Several research studies were focused to understand how grapevine cultivars respond to environment; nevertheless, the biological mechanisms tuning this phenomenon need to be further deepened. Particularly, the molecular processes underlying the interplay between clones of the same cultivar and environment were poorly investigated. To address this issue, we analyzed the transcriptome of berries from three "Nebbiolo" clones grown in different vineyards, during two ripening seasons. RNA-sequencing data were implemented with analyses of candidate genes, secondary metabolites, and agronomical parameters. This multidisciplinary approach helped to dissect the complexity of clone × environment interactions, by identifying the molecular responses controlled by genotype, vineyard, phenological phase, or a combination of these factors. Transcripts associated to sugar signalling, anthocyanin biosynthesis, and transport were differently modulated among clones, according to changes in berry agronomical features. Conversely, genes involved in defense response, such as stilbene synthase genes, were significantly affected by vineyard, consistently with stilbenoid accumulation. Thus, besides at the cultivar level, clone-specific molecular responses also contribute to shape the agronomic features of grapes in different environments. This reveals a further level of complexity in the regulation of genotype × environment interactions that has to be considered for orienting viticultural practices aimed at enhancing the quality of grape productions. Vitis vinifera is one of the most sensitive species to environmental variations and it shows a broad array of adaptation strategies  associated to clones can contribute to intra-specific variability in grapevine . In partarieties , shows garieties  associatarieties .Based on the above, it is worth to explore whether the molecular differences existing among clones of the same cultivar may play a role in events of environmental adaptation involving tissue-specific changes in turn affecting fruit quality.http://whc.unesco.org/en/list/1390). In order to dissect the whole complexity of the clone × environment (C x E) interaction and to identify the hub molecular changes controlled by clone, vineyard, phenological phase, or a combination of them, a multidisciplinary approach was followed. To this aim, transcriptomic data resulting from high throughput sequencing were integrated by: i) analysis of candidate gene expression by real-time PCR; ii) quantification of key secondary metabolite contents; iii) assessment of agronomical parameters; and iv) monitoring of climatic conditions in each growing site during the two consecutive years.To elucidate this point, we analyzed the molecular and metabolic variability of berries of the three sequenced "Nebbiolo" clones collected at three ripening stages, over two vegetative seasons, in three vineyards of the Langhe territory , a worldwide renowned Italian viticultural site , and planted at a spacing of 0.9 m (within the row) × 2.4 m (between rows) in parallel and contiguous rows.Trials were conducted in two consecutive vegetative seasons (2013 and 2014) on plants belonging to three Flavescence Dorée and Bois Noir, according to Before carrying out the experiments, virus detection was conducted by serological (ELISA) and molecular , were measured on the musts at the moment of commercial harvest in each vineyard according to the methods proposed by the International Organization of Vine and Wine . Bulk soil samples were collected after harvesting the grapes at a depth of 30–40 cm under the vine canopy. To avoid differences in soil composition, anomalous areas in terms of appearance were excluded from the sampling procedure as well as those 5 m from the vineyard edges or close to the end of the row. Analyses of soil samples were performed at an external laboratory service. Both collection of soil samples and related chemical analyses were carried out according to official methods as described in the Italian Ministerial Decree D.M 13/09/1999 . Climatic data representative of each vineyard were also collected over the 2 years  at spring, cluster number and weight, yield (kg of grapes per plant) at harvest, and juice composition . Technological ripening parameters, such as TSS (°Brix), pH, and TA  and metabolic  analyses (see below) were taken from three randomized field plots, each constituted by seven vines per clone, at three stages during ripening: E-L31, E-L35, and E-L38, respectively, referring to berry pea size, véraison, and harvest , as defined by the modified Eichhorn and Lorenz (E-L) system . The thrTotal RNA was extracted using the Spectrum™ Plant Total RNA extraction kit (Sigma Aldrich) starting from 100 mg of deseeded berries, and total RNA yield was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Integrity of RNA samples was assessed on an RNA 6000 Nano Labchip using a Bioanalyzer 1000  prior to library preparation. Only samples showing a RIN (RNA Integrity Number) value higher than 8 were submitted to sequencing and quantitative expression analyses. The cDNA libraries  using TopHat v.2.0.14, and an average of 82.3% reads per sample were mapped . Transcripts showing no expression (FPKM = 0) overall were discarded as well as those with FPKM > 0 but randomly expressed in only one biological replicate per thesis. After this filtering step, 22,280 expressed transcripts were obtained in total. All transcripts were grouped into functional gene categories .http://folk.uio.no/ohammer/past). Heat maps of transcriptional profiles associated with specific functional categories were generated with TMeV 4.9 (http://mev.tm4.org), using as input the average expression value (FPKM) of the three biological replicates.A dedicated data mining process, detailed in Expression changes of target transcripts were profiled by quantitative real-time PCR (RT-qPCR), as reported in Ubiquitin (VvUBI) and Actin1 (VvACT1), used as internal controls. Gene-specific primers are listed in HSD post hoc test for separating means when ANOVA results were significant (P < 0.05). Significant differences of pairwise comparisons were assessed by the Student’s t-test.Both RNA samples from the berries taken in 2013, also used for sequencing process, and RNA samples from the berries taken in 2014 were analyzed. Briefly, RNA samples were treated with DNase  and first-strand cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit . Real-time PCR assays were performed in a CFX Connect Real-Time PCR system , using the SYBR Green  method for quantifying amplification results. Thermal cycling conditions were as follows: an initial denaturation phase at 95°C for 2 min, followed by 40 cycles at 95°C for 15 s and 60°C for 30 s. Specific annealing of primers was inspected on dissociation kinetics performed at the end of each PCR run. Identity of amplicons was checked by sequencing when needed. Transcript expression levels were quantified after normalization to two endogenous genes, The SPSS statistical software package  and the GraphPad Prism software  were used to run the statistical analyses above reported and elaborate figure charts, respectively.-1 ethanol, 5 g L-1 tartaric acid, 2 g L-1 Na2S2O5, 22 mL L-1 NaOH 1 N) and incubated at 30°C for 72 h to allow extraction of skin phenolic compounds, as reported by Concentrations of total flavonoids, total anthocyanins, and anthocyanin profiles were determined on three biological replicates (obtained from the pool of 200 berries per clone) of 30-berry skin each that were further divided into 3 groups of 10 berries used as technical replicates. For each 10-berry sample, the skins were manually separated from pulp and seeds, then immersed in a pH 3.2 ethanol buffer  was estimated on a further sample of 50 berries per each clone, randomly selected from the pool of 200 berries (see above). After extraction in the same pH 3.2 buffer, skins were homogenized by means of an Ultraturrax system  and centrifuged for 5 min at 3,000 g at 20°C. The total content of anthocyanins determined by spectrophotometry on the obtained supernatants was used to evaluate the rate of skin anthocyanin extractability during maceration, as detailed in After a concentration step onto Sep-Pak C18 cartridges  to separate anthocyanins from other polyphenols and sugar contaminants, the berry skin extracts were eluted with methanol and analyzed by liquid chromatography to determine anthocyanin profiles, according to a previously published method .trans-resveratrol, and ε-viniferin were quantified by liquid chromatography on three biological replicates per clone starting from 500 mg of powdered berry skins, according to the procedure described in Contents of abscisic acid, We first addressed our survey to investigate the whole transcriptome reprogramming events occurring during the first year of trial and associated with the response of "Nebbiolo" clones to either different environments or ripening phase. To this aim, a dedicated data mining statistical approach  was applThere were classes of genes whose activation or inhibition at a specific phase induced metabolic changes essential for fruit ripening , stage-sSTS genes were most activated  included transcripts significantly affected by the environment . Despitectivated , and thectivated . It is cAs the studied "Nebbiolo" clones are characterized by a few genetic variants , accordiTable 1Measurements of agronomical parameters, quantity and quality features of the studied ‘Nebbiolo’ clones  grown in three different vineyards of the Langhe area  in 2013. Data are means ± standard error (n = 21). Results of ANOVA analysis are detailed below the table.These results provide molecular support for the intra-varietal variability previously observed at agronomic and physiological levels only , sustainThere were 16 clusters of transcripts (approximately 11% of expressed genes) for which a combined effect of both environment and clone was observed. In the cell growth and development category, a separation of the samples based on the growing site was evident at E-L38 and at E-L35; however, a clone-based distinction was also noticed at E-L31, for CVT71 and CVT185, respectively, in V1 and V3 . PCA disAll sequencing results discussed above were then used as the source to target the analysis of candidate genes and metabolites involved in the most important biological pathways affected by the studied variables, also highlighting changes between the 2 years of trial. The obtained information was thus integrated with the set of agronomical data collected in parallel over the two vintages.VvHT1 (VIT_00s0181g00010), the first HT member characterized in grapevine and localized at the plasma membrane (VvHT2 (VIT_18s0001g05570) and VvHT6 (VIT_18s0122g00850), both encoding tonoplast-localized transporters specific of the berry cells  was overexpressed at the onset of ripening in both the experimental years, in line with the patterns of ABA concentrations , which significantly differed based on environmental conditions and clone , can infTable 2n = 21). Results of ANOVA analysis are detailed below the table.Measurements of agronomical parameters, quantity and quality features of the studied ‘Nebbiolo’ clones  grown in three different vineyards of the Langhe area  in 2014. Data are means ± standard error  and VvAOMT (VIT_01s0010g03510), evidenced transcriptional changes associated with specific clone-vineyard combinations  more abundant over the whole year 2014 than 2013 , around véraison  in 2013, there were more precipitations  than during véraison  in 2014 , encoding a tonoplast-localized transporter of glucosylated anthocyanins (VvAM1 (VIT_16s0050g00930), encoding a vacuolar ANTHOMATE transporter involved in the delivery of acylated anthocyanins (VvGST4 (VIT_04s0079g00690), encoding a glutathione S-transferase mediating anthocyanin transport into the vacuole  and transport  well supported what observed at the metabolite level in terms of anthocyanin partitioning. Moreover, the fact that expression changes of acyltransferase and anthocyanin transporter genes were only partially confirmed in 2014 , among the most expressed genes of this functional category  , already characterized in grapevine  were similar among the three field sites . This represented another example of C x E interaction and was in agreement with previous reports attesting that the production of defense compounds can vary in function of grapevine genotype, soil, and climate  can affect the physiological and agronomical aptitudes of grapevine through fine transcriptional reprogramming events mainly linked to carbohydrate and secondary metabolic changes.Accordingly, despite transcripts involved in the anthocyanin biosynthesis are prevalently controlled by ripening stage—as well as sugar transport- and ABA biosynthesis-associated genes—the transcriptional regulation of anthocyanin acylation and transport and the related patterns of anthocyanin accumulation and partitioning strictly depend on the clone physiological adaptation to a specific growing site and to climatic variations occurring along the season.Development and stress defense responses are also processes transcriptionally controlled based on genotype, environment or their combination. For instance, the "V2 environment"-dependent up-regulation of stilbene synthase-encoding genes is most likely the upstream trigger for the increase in resveratrol and viniferin contents at véraison during the first year of trial.All these findings point to a complex and interactive molecular machinery that contributes to shape the final quality features of "Nebbiolo" grapes at harvest, and that, on a broader scale, can offer valuable support to orient viticultural practices aimed at enhancing the quality of grape productions in light of growing site and clone choice.Raw RNA-sequencing data and normalized expression values are available at the Gene Expression Omnibus (GEO) database with accession number GSE116238.CP performed most of the experiments, analyzed and compared all data and wrote the article. PB performed plant material collection, RNA extraction, and complemented the writing. WC carried out the metabolic analyses and complemented the writing. EC, AM, and MR carried out the sequencing analysis and bioinformatic elaboration of data. MS and PZ developed and applied dedicated statistical analyses on sequencing data. IP helped with interpretation of bioinformatics and gene expression data and revised the manuscript. DC, IG, and FM performed sample collection, carried out the agronomical measurements, analyzed the corresponding data, and revised the manuscript. LN helped with the metabolic analyses and analyzed the corresponding data. MP and MD supported sequencing analyses and critically revised the manuscript. GG and IG conceived the project. GG supervised all the experiments and wrote the article. All authors read and approved the manuscript.This work was supported by the Nebbiolo Genomics project, financed by Fondazione Cassa di Risparmio di Cuneo, and a research grant from the Italian Ministry of Education, University and Research (FIR project "The epigenomic plasticity of grapevine in genotype per environment interactions" No. RBFR13GHC5 to I.P).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Enzymatically concentrated anchovy oil (concentrate) is known to be much less stable than unconcentrated anchovy oil. However, we previously showed that concentrate surprisingly forms more stable microcapsules, when produced by complex coacervation, than does unconcentrated anchovy oil. Here we investigate the mechanism of this unexpected stability. We also investigate whether or not incorporation of concentrate can be used as an additive to improve the stability of unconcentrated anchovy oil microcapsules. Results showed that microcap stability increased as the amount of added concentrate increased. Decreased emulsion droplet size, lower positively charged zeta potential, and higher surface hydrophobicity were observed in the oil/water (O/W) emulsion, with the incorporation of concentrate in the oil phase, compared with the unconcentrated anchovy oil O/W emulsion. Both the decreased zeta potential and the increased hydrophobicity of concentrate in the mixed oil phase may improve droplet agglomeration, leading to enhanced oxidative stability of the concentrate-containing microcapsules. Decreased repulsive forces between droplets result in a more compact structure, thicker outer shell, and smoother surface, resulting in enhanced oxidation stability of the concentrate-containing microcapsules. Omega-3 polyunsaturated fatty acids (PUFAs) are essential fatty acids and have been shown to be associated with important nutritional and biological functions, including inflammation, membrane lipid composition, signal transduction, cellular metabolism, and the regulation of gene expression ,2, with Microencapsulation techniques, with the susceptible compounds as ‘core’ material entrapped within a surrounding ‘wall’ material, can be employed to protect bioactive compounds against adverse reactions such as oxidation , nutritiThermomyces lanuginosus lipase (TL 100L) [We previously produced acylglycerol concentrates by selective partial hydrolysis using TL 100L)  and subsTL 100L) . SurprisTL 100L) . HoweverAccelerated oxidation using Rancimat to test lipid oxidative stability is a fast and reliable analytical method  with gooAs shown in In order to further study whether this enhancement in the oxidative stability of the microcapsules was as a result of the separation process where the concentrate was extracted from the anchovy oil hydrolysis, the same hydrolysis (lipase blank) and separation process was also carried out on unconcentrated anchovy oil and the separated lipids are referred to below as ‘refined anchovy oil’. The refined anchovy oil (RAO) was microencapsulated in the same encapsulation process and its OSI was found to be slightly lower compared to the unconcentrated anchovy oil microcapsule , suggesting the separation process of the lipids did not contribute to the observed enhanced OSI value of the microcapsules.p < 0.01) than those of the unconcentrated anchovy oil microcapsules at the same homogenization speed. The OSI value of unconcentrated anchovy oil microcapsules prepared using the O/W emulsion homogenized at 22,000 rpm dropped dramatically, possibly due to air entrapped during homogenization. Wang et al. (2015) also reported the entrapment of the air within the microencapsulation system compromised stability, as measured by OSI, for some microcapsule [During microencapsulation, it was observed that the droplet size of the concentrate O/W emulsion was significantly smaller than that of the unconcentrated anchovy oil O/W emulsion . In order to investigate if the enhanced oxidative stability of the concentrate microcapsule was caused by the decreased size of the O/W emulsion droplets, both unconcentrated anchovy oil and concentrate O/W emulsions were prepared in a homogenization range of 10,000 to 220,000 rpm for 15 min, followed by microencapsulation using complex coacervation under the same conditions. The OSI values of the final dried microcapsules are shown in The impact of added concentrate on the oxidative stability of the microencapsulated mixed oil phase was investigated. Concentrate was mixed with unconcentrated anchovy oil from 0–100% (w/w) (concentrate/mixed oil phase), followed by homogenization and dehydration under the same conditions. The OSI values of the produced microcapsules are shown in The lipid class and fatty acids profiles of unconcentrated anchovy oil and concentrate were compared. Lipid class was analyzed using capillary chromatography , with anWaraho et al.  reportedOur previous research has shown that the EPA content in the unconcentrated anchovy oil and concentrate were similar, while the DHA content in concentrate was found to be significantly higher . In the Results from the characterization of the O/W emulsion during microencapsulation, stabilized by gelatin before complex coacervation—including emulsion droplet size, zeta-potential, and surface hydrophobicity—are shown in Zeta-potential of the O/W emulsion decreased with an increase in the concentrate content in the mixed oil phase, from 6.19 ± 0.35 mV in anchovy oil emulsion to 4.67 ± 0.43 mV in concentrate emulsion, probably because of less adsorbed positively charged gelatin at the O/W interface and a smaller O/W droplet size. Recent research indicates that surfactants and polymers will impact the zeta potential significantly in fine particle colloidal suspensions, and accelerate particle aggregation decreasing the absolute value of the zeta potential . Zeta poHowever, the surface hydrophobicity of the O/W emulsion droplets significantly increased with an increase in the concentrate content in the mixed oil phase, from 176.11 ± 10.15 in anchovy oil emulsion, increasing to 338.38 ± 15.98 in concentrate emulsion. This decreased O/W emulsion droplet resulted in more overall droplet surface area and therefore a lower ratio of gelatin to hydrophobic droplet surface area, which may explain the change in surface hydrophobicity.The physicochemical properties of the prepared microcapsules were investigated. The surface oil content, total oil, encapsulation efficiency, and microcapsule droplet size are shown in Both the decreased zeta potential and the increased hydrophobicity of concentrate in the mixed oil phase may improve droplet agglomeration leading to enhanced oxidative stability of the microcapsules. Droplet size is an important factor, but since a small amount of concentrate (3.13%) has the largest impact on droplet size  but not Thermomyces lanuginosus lipase (TL 100L) was obtained from Novozymes Australia Pty. Ltd. . Antioxidant Duralox Blend AN 110XT were provided by Kalsec Incorporation . Transglutaminase (Activa® KS–LS) was purchased from Ajinomoto  and stored at 4 °C before use. All other chemicals used were analytical grade and purchased from Sigma-Aldrich Australia .Anchovy oil was provided by DSM Nutritional Products  and stored at 4 °C before use. Enzymatic anchovy acylglycerol concentrate (concentrate) was prepared according to a previous reported method ,15 with Twenty g of oil was mixed with 150 g  gelatin dispersion and this mixture was stirred using a mechanical stirrer at 1200 rpm for 5 min. The mixture was then emulsified at 15,000 rpm for 15 min using a homogenizer  to prepare the oil-in-water (O/W) emulsion for microencapsulation. Then 2 g O/W emulsions were withdrawn and diluted 20 times using Milli-Q water to avoid their gelation, the emulsion was stored at 4 °C as the stock emulsions before their droplet size distribution, zeta-potential, and surface hydrophobicity determination.Microcapsules of anchovy oil, anchovy oil concentrate, and their mixture were prepared using gelatin-sodium hexametaphosphate complex coacervates as shell material based on our previous study ,15. AntiAccelerated oxidation tests of microcapsule were carried out using a Rancimat  following a previously described method . 2 g mic2SO4 and dried at 120 °C for 30 min. Once the lipid classes have been identified using standards, spot representing each lipid class was scraped off after being visualized under UV, resuspended in methanol for 12 h and then further analyzed by Iatroscan and GC. Each lipid class was identified using the SIC-480 II software for multiple chromatogram processing. Lipid class was separated by TLC. 500 mg of oil sample was suspended in 5 mL of heptane and carefully spotted onto thin layer chromatographic plates  (Merck). Plates were developed in hexane/diethyl ether/acetic acid (60:17:0.2) for 22 min and spots were visualized after spraying the plate with 60% HThe lipid classes of the native anchovy oil and concentrate were analyzed by capillary chromatography with flame ionization detector  based on our established method ,33. The The stock emulsions were further diluted 5-fold and the average droplet size and zeta potential values of the O/W emulsion droplets were analyzed using a NanoZS Zetasizer . sF and F0 are fluorescence intensity of protein-ANS conjugate and ANS alone, respectively. The surface hydrophobicity was expressed as the initial slope of the plot of RFI versus protein concentration (mg/mL).The surface hydrophobicity of the emulsion was measured according to previously methods with some modifications ,36. StocThe physical properties of microcapsules, including moisture content, surface oil content, total oil content, encapsulation efficiency (EE), payload (PL), and encapsulation yield (EY), were determined based on reported methods .d (µm), volume weighted average diameter, d (µm) and dispersion index (Span) were calculated using Equations (2), (3), and (4), respectively.in is the number of the droplets with the same diameter; id is the droplet size. The d(0.1), d(0.5) and d(0.9) are the average droplet size values corresponding to the cumulative distribution at 10, 50, and 90%, respectively.The particle size distribution of the final microcapsule powder was determined using a Malvern Mastersizer 2000 . The volume surface average diameter, All the experiments were carried out in triplicates and the SPSS statistical package  was used for the analysis of variance (ANOVA) to test the significant difference between the mean values.In the current study, we investigated the stabilizing effect of MAG and DAG on anchovy oil containing microcapsules. Possible reasons for the enhanced oxidative stability of concentrate microcapsules were investigated. The concentrate separation process and the O/W emulsion droplet size under various homogenization speeds were found not to be the most significant factors in stability enhancement. The involvement of concentrate during homogenization, specifically, the possible embedment of the DAG and MAG in the complex coacervate shell, may be partially responsible for the significantly improved OSI of the concentrate microcapsules. Moreover, the smaller size, lowered zeta-potential and higher hydrophobicity of the O/W droplets with high levels of concentrate content in the mixed oil phase may improve droplet agglomeration packing, leading to enhanced oxidative stability of the microcapsules. This more density compacted internal structure appears to also enable the formation of a thicker outer shell, further improving microcapsule stability.


Total phenolic content (TPC), reducing power (RP), superoxide radical scavenging (RS), and thiobarbituric acid reactive substances (TBARS) production inhibition were measured in raw and denatured aqueous extracts from sprouts and wheatgrass of einkorn and emmer obtained at increasing salinity. Grains were incubated and kept at 0, 25, 50, and 100 mM NaCl until either sprout or wheatgrass stage. Additionally, a recovery treatment was included, in which sprouts obtained at 100 mM NaCl were then transferred at 0 mM NaCl until wheatgrass stage. All parameters  increased with sprouting and were highest in wheatgrass. Salinity increased all parameters, but the effect varied with NaCl concentration, genotype, developmental stage, and plant material processing (raw or denatured). Overall, given the delay and limitation of growth at high NaCl concentration, the best compromise appears to be the application of a moderate salinity (25 to 50 mM NaCl). In denatured extracts, TPC, RP, and RS slightly decreased, and TBARS was not affected, which means that antioxidant activity was mainly related to compounds other than enzymes and peptides, and thus it can be assumed to remain after digestion. Thus, supplementing the human diet with einkorn or emmer sprouts and wheatgrass can actually benefit health.  Sprouted grains and wheatgrass of cereals are more and more widespread in the human diet, with a steep increase in western countries. Compared to grains, sprouts and wheatgrass can be consumed raw in salads or used to extract juices without any kind of cooking process, which preserves the integrity of molecules having high nutritional value. Cereals grains are rich in vitamins (E and C), phytochemicals such as polyphenols   and caroIn cereals, as in other species, the phytochemical content increases during germination and initial seedling growth ,6. BeninPhenolics and other phytochemicals are secondary metabolites that plants produce to attract pollinators through flower pigmentation, and they also play a major role in defence against biotic or abiotic stresses. For example, salinity causes oxidative stress and excess of ROS , and plaIn all these works, the polyphenol content was measured on alcoholic or hydro-alcoholic extracts for obtaining the highest amount of extractable molecules. However cereal sprouts and wheatgrass are consumed as salads or juices , i.e., tFinally, in most works on sprouts, the antioxidant activity is measured on crude extracts, but this does not necessarily correspond to that available after digestion because the passage through the stomach and intestine may alter antioxidant components. On one hand, the use of a crude extract as an additive needs to be considered so that the sensory properties of the food product are not adversely affected . On the Therefore, the objectives of this research were to study the polyphenol content and antioxidant activity of raw and denatured aqueous extracts from sprouts and wheatgrass of einkorn and emmer obtained at increasing levels of salinity in the germination substrate. This in order to (i) support sprouting under salinity as a means to increase the nutritional value of sprouts; and (ii) individuate the nature of the compounds  implicated in the antioxidant activity of sprouts. Triticum dicoccum cv Zefiro (TDiZ), where two days delay was recorded for S100 as compared to S0  S100 reached the wheatgrass stage, only 11% succeeded in TDiZ S100; the rest of them remained small and weak, and actually stopped growing  of raw extracts increased with the developmental stage in both TMoM A and TDiOn average over NaCl treatments, TPC of sprouts was over three-fold compared to seeds, and that of wheatgrass was over three-fold compared to sprouts. Salinity increased TPC in both sprouts and wheatgrass. The maximum TPC in both species was recorded for S50 in sprouts  and S100 in wheatgrass (+72% in TMoM and +42% in TDiZ). However, in TDiZ, NaCl concentrations lower than 100 mM decreased TPC compared to S0. In both species, S100-R showed TPC lower than S100, but the value was much lower for TDiZ. In denatured extracts B,D, the The reducing power (RP) of raw extracts increased with the developmental stage in both TMoM A and TDiOn average over the NaCl concentrations, RP of sprouts was about four and five times higher than in seeds for TMoM and TDiZ, respectively, and RP of wheatgrass was four times higher than in sprouts for both species.Concerning salinity, a slight increase of RP in sprouts of both species was observed only for S50, while the highest RP in wheatgrass was recorded for S50 in TMoM and S100 in TDiZ. As for TPC, RP in TDiZ wheatgrass decreased at NaCl concentrations lower than 100 mM compared to the control. In both species, S100-R showed RP lower than S100. In denatured extracts B,D, RP vAs for TPC and RP, the radical scavenging (RS) of raw extracts increased passing from sound seeds to sprouts to wheatgrass for both species A,C.On average over salt treatments, RS of sprouts in TMoM was +61% than in sound seeds, and RS of wheatgrass was almost triple than in sprouts. In TDiZ, RS doubled passing from seeds to sprouts and doubled from sprouts to wheatgrass. Salinity had a dual effect depending on the developmental stage and NaCl treatment. In sprouts of both species, RS fluctuated from high values of S0 and S50 and low values of S25 and S100. In wheatgrass of TMoM, RS was lowest in the unsalted control and increased markedly in all the other treatments, whereas in wheatgrass of TDiZ, only the increase recorded for S100 was significant and a marked drop was recorded in S100-R.In denatured extracts, RS values were generally lower than in raw extracts for any correspondent NaCl treatment B,D, exceThiobarbituric acid reactive substances (TBARS) production inhibition in raw extracts for both species was slightly affected passing from sound seeds to sprouts, whereas it greatly increased from sprouts to wheatgrass  A,C. The effect of salinity did not follow a clear trend. In sprouts, TBARS production inhibition was highest for S25 in TMoM and S50 in TDiZ , but was almost null for S25 in TDiZ. In wheatgrass, TBARS production inhibition showed little variations across salt treatments. In both species, S100-R showed a significant decrease compared to S100. TBARS production inhibition was not significantly reduced by denaturation in TMoM B, exceptIn both species, increasing salinity slowed germination and growth and, in case of TDiZ wheatgrass, 100 mM NaCl severely compromised biomass yield (data not shown) because most individuals stopped growing . This suThe increase of TPC observed in raw extracts A,C passiThe increase in TPC complies with the need of seedlings to prevent damages during early growth . In factThe decrease of TPC after denaturation is in line with literature B,D. HoweThe increase in RP of raw extracts observed with sprouting until the wheatgrass stage confirms evidences in wheat , green aAs far as RS is concerned, the lack of effect of salinity in sprouts (compared to the important effect on TPC) suggests that, besides polyphenols, other compounds assume key functions in the management of oxidative stress and that salinity allows to increase these compounds as well A,C. On t–  harvested from Central Italy crops were incubated in plastic trays containing solutions with 0, 25, 50, and 100 mM NaCl  according to a completely randomized block design with four replicates (trays). Parallel germination tests were performed on Petri dishes (two replicates of 50 grains each) to determine percent germination and time to reach 50% of germinated seeds. Once sprouts were obtained, part of them were harvested and part were kept on growing until wheatgrass stage. Additionally, part of the sprouts from S100 were transferred to 0 NaCl until wheatgrass stage as recovery treatments (S100-R). In order to guarantee constant water availability and prevent anoxia, grains were positioned on filter paper laid over glass balls immersed in the solution contained into the trays. The trays were weighed individually at the beginning of the experiment and distilled water was added daily to restore initial weight and keep NaCl concentration of each treatment constant, in the assumption that changes in plant weight during sprouting were risible as compared to those due to water evaporation [−2 s−1 [Grains of einkorn , Folin–Ciocalteu reagent, hypoxanthine, xanthine-oxidase from bovine milk, and nitrotetrazolium blue chloride (NBT) were purchased from Sigma Chemical Co., St. Louis, MO, USA.2CO3 200 mg g−1 and 803 μL of distilled water. Each sample had its own control, in which Folin–Ciocalteu reagent was replaced with the same quantity of distilled water. The mixture was incubated at room temperature for 30 min and the absorbance was measured at 700 nm. The values were computed by utilizing the dose-response obtained with gallic acid as standard. The total phenolic content was measured according to Singleton and Rossi  with pho−1 potassium ferricyanide and incubated at 50 °C for 20 min. An equal volume of 10 mg g−1 trichloroacetic acid was added to the mixture, which was then centrifuged at 6000× g for 10 min. A portion of the upper layer was mixed with distilled water and 1 mg g−1 FeCl3 at a ratio of 1:1:2. The absorbance was measured at 700 nm. Increased absorbance of reaction mixture indicated increased reducing power.The total reducing power was measured using potassium ferricyanide as reagent, according to Yen and Chen . The ext2EDTA 15 mM in buffer , 62 μL of 0.6 mM NBT in buffer, 37 μL of 3 mM hypoxanthine in 50 mM KOH, 6.25 μL of extract for samples, or distilled water for blanks, and 181 μL of buffer. Two 150-μL samples of reacting mixture were placed in two wells of 96-well microplates . 25 μL of distilled water were added to one well, while 25 μL of xanthine oxidase solution (1 unit in 10 mL of buffer) were added to the other well, causing the enzymatic reaction beginning. Then the reaction mixture was immediately incubated at 25 °C, and the absorbance at 550 nm was determined every 1 min in the first 5 min and then every 5 min up to 60 min using a plate reader . The capability to scavenge oxygen superoxide was calculated as inhibition % of oxygen superoxide according to the following equation:0 is the absorbance of control.Measurement of superoxide radical scavenging activity was achieved according to Kirby and Schmidt . The rea4)2SO4 2 mM in distilled water, according to Koracevic et al. [−1 were added only to control samples to stop the reaction, and 200 μL of Fe-EDTA solution and 200 μL of H2O2 were added to all samples, which were incubated for 60 min at 37 °C. Then, 1000 μL of acetic acid 200 mg g−1 were added only to the treated sample, and 1000 μL of thiobarbituric acid (TBA) 10 mM were added to all samples, which were then incubated at 100 °C in a boiling bath for 10 min and cooled in an ice bath. The absorbance was measured at 532 nm. The capability to inhibit TBARS production was calculated as inhibition % of TBARS production, with the Equation (1).A standardized solution of Fe–EDTA was prepared freshly by mixing equal volumes of EDTA (ethylenediamine tetraacetic acid) 2 mM in sodium phosphate buffer  and Fe, in order to allow comparisons between stages  and NaCl treatments. Average values of triplicate determinations for n = 2 independent replicates ± standard error are depicted. Means were compared by using the Fisher’s Least Significant Difference (LSD) at p = 0.05. Since plant material water content increased with the developmental stage and decreased with salinity, all data were normalized for a dry matter content of 130 mg gResults demonstrate that aqueous extracts from sprouts and wheatgrass of einkorn cv. Monlis and emmer cv Zefiro were rich in polyphenols and other antioxidants. This provides these foods with high levels of reducing power, radical scavenging, and TBARS production inhibition. Salinity increased polyphenols and antioxidant activity in sprouts and wheatgrass of einkorn cv Monlis, and in sprouts of emmer cv Zefiro, but not in its wheatgrass, which suffered from salt stress. In both genotypes, the effect varied with the salinity level and stage of development. Overall, considering that a high NaCl concentration  may delay germination and delay or limit growth, we can conclude that a moderate salinity (25 to 50 mM NaCl) may represent the best compromise to maximize polyphenol and antioxidant yield in salt tolerant genotypes. A relevant antioxidant activity remained after denaturation, which means it was related to compounds other than enzymes and peptides, and thus it can be assumed to remain available after digestion. This supports our deduction that supplementing human diet with einkorn or emmer sprouts and wheatgrass can actually benefit health.


Workers wore inadequate personal protective equipment (PPE). Mercury levels in indoor air varied within the building, with a maximum of 207.4 μg/m3 at floor level on the crushing platform, approximately eightfold higher than the ACGIH threshold limit value of 25 μg/m3  biologic exposure index of 20.0 μg/g creatinine in a facility worker. Twenty-four–hour urine samples were not obtained because of potential contamination at the work site during urine collection. Workers who received a diagnosis of mercury exposure were referred to an occupational health clinic for further evaluation. All workers were asked to participate in a survey that included employment history, symptoms of mercury toxicity, PPE use, and medical and social history.On May 12, 2017, the Wisconsin Division of Public Health, Public Health Madison & Dane County, and the Occupational Safety and Health Administration (OSHA) conducted a facility site visit to assess the work environment, interview workers, and perform environmental monitoring. Workers were advised to be tested for mercury exposure, and spot urine testing was offered at the time of the site visit. A case of mercury exposure was defined as a urine spot mercury level above the ACGIH biologic exposure index of 20.0 The 6,000–square-foot lamp recycling facility consisted of a large storage area with offices and kitchenette at the front and a break room at the back. A processing area with a drum-type crusher was located on one side of the storage area, and a bay door opened from the outside into the storage area on the opposite side. Ambient air sampling of the facility was conducted using a Lumex RA-915+ mercury vapor analyzer . Because of the timing of the unannounced visit, sampling was conducted when the facility was not processing; the bay door was open during sampling. Mercury vapors were measured just above floor level to assess spilled mercury and phosphor powder and at breathing height (approximately 4–5 feet above floor level) to assess worker exposure levels. The processing platform was approximately 4-feet high. All areas of the facility were sampled, including the facility entrance, reception area, office, kitchenette, hallways, bathroom, lockers, break room, and processing floor.Potential for take-home mercury exposure was assessed by wipe-sampling workers’ vehicle foot pedals on June 20. All workers declined assessment of their homes for mercury contamination. Wisconsin Department of Natural Resources sampled water and fish from two nearby ponds on May 25 and June 19 to evaluate potential contamination from the facility.Seven persons worked at the facility, including the owner-manager and six persons who worked in processing, administration, or as drivers. Workers’ mean age was 35 years (range = 23–50 years), six of seven workers were male, and mean duration of employment at the facility was 2 years (range = 0–5 years). Five workers had worked at the facility for a previous owner who had been cited by OSHA for elevated air mercury levels and failure to use respirators after an investigation on September 2, 2016. Appropriate respirators with mercury vapor cartridges were provided to workers after that investigation.μg/g creatinine (range: >23.8–71.2 μg/g creatinine). Follow-up during June–September 2017 for three workers evaluated at an occupational health clinic and one evaluated at a primary care clinic included repeat spot urines and 24-hour urine collections , followed by memory loss, irritability, insomnia, headaches, and weakness (three of four). No worker reported difficulty walking. One worker reported tremor, and another reported muscle twitches.Two of three processing workers wore rubber gloves, respirators, goggles, and disposable coveralls only while processing; the third wore only cloth gloves. Only one worker wore booties. One worker said he only started wearing PPE within the past month. No workers changed clothes or shoes before leaving the facility. Three workers attended an occupational health appointment with a physician during the 5 months after the initial investigation. One patient had no physical findings of mercury toxicity, one had tremor of the hands and head, and one had tremor of the fingers and a Mini Mental Status Exam score of 27/30 . No prior Mini Mental Status Exam score was available for comparison.μg/m3 outside of processing areas, with differences of up to 1 μg/m3 between ground level and breathing height measurements; higher mercury levels at the ground were reported, compared with breathing height (μg/m3 at the entrance and reached a maximum of 207.4 μg/m3 on the floor of the processing platform and 99.7 μg/m3 at breathing level on the processing platform ramp.Spot air sampling found mercury vapor concentrations of 0.2–6.8 g height . Inside Wipe samples from the cars of two workers determined the presence of mercury, indicating a risk for take-home exposure. Samples of water and fish from two nearby ponds found mercury levels consistent with regional freshwater mercury levels.Workers at the lamp recycling facility were exposed to mercury in the air, had elevated urine mercury levels, and experienced signs and symptoms of mercury toxicity. Previous investigations have reported that 33% of mercury is released from bulbs in the first 8 hours after breakage (The risk for mercury exposure in the manufacturing of fluorescent lamps has been known since the first investigation of a fluorescent lamp manufacturer in 1965 reported elevated urine mercury levels among glass blowers who made and repaired lamps (In this investigation, environmental measurements likely underestimated workers’ exposure to mercury because processing was suspended during the site visit and the bay door was open during sampling. Although the spot environmental mercury vapor concentrations measured in this investigation cannot be directly compared with the time-weighted averages used in OSHA (The risk for mercury exposure from manufacture of fluorescent lights has been known for many years; risks for exposure from recycling are not well documented.An investigation of environmental contamination at a fluorescent light recycling facility in Wisconsin in 2017 found elevated mercury levels among five of seven workers and clinical signs of mercury toxicity in two. Use of personal protective equipment was inconsistent, and mercury levels for inside air exceeded recommended thresholds.Employers at fluorescent light recycling facilities need to implement control technology, housekeeping, and exposure monitoring, and provide recommended PPE and training to their workers to reduce mercury exposures at their facilities.


A multidisciplinary approach was used, combining skeletal biology, molecular anthropology and archaeobotany with the aim of reconstructing the osteobiography of the alum miners buried at the site. Since 1460, the area of the Tolfa Mountains was significant for the exploitation of alum which was used for a wide range of purposes in the Middle Ages, ranging from woven production to medical practice. A total of 70 individuals (63 adults and 7 juveniles) were studied. The sex ratio of the community indicated a higher prevalence of males with respect to females. Morphological examination indicated occupational musculoskeletal stress markers, which might reflect the specific phase of alum production that each individual was occupied in. Dietary reconstruction was primarily performed through carbon and nitrogen stable isotope analysis with integration of the results obtained by microscopic, genetic and GC-MS investigations on dental calculus. The diet was omnivorous, indicating a reliance on C3-terrestrial protein and evidence for limited C4 consumption by some individuals. Herbivores, such as sheep and cattle, appear to have contributed to the diet more than pigs and chickens. Consumption of Fagaceae and Poaceae species was predominant; moreover, indicators of Brassicaceae and milk and its derivatives were abundantly recurrent in the population, followed by plant oils and theophylline. Furthermore, the detection of pharmacological alkaloids indicated the knowledge and application of medicinal plants by the community. The novel use of multiple techniques based on cutting-edge technologies has provided a unique window on the lifestyles of individuals from one of the first Italian settlements of alum workers.This research presents an in-depth study of the skeletal remains collected from the archaeological site of Allumiere (15 The exploitation of alum in Italy started in 1460, when Giovanni di Castro, a commissioner of the Pontifical State, identified the presence of alunite, a mineral from which alum could be extracted, in the territory of the Tolfa Mountains  . Alum isExtraction and processing of the alum required the involvement of specialized manpower. The production cycle of alum, described in detail by historical sources , 3, begaLa Bianca, several excavation campaigns have unearthed a Medieval cemetery and a church named Cappella dei Minatori (In the area of Minatori . It has Minatori .in situ nails (SU 296). A chronological sequence of burials was indicated by earlier burials were being laid in a N-S orientation and subsequent later individuals buried on an E-W axis.Archaeologically, the stratigraphic sequence of the site is challenging to define, as is common in Medieval cemeteries. In the majority, of the taphonomic observations, the burials were made up of simple earthen graves with individuals wrapped in shrouds, with the exception of a wooden coffin identified through the presence of doble excelente de la Granada and an excelente of the kings Fernando de Aragon and Isabel de Castilla and a Portuguese cruzado of King Giovanni II d'Aviz. There was also a duchy from Rhodes. The dating of the coins ranged between 1464–1523. The variety of coins and their origins are an important proof of the wealth of commercial traffic linked to alum.Items of jewelry and coins were recovered with a few individuals. In SU 110, three silver coins were found close to the left hand; SU 135 was buried with a religious medal, portraying the Immaculate Conception and SU 245 had eleven gold and two silver coins close to the left hand. All these artifacts confirmed the dating of the cemetery; in particular, the silver coins of SU 110 were dated to 1476–1483 [The present research aims to reconstruct the osteobiography of these alum miners of the Tolfa Mountains through a multi-proxy approach, combining skeletal biology, molecular anthropology and archaeobotany. Osteological analysis allows the reconstruction of the biological features of the Medieval workers, while molecular and archeobotanical analyses are used to shed light on the dietary patterns and medicinal habits of the community of Allumiere.13C) and nitrogen (δ15N) stable isotope analysis to human and animal bone collagen. Stable isotope analysis of bone collagen represents an established technique to identify the main protein sources in the diet of archaeological populations. This allows us to investigate marine and terrestrial and C3 and C4 sources of protein in addition to the trophic position an individual is feeding at [There is a general lack of information about the dietary habits of the lower social classes in historical texts from the Medieval period. Therefore, to better understand the dietary pattern of the studied community, we applied carbon   in a graMinatori . The arcLa Bianca . The preservation index for the individuals was calculated following the method proposed by Walker et al. [Seventy individuals were analysed, both adults and non-adults collected from the cemetery area of r et al. . The ager et al. –27, in tr et al.  and in tr et al. –30. Secor et al. –32 and or et al.  were alsr et al. –36, and r et al. . Moreover et al. .Sex diagnosis was performed only on adult samples, as proposed by Acsàdi and Nemeskèri  and reviOsteometrics was applied following the methods and standards proposed by Martin and Saller  and BorrParticular attention was paid to evidence of osteoarthritis, which was scored according to the standards proposed by Buikstra and Ubelaker . These aStatistical analyses were carried out using the statistical software R (v. 3.4.1) for the Student’s t-test and chi square test with Yates correction.Hystrix cristata, 1 Cervus elaphus, 2 Equus asinus, 2 Felix catus, 1 Canis lupus 2 Sus domesticus, 1 Equus caballus and 2 unidentified carnivores) in order to conduct carbon and nitrogen stable isotope analysis. Human samples comprised of rib bones, whereas animal samples were taken from the various skeletal elements that were available.Collagen extraction was carried out on 68 human and 12 faunal skeletal remains from the archaeological cistern . The solution was then frozen at -80°C for four hours and then freeze-dried for two days. A simultaneous extraction on modern bovine bone was performed and used as reference control. Approximately 0.8–1.2 mg of collagen was weighed and analysed in duplicate by EA-IRMS on a Sercon GSL analyser coupled to a Sercon 20–22 Mass Spectrometer at the University of York. The analytical error, calculated from repeated measurements of each sample, an internal laboratory control (fish gelatine), and international standards, was <0.2‰ (1σ) for both δ13C and δ15N. International Atomic Energy Agency (IAEA) standards were N-2, and 600 for nitrogen and International Atomic Energy Agency IAEA-600 and Iso-Analytical R006 for carbon. Isotope data are reported as delta (δ) values relative to V-PDB (Vienna Pee Dee Belemnite) for carbon and AIR (Atmospheric air) for nitrogen. Carbon content (%C), nitrogen content (%N), protein yield, and C/N ratios were checked to monitor the diagenesis of bone [Once the mineral component of the bone removed, samples were rinsed three times with ddH of bone  and dete of bone  and van  of bone .Dental calculus study was performed on 35 individuals, who were the only ones presenting maxillary and mandibular bones and teeth. Each analysis was carried out on different aliquots of calculus. Sodium hypochlorite 5% and UV exposure were employed to treat tools and working surfaces to restrict contamination during the analysis. In addition, different laboratories were used to carry out the various phases of this work . DepositAll criteria and precautions for the study and analysis of ancient DNA (aDNA) were applied –71. For http://www.bio.uniroma2.it/biologia/laboratori/lab-antropologia/DNAantico/DNA_antico/Facilities.htm) which has all the facilities to minimize potential contamination with extant DNA [For each sample, different polymorphic regions of mitochondrial genome of several animal species were amplified (35 PCR cycles), using pairs of primers that only amplified for the target species . For ovitant DNA . In ordehttps://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) was used to confirm the identity of the sequences found in the dental calculus. The presence of fish DNA was detected by polyacrylamide gel, using a molecular weight marker (GelPilot 50bp Ladder Qiagen).To verify the amplifications, PCR products were separated by electrophoresis on 1.5% agarose/Tris-acetate EDTA gel and the amplicons were then purified by enzymatic digestion with HT ExoSAP-ITVR (Affymetrix) following the manufacturer’s procedure and sequenced using the same PCR primers by an ABI PRISM 3130 Genetic Analyzer , as reported in Gismondi et al. . The BLAhilum and maximum width anywhere along the perpendicular axis) by the SuperAmpelo 2.0 program. Taxonomic identification of pollens, phytoliths and starch granules was carried out by direct comparison with a modern experimental collection  or data from literature, including a pollen Atlas [A protocol based on Hardy et al.  method wen Atlas .This analysis was carried out in qualitative and non-quantitative terms, according to Gismondi et al. . Ten mg La Bianca. The preservation index was calculated for each skeleton (ranging from a minimum of 0 to a maximum of 93%), showing an average preservation value of 59.4%. Individuals were sorted into 9 age classes. The composition by sex and age at death is summarized in 2, p<0.05).The present series of Medieval samples consisted of 70 individuals, 63 adults and 7 non-adults, sampled from primary burials in the cemetery area of For adult individuals, the highest mortality falls between the young adult and adult classes. Data on the juveniles’ mortality rate data should be considered cautiously due to the small sample size.p-value = 3.17x10-7). Post-cranial indices revealed high skeletal robusticity and skeletal asymmetry in males. In particular, the right side seemed to have been subjected to a higher biomechanical stress than the left. The results obtained for all the indices are shown in Living stature was estimated on 43 individuals . The mean stature for males was 169.22 ± 6.26 cm, while females exhibited an average stature of 156.70 ± 5.96 cm. A Student’s t-test indicated a statistically significant difference between the sexes (ante-mortem compound fracture which affected both tibia and fibula. This trauma (The highest frequencies were recorded for degenerative diseases (86%), Schmörl’s nodules (75%), periostitis (32%), and fractures (27%). Degenerative diseases were considered, jointly with enthesal changes, able to associate each individual to the most plausible phase in alum production. Fractures were relatively common within the community. For example, SU 245 demonstrated an s trauma  caused tante-mortem fracture.The analysis of musculoskeletal stress markers was conducted on male individuals as historical evidence indicates ,3 that tOn the basis of the development of occupational stress markers, the individuals seemed to cluster in four groups, corresponding to the four different phases of alum production  . These dOsteoarthritis confirmed all previous suppositions about musculoskeletal stresses and alum working activity of the individuals. The excavation phase caused a marked enthesal development of shoulders, elbows, hips and feet; in the calcination the most involved functional groups are shoulder, elbow, hip and knee; the individuals involved in moistening exhibit a high degree of biomechanical stress that affected shoulders, hips, knee and feet, while those employed in lixiviation show a great muscle development in the shoulders, forearms, hip and foot.m. pectoralis major , m. deltoideus  and m. biceps brachii (flexion of the forearm on the arm) could be related to the excavation phase according to the upper limb’s movements involved in the use of the picks. Even if, as mentioned, all phases of alum production cause a high biomechanical stress on muscles, the movement related to each one as well as the functional groups involved are different, and the excavation was observed to be the most strenuous phase involving the strain on both the shoulder and the elbow [All the working phases are characterized by well-developed musculoskeletal markers on the shoulders and the hip however, the combination of other functional groups significantly diverges amongst the other clusters. For example, the skeletons that show high robusticity of he elbow , 3.quadriceps tendon, whereas in the calcination m. vastus medialis should appear more distinctly stressed. The combined results of musculoskeletal stress markers and degenerative diseases are provided in Furthermore, a high degree of biomechanical stress on the knee could be observed in both moistening and calcination phases. However, moistening should involve a higher robusticity of the Unfortunately, a clear relationship between musculoskeletal markers, osteoarthritis and performed working activity could be identified only for twenty-one individuals because the others exhibited musculoskeletal stress markers or osteoarthritis patterns that could made them suitable for more than one working phase.Isotopic data and quality indicators are presented in Equus caballus), were excluded from the analysis due to their C/N ratio being outside the satisfactory range. The remaining samples were acceptable according to criteria proposed by Ambrose and Norr [13C versus δ15N isotope values for both faunal and human samples.Ten humans  and one animal  ranged from -21.4‰ to -21.1‰ (mean -21.3 ± 0.1‰), while δ15N values ranged between 3.5‰ and 5.9‰ (mean 4.5 ± 1.3‰). Omnivores  possessed values from 21.8‰ to -20.6‰ (mean -21.0 ± 0.7‰) for δ13C and from 3.6‰ to 4.7‰ (mean 4.1 ± 0.6‰) for δ15N. The carnivore  data ranged from -20.8‰ to -19.5‰ (mean -20.1 ± 0.46‰) and from 7.5‰ to 8.5‰ (mean 7.8 ± 0.4‰) for δ15N. Isotope data for all animals are in a range typically associated with the consumption of C3 plants. In terms of nitrogen, herbivores have δ15N values within the range found for primary consumers in the Mediterranean area [15N values are similar to those of the herbivores, which indicates that at Allumiere they consumed similar dietary resources, i.e. mainly plants with little/no animal protein. The Hystrix cristata (porcupine) show isotope values that are of an essentially herbivorous and it seems that any occasional consumption of insects and small vertebrates [15N values between the Sus domesticus and the other herbivores analyzed. During the Middle Ages, in some areas (including Lazio) pigs were free to roam in the fallow land close to the city [The δean area . Omnivortebrates  did not tebrates –88 amongtebrates , whereasthe city , where tthe city . Our resthe city  is probaLa Bianca (Allumiere) possessed δ13C values between -20.6‰ and -17.1‰ (mean -19.2 ± 1‰) and δ15N values between 5.9‰ and 12.1‰ (mean 8.6 ± 1.4‰). Both nitrogen and carbon stable isotope values for humans showed a high variability, in particular two individuals (SU 245 and 317) have elevated δ15N values of 12.1‰ and 11.9‰ and one (SU 388) has a particularly low δ15N value of 5.9‰. Despite the variability in isotopic values, no statistically significant difference was observed, between different sexes or age at death. The majority of humans demonstrate an enrichment of 15N  in comparison to the animals  indicates that animal protein made a greater or lesser contribution to the diet of particular individuals. On average, the human-animal offset in is 3.5‰, which is at the lower end of the accepted 3–5‰ between trophic levels [15N values of upwards of 9–10‰, animal protein will have had a major input and for the most elevated values, fish may also have made a contribution. High δ13C values (> 18‰) for some individuals  indicate the consumption of C4 plants or marine fish consumption. However, the fact that these individuals also tend to have amongst the lowest δ15N values would suggest that the former is most likely.The human remains from  animals , reflectc levels  and spacc levels . Therefo13C values, mostly with northeastern Italian sites , Cosa from entral Italy and Montella (AV) from Campania in southern Italy. For northeastern and central Italy sites, the consumption of C4 or marine resources were interpreted to be responsible for enriched δ13C values at these sites [15N with the sites of Siena and Montella. However, only the site of Siena [15N values, although this could also be due to the small sample size (N = 19). These differences describe a heterogeneous dietary landscape of the Medieval Italian communities probably related to geographical position and chronology.Comparisons of Allumiere with other Medieval published data –99 were se sites –96. As rof Siena  showed hAnalysis of aDNA from dental calculus was carried out to attempt to determine which animals were relied upon and to identify the possibility of fish consumption. Successful results were achieved for 19 individuals out of a total of 35 .La Bianca community the majority of individuals seemed to consume sheep and cattle products with a minimal amount of fish, pig and chicken  and to morphotype III .Moreover, unidentified remains showing no distinctive features were also recorded. The highest percentage of individuals presented starch of morphotype IX (Fagaceae), followed by miners who consumed morphotype I, II, VI and VII (Poaceae). Particular attention was paid to SU 198 individual, whose dental calculus presented an aggregate containing 220 starch granules, essentially ascribable to morphotype VII  or food categories , consumed in life at least once by the community of La Bianca were extrapolated, associating the detected compounds as reported in literature and scientific food databases [Camellia sinensis (L.) Kuntze. The detection of other plant chemical compounds  suggested the key role of plants  in the miners’ diet. Moreover, evidence of pharmacologically active alkaloids  supported the knowledge and application of medicinal plants  Trevis., Artemisia dracunculus, Conium maculatum L. and Securinega suffruticosa  Rehd).GC-MS analysis produced results for 23 individuals. The analysis was repeated for each sample at least 3 times and, in all cases, it always showed similar profiles. In atabases –104. GenLa Bianca (Allumiere) is relative in date to one of the first recorded human settlements in the area aimed to alum production. Musculoskeletal stress markers, combined with degenerative disease patterns provided important results in the reconstruction of miners’ working activity. Morphological changes depend on repeated daily exercises that stimulate bone remodeling at the attachment sites, increasing blood flow as a consequence [Archaeological evidence suggests that the archaeological site of sequence –106. Howsequence .moistening, could last up to 60 days. The last step was the lixiviation during which the moistened solution was purified by mixing for 24 hours. Even if all four phases  markedly involved the shoulder girdle, the different scheme of enthesal changes and osteoarthritis pattern observed on the specimens seemed to differ in relation to the biomechanical stress caused by the different tasks; thus allowing the classification of the individuals in four main clusters, each one corresponding to the working phase they were probably most involved in. It is known that osteoarthritis may be a result of traumas or infections, certainly the biomechanical stress suffered from the joint represents one of its causative factors [vs the value of 32% found in Allumiere. Similarly, degenerative diseases affected only 16.7% and 22.3% of the skeletal remains, in the two coeval cases, compared to 86% of La Bianca community. The morphological examination demonstrated peculiar and unique features on the analyzed sample showing that it is also one of the few Medieval examples that allows a clear identification of the social status of its components. The combined analysis of enthesal changes and osteoarthritis led to interesting results however the exact relationship between morphological variations on bone tissue and the activity carried out should be considered as a plausible hypothesis. It is known that age at death could also play an important role in the morphology of enthesal changes and that the different studies in literature underline the difficulties in finding a real correlation between entheses and activity patterns [The present work represents a unique case study, providing evidence of a relationship between occupational stress markers of bone tissue and working activities . This od factors  even if  factors . These d factors  and Sant factors  showed spatterns –113.13C values also suggested a contribution of C4 plants to the diet, as confirmed by archaeobotanical microscopy evidence. Indeed, the use of C4 plants as food, such as millet, has been documented in Italy since the Bronze Age [Stable isotope data supported a diet based on terrestrial proteins. The δonze Age , and cononze Age .DNA analysis revealed the consumption of animal proteins deriving from sheep and cattle meat, whereas pig and chicken use seemed to be negligible. Historical evidence indicates that the consumption of chicken remained low in the Middle Ages, corroborating our results . Similar3 Poaceae caryopses . Acorns, being rich in proteins, unsaturated fat, carbohydrates, minerals and vitamins, represented a food with high energy value [Triticum sp. and Myristica fragrans (nutmeg). This Asian spice was also detected in the dental calculus of other two individuals, suggesting the use of this plant species in the studied community. Nutmeg trades have been documented in the Mediterranean area since the 6th century, although archaeological findings are rare. It was usually grounded and mixed with red wine or used, in medicine, for its anti-inflammatory properties [Olea europaea fruits or acorns [Camellia sinensis (the tea plant). To a lesser extent, the use of foods containing cholesterol , Artemisia dracunculus  [Stachys officinalis  [Ephedra sp. (bronchodilator) [The dietary evidence presented by GC-MS and starch analysis will not be quantifiable in relation to the amounts consumed, however they do give an indication of the types of foods ingested during life, particularly when considered at the population level . The micgy value –118. Indgy value . The higoperties –121. GC-r acorns , 122–123perties) , Rosaceaedative)  and Ephedilator) . The detdilator) .Conium maculatum (hemlock) known to be poisonous but used in low doses in the Middle Ages, along with extract of S. officinalis and fennel seeds, as remedy against epilepsy, muscle spasms, impotence, tubercular adenitis and bites from dogs infected with rabies [Sorbus domestica, a small tree of the Rosaceae family used in the past for its fruits, called sorbs, rich in vitamin C and individual SU 110 showed evidence for Securinega suffruticosa, an oriental plant containing secondary metabolites that act by stimulating the central nervous system [S. suffruticosa, C. sinensis and M. fragrans, typical of Asian areas, could be explained by the existence of commercial trades with Eastern regions or the presence, among the miners, of individuals with Eastern origin, which probably introduced these medicinal species in the community. This latter hypothesis is in line with archaeological and historical data on alum production, a process widely developed in Turkey and more in general in the Eastern Europe [Individual SU 176 indicated the consumption of h rabies . Individs system . The pecn Europe . It is nn Europe .15N values >3.5‰ than the maximum animal measurements which indicated the consumption of protein enriched in 15N, perhaps from aquatic sources [15N value could be also associated to breastfeeding in SU 317  [15N values in tissues [A. dracunculus, a plant documented to be an anesthetic and anti-inflammatory [This present combined approach allowed us to obtain a more detailed appreciation of the diet. In particular, two individuals SU 317 and SU 245 showed an enrichment of δ sources –132, but sources . Analysiars old)  and possars old) , 135–137 tissues . Furtherammatory . It is rIn conclusion, applying an innovative and original multidisciplinary approach, we present a detailed osteobiography of the first Italian community of alum miners. In detail, according to morphological features, we hypothesized the working task of each individual and reconstructed their dietary patterns. Furthermore, archeobotanical analysis demonstrated that miners used spices and herbs endemic of Asia, for therapeutic purposes, revealing their knowledge about Eastern traditional medicine.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableNeoplasia (NP), Infectious diseases , trauma and stress markers , degenerative pathologies ) and congenital disorders (CO), and inflammation (IF).(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


MEMS-based skin friction sensors are used to measure and validate skin friction and its distribution, and their advantages of small volume, high reliability, and low cost make them very important for vehicle design. Aiming at addressing the accuracy problem of skin friction measurements induced by existing errors of sensor fabrication and assembly, a novel fabrication technology based on visual alignment is presented. Sensor optimization, precise fabrication of key parts, micro-assembly based on visual alignment, prototype fabrication, static calibration and validation in a hypersonic wind tunnel are implemented. The fabrication and assembly precision of the sensor prototypes achieve the desired effect. The results indicate that the sensor prototypes have the characteristics of fast response, good stability and zero-return; the measurement ranges are 0–100 Pa, the resolution is 0.1 Pa, the repeatability accuracy and linearity are better than 1%, the repeatability accuracy in laminar flow conditions is better than 2% and it is almost 3% in turbulent flow conditions. The deviations between the measured skin friction coefficients and numerical solutions are almost 10% under turbulent flow conditions; whereas the deviations between the measured skin friction coefficients and the analytical values are large (even more than 100%) under laminar flow conditions. The error resources of direct skin friction measurement and their influence rules are systematically analyzed. Skin friction is an important physical quantity in aerodynamics, and especially in hypersonic flow fields. Direct skin friction measurements in hypersonic wind tunnels mainly depend on various conventional skin friction balances ,2,3,4,5.M = 4 with low sensitivity, poor temperature stability and large volume, thus it couldn’t be used to accurately measure skin friction and its distribution for vehicles. Besides, the larger normal loads in hypersonic flow fields present many difficulties to design and fabricate feasible MEMS skin friction sensors.There are many studies on MEMS skin friction sensors, including various comb capacitance types and piezoresistive types, which are mainly used for skin friction measurements in low wind tunnels. A MEMS skin friction sensor designed by Jiang et al. in 2001 , adoptedA novel MEMS skin friction sensor was developed by us ,9, whichAiming at addressing the demerit of the test accuracy induced by fabrication errors, a novel fabrication scheme based on visual alignment is proposed, which mainly involves the optimization of the sensor-head and interface circuit, precise fabrication, micro-assembly based on visual alignment, prototype fabrication, static calibration, validation and exploration of its dynamic calibration in a hypersonic wind tunnel.The sensor-head of our MEMS skin friction sensor is optimized with one pair of differential capacitances, and its interface circuit is accordingly optimized with one set of AD7747  sensing components to minimize its encapsulation volume and reduce existing fabrication errors. The optimized overall scheme and sensor-head structure are shown in θ of vibratory electrodes induced by skin friction moment is given by [Combining material mechanics and electrostatics theories, the structure of the sensor-head and its parameters are optimized. The angle displacement given by :(1)θ=TsKC of unparallel rectangle electrodes with the torsional angle θ between the glass base and the silicon microstructure is:According to conformal transformation theory , the difC and the measured skin friction wτ.It can be seen from Equation (2) that there exists good linearity between the differential capacitance output ΔC is 0–2 pF. The resolution is 0.05 Pa/Ff, and the initial value of each sensing capacitance is 9 pF. Combined with the designed sensor indexes and the sensitivity model, the sensor-head is optimized, and its main structural parameters are achieved, as shown in The measurement range of the MEMS skin friction sensor is designed as 0–100 Pa, and the relevant differential capacitance output ΔBecause the sensor-head is optimized as one pair of differential capacitances, its interface circuit is accordingly optimized with one set of AD7747 sensing components , as showThe interface circuit not only needs to detect the weak differential capacitances of the MEMS skin friction sensor, but also needs to consider the high precision connections between the sensor-head and package shell. Printed Circuit Board (PCB) technology for interface circuits can’t satisfy the demands of sensor micro-assembly with high precision , thus prThe MEMS skin friction sensor can be divided into five key parts, including a silicon microstructure with single-crystal silicon material, a glass base with Pyrex glass material, an interface circuit with ceramic material, and a floating element and package shell made with aluminum alloy material. These key parts will be fabricated separately, and then assembled. The silicon microstructure and glass base will be bonded as a silicon-glass microstructure to output sensing signals, where the MEMS technologies are adopted. Micro-strip circuit technology is adopted for the interface circuit, and precision finishing technologies are adopted for the floating element and package shell.Silicon-glass microstructures are fabricated via MEMS technologies, and they mainly involve photolithography with double-sided alignment and deep reactive ion etching (DRIE) for the silicon microstructure, chromium-gold metal deposition and electrode fabrication for the glass base, silicon-glass anode bonding and wheel scribing, and other MEMS technologies. Silicon microstructures are fabricated from a single-crystal silicon wafer of 4 inches in diameter and 400 μm thickness, and mainly involves oxidation, photolithography and DRIE, as shown in .The MEMS process technologies of the glass base mainly involve metal deposition and electrode etching, and the MEMS processes are shown in The fabricated silicon microstructures and glass bases are aligned via double-sided alignment technology and the alignment masks fabricated in advance, and they are bonded together via anode bonding technology to keep their rigid connection. The bonded microstructures are separated by the wheel scribing method to maintain their 10 µm geometrical precision. The initial capacitances and dimensions of the separated silicon-glass microstructure are tested and selected, as shown in The ceramic-based interface circuit adopts precise micro-strip circuit technologies, which mainly involve substrate cutting, double side metal spattering, plating metal layer thickening, photolithography and etching, as shown in Limited by the fabrication errors of floating elements and package shells and the manual assembly process, the sensor prototypes showed poor assembly and package precision; thus, the accuracy of skin friction measurements couldn’t be estimated . As a reStudies show that the accuracy of skin friction measurements is very closely related to the system errors of sensors and the flow characteristics of the measured wall . HoweverThe locating flexures are adopted to implement preliminary alignment with 100 μm precision for silicon-glass microstructure and ceramic-based interface circuit, and then the two parts are fixed via an air-cured epoxy resin.The circular hole in the geometric center of silicon microstructure and the locating axis of floating element are used to implement their alignment with 10 μm precision, and then the two parts are fixed via an air-cured epoxy resin. As a result, the sensor-head assembly is finished.The package pedestal is placed in the locating flexure, and the package cover is installed. Then, the circular hole in the upper surface of the package cover is recorded by the machine vision camera, and its center position is identified via a canny edge detection method with ±5 μm precision .Move away the package cover, suck and place the sensor-head in the locating flexure. Record and identify the center position of its test-head via the camera and canny edge detection, and calculate the deviation of the center position between the circular hole in the package cover and the test-head. Then, carry and drive the sensor-head to match the center position of the circular hole in the package cover with ±20 μm precision.Lead the wires between the pads of the sensor-head and the interface circuit via a spot-welder. Install and fix the package cover, then the sensor micro-assembly are finished. Sensor prototypes are shown in These key parts mainly include a silicon-glass microstructure, ceramic-based interface circuit, floating element, package pedestal and package cover. Aiming at meeting the requirements of assembly precision and the structural features of key parts, a novel micro-assembly method based on visual alignment and micro-operating assembly is presented. It mainly involves five procedures, including silicon-glass microstructure and interface circuit assembly, floating element and silicon-glass microstructure assembly, sensor-head and package pedestal assembly, wiring, and final encapsulation. The micro-assembly technological procedures are shown The gaps between the floating element and the package cover of sensor prototypes based on visual alignment are almost 100 ± 30 μm, the gaps of the sensor prototypes assembled by hand are almost 300–500 μm, and the comparison of the package effect for the two-stage sensor prototypes is also shown in Because the volume of the MEMS skin friction sensor and the measured skin friction are very small, the conventional hanging weight calibration method used for skin friction balances is no longer feasible ,4. Thus,wτ = ck× △C. Several sensor prototypes were statically calibrated, each sensor prototype has 10 calibrated values in the 0–100 Pa region, and each calibrated value is tested seven times, as shown in wτ and the differential capacitance outputs △C can be linearly fitted, the fitted results and the calibrated indexes of sensor prototypes are shown in Given the structural parameters and the mass of the sensor-head and the distance between the fixing position of the sensor prototypes and the rotation center of the rotary loading platform, the centrifugal force equivalent to skin friction sensed by floating element can be determined. The relationship between skin friction and the differential capacitance output will also be obtained , viz. τwThe static calibration results indicate that most sensor prototypes have a fast response, good stability and zero-return; their measurement ranges are 0–100 Pa, their resolution is 0.1 Pa, and the repeatability accuracy and the linearity are both better than 1%. Compared with the preliminary sensor prototypes described in , the calValidation of the sensor prototypes in a hypersonic wind tunnel mainly involves prototype screening and repeatability tests. On these bases, dynamic calibration tests aiming at determining the accuracy of skin friction measurements are explored.Φ 0.5 m hypersonic wind tunnel of CARDC, as shown in M∞ = 6, the angle of attack for the model is α = 0°, and the detailed flow field conditions are presented in A flat-plate model is adopted to implement the sensor prototype validation. The length of the flat-plate model is 350 mm, its width is 210 mm, and the material is 45# steel. The model is divided into an upper cover plate and a fuselage. Sensor prototypes are imbedded in the fuselage, and the space between prototypes must be large enough to guarantee that they display no disturbances with each other. The two sensors have a 150 mm space from the sharp leading-edge of the model, and the sensors are symmetrically distributed with a distance of 80 mm along the neutral plane of model. Sensor prototypes and test facility are fixed in the working section of the P0 = 1.0 MPa conditions. Then, validations in laminar flow and turbulent flow conditions are implemented by changing the Reynolds number of the flow field. The test facility is placed below the nozzle exit and kept far away from the free jet region to reduce the shock effect of hypersonic wind tunnel start-stop. The test facility is rapidly sent into flow field uniform area to implement validation tests when the flow field is stable, and the corresponding Schlieren photo is presented in 3# and 5# sensor prototypes are selected to be validated via screening tests under P0 = 0.5 MPa conditions, seven output curves for the 3# and 5# sensor prototypes are separately presented in The repeatability tests under laminar flow are implemented at −4–9.10 × 10−4 range with a deviation from the analytic value of 7.25 × 10−4 in the 22.21%–25.52% range, and the repeatability accuracy is 1.52%. For the 5# sensor prototype, the measured skin friction values are in the 15.43–15.91 Pa range, the relevant skin friction coefficients are in the 20.06 × 10−4–20.86 × 10−4 range with a deviation from the analytic value of 7.25 × 10−4 in the 176.69%–187.72% range, yet its repeatability accuracy is 2.59%.The results indicate that the sensor prototypes have the advantage of rapid response, good stability and return-to-zero, and the dynamic pressure values of repeatable tests are in the 7.57–7.87 kPa range. For the 3# sensor prototype, the measured skin friction values are in the 6.84–7.06 Pa range the relevant skin friction coefficients are in the 8.86 × 10P0 = 2.0 MPa and P0 = 2.5 MPa conditions. The dynamic pressures of repeated tests under 2.0 MPa conditions are in the 31.97–32.23 kPa range, and six output curves for the 3# and 5# sensor prototypes are separately presented in Five repeatability tests under turbulent flow and one shock resistance test are separately implemented under −4–14.36 × 10−4 range, with deviations from the numerical solution of 112.74 × 10−4 under turbulent flow conditions in the 6.99%–12.79% range. The skin friction coefficients gradually increased during the previous five tests. The reason might be that the test-head gradually bulges out of the measured wall due to uncured epoxy resin in the micro-assembly, yet the repeatability accuracy is 2.83% in the last three tests. The measured skin friction value is 54.97 Pa under 2.5 MPa conditions, the related skin friction coefficient is 13.70×10−4, and the deviation from the numerical solution (12.26 × 10−4) is 11.75%. For the 5# sensor prototype, the measured skin friction values are in the 31.97–32.24 Pa range, the relevant skin friction coefficients are in the 11.45 × 10−4–12.12 × 10−4 range with deviations in the 4.87%–10.13% range from the numerical solution (12.74 × 10−4), and the repeatability accuracy is 3.20%. The measured skin friction value is 42.19 Pa under 2.5 MPa conditions, the related skin friction coefficient is 10.52 × 10−4 with a deviation of 14.19% from the numerical solution of 12.26 × 10-4.For the 3# sensor prototype, the measured skin friction values are in the 35.52–46.30 Pa range, the relevant skin friction coefficients are in the 11.11 × 10In total, the results of the repeatability tests indicate that the sensor prototypes possess rapid responses, good stability and return-to-zero, limited by uncured epoxy resin in the micro-assembly, yet the repeatability accuracy is better than 2% under laminar flow conditions and almost 3% under turbulent flow conditions. The deviations between the measured skin friction coefficients and the numerical solutions are almost 10% under turbulent flow conditions, whereas the deviations are large (even more than 100%) under laminar flow conditions, the reason might be the unstudied perceptibility of direct skin friction measurement, which will be discussed next.M∞ = 6, and the angle of attack for flat-plate model is α = 0°. Dynamic calibrations in laminar flow and turbulent flow conditions are implemented by changing the Reynolds number of the flow field, and the critical unit Reynolds number is about 1.0 × 107.The 3# and 5# sensor prototypes were also selected to explore dynamic calibration when the Mach number is The varying dynamic pressure method, viz. varying the stable section total pressure of the wind tunnel, is adopted to implement dynamic calibrations under laminar flow and turbulent flow conditions. The total dynamic calibration pressures under laminar flow conditions are in the 0.2–0.6 MPa range; and in the 1.5–2.5 MPa range under turbulent flow conditions. The detailed flow field conditions are presented in Five dynamic calibration tests under laminar flow conditions are implemented under 0.2–0.6 MPa total pressure, and five output curves for the 3# and 5# sensor prototypes are separately presented in The results indicate the deviations between the measured skin friction coefficients and the analytic values under laminar flow conditions are in the 17.89%–155.22% range for the #3 sensor prototype. Similarly, the deviations between the measured skin friction coefficients and the analytic values are in the 69.91%–162.39% range for the #5 sensor prototype. In brief, the deviations between the measured skin friction coefficients and the analytic values of the two sensor prototypes are almost in the 100%–160% range under laminar flow conditions, and they rapidly increase with increasing total pressure.Three dynamic calibration tests under turbulent flow conditions are implemented under 1.5, 2.0 and 2.5 MPa total pressure conditions. Three output curves for the 3# and 5# sensor prototypes are separately presented in The results indicate the deviations between the measured skin friction coefficients and the numerical solutions are in the 11.75%–23.60% range under turbulent flow conditions for the #3 sensor prototype. Similarly, the deviations between the measured skin friction coefficients and the numerical solutions are in the 0.53%–10.11% range for the #5 sensor prototypes. In brief, the deviations between the measured skin friction coefficients and the numerical solutions of the two sensor prototypes are almost 10% under turbulent flow conditions.Dynamic calibration results indicate that the deviations between the measured skin friction coefficients and the analytical values under laminar flow conditions are much larger than those seen under turbulent flow conditions. On the one hand, the skin friction coefficients under laminar flow conditions are very small, and equivalent to the sensor prototypes’ system errors induced by perceptibility. On the other hand, the skin friction coefficients under turbulent flow conditions are several times larger than those under laminar flow conditions, and they are much larger than the system errors. Studies indicate that system errors are closely related to many factors, including the flow regime and flow velocity, the shapes and sizes of floating elements, gaps surrounding floating elements, misalignment errors (intrusive or recessive) between floating elements and the measured wall, pressures acting on the lip and surface of floating elements and environmental factors in wind tunnel running. All of these factors are simply analyzed as follows.δ of the viscous sublayers (y+ = 6) for low-speed, low-shear flows and high-speed, turbulent flows is typically in the order of O (10−4 m) or greater, MEMS sensors have small feature sizes on the order of O (10−6–10−5 m).Thus, the gaps and the protrusions will typically fall within the viscous sublayers, and the flow is considered as being “hydraulically smooth” and the effect on the accuracy of skin friction measurements is negligible. However, the height δ of the viscous sublayers for high-speed, high-shear laminar flows  can be on the order of O (10−6 m) or even smaller, resulting in sensor features not being considered as hydraulically smooth, and this will greatly affect the accuracy of skin friction measurements. Besides, the main relative factors of flow regime and velocity for the flow field of wind tunnel are Reynolds number and Mach number; however, O’Donnell et al. [Studies indicate , that thl et al.  experimeDifferent shapes and sizes of floating elements will cause obvious flow disturbances to the flow near the measured wall, and also affect the accuracy of skin friction measurements. Four floating elements with different shapes and sizes are adopted to study the influence rule of the accuracy of skin friction measurements , thus flMisalignment errors between the floating elements and the measured wall mainly involve intrusive and recessive areas. Experiments are adopted to study the influence of misalignment errors on the accuracy of skin friction measurements by Meritt et al. . Resultsδ of the viscous sublayers for high-speed, high-shear laminar flows are on the order of O (10−6 m) or even smaller, resulting in the gaps surrounding the floating elements not being considered as hydraulically smooth, the accuracy of skin friction measurements is greatly affected. Under the same intrusive or recessive conditions, experiments are adopted to study the influence rule of the gaps surrounding floating elements on the accuracy [Because the height accuracy . Resultsaccuracy . AccordiZ = 0, the pressure surrounding the floating element is nearly constant without any influence on skin friction measurement. The pressure distribution will engender sharp variations when misalignment errors exist. When the floating element is recessive (Z/δ = −0.006), the lip pressure is sharp downstream around its trailing edge, and this pressure imbalance will produce lip-force opposing the direction of skin friction, thus reduce the output of the sensor. The influences are opposite for the intrusive (Z/δ = +0.018) test case and result in a large lip pressure at the leading edge of floating element. In short, recessive areas will bring negative lip-force contribution, and intrusive ones will bring positive lip-force contribution. Furthermore, the lip-force contribution is more significant when the gaps are smaller.Experiments indicate , that whThe output curves of sensor prototypes have already been analyzed in detail . The resFirstly, the sensor-head is optimized with one pair of differential capacitances; the interface circuit is accordingly optimized, and micro-strip circuit technology is adopted to implement its fabrication with miniaturization and high precision.Secondly, the MEMS skin friction sensor is divided into several parts. Their MEMS technologies and precision machining technologies are studied. The sample parts are fabricated with high precision. Micro-assembly based on visual alignment is adopted to fabricate the sensor prototypes. The precision of fabrication and assembly for sensor prototypes achieve the desired effect.Next, sensor prototypes are statically calibrated via the centrifugal force equivalence method, the measurement ranges are in 0–100 Pa, the resolutions are 0.1 Pa, and the repeatability accuracy and the linearity are better than 1%. In comparison, the calibration coefficients are greatly increased, and the repeatability accuracy and linearity are also improved.Finally, the sensor prototypes are validated in a hypersonic wind tunnel. Screening tests, repeatability tests and dynamic calibrations are implemented. The results indicate that sensor prototypes have the advantages of rapid response, good stability and return-to-zero in hypersonic flow; the repeatability is better than 2% under laminar flow conditions and almost 3% under turbulent flow conditions; the deviations between the measured skin friction coefficients and the numerical solutions are almost 10% under turbulent flow conditions, whereas the deviations are large (even more than 100%) under laminar flow conditions. Besides, the shapes of floating elements, the gaps surrounding floating elements, misalignment errors, lip-force contributions and other error sources of direct skin friction measurements under hypersonic flow are systematically analyzed, and their influence rules will be studied in depth to improve the accuracy of skin friction measurements under laminar flow conditions in the future.A novel fabrication method based on visual alignment for MEMS skin friction sensors is presented, and it mainly involves sensor optimization, precise fabrication of key parts, and sensor prototype micro-assembly based on visual alignment. On these bases, static calibrations and validations in a hypersonic wind tunnel are implemented. The main conclusions are: