Although malnutrition risk is well documented in elderly care institutions, few studies have been conducted to address concerns regarding community-dwelling elderly people. This study has been aimed to describe the nutritional status and its related socioeconomic and geriatric factors in community-dwelling elders with malnutrition.n = 326). Information on nutrition status (full MNA) and health information, like cognitive status (MMSE), daily functional status  and frailty was obtained. Multiple logistic regression analyses have been carried out, in order to identify the association of demographical and clinical factors with malnutrition.For this study, a randomized sampling among people aged 60 has been done , multiple physical disabilities  and chronic disease  were independently associated with malnutrition. Also financial independency , functional ability on ADL  and IADL , normal cognitive  and no frailty  independently were inversely associated with malnutrition. The model was adjusted for all socio- demographic and health variables that were significantly related in the previous models.Our results indicated a strong correlation between malnutrition and health status. Identifying predictive factors can potentially improve prevention and management strategies used for malnutrition in elderly. Malnutrition in the elderly is defined as undernourishment, described by inadequate food intake, poor appetite, and loss of muscle and weight . Health The prevalence of malnutrition in the elderly living in the community is relatively low 6–11%); however the rates are higher among hospitalized people or those in residential care centers 32–64%) ..5]. Esti–11%; howThis variation depends on the malnutrition diagnostic criteria, the country investigated, the population being rural or urban, residence type, having multiple chronic diseases, and the level of socioeconomic status. Despite these remarkable data, nutritional problems are still not recognized as a necessity in the management of the elderly. Evaluation and nutritional interventions are very important in this population with high prevalence of chronic diseases including depression, dementia, functional disorders, polypharmacy , 10. MalDespite the fact that previous studies have investigated determinants of malnutrition, research has not focused on a possible joint effect of multiple health factors that contribute to poor nutritional status among Iranian population. In addition to that, much of the previous researches on screening and prevention has largely been focused on the disabled elderly or resident in institutions; so the healthy elderly people were somewhat ignored. This study is aimed to determine the prevalence of malnutrition in community-dwelling elderly. The relationship between socio-demographic characteristics, as well as physical and mental health indicators and nutritional status has also been evaluated.The participants were 331 community dwelling elderly, aged 60 years and over, living in Babol city, from Mazandaran province, Iran. Babol is a northern City of Iran with 12 urban health care centers out of which, 6 centers have been selected randomly.Convenience sampling technique was used to choose among the elderly people attending the areas, serviced by each responsible health center, including elderly entertainment venues, parks and mosques. Considering these factors, 55 elderly citizens entered out study from each health center.Eligible elderly were not diagnosed with Alzheimer, intense mental disorders or serious diseases. Individuals would opt out from the study for the absence of consent or incomplete filled out questionnaire. Hence, 326 questionnaires were analyzed out of a total of 331 questionnaires. The questionnaires were completed by participants in the presence of the researcher .A standard multi-part questionnaire was used to assess nutritional status as an outcome variable and some other explanatory variables.Assessment of nutritional status of the subjects was conducted by a Persian version of the mini nutritional assessment (MNA) . The MNAThe Socio-demographic variables are composed of gender, age, place of living, marital status, education, number of children, occupation, residential home type, financial dependency, and adequacy of family income. All participants were covered by health insurance.Health status was described by chronic illnesses, type and amount of medications taken daily; as prescribed by a physician. We also looked for physical disabilities , psychological problems , smoking status and self-rated health.Functional ability was analyzed through Katz Index of Independence in Activities of Daily Living (ADL) and Lawton –Brody Instrumental Activities of Daily Living Scale (IADL) , 16. ADLIADL is an appropriate tool to analyze independent living skills. The skills that are assessed in this tool are more complex compared to the basic daily activities, as measured by Katz Index of ADLs. The instrument is most useful for identifying the way a person functions at the present time, and to identify any improvement or deterioration over time. There are eight areas of performance, measured by the Lawton IADL scale. Women’s performance is rated, in all eight areas. The areas of food preparation, housekeeping, laundering have been historically excluded for men. Clients are scored in each category according to their highest level of performance. For women a summary score ranges from 0, indicating dependence and low function, up to 8, which shows high function and independence. For men, the score ranges from 0 to 5. Full function is indicated by the scores of more than 7 and 4 for women and men respectively. Moderate impairment in women and men is shown by a score of 4–6 and 2–3, and severe functional impairment by the score of 3 and 1 or less, respectively.Cognitive status was described by the mini-mental state examination (MMSE). Since the cut-off point of 23 was reported for this test in the Iranian population , the resFrail status of the participants was assessed based on the five criteria by FRAIL scale items in AAH . The FRAEthics standards on human experimentation, as stated by the responsible institutional and national committee and the Declaration of Helsinki, were properly followed while carrying out procedures. Moreover, the study was approved by the ethics committee of Babol University of Medical Sciences (NO.:MUBABOL.REC.1388.1). All participants provided an informed written and signed consent form. All patients provided written informed consent.2 test was used to assess binary associations between socio-demographic variables and physical and mental health indicators and nutritional status. ANOVA test was also applied to compare means of numerical variables including age, number of children and physical disabilities with nutritional status classes. In addition to those, multiple logistic regression was used to determine independent variables related to nutritional status so that all independent variables were entered simultaneously in a full model. Nutrition status was considered as the dependent variable in two classes of poor  versus normal. The independent variables that were associated with nutritional status in the binary analysis (p ≤ 0.05) were included in these models. In Model 1, all socio-demographic predictors of nutritional status were entered at the same time. Model 2 presented health-related features . All variables that were significantly associated in the previous two models as independent variables were included in Model 3.Data were analyzed using the SPSS version 19.0. XThe study sample consisted of 115 elderly women and 211 men. Their mean age was 68.82 ± 7.15 years with the prevalence of malnutrition and risk of malnutrition 3.0 and 25.1%, respectively. Women’s nutritional status was considerably worse than men.p < 0.001). 46% of the participants who lived with their children were either malnourished or at risk of malnutrition, while it was lower in the elderly who lived alone (33.4%). Greater level of nutritional disorders was also observed amongst illiterate people (46%).Personal characteristics` binary analyses are depicted on Table p < 0.001). In regards to functional capacity (ADL and IADL), the findings showed functional decline in malnourished individuals and those at malnutrition risk compared to individuals with good nutritional status (p < 0.001). In addition to that, the largest proportions of frail subjects were at risk of malnutrition. Risk of malnutrition was found in 8.1% of the non-frail, compared to 37.3% in the pre-frail and 58.3% in the frail participants (p < 0.001). Furthermore, an association between cognitive function and nutritional status was demonstrated (p < 0.001). Normal cognitive performance is associated with decreased ratios of malnutrition and at risk individuals.Health status is shown in Table The prevalence of major diseases associated with frailty is shown in Fig. Table p < 0.001) was identified among poor nutritional status and several physical disabilities and comorbidity, perception better than individual health, impaired cognitive function, dependency on ADL and IADL and being frail compared to normal. The strongest nutritional risk factors were frailty (OR: 0.253), dependency vs total independency in IADL (OR: 0.319) and normal cognition vs low cognition (OR: 0.456).In model 2 , a relationship . The Cox & Snell RIn terms of health variables, multiple physical disabilities and comorbidities were also found to be highly correlated with lower nutritional status . Furthermore, elderly people with low cognition were 2.19 times more at risk of malnutrition or malnutrition. Being Independent for ADL was associated with 46.4% risk reduction of poor nutrition . Odds ratio as well, decreased with increased independency in IADL, rising moderate to total independency (0.782 to 0.319). Eventually, a strong and consistent association was demonstrated between nutritional status and frailty; frailty increased the risk of impaired nutrition 3.95 fold compare to normal people. However, the relation between poor nutritional status and pre-frailty was not significant.The present study used the full MNA tool to determine the prevalence of impaired nutritional status in elderly people of 60 years and above. This is one of the few studies to describe the prevalence of malnutrition and its associated socio-economic and geriatric factors in Iran. We found that 3.0% of the participants were suffering from malnutrition while 25.1% were at risk of malnutrition. The findings also revealed that single marital status , financial dependency, multiple physical disabilities and comorbidity, cognitive decline, impaired ADL and IADL function, and frailty were independently associated with the MNA in the multiple model.A systematic review of the prevalence of poor nutrition in different parts of Iran revealed that the prevalence of malnutrition or at-risk of malnutrition among the free-living elderly is between 3 and 10.6% and 32.7–46.7%, respectively. However, this rate was 3.2–53.6% and 38.7–68.8% among the elderly residents of nursing homes . HoweverIn terms of socio-economic status, financial independence had a strong inverse association with malnutrition; even after adjusting the potential confounding factors. In a study conducted on elderly of rural Bangladesh, low income was highly correlated with poor nutrition . Other rLiving alone was associated with a risk of malnutrition/malnutrition in our study. This finding is inconsistent with some previous studies showing that people who live alone are not at greater risk of malnutrition , 25. AltThe results of comprehensive geriatric evaluation in the present study showed that comorbidity, multiple physical disabilities, decreased cognition, impaired ADL and IADL function and frailty were associated factors of poor nutrition. Schilp et al.  evaluateAmong these geriatric conditions, a decline in cognitive functioning is itself a risk factor for malnutrition . In the Even mild cognitive decrease can be associated with dietary changes. A study carried out by Kimura et al.  suggesteIn the present study, ADL and IADL dysfunction also showed a significant relationship with malnutrition. Similar results were found in many of the previously conducted studies on outpatient elderly clinics , 20, 21.Frailty was found in 69.4% our study participants who were malnourished or at risk of malnutrition. Malnutrition prevalence was 4.5 folds higher in the elderly with frailty compared to healthy subjects. Nutritional status was closely associated with the degree of frailty, so that prevalence of poor nutrition diminished progressively in the pre-frail group and non-frail group. However, the relation between poor nutritional status and prefrailty was not significant. Our result is comparable to the Maseda study  and BoulRegardless of whether physical frailty or malnutrition occurs first, there is possibly a closed-loop cyclical association between the two in progression towards a combined frailty-malnutrition state. This association is reflected by the progressive increase in the prevalence of frailty across MNA-SF normal nutrition and at risk and malnourished groups, as well as the progressive increase in MNA-SF malnutrition prevalence from robust to prefrail to frail groups. However, this finding should be further investigated in prospective follow-up studies. The results of a study by Valentini et al.  indicateIt is widely believed that extensive physiological and psychosocial changes due to aging would make it more difficult for the elderly to meet their nutritional needs. However, those elderlies who suffer from geriatric problems such as physical dysfunction, cognitive decline and frailty, would face difficulty meeting their nutritional needs anyways. Meeting the nutritional needs of these people is very important for maintaining their health, functional independence and quality of life. In Iran, health treatment system and policies aim to make changes in the health care system in favor of the society; in its new policies, it is important to consider the nutritional needs of the elderly, especially those affected by geriatric problems. This is likely leads to an increase in independence and reduction in subsequent requests for social care and hospital admissions which leads to a less invasive and expensive health care.Incorporating multiple evaluation sets of potential socio-demographic and health determinants is an important strength of this study. These determinants have been analyzed to determine their possible association with presence or risk of malnutrition. However, this study includes several limitations, such as the cross sectional design by which establishing causality isn’t possible. In addition, information bias may be present because of cognitive status differences among individuals. Another limitation of this study is reliance on self-report for some measures including type and number of chronic diseases. Also, some remaining unidentified factors and residual bias may be present. Finally, we used non-probabilistic sampling, in which the samples might did not represent the actual population, properly.From this study, we conclude that elderly individuals who suffer from multiple physical disabilities, chronic illnesses and cognitive and functional decline, those who are financially poor, and those with a single marital status were at higher risk of malnutrition. Impaired nutritional status was clearly related to frailty. To clarify the findings, more research is needed. In addition to that, raising awareness of health professionals and caregivers about malnutrition needs to be addressed in the context of the demographic changes. Special training is required to strengthen the community-based knowledge about nutrition and to provide routine screening for vulnerable groups such as elderly with financial hardships, those without a spouse or those suffering from multiple diseases. Similarly, elderly people have to be provided with newly developed nutritional guidelines.


This work investigates a new generation structural health monitoring (SHM) system for fibre metal laminates (FML) based on an embedded thermoplastic film with compounded piezoceramics, termed piezo-active fibre metal laminate (PFML). The PFML is manufactured using near-series processes and its potential as a passive SHM system is being investigated. A commercial Polyvinylidene fluoride (PVDF) sensor film is used for comparative evaluation of the sensor signals. Furthermore, thermoset and thermoplastic-based FML are equipped with the sensor films and evaluated. For this purpose, static and dynamic three-point bending tests are carried out and the data are recorded. The data obtained from the sensors and the testing machine are compared with the type and time of damage by means of intelligent signal processing. By using a smart sensor system, further investigations are planned which the differentiation between various failure modes, e.g., delamination or fibre breakage. Resistance strain gauges ,8Fibre optic sensors, e.g., fibre Bragg grating (FBG) strain sensors ,10,11Active or passive piezoelectric sensors ,13,14Sensors using the electrical properties: resistance, impedance, dielectric, etc. ,16Non-destructive testing (NDT) is used to detect failures inside of polymer based lightweight structures, but this is usually only carried out offline and involves complex and cost-intensive methods, e.g., ultrasonic testing , eddy cuThe monitoring of the structural condition of lightweight structures can be carried out either by local sensor placement at positions with a high inclination to initial damage or by remote placement of the sensor in order to carry out an area measurement. Depending on the physical measurement principle and the type of structural monitoring, NDT methods can be divided into passive detection and active detection of structural integrity. The passive method only monitors structural/material properties or the occurrence of a damage event, whereas the second method actively interrogates the structure to derive a comprehensive structural behaviour.New approaches of passive sensing are based on online-capable SHM systems that combine advanced sensor technologies with intelligent algorithms and thus allow a diagnosis of the structural integrity of the monitored component ,18. In aThe use of such SHM systems is particularly relevant for classic highly stressed structures in mobile applications , which are increasingly being replaced by hybrid material systems based on metal/plastic pairings . The aimWith the increasing use of such hybrid material systems, however, production- and application-related requirements for the use of SHM systems are also changing. While less installation space is available for additional functions due to increasingly complex assemblies, the demand for increased function density is growing. To solve this contradiction, it is necessary to integrate a minimum number of discrete sensors into the structure, which monitor the structural integrity over a wide area ,22.A preliminary study showed that the PFML has potential for use as an SHM system, after which an approach for the feasibility of spectral energy density (SED) was investigated. It was observed that the SED of an undamaged PFML increases slightly independent of force and dispersion, whereupon the energy signals were analysed in more detail by means of descriptive statistics. It could be shown that a reproducible and characteristic trend of the standard deviation is obtained. The preliminary study was a proof of concept with a small number of samples and was therefore not published. Building on this, the present study analyses the signals with regard to a validation of the PFML as an SHM system. The PFML consists of a thermoplastic piezoelectric material which, in combination with the FML, creates a passive sensor system and therefore forms an integral part of the component. The basis for the mostly required large-scale production is thus a PFML, which is manufactured in a continuous production process ,18,22.an aluminium sheet (EN AW-6082 T4)a piezo-active sensor layera reinforcement layer of glass fibre-reinforced plastics (GFRP)The test specimens in this work are asymmetric hybrid laminates as shown in Two different types of sensor layers are investigated. The first type of sensor layer is based on a piezo-active compound, which is made from polypropylene ) filled with 70 wt.% piezoceramic lead zirconate titanate (PZT) powder ) and 0.5 wt.% carbon nanotubes ) ,24. Thisum sheet . Two copum sheet ,27. In dAfter functionalisation with the sensor layer, the aluminium sheet is combined with the reinforcement layer made of GFRP. It has a fibre volume content of ~45% and consists of bidirectionally aligned 0aluminium-based fibre metal laminate with PZT/PP film sensor layer and glass fibre epoxy reinforcement (PFML-PZT-EP)aluminium-based fibre metal laminate with PZT/PP film sensor layer and glass fibre polypropylene reinforcement (PFML-PZT-PP)aluminium-based fibre metal laminate with PVDF film sensor and glass fibre epoxy reinforcement (PFML-PVDF-EP)aluminium sheet with PZT/PP film sensor layer, but without fibre-reinforcement as reference specimen for static mechanical testing (AL-PZT)After joining the functionalised aluminium sheet with the reinforcement layer and curing the specimens (epoxy: 24 h at RT), they are cut to the outline dimensions of 150 mm × 40 mm. The average thickness is approximately 1.5 mm. The studies in this work started with the investigation of the PFML-PZT-EP specimen. This was the laminate type of the preliminary tests. Based on this, the active material of the sensor layer and the matrix material of the reinforcement layer were varied.The tests for static and dynamic testing of the laminates were carried out using a three-point bending test. The experimental set-up consists of a punch with a radius R = 5 mm and two supports with a radius r = 2 mm. The distance L between the supports is determined by:D7774-17  standardA total of three studies were conducted. The first, a case study, was the preliminary test to determine the applicability of the piezoceramic film. The second study was performed without audio measurement and the third was a repetition of the second study, but with audio measurement to be able to reference the signals clearly. The second and third study are the focus of this publication; they are further divided into a static and a dynamic test. For each of the three laminate types, three specimens were used for the static test and five specimens for the dynamic test. From the PFML-PVDF-EP test series, three of the static and one of the dynamic tests were conducted with audio measurement. In the dynamic PFML-PZT-EP and PFML-PZT-PP dynamic test series, the audio signals of two specimens in each series were recorded. In total, 24 specimens were investigated in studies 2 and 3.In the static test series, the punch moves in a path-controlled mode until the specimen fails. The test is aborted by the machine if the force change is less than 70% of the last force value. In the dynamic test series, a cyclical force curve was specified, which is shown in During mechanical testing, the mechanical load, the specimen deformation, the signals of the sensor layer and the acoustic emissions of the specimen are recorded. The punch force and the deflection of the specimen during the three-point bending test are recorded directly by the universal testing machine with a sampling rate of 50 Hz. The two integrated sensors of the test specimen, the additional microphone and the force output of the universal testing machine are connected to a data acquisition system . The two sensor channels and the microphone channel are recorded synchronously with a sampling rate of 50 kHz, while the force signal is recorded with a sampling rate of 100 Hz.As the data was recorded independently on the universal testing machine and the data acquisition devices with individual start times and sampling rates, data synchronisation is required. To achieve a comparable sampling rate, all recorded data from the universal testing machine and the data logger were interpolated to the sensor sampling rate of 50 kHz. Afterwards, the time offset of the signals was determined based on the force signal since this was recorded on both machines. To determine the time offset, the cross-correlation of the interpolated force signals and the offset of the global maximum were determined. Subsequently, the summed absolute difference of the force signals of both determined delays was calculated and represents the decision criteria for the correct time delay estimation. Consequently, all signals are displayed with a sampling rate of 50 kHz and synchronised by the force signal.In a first preliminary study, the possible use of the novel thermoplastic-based fibre-metal laminate in combination with spectral energy density (SED) was analysed for applications in the SHM sector. It was observed that the SED concentrated in a certain range independent of force and dispersion while the object is structurally healthy. For this reason, the energy signals were analysed using descriptive statistics as in state-of-the-art methods using the SED for structural health monitoring applications with piezoelectric sensors ,30,31. IS was decomposed into measurement windows of size k represents the maximum number of measurement windows. Each measurement window To analyse the feasibility of the SED, the sensor signal j is determined by:f of the Fourier transform of measurement window P was calculated using a sliding window of size 50. Furthermore, using the normalized SED leads to a lack of realisation of global intensity changes [The SED  changes , which sThe accuracy of the signal processing results depends on the window size However, most state-of-the-art methods use the wavelet packet-based energy index ,34,35 foj and in the frequency band i calculated on the basis of CWT. i. This adjustment is necessary due to the different specimen types used. The bridge bent-caps have a much higher potential energy leading to less natural vibration production, whereas the thermoplastic-based fibre-metal laminates used in this study represent lightweight specimens that tend to produce natural vibration, averaged in this way. The energy j of frequency band i is calculated by the CWT decomposition [A breaking of the glass fibres creates a vibration in the laminate, which in turn results in an energy change. This energetic change leads to a change in the statistical distribution of the measured values, which is quantified by the standard deviation allowing the difference from the normal state of the laminate to be observed. Furthermore, the resulting reduced stiffness of the specimens leads to a global increase in the voltage change in the piezoactive areas due to the increased bending of the laminate and thus to an increased energy level as long as no deformation occurs. The damage index methodology is comparable because it measures the energetic deviation from the normal state of the specimen caused by the same damage based vibrations. For this reason, the damage index is included in the analysis as a further indicator for the usability of the thermoplastic-based fibre-metal laminate for the SHM sector. The calculation of the damage index for measurement window g et al. , who useposition ,41 of SjIn this study, the Daubechies wavelet base db9 is used for CWT, leading to non-overlapping measurement windows due to the orthogonality of the Daubechies wavelet base. Since the same wavelet base was used by Song et al. , the comFrom the previous description, the following hypotheses are derived for the further investigation of the feasibility of the novel thermoplastic-based fibre-metal laminate in the field of SHM:Hypothesis 1 (H1).The standard deviation of the SED indicates the time of complete failure.Hypothesis 2 (H2).The standard deviation of the SED results in a characteristic trend formation.Hypothesis 3 (H3).The standard deviation of the SED indicates the damage type.Hypothesis 4 (H4).The damage index indicates the time of complete failure.Hypothesis 5 (H5).The damage index serves as an indicator for the health status of the specimen.S,D,For clarification and comparison of the tests, the results are divided into the sub-chapters static and dynamic tests. For comparison purposes, the maximum force An overview of the specific mechanical properties of thermoplastic and thermoset laminates is given in The comparison of all specimen types can be seen in the force–displacement diagram . As descThe failure of the two laminate types under static load is very different. This was recorded using a light microscope. The thermoplastic laminate fails mainly due to delamination in the bending legs . It is nThe failure of the thermoset laminates is mainly fibre cracking, which is shown in The analysis results of the standard deviation of the SED regarding a significant peak formation at the moment of complete failure for the static tests are shown in With regard to the damage index, it can be seen that this forms a significant peak for all specimens and window sizes from 256 to 2048 at the time of complete failure. Furthermore, the damage index results of laminate PFML-PVDF-EP show significant peak formation for all samples in all window sizes, except 32,768. On the contrary, one specimen of the laminates PFML-PZT-EP and PFML-PZT-EP with window sizes larger than 4096 does not form a significant peak at the time of complete failure.The trend results of the formation analysis are shown in On the other hand, the damage index forms a trend for all specimens for laminate PFML-PVDF-EP and PFML-PZT-PP, while just one specimen of laminate PFML-PZT-EP forms a characteristic trend in the best case. For both analysed methodologies, damage index and standard deviation of the SED, no best suited window size for characteristic trend formation can be determined, since PFML-PVDF-EP achieves the best results with a measurement window size greater than 1024, and PFML-PZT-PP at window size 512.Taking into account the complete failure type of the laminate specimens used in the static tests, it is seen that all specimens of PFML-PZT-PP are delaminations, while all specimens of PFML-PZT-EP result in fibre breakages. Laminate PFML-PVDF-EP delaminated once, while two specimens show fibre breakages. In terms of complete failure detection, no significant difference can be observed, since many configurations in all investigated samples lead to significant peak formation at the moment of complete failure. On the other hand, trend analysis shows that the delaminated specimen of laminate PFML-PVDF-EP forms a trend only at window size 8192 and standard deviation of the SED use, independent of the scaling. However, laminate PFML-PZT-PP forms no trend by linear scaling and standard deviation of SED use, but two out of three specimens form a characteristic trend at window size 4096 to 16,384 using the decibel scale. In case of laminate PFML-PZT-EP, only one specimen forms a characteristic trend at window size 4096 with linear scaling and at window sizes 4096 and 8192 with decibel scale use.The damage index forms a characteristic trend for the delaminated specimens of laminate PFML-PVDF-EP at all window sizes, except 4096. Furthermore, laminate PFML-PVDF-EP forms a characteristic trend for all specimens with fibre breakages at window sizes 1024 to 16,384. Including the results of laminates PFML-PZT-PP, all delaminated specimens form a characteristic trend at window size 512 and damage index use, which cannot be observed for the fibre breakages when considering the results of PFML-PZT-EP results.The dynamic tests were compared in terms of the maximum number of cycles that can be achieved and the associated force applied. Both, thermoplastic and thermoset laminates were used as specimens. In the case of the thermoset laminates, a distinction was made between the sensor type, piezoceramic film and PVDF . The theTo compare all specimen types, the results are shown in a bar chart for maximum force and the maximum cycles . As mentIn this experiment, no influence of the sensor type on the dynamic behaviour was found.The results of the dynamic tests for complete failure detection are shown in In contrast, laminate PFML-PZT-PP forms, in the best case, a significant peak at the moment of complete failure in four out of five specimens with linear scaling, independent of the measurment window size used. In contrast to the results obtained with laminates PFML-PVDF-EP and PFML-PZT-EP, laminate PFML-PZT-PP forms in forms significant peaks in fewer samples at the moment of complete failure when decibel scaling is used compared to linear scaling.The analysis of the damage index shows that all configurations of laminate PFML-PZT-EP show a significant peak at the moment of complete failure. In contrast, the amount of failed peak formations increases for laminates PFML-PVDF-EP and PFML-PZT-PP at window sizes larger than 2048. In the range of window sizes from 256 to 2048, the damage index forms a significant peak in all cases for laminate PFML-PZT-PP. However, in two cases the damage index forms a significant peak only for PFML-PVDF-EP and window size 256, while the use of window size 512 to 2048 results in four out of five specimens with significant peak formation at the moment of complete failure. Finally, there is a configuration where the damage index of laminates based on PZT leads to a significant peak formation in all cases, whereas in the best case four out of five specimens of laminate PFML-PVDF-EP show this peak formation.The trend analysis of the laminates used are shown in The trend analysis of the damage index shows only small differences across all laminates. For laminates PFML-PVDF-EP and PFML-PZT-EP at best two out of five specimens form a characteristic trend. Whereas PFML-PZT-EP shows a trend formation across all window sizes for two specimens, PFML-PVDF-EP only reaches these results in the range of 512 to 2048. PFML-PZT-PP, on the other hand, achieves at best three out of five specimens at window sizes 256 to 2048, beginning at window size 4096 the specimen quantity is reduced to two out of five. Across all laminates, the best results are achieved at window sizes 512 to 2048.Considering the complete failure type in terms of complete failure detection, no significant difference can be recognized, as there is at least one method and configuration for each laminate where all specimens form a significant peak. Equally, the complete failure type in terms of trend analysis shows no difference for the laminates on PZT basis since all specimen of PFML-PZT-PP delaminated and all specimen of PFML-PZT-EP resulted in fibre breakages. In contrast, PFML-PVDF-EP results in three delaminations and two fibre breakages.By analysing the standard deviation of the SED with linear scaling, one specimen of the delaminations of laminate PFML-PVDF-EP forms a characteristic trend independent of the window size used. Equally, one specimen of the fibre breakages only forms a characteristic trend with similar evaluation parameters at window sizes greater than 2048. Across all specimens of laminate PFML-PVDF-EP no characteristic trend was formed in the trend analysis using the standard deviation of the SED with decibel scale. The analysis results for the damage index are comparable, only one specimen of the delaminations and one specimen of the fibre breakages form a characteristic trend. One difference is represented by the applicable window sizes, in the case of delamination only the window sizes 512 to 2048 lead to a characteristic trend formation, while in the case of a fibre breakage the window sizes 256 to 16,384 show the trend formation.I represent sufficient methods to detect the time of complete failure. For each laminate at least one parameter set for both presented methods could be identified to detect the time of complete failure for all specimen in both static and dynamic tests scenarios. In the context of using the standard deviation of the SED, the results show that the linear scaling as well as the decibel scaling lead to detection improvements but also to decreases, depending on the specific laminate used. Therefore the scaling type must be considered for each laminate. It is also shown that it is necessary to determine the correct window size for each laminate as, depending on the test scenarios and the methods used, the best results could not be determined for a specific window size. Even though the window sizes from 512 to 2048 give sufficient results across all laminates and methods, corresponding to an observation period of 10 ms to 41 ms. Thus, in both cases the time can be determined by applying the threshold technique, only the determination of the healthy state and the corresponding reference value must be determined for each individual laminate. In summery, the complete failure detection in static test scenarios is going hand in hand with the results of the literature using carbon fibre-reinforced plastic [I and the standard deviation of the SED are applicable to determine the time of complete failure.The results show that both the standard deviation of the SED and the damage index  plastic ,42,43,44 plastic . ConsequWith regard to the characteristic trend formation, as observed in the case study, the results have to be differentiated. The formation of a characteristic trend in the static tests is observed for all specimens of laminates PFML-PVDF-EP and PFML-PZT-PP, but there was only one specimen for laminate PFML-PZT-EP which formed a characteristic trend by applying damage index methodology. With respect to the standard deviation of the SED only in case of laminate PFML-PVDF-EP all specimens form a characteristic trend, while PFML-PZT-PP forms a characteristic trend in two of three cases and PFML-PZT-EP only in one case. In the context of the damage index use, the results go along with the results from the literature ,33,34,39Consequently, Hypothesis H5 can be confirmed partially for laminates PFML-PVDF-EP and PFML-PZT-PP, since for both laminates one method results in a characteristic trend formation for all specimens. Regarding Hypothesis H2, only laminate PFML-PVDF-EP results in a characteristic trend formation for all specimens. Therefore H2 can be confirmed for this laminate only in connection with static test scenarios.Laminates PFML-PVDF-EP and PFML-PZT-EP are produced according to the same methodology, only the sensors used are different, but significant differences in characteristic trend formation are observed in the static test scenario. The difference in characteristic trend formation of laminates PFML-PVDF-EP and PFML-PZT-EP can be explained by three factors. The first factor, takes into account the type of complete failure, the static test scenario shows that three out of four delaminations form a characteristic trend when using the standard deviation of the SED. Furthermore, all delaminations result in a trend formation when using the damage index. However, no similar behaviour could be found for a glass fibre breakages independent of the methodology. Since all specimens of PFML-PZT-EP result in glass fibre breakages, the type of complete failure represents a possible reason for the observed difference. Secondly, the influence of the sensor type may explain the differences, since PZT and PVDF use the piezoelectric effect but are based on different chemical components leading to sensor specific outputs. The third factor is the amount of specimens, since just three specimens of each laminate have been tested, the effect can be explained by random distribution as well. In conclusion, both methods have the potential to reflect the characteristic trend in a static scenario as long as the parameters are determined and the possible expected type of complete failure is evaluated for the individual laminate.With regard to Hypothesis H3, the standard deviation of the SED gives the best results for delamination in the static test scenario. Thus, H3 can be partially confirmed for the static tests, but not conclusively clarified, since no direct inheritance comparison of PFML-PZT-EP and PFML-PZT-PP is possible due to a lack of specimens resulting in delamination in PFML-PZT-EP.In contrast, the results of the dynamic tests rarely show characteristic trend formations for all laminates. The highest amount of trend formation was achieved for laminate PFML-PZT-PP, where all specimens form a trend using the standard deviation of the SED, while laminate PFML-PZT-EP results in one of five and PFML-PVDF-EP in two of five specimens using the same methodology. With regards to the damage index use, laminates PFML-PZT-EP and PFML-PVDF-EP only form a characteristic trend in two of five cases, while laminate PFML-PZT-PP shows in three out of five cases. In consequence, hypotheses H2 and H5 are rejected in the context of the dynamic test scenario. In addition, the delamination results of the static test scenario could not be confirmed in the dynamic test scenarios. Furthermore, no characteristic trend formation for glass fibre breakage could be identified. With regard to Hypothesis H3, no indication of the damage type could be observed. So this hypothesis cannot be confirmed for the dynamic test scenario either.Nevertheless, in about two to three cases a trend could be observed for all laminates when applying the damage index. This is comparable to the results of the standard deviation of the SED, except for laminate PFML-PZT-PP, where the results of the damage index are behind the standard deviation of the SED. A clarification of the different results of using the damage index in the static and dynamic tests cannot be concluded from the observations made. Therefore, further analyses were conducted examining the observed differences in the damage index.The static test results show a general comparability with the results of the damage index by Song, but this is not evident in the dynamic tests. In most cases in literature, an active measurement method is used ,34,36, sFurthermore, an association of peak with a glass fibre breakage or delamination has to be ensured. For this reason, the measurements from study 3 are used as references, since here the audio signals were recorded with the cracks of the glass fibre breakages and delaminations. The recorded audio signals are labelled in such a way that each sample of an audible crack is marked as 1, all other samples as 0. However, since not every crack is audible, the force is used as an additional indicator for the static tests and the ratio of displacement to force for the dynamic tests. This distinction is made since in the static tests the displacement increases constantly and has no direct influence. For this reason, any drop in force corresponds to a change in the stiffness of the specimen caused by glass fibre breakage or delamination as it has been shown in ,42,44,47For this reason, in the following a further analysis is carried out to resolve the inconsistencies. The damage index signal has been calculated with a window size of 512 and filtered with a moving average filter of size 10. The results for one specimen of PFML-PVDF-EP and PFML-PZT-EP are shown in The same procedure was repeated for all specimens of the dynamic test scenario with a window size of 1024 and a moving average of length 15. This distinction was justified by the longer observation time in the dynamic test compared to the static test scenario. The results for one specimen of laminate PFML-PVDF-EP and PFML-PZT-EP are shown in However, these examples also show that the acoustic labels and the ratio of displacement and force alone are not sufficient to reproduce the process of destruction. An example are the peaks at sensor S2 at time 60 and 100 in Another observation is that the signal always returns to the healthy state after a damage occurs leading to peak formations and contradicting to the results from the literature ,33,34. OIn conclusion, the damage index provides the possibility to detect fibre breakages and delamination in static and dynamic scenarios. A distinction between the two types of damage cannot be conclusively clarified at present. In the static scenario, a characteristic trend formation was observed for all cases of delamination using the damage index, but this could not be confirmed in the dynamic scenarios. Due to the lack of delamination of laminate PFML-PZT-EP, no generalisation can be made with respect to delamination. Due to this reason, Hypothesis H5 can be partially confirmed. Thanks to the detectability of the occurred damages, which would lead to a complete failure in the future, an early warning system can be implemented. However, a direct characteristic trend formation cannot be identified at the current state of the data gathered.In a case study it was shown that the developed sensory hybrid laminate can be used for condition monitoring. Based on this, a second study with more specimens and variations of the sensor layer and the matrix material was designed. To back up the recorded signals, the second study was repeated in a third study with the recording of an audio signal. For the second and third study, three different aluminium-based PFML types were prepared. A novel piezo-active thermoplastic film based on polypropylene, lead zirconate titanate and carbon nanotubes was used as sensor layer for two PFML types. The matrix material of the glass fibre-reinforced plastic layer was varied between thermoplastic polypropylene and thermoset epoxy resin. The third PFML type uses polyvinylidene fluoride in the sensor layer and epoxy resin in the reinforcement layer. The specimens were produced under laboratory conditions and then tested statically and dynamically in a universal testing machine using a three-point bending test.The static mechanical properties show that a hybrid laminate with a thermoset matrix achieves the greatest reinforcing effect, but also has the lowest ductility. The failure of the thermoset hybrid laminate is mainly due to fibre breakage. In contrast, the thermoplastic hybrid laminate is more ductile in its failure and maintains a certain level of force over a greater bending distance. The thermoplastic hybrid laminate fails mainly due to delamination.The evaluation of the dynamic tests shows that the thermoplastic hybrid laminates fail due to delamination even at a low force and after a few cycles. This is attributed to the poor interfacial adhesion between polypropylene and aluminium. The thermoset hybrid laminates, on the other hand, achieve higher forces and withstand more cycles until they fail due to fibre tearing. In both investigations, static and dynamic, no influence of the sensor type on the mechanical behaviour could be determined.For the analysis of the applicability of the laminates for the SHM sector, two methods were analysed: First, the standard deviation of the spectral energy density (SED) was analysed, motivated by a preliminary study. Furthermore, the damage index as quantification of the energy changes based on wavelet transform was evaluated. The results show that both methods are suitable for detecting the time of complete failure of the laminates in both static and dynamic tests. Furthermore, the results show the necessity to determine an appropriate measurement window size for each individual laminate to ensure a correct failure detection. As an additional criterion, the scaling of the SED was examined, which shows the improvement or deterioration of the results depending on the laminate and measurement window size used.In addition, both methods were analysed for their suitability for predicting the time of complete failure by analysing the sensor signals for the formation of a characteristic trend. The results show that the damage index for the laminates PFML-PVDF-EP and PFML-PZT-PP forms a characteristic trend in the static test scenario. The same could be observed for the standard deviation of the SED method, only for laminate PFML-PVDF-EP in the static scenario. Furthermore, taking into account the type of damage, indicators were found showing the standard deviation of the SED has a characteristic trend formation in case of delaminations in static scenarios. This behaviour is not observable for the dynamic test scenarios. However, in a further analysis of the damage index, the possibility of detecting delaminations and glass fibre breakages is identified. These are represented by peaks, which are verified by temporal agreement with audible cracks from study 3 and by the correlation of force and displacement. Thus, the damage index could be confirmed as a method to identify damages in the laminates that would lead to a complete failure of the laminate in the future.In future work, open questions should be clarified. One of these questions is the differentiation of damage types, which was not possible in this studies due to the lack of delaminations for laminate PFML-PZT-EP and the lack of glass fibre breakages for laminate PFML-PZT-PP. For this purpose, it is necessary to identify possibilities to force specific types of damage in order to establish comparability between laminates. Furthermore, the described methods have to be verified for standard laminates such as GLARE, CAPAAL or CAPET  in order


An 85-year-old Caucasian female with past medical history of hypertension, hyperlipidemia, polymyalgia rheumatica, coronary artery disease, Osler-Weber-Rendu syndrome (diagnosed 18 years ago), intermittent epistaxis and pulmonary arteriovenous malformation (AVM) was referred to the clinic for abnormal chest imaging. She underwent multiple bilateral AVM embolization 17 years ago. She had minimal shortness of breath without epistaxis, hemoptysis, hematemesis or hematochezia. One month ago, her chest X-ray revealed multiple embolization coils bilaterally (A). Computed tomography (CT) chest scan without contrast showed multiple embolization coils, multiple tortuous vascular malformations in both lungs with enlargement of left lower lobe AVM (B). Magnetic resonance imaging (MRI) brain 17 years ago did not show brain vascular malformation. The diagnosis of progressive Osler-Weber-Rendu or hereditary hemorrhagic telangiectasia (HHT) was made. She was referred to HHT center and underwent coiling. After that, she has been clinically stable and a repeat CT chest on the follow-up visit showed smaller left lower lobe AVM (B), so conservative management was planned.


Recently, p21 has been reported to mediate inflammatory responses. As inflammatory responses are a feature of sepsis, our study investigated whether p21 has a role in cardiac dysfunction induced by sepsis and analyzed the mechanisms involved. To establish a mouse sepsis model, p21 global knockout (p21KO) and C57BL/6J wild-type (WT) male mice were treated with 5 mg/kg LPS intraperitoneally for 6, 24, or 48 h. After LPS stimulation, the level of p21 had significantly increased in the WT mice and in cardiomyocytes. Cardiac dysfunction induced by LPS was markedly aggravated in p21KO mice relative to that of WT mice. Downregulation of p21 expression exacerbated the LPS-mediated inflammatory response, and it increased oxidative stress as well as mitochondrial damage in the heart and in cardiomyocytes. In contrast, overexpressing p21 attenuated the increase of TNFα and promoted the increase of SOD2. Moreover, p21 regulated the LPS-induced autophagy activation; that is, the increase in autophagy was impaired when p21 expression was decreased, whereas the increase was significant when p21 was overexpressed. The autophagy inducer rapamycin partially rescued the cardiac deterioration caused by p21 downregulation in the LPS-stimulated groups. In addition, p21 regulated the autophagy level by interacting with LC3B. These results revealed that p21 controls LPS-induced cardiac dysfunction by modulating inflammatory and oxidative stress, and it is partially dependent on regulating the autophagy level. This study is the first to show that p21 could interact with LC3B to promote autophagy for the improvement of cardiac function during sepsis. If not treated promptly, it will proceed to multiple organ failure, and the heart is one of the main organs affected during sepsis. Some studies have shown that sepsis-induced heart dysfunction can be as high as 70%2. If there are cardiac complications, the patient’s condition deteriorates rapidly, and the mortality rate will be as high as 50–60%2.Sepsis is the most common systemic inflammatory response in the clinic. It is mainly caused by the release of large amounts of endotoxins from Gram-negative bacteria into the blood. The active chemical component of endotoxin-induced disease is mainly lipopolysaccharides (LPS)These data suggest that cardiac damage plays an important role in septic patients’ death. Echocardiography has also shown that a dilated left ventricular diameter (LVD) and decreased left ventricular ejection fraction (LVEF) occur in some patients with early septic shock, further suggesting that cardiac dysfunction may be present in patients with early septic shock. Until now, researchers and physician have generally recognized that cardiac dysfunction is closely related to patients’ prognosis. Therefore, improving cardiac function plays a crucial role in reducing mortality in patients with sepsis. However, the treatment for cardiac damage caused by sepsis is not ideal, because the mechanism of myocardial injury induced by sepsis is not clear.WAF1/CIP1 (hereinafter referred to as p21)3. It is also an important downstream target of the tumor-suppressor p534. p21 can regulate the cell cycle by inhibiting the binding of cyclin and CDK. p21 also plays an important role in cell senescence and apoptosis5. In mammals, p21 is rarely expressed in the embryonic and neonatal hearts but is highly expressed in the adult hearts.CDKN1A is an important member of the cyclin-dependent kinase (CDK) inhibitor family and belongs to the Cip/Kip family of CDK inhibitors, hence the name p217. The expression of p21 in doxorubicin-induced cardiotoxicity is similar to that of cardiac hypertrophy and has its two sides9. Recently, it has been reported that p21 plays an important role in inflammatory diseases independently of cell cycle regulation, but research on p21 and sepsis-induced cardiac dysfunction has not been conducted.A large number of studies have suggested that p21 is involved in the pathological process of myocardial injury, but the changes of p21 in cardiovascular disease and its effects are currently controversial. In different animal models of cardiac hypertrophy, the expression changes of p21 are inconsistent, and so are the corresponding effects. In Angiotensin II-mediated cardiac hypertrophy, p21 expression level is reduced, but in a variety of other cardiac hypertrophy models, such as spontaneously hypertensive rats and isoproterenol stimulation, p21 expression is continuously increasedIn this study, we investigated the role of p21 in LPS-induced cardiac dysfunction and its molecular mechanism. We found that LPS-mediated autophagy increases improved inflammation and oxidative stress was partially dependent on p21. In addition, the effects of p21 on autophagy were achieved by the protein–protein interaction between p21 and microtubule-associated protein 1 light chain3 (LC3).To explore whether p21 was involved in the cardiac stress response after LPS stimulation, we first compared the mRNA and protein levels of p21. After LPS administration, mouse cardiac p21 mRNA and protein levels were significantly increased at 6, 24, and 48 h Fig. . ImmunohWe first identified cardiac mRNA and protein expression of p21 in wild-type (WT) and global knockout (p21KO) mice Fig. . Next, wThe LPS-induced inflammatory response is one of the major causes of cardiac damage. To determine whether p21 deficiency worsened cardiac dysfunction by regulating inflammatory responses, we tested the mRNA levels of proinflammatory cytokines, including tumor necrosis factor-α (TNFα), interleukin-6 (IL6), and monocyte chemoattractant protein-1 (MCP1) in p21KO and WT mice under basal condition and in response to LPS Fig. . We founIn addition to the inflammatory response, oxidative stress has also been shown to play an important role in LPS-induced cardiac dysfunction. Our results showed that mRNA levels of SOD2 and NOX2 did not differ between WT and p21KO mice at baseline Fig. . After LMalondialdehyde (MDA) assays showed that the MDA content in the myocardial tissue of p21KO mice was significantly increased at 6 and 24 h after LPS stimulation compared to the WT group, while the MDA content in the myocardial tissue of WT mice stimulated by LPS was significantly increased at 6 h, and then it decreased to a normal level at 24 h Fig. . We alsoMitochondria are the main organelles involved in oxidative stress, and it has been reported that LPS induces mitochondria injury. Therefore, we observed mitochondrial changes between the WT and p21KO groups. Electron microscopy showed that, after LPS stimulation for 24 h and 48 h, in both the WT and p21KO mice we observed cardiac mitochondrial swelling and the ridge structure disappearing Fig. . CompareCollectively, these results indicate that a lack of p21 aggravates LPS-induced cardiac inflammation, oxidative stress, and mitochondria damage.To explore the molecular mechanism of how p21 regulates inflammation and oxidative stress, we compared the proteins involved in the above pathways between WT and p21KO mice. The results showed that antioxidative protein Nrf2, which was increased after LPS administration, did not differ between WT and p21KO mice Fig. . In addiThe above results showed that p21 deficiency could increase the number of damaged mitochondria. The increase in damaged mitochondria may be due to either too much production or not enough clearance. It has been proven that autophagy is one of the main ways to clear damaged mitochondria. Therefore, we observed the autophagy level between WT and p21KO mice at 24 h post-LPS stimulation. The results from electron microscopy showed that LPS caused an increase of autophagosomes in WT mice, while the increase was impaired in p21KO mice Fig. .Moreover, we also tested the protein levels of LC3 and p62. In line with the previous results, the cardiac level of LC3II was reduced and p62 was increased in p21KO mice compared to WT mice after LPS administration Fig. , and theNext, we examined whether the overexpression of p21 caused an increase in autophagy in CMs. The results showed that, compared with the control group, p21 overexpression could increase the level of LC3II in the basal state, and after LPS stimulation, it could activate an autophagy flux Fig. . In addiAll of these results indicate that p21 could regulate the level of LC3II and then influence the increased autophagy level caused by LPS.The expression level of LC3II is affected by many factors; thus we studied how p21 regulates LC3II. We first examined changes in the expression levels of the upstream kinases associated with LPS-induced autophagy. However, the data indicated that there were no differences in the protein levels of p-AKT, p-AMPK, or p-p38 between WT and p21KO mice Fig. . Then weIn this study, we identify that p21 could regulate the cardiac autophagy level, thus protecting the heart against LPS-induced inflammatory and oxidative injury Fig. . In addi11. It is well known that macrophages are proliferating cells, and the primary biological function of p21 is to regulate the cell cycle. Interestingly, p21 is also highly expressed in CMs, which are nonproliferating cells. This raises the question of whether p21 in CM, one of the target cells of inflammatory responses, change in response to inflammatory stimulation. Our experimental results showed that the expression level of p21 presented dynamic changes during LPS stimulation, but it was significantly higher than the basal state, which suggests that p21 might play a role in LPS-mediated cardiac injury. Moreover, the results of cardiac function tests further demonstrated that p21 was involved in LPS-induced cardiac dysfunction.It has been reported that the expression level of p21 in macrophages is significantly increased after LPS stimulation and that p21 is involved in the anti-inflammatory effect of macrophages12. In addition, p21 can also regulate macrophage polarization in an LPS second-stroke mice model11. These studies showed that p21 has anti-inflammatory effects, but they focused on its role in inflammatory cells, which are proliferating cells. We found that p21 knockdown promoted the expression of proinflammatory factors in vivo and in vitro, whereas p21 overexpression relieved their increased mRNA levels in primary CM treated with LPS, suggesting that p21 regulates the anti-inflammatory effect of nonproliferating cells as well. In addition, the activation of p65 may partly account for the inflammatory-regulation effects of p21, which is in accordance with previous research.The toxic effect of LPS is mainly induced by the inflammatory response. Previous studies and our studies have confirmed that LPS promotes the inflammatory response, and the absence of p21 aggravates the production of inflammatory factors. In the LPS-mediated inflammatory model, studies have shown that p21KO mice are more prone to endotoxin shock after LPS stimulation, because p21 in macrophages inhibits the formation of IL1β through its CDK-binding domain, thereby inhibiting inflammation and alleviating the deterioration of endotoxemia14. By examining the transcription level of antioxidant enzymes SOD2 and NOX2 in myocardial tissue and in CM stimulated by LPS, we found that p21 deletion could promote the expression of NOX2 and inhibit the transcription of SOD2. In addition, our detection of SOD activity and MDA content in cardiac tissue, as well as direct observations of ROS in CM, further supports the fact that p21 deficiency contributes to ROS production induced by LPS. However, p21 overexpression promoted increased SOD2 influenced by LPS, demonstrating that p21 has the ability to alleviate oxidative stress.Increased ROS is another important manifestation of heart dysfunction induced by LPS. LPS can promote the formation of ROS, which further aggravates myocardial injury16. It has been reported that, when fibroblasts and HCT-116 cells suffer oxidative stress injury, p21 directly binds to Nrf2 and promotes the antioxidant effect of Nrf2, which suggests that p21 could regulate oxidative stress injury17. However, in this study we found that p21 did not influence Nrf2, implicating that p21 regulate oxidative stress independently of Nrf2 pathway.Previous studies have shown that p21 could protect cells from the toxic effects of various compounds by inhibiting the production of ROS19. LPS inhibits the expression of CD3620 and the expression of lipid oxidation genes21, thereby reducing the formation of ATP and leading to cardiac dysfunction. In this study, we detected the transcriptional level of energy-related genes in heart tissue and found that p21 deletion had no effect on LPS-induced myocardial energy inhibition, indicating that deterioration of cardiac function caused by p21 deletion may not be related to energy metabolism.In addition to the direct damage of inflammatory cytokines and ROS to the heart, an insufficient energy supply in the heart accounts for an important aspect of LPS-induced cardiac dysfunction23. Notably, p21 also plays an important role in the regulation of autophagy. The DNA-damaging agent 5-methoxyflavone can induce apoptosis and autophagy in HCT-116 human colon cancer cells via a p21-dependent pathway24. In addition, the antimalarial drug quinacrine induces autophagy and apoptosis of breast cancer cells by regulating p21, thereby achieving the effect of antitumor cell proliferation25. All of these results suggest that p21 induces the formation of autophagy and exerts an antitumor effect. Our study found that, after LPS stimulation, autophagy levels gradually increased with the prolonged stimulation time in cardiac tissues and CM, but after reaching the peak, autophagy levels showed a decreasing trend, and the corresponding cardiac function also deteriorated significantly in the early stage but then gradually improved. After p21 deletion, compared with the normal group, the level of autophagy in the myocardial tissue and primary CM was decreased, and mouse cardiac function was always in a dysfunctional state. These results suggest that the absence of p21 may lead to insufficient autophagy in CM, which then cannot timely remove the harmful stimuli and damaged organelles from within the cells, thus preventing their recovery of cardiac function. The increasing autophagy level decreased the cardiac injury caused by p21 deletion, which further demonstrated that the effects of p21 on cardiac damage induced by LPS are partially mediated through regulating the autophagy level.Increasing evidences has suggested that sepsis induces increased autophagy in multiple organs, including the heart. Increased autophagy has been reported to play a protective role in septic cardiac dysfunction26. Studies have suggested that mTOR activation may suppress beneficial autophagy activity during severe stages of sepsis, but activation of autophagy in early sepsis may be independent of mTOR23, which is in line with our results in this study. Moreover, mTOR is a direct target of AMPKα, and AMPKα can promote the formation of autophagy by inhibiting the activity of the mTOR complex27. Our previous study has proven that p21 activates autophagy by regulating AKT and AMPKα in the hypertrophic heart28. In this study, we found that, after LPS stimulation, the levels of AMPKα were significantly decreased while the level of autophagy increased, indicating that the increase in LPS-induced autophagy was not mediated through the activation of AMPKα. In addition, our results also pointed out that p21 deletion did not affect the level of p-AMPKα and p-AKT after LPS stimulation. Recently, a novel mTOR-independent autophagy pathway has been reported. p38 plays an important role in autophagy regulation through p38 mitogen-activated protein kinase interacting protein (p38IP)29. In addition, it has been reported that Toll-like receptor 4 activation can improve liver injury by inducing increased autophagy through the p38 signaling pathway30. These results indicate that the p38 signaling pathway directly regulates intracellular autophagy levels. Our study confirmed that the expression level of p-p38 was significantly increased after LPS stimulation, but the expression level of p-p38 was not significantly changed after p21 deletion, suggesting that p21 does not regulate the level of autophagy through p38 pathway.To clarify the specific mechanism of the impaired autophagy increase caused by p21 deficiency, we first examined the expression of related kinases acting upstream of autophagy. mTOR is currently recognized as an important molecule in upstream inhibition of autophagy formation31. In this study, we also detected expression changes of Beclin1 and Atg7 in WT and p21KO mice after LPS stimulation, but there was no significant difference between the two groups, suggesting that p21 also does not act by regulating the initiation of autophagy. The excessive activation of autophagy lysosomes may also cause a decrease in autophagy level. However, the results of our study showed that p21 deletion resulted in aggregation of autophagy substrate p62 and had no significant effect on the expression of lysosomal-related protein Lamp1, indicating that p21 also does not affect the degradation of the autophagosome.p21 does not affect autophagy formation by affecting autophagy upstream kinases, thus we wondered whether p21 directly affects autophagy-related proteins. It has been reported that C2-ceramide could reduce the expression of the autophagy-associated proteins Beclin1 and Atg5 by p21, thereby inhibiting the level of autophagy in mouse embryonic fibroblasts32. Like other Atg proteins, LC3 is tightly regulated at the transcriptional, posttranscriptional, or posttranslational levels. According to some reports, transcription factors such as ATF4, CHOP, E2F1, FOXO 1/3, GATA1, and ZKSCAN3 have been found to regulate the LC3 expression level33. This is the first report to show that p21 directly binds to LC3, and the absence of p21 led to significantly impaired increase of LC3II in response to LPS stimulation, suggesting that p21 may directly regulate the formation of LC3II by binding to LC3. Notably, we did not explore how LC3 expression was regulated after p21 bound to LC3, which will be investigated in the future.In the process of autophagy, the formation and maturation of autophagosomes are regulated by several core proteins related to autophagy in a highly controllable manner. The LC3II level has become one of the gold standards for the detection of intracellular autophagyCIP1/WAF1 global knockout mice  were purchased from The Jackson Laboratory  and C57BL/6J WT mice were purchased from Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences . Male mice aged 8–12 weeks weighing 23–25 g were used. All procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health  and were approved by the Chinese Animal Welfare Committee as well as Wuhan Renmin Hospital.p2134, WT and p21KO mice were intraperitoneally injected with a single dose of 5 mg/kg Escherichia coli LPS  dissolved in sterile saline, and control mice received an equal volume of saline. p21 KO mice or WT mice were divided into four groups, and they are control group, LPS 6 h group, LPS 24 h group, and LPS 48 h group, with at least 20 mice in each group. Echocardiography was performed at 6, 24, or 48 h after LPS challenge to assess mouse cardiac function. To induce autophagy, the autophagy agonist rapamycin  was intraperitoneally injected 30 min before LPS treatment, as previously reported27. Mice were sacrificed by cervical dislocation, and their hearts were rapidly excised, weighed, and collected for later use. All animal experiments were replicated at least three times.To establish a mouse nonlethal septic cardiac dysfunction modelAn adenoviral vector carrying Rat p21 small hairpin RNAs (sh-p21) to knockdown p21 was generated by the OBIO Technology Corp., Ltd. , and scrambled shRNA (sh-NC) was used as the control. Adenovirus vector carrying Rat p21 (Ad-p21) to overexpress p21 and empty carrier control Ad-LacZ were constructed and amplified by the iBioscience Company .For constructing the pcDNA5-FLAG-p21 and pcDNA5-HA-LC3B recombinant plasmids, the full-length human p21 and LC3B-coding regions were amplified from the cDNA of HEK293T cells by polymerase chain reaction (PCR). The pcDNA5-FLAG and pcDNA5-HA plasmids were digested with BamH I  and Xho I . The PCR products of p21 and LC3B were ligated to the digested pcDNA5-FLAG and pcDNA5-HA plasmids, respectively, using the recombinase  according to the manufacturer’s instruction. The primers for amplifying the target gene are listed below: human-p21-F: TCGGGTTTAAACGGATCCATGTCAGAACCGGCTGGGGA, human-p21-R: GGGCCCTCTAGACTCGAGTTAGGGCTTCCTCTTGGAGAAGATCAG, human-LC3B-F: TCGGGTTTAAACGGATCCATGCCGTCGGAGAAGACCTT, human-LC3B-R: GGGCCCTCTAGACTCGAGTTACACTGACAATTTCATCCCGAACGT.35. NRCMs were incubated in serum-free medium for 12 h before LPS stimulation. Then the medium was replaced with Dulbecco’s modified Eagle’s medium (DMEM)/F-12  containing 15% fetal bovine serum , penicillin (100 U/mL), and streptomycin (100 mg/mL) , and NRCMs were challenged with LPS (10 µg/mL). The autophagy agonist rapamycin  was used 2 h prior to LPS exposure to induce autophagy36. All controls received appropriate vehicles. To knockdown or overexpress p21, NRCMs were transduced with the corresponding adenoviruses at a multiplicity of infection of 100 for 24 h before LPS administration.Primary CMs (NRCMs) were isolated from the left ventricle of neonatal SD rats and cultured as described previouslyHEK293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences  and cultured in DMEM  supplemented with 10% FBS. HEK293T cells were cotransfected with plasmids encoding HA-LC3B and/or FLAG-p21 according to the manufacturer’s instructions. After transfection for 24 h, HEK293T cells were harvested for IP. All cell experiments were repeated at least three times.20. Equal amounts of proteins were separated by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antibodies used were: anti-LC3B , anti-p62 , anti-p21 , anti-p-AKT , anti-AKT , anti-p-mTOR , anti-mTOR , anti-p-AMPKα , anti-AMPKα , anti-p-p38 , anti-p38 , anti-p-p65 , anti-p65 , anti-Beclin1 , anti-Atg7 , anti-Lamp1 , anti-cleaved-caspase3 , anti-caspase3 , anti-CDK2 , anti-Nrf2  and anti-GAPDH .For western blots, hearts and NCRMs were lysed in RIPA lysis buffer  containing phenylmethanesulfonylfluoride  and protease inhibitor pellets (Roche), and the protein was isolated as previously describedFor IP, NRCMs or HEK293T cells were lysed with ice-cold IP buffer containing Tris HCl , NaCl (137 mM), NP-40 (0.5%), EDTA (0.5 mM), and a protease inhibitor cocktail. Then the cell lysate was incubated with the indicated antibodies overnight at 4 °C. Subsequently, Protein A/G-agarose beads were added and incubated for 2 h. After IP, the immunocomplexes were washed with cell lysis buffer five times, then boiled at 100 °C for 5 min and subjected to immunoblotting.Total RNA was extracted from heart tissues or NRCMs using Trizol reagent , and then equal amounts of total RNA (2 μg) were reverse-transcribed into cDNA using a Transcriptor First-Strand cDNA Synthesis Kit , according to the manufacturer’s protocol. LightCycler 480 SYBR Green I Master Mix  was used to amplify and quantify the PCR products. Relative mRNA expression of the target genes was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Specific primer sequences used in the experiments are listed in Table For IHC, paraffin-embedded heart tissue sections were incubated with primary antibody against p21  overnight at 4 °C. Horseradish peroxidase-conjugated secondary antibody (1:200) was applied to the heart tissue and diaminobenzidine was used as the substrate.For immunofluorescence staining, NRCMs were cultured on cover slips. After different treatments, NRCMs were fixed with 4% paraformaldehyde and permeabilized with 0.03% Triton®X-100. Then NRCMs were incubated with specific antibody overnight at 4 °C. After washing three times, the NRCMs were incubated with a secondary antibody conjugated to Alexa-488 or -568 (1:200). Finally, 4′,6-diamidino-2-phenylindole was used to stain the nuclei. The NRCMs were observed under an Olympus fluorescence microscope.The activity of cardiac SOD was measured by a Total Superoxide Dismutase Assay Kit with WST-8 , and the content of cardiac MDA was determined by a Lipid Peroxidation MDA Assay Kit  according to the manufacturer’s instructions. ROS in the NRCMs were detected by a Reactive Oxygen Species Assay Kit . Briefly, NRCMs were incubated with 10 μM/L 2′,7′-dichlorodihydrofluorescein diacetate at 37 °C for 20 min. After washing three times, ROS was observed under an Olympus fluorescence microscope.28. Images were observed and acquired using a transmission electron microscope .Transmission electron microscopy was performed as described previouslyMMP of NRCMs were detected by a JC-1 Fluorescence Probe Kit  according to the manufacturer’s instruction. Briefly, NRCMs were incubated with 5 µg/mL JC-1  at 37 °C for 20 min. The fluorescence was observed and captured using an Olympus fluorescence microscope.t tests. For multigroup comparisons, analysis of variance followed by post hoc Tukey’s test was performed. The variances between the groups that are being statistically compared were similar. For animal studies, no randomization and blinding were used. A value of p < 0.05 was considered statistically significant.Data are presented as mean ± SEM. For direct comparisons, statistical significance was calculated by unpaired Student’s Supplementary Figure S1Supplementary Figure legend


The inhibitory effects of the drugs on hCA I and hCA II were determined with both the hydratase and esterase methods. The most potent inhibitors observed were propafenone (hCA I: 2.8 µM and hCA II: 3.02 µM) and captopril (hCA I: 1.58 µM and hCA II: 6.25 µM). Isosorbide mononitrate, propranolol, furosemide, and atorvastatin were also potent inhibitors. The inhibitor constant, Ki, value from the Lineweaver–Burk plot for propafenone was 2.38 µM for hCA I and 2.97 µM for hCA II. The tested cardiac drugs showed potent in vitro inhibition of the hCA I and II isozymes. Especially, in patients with atherosclerotic heart disease, these drugs may be preferred primarily due to the beneficial effects of carbonic anhydrase inhibition on atherosclerosis.Cardiovascular diseases are the leading cause of mortality worldwide. In recent years, the relationship between carbonic anhydrase inhibitors and atherosclerosis has attracted attention. In this study, we aimed to determine the  There are 15 human carbonic anhydrase (hCA) isoforms, and they all differ in their cellular/tissue localisation and enzymatic featuresIn recent years, the relationship between CA and atherosclerosis has attracted attention. Atherosclerotic cardiovascular diseases are the leading cause of death worldwideHowever, the potential role of CA inhibition related to cardiac drugs in atherosclerosis has not been sufficiently researched. Additionally, drug–enzyme interaction studies have recently gained great interest. Many adverse drug events may result from CA isozyme inhibition. Thus, CA has been increasingly studied by several scientists worldwide. Our laboratory has also specialised in this subject2.2.1.Sepharose 4B, L-tyrosine, protein assay reagents, phenol red, and chemicals for electrophoresis were obtained from Sigma-Aldrich Co. All other chemicals were of analytical grade and obtained from either Sigma-Aldrich Co. or Merck. Medications were provided by a local pharmacy. The study was approved by the local ethics committee .2.2.g for 20 min at 4 °C, and the supernatant was removed. The packed erythrocytes were washed three times with 0.9% NaCl and then were hemolyzed in cold water. The pH of the hemolysate was adjusted to 8.5 with the solid Tris base. The 25-ml hemolysate was applied to an affinity column containing y Sepharose 4B-ethylene diamine-4-isothiocyanato-benzenesulfonamide2HPO4 (pH 6.3) and 0.1 M CH3COONa/0.5 M NaClO4 (pH 5.6), which recovered hCA I and II, respectively.Blood samples (25 ml) from healthy human volunteers were collected. They were centrifuged at 10002.3.2 hydration2CO3/0.1 M NaHCO3 (pH 10.0), and phenol red was added as the pH indicator. Carbon dioxide-hydratase activity was calculated in enzyme units (EU) with the following equation: t0 – tc/tc, where t0 and tc are the times for the pH change of the nonenzymatic and the enzymatic reactions, respectively.CA activity was measured by the Maren method based on the determination of the time required for the pH to decrease from 10.0 to 7.4 due to CO50) was determined from the graphs. It was determined that DMSO had no effect on the enzyme activities in any of the tested concentrations.The drugs were triturated in a porcelain mortar pestle. Stock solutions of both drugs were prepared in dimethyl sulfoxide (DMSO). Different concentrations of the drugs were added to the enzyme activity determination medium with a total volume of 4.2 ml. The duration (in seconds) of the colour change from red to yellow in solution was measured in a 10-ml glass tube with a diameter of 1 cm. Control cuvette activity in the absence of the inhibitor was set at 100%. All compounds were tested in triplicate at each concentration used. For each inhibitor, an activity %–[inhibitor] graph was drawn (data not shown). The inhibitor concentration causing up to 50% inhibition  according to a method described in the literature3.in vitro effects of 35 frequently used cardiac drugs were investigated on human erythrocyte CA-I and CA-II. Affinity chromatography was used to purify hCA I and hCA II. SDS-PAGE was performed to determine the purity of the enzymes. The inhibitory effects of the drugs on hCA I and hCA II were determined with both the hydratase and esterase methods. The IC50 values were calculated from activity %–[I] graphs and are shown in In this study, the Ki value from the Lineweaver–Burk plot for propafenone was 2.38 µM (non-competitive) for hCA I and 2.97 µM (non-competitive) for hCA II (The most potent inhibitors were propafenone (hCA I: 2.8 µM and hCA II: 3.02 µM) and captopril (hCA I: 1.58 µM and hCA II: 6.25 µM). Isosorbide mononitrate (hCA I: 6.08 µM and hCA II: 5.5 µM), propranolol (hCA I: 1.25 µM and hCA II: 6.25 µM), furosemide (hCA I: 6.23 µM and hCA II: 4.95 µM), and atorvastatin (hCA I: 7.75 µM and hCA II: 9.85 µM) were also potent inhibitors. The r hCA II .CAs are drug-target enzymes. The inhibitors of these enzymes are important compounds for discovering new therapeutic agents and understanding enzyme–drug interactions in detail at the molecular level. Propafenone is a sodium channel-blocking antiarrhythmic drug. It works by blocking the activity of particular electrical signals in the heart that can cause arrythmias. It is used to restore a normal heart rhythm and maintain regular beats in supraventricular tachyarrhythmiasAtherosclerosis is a dynamic process that influences the aorta and its branches. Atherosclerotic plaques are a local thickening of the intima layer caused by cholesterol, hydroxyapatite and fibrous connective tissue accumulation, and proliferation of smooth muscle cells. The determinant step of athelerosclerosis is calcium precipitation, which traps cholesterol in the plaque precursor matrix, which includes calcium carbonate, triglycerides, lipoproteins, hydroxyapapatite, and calmodulinStudies have shown that both CA-I and CA-II play an important role in the aetiology of vascular calcification as a component of atherosclerosis. In a study by Oksala et al., CA-II was highly expressed in human atherosclerotic plaques in patients with advanced atherosclerosisAyari et al. detected overexpression of CA-II in human atheroma plaques compared with healthy arterial tissue from the same patient in a comparative genome-wide microarray expression analysis. When patients were compared with each other, CA-II was overexpressed more than 1.7 fold in the atherosclerotic plaquesYuan et al. studied the effects of CA on atherosclerosis in an atherosclerotic rat model and human aortic dissection and aneurysm caused by atherosclerosis. They demonstrated significantly higher CA-I levels in the animal atherosclerotic tissues, and CA-I levels increased in human atherosclerotic tissues. Moreover, methazolamide, a CA inhibitor, decreased CA-I levels in the animal model. These results suggest that the higher levels of CA-I are related to vascular calcification, and CA-I plays an important role in atherosclerosis and its progressionde novo cholesterol synthesis and decrease low-density lipoprotein (LDL). Their hypolipidemic effects result in stabilisation of atherosclerotic plaques, hence they are used for coronary and peripheral artery diseases50 value of 48 µMAtorvastatin belongs to a group of drugs known as statins, or HMG-CoA reductase inhibitors. Statins inhibit A recent study by Yuan et al. demonstrated that CA-I expression and CA-I-mediated calcification are significantly associated with atherosclerosis progression, and methazolamide significantly reduces atherosclerosis and suppresses CA-I expression. According to the results of our study, CA may be responsible for the atherosclerosis-reducing effects of statins as a secondary pathway in addition to the LDL-lowering effect50 value of 0.007 µMCaptopril is a competitive inhibitor of angiotensin converting enzyme (ACE). This enzyme is responsible for the conversion of angiotensin I to angiotensin II. Angiotensin II regulates blood pressure and is a key element of the renin–angiotensin–aldosterone system. Leppala et al. reported that captopril is an angiotensin I converting enzyme inhibitor with an IC50 value of propranolol was approximately 150 µM50 value of 4.4 mMBeta-blockers inhibit the sympathetic activity of beta-adrenergic receptors. Propranolol, a non-selective beta blocker, inhibits all beta receptors. This activity decreases cardiac contractility and heart rate. Propranolol is frequently used in the treatment of patients with ischaemic heart disease and hypertension. Sozzani et al. reported that propranolol is also an inhibitor of protein kinase C. The IC50 of 0.526 mMFurosemide is a loop diuretic that acts on the kidney. Furosemide inhibits the Na–K–2Cl cotransporter on the membrane of the epithelial cells of the thick ascending limb of the loop of Henle. The decreased sodium and chloride reabsorption results in diuresis and natriuresis. Furosemide is used to treat edoema in patients with heart failureIsosorbide mononitrate is a drug mainly used to treat angina pectoris. It relaxes the coronary arteries, thereby increasing the circulation in the ischaemic zone. Isosorbide mononitrate relaxes vascular smooth muscles through the formation of nitric oxide (NO). NO activates guanylyl cyclase, resulting in decreased blood pressure and relaxation of the veins and arteries. In our study, this drug inhibited CA isoenzymes at the micromolar level.50 value of 5.8 nM. The IC50 values of cardiac drugs used in our study were higher than the IC50 value for acetazolamide in a previous studyAcetazolamide is a classic CA inhibitor. It has been reported to show notable inhibitory effects on hCA I with an IC4.in vitro effects of 35 frequently used cardiac drugs were investigated on human erythrocyte CA-I and CA-II. In conclusion, the drugs showed potent inhibitory effects on hCA I and hCA II in vitro. Our results underline the potential of cardiac drugs to target atherosclerosis through hCAI-II inhibition. Especially, in patients with atherosclerotic heart disease, cardiac drugs that inhibit hCAs may be preferred primarily due to the beneficial effects of CA inhibition.In this study, the