically lower for ASA + C. This evidences that ASA + Cm is a competitive material in the design of light, stiff, and strongbeams or ties for applications where a minimum structural weight isrequired.A more detailed analysis of the mechanicalproperties of ASA, ASA+ C, and ASA + C26 which provokes a higher interlayer porosity when printing. Second,several pores are observed within the roads of the deposited material,likely due to the presence of cork. Although the cork particles arenot observed, the gaps found in the fracture surface match the sizeof the cork particles W/m K for pure ASA to (0.099 ± 0.015) W/m K and (0.098 ±0.017) W/m K for ASA + C and ASA + Cm, respectively. Thesevalues are slightly below those theoretically calculated for thesecomposites, where a thermal conductivity of 0.11 ± 0.01 W/m Kis predicted for ASA composites containing 5 wt % cork, accordingto the rule of mixtures. Different factors may have contributed tothis. On one hand, the interlayer porosity, characteristic of theFDM process, has created air bubbles inside the printed materials,which is expected to enhance their insulating behavior. This is supportedby the density results measured experimentally for the 3D-printedobjects, which are lower than the theoretical ones (see Table S2). On the other hand, the low densityvalues, together with the SEM results , alsom) were manufactured by solvent casting atroom temperature and subsequent extrusion in a single-screw extruderwith a short barrel, which minimizes the processing of cork at hightemperatures . This strategy is an alternativeto composite compounding via twin-screw extrusion, where longer residencetimes are typically required and the cork particles could be degradedto a higher extent. The densities of the filaments and objects manufacturedwere similar or lower than those predicted theoretically, suggestingthat there is no densification of the cork particles during the manufacturingprocess. Furthermore, the strategy proposed for the modification ofthe cork by surface polymerization of PBA greatly enhanced its compatibilitywith the ASA matrix as it was observed by SEM in the aspect of thefracture surface and in the increase of the mechanical propertiesof ASA + Cm when compared to ASA + C. ASA + Cm exhibited similar specific modulus and specific strength valuesto pure ASA, indicating that it is a good candidate for stiff andstrong beams and ties, where lightweight materials are required. ASA+ Cm composites showed significantly lower thermal conductivitythan ASA due to the air trapped as interlayer porosity and in thecellular structure of the cork. Thus, the combination of these propertiesmakes ASA + Cm a good candidate as a lightweight insulatingmaterial, allowing to valorize a residue and contributing to the circulareconomy.This research evidences the possibility of using cork particles as an additivein the development of composites suitable for their manufacturingvia FDM. The composites containing 5 wt % (22.54 vol%) of cork particles(either C or C