The Fuegians, ancient inhabitants of Tierra del Fuego, are an exemplary case of a cold-adapted population, since they were capable of living in extreme climatic conditions without any adequate clothing. However, the mechanisms of their extraordinary resistance to cold remain enigmatic. Brown adipose tissue (BAT) plays a crucial role in this kind of adaptation, besides having a protective role on the detrimental effect of low temperatures on bone structure. Skeletal remains of 12 adult Fuegians, collected in the second half of XIX century, were analyzed for bone mineral density and structure. We show that, despite the unfavorable climate, bone mineral density of Fuegians was close to that seen in modern humans living in temperate zones. Furthermore, we report significant differences between Fuegians and other cold-adapted populations in the frequency of the Homeobox protein Hox-C4 (HOXC4) rs190771160 variant, a gene involved in BAT differentiation, whose identified variant is predicted to upregulate HOXC4 expression. Greater BAT accumulation might therefore explain the Fuegians extreme cold-resistance and the protection against major cold-related damage. These results increase our understanding of how ecological challenges have been important drivers of human–environment interactions during Humankind history. Homo sapiens out of Africa during the Paleolithic1, human populations required adaptations to diverse climatic conditions2 achieved through morphological and cultural adjustments and metabolic mutations3, such as brown adipose tissue (BAT) upregulation in cold climates4. BAT is a highly heterogeneous energy-expending tissue that generates heat, characterized by extraordinary plasticity5. BAT helps maintaining core temperature in a cold environment without shivering. In humans, BAT is found primarily in infants and young children. Defined regions of functionally active BAT are present in some but not all adults (3–10%)7 in thermoneutral conditions (approximately 18–24 °C) and may be quantified noninvasively with the use of (18)F-fluorodeoxyglucose ((18)F-FDG) positron-emission tomographic and computed tomographic (PET-CT) scans. BAT negative individuals do not have measurable metabolically active BAT, provided that the temperature conditions the BAT measurement was carried out are given. For humans in typical living conditions  the contribution of BAT to total energy expenditure is believed to be very small. BAT can be activated with various levels of intensity depending on sex, age, and ethnicity by cold exposure8.Subsequently to the dispersal of 9. When Europeans reached Tierra del Fuego in 1520, it was inhabited by four ethnic groups: the Yamana/Yaghan, Haush and Kaweskar/Alakaluf, all hunter-fisher-gatherers spending a significant portion of their time in the canoes and swimming, and the Selknam, terrestrial hunters, altogether generally grouped under the term Fuegians10. These ethnic groups were identified as different communities in the XX century11. The descendants of Fuegians show a population continuity as supported by the genetic affinity of modern Yamana and Alakaluf with ancient individuals from their respective regions and a mixture of European ancestry reflecting post-colonial admixture14. What is known of the ancient inhabitants of Tierra del Fuego mostly comes from navigators and ethnographers’ reports16. All were nomadic hunter-gatherers, and they had an exceptional cold resistance: despite the extremely harsh climate, they did not use any closed clothing, and open fires and animal fat smeared on bare skin appeared inadequate means of protection to those describing them10.The interest in understanding the mechanisms of cold adaptation comes both from obvious anthropological and evolutionary implications, and from theories that attribute to populations with higher BAT activity an increased resistance to obesity and diabetes17: in Ushuaia they hardly surpass 9 °C (50 °F) in summers and average 0 °C (30 °F) in winters. Snowfall can occur in summer. The cold and wet summers help preserve the ancient glaciers.The climate in this region is inhospitable. It is a subpolar oceanic climate (Köppen climate classification Cfc) with short, cool summers and long, wet, moderate winters: the northeast is characterized by strong winds and little precipitation, in the south and west it is very windy, foggy, and wet for most of the year and there are very few days without rain, slush, hail or snow. Temperatures are steady throughout the year and across several centuries18, thyroid hormone levels variation20, high-fat and -protein diet21, BAT over-activity, and white adipose tissue browning23. In humans, BAT increases upon cold exposure24, and it generates heat via upregulation of uncoupling protein-1 (UCP1), increasing basal metabolic rate (BMR)25. Interestingly, in 1960 Hammel studied nine Fuegians belonging to the Alakaluf group, showing that their BMR was about 160% of that reported for the cold exposed "average white man" (p. 24)26. This report raised the suspicion that the Fuegians might have had increased BAT activity at the root of their amazing cold adaptation.Cultural and physiological mechanisms implicated in cold adaptation include clothing and shelter, and bodily changes such as peripheral vasoconstriction, muscle and subcutaneous fat-mediated insulation, lower surface area-to-mass ratio27 and femoral cross-sectional area (CSA)28. BAT volume parallels BMD in some29, although not all reports30, and predicts femoral CSA and cortical bone area (CBA)31. Moreover, BAT-impaired cold-exposed mice show more bone loss than WT via β2 adrenergic receptor activation, this loss being reduced through β2 adrenergic receptor pharmacologic blockade32; and UCP1 knock-out (KO) mice under permanent cold stress have lower bone formation and mineralization compared to WT33. These altered bone phenotypes are not seen in thermoneutral UCP-1 KO and WT mice, indicating that BAT may prevent, although not eliminate, an otherwise massive cold-induced bone loss33. Finally, it is well established that populations living in cold geographic areas have reduced bone density despite milder temperatures during summers36, and recent evidence shows that they also have increased BAT activity37.Beyond the interest in the role of BAT in thermoregulation and as a target for the prevention and treatment of obesity and type 2 diabetes, cold-activated BAT is also positively related to bone mineral density (BMD)To test the hypothesis that BAT could have contributed to the exceptional cold adaptation of the ancient Fuegians, and in the absence of more direct means to investigate BAT in this population, we leveraged the reported correlation between bone and BAT activation by evaluating bone morphology and density of the skeletal remains of the collection of Fuegians preserved in the Museum of Anthropology ‘G. Sergi’ of the Sapienza University of Rome . To deepen the possible link, we then analyzed genomes of this population available in public repositories for variants in genes involved in BAT modulation.6. In line with previous reports, BAT volume and activity were directly related to femoral CBA . Some genes with unknown biological functions were included if they had been associated with obesity in genome-wide association studies22. Coding and non-coding variants in these genes involved in BAT functional pathways were identified in the Fuegian genomes , were selected for the less striking cold adaptation leading to different lifestyle and habits, such as the use of warm clothing, compared to Fuegians, whose main protections from the harsh climate were open fires, animal fat smeared on bare skin and at most a small piece of fur over the shoulders.We therefore analyzed available genomes of this population, and we obtained reliable results for five of the 11 ancient Fuegian genomes retrieved, of which three were Yamanas, one was Selknam and one Alakaluf Dataset . All var22, the cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) rs75385144, was identified in four out of five Fuegians, with an allelic frequency of 0.4 . These results suggest that this variant might be implicated in cold adaptation, as the low frequency in African population suggested, but not in different ways across populations.Interestingly, a coding variant previously associated with cold adaptation40  rs2493270 and Homeobox protein Hox-C4 (HOXC4) rs190771160, both genes being involved in BAT differentiation41. Its variant rs2493270 was found in three out of five Fuegians and was not identified in controls (p = 0.002)  Table . This va46. Moreover, it is hard to determine to which extent the Fuegians resistance to cold lied in the bodily changes as opposed to sufficient cold protection behaviours , the latter more commonly a research issue in scientific literature. As previously mentioned, the habits of the ancient inhabitants of Tierra del Fuego were described by navigators and ethnographers, and the reports are charming but sometimes incomplete or contradictory. Again, further studies are warranted to investigate on these aspects. Second, for obvious reasons, there is small availability of Fuegian skeletons, and indeed the Roman collection is among the richest worldwide, together with collections housed in museums and research institutions of Argentina and Chile, making our sample generally small but actually wider than usual. Third, diagenesis, possibly hindering the bone analysis, cannot be ruled out. However, previously conducted analysis on the skeletal remains included in the present study showed and exceptional state of preservation as reflected by the collagen percentage and the stable carbon and nitrogen isotope ratios reported17. Fourth, the identified control populations for genomic comparison are not ideal, as they were living at a mean winter temperature approximately 10 °C lower than that of the Fuegians. However, the latter had little clothing to protect themselves as opposed to the inhabitants of subpolar northern regions, making them in our opinion the most suitable controls among other contemporary populations. Certainly, we cannot rule out that some population, better suited for such comparison, might be available, and further studies comparing the Fuegians with them would be ideal.We are aware that our approach suffers of some limitations. First, the studies on human subjects were conducted retrospectively: the availability of DXA scans performed at a similar time compared to the PET scan recorded for the study subjects was minimal, making our contemporary human sample small. Moreover, some parameters, such as smoking and alcohol habitual consumption, possibly affecting bone health were not available. Contrary to the association between bone geometry and BAT activity, which we were able to confirm in our BAT expressing living population , we were50.Overall, both the genomic data and bone analysis of the Fuegians suggest that this population might have been characterized by large BAT depots. However, the possibility that the Fuegians could have gained their resistance to cold through other functional pathways cannot be definitely ruled out. Likewise, we cannot exclude that factor such as muscle mass, whose positive correlation with bone mass is established, may have contributed to their better-than-expected BMDs. However, even if this was the case, our hypothesis could be maintained since the myokines derived from skeletal muscle after contraction or cold exposure (i.e. irisin) seem to have profound effects on enhancing bone mass and reinforcing the BAT phenotypeIn conclusion, although based on a small sample, our findings may confirm the hypothesis that the extraordinary cold-adaptation of the Fuegians, already noted by the nineteenth-century explorers, was due to an exceptional BAT accumulation. Our results can be framed as an example of biological adaptation as opposed to the cultural and technological aids that allowed populations to occupy new environments without having to first evolve biological adaptations to them. Whether this distinctive adaptive mechanism of the Fuegians to cold played some role in the human macroevolution deserves further studies.17.The selection criteria included the availability of femur and lumbar vertebras and estimated age of 25 years old or older as proof of achievement of adult bone structure. The collection was recovered by the Italian explorer captain Giacomo Bove. Bove recovered two skeletal collections of Fuegians in 1881 and 1883 stored respectively in the Museo di Storia Naturale in Florence and the Museo di Antropologia G. Sergi of Rome. Captain Bove recovered thirteen of the skeletons preserved in Rome during his second trip, while the further samples were later acquired by the Rome Museum through a donation. The history of the recovery and the composition of the assemblages are fully described in a paper by Marangoni et al.51. Both assemblages are described in Bove’s reports15, and were given to the captain by Yámana settled in the area of Yendegaia, near Ushuaia where in the 1860’s an Anglican Mission was established17. Considering Bove’s notes, those human remains refer to family members to whom the natives related suggesting a tentative attribution to the first half of the nineteenth century17. This fact suggests that the skeletons in the roman collection might belong to Fuegians who lived at a time when contacts with European and North American sealers became relatively more systematic although a steady systematic contact with Europeans would have taken place from 1843 onwards when the Chilean government sent an expedition whose aim was to establish a new city that would serve as a centre for the economic development of the region. Thus, we do not know whether, according with previous observations14, the remains of the roman collection belong to individuals modern enough to show a mixture of European ancestry .The skeletal remains of 12 adult Yàmanas  from Tierra del Fuego were selected from the collection of the Museum of Anthropology "G. Sergi" of the Sapienza University of Rome   18F-FDG uptake was in the paravertebral/cervical, supraclavicular, mediastinal, paravertebral/dorsal, and abdominal or perirenal areas, unrelated to muscle, joints and pathological findings; (2) 18F-FDG uptake had a maximum standardized uptake value (SUV) of 2.0 g/ml or greater (an indicator of 18F-FDG uptake intensity); and (3) the tissue corresponded to the density of adipose tissue on CT (– 250 to – 50 Hounsfield units)6. Among the 217 patients with BAT depots, individuals younger than 25 years old or who underwent PET/CT scans for malignant diseases and received the last treatment for malignant diseases within 1 year before the scan were excluded, as where subjects whose diagnosis and/or treatment timings were unknown. We also excluded those with diabetes, renal failure, and under steroids or osteoporosis treatment at the time of PET/CT. Moreover, a gender, age and BMI matched control population of BAT negative subjects was selected among the same population following the same exclusion criteria stated above. To control for BMI difference, given the reported lean body mass of the Fuegians, we only selected patients with a BMI ≤ 25 kg/m2.We retrospectively selected a population of 217 patients expressing F-FDG BAT among a total of 6454 patients who underwent 8004 consecutive 18F-FDG PET/CT scans from January 2007 to June 2010 at the Istituto Nazionale dei Tumori Regina Elena . The modality of BAT expression detection is described elsewhereAmong the patients enrolled in the present study, a subpopulation was subsequently selected to be age, BMI and gender matched to those estimated for the fuegian skeletal remains adopting the following criteria: available Dual energy X-ray absorptiometry (DXA) scan performed within 1 year of the PET scan with a Hologic scanner.18F- fluorodeoxyglucose (FDG) the patients rested at 24 °C for 1 h before undergoing the PET/CT scan. Data on gender, BMI, age, plasma glucose level and malignancy status  were obtained for all patients. Parameters measured from PET/CT scans included, in addition to BAT characteristics: cross-sectional area , cortical bone area , endocortical bone area , muscle area  taken at proximal-shaft diaphysis, 1 cm distal to the lesser trochanter. These were calculated with the use of ImageJ 1.52i, Wayne Rasband, National Institutes of Health, USA.PET/CT scans were performed using a Biograph 16 High Rez PET/CT scanner . Patients came from the temperate metropolitan area of Rome and were instructed to fast overnight for at least 12 h before the scan and to abstain from carbohydrates and very fatty foods consumption, nicotine, caffeine, or alcohol intake for the preceding 24 h. Room temperature water was allowed at all times. Since their arrival to the hospital, the patients were in an air-conditioned environment at about 22 °C. After the injection of 52. Briefly, height was calculated through Manouvrier tables using the femur, humerus, ulna and radius of the Fuegians, the mean of all estimations was taken as estimated height for each Fuegian skeleton. Then, CSA, CBA and EndA were divided by estimated height in the case of the Fuegian skeletons, and actual height in the case of the living population.For each Fuegian skeleton, the right femur was analyzed using a commercial CT scanner . Before the scan, femurs were oriented in standardized anatomical planes. Diaphyseal structural properties were calculated from DICOM files with the use of ImageJ 1.52i . Parameters measured included: cross-sectional area , cortical bone area , and endocortical bone area  taken at proximal-shaft diaphysis, 1 cm distal to the lesser trochanter. In order to control by body size, CSA, CBA and EndA were standardized by estimated height53.Lumbar spine , femoral neck (FN) and total hip (TH) BMD of Fuegians femur and lumbar vertebrae remains were measured through DXA . Before DXA scanning, femurs of Fuegians were oriented in standardized anatomical planes and the lumbar vertebrae placed in a rack that allowed them to be aligned, using rice as a soft tissue proxy39 were downloaded from http://www.cbs.dtu.dk/suppl/NativeAmerican/. Data corresponding to 8 Fuegians and 14 distinct Native Americans individuals exposed to cold temperatures were identified and extracted. Read statistics per sample and alignment methods are shown in the Supplementary Materials for Raghavan et al.39.Alignment data from Raghavan et al.39, samples with less than 5 million of mapper reads were removed prior to further analyses. The remaining 5 fuegian individuals included 3 Yamana (~ 47 M of average mapped reads and ~ 21% of endogenous content), 1 Selknam (81.5 M of mapped reads and ~ 16% of endogenous content) and 1 Alakaluf (~ 15 M of mapped reads and ~ 1.4% of endogenous content).Genotypes were called both per-sample and in a multi-sample approach by using GATK (v.4.1.2.0)54 and modelled after the GATK Best Practices Workflows and the parameters used in Raghavan et al.39. Briefly, variants were called per-sample using HaplotypeCaller in GVCF mode and prepared for filtering . Variants were extracted, filtered and ricalibrated for QualByDepth, RMSMappingQuality, MappingQualityRankSumTest, ReadPosRankSum, FisherStrand, ReadPosRankSumTest and HaplotypeScore using the HapMap 3.3, and dbSNP138 resources with priors 15 and 2, respectively.Since the individual Fuegians libraries have a variable endogenous content  and a low sequencing depth (average depth from 0.003 to 1.7)22, were selected and further investigated (S1).The filtered variants were imported and annotated using R packages vcfR (v.1.12.0), GenomicFeatures (v.1.42.1) and VariantAnnotation (v.1.36.0) and the gencode annotation v.19 as reference. SNPs located in coding, promoter and 5′/3′ UTR regions of a panel of 28 genes directly involved in BAT metabolism from Sazzini et al.55and RegulationSpotter56.These are online applications performing several in silico tests on both DNA and protein level ultimately estimating the impact of the identified variant, such as functional consequences of synonymous or intronic mutations up to amino acid substitutions, deletion and insertion of sequences, or variants within the intron–exon border.Candidate variants were further characterized based on possible functional effect as predicted by in silico analysis using MutationTaster6. The project had been approved by the Institutional Review Board of the Dipartimento di Prevenzione e Diagnostica Oncologica, UOC di Medicina Nucleare, Istituto Nazionale Tumori Regina Elena, Roma. The data were analyzed anonymously. Written informed consent was given by the patients or from the next of kin or caretakers for their information to be stored in the hospital database and used for research. All methods, including human remains handling and storage, were carried out in accordance with relevant guidelines and regulations. All efforts were made to minimize damage to human remains, and data will be made available upon reasonable request to allow critical re-examination of scientific findings57.Data relative to the study cohort of living humans was collected from a database previously used to report BAT prevalence in central Italyt-test or Wilcoxon test as appropriate. Relationships between variables were measured by Pearson’s correlation coefficient. Stepwise regression modeling was used to determine predictors of CBA. A chi-square test was performed to compare variant frequencies between Fuegians, Siberians/North Americans, and other modern populations from gnomAD database (https://gnomad.broadinstitute.org/). An α error of 0.05 was considered the threshold for statistical significance.Statistical analysis was performed using SPSS 25.0 . Data are expressed as mean ± standard deviation for normally distributed variables. Variables were tested for normality of distribution using Shapiro–Wilk test. Variables that were not normally distributed were log-transformed. Comparisons between groups were performed using the paired Student's Supplementary Information 1.Supplementary Information 2.


Ascophyllum nodosum, Undaria pinnatifida, Saccharina japonica and red alga Palmaria palmata) on some fecal bacterial populations and metabolites, fecal IgA and apparent total tract digestibility of nutrients. The different algal supplementations  did not have any significant effect on the selected fecal microbial parameters, intestinal immunity and nutrient digestibility. Further studies investigating higher dietary inclusions of intact seaweeds or their polysaccharide extracts are needed to gain a better understanding of the potential of these interesting marine resources in companion animal nutrition.Seaweeds represent a source of bioactive compounds that have recently drawn the attention of the scientific community for their possible application as health-promoting ingredients. In particular, their indigestible polysaccharides have exhibited promising prebiotic properties both in humans and in farm animals. The present study investigated for the first time in healthy adult dogs the effects of dietary supplementations with intact seaweeds . Seaweeds were added to food at a daily dose of 15 g/kg. The CD contained silica as a digestion marker. Each feeding period lasted 28 d, with a 7 d wash-out in between. Feces were collected at days 21 and 28 of each period for chemical and microbiological analyses. Fecal samples were collected during the last five days of each period for ATTD assessment. Dogs showed good health conditions throughout the study. The fecal chemical parameters, fecal IgA and nutrient ATTD were not influenced by algal supplementation. Similarly, microbiological analyses did not reveal any effect by seaweed ingestion. In conclusion, algal supplementation at a dose of 15 g/kg of diet failed to exert noticeable effects on the canine fecal parameters evaluated in the present study.The present study investigated in dogs the dietary effects of intact seaweeds on some fecal bacterial populations and metabolites, fecal IgA and apparent total tract digestibility (ATTD). Ten healthy adult dogs were enrolled in a 5 × 5 replicated Latin square design to evaluate five dietary treatments: control diet (CD); CD +  The crucial role that the intestinal microbiota plays in supporting host health is a well-established concept  and in rIn this context, marine macroalgae (seaweeds), plant-like multi-cellular organisms representing a substantial part of the ocean biomass and a traditional food for some Asian human populations, have recently drawn the attention of the scientific community for their possible application as health-promoting ingredients both in human diets  and animPhaeophyta), red (Rhodophyta) and green (Chlorophyta) algae according to their pigmentation. Their major components are minerals [Macroalgae comprise thousands of species that are classified as brown , Undaria pinnatifida (UP) and Saccharina japonica (SJ)) or one intact red seaweed ) on some fecal microbial populations and metabolites, fecal IgA and apparent total tract digestibility (ATTD) of nutrients in healthy adult dogs. We supposed that these intestinal functionality-associated parameters would be positively influenced by algal supplementation.The aim of the present study was to evaluate the effects deriving from dietary supplementations with different intact brown seaweeds . Concerning gender and reproductive status, there were eight spayed females, one castrated male and one intact male. Their average body weight ± standard deviation was 22.8 ± 6.1 kg. Their median body condition score was 4.5 (range from 4 to 5), according to the 9-point scale proposed by Laflamme et al. . During 0.75 [One commercial extruded and complete diet formulated for adult dogs , containing dehydrated poultry protein, rice, maize, wheat, animal fats, fish meal, linseed, sunflower oil, dehydrated eggs, sodium, phosphate, potassium chloride and sodium chloride, was used as the control diet (CD) for the study. Four different experimental diets were set up, through the supplementation of the CD with four intact seaweeds products. In particular, it was decided to use three brown seaweeds: AN, UP and SJ, and one red seaweed: PP. In order to remove any contaminants, the algae were washed with tap water and rinsed twice with deionized water. Finally, algae were freeze-dried and ground through a 1 mm screen. The algal supplements were added at a concentration of 15 g/kg of as-is diet. Since the seaweeds were in form of a powder, they were spread over the kibble together with a small amount of water to ensure their adhesion and total consumption. Dogs were fed twice a day. Consequently, the daily dose of supplements was divided and added equally to each of the two planned daily meals. No snack or other food was offered to the dogs during the whole study period. The CD did not include significant amounts of soluble fiber sources. Silica was included at a dose of 5 g/kg as a source of acid-insoluble ash to be used as digestion marker. The dogs’ daily ration was set up by considering the energy content of the CD (determined through the modified Atwater conversion factors) and the daily energy requirements, according to the recommendations for the maintenance of adult dogs with a moderate activity level: 120 kcal/kg BW0.75 . Prior tThe feeding trial was planned as a 5 × 5 replicated Latin square design. At the beginning of the study, all dogs were adapted to the basal diet for 30 days. During the crossover study, groups of two dogs received each of the five dietary treatments (CD and the four seaweed-supplemented diets). Each feeding period lasted 28 days, with a 7-day wash-out in between periods during which all dogs received the CD. On days 21 and 28 of each feeding period, a fresh fecal sample was collected within 30 min of defecation and then frozen at −80 °C for chemical (dry matter (DM), ammonia, short-chain fatty acids (SCFAs), branched-chain fatty acids (BCFAs) and biogenic amines) and microbiological analyses. A sub-sample of feces collected at both time-points (day 21 and 28) was freeze-dried for IgA measurement. During the last five days of each feeding period (from day 24 to 28), fecal samples were pooled and frozen at −20 °C and successively freeze-dried for nutrient analyses and apparent total tract digestibility assessment.The nutrients in the CD, intact seaweeds and freeze-dried fecal samples collected during the last 5 days of each feeding period were quantified according to AOAC International standard methods  , in order to accurately calculate the ATTD coefficients, the nutrient percentage in the diet was calculated by considering both the contribution of the CD and the seaweed supplements.w/v in distilled water). Ammonia was quantified through a commercial kit . SCFAs and BCFAs were determined using a 2 m glass column  of 10% SP-1000 + 1% H3PO4 on 100/120 Chromosorb W AW with nitrogen as the carrier. The chromatograph was a Fisons HRGC MEGA 2 series 8560 with a flame ionization detector. Both the injector and detector temperatures were 200 °C, and the oven was 155 °C. 2-ethylbutyric acid was used as the internal standard. For the measurement of biogenic amines, feces were diluted 1:5 w/v with perchloric acid (0.3 M); biogenic amines were later separated by HPLC and enumerated through fluorimetry, as described by Stefanelli et al. [Fecal pH was evaluated with a SevenMulti pH meter  in diluted fecal samples  using the Norgen Stool DNA Isolation Kit . Isolated DNA concentration (ng/μL) and purity were measured using a DeNovix DS-11 spectrophotometer . Template DNA was diluted to 50 ng/μL and stored at −20 °C until further analysis. Total bacteria , Firmicurmicutes , Bifidobcus spp. , Lactobalus spp. , Clostriluster I , Escherihia coli  and Faecusnitzii , were quThe qPCR assay was performed using a CFX96 Touch thermal cycler .Amplification was performed in duplicate for each bacterial group within each sample, while standard curves were run in triplicate.Firmicutes, bifidobacteria, enterococci, lactobacilli, Clostridium cluster I, Escherichia coli and Faecalibacterium prausnitzii. Cycle threshold values were plotted against standard curves for the quantification of the target bacterial DNA. Melting curves were checked after amplification to ensure the single product amplification of a consistent melting temperature.Briefly, the PCR reaction contained 7.5 μL 2× SensiFAST No-ROX PCR MasterMix , 4.8 μL of nuclease-free water, 0.6 μL of each 10 pmol primer and 1.5 μL of template DNA for a final reaction volume of 15 μL. The amplification cycle was as follows: initial denaturation at 95 °C for 3 min, 95 °C for 5 s, primer annealing at 55–64 °C for 10 s and 72 °C for 8 s. The cycle was repeated 40 times. A negative control (without the DNA template) was also run for each primer pair. Standard curves were constructed from eight tenfold dilutions for total bacteria, The determination of IgA content in lyophilized fecal samples was carried out by using a commercial kit , following the procedure described by Zannoni et al. .t-test. Since no significant difference was observed, mean values obtained from each dog at 21 and 28 d were considered for statistical analysis. Data were assessed for normality through a D’Agostino and Pearson omnibus normality test. When they were not normally distributed, a logarithmic transformation was applied to normalize the data distribution. Normalized data were analyzed by a general linear model with diet as a fixed effect and animal and period as random effects. The Dunnett test was used as the post-test. When microbiological analysis revealed unquantifiable outcomes, “zero” values were assigned.Firstly, for each parameter, data measured after days 21 and 28 of each feeding period were compared by a Student’s p ≤ 0.05 and 0.05 < p < 0.1, respectively. Statistical analyses were performed using Statistica 10.0 software .Levels of significance and tendency were set at Dog owners did not report any modification in body weight or fecal appearance and consistency of their animals during the study. All the dogs maintained good health conditions throughout the trial and entirely consumed the assigned daily ration and the algal supplements.The chemical parameters evaluated in fecal samples did not show any influence by the dietary supplementation with intact seaweeds .p = 0.07). In particular, compared with the CD, ATTD of Ca was improved when dogs received the AN diet   .Moreover, the concentration of fecal IgA did not significantly change among dietary treatments .The purpose of this study was to investigate the effect of dietary supplementations with intact seaweed-based products on the gastrointestinal environment of adult dogs through the determination of ATTD, fecal IgA and selected fecal bacterial populations and metabolites.In general, the results revealed a negligible influence exerted by the selected seaweeds on canine fecal microbiota. In particular, the lack of significant effects on bacterial metabolites such as SCFAs, BCFAs and biogenic amines reflected the absence of modulation in the microbial populations evaluated in fecal samples.Saccharina, Laminaria, Ascophyllum and Undaria spp.) or their major undigestible polysaccharide components  [Differently from the present study, several literature reviews, describing the results of both in vitro and in vivo studies, have recently emphasized interesting prebiotic effects deriving from dietary supplementation with intact seaweeds  ,16,17,18Laminaria spp. have shown positive changes in the intestinal microbiota, such as increased Lactobacillus and Bifidobacterium spp., decreased Enterobacteria, higher concentration of SCFAs, acidification of the pH and/or a reduction in ammonia in the hindgut [Laminaria hyperborean have shown interesting antimicrobial activity against E. coli, Salmonella typhimurium, Staphylococcus aureus and Listeria monocytogenes [Salmonellatyphimurium and Staphylcoccus aureus bacterial strains.Recently, a growing interest in the potential application of marine macroalgae or their extracts in farm animal nutrition has been observed. In pigs, for example, brown algae are considered promising dietary supplements , able to exert prebiotic and immunomodulatory effects on the intestinal environment ,40. In f hindgut . Moreoveytogenes . Likewisytogenes  describeCl. perfringens [Condrus crispus and Sarcodiotheca gaudichaudii (at doses of 5–10 and 20 g/kg of diet), positively affected the intestinal environment of layers hens, as higher concentrations of SCFAs and a higher abundance of Bifidobacterium longum and Streptococcus salivarius together with a reduction in C. perfringens was observed [A. nodosum at dietary doses up to 10 g/kg [Additionally, concerning red seaweeds, both as whole biomass or polysaccharide extracts , interesting prebiotic effects on gut microbiota have been reported ,44. In vfringens . Similarobserved . Neverthobserved  or intac 10 g/kg  in pigs As previously pointed out by other authors, the inconsistent results from studies on monogastric farm animals receiving seaweeds as “whole products” could be attributable to the different chemical composition of this type of supplements. In particular, their polysaccharide content can greatly differ in relation to algal species and environmental factors  ,50. In bMoreover, it must be underlined that in the present investigation only few of the main key taxa of the canine microbiome have been evaluated. This might represent a limitation for the study since possible changes in other bacteria could not have been detected.In general, for ethical reasons, according to the current European legislation, feces represent the only analyzable type of sample in studies investigating the gastrointestinal tract of companion animals. Actually, fecal composition is not fully representative of the gut environment , even thIn this study, fecal IgA analysis revealed no effect in dogs when they received the supplemented diets. Secretory IgA is the most important humoral immune factor in the intestinal tract and represents the expression of the mucosal immune response , limitinIt is important to emphasize that algal polysaccharides (glycans) have differences in their structure in comparison to homologous terrestrial compounds . Even thThe algal supplementations investigated in the present study did not influence nutrient ATTD. Only Ca digestibility displayed a tendency to be influenced by seaweeds , with an improvement of this last parameter when dogs received the AN diet.n-butyrate, in primis) could promote a better absorptive capacity of the intestinal epithelium [Better nutrient digestibility has been associated with a beneficial influence on the gut environment from seaweed consumption. In fact, a positive modulation of intestinal microbiota exerted by these marine substrates, favoring an improvement of the production of trophic metabolites such as SCFAs  during all five experimental periods. Nevertheless, it must be emphasized that the lack of influence on ATTD could be considered positively since the algal supplementations tested in the present study did not affect the relatively high digestibility of the basal diet.The present study showed that the ingestion of intact seaweeds at a dose of 15 g/kg of diet was well tolerated by the dogs and it did not alter nutrient ATTD. On the other hand, it failed to exhibit noticeable effects on fecal microbiota and immunological parameters such as fecal IgA.Despite the scientific evidence supporting a promising role for seaweeds and their extracts as “health-promoting ingredients” in farm animals, there is a limited understanding of the effective ability of canine gut microbiota to ferment algal polysaccharides since, to the authors’ knowledge, this research represents the first investigation on the effects of algal supplements on the intestinal ecosystem of the dog.Since it has been pointed out that intestinal microbiota must necessarily express specific enzymes to use algal polysaccharides as a carbon source for bacterial fermentation  and only


Climate change, environmental pollution and pathogen resistance to available chemical agents are part of the problems that the food industry has to face in order to ensure healthy food for people and livestock. One of the promising solutions to these problems is the use of cold atmospheric pressure plasma (CAPP). Plasma is suitable for efficient surface decontamination of seeds and food products, germination enhancement and obtaining higher yields in agricultural production. However, the plasma effects vary due to plasma source, treatment conditions and seed type. In our study, we tried to find the proper conditions for treatment of barley grains by diffuse coplanar surface barrier discharge, in which positive effects of CAPP, such as enhanced germination or decontamination effects, would be maximized and harmful effects, such as oxidation and genotoxic potential, minimized. Besides germination parameters, we evaluated DNA damage and activities of various germination and antioxidant enzymes in barley seedlings. Plasma exposure resulted in changes in germination parameters and enzyme activities. Longer exposures had also genotoxic effects. As such, our findings indicate that appropriate plasma exposure conditions need to be carefully optimized in order to preserve germination, oxidation balance and genome stability, should CAPP be used in agricultural practice. Cold atmospheric pressure plasma (CAPP) is relatively widely used in agriculture and food production for the treatment of plants and seeds and also as a more environmentally friendly way for water desalination or decontamination ,2,3 and There are many positive aspects of preharvest plasma treatment, e.g., surface decontamination ,14, germPlasma treatment efficiency is variable and can change according to the plasma source, working gas, treated seed type and morphology of their surface , and forHordeum vulgare L.) by CAPP generated in different gases , causing no harmful effects and, at the same time, maintaining the positive ones (such as increased germination). Since the main active plasma components are RONS, our attention was focused on indirect oxidative stress markers, namely superoxide dismutase (SOD) and guaiacol peroxidase (G-POX), which detoxify oxidative stress products from cells. Determining the amount of DNA breaks and the presence of oxidized bases in DNA were also used as indirect evidence of oxidative stress induced by CAPP.The aim of the present study was to identify treatment conditions for barley grains  significantly accelerated germination on the first day of culture compared to the untreated control . This trThe activity of protease was positively affected after 10, 20 and 30 s treatment of plasma  and its activity decreased significantly with prolonged plasma exposure. Interestingly, glucanase activity was significantly higher in all plasma treated variants (compared to the untreated control), peaked at 60 s plasma treatment, and then decreased sharply . Using tWhen monitoring the activity of antioxidant enzymes, lower plasma doses slightly stimulated superoxide dismutase activity (SOD), with statistically significant increases in SOD activity observed in variant A30. The activity of guaiacol peroxidase (G-POX) decreased slightly after 10 s treatment compared to the untreated control, peaked after 20 s treatment and subsequently began to decrease with increasing application dose .The genotoxic potential of CAPP treatment on barley grains was estimated by standard and modified alkaline comet assays and constant field gel electrophoresis (CFGE) on 7-day-old seedlings. As shown in The alkaline comet assay is the method used for the detection of DNA single- and double-strand breaks. Moreover, modification of this method using the formamidopyrimidine DNA glycosylase (Fpg) enzyme also enables the detection of oxidative damage of purines. In our study , we did As by comet assay, it is possible to detect both, single- and double-strand breaks, the CFGE method was used for double-strand break evaluation specifically. As shown in Hordeum vulgare L. cv. Maltz) was significantly accelerated after treatment with low doses of cold atmospheric pressure plasma (up to 30 s) on the initial days of cultivation compared to the untreated control. Park et al. [Fusarium and subsequent plasma treatment with low-pressure Ar/N2-O2 surface-wave microwave discharges for 3 min, Szöke et al. [Chenopodium album [It has been shown that plasma exposure affects seed germination differently depending on various factors including the type of the plasma source, working gas and exposure time ,29, plank et al.  state the et al.  noted ane et al. , wheat [ma up to  s on thee et al.  and ricee et al. —and dicoum album , pea [16um album , soybeanum album ,40 and tum album . As in oum album ,40,41,42um album , which fum album , and PAsum album . Severalum album ,38,46.We found that low doses of CAPP generated in various working gases slightly stimulate the activity of antioxidant enzymes (SOD and G-POX) involved in the detoxification of RONS. This is very important in managing oxidative stress. Moreover, our data show that it can also alleviate waterlogging stress, which is a major abiotic stress for barley . RestoraAs we mentioned before, plasma decontamination effects are probably caused by RONS, which can attack nearby cells and oxidize macromolecules, even DNA. It is the reason why we focused also on the genotoxic potential of plasma using three methods for oxidized bases, DNA single- and double-strand breaks detection. According to our results, we suggest that the least harmful working gas for barley grains treatment is nitrogen, where the number of primary DNA damages was the lowest one from all monitored working gases. However, these results differ from other studies where the same plasma source, diffuse coplanar surface barrier discharge (DCSBD), for various seeds’ treatment was used. In the study of Švubová et al. , plasma Hordeum vulgare L. cv. Maltz) grains used in the experiments were obtained from the Central Control and Testing Institute in Agriculture in Bratislava, Slovakia. The seeds were stored at 8 °C in the dark.The dried barley  . Unique 2) and volume (~80 W/cm3) power density resulted in short exposure times [Plasma is generated in a thin layer on the surface of ceramics with active thickness in the range of 0.3–0.5 mm. The size of the discharge area (8 × 20 cm) enables the plasma treatment of a high amount of material. The generated plasma is characterized by high surface  were sown on Petri dishes on sterile filter paper, watered and cultivated in dark conditions in an incubator at the temperature of 24 ± 2 °C for 5 days. During cultivation, the number of germinated grains was counted and, on the third day, material for biochemical analyses (enzyme activity assays) was collected. After 5 days, the length (cm) and weight (mg) of shoots and roots were measured and the number of adventitious roots of young seedlings was counted. These data were used to calculate Germination (%), Germination Potential (%), Germination Index (%) and Grain Vigor Index (%) according to Abdul-Baki and Anderson . Changesg), the supernatant was used for the determination of protein concentration according to Bradford [Samples (~1.5 g) were ground in liquid nitrogen with a mortar and pestle and suspended in 50 mM Na-Phosphate protein extraction buffer with 1 mM EDTA (pH 7.8). After 15 min centrifugation  BSA in 200 mM glycine-HCl buffer (pH 3.0) at 37 °C for 1 h. The reaction was stopped by the addition of 450 µL of 5% (w/v) trichloroacetic acid. Samples were incubated on ice for 10 min and centrifuged at 20,000× g for 10 min at 4 °C. The absorbance of the supernatant was measured at 280 nm by a Jenway 6705 UV/Vis spectrophotometer . One unit of proteolytic activity is defined as an increase of 0.001 in the absorbance at 280 nm per min [−1 mg−1 SP. As a standard, we used glucose. All chemicals were purchased from Sigma-Aldrich Co.Changes in the proteolytic activity in 3-day-old barley seedlings were determined by incubating 150 µL of an extracted protein sample with 150 µL of 2%  and ˙O2¯  were tested. The activity of guaiacol peroxidase (G-POX) was established according to Frič and Fuchs [−1mg−1 by molar extinction coefficient of tetraguaiacol (26.6). All chemicals were purchased from Sigma-Aldrich Co.The activities of enzymes that detoxify Hnd Fuchs , and thend Fuchs . One uni−1 for 15 min at 4 °C. Slides were then neutralized three times with 0.4 M Tris-HCl buffer solution (pH 7.5) and stained with fluorescent dye ethidium bromide  (Serva) for 5 min. DNA damage was observed using an OLYMPUS BX 51 fluorescent microscope with a green excitation filter UMWIG3 under 400× magnification. Plasma-untreated seedlings were used as a negative control.The alkaline comet assay is a method used for measuring DNA damage in eukaryotic cells. Using the alkaline comet assay and its modifications, it is possible to detect different DNA defects such as single-strand breaks, double-strand breaks, cross-links, apyrimidine and apurine (AP) sites ,70. Our Constant field gel electrophoresis (CFGE) is a genotoxicological method based on the incorporation of isolated nucleoids into agarose plugs that subsequently undergo lysis and electrophoresis at a constant and low voltage. In this way, it is possible to evaluate the relative amount of double-strand breaks produced by the action of a potentially genotoxic agent . Our expp ≤ 0.05. All experimental data in this work are from at least three independent experiments.Data were analyzed using Statgraphics Centurion XV v. 15.2.05  and Excel (Microsoft Office 2007). Treatment effects were analyzed by means of ANOVA single-step multiple comparisons of means using means of LSD tests or Tukey’s test and comparisons between the mean values were considered significant at Low plasma doses (up to 20 s for ambient air and nitrogen and up to 30 s for oxygen) have a positive effect on the germination dynamics of barley grains. This is probably due to significant stimulation of the activity of lytic enzymes that cleave the storage substances in the aleurone layers and in the starch endosperm. Slight stimulation of antioxidant enzyme activity improves the detoxification of emerging reactive oxygen species, leading to better production parameters and an overall improvement in the vitality of young barley seedlings. However, reactive oxygen species generated during plasma treatment could have negative effects on DNA molecules, as was proved in our study. Therefore, cold atmospheric pressure plasma application can represent a new green technology for the improvement of surface decontamination and germination of various seeds and grains, but its potentially harmful effects need to be further investigated.


The screening of prostate cancer (PCa), based on the serum prostate specific antigen (PSA), is characterized by a high number of false positives, leading to overdiagnosis of healthy men and overtreatment of indolent PCa. This clinical problem severely affects the quality of life of patients, who would benefit from more specific risk stratification models. By performing a mass spectrometry (MS) screening on urine samples collected prior to prostate biopsy, we identified novel biomarkers and validated them by ELISA. Here, we show that an upfront urine test, based on quantitative biomarkers and patient age, has a higher performance compared to PSA (AUC = 0.6020) and is a feasible method to improve the eligibility criteria for prostate biopsy, to detect healthy men (AUC = 0.8196) and clinically significant PCa, thereby reducing the number of unnecessary prostate biopsies.PCa screening is based on the measurements of the serum prostate specific antigen (PSA) to select men with higher risks for tumors and, thus, eligible for prostate biopsy. However, PSA testing has a low specificity, leading to unnecessary biopsies in 50–75% of cases. Therefore, more specific screening opportunities are needed to reduce the number of biopsies performed on healthy men and patients with indolent tumors. Urine samples from 45 patients with elevated PSA were collected prior to prostate biopsy, a mass spectrometry (MS) screening was performed to identify novel biomarkers and the best candidates were validated by ELISA. The urine quantification of PEDF, HPX, CD99, CANX, FCER2, HRNR, and KRT13 showed superior performance compared to PSA. Additionally, the combination of two biomarkers and patient age resulted in an AUC of 0.8196 (PSA = 0.6020) and 0.7801 (PSA = 0.5690) in detecting healthy men and high-grade PCa, respectively. In this study, we identified and validated novel urine biomarkers for the screening of PCa, showing that an upfront urine test, based on quantitative biomarkers and patient age, is a feasible method to reduce the number of unnecessary prostate biopsies and detect both healthy men and clinically significant PCa. Prostate cancer (PCa) is one of the most frequently diagnosed cancers worldwide and a prominent reason for tumor-related deaths in men . In pastUrine is an ideal clinical specimen for diagnostic testing. Its easy collection is completely non-invasive and it allows the processing of large volumes, compared to tissue, blood or other biological materials. This enables the detection of biomarkers at any time point during patient care and facilitates not only diagnosis, but also the monitoring of the disease. The detection of biomarkers in urine has been studied for a wide range of cancers with ultrasensitive screening methods, such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) ,8. SpeciThe prostate epithelium secretes cellular substances into the gland and prostate cancer cells can be shed into the prostatic fluids, where they exude into the urine ,11. SensThe aim of this study was to discover novel urine biomarkers for the detection of PCa and investigate their potential as an improved diagnostic test. The goal was to select, with high sensitivity, men with unspecifically elevated PSA from men who could benefit from prostate biopsy, which remains the standard of care for the diagnosis of PCa. Since low-grade PCas are generally considered indolent, the aim of the study was also to identify biomarkers for the selection of men harboring high-grade PCa. Thus, by improving the eligibility criteria for prostate biopsy, we would reduce the number of unnecessary prostate biopsies performed. Additionally, it might offer the possibility of non-invasive disease monitoring. Tests that rely on the quantification of single biomarkers are often limited in their power to predict cancer, a disease that is hallmarked by its heterogenic biology ,17. TherWe performed a MS screening on urine samples from 45 men with elevated PSA levels scheduled for prostate biopsy and identified 2.735 proteins across all samples, as well as potential biomarkers for the detection of all grades of PCa or high-grade tumors only. Top candidates were then validated by ELISA and a combinatory analysis predicted their performances as multiplexed diagnostic test for PCa screening.A total of 45 patients were enrolled in the study at the Urology Department of the University Hospital of Zürich . Samples were collected as first-morning urine from men not subjected to prostatic massage, with high serum PSA levels (≥2 ng/mL) and/or abnormal digital rectal examination (DRE) results, before the performance of the prostate biopsy. Sample aliquots were then stored at −80 °C until use. Patients’ recruitment, urine sample collection, and analysis were approved by the Ethics Committee of Kanton Zürich (BASEC n° 2016-00829).Mass spectrometry (MS) analysis was performed by Biognosys AG . All solvents were HPLC-grade from Sigma Aldrich  and all chemicals, if not stated otherwise, were obtained from Sigma Aldrich .After thawing, sample digestion was performed on single filter units  following a modified FASP protocol . Samples were denatured with Biognosys’ Denature Buffer and reduced/alkylated using Biognosys’ Reduction/Alkylation Solution for 1 h at 37 °C. Subsequently, digestion to peptides was carried out using 1 μg trypsin (Promega) per sample, overnight at 37 °C.Peptides were desalted using C18 Ultra Micro Spin columns (The Nest Group) according to the manufacturer’s instructions and dried down using a SpeedVac system. Peptides were resuspended in 17 μL LC solvent A ) and spiked with the Biognosys iRT kit calibration peptides. Peptide concentrations were determined using a UV/VIS Spectrometer .For HPRP fractionation of peptides, digested samples were pooled. Ammonium hydroxide was added to a pH value > 10. The fractionation was performed using a Dionex UltiMate 3000 RS pump (Thermo Scientific™) on an ACQUITY UPLC CSH C18 1.7 μm, 2.1 × 150 mm column (Waters). The gradient was 1% to 40% solvent B in 30 min, solvents were A: 20 mM ammonium formate in water, B: acetonitrile. Fractions were taken every 30 s and sequentially pooled to 12 fraction pools. These were dried down and resolved in 15 μL solvent A. Prior to mass spectrometric analyses, they were spiked with Biognosys’ iRT kit calibration peptides. Peptide concentrations were determined using a UV/VIS Spectrometer .m/z range of 350–1650 with a resolution of 60.000  and was followed by 15 data dependent MS2 scans with a resolution of 15.000 . MS2 acquisition precursor isolation width was 1.6 m/z, while normalized collision energy was centered at 27 (10% stepped collision energy) and the default charge state was 2+.For shotgun LC–MS/MS measurements, 2 μg of peptides per fraction were injected to an in-house packed C18 column  on a Thermo Scientific Easy nLC 1200 nano-liquid chromatography system connected to a Thermo Scientific™ Q Exactive™ HF mass spectrometer equipped with a standard nano-electrospray source. LC solvents were A: 1% acetonitrile in water with 0.1% FA; B: 15% water in acetonitrile with 0.1% FA. The nonlinear LC gradient was 1–52% solvent B in 60 min followed by 52–90% B in 10 s, 90% B for 10 min, 90–1% B in 10 s and 1% B for 5 min. A modified TOP15 method from Kelstrup was used [For DIA LC–MS/MS measurements, 2 μg of peptides and 1 IE of PQ500 reference peptides were injected per sample. For samples with less than 2 μg of total peptide available, the amount of reference peptides was adjusted accordingly. Peptides were injected into an in-house packed C18 column  on a Thermo Scientific Easy nLC 1200 nano liquid chromatography system connected to a Thermo Scientific Q Exactive HF mass spectrometer equipped with a standard nano-electrospray source. LC solvents were A: 1% acetonitrile in water with 0.1% FA; B: 15% water in acetonitrile with 0.1% FA. The nonlinear LC gradient was 1–55% solvent B in 120 min followed by 55–90% B in 10 s, 90% B for 10 min, 90–1% B in 10 s, and 1% B for 5 min. A DIA method with one full range survey scan and 22 DIA windows was used.The shotgun mass spectrometric data were analyzed using Biognosys’ search engine SpectroMine™, the false discovery rate on peptide and protein level was set to 1%. A human UniProt FASTA database  was used for the search engine, allowing for two missed cleavages and variable modifications , carbamylation (KR)). The results were used for generation of a sample-specific spectral library.HRM mass spectrometric data were analyzed using Spectronaut™ 14 software (Biognosys). The false discovery rate (FDR) on peptide and protein levels was set to 1% and data were filtered using row-based extraction. The spectral library generated in this study was used for the analysis. The HRM measurements analyzed with Spectronaut™ were normalized using global normalization.p-values were corrected for overall FDR using the q-value approach [For testing of differential protein abundance, MS1 and MS2 protein intensity information was used . Proteinapproach . The folValidation of mass spectrometry results was performed using commercially available ELISA kits and following the manufacturers’ protocols . Before n = 11 prostate adenocarcinoma cases, including periurethral tumor manifestations if available, was selected and stained for specific antibodies  were performed with the GraphPad prism software, version 9. Continuous variables were expressed as box-plots (from the 25th to the 75th percentile and median), with whiskers representing the minimum and the maximum values. Statistical significance was calculated with the unpaired non-parametric Mann–Whitney U test.For the characterization of single biomarkers, ROC curve analysis was performed applying the Wilson/Brown method, whereas for combinatorial analysis of non-correlated proteins, a multiple logistic regression was applied. The correlation matrix was assessed with the Pearson correlation method.https://huygens.science.uva.nl/VolcaNoseR/, accessed on 8 September 2021).An online tool was used to draw volcano plots  fractions from all 45 urine samples. Two samples showed a significant contamination with albumin, which led to the suppression of other peptide signals, and were therefore excluded from further analysis (data not shown). We identified a total of 38.454 precursors (peptides including different charges and modifications), corresponding to 23.059 unique peptides and 2.768 proteins across all 43 urine samples by using a false discovery rate of 1% B.For the identification of candidate biomarkers to detect healthy men, we compared the abundance of 2.768 proteins in samples from patients not affected by tumor and those with PCa. Significantly dysregulated proteins were identified by setting the q-value below 0.05, at an average fold change of more than 1.75, resulting in 351 biomarker candidates . StrikinA key selection criterion for the best target molecules from the screening was the ability to discriminate healthy patients (with high specificity and accuracy), achieving a negligible number of false negatives (sensitivity > 90%). For this reason, all proteins that were not detected in more than three samples were excluded from further analysis. Additionally, proteins with low diagnostic performances, displaying a receiver operating characteristic (ROC) area under the curve (AUC) smaller than 0.670 and a specificity of less than 10% at 100% sensitivity, were removed. This ranking resulted in 43 biomarkers, with the top 25 candidates listed in p value was a borderline result in terms of statistical significance for two biomarkers. The ROC plots  and ribonuclease A family member 2 (RNASE2) and used them as controls for ELISA quantification of the single biomarkers . ConsistDetection of high grade PCa has a relevant clinical impact, as it allows differentiation between patients who would benefit from active surveillance and those who need active treatments. We therefore also tested the potential of our biomarkers to discriminate also PCa GS ≥ 7. The quantitative analysis by ELISA shows that the seven biomarkers can detect high-grade PCa with high performance .When different biomarkers are normalized by the same controls, as in this study, their combinatory power is hampered by a highly correlated dataset (data not shown), driven by the identical normalization strategy. Hence, combinatorial analysis was performed by multiple logistic regression with non-normalized ELISA data. In this study, we excluded from the nomogram any clinical and demographic information with potentially high variability among individual clinics and cohorts. Prostate volume and digital rectal examination (DRE), for example, are known to be affected by the type of instrument used or by personnel expertise. We therefore included only the age of the patients as clinical variable to improve the predictive models. The Pearson correlation analysis of all variables is shown in Taken together, our data demonstrate that ELISA quantification of the biomarker candidates selected by MS is feasible and confirms the high diagnostic performance of the analytes, both as single and in combination for the detection of all PCa grades and clinically significant tumors (GS ≥ 7).To investigate the possible origin of the biomarkers, we performed immunohistochemistry analysis on prostate tissues from 11 men  that underwent radical prostatectomy. Because it was not possible to analyze prostate tissue from healthy patients, the healthy tissue areas of the prostate were used as control for each patient who underwent prostatectomy. The stainings were performed on tissue blocks, including benign and malignant areas of the prostate to compare biomarker expression levels. In concordance with the MS and ELISA data, KRT13 staining showed a distinct expression in benign and low expression in malignant tissue areas . We obseDespite continuous improvements in the reduction of overdiagnosis and overtreatment of men suspected of having PCa, the number of healthy men that are subject to invasive procedures remains high ,21. ThisThus, the aim of this study was to identify novel urine biomarkers to improve the eligibility criteria for prostate biopsy and to more specifically discriminate PCa at an early stage, reducing the number of unnecessary biopsies. Here, we demonstrated the feasibility of diagnostic tests for the screening of PCa relying on urine biomarkers that can be routinely quantified by standardized laboratory methods such as ELISAs.Urine samples were collected from patients before performing the biopsy and subjected to proteomic screening by mass-spectrometry (MS) to select biomarker candidates that are dysregulated when a prostate tumor is present. Although MS results showed promising results, the application of mass-spectrometry for urine analysis as routine diagnostic test is not feasible, due to the lack of a standard method to compare different batches of samples. A more practical approach is the implementation of quantitative immune-assays such as ELISA, which represents the gold standard for biomarker assessment and validation . ConsequPEDF showed the best performance as a single biomarker, with AUC of 0.8023 and specificity of 36.4% at 100% sensitivity A,B. On tThe proteomic content of urine is affected by many factors, such as individual life-style, diet and time of sampling. For this reason, absolute biomarker data need to be normalized with a different strategy compared to MS, in which normalization is based on the overall cohort protein content. A relevant portion of the proteins identified in our study has already been described in other mass-spectrometry analyses of urine and to a lesser extent, in urinary extracellular vesicles, plasma or prostate tissue of patients. The seven biomarkers validated in our study were chosen exclusively based on their ability to predict PCa prior to biopsy and not considering their biological function. Nevertheless, some of them have been reported to be related to cancer. Although signal reduction in case of tumor progression as described for the seven biomarkers might be surprising, both literature and tissue analysis performed in this study support these findings. Hornerin (HRNR), a member of the fused-type S100 protein family, was shown to be expressed and to play a role in different tumor types ,25,26. Ohttp://www.cbs.dtu.dk/services/SecretomeP/, accessed on 5 October 2021). PEDF, HPX, CD99, and CANX are expressed with signal peptides and potentially traffic through the classical pathway (Golgi apparatus), whereas membrane protein FCER2 was predicted to traffic through a non-classical pathway. Conversely, KRT13 and HRNR do not appear to be secreted. This suggests that the proteins detected may be present in urine due to either the presence of cellular debris or particles deriving directly from the prostate or through blood filtration.To investigate the possible origin of the biomarkers and their route to the urine, we performed a sequence-based analysis, predicting secretion pathways of proteins with the SecretomeP 2.0 server  has to be considered . Since oIn conclusion, here, we demonstrated that an upfront urine test based solely on the quantification of novel biomarkers is a feasible approach to improve eligibility criteria for a prostate biopsy and to detect the presence of high-grade PCa, independent of serum PSA, digital rectal examination, and clinical variables. The clinical implementation of a simple urine test represents one possible and safe way to reduce the overdiagnosis and overtreatment of PCa. Furthermore, since it is completely non-invasive, it could potentially be used for disease monitoring and active surveillance.This study was submitted for patent application . The patent application was submitted to the European patent office (application number: EP 21/215742.4).


A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 1011–8.7 × 1013 cp/mL for the nominally full rAAV5 samples and 3.4 × 1011–7 × 1013 cp/mL for the nominally empty rAAV5 samples with 3–8% and 10–37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC–MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples.Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC–MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS Viruses and their derivatives have many applications in biomedicine, biotechnology, and nanotechnology ,2,3,4. IAdenoviruses, retroviruses, lentiviruses, adeno-associated viruses (AAVs), and other human and animal viruses have been utilized for the development of gene therapies over several decades . In receRecombinant adeno-associated viruses (rAAV) are a class of viral vectors that are being investigated intensively in the development of gene therapies ,15. In oThese growing requirements warrant fit-for-purpose analytics and extended characterization through orthogonal use of multiple technologies. Accurate quantification and characterization of rAAVs can accelerate the development and advancement of gene therapies. Addressing these challenges requires extensive characterization of rAAV samples with multiple assays including measurements of capsid count, full–empty capsid ratio, particle size, aggregate formation, stability, genome release, and capsid charge . TypicalTechnologies such as dynamic light scattering (DLS), multiangle dynamic light scattering (MADLS), electrophoretic light scattering (ELS), size-exclusion chromatography multiangle light scattering (SEC–MALS), nanoparticle tracking analysis (NTA), isothermal titration calorimetry (ITC), and differential scanning calorimetry (DSC) can provide important information on the key analytical and quality attributes of viral vectors, enabling the characterization, comparison, and optimization of various parameters. DLS, MADLS, SEC–MALS, NTA, ITC, and DSC are label-free biophysical techniques which require minimal assay development and can be readily applied at all stages, strengthening the analytical workflow for gene therapy development. In this study, recombinant adeno-associated viruses were characterized to achieve convergence of evidence from multiple techniques. For the purposes of the reader, the techniques used in this work are summarized here. Further information can be found in the references listed for each technique.Dynamic light scattering (DLS) is a noninvasive technique that measures the time-dependent fluctuations in the scattering intensity arising from a particle dispersion or molecular solution. The intensity of the scattered light fluctuates due to the random movement of the particles or molecule undergoing Brownian motion. Analysis of these intensity fluctuations using auto correlation determines the translational diffusion coefficients and, subsequently, the hydrodynamic size using the Stokes–Einstein relationship ,27,28,29Multiangle dynamic light scattering (MADLS) allows for a higher-resolution size determination of multimodal samples, by using three different detection angles  and combining the information obtained into one angle-independent particle size distribution. An extension of MADLS is particle concentration measurements giving the total particle concentration and the particle concentration for each mode present in a sample ,31,32,33Electrophoretic light scattering (ELS) measures the frequency shift of scattered light from particles or molecules undergoing electrophoresis and enables the measurement of zeta potential. The zeta potential of a particle is the overall charge that the particle acquires in a particular medium, and it can be used to predict the stability of the dispersion and provide insights into the surface chemistry of the particle being investigated ,35,36,37Size-exclusion chromatography (SEC) is a technique that separates molecules according to their hydrodynamic radius as they enter and exit the pores of a porous gel packing matrix in a column. A range of advanced detectors, such as light scattering, UV, RI, and viscosity, allows for the measurement of absolute molecular weight, molecular size, intrinsic viscosity, branching, and other parameters .Differential scanning calorimetry (DSC) is an analysis technique used to characterize the stability of a protein or other biomolecule directly in its native form without the need for extrinsic or intrinsic fluorophores. It does this by measuring the heat capacity change associated with thermally induced processes, such as the molecule’s thermal denaturation, triggered by heating at a constant rate ,40.This paper discusses and demonstrates the applicability and value of various orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples.13 viral genome per mL (vg/mL) as calculated by qPCR. The disperse phase of the samples was PBS containing 0.001% Pluronic F-68 . The 100 nm polystyrene latex sizing standards (part number 3100A) were obtained from Thermo Scientific . Milli-Q® filtered water was obtained from Milli-Q Direct Water Purification System . Commercial rAAV samples (rAAV5) were purchased as nominally empty and nominally full rAAV5 from Virovek . From information provided by the supplier, the rAAV vectors were purified through two rounds of CsCl-gradient ultracentrifugation followed by sterile filtration of the nominally empty and nominally full fractions. The rAAV samples were used as received following the manufacturer’s specifications for purity and titer. These AAV capsids are referred to as empty and full throughout the rest of the paper. The full rAAV5 contained pFB-GFP ssDNA which consisted of 2544 nucleotides with a known molecular weight (Mw) of 785,000 g/mol. The sample had a defined viral titer of 2.5 × 10Chromatographic separation of the rAAV samples was achieved using a Superose 6 increase (10/300) with an isocratic flow rate of 0.8 mL/min and phosphate-buffered saline as the mobile phase. An OMNISEC system  consisting of an OMNISEC RESOLVE  and an OMNISEC REVEAL  was used to acquire the sample chromatograms. The samples were maintained at 4 °C in the autosampler prior to injection. The column oven and detector module were maintained at a constant 30 °C during this work.Following separation, the samples were processed using a compositional analysis designed to determine the concentration and molecular weight of two distinct components within a sample. For the compositional analysis to work, it is necessary to know the refractive index increment (dn/dc) and extinction coefficient (dA/dc) of both components. In this case, the dn/dc of the capsid and the ssDNA is well known. The dA/dc for the capsid can be measured using OMNISEC, and the dA/dc for the ssDNA is calculated from the sequence.Capsid is the concentration of the capsid in mg/mL as calculated, NA is Avogadro’s number, Mwcapsid is the molecular weight (g/mol) of the capsid as calculated, ConcDNA is the concentration of the DNA in mg/mL as calculated, and MwSeq DNA is the molecular weight of the ssDNA from the sequence. Therefore, using these calculated particle concentrations, the percentage of full rAAV5 in a sample can be easily derived. The compositional analysis of SEC–MALS data in this study is based on a simplified model where capsids are treated as empty or filled only.Total particle (Equation (1)), full particle (Equation (2)), and empty particle (Equation (3)) concentration can all be obtained using the following equations:Dynamic light scattering (DLS) thermal ramp measurements were made with a Zetasizer Ultra  using an He–Ne laser at a wavelength of 633 nm and maximum power of 10 mW. Three repeat measurements of each sample were made at each temperature using backscatter detection and a low-volume quartz batch cuvette . The instrument settings were optimized automatically by means of the ZS XPLORER software .The thermal ramps covered a temperature range of 45 to 75 °C with 1 °C increments, with size measurements collected at every 1 °C increase and a particle concentration measurement taken every 5 °C.Multiangle dynamic light scattering (MADLS) particle concentration measurements were carried out using a Zetasizer Ultra  equipped with an He–Ne laser at a wavelength of 633 nm and maximum power of 10 mW. All experiments were performed at 25 °C using a sample volume of 20 µL and a low-volume quartz batch cuvette . The instrument settings were optimized automatically by means of the ZS XPLORER software . A series of sample dilutions in PBS with 0.001% Pluronic F-68 buffer were prepared gravimetrically and measured to determine viral titer linearity. Each result reported was the average of 10 repeat measurements.Determination of dispersant viscosity was made by doping a small aliquot (2 µL) of a 100 nm polystyrene latex sizing standard  into 1 mL of each sample, assuming the viscosity of water and back calculating the correct viscosity to obtain the correct size for the polystyrene latex sizing standard ,42. The The optical properties used for the measurements were derived empirically through orthogonal light scattering technology. The values used in this study were particle refractive indices of 1.45 (empty rAAVs) and 1.50 (full rAAVs) with an absorption of 0.001. The scattering intensity of the dispersant was measured to be 68 kilocounts per second (kcps) and used during particle concentration measurements.Electrophoretic light scattering (ELS) measurements of the rAAV samples were measured on a Zetasizer Ultra instrument  equipped with an He–Ne laser at a wavelength of 633 nm and maximum power of 10 mW. Measurements were performed using the diffusion barrier method (DBM) ,44,45,4613 particles/mL and 4.3 × 1013 particles/mL, respectively. Mean molecular weight of the rAAV5 viral capsid protein was estimated on the basis of the reported 1:1:10 molar ratio for VP1:VP2:VP3 capsid proteins to be 64,900 g/mol. The protein monomer-based molar concentrations of the empty and full rAAV5 samples were established as 3.56 and 4.3 μM, respectively [MicroCal PEAQ DSC automated  was used for analysis of the thermal stability of empty and full rAAV5 samples. For the analysis, rAAV5 samples and matching buffer solutions at 325 μL aliquots were loaded onto a 96-well plate, covered with a silicon seal, and placed into the PEAQ DSC plate stacker thermostatically regulated at 10 °C. The empty and full rAAV5 samples were used at concentration of 3.6 × 10ectively .v/v Decon 90 solutions following a SCAN cleaning procedure, replicating the scanning conditions used in the measurements and including a thorough rinse with Milli-Q® filtered water.The thermal scans were performed in the range from 20 °C to 110 °C at a scan rate of 60 °C/h. In between the sample measurements, the sample and the reference cells of the PEAQ DSC instrument were automatically cleaned with 10% onset, Tm and enthalpy of transition, ΔHmain tr.The data were analyzed with dedicated PEAQ DSC Analysis software . For analysis, sample thermograms were normalized for the sample concentration and corrected for the instrument baseline by subtraction of the corresponding buffer–buffer scan. Lastly, the sample thermograms were automatically baseline-corrected following extrapolation of pre- and post-transition baselines with a spline function. The resulting normalized and baseline-corrected DSC traces of the full and empty rAAV5 samples were analyzed for TThe multi-detection chromatogram for empty rAAV5 is shown in As labeled in The triple detection chromatogram for full rAAV5 is shown in Using the compositional analysis method, the data in The data obtained from OMNISEC can be compared with the particle titer from the Zetasizer Ultra  and showThe key CQA parameter which can be calculated by OMNISEC is the percentage of full rAAV5 in the sample. In this case, the monomer was found to be 78% full rAAV5 with 22% empty rAAV5. Importantly, this analysis method assumes that the sample is either full or empty, whereas it is not possible to account for partly filled or overfilled particles.To confirm the analysis of full rAAV5 samples, the previously analyzed full and empty samples were mixed in controlled ratios. The capabilities of OMNISEC for detailed characterization of rAAV samples were further evaluated in the context of a limited stability study, where the rAAV5 sample was subjected to thermal ramps between 25 °C and 80 °C using the Zetasizer Ultra in backscatter detection, which has high sensitivity to minor changes in the aggregation profile of the sample.The hydrodynamic sizes ,26,27,28The 45 °C condition was selected for a limited stress stability study, followed by high-resolution multiple-detection SEC. rAAV5 samples were incubated at 45 °C, and aliquots were taken at 2, 5, 10, and 15 min for measurement by SEC–MALS. The chromatograms  show a cThe results obtained from repeat MADLS measurements of neat concentrations of empty and full rAAV5 samples are shown in 2 dilution range.To mimic viral vector titers typically seen during development, MADLS size measurements were conducted on a broad range of dilutions for both full and empty rAAV5 samples. The results are summarized in R2 values of 0.992 and 0.996, respectively.The results of MALDS particle concentration measurements for various dilutions of the empty and full rAAV samples are summarized in 13 and 7.0 × 1013 particles/mL respectively) were consistent with the concentrations reported by Virovek  filled and empty rAAV5 samples The temperature-induced process observed from about 50 °C only in full rAAV5 sample and extending up to 75 °C for around 30 min. This can be tentatively attributed to the kinetically controlled ejection of ssDNA in response to changes in capsid structure and stability due to thermal stress.(II)The pretransition process, which was most pronounced in the empty rAAV5 sample.(III)m value specific to AAV5 serotype.The main transition, whereby cooperative rAAV5 capsid disassembly was characterized by the T(IV)The additional transition only detected for the full AAV5 sample and tentatively attributed to melting of ssDNA.On the basis of the differences between thermal stability parameters such as T and Tm2  and over and Tm2  can be iCapsid size and titer;Full/empty ratio and genome titer;Aggregate content;Fragment content;Thermal stability;Charge;Capsid uncoating.In this study, the capabilities of DLS, MADLS, and SEC–MALS for the analyses of rAAV5 samples in a broad range of viral concentrations (titers) and at different levels of genome loading and sample heterogeneity were evaluated. Linear ranges for rAAV5 size and titer determination were established and reported along with %CV. In addition, the applicability and value of orthogonal and complementary techniques, which are label-free assays not requiring AAV calibration standards, provide information on the following important analytical and quality attributes of viral vectors:The SEC–MALS method described in this manuscript is a first-principles technique. It relies on the use of multiple detectors in one and the same run and does not require the use of a combination of separate methods to report AAV viral genome titer, capsid titer, and empty/full ratio. AAV samples are analyzed in their native state. MADLS provides a simple and rapid way to measure capsid titer. Unlike the UV-based methods , it doesThese label-free biophysical techniques require minimal assay development and can be applied from the capsid design stage through development to formulation development and extended characterization of the drug substance and drug product, strengthening the analytical workflow of in vivo gene therapy development.


Daphnia magna reached 15%. Growth of Sinapis alba L. was predominantly stimulated (73%), and Desmodesmus subspicatus Chodat was exclusively stimulated, possibly due to soil alkalinity and fertiliser-related nutrients. The hypothesis of a higher contamination of urban botanical gardens compared to urban parks was confirmed. However, urban parks can face a greater risk of soil ecotoxicity, hypothetically due to decreased activity of soil organisms resulting from adverse soil conditions caused by active recreation. The results highlight the need for an increased focus on botanical and ornamental gardens when assessing and managing UGS as areas potentially more burdened with contamination.Though botanical gardens are an important and widely visited component of urban green spaces (UGS) worldwide, their pollution is rarely studied. The aim of this study was to assess botanical garden soil contamination and ecotoxicity and to evaluate whether urban botanical gardens are more contaminated than urban parks. Soil assessments showed serious contamination with Cd, Pb and Zn, emitted predominantly by traffic, agrochemicals and past construction and demolition waste. The discovery of hazardous historical ecological burden in the UGS calls for the necessity of detailed surveys of such areas. Despite prevailing moderate-to-heavy contamination, the soil was only slightly ecotoxic. Maximum immobilisation inhibition of  Urban soil pollution by anthropogenic activities is an important research topic ,2. ReducUrban green spaces (UGS) and their soils are an essential component of urban ecosystems ,11,12. TWhen assessing UGS pollution with PTEs, great attention is paid to public urban parks  assessment of soil contamination with PTEs  in the botanical garden, (2) assessment of the spatial distribution of PTEs, (3) assessment of the soil ecotoxicity and (4) comparison of the contamination of botanical and ornamental gardens to urban parks.The studied botanical garden (and arboretum) is located in Brno, Czech Republic, at an altitude of 220–250 m a. s. l. in an area with an average total precipitation of approximately 550 mm/year and average annual temperature of 8.4 °C. The soil is primarily clayey with a high CaO content or artificial batch formed after clay mining. The arboretum was founded in 1938 on two hectares and was extended to eleven hectares in 1967. Until then, the area had a predominantly agricultural use. The lower section of the garden was adjacent to the construction landfill, and demolition waste remained after the bombing of the city during World War II. The northern section was bordered by an allotment and an agricultural area. Currently, a university campus, a dormitory, a sports area, an unmanaged green space, roads and tram tracks surround the garden. The garden grounds include an administrative building with classrooms, greenhouses and water features. It serves as a purpose-built educational facility and is also used by students for recreation and relaxation.A total of 37 samples were collected from the botanical garden topsoil (0–5 cm), and two comparative samples were collected near the road and tram stops outside the garden. The samples were taken from the regular sampling network with a 5 m spacing . Three saqua regia mixture (prepared from subboiled ultrapure acids) in a high-pressure, high-temperature microwave digestion system SW-4 . The procedure followed ISO 11466:1995 [For trace element analysis, about 1 g of dry milled soil sample was digested in 466:1995 . The dig75As, 52Cr, 60Ni, 65Cu, 66Zn, 111Cd and 208Pb, were measured in O2 reaction gas (0.29 mL/min) and He collision gas (4 mL/min) modes. The forwarded RF power was 1550 W, with a carrier gas flow rate of 1.07 L/min and integration time per isotope was 0.3 s. A calibration range of 0–100 µg/L for all elements was prepared by mixing from a 1.000 g/L single standard stock solution  in a matrix of 2% HNO3 (ultrapure) and 0.35% HCl (ultrapure). The internal standard solution was prepared in a final concentration of 100 µg/L as a mixture of Bi, Ge, In, Li, Sc, Tb, Y. Quality control was performed using reference materials (I) SRM 1640a Trace Elements in Natural Water  for instrument settings and calibrations and during measurement for control of signal stability, and (II) QCM Metranal 33 Clay-loamy soil  with a certified content of elements leachable by the aqua regia used for control of the whole procedure . Elemental determination was performed using a triple quadrupole inductively coupled plasma mass spectrometer ICP-MS/MS . Selected isotopes For ecotoxicological characterisation, water extraction in a Reax 20 rotary shaker  was carried out using 10.0 g of dry sieved soil sample and 100 mL of ultrapure water  for 24 h at laboratory temperature. Extracts were subsequently centrifuged by Universal 320 R benchtop centrifuge  for 20 min at the rotation speed of 4000 rpm and then filtered through a 5 µm paper filter. The filtered aqueous extracts’ pH was measured on a laboratory pH/Conductometer Orion 4 Star with a glass combination electrode. The pH measurement was performed according to ISO 10523:2010 .® cube .Total organic carbon (TOC) was measured using a TOC analyser soli TOCgeo), which assesses soil contamination by comparing the current and expected pre-industrial PTE contents. The index is calculated as follows [geo classes are as follows [geo ≤ 0: uncontaminated; 0 < Igeo ≤ 1: uncontaminated to moderately contaminated; 1 < Igeo ≤ 2: moderately contaminated; 2 < Igeo ≤ 3: moderately to heavily contaminated; 3 < Igeo ≤ 4: heavily contaminated; 4 < Igeo ≤ 5: heavily to extremely contaminated; Igeo > 5: extremely contaminated.The soil contamination assessment was carried out by Geoaccumulation Index (I follows :(1)Igeo=ý et al. , who con follows : Igeo ≤ Daphnia magna, freshwater green alga Desmodesmus subspicatus Chodat and the seeds of Sinapis alba L. The tests were performed on aqueous extracts of soil samples to evaluate the effect of water-leachable substances from soils on the organisms because of the greater importance of the bioavailable fraction of PTEs compared to the total soil PTE contents for risk assessment [Soil toxicity was assessed using acute ecotoxicity tests on three selected organisms representing different trophic levels: water planktonic crustacean sessment .D. magna crustacean test carried out according to ISO 6341:2013 [The 341:2013  determinD. subspicatus was performed in microtiter serological plates according to ISO 8692:2012 [The test on the alga 692:2012 . AqueousS. alba seeds were performed according to the Methodical Instruction of the Ministry of the Environment of the Czech Republic [Tests on Republic . The tesA vector point layer was created with the attribute table containing information on As, Cd, Cr, Cu, Ni, Pb and Zn soil contents. These sampling points were arranged into a regular net with a grid size of 0.1 × 0.1 km. Interpolation data analyses were performed using QGIS Desktop 3.4.13 software. Spline ,41 was cgeo of PTEs. The strength of the linear relationship between the PTEs and pH and TOC was measured by Pearson’s correlation coefficient. The principal component analysis (PCA) was implemented for reducing the number of variables of PTEs in used datasets. The comparison between the botanical garden and the park was made using the Tukey Honest Significant Difference (Tukey HSD) test at p = 0.05.Additional data processing and advanced statistical analysis were carried out using the statistical program R version 4.0.2. . Multiplgeo in some PTEs. Although As content levels were categorised as ‘uncontaminated’, indicating low enrichment from coal combustion [The results of the soil contamination assessment in the botanical garden are presented in mbustion ,45, Ni, geo assessment was also presented in the PCA , despite its overall low level, led to the immobilisation of D. magna in more than 70% of cases as it is a highly toxic element to crustaceans [D. subspicatus  less phytotoxic than in the park. The garden soil’s phytotoxicity was found in only 27% of samples with a maximum inhibition value of 23% (S. alba growth (Although the botanical garden was significantly more contaminated with PTEs , its soie of 23% . In the a growth . Finallya growth  could liTrees are critical components of UGS and their protection ensuring that people gain the benefits trees provide should be priority . The absS. alba indicated only slight soil phytotoxicity. Though few studies exist with similar findings and results are often disregarded, the hypothesis of a higher contamination of botanical and ornamental gardens compared to urban parks was confirmed. Because its indications appear globally, more attention needs to be paid to urban botanical and ornamental gardens as UGS potentially more burdened with contamination. Soil ecotoxicity of the botanical garden was low and not as risky as in the neighbouring urban park. The reasons for this need a more detailed investigation. Though the botanical garden soil assessments indicated serious contamination with Cd, Pb and Zn, all PTE contents were below the soil quality standards. The sources of contaminants were predominantly from traffic, historically deposited construction and demolition waste and agrochemicals. The soil does not pose a severe ecotoxicological risk to aquatic organisms, although the content of Ni, as a potentially hazardous element for crustaceans, should be monitored. Prevailing growth stimulation of the