The Editorial of the first issue of Adv. Lab. Med. included a description of the goals of this new scientific journal, which comprised achieving a significant presence in impact factor indices . The firThe quality and prestige of a scientific journal is measured by a range of quantitative bibliometric indices, which also suggest the difficulty in getting an article published in that journal. The most widely used bibliometric indicator is impact factor (IF), published by the Journal Citation Reports  on the basis of information available on Web of Science. IF is calculated as the average number of citations of items published in the journal during the preceding two years . Thus, tRegardless of the initial position achieved on bibliometric rankings, fulfilling this goal in such a short period of time was a remarkable success. Of note, our journal was indexed by IF and SJR starting with the first year of publication, which indicates that our journal was rapidly included in bibliometric indices. This success is the result of the large amount of effort and enthusiasm put by our editorial team, added to the rigor and quality required to all authors and, especially, the trust that authors have placed on this project. Of special mention is the major role of reviewers, whose voluntary work is essential to maintaining our journal to the highest standards.Fulfilling these goals will open new opportunities for the prestige or our journal to keep growing via the publication of good science. This will probably imply in the future adopting more strict requirements to ensure the general and methodological quality of the works published in our journal. The quality of the scientific production of a researcher is primarily measured by the number of publications in indexed journals. Therefore, the increased visibility of our journal, added to our IF, will encourage more and more authors to submit their works to us. We face a new era that will be even more stimulating than the first period of life of this journal.


Dioscorea villosa) was evaluated for its intrinsic physicochemical properties. From the results, essential metals such as K, Ca, P, and Fe were detected along with some nonessential heavy metals below the WHO permissible limits. Bulk density was 0.13–0.63 g/mL. The water absorption capacity, oil absorption capacity, swelling power, and solubility of the starch were pH-responsive. Thermal profiles showed onset temperature, To (59.21 °C), peak temperature, Tp (60.22 °C), endset temperature, Tc (63.12 °C), gelatinization enthalpy, ΔHgel (0.54 J/g), temperature range of gelatinization, R (3.91 °C), and peak height index, PHI (1.87 J/g °C). Exhibiting a crystallite size of 0.03 nm, absorption peaks of 15.3119°, 24.4120°, and 18.4170°, corresponding to interplanar d-spacings of 3.7500 Ǻ, 5.14000 Ǻ, and 4.954610 Ǻ, were obtained. Evidence of C–H at 1338.1 cm−1, C–O at 640.0 cm−1, C–H stretch at 2829.7 cm−1, and a strong and broad O–H group at 3291.2 cm−1 were obtained. The starch granules had low particle sizes, were homogeneous, and were aggregates of irregular shapes. At a lower pH (2-4), the wild yam starch studied could be a potential absorbent material in the production of disposable diapers and female napkins and as biodegradable films due to its high hydrophobicity at a high pH (8-12).Native starch extracted from wild yam ( Dioscorea , is a major home meal in West Africa, especially Nigeria [Dioscorea villosa).Yam, a species in the genus  Nigeria , 2. Alth Nigeria , only se Nigeria . HoweverWild yam is a perennial crop with nonfleshy but dried, narrow, and crocked roots, bearing horizontal branches of long creeping runners . Wild yaThe inedibility of wild yam species is due to the presence of antinutrients such as phenols, tannins, hydrogen cyanide, oxalate, amylase inhibitor activity, and trypsin inhibitor, which however, this can be eliminated through soaking, cooking, autoclaving, baking, and oil-frying, resulting in a good dietary source with high proportions of carbohydrate, fibres, essential amino acids, and low-level fats and protein –11.Starch is a highly versatile biopolymer that is widely used in both food and nonfood applications due to its exceptional bioavailability, biocompatibility, biodegradability, and low cost. It is composed of amylose and amylopectin chains and is readily available in various plant storage organs such as roots, tubers, corms, cormels, and legume grains. The functionality of starch in industrial products is influenced by its source and intrinsic physicochemical properties, including chemical composition, functional properties, morphology, thermal properties, and rheological properties –14.This research paper emphasizes the importance of analyzing the intrinsic physicochemical properties of wild yam starch to determine its potential for increased use as the demand for starch continues to grow.Dioscorea villosa) were obtained from a farm in Etsako-West Local Government Area, Edo State, Nigeria. The tubers were identified by the herbarium curator of the University of Medical Sciences, Ondo, Nigeria, having voucher details, UNIMED PBTH No. 012, and were deposited in the herbarium of the university. The reagents used in this investigation were of Analar quality and were utilized in their original form. All the methods were carried out following local and international institutional guidelines.Freshly harvested tubers of wild yam tubers of the wild yam species . After drying, grinding, and sieving, a 200 mg, 2.5 cm diameter pellet was formed with a pellet-pressing machine. The pellet was then exposed to radiation from a Cd-109 radioactive source for 2500 s while placed in the sample compartment. To correct for absorption, two measurements were taken using a liquid nitrogen-cooled Si (Li) detector. Different filters were used to ensure optimal detection of minerals , 16.The starch sample (1 g) was measured into a 25 mL graduated measuring cylinder and gently tapped on the bench top ten times from a height of 5 cm. After tapping, the volume of the sample was recorded. This process was repeated for 2, 3, 4, and 5 g weights of the same starch sample. Bulk density (BD) was calculated as the ratio of the weight of the sample to the volume of the sample after tapping .One (1) g of the starch sample was mixed with 10 mL of distilled water/oil using a Thermolyne Maxi mix II mixer (type 37600) for 30 seconds to obtain a slurry. The pH of the slurry was then adjusted to 2, 4, 6, 8, 10, and 12 by adding 0.1 M HCl and 0.1 M NaOH while stirring. After allowing the slurries to stand at 21 °C for 30 min, they were centrifuged at 5000 rpm for 30 min, and the volume of the supernatant was measured in a 10 mL graduated cylinder. The results were expressed on a dry weight basis, using densities of 1 g/mL and 0.89 g/mL for distilled water and Devon King's Vegetable Oil, respectively , 19.Six preweighed test tubes were taken, and 0.1 g of starch was dispersed in 50 mL of distilled water in each one. The pH of each solution was then adjusted to 2, 4, 6, 8, 10, and 12 by adding 0.1 M HCl or 0.1 M NaOH while stirring. The resulting slurries were heated in a water bath at 90 °C for 30 min and then allowed to cool down to 28 ± 2 °C. After centrifuging the slurries at 2500 rpm for 15 minutes, the supernatants were poured off. Finally, the swelling power was calculated using a specific equation . (1)SwelFor solubility, the supernatants were spread onto clean, preweighed Petri dishes and dried in an oven at 100 °C until a constant weight was achieved . (2)SoluThe thermal properties of the starch were analyzed using a differential scanning calorimeter . A sample of 3.00 ± 0.01 mg starch (mixed with water in a ratio of 1 : 3) was placed in an aluminium sample pan, which was then sealed and weighed. The pan was allowed to equilibrate at room temperature for an hour. The machine was conditioned at 75 °C for 2 hours, and the reference and indium pans were conditioned at a rate of 10 °C/min between 30-180 °C for 20 min. The sample was then tested for thermal properties by replacing the indium pan with the sample pans and heating at a rate of 5 °C/min from 20 to 100 °C. The DSC cell was purged with nitrogen gas at a flow rate of 100 mL/min, and the thermal profiles were generated using TA Instruments Universal Analysis software .α radiation. The resulting X-ray patterns were analyzed using peak positions to determine the starch sample's crystalline nature [k is the Scherrer constant (0.84), λ is 0.154 nm, B(hkl) = FWHM , and θ is the corresponding Bragg's angle to FWHM.The starch sample was equilibrated in a desiccator for three days above distilled water. It was then subjected to X-ray diffraction analysis using a D-5000 Siemens diffractometer with Ni-filtered Cu-Ke nature . (3)Crys−1 was used, and 32 scans were recorded for the sample [The functional groups present in the starch sample were identified using a Nicolet AVATAR 360 Fourier infrared spectrometer with KBR disks. Prior to testing, the sample was pressed into a pellet and dispersed in a KBR matrix of 100 mg, with a total weight of 1.0 mg. A spectral resolution of 4 cme sample .Gold-coated starch granules were positioned on an aluminium specimen stub and observed using a scanning electron microscope , which was equipped with a sputter coater . The sample morphology was analyzed at various magnifications, including 150 K, 500 K, and 1000 K .The statistical analysis of the data collected for mean comparison was conducted with SPSS 26.0 software. Duncan's least significant test and one-way analysis of variance (ANOVA) were utilized at a significance level of 5%.Bioaccumulation of K, Ca, P, Fe, Cr, S, Al, Si, Cl, Ti, and Mn in wild yam starch was studied . Fe, Cr,The bulk density is in a constant ratio of 1 : 1 with the weight of the starch sample studied . Thus, ip < 0.05) values as pH changes from 2 to 12  of 3.50 ± 0.01% is obtained at pH 10. As the pH increases from 2 to 10, OAC increases because the intermolecular bonds of the starch granules are weak; thus, the pores are enlarged, oil molecules are absorbed and retained in the interstitial network, and the starch granules behave oleophilic in nature . The impDioscorea villosa) starch studied at different pH of 2, 4, 6, 8, 10, and 12. The swelling and solubility of wild yam starch are pH-sensitive and vary significantly at different pH levels (p < 0.05). Swelling power (SP) is an indicator of the ability of the starch to hold water and form gels at temperatures close to or equal to its gelatinization temperature [Figures perature . The SP perature , 32. Staperature . Wild yaperature . As the perature . This phperature .To), peak temperature (Tp), and endset temperature (Tc) of gelatinization of the wild yam starch are 59.21, 60.22, and 63.12 °C, respectively  [Hgel) of 0.54 J/g compared to 17.0 J/g reported for potato and taro [R) obtained for the starch sample is 3.91 °C. R is the difference between To and Tc and significantly indicates the homogeneity or heterogeneity of starch granules. Homogeneity increases as R decreases [The onset temperature  . The sta59.8 °C) . This maand taro , 41. Theecreases . The peaB-type starch [The three major peaks used in characterizing the wild yam starch sample show absorption bands at 15.3119°, 24.4120°, and 18.4170°, which correspond to the interplanar d-spacings of 3.7500 Ǻ, 5.14000 Ǻ, and 4.954610 Ǻ, respectively . The date starch . The crye starch . Highly Discorea villosa) as a function of transmittance and wave number is depicted in −1 corresponding to transmittance values of 63.907, 68.330, 72.907, 68.095, 28.763, 56.052, 67.672, 80.119, 84.157, 86.432, 80.757, and 70.189%, respectively. The fingerprint representations at 928.1 cm−1 and 998.9 cm−1 are evidence of the presence of vinyl C–H out-of-plane bending in the starch sample studied. The transmittance peak at 1338.1 cm−1 represents CH in-plane bending, 1640.0 cm−1 is the carbonyl group, C–O, which can be oxidized to a carboxyl group, 2829.7 cm−1 is the C–H stretch of alkanes, and 3291.2 cm−1 represents a strong and broad O–H group, which is indicative of a strong hydrogen bonding between the starch molecules [The FTIR pattern of wild yam (olecules –47. The olecules . This unR) and PHI, as previously reported in studies [As shown in  studies , 21. The studies , 22.Discorea villosa) exhibits intrinsic physicochemical properties. The wild yam starch studied bioaccumulates some macro and heavy metals  in nonlethal proportions. The bulk density is not altered by a change in the weight of the starch sample, whereas the water absorption capacity, oil absorption capacity, swelling power, and solubility are pH-responsive. Swelling power is elevated in an acidic medium, while solubility is elevated in an alkaline medium. The granules of wild yam starch are thermally stable and homogeneous. Strong hydrogen bonding is identified between the molecules of the starch granules. Due to its intrinsic properties, the wild yam starch studied can serve as a suitable recipe for snack foods, food emulsifiers, and products such as mayonnaise. Its crystallite size is advantageous where penetration of the treatment agent into a starch matrix is required. At a lower pH (2-4), the wild yam starch studied could be a potential absorbent material in the production of disposable diapers and female napkins and as biodegradable films due to its high hydrophobicity at a high pH (8-12). At a pH level of 8, which is similar to the natural pH level of the body, wild yam starch is safe for human use as a diagnostic fluid in MRI and MPI recipes. The granules are shaped irregularly but are arranged in separate clusters.The native starch isolated from wild yam (


Typically, animal locomotion studies involve consecutive strides, which are frequently assumed to be independent with parameters that do not vary across strides. This assumption is often not tested. However, failing in particular to account for dependence across strides may cause an incorrect estimate of the uncertainty of the measurements and thereby lead to either missing (overestimating variance) or over-evaluating (underestimating variance) biological signals. In turn, this impacts replicability of the results because variability is accounted for differently across experiments. In this paper, we analyse the changes of a couple of measures of human leg stiffness across strides during running experiments, using a publicly available dataset. A major finding of this analysis is that the time series of these measurements of stiffness show autocorrelation even at large lags and so there is dependence between individual strides, even when separated by many intervening strides. Our results question the practice in biomechanics research of using each stride as an independent observation or of sub-selecting strides at small lags. Following the outcome of our analysis, we strongly recommend caution in doing so without first confirming the independence of the measurements across strides and without confirming that sub-selection does not produce spurious results. As a Sometimes, to avoid the extra challenges posed by correlated data, researchers choose to only use measurements from every two or three strides for their average estimate ,24. In eThe main objectives of this manuscript are, first, to underline that it is incorrect to not account for variability of measurements across strides in the analysis of data from locomotion experiments and, then, discuss the possible consequences of doing so. For these objectives, we concentrate on one relevant example and study it extensively, but we believe that our considerations are valid in general for terrestrial locomotion experiments which involve multi-stride data. We study the variability of human leg stiffness across strides during running on a treadmill at different velocities and use a publicly available dataset  to illusspring-mass/spring loaded inverted pendulum (SLIP)  = μ and Var(Y1) = σ2 is given by ρk the autocorrelation function at lags R is given byR depends on its parameters.The variance of a sample mean from ρk = ρ for k = 1, …, n. In this case for ρ = 0.05 and n = 101, we have R = 6, which means that, if we do not account for autocorrelation in the time series, we underestimate the variance of the sample mean by 6 times. Instead, if ρ = −0.01 and n = 11, then R = 0.9, which means that, without accounting for autocorrelation in the time series, the variance of the sample is overestimated by 10%. Note that the formula for R would not capture the full variation if ρk = ρ for every Suppose that ationary .ρk = ρ for one single k and zero otherwise. In this case, for ρ = 0.5, n = 80 and k = 20, we have R = 7/4, which means that, if we do not account for autocorrelation in the time series, the variance of the sample mean is underestimated by 75%. Instead, if ρ = −0.5 and n = 60 and k = 10, then R = 1/3, which means that, without accounting for autocorrelation in the time series, we overestimate the variance of the sample mean by 67%.Suppose that v = 2.5 m s−1 from our dataset and the measurements of kOLS (n = 75 strides), the only significant autocorrelation is at lag k = 14 with ρ14 = 0.2408617. If we use the formula for R with only one correlation lag different from zero, we get R = 1.391802. Therefore, neglecting in toto the autocorrelation structure of the time series would fail to account for nearly 40% of the variance of the average stiffness estimate across strides, even with one single lag showing significant correlation.If we consider subject 2 at v = 4.5 m s−1 from our dataset and the measurements of kmax (n = 89 strides), and consider only the largest significant lag in the first 40 lags, namely lag k = 8 with ρ8 = 0.2111892, we get R = 1.384412. Therefore, neglecting in toto the autocorrelation structure of the time series would again fail to account for nearly 40% of the variance of the average stiffness estimate across strides.If we consider subject 27 at v = 2.5 m s−1 from our dataset and the measurements of kOLS (n = 88 strides), and consider all possible lags, we get R = 0.4162655. Therefore, neglecting in toto the autocorrelation structure of the time series would overestimate the variance of the average stiffness estimate across strides by nearly 60%.If we consider subject 20 at v = 3.5 m s−1 (n = 79 strides) from our dataset and the measurements of kmax, we get the coefficient R = 0.3135405, which implies an overestimate of the variance of the average stiffness across strides of almost 70%.If we consider subject 26 at n, with n the sample size [We illustrated the risk of ignoring the correlation structure in the measurements using these several examples. The most direct consequence lies in mis-evaluating the magnitude of the variability of the estimate of the average stiffness across strides. Note that in the iid case, it is well known that the variance of the sample mean decreases on the order of 1/ple size . With cople size . Positivbatch effect and it is a major and interesting problem in machine learning. Often such a problem is addressed using transfer learning [One of the consequences of broad impact of the mis-estimation of the variance is the difficulty in combining the results of different laboratories. If the variance of an average measurement of a variable of interest in one experiment at one single laboratory is miscalculated, the effect could be catastrophic when results are combined across laboratories ,84. Thislearning –87, a sulearning ,88,89. MAltogether, this analysis underlines the necessity of determining the correlation structure across strides before modelling or performing hypothesis tests. Recall that if two random variables computed by different samples are dependent, then also the samples must be dependent . Therefo. 4.4a priori to throw away a significant portion of the data merely because there might be a statistically yet-to-be-confirmed dependency across strides or because one does not want to deal with the dependency structure. Even just considering the experimental effort and the cost in terms of time, it is important to get the most out of the data collected. As shown in this manuscript, the statistical tools to harness stride dependency and to give a correct uncertainty quantification of the estimate of the parameter of interest are available. We recommend assessing the dependency structure of the sample also in the case in which an investigator decides to choose one single stride per bout and consider multiple bouts, but also in every other estimation method used. Again, failing to correctly assess the variability of a measurement (e.g. stiffness) might result in spurious findings or in losing the possibility to uncover important biological information. The determination of what the greatest sources of dependency  are can have an important impact on the design of locomotion experiments and surely deserves further attention.In our analysis, we discussed to what extent autocorrelation between consecutive strides within a given trial of an experiment is present. We did not discuss extensively what happens to the variability of a measurement like leg stiffness in the case a researcher decides to estimate the parameter using a single stride per bout and to use multiple bouts. Using strides from different trials might require assessments such as: which stride of each bout shall we select? The one in the middle? Why? Is there a way to fairly compare bouts that have different numbers of strides? We believe that the answers to these questions require further data-driven research. Still, the strategy of using only one stride per bout risks to be sub-optimal because it does not seem reasonable to decide ρ between one observation and the next. In such a case, ρk = ρk, with k the lag considered. This means that the correlation predicted by the AR(1) model decays exponentially with the lag, but it is present at every lag. If, for example, the correlation is 0.7 at lag 1, at lag 2 the correlation is 0.49. The implication of this is that, removing only a bunch of strides from the sample might not remove in full the autocorrelation among the remaining strides. The case of correlation at large lags is a statistically more interesting case, but also very interesting from the biomechanical perspective because, although one might intuitively expect a relationship from one stride to the next, one might not expect to see a relationship between one stride and strides that take place 5 to 10 strides later.If multiple strides are available, it is really valuable information and, unless there has been some experimental failure owing to which some strides need to be excluded, it is important to keep them and evaluate them. We potentially understand the use of a single stride in the case in which the study is concentrated on that single stride, but even in such a case there is something to say. Suppose that a researcher wants to understand the behaviour of individuals during the first stride of a locomotor trial. Then, there is one single first stride in each experiment. Especially if the strides are independent, it is a reasonable choice to use only the first stride of the experiment for any estimate related to the first stride. However, the situation here is also tricky because if there is autocorrelation at lag 1, some information about the first stride is shadowed in the second stride, too. In a similar way, there is potentially some information about the first stride in other strides as well. Note that autocorrelation at lag 1 does not mean that stride 1 is correlated to stride 2 or that stride 2 is correlated to stride 3 on an individual stride-by-stride basis. Rather, it means that there is correlation between consecutive strides globally in the data. In the simplest autoregressive model AR(1) , there aIt is more common for data containing large numbers of strides to be available during running on a treadmill than overground or on uneven ground . TherefoIf relatively few strides are available (e.g. overground running), then the variance can be computed only using those strides, with calculations analogous to those in the examples of §4.3 . Still, if there is dependence between one stride and another, many strides can provide information about many others. As noted in our analysis, some strides might be outliers. If it is determined that a stride is an outlier via a careful verification with a test for outliers, the exclusion of that stride is recommended. Care must be taken, especially in the case in which only a few strides are available. A popular test for outliers is Grubbs’s test , but suc. 4.5in se, there is always the question: what stiffness are we really estimating? [model 1—kmax, we used a method which is biologically and mechanically well-motivated by the very definition of spring constant and elastic force. In our model 2—kOLS, we considered the stiffness as the least square estimates of a simple linear regression model without intercept [kmax and kOLS are good examples for the purposes of our paper, as they emerge from quite alternative perspectives . Incorporating a broad comparison of all possible methods for calculating stiffness is beyond the scope of this paper. Such an analysis would possibly encounter a similar across-strides-dependence problem and possibly lead to analogous results and consequences.For what concerns the estimation of stiffness imating? ,43. As fimating? ,30,80. Intercept . Note thNote that for several of the subjects, there is a possible outlier in the stiffness measurements of the first strides figures –12. We d. 5As far as we know, this is the first paper in the terrestrial biomechanics literature dedicated to discussing rigorously and extensively the effects of neglecting the correlation structure of consecutive strides. We found evidence of autocorrelation of the time series of human leg stiffness across strides during running on a treadmill and, thereby, evidence of the dependence of the time series of kinematic and kinetic data at different strides. Therefore, our paper has implications for the diffuse practice in biomechanics of assuming that individual strides within an experimental trial are independent and identically distributed. Lacking an understanding of stride dependence structure invalidates estimates of uncertainty of average measurements across strides, including the variance. Variability assessments are required for both building correct confidence intervals and valid hypothesis testing. The incorrect quantification of uncertainty might inflate biological signals in some cases or miss it in others. We caution against the practice of blindly assuming stride independence and recommend that researchers first test for stride independence before assuming it. If, after testing, the strides turn out to not be independent, we recommend exploiting the correlation structure. By contrast, retaining only one of every few strides is a suboptimal remedy because it decreases the sample size by an order of magnitude and risks to disrupt the correlation structure of the time series collected from the experiment. Having an incorrect estimate of the variability of biologically important quantities such as leg stiffness or any other measurement can negatively impact the replicability of the study. We argue that it is important not only to include measurements of uncertainty peculiar to an experiment in every study, but also to have a standardized methodology to assess uncertainty. Not only would this facilitate replicability and comparison, but it would also foster collaboration across research groups.


Rice flour is produced by various methods for use in the food industry, but little is known about how the structure of starch is affected during rice flour production. In this study, the crystallinity, thermal properties, and structure of starch in rice flour were investigated after treatment with a shearing and heat milling machine (SHMM) at different temperatures (10–150 °C). Both the crystallinity and gelatinization enthalpy of starch showed an inverse relationship with the treatment temperature; rice flour treated with the SHMM at higher temperatures showed lower crystallinity and gelatinization enthalpy than that treated at lower temperatures. Next, the structure of undegraded starch in the SHMM-treated rice flour was analyzed by gel permeation chromatography. A significant reduction in the molecular weight of amylopectin was observed at high treatment temperatures. Chain length distribution analysis showed that the proportion of long chains (degree of polymerization (DP) > 30) in rice flour decreased at temperatures ≥ 30 °C. By contrast, the molecular weight of amylose did not decrease. In summary, the SHMM treatment of rice flour at high temperatures resulted in starch gelatinization, and the amylopectin molecular weight decreased independently, due to the cleavage of amorphous regions connecting the amylopectin clusters. Wheat, the third most highly produced cereal grain in the world after maize and rice, is used worldwide as flour to prepare a variety of foods, such as bread, noodles, and confectioneries, and plays a paramount role in human nutrition . HoweverPreviously, an SHMM was used to investigate how different factors including shearing temperature, the gap between millstones, and grain moisture content, affect the crystallinity, physicochemical properties, and structure of starch . When poWhen the starch of completely amorphous rice flour was subjected to gel permeation chromatography (GPC) using a Sephacryl S-1000 column without any enzymatic degradation, the amylopectin peak was lower than that of crystalline rice flour, whereas the amylose peak was larger, indicating that high-molecular-weight amylopectin was converted into low-molecular-weight amylopectin . GPC perIn previous studies, when undegraded starch was separated by GPC and detected with a refractive index (RI) detector, it could be separated into only two peaks, one each for amylopectin and amylose ,15,16,17In this study, we investigated the crystallinity and gelatinization enthalpy of rice flour treated with an SHMM at various temperatures. Simultaneously, to determine the effect caused by SHMM treatment temperature on starch structure, the following three experiments were performed in this study: (1) the degree of reduction in the molecular weight of amylopectin was examined by the GPC of undegraded starch using a series of columns (Toyopearl HW75S × 2-HW65S-HW55S), (2) the ratio of amylose and amylopectin was examined by GPC of debranched starch using another series of columns (Toyopearl HW55S-HW50S × 3), and (3) further structural analysis of debranched starch was performed by capillary electrophoresis to identify the cleavage sites in starch molecules. The relationships among the crystallinity, gelatinization enthalpy, and structure of starch in rice flour sheared at different temperatures are discussed.A non-glutinous japonica rice cultivar, ‘Haenuki’, harvested in Yamagata (Japan) in 2018 was used in this study. ‘Haenuki’ is a staple rice cultivar developed in Yamagata Prefecture and is widely consumed as cooked rice, because of its good taste, at both domestic and commercial scales. Before milling, a commercial rice polishing machine  was used to polish ‘Haenuki’ grains until their weight was ca. 90% of that of unpolished rice grains. Six rice flour samples (A–E) were examined in this study . Sample The crystal diffraction pattern of the rice flour samples was examined by measuring wide-angle X-ray diffraction (WAXD) using an X-ray diffractometer , with a Cu-Kα radiation source at a wavelength of 0.154 nm. The tube voltage and current were maintained at 40 kV and 40 mA, respectively. Samples were scanned at room temperature from 5° to 35° at a scan rate of 10°/min. The crystallinity of the samples was measured using the peak separation method.The peaks obtained from the WAXD measurements were separated into crystalline reflection and amorphous scattering using the Peak Fit software . The peaks from crystalline reflection were defined as diffraction peaks that appeared at 15°, 17°, 18°, and 23°. The area of amorphous scattering was specified as a broad peak that covered the diffraction image. The crystallinity of a sample  was calculated using the following equation:o, onset temperature; Tp, peak temperature; Tc, conclusion temperature) and gelatinization enthalpy (ΔH) were determined.The thermal properties of rice starch were measured by differential scanning calorimetry (DSC) . Briefly, 10 mg of rice flour and 30 μL of deionized water were sealed in a stainless steel pan. The temperature was increased from 25 °C to 95 °C at a rate of 5 °C/min. Empty pans were used as a reference. The resulting endothermic peaks were analyzed using the Pyris software , and the gelatinization transition temperatures (TRice flour samples (20 mg) were suspended in 1.6 mL of distilled water in a 15 mL centrifuge tube and gelatinized with 400 µL of 5 M NaOH for 30 min at 37 °C. Then, 2 mL of distilled water and 2 mL of eluent (0.2% NaCl/0.05 M NaOH) were added to each sample, followed by filtration through a 5 µm Durapore polyvinylidene fluoride (PVDF) membrane . A 5 mL aliquot of the filtrate was used in a 4-column system  that was pre-equilibrated with the eluent, and carbohydrates were detected using an RI detector . The columns were incubated at 40 °C, and the samples were eluted at a flow rate of 1 mL/min using a DP-8020 pump (Tosoh Corp.).GPC analysis of the debranched starch extracted from the rice flour samples was performed using Toyopearl HW55S-HW50S × 3 columns , as described by Toyosawa et al. .The chain length distribution of starch in the rice flour samples was estimated by fluorophore-assisted capillary electrophoresis (FACE) with a P/ACE MDQ Carbohydrate System , according to the methods of O’Shea and Morell  and Fujio, Tp, and Tc of rice flour subjected to air flow milling were 62.5 °C, 69.0 °C, and 74.7 °C, respectively, and the gelatinizing enthalpy (ΔH) was 7.7 J/g  were slightly lower than those of the samples treated with air flow milling, consistent with the trend observed previously [o, Tp, and Tc of rice flour samples treated with the SHMM at 30 °C were similar to those treated with the SHMM at 10 °C (data not shown). The ΔH of rice flour treated with the SHMM above 100 °C was not detected  on the structure of starch in rice flour treated with SHMM, the undegraded starch in the SHMM-treated samples was separated by GPC using Toyopearl HW75S × 2-HW65S-HW55S columns and compared with starch in the rice flour subjected to air flow milling .Previously, undegraded starch extracted from the japonica rice cultivar ‘Nipponbare’ was separated into three peaks  using Toyopearl HW75S × 2-HW65S-HW55S columns ; Peaks 1Next, to compare the structure of debranched starch in rice flour treated with air flow milling and the SHMM at 10–150 °C, GPC was performed using Toyopearl HW55S-HW50S × 3 columns ; Table 2To analyze the differences in the fine structure of amylopectin in each rice flour sample, the chain length distribution of starch was analyzed by FACE A. The chsugary-1 (isoamylase 1 deficient mutant) rice lines, which partially accumulate phytoglycogen with highly branched and short side chains, are lower than those of wild-type rice, which accumulates starch [Starch crystallinity is governed by the molecular structure of amylopectin. For example, the crystallinity and ΔH of α-glucans in s starch ,22,23. Ts starch , and adjs starch . Therefos starch . The pol8, 3.4 × 107, and 8.4 × 105, respectively, with DP values of 19,480, 1120, and 27, respectively [Next, we attempted to separate the undegraded starch in the rice flour samples by GPC. Rice flour treated with the SHMM at a higher temperature showed a significant reduction in the molecular weight of amylopectin . Previouectively . Therefo2–3 chains connecting amylopectin clusters.Murakami et al.  separateThese results strongly suggest that the shearing-induced cleavage of amylopectin molecules does not occur within the amylopectin clusters, but rather in the amorphous regions connecting the clusters. Therefore, the SHMM treatment results in the reduction in the molecular weight of amylopectin molecules. It was also found that the shearing-induced reduction in the molecular weight of amylopectin was more severe at higher processing temperatures. The gelatinization of starch, which reduces crystallinity and ΔH, as well as the reduction in the molecular weight of amylopectin, are thought to occur simultaneously but independently, and the degree of both of these processes is greater at higher temperatures than at lower temperatures.A vast amount of research has been conducted on the processing of gluten-free bread using flour prepared from rice and other grains, except wheat . GeneralIn this study, we found that rice flour samples treated with an SHMM at different temperatures showed a lower degree of crystallinity and lower enthalpy, because of gelatinization, compared with the samples treated with air flow milling. The extent of the reduction in the degree of crystallinity and enthalpy of SHMM-treated samples was more pronounced at higher temperatures. Furthermore, the degree of degradation of amylopectin and amylose, which could not be clarified previously , was det


The research of starch as a matrix material for manufacturing biodegradable films has been gaining popularity in recent years, indicating its potential and possible limitations. To compete with conventional petroleum-based plastics, an enhancement of their low resistance to water and limited mechanical properties is essential. This review aims to discuss the various types of nanofillers and additives that have been used in plasticized starch films including nanoclays , poly-saccharide nanofillers , metal oxides , and essential oils . These reinforcements are frequently used to enhance several physical characteristics including mechanical properties, thermal stability, moisture resistance, oxygen barrier capabilities, and biodegradation rate, providing antimicrobial and antioxidant properties. This paper will provide an overview of the development of starch-based nanocomposite films and coatings applied in food packaging systems through the application of reinforcements and additives. The use of plastics is essential to many sectors of our economy  due to tTherefore, to minimize the impact of plastics, it is vital to develop new polymeric matrices that are biodegradable and eco-friendly. Polymers derived from renewable sources, such as starch, cellulose, or chitin, are an excellent approach to fulfill these conditions. However, the current transition rate from petroleum-based polymers to biodegradable polymers is rather low and still inconsistent. Companies prefer to recycle already-used plastics instead of investing in new polymer matrices due to the poorer mechanical and barrier properties that these newly developed matrices show ,7.To improve these limiting properties, a push toward the research and development of new and suitable matrices has to be made. In this context, starch-based polymers rise as one of the most promising alternatives to traditional petroleum-based polymers in the food packaging sector. The growing interest in sustainable plastics can be seen in Starch is a natural biopolymer extracted from conventional botanical sources such as potatoes, rice, or corn, but can also be extracted from non-conventional resources including roots, skins, and pits from vegetables and fruits, most of which are discarded and thrown away. Therefore, starch is abundant and can give value to what is commonly considered “waste”.Starch usage has been described for thousands of years, from the ancient Egyptians (~4000 BC) and later the Romans using it as an adhesive or the ancient Greeks incorporating it in medical preparations . NowadayIn the packaging sector, thermoplastic starch (TPS)-based films not only match the properties shown by conventional petroleum-based polymer films, but in some cases, they show a superior capacity to form colorless, tasteless, and translucent layers  as well Composite films consist of two phases: a continuous polymeric matrix and a discontinuous filler  . MoreoveThus, the objective of this review is to report an interesting scientific discussion on starch-based films for food packaging applications, focusing attention on the reinforcements and additives used and their main effects on starch-based film properties. In particular, after analyzing starch botanical sources and their extraction techniques, the properties of thermoplastic starch are discussed, including common preparation techniques such as solvent casting, extrusion, and film blowing. Then, the most typical organic and inorganic nanofillers used as reinforcements are described, detailing their effects on the mechanical response. Furthermore, additives such as essential oils and functional organic molecules such as carvacrol, eugenol, and cinnamic acid are reported to focus on their antioxidant and/or antimicrobial properties and their application in smart packaging. In the end, a short discussion of their perspective is provided from a circular economy point of view.The structure of starch is formed by the succession of α-D-glucose linked to each other through α (1 → 4) glycosidic bonds . Two difStarch can be extracted from multiple sources and, as a result, the ratio between amylose and amylopectin as well as the characteristics and properties of the obtained native starch can vary. Several methods have been used for starch extraction, of which two are the most commonly used. In the first method, starch extraction is carried out after treatment with a dissolution of hydrogen sulfite 0.1 M at 50 °C for 24 h, after which the substrate is ground in a laboratory grinder with distilled water to extract all the starch. After several screenings through different mesh sizes, the starch slurry is centrifuged several times and then dried in an oven for 12 h at 40 °C . Among tThe shape and size of the starch granules are characteristic of the source from which the starch was extracted. In corn, starch granules are angular shaped with a diameter of 11.5 ± 4.3 µm; potato starch granules exhibit large irregular and oval or cuboidal forms, with typical granule size for small and large potato starch granules between 1 and 20 µm. Meanwhile, rice starch granules present pentagonal and angular forms with a size of 3 to 5 µm . A wide Starch has received significant interest in the form of a thermoplastic polymer, namely thermoplastic starch, TPS. Even though starch is a poor choice to replace plastic as it does not exhibit true thermoplastic behavior, it can be transformed into a continuous polymeric-entangled phase through the addition of sufficient water and/or plasticizers . During The glass transition temperature (Tg) of starch is substantially higher than its decomposition temperature , therebyThe process of generating a starch film initiates with the application of heat to starch granules in an excess of water, thus facilitating the creation of a viscous solution. The viscoelastic behavior of TPS can be classified according to the quantity of plasticizer present: a glassy profile (hard and stiff but brittle) at low plasticizer concentrations, followed by a rubbery behavior (flexible) characterized by a glass-to-rubber transition as plasticizer content increases, concluding in a gel-like state at very high plasticizer content . The micTPS exhibits a few limitations in its utilization, including: a significant hydrophilic nature, making it susceptible to water; relatively poor mechanical attributes when compared to conventional polymers; and a significant variation in post-processing properties . In addiThere are three main techniques for the preparation of starch-based films; the casting procedure, the extrusion/thermocompression process, and the blow molding approach, which are briefly discussed in this review. Additionally, other alternative processing techniques based on the plastic industry have also been used for the elaboration of starch films, including injection molding, compression molding, foaming, and spinning  .The solvent casting technique is by far the most popular in starch film production and has been widely documented in the academic literature. The technique was developed at the end of the 19th century to manufacture thin plastic films for the emerging industry of movies and photography .The casting of starch-based films typically includes four steps: solution preparation, gelatinization, casting, and drying. As a general method ,60, the Despite its simplicity and popularity, the long drying phase makes this method slow and discontinuous, producing small quantities of material after a long process. Moreover, the casting methodology presents certain drawbacks: difficulty when combining materials of different natures; separating the film from the underlying support can be challenging; and limited capacity of scaling up the technique from an experimental setup to industrial production .Tape casting was first introduced by Glenn Howatt in the 1940s, to produce capacitor dielectrics, and, to this day, continues to be one of the main applications for this technology . The tecThe tape casting method allows the casting of the film-forming solution on large supports (batch process) and on continuous belts (continuous process), where the film thickness can be modulated by an adaptable blade positioned at the base of the spreading device , as seenFrom an industrial standpoint, the manufacturing of TPS demands a highly effective mass-production technique; a simple solution is using processes with the standard equipment used in the manufacture of synthetic polymers. Considering this, hot extrusion presents itself as an answer to scale up biopolymeric film production.Throughout the extrusion process, starch is subject to elevated pressure and temperature, as well as the application of mechanical shear forces, leading to a range of chemical and physical reactions, disintegrating the starch granules . The plaGonzález-Seligra et al.  studied The process of film blowing is a technique frequently used to manufacture self-supporting plastic films, which involves extruding a hollow tube and then expanding it into a bubble by raising the pressure within the tube . The popRejak, A. and Mosciki, L.  processeNonetheless, TPS production by film blowing may result to be complex due to the low melting tenacity of starch . TherefoThe hydrophilic characteristic of starch results in elevated water vapor permeability (WVP) and low tensile strength and Young’s modulus in contrast to synthetic materials of manufactured products. A feasible approach to enhance these attributes involves the integration of reinforcement fillers to create starch-based nanocomposites.The incorporation of nanoparticles as fillers in polymers is a widely recognized technique to enhance the properties of polymeric materials while using less product. For an equal amount of filler, if the dispersion exhibits homogeneity, the influence on the macroscopic characteristics will be more pronounced for nanoparticles of a smaller size, attributable to the augmented polymer/filler interface . HoweverNanoparticles can be grouped by their nature into inorganic nanofillers  and organic nanofillers  . AlternaRecently, Wolf et al.  compared2 has approval for use in human food, medicines, cosmetics, and food contact products by the US FDA [2 NPs, the European Commission in 2022 banned the use of TiO2 as a food additive and encourage its replacement in medicines over health safety concerns [As nanotechnology innovations make their way into different industrial sectors, many countries are establishing controls over the use of nano-based products. However, regulation frameworks applied to control and ensure the safe use of nanotechnologies differ across countries and regions . For exae US FDA . Howeverconcerns .In the EU, nanomaterials are covered by the same regulatory framework that ensures the safe use of all chemicals and mixtures, i.e., the REACH and CLP regulations. The EU has added regulatory measures for nanomaterials in the food and feed chain, together with risk assessment guidance. Current EU legislation includes Regulation (EC) No. 1169/2011 on the provision of food information to consumers, which defines ‘engineered nanomaterial’ as any intentionally produced material with at least one or more dimensions of the order of 100 nm or less, including structures, agglomerates and aggregates which may exceed this size but retain properties of the nanoscale . EU ReguIncorporating inorganic nanoparticles is a highly effective form of enhancing the mechanical and physical properties of starch-based films. The incorporation of these additives in polymeric nanocomposite causes an improvement in mechanical and barrier qualities, including enhanced gas barrier qualities, and stability across a range of temperature and moisture levels . FurtherAn interesting class of nanofillers that solves the issues of starch matrices previously mentioned is nanoscale lamellar silicates, commonly referred to as nanoclays. The economic cost, biocompatible nature, and widespread availability of nanoclays have seen them being frequently utilized for the production of biodegradable films and coatings, as well as synthetic films to improve their barrier and thermal stability properties . AccordiTypically, within a polymer matrix, nanoclays can display three distinct morphologies of nanoclay, depending on the dispersion state of the nanoclay, and the degree of separation between the silicate layers, as well as the level of polymer penetration between them. These morphologies are: aggregated, intercalated, and exfoliated , as seenThe impermeable properties of nanoclays dispersed within the film oblige water molecules to travel through a convoluted path to cross the matrix, improving barrier properties to water vapor . IncreasOsman et al.  demonstrInterestingly, Müller et al.  comparedMontmorillonite (MMT) is a relatively cheap and widely available natural clay derived from volcanic ash/rocks , popularIntegration of MMT NPs as a filler in starch-based films is frequently used to overcome low mechanical resistance and poor barrier properties . Huang eKaolinite is an abundant 1:1 clay with little research into starch composites; moreover, kaolinite-starch interactions are considered very weak, as kaolinite clay sheets present strong hydrogen bonds complicating dispersion in aqueous solutions and polymeric matrices . HoweverRen et al.  prepared2+ ions affects the numerous processes that Ag uses. These are the suggested mechanisms:(1)2+ interacts with sulfhydryl groups in the cell wall to generate insoluble compounds that are parts of several enzymes involved in the production of transmembrane energy and the transport of electrolytes.Ag(2)2+ inhibits the cytochrome oxidase and NADH-succinate-dehydrogenase regions of the bacterial respiratory chain.Ag(3)2+ enters the cell and interacts with the bacterial DNA. This reversible binding to the nucleotide bases results in denaturation by breaking the hydrogen bonds between the neighboring purines and pyrimidines [Agimidines , as seenLooking at the use of metallic nanoreinforcements, a variety of metal nanoparticles have been incorporated in multiple polymeric nanocomposites for a wide range of applications, for example, gold, copper, silver, palladium, and cobalt ; howeverAg NPs biosynthesized from plant extracts have demonstrated the capacity to scavenge free radicals in addition to their antibacterial capabilities. This is primarily because phenolic chemicals are present in the structure of Ag NPs .S. asoca leaf extract; the obtained Ag NP/starch films exhibited lower water solubility and WVP with impending antibacterial activity. The addition of Ag NPs to starch and chitosan coatings on paper surfaces upgraded the barrier and mechanical properties and repressed the growth of S. enteritidis and E. coli [In thermoplastic starch matrices for food packaging Ag nanoparticles have been used as an important additive highly increasing the antimicrobial properties of the matrix, more than essential oils and nano-emulsions. Multiple edible films containing starch as a matrix and essential oil or silver nanoparticles were obtained via solvent casting. The antimicrobial activity of these films was measured proving that edible films containing silver nanoparticles showed almost a three-times-bigger diameter of the inhibition zone . The ant E. coli . Similar E. coli .Compared to other metal oxide NPs, ZnO has received the highest attention from researchers. Among its many attributes are biocompatibility, nontoxic, chemically stable under exposure to both high temperatures and UV light, and the recognition by the US Food and Drug Administration (FDA) as a generally recognized as safe substance (GRAS) ,128.L. monocytogenes and preserved the quality of fresh-cut mushrooms throughout storage [ZnO NPs’ inclusion in buckwheat starch films enhanced physical and water barrier qualities as well as their antimicrobial activity. The 3% ZnO NPs film in particular showed antibacterial action against  storage . Anugrah storage  successfS. aureus, E. coli, and A. flavus with results showing a significant improvement as the concentration of ZnO and FEO increased [In another study, ZnO nanoparticles and fennel essential oil (FEO) were combined on potato starch films produced by the casting method. The study reveals an improvement in barrier properties, a decrease in water vapor and oxygen permeability, and tensile strength. The antimicrobial activity of starch films was tested against ncreased .E. coli., and with ZnO migration below the overall migration legislative limits; however, ZnO NPs over 7% led to sharply decreased transparency. In a similar manner, mangos were directly coated with starch and ZnO NPs to prevent post-harvest degradation caused by C. gleosporides, prolong shelf life, and preserve the quality of the fruit while being stored [Ni et al.  developeg stored .2 is commonly used as a white pigment in the paint and coating industries due to its high refractive index and low absorption, resulting in a high light scattering that renders the materials opaque with outstanding thermal stability [2 NPs have been used to modify the properties of different biopolymer films including starch-based nanocomposites [2 has antimicrobial properties that destroy microbial cells through the production of active oxygen species (ROS) under adequate light energy [2 has been widely tested on a variety of microorganisms, including E. coli, S. aureus, P. putida, and L. innocua on multiple surfaces [TiOtability . In the mposites . In addit energy , which csurfaces ,137.w/w TiO2 into starch films and managed to extend the shelf life of fresh fruits over petrol-based films. Moreover, the composites decomposed under 14 days leaving no trace of NP residue in the soil. However, higher concentrations of NPs led to brittleness and fracture of the packaging. Ostafinska et al. [2 NPs and titanium nanotubes in TPS films using a combination of solvent casting and melt mixing procedures to increase dispersion. Although individual nanotubes were smaller than the TiO2 NP, they tended to form big agglomerates, whereas TiO2 NPs exhibited better dispersion and higher interface surface than agglomerated titanium nanotubes, and as a result, the highest increase in shear moduli.Tunma et al.  incorpora et al.  compared2 NPs in sago starch-based film which presented excellent antimicrobial activity against E coli, S. typhimurium, and S. aureus. On the other hand, the additives caused colliding effects in mechanical and barrier properties, a higher TiO2 NP concentration increased tensile strength and Young’s modulus, and lowered elongation at break, WVP, and oxygen permeability, while CEO had the opposite change. The developed films allowed an increase in shelf life and better preservation of fresh pistachios due to the antioxidant and antimicrobial properties of CEO and TiO2 combined.Arezoo et al.  combinedw/w starch as a plasticizer and different amounts of CuO concentrations (0.05–2 wt%). These nanoreinforced blends present higher mechanical properties (TS increased 29.1% and EB increased 67% over the PBAT/TPS film), increased vapor barrier properties, and an antimicrobial rate of over 99% against E. coli, as well as presenting great antimicrobial activity against bacteria (gram-positive and gram-negative bacteria) [Copper oxide nanoparticles do not only act as reinforcements for the polymeric matrix. Bumbudsanpharoke et al.  studied acteria) , and alsacteria) , virusesacteria)  and algaacteria) .Due to their small size and large specific surface area of CuO, nanoparticles have remarkable antibacterial potential because they can interact intimately with cell membranes . Copper Numerous studies have reported that edible films containing CuO nanoparticles presented superior properties to other films in terms of their antioxidant qualities. For example, Rehana et al.  reportedWhen used in thermoplastic starch matrices, they not only produce excellent antimicrobial  responseE. coli and S. aureus, and decreased solubility, moisture content, and WVP [E. coli and S. aureus with an inhibition zone diameter of 25.60 mm and 27.50 mm, respectively. Moreover, the lower WVP and oxygen permeability of the reinforced films proved to prolong the shelf life of various fruits, as they reduced weight loss of various fruits, while the reduced enzyme activity and inhibition of the microbial growth on surfaces delayed the decline in firmness.The incorporation of magnesium nanoparticles (MgO NPs) in starch films resulted in increased thickness, enhanced antioxidant activities and antimicrobial activity against  and WVP . In the  and WVP  and exhi2 NPs) combined with encapsulated essential oil increased the antioxidant, antibacterial, and mechanical properties of potato starch and apple peel composite films [Zirconium oxide nanoparticles  and starch nanoparticles (SNP) . SNC are2SO4 solution (15% w/v), at 40 °C under mechanical stirring for 5 days. The remaining suspension is repeatedly washed with distilled water and separated using centrifugation at 10,000 rpm and 4 °C until a neutral pH is reached. Finally, the suspension is submitted to mechanical treatment using an Ultra Turrax to avoid aggregates before being freeze-dried.Starch nanocrystals (SNC) constitute crystalline fragments produced through the disintegration of amorphous sectors in starch granules and exhibit nanometer scale dimensions (<100 nm) in at least one dimension. SNC can be obtained from different sources and multiple approaches, with acid hydrolysis with sulfuric acid being the most frequent procedure. In this method ,162, sta2SO4 [During the initial stage of acid hydrolysis (days 0–2), the amorphous regions, which are more susceptible to acid, are quickly degraded resulting in an increase in crystallinity. The second phase (days 2–5) consists of slow hydrolysis of the semi-crystalline rings, breaking the starch granules into nanocrystals ,163. Aci2SO4 . When st2SO4  discoverNevertheless, the application of SNC generated through traditional acid hydrolysis is restricted by their susceptibility to aggregation, combined with long elaboration periods and low extraction yields . Moreove−10 to 2.72 × 10−10 g m−1 s−1 Pa−1) of starch nanocomposites [Starch nanomaterials have successfully been used as reinforcement fillers in many natural and synthetic matrices. The incorporation of SNC into starch-based films increased mechanical and water vapor barrier properties  and thermposites .Cellulose is the most abundant natural polysaccharide, composed of linear chains of β-1,4 linked D-glucose units, which form strong intramolecular hydrogen bonds which shape its crystalline characteristics . CelluloThis polysaccharide has received substantial attention in various forms of cellulose nanomaterials (CN), including cellulose nanocrystals (CNC), cellulose nanofibrils (CNF), and bacterial nanocellulose (BNC) , as seenCellulose Nanocrystals (CNC), also called nanowhiskers , are a vw/w) sulfuric acid aqueous solution with the ratio of 1:8 to 1:20 (w/v) of raw material containing cellulose vs. acid at 45 °C, followed by centrifugation washing to a neutral pH, homogenization, and freeze-drying [Similar to starch, cellulose is commonly treated by acid hydrolysis to form CNC, but through a two-step procedure. First, a pre-treatment of the source material is needed to eliminate the hemicellulose, lignin, and other components of the matrix, normally through chemical treatment (pulping) or steam explosion . In the e-drying , as seene-drying .The addition of small amounts of wood CNC (1.5–2.5%) to extruded native potato starch formed strong hydrogen-hydroxyl bonds, which improved the mechanical properties while reducing swelling and enzymatic degradation . In contE. coli and S. aureus [The use of cellulose nanocrystals (CNC) can be impeded by their intrinsic tendency to self-aggregate and hydrophilic behavior. Consequently, several chemical modifications and copolymerization have been documented as strategies to address these deficiencies , and to . aureus .Cellulose nanofibers (CNF) consist of cellulose chains that are laterally stabilized by strong hydrogen bonds that form between the oxygen and hydroxyl groups of neighboring molecules . The ISOProduction of CNF using mechanical processes is very energy-intensive; however, pretreatment methods offer an efficient approach to reduce energy input ,210. Exaw/w) proved to be UV resistant and have increased water barrier properties [Nanocomposite films based on potato starch and pineapple leaf CNF -linked 2-amino-deoxy-b-D-glucan, as seen in B. cereus and S. aureus) bacteria compared to gram-negative (E. coli and S. typhi) bacteria; a similar conclusion was reached on chitosan NP/tara gum edible films [The chemical similarity between the starch matrix and chitin/chitosan NPs creates a strong interfacial interaction, resulting in a reinforcement effect, visible through an increase in tensile strength and decreased elongation at break; an improvement in the intermolecular interaction increasing thermal stability as Tg shifts to higher temperatures; and the creation of a more tortuous path that lowers WVP ,242. Addle films .As mentioned before, the properties of materials based on a thermoplastic starch matrix, despite their many benefits, have some well-known drawbacks. Therefore, it is necessary to incorporate additives to improve the material’s intrinsic characteristics or even introduce some new functional properties, such as antimicrobial or antioxidant activity or even some mechanism that can detect the state of degradation of the material . The claA plasticizer is a polymer additive, with a low molecular weight and non-volatile nature, that is added to the polymeric matrix to provide elasticity to the base polymer . PlasticThe introduction of plasticizers together with elevated temperatures and shear increases the flexibility of native starches similar to conventional thermoplastic polymers . The mosw/w) of waste glycerol [In packaging applications, glycerol is the most utilized plasticizer in starch-based films, due to its stability and compatibility with the hydrophilic bio-polymeric packaging chain . The effglycerol . TherefoWhen comparing glycerol and sorbitol, starch-glycerol films presented lower tensile strength and elastic modulus values and higher elongation compared to starch-sorbitol films, indicating the pronounced plasticizing influence of glycerol ,256. AddSimilarly, Maniglia et al.  studied Ma et al.  comparedDue to the many characteristics of these additives, essential oils have recently captured the interest of many researchers in the field. In general, essential oils come from a variety of aromatic plant parts, including seeds, bark, flowers, peel, fruit, roots, leaves, and wood. Their biological actions are caused by complex combinations of more than a hundred molecules, such as terpenes and isoprenoids, which are examples of volatile compounds and lipophilic compounds . The EOsAdditionally, EOs have the ability to neutralize a variety of bacteria , as wellTerpenes: With more than 40,000 distinct chemical structures, terpenes are one of the principal constituents of essential oils and one of the most abundant and diverse categories of plant natural products . There aHemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesterpenes (C25), triterpenes (C30), and tetraterpenes/carotenoids (C40) are the different categories of terpenes based on the number of times this structure is repeated .Polyphenols: These molecules can be divided into different classes depending on their chemical structure. Polyphenols are found in nature as a product of the secondary metabolism of most plants . Two maiEOs’ constitutions, and therefore their method of action, determine their antibacterial activity . The bacRosemary essential oil (REO) has been widely studied in various matrices, such as PLA. Fiore et al.  successfBio-chitosan films were produced via the solvent casting method, using montmorillonite as reinforcement and REO as well as ginger essential oil to add some antimicrobial and antioxidant behavior to the matrix . The meaw/w starch) and the EOs’ nanoemulsion concentrations . As a result, it was observed that these essential oils caused a plasticizing effect on the matrix that allowed higher transparency and elongation at break.REO has also been used as an additive in starch-based films. In particular, Restrepo et al.  formed eS. epidermidis, S. aureus, and B. subtilis), gram-negative bacteria , and fungi  [Moreover, REO has been proven to have high antimicrobial activity against gram-positive bacteria (. niger)  and to i. niger) . The pri. niger) .Due to all these antimicrobial characteristics, REO has been one of the most used in starch matrices. In particular, Tafa et al.  obtainedAs mentioned before, REO can act as a plasticizer, allowing for greater transparency and elongation at break, both of which are desired in the food sector and raise water vapor permeation values. However, this impact appears to be offset by the EOs’ hydrophobic properties .w/w starch) and varying the bioactive compound [Cinnamon essential oil (CEO) is one of the most significant and promising EOs being used in active food packaging due to its antibacterial and antioxidant capabilities. In general, cinnamaldehyde has been primarily involved in CEO’s excellent antibacterial and antioxidant activities. CEO is considered one of the most common EOs in starch matrices for packaging applications because of its great antioxidant properties. In particular, the addition of CEO reduces the moisture content of the films and the water solubility due to its hydrophobic behavior and provides good thermal stability and antioxidant capacity as shown in a recent article where films were obtained via solvent casting, using glycerol as a plasticizer  is also one of the most promising and widely used essential oils, due to the number of residues that can be used to obtain it and its antibacterial properties against all types of bacteria. TEO has been used in direct contact with foods such as lettuce, meat and milk, showing good results against pathogens of food origin, but less efficacy and less antioxidant activity in food matrices than by itself .Nieto et al. , studiedBotryodiplodia theobromae Pat and Colletotrichum gloeosporioides Penz on the surface of mango fruit. In another study, a starch matrix nanocomposite activated with TEO was observed to improve the shelf life of vegetables, in this case baby spinach leaves. For this, starch/clay nanocomposite films (montmorillonite) and TEO were prepared by the solvent casting method and used as packaging for spinach leaves. As a result, the film could improve the acceptability of leaves by around 8 days by controlling the growth of bacteria such as E. coli and S. Typhimurium and improving the microbiological quality of baby spinach leaves during refrigerated storage. [TEO is widely studied as an additive in biodegradable polymers for food packaging, including starch matrices, due to its non-toxic, antimicrobial and antioxidant nature. Cai et al.  studied storage. . Howeverstorage. .TEO is formed by 52 distinct constituents that have been identified. Moreover, it was discovered that thymol and carvacrol were the 2 most prevalent chemicals along with terpineol, linalool, p-cymene, and other substances . Due to Aldehydes, ketones, and carboxylic acids are produced at the end of the polymer chain by photooxidation. Antioxidants stop these chain reactions and prevent subsequent oxidation processes by eliminating the free radical intermediates . To perfThe majority of the components included in this category, such as carvacrol, ferulic acid, and cinnamic acid, can also be found in essential oils, although their usage as single molecules is different  or even The number of procedures required for the purification of such components is its principal drawback. These molecules are often derived from the essential oils collected from plant resources; as a result, the purifying process entails an increase in overall cost and difficulty.Carvacrol , a phenoThe antimicrobial activity of carvacrol is well known and studied. Carvacrol has free phenolic hydroxyl groups that can form hydrogen bridges and release protons that can damage the membrane structure and lead to bacterial cell death .Thymbra spicata leaves to radiation (at a wavelength of λ > 200 nm), which caused the OH groups to break apart and the H atom released in the orto- or para-position to recombine. However, as different types of cell walls have distinct reactions to UV light, the activity varies between cell walls.Eliuz et al.  exposed In starch matrices, enhancement of antimicrobial and antioxidant properties via the addition of carvacrol has been widely reported. Edible starch films were obtained using carvacrol and thymol as a method of reducing the incidence of anthracnose symptoms in mango and papaya, enhancing fruit quality as long as possible by an additive effect of both molecules . Mao et The primary ingredient in some essential oils, such as clove essential oil, which has a phenol content ranging from 76% to 82%, is eugenol  306,307307.The free hydroxyl groups of this molecule are responsible for most of its antibacterial action, as it can damage cell membranes, increasing nonspecific permeability, which disrupts intercellular ATP and ion transport . MoreoveE. coli, S. aureus and B. subtilis and improved mechanical properties. In a second example [The addition of eugenol in starch matrices has been reported in several articles to increase antimicrobial properties. In a study , eugenol example , eugenolIn relation to the mechanical properties response, these systems show a big dependence on the plasticizer due to the possible hydrogen bond interactions between eugenol and the plasticizer. In particular, Cheng et al.  reportedCinnamic acids (CA) and their derivatives  are a wiThe exact molecular mechanisms by which CA acts are unknown. However, two possible mechanisms have been proposed: CA blocks the post-translational modification of proteins that control cell growth, such as some p21ras proteins, thus preventing protein prenylation by blocking the synthesis of residues derived from mevalonate, or CA inhibits the expression of genes that promote cell proliferation . CA exhiw/w of ferulic and cinnamic acid, respectively. It was only observed that antimicrobial inhibition in films containing 2% acid was more effective than the ones with just cinnamic acid. There are still no studies that imply any antioxidant effect or modification of mechanical properties by the addition of CA in food packaging applications.Moreover, the antimicrobial activity of CA in starch matrices has been reported. In particular, Ordoñez et al.  incorporA new application in food packaging that has been gaining interest from researchers is food safety and quality monitoring, known as smart or real-time packaging. There are three main types of smart packaging, including indicators, data carriers, and sensors. In this regard, food packaging containing an indicator film can detect changes in the quality or freshness of the product, and respond by providing a visual warning to consumers. Briefly summarized, food spoilage can be tracked by alterations in pH as a reaction to the growth and metabolism of microorganisms or the development of organic acids and/or volatile amines  . CommonlIn terms of natural pH indicators in starch matrices, anthocyanins are by far the most studied, as seen in Fotouhi et al.  developeCurcuma longa, and is yellow-orange in its natural state [S. aureus, B. subtilis, and E. coli.Besides anthocyanin, curcumin has also been proposed as a pH indicator for smart packaging applications. While curcumin does not possess the wide color range of anthocyanin, it presents a clearer reaction to alkaline environments . Curcumial state . Liu et al state  presenteIn regards to film elaboration, the use of solvent casting and extrusion techniques has been frequently reported, as seen in In recent years, biopolymeric materials have been gaining popularity as an alternative to traditional plastic packaging. Moreover, stricter regulations on plastic packaging and the recent interest in the revalorization of agro-alimentary and industrial waste may increase the demand for biopackaging in the near future. Among biopolymers, starch is an abundant, sustainable, and worldwide-available source with interesting properties, that possesses plastic-like behavior in its thermoplastic form. However, the poor mechanical properties and hydrophilic nature make the processed material vulnerable and of poorer quality than its synthetic counterpart.The integration of nanofillers into starch-based films has been studied and found to enhance several of their physical characteristics, including mechanical properties, thermal stability, moisture resistance, oxygen barrier capabilities, and biodegradation rate. Nevertheless, excessive nanofiller content can lead to negative effects due to the agglomeration and formation of rigid regions within the polymer matrix, and the consequent decline in the interfacial surface area. On the other hand, incorporating additives, plasticizers, essential oils, and organic molecules into the starch films offers interesting functional properties such as antimicrobial, antioxidant or pH-indicative properties. Nevertheless, the addition of nanofillers in nanocomposites is not limited to one element. Multiple research papers have proposed a combination of reinforcement and additives to obtain the benefits of each element while minimizing the downside effects. In summary, from a circular economy point of view, the key point is that thermoplastic starch biocomposites can challenge synthetic packaging when modified with adequate additives and nanofillers.Therefore, the objective of this review is to discuss starch-based films for food packaging applications. In particular, after analyzing different starch botanical sources and their extraction techniques, the properties of thermoplastic starch are considered, including the main preparation techniques such as solvent casting, extrusion, and film blowing. Then, the most typical organic and inorganic nanofillers used as reinforcements are described, detailing their effects on the mechanical response. Furthermore, additives, plasticizers, essential oils, and functional organic molecules are reported to focus on their antioxidant and/or antimicrobial properties and their application in smart packaging.


Anterior cruciate ligament injuries are common in high school and college with an estimated 120,000 cases per year in the United States. Most sports injuries occur without direct contact, and knee valgus with external rotation of the foot is the most common movement. This movement may be related to the injury of the anterior oblique ligament located in the anteromedial quadrant of the knee. This technical note presents anterior cruciate ligament reconstruction with extraarticular anteromedial reinforcement using hamstring and the anterior half of the peroneus longus grafts. The procedure is performed with the patient in the supine position on the surgical table. Harvest of the tendons of the semitendinosus and gracilis muscles is preferentially performed in the injured limb. A single longitudinal incision of approximately 3 cm is made in the posterolateral region of the fibula over the peroneus longus. After the subcutaneous tissue is separated, the peroneus longus is identified and isolated with the aid of a hemostatic forceps. Both peroneal tendons are then brought together in the most distal region of the incision using single sutures with Vicryl thread. After the tendons are unified with sutures, the peroneus longus is divided in anterior and posterior with a scalpel, the anterior half is repaired with Vicryl thread and removed to its proximal insertion with the aid of a tenotome. The hamstrings grafts are folded to form a single quadruple graft. Subsequently, the peroneus longus graft is incorporated into the hamstrings grafts without folding, forming a quintuple graft in the most proximal region and a single, more distal graft, comprising the remainder of the length of the peroneus longus. Reconstruction is performed through outside-in anatomic positioning to create the femoral and tibial tunnels. The graft is pulled through the tibial tunnel, and an interference screw is fixed to the femur. The next step is to fix the inferior end of the quintuple graft to the tibia after pretensioning. After tibial fixation, the remaining PL is passed through the subcutaneous, in the medial side, and is passed through the anterior oblique ligament in the femur. With the aid of radioscopy, the medial epicondyle is identified, in the lateral view. At this point, a guidewire is passed through the femur, starting at the medial epicondyle anterior portion, in the anterior direction, and a bone tunnel is constructed. The ligament is fixed under traction, neutral rotation, and extension with an interference screw. More than 120,000 anterior cruciate ligament (ACL) injuries occur every year in the United States, mostly during the high school and college years.The use of hamstring tendons in ACL reconstruction is widely accepted, and the results comparing with the patellar tendon, even in athletes, are the same,The most ACL lesions in sports occur without direct trauma, and the valgus knee and foot external rotation are the most common biomechanical movements.,The anterolateral ligament (ALL) is a distinct structure in the anterolateral portion of the knee. After the study of Jorge et al.,The procedure is performed with the patient in the supine position on the surgical table. A pneumatic tourniquet is placed proximally on both lower limbs and inflated after the placement of sterile drapes.Harvest of the tendons of the semitendinosus and gracilis (STG) muscles is preferentially performed in the injured limb. First, a longitudinal incision of approximately 3 cm is made 4 cm distal to the knee joint. A new transverse incision is made in the fascia of the sartorius muscle, and the STG is isolated. The STG is repaired with no. 1-0 Vicryl thread , and its proximal insertions are removed with the aid of a tenotome.A single longitudinal incision of ∼3 cm is made in the posterolateral region of the fibula over the PL . The incThe STG grafts are folded to form a single quadruple graft. Subsequently, the PL graft is incorporated into the STG grafts without folding, forming a quintuple graft in the most proximal region and a single, more distal graft comprising the remainder of the length of the PL, as shown in Standardized anterolateral and anteromedial portals are used. We opt for a high anterolateral portal close to the patellar edge and flush with the patellar tendon. The anteromedial portal is created with care taken to form a good angle of inclination with the lateral intercondylar wall.Reconstruction is performed through anatomic positioning to create the femoral and tibial tunnels. The femoral tunnel is positioned in the anatomical position. Guides  are used to make the tunnels using the outside-in technique.With the aid of radioscopy, the medial epicondyle is identified, in the lateral view. At this point, a guidewire (Smith & Nephew) is passed through the femur, starting at the medial epicondyle anterior portion, in the anterior direction, and a bone tunnel is constructed under radioscopic visualization.The graft is pulled through the tibial tunnel and passed through the femoral tunnel with the aid of a no. 5.0 Ethibond thread . The single portion of the graft remains out of the tibial tunnel until the quintuple graft portion occupies both tunnels. The graft is then pulled, and an interference screw  is fixed to the femur. The next step is to fix the inferior end of the quintuple graft to the tibia after pre-tensioning. Fixation is performed with the graft tensioned and the knee in total extension and after performance of the posterior drawer maneuver. After tibial fixation, the remaining PL is passed through the subcutaneous, in the medial side, and is passed through the Anterior Oblique Ligament in the femur . The ligFootball players sustain the greatest number of ACL injuries  with skiers and gymnasts also at high risk.Adequate treatment of all concomitant injuries, aiming at joint stability, and extra-articular ligament reinforcement can lead to better results. Lateral extra-articular procedures (LEAPs) in the anterior cruciate ligament injured knee were widely abandoned in the 1990s but have seen a recent resurgence. The LEAPs associated with ACL reconstructions seem to have a great importance in the surgery survival. In a recent study, by the Santi Group, patients who underwent combined ACL+ALLR  experienced significantly better long-term ACL graft survivorship, lower overall rates of reoperation, and no increase in complications compared with patients who underwent isolated ACLR. Further, patients who underwent isolated ACLR had a >5-fold increased risk of undergoing revision surgery at a mean follow-up of 104.3 months.A question that arises: Would the ALL be the answer for all?,Most ACL injuries occur without physical contact between athletes—referred to as noncontact ACL injuries,By the Theory of Tibial Quadrants,Our group believes that, in almost all ACL ruptures, the extra-articular reinforcement is important, and in the future, it will be routinely done. But we also believe that the anterolateral reinforcement is not the answer to all cases. The patients that don’t have grade 2 pivot shift, the patients that have a mild medial instability (not treated in past), and the patients who have a history of valgus and external rotational trauma, can benefit from anteromedial reinforcement in the AOL topography.,,And for this, the use of peroneal longus tendon, with hamstrings, can be a good and secure option.


Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disorder that produces a broad spectrum of clinical conditions such as dementia, upper motor neuron involvement, extrapyramidal symptoms, and neuropathy. Some studies have reported ophthalmological conditions associated with the disease; however, the details of these conditions remain unclear.We report a 63-year-old Japanese female with cognitive decline, blurred vision, photophobia, and color blindness at 52 years of age who was diagnosed with cone dystrophy. She also had anxiety, insomnia, depression, delusions, hallucinations, a wide-based gait with short steps, and urinary incontinence.NOTCH2NLC gene analysis revealed abnormal GGC expansion; therefore, NIID was diagnosed.Magnetic resonance imaging revealed diffuse cerebral white matter changes and subcortical hyperintensity on diffusion-weighted imaging. Skin biopsy showed p62-positive intranuclear inclusions in sweat glands. NOTCH2NLC mutation-positive NIID may be associated with retinal dystrophy. Brain magnetic resonance imaging and skin biopsy are helpful diagnostic clues, and gene analysis is crucial for accurate diagnosis and appropriate management. Recent studies have revealed diagnostic skin biopsy findings, that is, intranuclear inclusions in sweat glands, and demonstrated that NOTCH2NLC gene analysis is crucial for diagnosing the condition. Some studies have mentioned ophthalmological conditions associated with the disease but the details of these conditions remain unclear.Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disorder that produces a broad spectrum of clinical conditions such as dementia, upper motor neuron involvement, extrapyramidal symptoms, and neuropathy.Herein, we report a rare case of NIID with retinal dystrophy and a literature review.A 63-year-old Japanese female was referred to our clinic with cognitive decline. She had been well until she developed blurred vision, photophobia, and abnormalities of color perception at the age of 52 years and was diagnosed with cone dystrophy. By the age of 61 years, she also had anxiety, insomnia, depression, jealous delusions, suicidal wishes, visual hallucinations, and urinary incontinence. She became forgetful at the age of 63. She had a history of bronchial asthma, hypertension, and dyslipidemia, which were treated continuously with medications. She had a history of smoking and alcohol drinking between the ages of 20 and 25 but had ceased these habits. The patient’s family history was unremarkable.n-isopropyl-(123I)-p-iodoamphetamine revealed unremarkable changes (data not shown). A nerve conduction study demonstrated a mild reduction in tibial nerve velocity. A skin biopsy sample from the right forearm showed p62-positive intranuclear inclusions in sweat glands, together with fibroblasts and adipose cells  and decreased visual acuity. The patient experienced gait difficulties . Brain magnetic resonance imaging (MRI) showed diffuse hyperintensity in the cerebral white matter on T2-weighted imaging and fluid-attenuated inversion recovery imaging and subcortical hyperintensities in the frontal and parietal lobes on diffusion-weighted imaging  and 0.2 in the left eye (+1.0 dpt), respectively. A color perception examination involving the panel D-15 test, standard pseudoisochromatic plates, and Ishihara test revealed total color blindness. Her light reflex was prompt. Goldmann perimeter test showed a central scotoma and a relatively narrow peripheral visual field Fig. A. FundosThe patient received regular follow-up but her visual field and acuity deteriorated progressively (hand motion: 50 cm in both eyes at 65 years of age).Written informed consent was obtained from the patient and her legal guardian/next of kin.NOTCH2NLC-mutated NIID with retinopathy have been reported to date  in the acceleration of retinal degeneration in NIID.Some neurodegenerative disorders, such as triplet repeat diseases, show repeat length-dependent severity,NOTCH2NLC gene were found to be associated with NIID in patients of Japanese descent, but only a single European case harboring a pathogenic repeat expansion with a distinct haplotype structure was identified in a study of 11 European NIID cases based on whole-genome sequencing data from 20,536 participants in the 100,000 Genomes Project. Other studies have reported cases of NIID with retinopathy in Taiwan and China that involved repeated expansion of the NOTCH2NLC gene.,9 It should also be noted that fragile X mental retardation 1 mutations produce similar MRI abnormalities and intranuclear inclusions in skin biopsy samples and therefore, accurate diagnosis and classification based on genetic analysis are required.A recent study revealed that NIID is genetically heterogeneous; repeat expansions in the NOTCH2NLC mutations remain unknown. Electrophysiological studies have shown rod-cone dysfunction in most cases; thus, the suggestion of Nakamura et al that photoreceptors are the primary sites of NIID defects seems reasonable. In contrast, Hsia et al described a case involving damaged ganglion cells and photoreceptors. Some variations or inconsistencies in the clinical features of NIID could be partly explained by differences in the clinical stage because NIID is a progressive disorder.The histopathological features of retinopathy in NIID cases with Ophthalmological abnormalities observed in NIID seem to have received insufficient attention, and it is important to perform appropriate evaluations of visual function in patients with NIID. NIID is thought to be a rare disorder; however, the number of reported cases has been growing in recent years because of diagnostic advances in MRI, skin biopsies, and genetic tests. Early and accurate diagnosis, better genetic counseling, and more innovative treatments are required.We thank the patient and her relatives for agreeing to the publication of this report. We also thank Professor Hidetoshi Takei  and the staff of the Department of Pathology at Hokkaido University for performing pathological examinations.Conceptualization: Takayuki Katayama.Data curation: Kae Takahashi, Jun Sawada, Ken-ichi Ishida, Asuka Asanome, Hisako Endo, Tsukasa Saito, Mari Kishibe, Harumi Kanno, Satoshi Ishiko, Jun Sone.Project administration: Takayuki Katayama, Osamu Yahara.Supervision: Osamu Yahara, Naoyuki Hasebe, Satoshi Ishiko.Writing – original draft: Takayuki Katayama.Writing – review & editing: Osamu Yahara, Naoyuki Hasebe, Satoshi Ishiko.


Aging is an important risk factor for cardiovascular disease and is associated with increased vessel wall stiffness. Pathophysiological stiffening, notably in arteries, disturbs the integrity of the vascular endothelium and promotes permeability and transmigration of immune cells, thereby driving the development of atherosclerosis and related vascular diseases. Effective therapeutic strategies for arterial stiffening are still lacking.Here, we overview the literature on age-related arterial stiffening, from patient-derived data to preclinical in-vivo and in-vitro findings. First, we overview the common techniques that are used to measure stiffness and discuss the observed stiffness values in atherosclerosis and aging. Next, the endothelial response to stiffening and possibilities to attenuate this response are discussed.Future research that will define the endothelial contribution to stiffness-related cardiovascular disease may provide new targets for intervention to restore endothelial function in atherosclerosis and complement the use of currently applied lipid-lowering, antihypertensive, and anti-inflammatory drugs. The risk of developing cardiovascular disease is higher upon older age, with a majority of cardiovascular disease-related deaths occurring at an age of 70 years or older ,2. The hThe onset of atherosclerosis often occurs asymptomatically and it is difficult to predict the stage of atherosclerotic disease based on current traditional cardiovascular disease risk factors. Hypertension is a key cardiovascular risk factor, which precedes clinically identifiable atherosclerosis. Arteries undergo structural and functional changes upon aging and one of the main alterations is wall stiffening ,4. Arter▪▪]. A feedback loop of increased vascular stiffness and endothelial dysfunction promotes atherogenesis and its targeting has the potential to restore overall vascular homeostasis and counteract arterial stiffening.Currently applied therapeutic interventions already significantly reduce cardiovascular risk. Targeting the vascular response to stiffening, if used as combination therapy, could further improve such existing treatments . Researc22This review provides an overview of current findings on arterial stiffening, from clinical studies to preclinical in-vivo and in-vitro experiments. We will overview the common techniques that are used to measure arterial stiffness and discuss patterns, and discrepancies, in the observed pathological and age-related arterial stiffening values. Finally, the endothelial responses to arterial stiffening, and current therapeutic strategies to attenuate them, are briefly mentioned. As effective therapeutic strategies for arterial stiffening are still lacking, improved models to better study and understand the endothelial role in arterial stiffness-related cardiovascular diseases will pave the way for new potential therapeutic strategies. Arteries are composed of three layers, namely the tunica intima, media, and adventitia layer. The intimal and medial layers, and the medial and adventitial layers are separated by the internal and external elastic membranes, respectively. The tunica intima consists of the endothelium, a monolayer of cells that lines the inner surface of the blood vessel, and a thin subendothelial layer of extracellular matrix, called the basement membrane. The basement membrane is composed of ECM proteins, such as laminins and collagen IV . During The tunica media is comprised of multiple layers of VSMCs and ECM. Two important constituents of the ECM in the media are the elastin sheets and collagen fibers. Elastin contributes to the elastic properties of the blood vessel and its resilience to pulsatile pressure; whereas collagen fibers are not elastically deformable and determine the expansibility of the blood vessel . VSMCs iThe tunica adventitia contains a variety of cell types, including immune cells and fibroblasts. The ECM of the adventitia consists of fibrillar collagens and proteoglycans, like versican, that contribute to the compressibility of the vessel wall. In healthy arteries, versican is abundant in the adventitia, but during atherogenesis versican expression is also expressed by VSMCs and endothelial cells and contributes to the pathological retention of lipids in the vessel wall . FinallyThe structural remodeling and stiffening of the arteries during aging is a strong prognostic indicator for cardiovascular risk, but it is not yet incorporated in routine clinical practice. One reason for this may be the wide variety of devices and methods that are being used to measure arterial stiffening. In this section we highlight pulse wave velocity (PWV), an ultrasound-based technique that is the golden standard in the clinic to assess arterial stiffness. The PWV is the speed of arterial pressure waves traveling along the arteries and is determined by taking the distance between two recording sites over the pulse transit time . The pulThe PWV provides an estimate of the average artery elasticity between the two recording sites. However, arterial stiffness is known to develop heterogeneously throughout the vascular system ,37. UltrUpon a decrease in the elasticity of arteries (such as in old and stiff arteries), the propagation speed of the pulse wave increases resulting in higher PWV values. PWV values increase by age and is thus shown to be an independent predictor for cardiovascular risk. Current European guidelines point to a carotid-femoral PWV of more than 10 m/s as indicative of significant alterations of the vessel wall and high cardiovascular risk . ClinicaIn clinical practice, arterial stiffness is effectively synonymous to PWV. PWV levels are inversely related to the distensibility of arteries. The distensibility of an artery represents the relative change in cross-sectional area in response to pressure . The disTo study the soft material properties of arteries in the context of vascular stiffening during aging and the progression of atherosclerosis, a variety of mechanical tests can be used. The principle underlying these techniques is that a certain force is applied to the artery (compression or extension force) and subsequently its deformation is monitored. Depending on the technique, material properties can be examined from macroscale to the nanoscale, providing insight from bulk mechanics of the artery to layer-specific mechanical properties.The mechanical properties of atherosclerotic tissue has been investigated for quite some time. The first study that directly measured the mechanical properties of atherosclerotic lesions was reported in 1966 . Since tHere, we provide a summary of uniaxial tensile studies that have reported the ultimate tensile stress of human atherosclerotic plaques. The ultimate tensile strength represents the maximum stress the artery can withstand while being stretched before rupture of the tissue. Two studies that investigated human carotid arteries have reported similar rupture stresses of noncalcified plaques to be around 342 ± 160 kPa  and 366.in situ. The media and intima layers increased in stiffness during plaque progression and have a comparable stiffness in the advanced plaque  and the Amsterdam Cardiovascular Sciences institute.There are no conflicts of interest.