Here, active Mpro was obtained as a 34.5 kDa protein by overexpression in E. coli BL21 (DE3). The optimal pH and temperature of Mpro were 7.5 and 37 °C, respectively. Mpro displayed a mK value of 16 μM with Dabcyl-KTSAVLQ↓SGFRKME-Edans. Black garlic extract and 49 polyphenols were studied for their inhibitory effects on purified Mpro. The IC50 values were 137 μg/mL for black garlic extract and 9–197 μM for 15 polyphenols. The mixtures of tannic acid with puerarin, daidzein, and/or myricetin enhanced the inhibitory effects on Mpro. The structure–activity relationship of these polyphenols revealed that the hydroxyl group in C3′, C4′, C5′ in the B-ring, C3 in the C-ring, C7 in A-ring, the double bond between C2 and C3 in the C-ring, and glycosylation at C8 in the A-ring contributed to inhibitory effects of flavonoids on Mpro.The main protease (M Due to rapid dissemination and deaths, COVID-19 was declared a pandemic for the first time by WHO on 11 March 2020 [A new coronavirus disease, termed coronavirus disease 2019 (COVID-19), was first identified in Wuhan, China in December 2019 . COVID-19 has spread to 235 countries resulting in 81,947,503 confirmed cases and 1,808,041 confirmed deaths by 2 January 2020 according to the World Health Organization (WHO) (pro) or 3C-like protease and papain-like protease [pro is considered one of the important targets for blocking viral replication. At present, there are no antiviral drugs on the market for COVID-19 treatment [clinicaltrials.gov) (accessed on 11 October 2020).COVID-19 is caused by a novel severe acute respiratory syndrome-related coronavirus (SARS-CoV-2)  which beprotease . Thus, Mreatment ,11. Morepro. Black garlic has a higher total phenolic and total flavonoid content than raw garlic [pro of SARS-CoV [pro sequence identity between SARS-CoV and SARS-CoV-2 was found to be 96% [pro of SARS-CoV-2 were confirmed [pro and their structure-activity relationship. In this study, the Mpro of SARS-CoV-2 was overexpressed in E. coli BL21(DE3) using IPTG as an inducer and purified using Ni-NTA chromatography. Its proteolytic activity was confirmed by using Dabcyl-KTSAVLQSGFRKME-Edans (FRET) as a substrate. The optimum pH, pH stability, optimum temperature, and temperature stability of SARS-CoV-2′s Mpro were studied. Also, the Michaelis–Menten constant (mK) and the effect of DMSO concentration on Mpro activity were studied. Then, the inhibitory effects of black garlic extract and 49 polyphenols on Mpro and their structure–activity relationships were investigated. The 50% inhibitory concentration (IC50) of black garlic and 15 polyphenols were determined. In addition, the effects of mixtures of polyphenols  on Mpro activity were studied.Nutraceuticals are foods that provide medical or health benefits such as avoiding side effects, protecting human health against chronic diseases, improving human health, and are economically affordable ,13,14,15w garlic . Black gw garlic . Besidesw garlic ,19,20,21o be 96% . Also, tonfirmed . Therefopro were optimized for expression in E. coli through a custom gene synthesis service of Genscript. It was expressed in E. coli BL21(DE3) by using 1.0 mM IPTG at 16 °C for 12 h, purified using Ni-Sepharose chromatography, and confirmed by SDS-PAGE  in 50 mM Tris-HCl pH 7.5. The purified Mpro reached maximum activity at pH 7.5  DMSO (v/v). At 50% (v/v) DMSO, Mpro activity was not detected (Dimethylsulfoxide (DMSO) is a potent organic solvent that dissolves a variety of organic compounds due to its high dielectric constant and stereochemistry . Althoug studied . Mpro wa/v) DMSO . Then, idetected .pro activity at 0.5 mg/mL. The IC50 value of extracted garlic acid was 137 ± 10 μg/mL , vitexin, daidzein, puerarin, resveratrol, tannic acid, chlorogenic acid, and caffeic acid showed over 50% inhibitory activity on Mpro. The compounds exhibiting more than 50% inhibitory activity on Mpro were selected to determine IC50. Their IC50 values were from 9 to 197 μM  substitution in the B-ring on Mpro inhibitory effects were evaluated. The inhibitory effects of quercetin, which has 3′-OH and 4′-OH at the B-ring, and kaempferol, which has 4′-OH group at the B-ring, were lower than that of myricetin which has 3′-OH, 4′-OH, and 5′-OH groups at the B-ring. The absence of 3′-OH and 4′-OH groups at the B-ring was the reason for the lower inhibitory activity of kaempferol and quercetin than myricetin. When quercetin was glycosylated at C4′ in the B-ring (quercetin-4′-O-α-glucopyranoside) and C3 in the C-ring (rutin), its inhibitory effect was decreased. The presence of the OH group at C6 in the A-ring of quercetagenin decreased the inhibitory activity compared to quercetin. Therefore, the OH at C6 in the A-ring, the C3′, C4′, C5′ in the B-ring, and the C3 in C-ring affected Mpro inhibitory activity. Ampelopsin is dihyromyricetin. The existence of double bonds between C2 and C3 in the C-ring of myricetin was the reason for the higher inhibitory activity of myricetin than ampelopsin.First, the inhibitory activity of compounds in the sample group was compared. In the flavonol group, the order of Mpro inhibitory activity was as follows: naringin < hesperidin < naringenin. Naringenin is glycosylated naringin. However, its inhibitory activity was 3.2-fold higher than that of naringin. Hesperidin that contained glycosylation at 7-OH at the A-ring like the naringenin and the methoxy group at position 5′ of the B-ring was shown to have higher inhibitory activity than that of naringin but lower inhibitory activity than that of naringenin, indicating that glycosylation at the C7 position enhanced the Mpro inhibitory effect. In contrast, the methoxy group at C5′ in the B-ring reduced its inhibitory activity.In the flavanone group, the order of Mpro inhibitory activity than that of the remaining compounds , suggesting that the OH group at C3′, C4′, C5′ in the B-ring increased the inhibitory activity against Mpro, similar to the compounds in the flavonol group. EGCG, GCG, CG, and ECG, which have a galloyl moiety at C3 in the C-ring, showed higher Mpro inhibitory activity, suggesting that galloyl moiety at C3 of the C-ring increased the inhibitory activity on Mpro. Thus, the inhibitory activity of gallic acid on Mpro was studied. Gallic acid inhibited 6.9% Mpro activity at 200 μM. Besides gallic acid, we also tested the inhibitory effects of tannic acid composed of 10 galloyl moieties. Tannic acid inhibited 100% Mpro activity and its IC50 value was 9 μM. Compared to myricetin, EGCG, which has the same three OH groups in the B-ring, galloy moiety at C3 in the C-ring, no double bonds between C2 and C3, and no C4 = O in the C-ring, displayed decreased inhibitory activity on Mpro.In the flavan-3-ols group, the order of the inhibitory effect was as follows: epicatechin (EC) < catchin < epicatechin gallate (ECG) < catechin gallate (CG) < epigallocatechin (EGC) < gallocatechin gallate (GCG) < epigallocatechin gallate (EGCG) . EGCG, Gpro was as follows: chrysin < apigenin < luteolin < vitexin. Chrysin, lacking the OH group in the B-ring, exhibited the lowest inhibitory activity on Mpro. Compared to the three compounds , we found that the more the OH group increased in the B-ring, the more the inhibitory effects on Mpro increased. While with attacked glucosyl moiety at C8 in the A-ring, the inhibitory activity on Mpro increased by 2-fold that of apigenin. The difference between flavone and isoflavone is the linked position of B-ring to C-ring. Compared to apigenin, which contained the same OH group in the B-ring but the B-ring was linked in position C3 in the C-ring and lacked the OH group at C5 in the A-ring, daidzein showed an increased inhibition effect on Mpro. Puerarin, which contained the 8-C-glucoside of daidzein, exhibited a slightly higher inhibitory effect on Mpro than that of daidzein. From the inhibitory effect of compounds from flavone and isoflavone, we concluded that the glycosylation at the C8 position increased the inhibitory effect on Mpro.In the flavone group, the order of inhibitor activity on Mpro than bisdemethoxycurcumin which lacked the methoxy group. However, its inhibitory activity was lower than that of dimethylcurcumin, which contained one methoxy group in C2′.In the diarylheptanoid group, the order of inhibitory effects was as follows: bisdemethoxycurcumin < curcumin < dimethylcurcumin. In this group, curcumin contained two methoxy groups (C2′ and C4”) and showed higher inhibitory activity on Mpro SARS-CoV2 but also transmembrane protease serin 2 (TMPRSS2) [pro activity. The concentration of tannic acid was fixed at 5 μM, while the concentrations of myricetin, puerarin, and daidzein were fixed at 20 μM. The inhibitory effects of mixing tannic acid with myricetin, puerarin, and/or daidzein on Mpro are shown in pro were 30 ± 1% at 5 μM tannic acid, 23 ± 5% at 20 μM puerarin, 27 ± 3% at 20 μM daidzein, and 34 ± 1% at 20 μM myricetin. The inhibitory effects increased to 41 ± 2% at 5 μM tannic acid with 20 μM puerarin, 50% at 5 μM tannic acid with 20 μM daidzein, and 58 ± 2% at 5 μM tannic acid with 20 μM myricetin. The Mpro inhibitory activities of flavonoids at 45 μM were 50.8 ± 2% for puerarin, 43% for daidzein, and 50.5 ± 4% for myricetin, while they were 62 ± 6% at 5 μM tannic acid with 20 μM puerarin and 20 μM daidzein, 69 ± 2% at 5 μM tannic acid with 20 μM puerarin and 20 μM myricetin, and 69 ± 2% at 5 μM tannic acid with 20 μM puerarin and 20 μM myricetin. The highest inhibitory activity on Mpro (77 ± 1%) was achieved at 5 μM tannic acid with 20 μM puerarin, 20 μM daidzein, and 20 μM myricetin.Herbal medicines have been used to treat COVID-19 in China . AccordiTMPRSS2) . AlthougTMPRSS2) ,30,31. Tpro is one of the best-characterized targets for antiviral drug discovery. It plays an essential role in virus replication by digesting the viral polyproteins at more than 11 sites on the large polyprotein 1ab. The recognition sequence at most sites is LQ↓ (the cleavage site is indicated by ↓) [pro [pro activity will block viral replication, we selected SARS-CoV2 Mpro as the target protein and Dabcyl-KTSAVLQ↓SGFRKME-Edans as its commercially available substrate. The biochemical characterization of Mpro showed that the optimum temperature, pH, and mK value were 37 °C, pH 7.5, and 16 μM, respectively. The mK value of the purified Mpro was similar to that of the SARS-CoV 3CLpro expressed in Pichia pastoris with 15 ± 1 μM [pro expressed in E. coli with 17 ± 4 μM [v/v). At up to 10% (v/v), we found no significant effects on SARS-CoV2 Mpro activity, but over 10% (v/v), DMSO significantly inhibited Mpro activity. At 50% (v/v) DMSO, Mpro lost its activity. No significant effect of 10% (v/v) DMSO on SARS-CoV2 Mpro activity was observed, similar to that on SARS-CoV 3CLpro activity [The SARS-CoV-2 Med by ↓)  similar  ↓) [pro ,34. Sinc5 ± 1 μM  and to t7 ± 4 μM . We testpro [50 of 411 μg/mL, reduced the production of pro-inflammatory cytokines, and affected particle morphology of the virus [pro as well as their structure–activity relationship. Garlic is a popular medicinal food owing to its numerous health benefits  [pro was possibly owing to its phenolic acids  and flavonoid compounds  [Polyphenols are a large family of phytochemicals with great chemical diversity as well as potential therapeutic diversity. Many polyphenols act as multi-target agents of high biochemical specificity and chemical diversity with lower cost, more covering mechanism, and minimal side effects. As one of the plant derivate polyphenols, curcumin down-regulated the coactivator of HBV transcription, peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC-1α), and inhibited HBV gene replication and expression . In anotpro . Thus, ppro ,38,39. Tpro ,40,41. Hpro . It inhienefits) . Howeverenefits) , overcamenefits) ,44. Alsoenefits) . From theratrol) .pro, their efficiencies against Mpro need to be confirmed by in vitro and in vivo experiments. To the best of our knowledge, among our tested polyphenols, EGCG and tannic acid have been reported to have inhibitory effects on Mpro with IC50 values of 7.6 μg/mL and 2.1 μM, respectively [pro with IC50 values of 171 μM and 9 μM, respectively , hesperidin , naringin , EGCG , catechin , myricetin , and caffeic acid , had the highest binding energy and hydrogen-bonding interactions with key amino acid residues of Mpro [pro  by Genscript . Mpro was inserted into the pET28a vector (pET28a-Mpro)  for overexpression of Mpro protein with polyhistidine tags. pET28a-Mpro was transformed into E. coli BL21(DE3) using the heat shock method and then spread on LB agar containing (50 μg/mL) (Sigma). The plate was inoculated at 37 °C overnight. LB supplemented with kanamycin was used for a single colony grown at 37 °C. One mM isopropyl β-d-1-thiogalactopyranoside (IPTG) (Sigma) was used to induce Mpro when the optical density (OD600) became 0.5. Induced cells were cultured at 16 °C with shaking (200 rpm) for 12 h. Cells were harvested by centrifugation (8000× g for 30 min at 4 °C), resuspended in 50 mM Tris-HCl pH 7.0, and lysed by sonication . After sonication, cell lysate was obtained by centrifugation  and then loaded onto 12 mL of Ni-Agarose resin . A buffer containing 50 mM Tris-HCl, 100 mM NaCl, 300 mM imidazole, pH 8.0 was used to elute proteins from the Ni-Agarose column. Fractions containing purified proteins were obtained and dialyzed against a 50 mM Tris-HCl buffer (pH 7.5). Protein concentration was determined using a protein determination kit , and 12% SDS-PAGE was used to confirm the purity of the protein.SARS-CoV-2 Mpro was measured by using fluorogenic peptide (Dabcyl-KTSAVLQ↓SGFRKME-Edans) (FRET) as a substrate . The reaction mixtures composed of 20 μM of FRET substrate and 8 μg of Mpro in 50 mM Tris-HCl (pH 7.5) were run at 37 °C for 30 min. Relative fluorescence units (RFUs) were verified with SpectraMax M3  at excitation and fluorescence emission wavelengths of 355 and 538 nm, respectively.Proteolytic activity of Mpro activity was studied at different pHs ranging from pH 3.0 to 11.0 using the following buffers: glycine-HCl , sodium acetate buffer , Tris-HCl buffer , and glycine-NaOH buffer  [pro in each buffer for 30 min using 20 µM of FRET as a substrate at 37 °C for 30 min. RFUs were recorded as described above.The effect of pH on M.5–11.0) . The optpro was carried out by incubation of Mpro for 24 h in the chosen buffers at 4 °C. Then, the pH of Mpro was accustomed to 7.5 by membrane dialysis. Mpro activity was carried out with 20 µM of FRET at 37 °C for 30 min. RFUs were recorded as described above.The pH stability of Mpro activity was performed by reacting a reaction mixture composed of Mpro enzyme and 20 µM of FRET in 50 mM Tris-HCl (pH 7.5) at various temperatures from 4 to 70 °C. RFUs were recorded as described above.The effect of temperature on Mpro temperature stability was performed by keeping the enzyme from 4 to 60 °C for 24 h. Then, the enzyme was added to the reaction digest containing 20 µM of FRET in 50 mM Tris-HCl (pH 7.5). The reactions were done at 37 °C for 30 min. RFUs were verified with SpectraMax M3 as described above.Mpro activity was measured by adding different concentrations of DMSO from 0 to 50% (v/v) to the reaction mixture comprising Mpro in 50 mM Tris-HCl pH 7.5. The FRET substrate was added to initiate the reaction. The control was a reaction without adding DMSO. The reactions were conducted at 37 °C for 30 min. RFUs were monitored as described above.The effect of dimethyl sulfoxide (DMOS) on Mpro was added to different concentrations of FREP substrate (2.5–45 μM) to the reaction mixture composed of Mpro in 50 mM Tris-HCl pH 7.5. The reactions were run at 37 °C for 8 min. Reaction results were linear within this time. The Michaelis–Menten constant (mK) was obtained from the Lineweaver–Burk plot using the Sigmaplot program .The kinetic parameter of Mpro inhibitory activity are listed in pro and a 200 μM inhibitor or 0.5 mg/mL of black garlic extract in 50 mM Tris-HCl (pH 7.5) to start a reaction. The reaction was run at 37 °C for 15 min and RFUs were recorded as above. The inhibition activity of Mpro was calculated as follows:o and C were the RFUs of the control  after 0 and 15 min of reaction and So and S were the RFUs of the assay sample  after 0 and 15 min of reaction. Fifteen compounds and black garlic extract were selected for the 50% inhibitory concentration (IC50) determination. IC50 was the Mpro inhibitor concentration necessary to reduce 50% Mpro activity compared to the reaction without adding an inhibitor. To determine IC50, 16 μL FRET substrate was added to the reaction digest containing Mpro and different concentrations of inhibitors (1–400 μM) in 50 mM Tris-HCl (pH 7.5) to start the reaction as described above.Black garlic extract and 49 compounds that were selected for Mpro in 50 mM Tris-HCl (pH 7.5) as described above.The combinative inhibitory effects of tannic acid, puerarin, daidzein, and myricetin were performed by adding 5 μM of tannic acid with 20 μM of puerarin, and/or daidzein, and/or myricetin to a reaction mixture containing a 16 μM FRET substrate and Mp < 0.05.All experiments were performed in triplicate, and results were expressed as the mean ± standard deviation. Statistical comparisons were made by one-way analysis of variance followed by Tukey’s comparison tests on GraphPad Prism 8 . Values were considered to be significant when pro. The purified Mpro was used to study the inhibitory effect of black garlic extract and 49 polyphenols. Black garlic extract and 15 polyphenols showed the inhibition of Mpro activity with IC50 values of 137 μg/mL and from 9 to 197 μM, respectively. From the structure–activity relationship of these polyphenols, we found that the hydroxyl group in C3′, C4′, C5′ in the B-ring, C3 in the C-ring, C7 in the A-ring, the double bond between C2 and C3 in the C-ring, and glycosylation at C8 in the A-ring contributed to the inhibitory activity of flavonoids on Mpro. Different combinations of tannic acid with puerarin, daidzein, and/or myricetin also increased inhibitory effects on Mpro. Polyphenols are products extracted from plants and found in foods and beverages such as fruits, vegetables, tea, wine. Therefore, further studies of foods containing these polyphenols are needed.Here, we reported the overexpression and biochemical characterization of an active SARS-CoV-2 M


Horse hoof possesses a micro-flora that may promote the development of secondary infections following wounds or surgery. For this reason, in vitro and in vivo experiments were used to verify and compare the bactericidal power of two well-known disinfectants, Povidone-iodine (PI) and Chlorhexidine (CHx), and a new stable colloidal suspension of iron oxide, called Iron Animals (IA). In vitro, IA was able to completely suppress the bacterial growth on all of the tested microorganisms. In vivo, PI alone possessed the lowest efficacy for hoof disinfection; CHx resulted as the best treatment in 18% of cases. CHx + IA emerged as the best disinfection protocol for equine hoof achieving the lowest bacterial load in 55% of cases. The addition of IA, after PI or CHx, improved both disinfectants’ effectiveness, leading to the highest bactericidal activity in 82% of cases. IA would deserve a chance as a possible solution in pre-surgical disinfection of the equine hoof, reducing the risk of infection. In vitro, IA completely suppressed the bacterial growth of all the tested microorganisms, resulting in effectiveness also against CHx-resistant bacteria, such as Staphylococcus aureus. In vivo, PI emerged as an ineffective protocol; CHx was effective in 18% of cases, but with the addition of IA (CHx + IA) its use emerged as the best disinfectant protocol for horse hoof, achieving the lowest bacterial load in 55% of cases. The addition of IA, after PI or CHx, improves the effectiveness of both disinfectants leading to the highest bactericidal activity in 82% of cases. The presence of bacteria of various origins on horse hoofs enables the onset of infections following trauma or even post-surgical wounds. Thus, the analysis of new antibacterial substances is of fundamental importance. In this study, the antibacterial efficacy of Iron Animals (IA), a stable colloidal suspension of iron oxide, organic acids, and detergents, was tested in vitro and in vivo. In vitro assays were performed to test the unspecific inhibitory effect of IA on both gram-positive and gram-negative bacteria monitoring the microorganism growth by spectrophotometry  at 37 °C for 24 h. In vivo test consists on the quantification of the bacterial load in colony forming units per gram (CFU/g) of specimens collected from the frog region of the anterior hooves of 11 horses. Sampling followed the application of four disinfectant protocols consisting of two consecutive 3 min scrubs with 50 mL of 10% Povidone-iodine (PI) or 4% Chlorhexidine (CHx), with or without an additional application for 15 min of 10 mL of Iron Animals (PI+IA and CHx+IA).  Staphylococcus epidermidis, S. aureus, α-Streptococcus viridans, Micrococcus spp., Bacillus spp., and Corynebacterium spp.); (2) gram-negatives of fecal origin ; (3) hoof-capsule residents (Acinetobacter spp.) [The equine hoof is exposed to a variety of microorganisms, which can be subdivided into 1) skin inhabitants, gram-positive bacteria ( skin inhm spp.);  gram-negxytoca);  hoof-caper spp.) . The disinfection of a wound or a surgical site allows the reduction of the microflora aimed at diminishing the risk of secondary infections. However, hoof disinfection is difficult for the presence of foreign materials, crevices, and the periople, the external lipid-rich layer of the hoof ,5. MoreoThe most widespread hoof disinfectants remain Povidone-iodine (PI), Chlorhexidine (CHx), iodine tincture, and Lugol solution ,6,8,9,10The final aim of this study is to evaluate the effectiveness of colloidal iron oxides on the enhancement of the bactericidal activity of two common disinfectants (PI and CHx) on the horse hoof.Staphylococcus aureus ATCC 29213, S. aureus 393, Pseudomonas aeruginosa L2, and P. aeruginosa L4L were used for the experiments. The strains were plated on Mueller Hinton Agar  and incubated at 37 °C for 19 h. Pure colonies were suspended in 10 mL Mueller Hinton Broth  and diluted until the concentration was 5.0 Log10 colony forming unit/mL (CFU/mL). The microbroth dilution technique of antimicrobial susceptibility was performed to test the inhibitory effect of the formulations on the bacterial growth in comparison to controls. Dilutions were carried out using sterile deionized water in 96-well flat-bottom microtiter plates and were incubated with standardized inoculum (100 mL). Control experiments on bacteria were carried out by simple addition of sterile deionized water. The microtiter assays were incubated at 37 °C and the optical density (OD600), namely the UV-vis absorbance (ABS) at 600 nm, was continuously monitored by spectrophotometry  for 24 h. Three biological replicates were performed for each strain and two different experiments were duplicated.Standardized inoculum of 2 was collected using a sterile scalpel for microbiological study and then placed in a test tube containing 5 mL of sterile saline solution (0.9% NaCl). In the laboratory, the sample was shaken with vortex and 50 μL of the suspension was used to perform microbiological analysis. The suspension was plated on Nutrient Agar  and incubated at 37 °C for 24 h. In the light of the obtained results, which will be discussed subsequently in A preliminary experiment was made to set up the study and test the efficacy of Iron Animals (IA) in eliminating microorganisms on the horse hoof. The front and rear hooves of one horse were superficially cleaned to remove the dirt mechanically. A fragment of the left front hoof sole was sampled as control and was subjected to microbiological analysis. The right front hoof sole was treated with two consecutive 3 min scrubs with PI, simulating a pre-surgical scrub. After the scrubs, the sole was rinsed with saline solution and dried with sterile gauze before sampling and analyses. The same experiment was made on the rear hoof replacing the PI scrubs with the application of a gauze soaked in 10 mL of IA on the hoof sole for 15 min. A tissue sample of 2 cmThe study involved 11 horses of different breeds (7–19 years old). The tissues were collected from the frog of the front hooves which, together with the sole, is the most contaminated site of the equine hoof . Horses 2 was taken from the frog region with the use of a sterile scalpel for microbiological study (protocol PI). After the sampling, 10 mL of Iron Animals  were applied and left in place. The sole and the hoof were covered with sterile gauzes, wrapped with Vetrap, and put inside a plastic bag. The top of the bandage was sealed with adhesive tape to avoid any external contamination. Until the application of the bandage, the hoof was held off the ground. After 15 min, the dressing was removed and the sole was rinsed and dried as previously described. The second tissue sample was taken from the frog region (protocol PI+IA). The same protocol was accomplished by replacing the PI with 4% CHx  on the right hooves, setting up protocol CHx (2 scrubs with CHx) and CHx+IA .The left front hoof of each animal was cleaned with a hoof pick and trimmed; then, two consecutive 3 min scrubs were made using a firm sterile brush with 50 mL of 10% PI . At the end of each scrub, the hoof was rinsed with sterile saline solution (0.9% NaCl). The sole was then dried with sterile gauzes and a tissue sample of about 2 cmThe collected tissue samples were placed in individual test tubes previously weighed and containing 5 mL of sterile saline solution (0.9% NaCl). In the laboratory, the sample weights were calculated by carrying out a second weighing using a scientific balance  and evaluating the difference in grams between the first weighing of the tube and the second one . Each saEach sample, contained in 5 mL of sterile saline solution, was mixed by vortex to spread all the microorganisms in the solution and serial 10-fold dilutions were performed. An aliquot of 100 μL from each dilution was plated on the Nutrient Agar  and incubated for 24 h at 37 °C to determine the bacterial counts as the number of CFU/g of the specimen. p < 0.05 was considered to be statistically significant.For the preliminary study and in vitro test, descriptive observations were reported. Data obtained by the in vivo sampling and laboratory analyses of quantitative microbiology were performed using the SAS 9.4 software . Comparisons among protocols were performed using the non-parametric Kruskal-Wallis test, due to a not-normal distribution of CFU/g. For simplicity, the average CFU/g values obtained from the protocols PI and CHx, in the presence and absence of IA, were normalized for the residual bacterial load achieved in PI protocol, which was used as a reference procedure and was referred to as 100%. The number of cases in which one protocol was better than the others in reducing the residual bacterial load for each horse was analyzed using the Chi-square comparison test between percentages, using the Marascuilo procedure. Staphylococcus aureus ATCC 29213, S. aureus 393, Pseudomonas aeruginosa L2, P. aeruginosa L4 are presented as controls (broth plus water). It is noteworthy that the application of IA was able to completely suppress the bacterial growth on all the tested microorganisms  . Protocol CHx + IA resulted as the treatment that allows the major reduction of the bacterial load of the equine hoof. Moreover, it resulted in a significant difference from PI treatment (p = 0.03).As expected, CHx resulted in a significant and more efficient bactericidal activity, leading to seven times lower bacterial load, in comparison to PI (= 0.006) . InteresA graphical representation of the number of cases, in which each protocol achieved the lowest bacterial load, is presented in Given the characteristics of hoof structure and the surrounding environment, the pre-surgical disinfection or cleaning of a wound resulted extremely important. Indeed, the presence of microorganisms on the operative surface or the wound bed is one of the most significant risk factors for the development of secondary infections ,5. The mStaphylococcus aureus, gram-positive bacteria was chosen, as it is a matter of concern in the veterinary field and because of its resistance to CHx, which is one of the most common disinfectants in clinical practice. Pseudomonas aeruginosa (gram-negative) was chosen to demonstrate the specific mechanism of IA and its efficiency on both gram-positive and gram-negative bacteria. It is noteworthy that, both these species, are commonly associated with tissue infections [In the in vitro study, fections . The res5 microorganisms/g of tissue for surgical skin wounds [PI is the most common disinfectant reported in the literature and used in practice for the disinfection of the equine hoof, followed by CHx ,22. On tn wounds ,23, but Staphylococcus aureus. Infections by methicillin-resistant Staphylococcus aureus are frequent in the veterinary field and enterococci have been isolated from the hands of veterinary surgeons [The protocol that involves the addition of Iron Animals to two consecutive 3 min scrubs with Chlorhexidine obtains the highest bactericidal power. This result is statistically significant in comparison to PI scrubs, which represent one of the most used protocols for disinfection during veterinary clinical activity ,8. Most surgeons . Indeed,surgeons , alreadysurgeons . The actsurgeons ; (2) a psurgeons ; (3) A dsurgeons . In addiIn light of the results obtained so far, further studies are desirable to (i) assess the potential of IA formulation when used alone and against other microorganisms, such as mycosis or yeasts; (ii) optimize the use of Iron Animals both in terms of time and methods of application; (iii) evaluate its use for the antisepsis of surgeon hands that still represent a source of bacterial contamination. Currently, 80% of veterinary surgeons use PI or CHx for pre-surgical hand disinfection and the search for new products, less harmful to the skin, becomes mandatory ,27.Staphylococcus aureus, represents a matter of concern that is not solved by the application of common disinfectants such as CHx. In the present study, IA displayed the ability to eliminate S. aureus, as substantiated by the in vitro tests. In this light, IA, thanks to its localized and unspecific action, may represent a valid alternative to substances currently used for animal skin disinfection, such as antibiotics. This aspect is of crucial importance considering the implications correlated to the development of microbial resistance. Nevertheless, PI and CHx were used to respect common pre-surgical practices, the observed results stimulate deeper investigations and encourage: (i) the development of protocols based on the sole use of IA, to reveal the actual potential of the proposed approach; (ii) further study on IA in term of time and methods of application.Besides potentiating both PI and CHx, the treatment with IA led to the lowest bacterial load in 82% of cases. An application of IA  after two consecutive scrubs with CHx achieved the best bactericidal effect, overcoming the efficiency of the sole CHx. It is important to stress that the resistance of some bacteria, such as


Multivariate analysis indicated that viscosity index and consistency were the top parameters affected by THY. The authors concluded that THY and VAN are promising stabilizers for ice-cream clean production.Using natural multi-function phytochemicals could be one of the best solutions for clean-label production. In this study, dairy ice creams were prepared containing 14% egg yolk and 0.1% of thymol (THY), trans-cinnamaldehyde (TC), menthol (MEN), or vanillin (VAN). Then, the physical, chemical, and structural characteristics were evaluated. Magnetic resonance imaging (MRI) analysis  was carried out to evaluate the water distribution. A multivariate analysis was conducted among all studied variables. According to the results, the overrun of the MEN ice cream was significantly increased as compared to the control sample. The density was also reduced in the MEN sample. Meanwhile, the spreadability (%) of VAN was significantly increased after 6 min as compared to the control treatment. MRI analysis revealed that water distribution was significantly changed in the THY group. The firmness and viscosity of THY samples were significantly increased ( Using proper stabilizers is one of the major challenges in the ice cream industry because of the difficulty to enhance the texture properties with a single stabilizer. Moreover, selecting and applying the proper amount of stabilizers in ice cream formulation depends upon the total solids and fat levels of the mass . HoweverAs a multifunctional agent, egg yolk has a significant effect on ice cream texture, i.e., enhancing water retention and viscosity that prevent ice cream separation . In addiUsing multivariate analysis to solve the problems related to specialized computer software has become one of the promising approaches in agricultural product evaluation and characterization ,12. AddiSeveral analyses can be used to evaluate the physical and chemical changes and problems in the ice cream industry. For instance, overrun (%) is one of the most important factors correlated with ice cream texture properties. The differences in the ice cream texture and rheological characteristics could be due to crosslink protein interactions and changes in its dairy and yolk protein structures ,15. GoudThis study aimed to evaluate the physical and structural effects of four safe natural alternatives to the synthetic substances representing mono-terpenes and phytochemical groups  mixed with egg yolk in producing different types of dairy ice cream products. Firmness, consistency, cohesiveness, viscosity, yield stress, microstructure by MRI, overrun, density, spreadability, and color characteristics were evaluated to provide evidence of their applicability as an alternative stabilizer in the design of ice cream formulations and consider their positive effects in the production of ice cream. In addition, a multivariate analysis of data was carried out to evaluate the physical and structural effects, providing evidence of integrating these analytical methods in the field of ice cream engineering.Grade A eggs were purchased from the Huazhong Agricultural University , 3.5% fat milk was purchased from Weidendorf Ltd. , and 35% fat whipping cream was purchased from Nestle Co. Ltd. . Gelatin was purchased from Henan En Mian Food Co. Ltd.  and sucrose fine sugar powder was purchased from Guangdong Xiang Food Co. Ltd. . Thymol , and vanillin  were purchased from Sinopharm, Co. Ltd. , trans-cinnamaldehyde  was purchased from Aladdin Industrial Co. Ltd.  and menthol  was purchased from Tokyo Chemical Industry Co. Ltd. .The eggs were manually broken, and yolks were separated from the albumen. Each yolk was carefully rolled on a filter paper  to remove traces of albumen that adhered to the vitelline membrane. The vitelline membrane was then disrupted with a scalpel blade, and the yolk was collected in a cooled beaker. Five egg yolk mixtures were prepared following the protocol indicated by Gouda et al.  as the hFor ice cream preparation, concentrations of the following materials were used: 35% milk (3.5% fat), 35% cream (35% fat), 15% sugar, 14% egg yolk mixture, and 1% gelatin . Milk, cAn aliquot of 100 g from the control and prepared ice cream samples was put in a standard-size back extrusion container (50 mm diameter) for measuring the texture by TA-XT-Plus texture analyzer . Compression test was carried out with a load cell of 5000 g, and 35 mm diameter cylindrical probe was used a texture profile analysis and a pre-speed of 2 mm/s, test-speed of 2 mm/s, penetration depth 15 mm, and trigger force 5.0 g to characterize 80 g of samples. Firmness, cohesiveness, consistency, and index of viscosity were calculated from the TPA software system. The average value of three replications was used for the data analysis .−1 was obtained for measuring viscosity and yield stress. The experimental time was adjusted to 120 s. A total of 32 points was collected with linear distribution and duplicates presented differences lower than 10%. Data obtained were adjusted to the Power law and Herschel–Bulkley models by using Trios Software Version: 3.1.0.3538.Rheological measurements were carried out according to the flow behavior of the samples after 60 min at 5 °C by using a discovery hybrid rheometer  with a parallel geometry sensor  and 1 mm gap following the method of Gouda et al.  with somMRI was used to analyze the internal water distribution state, mobility, and changes of the ice cream samples by an imaging system . The expThe experimental conditions were: FOV of 100 mm/100 mm, width of slice of 2.5 mm, gap of 0.5 mm, no. 4, size of reading of 256, size of phase of 192, T1 weighted image TE of 19 ms and TR of 300 ms, and T2 weighted image TE of 50 ms and TR of 1600 ms. Three layers of images of each sample are taken . The gradient colors from blue and red represent the highest amplitude whereas orange color represents the lowest amplitude. The surface water is characterized by higher amplitudes colors.Overrun was determined according to the method described by Adhikari et al. . First, Density was determined according to the modified method described by Pourashouri et al. . First, 3. Then the samples were frozen at −20 °C for 12 h. Then, the plastic containers were placed in a tray along with a 30 cm rule. The ice cream cube samples were put next to a ruler with a lab glass plate and a weight of 100 g on top at 21 °C. After that, the diameter of the examined samples was taken every 2 min for 12 min, six times for each ice cream treatment [A weight of 20 g from the ice cream samples was placed separately on plastic cubes of 2 × 2 × 2 cmThe color of the samples was expressed using the CIELAB system: L* , a* (redness or greenness), and b* (yellowness or blueness) . The meap-value ≤ 0.05) [p-value < 0.05. The least significant differences (LSD) and Duncan tests were used to determine significant differences among the tested samples, in which, mean ± SD with different Roman alphabet superscripts indicates that the values differ significantly  and (a < b < c < d …). In addition, * and ** asterisks were used as the column differs significantly (p ≤ 0.05) and (p ≤ 0.01), respectively). The Pearson correlation coefficients (r2) for the relationships between the properties were also calculated.Data statistical analyses was performed using SPSS 16.0 , Origin 2021 , MetaboAnalyst software , and Matlab R2017b . Principal component analysis (PCA) and other multivariate discriminant analyses (DA) were analyzed using the Partial Least Squares (PLS) model. Clustering of treatments was assessed with PCA score plots and heatmeat projections. The relevance of each analysis in the experimental data variance was evaluated using the variable importance in the projection (VIP). The different colored boxes in the heatmap and the VIP represent significant differences ( ≤ 0.05) . Analysip < 0.05) as compared to the control (135.85 ± 27.61 g). El-Sayed et al. [Ice cream texture properties are the most important parameters in the dairy industry . The effd et al.  mentioned et al. .p < 0.05) in the cohesiveness (369.69 ± 30.64 g)  c and con30.64 g) b, as com30.64 g) a,d.The increase in THY and VAN cohesiveness and consistency might be due to the changes in the hydrophobic core of yolk granular molecules in the ice cream. Moreover, the increase in texture levels of ice cream is generally related to the reduction in the growth rate of ice crystals in the ice cream . JardineThe effects of TC, THY, MEN, or VAN on the ice cream rheological properties such as viscosity and stress yield are shown in p < 0.05) increased (27.92 ± 5.25 Pa·s) as compared to the control (21.24 ± 0.15 Pa·s). Conversely, the viscosity of the MEN group was significantly decreased to 15.68 ± 1.24 Pa·s in relation to the control sample. Moreover, a high positive correlation (r2 = 0.90) was found between the Index of viscosity and cohesiveness.According to the Power-law model, the viscosity of the THY group was significantly (p < 0.05) increased (30.88 ± 5.80 Pa·s) as compared to the control (20.47 ± 6.08 Pa·s). Differently, the viscosity of the MEN group was significantly decreased (17.46 ± 1.37 Pa·s). Additionally, a positive correlation (r2 = 0.77) was found between stress yield and consistency. Anjo et al. [According to the Herschel–Bulkley model, the yield stress of the THY group was significantly  higher than observed for the control sample (16.61 ± 2.39%). The authors of [The overrun represents the air content in ice cream, which is an important indicator that affects the melting behavior, structure, texture, and sensory properties of ice cream . The resthors of  proved tthors of . In linethors of  mentionethors of , which m2 = −0.65) was found between stress yield and overrun (A negative correlation (r overrun d. Similap < 0.05) decreased (0.84 ± 0.03 g/cm3) as compared to the control (0.94 ± 0.01 g/cm3). However, the density of the ice cream in the MEN group was significantly decreased to 0.71 ± 0.00 g/cm3 as compared to the control (0.81 ± 0.02 g/cm3). Ismail et al. [The density results of mixtures and ice cream samples are shown in l et al.  mentionel et al. .p < 0.05) from the control. After 2 min, the control group was significantly higher (p < 0.05) than other groups. While after 4 min, all five groups had no significant differences among them. After 6 min, significant differences among groups were observed, but the THY and TC groups had similar spreadability in comparison to the control ice cream.As seen in p < 0.05) higher than that observed in the control sample until 12 min. These results are negatively attributed (r2 = −0.80) to the results of ice cream density (The spreadability of the MEN and VAN groups was significantly ( density d. Therefp < 0.05) increased the lightness of the ice cream in comparison to the control samples. Regarding the yellowness, the b* values for the MEN and VAN treatments were significantly lower than those obtained from the control . One possible explanation for changes in the color parameters  is due to the fact that surface hydrophobicity changes with the amount of fat in the surface, which has the majority of yolk hydrophobic pigments in the ice cream [The variation in color parameters, lightness (L*), redness (a*) and yellowness (b*) is shown in ce cream ,31.p < 0.05) different from the control. Meanwhile, from VIP and heatmap (p < 0.05) increased the cohesiveness and consistency of ice cream  among all studied groups is presented in  heatmap b,c, it wce cream b. This o2) heatmap among all studied parameters. A positive correlation (r2 = 0.75) was found between L* value and overrun. The b* value was negatively correlated (r2 = −0.93) with spreadability after 8 min to 12 min (2 = 0.90) was found between the Index of viscosity and cohesiveness. Our results are in agreement with Harlina et al. [o 12 min d. Moreova et al. , who foua et al. .In this study, four bioactive compounds with egg yolk  were evaluated for obtaining a high-quality ice cream product. These four compounds showed different effects on the functional properties of ice creams. Different significant responses were observed in the following analyses . Therefore, it can be concluded that natural phytochemicals  can play an important role with egg yolk as a natural stabilizer in the design of ice cream formulations. The advances in the pretreatment with bioactive terpenes for ice cream production came from the changes in the yolk functionality, which can direct the production without any further chemical or physical assistance. This study can support the applicability of natural bioactive molecules as a simple “clean label” strategy. Furthermore, large-scale studies are needed to optimize the used formulations together with the other processing conditions in view of their industrial applications.


Instrumentation of the lumbosacral region is one of the more challenging regions due to the complex anatomical structures and biomechanical forces. Screw insertion can be done both navigated and based on X-ray verification. In this study, we demonstrate a fast and reliable open, low exposure X-ray-guided technique of iliac screw placement.Between October 2016 and August 2019, 48 patients underwent sacropelvic fixation in tear-drop technique. Screw insertion was performed in open technique by using an X-ray converter angulated 25-30° in coronal and sagittal view. The anatomical insertion point was the posterior superior iliac spine. Verification of correct screw placement was done by intraoperative 3D scan.In total, 95 iliac screws were placed in tear-drop technique with a correct placement in 98.1%.The tear-drop technique showed a proper screw position in the intraoperative 3D scan and therefore may be considered an alternative technique to the navigated screw placement. Instrumentation of the lumbosacral region is one of the more challenging regions due to the complex anatomical structures and biomechanical forces , 14. EspBetween October 2016 and August 2019, 48 patients underwent sacropelvic fixation in tear-drop technique. Between 2016 and 2019, all patients who received iliac screw placement were included. Only in one case, iliac screw placement was achieved in navigated technique (due to neoplastic sacrum deformity). This case was excluded. To indicate surgery, we applied generally accepted standards in every specific case. The preoperative diagnostic procedure included a thorough anamnesis and clinical examination. The preoperative imaging included an MRI and CT of the lumbar spine. Intraoperative screw position was verified with a 3D X-ray scan with c-arm. Follow-up X-ray was performed before discharge and 3 months after surgery. The data was collected prospectively. The chart review was done retrospectively. The statistical analysis was done by using Excel and SPSS for Windows.All surgical procedures were performed by our senior spine surgeon and senior author (J.-U. Müller). Our spinal instrumentation system and pedicle screws used in all cases of this study are from Signus Medizintechnik .Surgery is performed in general anesthesia with the patient being in prone position. Spinal exposure is obtained via a standard midline incision from the lumbar spine to the sacrum. The distal extent of the incision should be lower than the line between the posterior superior iliac spine (PSIS). The traditional anatomical landmark for iliac screw fixation is the PSIS . The bonBetween October 2016 and August 2019, we included 48 patients. A total of 27 of them were female and 21 male. The mean age was 64 years (27-81 years). In all cases, bilateral iliac screw positioning was performed. In total, 95 iliac screws were placed in tear-drop technique. Only in one side (1.04%), navigated placement was performed due to tumor which deformed the iliac anatomy. The mean follow-up was 9.8 months, ranging from 7 days to 27 months. Spinal instrumentation was performed due to degenerative spinal scoliosis in 22 cases (45.8%). In an equal number , a revision surgery after previous spinal instrumentation needed spinopelvic fusion. Of that group, there were 7 cases with pseudarthrosis of L5/S1 (14.6% from the whole cohort). All cases received a multilevel spinal instrumentation. Pseudarthrosis of the sacroiliac joint after multilevel spinal instrumentation was seen in 3 cases (6.2% of the whole cohort). Spinal trauma involving the sacrum and pelvic tumor infiltration contributed 2 cases each (4.2%). On average, 8 X-ray images were needed to adjust the tear-drop sign, make the pre-drilling, and place the screw on one side. The radiation exposure depends on the X-ray converter used and the technical settings applied by the surgeon. On average, our X-ray converter takes 83kV and 9.1mA. In all cases but one, the 3D X-ray scan after tear-drop screw placement confirmed a correct position of the screw (98.9%). In one case, there was a lateral breach of the screw head. Prior to the 3D X-ray scan, no redirection of a screw was needed. In the 3-month follow-up, there were no complications regarding the iliac screws. In the overall follow-up time, there were no complications involving the iliac screw, up until today. Other surgical complications included postoperative infection in 4 cases (8.3%), non-fusion of the upper and mid-level in 4 cases (8.3%), and fractured pedicle screw on level L2 in one case (2.1%).The addition of iliac fixation should be considered in multilevel spinal fusion including the lumbosacral crossover in adult deformity and instability to reduce the risk of lumbosacral pseudarthrosis , 11. In The radiographically guided tear-drop technique combines the advantages of both free-hand and navigated placement techniques. The exposure level is minimal, as we use the existing surgical corridor for spinopelvic fusion. Only a minimal exposure of the PSIS will be performed for visualization and preparation of the entry point. Additionally, the radiological exposure level will be low. The tear-drop sign must be adjusted on the X-ray monitor and the screw placed under radiological guidance. We recommend, if available, a 3D scan directly after screw placement in open as well as in navigated placement to ensure a correct screw position without breach.The technique necessitates at least a c-arm X-ray apparatus and a carbon surgical table to ensure visualization. Also, it is important to align the tear-drop sign in the central X-ray beam as otherwise a visual aberration can lead to wrong screw position leading to screw breach. A recent meta-analysis comparing iliac screw placement with S2 ala-iliac screw placement detected mechanical complications in up to 27.9% of the cases vs. 14.2% respectively . Our ratWhen comparing to other free-hand techniques, as for example proposed by Fridley et al., we achieve comparable results . In theiThe tear-drop technique for iliac screw placement in spinal surgery is a reliable and safe method only utilizing standard X-ray imaging and minimal exposure of the surgical field.


A novel scaling-up computer system for single screw extrusion of polymers has been developed. This system makes it possible to scale-up extrusion process with both starve feeding and flood feeding. Each of the scale-up criteria can be an objective function to be minimized, represented by single values or functional dependencies over the screw length. The basis of scaling-up is process simulation made with the use of the GSEM program . Scaling-up is performed using the GASES program  based on Genetic Algorithms. Scaling-up the extrusion process has been performed to increase extrusion output according to the scaling-up criteria defined by the single parameters of unit energy consumption, polymer plasticating rate and polymer temperature, as well as by the process parameters profiles of the temperature and plasticating. The global objective function reached the lowest value for the selected process parameters, and extrusion throughput was significantly increased. Computer modeling is widely used for designing polymer processing. However, CAD/CAE systems, while useful, do not make it possible to optimize the process. Extrusion optimization is a complex issue due to the multiplicity of potential optimization criteria, often contradictory, as well as a very large number of process data: material data, geometry, and operation parameters.Optimization consists in creating a multidimensional response space of process parameters based on input parameters and searching for extreme values in this space, maximum or minimum. Data for optimization can be obtained on the basis of experimental or simulation tests, but optimization based on simulation data is more effective.There are many different optimization methods which can be classified into: analytical, stochastic, and enumerative ,7,8,9,10Optimization methods based on Genetic Algorithms are of particular importance in the case of polymer extrusion ,24,25,26-Parameters of the optimization task are processed in the coded form,-Searching the solution of the optimization task is performed from a randomly selected population, which avoids local extremes,-The rules of selection of population are probabilistic,-A new area of searching is determined using previous experiences,-Only the values of the objective function are required for searching the extremes, the derivatives are not needed.In general, Genetic Algorithms are characterized by the following features :-ParametThese methods have been used to optimize most of extrusion processes, i.e., classical flood fed single screw extrusion ,18,19,20An important method of designing physical processes is scaling, i.e., changing the scale of the process according to the selected criteria while maintaining the parameters of the scaled process at a level as close to the parameters of the reference process as possible.When scaling-up the extrusion process we define the screw geometry and operating conditions of an extruder under designing (the target extruder) that should replicate the operation of the reference extruder. Scaling-up enables to design large extruders based on the studies on the laboratory scale.Over the years, a number of various scaling-up concepts were proposed. These were discussed in books ,34,35,36X1, X2 are the small and large parameter; D1, D2 are the small and large screw diameter; s is the scaling-up factor.Most scaling-up concepts were based on the analytical models and consisted in correlating the large and small primary scaling parameters  in terms of the exponent of the ratio of the target screw diameter and the reference screw diameterCarley and McKelvey  were theRauwendaal ,46 analy-Can tackle the single scale-up criteria only, e.g., plasticating rate or pumping rate and the single step of the process, e.g., polymer plasticating or melt flow;-Can take into account only a few geometry or process parameters, e.g., screw rotational speed, screw diameter, screw channel depth, screw length;-Use the simple mathematical models;-Are not flexible.This analysis was extended by Covas and Cunha  who conc-To consider simultaneously several criteria;-To select the single parameters or functions as the scale-up criteria;-To freely choose and define the criteria.Thus, the more effective scaling-up methods are needed which would use the accurate mathematical models, and would allow:Covas and Cunha concluded  that the-Simulating the process to have the response data of the reference extruder at a specific set of input data;-Defining the scale-up criteria;-Specifing the fixed parameters of the target extruder;-Performing the scaling-up by minimizing the differences between the selected parameters of the target and reference extruders.The implementation of these new scale-up concepts requires:Selection of the scale-up criteria is fundamental for the quality of scaling-up. Usually, the scale-up criteria include the shear rate, the rates of feeding or pumping, the rate of polymer plasticating, the residence time, and the power consumption as proposed by Rauwendaal  and Pote-Ratio of the total/drag flow rate;-Specific mechanical energy;-Pressure variation over the unit screw channel;-Ratio of the screw length required for melting/total screw length (relative melting length);-Average shear rate;-Average shear stress;-Overall vsicous dissipation;-Average total strain (WATS), which is the measure of mixing degree.Covas and Cunha  proposedMost of the scale-up criteria are represented by single values but, in some cases, it might have a sense to consider the profiles of them over the screw length. A good example is the solid bed profile (SBP) which is the ratio of the solid bed width to the screw channel width.iF to be minimized, for the single values or functional dependencies, in the form [iF is the i-criterion fitness; i-criterion for the target extruder and reference extruder; i-criterion at the k-location over the screw length for the target extruder and reference extruder.Each of the scale-up criteria may be considered as an objective function the form (2)Fi=|CThe simple way to do a multi-objective optimization is to take into account the global objective function that includes individual objectives with the use, for example, simple scalar function as was done by Covas and Cunha in optimization of extrusion ,21,22,23Using this approach, Covas and Cunha  carried Recently, Vergnes et al.  solved tThe use of optimization methods for scaling-up, including Multi-Objective Evolutionary Algorithms (MOEA), were also proposed by Covas and Cunha ,52,53.Summarizing, scaling-up the extrusion process based on the process modeling is limited, up to now, to the classical single screw extrusion with flood feeding and to the co-rotating extrusion ,52,53,54In this paper, a novel scaling-up computer system for single screw extrusion with both flood feeding and starve feeding is presented. Each of the scale-up criteria can be an objective function to be minimized, represented by single values (Equation (2)) or functional dependencies over the screw length (Equation (3)). The basis of scaling-up is the process simulation made with the use of the GSEM program  ,58,59. SExtrusion process can be performed with flood feeding or metered feeding. When flood feeding, the screw is fully filled with polymer a, while Extrusion with metered feeding, also called extrusion with starving, has some advantages over extrusion with flood feeding ,63,64,65Single screw extrusion with flood feeding was widely studied, while little studies were performed on extrusion with metered feeding. The state-of-the-art on these issues was discussed in books ,67,68,69Wilczyński et al. made extensive experimental study ,78, and Modeling of starve fed extrusion differs substantially from modeling of flood fed extrusion, and different computation algorithms have to be applied, here. This has been discussed in details in .Computation algorithms for single screw extrusion with flood feeding are well known ,84,85,86Computation algorithms for single screw extrusion with metered feeding are less known ,58,59. TUsing these backward algorithms, the models for counter-rotating extruders have been built by the authors ,28,29, aRecently, the authors built the program GSEM  ,58,59,91In the study, a scaling-up program GASES  has been developed. The source of parameters for scaling-up are computations carried out with the program GSEM  ,58,59,91255 values. A “roulette wheel” is applied as a method of selection. An operation scheme of the “roulette wheel” is shown in iF = 0.9904, i.e., the highest value of the reciprocal of the objective function iF = 8.9431, i.e., the lowest value of the reciprocal of the objective function GASES scaling-up program, cooperating with the program GSEM, allows for scaling-up the extrusion process with various number of process parameters, with various scale-up criteria specified by the single parameters, e.g., specific energy consumption, the rates of solids conveying, plasticating or pumping, as well as by the process parameters profiles, e.g., plasticating profile, temperature profile, etc. The search accuracy of the response surface is determined by the number of divisions of the data range, which results from the length of writing these numbers in binary form. The length of the binary series is regulated, and its maximum length is 255 characters. This makes it possible to divide the range of each parameter into 2In the GASES program, scaling-up is defined by the number of scaling variables, the size of initial population, the length of chromosomes, the probability of crossover, the point of crossover, and the probability of mutation. We have not studied the influence of the GA parameters on the results. However, we observed that this influence is rather not important. We observed that GA parameters affect the computation time. We established these parameters based on the literature  and our Scaling-up was made for single screw extrusion to increase the process output according to the scale-up criteria defined by the single parameters of unit energy consumption, polymer melting rate, and polymer melt temperature, and by the process parameters profiles of temperature and melting.r = 45 mm to the level of the target extruder with the screw of diameter Dt = 60 mm while maintaining the ratio of the length/screw (Lr/Dr) = (Lt/Dt) = constant. The extrusion with flood feeding and metered feeding were investigated.The research program included a scale-up of the extrusion process from the level of the reference extruder with the screw of diameter Dr = 45 mm, and length/diameter ratio (Lr/Dr) = 27 was applied as a reference screw configuration. It has zones of feeding (F), compression (C), and metering (M) with length/diameter ratios equal to (L/D)F = 10.78, (L/D)C = 7.11, and (L/D)M = 9.11. The compression ratio, that is the ratio of the channel depth (HF) in the feeding zone to the channel depth in the metering zone (HM), CR = HF/HM, was equal to CR = 2.66 . The die for extrusion of rods of diameter Ddie = 5 mm was used. Screw geometry of the reference and target extruder are presented in A classical three-zone screw of diameter DHigh density polyethylene (HDPE) Rigidex 6070EA  was used in the study. Material properties are presented in T is temperature, A0, A1, A11, A12, A2, A22 are parameters of the Klein equation, A0 = 10.9183, A1 = −0.2184, A11 = −0.0368, A12 = 0.0010, A2 = −0.0226, A22 = 0.000021.Rheological properties of the polymer (HDPE) were determined with the use of the high-pressure capillary rheometer RG-25  at temperatures: 180 °C, 190 °C, and 200 °C. The model of Klein was used to describe the viscosity as a function of temperature and shear rate.maxQ, minimum unit energy consumption s minE, and minimum polymer melt temperature at the die outlet meltTmin, in the range of the screw rotational speed N = 20 ÷ 80 rpm and the barrel temperature in the subsequent sections of the extruder: T1 = 150 °C, T2 = 150 ÷ 240 °C, T3 = 150 ÷ 240 °C, T4 = 150 ÷ 240 °C.Scaling-up was performed with reference to the extrusion process, the operation parameters of which were determined as a result of optimization. These parameters were screw rotational speed and barrel temperatures. The optimization of the reference process was carried out according to the criteria of maximum throughput The global objective function was defined as,iFo is a global objective function, i_normQ is a normalized flow rate, s i_normE is a normalized specific energy consumption, melt i_normT is a normalized melt temperature at die outlet, i is a number of the next value from the data set.The output variables (optimization criteria) were normalized as,N = 79.53 rpm and the barrel temperatures: T1 = 150 °C, T2 = 192.51 °C, T3 = 180.47 °C, T4 = 180.47 °C. These optimal parameters, according to the assumed optimization criteria of maximum throughput, minimum unit energy consumption, and minimum polymer melt temperature at the die outlet, correspond to the process output parameters of mass flow rate Q = 27.10 kg/h, unit energy consumption: sE = 489.36 kJ/kg, polymer temperature meltT = 254.88 °C, and the relative “melting length” meltingL = 0.796, i.e., the ratio of the screw length necessary for melting of polymer to the total screw length.The highest value of an objective function was obtained at the screw rotational speed SBP), and the profile of screw filling (FF).Simulations for the reference extruder at the optimal operation parameters are presented in N = 20 ÷ 80 rpm, and the barrel temperature in the subsequent sections of the extruder: T1 = 150 °C, T2 = 150 ÷ 240 °C, T3 = 150 ÷ 240 °C, T4 = 150 ÷ 240 °C.With regard to such an optimized reference process, the extrusion scaling-up was performed in the same range of input data of the screw speed Es, the polymer melt temperature meltT, and the relative “melting length” meltingL, i.e., the ratio of the screw length necessary for polymer melting to the screw length, and to the functional criteria of the temperature profile and the plasticating profile, i.e., the solid bed profile (SBP).Scaling-up was carried out according to the single-parameter criteria of the unit energy consumption i sF is the global objective function for scale-up, s AE is the specific energy consumption for reference extruder, s BiE is the specific energy consumption for target extruder, meltTA is the melt temperature at die outlet for reference extruder, melt BiT is the melt temperature at die outlet for target extruder, melting AL is the relative melting length for reference extruder, melting BiL is the relative melting length for target extruder, AT is the melt temperature in reference extruder, B iT is the melt temperature in target extruder, ASBP is the polymer melting profile for reference extruder, BiSBP is the polymer melting profile for target extruder, i is a number of the next value from the data set, n is a number of the next value in the profile.The global objective function was defined as,N = 57.14 rpm for the barrel temperatures: T1 = 150 °C, T2 = 200 °C, T3 = 197.85 °C, T4 = 190.71 °C. These parameters correspond to the process output parameters of the flow rate Q = 44.20 kg/h, the unit energy consumption Es = 457.76 kJ/kg, the polymer melt temperature melt =T 254.93 °C, and the relative “melting length” melting =L 0.793. The differences between the parameters of the reference process and the target process are small  were normalized as,N = 80 rpm and the barrel temperatures: T1 = 150 °C, T2 = 234.33 °C, T3 = 228.66 °C, T4 = 222.99 °C, and the feeding rate STQ = 27.32 kg/h. These optimal parameters, according to the assumed optimization criteria of maximum throughput, minimum unit energy consumption, and minimum polymer melt temperature at the die outlet, correspond to the process output parameters of mass flow rate STQ = 27.32 kg/h, unit energy consumption sEST = 453.84 kJ/kg, polymer temperature melt STT = 223.31 °C, and the relative “melting length” meltingL = 0.617, i.e., the ratio of the screw length necessary for melting of polymer to the total screw length.The highest value of the objective function was obtained at the screw rotational speed Simulations for the reference extruder at the optimal operation parameters are presented in N = 20 ÷ 80 rpm, the barrel temperature in the subsequent sections of the extruder: T1 = 150 °C, T2 = 150 ÷ 240 °C, T3 = 150 ÷ 240 °C, T4 = 150 ÷ 240 °C, and the feeding rate QST = 36 ÷ 42 kg/h.With regard to such an optimized reference process, the extrusion scaling-up was performed in the same range of input data of the screw speed s STE, the polymer melt temperature melt STT, and the relative “melting length” melting STL, i.e., the ratio of the screw length necessary for polymer melting to the screw length, and to the functional criteria of the temperature profile and the plasticating profile, i.e., the solid bed profile (SBP).Scaling-up was carried out according to the single-parameter criteria of the unit energy consumption ST i sF is the global objective function for scale-up, Es ST A is the specific energy consumption for reference extruder, s ST BiE is the specific energy consumption for target extruder, melt ST AT is the melt temperature at die outlet for reference extruder, melt ST BiT is the melt temperature at die outlet for target extruder, melting ST AL is the relative melting length for reference extruder, melting ST BiL is the relative melting length for target extruder, ST AT is the melt temperature in reference extruder, ST BiT is the melt temperature in target extruder, ST ASBP is the polymer melting profile for reference extruder, ST BiSBP is the polymer melting profile for target extruder, i is a number of the next value from the data set, n is a number of the next value in the profile.The global objective function was defined as,N = 47.67 rpm for the barrel temperatures: T1 = 150 °C, T2 = 204 °C, T3 = 194.33 °C, T4 = 193 °C, at the feeding rate STQ = 39.17 kg/h. These parameters correspond to the process output parameters of the unit energy consumption s STE = 360.32 kJ/kg, the polymer melt temperature melt ST =T 200.67 °C, and the relative “melting length” melting ST =L 0.571. The differences between the parameters of the reference process and the target process are small (The results of scaling-up are presented in re small . Thus, iA novel scaling-up computer system for single screw extrusion of polymers has been developed. This system makes it possible to scale-up extrusion process with both starve feeding and flood feeding. Each of the scale-up criteria can be an objective function to be minimized, represented by single values or functional dependencies over the screw length. Scaling-up the extrusion process has been performed to increase extrusion output according to the scaling-up criteria defined by the single parameters of energy unit consumption, polymer melting rate, and polymer temperature, as well as by the process parameters profiles of temperature and melting. The global objective function reached the lowest value for the selected process parameters, and extrusion throughput was significantly increased. The use of functional scaling-up criteria in addition to the single-parameter criteria increased the accuracy of scaling-up.It is worth noticing that the surface of the “roulette wheel” assigned to the individual genotypes is inversely proportional to the objective function values generated by these. When the genotype has the lowest value of the objective function, i.e., the highest value of the reciprocal of the objective function, the largest area of the “roulette wheel” is covered. When the genotype has the highest value of the objective function, i.e., the lowest value of the reciprocal of the objective function, the smallest area of the “roulette wheel” is covered.So far, there is a lack of optimizing and scaling-up studies on the counter-rotating twin screw extrusion. However, the global models of this process are available. Thus, it seems to be reasonable to apply the Genetic Algorithms to solve this task, as for the co-rotating twin screw extrusion. Defining the screw geometry for the counter-rotating extrusion is similar to that for the co-rotating one, and differs from that for the single screw extrusion. In the former, the screw configuration is determined by choosing the screw elements from a set of elements available and locating them along the screw. In the latter, the geometry variables can vary continuously within a prescribed range.


TTN are the most frequent. Proteomic and functional characterizations are required to elucidate the origin of the disease and the pathogenic mechanisms of TTN-truncating variants.Dilated cardiomyopathy (DCM) is associated with mutations in many genes encoding sarcomere proteins. Truncating mutations in the titin gene TTN mutations and measured the Ca2+ sensitivity of force and its length dependence. Simultaneous measurement of force and adenosine triphosphate (ATP) consumption in skinned cardiomyocytes was also performed. Phosphorylation levels of troponin I (TnI) and myosin binding protein-C (MyBP-C) were manipulated using protein kinase A and λ phosphatase. mRNA sequencing was employed to overview gene expression profiles. We found that Ca2+ sensitivity of myofibrils carrying TTN mutations was significantly higher than in myofibrils from donor hearts. The length dependence of the Ca2+ sensitivity was absent in DCM myofibrils with TTN-truncating variants. No significant difference was found in the expression level of TTN mRNA between the DCM and donor groups. TTN exon usage and splicing were also similar. However, we identified down-regulation of genes encoding Z-disk proteins, while the atrial-specific regulatory myosin light chain gene, MYL7, was up-regulated in DCM patients with TTN-truncating variants.We isolated myofibrils from DCM hearts carrying truncating -truncating mutations lead to decreased length-dependent activation and increased elasticity of myofibrils. Phosphorylation levels of TnI and MyBP-C seen in the left ventricles are essential for the length-dependent changes in Ca2+ sensitivity in healthy donors, but they are reduced in DCM patients with TTN-truncating variants. A decrease in expression of Z-disk proteins may explain the observed decrease in myofibril passive stiffness and length-dependent activation.Titin  Truncation mutations have been discovered in up to 25% of familial DCM.TTN-truncating variants (TTNtv) has been associated with DCM in this cohort of patients.TTN mutations and how they might lead to the development of DCM is not well understood. A further complication is the observation that TTN-truncating variants (TTNtv) have also been detected in the healthy population (0.6–2.9%),,,The 2+] changes. The sensitivity of myofilaments to Ca2+ is modulated by phosphorylation of troponin I (TnI) at Ser 23 and 24, and of myosin binding protein-C (MyBP-C) at Ser 275, 284, and 304 by protein kinase A (PKA) in response to adrenergic2+- and phosphorylation-dependent changes in contractility that occur during DCM are poorly understood.The sarcomere is the basic contractile unit of striated muscles, including cardiomyocytes. Cardiac sarcomeres are ∼1 µm in diameter and 2.2 µm long. Myofibrils consist of repeating sarcomeres. Z-disks mark the ends of the sarcomere and are the structure that links adjacent sarcomeres together. Actin-containing thin filaments and myosin-containing thick filaments are connected to the Z-disk by α-actinin and titin. Powered by the energy of adenosine triphosphate (ATP), myosin heads interact with actin filaments to produce force. Actin filaments are associated with the troponin complex and tropomyosin that control this process in response to sarcomeric nH/(2+]nH), where F is steady-state developed force, maxF is the maximum saturated value F can attain, EC50 is the concentration of Ca2+ at which F attains 50% of maxF, and nH is the Hill coefficient. Information on myofibril kinetics is provided in Methods. Measurement of passive force was performed as described previously.The apparatus for the measurement of force and passive stiffness in single myofibrils has been previously described.2+) and activating (0.4–15.8 µmol/L free Ca2+) solutions contained (in mmol/L): 10 MOPS (pH 7.0), 5 Mg-ATP, 1 free Mg2+, 5 DTT, 10 phosphocreatine, 0.5 mg/mL creatine kinase, 1 unit/mL bacterial purine nucleoside phosphorylase, and 0.5 7-methylguanosine. The Ca-EGTA:EGTA ratio was set to obtain 10 mmol/L total EGTA and the desired free [Ca2+]. K-propionate and Na2SO4 were added to adjust the ionic strength of the solution to 200 mmol/L. The relaxing solution in the bath chamber (0.01 µmol/L free Ca2+) was supplemented with (in μmol/L): 10 chymostatin, 5 pepstatin, 40 leupeptin, 10 E-64, and 200 PMSF.Relaxing , and estimated means are reported. In this case, data are shown as estimated means ± SEMs. P-values of <0.05 were considered statistically significant. The Wald test was used to generate P-values and log2 fold changes in gene expression. Genes with an adjusted P-value <0.05 and absolute log2 fold change >1 were called differentially expressed genes.Statistics analysis and graphs were prepared using Prism 7 . Data are expressed as means ± SEMs. One-way ANOVA followed by Fisher’s least significant difference multiple comparison test was used for multiple group comparison. The Student’s TTN mutations  result in premature termination of mRNA translation. We performed mRNA sequencing on the samples of left ventricular heart tissue used for the functional measurements to examine gene expression patterns and TTN alternative exon usage. A stop codon can lead to non-sense-mediated decay of the mRNA or production of truncated titin proteins. However, we did not find significant down-regulation of the TTN gene in the DCM samples (n = 4) with frameshift mutations compared to healthy donor heart samples  was up-regulated  in the left ventricle of the DCM patients with TTNtv. In contrast, the expression of MYH6 encoding alpha heavy chain subunit of cardiac myosin, which is also expressed predominantly in atrial tissue, decreased 12.4-fold.The four heterozygous s n = 6; Table S3.r hearts Table S4 FLNC , MYOT , PALLD , XIRP2 , ZYX , CRYAB , MYOZ1 , and LMCD1 . FHL1  and KLHL40 (kelch-like family member 40) were down-regulated 2.4 and 3.6-fold, respectively. The gene LMOD2 (leiomodin-2),MYOM1 and MYOM2 (myomesin-1 and -2) were 2.1- and 3-fold up-regulated in DCM patients with TTNtv. Myomesin is located in the M-band and links titin to myosin filaments. Overexpression of myomesin was associated with sarcomere damage.HSPB1 (heat shock protein beta-1), involved in mechano-transduction,MYOC , FMOD , OGN , and SOD3 . The pro-inflammatory protein genes S100A8 and S100A9  were down-regulated 12.5-fold and 15.6-fold, respectively. A marker of endoplasmic reticulum stress, HSPA5 (heat shock protein family A member 5) was down-regulated 2.2-fold. The full list of the significantly impacted genes is shown in the The expression levels of many central genes encoding Z-disk structural proteins were significantly down-regulated in DCM patients with TTNtv compared to healthy donors: Figure ). Western blots with anti-TnI antibodies showed three bands corresponding to bis-phosphorylated (at Ser 23 and 24), monophosphorylated and unphosphorylated protein . MyBP-C contains three accessible phosphorylated sites  per molecule.,Figure ). The intensity of the fourth band (3P) was weak and seen only in the highly phosphorylated control sample NM. All other studied donor heart samples showed only three bands . The level of phosphorylation of TnI, MyBP-C, and MYL2 was significantly decreased in DCM patients with TTNtv compared to healthy donors . The level of phosphorylation of TnI but not MYL2 was also reduced in DCM samples without TTNtv .We performed Phos-tag gel electrophoresis, in which proteins are separated according to their phosphorylation level, and western blotting to determine the level of TnI, MyBP-C, and MYL2 phosphorylation in the samples . This suggests that the TnI phosphorylation level in non-diseased hearts may naturally decline with age. DCM samples with mutations in the TTN gene had significantly lower levels of phosphorylation of TnI: 0.66 ± 0.12 vs. 1.52 ± 0.06 mol Pi/mol TnI . The level of phosphorylation of MyBP-C and MYL2 in DCM samples with TTNtv was 0.62 ± 0.18 vs. 1.55 ± 0.08 mol Pi/mol MyBP-C  and 0.06 ± 0.11 vs. 0.18 ± 0.02 mol Pi/mol MYL2 .We observed a significant negative correlation between TnI phosphorylation and donor age . Treatment with λ phosphatase  resulted in fully dephosphorylated TnI and MyBP-C , and a decrease (∼27%) in MYL2 phosphorylation .In the diseased myocardium, the β adrenoceptor is often down-regulated via receptor phosphorylation and β-arrestin binding.2+ sensitivity of force production, the rates of force development and relaxation and their modulation by sarcomere length, and TnI and MyBP-C phosphorylation levels. The maximum force, length dependence of maximal force, kinetics of muscle contraction and relaxation, and passive stiffness have been studied previously in DCM heart samples with truncating mutations.2+ sensitivity of force development, its length dependence and the possible functional role of changes in phosphorylation of contractile proteins in the disease. We also verified that myofibrils isolated from an additional DCM sample with TTNtv, D28, had decreased passive stiffness. The Young’s modulus was 45.6% lower in D28 compared to donor heart myofibrils, similar to the values found with the other TTNtv DCM samplesMyofibril contractility can be described by several parameters: the maximum force of isometric contraction, the CaFour DCM samples with TTNtv were compared with left ventricular samples of four donor hearts: NM, young adult (23 years old); KN1, middle-aged adult (47 years old), NH (48 years old), and KN2 (61 years old). TnI and MyBP-C were highly phosphorylated in donor NM (1.80 ± 0.05 mol Pi/mol TnI and 1.62 ± 0.06 mol Pi/mol MyBP-C) and NH (1.6 ± 0.1 mol Pi/mol TnI and 1.56 ± 0.11 mol Pi/mol MyBP-C), but not in KN1 (1.18 ± 0.04 mol Pi/mol TnI and 0.80 ± 0.11 mol Pi/mol MyBP-C). These differentially phosphorylated healthy donor samples were used to distinguish the effect of dephosphorylation of TnI and MyBP-C regulatory proteins in DCM samples from other effects caused by mutations. The relatively low phosphorylated control sample KN1 was used in functional measurements for the donor–patient comparison. Furthermore, we treated isolated myofibrils with λ phosphatase and PKA to change phosphorylation levels of TnI and MyBP-C.2+] was fit with the Hill equation to calculate the EC50, [Ca2+] required to reach half-maximal force response. The EC50 for donor heart myofibrils was consistent with the TnI phosphorylation level. Myofibrils with a lower TnI phosphorylation level had a higher Ca2+ sensitivity (lower EC50). The EC50 values for donor heart samples NM, NH, KN1, and KN2 were 2.24 ± 0.06, 1.87 ± 1.14, 1.48 ± 0.04, and 1.77 ± 0.07 μmol/L, respectively . The Ca2+ sensitivities of DCM samples with truncating mutations in the TTN gene were significantly higher than those of donor hearts NM  and NH (P < 0.038), but with no significant difference compared to KN1 . The EC50 values for DCM with TTNtv were 1.36 ± 0.05 μmol/L for D6, 1.46 ± 0.04 μmol/L for D7, 1.53 ± 0.08 μmol/L for D9, and 1.36 ± 0.06 μmol/L for D28.The maximum tension response to different ; the National Institutes of Health grant [HL-PO1 HL62426 to P.d.T.]; the Fondation Leducq [17CVD04 to M.H.Y.]; and by the Magdi Yacoub Institute [Charity number 1082750].The data underlying this article are available in the article and in its online cvaa316_Supplementary_DataClick here for additional data file.