PEPC is differentially expressed at different stages of plant development, mostly in leaves, but also in developing seeds. PEPC is known to show tissue-specific distribution in leaves and in other plant organs, such as roots, stems, and flowers. Plant PEPC undergoes reversible phosphorylation and monoubiquitination, which are posttranslational modifications playing important roles in regulatory processes and in protein localization. Phosphorylation activates the PEPC enzyme, making it more sensitive to glucose-6-phosphate and less sensitive to malate or aspartate. PEPC phosphorylation is known to be diurnally regulated and delicately changed in response to various environmental stimuli, in addition to light. PEPCs belong to a small gene family encoding several plant-type and distantly related bacterial-type PEPCs. This paper provides a minireview of the general information on PEPCs in both C4 and C3 plants.Phosphoenolpyruvate carboxylase (PEPC) is a ubiquitous cytosolic enzyme, which is crucial for plant carbon metabolism. PEPC participates in photosynthesis by catalyzing the initial fixation of atmospheric CO PEPCs pate (Pi) , such asate (Pi) ,5.4 and crassulacean acid metabolism (CAM) plants, or non-photosynthetic, such as in C3 plants [4-plant PEPCs, with information on higher-plant PEPCs provided for comparison.Two classes of plant PEPCs have been identified in a number of studies ,7. Class3 plants ,8. In th4 and C3 species of the genus Flaveria. A sequence alignment with the maize enzyme [4 species F. trinervia, while arginine was found at this site in the C3 species F. pringlei and in the C3–C4 intermediate species F. pubescens. Moreover, at position 774, C4 Flaveria species have a serine residue, while C3 and C3–C4 intermediate plants contain alanine [In a study by Westhoff and Gowik , differee enzyme  showed t alanine .4-cycle enzymes, such as PEPC, is largely due to transcriptional control [4 species Bienertia sinuspersici, differential expression of PEPC isozymes has been observed at different developmental stages [B. sinuspersici showed that PEPC3 was predominantly expressed in the early stage, while PEPC1 and PEPC2 were expressed in the intermediate and mature stages of leaf development, respectively  was detected on immunoblots of extracts from young, intermediate, and mature leaves and was found to be more apparent in mature leaf samples [PEPC distribution can vary in different tissues and in specific areas of a particular tissue. As in the case of PEPC4 of  samples .Furthermore, immunoblotting of extracts obtained from three regions of mature leaves, namely, the base, middle part, and tip, showed that the p107 PEPC polypeptide was highly expressed in the tip portion of the mature leaf but not in the base and middle portion . These f4 plant, throughout a 45-day period. Conversely, it was found that the PEPC protein attained a high level in sorghum roots, which peaked at approximately 14 days after seed germination, suggesting that the PEPC present in roots may play a significant role in plant development. In a further study in sorghum, Abergel and Glick [3 PTPCs  and a C4 photosynthetic PTPC (SbPPC1). SbPPC4 were detected at higher levels during the early seed development shown to be translated into p107 PTPC subunits. It is deemed to play a major role during the early stage of development known as the phase of cellularization. Conversely, SbPPC2 and SbPPC3 have higher levels of detection throughout the life cycle with SbPPC2 present as a monoubiquitinated p110 subunit in the embryo of both dry and post-imbibed seeds. SbPPC3 was the only one detected in the aleurone or endosperm and it was highly abundantly in the embryo. SbPPC5 was detected only as p110 polypeptides in stage I of sorghum seed development [A study by Penrose and Glick  showed tnd Glick  observednd Glick ,20. Furtnd Glick  in sorghelopment .Ricinus communis PEPC gene RcPPC4 appeared more prominent in budding and expanding leaves than in mature ones, while those of the PEPC gene RcPPC3 were higher in the integument and hypocotyl of the flower than in the entire male flower. It was also observed that RcPPC3 and RcPPC4 co-expressed in the tissues of developing seeds, flowers, and leaves, which was interpreted as a necessary interaction for the formation of a novel hetero-octameric Class-2 PEPC complex [Tissue-specific distribution of PEPC was also observed in a castor oil plant by O’Leary and colleagues . The exp complex ,22. Arabidopsis. Except for AtPPC4, these molecules are known to be plant-type PEPCs, while AtPPC4 has been reported to encode a PEPC without any phosphorylation motif. Tissue specificity was demonstrated for these PEPCs based on differential expression of their corresponding genes in Arabidopsis organs. The AtPPC1 and AtPPC4 genes were expressed in both roots and flowers; the AtPPC3 gene was only expressed in roots, while AtPPC2 transcripts were detected in all Arabidopsis organs, which suggests that the latter is a housekeeping gene. The PEPC activity was the highest in the roots, in which all of the four genes were typically found to be expressed [Arabidopsis, and the results showed differential induction of PEPC gene expression in the roots, with AtPPC4 having shown the highest levels of induction in response to both stresses [Sanchez and colleagues  identifixpressed . In thisstresses . These fstresses  reportedGmppc1, Gmppc2, Gmpppc3, Gmpppc7, Gmpppc16, and Gmppc17) which exhibited relatively high levels of expression in ELSC. Among these, Gmppc1 and Gmppc7 were known to predominantly express and exhibit the highest gene expression levels among the analyzed samples. It was also found out that while Gmppc1 and Gmppc7 are highly expressed in the ELSC, on the contrary, the isogenes Gmppc2 and Gmppc3 are highly expressed in the developing whole seeds [In a recent study of Yamamoto and colleagues, PEPC expressed in the external layer of seed coat (ELSC) during seed formation in soybean was investigated. Results in this study showed that there are likely six PEPC isogenes , which binds to C4 PEPC. In a study by Monreal and colleagues [Moreover, PEPC enzymes are allosterically regulated by positive or negative effectors. These effectors vary in different species and exert different effects on kinetic properties of PEPCs, including among isoforms in the same organisms ,32,34,35 PEPC-PK . It is t of PEPC . These e of PEPC . Other klleagues , the addlleagues . PA actslleagues ,36,37.4 PEPC inhibition by various phospholipid entities is direct and is deemed to be independent of the phosphorylation status or known allosteric regulators, as confirmed by the separate addition of Glu-6-P and L-malate to media with PA. The data showed that the addition of allosteric regulators and phosphorylation of the PEPC enzyme had no effect on the PEPC-PA interaction [4 PEPC protein [This Ceraction . Law anderaction  found theraction . In addi protein . A summa4 plants has been shown to be regulated at the transcriptional level [4 B. sinuspersici plant. Immunoblotting of samples exposed to light showed a prominent phosphorylated band of approximately 107 kDa. C4 PEPCs are thus modulated by light via phosphorylation [4 plants [PEPC phosphorylation has been known to be diurnally regulated and delicately changed in response to various environmental stimuli, in addition to light ,37. The al level ,40. Diurrylation . The mecrylation , who ela4 plants . It was 4 plants ,42.4 PEPC from leaves of Amaranthus hypochondriacus. The results of this study showed that the peak of PEPC activity, which may indicate an increase in the activation by Glu-6-P or a decrease in malate sensitivity, was always preceded by a maximum light intensity and coincided with a high temperature. This consistent trend was observed throughout the year, thus indicating an apparently strong relationship between the modulation of PEPC activity by malate/Glu-6-P and light or temperature. The PEPC activity was found to be higher during a light period when it was assayed at 0.05 mM PEP and 0.05 mM NaHCO3 than at considerably higher concentrations. It is possible that light illumination increases the affinity of PEPC for PEP or HCO3−, making the enzyme function more efficiently, including at a low substrate concentration [Avasthi and colleagues  elucidatntration ,43,44. 4 PEPC [A. hypochondriacus was also determined on a typical day in May and December. It was concluded that the phosphorylation status and not the mRNA or protein level was more crucial for the daily and seasonal patterns of PEPC activity and regulatory properties [Further studies revealed that light had a greater influence on PEPC activity, while temperature exerted a much greater effect on the regulatory properties of the C4 PEPC . In this4 PEPC . The maxoperties .Arabidospsis rosettes under controlled CO2 and O2 mole fractions showed a slight decrease in phosphorylation of the PEPC1 isoform at high concentrations of CO2 and low fraction of oxygen (1000 umol mol−1 and 0.03% respectively). This may imply a downregulation of PEPC activity at higher levels of photosynthetic rates [Moreover, phosphoproteomics conducted on ic rates ,46.B. sinuspersici leaf samples [B. sinuspersici PEPC from the tip part of mature leaves showed the hydrolysis of the 110-kDa band into a 107-kDa band upon treatment with a deubiquitinating enzyme. Other studies suggested that the monoubiquitinated form of PEPC is significantly more sensitive to malate and aspartate inhibition than the deubiquitinated one is [Monoubiquitination is another reversible PTM that mediates a number of protein–protein interactions by recruiting the ubiquitin-binding domain of client proteins and that mediates protein localization to help control processes such as gene expression, endocytosis, transcription and translation, DNA repair, and signal transduction ,47,48. T samples . ImmunobConversely, in germinating sorghum seeds, p107 is monoubiquitinated to form p110 at Lys-624, which is known as a conserved residue in vascular plant PEPCs and is proximal to a PEP-binding or PEPC catalytic domain . Further3-type PPC5 and the bacterial-type PPC6 was not detected in leaves and in roots but NH4+ has been shown to increase the phosphorylation state of PEPC and PEPC-kinase activity in the leaves both in the presence and absence of light [+ also increased the amount of monoubiquitinated PEPC in sorghum roots; hence, in such a case, although the roles of phosphorylation and monoubiquitination seem to be opposite in manner, both PTM’s co-exist in leaves and in roots, respectively. An increase in phosphorylation was a consequence of favorable PPCK1 expression in leaves, while PEPC activity in roots augmented due to the increase in PPC3 expression thus increasing the capacity of sorghum to cope with the ammonium stress [4-PEPC proteolysis in the presence of pC19  was prevented by the PEPC allosteric effector glucose 6-phosphate (Glc-6P) and by PEPC phosphorylation [Moreover, in a study of PEPC in ammonium-stressed sorghum, the expression of the Cof light . NH4+ alm stress . Recent m stress  on sorghrylation . Thus, irylation . Anotherrylation . 3 plants, a study by Fukayama et al. [Monochoria vaginalis, and Sagittaria trifolia. In these plants, the phosphorylation levels at night were higher than those during the day. Among 24 other C3 species studied, 12 species showed diurnal phosphorylation, and 12 showed almost no changes in the phosphorylation levels between the day- and nighttime [M. vaginalis, have a PEPC protein inside the chloroplast in addition to the cytosol, which suggests a relationship between the presence of the chloroplastic PEPC and nocturnal phosphorylation of the cytosolic enzyme [In the case of Ca et al.  found noighttime . Two of c enzyme . Plant PEPCs belong to a small gene family encoding several PTPCs and distantly related BTPCs ,22. The B. sinuspersici, another type of PEPC, termed PEPC4, was found to be highly expressed in young leaves (unpublished data), in addition to PEPC3 [B. sinuspersici, the BTPC component is thought to be a 117-kDa tetraenzyme (B. sinuspersici leaf development stages is shown in In to PEPC3 . PEPC4 ito PEPC3 , upregulto PEPC3 . In the B. sinuspersici, the Class-1 PEPC is expressed in both intermediate and mature leaves, based on the presence of the phosphorylated p107 and monoubiquitinated p110 PTPCs. The close interaction between BTPCs and PTPCs in a Class-2 PEPC complex during early leaf development may indicate that BTPCs have a significant regulatory role that is commenced during the early stage but is no longer needed later, as other mechanisms, such as PTMs, can provide further regulation at a more advanced stage of leaf development.While the hetero-octameric Class-2 PEPC is highly expressed in young leaves of B. sinuspersici, BTPC acts as a regulatory enzyme, which provides components that may be critical for PEPC catalysis and heterologous expression, such as in green algae, or for PEPC activity, such as in the case of a COS BTPC [Hence, from a single cell during the early leaf development of COS BTPC ,55. Howe2 assimilation in C4 and CAM leaves, during rapid nitrogen assimilation following NH4+ or NO3− resupply to nitrogen-limited cells, or during photosynthate partitioning to storage end-products in developing COS [This activation of Class-1 PEPCs by phosphorylation has mainly been observed in plant tissues in which a high and tightly controlled flux of PEP to malate plays a well-known metabolic role, e.g., upon nutritional Pi starvation during COping COS ,57,58,59SbPPC6 genes encodes the distantly related BTPC. However, transcript of this gene was low during the development and germination of seeds [Moreover, in the case of sorghum seeds, of seeds .3 plants, such as lily in a study by Igawa and colleagues [Arabidopsis, but there was a difference in the initiation of the expression of each BTPC during pollen development. LIBTPC started to accumulate following GC formation after pollen mitosis I, whereas AtBTPC expression started immediately following sperm cell formation after pollen mitosis II [In Clleagues , a BTPC,tosis II . Meanwhitosis II . B. sinuspersici leaves, castor oil plant leaves, sorghum seeds, Arabidopsis root and rosettes, and soybean seeds. This differential expression can also be tissue specific, and at large, varies depending on plant organs or tissues. Various PEPC isoforms may also differ in PEPC activity depending on conditions either in vivo or in vitro. Factors such as salinity, light, temperature, and ammonium-stress may also regulate PEPC activity by simply affecting post-translational control mechanisms such as phosphorylation and/or monoubiquitination. Although phosphorylation and monoubiquitination seem to be exclusive and work in an opposite pattern, studies in other plants such as in the case of sorghum roots showed that both PTMs occur simultaneously to increase PEPC activity. Both PTMs regulate Class-1 PEPC activity in different plant tissues under various physiological conditions. Other regulating mechanisms also include changes in intracellular pH, and presence of allosteric effectors. However, in the castor oil plant, monoubiquitination is more widespread than phosphorylation and appears to be a more predominant PTM of the Class-1 PEPC. While most PEPCs encode PTPCs, inclusion of the distantly related BTPCs into the Class-1 PEPCs will comprise Class-2 PEPC which was regarded as a 910 kDa hetero-octameric complex in castor plant. Within the Class-2 PEPC complex, both PTPC and BTPC subunits exhibit PEPC activity in developing COS. With the vast potential of regulatory mechanism affecting PEPC activity, an understanding of the intricate interaction between these post-translational mechanisms as well as the occurrence and function of such in different plant tissues in various stages of development both in normal and stressed conditions will be a challenge worthy to notice for one to fully describe the roles and metabolic control of plant PEPC. Such an undertaking, if considered, may further elucidate the crucial role of PEPC in plant carbon metabolism.PEPC plays a crucial part in plant carbon metabolism. Different stages of plant development portray differential expressions of PEPC activity as in the case of


This study was conducted to estimate multi-trait genetic parameters for somatic cell score (SCS), milk yield and type traits in Nigerian Dwarf (ND) goats from the United States.2), MILK, FAT, PROT, and 14 type traits was applied. A factor analytic approach (FA1) in ASReml software was used to obtain convergence.Data from 1,041 ND goats in the United States with kiddings in 95 herds were used to estimate multi-trait genetic parameters for SCS, milk (MILK), fat (FAT), and protein (PROT) yields, and 14 type traits. An 18-trait mixed linear animal model for lactation mean SCS (Log2), and were 314±110.6, 20.9±7.4, and 14±4.9 kg, respectively, for MILK, FAT, and PROT. Heritabilities for SCS, MILK, FAT, and PROT were 0.32, 0.16, 0.16, and 0.10, respectively. The highest heritabilities for type traits were for stature (0.72), teat diameter (0.49), and rump width (0.48), and the lowest estimates were for dairyness (0.003) and medial suspensory ligament (0.03). Genetic correlations of SCS with MILK, FAT, and PROT were positive but low . Genetic and phenotypic correlations between MILK, FAT, and PROT were high and positive (≥0.66). Absolute values of genetic correlations involving SCS with type traits were generally low or no different from zero. Most of the phenotypic correlations involving SCS with type traits were low. No serious unfavorable genetic correlations between milk yield traits and SCS or between milk yield traits or SCS and type traits were found.Averages for SCS were low (2.85±1.29 LogGenetic variation exists in the ND breed for most studied traits. The development of selection programs based on these estimates may help accelerate favorable multi-trait genetic changes in this breed. Reliable estimates of genetic parameters, including heritability and genetic (co)variances, are crucial to the establishment of efficient genetic improvement programs and development of multi-trait selection indices . Multi-tFew joint multi-trait quantitative genetic analyses of somatic cell score (SCS), milk and type traits have been conducted simultaneously in goats . Data onIn this analysis, we used data from 1,041 ND goats registered by the ADGA with kiddings in 95 US herds from 2005 to 2015. Animals were evaluated for the following 14 type traits: final score (FIS), stature (STA), strength (STR), dairyness (DAI), rump angle (RUA), rump width (RUW), rear legs (REL), fore udder attachment (FUA), rear udder height (RUH), rear udder arch (RUC), medial suspensory ligament (SUS), udder depth (UDD), teat placement (TEP), and teat diameter (TED), using the ADGA appraisal system. A scale of 50 to 99 points was used for FIS, while a scale of 1 to 50 points was used for all other (linear) type traits ,7.2 +3 [Milk samples were collected as part of the official national milk recording system used by ADGA. Individual data on milk yield were obtained monthly during the complete lactation by using equipment built into the milking system according to Dairy Herd Improvement Association standards. Milk analysis included SCC using Soma Count laser-based flow-cytometry equipment calibrated with cow milk . Fat and0,000)+3 ,9. For S0,000)+3 . All typ2 SCC/0,000+3 [Animals with incomplete information were eliminated. Only one record was retained for each animal in the final file, since the small numbers of animals with repeated records do not allow for an accurate estimate of a permanent environmental effect, which could have compromised the unbiasedness of the genetic parameter estimates. Records from animals with less than three test day records for milk traits and SCS and from herds with less than three records were also deleted.Pedigree was verified and ordered and was pruned to keep only connected ancestors up to two generations back from animals with records. The final file contained a total of 2,762 ND animals. Excluding the base animals, 2.7% of sires and 4.9% of dams were unknown.2), MILK, FAT and PROT [The model used in the analysis was an 18-trait mixed linear animal model for mean SCS  and (co)variance matrices (Var) of the model effects are defined in the following equations:A is the numerator additive relationship matrix, ⊗ is the Kronecker product operator and ija, σijh and σije are scalars.Where: 2), MILK, FAT, and PROT included fixed effects of year of kidding, lactation number , kidding season . A Legendre regression polynomial of order 3 for average days in milk was applied as fixed effect for SCS only. The model for type traits included the fixed effects of the linear and quadratic effects of age at appraisal, plus the effect of month of appraisal which included five levels from May to September. Some data from April and October were classified together with the May and September levels, respectively. Random correlated herd-year, animal and error terms were fitted for all traits. The inverse of the numerator additive genetic relationship matrix A was used to estimate the variances and covariances in the model. Genetic parameters were obtained using the sum of animal and error variance as estimates of phenotypic variance similar to García-Peniche et al [The model for SCS  with values between 0.18 and 0.25 . The genModerate negative values of genetic correlations were found between FIS with SUS, RUW, STA, and DAI (−0.36 to −0.46), and a positive genetic correlation was observed with RUH (0.33) . PositivHigh and positive phenotypic correlations were found between milk yield traits (≥0.84), with moderate to low correlations between FIS, STA, DAI, RUW, RUC, SUS, and FUA with milk yield traits (from 0.12 to 0.33) and negative correlations between UDD with milk yield traits (approximately −0.26). Other moderate and positive relationships were observed between FIS with DAI, FUA, RUC, RUH, TEP, and SUS, with values ranging from 0.24 to 0.45, and we found negative correlations between DAI with STR (−0.39) and UDD with FUA (−0.24), RUC (−0.23), and SUS (−0.17).The results obtained are the first report for this breed for the traits included in this study. A FA1 was used to facilitate the simultaneous analysis for a 18-trait model by using single summary lactation records for SCS, MILK, FAT, and PROT. Here we adopted an approach based on summary lactation information for SCS and milk yield traits. From a practical point of view, lactation summary information is a logical approach to the genetic evaluation of dairy goats, similar to that used by the USDA for the genetic evaluation of dairy goats in the United States ,15.3 cell/mL for Nubian and Toggenburg breeds in the United States [Averages for SCS of ND were low, compared to other breeds of dairy goats ,16,17. Ad States .For MILK, FAT, and PROT, ND productions were low compared to other US goat breeds, whose minimum and maximum values ranged from 874 to 1,169 kg for MILK, 37 to 42 kg for FAT and 27 to 35 kg for PROT . The meaHeritabilities for milk yield traits were lower compared to those estimated in other goat populations ,23,24, b2 of SCC/1,000) in mixed dairy goats using a random regression model was 0.12 during the first month of lactation and increased to 0.25 at the end of lactation [2 (SCC) from each flock-test, in dairy goats also from New Zealand.Heritability for SCS in ND was slightly higher than those obtained in Alpine and Saanen goats in France 0.20 and 0.24) , and 0.2 and 0.24actation . Scholteactation  obtainedThe estimated heritabilities for type traits in the goats of this study were not notably different from those obtained by Luo et al  and CastGenetic correlations between SCS with milk yield traits similar to those estimated in Alpine and Saanen breeds in France (from −0.13 to 0.12) , and f oIn this study, the values of genetic and phenotypic correlations between milk yield traits were high as is usual in dairy goats ,21,26,27Because of low genetic correlations between SCS and type traits, options for indirect selection are limited; however, the heritability for SCS was moderate (0.32), therefore direct selection for this trait may be successful.McLaren et al  estimateFinal score is an important trait because it evaluates the overall type of an animal and therefore is related to linear type traits because it is calculated on the basis of four major categories: general appearance, dairy character, body capacity, and mammary system ,7. FinalMcLaren et al  generallLuo et al  found thThe phenotypic correlations estimated by Luo et al  among tyMontaldo and Martínez-Lozano  studied In another study, Valencia-Posadas et al  estimateConsiderable multi-trait genetic variation exists in the ND breed for most of the traits considered in this study, which could be expected insofar as it is a breed that has only recently started systematic breeding. However only moderate heritability values were found for MILK and FAT and lower for PROT. It is difficult to determine the reason for these particular heritability values for milk yield traits, but the data structure and the model may have contributed. Strong and positive genetic correlations were found between MILK, FAT, and PROT with STA, SUS, DAI, and RUW, and moderate to highly negative correlations were observed between the same milk yield traits with RUH and RUA, suggesting that our results are different from those found in other dairy goat populations in the US, since some type traits in ND goats are related to milk yield traits. These relationships will need to be confirmed in further studies. The development of selection indices to weight the different traits on a rational basis may help accelerate favorable changes in this breed. The development of genetic programs in this breed could be a factor favoring their increased use in commercial lines of dairy goats to avoid losses of genetic diversity.In conclusion, substantial genetic variation was observed in many of the studied traits, including SCS, therefore selection can be used for their improvement. Genetic and phenotypic correlations between milk traits were very high as is typical in dairy goats and absolute values of genetic correlations involving SCS with type traits were moderate to low. High and positive genetic correlations were found between DAI, RUW, SUS, and STA with milk yield traits, and high but negative correlations were identified between RUH and FIS with the same milk yield traits. Most of the phenotypic correlations obtained in this study were low.


This research project aimed to investigate the physico-chemical, sensory, hygienic and safety characteristics of raw goat milk, whey, brine and traditional goat cheese during the ripening period of 28 days. Physico-chemical parameters included the determination of dry matter, fat, ash, protein, pH, water activity and NaCl content. The presence of Enterobacteriaceae and fungi was estimated on milk and cheese samples, and a sensory panel evaluated the products’ features and acceptability during ripening. The results show that the cheese under study belongs to the acid full-fat cheese group. A consumer panel attributed high scores to the goat cheese, until the 21st day of ripening. After this period, the overall features altered significantly, including augmented bitterness, odor intensification and the development of molds on the surface. The presence of fungi, associated with Enterobacteriaceae, suggests that the hygiene of the production processes needs to be improved. Regarding microbial safety, the detection of putative pathogens and antibiotic resistances recommend an active surveillance of traditional foods to avoid foodborne infections and/or the dissemination of resistant microorganisms along the food chain. Fermented foods are a very important part of the human diet, and fermentation has been used to improve the shelf life, safety, digestibility and nutritional value of diverse foods . WorldwiMembers of the Enterobacteriaceae family are an important members of the microbial communities present in raw milk and cheeses (such as Montasio and Urfa cheeses) ,8,9. TheAntimicrobial drug resistance (AMDR) is one of the top 10 global public health threats facing human and animal health in the 21st century, due to the lack of therapeutic options . In addiIn this context, the present study evaluated the physico-chemical properties of Serbian goat milk, cheese, brine and whey as well as the presence of Enterobacteriaceae and molds. Moreover, the sensory features of the traditional goat cheese were also assessed during 28 days of ripening. To further elucidate the safety of the foods under study, enterobacteria were evaluated regarding antibiotic resistance profiles.The cheese under study was manufactured in a countryside household in the Pajsijević village  in spring of 2021. Fresh hand-milked goat milk (pH 6.6) was collected after morning and evening milking in an enameled pot and stored at 10–12 °C (temperature in cellar). The raw milk was filtered through gauze into an enameled pot and heated to 32 °C, and a liquid rennet Sirela  (85% chymosin and 15% pepsin) was added in the amount of 28 mL for 10 L of milk, with constant stirring. After 30–60 min, the whey separated, and the clotted mass was cut into cubes using a knife. The cheese was mixed with a wooden spoon by turning it over  and covered with a gauze. After a rest time of 15 min, the entire contents were transferred to a new, large gauze and left to rest overnight, to enable whey removal. The next morning, the cheese was salted  and cut into cubes. The cubes were placed in wooden boards and poured with brine ). A cotton cloth was placed on top, which was then covered with a wooden board and finally with a 1 kg marble stone and stored in a cellar (ripening period) at 15–16 °C, for 28 days. Ten liters of milk were needed to produce 2.65 kg of cheese. Generally, the cheese is consumed fresh, i.e., immediately after production. This study investigated the maximum shelf life of cheese during ripening in described conditions in which the cheese could be safely consumed.For the present study, cheese samples (200 g) were collected from the container immediately after manufacturing, i.e., immediately before storage (day 0) and during the next 28 days, i.e., at the 7th, 14th, 21st and 28th day of ripening; aseptically transported to the microbiology laboratories at the Department of Science, Institute for Information Technologies, University of Kragujevac and at the Faculty of Technology, University of Novi Sad; and kept at 4 °C until processing. The analysis occurred in the next 24 h after sampling. Goat’s milk, brine and whey were also sampled.Kjedahl [aw value) by using LabSwift-aw device ; NaCl content was determined according to Carić et al. [Chemical analysis of goat cheese was performed by using standard methods: dry matter content after drying cheese samples at the temperature of 105 ± 1 °C ; milk faKjedahl ; pH valuKjedahl ; water ać et al. , and fatć et al. . Milk anć et al. . The pH 0*−L*)2 + (a0*−a*)2 + (b0*−b*)2}1/2, where L0*, a0* and b0* are the value of cheese color components after production, and L*, a* and b*are the value of cheese color components during ripening [Milk and cheese color were evaluated using the photoelectric tristimulus colorimeter  with a CR-A33F special extension for the determination of cheese color parameters. The working principle was based on the measurement of reflected color and differences in color values on different cheese surfaces, including whiteness (L*), red/greenness (a*), and yellow/blueness (b*), and dominant wavelength (λ). All assays were performed in triplicate. The total color difference (ΔE) was calculated between measurements of cheese sample after production and cheese samples during ripening according to ΔE = { sodium citrate, previously sterilized and heated at 45 °C, and thoroughly mixed in the vortex until complete homogenization. Subsequently, successive ten-fold dilutions in 2% (w/v) sodium citrate were prepared and inoculated on selective media for Enterobacteriaceae counts, specifically Violet Red Bile Agar  [2S, TDA and VP assays) and growth on selective media, such as citrate agar  and HiChrome coliform agar . Presumptive Enterobacteriaceae were further analyzed using Microgen GN-A and Microgen GN-B test , and results were interpreted according to Microgen ID (software version 2.0.8.33). Lastly, identification was confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using the corresponding values to match manufacturer suggested results ≥ 2.00 [Samples were collected from the center of the goat cheese container, using a sterile spoon, on days 0, 7, 14, 21 and 28. The working section (10 g) was added to 90 mL of 2%  ,30,31. Ints, UK) . BrieflyThe total number of aerobic mesophilic bacteria was also enumerated in goat milk and cheese samples according to the standard method .Bacillus subtilis ATCC 6633 was used as a positive control, while E. coli ATCC 25922 was used as a negative control.The proteolytic activity was examined according to Harrigan and McCance , with slTo determine antibiotic resistance profiles, the disk diffusion method was applied following the methodology described by European Committee on Antimicrobial Susceptibility Testing (EUCAST). Four antibiotics, representing distinct classes and modes of action, were selected . According to EUCAST breakpoints  zones ofE. coli O157 rapid latex agglutination test  is designed to confirm the identity of E. coli serotype O157 isolated from samples of human origin or foods [E. coli serotypes. Latex particles are coated with antibodies raised against the somatic O157 lipopolysaccharide antigen of E. coli O157. When sensitized latex particles are mixed with an E. coli suspension containing the antigens O157, a sensitive and specific immunochemical reaction takes place, causing the finely dispersed latex particles to agglutinate into aggregates that are easily visible to the naked eye.or foods . The metAcremonium, Alternaria and Cladosporium, Geotrichum genera were subcultured on Sabouraud Maltose Agar (SMA) , while presumptive Aspergillus, Penicillium and Talaromyces genera were subcultured on Czapek Yeast Autolysate Agar  and incubated for 7 days at 25 °C ± 2 °C. The identification of molds was performed based on macroscopic  and microscopic  characteristics, according to criteria described by Samson and Frisvad [The total number of molds present in milk, whey, brine and cheese was evaluated according to standard procedures : 20 g of Frisvad  and Pitt Frisvad .t-test (Statistica 9.0) was applied to determine the significance of the differences observed.Results obtained are presented as mean ± standard deviation, and the The results observed for goat’s milk, whey and cheese during 28 days of ripening are displayed in The goat cheese colors were measured using the CIE Lab color system. It was evident that goat cheese samples  presenteThe goat cheese sensory features were analyzed during 28 days of ripening, using the 5-point hedonic scale, based on the evaluation of appearance, color, texture, cross-section, odor and taste. The results obtained are shown in 4 CFU/g (day 0) and 1.24 × 108 CFU/g (14th day) of cheese, whilst in milk the levels were similar to those observed at cheese day 0. As aforementioned, after 28 days of ripening molds developed on the surface of the cheese, which prevented the isolation of enterobacteria for further analysis.The counts of total enterobacteria present in goat’s milk and cheese samples were assessed using the VRBG medium . Briefly6 CFU/g (day 0) and 5.24 × 1011 CFU/g (14th day) of cheese. It could be noticed that the initial number of aerobic mesophylic bacteria was lower compared to the total number of enterobacteria in milk. However, in cheese samples, the number of aerobic mesophylic bacteria was higher compared to the total number of enterobacteria.The total number of aerobic mesophilic bacteria present in goat’s milk and cheese samples were addressed using a nutrient agar . MicrobiEscherichia coli (54 isolates), Proteus mirabilis (6 isolates), Rahnella aquatilis (1 isolate), Pseudomonas sp. (1 isolate), Enterobacter cloacae (2 isolates) and Enterobacter asburiae (1 isolate). It was observed that E. coli is the most dominant species in cheese, while P. mirabilis is the dominant in goat milk  and submitted to conventional microbiological characterization. All Gram-negative, catalase-positive and oxidase-negative bacteria were further analyzed by biochemical tests and with Microgen GN-A and GN-B tests . All isooat milk .P. mirabilis GM-6 and R. aquatilis GM-9, both isolated from milk, which showed proteolytic activity.The results of the investigation of the proteolytic and lipolytic activity of isolated bacteria from goat’s milk and cheese indicated that none of the isolates possess proteolytic or lipolytic activities . The excEscherichia showed resistance to gentamicin, but they were sensitive to amoxicillin, chloramphenicol and tetracycline. E. asburiae and R. aquatilis isolates were sensitive to all tested antibiotics, while E. cloacae isolates showed resistance to amoxicillin and tetracycline. Isolates from the genus Proteus showed sensitivity to gentamicin and chloramphenicol, but they were resistant to amoxicillin and tetracycline. Pseudomonas sp. showed resistance to tetracycline and amoxicillin. In general, there was no isolate that showed full resistance to antibiotics.Goat’s milk and cheese isolates showed different sensitivity to the tested antibiotics . All isoE. coli O157 rapid latex agglutination test, none of the tested E. coli isolates belong to the serotype E. coli O157.According to the Alternaria alternata and Geotrichum candidum. In whey, the total number of molds was 100 CFU/mL, while the identified species corresponded to Acremonium strictum, Penicillium brevicompactum, Penicillium chrysogenum, Penicillium expansum, Penicillium glabrum and Talaromyces albobiverticillus. The total number of molds in brine matched 80 CFU/mL, and the identified species were Acremonium strictum, Penicillium aurantiogriseum, Penicillium glabrum and Penicillium thomii. On the first day of cheese ripening (day 0), the total number of molds was 200 CFU/g, and the following species were identified: Cladosporium macrocarpum and Penicillium aurantiogriseum. On the 7th and 14th day of ripening, the total number of molds was less than 100 CFU/g, whereas in the 21st day, the total number corresponded to 5.4 × 105 CFU/g, and Aspergillus flavus was the dominant species  of cheese samples decreased, while b values increased during the 21 days of ripening. These results could be related with the cheese’s yellowness caused by moisture loss. The highest changes in cheese color occurred after 14 days of ripening, as a result of the microbiological activity of non-starter lactic acid culture and complex biochemical processes  [The sensory characteristics and color of goat cheese are fundamental to consumer acceptance. Cheese color and flavor depend on the presence of fat globules and casein micelles. Our results show that goat cheese samples have significantly higher L*, a and b values compared to goat’s milk. Regarding goat milk and cheese samples, lightness values ranged from 82.44 (goat milk) to 88.49  ,43.A. niger appeared, no sensory evaluation was performed on the 28th day of ripening, and the color of the cheese was not assessed.Regarding the organoleptic features and consumers’ acceptance of the goat cheese under analysis, the results show that Serbian traditional cheese presented excellent sensory characteristics until the 3rd week of ripening, after which the smell and bitter taste started to be noticeable. This perception reflected on the points attributed by the sensory panel, which decreased from the 14th day onwards. These undesirable features could have resulted from complex biochemical transformations attributed to specific production steps  and/or to the microbial communities present . Raw milFor consumers’ acceptance it is fundamental to trust the microbiological quality and overall safety of the cheeses produced under traditional domestic conditions, at the farm level. The microbial communities present in dairy foods, produced from raw milk, rely primarily on the microorganisms from the raw materials, the hygiene during production and the possibility of cross contamination during manufacture or storage . In fact4 to 1.24 × 108 CFU/g of cheese, from the first to the last day of ripening, respectively. The number of enterobacteria depends on the pH value of the cheese; as it becomes more acidic, the number of Enterobacteriaceae decreases [Coliform bacteria, such as members of the Enterobacteriaceae family, are considered fundamental indicators of the hygiene practices during the production processes . Specifiecreases . In the Bacterial identification on the species level is a very important aspect of microbiology. The VRBG agar was used for isolation of enterobacteria. Further, Gram-negative, catalase-positive and oxidase-negative isolates were submitted to biochemical tests and growth on selective media, such as citrate agar and HiChrome coliform agar  in order to detect presumptive enterobacteria. Isolates were further analyzed using Microgen GN-A and Microgen GN-B tests. These are the basic tests used for Enterobacteriaceae identification . HoweverSerratia, Enterobacter and Raoutella) from pasteurized milk samples. The authors indicated that diverse bacteria found in pasteurized milk, exhibiting phenotypic characteristics such as production of lipolytic and proteolytic enzymes, can result in milk spoilage. Similar results were verified in the present study since R. aquatilis showed proteolytic activity. Although Pseudomonas spp. represents the genus of psychotropic microorganisms most related to proteolytic activity in raw milk [Enterobacter and Klebsiella, usually do not display this trait [Enterobacteriaceae capable of synthesizing proteolytic and lipolytic enzymes are largely responsible for the deterioration of milk and dairy products, which may cause various issues in the dairy industry ,57. In craw milk , the goais trait , which wProteus mirabilis was predominant in milk, while E. coli was the most frequently recovered species from cheese. After the agglutination test, E. coli isolates were confirmed as not belonging to O157 serotype, which can be interpreted as a relevant result, as it points towards the microbial safety of the final product.The presence of Gram-negative bacteria in dairy foods is common ,47,60,61E. coli, Enterococcus faecalis and Salmonella spp. are not only resistant to multiple antibiotics given to animals but also to antibiotics made available to humans [E. cloacae strains are often isolated from dairy products. However, studies of the virulence factors of these strains and their MDR potential are still scarce. Nonetheless, in our research low levels of resistance were observed. All isolates showed susceptibility to chloramphenicol. E. coli isolates were resistant to gentamicin and susceptible to other tested antibiotics. E. cloacae, P. mirabilis and Pseudomonas spp. isolates showed resistance to amoxicillin and tetracycline, while E. asburiae and R. aquatilis isolates were susceptible to all antibiotics.Antibiotics have been extensively used in human and veterinary medicine, and in agricultural settings, for the treatment of infections, growth enhancement and prophylaxis, potentially leading to the selection of drug- and multidrug-resistant bacteria ,66,67. Io humans . These bo humans . Hence, o humans . AdditioMold contamination in cheese can affect its organoleptic properties, influence the cheese quality and potentially produce secondary toxic metabolite mycotoxins, and therefore poses a potential health risk to consumers. Mycotoxin cheese contamination can occur indirectly via milk contamination or directly through mycotoxin producing spoilage molds . HoweverPenicillium, Geotrichum, Aspergillus, Mucor and Fusarium [Fusarium merismoides, Penicillium glabrum, Penicillium roqueforti, Aspergillus fumigatus, Engyodontium album, as well as species of the Cladosporium and Torrubiella genera, are common [Penicillium species, while Alterinaria alterinata, Geotrichum candidum and Cladosporium macrocarpum were less dominant. The presence of the aflatoxigenic species Aspergillus flavus was observed on the cheese on the 21st day of ripening. According to the obtained results the molds found in cheese samples may indicate poor hygienic conditions during cheese production and/or storage, such as Cladosporium macrocarpum, or from the brine, such as Penicillium aurantiogriseum. Since the toxigenic mold Aspergillus flavus was found only in the cheese sample, after the 21st day of ripening, it is most likely that its occurrence resulted from cross-contamination.The presence of fungi in raw milk can be influenced by the physiological state of the animal, as well as the weather, feeding and season ,65. FungFusarium . At the e common . In the The present investigation evaluated the physico-chemical, sensory, hygienic and safety characteristics of raw goat milk, whey, brine and traditional cheese during the ripening period of 28 days. The results show that the cheese under study belongs to the acid full-fat cheese group. A consumer panel attributed high scores to the goat cheese, until the 21st day of ripening. After this period, the overall features altered significantly, including bitterness, odor intensification and the development of molds on the surface. The presence of fungi, associated with Enterobacteriaceae, suggest that the hygiene of the production processes needs to be improved. The results indicate the need for better hygiene during milking and cheese production. Additionally, efforts are being made to control the production of traditionally made cheese, i.e., to place it under industrial conditions and to use pasteurized milk or starter cultures previously isolated from the same cheese in order to attain a standard of uniform quality and safety. Regarding microbial safety, the detection of putative pathogens and antibiotic resistances is recommended for an active monitoring of traditional foods to avoid foodborne infections and/or the dissemination of resistant microorganisms along the food chain.


Patients with a spinal cord injury (SCI) frequently experience sudden falls in blood pressure during postural change. Few studies have investigated whether the measurement of blood flow velocity within vessels can reflect brain perfusion during postural change. By performing carotid duplex ultrasonography (CDU), we investigated changes in cerebral blood flow (CBF) during postural changes in patients with a cervical SCI, determined the correlation of CBF change with presyncopal symptoms, and investigated factors affecting cerebral autoregulation. We reviewed the medical records of 100 patients with a cervical SCI who underwent CDU. The differences between the systolic blood pressure, diastolic blood pressure, and CBF volume in the supine posture and after 5 min at 50° tilt were evaluated. Presyncopal symptoms occurred when the blood flow volume of the internal carotid artery decreased by ≥21% after tilt. In the group that had orthostatic hypotension and severe CBF decrease during tilt, the body mass index and physical and functional scores were lower than in other groups, and the proportion of patients with a severe SCI was high. The higher the SCI severity and the lower the functional score, the higher the possibility of cerebral autoregulation failure. CBF should be assessed by conducting CDU in patients with a high-level SCI. Significant cardiovascular and autonomic dysfunction is a common consequence of high-level spinal cord injuries (SCIs) . PatientPossible mechanisms underlying OH in SCI patients include changes in sympathetic activity, altered baroreflex function, lack of skeletal muscle pumping activity, cardiovascular deconditioning, and altered salt and water balance ,5. HowevFew studies have investigated whether the measurement of blood flow velocity within vessels can reflect brain perfusion during postural change. The blood flow velocity in the middle cerebral artery (MCA) is highly correlated with CBF under conditions of varying mean arterial pressure and cerebrovascular conductance ,8. We beFew studies have measured CBF in patients with an SCI and evaluated cerebral hemodynamics during postural changes. In this study, we assessed CBF by using CDU to measure changes in the internal carotid blood flow during postural changes.The purpose of this study was to investigate, with CDU, the changes in cerebral blood flow volume (CBFV) after postural change, using a tilt table in patients with tetraplegia due to a cervical spinal cord injury (CSCI). We aimed to confirm the correlation of CBFV with symptoms of OH and evaluate the necessity of applying this test in actual clinical practice. In addition, we aimed to determine whether the degree of the SCI was related to changes in CBF when standing, and to elucidate the factors that affect CA.This study was a retrospective review of the medical records of all patients who underwent CDU after a CSCI at the Department of Rehabilitation Medicine, Chungnam National University Hospital, from 1 January 2018 to 31 December 2019.Patients with complete or incomplete tetraplegia due to a CSCI and who were 18 years of age or older were included in the study. We excluded cases with insufficient medical records or insufficient test results to explain hemodynamic changes, cases with history of fatal cardiovascular disease, and cases of unstable SCIs. Finally, the medical records of 100 patients were reviewed.2) × time-averaged mean velocity (cm/s). The peak systolic velocity, end diastolic velocity, time-averaged mean velocity, vessel diameter, and vessel area were all measured during the test. All patients were allowed to rest on the examination table for 5 min before the test. CBFV, BP, and heart rate (HR) were measured in the supine position, immediately after the patient was tilted by 50°, and 5 min after the tilt. To increase the reliability of the test, CBF was measured three times in each position, and the mean value was used. In addition, the presence or absence of presyncopal symptoms, such as dizziness, light-headedness, nausea, and blurry vision, in each position, was recorded.We conducted CDU during head-up tilt in patients with a CSCI. The ICA was studied on both sides, and intravascular flow volumes were calculated using a 9-MHz linear array transducer. BFV measurements were automatically calculated by the built-in software of the ultrasound device . For flow-volume measurements, the head was turned 25°–40° to the contralateral side, and a straight segment of the ICA, at least 2 cm above the carotid bulb, was selected . MeasureThe data obtained during the ICA Duplex ultrasonography were systolic blood pressure (SBP), diastolic blood pressure (DBP), HR, average BFV, and presence of OH symptoms in each position. In order to determine whether the patient experienced OH symptoms when BFV (L/min) was reduced to some extent during the tilt, after obtaining the BFV difference (BFV difference (%) = BFV(supine) − BFV(tilt 50° or 5 min)/BFV(supine) × 100) between the value measured in the supine posture and the value measured after 5 min at 50° of tilt, the point with the highest sensitivity and specificity was obtained by applying the receiver operating characteristic (ROC) curve to the relationship between the BFV difference and the presence of symptoms. It was assumed that there would be symptoms of OH when CBF fell below that point.We calculated the difference between the SBP, DBP, and CBFV values measured in the supine position and the values measured after 5 min at 50° tilt. Based on the presence of OH or ΔCBF, the patients were divided into four groups. The presence of OH was determined according to criteria defined by The American Autonomic Society and the American Academy of Neurology (OH was defined as a decrease in SBF of at least 20 mm Hg or a decrease in DBF of at least 10 mm Hg within three minutes of standing up). If there was a difference in BFV beyond the set point of the presyncopal symptoms mentioned above, it was marked as ΔCBF+; otherwise, it was marked as ΔCBF−. As shown in Data on age, height, body mass index (BMI), duration of injury (DOI), American Spinal Cord Injury Association impairment scale (AIS) grade, sex, neurological level of injury (NLI), and underlying disease (such as diabetes mellitus (DM) and hypertension (HTN)) were collected in each group. To confirm the relationship between functional status and CA, the motor score (MS), sensory score (SS), and Korean spinal cord independence measure (K-SCIM) score were assessed. In addition, the presence or absence of presyncopal symptoms after 5 min at 50° tilt was noted in each group.p < 0.05.The ROC curve was used to determine whether the patient experienced symptoms when the BFV (L/min) decreased to a certain extent at 5 min after 50° tilt. A one-way analysis of variance was used to compare differences in age, height, BMI, DOI, MS , MS , total MS, SS , SS , total SS, and K-SCIM among the four groups. For post-analysis, the Scheffe test was used to determine whether the differences among the groups were significant. The data are reported as mean ± standard error. Crossover analysis and chi-squared test were used to evaluate differences in distributions of AIS grades, sex, NLI, DM, HTN, and the presence of presyncopal symptoms among the four groups. All statistical analyses were performed with IBM Statistical Product and Service Solutions software, version 26.0 . Statistical significance was set at Of the 100 patients included in the study, 40 complained of presyncopal symptoms during tilt. The ROC curve of the difference in CBFV after tilt in the presence of presyncopal symptoms is shown in p-value < 0.05, 2 (p = 0.006) higher than that in G1. MS (UE), MS (LE), MS , and K-SCIM scores were lower in G1 than in the other groups, and the differences were statistically significant. SS (LT), SS (PP), and SS  values in G1 were also significantly lower than those in G4 (p < 0.05).G1  had 32 patients, G2  had 18, G3  had 10, and G4  had 40 patients, as shown in Presyncopal symptoms were found in approximately 96.88% of patients in G1, 5.56% in G2, 40% in G3, and 10% in G4 . In G2, Presyncopal symptoms occurred when the BFV of the ICA decreased by ≥21% after tilt in patients with a CSCI.In G1, i.e., patients who had OH and severe CBF decrease during tilt (because CA did not occur), the BMI was lower than that in G4 (patients who had neither OH nor CBF decrease); physical and functional scores such as MS (UE), MS (LE), MS , SS (LT), SS (PP), SS , and K-SCIM scores were low; and the proportions of AIS grades A, B, and C were high.In G2, i.e., the group of patients who had no decrease in CBF even though there was OH during tilt (because CA occurred), presyncopal symptoms rarely occurred (5.56%).To our knowledge, this is the first study to use CDU to investigate changes in CBF during postural change in patients with a CSCI. The main findings are as follows:The mechanism that underlies OH after an SCI remains unclear. According to some studies, if there is a problem in the pathway from the motor center to the sympathetic nerves due to a high-level SCI, the activation of the sympathetic nervous system through baroreceptors and chemoreceptors fails. Therefore, when external factors that can affect BP, such as postural changes, occur, the mechanism of maintaining normal BP does not work. Therefore, in the early stages of an SCI, OH is a result of decreased sympathetic nerve response, venous vasodilation, abdominal muscle paralysis, and inadequate secretion of hormones ,13. HoweIn 1991, a study using TCD suggested that the difference in CA, which regulates CBF, rather than changes in BP, would be involved in symptomatic improvement in patients with sympathetic nervous system abnormalities due to an SCI . Since tIn the aforementioned studies of CBF in patients with an SCI, TCD was used to assess the flow velocities, but the cross-sectional areas of blood vessels could not be evaluated; therefore, there is a limit to measuring the exact CBFV with TCD. For this reason, we used CDU to measure blood flow, and we measured ICA BFV as an estimate of CBF. Previous studies have measured ICA BFV to estimate CBF ,10,11,12CDU was used to assess CBF in this study. Previous studies have measured regional CBF according to changes in BP using TCD, and some studies have shown that the ICA/MCA region is more sensitive to orthostatic challenges than the vertebral artery/posterior cerebral artery region ,27,28. FPatients with an SCI suffer from voiding problems, for which most of them take medications such as α-blockers, which can cause OH. This study did not find out whether patients were taking such medications, and this should be considered in future studies. Another limitation is that only the presence or absence of presyncopal symptoms was noted; their severity, which would be more useful for determining the degree of orthostatic tolerance, was not considered. In order to obtain a more accurate conclusion, future studies should include more patients and have a longer follow-up period. There is no precise guideline on whether to administer an anti-OH drug if the SBP falls but the CBF is preserved. In a future study, the follow-up of changes in CBF and prognosis after treatment of OH will be helpful in establishing guidelines for further treatment.This study used CDU to confirm changes in CBF during postural changes in patients with a CSCI. If CBFV decreased by more than 21%, presyncopal symptoms occurred. However, even in the presence of OH, if CBFV was preserved , patients were less symptomatic. The higher the SCI severity and the lower the functional score, the higher the possibility of CA failure. It is, therefore, necessary to use CDU to assess CBF in patients with a high-level SCI, in order to ensure proper administration of drugs and smooth rehabilitation, and to ultimately improve the quality of life of patients.


Further research is needed to confirm these data and to maximize the developmentally positive effects of videogames and preventing the negative consequences.Videogames use has constantly increased among children and adolescents, with uncertain consequences on their health. This study aimed to assess the prevalence of videogames use and addiction in a sample of Italian children during the COVID-19 pandemic and their association with anxiety symptoms. One hundred and sixty-two children  completed the Videogame Addiction Scale for Children (VASC), the Test of Anxiety and Depression (TAD) and the Children’s Anxiety Meter—State (CAM-S). Demographic variables and data on the access to electronic tools and games preferences were also collected. Overall, 96.3% of the study participants stated to have access to one or more device. They reported a low risk of videogame addiction (VASC score (mean ± SD): 46.7 ± 15.4), a moderate level of trait anxiety (TAD score (mean ± SD): 135 ± 16.8) and a low state anxiety (CAM-S score (mean ± SD): 2.2 ± 2.1). Males reported to spend more time on videogames, to perceive higher self-control and to be more influenced by reinforcement mechanisms; females described higher levels of trait anxiety. In the regression analysis, state anxiety was a predictor of videogame use and addiction ( Over the last decade, the amount of time that children and adolescents spend online playing videogames has constantly increased . LockdowThe consequences of online videogame use depend on frequency and duration. Previous studies found positive effects of online gaming in children, including distraction from pre-operative anxiety ,4, motivBy contrast, other studies found a correlation between excessive and compulsive use of videogames and retreat, social problems, attention difficulties and criminal and aggressive behaviors ,19,20. RAddictive behaviors towards playing online have been linked to a possible diagnosis of Internet Gaming Disorder (IGD) . The DiaRecent research documented the association between IGD and psychosocial variables in adolescents, including anxiety, depression, OCD, somatization and social difficulties ,25,26. MA recent study  has provIn this respect, anxiety can be defined as an alarm reaction to a stimulus perceived as dangerous, with the feeling that something bad is going to happen ,29. It rAccording to the DSM 5, anxiety disorders include conditions that share features of excessive fear and anxiety and related behavioral disturbances, leading to an impairment in daily functioning . A numbeWe conducted a cross-sectional study to assess the prevalence of videogames use and videogames addiction in a sample of Italian children during the COVID-19 pandemic. We also investigated the prevalence of trait and state anxiety symptoms, given their possible increase related to the COVID-19 pandemic, and their association to videogames use.We recruited a sample of children in three primary schools in the province of Catania ; all children were attending school in presence in the last three months . After discussing with the school heads and receiving their consent to the administration of the tests during school hours, we sent written information to all parents or legal guardians of children attending these primary schools, being available for face-to-face contacts but also to discuss the study and its characteristics by phone or messaging systems. After that, all parents or legal guardians signed an informed consent. In total, 162 children , 8–10 years old , were included in this study: 24.7% of them were an only child, while 75.3% had brothers or sisters.We administered an ad hoc questionnaire to explore the demographic variables , the access to electronic tools that could be used for playing and the games usually played.(1)the Videogame Addiction Scale for Children (VASC) : specifi(2)the Test of Anxiety and Depression (TAD) : this is(3)the Children’s Anxiety Meter—State (CAM-S) : this isMoreover, each child completed three questionnaires:Statistical analysis was performed using IBM SPSS Statistics 26 for Windows.Ethical approval for the study was granted by the Ethic committee of the Department of Education Science, University of Catania.Overall, 96.3% of the 162 children recruited reported access to one or more devices used to play videogames (console (65.4%), smartphone (56.2%), tablet (48.1%) or personal computer (36.4%)). Their favorite games were Fortnite (21.6%), FIFA (10.5%), Minecraft (7.4%), SuperMario (6.2%) and Just Dance (4.3%).We found an average score at the VASC of 46.7 (SD: 15.4), indicating a low risk of videogame addiction; the average score at the TAD was 135 (SD 16.8), indicating a moderate level of pathological trait anxiety; as for the CAM-S, a low level of state anxiety was reported .p < 0.001), declared higher self-control  and had a higher level of reinforcement mechanisms , but also of trait anxiety .There were significant differences comparing males and females . Despiter = 0.19; p < 0.05), reinforcement mechanisms  and involvement in videogames use , while we found no significant correlations between trait anxiety levels and the VASC score or sub-scores. Analyzing males, state anxiety had a positive correlation with videogame involvement , while trait anxiety correlated with more frequent use of videogames , problems with life-style and time-use  and self-control . As for females, state anxiety was positively related to videogames use  and with reinforcement mechanisms , while trait anxiety correlated with videogames use , reinforcement mechanisms  and involvement in videogames .The correlation analysis showed in the whole sample that state anxiety was positively related to videogames use (p = 0.01) was a possible predictor of videogame use and addiction, while trait anxiety was only “nearly significant” .We also conducted two linear regression analyses with the enter method between a dependent variable (VASC) and two independent variables (TAD and CAM) on the whole sample. They showed that state anxiety , in a sample of 780 school children before the COVID-19 pandemic 7, in a s.To date, there is still an ongoing debate about the proper conceptualization of IGD ,36. VideThe existing literature has focused on five aspects to be considered to assess the effect of videogames on children: the amount of time spent playing , the content of the videogame, the game context, the game structure and the game mechanics ,35. VideMoreover, a large body of research clearly documented the association between IGD and anxiety symptoms in adolescents. In this regard, previous studies showed how videogames may be used as a treatment tool for internalized anxiety ,42,43 whRegarding gender differences, previous studies found that an increased amount of time spent playing with videogames was associated with higher anxiety symptoms in female adolescents. By contrast, it was associated with decreased anxiety levels in males ,43. SociOur data are relevant to help children and caregivers to be aware of the risk of developing addiction to videogames . Of noteOur study has some limitations. First, its cross-sectional design does not allow to understand the evolution of the relationship between anxiety and videogaming. Second, children were recruited during the so-called “second wave” of the SARS-CoV-2 diffusion in Italy, a fact that could have increased the reported levels of anxiety. Third, we used only self-administered questionnaires, a fact that could limit the interpretation of our results. Specifically, we assessed IGD and anxiety symptoms. Only a detailed clinical assessment can identify IGD and anxiety disorders. Fourth, the sample size was relatively small; therefore, our data should be replicated on larger samples. In fact, the number of children recruited is limited and could therefore be insufficient to represent the population of subjects of the same age range.Notwithstanding these limitations, this study provides information about an emergent important issue in children in the context of a highly stressful and unique situation, such as the COVID-19 pandemic. Recently, a possible use of active video games for improving mental health and physical fitness during isolation periods was reported ; however


Computed Tomography (CT) is widely used in oncology for morphological evaluation and diagnosis, commonly through visual assessments, often exploiting semi-automatic tools as well. Well-established automatic methods for quantitative imaging offer the opportunity to enrich the radiologist interpretation with a large number of radiomic features, which need to be highly reproducible to be used reliably in clinical practice. This study investigates feature reproducibility against noise, varying resolutions and segmentations (achieved by perturbing the regions of interest), in a CT dataset with heterogeneous voxel size of 98 renal cell carcinomas (RCCs) and 93 contralateral normal kidneys (CK). In particular, first order (FO) and second order texture features based on both 2D and 3D grey level co-occurrence matrices (GLCMs) were considered. Moreover, this study carries out a comparative analysis of three of the most commonly used interpolation methods, which need to be selected before any resampling procedure. Results showed that the Lanczos interpolation is the most effective at preserving original information in resampling, where the median slice resolution coupled with the native slice spacing allows the best reproducibility, with 94.6% and 87.7% of features, in RCC and CK, respectively. GLCMs show their maximum reproducibility when used at short distances. Nonetheless, since the early nineties2, radiologists have benefited from computer-aided systems exploiting a large number of features, developed by specialized research groups. The recent increase in high-performance computing resources in entry-level workstations and the growth of automatic tools for radiomic analyses, has made them popular and accessible to research groups. Accordingly, the number of radiomic studies has exploded, a large number of features is analysed to measure macroscopic tumour or tissue characteristics or to find latent properties3. The automatic quantification of tissue features based on radiomic approaches has shown improvements in terms of both reproducibility, discrimination and classification capability4, thus increasing the number of candidate imaging biomarkers3. However, the plethora of software packages available for radiomic analyses used by groups, with different degrees of expertise, has highlighted the urgent need for standardisation of methodology and measurements6. In fact, many factors are known to induce variability in radiomic features including noise7, heterogeneous voxel size8 and other CT parameter settings9, Region Of Interest (ROI) segmentation13, as well as tumour phenotype14. Despite its importance, only a few studies perform a dedicated analysis of the robustness and reproducibility of radiomic studies. Some of them use phantoms to explore the effects of variable acquisition parameters, such as tube current7, or voxel size15. Other radiomic studies assess the effects of varying segmentations on first or second order texture features, for instance, in non-small cell lung cancer (NSCLC)16, and rectal cancer17. A different approach has been recently proposed18, where the authors test different perturbation chains on NSCLC and head and neck cancer datasets, to find the chain better reproducing the outcome of a test-retest procedure, to be used when such method is not applicable. All these studies, carried out on different tumours, analyse the reproducibility mainly against varying ROI segmentation, or a set of perturbations.Computed Tomography (CT) is one of the most widely used technologies for morphological imaging and the standard of care adopted in oncology for diagnosis, staging and treatment follow-up. It is still based on visual lesion detection and morphological measurements , often performed with the aid of software tools19, thus improving standardisation of radiomic studies.To the best of our knowledge, this is the first work assessing robustness of first order (FO) and 2D and 3D second order texture features in CT imaging of renal cell carcinoma (RCC) and normal kidney (CK), by addressing three types of perturbations induced by Added White Gussian Noise (AWGN) (N), different voxel-size (V) and varying ROI (R). In addition, we perform a comparative analysis to select the best interpolation methods to be preliminarily applied, if needed, before any feature extraction procedure. Finally, results can provide practical operating guidelines to choose the proper voxel size in case of datasets with heterogeneous in-plane resolutions and to aggregate information derived from grey level (GL) co-occurrence matrices (GLCMs)This study included 98 patients with RCC imaged at a single institution. CT acquisition parameters are provided in Table https://www.microsoft.com/en-us/research/project/medical-image-analysis/) by a medical doctor and clinical researcher with three years experience in renal imaging. The structures were segmented in all slices at the original scan resolution . Polygonal ROIs were exported as DICOM RTSTRUCT and imported in MATLAB  to generate binary segmentation masks for RCC and CK. In particular, CK and RCC were segmented on the first and last slice where they were visible and contours for every other slice were interpolated20. Manual corrections were applied to sub-optimally segmented slices, leading to an iterative re-calculation of the remaining interpolated slices. The segmentation of CK included the renal cortex and medulla but not the the collecting system and hilar fat.ROIs of RCC and CK were semi-automatically outlined using the MICROSOFT RADIOMICS TOOL  were computed on the original and perturbed CT images, and their reproducibility was studied. All the procedures were implemented in MATLAB.After CT image acquisition and segmentation, an image interpolation procedure was needed to standardize the different resolutions of the dataset. After discarding the highest and lowest 2.5% of grey values according to a simple and standard outlier removal procedure, three well-known interpolation methods were analysed and compared, to find the method best preserving the statistical properties of the original images, to be used for the image perturbation by voxel-size resampling. The comparison was carried out by exploiting the Enhancement Measurement Error-Image Quality (EME-IQ)8. Hence, applying a resampling procedure is needed, to achieve one voxel size15. However, any resampling method relies on interpolation techniques that may potentially alter latent image properties, thus affecting the reproducibility of the features extracted from the resampled image and from the original22. In radiomic analyses, the interpolation method must be conceived for quantitative imaging and preserve the original properties of the CT GL distributions, rather than to simply yield visually pleasant images. In this work, we analysed and compared three well-known interpolation methods, that is, linear interpolation, largely employed in most radiomic studies15 thanks to its low complexity and computational cost, Akima cubic Hermite spline23 and Lanczos24 interpolation, the latter mostly used in computer vision and quantitative imaging. The comparison was performed by resampling the original CT images at the best and worst resolutions, corresponding to the smallest  upsampling the whole dataset to 1. Similarly to what done in29 and18, we simulated such variability, considering volume variations up to 20%, by ROI enlargement and shrinking. Actually, while ROI erosion just implies missing some tissue of the same type, ROI dilatation means including different tissues. Consequently, ROIs were shrunk by 10%, 15% and 20% or dilated by 10%. This procedure was carried out through binary morphological dilation and erosion, with a 3 × 3 pixel square structuring element (SE), according to a pixel-based random contourization procedure. Of course, it is unlikely to achieve the exact percentage variation, therefore the exceeding pixels were randomly removed to attain the expected percentage.One of the causes affecting the clinical reliability of radiomic features as predictive or prognostic biomarkers is the lack of reproducibility of quantitative measurements, depending on the variability of intra- and inter-observer ROI segmentation30. FO and GLCM-based texture features are very attractive, also because of their low computational complexity, they are computed in all radiomic packages31 and also employed for building predictive models in renal diseases4. In this study, we included 13 FO and 19 GLCM features computed in both 2D (GLCM2D) and 3D (GLCM3D), since no agreement exists yet on how to aggregate GLCM information to extract single representative features6. Hence, GLCM2D were computed in four directions, 32, with five odd distances, from 33 of 32 quantization levels was adopted for the GLCM computation. GLCMs were also symmetrized and direction-weighted. In GLCM2D, features were first computed on each slice and then averaged. In all, 108 radiomic features were computed on the original and perturbed CT images, for RCC and CK separately.Radiomic features commonly employed to depict tumour heterogeneity can be grouped into first, second, and higher order statistical descriptors. In particular, FO features measure the statistical properties of the GL distribution and ignore its spatial relationships within the ROIs, whilst texture features (i.e. second and higher order ones) investigate the relationships between neighbouring grey levels at pixel- or region-levelTable 18. Radiomic features were considered as being robust (r) if ICC 95nr) if CI < 0.90, and with indeterminate robustness (i) otherwise (i.e. with 0.90 strictly included in CI). In total, 29 perturbations were assessed, one arising from N, 24 combinations of V, and four from R, as detailed in Table All the extracted features were analysed for both RCC and CK and robustness was assessed using the Intraclass Correlation Coefficient (ICC) with 95r, nr, and i features were reported for each perturbation type. Moreover, the proportional contribution given by each feature class to the global robustness was investigated, together with the contribution of the single features. In practice, robustness was assessed (i) for all feature classes  against all image perturbations, (ii) for each feature class against each perturbation type , and (iii) for each feature against all image perturbations.First, the robustness of all radiomic features together was investigated against all perturbations, to have an overview of features behaviour depending on the tissue phenotype (i.e. RCC or CK) only. Mean percentage of p-value Finally, this study assessed the real need for having GLCMs computed at multiple Figure entclass1pt{minimaentclass1pt{minimar, nr, and i features against each perturbation for RCC , or at worst i (i-CK: 0.4%). As regards V perturbations, whilst in RCC the percentage of r and nr features was 73.0% and 14.5%, in CK values are lower, with r-CK: 50.8% and nr-CK: 30.3%. Despite this difference, RCC and CK showed a common behaviour against all perturbations at fixed r features  if compared with those at fixed r features in RCC, mainly referred to z-direction. This couple was also the best in CK, with 87.7% of r features. As regards R perturbations, whilst in RCC there was 94.6%, 76.9%, and 70.4% of r features at R-10, R-15, and R-20, respectively, in CK they were 8.9%, 4.9%, 2.5%, respectively. Finally, as regards R+10, RCC showed 19% of r features, while CK had only 4% of them.Figure RCC Fig. a and CK RCC Fig. b, respecRCC Fig. . SummariFigure r features, in both RCC and CK, for V , R , and N (RCC and CK: 100.0%) perturbation types. GLCM2D and GLCM3D features achieved comparable results in both RCC and CK. In fact, the percentage of r features averaged over all perturbations was GLCM2D: 80.9% and GLCM3D: 78.6% in RCC, and GLCM2D: 47.4% and GLCM3D: 46.4% in CK. In particular, robustness against V and R was always higher in RCC than in CK. In particular, R perturbation showed the greatest difference, with GLCM2D-R: 83.2% and GLCM3D-R: 76.1% in RCC and GLCM2D-R: 3.9% and GLCM3D-R: 2.5% in CK. Finally, analysing how each of the 29 perturbations affected each feature class , in RCC 13 and 10 perturbations showed at least 60% of r features, in GLCM2D and GLCM3D, respectively, but only 7 of them were shared.FO features hold the highest percentage of r features in RCC in at least m, M, max, m90th, M90th, iqr, u, e, and lCV) were confirmed in CK too. As far as second order features are concerned, 48 GLCM2D and 53 GLCM3D resulted r features in RCC in at least r features in at least 60% of perturbations and 21 of them were shared. Finally, as regards r features shared between RCC and CK, they were autoc, entro, savgh, sentro, sosvh, svarh. It is worth mentioning that cprom, cshad, energ, inf1h, and maxpr were found in RCC only.All the FO features were p-valuessosvh, computed in RCC at multiple sosvh for all p-valueentro and sentro representing local tissue heterogeneity showed significant differences between p-valuecprom, maxpr, and inf1h in RCC were statistically equivalent for p-valueGLCM features have an intrinsic redundancy, since they were computed at multiple 14, hence, this study focuses on the need for analysing feature robustness on RCC in CT using one of the largest datasets22. In addition, the analysis is extended to CK to determine which features might be robust enough to assess, for instance, diffuse renal diseases. In particular, in this study we analyse the robustness of radiomic features against some of the most frequent sources of variability, which are noise, heterogeneous scan voxel size and varying segmentation. Moreover, this study offers an in-depth analysis of three known interpolation methods aiming at supporting researchers in choosing the most appropriate one when resampling CT images. Results show that Lanczos interpolation outperforms the other methods in both upsampling and downsampling procedures. In particular, this study highlights one of the major limits of linear interpolation, probably the method most widely employed in radiomic studies for feature stability assessment in heterogeneous datasets and suggested by the Imaging Biomarker Standardization Initiative (IBSI)6. More specifically, if on the one hand resampling based on linear interpolation improves visual images’ perception, on the other hand it smooths tissue edges and texture variation, thus limiting quantitative information34. Some authors are aware of the importance of the interpolation methods, which may influence feature robustness. For instance, Whybra et al.30 carried out a comparison of feature robustness, after linear and spline interpolations. The authors concluded that the two methods were equivalent since no difference existed in terms of feature stability, albeit in the presence of large numerical variations. However, although those features may be reproducible, this does not ensure that the features are correctly representing the original CT image information. For this purpose, we recommend a preliminary analysis to assess that the resampling procedure does not affect the properties of the GLs distribution. It is worth noting that the upsampling procedure, although adding artificial information, improves the original image quality (EME-IQ = 2.87) with all methods considered . Even more relevant, when downsampling, while linear interpolation degrades (EME-IQ = 2.71) original image quality, Lanczos, performing the best (EME-IQ = 2.85), preserves the EME-IQ score of the original image.Assessing the robustness of radiomic features has become necessary to determine feature reproducibility before translating predictive and prognostic radiomic biomarkers into clinical practice. Feature robustness depends on the tumour phenotype and is not generalizabler features in RCC (65.6%) than in CK (39.0%), although both RCC and CK show an excellent robustness against N perturbation  are robust in RCC as well. Besides the well-established statistical descriptors , there are both lCV and e, two common indicators for measuring local heterogeneity or irregularity, that is also one of the changing properties of normal tissues while shifting into tumour ones35. The remaining 4/13 FO features resulted robust in RCC only, thus showing a higher specificity for tumour tissues, which could be useful for specific tumour-related clinical questions. It is worth noting that also all GLCM features (both 2D and 3D) that prove to be robust in CK, are robust in RCC too, while other features, measuring local asymmetries of GLCMs, are more tumour-specific.Our robustness analysis finds the FO features are definitely the most reproducible ones, confirming what was reported in the review of the most recent research works regarding feature repeatability and reproducibility by Traverso et al.r features show a difference in RCC when computed at 36. Our results show that the overall robustness of GLCM features computed in 2D or 3D is similar. However, our findings show a higher number of r features for GLCM2D and, at the same time, a higher number of perturbations not affecting robustness of GLCM2D features. Therefore, GLCM2D texture features should be preferred.This research also investigates the well-known phenomenon of the high correlation of GLCM-based texture features computed at different distances, to see whether and to what extent using higher distances is worth. In practice, almost all features are shown to be equivalent when computed at distances from 3 to 9. Five The main limitation of this study is that only first and second order features are considered. However, these are the first features whose robustness has been analysed in radiomic studies based on CT images of renal disease. In addition, our methodological approach can be exploited to extend the study to include more feature classes. An additional limitation arises from having considered 10% as the lowest volume variation bound. This threshold might yield overestimated inaccuracies of radiologist’s segmentations. Therefore, the results reported in this study can be considered as the worst scenario. Finally, another limitation is the analysis of all RCC subtypes together. However, an analysis of feature robustness across different RCC subtypes would have been beyond the scope of this study.This work aimed at assessing the robustness of radiomic features against some of the most common sources of variability. Our findings allowed drawing some concluding remarks that could be useful guidelines for radiomic studies. In particular, texture features should be used at very short distances, heterogeneous CT datasets have to be resampled at the median slice resolution, whilst should not be interpolated along the cranial-caudal direction and, Lanczos should be used as the interpolation method.Supplementary Information.


Among the enrolled patients, during follow-up, we did not record new cases of vascular diseases. Attention to patients’ clinical evolution, monitoring of the INR, and timely and frequent adjustment of dosages represent the cornerstones of the safety of care for patients administered fibrinolytic drugs as a single treatment or in pharmacological combination. Therefore, we can conclude that the use of nattokinase represents an efficient and safe treatment able to both prevent and treat patients with vascular diseases.Nattokinase (NK) is a serine protease enzyme with fibrinolytic activity. Even if it could be used for the treatment of several diseases, no data have been published supporting its use patients who underwent vascular surgery. In this study, we evaluated both the efficacy and the safety of nattokinase (100 mg/day per os) in patients admitted to vascular surgery. Patients were of both sexes, >18 years of age, with vascular diseases , and naïve to specific pharmacological treatments (anticoagulants or anti-platelets). Patients were divided into three groups. Group 1: patients with deep vein thrombosis, treated with fondaparinux plus nattokinase. Group 2: patients with phlebitis, treated with enoxaparin plus nattokinase. Group 3: patients with venous insufficiency after classical surgery, treated with nattokinase one day later. During the study, we enrolled 153 patients (age 22–92 years), 92 females (60.1%) and 61 males , and documented that nattokinase was able to improve the clinical symptoms ( Bacillus subtilis natto [Nattokinase (NK) is a serine protease enzyme discovered in 1980 and extracted from natto, a traditional Japanese food, made by boiling or steaming soybeans and fermenting them with the The fibrinolytic activity of nattokinase is related to a complex mechanism of action: the enzyme  breaks dThe effects of nattokinase on the cardiovascular system have been reported in both clinical and experimental studies. Its anti-hypertensive ,6, anti-Nattokinase has high gastrointestinal stability  and presents a discrete absorption in the intestinal tract ,5. In a A fundamental feature for nattokinase is that it is used in several clinical manifestations in many other vascular diseases  without causing the development of adverse drug reactions or drug interactions .However, to date, no data have been published supporting the use of NK in vascular surgery as sequential therapy. In this regard, in the present study, we evaluated both efficacy and safety of nattokinase (100 mg/days) as sequential therapy in patients admitted to vascular surgery.We performed an observational open-label clinical trial on patients referred to the Vascular Surgery Unit of the “Pugliese Ciaccio” Hospital of Catanzaro, from 1 April 2019 to 1 April 2020. The Local Ethics Committee approved our study protocol (120/May 2018). It was conducted in compliance with the Institutional Review Board/Human Subjects Research Committee requirements and according to the Declaration of Helsinki and the Guidelines for Good Clinical Practice criteria. Before the beginning of the study, all patients signed an informed consent.Inclusion criteria: patients of both sexes, >18 years of age, with vascular diseases , and naïve to specific pharmacological treatments (anticoagulants or anti-platelets) that signed the informed consent.Exclusion criteria: patients < 18 years of age, with cancer and severe blood disorders, bleeding, who did not sign the informed consent or used drugs or nutrients with anticoagulant or anti-platelet activity.After clinical, laboratory, and instrumental evaluation, the admitted patients were enrolled into three groups:Group 1: patients with deep vein thrombosis, treated with fondaparinux (7.5 mg/day subcutaneous) for 30 days (T1) and then nattokinase  (T2).Group 2: patients with superficial vein thrombosis or phlebitis, treated with enoxaparin  (T1). Nattokinase  (T2). Group 3: patients who underwent classical surgery . Patients suffered from venous insufficiency (T1). After surgery, nattokinase  was started (T2).In agreement with international guidelines , during Furthermore, in agreement with our previous studies ,19,20,21p < 0.05) of clinical symptoms in patients 30 days after the beginning of nattokinase administration (100 mg/day) (T2), for (T1). The second endpoint was the absence of adverse drug reactions or drug interactions in patients treated with nattokinase recorded at the end of the study concerning the admission and follow-ups.The first endpoint was the statistically significant improvement . Mean and standard deviation (SD) were taken into consideration for the analysis. For normality, data parameters were evaluated using the Shapiro–Wilk normality test. The Anova test was used to evaluate the difference in each group, while the Student’s p = 0.632, mean −1.9 ± 17.9, CI 95% −9.1, 5.2).During the study, we enrolled 153 patients , 92 females  and 61 males , without difference between the groups , age 32–78 years (mean 55.2 ± 14.2 years) without differences by gender .Group 2: 57 patients , age 22–92 years (mean 60.7 ± 19.7 years) without differences by gender  or with respect to body mass index, drugs used, or co-morbidity. Group 3: 46 patients , age 42–67 years (mean 56 ± 7.8 years), without differences by gender  (p = 0.9) .p < 0.01) and an improvement in vascular echo-color Doppler analysis. The 30-day treatment with nattokinase induced complete remission of the clinical symptoms (p < 0.01).All patients enrolled in Group 1 were treated with fondaparinux (7.5 mg/day) for 30 days (T1) and presented a significant decrease in clinical symptoms  and an improvement in vascular echo-color Doppler analysis. In these patients, nattokinase was started after the discontinuation of enoxaparin. In this group, there was a complete remission of the clinical symptoms (p < 0.01)  and showed a significant decrease in the clinical symptoms  .p < 0.01)  was started one day after vascular surgery and induced complete remission of the symptoms in all treated patients ( < 0.01) .In patients enrolled in these groups, we did not record the development of ADRs or drug interactions.In this study, we evaluated the efficacy of nattokinase in patients with DVT, SVT, phlebitis, or VI referred to the vascular surgery unit for clinical evaluation.Nattokinase is an enzyme with thrombolytic and anticoagulant activity , documenMoreover, it has also been reported that nattokinase induces protection against brain ischemia through its antioxidant and anti-inflammatory activity  ,28,29. MIn previous studies, we documented that several compounds could modulate the expression of matrix metalloproteinases ,32,33,34These findings support our data documenting that nattokinase was able to prevent the development of thrombosis in enrolled patients.In a previous study, Zhang et al. , using aBacillus natto or Bacillus subtilis is rich in the ACE inhibitor peptides VAHINVGZK and YVWK [It has been reported that soybean fermented with and YVWK .In this regard, Kim et al.  randomizIn agreement with this study, Jensen et al.  documentAnother surprising observation is that fermented soybean products contain other soy peptides that prevent the onset of type II diabetes ,38. In _As reported in international guidelines , the firFinally, another important observation is that we did not record the development of adverse drug reactions, including drug interactions.These data agree with literature data. In fact, experimental toxicology studies reported the safety of the oral consumption of nattokinase also in humans .However, this study has some limitations, for example, the absence of a placebo control group. In fact, we compared the effect of nattokinase administered alone and as an add-on to anticoagulant compounds but not with respect to an untreated or placebo-treated group; therefore, these data must be validated in a placebo-controlled trial.Nattokinase (NK) was first presented as a natural drug that can be administered as a mild anticoagulant. It is a natural drug that can be administered both as a single agent and in pharmacological combinations. Although the positive effects and advantages of the drug are greater than the possible described complications, patients under treatment must always be monitored for laboratory parameters to maintain the balance between hyper-coagulation and hypo-coagulation. In fact, patients on a monotherapy with nattokinase or on pharmacological combinations in which one of the drugs prescribed is nattokinase are at risk of thrombus formation or bleeding and hemorrhage. However, to establish the safety of pharmacological treatments of this type, it is necessary to monitor the coagulation parameters and ensure their stability in time, even for drugs known to have mild or no side effects. Coagulation profile, renal function, diet, clinical condition, weight, and age of the patient should be considered as reference parameters. Monitoring implies both constant checks of the laboratory parameters and availability of the results within a few hours from the collection of the blood sample to allow the pharmacological corrections necessary to ensure patient’s safety. In fact, healthy values outside the reference range increase the risk of adverse events. In conclusion, the available evidence suggests that NK is a unique natural compound and represents an effective and safe treatment for patients with vascular disease with medical or surgical indications. The indication of nattokinase could be useful for patients chronically treated with heparin.