The distribution of the absolute errors of the model, after discarding outliers, has a normal distribution as in the development of other models of this type, which was confirmed by appropriate statistical tests. On the basis of the tests and calculations carried out, it was concluded that, in order to increase the precision of the predictive models, it is advisable to reduce the measurement uncertainty of the bitumen complex shear modulus G*. For the developed model A, the limiting values of the stiffness modulus |E*| are also shown, within which the determined stiffness modulus should fall.The main purpose of this paper is to present the development of a new predictive model intended for the calculation of stiffness modulus |E*| determined by a four-point bending beam test (4PBB or 4PB-PR). The model developed, called model A, was based on the Witczak model, which was developed for the dynamic-modulus (DM) method. Most of the asphalt mixtures used to develop the model were high-modulus asphalt concrete (HMAC). The most commonly used methods for determining the stiffness modulus |E*| of asphalt mixtures were also discussed. The paper presents the results of the study for 10 asphalt mixtures but 8 of them were used to develop the predictive model. In addition, the results of complex shear modulus G* tests on neat and modified bituminous binders carried out in a dynamic shear rheometer (DSR), necessary for the development of a predictive model, are presented. The tests carried out in the dynamic shear rheometer had significant measurement uncertainties. The results of the volumetric parameters of the asphalt mixtures are also reported. The developed model A has maximum absolute errors e = 1930 MPa ( The stiffness modulus |E*| of an asphalt mixture is an important parameter characterizing its properties. Different values of stiffness modulus |E*| appear at particular stages of asphalt pavement design and realization. The stiffness modulus |E*| is used in the designing process of pavements through mechanistic methods. It is also determined in the case of asphalt mixture composition designing as well as the factory production-control process .The stiffness modulus |E*| is a physical property describing the relationship between stress and strain in the case of linear viscoelastic material as it is loaded and unloaded. When the stress and stiffness modulus are known, the strain and displacement can be calculated. The stiffness modulus |E*| extensively describes the asphalt mix, due to its dependence on many factors. These factors are the composition of an asphalt mixture, the content of air voids, shape, lay down, and composition of aggregate grains in an asphalt mixture. The values of the stiffness modulus |E*| change substantially along with the temperature and loading frequency ,3 which Mechanistic—Empirical Pavement Design Guide (MEPDG) ;E|G*|—shear modulus of binder [psi];δ—phase angle of binder [°];beff—effective binder content expressed by volume (v/v) [%];Va—air-voids content [%];V200—percentage of aggregate passing the No. 200 sieve (#0.075 mm) [%];P4—cumulative percentage retained on the No. 4 sieve (#4.76 mm) [%];P38—cumulative percentage retained on the 3/8 in sieve (#9.5 mm) [%];P34—cumulative percentage retained on the 3/4 in a sieve (#19 mm) [%].PHowever, the form of the model equation (Equation (1)) has undergone some modifications, also aimed at simplification. Some quotients (four) with the squares of the variables characterising the aggregate grain-size distribution were removed from the Witczak–El-Badawy model equation but other quotients with the variables characterizing the aggregate-size grain distribution were still left. The decision to remove some quotients from the equation was based on the results of the sensitivity analysis carried out for the Witczak–Bari model presented in . This an4PB—stiffness modulus determined by four-point bending beam test [MPa];E|G*|—shear modulus of binder [MPa];δ—phase angle of binder [°];beff—effective binder content expressed by volume (v/v) [%];Va—air-voids content [%];V0—percentage of aggregate passing the 0.063 mm sieve [%];P4—cumulative percentage retained on the 4.0 mm sieve [%];P8—cumulative percentage retained on the 8.0 mm sieve [%];P16—cumulative percentage retained on the 16.0 mm sieve [%];PA1–A17—optimized equation coefficients [-].4PB and the phase angle Φ representing the results of determinations made on the eight HMA mixtures were used to develop model A. The range of variability of the input data for the optimization process, i.e., the variables in the models, is given in As already stated, 471 values of the stiffness modulus EOptimization of the coefficients in the equations of the models was carried out using the lsqnonlin command in Matlab, which returns the solution of the nonlinear least-squares method task. The least-squares method is a method of approximating a function of a given type, to a set of empirical points. The method consists in choosing the parameters of the function being approximated in such a way that the sum of squares of the deviations of the empirical points from the value of this function is as small as possible.i2), the limiting value of relative errors (P95(re)) and the distribution of absolute errors  were explained under Equation (2).4PB stiffness moduli are greater than the calculated ones, while the opposite is true for the HMA 2 and HMA 6 mixes. The influence of the type of aggregate and, more specifically, the silica content (SiO2) in the aggregate, which affects the adhesion of the asphalt to the aggregate, can be seen here. For mixtures with more alkaline aggregates (HMA 7 and HMA 8), the determined stiffness moduli of E4PB are larger than calculated. Therefore, at the stage of further work on the predictive models, the introduction of an additional variable characterizing the silica content of the aggregate used to make the HMA can be considered in the models. In 4PB were obtained, and for low temperature, higher values of the stiffness modulus E4PB were obtained, confirming the generally known relationship and confirming the correctness of the developed model A. It can be noted, however, that for the analysed HMA, the values of stiffness moduli E4PB obtained at 10 °C are only slightly smaller than those obtained at 0 °C. In Evaluating the model according to the graphs  and Tabl4PB stiffness modulus, a normality analysis of the distribution of the model’s absolute errors was performed. The analysis of the normality of the distribution was performed by plotting histograms and performing multiple statistical tests for the normality of the distribution, with a significance level of α = 0.05. The D’Agostino–Pearson test was used as the binding test for the normality of the distribution due to the fact that the power of this test classifies at the medium level. Before analyzing normality, deviating absolute error values were rejected using the Hampel statistical test.To further confirm the validity of the developed model for calculating the Ep statistic > 0.05. According to all applied tests, the distribution of absolute errors can be considered a normal distribution. Almost all outliers, with few exceptions, are values calculated for results obtained at 0 °C.After applying the Hampel statistical test to eliminate outliers from the set of absolute errors for model A, 65 outliers were eliminated. The histogram  indicate4PB stiffness modulus determinations were carried out. The highest values of absolute errors are shown by determinations of the E4PB stiffness modulus made at 0 °C, while the lowest values of absolute errors are shown by tests carried out at the highest temperature of 40 °C. Such error distributions are consistent with the achieved E4PB stiffness modulus values. The largest relative error values are shown by the E4PB stiffness modulus determined at 40 °C and 30 °C, where the values are so small that even a small difference will result in a significant relative error. Medium relative error values were found in the results of tests carried out at 0 °C. The smallest relative error values were found at 10 °C and 20 °C. The relative error values at 10 °C are within approx. 30% and at 20 °C within approx. 20%, which can be considered satisfactory.According to the study and its analysis, further search for a better formulation of the model equation may not yield significant results. However, the introduction of an additional variable into the models to determine the silica content of the aggregate used to make the HMA or variables characterizing the shape of the aggregate grains could be considered. Further efforts to increase the accuracy of predictive models should focus on reducing the measurement uncertainty of the variables used in the models, which would be in line with the work described in .4PB) and the values obtained from the model (Predicted E4PB) is very high, the coefficient of determination being R2 = 0.936.It is also very important to assess predictive models in terms of relative errors. It can be concluded that in the literature on these models, information containing relative error analysis is very scarce. Among many publications analyzed, the authors found information on relative errors only in the article , where t4PB at 0 °C, confirming the results of the work of Bari and Witczak [Model A shows the greatest inaccuracy in calculating the stiffness modulus E Witczak . Similar4PB of HMA itself. A more precise procedure for determining the stiffness modulus E4PB would contribute to a reduction in uncertainty. The conditions for thermostating the specimens prior to the low-temperature determination of the stiffness modulus E4PB of HMA need to be defined more precisely in order to further reduce the possible influence of physical hardening. The algorithm for calculating the stiffness modulus E4PB of HMA also needs to be clearly defined.The authors are of the opinion that, in order to increase the accuracy of model A, a number of measures are needed to reduce the uncertainty of both the input variables of the model in question  and the stiffness modulus E4PB, it is also proposed to calculate the limiting values at the significance level α = 0.05 within which the result of the determination should fall, i.e., the uncertainty interval. These limits are proposed to be calculated using the P95(re) statistic. This would be done by subtracting and adding 50% of this value from the calculated value of the stiffness modulus E4PB of the HMA; in this case, it would be known in which interval with 95% probability the laboratory-determined value of the stiffness modulus E4PB should fall.Using model A, it is proposed that, in addition to calculating the value of the HMA stiffness modulus E


In January, 2023, the Science and Security Board of the Bulletin of the Atomic Scientists moved the hands of the Doomsday Clock forward to 90 s before midnight, reflecting the growing risk of nuclear war.Current nuclear arms control and non‐proliferation efforts are inadequate to protect the world's population against the threat of nuclear war by design, error, or miscalculation. The Treaty on the Non‐Proliferation of Nuclear Weapons (NPT) commits each of the 190 participating nations”to pursue negotiations in good faith on effective measures relating to cessation of the nuclear arms race at an early date and to nuclear disarmament, and on a treaty on general and complete disarmament under strict and effective international control”.Any use of nuclear weapons would be catastrophic for humanity. Even a “limited” nuclear war involving only 250 of the 13,000 nuclear weapons in the world could kill 120 million people outright and cause global climate disruption leading to a nuclear famine, putting 2 billion people at risk.http://www.ippnw.org).The health community has had a crucial role in efforts to reduce the risk of nuclear war and must continue to do so in the future.In 2007, the IPPNW launched the International Campaign to Abolish Nuclear Weapons, which grew into a global civil society campaign with hundreds of partner organisations. A pathway to nuclear abolition was created with the adoption of the Treaty on the Prohibition of Nuclear Weapons in 2017, for which the International Campaign to Abolish Nuclear Weapons was awarded the 2017 Nobel Peace Prize. International medical organisations, including the International Committee of the Red Cross, the IPPNW, the World Medical Association, the World Federation of Public Health Associations, and the International Council of Nurses, had key roles in the process leading up to the negotiations, and in the negotiations themselves, presenting the scientific evidence about the catastrophic health and environmental consequences of nuclear weapons and nuclear war. They continued this important collaboration during the First Meeting of the States Parties to the Treaty on the Prohibition of Nuclear Weapons, which currently has 92 signatories, including 68 member states.We now call on health professional associations to inform their members worldwide about the threat to human survival and to join with the IPPNW to support efforts to reduce the near‐term risks of nuclear war, including three immediate steps on the part of nuclear‐armed states and their allies: first, adopt a no first use policy;The danger is great and growing. The nuclear armed states must eliminate their nuclear arsenals before they eliminate us. The health community played a decisive part during the Cold War and more recently in the development of the Treaty on the Prohibition of Nuclear Weapons. We must take up this challenge again as an urgent priority, working with renewed energy to reduce the risks of nuclear war and to eliminate nuclear weapons.Respective authors were paid by their employers. Chris Zielinski's time was funded by International Physicians for the Prevention of Nuclear War.KB‐D is a full‐time employee of the American Medical Association, working as the Editor‐in‐Chief of JAMA and the JAMA Network. AH is principal investigator of the Pathfinder Initiative 2020–2025, co‐investigator of Sustainable Healthy Food Systems research programme 2017–2023, and co‐investigator of Complex Urban Systems for Sustainability and Health 2017–2023, all funded by the Wellcome Trust, with additional funding from the Oak Foundation for the Pathfinder Initiative, and he reports royalties from Cambridge University Press for the coauthored book Planetary Health; consultancy fees paid to his institution from the Wellcome Trust for his role as Senior Advisor on Climate Change and Health in 2021; travel/meeting support from WHO and Human Frontiers Science Program; and he is a member of the Cool Roofs trial steering committee Nouna Research Centre, Burkina Faso/University of Heidelberg, is Co‐chair of the International Advisory Committee, NIHR Clean‐Air (Africa) Global Health Research Unit, is a member of the Independent Advisory Group, Collaboration for the Establishment of an African Population Cohort Consortium, and he was Co‐chair of the InterAcademy Partnership, Climate Change and Health Working Group 2019–2022 and Co‐chair of the Academy of Medical Sciences/Royal Society working group on ‘A healthy future—tackling climate change mitigation and human health together’ 2020–2021 . IH reports honoraria for several speaking engagements, all donated to Back from the Brink, the International Physicians for the Prevention of Nuclear War, or Physicians for Social Responsibility; travel/ meeting support for Nobel Peace Laureates' Summit, the World Federation of Public Health Associations World Congress, and the UN Human Rights Commission Youth Summit; and he is a member of the steering committee of Back from the Brink and the International Steering Group of the International Campaign to Abolish Nuclear Weapons, a Board member of the International Physicians for the Prevention of Nuclear War and Physicians for Social Responsibility, and a Trustee of the Phillips Exeter Academy . MGMOR reports research grants from the Dutch Research Council, NOW (grant number COMPL.21COV.001) and from the Netherlands Organisation for Health Research ZonMw (grant number 09120012010063) and he is Chair of the Dutch guideline committee on cognitive impairments and dementia. TR reports a contract with the Institute for Energy and Environmental Research (USA) for papers addressing the health and environmental consequences of nuclear testing in multiple locations, including Australia, French Polynesia, central Pacific, and China; honorarium from The Choisun Ilbo media group in South Korea for a lecture on nuclear weapons in 2022 and for nuclear weapons presentations from Hyogo Medical Practitioners Association (Japan), Peace Boat (Japan), and the University of Sydney; he was an expert witness on radiation and health for Environmental Justice Australia acting for Mine‐Free Glenaladale regarding proposed Fingerboards Mineral Sands Mine to the Victorian Government Fingerboards Inquiry and Advisory Committee; he is a member of RV3 Rotavirus Vaccine Development Scientific Advisory Board, Murdoch Children's Research Institute/ Royal Children's Hospital; he is a member of the Committee of International Campaign to Abolish Nuclear Weapons Australia; he is a member of the Internet Peace Prize Award Committee ; he was a member of the Victorian International Humanitarian Law Advisory Committee, Australian Red Cross; he is a Board member of the Initiative for Peacebuilding, Faculty of Arts, University of Melbourne; he is an At‐large Board member of the International Physicians for the Prevention of Nuclear War; he was Co‐president of the International Physicians for the Prevention of Nuclear War 2012–23; and he is Honorary Principal Fellow, Melbourne School of Population and Global Health, University of Melbourne. PY reports grants from Atea Pharmaceuticals; honoraria for lectures, presentations, and educational events from bioMérieux and Pfizer Pharmaceuticals; fees for participation on an advisory board from Pfizer Pharmaceuticals; and he is a member of the Antimicrobial Stewardship Study Group Executive Committee (2022–2024) and the Clinical Practice Guideline Panel on Vaccinations in Immunocompromised hosts for the European Society of Clinical Microbiology and Infectious Diseases. CZ reports consulting fees for his role as senior adviser on the international journals project from the International Physicians for the Prevention of Nuclear War. All the other authors declare no competing interests.


Clostridium sp AF 23-8 correlating to inflammatory lesion counts, while Actinomyces naeslundii str Howell 279 correlated to non-inflammatory lesions. Overall, measures of stress and circulating hormones correlate to skin biophysical properties and acne lesion counts. Also, different gut bacteria correlate with either inflammatory or non-inflammatory lesion counts. We hope that our findings stimulate further work on the gut–mind–stress–skin axis within acne.Acne vulgaris is a common inflammatory condition that is multi-factorial and impacted by both intrinsic and extrinsic features. Several previous studies have assessed for correlations between factors such as circulating hormones, stress, or the microbiome. However, there have not been any correlations specifically against lesion counts or differentiating correlations between inflammatory and non-inflammatory lesion counts. Here, we correlate several factors against acne lesions. Twenty men and women with mild to moderate acne were recruited, and their hormonal levels and their gut microbiome were collected and correlated against their inflammatory and non-inflammatory lesions of acne. Facial non-inflammatory lesions were weakly correlated to sebum excretion rate and weakly inversely correlated to forehead and cheek hydration. We examined stress through the use of a normalized peak-to-trough ratio (higher numbers indicated less stress), which correlated with skin hydration and inversely correlated with sebum excretion rate. Sebum excretion rate was weakly correlated to testosterone levels, and facial hydration correlated with estradiol levels. Correlations with the gut microbiome showed differential correlations with inflammatory and non-inflammatory lesions, with  Cutibacterium acnes (C. acnes), and inflammation [Acne vulgaris affects over 85% of adolescents and young adults and has a major impact on both physical and mental health . Acne paammation . Typicalammation ,4.Recent evidence shows that systemic and distant factors may have an influence on acne as well. Some examples include noted associations with circulating androgens ,6,7, shiThe gut microbiome appears to have an influence on acne as well. For example, shifts in the gut microbiome have also been associated with either the presence or absence of acne ,22. In fWhile these previous studies have looked at correlations in relation to either the presence or absence of acne, there have not been any specific correlative studies that have correlated specifically against inflammatory or non-inflammatory lesion counts or against the skin’s biophysical features. Here, we explore the association between circulating hormones and the gut microbiome to acne and correlate it specifically to the presence of either non-inflammatory or inflammatory lesions of acne to better understand the correlations to acne.This study was conducted between March 2022 and December 2022 as an 8-week open-label study. Institutional Review Board approval was received on 5 March 2022, by Allendale, and the study was listed on clinicaltrials.gov (NCT05271487). Written informed consent was received from all participants prior to enrollment. Subjects from the greater Sacramento region were recruited. Inclusion criteria included males and females between the ages of 12 to 45 years old, with mild-to-moderate acne classified by an investigator global assessment (IGA) of 2 or 3, along with the presence of at least 10 inflammatory lesions and at least 15 total acne lesions. Subjects who had more than two nodules were excluded. Those with severe acne (IGA ≥ 4), women who were pregnant or breastfeeding, those who were current smokers or had a smoking history of >10 pack years, those unwilling to discontinue facial products except for what is provided in the study, and those who changed their hormonal based contraception within 3 months prior to enrollment were excluded from the study. Those who had isotretinoin use within the 3 months prior to joining the study and those who were unable to discontinue oral antibiotics, probiotics, topical antibiotics, and topical benzoyl peroxide use were also excluded from the study.Written consent and assent were obtained prior to enrollment. Subjects were asked to undergo a 2-week washout from topical antibiotics or benzoyl peroxide use, or a 4-week washout for oral probiotic supplements or oral antibiotic use. The main study reported previously consisted of a total of 4 visits  . This ma® , Vapometer® (Delfin Technologies) and the Sebumeter® , respectively.Facial and truncal lesion counts for inflammatory and non-inflammatory lesions were performed by a trained doctor or board-certified dermatologist. During these visits, biophysical features such as stratum corneum hydration, trans-epidermal water loss, and sebum excretion rate were obtained using the MoisturemeterSCCirculating hormones were assessed with kits supplied by ZRT Laboratories ; measurements included a four-point salivary cortisol test, testosterone, progesterone, estradiol, and dehydroepiandrosterone sulfate (DHEA-S). A normalized peak-to-trough difference in the cortisol was utilized to measure cortisol variability, where the absolute peak-to-trough difference in the cortisol levels was normalized against the peak cortisol level.Stool samples were collected with sample collection kits, and the stool was stored at −80 °C until they were shipped for analysis on dry ice. Taxonomic and functional analyses of the WGS stool samples were performed by CosmosID.DNA from fecal samples was isolated using the QIAGEN DNeasy PowerSoil Pro Kit, according to the manufacturer’s protocol. Extracted DNA samples were quantified using Qubit 4 fluorometer and Qubit™ dsDNA HS Assay Kit . Isolates were stored at −80 °C until they were sequenced.DNA libraries were prepared using the Nextera XT DNA Library Preparation Kit  and IDT Unique Dual Indexes with total DNA input of 1 ng. Genomic DNA was fragmented using a proportional amount of Illumina Nextera XT fragmentation enzyme. Unique dual indexes were added to each sample, followed by 12 cycles of PCR to construct libraries. DNA libraries were purified using AMpure magnetic Beads  and eluted in QIAGEN EB buffer. DNA libraries were quantified using Qubit 4 fluorometer and Qubit™ dsDNA HS Assay Kit . Libraries were then sequenced on an Illumina HiSeq 4000 platform 2 × 150 bp with a read depth of 3 million reads per sample.Unassembled sequencing reads were directly analyzed by CosmosID-HUB Micro-biome Platform  as described elsewhere for multi-kingdom microbiome analysis, the profiling of antibiotic resistance and virulence genes, and quantification of organisms’ relative abundance ,27,28,29p-values. The correlations were performed in Python and used a set of nested loops with the actual Pearson Correlation and p-values calculated using SciPy.Stats and the pearsonr function. . Because of the pilot nature of this study, we stratified the p-values to seek trends in addition to statistical significance. A p-value of less than 0.3 was determined to be a weak correlation, a p-value less than 0.2 was considered a moderate correlation, and a p-value less than 0.1 was considered to be a strong correlation.The statistical analysis was performed using Pearson Correlations and associated Twenty-four participants were screened, and twenty participants completed all baseline visits and lab collection procedures. Three participants did not complete the lab collection procedures per protocol, and a total of seventeen were analyzed. The majority of participants were female, with a mean age of 24.4 ± 7.3 years. The mean acne severity at enrollment was graded to be an IGA of 2.7.p-value: 0.28), inversely weakly correlated to forehead hydration , and inversely weakly correlated to cheek hydration .Facial non-inflammatory lesions were weakly correlated to the cheek sebum excretion rate , inversely correlated to the progesterone/testosterone ratio , and inversely weakly correlated to the normalized peak-to-trough cortisol difference .The facial sebum excretion rate was weakly correlated to the circulating levels of testosterone  and with the normalized peak-to-trough cortisol difference .The facial hydration was correlated with circulating levels of estradiol  and Intestinibacter bartlettii , while the bacterium that was most inversely correlated was Adlercreutzia equolifaciens , which is an equol (isoflavone)-producing bacterium [Multiple gut bacteria either inversely or directly correlated with non-inflammatory lesions . Two patacterium .Clostridium , Butyrivibrio crossotus , Pseudoruminococcus massiliensis , Sutterella faecalis , Megamonas rupellensis , Akkermansia muciniphila , and Methanobrevibacter smithii .In regard to the inflammatory lesions of acne, several bacteria were correlated with the presence of acne  such as Actinomyces naeslundii str Howell 279, Bifidobacterium dentium, Intestinibacter bartlettii DSM 16795, and Eubacterium sp AM28-29. The following strains were inversely correlated to non-inflammatory lesions  but had a stronger correlation with the presence of inflammatory lesions . Furthermore, we noted an association for the presence of inflammatory lesions with Methanobrevibacter smithii, which is a known pathogen associated with gut dysbiosis such as Small Intestinal Bacterial Overgrowth (SIBO) and Intestinal Methanogen Overgrowth (IMO) [Our results expand on the correlations of the gut microbiome to the presence of acne by specifying the correlations between specific species and strains against specific acne lesion types. One example is the ith acne . Anotherchildren . Anotheremphigus , but ourove acne , and it ic genus , althougith acne , which ath (IMO) . While Sth (IMO) ,49, our Akkermansia mucihiphila and inflammatory lesions. Although A. muciniphila presence has typically been associated with decreased inflammation [A. muciniphila as well [A. muciniphila is associated with inflammatory lesions as a causative effect or as a reactionary mechanism by the gut microbiota to reduce inflammation.A notable finding is that we identified an inverse correlation between the gut presence of ammation ,51, seveProteobacteria/Actinoobacteria ratio and a decrease in the Lactobacillus, Bifidobacterium, and Butyirococcus genera are associated with the presence of acne [Clostridia, Clostridiales, Lachnospiraceae, and Ruminococcaceae genera in those with acne [Clostridum species positively correlated with inflammatory lesions and, in particular, the strain of Clostridium Clostridium sp AF 23-8 was correlated to the presence of inflammatory lesions. The reason for the differential results is not clear, but may reflect that there could be a strain-to-strain level variation that may not be captured at the species level. Further studies will help elucidate the contributions of gut bacterial species and strains.As the evidence for the role of nutrition in acne continues to grow, there has been increased interest in understanding how the gut microbiome correlates with the presence of acne. Previous gut microbiome analyses have indicated that an increase in the  of acne . Anotherith acne . In bothOur findings suggest that multiple factors may correlate with shifts in the skin’s biophysical properties and with acne lesion counts. In particular, the normalized peak-to-trough cortisol ratio may correlate with facial hydration and inversely correlate with sebum excretion rate. Finally, the gut microbiome correlated with acne with differential correlations between different species with inflammatory and non-inflammatory lesions.


This study sought to evaluate starch from black and red rice modified by heat–moisture, investigating the extraction yield, starch and amylose content, color, and phenolic compounds. The water and oil absorption capacity, whole milk and zero lactose absorption index, syneresis index, and texture were also analyzed. Microstructural analysis included Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy. The heat–moisture treatment (HMT) reduced the extraction yield and the starch and amylose content, with native black rice starch having the highest values for these parameters. The modification also affected the color and phenolic compounds of the starch, making it darker and changing its appearance. The modification improved the absorption of water, oil, and milk, reducing syneresis and increasing stability during storage. The starch surface was altered, especially for modified black rice starch, with larger agglomerates. The type of starch also changed from A to Vh, with lower relative crystallinity. The textural properties of modified red rice starch were also significantly altered. The HMT proved to be a viable and economical option to modify the analyzed parameters, influencing the texture and physicochemical properties of pigmented rice starch, expanding its applications, and improving its stability during storage at temperatures above 100 °C. The National Supply Company (CONAB)  predicts that Brazil’s rice harvest for the 2022/23 period will be 7.2% smaller than the previous harvest, estimated at 10 million tons [The grain of rice is constituted mainly by starch, which can reach up to 90% of the grain. In this way, rice becomes an option in the industry for the production of various products that have starch as an important constituent . Starch Generally, starch chain structures can be altered through biological, thermal, non-thermal, hydrothermal, and chemical methods to achieve the desired technological, functional, and nutritional properties . PhysicaBlack rice stands out among pigmented rice varieties because it has good sensory characteristics and high nutritional value. It has higher content of amino acids, organic acids, fatty acid methyl esters, and free fatty acids, and, being gluten-free, it becomes an excellent raw material for the development of products for patients with celiac disease or gluten sensitivity. Some of the polyphenols found in black rice grains include anthocyanins, proanthocyanidins, ferulic acid, ellagic acid, and quercetin .Ito & Lacerda,  found thHeat–moisture treatment (HMT) promotes physicochemical modifications in the granule without changing its molecular composition, where it is carried out at a temperature above the gelatinization temperature, with an insufficient amount of water content to gelatinize the biopolymer . HMT canThe most recent applications of HMT-modified starches are ensuring the quality of 3D-printed wheat starch gels , the con®, Timbaúba, Brazil) were purchased from local companies in the city of Campina Grande, Paraíba, where they were taken to the Drying Laboratory (UAEALI) of the Federal University of Campina Grande (UFCG) for the removal of impurities and manual selection. Until the rice starch extraction, the samples were stored at room temperature (25 ± 2 °C).Grains of black and red rice , sunflower oil , ethyl alcohol, 3,5-dinitro salicylic acid , sodium acetate , acetic acid , resublimated iodine , soluble starch , pectin , sodium metabisulfite and dimethylsulfoxide—DMSO , and potassium iodide .To carry out this research, the following materials were acquired: lactic acid , whole milk and zero lactose (Betâniaw/v) for 72 h at a temperature of 7 °C. Subsequently, it was rinsed in running water for 3 min, thus proceeding to the crushing stage, which occurred by adding distilled water in the proportion of 1:2 (w/v) with the aid of an industrial blender  over 5 min. After this process, filtering through an organza mesh (0.3 mm) was performed to obtain the starch suspensions. Again, the residues were ground and filtered four times to increase the extraction yield. From this point on, the starch suspension was passed through a 32-mesh sieve and was decanted five times every 12 h in a domestic refrigerator  at 7 °C to avoid the occurrence of enzymatic or fermentative action during the sedimentation process. Vacuum filtration was used to remove the remaining water and thus optimize the process.To perform starch extraction, the method initially described by Bento et al.  and late−1, followed by grinding with a knife mill  [The final water content of 20% was achieved by adding the black and red rice starches (3 g) to distilled water in conical flasks. After this, these flasks were sealed and heated at a temperature of 110 °C for 3 h (HMT3) with the aid of an electric oven  with dimensions of 25 × 41.5 × 32.2 cm . Subsequ−1) of α-amylase diluted in 0.016 M sodium acetate buffer pH 6.0 was added. For 30 min, the samples were incubated at 75 °C and, after this, they were cooled to 50 °C and then 1.0 mL of a solution comprising 70.0 U g−1 of amyloglucosidase in 100 mM (pH 4.5) sodium acetate buffer was added. After this, the samples were incubated for 30 min at 50 °C and centrifuged . Afterward, the starch was measured by quantifying the glucose released in the process according to the methodology proposed by Miller [Starch determination was performed using a methodology adapted from Amaral et al. . First, y Miller .−1 of starch on a dry basis.In agreement with the descriptions by Nadiha et al.  and McGrThe CIELAB color scale CIELAB  was chosen for the measurement of starch color. The registration of the color values was based on the average of the values of five photographs, where the parameters included L* = brightness , a* , and b * .The starch color was determined by measuring the coordinates L* (lightness), a* (redness), and b* (yellowness), using a digital colorimeter (X). For the reading, the following conditions were set: illuminant C, viewing angle 8°, standard observer angle 10°, specular included, according to the specifications of the Commission Internationale de L’éclairage . Before Y is equivalent to the extraction yield, dbW to the weight of starch in the grains on a dry basis (g), and tW to the weight of the grains (g).The extraction yield was established from the ratio between the starch weight and the seed weight, both expressed on a dry basis using Equation (1):−1 for 17 min. As for the composition of the mobile phase, it consisted of 1% acetic acid (v/v), ethanol (1.5:1) (phase A), and HPLC-grade acetonitrile (phase B). A volume of 20 μL of the samples was applied to the system, and the run occurred using the following gradients: from 100% to 70% of phase A (0–5 min), followed by 70% to 30% of phase A (5–10 min) and 30% to 0% of Phase A (10–12 min) [−1). Next, identification was carried out under the same treatment conditions as the respective standards, and the quantification of the phenolic compounds was based on the retention time  and peak area (A).Phenolic compounds were obtained by using 0.1 g of starch diluted in 5 mL of acetic acid and ethanol solution (1.5:1). There was filtration through a 0.22 µm membrane followed by HPLC analysis using the SPD-10AV-VP . Separation was performed on a Shim-Pack CLC-ODS C18 column  (4.6 mm × 15 cm) at a temperature of 25 °C, with a mobile phase draining at a flow rate of 0.6 mL min–12 min) . Finallyg. Absorption was calculated according to Equation (5):CAA represents the water absorption capacity expressed in (g 100 g−1), Ms is the initial mass of starch on a dry basis (g), Mw is the mass of water (g), and Ew is the mass of excess water (g).The water absorption capacity was determined from the method Beuchat, , in whicg for 15 min. After this process, excess oil was removed and weighed. The calculation of the amount of oil retained per gram of sample was performed according to Equation (6) [A combination of 1 g of rice starch and 10 mL of sunflower oil was created in centrifuge tubes. Samples were kept at 24 °C, manually shaken for 5 min, and then centrifuged  at 15,000× tion (6) :(6)CAO=(CAO corresponds to the absorption capacity of oil expressed in (g. 100 g−1), Ms is the initial mass of starch on a dry basis (g), Mo is the mass of oil (g), and Eo is equivalent to the mass of the oil excess (g).Here, g for 15 min . Soon after centrifugation, the supernatant was transferred to a Petri dish of known mass, followed by the weighing process. The milk absorption index, which corresponds to the mass of the gel obtained after removing the supernatant, was acquired by Equation (7) [IAL is the milk absorption index (g. 100 g−1), Ms corresponds to the mass of starch on a dry basis (g), Mrg to the mass of the centrifugation residue (g), and Msu to the supernatant mass (g).Starch (2.5 g) was added to 30 mL of milk at a temperature of 25 ± 2 °C for 30 min and then centrifuged at 3000× tion (7) :(7)IAL=(w/v) at a temperature of 80 °C for 12 min, controlling the temperature with a thermostatic bath . The samples spent 72 h stored at 7 °C and, subsequently, there was 15× g centrifugation  at 3000× g for 10 min. The analysis was performed in triplicate and the syneresis index was calculated according to Equation (8).fm corresponds to the mass of water separated from the gel after centrifugation (g) and im is the initial mass of the gel (g).The determination of the syneresis index occurred according to the methodology proposed by Farnsworth et al. . Starch −1, the Cary 630 equipment  was used, with a resolution of 4.0 cm−1 and 32 scans [−1, 1022 cm−1, and 995 cm−1 was performed by recording the absorbance height to characterize the molecular order [To obtain FT-IR spectra in the region of 650–4500 cm32 scans . To decoar order .IC is the crystallinity index, cA is the crystalline area below the peaks, and tA is the total area.The process of determining the X-ray crystallographic patterns of the samples was carried out with the aid of an X-ray diffractometer  equipped with Cu-Ka radiation (λ = 1.5406 Å) at 80 mA and 40 kV. A scan was performed at a 0.02° step, with a counting time of 2 s from 10° to 40° at room temperature. Intending to convolve the diffractogram and reveal the amorphous area, the Gaussian response function was used. Soon after, the crystallinity of the samples was established by the area method using Equation (9) :(9)IC(%)http://rsbweb.nih.gov/ij/ (accessed on 7 September 2023).The samples were analyzed before and after hydrothermal modification by scanning electron micrographs . Then, they were evenly dispersed on the sample table, to which a conductive double-adhesive carbon tape was attached and covered with a thin layer of gold. Then, 1000× magnification was used with an acceleration potential of 10 kV and the measurement of the granule size was performed with the help of the Image J 1.0 software The preparation of red rice starch pastes occurred with the proportion of (1:10) starch–water that was heated at a temperature of 80 ± 2 °C until pastes were formed in approximately 30 min. The samples were stored at a temperature of 8 °C in a refrigerator  for 24 h; they were then placed at room temperature 25 ± 2 °C) and examined using the TPA method, which associates textural parameters with sensory analysis  ± 2 °C a.p < 0.05), with data processing using Statistica® v.7 .The data were expressed as mean ± standard error (M ± SD) calculated in triplicate for each analysis, where they were subjected to ANOVA using the Tukey test  and modified red rice starch (AVHMT) had lower yields . The starch extraction yield was affected by HMT, as, in the steam processes, the high moisture content and high condensation temperature of the steam on the starch surface resulted in partial gelatinization, making extraction more difficult [Regarding the starch extraction yield , it was ifficult . Almeidaifficult  observedifficult  found a For both starches, a significant reduction in starch content was noted after HMT. HMT can also cause the breaking of glycosidic bonds in starch, leading to the fragmentation of starch molecules into smaller parts. This can contribute to a reduction in starch content .According to Ramli et al.  and KumaThese results highlight the predominance of amylopectin in the black and red rice starches used in this research, as the relatively high proportion of amylopectin in relation to amylose can influence the functional properties of these starches, such as the gelatinization capacity, texture stability, and thickening ,45.HMT samples, indicating the independence of shades in the appearance of starches. The color analysis results revealed significant differences between black (AP) and red (AV) rice starch formulations. Regarding brightness (L*), AP starch obtained an average value of 62.71, indicating a darker hue, while AV starch had an average value of 71.16, indicating a lighter hue. As for the chroma a*, AP starch obtained an average value of 4.92, while AV starch obtained an average value of −0.78, indicating a redder hue for AP starch. On the other hand, chroma b* showed mean values of 6.52 for AP starch and 10.16 for AV starch, indicating differences in the yellow/blue hue between the formulations.We then considered the color parameters (L (brightness), a* (+a) red, (−a) green, b* (+b) yellow, and (−b) blue) among the starch formulations. Chroma a* showed a difference and chroma b* showed the same behavior under background conditions for AP and APThe luminosity of the products showed a difference between their values that may have resulted from the coloring of the starches. In These results confirm the differences in the color properties of black and red rice starches, highlighting their potential applications in various industries that require specific color characteristics in their products. These differences can be attributed to the chemical composition and intrinsic characteristics of the black and red rice grains, as well as the processing conditions used to obtain the starches. According to Elias et al. , the proNative and HMT-modified rice varieties showed differences in all phenolic compounds found: gallic acid, proanthocyanidins, and quercetin . It was −1 and 0.91 mg of gallic acid L−1, respectively. On the other hand, HMT-modified starches (APHMT and AVHMT) showed lower values, with 1.52 mg of gallic acid L−1 and 0.63 mg of gallic acid L−1, respectively. These results indicate that HMT processing resulted in a significant reduction in the amount of gallic acid in the starches. The values of gallic acid obtained in this study are in agreement with previous studies that also reported similar levels of this compound in rice. Mira et al. [−1. According to Tyagi et al. [Regarding gallic acid, the native starches of black (AP) and red (AV) rice showed higher values, with 2.49 mg of gallic acid La et al.  obtainedi et al. , black aHMT) showed lower values, with 0.71 mg of catechin L−1 and 0.48 mg of catechin L−1, respectively. Red rice starches (AV and AVHMT) showed higher values, with 1.37 mg of catechin L−1 and 1.02 mg of catechin L−1, respectively. These results demonstrate significant differences in proanthocyanidins between different types of starch, indicating that HMT processing can affect the presence of this compound. The proanthocyanidin values became consistent when comparing both starches (black and red). According to Mackon et al. [In the case of proanthocyanidins, black rice starches  showed lower values, with 2.58 mg of quercetin L−1 and 1.20 mg of quercetin L−1, respectively. These results show that HMT processing also resulted in a reduction in the amount of quercetin in the starches.In the case of quercetin, the native starches of black (AP) and red (AV) rice showed higher values, with 2.96 mg of quercetin LThese data indicate that modification by HMT affected the presence of phenolic compounds in black and red rice starches. The reduction in the levels of gallic acid, proanthocyanidins, and quercetin in modified starches may have nutritional and functional implications since these compounds have bioactive properties. It is important to consider these changes when using black and red rice starches in food and industrial applications, taking into account the properties of phenolic compounds and their potential effects. Furthermore, the reduction in the amount of quercetin in starches modified by HMT is in agreement with studies that show a decrease in this compound in vegetables subjected to temperature increase processes, according to the research by Harnly et al. , who cla−1) (p < 0.05), and there was also an increase compared to the native starches, with APHTM showing a 9.87 g 100 g−1 increase. The application of starch modification techniques, such as HMT, can interfere with characteristics related to the solubility index and functional properties involving water absorption, gelation, and pasting [Among the native starches, red rice starch showed the highest water absorption capacity (70.01 g 100 g−1) . This wa pasting .HMT) has the highest oil absorption (72.12 g 100 g−1) among the formulations. Native red rice starch (AV) shows the lowest oil absorption (58.52 g 100 g−1), while HMT-modified black rice starch (AVHMT) and HMT-modified red rice starch (AVHMT) have intermediate values of 65.96 and 65.96 g 100 g−1, respectively. Almeida et al. [HMT-modified black rice starch  has the highest absorption from lactose-free milk (74.24 g 100 g−1), followed by modified black rice starch (APHMT) (73.58 g 100 g−1). The opposite is verified for the absorption of whole milk, where APHMT presents the highest value (73.93 g 100 g−1). When evaluating enzymatically modified native red rice starch, Almeida et al. [−1 and a whole milk absorption value of 50 g 100 g−1.Modified red rice starch , possibly due to the presence of a higher amount of amylose in its composition compared to modified red rice (AVHMT).Regarding the morphological part of the AP A and AV The starch present in rice can be spongy and have undefined shapes. It is also possible to observe that the black and red rice starch granules present regular formation of their shapes . ZieglerHMT, and AVHMT showed larger mean diameters . These results suggest that the HMT modification process resulted in larger starch particles. According to Shi et al. [Regarding the average diameter  of the si et al. , the avei et al. . The difHTM, which is of the Vh type, due to the flattening of the peaks at 17° and 18° and the presence of the peak at 19.37°. According to Fonseca et al. [It is evident that the XRD profiles of the rice starch samples exhibit type A diagrams, commonly found in cereal starches , except a et al.  diffracta et al.  indicateHMT and AVHMT show greater differences in peak intensity within the range of 20°, with APHMT representing a greater absorption spectrum compared to the other samples.It is possible to observe that the intensity between the bands present in the diffractograms in HMT having greater dispersibility of these granules. Relating the relative crystallinity (RC) data  data , it is oHMT, and AVHMT presented lower relative crystallinity values , suggesting that the HMT modification process may result in a decrease in starch crystallinity.Regarding the relative crystallinity, it is observed that the AP and AV samples presented higher values , indicating a greater proportion of crystalline regions in the starch. On the other hand, the samples modified by HMT, AP−1, related to the stretching of O–H groups, bands referring to the region of 2927 cm−1 that involve C–H, and those at 1643 cm−1 and 1327 cm−1 that may be associated with water bound in starch and the vibrations of CH2 groups, respectively , the starches modified by HMT in black and red rice did not differ statistically, regarding AP and AV, demonstrating that the functional groups and the chemical bonds that constitute the modified rice starch remained constant. However, when relating wavelengths at 995/1022 cm−1, both native and modified starches differed from each other (−1) values, differences between the samples could be noticed. For IR (1022/1047 cm−1), AP and AV samples showed intermediate values of 1.39 and 1.45 cm−1, respectively, while APHMT and AVHMT showed slightly lower values of 1.34 and 1.33, respectively. As for IR (995/1022 cm−1), the APHMT (1.08 cm−1) and AVHMT (1.28 cm−1) samples showed lower values, while AP and AV showed intermediate values of 1.39 and 1.45 cm−1, respectively. These results indicate that the modification process by HMT can lead to changes in the molecular structure of starch.Regarding the values obtained for IR , while the modified AVHMT starch had the lowest firmness (0.34 N). This indicates that the modification process can lead to a softer and less firm texture.It can be seen that there were no significant differences in adhesiveness for the AP and APHMT formulation shows a lower rate of syneresis.The analysis of texture parameters in native and HMT-modified starches is essential to understanding how structural changes can influence the physical characteristics of these polymers. Modification by HMT can change properties such as adhesiveness, firmness, gumminess, and cohesiveness, affecting the behavior of starch in various applications, including food and materials . These rHMT starch had the lowest gumminess (0.22 N). According to Silva et al. [Gumminess, which is related to the sensation of chewing and the release of flavor during consumption, also showed a decrease in values as the modification by HMT occurred. The native AP starch had the highest gumminess (0.42 N), while the modified AVa et al. , the gela et al. .HMT starch had the lowest cohesiveness (0.52 N). The values demonstrated in this study for the texture parameters indicate that black and red rice starch form harder gels from the long chains of amylopectin, as stated by Ramos et al. [Cohesiveness, which is related to the ability of starch to hold together during mastication, also shows a tendency to decrease in values with modification by HMT. The native AP starch had the highest cohesiveness 0.74 N), while the modified AVs et al. . Cohesivs et al. . High co N, whileHMT starch had the lowest adhesiveness (0.35 ± 0.01 N.m). High adhesiveness can bring an undesirable rubbery mouthfeel [Adhesiveness, which is related to the ability of starch to adhere to teeth and oral tissues, shows a decrease in values with modification by HMT. The native AP starch had the highest adhesiveness (0.49 ± 0.02 N.m), while the modified AVouthfeel .−1 for the first two parameters, respectively. As for the amylose content, native red rice starch (AV) stood out (15.46 g 100 g−1).Native and modified black and red rice starches showed different characteristics when compared according to the studied parameters. The hydrothermal modification by heat–moisture (HMT) decreased the extraction yield and the starch and amylose content, where the native black rice starch (AP) presented the highest values of 52.06% and 82.06 g 100 gThe HMT also influenced the results of the color and phenolic compounds of the starch, with a darker appearance due to the temperature to which the starch is subjected during processing, with a decrease in coordinate (L) and an increase in (a* and b*). The AP showed the highest value for gallic acid and quercetin and the AV for proanthocyanidins. Overall, the modification decreased syneresis, thus showing the efficiency in modifying the pigmented rice starch structure and greater stability during storage.HMT, where most granules were gelatinized, and the average diameter was greater. Likewise, the type of starch was modified from type A to type Vh, with lower relative crystallinity (15.08%) and lower intensity of peaks in the FT-IR. Checking the textural parameters, the modified red rice starch was significantly altered by the hydrothermal treatment, showing lower values of firmness, gumminess, cohesiveness, and adhesiveness. In general, HMT proved to be a viable and low-cost hydrothermal treatment to modify the analyzed parameters due to changes in the amylose and amylopectin chains, which influence the texture and physicochemical properties of pigmented rice starch. This modification aims to expand the range of applications of rice starch and present greater stability during storage when subjected to temperatures above 100 °C.The surface appearance was significantly altered with larger agglomerates, being more visible in the AP


The Stanford Integrated Psychosocial Assessment for Transplantation (SIPAT) is a comprehensive psychosocial assessment proven useful for predicting the outcomes of organ transplantation that is expected to be useful in Japan. However, the characteristics of organ-specific SIPAT scores for organ transplant recipient candidates in Japan are unclear and, to date, the SIPAT has not been properly utilized in clinical practice. The purpose of this study was to present basic data that can be used to establish the relation between SIPAT scores and post-transplantation psychosocial outcomes as well as organ-specific outcomes.This study included 167 transplant recipient candidates  who completed a semi-structured interview based on the Japanese version of SIPAT (SIPAT-J) prior to transplantation. The differences between organs in terms of SIPAT scores and differences in SIPAT scores based on demographic data were comparatively analyzed.The total SIPAT scores were higher for liver recipient candidates than for heart recipient candidates (P = .019). Regarding the subscales, SIPAT B  scores were higher for liver and kidney recipient candidates than for heart recipient candidates (P = .021), whereas SIPAT C  scores were higher for liver recipient candidates than for kidney recipient candidates (P = .002). Recipient candidates with a history of psychiatric treatment and those who were unemployed had higher SIPAT scores, regardless of the transplant organ, than recipient candidates without a history of psychiatric treatment and those who were employed .There were notable differences in the total SIPAT-J and subscale scores among the liver, heart, and kidney recipient candidates. Each organ was associated with specific psychosocial issues that should be addressed before transplantation. Interventions such as information provision and patient education based on SIPAT assessment results for each organ may improve recipient post-transplant outcomes. Although organ transplantation improves the prognosis of patients with end-stage organ failure, post-transplant physical and psychological outcomes are influenced by the recipient’s psychosocial factors prior to transplant surgery –5. ThereOne tool for the psychosocial assessment of recipients of both solid organ and hematopoietic cell transplants is the Psychosocial Assessment of Candidates for Transplant (PACT) , 8. Low Maldonado et al.  developeTransplantation care in Japan is unique compared with that in other countries. In Japan, there are very few organ donations from patients after brain and cardiac death, and the waiting period for recipients is long . AdditioThis exploratory research aimed to present the SIPAT scores of organ transplant recipient candidates in Japan and to examine the differences in the scores among different organs. Additionally, by comparing the SIPAT scores using organ-specific demographic data, we aimed to determine the characteristics of SIPAT scores for each organ. Thus, this study presents basic data that could be used to establish the relation between SIPAT scores and post-transplantation psychosocial outcomes as well as organ-specific outcomes.Candidates for organ transplant at the Tokyo Women’s Medical University Hospital were eligible for participation. All candidates underwent a psychosocial assessment interview before transplant surgery. Consecutive heart (26), liver (72), and kidney transplant (83) recipients who underwent pre-transplant psychosocial assessment from September 2018 to December 2021 were eligible. Of the 181 candidates, consent to participate in the study was not obtained from nine, one did not speak Japanese, and four under 20 years of age were unable to obtain consent from a surrogate; thus, they were excluded. There were no candidates judged ineligible for transplantation due to poor psychosocial factors during study period. However, we cannot rule out the possibility that some cases were deemed ineligible by the transplant team before they were referred to our pre-transplant interviews.The SIPAT has demonstrated excellent inter-rater reliability (Pearson’s correlation coefficient = 0.85) and predictive ability for outcomes in previous studies . The relFor all organs, candidates were deemed eligible for transplantation, informed consent was obtained, a psychiatric interview was requested, and the SIPAT was conducted. The evaluators, including one psychiatrist and three clinical psychologists, involved in the transplant care of the patients, independently and blindly applied SIPAT-J to the medical records of anonymized transplant recipient candidates. The evaluators were provided anonymized records, including the results of interviews with psychiatrists, clinical psychologists, and transplant coordinators.The level of significance for the statistical analysis was set at P < .05 (two-sided). Differences in total SIPAT scores, SIPAT sub-scores, and SIPAT scores for specific organs were compared using the Kruskal–Wallis test. Multiple comparisons were made between groups on measures for which significant differences were found using the Dunn–Bonferroni method. Kruskal–Wallis and Mann–Whitney U tests were used for comparisons of SIPAT scores by demographic data. Nonparametric tests were used in this study. The calculation of the sample size was based on the calculations in the ANOVA. The total sample size was 159, calculated with a number of groups of 3, significance level of 0.05, power of 80%, and effect size of 0.25. Statistical analyses were performed using the IBM SPSS Statistics version 28 .The sample consisted of 167 transplant recipient candidates . The demographic information of the eligible participants is shown in Table Table Table Table The purpose of this study was to present basic data that can be used to establish the relation between SIPAT scores and post-transplantation psychosocial outcomes as well as organ-specific outcomes. To achieve this, we determined the distribution of SIPAT scores among organ transplant recipient candidates, examined the differences in scores by organ, and compared the SIPAT scores for each organ according to demographic data. We found that the liver recipient candidates had higher scores than heart recipient candidates. Regarding subscale scores, the liver and kidney recipient candidates had higher scores than heart recipient candidates on SIPAT B . The liver recipient candidates had higher scores than kidney recipient candidates on SIPAT C . Additionally, recipient candidates with a history of psychiatric treatment and those who were not employed had very poor overall SIPAT scores. However, there were no differences in SIPAT scores by age, sex, or educational background.Previous studies on SIPAT presented data from several countries including the United States, Spain, Italy, and Thailand. However, many previous studies described only the total SIPAT scores or score distributions , 21, 26 A comparison of SIPAT scores based on demographic data showed that scores of recipient candidates with a history of psychiatric treatment for all organs were higher than the scores of recipient candidates with no history of psychiatric treatment. In a study of kidney transplant recipients in the United States, men had higher scores than women, those with renal impairment secondary to hypertension had higher scores than those with renal impairment because of other causes, and those with low education levels had higher scores than those with high school education . In thisThe SIPAT subscales are classified into four domains . As withThis study has several limitations. First, this was a single-center study. Although the facility performs a large number of organ transplantations, there are many other transplantation facilities in Japan. There may be differences in patient education among the facilities, and these differences may affect the SIPAT scores. Furthermore, our analysis did not consider differences by facility, including regional differences. Second, our analysis only included heart, liver, and kidney transplant recipient candidates; therefore, the results may not be applicable to other organs. Considering the application of SIPAT in recipient candidates for a wide range of organs, it is necessary to clarify the characteristics of SIPAT for organs that were not included in this study. In addition, many heart recipient candidates in Japan are required to wait for long periods, during which time they may undergo implantation of ventricular assist devices , 32. TheOur study presented the SIPAT scores of organ transplant recipient candidates in Japan and evaluated the differences in the scores by each organ. Once it is clear that SIPAT-J predicts post-transplant outcomes, the early evaluation of recipient candidates followed by timely interventions can improve post-transplant outcomes. Larger datasets are needed to clarify the association between SIPAT and post-transplant outcomes. Finally, although previous studies have used a four-point scale based on the total SIPAT score, it is not clear whether this evaluation method is available in Japan. Comparisons with other countries showed different mean scores and distributions; therefore, the cut-off scores of SIPAT-J need to be confirmed for the Japanese population.This study is the first to present the SIPAT-J scores, which will provide important basic data for future SIPAT studies. The results showed differences in the SIPAT-J total and subscale scores among the transplant organs, indicating that specific psychosocial issues should be addressed before the transplantation of specific organs. Interventions such as information provision and patient education based on SIPAT assessment results for each organ may improve the post-transplant outcomes of recipients. Future prospective studies should clarify whether SIPAT assessment results predict physical and psychosocial outcomes by organ type. In addition, organ-specific cut-off values need to be established for the Japanese population.


Naganishia liquefaciens reveals the general attributes of fungal BRCA2 and its essential role in Rad51-mediated homologous recombination.Identification of the BRCA2 homolog in the Basidiomycota yeast  BRCA2 tumor suppressor plays a critical role in homologous recombination by regulating RAD51, the eukaryotic homologous recombinase. We identified the BRCA2 homolog in a Basidiomycota yeast, Naganishia liquefaciens. BRCA2 homologs are found in many Basidiomycota species but not in Ascomycota species. Naganishia BRCA2  is about one-third the size of human BRCA2. Brh2 carries three potential BRC repeats with two oligonucleotide/oligosaccharide-binding domains. The homolog of DSS1, a small acidic protein serving as an essential partner of BRCA2 was also identified. The yeast two-hybrid assay shows the interaction of Brh2 with both Rad51 and Dss1. Unlike human BRCA2, Brh2 is not required for normal cell growth, whereas loss of Dss1 results in slow growth. The loss of Brh2 caused pronounced sensitivity to UV and ionizing radiation, and their HR ability, as assayed by gene-targeting efficiency, is compromised. These phenotypes are indistinguishable from those of the rad51 mutant, and the rad51 brh2 double mutant. Naganishia Brh2 is likely the BRCA2 ortholog that functions as an indispensable auxiliary factor for Rad51.The  BRCA2 gene are associated with a high risk of developing breast, ovarian, and other types of cancers  and Naganishia  diverged from a common ancestor about 450 million years ago  and Dss1 in N. liquefaciens genome using the U. maydis Brh2 sequence as a query . Its cDNA was sequenced, and the gene structure was determined . The coding sequence encodes a protein consisting of 1,193 amino acids  and human BRCA2 is limited to its C-terminal half, where major characteristic domains are clustered, including the helical domain and two oligonucleotide/oligosaccharide-binding domains (OB1 and OB2)  . OB2 disr domain . On the r domain  [ASV] to Brh2  Fig 1B,,Fig 1B. Naganishia Brh2 as a query, we searched for BRCA2 homologs. Along with metazoa and viridiplantae (green plants), ∼680 BRCA2 homologs were identified within the fungal kingdom  , two sequences were identified [ASV] [ASV] , Um_BRC 12)[ASV] . The BRC12)[ASV] . In the 12)[ASV] . Howeverlago BRC . It is pNaganishia Brh2 and human BRCA2  were generated using AlphaFold2  revealed that two acidic regions are highly conserved across these species   showed interaction with Rad51 and Dss1 fused to the Gal4 transcription activation domain (GAD), and GAD-Brh2 also showed interaction with DBD–Rad51 and DBD–Dss1 . These isection) . It is isection) .N. liquefaciens, these genes were deleted, and the null mutants were subjected to further analysis. In the absence of Dss1, cells exhibited a marked reduction in growth  (rad51 or brh2 mutant was combined with rad52.We previously demonstrated that the relatively low level of gene targeting in y (NHEJ) . Indeed,y (NHEJ)  right. Tku70 mutant background, the absence of Rad51 or Brh2 marginally reduced gene targeting, by about 10%. In the absence of Rad52, regardless of the presence of Rad51 and Brh2, gene targeting was completely abolished (We then examined the gene-targeting conditions where targeting DNA has shorter homology arms (80 bp), a context where Rad52 is essential, whereas the Rad51 requirement is limited . The absbolished  right.Collectively, these genetic findings strongly support that Rad51 and Brh2 function together in HR-mediated gene targeting.N. liquefaciens. We propose that the Naganishia Brh2 homolog functions as the BRCA2 ortholog, substantiated by several lines of evidence. First, the C-terminal region, spanning from the helical domain to OB1 and OB2, exhibits significant similarity with BRCA2 orthologs from diverse species, including vertebrate BRCA2. Notably, the structural model of the C-terminus of Naganishia Brh2 shares remarkable resemblance with those of human and mouse BRCA2 proteins. This is particularly evident in the distinctive tower domain protruding from OB2, implying conserved functional attributes. Furthermore, we identified the presence of the BRC repeat, a sequence motif characteristic of BRCA2, occurring in triplicate. Our investigation using the yeast two-hybrid assay demonstrated a robust interaction between Brh2 and Rad51, along with Dss1, an essential partner facilitating BRCA2 function.In this study, we present the discovery of a BRCA2 homolog within the Basidiomycota yeast, brh2 mutant and both the rad51 mutant and the rad51 brh2 double mutant. Notably, all three mutants displayed a comparable phenotype, characterized by compromised DNA damage repair efficiency. Sensitivity to UV and γ-ray exposure was observed at similar levels, further exacerbated in the context of an additional rad52 mutation. Similarly, our assessment of HR, as evaluated through gene targeting, unveiled a parallel reduction in efficiency within the brh2, rad51, and rad51 brh2 double mutants. This convergence of phenotypes strongly suggests that Rad51’s functionality in HR is contingent upon the presence of Brh2 in vivo. Consequently, Brh2 is deemed an indispensable auxiliary factor for Rad51 in N. liquefaciens. This study represents the first rigorous comparison of phenotypes resulting from the absence of Rad51 or BRCA2 with those stemming from the absence of both.Genetic analysis showcased parallels between the S. cerevisiae and S. pombe, both of which lack BRCA2. Instead, Rad52 assumes the role of a crucial auxiliary factor for Rad51. In these yeasts, Rad52 acts as a primary mediator, facilitating the replacement of ssDNA occupied by RPA with Rad51  exclusively relies on Rad52 in N. liquefaciens  is available. However, Rad52 becomes indispensable when homology is limited to 80 bp. This relationship between Brh2 and Rad52 closely parallels the relationship between Rad51 and Rad52, establishing that Brh2 functions exclusively with Rad51 in the HR pathway when NHEJ is impaired.We previously demonstrated that the NHEJ pathway predominates in DSB repair in ganishia . In U. mand Brh2 . In contU. maydis , and frog (Xenopus laevis) BRCA2, which have sizes of over 3,000 amino acids. Consistent with observations in other species, the sequence similarity of Basidiomycota BRCA2 homologs primarily resides in their C-terminal regions, encompassing the helical domain, OB1, and OB2. Notably, fungal BRCA2 proteins lack the OB3 domain commonly found in higher eukaryotes. Structural insights derived from the crystal structure of the mouse BRCA2 C-terminus, in complex with Dss1 and ssDNA, reveal that ssDNA resides at the base of the tower domain, making contact with OB2 and OB3  and RAD51, the interaction is mediated by two tetrameric clusters of hydrophobic residues in BRC4, each occupying distinct hydrophobic pockets of RAD51  exhibits FxxV  and grown for 4–6 h until reaching the log phase (1–2 × 107 cells/ml). Cell harvesting was achieved through centrifugation . The pelleted cells underwent washing with ice-cold water and electroporation buffer . The cells were resuspended in 10 ml of EB containing 4 mM DTT. After incubation on ice for 15–30 min, cells were collected, washed with EB, and resuspended in 200 μl of EB. The cell suspension (45 μl) was mixed with 5 μl of DNA (3 μg) in a 0.2-cm electroporation cuvette (Bio-Rad) and used for electroporation . Subsequently, electroporated cells were suspended in 1 ml of YPD and incubated at 30°C for 2 h before plating onto the appropriate selection medium. Typically, plates were incubated for 3 d at 30°C.A fresh colony of HIS3 were first amplified by PCR using a set of primers  and N. liquefaciens N6 genomic DNA as the template. Subsequently, the 5′ and 3′ homologous arms were fused to a fragment carrying a drug-resistance marker through overlap extension PCR. PCR products were purified using the MonoFas DNA purification kit (GL Sciences).Gene-targeting DNA with 80-bp and 1-kb homology arms was prepared as previously described . In briebrh2::NAT, targeting DNA was obtained through PCR with primers (Pr-401-Pr-402) and pBS-Ptef1:NAT as the template. In the case of brh2::NEO, primers (Pr-320-Pr-321) and pBS-Pact1:NEO were employed. Gene targeting was facilitated by pM101-gBRH2, which was created by cloning the annealed primers  at the BbsI site of pM101. Verification of successful gene targeting was performed through diagnostic PCR with primers (Pr-172-Pr-173). Similarly, for constructing rad52::NAT, gene-targeting DNA was obtained through PCR with primers (Pr-403-Pr-404) and pBS-Ptef1:NAT as the template. Gene targeting was conducted with pM101-gRAD52 , and correct targeting was confirmed using primers (Pr-174-Pr-175). The construction of dss1::NAT involved the preparation of two PCR fragments (Pr-140-Pr-112 and Pr-141-Pr-111), both using pBS-Ptef1:NAT as the template. The mixture of fragments was then used for gene targeting, and correct targeting was confirmed using primers (Pr-146-Pr-147). The creation of strain MP143 (brh2::NAT ku70::NAT) was achieved by transforming MP127 (brh2::NEO ku70::HYG) using a mixture of pM101-gNEO , the brh2::NAT donor fragment, pM101-gHYG  targeting the HYG cassette, and the ku70::NAT donor fragment. Nourseothricin-resistant clones sensitive to both hygromycin and G418 were selected, and gene replacement was confirmed through PCR . Strain MP147 (brh2::NAT rad52::HYG ku70::NAT) was constructed by transforming MP143 (brh2::NAT ku70::NAT) with a mixture of pM101-gRAD52 targeting RAD52 with the rad52::HYG donor fragment. Hygromycin-resistant clones were selected, and gene replacement was confirmed through PCR (Pr-174-Pr-175 for rad52::HYG). Construction of rad51::NAT, rad51::HYG, rad52::NAT, rad52::NEO, ku70::HYG and ku70::NEO was described previously (Gene replacement or editing was accomplished using the one-step replacement method or CRISPR/Cas9 technique. The latter employs pM101 that expresses Cas9 and the guide RNA scaffold . For coneviously .8 cells at the late log phase using the NucleoSpin RNA kit (Macherey–Nagel). cDNA was synthesized using the ReverTra Ace-α- kit (Toyobo) as per the manufacturer’s instructions. For this, 0.25 μg of total RNA was employed with the primers , or alternatively, 10 pmol of oligo (dT) 20 was used. The first strand synthesis was carried out at 42°C for 20 min. Subsequently, cDNA for BRH2 or DSS1 was PCR-amplified using primers listed in Table S2: BRH2, Pr-460-Pr-461; DSS1, Pr-567-Pr-568. The amplified DNA was sequenced, and the cDNA was cloned into pBluescript II SK(+) at the NdeI and BamHI sites, yielding pBS-BRH2 and pBS-DSS1, respectively.Total RNA was extracted from ∼1 × 10t test (Graphpad Prism).Gene-targeting efficiency was determined as the ratio of the number of transformants displaying auxotrophic phenotypes to the total count of antibiotic-resistant transformants. Statistical analysis of differences in gene-targeting efficiencies between strains was performed using unpaired two-tailed 6 cells/ml), and growth was continued until the cells reached the log phase. For spot tests, the cell suspension was adjusted to 1 × 107 cells/ml and serially diluted by 10fold. Next, 5 μl of each dilution was spotted onto YPD plates. For the clonogenic assay, cells prepared as described were spread onto YPD plates and subjected to UV or γ-ray irradiation at specified doses. Colony counting was conducted after 3 d of incubation at 30°C, and the survival efficiency was calculated as the percentage of colonies on the irradiated plate relative to those on the unirradiated plate.Cells were cultivated in 2 ml of YPD medium for ∼24 h at 30°C. This culture was then used to inoculate 2 ml of fresh YPD medium  was used for the yeast two-hybrid analysis . The ind


Today, with the globalization of the food trade progressing, food safety continues to warrant widespread attention. Foodborne diseases caused by contaminated food, including foodborne pathogens, seriously threaten public health and the economy. This has led to the development of more sensitive and accurate methods for detecting pathogenic bacteria. Many signal amplification techniques have been used to improve the sensitivity of foodborne pathogen detection. Among them, hybridization chain reaction (HCR), an isothermal nucleic acid hybridization signal amplification technique, has received increasing attention due to its enzyme-free and isothermal characteristics, and pathogenic bacteria detection methods using HCR for signal amplification have experienced rapid development in the last five years. In this review, we first describe the development of detection technologies for food contaminants represented by pathogens and introduce the fundamental principles, classifications, and characteristics of HCR. Furthermore, we highlight the application of various biosensors based on HCR nucleic acid amplification technology in detecting foodborne pathogens. Lastly, we summarize and offer insights into the prospects of HCR technology and its application in pathogen detection. Salmonella. Enteritidis (S. enteritidis), Salmonella Typhimurium (S. Typhimurium), Campylobacter, Staphylococcus aureus (S. aureus), Listeria monocytogenes (L. monocytogenes), and Escherichia coli O157:H7 (E. coli O157:H7) [With the continuous development of international food trade and the globalization of the food market, food safety is still worthy of extensive attention. As we all know, foodborne pathogens  widely exist in human daily life. They quickly cause foodborne diseases by contaminating food in food production, processing, storage, and transportation, which poses a significant threat to public health, personal health, and the national economy ,2. AccorO157:H7) ,7. It isO157:H7) . Early dO157:H7) .For the detection of pathogenic bacteria, the traditional culture method needs to go through the procedures of pathogenic bacteria cultivation, isolation, recovery, and identification, which often takes 3–7 days and has the disadvantages of requiring professional staff, being time-consuming, and having a complicated operation process, as well as being a false negative and lacking sensitivity, which is challenging to adapt to the development of rapid, accurate, and sensitive detection technology. With the development of immunology and molecular biology, enzyme-linked immunosorbent assays (ELISA) and polymerase chain reaction (PCR) have become the gold standard for detecting foodborne pathogens. However, both methods have their drawbacks. ELISA, which is based on the immune response, is limited by the complexity of antibody preparation and lot-to-lot variations. Based on nucleic acid amplification, PCR has high sensitivity but requires specialized personnel and expensive equipment and is prone to cross-contamination and false positives ,11,12,13Some bacteria (such as emetic Bacillus cereus) are non-antigenic and lack antibodies. In addition, proteins themselves cannot be amplified, such as antibodies or antigens. Therefore, nucleic acid-based signal amplification has more significant potential advantages and generally falls into two categories: thermal cycle amplification techniques, such as PCR and ligase chain reaction (LCR) ; and isoHCR, first proposed by Dirks and Pierce in 2004, is an enzyme-free, entropy-driven, instrument-independent isothermal amplification technique with simultaneous recognition and signal amplification capabilities, producing ladder-shaped products with fragments of different sizes, the molecular weight of which is usually inversely proportional to the size of the initiator . EssentiThere are two main types of HCR: linear HCR and non-linear HCR . TypicalPiece et al. 2007 proposed an improved type of migrating HCR that significantly reduces the possibility of non-specific initiation and relaxes sequence design requirements. However, there is still the problem of low sensitivity due to the limited amplification of linear HCRs. For this reason, continuous efforts have been made to develop non-linear HCR types with their characteristics. Some are named after the unique hairpin design and generation of high-molecular products, such as dumbbell HCR (DB-HCR), branched HCR (B-HCR), and dendritic HCR (D-HCR); some are based on the characteristics of the application, such as multiplex HCR (multi-HCR), with a large surface area of nanoparticles loaded with a large number of starting chains, and in situ-HCR for bioimaging. In addition, it is worth noting that the efficiency of HCR hybridization in a solution environment is influenced by the collision rate of molecular monomers at a local scale. Therefore, in addition to the focus on improving HCR design to increase HCR sensitivity, several methods to increase collision frequency by appropriately increasing probe concentration and using unique DNA structures  and multi-technology combinatorial strategies in the form of “HCR + X” are also being developed, driving the broader development of HCR.HCR technology has its unique attractions and features. The primary feature is that it generates long dsDNA polymers with repeating units for signal amplification based on the extension of the target sequence rather than making copies of the target, which avoids the problems of false positive results and cross-contamination that often occur with PCR, LAMP, and other techniques using universal primers . SecondlThe ideal pathogen detection method should be rapid, sensitive, specific, economical, equipment-free, and intuitive. Therefore, developing new detection methods that meet the comprehensive performance requirements of quantification, sensitivity, specificity, and economy is urgently needed. Among them, HCR has attracted much attention due to its advantages, such as being enzyme-free, having isothermal conditions, its self-assembly, and excellent amplification efficiency. In addition, new technology for pathogen detection often needs to have three parts: identification, transduction or amplification, and output signal. Among them, HCR is based on the specific recognition and triggering characteristics of target nucleic acid and has both easy modification of signal molecule transduction  in milk using HCR loaded with horseradish peroxidase (HRP) [E. coli O157:H7, a “pAb/E. coli O157:H7/mAb-AuNP-DNA1” sandwich complex was formed on the ELISA substrate, followed by the addition of biotin-modified hairpin probes . Triggered by DNA1, HCR generated long-nicked dsDNA rich in biotin sites. After the addition of horseradish peroxidase (HRP-SA) anchored with streptavidin and chromogenic substrate (TMB + H2O2), this sandwich ELISA method had a detection limit of 1.08 × 102 CFU/mL in the concentration range of 5 × 102–1 × 107 CFU/mL of E. coli O157:H7, which was 185 times lower than that of the traditional ELISA  . A gold al ELISA A.Furthermore, not only can nucleases (DNAzyme) solve the unstable and expensive problems of natural enzymes and the editable properties of nucleic acids with the compatible rivalry of HCR’s editable nature, but HCR-DNAzyme can be developed into a universal paradigm. Tian et al. proposed an ultrasensitive colorimetric dual-target detection platform based on HCR-G-quadruplex/hemin DNAzyme (HCR-GQH DNAzyme) . First, 5 CFU/mL [In addition, AuNP is also popular for its property of changing the color of the solution due to aggregation and dispersion. However, the sensitivity of sensors based only on unlabeled aptamers and AuNP has been reported to reach only 105 CFU/mL . Therefo5 CFU/mL ,37, or iE. coli from tap water and milk samples. Sun et al. innovatively combined two mechanisms to develop a highly sensitive dual-signal amplification colorimetric strategy  based onHowever, single-color colorimetric methods continue to face the problem of being difficult to distinguish and having low color resolution. With the development of nanoscience, various AuNP-derived structures have emerged  that break the shackles of a single color. Due to their sharp morphology, small changes at hot spots easily lead to sensitive changes in extinction coefficients, resulting in rainbow-like color changes, promoting the broader development of colorimetric methods.mecA gene) in milk based on the multi-HCR of AuNPs and the alkaline phosphatase (ALP)-mediated in situ growth of gold nanobipyramid particles (AuNBPs)  A. In theL. monocytogenes specific genomic sequences are present simultaneously is the HCR reaction triggered to form a long dsDNA that is enriched with glucose oxidase (GOx) and is HRP-rich. GOx converts glucose to gluconate and produces hydrogen peroxide, allowing it to further mediate the oxidation of TMB by HRP, producing a highly specific visual color signal output for enzyme cascade amplification with a detection limit of 1.12 nM [At the same time, the powerful editable capability of HCR not only significantly enhances specificity but also enables cascade amplification of the enzyme. Yang et al. by using a DNA logic gate strategy and combining Dig-Probe and FITC-probe strategies B. Only w 1.12 nM , and sucFluorescence (FL)-based methods have a higher sensitivity compared to colorimetric methods, with fluorescence spectra as the resultant response. The current fluorescence strategies using HCR for pathogen detection are broadly divided into two types : (1) lab2, MXene,) to obtain an accurate and sensitive fluorescence signal.The first type is more cost-effective than the others and is commonly used, but the sensitivity is somewhat reduced by the use of a single fluorescent group or dye with its high background fluorescence interference. Therefore, it is often necessary to separate or adsorb the background fluorescence with the aid of various nanomaterials  invA gene in lettuce based on HCR using MBs as a separator [1 CFU/mL in buffer and 6.9 × 102 CFU/g in spiked lettuce   is commo in milk B.1 CFU/mL in buffer and 7.2 × 102 CFU/mL in milk  have been proposed. Liang et al. coupled variable temperature amplification with isothermal amplification techniques to establish a novel PCR-HCR dual-signal amplification fluorescence method , effecti in milk A. Zhang  in milk B.S. aureus 16S rRNA with a LOD of 50 pM  and FAM fluorophores to produce a fluorescence synergy effect to enhance the fluorescence signal due to the generation of double-stranded products and ease of modification. Tang et al. combined the “FAM-SYBR Green I” fluorescent synergy effect with the “HCR-GO” platform and achieved sensitive detection of of 50 pM C. It was2 CFU/mL . The Lu rategies ,50.Vibrio parahaemolyticus (V. parahaemolyticus) ssDNA using a four-way migration HCR [V. parahaemolyticus detection strategies have also been investigated based on the formation of dsDNA by triple oligonucleotide (TFO) insertion [FRET can effectively reduce its background fluorescence by bringing the donor-acceptor fluorophores closer together without the aid of additional nanomaterials, which can be easily achieved by HCR self-assembly. Ren et al. proposed a simple FRET-based fluorescence sensor for the detection of tion HCR . When ssnsertion . Salmonella, CRISPR-Cas12a cannot be activated, and the initiator is intact, triggering the TDN-hHCR process and promoting FRET. This method has been applied to actual samples, achieving Salmonella sensitive detection of 17 CFU/mL in milk and 25 CFU/mL in egg white  to investigate a more sensitive and accurate FRET output strategy [1 CFU/mL, significantly lower than that of the CQ hairpin-based immune HCR (3.28 × 103 CFU/mL) and the FRET hairpin-based immune HCR (6.49 × 104 CFU/mL), which were 93-fold and 1854-fold higher, respectively. But there is no doubt that it also increases costs and expenses -excited upconversion fluorescence strategies represented by lanthanide-doped upconversion nanoparticles (UCNPs) are receiving much attention in the fields of cell imaging and tumor therapy . RecentlElectrochemical biosensors typically employ three main kinds of signals as their readout, namely current, voltage, and impedance signal, to reflect the concentrations of analytes ,59,60,61S. aureus in milk and pear juice based on DB-HCR was proposed by Wu et al  and CRISPR-associated proteins (Cas) systems have been widely reported in the field of biosensing due to their high-turnover non-specific endonuclease activity ,74. Howe in milk . In the U/mL LOD D.Clostridium perfringens (C. perfringens) [C. perfringens at a minimum concentration of 1 CFU/g in meat products  through a multi-modal signal output approach.To address the criticism of electrochemistry’s inherent instability and poor reproducibility , Wang etringens) . The metSurface-enhanced Raman spectroscopy (SERS) is also used in bioanalysis, including pathogen detection, because of its fingerprinting and interference-resistant, rapid, and high-resolution advantages . So far,S. aureus in milk with a three-part system, Apt and cDNA modified gold magnetic receptive probes (Au-MNPs-dsDNA), 4-ATP modified gold core–silver shell signal response probes (Au@Ag/4-ATP) and an integrated PDMS conical sensing array [6 CFU/mL for S. aureus  and low detection limit (4 CFU/mL) for S. Typhimurium were achieved  to influence the extent of Raman molecular aggregation. The oxidation of L-cysteine to cystine (thiol oxidation to disulfide) is catalyzed by the HCR-generated GQH DNAzyme, which affects the L-cysteine-mediated aggregation of 4-NTP-modified AuNPs, increases the electromagnetic field, and amplifies the Raman signal. A wide linear range  as well as professional staff and laboratory conditions, etc. Therefore, developing low-cost, simple-to-operate, portable HCR-based high-sensitivity point-of-care (POC) solutions would benefit a wider region and population.Salmonella ssDNA in 2016 [Salmonella is 3 × 103 CFU/mL  for detecting  in 2016 . This me3 CFU/mL A.S. enteritidis 16S rRNA in milk  with LODs of up to 250 and 400 CFU/mL in buffer and milk samples, respectively [S. aureus is 4 CFU/mL; under Mode 2, the LOD for Salmonella is 8 CFU/mL  for foodborne pathogen detection are gradually being proposed and still hold great promise for research.S. aureus, 57 CFU/mL for Klebsiella pneumoniae, and 61 CFU/mL for E. coli, respectively  cyclical cleavage and HCR amplification . When taectively A.2O2 to produce a CL signal. This protocol achieved a detection limit of 50 CFU/mL in spiked milk samples 32+ further enhances the ECL signal, achieving a low LOD of 38 CFU/mL and successfully detecting E. coli O157:H7 in milk samples  is first briefly described. The basic principles, types, and technical characteristics of HCR are then described. Finally, the application of HCR amplification techniques for detecting foodborne pathogens over the last five years is discussed, with a focus on specific examples, summarized in The primary challenge is the rational design of HCR hairpins . The lenHCR is still more focused on bacterial DNA/RNA detection in foodborne pathogen detection due to its amplification properties based on nucleic acids. However, this often involves tedious operational steps such as cell lysis, target extraction, and preservation. With the continuous development of aptamer technology, various types of aptamers with particular recognition of foodborne pathogenic bacteria have emerged, making HCR more convenient, effective, and promising for the direct detection of whole bacterial cells, and further research should focus more on the detection of intact target bacteria to reduce complex operations and shorten detection time, and to extend the application to a broader range of pathogenic bacteria and actual samples.In food testing, the main focus is still on the rapid, accurate, and sensitive identification of foodborne pathogens. Sensitive “real-time monitoring” and “timely eradication” of pathogen loads during food production, processing, and distribution  is essential to ensure food quality, shelf-life stability, economic cost savings, and protection of human life and health. The programmable and easily modifiable capabilities of HCR, combined with the evolving nanomaterials technology for sensitive identification, real-time monitoring, and timely eradication, are also areas of great potential for future exploration, requiring pioneers to continue to forge ahead.The simultaneous detection of multiple targets is critical in samples where more than one bacterium is often present. At present, HCR technology for pathogen detection is still dominated by a single strain. Although multi-target simultaneous detection methods have been proposed—for example, Lai’s group proposed a fluorescent HCR-ELISA method  based onFinally, the amplification capacity of HCR is improving and even surpassing the amplification capacity of PCR technology. Due to its enzyme-free and mild conditions, it has a vast market and research potential in POC strategy and product development. It is important to note that the detection of pathogenic bacteria usually involves three steps: identification, signal amplification, and signal output. In contrast, HCR can act as an intermediate bridge, unifying the magnetic enrichment pre-processing technology with various signal transduction output platforms. Powered by microfluidic technology, it shows extraordinary potential for extracting and separating analytes from complex sample matrices for high-throughput one-plot or full-process detection. Sample-answer assays are bound to be a trend in the future and therefore require attention to research into miniaturized, integrated, and automated analytical systems. We expect more HCR-based POC strategies to emerge for foodborne pathogen detection for food safety.


Human hands are complex biomechanical systems that allow for dexterous tasks with many degrees of freedom. Coordination of the fingers is essential for many activities of daily living and involves integrating sensory signals. During this sensory integration, the central nervous system deals with the uncertainty of sensory signals. When handling compliant objects, force and position are related. Interactions with stiff objects result in reduced position changes and increased force changes compared to compliant objects. Literature has shown sensory integration of force and position at the shoulder. Nevertheless, differences in sensory requirements between proximal and distal joints may lead to different proprioceptive representations, hence findings at proximal joints cannot be directly transferred to distal joints, such as the digits. Here, we investigate the sensory integration of force and position during pinching. A haptic manipulator rendered a virtual spring with adjustable stiffness between the index finger and the thumb. Participants had to blindly reproduce a force against the spring. In both visual reference trials and blind reproduction trials, the relation between pinch force and spring compression was constant. However, by covertly changing the spring characteristics in catch trials into an adjusted force-position relation, the participants’ weighting of force and position could be revealed. In agreement with previous studies on the shoulder, participants relied more on force sense in trials with higher stiffness. This study demonstrated stiffness-dependent sensory integration of force and position feedback during pinching. Accurate control of hands and fingers is essential for many activities of daily living. Everyday tasks such as tying a knot, writing a note, and playing the piano require dexterity and involve many degrees of freedom of the fingers. Adaptation of internal models is essential for human motor control, i.e. control signals are adjusted due to dynamics and loads . Eventually, one participant was excluded from the study due to technical issues with the equipment, leaving ten participants for analyses . Participants held a response button in their non-dominant hand. Pressing the button triggered a recording of 100 ms of applied force and the pinch position (measured aperture between thumb and index finger).Each participant performed three sixty-trial blocks. In every block, the haptic manipulator rendered a specific stiffness, see Table Before every block, participants familiarized themselves with the stiffness in a training session of at least 15 trials, including visual feedback of the applied force. In each sixty-trial block, the participants executed 30 duos of alternating trials: trials with visual feedback  were alternated with trials without visual feedback . To reveal the weighting of the participants out of each two trials without visual feedback one was a blind trial and one was a catch trial in random order. In a catch trial, the  regular spring was replaced by an adjusted spring. The three trial types are elaborated on in Table The aperture required to reach a target force is defined by the target force and the stiffness of the spring, entclass1pt{minimaFor each trial, the response button triggered the recording, and the aperture and force were determined from the recorded signals (averaged over 50 ms before till 50 ms after button press). One (reference) trial was discarded as the button was unintentionally pressed too early, i.e. within 50 ms from the start of the trial. The total dataset thus consists of 1799 trials instead of 1800: ten participants who performed sixty trials in each of the three stiffness conditions. The dataset is available via 4TU. Centre for Research Data (10.4121/21400218).For each stiffness level and for each participant, the apertures were averaged over all repetitions grouped by trial type. The difference between the apertures during the blind and catch trials  , the aperture error is larger for low stiffness than for high stiffness, explaining the higher aperture variance in the low stiffness block. Whether the scaling of visual feedback affected weighting is unknown.In our experiments, the three stiffness blocks were equal for all participants. However, their strength varied considerably and where some indicated the high stiffness level was challenging, others could probably have handled higher stiffness without fatiguing. The stiffness range of the manipulator was limited, thus reducing the experimental design options. A setup with a more extensive range would allow for additional stiffness blocks to fit a wider range of individual crossover points.Despite the normal distribution of the data, non-parametric tests were used to account for the small sample size. Friedman test is often seen as the non-parametric counterpart of repeated-measures ANOVA. However, it is a generalization of the sign test that handles both normal as well as many non-normal distributions and offers lower statistical power than repeated-measures ANOVA  compared to medium and high stiffness conditions . This study demonstrated stiffness-dependent sensory integration of force and position feedback during pinching.