International Journal of Biometeorology has published a total of 2183 papers that broadly consider human health and its relationship with climate. In this review, we identify a total of 180  of these papers that specifically focus on the intersection between meteorological variables and specific, named diagnosable diseases, and explore the publication trends thereof. The number of publications on climate and health in the journal increases considerably since 2011. The largest number of publications on the topic was in 2017 (18) followed by 2021 (17). Of the 180 studies conducted, respiratory diseases accounted for 37.2% of the publications, cardiovascular disease 17%, and cerebrovascular disease 11.1%. The literature on climate and health in the journal is dominated by studies from the global North, with a particular focus on Asia and Europe. Only 2.2% and 8.3% of these studies explore empirical evidence from the African continent and South America respectively. These findings highlight the importance of continued research on climate and human health, especially in low- and lower-middle-income countries, the populations of which are more vulnerable to climate-sensitive illnesses.The climate-health nexus is well documented in the field of biometeorology. Since its inception, Biometeorology has in many ways become the umbrella under which much of this collaborative research has been conducted. Whilst a range of review papers have considered the development of biometeorological research and its coverage in this journal, and a few have reviewed the literature on specific diseases, none have focused on the sub-field of climate and health as a whole. Since its first issue in 1957, the The online version contains supplementary material available at 10.1007/s00484-023-02466-8. International Journal of Biometeorology  formalized the academic theme of medical meteorology Tromp . Rather Holistic values underscored an organic perspective on man as a patient, creating opportunities for natural healing, for example, climatotherapy Bynum . The theInternational Society of Biometeorology and its flagship journal in 1956 and 1957 respectively  of this total, whilst publications assessing balneotherapy account for 5.4% (n = 117). There have been special issues in the journal dedicated to Balneology  and the Universal Thermal Climate Index . There is an overall increase in publications on climate and human health in the journal over the period 1957–2012. This coincides with changes in the total number of papers in the journal  focused on a specific disease in the context of a meteorological variable  was for 1988 . On the scale of individual countries, the greatest number of studies have been conducted in China , followed by Japan . In 2008, the CHC held a meeting in Japan, and in 2019 the International Journal of Biometeorology dedicated a special issue to Asian Biometeorology (Fdez-Arroyabe and Robau n = 75), genetic disorders , and vector-borne diseases   and Lithuania . European studies particularly dominated the earlier volumes of the International Journal of Biometeorology (Table n = 180) of papers on climate and health in the International Journal of Biometeorology.The second largest continental representation in the published research is in Europe (35.6%). This may be because researchers were predominantly based in Europe before the ISB expanded to other parts of the world. Studies conducted in Europe have been concentrated in Spain  of all papers in the journal in total. However, whilst this fits proportionately with the journal figures , with research on all six of the habitable continents, including both chronic respiratory conditions such as asthma and acute respiratory conditions such as influenza. As there has been a lot of research conducted on respiratory diseases and it is well-understood that climate and weather influence their transmission dynamics. These studies, similar to the overall trends, were concentrated in the Northern Hemisphere, with 25 papers on respiratory health from Asia and 23 from Europe  of the studies published in the International Journal of Biometeorology examined strokes.Across the 180 papers (1957–2022), a total of 14 disease groups were explored: neurological, cardiovascular, skin, rheumatic, intestinal, metabolic, psychiatric, vector-borne, genetic, cerebrovascular, respiratory, renal, and bacterial diseases and others such as measles Fig.  respiratope Fig. . Accordin = 180). This is likely as a result of an increase in research on non-communicable diseases in the twenty-first century and the fact that cardiovascular diseases are the major cause of mortality . This is likely due to the ageing population of the region, heightening the vulnerability to these diseases  of the papers being investigated in this review report results that are not statistically significant . This makes the comparison of results from studies difficult to perform. So, whilst statistical analyses of empirical case data are important, they do not negate the importance of laboratory work in understanding aetiology, and qualitative work on experiential components of seasonality. An important future direction, in this regard, is to employ a less reductive approach and to focus more holistically on the physiological reasons that underpin any relationships found and reported from the data. This would be most effectively achieved through greater collaboration between climatologists, healthcare practitioners, and data scientists. Indeed, this was one of the key aims of the society at its inception Tout .International Journal of Biometeorology. Over the full period of publication of the journal, studies dedicated to the intersection between specific, diagnosable diseases and meteorological variables account for only 4.1% of the 4350 works of the journal to date. Although the criteria for this review were specific, the 180 papers analysed are an indication that the International Journal of Biometeorology has indeed stuck to its scope in documenting such studies. Researchers in Europe and Asia have done an excellent job in documenting their studies in the journal since its inception, contributing a combined 77.8% to the total 180 studies. It is important to have more studies explore the extent to which diseases are climate sensitive. Of the 180 papers reviewed, only four (2.22%) studies were based in Africa. A total of 15 (8.3%) studies were based in South America. This means that regionally endemic conditions such as Ebola, yellow fever, and malaria are largely unstudied in this journal. Given a change in climatic conditions, continents such as South America and Africa, which are affected greatly by climate change have minimal to no studies on climate and health. In Africa and South America, dysfunctional health systems and the collision of epidemics of communicable and non-communicable diseases have exacerbated this. The paucity of research on human biometeorology conducted in Africa and South America was exposed by this systematic literature review, which also identified shortcomings that may serve as the basis for future studies. The study had the advantage of making it clear which sectors should receive the majority of the funding for research in the near future. In this way, gaps in publication production can promote the expansion of research in specific subregions of South America and Africa.A significant increase in human health papers has been published in the last decade in the Supplementary file1 (DOCX 46 KB)Below is the link to the electronic supplementary material.


A multivariate analysis showed that the PDRI, donor hemoglobin A1c values, and pancreas transplantation alone significantly predicted 1-year pancreas graft survival . Spline curve analysis showed that the PDRI was incrementally associated with an increased risk of 1-year graft failure. In the group with a PDRI ≥ 2.87, 8/56 patients had graft failures within 1 month, and all were due to graft thrombosis. The PDRI is a prognostic factor related to the 1-year graft survival rate. However, pancreas transplantation from high-PDRI donors shows acceptable results and could be an alternative when the donor pool is insufficient.Pancreas transplants from expanded criteria donors are performed widely in Japan because there is a shortage of brain-dead donors. However, the effectiveness of this strategy is unknown. We retrospectively studied 371 pancreas transplants to evaluate the possibility of pancreas transplantation from expanded criteria donors by the Pancreas Donor Risk Index (PDRI). Patients were divided into five groups according to quintiles of PDRI values (Q1–Q5). The 1-year pancreas graft survival rates were 94.5% for Q1, 91.9% for Q2, 90.5% for Q3, 89.3% for Q4, and 79.6% for Q5, and were significantly lower with a lower PDRI (  Pancreas transplantation enables insulin withdrawal in patients with insulin-dependent diabetes and considerably improves patients’ survival and quality of life –4. HowevIn pancreas transplantation, the Pancreas Donor Risk Index (PDRI), which was reported by Axelrod et al. in 2010, is currently used to predict 1-year pancreas graft survival as a pre-procurement scoring system . AdditioA high percentage of grafts are discarded because pancreas grafts are often evaluated under relatively strict criteria , 20. In A total of 400 pancreas transplants performed at 18 certified pancreas transplant centers in Japan between January 2001 and July 2019 were included in this study. Of these, 371 cases were included after excluding 27 cases of living pancreas transplantation and two cases of incomplete data. The primary disease was type 1 diabetes mellitus in all cases.The following clinical data were retrospectively extracted from the national database administered by the Japan Society for Pancreas and Islet Transplantation: transplantation type, recipient age, recipient sex, recipient height, recipient BMI, duration of type 1 diabetes mellitus, episode of preoperative dialysis, duration of dialysis, donor age, donor sex, donor height, donor BMI, donor HbA1c concentrations, cause of death, episode of cardiopulmonary resuscitation, SCr concentrations, total ischemic time of the pancreas graft, pancreas graft position, ductal management, type of venous drainage, artery reconstruction, gastroduodenal artery reconstruction, and portal vein extension. Written informed consent was obtained for enrollment in the registry of the Japan Society for Pancreas and Islet Transplantation. The application and approval of the institutional review board were exempt because all data and information used in this study were de-identified. This study was conducted in accordance with the principles of the Declaration of Helsinki and Istanbul.The PDRI of Japanese patients with a pancreas transplant was calculated according to the formula reported by Axelrod et al.  Several 2 test, as appropriate. Continuous variables are presented as the mean ± standard deviation and were analyzed using the Mann–Whitney U test. Kaplan–Meier curves with log rank tests were used to examine graft and patients’ survival. Bonferroni correction was used to adjust for multiple comparisons. Potential risk factors for 1-year pancreas graft survival were assessed using univariate and multivariate Cox proportional hazards analyses. Restricted cubic spline curves were plotted to describe the multivariable-adjusted association between the PDRI and the hazard ratio (HR) with the 95% confidence interval (CI) for graft survival. The cutoff value of the PDRI calculated from receiver operating characteristic curve analysis was chosen as the reference for the spline plot. Statistical significance was set at p < 0.05. All statistical analyses were performed using JMP 16.0.0 , EZR (Easy R) version 1.54  [Categorical variables are presented as the count (percentage) and were compared using Fisher’s exact test or the χ, Japan) , and R vThe distribution of the mean PDRI of the 371 patients in Japan is shown in p < 0.001), height (p < 0.01), BMI (p < 0.001), cause of death (p < 0.001), and TIT of the pancreas graft (p = 0.01), which are factors that constitute the PDRI, were found between the five groups. Other than the factors constituting the PDRI, significant differences were observed in HbA1c values (p < 0.001), cardiopulmonary resuscitation (p < 0.01), and graft position (p = 0.01) between the groups. The 1-year pancreas graft survival rates were 94.5% for Q1, 91.9% for Q2, 90.5% for Q3, 89.3% for Q4, and 79.6% for Q5, which were significantly lower with a lower PDRI  time to pancreas graft failure was 14 days (2.75–113.25 days). The causes of graft were thrombosis in 22 cases, rejection in seven cases, graft duodenal perforation in seven cases, non-adherence in two cases, recurrent type 1 diabetes in one case, and unknown in one case. Patients were divided into five groups (Q1–Q5) according to the quintile of the PDRI value . Signifi2, height of 173 cm, cause of death is not CVA, total ischemic time of 12 h for the pancreas graft, no donation after circulatory death, and creatinine concentrations < 2.5 mg/dL [Axelrod et al. defined the following as reference donors with a PDRI = 1: male sex, 28 years old, non-black, non-Asian, BMI of 24 kg/m.5 mg/dL . Table 2A Cox proportional hazards model was used to identify the factors associated with 1-year pancreas graft failure. The univariate analysis showed that the PDRI, donor HbA1c values, cause of death, PAK, and PTA were independent factors that significantly predicted 1-year pancreas graft survival . The mulp < 0.001), age (p < 0.001), height (p < 0.01), BMI (p < 0.001), HbA1c level (p < 0.01), death at CVA (p < 0.001), and PAK (p = 0.03) between the two groups (p < 0.01, The range of PDRI values evaluated by Axelrod et al. ranged from 0.64 to 2.86 , and reso groups . When we16 in the UK with a median PDRI of 1.30,11 in Germany with a median PDRI of 1.30 [In Japan, pancreas transplants from expanded criteria donors are frequently performed owing to the unique shortage of brain-dead donors. In this study, the mean and median PDRI values were 2.01 and 1.88, respectively. The reason for this finding is that the donors were older, and the cause of death was often cerebrovascular disease . These d of 1.30 , and in  of 1.30 . Despite of 1.30 , 16, 17  of 1.30 . This fiIn the multivariate analysis of factors involved in the 1-year graft prognosis, the PDRI, donor HbA1c levels, and PTA were prognostic factors. This analysis confirmed the validity of evaluating the PDRI using the Japanese data. There have been two types of reports on the effectiveness of PDRI as a prognostic factor. Some reports showed that the PDRI was effective –14, wherIncreasing the donor pool is not a problem that can be accomplished in the short term, and donors with high PDRIs must also be used. However, the acceptable range of the PDRI must be discussed. In this study, as shown by the spline curves in In the study by Axelrod et al., PDRI values were validated only up to 2.87 . In thisA high rate of thrombosis occurs in transplants from donors with a high PDRI, which leads to graft failure. Donor risk factors for thrombosis are age –26, cereA strength of this study is that, to the best of our knowledge, this is the first report on the evaluation of the PDRI against a background of data from a large number of expanded criteria donors. We were able to show the results of patients with extremely high PDRI values, which have rarely been previously verified. Additionally, external evaluation of the PDRI has mainly been conducted in Western populations, and whether the PDRI is effective in Asian populations is unknown . The preIn conclusion, the PDRI is an effective evaluation tool for pancreas transplantation in Japan. Pancreas transplantation from donors with a high PDRI can be performed with acceptable results as an alternative until the donor pool is increased. However, the early development of thrombosis should be noted in cases of an extremely high PDRI.


There is an urgentneed for improved outcomes in the treatmentof pelvic organ prolapse (POP). Success of primary surgery relieson the load bearing capacity of plicated connective tissue underneaththe vaginal wall, which is compromised due to an altered vaginal fibroblastfunction and collagen composition. There is an important factor inconnective tissue repair that relates to changes in stiffness of thevaginal fibroblast microenvironment, which influences cell activitythrough cellular mechanosensing. The aim of this study is to investigatethe effect of stiffness changes on vaginal fibroblast functions thatrelate to connective tissue healing in prolapse repair. The substratestiffness was controlled by changing the polymer concentration inthe fibrous and strongly biomimetic polyisocyanide (PIC) hydrogel.We analyzed stiffness during cell culture and assessed the consequentialfibroblast proliferation, morphology, collagen deposition, and contraction.Our results show that increasing stiffness coincides with vaginalfibroblast alignment, promotes collagen deposition, and inhibits PICgel contraction. These findings suggest that the matrix stiffnessdirectly influences vaginal fibroblast functionality. Moreover, weobserved a buildup in stiffness and collagen, with an enhanced fibroblastand collagen organization on the PIC-substrate, which indicate anenhanced structural integrity of the hydrogel-cell construct. An improvedtissue structure during healing is relevant in the functional repairof POP. Therefore, this study encourages future research in the useof PIC gels as a supplement in prolapse surgery, whereby the hydrogelstiffness should be considered. However,the load bearing capacity of the pelvic floor is compromised, contributingto a high recurrence rate (32–45%).2 The fibroblast function in the vaginal connective tissue of POPpatients is altered with impaired production and remodeling of collagen.4 Studies report that the imbalance in collagen metabolism associateswith changes in vaginal stiffness, suggesting an affected mechanicalintegrity of the tissue.7In patients with pelvicorgan prolapse (POP), one or more pelvicorgans  descend into the vagina dueto weakening of supportive tissues. POP can be treated by surgery,which is indicated for one out of 10 women during a lifetime.10 Earlier work showed that cytoskeletal tension viacell-matrix connections  affects cell morphologyand activates mechanosensitive responses.12 For fibroblasts, these responses may result in altered collagendeposition that further changes the stiffness of the ECM.7 In prolapse repair, such changes in tissue metabolismmay result from the insertion of biomaterials with incompatible stiffnessproperties.14 Despite the relevance of stiffnessas a functional outcome in connective tissue healing and its influenceon the cell microenvironment, only a few in vitro studies address the effect of stiffness changes on the vaginal fibroblastresponse.15 Furthermore, none of these studies involvethe use of biomaterial stiffness to stimulate the regenerative behaviorof vaginal fibroblasts for prolapse repair.Stiffness is an importantparameter in functional healing of vaginalconnective tissue, not only as a contributor to being load bearingbut also as a property of the vaginal fibroblast microenvironment.Changes in extracellular matrix (ECM) stiffness influence the fibroblastbehavior.in vitro.18 Recently, we were able to stimulate vaginal fibroblast activitywith respect to ECM deposition and remodeling through adjustmentsin ligand density in the PIC gel.20 Based on theseresults, we hypothesize that we can manipulate the vaginal fibroblastfunctions and improve relevant parameters in prolapse repair by changingthe PIC gel stiffness in the cell microenvironment. Understandingthe effects of stiffness on fibroblast function may help in initiatingsuch tissue regenerative strategies. We evaluated stiffness of vaginalfibroblast-hydrogel constructs during cell culture and investigatedthe cell behavior in terms of proliferation, morphology, and collagendeposition. Moreover, as fibroblasts contract the hydrogel, we monitoredthe hydrogel contraction.21In our opinion,changing biomaterial stiffness is an effectiveway to trigger fibroblast activity and collagen metabolism in vaginalconnective tissue. In this study, we aim to investigate the vaginalfibroblast function by changing the stiffness of a polyisocyanide(PIC)-hydrogel. A PIC gel is a mechanically biomimetic and thermosensitivematerial that shows promise for regenerative purposes by its potentialto promote vaginal fibroblast activity, steer stem cell differentiationand secretome, and facilitate neovascularization 2Three PIC hydrogelsof different stiffnesses were obtained by increasingthe concentration in PIC polymer solutions to 0.2% w/v (PIC-0.2%),0.4% w/v (PIC-0.4%), and 0.6% w/v (PIC-0.6%).2.121 Briefly, isocyanidemonomers were randomly copolymerized with a 3.3% feed of isocyanidescontaining azide (N3) groups. The viscosity average molecularweight was determined as described previously.22 The RGD peptides were conjugated to a DBCO-PEG4-NHS linker. Via click chemistry, RGD was conjugated to obtain thePIC-RGD polymer in a concentration of 55 μM . PIC-RGD was sterilized with a 10-min exposureto UV-light and dissolved at a concentration of 6 g L–1 (PIC-0.6%) in cell culture medium at 4 °C on a roller for 2days. PIC-0.6% was further diluted in cell culture medium to obtain4 g L–1 (PIC-0.4%) and 2 g L–1 (PIC-0.2%) concentrations. Gelation involved bundle formation ofthe polymer, which was induced by increasing the temperature beyond15 °C.19A PIC polymerdecorated with RGD for cell adherence was synthesized using methodsas previously described.2.22.2.1G′as a function of temperature T was measured usinga temperature ramp from T = 5 to 37 °C . The differential modulus K′ was measured under a small oscillatory stress(ω = 62.8–0.628 rad s–1), superposedon a much larger prestress (0.4–200 Pa). For each gel, G′ was determined at 37 °C.Hydrogel mechanics were characterized using arheometer  with a steel parallel plate . The storage modulus 2.2.2Eeff) on day 0. PIC-gels wereindented in 96-well plates at 37 °C in a noise-canceling box.20 Thin piezos with low-stiffness probes were used and indented the gel surface during a matrix scan of minimally6 points (100 μm per step). The indentation depth was 10 μm,and the Hertzian model was chosen.23Stiffnessdifferences between the hydrogel surfaces were assessedby nanoindentation  in terms of the effective Young’smodulus  forfluorescent morphology and collagen imaging. The culture plates wereincubated at 37 °C for gelation. Vaginal fibroblasts were culturedin Dulbecco’s modified Eagle’s medium (DMEM) supplementedwith 10% fetal bovine serum (FBS) and 2% penicillin (100 U mL–1)/streptomycin (100 mg/mL)  up to passage 4 and seeded on the hydrogels ata density of 3 × 103 cells cm–2.Culture plates containing hydrogels with cells, further referred toas (cell-hydrogel) constructs, were incubated at 37 C and 5% CO2 in a humidified environment. Medium was refreshed once every3 days on a heating plate. The experiments were independently executedthree times in triple.Vaginalfibroblasts were isolated from the anterior vaginal wall of an informedand consented patient undergoing POP surgery as previously described.2.3.2Eeff) during culture was determinedon days 14, 21, and 28. The construct surfaces were indented followingthe specifics as described in Stiffnessof the cell-hydrogel constructs by means of the effective Young’smodulus  at 605 (reference) and 440 nm (formazan).2.3.42) and spreading area on day 1 and the distribution of theactin filament orientation on day 21 .The cytoskeletonand nuclei of the vaginal fibroblasts were imaged by fluorescent microscopyon days 1, 3, 7, and 21. All steps were performed at 37 °C. Thesamples were fixed with 4% paraformaldehyde (PFA) for 20 min. Thecells were permeabilized with 0.1% Triton X-100. After being washedwith PBS, actin filaments were stained with 100 nM Phalloidin TexasRed-X  and incubated for 90 min. The samples werewashed twice with PBS, and nuclei were stained with 20 nM Hoechst for 30 min. The samples were imaged with a LeicaDM5000B fluorescent microscope . Morphology was further analyzed by meansof individual fibroblast circularity on days 14, 21, and 28. PIC gels were liquefied by placing the well-plateson ice for 20 min. Liquid PIC solutions from 8 wells containing cellsand ECM were pooled in one centrifuge tube. The tubes were centrifugedfor 5 min at 2500 g at 4 °C to remove PIC from the precipitatedECM. The ECM was stored overnight in a polyethylene glycol solutionat 4 °C. The samples were washed by 5 min of centrifugation at3000 g and stained with Picrosirius red for 20 min. The stained precipitatewas washed with PBS by a 5-min centrifugation at 3000 g and extractedwith buffer (Chondrex). Absorbance was measured at 540 nm by theSynergy H1 microplate reader (Biotek Instruments Inc.). Values weresubtracted with background (only PIC gel) and normalized to a standardrat collagen-I standard solution .25 Fibroblasts on PIC-gels werefixed in 4% PFA. Samples were washed with PBS and stained with CNA,which was diluted in PBS at a ratio of 1:50, and incubated overnight.After being washed three times with PBS, fibroblast nuclei were stainedwith Hoechst. Images were taken using the Leica DM5000B fluorescentmicroscope .Fluorescent imaging of deposited collagenwas performed on days 14, 21, and 28 using the CNA35-OG488 dye (CNA),which specifically binds to collagens, such as collagen types I, III,and IV.2.3.626 Thecontracted hydrogel region was determined by the perimeter that followedcontracting fibroblasts at the hydrogel edge .Hydrogelcontraction, due to contracting fibroblasts, was determined on days14, 21, and 28 from photographs of PIC gels through a microscopy lensat 5× magnification. The contraction was calculated based onthe area percentage of the well occupied by hydrogel using ImageJsoftware as previously described.2.4p < 0.05.Data were presentedand analyzed using the Graphpad Prism version . If data passed the D’Agostino-Pearson normalitytest, results were presented by the mean ± the standard deviation(SD). A two way ANOVA was performed with Bonferroni’s posthoctest to compare between time points and PIC-gels. For data that concernedonly one time point, one way ANOVA was selected with Bonferroni’sposthoc test. In the case of skewed data, results were presented inmedian and interquartile range (IQR). Then, a Kruskal–Wallistest with Dunn’s test was used to compare between time pointsand hydrogel-groups. Differences were considered statistically significantif 33.13.1.1Table S1. Figure S1 illustratesthe chemical structure of the PIC-RGD polymer. Rheology revealed thermoresponsiveand stress-responsive behavior. The thermosensitivity of the gelsis shown by the relation between the storage shear modulus G′ and temperature (T) . With regard to G′ at 37 °C,the rheology results display an increasing trend with an increasingpolymer concentration  with an increasing polymer concentration (Table S1), confirming differences in substratestiffness at the start of the cell culture (Nanoindentations across the hydrogel surfaces onday 0 (37 °C)show an increasing effective Young’s modulus ( culture 1C. Examp culture 1D.3.1.3Eeff) over time revealed an increase for PIC-0.4%(day 14 vs day 28: p = 0.003). In contrast, Eeff values of the other cell-hydrogel constructswere constant in time  and PIC-0.4%(day 14: p < 0.05). However, on day 28, the PIC-0.4%construct modulus increased significantly as compared to PIC-0.2%(p < 0.01).When looking per time point, the surface stiffnessincreased withincreasing PIC concentration on days 14 and 21. Specifically, thePIC-0.6% construct exhibited a significantly increased modulus onthese days with respect to PIC-0.2% (days 14 and 21: 3.23.2.1p <0.05). After day 7, the proliferation increase was significant betweentime points for fibroblasts on all hydrogels (p <0.001). Comparing between the PIC gels on day 7 revealed that withhigher stiffness, fibroblast proliferation tended to increase , albeit insignificantly. The hydrogel stiffness did not affectfibroblast proliferation on the other time points.Vaginal fibroblastproliferation increased on all three hydrogel stiffnesses over time3A. Howev3.2.2p < 0.05, Figure S3). Fluorescentimages of actin filaments over time showed that vaginal fibroblastsattached and spread on all gels 3B. Compa3.33.3.1p < 0.05; PIC-0.4%: p < 0.001; PIC-0.6%: p < 0.01; p < 0.05).Semiquantitativecollagen data shows a significant increase over time for all cell-hydrogelconstructs . Contraction started for the softestPIC-gel (PIC-0.2%) on day 14 (p < 0.05) andPIC-0.4% (day 14 vs 21: p < 0.001 and day 21vs 28: p < 0.05). Contraction decreased with anincreasing initial gel-stiffness, which reached significance betweenconstructs on day 28 Contractionwas characterized by the appearance of a hydrogel edgewith high densities of elongated vaginal fibroblasts S2. Contr4in vitro changed noticeably. A stiffness increase of the gel-cell constructcoincided with alignment and directionality of vaginal fibroblasts,an enhanced collagen deposition, and inhibition of hydrogel-contraction.In this study, we manipulatedthe microenvironment of vaginal fibroblaststhrough alterations in PIC-gel stiffness to influence cell functions.Parameters that relate to a regenerative response 27 The shear storage and effective moduli were of the same order ofmagnitude, despite the experimental differences in deformation modeand length scales.28 We note that PIC gels,analogous to biological gels such as collagen and fibrin, displayunique nonlinear mechanical responses under complex multiaxial deformation(like nanoindentation), which further emphasizes the biomimetic characterof the PIC gels.30 Furthermore, we note that itis challenging to obtain consistent nanoindentation measurements onsoft substrates due to increased sensitivity to noise during indentation,as reflected by the presented variations in the modulus. Additionally,during cell culture, the nanoindenter measures across an uneven PIC-gelsurface with a heterogeneous distribution of cells and ECM.The bulk and surface measurements of the selected PIC-gels werecorrelated with the polymer concentration. The stiffness outcomeswere within range of soft tissues and PIC-substrates that facilitatea sustained fibroblast activity, which supports our choice of theselected PIC-concentrations.31 During proliferation, these cell activitiesadditionally change the microenvironment, which in turn may affectthe cell responses, suggesting the potential involvement of a feedbackloop.34 In this study, an effect of stiffness on vaginalfibroblast behavior was possibly reflected in the cell morphologies,where an initially higher stiffness slightly increased spreading areaon day 1. Furthermore, stiffer PIC-gels promoted fibroblast alignmentand directionality, which can be explained by traction on fibroblastfocal adhesions at an increased substrate stiffness.35 The fibroblasts contract in response, which induces alignmentalong directions of increased tension.36 The consequentialchanges in the cytoskeletal configuration may promote proliferationthrough cell spreading, cell motility, and regulation of mitogenicsignaling pathways.39 This effect might explain the significant proliferationincrease on the stiffest PIC-gel on day 7 as compared with day 1.Initial differences in substrate stiffness may influence fibroblastfunctions, such as proliferation, contraction, and ECM synthesis.40 Furthermore, the organization of collagenlikely followed the fibroblast arrangement, which seemed to alignmore when the PIC-gels were stiffer. In addition to the initial hydrogelstiffness, the consequential fibroblast and collagen structure mayaid in the stability of the construct and could limit contraction.42 Indeed, increasing PIC-gel stiffness delayed contraction by thefibroblasts. As stiffness increases, the constructs logically contractat later time points when fibroblast proliferation further progresses,as more work by actin filaments is needed to deform the gel.21 In contrast, the softest gel contracted earlierin the presence of little collagen, as lower contraction forces byproliferating fibroblasts were sufficient for deformation of the construct.The enhanced stiffness, collagen deposition, and fibroblast alignmentduring culture of the intermediate stiffness gel might be attributedto a concurrent action of hydrogel stiffness that applies a mechanicalcue to the respective fibroblast functions.34 Hydrogelcontraction may further contribute to this action by promoting stiffnessthrough the deformation of PIC to a denser configuration.43 We speculate that the coinciding collagen, contraction,and stiffness increase could be the result of the aforementioned positivefeedback; the vaginal fibroblast activities with respect to collagenproduction and contraction increase stiffness, which further triggerfibroblast responses. Surprisingly, the stiffening could not be confirmedfor the softer gel, despite the strong contraction behavior. Thisis likely a result of the high probe sensitivity to noise and heterogeneoussurfaces on low stiffness substrates, which could hinder obtainingsignificant differences between time points.Nanoindentation data showed that the stiffness increase over timeof the gel-cell construct in the initial intermediate stiffness range reached statistical significanceon day 28, in contrast to PIC-0.2% and PIC-0.6%. Furthermore, on thisday, the former construct exhibited a significantly higher collagenamount compared with the other hydrogels. There could be an effectof stiffening of PIC-0.4% on collagen deposition in line with previouswork which demonstrates that an increasing stiffness enhances collagenexpression by fibroblasts, which involves mechanically activated signalingpathways.in vitro regeneration for pelvicfloor repair. Stiffness influences vaginal fibroblast behavior inthe vaginal wall and plays a role in tissue functioning, evidencedby the affected tissue stiffness associated with POP, menopause, andfibrosis.45 Although thereis consensus that vaginal fibroblast behavior is critical in pathophysiologyand treatment of prolapse, there are few in vitro studies that consider stiffness as variable during regenerationin biomaterial development for prolapse repair.46 Using a biomimetic injectable gel as a substrate in thecontext of prolapse repair aids the novelty. With regard to prolapserepair, the current results are encouraging in terms of the potentialof PIC-gel to allow for vaginal fibroblast spreading, deposition ofcollagen, and buildup of stiffness. A controlled ECM deposition accompaniedby a stiffness increase in the connective tissue could improve thestructural and mechanical integrity of the repair.48 PIC-gel inthe intermediate stiffness range performed best in this context asthe corresponding cell activity resulted in increased collagen productionand effective Young’s modulus over time. Furthermore, the increasedconcentration of PIC, limited contraction, and increasing collagenand modulus indicate enhanced stiffness and structural integrity ofthe construct. Moreover, in vivo, an improved hydrogelintegrity aids to surgical applicability as it is beneficial for handlingand improves the contact surface with tissue.50 Therefore, we intend to use PIC-gel of 0.4% w/v in vivo to study the potential of PIC-hydrogel as a healing supplement inPOP surgery. We acknowledge that in vitro modelsfall short to simulate in vivo physiology, implyingthat the impact of the gel stiffness on the fibroblast response canonly further be emphasized in vivo due to the presenceof immune components and applied mechanical forces. Still, our resultsimply that stiffness in the vaginal fibroblast microenvironment affectsvaginal fibroblast morphology and collagen metabolism. In turn, thevaginal fibroblast activity influences PIC-gel behavior in terms ofa stiffness increase and contraction. Therefore, we emphasize that in vitro research to biomaterial solutions in prolapse repairshould include stiffness as a variable that affects biomaterial performance.The strength ofthis study lies in the effort to investigate stiffnessas a parameter in 5In this study, we modifiedthe PIC-gelstiffness in the vaginalfibroblast environment, which accompanied an altered cell function.In the intermediate stiffness range, the hydrogel-fibroblast constructstiffened during cell culture and promoted collagen production comparedto an initially higher or lower substrate stiffness. Thus, dependingon the initial substrate stiffness, PIC-gel might trigger an increasedregenerative response of vaginal fibroblasts with a buildup in stiffness.The outcome of this study underlines the need to consider mechanoresponsivecell behavior in selecting PIC-gel compositions and other biomaterialsfor regenerative purposes, such as prolapse repair.


The search for biomarkers for clinical use in kidney transplant monitoring sometimes seems a tantalizing torment.The first hint about the potential application of donor-derived cell-free DNA (dd-cfDNA) in post-transplant monitoring dates back 25 years . SpecifiThe advancement of more comprehensive and scalable DNA sequencing technologies, coupled with the easy accessibility and short half-life of cfDNA, has paved the way for the development of commercially available assays for measuring dd-cfDNA in the plasma of transplanted recipients. In the past 10 years, after demonstration of analytical robustness, these assays have been validated in clinical practice for kidney transplant recipients, and consistently demonstrated a significant correlation between plasma dd-cfDNA levels and graft damage –5.These promising findings have led to the clinical adoption of dd-cfDNA assays for monitoring the occurrence of graft rejection and injury in kidney transplant recipients in the Unites States. This was boosted by Medicare reimbursement in 2017, and positive coverage decisions from several commercial payers. In Europe, however, the adoption of dd-cfDNA assays in clinical practice lags behind, due to cost concerns of the test and a lack of data demonstrating their clinical utility and context of use.In two prospective single-center studies in this journal, Benning et al. and Mantios et al. report on the diagnostic performance of dd-cfDNA% in discriminating kidney transplant recipients experiencing graft rejection from rejection-free patients, at the time of clinically indicated biopsies , 7. The A major added value of the study by Benning et al  is the rHowever, a NPV of 77% with the best cut-off (0.57%) is far from excluding all rejection cases. As outlined by the authors, the lower sensitivity of dd-cfDNA for borderline changes, could be a major downside of the test at time of graft dysfunction. Borderline changes in indication biopsies are considered as clinically meaningful  and are Instead of proposing single thresholds, more work is needed to identify the thresholds below which rejection (including borderline changes) can be safely excluded, and to calculate how many biopsies could be avoided with such test. This would allow for establishing the true clinical benefit of dd-cfDNA% testing at the time of clinical suspicion of injury/rejection and help calculate cost-effectiveness in such a context.In addition, these two studies , 7 highlFirst, while typically higher in severe active rejection, dd-cfDNA% shows considerable variability within specific rejection categories, correlation with both active and chronic lesions, and possibly increased levels in case of rejection-free graft injuries . These observations suggest that although dd-cfDNA may be used as an intuitive biomarker of graft injury, what exactly is being measured at the biological level has not yet been elucidated. Coupling plasma dd-cfDNA and biopsy gene expression data, a weak association between dd-cfDNA and injury as well as atrophy-fibrosis gene sets was noted. This supports the idea that dd-cfDNA correlates with unspecific parenchymal injury and not primarily with alloimmune mediated inflammation .per se. By being more comprehensive, such non-invasive biomarker could indicate invasive confirmation of a potentially treatable condition, in addition to rejection. Nonetheless, larger prospective studies, including heterogeneous real-life kidney transplant populations and integrating multiple layers of information  are needed to untangle dd-cfDNA biology in renal allograft recipients and eventually extend the applicability of dd-cfDNA testing in post-transplant monitoring.Such non-specificity of dd-cfDNA for graft rejection is not necessarily a disadvantage Second, beyond its value at time of clinical suspicion by avoiding some biopsies, timely detection of subclinical and/or incipient immunological activation is an even greater unmet need in post-transplant monitoring. Besides protocol biopsies, which cannot be performed as a serial testing approach, there are not many other options available for frequent surveillance of kidney transplant status and identification of subclinical rejection or graft injury , 12. WheIn conclusion, the graft-specificity and apparent intuitive use of dd-cfDNA% resulted in an acceleration of its clinical implementation in the United States. Despite a large body of research been done and slowly advancing insights into its added value, many questions and confusion remains, hindering more global implementation of dd-cfDNA% as biomarker in kidney transplantation. We must remain critical and focus on intrinsic biology and the best context of use. Especially the latter will be very important for reimbursement discussions with payers in, e.g., European countries.Moving from a promising biomarker to a widely used standard biomarker goes through larger prospective studies in real-life patient populations, and even randomized trials with clinically meaningful endpoints, such as the number of biopsies that can be avoided with non-invasive monitoring. This requires tireless efforts to integrate current monitoring practice with the results of dd-cfDNA measurements in a wide range of clinical scenarios.Unlike Tantalus, who is eternally close to food and water without ever reaching them, we are rapidly closing the knowledge gap around dd-cfDNA testing for kidney transplantation. Well-conducted studies evaluating clinical utility and context of use are needed to implement dd-cfDNA testing in routine clinical care in Europe.


Q. pyrenaica (0.1 and 460 MPa for 20 min) and Q. robur (0.1 and 333 MPa for 17.4 min) acorn starches to obtain high-valued ingredients. Pressure significantly altered the span distribution and heterogeneity of the acorn starch granules depending on the species, but their morphology was unaffected. Pressurization increased the amylose/amylopectin ratio and damaged starch contents, but the effect was more prominent in Q. pyrenaica than in Q. robur. However, the polymorphism, relative crystallinity, gelatinization temperatures, and enthalpies were preserved. The pressure effect on the starch properties depended on the property and species. The solubility, swelling power, and acorn gels’ resistance towards deformation for both species decreased after pressurization. For Q. pyrenaica starch, the in vitro digestibility increased, but the pseudoplastic behavior decreased after pressurization. No differences were seen for Q. robur. Regarding the commercial starch, acorn starches had lower gelatinization temperatures and enthalpies, lower in vitro digestibility, lower resistance towards deformation, superior pseudoplastic behavior, and overall higher solubility and swelling power until 80 °C. This encourages the usage of acorn starches as a new food ingredient.Despite being rich in starch, over half of acorn production is undervalued. High hydrostatic pressure was used to modify the properties of  Quercus spp. range from temperate to tropical latitudes in the Americas, Asia, Europe, and North Africa. They are evergreen, semi-deciduous, or deciduous trees that bear a small fruit called acorns [Native to the Northern Hemisphere, oak trees of the d acorns . There ad acorns . Despited acorns .Native starch is formed by granules that are almost exclusively formed by amylose and amylopectin. Amylose is a linear chain of α-D-1,4)-glucose residues, but amylopectin is highly branched since it has additional α-D-1,6)-glucose bounds -glucose . To endo-glucose Q. pyrenaica, and Q. robur acorn starch and compare them to commercial corn starch.High hydrostatic pressure (HP) is a non-thermal physical starch modification technology that endows starch properties from several botanical sources by using water and applying pressure levels from 100 to 600 MPa for 2 to 30 min at room temperature . HP can Q. pyrenaica and Q. robur species, as previously observed for lily, chestnut, rice, and maize starches up to 500 MPa [Commercial starch granules occur in a range of shapes, as shown in  500 MPa ,23. Acco 500 MPa . Acorn s 500 MPa ,16,25.The size and shape of starch granules are related to their botanical origin and are genetically controlled. During their biosynthesis in amyloplasts or chloroplasts, the physical structures of plastids can give a certain shape to the granules and also affect the arrangement of amylose and amylopectin . Hence, Q. pyrenaica, the commercial and acorn starches (P0.1/t20 and P460/t20) had a non-normal distribution (In the case of ribution . While tribution . Howeverribution .p < 0.05)  . These rribution . The sigribution , indicatribution ,31.Q. robur, the commercial and acorn starches (R0.1/t17.4 and R333/t17.4) had a non-normal distribution  and Q. robur  were comparable to those found for Q. wutaishanica  [The percentage of very small, small, medium, and large granules found for  14.14%) .Regarding amylose, the commercial starch had a significantly lower content when compared to the P0.1/t20 and R0.1/t17.4 starches .Q. pyrenaica and Q. robur starch increased significantly by 27% from P0.1/t20 to P460/t20 and 13% from R0.1/t17.4 to R333/t17.4, respectively. Previous authors also observed an increase in the amylose starch content after pressurization for litchi, maize, mango, potato, sweet potato, buckwheat, and sorghum starches [Q. pyrenaica  and Q. robur starches  were similar to those reported for Q. rotundifolia (53.7–54.5%) and Q. suber (57.9–59.4%) starches, respectively [Q. ilex (25.8%) [Q. palustris (31.4%) [Q. serrata (27.1%) [Q. suber (24.4%) [Q. calliprinos (29.2%) [Q. suber (34.4%) [Q. acutissima (30.6%) [Q. ilex (31%) [Q. wutaishanica (31.4%) [Q. ilex (39.0%) [Q. pubescens (19.5%) [Q. rotundifolia (41.7%) [Q. leucotrichophora (15.6%) [Q. brantii (~18%) [Q. coccifera (36%) [Q. suber (48.9%) [Q. pyrenaica and 43% from R0.1/t17.4 to R333/t17.4 for Q. robur, respectively  , Q. palu (31.4%) , Q. serr (27.1%) , Q. sube (24.4%) , Q. call (29.2%) , Q. sube (34.4%) , Q. acut (30.6%) , Q. ilexex (31%) , and Q.  (31.4%) , Q. ilex (39.0%) , Q. pube (19.5%) , Q. rotu (41.7%) , Q. leuc (15.6%) , Q. brani (~18%) , Q. coccra (36%) , and Q.  (48.9%)  starches (48.9%) . Indeed,ectively .Q. pyrenaica, both P460/t20 and commercial starches had a significantly higher content when compared to the P0.1/t20 starch  were more similar to the spectrum of the commercial starch than the corresponding native acorn starches (P0.1/t20 and R0.1/t174) .Q. pyrenaica, both commercial and P460/t20 starches had similar but significantly lower 1047/1022 ratios when compared to P0.1/t20 starch  were more similar to those previously reported for Q. wutaishanica (1.049) [Q. variabilis starches (1.082) [Q. robur starch  found in the present paper.Regarding the degree of short-range molecular order of 0 starch . Furthertructure . The 104 (1.049)  and Q. vQ. pyrenaica, the commercial starch had statistically lower 995/1022 ratios than the P0.1/t20 and P460/t20 starches  and Q. robur  starches were following the Q. wutaishanica (24.3%) [Q. glandulifera (23.5%) [Q. palustris (22.3%) [Q. variabilis (30.6%) [Q. rotundifolia (43.1–46.6%) and Q. suber (43.1–44.0%) [Q. fabri (48.2%) [Q. brantii (47.8%) [Regarding relative crystallinity, commercial starch showed a significantly higher value than acorn starches, but pressurization had no impact on acorn starch from Q. robur . The rel (24.3%) , Q. glan (23.5%) , and Q.  22.3%)  starches1–44.0%) , Q. fabr.3%  star2 < 0.96, p > 0.05), showing that solubilization and granular swelling occurred simultaneously, as previously reported for Q. rotundifolia and Q. suber [The solubility values of the different starches increased with the corresponding swelling power values  and Q. robur  starches diverged from those of Q. brantii  [Q. fabri  [Q. brantii  [Q. rotundifolia (9.1 g/g) [Q. suber (9.0 g/g) [Q. ilex  [The solubility and swelling power values of ctively) , Q. branctively) , Q. rotu9.1 g/g) , Q. sube9.0 g/g) , and Q. ctively) .It was verified that starch gelatinization is an endothermic phenomenon characterized by a positive increase in the enthalpies of the system  and Q. robur  starches were generally lower to those of Q. wutaishanica  [Q. fabri  [Q. palustris  °C, respectively) [Q. brantii  [Q. acutissima  [Q. glandulifera  [Q. ilex  [Q. rotundifolia  [Q. suber  [Pressurization did not affect the Tut flour . The To,ctively) , Q. fabrctively) , Q. paluctively) , Q. branctively) , Q. glanctively) , Q. ilexctively) , Q. rotuctively) , and Q. ctively)  starchesSince the gelatinization temperatures were not affected after pressurization, this reinforces the hypothesis that the decrease in solubility and swelling power of starches after pressurization could have been due to the formation of lipid–amylose complexes, as well as the scenario that the observed limited swelling power values were attributed due to strong interactions between amylose and/or amylopectin and/or the presences of amylose–lipid complexes ,35.Q. pyrenaica  and Q. robur  starches are in accordance to those found for the Q. brantii (14.9 J/g) [Q. acutissima (9.7 J/g) [Q. fabri (10.9 J/g) [Q. palustris (20.8 J/g) [Q. suber (4.2–4.3 J/g) [Q. wutaishanica (4.3 J/g) [Q. glandulifera (4.3 J/g) [Q. rotundifolia (4.2–4.3 J/g) [Regarding gelatinization enthalpies, the commercial starch also showed significantly higher gelatinization enthalpies than the acorn starches . This sh9.7 J/g) , and Q. 0.9 J/g)  acorn st0.8 J/g) , Q. sube4.3 J/g) , Q. wuta4.3 J/g) , Q. glan4.3 J/g) , and Q. 4.3 J/g)  starchesIt is thought that the commercial starch would have shown a greater relative crystallinity than the acorn starches since it had a lower amylose/amylopectin ratio . In otheQ. pyrenaica, both P460/t20 and commercial starches were shown to have statistically more digestible starch than P0.1/t20 starch  and Q. robur  starches diverge from those of Q. wutaishanica  [Q. variabilis starches  [Pressurization may have stabilized amylopectin and double-amylose molecules by increasing the content of olecules . These mctively)  and Q. vQ. pyrenaica  and Q. robur  had a similar order of magnitude to that found for Q. brantii (0.011 Pa.sn) [For 1 Pa.sn) .Q. pyrenaica, the commercial and P460/t20 starches showed a similar index of flow behavior when compared to P0.1/t20 starch. Pressurization from P0.1/t20 to P460/t20 led to a significant increase in the flow behavior index by 10%. The same effect was observed for mango, maize, and quinoa starches [Concerning the index of flow behavior, all analyzed starches had values lower than 1, indicating that starches were pseudoplastic fluids . For Q. starches ,32. ThisQ. pyrenaica starch showed a much greater increase in amylose after pressurization than Q. robur starch  and Q. robur  was in agreement with that found for Q. brantii (0.780) [Since r starch , the α-D (0.780) .H values, which corresponds to the linearization slopes for each of the starches. These values show how the complex viscosity decreases with frequency among the different starches  and Q. robur  were similar to Q. brantii (−0.94) [starches . Regardiic fluid . It is t (−0.94) .Q. pyrenaica, the complex index of the commercial starch was significantly lower than that presented by the P0.1/t20 and P420/t20 starches. However, pressurizing from P0.1/t20 to P420/t20 led to a significant increase in the complex index by 23%. This represents an increase in the dependence of the complex modulus with frequency, which, in other words, translates to a decrease in resistance towards deformation. For Q. robur, the complex index of the commercial starch was also significantly lower than that presented by the R0.1/t17.4 and P333/t17.4 starches. Pressurizing from R0.1/t17.4 to P333/t17.4 significantly increases the complex index by 8%. These results are lower than those found for quinoa [Q. pyrenaica than in Q. robur, it is considered that amylose retrogradation may have been facilitated more in Q. pyrenaica than in Q. robur, leading to a distinct increase in the complex index  and viscous (G″) components of the materials. The former is the elastic modulus and measures the material’s ability to store energy, whilst the latter is the dissipation module and measures the ability to dissipate energy [G′ > G″, with no crossover  and Q. robur  was higher than that found for Q. brantii (0.05) [Regarding the elastic moduli, they behaved as the complex moduli counterparts . In the behavior . Hence, i (0.05) .Q. pyrenaica, the viscous index value of the commercial starch was similar to the one presented by P0.1/t20. However, pressurizing from P0.1/t20 to P420/t20 led to a significant and sharper increase of the viscous index by 47%. For Q. robur, the viscous index value of the commercial starch was also similar to the one presented by R0.1/t17.4. Pressurizing from R0.1/t17.4 to P333/t17.4 also led to a significant and sharper increase of the index by 18%. This increase represents an increase in the dependence of the viscous modulus with frequency, which translates into a higher ability to dissipate energy. The viscous index of Q. pyrenaica  and Q. robur  was much lower than that found for Q. brantii (0.45) [Concerning the viscous modulus, it behaved differently than the elastic moduli . In the i (0.45) .Q. pyrenaica and Q. robur acorns under optimal conditions led to significant structural and property changes, depending on the species. Pressure affected the span distribution and uniformity of acorn starch granules, but the granular morphology remained unaffected. Pressurization increased the amylose/amylopectin ratios and damaged starch contents, particularly in Q. pyrenaica. However, relative crystallinity, polymorphism, gelatinization temperatures, and enthalpies remained unchanged. The properties of acorn starches were affected by pressurization, with decreased solubility and swelling power due to amylopectin depolymerization and increased complex resistance to deformation. Pressurization increased in vitro digestibility and decreased pseudoplastic behavior in Q. pyrenaica starch, while no differences were observed in Q. robur. Acorn starches may be more advantageous than commercial corn starch due to lower gelatinization temperatures, enthalpies, in vitro digestibility, superior pseudoplastic behavior, and lower resistance to deformation, which encourages acorn starch to be used as a food ingredient or additive food product, thus valorizing acorn starches.Pressurized starch extraction from Q. pyrenaica and Q. robur species were harvested in Parque Nacional da Peneda-Gerês, Portugal, in Assento, Terras de Bouro, and Braga. The Q. robur oaks were located at Parque Cerdeira  and the Q. pyrenaica oaks were found next to Rio Homem . Acorns were hand collected from the entire area of ground covered by the canopy of various oak trees according to their quality . Acorns were brought to the laboratory facilities the same day in thermal plastic bags, washed with tap water to remove soil and foliage, cleaned with a fabric cloth, and stored at −20 °C until further use.On 22 November 2018, acorns of the ®, Zamora, Spain) was purchased from a local supermarket.A commercial corn starch  acorn flour suspension was prepared with deionized water in low-permeability polyamide-polyethylene bags . Bags were manually heat sealed with minimum air and placed inside the high hydrostatic pressure vessel with an inner diameter of 200 mm and 2000 mm in length . The industrial equipment had a maximum operating pressure of 600 MPa and was connected to a refrigeration unit  to control the temperature of the input water used as a pressurizing fluid. Starch extraction conditions were optimized using different pressure levels  and extraction times  by response surface methodology. The compression rate used was 250 MPa/min and the decompression was instantaneous. Suspensions were centrifuged at 3000× g for 20 min, and the pellets were screened using 180 and 40 μm diameter mesh sieves, thoroughly washed with water, and left to stand at 4 °C overnight. The starchy material was collected and dried at 45 °C in a ventilated oven-drying chamber until constant weight.An 8%  scanning electron microscope (SEM). Samples were placed on top of the observation pins covered with double-sided adhesive carbon tape . Afterwards, the samples were coated with gold/palladium on a sputter coater  and visualized. SEM analyses were performed with the equipment operated at a high vacuum and an accelerating voltage of 20 kV using the secondary electron detector. Morphology was accessed according to the literature .10, D50, and D90 represented the particle sizes at which 10, 50, and 90% of all the particles by volume were smaller, respectively, and the Sauter and De Brouckere mean diameters  were recorded. Uniformity dispersion and span values were also registered. Granules were classified as large (>25 µm), medium (10–25 µm), small (5–10 µm), and very small (<5 µm) [Measurements were performed in triplicate using a Mastersizer Hydro EV 3000  by laser diffraction with a constant paddle rotation of 2100 rpm. The absorption index used was 0.10 and the refractive indices of water and starch were 1.33 and 1.53, respectively. The D (<5 µm) .Damaged starch was quantified in duplicate using a commercial kit from Megazyme, Wicklow, Ireland (REF: K-SDAM) based on the hydration of damaged granules and hydrolysis into maltosaccharides and α-limit dextrins with fungal α-amylase. Results were expressed in percentage  on a dry basis.Amylose was quantified in duplicate using a commercial kit from Megazyme International, Wicklow, Ireland (REF: K-AMYL) based on amylopectin precipitation by concanavalin A lectin. Amylopectin was computed by the difference between the total starch and the amylose content. Results were expressed in percentage  on a dry basis.−1 using a Spectrum 100 FT-IR Spectrometer . The measurement was conducted at room temperature, and spectra were recorded in the 400–4000 cm−1 range at 64 scans.Measurements were performed in triplicate in the mid-infrared region with a resolution of 4 cmaC is the crystalline area and aA is the amorphous area.Diffractograms were obtained in duplicate using a MiniFlex 600 diffractometer  with Cu-Kα radiation using a voltage of 40 kV and a current of 15 mA. Starches were scanned from 3 to 70° at a rate of 3°/min at a 0.01° step. Relative crystallinity (RC) was computed according to Equation (1), where w/v) was incubated in a water bath for 30 min from 50 to 100 °C. Suspensions were then cooled and centrifuged at 4100× g for 30 min. The supernatants were freeze dried using a vertical freeze dryer BK-FD12P  and weighed, whilst gels were weighed as is. Solubility and swelling power were computed according to Equations (2) and (3), respectively, where LSW is the weight of the lyophilized supernatant (g), SW is the starch weight in dry basis (g), and GW is the gel weight (g). Solubility and swelling power results were expressed in percentage  and gram of gel per g of non-solubilized starchy material on a dry basis (g/g SM), respectively.Solubility (S) and swelling power (SP) were measured in duplicate according to . A starco; °C), peak , and conclusion  temperatures, and gelatinization enthalpy (∆H) were obtained using the Pyris software (v13.4.0).Thermal characteristics were determined in triplicate using Diamond differential scanning calorimetry equipment  . The equThe rapidly digestible starch (RDS), slowly digestible starch (SDS), total digestible starch (TDS), and resistant starch (RS) fractions were quantified in duplicate using a commercial kit from Megazyme, Wicklow, Ireland (REF: K-DSTRS). Total starch content was defined as the sum of the total digestible (TDS) and resistant starch (RS) fractions. Results were expressed in percentages  on a dry basis.w/v) was prepared in triplicate using deionized water and heated to 90 °C in a water bath for 30 min. Suspensions were cooled down in a water bath until room temperature [−1 in 50 s and the experimental data were fitted into a power-law model using Equation (4), where σ is the shear stress (Pa), K is the consistency coefficient (Pa.sn), −1), and n is the flow behavior index (unitless).A starch suspension  was prepared in triplicate using deionized water and heated to 90 °C in a water bath for 30 min. Suspensions were cooled down in a water bath until room temperature [G*), elastic modulus (G′), viscous modulus (G″), and complex viscosity (η*), were recorded as function frequency (ω) and fitted using Equations (5)–(8), where K*, K’, and K″ are the consistency coefficients (Pa.sn) and n*, n′, and n″ are the index values (unitless).A starch suspension (8% d.b., perature . The gelEach sort of test involved the analysis of samples at random. After the Shapiro–Willk test confirmed that the data were normal, a one-way ANOVA was conducted to determine the statistical differences between the starches in all of the data. Data were reported as mean standard deviation, keeping the significant values permitted by the size of the standard deviation, and statistical tests were run using Tukey’s test at a 5% significance level.


Targeted biopsies of suspicious lesions on multiparametric magnetic resonance imaging (mpMRI) improve the sensitivity of systematic biopsies for clinically significant prostate cancer (csPCa) detection. However, systematic biopsies are still recommended, since a small, albeit significant, percentage of csPCa is only detected in them. In a contemporary series, 13.9% of csPCa cases were only detected in systematic biopsies, which represented 6.7% of its overall detection rate. The finding of independent predictive factors for csPCa detection in targeted biopsies allowed the possibility to develop a predictive model for avoiding systematic biopsies with a low risk of missing csPCa.The primary objective of this study was to analyse the current accuracy of targeted and systematic prostate biopsies in detecting csPCa. A secondary objective was to determine whether there are factors predicting the finding of csPCa in targeted biopsies and, if so, to explore the utility of a predictive model for csPCa detection only in targeted biopsies. We analysed 2122 men with suspected PCa, serum PSA > 3 ng/mL, and/or a suspicious digital rectal examination (DRE), who underwent targeted and systematic biopsies between 2021 and 2022. CsPCa (grade group 2 or higher) was detected in 1026 men (48.4%). Discrepancies in csPCa detection in targeted and systematic biopsies were observed in 49.6%, with 13.9% of csPCa cases being detected only in systematic biopsies and 35.7% only in targeted biopsies. A predictive model for csPCa detection only in targeted biopsies was developed from the independent predictors age (years), prostate volume (mL), PI-RADS score (3 to 5), mpMRI Tesla (1.5 vs. 3.0), TRUS-MRI fusion image technique (cognitive vs. software), and prostate biopsy route . The csPCa discrimination ability of targeted biopsies showed an AUC of 0.741 (95% CI 0.721–0.762). The avoidance rate of systematic prostate biopsies went from 0.5% without missing csPCa to 18.3% missing 4.6% of csPCa cases. We conclude that the csPCa diagnostic accuracy of targeted biopsies is higher than that of systematic biopsies. However, a significant rate of csPCa remains detected only in systematic biopsies. A predictive model for the partial omission of systematic biopsies was developed. Prostate cancer (PCa) is the leading malignant tumour in men from industrialised countries . The EurThe Barcelona predictive models and risk calculators were developed to predict the risk of csPCa detection just after PCa suspicion to avoid unnecessary mpMRI exams and before mpMRI to improve candidate selection for prostate biopsy. Both predictive models were developed at our centre and externally validated in the metropolitan area of Barcelona ,18. CurrAmong 3.557 men with suspected PCa, due to serum PSA > 3 ng/mL and/or a suspicious DRE result, enrolled in a prospective trial for the validation of the Barcelona risk calculators ,18 at 10MpMRI exams were always performed at each participant centre and reported with PI-RADS v.2.0 by an experienced radiologist. A pelvic phased-array surface coil was always used as a 1.5 or 3.0 Tesla MRI magnetic field strength.MRI-TRUS fusion images were obtained using the cognitive or software technique. TRUS-assisted prostate biopsy was performed via the transrectal or transperineal route. Targeted biopsies of suspected lesions (PI-RADS ≥ 3) were performed, obtaining between 2 and 6 cores, and 12-core TRUS systematic prostate biopsy was also conducted. Prostate biopsies were performed by an experienced urologist at each centre.Biopsy material was analysed in the pathology department of each participant centre by an experienced pathologist using the International Society of Urologic Pathology grade group (GG) to classify PCa. CsPCa was reported when GG was 2 or higher .CsPCa detection in systematic and targeted biopsies was the outcome variable for this study.The candidate predictive variables for the concordance between the systematic and targeted biopsies were age (years), serum PSA level (ng/mL), the type of biopsy , PCa family history (no vs. yes), DRE , prostate volume (mL), PI-RADS v.2 score (3 vs. 4 vs. 5), the magnetic field strength of mpMRI (1.5 vs. 3 Tesla), TRUS-MRI image fusion technique (cognitive vs. software), and the type of biopsy route . If independent predictive variables were found, the clinical utility of a predictive model developed to predict csPCa in targeted biopsies could be ve-rified.p values were lower than 0.05. SPSS v.25 was used to perform this statistical analysis.A statistical analysis was carried out through the harmonisation of the anonymised data sets provided by each participant centre. Quantitative variables are defined as medians and interquartile ranges (IQR: 25–75 percentile). Qualitative variables are defined as percentages. The recodification of quantitative to qualitative variables was performed to estimate the complementarity of the systematic and targeted biopsies. Odds ratios (ORs) and 95% of confidence intervals (CIs) were calculated. The concordance degree of the systematic and targeted biopsies was assessed with the Pearson chi-square test. Independent predictive variables of the concordance between the systematic and targeted biopsies were assessed in a binary logistic regression analysis. A predictive model for the ability to detect csPCa in targeted biopsies was developed from the identified independent predictors of csPCa detection in targeted biopsies. A receiver operating characteristic (ROC) curve was generated, and the area under the curve (AUC) was calculated to assess the discrimination ability of targeted biopsies to detect csPCa. The corresponding specificities to 100, 97.5, and 95% sensitivity threshold likelihoods were assessed, and the rates of avoided unnecessary systematic biopsies for csPCa were undetected. Significant differences were considered when The characteristics of the men with suspected PCa selected for this study are presented in PCa was detected in 1424 men (67.1%), with csPCa in 1026 (48.4%) and insignificant PCa in 398 (18.7%).p < 0.001. Discordance between the results reported in the systematic and targeted biopsies was observed in 509 cases (24%). CsPCa was detected only in targeted biopsies in 366 cases (17.3%), whereas it was only detected in systematic biopsies in 143 cases (6.7%), p < 0.001.The overall concordance–discordance between the systematic and targeted biopsies reporting csPCa is presented in p = 0.129.Considering the 1026 cases of detected csPCa, in 517 cases (50.4%), csPCa was detected in both types of biopsies. In 366 (35.7%) cases, csPCa was detected only in targeted biopsies, whereas in 143 (13.9%) cases, csPCa was only detected in systematic biopsies. The ISUP-GG of csPCa detected only in targeted biopsies corresponded to GG 2 in 233 cases (60.9%), GG 3 in 68 (18.6%), GG 4 in 51 (13.9%), and GG 5 in 24 (6.6%). The ISUP-GG of csPCa detected only in systematic biopsies corresponded to GG 2 in 99 cases (69.2%), GG 3 in 21 (14.7%), GG 4 in 15 (10.5), and GG 5 in 8 (5.6%). After Recoding the ISUP-GG in high-grade csPCa (GG ≥ 3) resulted in 44 cases (30.8%) being detected only in systematic biopsies and 143 cases (39.1%) being detected only in targeted biopsies, p < 0.001; prostate volume (≤53 >mL), p < 0.001; the MRI Tesla scanner (1.5 vs. 3.0 Tesla); PI-RADS score (3 vs. 4 vs. 5), p < 0.001; the TRUS-MRI image fusion technique (cognitive vs. software), p < 0.001; and biopsy route , p < 0.001.The odds ratios and 95% confidence intervals of the candidate predictive variables for the concordance between the systematic and targeted biopsies are presented in p < 0.001. Prostate volume (ref. ≤53 mL) presented an OR of 0.582 (0.480–0.720), p < 0.001. The MRI Tesla scan (ref. 1.5 Tesla) presented an OR of 1.463 (1.148–1.865), p = 0.002. The PI-RADS score (ref. 3) presented an OR of 1.590 (1.352–1.870), p < 0.001. The TRUS-MRI image fusion technique (ref. cognitive) presented an OR of 1.333 (1.046–1.701), p = 0.020. The biopsy route  presented an OR of 1.335 (1.052–1.694), p = 0.018. Age and prostate volume were clinical characteristics of suspected PCa in men for predicting concordance between systematic and targeted biopsies. The magnetic field strength of MRI and the PI-RADS score were characteristics of MRI, whereas the TRUS-MRI fusion technique and biopsy route were characteristics associated with prostate biopsy.The binary logistic regression confirmed that age (ref. ≤68 years) presented a concordance OR of 1.480 (1.182–1.803), p < 0.001. However, from a prostate volume ≤53 mL to >53 mL, the discordance rate decreased from 29 to 18.9%, p < 0.001 . After assessing the thresholds with 100, 97.5, and 95% sensitivity thresholds for csPCa detection, the corresponding specificities were 5.8, 22.5, and 27.4%. Using these parameters, we observed that 11 systematic biopsies were avoided without missing csPCa at 100% sensitivity. In total, 312 systematic biopsies were avoided (14.7%) and 25 csPCa cases were undetected (2.4%) in the targeted biopsies at the 97.5% sensitivity threshold. At 95% sensitivity, 368 systematic biopsies were avoided (18.3%) and 47 csPCa cases (4.6%) were undetected .The present study confirms that targeted biopsies are more sensitive than systematic biopsies for detecting csPCa. However, 6.7% of csPCa cases were detected only in systematic biopsies, whereas 17.2% of csPCa cases were detected only in targeted biopsies. Among 1026 csPCa cases, 13.9% were identified only in systematic biopsies, 35.7% were detected only in targeted biopsies, and 50.4% were identified in both. CsPCa cases detected only in targeted biopsies showed an increasing trend of higher GG, but this was not significant regarding those observed in systematic biopsies. These results are in line with other results previously reported, which supports the need for systematic biopsies ,25,26,27The detection of csPCa in systematic biopsies alone is mainly due to non-visible MRI lesions containing this type of tumour . This isIn 2009, Ahmed et al. suggested that men undergoing systematic biopsy are at risk of the underdiagnosis of csPCa and overdiagnosis of clinically insignificant PCa . In 2017Brisbane et al. observed that, in 2048 men with suspected PCa and PI-RADS ≥ 3, from two US institutions, 90% (95% CI 89–91%) of the csPCa cases detected were located within a radius of 10 mm from the nearest lesion. In total, 65% (95% CI 63–67%) of csPCa cases were detected within the region of interest identified on mpMRI, whereas 26% (95% CI 24–27%) of csPCa cases were detected in the perilesional area . RecentlOur study reported predictive variables for the concordance between systematic and targeted biopsies in csPCa detection. We observed that older age and a larger prostate volume increased the discordance between systematic and targeted biopsies, as also observed by Williams et al. . RegardiBecause independent predictive factors of the csPCa discrimination ability of targeted biopsies were found , a model for the prediction of csPCa in targeted biopsies was developed. The AUC of the model was only 0.741 (95% CI 0.721–0.762); however, the ROC curve presented good performance in the high-sensitivity zone, which allowed for 11 systematic biopsies (0.5%) to be avoided without missing csPCa at 100% sensitivity, whereas 14.7% of systematic biopsies were avoided, missing 2.4% of csPCa at 97.5% sensitivity, and 18.3% systematic biopsies were avoided, missing 4.6% of csPCa at 95% sensitivity. No predictive model for avoiding systematic biopsies in men with suspected PCa and PI-RADS ≥ 3 has been reported previously. We summarise that, currently, targeted biopsies are more sensitive than systematic biopsies for detecting csPCa. However, systematic biopsies are still needed to detect all csPCa cases. The newly developed predictive model could increase the avoidance rate from a small number of systematic biopsies without missing csPCa to up to 18.3% of systematic biopsies missing less than 5% of csPCa.The main limitation of our study is the definition of csPCa in prostate biopsies, which may not represent the true pathology of the entire prostate gland. The recent recruitment of selected men prevented knowing the evolution of detected tumours. This study is a post hoc analysis of a cohort of men who underwent systematic and targeted biopsies in a prospective trial designed to validate the Barcelona risk calculations in Catalonia. The prediction of the csPCa discrimination ability of targeted biopsies could be improved through other independent predictive variables not included in our study .68PSMA-PET (Ga68PSMA-PET-MRI) is promising. The metabolic activity detected in MRI-visible lesions or in areas without visible lesions can improve the effectiveness of targeted biopsies. The specificity of MRI could also be increased by differentiating visible lesions with no or low metabolic activity [Improvements in MRI, based on future increases in magnetic field strength, could enhance the visibility of csPCa. The combination of MRI with Gaactivity ,36. Addiactivity .Targeted biopsies currently remain more sensitive than systematic biopsies for detecting csPCa. However, 13.9% of csPCa cases were only detected in systematic biopsies, which represented 6.7% of the overall csPCa detection rate. The disagreement between the systematic and targeted biopsies for csPCa detection was multifactorial, with age, prostate volume, MRI magnetic field strength, PI-RADS score, TRUS-MRI image fusion technique, and biopsy route being independent predictive variables. The predictive model developed for csPCa detection in targeted biopsies was able to increase the omittance rate of systematic biopsies from 0.5% without missing csPCa to 18.3% missing 4.6% of csPCa cases.


We aimed to explorethe relationship between pulse wave velocity and urinary proteomics.We included 919 apparently healthy (no chronic illnesses) Black andWhite men and women  between 20 and 30 yearsfrom the African-PREDICT study. Capillary electrophoresis time-of-flightmass spectrometry was used to analyze the urinary proteome. We measuredthe carotid-femoral pulse wave velocity to estimate arterial stiffness.In the total group, pulse wave velocity correlated positively withcollagen-derived peptides including collagen types I, II, III, IV,V, and IX and inversely with collagen type XI . Regarding noncollagen-derived peptides, pulse wave velocitypositively correlated with polymeric immunoglobulin receptor peptides( Pulsewave velocity associated positively with collagentype I-derived peptides in a young and healthy population.Collagen type I is one of the main collagenproteinsfound in the vascular ECM and is responsible for the stability andfunction of blood vessels.The dysregulationof collagen types I and III turnovermay lead to increased arterial stiffness.1 and increased arterialstiffness, especially at younger ages, may reflect early vascularaging.2 Pulse wave velocity is a strongand independent predictor for increased cardiovascular disease (CVD)risk and all-cause mortality,4 even at young ages.5 Increased arterial stiffness with aging may be accelerated by factorssuch as oxidative stress, inflammation, endothelial dysfunction, andhemodynamic forces7 and may reflect subclinical organdamage.9 The molecular mechanisms underlying thesepreclinical changes are largely unexplored but remain important froma personalized medicine perspective. The use of omics-based biomarkersand, in particular, urinary proteomics may be useful in this regardsince it provides more insight into the structure and function ofa biological system than genomics.11Aortic pulse wave velocity (PWV) is the gold standard measurementof arterial stiffness,12 Various proteomics studies have successfully identified biomarkersunique to diseases such as coronary artery disease,14 chronic kidney disease,16 and heart failure.17 Previous proteomics studies focusing on arterialstiffness were limited to very small sample sizes,18 which were performed in older and diseased populations19 or in plasma samples from unhealthy patients with low and high PWV (mean age 65.5years old)20 or arterial tissue samplesin young and healthy adults (18–26 years old).18 To the best of our knowledge, no urinary proteomics studyhas focused on the early molecular phenotype of arterial stiffnessin young adults.Proteomicsis a rapidly growing field in “omics”that provides the ability to study the function, structure, and interactionsof proteins at a certain point in time.21 we compared the urinarypeptide abundances betweenlow, medium, and high CVD risk groups and found that collagen typeI and III- derived peptides were lower in the high compared to thelow CVD risk group, suggesting potential early alterations in thevascular extracellular matrix.21 Therefore,we aimed to determine whether a measure of large artery stiffness(PWV) is associated with a urinary proteomics profile in young adults,with a specific focus on vascular-specific extracellular matrix proteinssuch as collagen type I and III.In a recent study on urinary proteomics andearly CVD risk,22 Participants were recruited from the North-WestProvince of South Africa. If the participants met the inclusion criteriaof the screening phase ), they were invitedto join the research phase of the study. For this study, we included964 participants with complete urinary peptidomics data. All participantswith incomplete PWV data (n = 45) were excluded,leaving a total group of n = 919. Participants signeda written informed consent form to participate in both the screeningand research phase of the study. Both the African-PREDICT study (NWU-00001-12-A1)and this substudy (NWU-00495-19-A1) were approved and registered bythe Health Research Ethics Committee of the North-West University.Thisstudy forms part of The African Prospective Study on the Early Detectionand Identification of Cardiovascular Disease and Hypertension (African-PREDICT).The African-PREDICT study has a longitudinal design, aiming to characterizethe development of hypertension over a follow-up period of 10 years.Baseline data included 1202 healthy adults (aged 20–30 years).21A detailed description of questionnaires,physical activity monitoring, and anthropometric, cardiovascular,and biochemical measurements were previously described.23 ActiHeart physical activity monitors  were used to measure energy expenditure and to record the participant’sheart rate variability. The International Society for the Advancementof Kinanthropometry24 guidelines were followedto perform anthropometric measurements with the use of the followingapparatuses: a SECA 213 Portable Stadiometer to measure height, a SECA 813 Electronic Scales with a weighing capacityup to 200 kg  to measure weight (kg), anda Lufkin Steel Anthropometric Tape  tomeasure waist circumference (cm). Body mass index (BMI) was calculatedby dividing the weight (kg) by height (m2). A Dinamap Procare100 Vital Signs Monitor  withan appropriate sized cuff was used to calculate systolic and diastolicblood pressure as well as heart rate. Carotid-femoral pulse wave velocitywas measured noninvasively with the use of a SphygmoCor XCEL device. Participants were ina supine position and relaxed before the measurements took place.Pulse wave velocity was performed by placing a brachial cuff on theright upper-arm and measured in duplicate along the descending thoraco-abdominalaorta using a foot-to-foot velocity method.Briefly, a general health and demographicquestionnaire was completed for each participant. The Kuppuswamy’sSocioeconomic status scale was used to calculate each participant’ssocio-economic score for a South African environment.25 A detailed description on the biochemical analysis,sample preparation, and identification of urinary peptidomics waspublished previously.21Regarding biochemicalanalysis, blood sampling and an early morningspot urine sample were taken after the participant fasted for a periodof 8 h. Basic biochemical measurements included serum total cholesterol,low-density lipoprotein cholesterol (LDL-c), high-density lipoproteincholesterol (HDL-c), triglycerides, glucose, gamma-glutamyl transferase(GGT), glycated hemoglobin (HbA1c), C-reactive protein (CRP) , and serum cotinine . With regard to urinary peptidomics analyses,capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS)was performed using a P/ACE MDQ capillary electrophoresis system  coupled with a microTOF mass spectrometer as previously described.26 IBM SPSS Statistics version 25 software , and G*Power version 3.1.9.3 software 27 to perform statistical analyses.We used R version 3.6.0 software,n = 919)  were logarithmically transformed. We performeddescriptive analyses to summarize the characteristics of the totalgroup (n = 919) 1.2) transformed to obtaincomparable intensity ranges. In this study, we further explored whetherPWV correlated with the peptides previously identified as being potentiallyassociated with cardiovascular disease risk (n =147)21 by performing partial regressionanalysis ) and multivariableadjusted regression analyses (backward elimination regressions) todetermine independent relationships between PWV and the urinary peptides.For partial regression analysis, we also adjusted for multiple comparisons(Benjamini-Hochberg) . Thecovariates considered for entry in the multiple regression modelswere age, sex, ethnicity, MAP, heart rate, BMI, physical activity, LDL-c, GGT, cotinine, HbA1c, and CRP.With regardto peptide data, all peptides with >45% missing orundetectable values were excluded for further data analyses. The remainingpeptides were logarithmic  enrichment analysisis determined by a hypergeometric test followed by a false discoveryrate (FDR) distribution. The pathways were sorted according to anFDR ≤ 0.05, which describes how likely the pathway enrichmentis by chance.We used STRING database v11.5n = 919) are described in The general characteristicsof the study population .Pulse wave velocity also correlated positively with collagen alpha-2(I)(COL1A2)-derived peptides (represented by three peptides)  as well as collagen alpha-3(I) (COL3A1)-derivedpeptides (represented by six peptides) and negatively with peptidese06961 and e10876  (Supplementary Table 1).In thetotal group, after adjustment for mean arterial pressure, pulse wavevelocity correlated positively with several collagen alpha-1(I) (COL1A1)-derivedpeptides (represented by 12 peptides) and negatively with peptidese01100, e04169, and e06978 .Regarding noncollagen peptides, PWV positively correlated with a polymericimmunoglobulin receptor (PIGR) (represented by two peptides)  (Supplementary Table 1).Pulse wave velocity correlated positively withcollagen alpha-1(II)(COL2A1) (represented by one peptide), collagen alpha-2(IV) (COL4A2)(represented by one peptide), and collagen alpha3(V) (CO5A3) (representedby one peptide) and inversely with collagen alpha-2(XI) (COL11A2)(represented by one peptide) , PWV associated positively and independently withCOL1A1 (represented by five peptides) and COL1A2 (represented by onepeptide) as well as with PIGR (represented by one peptide) .In multivariable adjusted regression analysis (n = 3 types of peptides) , we identified two pathways for molecular functions (Supplementary Table 3). The functional annotationsof the identified urinary peptides revealed that the main Gene Ontology(GO) terms of molecular functions wherein the majority of urinarypeptides overlapped (COL1A1 and COL1A2) were (i) extracellular matrixstructural constituent conferring tensile strength and (ii) platelet-derivedgrowth factor binding. Also, in STRING database (Supplementary Table 3), Reactome pathways involved in thevascular ECM included (i) anchoring fibril formation, (ii) plateletadhesion to exposed collagen, (iii) cross-linking of collagen fibrils,(iv) platelet aggregation (plug formation), (v) collagen chain trimerization,(vi) collagen degradation, (vii) ECM proteoglycans, (viii) integrincell surface interactions, and (ix) cell surface interactions at thevascular wall.After performing pathway analysiswith the peptides that associated with PWV in multivariable adjustedanalysis . We found that the gold standard measure of arterialstiffness, PWV, was associated significantly and independently withseven urinary peptides. The majority were collagen type 1-derivedpeptides. Moreover, in STRING analysis, we also identified molecularfunctions and Reactome pathways associated with the vascular ECM.We suggest that if these pathways are dysregulated, it may lead tothe earlier development of arterial stiffness and should thereforebe closely monitored in high risk individuals from a young age.19 However, proteomicsstudies focusing on arterial stiffness are limited and it remainschallenging to compare results across different age groups and varioussample matrices in either healthy or diseased populations.20 Recently, a study developed a PWV urinary proteomics score in theFlemish Study on Environment, Genes, and Health Outcomes population(mean age 50 years old) and found it to prospectively associate withall-cause mortality and cardiovascular outcome over a period of 9.2years.19 In the latter study, the majorityof the peptides in the urinary proteomic profile consisted of collagentype I and III fragments and were mostly associated negatively withPWV. This is in contrast with our findings of positive associationsbetween PWV and urinary peptides. We suggest that with advancing age,the impact on collagen generation may change with increasing collagenaccumulation, increased nonenzymatic glycation and collagen cross-linking,29 and consequently less collagen degradation.Nonetheless, our findings add to this since we identified collagentype I fragments to be associated with PWV in a large young and healthypopulation without end-organ damage, which may suggest that the dysregulationof collagen type I turnover may lead to increased arterial stiffnessin the setting of early vascular aging.The urinary peptides associated with PWV in this study are similarto previous proteomics studies in the field of arterial stiffness,such as collagen types I, II, and III.30 Collagens type I and III are found in the tunicaadventitia of the arterial wall and play a pivotal role in the mechanicaland tensile strength and contractility of arteries,30 whereas elastin, found in the media of the arterial wall,is responsible for vascular elasticity.32 As such, anunbalanced vascular ECM turnover will stimulate vascular remodelingand may consequently induce early vascular aging and increased arterialstiffness.Collagen type I and III are the main collagen proteins in the vascularECM with collagen type I being the most prominent of collagen fibrils.33 In addition to the respective peptides, GGT,a marker associated with vascular inflammation,34 also contributed significantly to the variance in PWV.With an increase in vascular inflammation, matrix metalloproteinases(MMPs), which are responsible for the degradation of proteins in theECM, are also up-regulated.35 IncreasedMMPs, such as MMP2 and MMP9, are associated with arterial stiffness,even in younger and apparently healthy subjects.37 We therefore propose that if vascular inflammation increases, proteaseactivity may also increase, leading to early changes in the vascularECM, including degraded and fragmented elastin and increased collagendegradation.38 This may lead to the up-regulationof collagen biosynthesis39 and depositionto counteract the potentially harmful effects of inflammation andMMPs on the vascular ECM structure, which may over time enhance collagencross-linking and increase arterial stiffness.40In these young and healthy subjects withoutend-organ damage, thereis no evidence of chronic pathological processes on collagen cross-linking.We propose that if inflammation and protease activity increase asseen with increasing arterial stiffness, urinary collagen fragmentswill also increase, hence the positive associations between PWV andurinary peptides. In pathway analysis, we showed that collagen typeI-derived peptides are involved in processes that are related to thevascular ECM, such as platelet aggregation, collagen cross-linking,and degradation. Research has shown that platelets are recruited toinflamed vessels or to the site of injury and can perform pro- oranti-inflammatory reactions, depending on the cause of inflammation.42 This may lead to theup-regulation of collagen biosynthesis and deposition to maintainvascular ECM homeostasis in the midst of higher mechanical strainexerted on the arterial walls, which may ultimately result in poorlyorganized nonenzymatically collagen cross-linking,32 contributing to increased arterial stiffness. We thereforepropose that collagen type I-derived peptides may play a key rolein our understanding of structural vascular ECM changes, leading tohigher arterial stiffness and early vascular aging. Regarding noncollagenpeptides, PWV associated significantly with PIGR; however, more researchis needed to explore the possible role between PIGR and arterial stiffness.In addition to the respective peptides, MAP (as expected)alsocontributed significantly to the variance in PWV. Since we found positiveassociations between PWV and collagen type I-derived peptides, wepropose that with increased blood pressure, mechanical stress in bloodvessels will be elevated.To the best of our knowledge,this is the first urinary proteomicsstudy to show independent associations between PWV and vascular ECMspecific peptides involved in pathways related to extracellular matrixorganization in a young and healthy population. Even though PWV iswithin normal ranges in this study, the impact of PWV on urinary peptidesis significant and consistent. This study did not include MMP data,and further investigation is proposed to test our hypotheses withregard to the role of MMPs with urinary peptides in the early changeswithin the vascular ECM. Data collection for the first follow-up phaseof the African-PREDICT study is continuing, which will enable hypothesistesting in a longitudinal setting, to explore the development of arterialstiffness and early vascular aging over time, as well as the predictivevalue of the identified peptidome in young asymptomatic adults.In conclusion, in healthy young adults, we found positive and independentassociations between PWV and collagen type-I derived peptides. Ourfindings likely reflect early mechanisms for early vascular agingassociated with the structural integrity and function of the vascularECM as described by peptides that indicate collagen turnover, hightensile strength under mechanical strain, and cell signaling for vascularremodeling.