Generally, alpha-1 antitrypsin deficiency (AATD) is suspected in young patients with pulmonary emphysema or chronic obstructive pulmonary disease (COPD). Patients often suffer from diagnostic gaps and are misdiagnosed with chronic obstructive pulmonary disease (COPD), asthma, and airway hyperresponsiveness (AHR), as AATD may present with nonspecific respiratory symptoms. It is never too late to suspect AATD, especially in a patient with an unusual medical history. In recent years, evidence is beginning to emerge that there may be value in identifying and treating patients who do not already have deterioration of functional parameters.1) between 30 and 65%, the patient was treated with alpha-1 antitrypsin (AAT) augmentation therapy and achieved clinical and functional improvement.We describe a case of a 69-year-old Caucasian female patient, late diagnosis of AATD, with both severe bronchial hyperreactivity and numerous exacerbations due to the peculiar clinical history and the presence of a rare mutation; although not presenting forced expiratory volume in 1 second (FEV1 between 30 and 65%. It may be useful to evaluate and treat patients based on clinical symptoms, even outside the established parameters, in particular cases.AATD should always be suspected. The Alpha-1 Foundation recommendations for the diagnosis and management of AATD in adult patients indicate that treatment should be provided for patients with FEV AATD is a rare lung disease, with a progressive clinical course, and early diagnosis enables patients to make lifestyle changes and initiate therapy to decelerate or prevent further loss of lung tissue. However, AATD goes largely unrecognized; evidence from screening programs in the United States shows that less than 10% of individuals with AATD are clinically diagnosed with the disorder [ionality . Emphysepatients . TherapeWe describe the case of a 69-year-old Caucasian female patient, housewife and mother of three daughters, in apparent good health, who had never smoked. The patient reported six episodes per year of exacerbation of respiratory failure documented over the previous 10 years. Respiratory symptoms were resolved with steroids and antibiotics. The patient had been hospitalized twice in the last 3 years for acute respiratory failure. She was discharged with a diagnosis of "acute respiratory failure exacerbated by COPD.”Due to persistence of respiratory symptoms characterized by dyspnea and a dry cough, the patient visited the pulmonary clinic of our department, and a functional assessment was carried out. At a young age, she reported occasional wheezing, sporadic episodes of respiratory exacerbation, especially during the three pregnancies, and intense dyspnea and asthma-like symptoms. She had undergone spirometry several years earlier, for which no written documentation was generated. The patient was being treated with an inhaled corticosteroid and long-acting β2-agonist twice daily, long-acting muscarinic antagonist twice daily, theophylline 200 mg orally twice a day, and montelukast 10 mg per day orally.1 1.64 (113%) and diffusing capacity for carbon monoxide (DLCO) of 4.11 mmol/(minute KPa), 80%. Spirometry performed at the second visit showed FVC 1.95 (100%), FEV1 1.44 (100%), Tiffeneau index of 75% and DLCO 4.11 mmol/(minute KPa), 80%. The six-minute walk test was normal. She was subjected to a methacholine challenge test, which was positive for severe hyperreactivity. Allergic skin tests for inhalants and foods were negative. A paper radioimmunosorbent test (PRIST) showed a level of 39 U/mL. The blood tests were normal except for serum alpha-1 antitrypsin (AAT) of 67 mg/L . Chest computed tomography scan revealed the presence of small areas of air trapping and diffuse bronchiectasis  of 2.17 (120%), FEVLowell.Genetic screening was performed for the AATD inheritance and showed compound heterozygosity for deficient alleles pis and PiAfter informed consent was obtained from the patient, we started intravenous augmentation therapy with human AAT at a dosage of 60 mg/kg every 7 days. At the request of the patient and her family, and after having monitored serum AATD, therapy was performed every 14 days. At 20 months of treatment, the patient showed no exacerbation, dyspnea exertional resolution and stable improvement in respiratory parameters.After subsequent revaluation diagnostics, this patient, with a rare double heterozygous mutation, was treated with augmentation therapy, obtaining clinical and functional improvement without exacerbation over 2 years of treatment.1 [A review by Eden  examined1 , 10.Lowell mutation. The evolution of lung disease in people with AATD would appear to be aggravated by the presence of gene interactions that are not yet clearly understood. Presumably, atopy, modifier genes and extrinsic influences, environmental factors and cigarette smoking work together to increase airway hyperreactivity and asthma, contribute to the decline of lung function in AATD, and play a role in exacerbation [For over 10 years, the patient had respiratory symptoms variously diagnosed as episodes of acute bronchitis or chronic bronchitis. There were no professional or lifestyle risk factors, and the patient had never smoked; thus AATD was never suspected. However, despite maximizing drug treatment, the clinical conditions worsened, with an increase in the frequency of episodes of bronchial exacerbation. In the most recent few months, exertional dyspnea had arisen, but the basal functional evaluation and allergology tests carried out on the occasion of different re-evaluations determined nothing pathological. At the time of our first evaluation, the finding of lower than normal serum AAT level necessitated a genetic study documenting the heterozygous presence of a pis and Pierbation .It is interesting to observe that in our case, the patient at the second evaluation showed a significant reduction in respiratory function compared to her previous parameters, while remaining in the normal range of respiratory function. During the months of augmentation therapy, our patient did not show any bronchial exacerbation; the dyspnea index was reduced as assessed by the six-minute walk test and modified Medical Research Council (mMRC) scale Table . The rolThese data strongly support a recommendation to always suspect AATD in the presence of respiratory symptoms at any age, and above all to promote greater awareness of this genetic condition, which is still under-recognized more than 50 years after its discovery. The exclusion of a genetic component linked to AAT is indicated in all patients with respiratory symptoms, asthma or COPD, and it is important to evaluate each case for the possible indication and potential advantages of AAT augmentation therapy.1 of 30–65% [The latest Alpha-1 Foundation recommendations for the diagnosis and management of AATD in adult patients supports replacement therapy in individuals with FEVf 30–65% , but emef 30–65% . Data on


Here, we report a unique optical photoluminescence (PL) characteristic of native, white PA2200, in which it yields a persistent, phosphorescence-type emission. An analysis of luminescence imaging data with emission measurements demonstrated that the anatase phase of the titanium dioxide additive is the source of the persistent PL properties. This characteristic of PA2200 enables advanced optical imaging applications, as demonstrated by luminescence imaging of an anatomical rat skeleton and a novel Derenzo-type phantom on a commercial image station. In summary, the light emission properties of PA2200 induced by the presence of anatase titanium dioxide open the door to a vast new array of complex optical applications, including the generation of imaging phantoms for training, calibration, and quality control.Selective laser sintering (SLS) is a prominent 3D printing modality that typically uses a polyamide (PA) powder as the substrate. One commercially available SLS material is known as PA2200, which is comprised of nylon 12 and titanium dioxide (TiO Unlike traditional methods of fabrication where material is removed (reductive manufacturing) or fit into a predefined space (molding), 3D printing is an additive process in which material is deposited with high spatial resolution in layers that are built up vertically. A myriad of 3D-printing techniques exists, which are generally classified by the way the material is delivered and bonded. Common examples include stereolithography (SL) and PolyJet printing, which use light to polymerize a liquid resin; fused deposition modeling (FDM), where molten filament is extruded and hardens in place via cooling; and sintering, where a laser selectively traces and fuses layers of powder resin (SLS) . In part2 is incorporated into PA2200 to improve its flowability and absorption properties [Titanium dioxide is a white powder that has widespread use as a pigment in dyes due to its brightness, high refractive index, and resistance to discoloration . In the operties ,6. The goperties . As a pioperties . Additiooperties . In natuoperties . Both ruoperties .Optical imaging techniques that make use of light in the visible to near-infrared window have been used for a variety of research and clinical applications. In the pre-clinical research setting, bioluminescence (BLI) and fluorescence (FLI) imaging modalities have been developed to non-invasively monitor disease state progression and/or small molecule biodistribution in living mice ,9. In thHere, we present the unique photoluminescence (PL) properties of PA2200, which is comprised of polyamide-12 doped with titanium dioxide to yield a white SLS substrate. PL acquisitions were gathered to determine if PA2200 would photoluminesce with delayed emission of optical wavelength photons after “charging” with white light. The sustained emission of the material emulates a typical bioluminescence imaging (BLI) signal that is obtained by integrating several minutes of exposure on a charge-coupled device (CCD) chip, thus making PA2200 suitable for phantoms with this imaging modality. PA2200 displayed sufficient PL to detect with mainstream imaging equipment, and upon further analysis, it demonstrated very unique emission in the near infrared zone (≈800 nm). This PL signal was discovered to be long lasting, demonstrating ultra-long persistent luminescence on the order of 10s of minutes. To further showcase the breadth of capabilities of this material, a complex anatomical phantom was 3D printed out of PA2200 and photoluminescently imaged as a potential training and/or quality control aid. A Derenzo-type phantom, a common characterization and calibration phantom type in nuclear medicine imaging , was alsIn summary, the optical properties of PA2200 described here, combined with its robust physical properties and mainstream use for 3D printing applications, make it a tremendously valuable material that can be utilized to create complex calibration and training phantoms for optical imaging devices in the lab and clinic.Three-dimensional (3D) printed objects comprised of native PA2200 were obtained via a third party, Shapeways . Objects were exported as an “.stl” file, uploaded to Shapeways, and 3D printed using the SLS nylon (referred to as “versatile plastic”) in the polished white color. Shapeways, a private corporation, does not publicly share their printing parameters.To generate a bulk sample of PA2200 for testing, a square tube with 10 mm × 10 mm base and 40 mm length was created using the SolidWorks Professional software  and printed as above. To showcase the training applications for PA2200 within optical imaging, a 3D-printed rat skeleton was produced as previously reported .To demonstrate the ability of PA2200 as an optical imaging calibration phantom, a “Derenzo-style” phantom was created and manufactured. Using SolidWorks, a solid circular object was designed with six triangular subsections. Each subsection consisted of uniform, equidistant holes, ranging in diameter from 0.4 to 2.4 mm. The circular phantom was milled out of a flat sheet of black polypropylene  to be placed underneath, such that radiant photons would pass through the subsections of holes to be detected by CCD detector from above. Samples of PA2200, anatase, and rutile titanium dioxide , and Nylon-12 (Sigma Aldrich) were charged for 10 s with ambient white light and placed in the light tight chamber of a Spectral advanced molecular imaging high throughput system . Then, PL images were acquired with the following parameters: 30 s exposure time, 250 mm field of view, fstop of 1.2, focal-plane of 0 mm, open emission, and 4 × 4 binning. Then, the anatase titanium dioxide and the PA2200 were imaged under similar parameters with the exception of varied emission filters  to create an emission spectrum. All data were plotted on GraphPad Prism version 7 .Both the training and calibration phantoms were imaged and tested for PL characteristics. The complex rat skeleton was charged with ambient light for 10 s and then imaged with the Spectral AMI HT with the following parameters: 30 s exposure time, 250 mm field of view, fstop of 1.2, a focal plane of 0 mm, open emission, and 4 × 4 binning.The white PA2200 disk was charged for 10 s with ambient light, inserted into the Derenzo-type phantom, and imaged under the same parameters as the rat skeleton, apart from the binning. Four images were taken with varied binning parameters of none, 2 × 2, 4 × 4, and 8 × 8, while keeping all other parameters constant. All sets of images were analyzed for image resolution using the ImageJ software package, which was also used to make montages.2 powder, (3) rutile TiO2 powder, and (4) a sample PA2200 part. Each material was imaged with the open filter in an AMI HT imaging station after being charged with ambient room light. Results showed that anatase TiO2 yielded significant PL, while the rutile form did not. Sintered PA2200 yielded about 50% of the PL intensity of anatase TiO2, while the native nylon 12 displayed no optical signal  nylon 12 polymer, (2) anatase TiOl signal .2 powder was also imaged with the same parameters and showed an almost identical spectrum as noted with the PA2200. These results confirmed that anatase TiO2 is the photoactive species that gives PA2200 its optical properties.The PA2200 SLS sample part exhibited sustained PL during optical imaging experiments on a commercial, CCD-based image station. A subsequent analysis was undertaken to determine the emission wavelength with strongest optical output. PA2200 was charged for 10 s with ambient room light and imaged in BLI mode with varying emission filters in front of the CCD to determine the wavelength with maximum optical output. The intensity values in each emission wavelength were plotted to yield a spectrum that revealed strong signal in the near infrared region . AnataseA rat skeleton was 3D printed in PA2200 as an optical phantom for potential use as a training aid and/or a quality control device. The modern SLS 3D printing technology deployed by third party Shapeways successfully created the complex object, which was robust enough to ship without damage a. This aTo display the potential use of PA2200 as a calibration tool, a Derenzo-style phantom was designed to test the resolution capabilities of PL image collecting instrumentation. A disk with 5 mm thickness and 60 mm diameter was machined with six sets of hole patterns ranging from 0.4 mm (with 0.4 mm spacing) to 2.4 mm (hole and spacing). A disk comprised of white PA2200 was positioned underneath the hole grid such that light would only pass through the holes to be detected by the CCD detector. Optical images were collected at different bin states to demonstrate the relationship between binning and resolution . The pha2 is commonly produced in two forms, anatase and rutile, the source of the optical behavior found in PA2200 was studied via component analysis. Imaging of PA2200 components confirmed that its long-lived PL characteristics were due to the anatase derivative of TiO2. While multiple crystal forms of TiO2 display photoluminescent properties spanning the blue to the NIR, only bulk anatase exhibits long-lived multiexponential decay of luminescence consistent with the slow migration of electrons through the bulk anatase TiO2 to electron traps [3+ and oxygen vacancies), NIR light is emitted [2 and PA2200 further supported that anatase TiO2 is the component that imparts PL properties to PA2200. Although both anatase and rutile TiO2 have tetragonal crystal structures and both can display photoluminescence, the mechanisms differ for the two polymorphs. Pallotti et al. provide detailed analysis of the differing photoluminescence mechanisms of anatase and rutile [Published reports indicated that PA2200 is a mixture of Nylon-12 and titanium dioxide, and the material derives its white color from the latter . Given ton traps ,30,31,32 emitted . Additiod rutile , which iIt is uncommon to find 3D printable materials with photoluminescent properties that simulate an imaging modality. The discovery of near infrared PL properties of PA2200 was serendipitous, as these enable a wide array of phantoms for training and calibration. Here, two different phantoms were created as examples. First, since small animal optical imaging has robust usage in the pre-clinical research space, a rat skeleton was 3D printed with PA2200 and its luminescence captured on the Spectral AMI HT system. Since many training sessions on this type of machine would be conducted with live animals, this phantom provides a worthy substitute to reduce animal usage. A Derenzo-type phantom was also created and imaged for optical resolution of the AMI HT system. As noted in the Three-dimensional (3D) printing is finding expanded use in creating affordable and facile prototypes and products in a range of fields, including medical imaging. Within luminescent imaging, optical properties can dictate appropriate material choices for a given application. Here, we present the photoluminescent properties of a common 3D printing material, PA2200, which is amendable to selective laser sintering. To demonstrate the use of these PL properties, a complex anatomical imaging phantom was printed with the white PA2200. This phantom demonstrated near infrared PL properties, enabling its use as a training or teaching device. The Derenzo-style phantom demonstrated the use of PA2200 for calibration purposes, even showing tangible results when uncovering an artifact within the AMI HT system. With the dual ability to create complex architectures that also display a PL signal in the near infrared zone, intricate phantoms can be custom made for individualized training that caters to the specific needs of the industry in which it is used. This opens the doors to creating unique phantoms for research and clinical use, allowing for the potential expansion of research within the PL imaging sector as a whole. Finally, the search for additional common 3D printing materials that may also have desirable optical properties that make them well suited as phantom materials for a range of PL imaging techniques can be opened up to the scientific community, enabling further possibility of research that intertwines the worlds of biological imaging modalities and 3D printing technology.


Due to climate change sorghum might gain widespread in the Western countries, as the grain is adapted to hot climate. Additionally sorghum contains a notable amount of health-promoting nutrients. However, Western countries do not have a long history of sorghum consumption, and thus little experience in processing it. Milling systems in these areas were mostly developed for wheat or rye milling. In the present work, the effectiveness of sorghum milling when using a stone and a roller milling system  was investigated as well as its impact on the chemical and physical properties of the obtained flour fractions and whole-grain flours. Results showed that both milling systems could be successfully adapted to producing chemically and physically distinct flour and bran fractions from the small sorghum kernels. Fractions with increased bran material that contained higher amounts of ash, protein, fat, total dietary fiber, and total phenolic content but less starch, showed enhanced water absorption indices and water solubility indices. Interestingly, no significant difference was found in the ash and fat content of the different fractions obtained from stone milling. Overall, the study provided information on the production and composition of distinct flour fractions, which offer a wider range of future food applications. Sorghum is the fifth most produced cereal worldwide and it is mostly harvested in Africa, followed by America and Asia . TraditiWhen using cereals for food processing, flour production is often the first step. As flour properties and composition greatly affect end-product quality, milling, and fractionation processes play a crucial role. Knowledge on sorghum processing has been mostly gained in Africa and Asia. Some studies have described grinding of sorghum (whole-grain) flour using lab scale mills or local milling systems, e.g., hammer mills ,6. MoreoIn Austria, different varieties of sorghum are cultivated in very small quantities. The harvested grain is used for animal feed and bioethanol production, but not for human nutrition. Therefore, these sorghum varieties have neither been investigated for their food processability nor been chemically analyzed before. In order to assess the potential of these Austrian grown sorghum varieties, the aim of this study was (1) to produce sorghum whole-grain flours, as well as flour fractions; and (2) to characterize all the obtained flours and fractions for their chemical and physical properties. Milling and fractionation of sorghum was carried out using two pilot scale milling systems (stone mill and roller mill). For comparison purposes, an industrial scale whole-grain flour, milled by a roller milling system was included. Chemical and physical analyses were performed to evaluate on the other hand, whether the (wheat) pilot scale stone mill and/or the roller milling system can be used for sorghum milling and fractionation. For roller milling, all single fractions were collected separately, in order to follow the performance and effectiveness of this rather complex milling system see . The stuArmorik), where grown on an experimental farm in Hörsching, Austria and harvested in 2019. They were obtained from Caj. Strobl Naturmühle GmbH  and were used for the milling trials  was obtained.DFS-PSM : Several pre-trials were performed to select the appropriate equipment and settings for stone milling. Based on these results (data not shown) a pilot scale dry flake squeezer  and a pilot scale stone mill  produced a WSF, which was separated by a sifter  into three flour fractions with particle sizes >400 µm, 400–180 µm, and <180 µm. The purpose of squeezing the kernels prior to stone milling is to increase the flour yield. As a first step, the sorghum kernels were squeezed in the dry flake squeezer at 1000 kg/h, then they were ground in the pilot scale stone mill (PSM), set with the smallest possible gap between the stones, 507 revolutions per minute and an hourly rate of 100 kg/h. The gap between the stones was estimated 0.2–0.5 mm, which could not be quantified more accurately. One stone of the mill veered and the other one backed. For fractionation, the whole-grain sorghum flour was first sieved with a 400 µm sieve at 2000 kg/h. The <400 µm fraction was further sieved through a 180 µm sieve, thus obtaining two further fractions (400–180 µm and <180 µm). DFS-PSM was performed from 100 kg of sorghum grain.PRM : The second milling trial applied a pilot scale roller milling plant made of several roller mills  and plansifters  after each passage. Milling was performed with an hourly rate of 150 kg/h. The configuration and settings of this pilot scale roller milling plant were established according to pre-trials on Bühler MLU-202 , which is a lab scale roller milling system (results not presented here). The process of  roller milling/sifting is based on various breaking and reduction steps see . UsuallyAll produced WSFs, as well as all defined flour fractions were analyzed, as described below. From the 14 flour fractions obtained in the second milling system five fractions were selected, representing different sections of the sorghum kernel: bran fine (outer layer), B1, C1 (white endosperm flour), C4, C5/C6/B4/B5 (dark endosperm flour), as well as a reconstituted WSF. The selected fractions were chosen in order to follow if fractions from different parts of the kernel were characterized by different chemical or physical properties.Color analyses were carried out as described in Bender et al. . A DigiEg. Supernatants were separated from the sediment and the liquid phase was collected. After adding 10 mL ethyl acetate to both, the solid and the liquid phases, the mixtures were shaken for 5 min and afterwards centrifuged for 2 min at 3092× g. For these analyses ethyl acetate was chosen as extraction aid in order to extract the liberated phenolics, as well as the previously unesterified phenolics, from the aqueous extract. Ethyl-acetate is a suitable solvent for the phenolics and, in contrast to more polar solvents like methanol or ethanol, can be separated from the aqueous phase via centrifugation. The supernatants (ethyl acetate phases) of the solid and the liquid phases were collected together in a shaded round bottom flask. The extraction with ethyl acetate was repeated twice. Samples were concentrated using a rotavapor  and dissolved in 4 mL of 50% methanol. For TPC determination 100 µL of the extracts were mixed into 0.5 mL of freshly prepared 10-fold diluted Folin-Ciocalteu reagent. After 2 min waiting time 0.8 mL sodium carbonate (75 g/L) was added. The mixtures were incubated for 5 min at 50 °C. The absorbances were read at 755 nm against a blank. The results were expressed as mg ferulic acid equivalent (FAE) per 100 g dry substance. All chemical analyses were carried out in triplicate.Moisture content was determined by ICC standard method Nr. 110/1 . Ash cong for 10 min and residues were weighed for WAI calculation. Supernatants were collected in aluminum dishes and evaporated for 4 h in a drying cabinet at 103 °C. Dried solids were used for WSI calculation. WAI was expressed as absorbed water in g/g dry sample, WSI was expressed as percentage of dry solids/2.5 g dry sample. Measurements were carried out in triplicate.The water absorption index (WAI) and the water solubility index (WSI) were measured based on the method from Anderson et al. . BrieflyStatistical data analyses were carried out using Statgraphics Centurion 18, Version 18.1.13 . Significant differences were evaluated by performing an ANOVA and Fisher’s least significant test with a significance level of α = 0.05. Results were expressed as mean ± standard deviation and significant differences between results were expressed by different superscript letters.Milling yields obtained from the two investigated milling systems are visualized in The most abundant flour fraction in DFS-PSM, was the flour with a particle size of <180 µm, 43.9%, followed by almost equal parts of the remaining two fractions >400 µm, 27.5%, and 400–180 µm, 28.6% see .As described in the methodology, all PRM passages were collected separately. As for the PRM milling system, one-fifth of the flour fractions ended in bran material (bran coarse and bran fine) and further 8% in the bran-rich animal feed flour. As expected, the flour fraction B1 was extracted to a very low extent, as it is the first endosperm flour obtained after grain breaking. Higher yields were obtained during the reduction milling system (C1–C5). The flour fraction C1 showed a milling yield of 11%. However, the highest amount within the flour fractions C and B was extracted after the C5/C6 passage, and after the B4/B5 passage, respectively. Total endosperm flour accumulated to 72%, while the remaining 28% were bran and animal feed. Only five flour fractions were selected from this milling system for subsequent chemical and physical analyses and are highlighted in Overall, both milling trials were successful to produce not only whole-grain flour, but also different flour fractions. Although the sorghum kernel differ greatly from wheat or rye kernels in size and shape, both the stone mill and the roller milling system could be used and adapted for production of sorghum milling fractions. Endosperm flour was successfully extracted in both cases, as could visually be observed, and will be described by the color and chemical data in the subsequent sections, Well configurated industrial scale mills with even higher number of passages are able to extract up to 80% of endosperm flour in wheat milling.In In the DFS-PSM milling fractions lightness  increased with decreasing particle sizes. They were also less red and yellow, which indicated a lower content of bran in the finer fractions.In the PRM milling fractions, the bran fraction had the highest a*-values (red) and b*-values (yellow), as expected. These value trends are further in line with previous findings . HoweverThe results of the chemical analyses are displayed in The dry matter content of all analyzed sorghum flours ranged between 88.73 annd 91.72%. Among the sorghum flour fractions obtained from DFS-PSM, no significant differences in ash and fat content were observed. This was also seen in a previous study, which stated no differences in ash, fat, and protein in sieving fractions of whole-grain wheat . The floBased on protein, starch, TDF, and total polyphenol content, it seemed that nutritional fractionation within the DFS-PSM sieving took place; although, ash and fat contents did not differ significantly among different fractions. Both, fat and ash, are the predominant nutrients in the germ ,26. An eAs described in type W480 according to the Austrian codex Alimentarius [The C1 passage flour fraction showed similar results to the B1 passage fraction flour. On the basis of ash, protein, starch, fat, TDF, and TPC values, it can be concluded that the C1 fraction flour corresponds to a rather pure endosperm flour. In wheat milling, the flour obtained after the C1 passage milling corresponds to a very white endosperm flour, which is categorized as entarius . The redAs mentioned previously, milling has a great effect on the resulting flour properties. The effect of the two applied milling systems on flour quality can be followed when comparing the whole-grain sorghum flours produced in this study. For comparison, a commercially available whole-grain sorghum flour was included . Again, it has to be considered that PRM WSF was reconstituted. Ash was the only component which did not differ significantly between the three whole-grain sorghum flours. The highest protein value was observed for PRM WSF, followed by DFS-PSM and IRM. An explanation for the higher protein content in PRM could be an improper reconstitution of WSF based on the fraction yields. Endosperm flour yields may have been underestimated, as an undefined amount of flour residue inevitably remained in the mill. So far, lower protein contents in tartary buckwheat flours obtained from a roller mill compared to a stone mills have been reported , but no Generally, rather low and insignificant differences were observed between the stone milled and roller milled flours. Pagani et al.  also didResults of water absorption index (WAI) and water soluble index (WSI) are given in Flours obtained from DFS-PSM decreased in WAI with decreasing particle size. Al-Rabadi et al. (2012) evaluated comparable results on sorghum flour for similar particle sizes. However, the authors reported no consistent WAI trend in flours of different particle sizes . Cadden Water solubility indices (WSI) of DFS-PSM flour fractions were lower with decreasing particle size. Looking at the PRM flour fractions, the bran had the highest WSI. Flour fractions from B and C fractionation showed similar WSI ranging from 2.42 to 3.7 1%. Comparing the whole-grain flours, WSI from PRM milling had a significant higher WSI than from PSM or IRM milling. This might also be related to the higher ratio of bran in the reconstituted whole-grain flour of PRM.Considering the chemical data in combination with the physical properties, it can be stated that the obtained sorghum flour fractions offer different food uses, both, either in terms of intended nutritional enrichment, and in terms of specific processing needs. Some bakery products require very defined physical or rheological conditions of flours, e.g., fine bakery products or wafers. Replacing wheat flour with any other flour therefore has major effects on the end-product quality and requires thorough adaptation of recipe and processing parameters. Detailed knowledge on flour properties is one of most important factors to be considered in this respect. The data obtained in this study for sorghum flour fractions provide a step forward to future use of sorghum and incorporation into Western type bakery products.Results of this research indicated that stone and roller milling, both typically applied for wheat milling, were effective for milling sorghum and could successfully produce defined flour fractions, as well as whole-grain flours.With regard to stone milling, the chemical and physical properties of the obtained flour fractions were clearly distinct, demonstrating that fractionation was not only based on their particle sizes , but also on their chemical profile.In the roller milling system, flour fractions were not separated by sieving only, but by a typical sieving/sifting combination after each roller mill passage. Although the size and shape of sorghum kernels differed to wheat, roller milling settings could be adapted to sorghum milling. Chemical and physical properties of defined roller milled flour fractions indicated that sorghum grains were separated into different anatomical parts. Considering the chemical data, it can be concluded that the B1 and C1 flour fractions were highly concentrated from endosperm parts. The C4 and C5/C6/B4/B5 fractions can be defined as dark endosperm flours, which was confirmed by a lower starch content, but a higher ash, protein, fat, and TDF content compared to B1 and C1 flour fractions. As the bran fraction was shown to be largely composed of outer kernel layers and germ, it was confirmed, that it could successfully be separated from the endosperm. The WSF from the roller milling system was significantly higher in protein and TPC, but lower in starch compared to the WSF from stone milling. This was likely the result of an improper reconstitution of WSF based on the fraction yields.Overall, the results of the chemical and physical analyses of the flour fractions, obtained from stone or roller milling systems, seem to confirm that sorghum milling can be carried out on typical wheat milling systems without detailed or complex technical adaptations. Both milling systems were successful in producing flour fractions with distinct chemical and physical properties. The two milling systems differed in their ability to produce distinct fractions, with roller milling being superior to stone milling. Depending on the intended food use, the availability of defined flour fractions can be advantageous for selected food applications ,9 as somThis study provided information on defined sorghum flours and flour fractions that might facilitate the future use of sorghum in the Western diet.


There are variations in the values reported for aerosol drug delivery across in vitro experiments throughout the published literature, and often with the same devices or similar experimental setups. Factors contributing to this variability include, but are not limited to device type, equipment settings, drug type and quantification methods. This study assessed the impact of head model choice on aerosol drug delivery using six different adults and three different paediatric head models in combination with a facemask, mouthpiece, and high-flow nasal cannula. Under controlled test conditions, the quantity of drug collected varied depending on the choice of head model. Head models vary depending on a combination of structural design differences, facial features (size and structure), internal volume measurements and airway geometries and these variations result in the differences in aerosol delivery. Of the widely available head models used in this study, only three were seen to closely predict in vivo aerosol delivery performance in adults compared with published scintigraphy data. Further, this testing identified the limited utility of some head models under certain test conditions, for example, the range reported across head models was aerosol drug delivery of 2.62 ± 2.86% to 37.79 ± 1.55% when used with a facemask. For the first time, this study highlights the impact of head model choice on reported aerosol drug delivery within a laboratory setting and contributes to explaining the differences in values reported within the literature. In vitro experiments play a hugely valuable role in predicting and characterizing aerosol drug delivery across a range of device and interface combinations. The information and data reported are vital in the optimisation of device design, and a device’s clinical application. A wide range of head models is reported within the literature as a means to assess aerosol delivery to simulated spontaneously breathing adult and paediatric patients. Detailed information on make, model and material is often descriptive, but limited ,2,3. Rep®, Craigavon, Ireland) and Nasco Healthcare  feature anatomical landmarks, such as the teeth, tongue, nasal mucus, and skin-like surface material to closer resemble “real-life” humans. In addition, other representative head models, such as CPR and head simulation models are commonly available and utilised for aerosol drug delivery experiments. It has been our group’s experience that with the range of options available, a head model choice can be a significant contributing factor to the range of results reported across the published literature.It is generally accepted that 3D printed head models, generated from an individual CT scan are valuable in estimating aerosol dose delivery. Moreover, the availability of realistic airway management head models, also based on real life CT Dicom data, may further advance the sophistication of head models available in estimating aerosol drug delivery. In comparison to 3D printed head models, realistic airway management head models developed by companies, such as TruCorp  varied across the different head models. The adult airway management trainer and 3D printed head models contained intricate and anatomically correct geometries. These head models offered a more “real-life” representation compared to CPR and head simulation head models  which contained a single oropharyngeal airway with a simple cylindrical airway geometry.The 3D printed paediatric nasopharyngeal and oropharyngeal head models also contained singular airways due to the lack of oral and nasal cavities. However, these airways were anatomically correct in contrast to the simplistic airways observed in the CPR and head simulation head models.® Adult Airway Management Trainer, Head Only, SKU: LF03603, Saugerties, NY, USA) and the TruCorp AirSim Advance X (AA91100X) and TruCorp AirSim Child X (AC 10006X). Similarly, the internal geometry of the airway management trainer models were generated from individual CT scans. Despite the anatomical correctness, these head models varied in shape, size and internal volume, which may be expected considering real life population heterogeneity.The 3D printed head models have exact replicas of nasal and airway geometries, however, they lacked additional anatomical markers, such as teeth, tongue and external facial features present on the airway management head models, LUCY  oropharyngeal head model was used for facemask and mouthpiece testing only due to complete occlusion of the nasal opening while the 3D printed paediatric nasopharyngeal head model was used for high flow nasal cannula testing only due to occlusion of the oral cavity. The LUCY, TruCorp AirSim Advance X and the TruCorp AirSim Child X head models were tested across all three patient interfaces considered in the study. To ensure consistency across test set-ups, the oral cavity of the TruCorp AirSim Advance Child X was occluded to align with the 3D printed nasopharyngeal (1688) paediatric head model for the nasal cannula interface experiments. Similarly, the nasal cavity of the TruCorp AirSim Advance Child X was occluded to allow for comparison with the 3D printed  oropharyngeal head model for facemask and mouthpiece interface testing. Likewise, the oral cavity of the TruCorp AirSim Advance X, LUCY, and oropharyngeal airway of the 3D printed adult head model were occluded for the high flow nasal therapy testing whilst the nasal cavity of these same models was occluded during mouthpiece testing.t) of 500 mL were utilised to simulate adult breath settings (ISO 27427) [t: 300 mL, BR: 20 BPM, I:E 1:2).O 27427) . ApproprFor all tests, 4000 µg (2 mL of 2 mg/mL) Albuterol Sulfate  was aerosolised using a vibrating mesh nebuliser (VMN) . Five test runs were completed for each head model and device interface combination.The VMN was used in combination with an aerosol holding chamber  and either a facemask or mouthpiece. The droplet size  produced by the Aerogen Solo was 4.5 microns and was characterised using laser diffraction as previously described . SupplemFor High Flow Nasal Therapy (HFNT) the VMN was placed at the humidifier using the Optiflow system . The appropriate nasal cannula (Adult: OPT946 (large), Paediatric: OPT318) and circuit  were used to deliver aerosol at gas flow rates of 10 & 50 LPM for simulated adult and 3, 5 & 10 LPM for simulated paediatric models, respectively.t-tests were completed to compare % tracheal dose between paediatric head models for facemask, mouthpiece, and high flow nasal canula testing. For all tests, differences were considered statistically significant at p ≤ 0.05.For each test run the aerosolised drug captured on the tracheal filter  was eluted from the filter using 10 mL deionised water. The drug mass was measured using UV spectroscopy at 276 nm and interpolation on a standard curve as previously described . Resultsn = 5, for a total of 180 test runs) for each of the adult head models used in this study. The aerosol was delivered from the Aerogen Ultra to the respective head models using a facemask or mouthpiece at supplemental gas flow rates of 0, 2 and 6 LPM. Aerosol from the high flow system was delivered at 10 and 50 LPM. The data presented in p ≤ 0.05.n = 5, for a total of 90 test runs) for the paediatric head models. Aerosol was delivered from the Aerogen Ultra at supplemental gas flow rates of 0, 2 and 6 LPM to the respective models using either a facemask or mouthpiece. Aerosol was delivered from the high flow system at flow rates 3, 5 and 10 LPM respectively via the appropriate nasal cannula. All paediatric models were constructed based on real-life CT scans, and therefore, contained anatomically correct airway geometries. The shape, size and internal volume measurements varied between head models. The 3D printed oropharyngeal head model facilitated more aerosol deposition onto the tracheal filter compared to the TruCorp AirSim Child X when used in combination with a facemask at 0 LPM (31.86 ± 3.67% vs. 5.24 ± 1.53%). In contrast, the TruCorp AirSim Child X facilitated more aerosol deposition onto the filter compared to the 3D printed oropharyngeal head model when used in combination with a mouthpiece at 0 LPM (20.42 ± 3.58% vs. 3.88 ± 0.70%). Both head models, however, were comparable when used in combination with a facemask/mouthpiece at 2 and 6 LPM. With regards to aerosol dose delivery using the high flow nasal cannula, both the TruCorp AirSim Child X and the 3D printed nasopharyngeal head model were comparable at gas flow rates of 3 and 5 LPM. However, at a flow rate of 10 LPM the 3D printed nasopharyngeal head model delivered a significantly higher percentage tracheal dose compared to the TruCorp AirSim Child X (6.28 ± 0.22% vs. 4.00 ± 0.63%). Results were considered statistically significant for p ≤ 0.05.The current findings demonstrate the impact of head model choice on aerosol drug delivery using the same device and test parameters and highlight a lack of standardisation across testing for in vitro studies in this area. Head model variation and on occasion, limited access to specific head model information impact the ability to accurately compare aerosol delivery measurements in the published literature. To our knowledge, this is the first study to assess the impact of head model choice on the assessment of aerosol drug delivery.Overall size and large facial features, such as the nose and mandible, influenced interface fit when using the TruCorp AirSim Advance X in combination with a facemask. It is proposed that this may have influenced the lower aerosol drug delivery measured using this head model and interface combination. Facemask fit improved and was more easily managed in smaller airway management head models (LUCY), and CPR and head simulation models  (range at 0 LPM for example was 2.62 ± 2.86% to 37.79 ± 1.55%).It is likely that the mask fit combined with both a single simplified airway geometry and small internal volume contribute to the higher percentage tracheal dose observed in FRANK when used in combination with a facemask. Aerosol is less likely to get trapped in the absence of anatomical features, such as the tongue and teeth and in the presence of simplified cylindrical airway geometries.3 vs. 27 cm3) which, along with occlusion of the nasal cavity may contribute to the lower aerosol drug delivery observed when using this model in combination with a facemask. Whilst similarly constructed, the airway geometry for ANNE contained a sharp bend as opposed to an elongated curvature of the throat as seen in FRANK and other head models. This may have influenced aerosol delivery in this head model.Although, CPR head model ADAM contained a simple airway geometry, the internal volume in this head model was larger compared to FRANK  and the presence of one  as opposed to two airways may have also enhanced aerosol delivery in the 3D printed oropharyngeal head model vs. the TruCorp AirSim Child X.For paediatric head models, a small internal volume . Furthermore, this opening was partially sealed and rigid, which created challenges in aligning the mouthpiece, oral opening and cylindrical airway. Similar to the facemask interface, it is likely that an altered airway geometry also impacted aerosol delivery for ANNE when used in combination with a mouthpiece.The solid oral opening of the 3D printed oropharyngeal paediatric head model was immobile and smaller in width (3.5 cm vs. 4.0 cm) compared to the adjustable opening of the TruCorp AirSim Child X. It is possible that alignment of the mouthpiece with the airway contributed to the low delivered dose observed with this head model and interface combination when compared to the TruCorp AirSim Child X (3.88 ± 0.70% vs. 20.42 ± 3.58% at 0 LPM). This low dose increased with increasing supplemental gas flow rates, where the very low and highly variable dose at 0 LPM increased at 2 and 6 LPM and was closer to the TruCorp Air-Sim Child X. This would support the above hypothesis that aerosol entry into the oral cavity facilitated larger doses, where here, the supplemental gas flow carried the aerosol into the oral cavity, making more available for inhalation onto the tracheal filter. The structure of the 3D printed adult head model allowed direct access to the airway, which eliminated these challenges.When considering these models’ ability to predict in vivo performance in spontaneous breathing patients, there is a paucity of direct comparisons in the literature, and only adult data is available. However, it should be noted that each of the TruCorp AirSim Advance X, LUCY and the 3D printed head model all recorded similar delivered doses to that reported in a comparative scintigraphy imaging trial in healthy adult volunteers that also used the Aerogen Ultra in combination with a mouthpiece at 0 LPM  .3 vs. 21 cm3), aerosol delivery was comparable at gas flow rates of 3 and 5 LPM. The 3D printed nasopharyngeal head model delivered a significantly greater aerosol dose at a gas flow rate of 10 LPM only.With regards to high flow nasal therapy, the fit of the adult nasal cannula was visibly tighter in the LUCY and 3D printed adult head models compared to the TruCorp AirSim Advance X, which measured at least 1 cm larger in length and width of the dorsum nasi and nasal opening. The width of the nasal orifices for the two paediatric head models was similar  and thus the nasal cannula was considered a good fit in both models. Whilst the internal volume for the TruCorp AirSim Child X was larger than that for the 3D printed nasopharyngeal head model  to that reported in a scintigraphy imaging trial in healthy volunteers that used the Aerogen Solo and Optiflow system with 50 LPM gas flow rate .A degree of variation is expected between head models constructed from individual CT scans, however, the anatomical correctness of these models helps predict aerosol delivery in “real-life” scenarios with greater accuracy. Combined with advanced features, such as teeth, tongue, nostril mucus and “skin”, the airway management models were more intricately representative of “real-life” patients. Nevertheless, 3D printed head models remain a reliable resource for estimating aerosol dose delivery. Javaherin et al. compared an idealised geometry with 10 realistic infant geometries and found agreement between the different models, which supports the use of an idealised 3D printed model for predicting aerosol delivery . Additio®  are also based on CT Dicom data, however, these models are constructed of silicone skin  and latex based airways . It is possible that the material composition and construction methods may further influence aerosol delivery through the airways of varying head models.Although anatomically correct, the material and construction methods for head models based on CT scans also varied. The 3D printed head model described by Golshahi et al.  was consNot addressed by these models, and consequently by our analysis, are several features that exist in humans, such as the variable position of the tongue, jaw and glottal aperture. This, combined with the effect of the airway lining liquids and mucus will impact aerosol deposition, and ultimately aerosol distribution throughout the airways. Additionally, these upper airway models, and despite some being derived from CT scans, are not representative of the entire human adult or paediatric populations. As such, reported aerosol doses should always be considered indicative and no more. Attempts have been made to address these limitations in models as tools, but there are still none that meet all the needs of the research field. For example, the Alberta Idealized Throat model  was deveFurthermore, as noted above, interface (mask/mouthpiece/cannula) fit against the face/head model is a critical determinant of aerosol delivery. Whilst not the focus of our studies, this interface “fit” element is deserving of further investigation in future studies. This may lead to complexity in interface options, e.g., several sizes need to be designed or made available, but the resulting improvement in aerosol delivery and the consequent healthcare outcomes are deserving reasons.A considerable amount of work is required in order to close these gaps but until that happens, the commercially available head models, such as those described herein remain a valuable research tool, albeit with their own limitations.A range of commercially available head models is described within studies pertaining to aerosol inhalation and drug delivery across the published literature. These head models vary depending on design, structure, volume, and geometrical complexities and, thus, impact aerosol measurements when used in combination with a range of interfaces. With significant variability noted across combinations of head model type, interface and supplemental gas flow rate, this study highlights the need for increased awareness on the impact of head model choice on reported aerosol drug delivery. As such, the suitability of and ability to predict in vivo aerosol performance of the head model under test needs to be considered carefully. Here, based on limited in vivo data reported in the literature, we found the TruCorp AirSim Advance X, LUCY and the 3D printed head model the most predictive of in vivo aerosol delivery performance for both mouthpiece and nasal cannula in a model of the adult patient. Unfortunately, due to the absence of published scintigraphy data using the same nebuliser in paediatric patients, we were unable to determine the most predictive of in vivo performance in a model of the paediatric patient. Comparisons with reported nonhuman primate data where the same nebuliser was used, for example, Réminiac et al.  are not


The study aimed to investigate the efficacy of new mycotoxin adsorbents based on purified and activated bentonites combined with yeast and phytogenic compounds in fattening pigs. The experiment involved 96 pigs (31.2±2.4 kg). Control (C) group was fed a diet naturally contaminated with mycotoxins  without an adsorbent. Treated groups received the feed with mycotoxin adsorbents: purified and activated bentonite (T1), purified and activated bentonite, yeast derivatives, phytogenic substances (T2), and purified, activated, and sulphurated bentonite with phytogenic substances (T3). Evaluated parameters involved growth performance, organ weight, small intestine and liver histopathology, complete blood count, serum biochemistry, antioxidant status of the organism and total and free DON content in urine. In all treated groups, an significant increase in intestinal GSH and GSH/GSSG ratio was observed when compared to C. No significant effects on liver and kidney weight, complete blood count, serum or intestinal malondialdehyde concentration, or total/free DON content in urine were observed. All adsorbents improved histopathological findings in the liver when compared to C. Moreover, T1, and T2 groups showed no presence of inflammatory reaction or necrotic changes in the livers. Although, mycotoxin adsorbents investigated in this study had no significant impact on pig growth performance, they reduced the oxidative stress, and on the tissue level they protected the jejunal tissue and liver parenchyma under deoxynivalenol challenge. Mycotoxins are a persistent global issue and cause significant production losses in pigbreeding. Furthermore, recent climate change is responsible for the changing spectrum of mycotoxins in food and feed ,2. DespiPigs are considered very sensitive to mycotoxins’ effects ,4. DepenThe economic impact of DON contamination includes pig mortality, increased veterinary costs, and reduced meat production. As a result, there is ongoing research and development of strategies to eliminate or inactivate DON and other mycotoxin production or reduce their occurrence in feed . A commoThe most significant criteria for evaluating adsorbents include the stability of the sorbent-toxin bond and their efficacy over a wide pH range, as the product is expected to function in the gastrointestinal tract . The mosHowever, the effectivity of clay minerals in reduce DON adsorption is limited. Despite this, DON possesses some structural features that could improve its reactivity—the epoxy ring, hydroxyl groups, and an α,β-unsaturated carbonyl group. In this context, clay minerals can be modified to increase their binding capacity . NumerouMore recent attention has focused on the application of yeast cell walls for DON elimination ,23. MainIn addition, several phytoactive compounds such as essential oils, flavonoids or organic acids have been discovered to prevent cytotoxicity caused by DON –28. A meThe study´s aim was to verify the impact of purified and activated bentonite combined with yeast and phytogenic substances on fattening pigs’ growth performance and health status. In our study, mycotoxin adsorbents based on bentonite subjected to a several-step activation and purification were used. This process results in the change of the original physicochemical properties and an increase in the number of binding sites and adsorption capacity of the final product. We examined the combination with yeast and phytogenic substances for further increase its efficacy.The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of Expert Commission for Ensuring the Welfare of Experimental Animals of Mendel University in Brno . A total of 96 experimental pigs  with an average weight 31.2 kg were housed in 36 identical pens measuring 2.43 x 1.46 m. The animals were exposed to the artificial lighting sources with light intensity of 45 lux in the mode 12 hour-light and 12-hour dark cycle. According to the pre-fattening pigs’ requirements, the microclimatic conditions of pigs’ housing were maintained by an artificial computer-controlled ventilation system. The basic microclimatic standards were maintained following the requirements of the Act on the protection of animals against cruelty (No. 246/1992 Coll.).The composition and nutritional values of the feed ration are shown in Tables All experimental animals were provided ad libitum access to feed and drinking water. Also, the unconsumed feed mixture was monitored. In the experimental and control groups, the feed ration was composed of wheat in which high concentrations of mycotoxins were detected . The natA total of 96 pigs were included in the experiment at an average weight of 31.2 ± 2.4 kg. Animals were fed the experimental diets for 35 days and were divided into six groups of 24 animals (6 pens x 4 pigs). The control group (C) was fed a naturally contaminated wheat diet with high levels of DON without adsorbents. Other treated groups were fed with the same diet with the addition of the tested mycotoxin adsorbents. All mycotoxin adsorbents were purchased from Addicoo group, s.r.o., Czech republic. Treated group T1 was supplemented with 100% purified and activated bentonite  at a dose of 1.5 kg/ton. T2 group was supplemented with an adsorbent containing 75% purified and activated bentonite, 17.8% yeast cell wall derivatives, and 7.2% phytogenic substances  at a dose of 2 kg/ton. T3 group was supplemented with mycotoxin adsorbent based on 95% purified, activated, and sulphurated bentonite with 5% phytogenic substances at a dose of 3.5 kg/ton.The weight of the animals was monitored at regular weekly intervals. The average daily feed intake and feed conversion were monitored for the periods: 1–14 days; 15–28 days; 29–35 days. Six randomly selected pigs from each group ranging in final weight from 62.4 to 76.5 kg were slaughtered on the same day. The animals were left to rest and fast for about two hours before slaughtering. They were then slaughtered by electrical stunning  and exsanguination. In each slaughtered pig, kidney and liver weight were determined as percentages of the pigs’ live weight. After slaughter, blood samples were collected via the jugular vein into EDTA tubes and 10 mL heparinized vacuum tubes, then aliquots were centrifuged at 3000 x g for 10 min to collect plasma, which was then frozen at -20°C until analysis; reduced and oxidized glutathione (GSH/GSSG), malonyldialdehyde (MDA), glutathion peroxidase (GPx) and biochemical parameters). Liver and intestine samples were collected, washed in PBS buffer and placed in 4% formaldehyde until histopathologic evaluation. Urine was collected by punction of the bladder and frozen at -20°C until total and free DON analysis. Middle jejunum  samples were collected and frozen at -20°C until analysis (MDA).For documentation of the activated bentonite structure , a scannAll feed samples were oven-dried at a temperature of up to 50°C, then ground with a grinder to a particle size of 1 mm and analyzed for the basic nutrient content. The fiber was determined on an A200 Fiber Analyzer , nitrogenous substances according to the Kjeldahl method (N × 6.25), and fat (by direct extraction according to Soxhlet method) . The ash2. Each dried residue was dissolved in 60 μL water and 40 μL ethanol/pyridine (4:1). Five μL ethyl chloroformate was added to the mixture. Finally, 150 μL of chloroform was added.The method for AAA was adopted from Husek and Sweeley et al. Briefly, 5 g of sample was hydrolyzed according to . 1 mL ofDerivatized samples were analyzed using the GC system  equipped with an FID. Separation of compounds was conducted on a 10 m CP-Sil 19 capillary column  using nitrogen 5.0 as the carrier gas . The injection volume was 1 μL, and the flow rate was set at 0.7 mL/min. The injector temperature was 250°C with a split ratio of 50:1, and the FID temperature was 250°C. The oven temperature was programmed as follows: the column was held initially at 140°C, then increased to 280°C at 40°C/min and held for 3 min. Chromatographic data were recorded and integrated using Clarity software  .Urinary DON and DON-GlcA concentrations were measured using a two-step process. Stored urine samples were allowed to thaw and were then centrifuged at 2000 g. Supernatant pH was adjusted to pH 6.8 with the dropwise addition of KOH or HCl. An aliquot (250 μL) m was digested using β-glucuronidase (20 000 U/mL) in a thermoshaker at 37°C for 18h, ensuring a gentle mixing. Digested and native urine samples were passed through IAC columns  with 2 mL of methanol. Free and total DON were analyzed by ELISA commercial kit  according to manufacturer protocol. Creatinine was measured by a commercial kit  according to manufacturer protocol.Blood samples were collected from all experimental animals to determine haematological  and biochemical parameters , gamma-glutamyltransferase (GGT), glucose, total bilirubin and protein, urea). Spectrometric analyses were performed by a Konelab T20xt biochemical analyzer (Thermo Fisher Scientific) and commercially available reagents, according to . FurtherFor the optical density reading, the Synergy HTX Microplate reader  was used.Tissue samples of the liver  and the intestine  were collected and immediately fixed in 4% formaldehyde solution to investigate and evaluate pathomorphological changes. Fragments of tissues were cut at 3.0 μm, then positioned onto Superfrost Plus slides  with the orientation core placed up on the slide. All tissue blocks were oriented the same way; then, the entire tissue block was cut with the remaining sections dipped in wax and stored at room temperature. The sections were stained with hematoxylin and eosin according to standard procedures. Pictures were taken using an inverted Olympus microscope IX 71S8F-3  at the magnification 10–20 x for liver samples and 10x magnification for jejunum.The data were analyzed using STATISTICA.CZ, version 12.0 (Czech Republic). The results were expressed as a mean from all samples ± standard deviation. Experiments were performed in triplicate. Statistical significance was determined by examining the basic differences among groups using ANOVA and Schaffer’s method for all parameters. The differences with p<0.05 were considered significant.The average scores of feed intake, conversion and daily weight gain were compared in order to evaluate feed efficiency. During all three monitored periods , no statistically significant differences in efficiency between the control and experimental groups were observed. All results of pig’s efficiency traits are presented in During dissection, the liver and kidneys were removed . The weiThe first set of analyses examined the impact of mycotoxins (C group) on the antioxidant status of pigs and adsorbent ability (T1 –T3 groups) to reverse this effect. The results are present in All haematological parameters were within the physiological range, and no significant differences indicating a health problem were observed between groups. .To assess health status and liver function, the selected enzymes’ activities were compared between C group and treated groups . No significant differences were found in all studied parameters; albumin, AST, GGT, CREA, Urea, GLUC and SDH .The average levels of unmetabolized free DON and total DON after enzymatic cleavage were evaluated in the urine. No significant differences between C and T1, T2 and T3 were observed. However, all treated groups showed a slightly decreased level of free and total DON compared to C .Pathological changes were observed in all samples of animals in the experimental groups, with various intensities. For histopathological evaluation of the intestine, the samples were taken from the middle jejunum. The C group had predominantly marked lymphoplasmacytic inflammatory infiltrate in the mucosa with abundant eosinophils; occasional macrophages; focal hyperemia; and predominantly mild, moderately focal edema of lamina propria. Moderate lymphoplasmacytic infiltrate, lymphangiectasia of some villi, shortening and fusion of some villi, and submucosal edema have been observed . The meaHistopathological evaluation of the liver showed focal hepatocyte necrosis and focal proliferation of fibrovascular granulation tissue in the C group . SignifiThe main objective of the study was to evaluate the effectivity of purified and activated bentonite adsorbents enriched with phytoactive compounds and yeast cell walls on pigs´ health status and fattening performance under higher DON occurrence in feed. Three mycotoxin adsorbents  were tested and compared to the control group fed with contaminated feed.T1 group was supplemented with an adsorbent based on purified and activated bentonite. However, bentonites have been reported to show weaker binding capacity for DON , as theyT2 group was supplemented with an adsorbent containing purified and activated bentonite, yeast derivatives, and phytogenic substances, which help the body to cope with some of the toxic effects of DON . Yeast wT3 group was supplemented with mycotoxin adsorbent based on purified, activated, and sulphurated bentonite with phytogenic substances. In pig nutrition, it has been observed that the enrichment of bentonites by sulphur compounds such as sodium metabisulfite or sodium sulphite have shown positive ability to mitigate DON effects on pigs . Tran, eIn our study, DON concentration in contaminated feed mixture was 5 mg/kg of dry matter, which is the concentration that occurs very frequently in feed contaminated by moulds . Other mIt has been observed that DON exposure could affect hematologic results and the antioxidant status of pigs. The proposed mechanism of action is via activation of several mitogen-activated protein kinases (MAPKs) responsible for cell apoptosis, inflammatory response and oxidative stress . HoweverAnimal studies demonstrated that exposure to DON could be measured in urine. Experimental evidence indicated unmetabolized DON, and its conjugates with glucuronic acid  and glucuronidation products in urine samples in pigs –65. In oSome studies investigating DON effects on organisms have reported histopathological changes of the gastrointestinal tract such as inflammatory infiltration, necrotic changes in the intestinal villi, edema of lamina propria, a decrease in the number of goblet cells in the jejunum and the ileum, intensification of apoptosis and degeneration of lymphoid cells in the gastrointestinal tract ,68. ConsThe liver, the main detoxifying organ, is strongly exposed to toxic and foreign substances. In our study, the most considerable morphological changes of liver influenced by treatment  were observed compared to C group. C group showed, in addition to degenerative changes in the liver parenchyma, the presence of necrotic deposits, inflammatory reactions and a reparative process in the localization of these lesions. The occurrence of regressive lesions in the liver, i.e., parenchymatous and vacuolar degeneration of individual hepatocytes was confirmed by detailed histopathological study after DON administration to pigs at the dose ranged from 0.2 to 0.4 mg/kg/BW per day . SimilarThis study set out to determine the influence of three groups of mycotoxin adsorbents based on purified and activated bentonites and/or sulphurated or with the addition of yeast cell derivates, phytoactive compounds a on pig health status and performance under deoxynivalenol challenge. In our study, we did not observe significant changes of pigs’ growth performance within either haematological and biochemical parameters. Slightly upward trends have been observed in GSH level and GSH/GSSG ratio in the treated groups compared to C group. The most visible changes were observed between C and treated groups in histopathological examination, which showed that the combination of activated, sulphonated bentonites, yeast extract and phytoactive compounds had a protective and improving effect at the tissue level.


Therefore, it is of great significance to investigate the effects of corresponding compound stimulation on cell behaviors. Herein, a fluid shear stress (FSS) plus ultrasound stimulation platform suitable for cell studies based on a microfluidic chip was constructed and bone marrow mesenchymal stem cell (BMSC) was chosen as a model cell. The proliferation and osteogenesis of BMSCs under the compound stimulation of FSS plus ultrasound in growth medium without any soluble induction factors were firstly investigated. Single FSS stimulation and static culture conditions were also examined. Results illustrated that suitable single FSS stimulation (about 0.06 dyn/cm2) could significantly enhance cell proliferation and osteogenesis simultaneously when compared to the static control, while greater FSS mitigated or even restricted these enhancing effects. Interestingly, ultrasound stimulation combined with this suitable FSS stimulation further accelerated cell proliferation as the intensity of ultrasound increasing. As for the osteogenesis under compound stimulation, it was relatively restricted under lower ultrasound intensity (about 0.075 W/cm2), while promoted when the intensity became higher (about 1.75 W/cm2). This study suggests that both the cell proliferation and osteogenesis are very responsive to the magnitudes of FSS and ultrasound stimulations and can be both significantly enhanced by proper combination strategies. Moreover, these findings will provide valuable references for the construction of effective cell bioreactors and also the treatment of orthopedic diseases.Bone tissue with strong adaptability is often in a complex dynamical microenvironment  Many factors, such as congenital defects or acquired injuries can lead to bone tissue disease or defect, which will seriously affect the living qualities of patients. With the pluripotent capability, stem cell, especially bone marrow mesenchymal stem cells (BMSCs), combined with proper tissue scaffolds, has become a promising tissue engineering strategy for the treatment of bone and cartilage defects . In ordein vivo are often in a complex dynamical microenvironment combined with various stimulations, such as fluid shear stress (FSS), compression stress and hydrostatic pressure caused by motions. So, cells in the bone marrow, such as BMSCs, will inevitably experience the relevant compound stimulation . cDNA synthesis and fluorescence quantitative testing were carried out by Shanghai Daixuan Biotechnology Co., Ltd. Data were analyzed by using the 2-ΔΔCt method, and β-actin was chosen as the housekeeping gene. The primers used in this study are listed in Reverse transcriptase-polymerase chain reaction (RT-PCR) was applied to measure the expression of osteogenic related genes. After 7 days of culture in the microchannels, the total RNA of all samples  was isolated by the MagneSiln = 3 for each group, unless otherwise indicated. One-way ANOVA analysis was applied to evaluate the differences between indicated groups. A difference was regarded as significance when P < 0.05 as usual.All of the statistical data were presented by mean value ± standard deviation, and The microchannel was designed with indicated curve features to increase the investigation region at some extent so that more cells could be observed, as shown in 2) in the microchannel could be calculated by the equation [3/s) is the velocity of perfusion liquid, As the culture medium flowing through the microchannel with varied perfusion rates, different FSS will be applied to the cells cultured on the bottom of the channel. The FSS equation  τw=6μQ/(2) in our study.It is important to make sure that the cells will not be detached from the substrate under perfusion condition when investigating the FSS effect on cell behaviors. In order to promote cell adhesion in the microchannel and hence increasing the range of investigated FSS, the inner surface of all the microchannels were incubated and coated with 25 μg/ml fibronectin solution for 12 h in the incubator. It indicated that after the initial 3 h perfusion in the microchannel, the cell density showed no significant difference between different perfusion conditions and the static control, as shown in In addition to the mentioned microfluidic chip for cell culture and perfusion system for adjusting FSS intensity, the whole compound stimulation platform was consisted of another independent part, which is the ultrasound system, as schematically illustrated in 2, respectively. The cells cultured under static condition were set as control.In order to better understand the role of FSS-ultrasound compound stimulation on stem cell behaviors, the effects of single FSS stimulation on the adhesion, proliferation and osteogenesis of BMSCs in growth medium were firstly investigated. In this study, three typical perfusion rates  were chosen to stimulate the cultured stem cells. Corresponding FSS applied on the cultured cells were about 0.06, 0.19 and 0.56 dyn/cmAfter 4 days of culture under different single FSS stimulations, cell cytoskeleton (F-actin) and nuclei were both stained. Typical fluorescence micrographs of different groups were shown in 2 FSS condition is much higher, as clearly shown in 2) stimulation could effectively promote BMSCs proliferation. However, as the intensity of FSS further increased, cell proliferation was gradually reduced, and even seriously inhibited under the condition of 0.56 dyn/cm2. It is probably because that the cell viability would be seriously reduced under higher FSS intensity. Similar inhibition of cell proliferation by higher FSS stimulation have also been reported by other literatures [Compared with the static control, the cell density under 0.06 dyn/cmeratures , 66.In addition, the cell spreading under tested FSS stimulations were all significantly increased when compared with the static culture condition see , while tet al. [2), while increased obviously under 0.06 dyn/cm2 FSS stimulation, as shown in 2) due to its optimal condition for cell proliferation , the expression of ALP was significantly increased, therefore presented a dark blue color (see 2) when compared to static control, as shown in 2 FSS was almost similar to that of the 0.06 dyn/cm2 FSS (no significant difference), both significantly upregulated when compared to the static control, while the group of 0.56 dyn/cm2 FSS presented similar level to the static control. As for the expression of Col I, the groups with higher FSS stimulations (0.19 and 0.56 dyn/cm2) were both obviously downregulated when compared to the static control, while the group of 0.06 dyn/cm2 FSS was still significantly upregulated. Taken together, proper FSS stimulation (0.06 dyn/cm2) has obviously enhanced stem cell osteogenesis, as the intensity of FSS further increased, the expressions of osteogenic specific genes were gradually decreased.The staining results of ALP and statistical results of specific genes expression were presented in olor see . In addi2 FSS (see 2). Under the stimulation of 0.19 dyn/cm2 FSS, the cell density and cell–cell contact were relatively lower than that of the 0.06 dyn/cm2 FSS group, while the spreading area of the cell was similar. Consequently, the expression of characteristic gene became lower. Under the stimulation of 0.56 dyn/cm2 FSS, cell proliferation was significantly inhibited . So, the perfusion condition was fixed as 5 μl/min to investigate the effects of FSS plus ultrasound stimulations on the adhesion, proliferation and differentiation of stem cells. The intensities of combined ultrasound in this study were 0.075, 0.5 and 1.75 W/cm2, and corresponding compound stimulation groups were denoted as I, II and III, respectively.Cells in the body are often exposed to a complex microenvironment with varied stimulations. Herein, FSS plus ultrasound stimulations were selected as a model to investigate the effects of compound stimulation on stem cell behaviors for the first time. Based on the evaluation of single FSS stimulation, it was found that both the proliferation and osteogenic differentiation could be significantly enhanced under the proper intensity of FSS stimulation (0.06 dyn/cm2), the combination of ultrasound stimulation further increased cell proliferation as the ultrasound intensity increased, as shown in Typical fluorescence micrographs of stem cells and corresponding statistical results of cell density, cell spreading and cell aspect ratio distribution after 4 days of culture with compound stimulations were shown in 2), cell spreading showed no further significant changes under the compound stimulation , as shown in 2.In order to reveal the effect of compound stimulation on osteogenic differentiation of stem cells, the expressions of OPN and Col I were both characterized after 7 days of culture in growth medium under FSS plus ultrasound stimulations. Relative gene expressions were illustrated in 2, the gene expression could be improved. Therefore, combined ultrasound stimulation in this study may afford another independent factor (different from cell spreading and cell–cell contact) for comprehensively regulating the osteogenic differentiation of stem cells. Under relatively lower ultrasound intensity, it will inhibit the cell osteogenesis to a certain extent, while at a relatively higher stimulation intensity it will act as a promoter for stem cell osteogenesis.Compared with the single FSS stimulation, the cell density and cell–cell contact were increased as the ultrasound intensity increased under compound stimulation, while cell spreading area presented no obvious changes. In addition, when compared to the static group, both the cell spreading and cell–cell contact were increased in the compound stimulation groups. From the perspective of the cell–cell contact and cell spreading effects on BMSCs osteogenesis reported before , the ost2) of single FSS stimulation was found to be beneficial both for cell proliferation and osteogenesis, while this kind of enhancing effect gradually attenuated or even evolved into negative effect as the FSS intensity further increased. Based on this suitable FSS stimulation (0.06 dyn/cm2), stem cell proliferation was further enhanced with the increase in the intensity of combined ultrasound stimulation. In addition, stem cell osteogenic differentiation could also be further promoted under the compound stimulation with a higher ultrasound intensity. Corresponding results illustrated that both the FSS and ultrasound stimulations could profoundly regulate stem cell behaviors in an intensity-dependent manner. Under proper combination strategies, both the proliferation and differentiation of stem cell could be obviously enhanced. The construction of compound stimulation platform and corresponding revealed effects on stem cell behaviors are valuable for guiding the design of novel cell bioreactors. Furthermore, these findings might also be meaningful for the prevention and treatment of bone tissue related diseases or defects.In summary, we have constructed a compound stimulation (FSS plus ultrasound) platform suitable for cell studies based on a microfluidic chip. Compared to static culture condition, a suitable intensity (0.06 dyn/cmREGBIO online.rbab066_Supplementary_DataClick here for additional data file.


ZBED9 associated with HTN in the genome-wide borderline threshold after adjusting for different sets of environmental predictors. Moreover, strong signals on ABHD17C and suggestive signals on FBN1 were detected for DBP and SBP, respectively, while these signals were not consistent in different GWA analysis. Our finding on ZBED9 was confirmed for all BP traits by linkage analysis in an independent sample. We found significant associations with similar direction of effects and allele frequency of genetic variants on ZBED9 with DBP (genome-wide threshold) and HTN  in GWAS summary data of UK Biobank. Although there is no strong evidence to support the function of ZBED9 in blood pressure regulation, it provides new insight into the pleiotropic effects of hypertension and other cardiovascular diseases.High blood pressure is the heritable risk factor for cardiovascular and kidney diseases. Genome-wide association studies(GWAS) on blood pressure traits increase our understanding of its underlying genetic basis. However, a large proportion of GWAS was conducted in Europeans, and some roadblocks deprive other populations to benefit from their results. Iranians population with a high degree of genomic specificity has not been represented in international databases to date, so to fill the gap, we explored the effects of 652,919 genomic variants on Systolic Blood Pressure (SBP), Diastolic Blood Pressure (DBP), and Hypertension (HTN) in 7694 Iranian adults aged 18 and over from Tehran Cardiometabolic Genetic Study (TCGS). We identified consistent signals on  In the analysis of world data, one in three adults is expected to be affected by high blood pressure up to 2025, while macro or microvascular complications are not limited to extreme ranges of systolic and diastolic blood pressures (SBP and DBP)3. Elevated BP is categorized into primary and secondary hypertension based on disease etiology. While the latter is secondary due to other diseases, some medications, and related side effects, the former is known as essential hypertension and is prevalent in all populations5.High blood pressure as a heritable risk factor with direct and indirect effects on the incidence of cardiovascular diseases (CVD), stroke, and chronic renal failure is the leading cause of morbidity and mortality worldwide7. Emerging new genotyping technologies has facilitated studies with Genome-Wide Association (GWA) design in non-communicable and infectious diseases to detect risky or preventive genetic variants after adjusting for environmental risk factors. Promising results from these hypothesis-free studies addressing different pathophysiologic pathways and gene targets for future pharmacologic interventions in complex diseases8.Genetic and environmental risk factors such as age, obesity, and lifestyle are attributed to the heterogeneity of essential hypertension risk in the general population10.The majority of the genomic findings on BP traits show pleiotropic associations. Until recently, these findings were predominantly from Europe-centric studies, have not been translated into understanding of underpinning molecular pathways, new drug development and personalization of HTN treatment in diverse populations with different genetic backgrounds11. A recent review addressed evidence concerning the origin of non-generality of GWAS findings on BP traits in different ethnic groups. Accordingly, a large proportion of GWAS was conducted in individuals with European ancestry, and it was introduced as a major cause of low reproducibility of findings in other populations12.From the viewpoint of genomic architectures, different distribution of risk allele frequencies are contributed to the non-generality of findings between ethnicities14. High blood pressure is main risk factor for CVD events in the Iranian population and its prevalence among CVD cases aged over 18 years is between 17.3% to 20%16. In comparing super populations in 1KG project including, Europeans, East Asians, South Asians, Africans and Hispanics, a large number of novel variants with a higher degree of genomic specificity were identified in the Iranian population17. Heritability estimations on BP traits provide evidence to support role of genetic variants are playing on BP regulation in this population18. However, the Iranian population has not been represented in international genomic databases to date, so there is no evidence concerning the effect(s) of genetic variants in this population. To fill the gap, we conducted the first study using GWA design to investigate the replication or discovery of genetic variants on the Iranian population's BP traits.Iran has experienced the epidemiological transition from communicable to non-communicable diseases during the last four decades, and higher prevalence rates of pre-hypertension (47.3%) and hypertension (22.6%) have been reported in both adults and adolescents in recent years20.The Tehran Cardiometabolic Genetic Study (TCGS) is a family-based genetic analysis of the Tehran Lipid and Glucose Study (TLGS) as the oldest Iranian cohort. In total, more than 20,000 participants are followed in 7 phases since 1999 and underwent a clinical examination on more than 230 metabolic-related traits in each phase of the study. The Medical Ethics Committee of Shahid Beheshti University of Medical Sciences approved this study and all methods were performed in accordance with the relevant guidelines and regulations. All participants gave written informed consent to participate in the original cohort and TCGS. In the case of younger participants, written formal consent was obtained from the parents or guardians. Principles for clinical investigations could be found in the original TCGS and TLGS papersIn the present study, 17,462 participants from the five phases of TCGS (1999–2014) were recruited to investigate hypertension. More details on DNA sample collection, genotype quality control process, phenotype measurements, covariate imputation, case definition, and selection criteria are explained in the supplementary information 1.21 . Moreover, related subjects with Identity By Decent (IBD) ≥ 18.5% were excluded from the study. In the genotype level, variants with minor allele frequency (MAF) < 1%, missing genotype calls > 5%, and Hardy Weinberg Equilibrium (HWE) P value < 1e−6 for quantitative traits, P value < 1e−10 in the HTN cases and P value < 1e−6 in controls were filtered out  of genotyping data was implemented on a per-individual basis before per-marker using PLINK version 1.9 and R2 > 0.8), linear and logistic regression tests were performed. To explore any associations between BP traits and genetic variants, the covariates and top 5 Principal Components(PC) were adjusted and additive and overdominant inheritance models on autosomal chromosomes were checked for GWAS on the quantitative and binary traits in PLINK v1.9, respectively. A conventional genome-wide threshold of 5 × 10–8 considered for a significant P value. The genomic inflation factor was computed for each analysis, and observed versus expected P values were highlighted in the Q–Q plots to check for population stratification. Finally, four regression-based multivariate analyses evaluated the predictive accuracy of initial GWAS outputs for quantitative traits on discovery dataset and also an independent sample of TCGS (N = 2799)24. Accordingly, Polygenic Risk Score (PRS) was calculated after adjusting for the covariates and top 5 PCs, to evaluate proportion of variance, which is explained by genomic variants (R2). We used a standard pipeline to evaluate R2 through calculating PRS on our GWAS summary data. Moreover, discrimination of best fit PRS was assessed for each analysis according to sex. The PRS was computed in the following steps by PLINK and R:i.2 > 0.25 were filter out,Pruning: A window size of 200 variants, sliding across the genome with step size of 50 variants at a time were considered, and any SNPs with rii.2 > 0.2) with P value ≤ 1 and only index SNP captured,Clumping: SNPs within 250 k of the index SNP were considered for clumping. Then, all SNPs are correlated with each other were removed  for inclusion of SNPs in the PRS was extracted,Seven iv.P values for SBP and DBP,PRS generated in different ranges of v.P values thresholds,To find "best-fit" score for the quantitative traits, PRS calculated in different ranges of vi.2 as an index of trait variance explained by genomic variants calculated in different ranges of P values thresholds for two sets of covariates, ,Rvii.2 was plotted by different ranges of P values using "ggplot2",Calculated Rviii.2 by sex using "ggplot2".The best-fit PRS was plotted against quantitative traits based on best result of RAfter checking collinearity for all covariates  with a r2 > 0.2, the effects of significant independent SNPs in the initial GWAS were tested in the presence of the same covariate sets using two-level Haseman-Elston regression model by SAGE version 6.426. This model-free linkage analysis is mathematically equivalent to the likelihood-based score test in variance component linkage analysis, which compute a random effect for genetic variants with T score test.The confirmation study was conducted on 1618 participants in 210 selected TCGS families with an age range of 1 to 93 and familial aggregation of two or more affected (HTN) casesP values less than 1 × 10–4. In the first step, chromosomal coordinates, genes, transcripts, and variants on protein sequence were annotated in Ensemble Variant Effect Predicator27. In the second step, GWAS catalog information was retrieved to identify the association of specified loci on BP traits28. Moreover, we sought to map known BP loci by assessing these loci's functional consequences in Ensemble29. Functional analysis was performed for those variants with consistent results and at least a P value less than 5 × 10–7 after adjusting for different covariate sets. In the case of a similar locus, ldlink browser by National Cancer Institute and LinDA (LINkage Disequilibrium-based Annotation) browser were checked for LD of detected and previously reported variant(s) in all populations using the website http://analysistools.nci.nih.gov/LDlink/ and http://linda.irgb.cnr.it/, respectively. In the third step, a list of detailed information on all loci was retrieved separately in Open Targets POST GWAS, including disease associations, protein interactions, pathways, similar targets based on diseases in common, RNA, and protein baseline expression by the anatomical system and organ30. In the final step, the overall association score was retrieved for each locus and other genes whose protein products interact with new loci through protein interaction networks32. Open Target Platform provides the score from 20 data sources, is ranged from 0 to 1, that the former implies no evidence and later corresponds to the most reliable evidence supporting evidence based on frequency, severity, and significance of association33.Four consecutive steps were followed to explain probable functions and pathophysiologic pathway(s) of discovered variants' effects on BP with Table ABHD17C detected for DBP corresponding to the genome-wide significance threshold. Moreover, suggestive signals within 6p22.1 (ZBED9) and FBN1 with borderline P values detected with the HTN and SBP traits, respectively . The associations of other genetic variants on ZBED9 with QRS amplitude and interval in Europeans and also ABHD17C and FBN1 with BP traits or cardiovascular diseases in Europeans and East Asians were previously reported in GWAS literature. However, there is no correlation (LD) between detected and previously reported genetic variants in the same locus  and HTN  in UK Biobank, while the associations were not significant  in Japanese population (Table 2 > 0.4) and BP traits in the region. Except for Africans, allele frequencies of detected variants on ZBED9 are similar in Europeans, South Asians, Middle East(Qatari) and Iranian population, while the allele frequencies of detected variants on FBN1 and ABHD17C is less than 1% in Europeans  of detected variants on on Table . We chec2 in the independent sample of TCGS was much lower compared to discovery dataset (≤ 1%) and there was low correlation between the PRS and BP traits by sex . In further investigation via sentinel variants, we found rs853684 on ZSCAN31 as a missense consequence in moderate LD (r2 > 0.60) and five missense variants in perfect LD (r2 = 1.0) with detected variants on ZBED9 . Further, the protein interaction network suggests gene subnetworks for ZBED9 with relatively low association with BP and cardiovascular diseases , respectively.The cumulative evidence for association with BP traits for three detected loci was retrieved in the Open Targets POST GWAS. The overall association scores were summarized in Fig. ZBED9, FBN1, and ABHD17C, associated with HTN, SBP, and DBP, respectively. We found significant associations with similar direction of effect and allele frequency of detected variants on ZBED9 with DBP (genome-wide threshold) and HTN  in UK Biobank. However, There is no LD between detected and previously reported genetic variants on three loci in Europeans and other populations including, South Asian, Hispanic or Latin American, East Asian, African American, and Afro-Caribbean populations. The detected signals were not consistent after adjusting for two different sets of covariates on FBN1 and ABHD17C. On the other hand, the association of genetic variants on ZBED9 and BP traits has not been previously reported in other populations, while their effects were consistent after controlling for environmental factors by two different GWA analysis. Further, the confirmed association of genetic variants on ZBED9 in discovery study with SBP, DBP, and HTN in an independent sample of TCGS families substantially increase our knowledge of its effects on HTN in the Iranian population.To our knowledge, this is the first GWAS to examine genetic associations with BP traits in the Iranian population. This study identified suggestive and strong signals on 30.In an effort to explain the function of new loci by RNA and protein baseline expression, the rate of expression for all detected loci was in the range of low to moderate in the circulatory system, including blood, endothelial cells of the umbilical vein, aorta, coronary artery, left heart ventricle, tibial artery, atrium auricular region, and heart muscle, provided by Human Protein Atlas and Expression AtlasZBED9 on BP regulation in the literature. However, GeneHancer-gene association results indicate some known BP loci, including EBF1, NR2F2, SOX5, and PRDM6, are likely acting as transcription factor binding sites or gene targets for ZBED946. In this way, significant association with QRS interval and amplitude as a surrogate of myocardial mass in European population41 and replication with SBP, DBP and also HTN using different criteria based on reference adjusted curves in the Iranian children and adolescents47, who was included in TCGS confirmation study is a signal for further investigations on the functional role of ZBED9 in BP regulation pathway in other populations.There is no evidence to support the functional effects of 48. Accordingly, gene-set-based analysis by Open Target Post GWAS highlighted the probable pleiotropic effects of detected loci, which are likely acting as a common genetic etiology for BP and cardiovascular diseases. Accordingly, the lethal cardiovascular diseases may affect allele frequency of effective variants with advancing age in our study due to survival reduction, so overlook the effect of functional variants in older groups as a competing risk50.High blood pressure is directly associated with vascular mortality due to stroke, cardiomyopathy, aneurysm, cardiac hypertrophy, aorta stenosis, sudden cardiac arrest, and myocardial infarction51. However, there was inadequate statistical power to conduct GWAS by sex after removing related individuals in our study. Moreover, the calculated PRS for the quantitative traits showed similar patterns by sex. Second, despite the high predictive accuracy of PRS by genomic variants and confirm significant SNPs on ZBED9 with family-based regression analysis, it was a case for overfitting due to selecting individuals with similar genetic background from the TCGS cohort. Accordingly, our findings validated in an independent sample of the Iranian population. However, our results still needs to be cross-validated in a different sample of the Iranian population as well52. Third, using a family-based design for GWAS has the advantages of complete robustness against genetic heterogeneity. However, family-based designs are likely biased due to population substructures, and association tests yield at the price of inflated type I error and reduce statistical power54. In this way, we included independent samples from a family bases cohort.We acknowledge that some limitations are evident in our study. First, there is evidence for significant differences in the magnitude of genetic variants effects on high blood pressure between men and women55. Finally, we found signals which were not in LD with previously reported genetic variants on BP traits. Further investigation is necessary to fine mapping of these regions when imputed variants on the Iranian genome become available56.Additionally, there was no association between autozygosity and BP quantitative traits in an international meta-analysis, including TCGS families with a relatively high inbreeding rateZBED9 and BP traits has not been previously reported in other populations. Their effects were consistent after controlling environmental factors by two different GWA analyses and confirmed in a family-based linkage study. Although there is no strong evidence to support the function of ZBED9 in blood pressure regulation by Open Target Post GWAS, its association with QRS interval and QRS amplitude as surrogates of myocardial mass may provide new insight into pleiotropic effects of hypertension and other cardiovascular diseases.We identified three loci, of which two loci were previously reported in individuals with European and Non-European ancestries. The association of genetic variants on Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.